H O M E O S TAT IC C O N T R O L

O F B R A I N F U NC T I O N
H O M E O S TAT IC C O N T R OL
O F B R A I N F U NC T IO N

EDITED BY

D E T L E V B O I S O N Ph D
Director of Basic and Translational Research
Legacy Research Institute
Portland, OR

SUSA NA.M ASINO

Professor, Neuroscience and Psychology
Trinity College
Hartford, CT

1
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Homeostatic control of brain function/edited by Detlev Boison and Susan Masino.
p.;cm.
Includes bibliographical references andindex.
ISBN 9780199322299 (alk.paper)
I. Boison, Detlev, editor. II. Masino, Susan, editor.
[DNLM:1.Brainphysiology. 2.Homeostasisphysiology. 3.Brain Chemistry. WL300]
QP376
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2015011860

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CONTENTS
Contributors vii
Prologue xi
Abbreviations xiii
PART I: Homeostatic Regulators: Molecules
andIons
1. Microdynamics of Water and Ion
Homeostasis in the Brain:Role
of Aquaporins and Ion Channels of
Astroglial Cells 3
Valentina Benfenati
and Stefano Ferroni
2. Homeostatic Control of Adenosine
Levels and Functions in the Brain 31
Xuesong Chen, Mahmoud L.Soliman,
Liang Hui, and Jonathan D.Geiger
3. Glutamate Homeostasis as a Regulator
of Neurotransmitter Recycling and
Synaptic Function 44
Mary Ellen Kelly
and Douglas a. Coulter
4. Homeostasis of Neuronal Excitability
Via Synaptic and Intrinsic Inhibitory
Mechanisms 51
Jochen Meier, Marcus Semtner,
and Jakob Wolfart
PART II: Homeostatic Control:Systems
andCells
5. Role of Astrocytes in Sleep and Epilepsy 75
Jerome Clasadonte
and Philip G.Haydon
6. AstrocyteNeuron Interactions 98
Angus M. Brown and Bruce R.Ransom
7. Homeostatic Synaptic Scaling
at Central Synapses 108
Niraj S.Desai and
Elisabeth C.Walcott
8. Homeostatic Role of Heterosynaptic
Plasticity 124
Nicholas M.Bannon,
Marina Chistiakova,
and Maxim Volgushev
9. The Blood-Brain Barrier 143
Yonatan Serlin, Alon Friedman,
and Uwe Heinemann
10. Inflammation and Immunomodulation
in Epilepsy and Its Comorbidities 155
Andrey Mazarati and
Annamaria Vezzani
11. Neuroplasticity 175
HiroyukiOkuno
12. Epigenetics 187
Katja Kobow and Ingmar Blmcke
13. Adult Neural Stem Cells and Brain
Homeostasis 202
Ashok K.Shetty
PART III: Homeostatic Manipulators:
Preventative and Restorative Opportunities
14. Systems (Network) Pharmacology
for Brain Functionality Restoration 231
Doru Georg Margineanu
15. Ketogenic Diets for Neurological
Disorders 248
Lindsey B.Gano, Manisha Patel,
and JongM.Rho
vi Contents
16. Dietary Manipulations 271
Athanasios Evangeliou and
Martha Spilioti
17. Exercise 298
MarkusDworak
18. Sleep 314
Kristina Simeone, Chaz Johnson,
Kaeli Samson, Harrison Roundtree,
Tim Simeone, and LeilaTarokh
19. Botanicals 333
Dana Ekstein and
Steven C.Schachter
20. The Role of Acupuncture in Nociception
Homeostasis 341
Nanna Goldman, Takahiro Takano,
Benjamin T.Kress, and
Maiken Nedergaard
21. Meditation 353
Galle Desbordes
22. Neurotrophic Regulation in
Neurorestoration of the Brain 371
Caixin Su, Michel Rathbone,
and ShucuiJiang
PART IV: Homeostatic Therapies
for Disease and Dysfunction
23. Epilepsy 401
DetlevBoison
24. Traumatic Brain Injury 420
Raj G.Kumar and Amy K.Wagner
25. Adenosine and Alzheimers
Disease:APossible Epigenetic Link 453
David Blum, Ursula S.Sandau,
Olivier Bousiges, Luisa V.Lopes,
Vanessa Flaten, Emilie Faivre,
Luc Bue, Anne-Laurence Boutillier,
and DetlevBoison
26. Brain Homeostasis and
Parkinsons Disease 481
DeeptiLall
27. Brain Homeostasis and Addiction 509
Letisha R.Wyatt
28. Anxiety and Stress Disorders 535
Eva Mara Marco and
Mara-Paz Viveros
29. Malignant Brain Cancer Management
with Metabolic Therapy 553
Thomas N.Seyfried and
Purna Mukherjee
30. Obesity and Diabetes:Nature,
Nurture, and Beyond 570
CharlesMobbs
31. Autism Spectrum Disorder
and Homeostasis 586
Susan A.Masino, Jessica A.Fortin,
Michelle I.Murphy, Lisa Saa,
and David N.Ruskin
Index 611
C O N T R I BU T O R S
Nicholas M.Bannon
Department of Psychology
University of Connecticut
Storrs,CT
Valentina Benfenati
Consiglio Nazionale delle Ricerche
Istituto per la Sintesi Organica e la
Fotoreattivit (ISOF)
Bologna, Italy
DavidBlum
Inserm
Universit deLille
Jean-Pierre Aubert Research Centre
Institut de Mdecine Prdictive
et de Recherche Thrapeutique
CHRU-Lille
Lille,France
Ingmar Blmcke
Department of Neuropathology
University Hospital Erlangen
Erlangen, Germany
Olivier Bousiges
Laboratoire de Neurosciences
Cognitives et Adaptatives(LNCA)
Universit de Strasbourg
Strasbourg,France
Anne-Laurence Boutillier
Laboratoire de Neurosciences
Cognitives et Adaptatives(LNCA)
Universit de Strasbourg
Strasbourg,France
Angus M.Brown
School of Life Sciences
University of Nottingham
Nottingham, England
LucBue
Inserm
Lille,France
Universit deLille
Jean-Pierre Aubert ResearchCentre
Institut de Mdecine Prdictive
et de Recherche Thrapeutique
CHRU-Lille
Lille,France
XuesongChen
Department of Basic Biomedical Sciences
University of North Dakota School
of Medicine and Health Sciences
Grand Forks,ND
Marina Chistiakova
Department of Psychology
University of Connecticut
Storrs,CT
Jerome Clasadonte
Department of Neuroscience
Tufts University School of Medicine
Boston,MA
Douglas A.Coulter
Departments of Pediatrics and
Neuroscience
Perleman School of Medicine
University of Pennsylvania and the
Division of Neurology
Childrens Hospital of Philadelphia
Philadelphia,PA
Niraj S.Desai
Center for Learning andMemory
The University of Texas atAustin
Austin,TX
viii Contributors

Galle Desbordes Lindsey B.Gano

Massachusetts General Hospital & Department of Pharmaceutical Sciences
Harvard MedicalSchool School of Pharmacy
Boston, MA University of Colorado
Denver,CO
MarkusDworak
Institute for Neuroscience Jonathan D.Geiger
German Sport University Cologne Department of Basic Biomedical Sciences
Cologne, Germany University of North Dakota School of Medicine
and Health Sciences
Dana Ekstein Grand Forks,ND
EpilepsyCenter
Department of Neurology Nanna Goldman
Hadassah-Hebrew University Medical Center Center for Translational Neuromedicine
Jerusalem,Israel University of Rochester MedicalCenter
Rochester,NY
Athanasios Evangeliou
Philip G.Haydon
Aristotle University of Thessaloniki
Department of Neuroscience
Department of PediatricsIV
Tufts University School of Medicine
Thessaloniki, Greece
Boston,MA
EmilieFaivre Uwe Heinemann
Universit deLille Institute of Neurophysiology and Neurocure
Jean-Pierre Aubert ResearchCentre ResearchCenter
Institut de Mdecine Prdictive et de Recherche Charit Universittsmedizin
Thrapeutique Berlin, Germany
CHRU-Lille
Lille,France LiangHui
Department of Basic Biomedical Sciences
Stefano Ferroni University of North Dakota School of Medicine
Department of Pharmacy and Biotechnology and Health Sciences
University of Bologna Grand Forks,ND
Bologna,Italy
ShucuiJiang
VanessaFlaten Department of Surgery (Neurosurgery,
Inserm Neurobiology)
Lille,France Hamilton NeuroRestorative Group(NRG)
Universit deLille McMaster University
Jean-Pierre Aubert ResearchCentre Health SciencesCentre
Institut de Mdecine Prdictive et de Recherche Hamilton, ON,Canada
Thrapeutique Chaz Johnson
CHRU-Lille Creighton University School of Medicine
Lille,France Department of Pharmacology
Jessica A.Fortin Omaha, NE
Neuroscience Program Mary EllenKelly
Trinity College Departments of Pediatrics and
Hartford,CT Neuroscience
Alon Friedman Perleman School of Medicine
Department of Physiology and Cell Biology University of Pennsylvania and the
Zlotowski Center for Neuroscience Division of Neurology
Ben-Gurion University of theNegev Childrens Hospital of Philadelphia
Beer-Sheva,Israel Philadelphia,PA
Department of Cognitive and Brain Sciences KatjaKobow
Zlotowski Center for Neuroscience Department of Neuropathology
Ben-Gurion University of theNegev University Hospital Erlangen
Beer-Sheva,Israel Erlangen, Germany
 contributors ix

BenjaminKress Maiken Nedergaard

Center for Translational Neuromedicine Center for Translational Neuromedicine
University of Rochester MedicalCenter University of Rochester MedicalCenter
Rochester,NY Rochester,NY
Raj G.Kumar HiroyukiOkuno
Physical Medicine and Rehabilitation Medical InnovationCenter
University of Pittsburgh Graduate School of Medicine
Pittsburgh, PA Kyoto University
DeeptiLall Kyoto,Japan
Board of Governors Regenerative Medicine ManishaPatel
Institute Department of Pharmaceutical Sciences
Cedars-Sinai MedicalCenter School of Pharmacy
Lost Angeles,CA University of Colorado
Luisa V.Lopes Denver,CO
Instituto de Medicina Molecular Bruce R.Ransom
Lisbon, Portugal Department of Neurology
Eva MaraMarco University of Washington
Departamento de Fisiologa (Fisiologa Seattle,WA
AnimalII) Michel Rathbone
Facultad de Biologa Department of Medicine (Neurology,
Universidad Complutense Neuroscience)
Ciudad Universitaria Hamilton NeuroRestorative Group(NRG)
Madrid,Spain McMaster University
Doru Georg Margineanu Health SciencesCentre
Department of Neurosciences Hamilton, ON,Canada
Medicine and Pharmacy JongM.Rho
University ofMons Departments of Pediatrics and Clinical
Mons, Belgium Neurosciences
Andrey Mazarati Alberta Childrens Hospital Research Institute
Department of Pediatrics for Child and MaternalHealth
Childrens Discovery and Innovation Institute University of Calgary
David Geffen School of Medicine Calgary, Alberta,Canada
University of California Harrison Roundtree
Los Angeles,CA Creighton University School of Medicine
JochenMeier Department of Pharmacology
Technical University Braunschweig Omaha, NE
Zoological Institute David N.Ruskin
Division Cell Physiology Neuroscience Program
Braunschweig, Germany Psychology Department
CharlesMobbs Trinity College
Neuroscience, Endocrinology, and Geriatrics Hartford,CT
Mount Sinai School of Medicine LisaSaa
NewYork,NY Neuroscience Program
Purna Mukherjee Trinity College
Biology Department Hartford,CT
Boston College KaeliSamson
Chestnut Hill,MA UrsulaSandau
Michelle I.Murphy Legacy Research Institute
Neuroscience Program Portland,OR
Trinity College
Hartford,CT
x Contributors

Steven C.Schachter TakahiroTakano

Marcus Semtner Center for Translational Neuromedicine
Max Delbrck Center for Molecular Medicine University of Rochester MedicalCenter
Berlin, Germany Rochester,NY
YonatanSerlin LeilaTarokh
Department of Physiology and Cell Biology Creighton University School of Medicine
Zlotowski Center for Neuroscience Department of Pharmacology
Ben-Gurion University of theNegev Omaha, NE
Beer-Sheva,Israel Annamaria Vezzani
Thomas N.Seyfried IRCCSIstituto di Ricerche Farmacologiche
Biology Department MarioNegri
Boston College Milano,Italy
Chestnut Hill,MA Mara-Paz Viveros
Ashok K.Shetty Departamento de Fisiologa (Fisiologa
Institute for Regenerative Medicine AnimalII)
Texas A&M Health Science Center College of Facultad de Biologa
Medicine at Scott andWhite Universidad Complutense
Temple,TX Ciudad Universitaria
Research Service Madrid,Spain
Olin E.Teague Veterans MedicalCenter Maxim Volgushev
Central Texas Veterans Health careSystem Department of Psychology
Temple,TX University of Connecticut
Department of Molecular and Cellular Medicine Storrs,CT
Texas A&M Health Science Center College of
Medicine Amy K.Wagner
College Station,TX Physical Medicine and Rehabilitation
University of Pittsburgh
Kristina Simeone Pittsburgh,PA
Creighton University School of Medicine
Department of Pharmacology Elisabeth C.Walcott
Omaha, NE Department of Psychological Sciences
University of SanDiego
Tim Simeone San Diego,CA
Creighton University School of Medicine
Department of Pharmacology Jakob Wolfart
Omaha, NE Oscar Langendorff Institute of Physiology
University of Rostock
Mahmoud L.Soliman Rostock, Mecklenburg-Vorpommern, Germany
Department of Basic Biomedical Sciences
University of North Dakota School of Medicine Letisha R.Wyatt
and Health Sciences R.S. Dow Neurobiology Laboratories
Grand Forks,ND Legacy Research Institute
Portland,OR
Martha Spilioti
Aristotle University of Thessaloniki
Department of PediatricsIV
Thessaloniki, Greece
CaixinSu
Department of Surgery (Neurosurgery,
Neurobiology)
Hamilton NeuroRestorative Group(NRG)
McMaster University
Health SciencesCentre
Hamilton, ON,Canada
PROLOGUE
The brain is a complex and metabolically-
demanding organ. The loss of homeostatic con-
trol of brain function is expressed as a diverse
array of neurological disorders. When current
therapies are inadequate or ineffective, restor-
ing or maintaining homeostatic functions can
improve brain health, regardless of the disorder.
There is also evidence that targeting homeostatic
regulatory systems may delay or avert dysfunc-
tion. This book offers a broad overview of brain
health and treatments for brain disease via home-
ostatic control systems such as mitochondria, the
immune system, and epigenetic changes as well
as via regulatory molecules such as ions, neuro-
peptides, and neuromodulators. We also high-
light emerging research on non-pharmaceutical
approaches such as botanicals, meditation, diet
and exerciseall of which have been shown to
impact brain function. Altogether we provide
high-quality science from molecules to disor-
ders, alongside emerging practical informa-
tion for improving homeostasis. This book will
convey the main message that the health of our
brains significantly depends on the ways we live
and what we eat or drink. In an age of an increas-
ing personal and social burden of brain diseases
such as Alzheimers or Parkinsons, the realiza-
tion of homeostatic mechanisms that could be
exploited to promote brain health is an emerging
and timely topic of broad significance.
The volume targets scientists, clinicians and
researchers at all levels as well as undergraduate,
graduate and medical students, and a sophisti-
cated public audience. The volume covers a very
broad topic which applies to a wide variety of
disciplines.
Understanding and promoting homeostatic
regulation of brain health is an urgent and
emerging topic. At this time, highlighting this in
a specific volume which includes basic research
and clinical content will help to coalesce this
concept for therapeutic potential and incorpo-
rate new ideas into existing paradigms. With this
concept in mind, the volume is unique in its col-
lection of chapters and sets the book apart from
otherworks.
A B B R E V I AT I O N S
5-HT 5-hydroxytryptamine (serotonin)
5-HTT serotonin transporter
ACh acetylcholine
AD Alzheimers disease
ADK adenosinekinase
AMPA alpha-amino-3-hydroxy-5-
methyl-4-isoxazolepropionicacid
AQP4 aquaporin-4
BBB blood-brain barrier
BDNF brain-derived neurotrophicfactor
CNS central nervoussystem
COX-2 cyclooxygenase-2
CPA conditioned place avoidance
CPP conditioned place preference
CPu caudate-putamen
CTA conditioned taste aversion
DA dopamine
DAergic dopaminergic
DAT dopamine transporter
DHEAS dehydroepiandrosterone sulfate
DR dopamine receptors
EAAT excitatory amino acid transporters
EE environmental enrichment
EGF epidermal growthfactor
EIF4G1 eukaryotic translation initiation
factor 4 gamma1
ERK extracellular signal-regulated
proteinkinase
FBXO7 F-box only protein7gene
FDA Food and Drug Administration
GABA gamma aminobutyricacid
GBA glucocerebrosidase
GDNF glial cell line-derived
neurotrophicfactor
GFAP glial fibrillary acidic protein
GLT-1 glutamate transporter-1
GPi globus pallidus
HPA hypothalamic-pituitary-adrenalaxis
ICSS intracranial self-stimulation
IFN interferon
iGluRs ionotropic glutamate receptors
IL interleukin
iNOS nitric oxide synthase
LB Lewybody
L-DOPA L-3,4-dihydroxyphenylalanine
LID levodopa-induced dyskinesia
LRRK2 leucine-rich repeat kinase2
LSD lysergic acid diethylamide
LTP long-term potentiation
MAO-B
inhibitors monoamine oxidase inhibitors
MDMA 3,4-methylenedioxy
methamphetamine
mGluR metabotropic glutamate receptor
MPTP 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine
MR muscarinic receptor
NAC N-acetylcysteine
NAc nucleus accumbens
nAch neuronal acetylcholine
nAChR nicotinic receptors
NE norepinephrine
NF-kB nuclear factor
kappa-light-chain-enhancer of
activated Bcells
NGF nerve growthfactor
NIDA National Institute on DrugAbuse
NMDA N-methyl-D-asparticacid
NTF neurotrophicfactor
OHDA 6-hydroxydopamine
PD Parkinsons disease
PF11 psuedogensenoside-F11
PFC prefrontalcortex
PI-3K phosphatidylinositol-3-kinase
PINK1 phosphatase and tensin homolog
(PTEN)-induced putative kinase1
PLA2G6 phospholipase A2, groupVI
PLC phospholipaseC
xiv Abbreviations
PPN pedunculopontine nucleus
PTZ pentylenetetrazol
ROS reactive oxygen species
SE status epilepticus
SN substantianigra
SNCA alpha-synuclein
SNpc substantia nigra pars compacta
SPNs spiny projection neurons
SSRI selective serotonin reuptake inhibitor
Tfam mitochondrial transcription factorA
TGF transforming growth factor-alpha
THC tetrahydrocannabinol
TLR Toll-like receptor
TNF tumor necrosisfactor
TNF- tumor necrosis factor-alpha
UPS ubiquitin-proteasomesystem
VPS35 vacuolar protein sorting35
VTA ventral tegmentalarea
PARTI

Homeostatic Regulators

Molecules andIons
1
Microdynamics ofWater and Ion
Homeostasis intheBrain
Role of Aquaporins and Ion Channels of AstroglialCells
VA L E N T I N A B E N F E N AT I A N D S T E FA N O F E R R O N I
ASTROCYTES AND
B R A I N H O M E O S TA S I S
Maintaining constancy in the internal medium
is also essential for the proper functioning of the
central nervous system (CNS), whose activi-
ties rely on the unequal distribution of solutes
(organic molecules, ions, and neurotransmitters)
and water between the intra- and extracellular
compartments (Simard and Nedergaard, 2004).
The brain is constrained by the rigidity of the
skull, which can lead to a deleterious increase in
intracranial pressure when brain swelling, initi-
ated by a pathological redistribution of water and
electrolytes in the brain parenckyma, occurs. The
mechanisms underlying the ion and water trans-
port across plasma-membrane of the cells that
face and delimit fluid-filled compartments of
the brain have a central role in brain physiology
and pathophysiology and are becoming attrac-
tive pharmacological targets to counteract the
neuronal damage caused by the dysregulation
of brain homeostatic processes (Kimelberg and
Nedergaard,2010).
Three physical barriers delimit and regulate
the exchange between the microvasculature and
the fluid compartments of the brain. Two of these
are epithelia that separate the blood from the ven-
tricular cerebrospinal fluid (the choroid plexus),
and from the subarachnoid cerebrospinal fluid
(arachnoid epithelium). The third one (the brain
blood barrier, BBB) is formed by the endothelial
cells lining brain microvessels and demarcates
the boundary between the blood and the intersti-
tial fluid of the cerebral tissue. The BBB enables
a selective, dynamic bidirectional communica-
tion between the brain microenvironment and
the blood, thereby tightly controlling the water
and osmolyte concentrations in the interstitial
space (Abbott et al., 2006). The processes that
we define here as microdynamics, are mediated
by the so-called neurovascular unit, a modular
dynamic ensemble formed by neurons, astroglial
cells and vascular endothelia of the BBB. It is now
well known that the neurovascular unit is essen-
tial for CNS physiology by providing and regu-
lating local blood supply and integrating CNS
function (Iadecola and Nedergaard, 2007; Filosa
and Iddings, 2013). In this scenario, astrocytes
are central players as several pieces of in vivo
and in vitro evidence have established their piv-
otal function in homeostatic regulation of water
and ion concentrations, as well as in the control
of neurotransmitter dynamics in the interstitial
fluid (Anderson and Swanson, 2000; Kofuji and
Newman, 2004; Simard and Nedergaard,2004).
The term astrocyte (astroglial cell) was first
proposed by Michael von Lenhossek describing
cells with cylindrical processes departing from a
defined round cell body, presenting a star-like
morphological phenotype (Somjen, 1988). The
complexity and heterogeneity of astrocytes were
then defined by their location in the white versus
grey matter, classifying them into fibrous and
protoplasmatic astroglial subtypes (Freeman,
2010). Fibrous astrocytes are abundant in the
white matter. They display numerous filaments
that stain with the glial fibrillary acidic protein,
which is the typical glial marker. Protoplasmic
astrocytes populate the grey matter and have
more irregular processes and few glial fila-
ments (Nedergaard et al., 2003). They contact
and cover synapses by extending thousands of
thin processes, with typically only one or two
in contact with blood vessels or CNS bounda-
ries (Bushong, 2002; Filosa and Iddings, 2013).
Two types of specialized astroglial cells are also
4 Part I: Homeostatic Regulators
recognized in the CNS: Bergmann glia and
Mller cells that are present primarily in the cer-
ebellum and the retina, respectively. Bergmann
glia cell bodies lie within the Purkinje cell layer,
and their processes extend into the granule cell
layer to end at the pial surface (Hoogland and
Kuhn, 2010). Retinal Mller cells are oriented
radially, spanning from the photoreceptor layer
to the inner retinal surface (Reichenbach and
Bringmann, 2013).
Over recent years, glioanatomy studies based
on electron microscopy, confocal imaging, and
3D computational reconstruction, as well as
two photon imaging analyses on enhanced
green-fluorescent protein-expressing astrocytes
(Bushong et al., 2002; Nedergaard et al., 2003;
Mathiisen et al., 2010), have provided indica-
tion against the view of a random distribution
of astrocytes throughout the adult CNS. It is
remarkable that the morphology of astrocytes
resembles more a polyhedron rather than a
star-like shape, with major processes that ramify
into thinner ones and are uniformly distributed
in leaflet-like appendages through which each
astrocyte, filling a specific and limited volume
and space, defines its own anatomical (functional)
domain. Each protoplasmic astrocyte endfoot
occupies its own district and covers all sur-
rounding neuronal elements, parcelling the grey
matter into specific subdomains (Bushong etal.,
2002). Therefore, this highly and orderly organ-
ized astrocyte cytoarchitecture (Figure 1.1A)
defines the precise modular geometry of the
entire neuropil of the grey matter (Nedergaard
etal., 2003; Kettenmann and Verkhratsky, 2008).
This picture is even more relevant considering
that a loss of such a compartmentalized organi-
zation is now believed to be involved in a variety
of brain pathologies (Seifert etal., 2006; Chvtal
et al., 2008; Takano et al., 2009; Steinhauser
et al., 2012). Astroglial microdomains are also
highly organized and compartmentalized at the
interface with the blood vessels, with each astro-
cyte endfoot enveloping the endothelial cells in a
specific district, thus providing a contiguous but
nonoverlapping sheath around the capillary bor-
dering (Simard etal.,2003).
The evidence on astroglial cytoarchitecture
and distribution have supported for years the
plausible hypothesis of a nutritional and trophic
role of astrocytes, as it was first proposed by
Golgi (Somjen, 1988). Over the past decades,
however, this view has radically changed as
numerous studies have clearly shown that astro-
cytes also serve as metabolic and homeostatic
regulators of the brain function. The homeo-
static control is performed by astrocytes in a
stratified and ordered manner with dynamics
involving cellular and molecular events (micro-
dynamics) with both processes occurring
throughout the life of the brain, from its neu-
rogenesis to development and aging (Molofsky
etal., 2012; Nagelhus etal., 2013). Astroglia are,
in fact, intimately involved in CNS formation
as both stem cell elements and regulators of
neurogenesis. During development, astrocytes
define the pathways regulating migration of
neural cells and control synaptogenesis/synap-
tic pruning, thereby shaping microarchitecture
of the grey matter (structural homeostasis)
(Nedergaard et al., 2003; Bernardinelli et al.,
2014). Astrocytes store glycogen and deliver
glucose and metabolic substrates such as lactate
(metabolic homeostasis) to neurons (Pellerin
etal., 2007; Stobart and Anderson, 2013). They
also control and maintain local ion and water
homeostasis, and provide clearance of cat-
abolites and neurotransmitters/neuromodu-
lators released during synaptic transmission,
which are sequestered through active uptake
(microdynamic homeostasis) (Simard and
Nedergaard, 2004; Benfenati and Ferroni,
2010). Moreover, astrocytes regulate systemic
homeostatic responses as they are also able to
perceive systemic fluctuations in CO2 and pH
and variations in sodium (Na+) and water con-
tents (osmolarity) and then elaborate the appro-
priate responses at molecular and organ levels
(Simard and Nedergaard, 2004; Kimelberg and
Nedergaard,2010).
To fully understand the homeostatic role of
astrocytes, one should refer to their compart-
mentalized and polyhedral morphology and
envision them as multifunctional units regu-
lating separated fluid compartments through
various functional microdomains located in
their process endfeet (Simard and Nedergaard,
2004; Benfenati and Ferroni, 2010). At the
neuronastrocyte interface the astroglial micro-
domain take ups and secretes ions, organic mol-
ecules, and water (Simard and Nedergaard, 2004;
Kimelberg and Nedergaard, 2010). To ensure ion
and water homeostasis of the interstitial fluid,
different processes occur at the endfeet facing the
vasculature and at those of astroglia contacting
the cells lining other fluid-filled compartments
(Amiry-Moghaddam and Ottersen, 2003; Kofuji
and Newman, 2004; Simard and Nedergaard,
2004). Aprerequisite for their homeostatic func-
tion is the abundant interastrocytic connection
 Microdynamics of Water and Ion Homeostasis in the Brain 5

(a) (b)

FIGURE 1.1: Structural and functional relationships of astroglia in the regulation of brain
homeostasis. (A) Spatial interaction between astrocytes and neurons in the rat cortex. Neurons are labeled
with antibody to microtubule-associated protein 2 (MAP-2), whereas astrocytes are the cells expressing enhanced
green-fluorescent protein (eGFP). eGFP-positive astrocytic endfeet are abutting blood vessel. (B) Hypothetical
diagram of the microdynamics involved in astrocyte-mediated extracellular homeostasis. Astrocyte morphology
is characterized by an irregularly shaped cell body from which depart many leaflet-like processes. The endfeet of
the processes contact the neuronal soma (arrow a) and cover most synapses (arrow b) defining nonoverlapping
districts. In a given astrocyte other endfeet make contact with the surface of the brain or abut the blood vessels,
thereby forming a perivascular sheet (arrow c). Astrocytes are coupled by gap junctions (arrow d) that allow
synchronization of distal cell responses in the astroglial syncytium. The astroglial architecture is functional to
the homeostatic role of astrocytes, which is crucial for the maintenance of physiological neuronal activity. As a
consequence of the propagation of an action potential (1), neurotransmitters and ions are released in the inter-
synaptic space and perineuronal milieu (2). Astrocytes, by forming the neuro-glial unit, recover and maintain
the extracellular potassium (K+) and glutamate (Glu-) concentrations at optimal levels to ensure proper function
of the neuronal network (3):Astroglial Na+-coupled Glu- transporter (GLT-1) is responsible (in conjunction with
GLAST transporter) for Glu- uptake. Among the large variety of K+ channels expressed by astrocytes, the inwardly
rectifying K+ channel (Kir4.1) is crucially involved in local K+ uptake from the extracellular space. (4)Excess of
intracellular K+ is redistributed toward the vascular system, and gap junctions shunt K+ from perineuronal space
to distal vascular districts. (5)Finally, K+ is secreted at gliovascular interface mainly via Kir4.1, even though out-
wardly rectifying K+ channels (Kv) and calcium (Ca 2+)-activated K+ channel (BKCa) could also play a role. During
the spatial buffering of K+, the transient osmotic gradients created are rapidly counteracted by water flux. An
exclusive role for osmotic water movement is mediated by AQP4, which is largely distributed at astroglial endfeet
surrounding blood vessels and contacting the pial surface. The transport of chloride (Cl) ions is also critical for
the regulation of astroglial functions. The Na+-K+-2Cl- cotransporter and the Cl-/HCO3 anion exchanger (not
shown in the scheme) play a major role for pumping Cl into astrocyte. The role of Cl channels of the ClC fam-
ily that have been described in astrocytes is still unclear. Volume-regulated anion channels (VRACs) have been
identified in vitro and might contribute to cell volume homeostasis. However, whether they are also expressed and
operative in vivo is still unclear. The expression of the Ca 2+ permeable channel TRPV4 has been reported both in
situ and in vitro. For clarity, pumps and transporters that contribute to the transmembrane movement of ions have
not been included in the scheme.
Panel Amodified from Nedergaard etal. [2003], panel B modified from Benfenati and Ferroni [2010].

and coupling that allow astrocytes to act as a turn, the efficacy of the homeostatic processes
functional syncytium (Nagy and Rash, 2000). (Scemes and Giaume,2006).
This enables rapid and coordinated communi- It is widely accepted that astrocytes regu-
cation within the astroglial tissue increasing, in late the extracellular fluid homeostasis by using
6 Part I: Homeostatic Regulators
a variety of transmembrane proteins that drive
passive and active transport of ions, organic
osmolytes, and osmotically obliged water
(Pasantes-Morales and Cruz-Rangel, 2010).
Envisaging the astrocyte as a multifunctional
unit regulating separated fluid compartments,
it is not surprising that ion and water channels
display a polarized distribution (Figure 1.1B)
and that, in order to mediate specific functional
processes, they co-localize in cellular domains
(Benfenati and Ferroni, 2010). In the follow-
ing sections, microdynamics regulating ion and
water homeostasis are described, addressing the
specific ion and water channels involved and
the interplay of microdomains engaged in these
homeostatic processes.
MICRODY NAMICS
R E G U L AT I N G
EXTR ACELLULAR
P O TA S S I U M
H O M E O S TA S I S
A fundamental homeostatic function of astro-
cytes is the maintenance of the low extracellular
concentration of potassium ion ([K+]o). In neu-
rons the relative concentrations of K+ and Na+
inside and outside the cell define the resulting
chemical gradients across the plasma membrane,
which are crucial to the genesis and propagation
of the action potentials. Since information pro-
cessing through action potential firing is medi-
ated by Na+ influx and K+ efflux through the cell
membrane, it follows that after neuronal activ-
ity, increments in [K+]o do occur and should be
considered significant, if correlated to the lim-
ited volume of the extracellular space in the
CNS (Nicholson and Sykov, 1998). If a rise in
[K+]o remained unbalanced, the neuronal rest-
ing membrane potential would be altered and
the activity of plasma membrane proteins form-
ing channels, receptors, and transporters would
be affected with consequent modifications of a
variety of neuronal processes, including synap-
tic transmission and electrogenic transport of
neurotransmitters, which would lead to a com-
promission of the functionality and integrity of
the CNS (Seifert etal., 2006; Takano etal., 2009;
Steinhauser etal., 2012). In the brain the [K+]o is
tightly controlled and regulated to be kept close
to the physiological value of 3 mM. However, in
vivo studies have demonstrated that under physi-
ological conditions characterized by high neu-
ronal activity, [K+]o can transiently augment to
ceiling levels of 1012 mM (Kofuji and Newman,
2004)and reach values as high as 60 mM during
pathological conditions such as spreading depres-
sion (Somjen, 2001). The principle mechanisms
responsible for balancing [K+]o during neuronal
activity are called K+ uptake and spatial K+ buff-
ering (Figure 1.2) (Odette and Newman,1988).
In the K+ uptake mechanism, the excess of
[K ]o is cleared by the coordinated action
+
of Na+-K+-2Cl cotransporter (NKCC) and
Na+/K+-ATPase pump (Walz and Hertz, 1982;
Walz and Hinks, 1985). Whereas both are likely to
contribute significantly to K+ uptake under physi-
ological [K+]o variations, when [K+]o increases
above ceiling levels, as it occurs in ischemia and
trauma, the NKCC would become predominant
(Walz and Hertz, 1984). In this context, it was
shown that the NKCC inhibitor bumetanide was
effective in limiting edema in ischemic condi-
tions in vivo (Yan etal., 2003), a result that was
also observed in NKCC1 knock-out (KO) mice
(Chen et al., 2005). Simulated ischemic condi-
tions in vitro also caused astrocyte swelling,
which was blocked by bumetanide (Su et al.,
2002). Moreover, in vitro as well in vivo studies
on rat optical nerve indicated that the rise in [K+]o
induced astrocyte swelling, which was reversibly
depressed by furosemide that also inhibits NKCC
(MacVicar et al., 2002). In contrast, available
information suggests that Na+/K+-ATPase activ-
ity could play a prominent role in the clearance
of the stimulus-evoked increment in [K+]o when
its level is 1012 mM, as determined by the obser-
vation that under conditions of physiological
neuronal activity pharmacological inhibition of
Na+/K+-ATPase caused a strong delay in K+
clearance (Ransom et al., 2000; DAmbrosio
etal.,2002).
Another mechanism that appears to play a
relevant role in [K+]o homeostasis under physi-
ological conditions is the so-called spatial K+
buffering. This is a mechanism involved in
local [K+]o uptake and K+ dissipation through
the astroglial tissue, which is regulated by the
K+ equilibrium potential and the gradient in
syncytial membrane potential (Kofuji and
Newman, 2004). The first evidence supporting
the K+ spatial buffering was provided by Orkand
etal. (1966), demonstrating that stimulation of
the amphibian optic nerve led to slow depolari-
zation and repolarization of the glial cells sur-
rounding the nonmyelinated axons. According
to the Orkand model (Orkand, 1986), at the
neuronal site, where [K+]o is locally increased
by neuronal activity, the resting membrane
potential of astrocytes is negative with respect
to the Nernst equilibrium potential for K+.
 Microdynamics of Water and Ion Homeostasis in the Brain 7

1 EK = Vm = 90 mV; [K+]o = 3 mM

2 3
[K+]i = 108 mM Spatial
Net
Uptake Buffer

[K+]o = 12 mM [K+]o = 4 mM
EK = Vm = 56 mV; [K+] o = 12 mM Na+
K+

K+
K+ [K+]i = 110 mM [K+]i = 108 mM
K+
Na+ K+
K+ Cl Na+
50 50 EK
EK = Vm = 56 mV K+ in
mV 70 mV 70 Vm K+ out

90 90
Distance Distance

FIGURE 1.2: Mechanisms of extracellular potassium homeostasis by the astroglial syncytium.

(1) Under conditions in which [K+]o is 3 mM the astroglial syncytium has a resting membrane potential (Vm)
close to the Nernst equilibrium potential for K+ (EK -90 mV). (2)K+ uptake mechanism:when [K+]o is increased
up to 12 mM as a consequence of neuronal activity, astrocytes accumulate [K+]o prevalently by the activity of a
Na+/K+ATPase and, to a lesser extent, by the Na+-K+-2Cl- cotransporter. Under these conditions the membrane
potential in the astroglial syncytium is estimated to be spatially uniform at a value close to that of EK of 56 mV.
(3)K+ spatial buffering mechanism:local increases of [K+]o produce a glial depolarization that spreads through the
glial syncytium. The local difference between the astrocyte Vm and EK causes the K+ inflow in regions of elevated
[K+]o and K+ outward efflux at distant regions. The result is a net flux of K+ away from the region where it is accu-
mulated extracellularly.The graphs show the distribution of EK and Vm as a function of distance along the astroglial
syncytium for the two mechanisms.
From Orkand,1986.

This difference drives the inflow of K+ into astro-
cytes compartments exposed to the transient
[K+]o elevation, causing a local astrocytes depo-
larization that propagates through electrically
coupled astrocytes and allowing the movement
of K+ toward regions and districts where [K+]o
is lower. In certain CNS regions like the retina,
K+ currents flow within single Mller glial cells
rather than through a cell network by a mecha-
nism called K+ siphoning (Newman etal., 1984).
To satisfy the Orkand model, two conditions are
essential: (a) astroglial cells should be highly
permeable to K+; and (b) astrocytes should be
electrically coupled to form a syncytium in
which K+ currents can traverse relatively long
distances. Moreover, as recently pointed out, the
overall mechanism implies osmotically driven
water movement across the plasma membrane
(Nagelhus et al., 2004). Several lines of in vivo
evidence in the past decades have indicated
that spatial buffering and siphoning depend on
proper distribution and function of astrocytic
K+ channels, gap junctions, and their interplay
with the water channels aquaporins (AQPs)
(Benfenati and Ferroni,2010).
Potassium Channels Involved
inAstrocyte-Mediated
Homeostasis ofExtracellular
Potassium
The use of tools and technologies in the field
of molecular biology (cloning, expression, and
mutagenesis), genetics (full and conditional
KO mice with cell-specific gene deletion), elec-
trophysiology (path-clamp), and microscopy
(immunogold electron microscopy, laser scan-
ning confocal microscopy) have enabled the
acquisition of functional and molecular insights
into the channel proteins that play a role in astro-
glial K+ homeostasis through the mechanism of
spatial buffering. Astrocytes are highly permeable
to K+ owing to a variety of K+ channels expressed
in their plasma membrane (Verkhratsky
and Steinhauser, 2000). Inward rectifying
8 Part I: Homeostatic Regulators
K+ channels (Kir) underpin the dominant astro-
glial K+ conductance and they are the only K+
channels to date for which clear physiological and
pathological roles have been established (Horio,
2001; Olsen and Sontheimer, 2008). After being
cloned and studied in a heterologous expression
system for the first time (Kubo etal., 1993), over
20 genes are currently known to encode various
Kir channel subunits (Hibino etal., 2010). Among
the various subfamilies cloned at least five of
these (Kir 2.1, 2.2, 2.3, 4.1, 5.1) have been iden-
tified in astrocytes (Higashi etal., 2001; Hibino
etal., 2004). Apeculiar biophysical feature of Kir
channels is their rectification property, which
implies that at any voltage applied, K+ inward flow
is greater than outward flux caused by an oppo-
site driving force (Nichols and Lopatin, 1997).
This behavior depends on channel blockade by
cytoplasmic magnesium (Mg2+) and polyamines,
which limits K+ outflow (Matsuda, 1991; Lopatin
et al., 1994). Another typical feature of the Kir
channels is their dependence of gating on [K+]o,
which results in an increase in membrane K+ per-
meability upon augmentation of [K+]o (Nichols
and Lopatin, 1997). Astroglial Kir channels are
characterized by a high sensitivity to extracellu-
lar exposure to submillimolar barium (Ba2+) and
caesium (Cs+) (Ransom and Sontheimer,1995).
Numerous in vitro and in situ molecular and
functional evidence have shown that Kir4.1 under-
lies the majority of the astrocytic Kir conductance
(Li et al., 2001; Olsen et al., 2006; Butt and Kalsi,
2006). The Kir4.1 drives bidirectional movement
of K+ depending on the transmembrane K+ gradi-
ent because it is weakly rectifying. This property is
essential for the K+ buffering and siphoning occur-
ring in specific astroglial districts (Kofuji and
Newman, 2004; Butt and Kalsi, 2006). The contri-
bution of Kir to the astrocytic K+ conductance has
been addressed in in vitro and in situ biophysical
and pharmacological analyses, showing that a cur-
rent inhibited by relatively low concentrations of
Ba2+ was expressed in cultured astrocytes and in
Mller cells (Barres etal., 1990; Ferroni etal., 1995;
Ransom and Sontheimer, 1995; Olsen etal., 2006;
Benfenati etal., 2006). Molecular analyses studies
have shown that whereas Kir2.1 is expressed mainly
in neurons, the Kir4.1 subtype is depicted almost
exclusively in different subtypes of macroglial cells
(Butt and Kalsi, 2006; Olsen and Sontheimer, 2008),
reinforcing the hypothesis of its primary role in
homeostatic astroglial function. Notably, in mouse
neocortex Kir4.1 was reported to form a heterote-
tramer with Kir5.1 whereas homomeric Kir4.1 was
expressed by hippocampal astroglia (Hibino etal.,
2004). In Mller cells, expression of Kir2.1 was
shown to accompany that of Kir4.1 (Kofuji et al.,
2002). The functional relevance of this differential
channel compositions is unknown.
The pivotal role of Kir4.1 in spatial buffer-
ing has been confirmed in experiments per-
formed in mice lacking the gene encoding for
Kir4.1 (KCNJ10). Genetic inactivation or condi-
tional deletion of Kir4.1 was shown to produce
a strong depolarization of the resting mem-
brane potential of Mller cells (Kofuji et al.,
2000)and hippocampal astrocytes (Djukic etal.,
2007; Chever et al., 2010). In Mller cells this
effect was accompanied by a 10-fold increase in
the membrane input resistance and an altered
response to light-evoked electro-retinogram,
which is dependent on K+ influx (Kofuji et al.,
2000). In hippocampal astrocytes the lack of
Kir4.1 impaired the K+ and glutamate uptake
and increased short-term synaptic potentiation
(Djukic et al., 2007). The role of Kir4.1 in [K+]o
and glutamate homeostasis by astrocytes was
also investigated in an in vitro study showing
that the knockdown of Kir4.1 protein by RNA
interfering caused a decrease in Kir4.1 expres-
sion that was paralleled by a diminution in glu-
tamate uptake capability (Kucheryavykh et al.,
2007). Kir4.1 deletion was also shown to alter the
[K+]o homeostasis in the respiratory network but
did not affect the network activity (Neusch etal.,
2006). Mutations causing loss of function of the
KCNJ10 gene result in a patient phenotype with
multisymptomatic syndrome, called SeSAME
syndrome, characterized by epilepsy, sensorineu-
ral deafness, ataxia, and electrolyte imbalance
(Bockenhauer etal., 2009; Scholl etal.,2009).
A major issue in the evaluation of the Kir4.1
role in astrocytes in vitro is that its molecular
and functional expression is rare without phar-
macological manipulation (Ferroni et al., 1995;
Guadagno and Moukhles, 2004; Benfenati etal.,
2006; Noell etal., 2007). The setup of experimen-
tal models in which astrocytes in vitro resemble
more the functional and molecular phenotypes
in vivo remains challenging, and it is currently
the target of novel emerging tools and technolo-
gies (Benfenati etal., 2010; Benfenati etal., 2013;
Benfenati etal.,2014).
The process of spatial redistribution of K+ in
the astroglial syncytium mediated by Kir chan-
nels assumes that these channels are compart-
mentalized in specific astrocytic membrane
regions facing liquid compartments where
 Microdynamics of Water and Ion Homeostasis in the Brain
oscillations in [K+]o occur. Kir4.1 was shown
to be enriched at the endfeet of astrocytic pro-
cesses enwrapping synapses and facing blood
vessels and the pia mater (Higashi et al., 2001;
Hibino et al., 2004; Nagelhus et al., 2004) or
in perivascular processes of Mller glial cells
(Nagelhus et al., 1999; Kofuji et al., 2002). The
pattern of Kir4.1 expression suggests that the
microdynamics regulating [K+]o homeostasis
could be mediated by plasma-membrane pro-
teins arranged in a microdomain forming mul-
timolecular functional complexes. The analysis
of Kir4.1 indicates that its C-terminus harbors
a PDZ-binding motif for the interaction with
proteins containing PDZ domains (Connors
et al., 2004). Kir4.1 was found to colocalize
with syntrophins, a group of proteins belonging
to the dystrophin-associated protein complex
(DAPC), and to form a macromolecular assem-
bly with dystrophin (Dp71), -dystroglycan,
-dystroglycan and -syntrophin both in
Mller cells and astrocytes (Ishii et al., 1997;
Guadagno and Moukhles, 2004; Hibino et al.,
2004; Nol et al., 2005; Connors and Kofuji,
2006). Alpha-syntrophin was shown to be cru-
cial for DAPCKir4.1 interaction in brain cortex
and retina, depicting a critical role of scaffold-
ing proteins in [K+]o homeostasis (Connors etal.,
2004; Connors and Kofuji, 2006). Mice deficient
of the syntrophin gene display Kir4.1 dis-
tribution comparable to that of wild-type ani-
mals. However, the absence of the syntrophin
causes a delay in [K+]o clearance elicited by
sustained neuronal activity due to mislocaliza-
tion of the water channel aquaporin-4 (AQP4)
(Amiry-Moghaddam etal., 2003). Moreover, two
other anchoring proteins, PSD95 and SAP97,
were reported also to colocalize with Kir4.1 in
Mller cells, thereby providing indirect evidence
of their involvement in channel compartmental-
ization (Horio etal., 1997). The crucial role of the
patterned distribution of Kir4.1 in [K+]o homeo-
stasis was also confirmed by works showing
that its deletion caused an epileptic phenotype
(Haj-Yasein etal., 2011). Notably, in the sclerotic
hippocampus of patients with mesial temporal
lobe epilepsy, a loss of perivascular Kir4.1 expres-
sion was associated with loss of dystrophin and
-syntrophin but not with that of -dystroglycan
(Heuser et al., 2012). These data suggest that
the perturbation of the dystrophin-associated
protein complex, by altering Kir4.1 expression,
could modify [K+]o buffering and clearance, thus
contributing to the epileptiform activity. Other
9
studies have shown that Kir4.1 is developmen-
tally regulated as Kir4.1 transcript and protein
rise up during the first postnatal days, thereby
leading to a large increase in astrocyte inward
K+ current density underlying the passive cur-
rent pattern of hippocampal astrocytes (Seifert
etal.,2009).
It should be pointed out that, even though
Kir4.1 has a major role in [K+]o homeostasis,
other K+ channels are emerging as possible can-
didates contributing to this process. Of potential
relevance could be the members belonging to
the two-pore domain K+ (K 2P) channels family
(Patel and Honor, 2001). The TREK and TWIK
subfamilies have been recently characterized in
astrocytes in vitro (Gnatenco etal., 2002; Ferroni
etal., 2003)and in situ (Seifert etal., 2009; Zhou
etal., 2009; Benesova etal., 2012). These channels
are open in a large range of membrane poten-
tials, providing a substantial contribution to the
high astrocyte passive K+ conductance in situ
(Zhou et al., 2009). A strong molecular interac-
tion mediating TWIK-1/TREK-1 heterodimers
was shown to underlie astrocytic passive con-
ductance and cannabinoid-induced glutamate
release from astrocytes (Hwang et al., 2014).
Immunofluorescence studies showing that K 2P
channels are particularly enriched at the astro-
cytes endfeet support the view of their poten-
tial role in [K+]o homeostasis (Zhou etal., 2009;
Seifert et al., 2009). In this context, K 2P chan-
nels were suggested to be involved in K+ uptake
in the hippocampus (Psler et al., 2007). It also
remains to be defined whether outward K+ cur-
rents mediated by voltage-gated (Kv) and cal-
cium (Ca 2+)-activated K+ channels that have been
described in astrocytes both in vitro and in situ
have a role in [K+]o homeostasis (Tse etal., 1992;
Bordey and Sontheimer, 1999; Bekar etal., 2005;
Filosa etal., 2006). Interestingly, it was reported
that Ca2+-activated K+ channels (rSlo) and
the voltage-gated K+ channel Kv1.5 are highly
expressed at astroglial endfeet enwrapping
microvasculature which are also rich in AQP4
(Roy etal., 1996; Price etal., 2002). Another issue
that remains to be addressed in the context of
[K+]o homeostasis mediated by the spatial buff-
ering mechanism is the fact that the expression
of K+ channels is dynamically regulated, and
changes in K+ conductance have been reported
in various experimental paradigms of pathologi-
cal conditions in situ and in vitro (MacFarlane
and Sontheimer, 1997; Anderov et al., 2004;
Pivonkova etal.,2010).
10 Part I: Homeostatic Regulators
Role ofAquaporins
inAstroglial Potassium
Homeostasis
There is experimental evidence that in the ret-
ina, as well as in other regions of the CNS, the
increase in [K+]o caused by neuronal activity pro-
duces a diminution in extracellular space owing
to a transient increase in volume of glial endfeet
mediated by the high rate of fluid accumulation
accompanying K+ uptake (Holthoff and Witte,
2000; stby etal., 2009). Notably, AQP4, which
is the predominant water channel in astrocytes,
has been proposed as the molecular partner of
Kir4.1 in spatial buffering of K+ by facilitating
the osmotic water movement through the plasma
membrane (Nagelhus et al., 1999; Nagelhus
etal.,2004).
AQP4 is one of the members of the large
family of water channels called aquaporins,
which in mammals is composed of 13 members
(Papadopoulos and Verkman, 2013). The first
member of the family, called AQP1, was identi-
fied by Agre and coworkers who discovered that
the protein CHIP28 is a bona fide water chan-
nel when expressed in Xenopus oocytes (Preston
et al., 1992). The functional subunit of AQP is
a monomer composed of six transmembrane
domains in which two short helical segments
that surround cytoplasmic and extracellular ves-
tibules, connected by a narrow aqueous pore, can
be identified (Ho et al., 2009). The pore allows
the transport of only 1 ion for 109 water mol-
ecules (Pohl, 2004). The functional channel is a
tetrameric assembly, implying that four conduit
pathways are present in each AQP protein (Walz
etal., 1994; Cheng etal.,1997). and -syntrophin-mediated binding to DAPC
Expression pattern of AQPs in the mamma-
lian brain is characterized by a dominant pres-
ence of AQP4 and AQP1 (Amiry-Moghaddam
and Ottersen, 2003; Papadopoulos and Verkman,
2013). The expression of AQP9 has also been
reported (Badaut et al., 2004), but its localiza-
tion is still controversial (Amiry-Moghaddam
et al., 2005). In rodent AQP1 is expressed in
the epithelium of the choroid plexus facing the
cerebrospinal fluid, whereas AQP4 is primar-
ily expressed in the astrocytic processes of the
glia limitans facing the brain surface and in
the endfeet of perivascular astrocytes (Badaut
etal., 2002). AQP4 is also expressed in ependy-
mal cells of the glia limitans and in perivascular
processes of Mller cells (Hamann etal., 1998).
In the mouse hippocampus, AQP4 was shown
to be developmentally regulated and expressed
in astrocytes prominently in the CA1 stratum
lacunosum-moleculare and the molecular layer
of the dentate gyrus (Hsu et al., 2011). AQP4
can assemble in supramolecular assemblies
in the plasma membrane, called orthogonal
arrays of particles (OAPs). Immunogold stain-
ing by anti-AQP4 specific antibodies and analy-
sis by freeze-fracture electron microscopy have
allowed the earliest description of OAP visu-
alization in brain astrocytes (Rash etal., 1998),
a result confirmed by live imaging performed
by using super-resolution microscopy (Rossi
etal., 2012). While the physiological role of such
highly hierarchically organized supramolecular
structure is still unclear, a crucial role for AQP4
OAPs has been demonstrated in the pathophysi-
ology of neuromyelitis optica (NMO). NMO
patients are characterized by the presence of IgG
autoantibody (NMO-IgG), which binds to AQP4
(Jarius et al., 2008). This antibody recognizes
AQP4 when it is aggregated into OAP square
arrays (Nicchia etal., 2009). Aspecific nontoxic
inhibitor for AQP4 is still lacking, but experi-
ments performed on different KO mice models
have provided clear evidence on the crucial role
of AQP4 in brain water microdynamics, astro-
cyte migration, neurodevelopment, neuron exci-
tation, and synaptic plasticity (Papadopoulos
and Verkman,2013).
AQP4 has been shown to be associated
with DAPC by interacting with -syntrophin
(Amiry-Moghaddam et al., 2004), and in
vivo evidence using different pathophysi-
ological models supports the view that the
molecular interplay between AQP4-Kir4.1
is essential to K+ clearance in pathohysiologi-
cal conditions (Amiry-Moghaddham et al.,
2003). Data regarding the interactions of
AQP4 with Kir4.1 in the physiological con-
text are more controversial. Brain astrocytes
in which Kir4.1 was knocked down by siRNA
did not display alteration in water perme-
ability (Zhang and Verkman, 2008). Genetic
ablation of AQP4 did not alter Kir4.1 distribu-
tion and functional properties in Mller cells
(Ruiz-Ederra etal., 2007), but caused a slowed
dynamics in [K+]o homeostasis in rodent brain
(Binder et al., 2006; Strohschein et al., 2011).
Electrophysiological studies in hippocampal
slices from AQP4 KO mice evidenced a marked
alteration of stimulus-evoked K+ uptake due to
the impairment of the astroglial Na+/K+-ATPase
(Strohschein etal., 2011), which was also shown
 Microdynamics of Water and Ion Homeostasis in the Brain
to interact with AQP4 (Illarionova etal., 2010).
Dysregulated water and [K+]o homeostasis have
been linked to epilepsy (Binder and Steinhuser,
2006; Binder et al., 2012). A pronounced
decrease in AQP4 expression was observed
in the early phases of epilepsy in a model of
kainate-induced seizures (Lee etal., 2012)and
immunogold analysis showed that in human
epileptic tissue the impaired buffering of [K+]o
was associated with a loss of AQP4 in perivas-
cular astrocyte endfeet (Eid etal., 2005). Based
on these results, it could be envisaged that the
characterization of microdomains serving the
process of water homeostasis would be useful
to unravel the pathophysiology of unbalanced
[K+]o microdynamics.
Role ofConnexins and
Pannexins inAstroglial
Potassium Homeostasis
A prerequisite for the homeostatic function of
astroglia is the abundant interconnection and
coupling to work as a functional syncytium.
Gap junction protein subunits, called connex-
ins (Cxs), are the membrane channels distrib-
uted along the astrocytic processes that provide
unique direct conduit between astrocytes. Gap
junctions play a crucial role in enabling the
synchronization of cellular processes in astro-
cytes, such as intercellular Ca 2+ waves propa-
gation and metabolite trafficking and delivery
to neurons (Giaume et al., 1997) but also for
signaling at the gliovascular interface (Simard
et al., 2003). They mediate the passage of ions
(K+), amino acids, glucose, glutathione, ATP,
and small signaling molecules (Theis et al.,
2005). The main gap junction protein isoforms
expressed by astrocytes are Cx30 and Cx43
(Nagy and Rash, 2000; Theis etal., 2005). Cx43/
Cx30 double-KO mice have only minimal gap
junction communication between astrocytes
(Wallraff et al., 2006; Rouach et al., 2008),
suggesting that both proteins are the main
components of functional astroglial gap junc-
tion channels. There is evidence that astrocyte
Cxs could also form hemichannels that open
between the cell interior and the extracellular
space both in physiological and pathological
conditions, allowing exchange of small mol-
ecules between the cytoplasm and the extracel-
lular environment (Bennet etal.,2003).
Several findings indicate that gap junc-
tions play a major role in the coordination
and synchronization of astroglial signaling
11
underpinning [K+]o homeostasis (Scemes and
Spray, 2012). High [K+]o was shown to increase
coupling between cultured spinal cord astro-
cytes (Enkvist etal., 1994). The major evidence
of gap junction implication in [K+]o homeo-
stasis has been provided recently by use of
K+-selective microelectrodes in double-KO mice
for astroglial Cx30 and Cx43 (Wallraff et al.,
2006). It was shown that in the hippocampus,
gap junctional communication contributed to
the radial dissipation of [K+]o in the stratum
lacunosum-moleculare but not in hippocampal
astrocytes located in the stratum radiatum. The
mechanism underpinning the role of Cx and gap
junction communication in [K+]o homeostasis is
not clearly understood. It has been shown that
changes in phosphorylation state of Cx43 are
likely to be involved in cell-coupling increase
following a challenge with high [K+]o (De
Pina-Benabou et al., 2001; Scemes and Spray,
2012). Several pieces of in vivo evidence have
indicated that astroglial Cx expression could
be modified in many diseases and pathological
conditions characterized by alteration in [K+]o
homeostasis (Eugenin et al., 2012; Steinhauser
etal., 2012). However, mainly because of differ-
ences in animal models, types of experimen-
tal paradigm used, and parameters analyzed,
inconsistencies and contradictions about Cx
role in neuronal excitability have been reported.
Some studies have explored the effect of phar-
macological disruption of gap junction commu-
nication on seizure activity (Medina-Ceja et al.,
2008; Voss et al., 2009). Notably, most of these
researches investigated the effect of Cx modula-
tors such as carbenoxolone, which, however, is
now known to affect also other plasma mem-
brane ion channels at least in cultured astro-
cytes (Ye et al., 2009; Benfenati etal.,2009).
Concerning the mechanisms of [K+]o homeo-
stasis, it was reported that AQP4 knockdown
by siRNA induced the downregulation of Cx43
protein expression and junctional coupling in
primary cortical astrocytes, suggesting that a
functional interaction exists between AQP4 and
Cx43 in astrocytes in vitro (Nicchia etal., 2005).
However, recent evidence has revealed improved
K+ spatial buffering occurring in AQP4-deficient
mice that might result from the increase in gap
junction coupling (Strohschein et al., 2011).
Further studies will elucidate whether a molecu-
lar or functional interplay occurs between AQP4
and Cxs to serve the microdynamic regulation of
[K+]o homeostasis.
12 Part I: Homeostatic Regulators
Pannexin 1 (panx1) is a protein that func-
tions as a large-pore membrane channel per-
meable to relatively large molecules including
ATP (MacVicar and Thompson, 2010). In the
CNS it is expressed in neurons as well as in
astrocytes (Thompson and MacVicar, 2008).
In astrocytes-neuron cocultures it was shown
that high [K+]o activates panx1 channels by
voltage-independent mechanisms (Scemes and
Spray, 2012). In a mouse model of epilepsy, panx1
blockade was reported to reduce seizure activity,
suggesting that it may contribute to its develop-
ment (Santiago et al., 2011). However, the pre-
cise role of panx1 in [K+]o homeostasis remains
largely unknown.
MICRODY NAMICS
OFCELL VOLUME
H O M E O S TA S I S
Brain homeostasis is also characterized by the
ability of brain cells to maintain their volume
in response to physiological stimuli. This is
possible because they can constantly counter-
act the increase in cell volume induced by the
formation of transient and spatially-defined
osmotic gradients with a process called regula-
tory volume decrease (RVD). Since the osmotic
gradients are generated by the release and
uptake of osmolites at the neuron-astrocyte and
astrocyte-endothelium interfaces, it results that
astrocytes are central players in brain volume
homeostasis. It is also well known that under
several acute and chronic pathological condi-
tions larger and more sustained cell volume
increments are observed, which lead to dynamic
changes in the extracellular space volume (ESV).
The astrocyte volume increase promoted by the
redistribution of water and osmolytes between
intra- and extracellular compartments is called
cytotoxic edema (Liang etal., 2007), Cytotoxic
swelling affects ESV and geometry, by creating
diffusion barriers for signaling molecules that
contribute to neuron-glia communication and
hence can alter the excitability of the neuronal
tissue (Sykov and Nicholson, 2008). It devel-
ops as consequence of hyponatremia, ischemia,
and hepatic encephalopathy but also as result of
head trauma and epilepsy (Liang et al., 2007).
Accumulation of [K+]o, glutamate and ammo-
nia, as well as an augmentation of products of
oxidative stress, have been shown to regulate
the cellular processes controlling the cell vol-
ume homeostasis and to have a pathogenetic
role in the development of cytotoxic edema
(Kimelberg, 2005; Hussinger and Grg, 2010).
The cytotoxic swelling in isotonic conditions
causes the movement of ions and water through
brain capillaries, which leads to the formation of
ionic edema. If these dynamic changes develop
with the rupture of the BBB, fluid, proteins
and cells enter the brain parenchyma, promot-
ing the accumulation of fluid and the develop-
ment of vasogenic edema (Simard etal., 2007a).
Vasogenic edema occurs at late stages of various
neurological insults in which cytotoxic edema
are observed, such as ischemic insults, trau-
matic injuries, and brain tumors (Marmarou,
2007) but can also develop as a result of men-
ingitis (Nau and Brck, 2002). Transient vari-
ations in ESV also occur under physiological
conditions in response to high neuronal activ-
ity (MacVicar and Hochman, 1991; Holthoff
and Witte, 1996). Measurements of changes in
cellular volume obtained from optical and dif-
fusion properties in the brain suggest that they
were caused by alterations of astrocytic vol-
ume (Holthoff and Witte, 1996). Even though
it was reported that in situ transient swelling of
astrocytes promoted the outflow of glutamate,
which modulated neuronal excitability (Takano
etal., 2005), the functional consequences of this
physiological volume increase remain to be fully
elucidated.
Plasma Membrane Channels
Involved inCell Volume
Regulation ofAstroglia
In the past decade, significant efforts have been
made to uncover the molecular underpinnings
that contribute to cell volume regulation in
physiological conditions and whose dysfunction
underlies the development of cytotoxic edema. In
this context, plasma-membrane transporters, ion
channels, and water proteins have been reported
to be key effectors of physiological and patho-
physiological stimuli that promote astroglial
swelling (Pasantes-Morales and Vzquez-Jurez,
2012). The role of astrocytic transporters in cell
volume regulation is well known (Kahle et al.,
2009), and therefore we review here only the pos-
sible relevance of ion and water channels, some
of which have been characterized just recently
(Figure 1.3). The functional modulation of these
proteins, as well as alterations in their expression
and distribution in astrocyte microdomains,
have been linked to the development of cyto-
toxic edema and therefore could become inter-
esting therapeutic candidates to counteract the
cell swelling that occurs in various neurological
disorders.
 Microdynamics of Water and Ion Homeostasis in the Brain 13

H2O (AQP4) 5 Ca2+(TRPV4) (?)

Glu Na+ 1
3
H2O Na+ (?)

2
3 [Ca2+]
H2O 1 [osm]
4

1 K+ Cl, Tau, EAA

5 (VRAC)

H2O 4
K+

1) Rise in intracellular osmolyte concentration

2) Water influx and osmotic swelling
3) Volume sensing and osmotransduction
4) Inorganic and organic osmolyte efflux
5) Osmotic water efflux and volume recovery

FIGURE 1.3 Astroglial swelling and possible routes for cell volume recovery. Under physiological
conditions astrocyte volume remains stable owing to the osmotic equilibrium between intra- and extracellu-
lar compartments. However, cell volume homeostasis can be locally compromised by the transient increment of
intracellular osmolarity. As consequence of the neuronal activity K+, Na+, and Glu- uptake occurs at the endfeet
of astroglial cells (1). This increase in intracellular osmolytes is accompanied by osmotically driven water influx
through diffusion or by means of AQP4 (2). The mechanism underlying astroglial volume sensing, which initi-
ates the series of events leading to the process of cell volume recovery, called regulatory volume decrease (RVD),
remains to be determined unequivocally. Swelling-induced, intracellular Ca 2+ elevation ([Ca2+]i) might play a criti-
cal role at least in vitro (3). In addition to Ca 2+ release from intracellular stores, members of the transient receptor
potential (TRP) channel superfamily are plausible molecular candidates for osmotransduction. The osmosensitive
Ca 2+ channel TRPV4 is expressed in astrocytes in vitro and in situ and may be involved. The ensuing process of
RVD is based on the extrusion of intracellular solutes paralleled by obliged water efflux. The volume-regulated
anion channel (VRAC), by permeating inorganic (Cl) and organic osmolytes such as taurine and excitatory
amino acids could be an effector of RVD in vitro and perhaps also in vivo. Acontribution of K+ efflux is also likely,
but which are the specific volume-sensitive K+ channels involved is still unclear (4). The osmolyte outflow creates
the gradient for water efflux through diffusion and AQP4, and promotes RVD to recover the initial volume (5).
The diagram highlights the role of ion channels and does not consider the relevant role of various transporters.
Modified from Benfenati and Ferroni [2010]

Water Channels produced an augmentation of the rate of cyto-

It is now widely accepted that AQPs are involved
in pathological brain swelling and have been
postulated to be relevant pharmacological tar-
gets (Pasantes-Morales and Cruz-Rangel, 2010;
Badaut et al., 2011; Verkman, 2012). The role
of AQP4 in cytotoxic edema has been extrapo-
lated by studies in AQP4-KO mice subjected to
water intoxication and ischemic stroke (Manley
et al., 2000). In these mice, toxic hypotonic
challenge caused a reduced swelling of perivas-
cular endfeet and displayed a less severe neu-
rological phenotype and minor mortality. By
contrast, in mice in which AQP4 was overex-
pressed in astrocytes, acute water intoxication
toxic swelling, which was associated with an
increase in intracranial pressure (Yang et al.,
2008). Results confirming the role of AQP4 in
cytotoxic edema were obtained in dystrophin-
and -syntrophin KO mice, in which the dis-
ruption of the macromolecular complex with
AQP4 caused a mislocalization of astroglial
AQP4 and delayed the development of brain
edema (Vajda etal., 2002; Amiry-Moghaddam
et al., 2004). The mislocalization of AQP4 at
astrocytic endfeet has been proposed to be of
possible therapeutic value to counteract the
early formation of cytotoxic edema in ischemic
mice (Steiner etal.,2012).
14 Part I: Homeostatic Regulators
Variations in functional assembly of AQP4 in
target astrocytic regions could also be involved
in cell volume regulation by affecting the efficacy
of the process of [K+]o homeostasis. High [K+]o
was reported to induce astrocytic swelling in situ
(Anderov etal., 2001), and, in the human brain,
high [K+]o correlates with an increase in intrac-
ranial pressure in patients with severe traumatic
brain injury (Reinert et al., 2000). AQP4 is also
upregulated in pathological conditions associ-
ated with the development of cytotoxic edema
(Vizuete et al., 1999; Taniguchi et al., 2000;
Saadoun etal., 2002; Aoki etal., 2003; Aoki etal.,
2005), and reduction of AQP4 expression was
shown to be protective (Zeng etal., 2010; Fukuda
etal., 2013; Rama Rao etal.,2014).
It must be pointed out that the role of AQP4
in the regulation of brain volume homeostasis
seems completely different in vasogenic edema.
Since AQP4 permits the bidirectional movement
of water, in vasogenic edema it could facilitate
the removal of water from brain parenchyma.
To support this view it was shown that in
AQP4-deficent mice, infusion of an isotonic
solution augmented the intracranial pressure
owing to fluid accumulation (Papadopoulos
etal., 2004). The same result, accompanied by a
worsening of neurological scores, was obtained
upon genetic ablation of AQP4 in a model of
tumor-induced vasogenic edema (Papadopoulos
et al., 2004), upon induction of brain abscesses
(Bloch etal., 2005), and as a consequence of sub-
arachnoid hemorrhage (Tait et al., 2010). In the
context of brain volume homeostasis, the abil-
ity of AQP4 to remove the excess of water from
brain compartments could also influence the
development of hydrocephalus. Hydrocephalus
is the result of the imbalance between produc-
tion and absorption of cerebrospinal fluid that
leads to fluid accumulation in the ventricular sys-
tem and an increase in intracrananial pressure.
Obstructive hydrocephalus, which can be caused
by trauma, tumor, hemorrhage, and infection, is
determined by a diminution of the cerebrospinal
fluid draining into the subarachnoid space. In an
experimental mice model of progressive obstruc-
tive hydrocephalus, the deletion of AQP4 pro-
duced a strong increase in intracranial pressure
and an enlargement of lateral ventricles (Bloch
etal., 2006). This finding again supports the tenet
that the modulation of AQP4 expression and/or
function could be exploited for favoring water
removal from brain compartments in conditions
of fluid accumulation.
Potassium Channels
As already stated, a close correlation exists
between astrocytic volume regulation and the
ability of astrocyte to remove extracellularly
accumulated K+, thereby suggesting the presence
of closely coupled mechanisms. As described
in the previous section, Kir4.1 channels are the
predominant pathway of K+ uptake in astroglial
cells. The co-expression of Kir channels and
AQP4 in astrocyte endfeet suggests that K+ and
water could be cotransported through the syn-
cytium and contribute to the activity-dependent
volume changes of the extracellular space
(Nagelhus et al., 2004). Upregulation of both
proteins has been observed in human brain
astrocytes following several pathological states
(Saadoun etal., 2003). The K+ flux through Kir4.1
was reported to regulate swelling of astroglial
processes in experimental spinal cord edema
and in other regions of the CNS challenged
with an hypotonic stimulus (Dibaj et al., 2007;
Hirrlinger etal.,2008).
Recent studies in situ have shown that in slices
subjected to oxygen glucose deprivation, astro-
cytic swelling is modulated by putative inhibi-
tors of K2P channels, suggesting that they might
play a pivotal role in cell volume responses dur-
ing ischemia (Benesova etal., 2012). Some mem-
bers of the TREK subfamily of K 2P contribute to
the K+ conductance in hippocampal astrocytes in
situ and are particularly enriched at the process
endfeet (Zhou etal., 2009; Seifert etal., 2009). In
cultured astrocytes, two members, TREK-1 and
TREK-2, have been identified and functionally
characterized (Gnatenco etal., 2002; Ferroni etal.,
2003). Simulated ischemia in vitro was shown
to promote an increment in expression of both
channels (Kucheryavykh etal., 2009; Wang etal.,
2012; Wu etal., 2013). However, only studies per-
formed in KO animals will provide a clear answer
about their putative role in cell volume regulation.
In epithelial cells, Ca2+-activated K+ channels of
intermediate/small conductance and large con-
ductance have been associated with cell volume
regulation (Barfod et al., 2007). In astrocytes in
situ, large-conductance Ca2+-activated K+ chan-
nels of the rSLo subtype have been reported to be
regionally distributed in AQP4-expressing astro-
cyte endfeet that abut blood vessels (Price etal.,
2002). Although the activity of these K+ channels
has been linked to neuron-mediated regulation of
vascular tone (Filosa etal., 2006; Girouard etal.,
2010), a role in cell volume regulation, especially
at the endfeet, cannot be ruledout.
 Microdynamics of Water and Ion Homeostasis in the Brain
Chloride Channels
In virtually all mammalian cells volume control
is achieved through tightly regulated activation
of chloride (Cl) and cation channels and the con-
tribution of transport mechanisms (Hoffmann
et al., 2009). Whereas in cell swelling and the
ensuing RVD the role of different types of cat-
ion channels depends on their relative expres-
sion levels and on their gating process, Cl fluxes
through a specific volume-sensitive ion channel
can contribute to both cell swelling and volume
recovery owing to the dynamics of the electro-
chemical driving force. In neurons, prolonged
glutamate challenge was reported to generate
the formation of varicosities with the contri-
bution of Cl influx through volume-sensitive
anion channels. The same channels were also
involved in its extrusion, leading to volume
compensation upon glutamate washout (Inoue
and Okada, 2007). Astrocytes in vitro display a
very low background Cl permeability (Walz,
2002), and previous studies have shown that
Cl actively accumulates in astrocytes reaching
an equilibrium potential more positive than the
resting membrane potential (Kettenmann etal.,
1987; MacVicar et al., 1989). Cultured astro-
cytes respond to a hypotonic challenge with an
augmentation of Cl conductance promoted by
the activation of volume-regulated anion chan-
nels (VRACs) (Parkerson and Sontheimer 2004;
Benfenati et al., 2007a; Benfenati et al., 2009).
Asimilar current was also reported to be stimu-
lated by changes in astrocyte morphology and
biochemical alteration of the actin cytoskeleton
(Lascola et al., 1998). The molecular identity of
VRAC, or part of it, has recently been unveiled
(Qiu etal., 2014; Voss etal., 2014)but it remains
to be determined whether it also underlies
astrocyticVRAC.
VRAC stimulation in cultured astrocytes
has been associated with RVD (Parkerson and
Sontheimer, 2003). Interestingly, a downregula-
tion of AQP4 in cultured cortical astrocytes led
to a strong diminution in VRAC activity, adding
further support to its role in cell volume regula-
tion (Benfenati et al., 2007a). Pharmacological
analysis showed that a putative inhibitor of VRAC,
tamoxifen, was neuroprotective in a rat model of
focal ischemia (Kimelberg etal., 2003). The same
effect was reported with another specific VRAC
blocker, DCPIB, in a model of ischemia induced
by middle cerebral artery occlusion (Zhang etal.,
2008). However, neuroprotection was not attrib-
uted to cell volume modulation through VRAC
15
inhibition but to the diminution of excitotoxic
amino acids release through VRAC (Abdullaev
et al., 2006). Another Cl conductance, the
hyperpolarization-activated, inward rectifier Cl
current (IClh), has been described both in vitro
and in situ (Ferroni et al., 1995, Ferroni et al.,
1997, Makara etal., 2003). There is evidence that
IClh is mediated by ClC-2 (Makara et al., 2003),
which is a member of the growing family of the
Cl channels and transporters called ClC (Jentsch
etal., 2002). ClC-2 is ubiquitously expressed and
abundantly present in the brain (Thiemann etal.,
1992). In astrocytes in situ ClC-2 was found in the
CA1 and CA3 regions of the hippocampus and
in brain slices (Sk et al., 2000; Benesova et al.,
2012). A detailed analysis showed that ClC-2
is expressed at the endfeet of astrocytes facing
blood vessels (Blanz etal., 2007). The fact that IClh
favours the outflow of Cl at membrane poten-
tials negative to the equilibrium potential for Cl
makes this conductance an actractive candidate
as one of the RVD effectors. Notably, recombi-
nant ClC-2 activity was shown to be upregulated
by hypotonic challenge (Grnder etal., 1992)and
in various cell types it was reported to contrib-
ute to cell volume regulation (Furukawa et al.,
1998, Xiong et al., 1999). Deletion of ClC-2
causes white matter spongiform vacuolation in
mice brain and spinal cord, similar to that seen
in humans with leukoencephalopathies (Blanz
et al., 2007). Recent work has evidenced that in
patients with leukoencephalopathies character-
ized by white matter edema, ClC-2 is mutated
(Depienne et al., 2013). The IClh is highly sensi-
tive to mild extra- and intracellular acidification
(Ferroni etal., 2000), which are conditions asso-
ciated with the development of brain ischemia.
In cultured astrocytes there is no correlation
between ClC-2 expression and functional activ-
ity (Parkerson and Sontheimer, 2004), and IClh is
stimulated only upon astrocyte morphological
differentiation with a long-term treatment with
cAMP analogs (Ferroni etal., 1995). Even though
other isoforms of the ClC family have been found
in astrocytes, their relevance to cell volume regu-
lation is still questionable. Immunostaining anal-
ysis has shown that in cultured astrocytes other
members of the ClC family, namely ClC-3, ClC-5,
and ClC-7, were also expressed at the cell surface
but were not functionally identified (Parkerson
and Sontheimer, 2004). Moreover, astrocytic iso-
forms of the skeletal muscle ClC-1 channels have
also been described but not yet characterized at
a functional level (Zhang et al., 2004). Finally,
16 Part I: Homeostatic Regulators
bestrophin-1, a gene-encoding calcium-activated
Cl channel, was identified in astrocytes in vitro
and in situ (Park et al., 2009). Interestingly, the
fruit fly (Drosophila melanogaster) ortholog of
bestropin-1 was reported to be independently
activated by intracellular Ca2+ elevation and
extracellular hypotonicity, therefore suggesting
that it might play a role in cell volume homeo-
stasis (Chien and Hartzell, 2007). However, its
volume sensitivity in mammalian cells is still
controversial (Fischmeister and Hartzell, 2005),
and also the involvement of bestrophin-1 in the
regulation of astrocytic volume remains to be
addressed.
Cation Channels
It is now widely accepted that cationic channels
play an important role in volume regulation in
mammalian cells (Hoffmann et al., 2009). It is
also well known that cation entry in astrocytes,
by promoting osmotically driven water influx,
are critically involved in triggering cytotoxic
swelling (Liang et al., 2007). Na+ and Ca 2+ have
favorable electrochemical gradients for moving
into astrocytes when cation channels and tras-
porters are activated. Whereas the massive entry
of Na+ into the cells creates the osmotic gradient
that drives water influx, the cytosolic rise of the
second messenger Ca 2+ could influence cell vol-
ume through more complex biochemical path-
ways (OConnor and Kimelberg, 1993; Akita and
Okada,2011).
The presence of different subtypes of
voltage-gated Na+ channels has been reported
in cultured cortical and spinal cord astrocytes
(Sontheimer et al., 1991) as well as in slices of
different CNS regions (Bordey and Sontheimer
2000; Chvtal et al., 1995). The expression of
voltage-gated Na+ channels is heterogeneous in
terms of sensitivity to the specific blocker tetro-
dotoxin and biophysical properties (Sontheimer
et al., 1991; Thio and Sontheimer, 1993), and
it is also developmentally regulated (Kressin
et al., 1995; Bordey and Sontheimer, 1997).
Differences in expression level have also been
observed in gliotic astrocytes in situ and in vitro
(Jabs et al., 1997; MacFarlane and Sontheimer,
1998). The estimated densities and the biophysi-
cal properties of the channel subtype expressed
provides some clues to their functions that
are not related to the genesis of action poten-
tials (Thio and Sontheimer, 1993). It has been
reported that voltage-gated Na+ channels cre-
ate the route for the small, sustained Na+ influx
necessary to keep Na+/K+-ATPase operative
and therefore could influence [K+]o homeosta-
sis (Sontheimer etal., 1994), but their relevance
in cell volume regulation has not been demon-
strated. It must be emphasized that Na+ could
also permeate into astrocytes through nonse-
lective cation channels. One channel that has
recently attracted great interest is the NCCa-ATP,
which was identified in freshly dissociated adult
astrocytes (Chen and Simard, 2001). This chan-
nel is permeable to inorganic monovalent cati-
ons but not to Ca 2+ and Mg 2+. Importantly, its
opening is dependent on the presence of intra-
cellular nanomolar free Ca 2+ and the absence
of ATP, therefore suggesting that it could be
operative in conditions of metabolic failure.
The NCCa-ATP channel is made of a pore-forming
subunit and a regulatory subunit, the sulfonylu-
rea receptor 1 (SUR1), which is also the regula-
tory subunit of the K ATP channels in pancreatic
beta cells (Chen etal., 2003). The pore subunit is
formed by the transient receptor potential mel-
astin 4 (Woo etal., 2013). The NCCa-ATP activity
is downregulated by sulfonylurea compounds
such as tolbutamide and glibenclamide which
inhibit the SUR1-regulated K+ channel (K ATP).
Interestingly, this channel is not constitutively
expressed, but it becomes apparent following
hypoxia or injury (Chen and Simard, 2001),
causing astrocyte depolarization and the devel-
opment of cytotoxic edema. Its blockage in vivo
by glibenclamide promotes a strong reduction in
the formation of cerebral edema (Simard etal.,
2006). Other ion channels permeable to cations
that are likely to play a role in cell volume home-
ostasis include those belonging to the transient
receptor potential (TRP) superfamily (Montell,
2001). The first TRP channel was described in
the photoreceptor of Drosophila melanogaster
(Montell and Rubin, 1989). More than 20 TRP
genes have been identified in mammals (Nilius
and Owsianik, 2011). From protein homol-
ogy, members of the TRP channel family are
grouped into seven subfamilies (Wu etal., 2010).
Few members are selectively permeable to Ca 2+,
whereas most are non-selective cation channels.
Their activation mechanisms are very different,
and, interestingly, some of them are stimulated
by conditions which are relevant to ischemia
and lead to the development of brain edema
such as pH acidification, osmotic changes, and
membrane stretching (Simard et al., 2007b).
The expression and functional characterization
of the astrocytic TRP subtypes involved in cell
 Microdynamics of Water and Ion Homeostasis in the Brain
volume homeostasis is still incomplete. Cultured
astrocytes express mRNAs and proteins for
various subtypes of TRPC subfamily (C stands
for canonical) (Pizzo etal., 2001; Beskina etal.,
2007) which appear to be developmentally
regulated (Malarkey et al., 2008). TRPCs have
variable cation permeability ratios and have
been implicated in mediating store-operated
Ca 2+ entry in astroglial cells (Malarkey et al.,
2008; Akita and Okada, 2011). Of note, TRPC1,
which is permeable to both Ca 2+ and Na+, was
shown to be activated by mechanical stress
(Hua et al., 2004; Malarkey et al., 2008; Reyes
et al., 2013), a stimulus that triggers RVD in
astrocytes (Hua et al., 2010). A role of TRPC1
in cell volume regulation, therefore, cannot be
ruled out but deserves further investigation.
Additional TRP candidates that could play a
role in cell volume regulation are those of the
TRPV subfamily (V stands for vanilloid). This
subfamily is composed of 6 members which
are activated by various chemical and physi-
cal stimuli and display large Ca 2+ over mono-
valent cations permeability ratios (Owsianik
etal., 2006). TRPV4 was shown to be involved in
osmoregulation and hence could function as an
osmosensor (Mizuno etal., 2003; Liedtke etal.,
2003). Cortical and hippocampal rat astrocytes
in vitro and in situ express functional TRPV4
(Benfenati et al., 2007b; Bai and Lipski, 2010;
Butenko etal., 2012). High-resolution immuno-
gold analysis showed that TRPV4 expression in
situ was largely confined to astrocytic endfeet
enriched in AQP4 and abutting blood capillaries
and the pial surface (Benfenati etal., 2007b). It
is noteworthy that in cortical astrocytes TRPV4
was reported to interact physically with AQP4
and the macromolecular complex was demon-
strated to be necessary for the regulation of cell
volume because manipulation of the expression
of TRPV4 and AQP4 led to a dramatic altera-
tion in RVD following hypotonicity challenge
(Benfenati etal.,2011).
ROLE OFINTR ACELLULAR
CALCIUM AND SODIUM
DY NAMICS INASTROCY TE
R E G U L AT I O N O F B R A I N
H O M E O S TA S I S
It is now widely accepted that in astroglial cells
the intracellular Ca2+ concentration ([Ca 2+]i) can
oscillate with temporal and spatial dynamics that
are critical for brain homeostasis. The first evi-
dence of [Ca2+]i dynamics came from experiments
17
in cultured astrocytes in which it was shown that
stimulation with the neurotransmitter glutamate
or application of a mechanical stimulus caused
transient elevations in [Ca 2+]i that propagated as
[Ca2+]i waves through the astrocyte monolayer
(Cornell-Bell et al., 1990; Charles et al., 1991).
Importantly, [Ca 2+]i waves were reported also
in vivo, a result that corroborates the view that
they are physiologically relevant (Kuga et al.,
2011). In addition to Ca2+ permeation through
ion channels, [Ca2+]i rise in vitro and in situ can
be triggered by stimulation of various receptors
that allow Ca2+ entry or its release from intra-
cellular stores (Verkhratsky et al., 2012). There
are indications that [Ca2+]i signaling modu-
lates neuronal excitability (Araque et al. 2001;
Volterra and Steinhuser, 2004), even though
this view has recently been questioned (Agulhon
etal., 2010). Astrocytic [Ca 2+]i dynamics are also
involved in the regulation of cerebral vascular
tone (Filosa and Idding, 2013). It has been dem-
onstrated that the [Ca2+]i elevation can promote
the astroglial release of signaling molecules (gli-
otransmitters) through vesicular mechanisms
in vitro and in situ (Parpura and Zorec, 2010),
but its physiological relevance is still unclear
(Hamilton and Attwell, 2010). In cultured astro-
cytes it was reported that hypotonic challenge
caused an increase in [Ca 2+]i, which was neces-
sary for RVD (OConnor and Kimelberg, 1993),
and therefore a role of [Ca 2+]i dynamics in cell
volume homeostasis in vivo cannot be ruled out.
As second messenger molecule, [Ca2+]i signaling
could modulate astrocytic functions through
different mechanisms (Verkhratsky etal., 1988).
However, which are those involved in the modu-
lation of ion channels that regulate cell volume
in vitro is still unclear. Previous work has shown
that in cultured astrocytes extracellular ATP
released through multidrug resistance protein
could modulate volume-sensitive Cl current in
an autocrine fashion via activation of purinergic
metabotropic receptors coupled with intracellular
Ca 2+ mobilization (Darby etal., 2003). The hypo-
tonically activated Cl current was also abolished
by broad-spectrum inhibitors of calcium/calmo-
dulin protein kinase (Li etal., 2002; Olson etal.,
2004). Unfortunately, the lack of unequivocal
evidence about the presence of Cl channels acti-
vated by hypotonicity in situ has hampered the
possibility of defining the relevance of this in vitro
findings. In this context, the bestrophin channel
could deserve closer inspection. The astrocytic
bestrophin-1 channel is, in fact, activated by a rise
18 Part I: Homeostatic Regulators
in [Ca2+]i mediated by activation of Gq-coupled
receptors (Park et al., 2009) and is expressed in
CA1 hippocampal astrocytes in situ. However, it
remains to be investigated whether it is also pre-
sent in astrocytes of other brain regions and what
is its localization in specific cell microdomains.
In cultured astrocytes two types of Ca2+-activated
K+ channels have been reported to be stimulated
by metabotropic glutamate receptors coupled to
Ca2+ mobilization (Gebremedhin etal., 2003)and
in situ the presence in the endfeet of intermedi-
ate and large-conductance Ca 2+-activated K+
channels has been described (Filosa etal., 2006;
Longden etal., 2011). Their relevance in cell vol-
ume regulation in these spatially restricted cell
regions is still unknown and warrants further
investigations.
Another ion that has recently been associ-
ated with intracellular signaling is Na+. It has
been reported that in vitro and in situ tran-
sient changes in cytosolic concentration of Na+
([Na+]i) occur as a result of synaptic stimulation
(Kirischuk etal., 2007; Langer and Rose, 2009),
and [Na+]i signaling can propagate as Na+ waves
through the astroglial syncytium (Rose and
Ransom, 1997; Langer and Rose, 2009). Chemical
and mechanical stimulations can also promote
astrocytic [Na+]i transients in situ and in vitro
(Rose and Karus, 2013). These [Na+]i dynamics
are mediated by Na+ influx through ion channels
or Na+-coupled secondary transporters for gluta-
mate and gamma-aminobutyric acid (Rose and
Karus, 2013). It is also worth noting that the Na+
and Ca2+ dynamics are tightly linked because of
the presence of plasmalemmal Na+/Ca2+ exchang-
ers and ion channels that are permeable to both
Na+ and Ca2+, such as TRP channels and several
ionotropic receptors.
The amplitude and spatial dynamics of
[Na+]i transients in astrocytes are governed
by the cellular distribution of the diverse Na+
influx and efflux pathways. For example, the
Na+-dependent glutamate transporters are more
expressed at the endfeet facing the synapses
than those abutting blood vessels (Chaudhry
et al., 1995; Lehre et al., 1995). An important
factor that influences [Na+]i dynamics is also
the fact that accumulated Na+ does not appear
to be readily buffered, and there are no known
specific intracellular binding proteins. This cer-
tainly helps explain the general observation that
[Na+]i dynamics develop with a much slower
time course as compared to [Ca 2+]i oscillations
(Paemeleire and Leybaert, 2000; Langer et al.,
2012)and can last tens of seconds (Bennay etal.,
2008). The relevance of [Na+]i transients in the
context of astrocyte regulation of brain homeo-
stasis in physiological conditions is linked, to
a large extent, to the capacity of accumulated
Na+ to be exported through the Na+/K+-ATPase,
hence contributing to the uptake of extracel-
lular K+ (Sontheimer et al., 1994; DAmbrosio
etal., 2002). Less clear are the functional conse-
quences of pathological [Na+]i increases such as
those occurring during energetic failure (Silver
etal., 1997). An increase in [Na+]i mediated by
the NKCC1 transporter was shown to contrib-
ute to the development of cerebral edema fol-
lowing ischemia (Chen and Sun, 2005; Kahle
et al., 2009). Clearly, since the unravelment of
[Na+]i signaling in astrocytes is relatively recent,
more work is warranted to uncover the molecu-
lar mechanisms through which [Na+]i dynamics
can regulate astrocyte homeostatic processes,
especially in pathological settings. In this con-
text, it was recently reported that in cultured
astrocytes the activity of VRAC was negatively
regulated by pathological elevation of [Na+]i
(Minieri etal., 2015).
CONCLUDING REMARKS
There is clear evidence that alterations of the
mechanisms regulating ion and volume homeo-
stasis are involved in the cytotoxic edema of the
astrocytic syncytium that develops upon vari-
ous brain injuries (Liang etal., 2007). Astrocyte
swelling occurs as result of a variety of biochemi-
cal and molecular changes that affect the activity
of the ion channels and transporters which regu-
late the movements through the plasma mem-
brane of organic and inorganic osmolytes as well
as water (Pasantes-Morales and Vzquez-Jurez,
2012). Since cytotoxic edema has a causal role in
the secondary injury of neuronal tissue, the pre-
vention of astrocyte swelling should be a primary
goal that novel pharmacological approaches shall
address for the development of effective therapeu-
tic strategies (Kimelberg and Nedergaard, 2010).
However, the pharmacological tools available so
far are still relatively poor in terms of specificity
and knowledge of the underlying action mecha-
nisms. Previous work has shown that tamoxifen,
a known inhibitor of VRAC channels (Voets
et al., 1995), was neuroprotective in a model of
focal ischemia (Kimelberg etal., 2000; Kimelberg
etal., 2003), but tamoxifen has a large spectrum
of known targets, including other ion channels
(Rivera-Guevara and Camacho, 2011). The puta-
tive, more specific inhibitor of VRAC channels,
 Microdynamics of Water and Ion Homeostasis in the Brain
DCPIB, was shown to reduce infarct size in the
ischemic cortical penumbra in a model of mid-
dle cerebral artery occlusion (Zhang etal., 2008),
but recently the same molecule was shown to
modify the activity of other targets that are likely
to play a role in cell volume regulation (Minieri
et al., 2013; Bowens et al., 2013). Perhaps the
more attractive pharmacological candidate so
far is the SUR1 inhibitor glibenclamide that
blocks the SUR1-regulated NCCa-ATP channel and
ameliorates edema formation and tissue dam-
age (Simard et al., 2012). The pharmacology
issue is further complicated by the fact that it is
not known whether the changes in cell volume
regulation observed in pathological conditions
could also be due to a dysfunction of ancillary or
regulatory subunits that interact with the known
targets. In this view, albeit in another context, it
was shown that the aberrant astrocytic volume
control involved in megalencephalic leukoen-
cephalopathy with subcortical cysts, a genetic
disease characterized by chronic white matter
edema (van der Knaap et al., 2012), was due to
mutations of a protein called GlialCAM, which
interacts with the Cl channel ClC-2 (Jeworutzki
etal.,2012).
Even though the amount of information now
available on the physiology and pathophysiol-
ogy of astrocytes concerning water and ion
homeostasis is substantial, much work remains
to be done to obtain a more exhaustive picture
of the underlying mechanisms. In this view, the
possibility of exploiting complementary inter-
disciplinary approaches could be essential to
identify new perspectives for addressing these
homeostatic processes. In the past two decades
the development of technologies concerning cell
isolation and manipulation, coupled with the
discovery and implementation of electrophysio-
logical, molecular biology, and cell biology tools,
has enabled functional and molecular identifi-
cations of plasma-membrane proteins forming
receptors and voltage-dependent ion channels
in astroglia that were thought to be uniquely
expressed in neurons (Barres, 1990; Verkhratsky
and Steinhuser, 2000). Furthermore, the appli-
cation of optical technologies for monitoring
ion dynamics in vitro and in situ has provided
new insights into astrocytic functions (Maschio
etal., 2012). It can be envisaged that in the near
future the emerging interface between biology,
nano-materials, and bioelectronic technologies
(Benfenati etal., 2010; Benfenati etal., 2013)will
provide novel tools to discover unexpected roles
19
of astroglial cells and new avenues to manipulat-
ing their physiology.
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2
Homeostatic Control ofAdenosine Levels
and Functions intheBrain
XUESONG CHEN, M AHMOUD L .SOLIM A N, LIA NG HUI,
A N D J O N AT H A N D . G E I G E R
INTRODUCTION
Applying the concept of homeostasis, as intro-
duced and elaborated on by Claude Bernard and
Walter Cannon, to the regulation of the levels
and actions of the purines adenosine and ATP
in the brain is both complex and interesting. Of
course there are many homeostatically regulated
physiologically-important brain functions, and
the levels and actions of a variety of important
physiologically active ions, gases, chemicals,
and compounds help control these functions,
including ATP and adenosinethe focus of this
chapter.
When thinking about the homeostatic regu-
lation of purines, one needs to pay close attention
to not only normal fluctuations in their levels
and actions, something akin to noise (Woods
and Wilson, 2013)but also to signals caused by As a neuromodulator and a homeostatic regula-
increased levels and actions of ATP and adenosine
as occurs during neuronal stimulation, depolari-
zation, metabolic insults, and pathological states.
It is in this latter context of signals that Andrew
Newby coined the term retaliatory metabolite
(Newby et al., 1990); although not specifically
discussed at that time some 25years ago, inher-
ent in that term are the concepts of homeostatic
regulation and negative feedback.
Adenosine is produced largely from the
metabolism of the excitatory purine ATP, and the
largely inhibitory actions of adenosine (certainly
those mediated through adenosine A1 receptors)
help to provide negative feedback (retaliation)
against initial brain stimuli such as increases
in energy demand and decreases in energy sup-
plies. However, understanding the homeostatic
regulation of purine levels and actions is com-
plicated because adenosine can be formed both
intracellularly and extracellularly; there are
many different anabolic and catabolic enzymes
and transporters that help regulate the levels;
there are multiple receptor systems upon which
the purines act to elicit their actions; multiple
cell types even in the brain are involved; and all
of these sites of actions are subject to transcrip-
tional, translational, and epigenetic modifica-
tions. Indeed, it seems reasonable to propose that
these regulations and modifications are, in fact,
homeostatic mechanisms. In this chapter we dis-
cuss issues important to understanding where,
when, and how these homeostatically regulated
brain energy metabolites act in the brain with a
focus on adenosine.
BR AIN LEVELS
OFADENOSINE
tor, adenosine is involved in a variety of impor-
tant central nervous system (CNS) functions,
including bioenergetics, sleep and arousal,
learning and memory, and motor control. These
actions of adenosine depend not only on the
levels of extracellular adenosine but also on its
access to and the distribution of adenosine recep-
tor subtypes through which the actions of adeno-
sine are mediated at the cellularlevel.
Under physiological conditions, extracel-
lular adenosine concentrations in the brain are
in the 30 to 200 nanomolar range (Fredholm
et al., 2011). These levels reflect a balance and
homeostatic regulation of the production,
release, metabolism, and uptake of adenosine
and its precursors, as shown in Figure 2.1. In the
brain, purine salvage is key to the maintenance
of purine levels because the de novo synthe-
sis of adenine nucleotides is limited and the de
novo synthesis of adenosine is absent. Adenosine
32 Part I: Homeostatic Regulators

FIGURE 2.1: Adenosine formation, metabolism, and transport. Neurons and astrocytes express
enzymes that catalyze the synthesis and removal of adenosine. Adenosine can be formed intracellularly from
catabolism of ATP and S-adenosylhomocysteine (SAH) or extracellularly from breakdown of released ATP by
ecto 5-nucleotidase (5N). Equilibrative nucleoside transporters (ENTs) are bidirectional and facilitate the move-
ment of adenosine into or out of cells along adenosine concentration gradients.

levels are regulated intracellularly and extracel-
lularly by enzymes that catalyze its synthesis,
5-nucleotidases and S-adenosylhomocysteine
hydrolase, and enzymes that catalyze its removal,
S-adenosylhomocysteine hydrolase, adenosine
deaminase (ADA), and adenosine kinase (ADK;
King eta l., 2006; Latini and Pedata, 2001; Peng eta l.,
2005). Intracellularly, adenosine formation is cat-
alyzed from 5-AMP by cytosolic 5-nucleotidases
and from S-adenosylhomocysteine by
S-adenosylhomocysteine hydrolase. Extra
cellularly, adenosine formation from ATP
and cAMP is catalyzed by ATPase, apyrase,
and ecto-5-nucleotidase. Adenosine catabo-
lism appears to occur mainly intracellularly
where S-adenosylhomocysteine hydrolase
catalyzes the conversion of adenosine to
S-adenosylhomocysteine, ADA catalyzes the
conversion of adenosine to inosine, and ADK
catalyzes the conversion of adenosine to 5-AMP.
The activities and expression levels of these
enzymes are themselves homeostatically regu-
lated by various influences, including the levels
of adenine nucleotides and adenosine (Cui etal.,
2009; Kowaluk and Jarvis, 2000; Orford and
Saggerson,1996).
In addition to the aforementioned enzymes,
nucleoside transporters help regulate the
intracellular and extracellular levels of adeno-
sine (King et al., 2006; Latini and Pedata, 2001;
Peng et al., 2005). Equilibrative and concentra-
tive (sodium-dependent) nucleoside transporters
are present in the brain (Geiger and Fyda, 1991;
Parkinson et al., 1994). Equilibrative nucleoside
transporters (ENTs) are bidirectional and facili-
tate the movement of adenosine into and out of
cells along adenosine concentration gradients
(Baldwin et al., 2005). In contrast, adenosine
movement into and out of cells through con-
centrative nucleoside transporters follows Na+
electrochemical gradients. Of these nucleoside
transporters, ENTs play a major role in adeno-
sine transport (Geiger and Fyda, 1991; Parkinson
etal., 1994). Not only do these transporters par-
ticipate in the homeostatic control of adenosine
levels, but the transporters themselves are home-
ostatically controlled by brain levels of adenosine
(Sebastio, 2011; Delicado etal., 1994; Hertz and
Matz,1989).
Adenosine levels can also be controlled
through action potential dependent release
mechanisms. Unlike more traditional neu-
rotransmitters, adenosine is not packaged in
secretory vesicles but, as mentioned, can be
released through ENTs. Under conditions that
increase neuronal metabolic load by, for instance,
 Homeostatic Control of Adenosine Levels and Functions in the Brain 33

neuronal firing, the intracellular catabolism of
ATP can increase thus causing a concentration
gradient-dependent rapid efflux of adenosine
through ENTs. Alternatively, ATP is packaged
in secretory vesicles and can be released where-
upon it can be metabolized to adenosine albeit
somewhat more slowly (Wall and Dale, 2013).
Activation of excitatory neurons can result in
glutamate release whereupon it can activate
excitatory amino acid receptors and increase lev-
els of intracellular Ca2+ and increased exocytotic
release of ATP that can then be metabolized to
adenosine (Pankratov etal., 2007). Extracellular
adenosine once formed may be preferentially
taken up into astrocytes via ENTs because astro-
cytes maintain very low levels of intracellular
adenosine due to high activity levels of ADK, an
enzyme that catalyzes the conversion of adeno-
sine to 5-AMP (Boison etal.,2010).
ADENOSINE RECEPTORS
AND DISTRIBUTION
There are four subtypes of cell surface adeno-
sine receptors; A1, A 2A, A 2B, and A3 (Benarroch,
2008; Boison etal., 2010; Dunwiddie and Masino,
2001; Fredholm et al., 2005), all of which are
seven-transmembrane G-protein coupled recep-
tors (Figure 2.2). Adenosine receptors exhibit dif-
ferent binding affinities for adenosine (Dunwiddie
and Masino, 2001; Olah and Stiles, 1995); A1
receptors have the highest affinity (70 nM),
A 2A receptors have high affinity (150 nM); and
A 2B and A3 have lower affinities ( 5000 nM). In
the CNS, expression levels of adenosine recep-
tors vary between regions: A1 receptors are the
most abundant and widespread with enrich-
ment in hippocampus, cerebellum, and cerebral
cortex; A2A receptors are highly concentrated
in basal ganglia and limbic areas; and A2B and
A3 receptors are expressed at much lower levels
(Fredholm etal., 2005; Gomes etal., 2011). At the
cellular level, expression levels of A1 receptors
are higher in neurons than in glial cells (astro-
cytes, microglia, and oligodendrocytes), whereas
A 2A receptors are expressed on neurons, glial
cells, and endothelial cells. For neurons, A1 and
A 2A receptors are densely enriched in presynap-
tic active zones as well as in postsynaptic den-
sities, but for striatal neurons A 2A receptors are
predominantly located at postsynaptic densities

FIGURE 2.2 Adenosine as a neuromodulator and homeostatic regulator of brain bioenergetics.

All four subtypes of adenosine receptors are expressed on neurons, astrocytes, and endothelial cells that make up
tripartite synapses. With their direct contacts with neuronal synapses and cerebral vasculature, astrocytes are
uniquely situated to regulate the involvement of adenosine in the homeostatic regulation of brain bioenergetics
and functions. On one hand, adenosine derived from astrocytes regulates both neurotransmitter release and syn-
aptic integrity, thus playing a major role in establishing set points of inhibitory tone within neuronal networks. On
the other hand, astrocyte-derived adenosine can regulate blood flow and blood-brain barrier integrity and modu-
late the entry of energy-yielding nutrients into the brain, thus regulating brain bioenergetics homeostatically.
34 Part I: Homeostatic Regulators
(Cunha, 2005). The effects of adenosine on brain
functions are regulated mainly through activa-
tion of A1 and A 2A receptors because the normal
levels of extracellular adenosine preferentially
activate A1 and A 2A receptors, because A1 and A2A
receptor expression levels are relatively high, and
because manipulation of A 2B and A3 receptors has
been shown to have relatively modest impacts on
brain function (Cunha, 2005; Fredholm et al.,
2005; Gomes etal., 2011). This contrasts with the
actions of adenosine under pathological condi-
tions when adenosine levels rise dramatically and
activate lower affinity A2B and A3 receptors.
ADENOSINE RECEPTOR
SIGNALING
For many years adenosine receptor signaling
was thought to be restricted to cAMP formation,
with adenosine A1 and A3 receptors coupling to
Gi proteins that once activated inhibit adenylate
cyclase and adenosine A2A and A2B receptors cou-
pling to Gs proteins that once activated activate
adenylate cyclase. However, adenosine receptor
signaling is far more complex than this; depend-
ing on sites and degree of activation, adenosine
receptor subtype activation can lead to coupling
to more than one G protein and to different sig-
nal transduction systems. For example, A1 recep-
tors can couple to Gi1/2/3 and Go; A2A receptors
can couple to Gs, Golf, G15, and G16; A2B recep-
tors can couple to Gs and Gq/11; A3 receptors can
couple to Gi/2,3 and Gq/11 (Fredholm etal., 2001;
Fredholm et al., 2011); and adenosine receptors
can couple to G subunits (Schwindinger etal.,
2010; Yao etal.,2002).
Adenosine receptor signaling is further
complicated by the degree to which A1 and A2A
receptors can oligomerize as homodimers and
their ability to form heterodimers with, for
example, ATP, dopamine, and glutamate recep-
tors (Ciruela etal., 2012; Fredholm etal., 2011).
A1 receptors form heterodimers with P2Y1- and
P2Y2-purinergic receptors (Suzuki et al., 2006;
Yoshioka et al., 2001), dopamine D1 receptors
(Ismayilova et al., 2004), and metabotropic glu-
tamate type 1 receptors (mGluR1; Ciruela etal.,
2001). A2A receptors form heterodimers with
dopamine D2 receptors (Hakansson etal., 2006;
Higley and Sabatini, 2010), dopamine D3 recep-
tors, metabotropic glutamate type 5 receptors
(mGluR5; Nishi et al., 2003), and cannabinoid
CB1 receptors (Carriba et al., 2007). Such oli-
gomerization can enable the fine-tuning of
adenosine signaling, and such aspects of sign-
aling can add to the homeostatic regulation of
adenosines functions. Moreover, prolonged acti-
vation of adenosine receptors can lead to aden-
osine receptor desensitization and changes in
adenosine receptor trafficking. In addition, and
depending on the subtype of adenosine receptor,
trafficking kinetics can be differently regulated
(Klaasse et al., 2008; Mundell and Kelly, 2011).
Such activity-dependent desensitization and traf-
ficking mechanisms add another layer of homeo-
static control of adenosine signaling.
ADENOSINE ASA
H O M E O S TAT I C
R E G U L AT O R O F B R A I N
BIOENERGETICS
The brain in general and neurons in particu-
lar require a large and almost constant supply
of energy (Rolfe and Brown, 1997). Because the
brain has a very limited capacity to store read-
ily useable bioenergetic molecules, it requires
a continuous supply of blood containing oxy-
gen and energy-yielding nutrients. Thus even
very brief interruptions (seconds) in blood flow
and/or absence of oxygen results in dramatic
losses of cellular energy, loss of consciousness,
and profound abnormalities in brain function
up to and including increased susceptibility to
insult-induced neuronal celldeath.
ATP is not only a readily available source
of adenosine; it is also essential for a variety of
neuronal functions, notably the maintenance
of transmembrane ion gradients, cAMP forma-
tion, kinase-mediated phosphorylation, vesicu-
lar storage of neurotransmitters, and glutamate
metabolism. Brain levels of adenosine are nearly
10,000-fold lower than ATP (Delaney and Geiger,
1996; Pazzagli et al., 1993; Van Wylen et al.,
1986), which means that huge increases in adeno-
sine levels can originate from minor fluctuations
in ATP levels. In general, adenosine inhibits neu-
ronal activity and in so doing allows target cells
like neurons and astrocytes to homeostatically
regulate their energy demands to match energy
supplies (Meghji and Newby, 1990; Newby etal.,
1990). However, it appears clear that the ability of
adenosine levels to increase as brain energy lev-
els decrease is quite different depending on the
severity of the stimulation (Shepel et al., 2005).
With no significant external excitatory stimula-
tion, ATP levels remain within a physiological
range and adenosine increases modestly to lev-
els where adenosine A1 receptors would be pref-
erentially activated. With significant external
excitatory stimulation as occurs during excito-
toxicity, ATP levels decrease below physiological
 Homeostatic Control of Adenosine Levels and Functions in the Brain
thresholds and adenosine increases to levels
capable of upregulating and activating adeno-
sine A2A receptors. When the brain is exposed
to more drastic insults such as global ischemia,
ATP levels drop dramatically and adenosine lev-
els increase markedly as the brain attempts to
homeostatically prevent cell death (Atkinson and
Walton, 1967) by activating low affinity adeno-
sine A3 receptors and mobilizing tissue repair
mechanisms (Abbracchio etal., 1997; von Lubitz
etal.,1999).
Astrocytes can outnumber neurons 9:1
(White and Venkatesh, 2011)and are one of the
major sites for adenosine formation (Pascual
etal., 2005). Activation of excitatory amino acid
receptors on astrocytes can lead to increased
synaptic levels of adenosine originating from
ATP released through vesicles or hemichannels
(Iglesias etal., 2009; Kang etal., 2008). Astrocytes
can also increase extracellular levels of adeno-
sine originating from ATP released through
volume-regulated ion channels (Darby et al.,
2003). In addition, adenosine can be released via
nucleoside transporters (King etal., 2006; Latini
and Pedata, 2001; Peng et al., 2005). Astrocytes
may also play an important role in enzyme-linked
regulation of adenosine levels because ADK is
mainly expressed in astrocytes and is known
to catalyze the salvage of adenosine to 5-AMP
(Boison etal., 2010; Studer etal.,2006).
With their direct contacts with neuronal syn-
apses and cerebral vasculature, astrocytes are
uniquely situated to regulate the involvement of
adenosine in the homeostatic regulation of brain
bioenergetics and functions. Astrocytes, neurons,
and endothelial cells make up tripartite synapses,
and together these cells function to coordinate
synaptic activity and networks. Moreover, aden-
osine derived from astrocytes in the functional
vicinity of tripartite synapses appears to play a
major role in establishing set points of inhibitory
tone within neuronal networks (Figure 2.2). For
example, in actively functioning cells, energy lev-
els decline and adenosine levels increase to levels
that preferentially activate inhibitory cell surface
adenosine A1 receptors. Thus at a very local level,
adenosine formation provides a rapidly acting
negative feedback mechanism that prevents fur-
ther brain energy demands and homeostatically
regulates brain energy metabolism. Alternatively,
endfeet of astrocytes are integral elements of the
blood-brain barrier where astrocyte-derived
adenosine can homeostatically regulate blood
flow, maintain blood brain barrier integrity,
and modulate the entry of energy-yielding
35
nutrients into the brain (ORegan, 2005;
Pelligrino etal.,2011).
Clearly, functional coupling between neu-
ronal activity and blood flow is central to the
homeostatic regulation of brain bioenergetics.
For example, when neurons are activated, cer-
ebral blood flow increases in localized brain
regions thereby increasing temporally and spa-
tially the supply of energy-yielding nutrients.
Mechanistically this occurs because neuronal
stimulation leads to glutamate release, the release
of ATP from astrocytes, the metabolism of ATP
to adenosine, and the activation of adenosine A2A
receptors that are highly expressed on cerebral
blood vessels; the end result is vasodilation and
increased cerebral blood flow (Mills etal., 2011;
ORegan, 2005; Pelligrino etal., 2010). In addition
to neurovascular coupling, astrocyte-derived
adenosine can affect the permeability of the
blood brain barrier through coordinated activa-
tion of A1 and A2A receptors, both of which are
expressed on brain endothelial cells (Carman
etal.,2011).
As such, adenosine is able to homeostatically
regulate brain microenvironments for optimal
functioning of neuronal networks. Moreover,
these same homeostatically-controlled mecha-
nisms and set points are activated in a variety of
acute and chronic neurological and neuropsy-
chiatric disorders such as brain ischemia, sleep
deprivation, epilepsy, Huntingtons disease,
Parkinsons disease, Alzheimers disease, depres-
sion, bipolar disorder, and schizophrenia (Gomes
etal., 2011), and altering adenosine signaling may
therefore be exploited therapeutically against
these acute and chronic neurological disorders
(Cunha, 2005; Gomes etal.,2011).
ADENOSINE ASA
N E U R O M O D U L AT O R
As a neuromodulator, adenosine working
through A1 and A2A receptors helps tune and fine-
tune synapses, in part, by homeostatically con-
trolling inhibition and excitation (Ribeiro and
Sebastiao, 2010; Sebastiao and Ribeiro, 2009a,
2009b). An important aspect of this synaptic
tuning is the control of neurotransmitter release
and synaptic plasticity through the coordinated
actions of inhibitory adenosine A1 and excita-
tory adenosine A2A receptors (Dias et al., 2013).
Indeed, adenosine has been shown to modulate
excitatory neurotransmitters, including gluta-
mate, acetylcholine, and serotonin, as well as
the inhibitory neurotransmitter GABA. Such
actions underlie critical physiological functions
36 Part I: Homeostatic Regulators
of adenosine, including sleep/arousal (Brown
etal., 2012), learning and memory (Prediger and
Takahashi, 2005; Wei et al., 2011), and motor
control (Ciruela et al., 2012; Cunha, 2005; Fuxe
et al., 2010). Here we focus on how adenosine
adjusts dynamically the strength of excitatory
and inhibitory inputs reaching a given neuron
thus controlling neuronal firingrates.
The tonic release of adenosine via neuronal
ENTs can activate presynaptic A1 receptors and
inhibit the presynaptic release of glutamate by
inhibiting calcium influx through voltage-gated
calcium channels, including N-type and
P/Q-type calcium channels (Ambrosio etal., 1997;
Brambilla etal., 2005; Gundlfinger etal., 2007).
Such tonic release can also activate postsynap-
tic A1 receptors, inhibit N-methyl-D-aspartate
(NMDA) receptors (de Mendonca et al., 1995;
Hartwick et al., 2004) and activate potas-
sium channels (including G protein-coupled
inwardly rectifying potassium channels [GIRKs],
ATP-sensitive potassium channels, and small
conductance calcium-activated potassium chan-
nels), thus leading to postsynaptic neuronal
hyperpolarization (Chung et al., 2009; Clark
et al., 2009; Hosseinzadeh and Stone, 1998;
Kawamura etal., 2010). On the other hand, aden-
osine derived from the release of ATP can activate
A2A receptors abundantly located in presynaptic
active zones, where it can facilitate glutamate
release (Ciruela etal., 2006)and inhibit glutamate
uptake into astrocytes (Matos etal., 2012). A1 and
A2A receptors are expressed pre- and postsynapti-
cally, where these receptors can heterodimerize
and help functionally tune adenosine and gluta-
mate signaling. At low adenosine levels, adeno-
sine preferentially activates A1 receptors, and at
higher levels it activates A 2A receptors within A 2A/
A1 oligomers. Such a hierarchical activation of A1
receptors or A2A receptors allows functional tun-
ing of adenosine signaling and the control of glu-
tamate release (Dias etal.,2013).
Compared to glutamatergic transmis-
sion, much less is known about how adenosine
affects GABAergic transmission. It is generally
accepted that adenosine does not affect directly
the presynaptic release of GABA (Thompson
et al., 1992) and that the effects of adenosine
on GABA synaptic transmission are mainly
achieved by inhibiting the excitatory synaptic
inputs to GABAergic neurons via A1 receptors
(Yang et al., 2013). Recent studies indicate that
adenosine regulates GABA levels directly at the
synaptic cleft by altering GABA transporter
expression on astrocytes, where activation of A1
receptors within A2A/A1 oligomers decreases the
expression of GABA transporters thus inhibiting
GABA transport into astrocytes. On the other
hand, activation of A 2A receptors within A 2A/
A1 oligomers increases the expression of GABA
transporters thus facilitating GABA transport
into astrocytes (Cristovao-Ferreira et al., 2013).
In addition, adenosine could affect GABA recep-
tor signaling at postsynaptic densities because
A1 receptor-dependent activation of potassium
channels increases cell membrane conductance
and thus has a shunting effect on GABA AR cur-
rents (Ilie etal.,2012).
Such actions of adenosine on the strength
of excitatory and inhibitory input may play an
important role in the regulation of sleep/arousal,
quite possibly the least well-understood homeo-
static mechanism of any organism (Brown etal.,
2012). Prolonged wakefulness can increase
adenosine levels (Kalinchuk et al., 2011) and
might promote sleep through activation of A1
and A2A receptors in specific brain regions. In
basal forebrain, activation of A1 receptors, which
are upregulated during prolonged wakefulness
(Basheer etal., 2007), inhibit glutamatergic neu-
rons and wake-promoting cholinergic neurons
(Yang et al., 2013). In the tuberomammillary
hypothalamic nucleus, activation of A1 recep-
tors results in inhibition of wake-promoting
histaminergic neurons (Hong et al., 2005). In
the ventrolateral preoptic area, activation of A2A
receptors activates sleep-promoting neurons and
inhibits arousal-promoting regions (Urade and
Hayaishi, 2011). In addition, A 2A receptors in the
nucleus accumbens may play a role in sleep-wake
regulation, especially caffeines wake-promoting
effects (Lazarus etal., 2011; Zhang etal.,2013).
Through coordinated activation of high-
affinity inhibitory A1 and excitatory A 2A recep-
tors, adenosine has been recognized as an impor-
tant homeostatic regulator of synaptic plasticity
(Dias etal., 2013)with a unique ability to allow
neurons to dynamically modify their synaptic
structure and function as a result of activity.
Here we discuss how adenosine affects synaptic
plasticity of glutamatergic and dopaminergic
systems.
Adenosine has been shown to provide
homeostatic regulation of glutamatergic sys-
tems, essential for two well-described forms
of synaptic plasticity: long-term potentia-
tion (LTP) and long-term depression (LTD;
Abraham, 2008; Queenan et al., 2012). LTP is
 Homeostatic Control of Adenosine Levels and Functions in the Brain
a form of activity-dependent plasticity that
results in a persistent enhancement of synap-
tic efficacy, whereas LTD is a long-lasting and
activity-dependent decrease in synaptic efficacy.
LTP and LTD are expressed by synapse-specific
regulation by AMPA and NMDA glutamate
receptors (Lee et al., 2010). Tonic release of
adenosine through ENTs can activate postsyn-
aptic A1 receptors, GIRK channels, and home-
ostatically control LTP (Chung et al., 2009).
Activation of postsynaptic A1 receptors also
affects LTD. Activation of A1 receptors blocks
mGluR1-mediated LTD by decreasing the
ligand sensitivity of mGluR1 (Kamikubo etal.,
2013), possibly through direct interactions
between A1 receptors and mGluR1 in the form
of A1/mGluR1 heterodimers (Ciruela et al.,
2001). High-frequency bursts of stimulation, as
occur in the induction of LTP, can trigger the
release of ATP, and adenosine levels formed
from the released ATP are high enough to acti-
vate postsynaptic A 2A receptors (Cunha, 2001).
Such activation of postsynaptic A 2A recep-
tors can induce different forms of short- and
long-term synaptic plasticity by enhancing the
surface expression of GluR1 (Dias etal., 2012).
Activation of A 2A receptors can also potentiate
NMDA receptor-mediated LTP (Rebola et al.,
2008), possibly through co-activation of A 2A and
mGluR5 in the form of A 2A/mGluR5 heterodi-
mers (Tebano et al., 2005). Thus, homeostatic
regulation of synaptic plasticity in the form of
LTP and LTD by adenosine suggests that aden-
osine plays an important role in learning and
memory. Indeed, altering A1 and A 2A receptor
signaling affects learning and memory in ani-
mal models (Prediger and Takahashi, 2005; Wei
et al., 2011), and this opens the door to possi-
ble exploitation of therapeutic interventions
against such conditions as Alzheimers disease
where memory is often seriously impaired.
Adenosine also homeostatically regulates
dopaminergic systems in basal ganglia where A1
and A 2A receptors are highly expressed (Ciruela
et al., 2012; Cunha, 2005; Fuxe et al., 2010).
Dopamine D1 receptors are primarily expressed
on striato-nigral neurons of the direct pathway
where they couple to stimulatory G s proteins,
and dopamine D2 receptors are highly expressed
on striato-pallidal neurons of the indirect path-
way where they are coupled to inhibitory G i
proteins. Dopamine induces motor activation
by activating the direct pathway through D1
receptors and depressing the indirect pathway
37
through D2 receptors. In general, adenosine
exerts antagonist effects on dopamine recep-
tors. In direct pathway neurons, A1 receptors
expressed at presynaptic dopaminergic nerve
terminals exert an inhibitory modulation
on dopamine release (Ferre et al., 1997). At a
postsynaptic site, A1 receptors form heterodi-
mers with D1 receptors, thus negatively affect-
ing the binding characteristics of D1 receptors
(Ismayilova et al., 2004). Behaviorally, adeno-
sine A1 receptor antagonists potentiate the
motor effects of D1R agonists through antago-
nistic A1D1 receptor interactions (Popoli etal.,
1996). In direct pathway neurons, adenosine
A 2A receptors are more densely expressed in
the striatum than anywhere else in the brain
(Cunha, 2001; Cunha, 2005), and striatal post-
synaptic A 2A receptors are selectively localized
in the dendritic membranes of medium spiny
neurons in the indirect-pathway, where they
can form heterodimers with D2 receptors and
exert strong functional antagonistic interac-
tions with D2 receptors (Hakansson etal., 2006;
Higley and Sabatini, 2010). Stimulation or
blockade of striatal postsynaptic A 2A receptors
counteracts and potentiates, respectively, the
motor-activating effects of D2 receptor stimula-
tion (Ferre etal., 1997; Schiffmann etal., 2007).
In addition, A1 receptors expressed in neurons
of indirect pathway could affect the function
of A 2A receptors though A 2A-A1 heterodimeri-
zation (Karcz-Kubicha etal., 2003). Such find-
ings suggest that the coupling of adenosine and
dopamine receptors is a key mechanism for
the homeostatic regulation of the dopaminer-
gic system (Stromberg etal., 2000). Given that
dopamine plays an important role in control-
ling locomotor activity, motivation, and addic-
tion (Arias-Carrion et al., 2010; Beninger,
1983; Salamone and Correa, 2002), the ability
of adenosine to homeostatically regulate dopa-
minergic systems reinforces the concept that
adenosine levels and/or signaling plays a role in
the pathogenesis of Parkinsons disease, schizo-
phrenia, and drug addiction (Fuxe etal.,2010).
ADENOSINE-BASED
THER APEUTICS
Because adenosine, as a neuromodulator and a
homeostatic regulator, is involved in a variety
of important CNS functions, including bioener-
getics, sleep and arousal, learning and memory,
and motor control, and because altered adeno-
sine signaling is involved in a number of acute
38 Part I: Homeostatic Regulators
and chronic neurological disorders, includ-
ing brain ischemia, sleep deprivation, epilepsy,
Huntingtons disease, Parkinsons disease,
Alzheimers disease, drug addiction, depres-
sion, bipolar disorder, and schizophrenia (Gomes
et al., 2011), adenosine-based therapies might
be developed. Such adenosine-based thera-
pies include strategies to alter adenosine levels
(Boison, 2013; Chu etal., 2013)and/or adenosine
receptor signaling (Fishman etal., 2012; Gomes
etal., 2011; Sachdeva and Gupta,2013).
With respect to the development of phar-
macological inhibitors aimed at augmenting
the effect of endogenous adenosine, ADK has
received considerable attention (Boison, 2013;
Boison et al., 2002). However, ADK inhibitors
have been linked to increased risks of liver
toxicity (Boison etal., 2002)and brain hemor-
rhages (McGaraughty et al., 2005). Therefore,
there has been a growing effort to manipulate
ADK and adenosine levels site and event specif-
ically by transplanting stem cells with deletions
of ADK and the use of gene therapy vectors
to downregulate ADK expression (Shen et al.,
2011; Theofilas et al., 2011). Another approach
might be to alter extracellular adenosine levels
by manipulating ENTs. Indeed, blocking ENTs
with, for example, dipyridamole decreased
adenosine efflux in neurons but not astrocytes
in a cell culture model of ischemia (Parkinson
and Xiong, 2004), and ENT1 knockout mice
have decreased motor incoordination and
hypnosis after ethanol administration (Choi
etal.,2004).
With regard to modulating adenosine
receptor signaling, subtype-specific adenosine
receptor agonists and antagonists have been
examined for their use for neurological diseases.
Neuroprotective effects of adenosine are mostly
attributed to A1Rs that decrease excitotoxic-
ity and reduce cellular metabolic needs. On the
other hand, activation of A 2ARs can exacerbate
neuronal injury (Fredholm etal., 2005). Thus A1R
agonists and A2AR antagonists are neuroprotec-
tive (Blum etal., 2002; Delle Donne and Sonsalla,
1994; MacGregor etal.,1997).
Finally, for homeostatic regulations by
adenosine it is important to consider duration-
dependent effects of adenosine receptor manipu-
lation as well as age-dependent effects (Fredholm
et al., 2005). Indeed, effect inversion has been
observed where, for example, acute activation of
A1Rs is neuroprotective while chronic treatment
with A1R agonists is associated with neuronal
cell death (von Lubitz etal., 1994). Furthermore,
homeostatic regulation by adenosine appears
to be different in newborns and adults (Bona
etal., 1995; Mody and MacDonald, 1995; Turner
etal.,2004).
SUMMARY
Homeostatic regulation of the levels and
actions of ATP and adenosine in the brain
is complex. There are normal f luctuations
(noise), and there are increased levels and
actions of purines (signals) as occurs during
neuronal stimulation, depolarization, meta-
bolic insults, and pathological states. Some
25 years ago, adenosine was described as act-
ing like a retaliatory metabolite because
its largely inhibitory actions (certainly those
mediated through adenosine A1 receptors)
help to provide negative feedback (retali-
ation) against initial brain stimuli such as
increases in energy demand and decreases in
energy supplies. However, understanding the
homeostatic regulation of purine levels and
actions is complicated because of the many
factors that regulate the intracellular and
extracellular levels of adenosine including
the transcriptional, translational and epige-
netic regulation of adenosines anabolic and
catabolic enzymes, transporters; and receptor
systems. Nevertheless, there remains value in
understanding all of this further because of
the need to understand better pathological
mechanisms as well as the need to create new
and/or better therapeutics.
ACK NOWLEDGMENTS
Our research activities in this area have been
supported consistently by grants awarded by the
Canadian Institutes of Health Research (Canada)
and the National Institutes of Health (United
States), including R01NS065957, P30GM103329,
R01MH100972 and R01MH105329.
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3
Glutamate Homeostasis asa Regulator
ofNeurotransmitter Recycling
and Synaptic Function
M A R Y E L L E N K E L LY A N D D O U G L A S A . C O U LT E R
INTRODUCTION
To ensure appropriate temporal fidelity and
maintenance of maintain proper synaptic trans-
mission, neurotransmitters must be removed
rapidly from the leave the synaptic cleft follow-
ing release, and metabolic intermediates must be
returned to presynaptic terminals for subsequent
regeneration of neurotransmitters to sustain
subsequent synaptic function. For the central
nervous system (CNS)neurotransmitters gluta-
mate and GABA, these requirements are fulfilled
through partitioned and segregated transporter
and enzyme expression and function in neurons
and astrocytes. Unlike monamine and acetyl-
choline uptake, the majority of neurotransmitter
transporter expression (and associated trans-
mitter uptake) at glutamatergic and GABAergic
synapses is evident on perisynaptic astrocytic
processes (Danbolt, 2001; Schousboe, 2000).
Once taken up into astrocytes, GABA and gluta-
mate are metabolized, and metabolic precursors
for subsequent glutamate and GABA re-synthesis
are shuttled to neuronal terminals to allow main-
tenance of synaptic transmission.
Specialized machinery exists to synthesize
and package neurotransmitter in membrane-
delimited packets (synaptic vesicles) for use in
intercellular communication. Since the early cal-
culations of molar content of cholinergic vesicles
over 30years ago (Kuffler and Yoshikami, 1975),
it has been known that vesicular content of neu-
rotransmitter is extremely high, approaching the
physical limits for packaging sufficient numbers
of molecules within the limited lumenal space
of a vesicle. Several structurally related proteins
have been identified for mediating small mole-
cule neurotransmitter vesicle packaging, includ-
ing vesicular transporters for glutamate, GABA,
monoamines, and acetylcholine, among others
(for review, see Eiden, 2000). The GABA vesicu-
lar transporter (VIAAT), like other vesicular
transporters, relies on the proton gradient within
synaptic vesicles to transport GABA against its
concentration gradient. Evidence has emerged
demonstrating complexity in this process of
sequestering GABA. VIAAT activity may be
tightly linked to local GABA synthesis, due to the
fact that it may participate in a supramolecular
complex including glutamic acid decarboxylase
(GAD), and Ca2+/calmodulin-dependent pro-
tein kinase II (Jin etal. 2003). This suggests that
glutamate, in addition to serving as an excita-
tory neurotransmitter in principal neurons, may
be the critical precursor in inhibitory synaptic
terminals, regulating intravesicular GABA lev-
els. Two patch clamp recording studies have lent
support to this hypothesis (Sepkuty et al. 2002;
Mathews and Diamond,2003).
T H E G L U TA M AT E - G L U TA M I N E
CYCLE AS A LOCAL , R APID
ECONOM Y IN SY NAPSES
Much of what we know about the various stages
of neurotransmitter recycling comes from bio-
chemical studies in tissue homogenates, or stud-
ies utilizing sampling of extracellular amino
acid concentrations in labelled tissue sam-
ples. Although this confers information about
global transfer of amino acids through the
glutamate-glutamine cycle in brain regions in
general, it misses significant information con-
cerning the local recycling of neurotransmitter in
synaptic compartments, which are isolated and
remote from cellular metabolism occurring in
neuronal somata. Synapses can be quite distant
in both space and time relative to overall cellular
 Glutamate Homeostasis as a Regulator of Neurotransmitter
metabolism of amino acids. There are signifi-
cant temporal delays imposed by the necessity of
transferring metabolic substrates from the cell
somata to the synaptic compartment. Synapses
overcome this constraint by conducting the
majority of their neurotransmitter metabolism
locally, within the tripartite compartment cre-
ated by the perisynaptic, sastrocyte process and
the pre- and postsynaptic cellular components.
Within this tripartite necessary there is a
tightly coupled linkage between glutamate uptake
in astrocytes and subsequent release and transfer
of glutamine to neurons (Uwechue etal., 2012). As
a primary contributory mechanism to this cou-
pled function, both the glutamate transporter-1
(GLT-1) and the System N glutamine trans-
porter (SN1) are colocalized on the fine astro-
cytic processes ensheathing synapses (Boulland
etal., 2002, Chaudry etal., 1995; Danbolt, 2001).
Possibly interacting with the precise colocaliza-
tion of these astrocytic transporters, the milli-
second precision in coupling between glutamate
uptake into astrocytes and glutamine resupply
to neurons (Uwechue etal., 2012)may be gener-
ated through the interacting stoichiometry of the
various transporters involved. Glutamate uptake
via GLT-1 is linked to the cotransport of three
sodium ions and a proton and reverse transport
of a potassium ion and so is accompanied by a
rapid rise in intracellular sodium within astro-
cytes (Langer and Rose, 2009; Langer etal., 2011;
Uwechue et al., 2012). Glutamine transport out
of astrocytes is mediated by System N transport-
ers, which cotransport a sodium ion with glu-
tamie and countertransport a proton, resulting in
electroneutral release of glutamine (Broer et al.,
2002). Because System N transporters operate in
a near equilibrium, the sodium influx occurring
during glutamate uptake in astrocytes (with a 3:1
stoichiometry) may be sufficient to couple GLT-1
function to the rapid initiation and amplification
of glutamine efflux via System N transport (with
their 1:1 stoichiometry for Na+ and glutamine;
Broer et al., 2002). This rapid coupling may be
enhanced by its occurrence within the fine astro-
cytic processes investing synapses, which will
generate a much larger increase in net sodium
concentration during glutamate uptake due to
the small cytoplasmic compartment in which it
occurs.
SOURCES
OFPRESY NAPTICGABA
In addition to serving as a neurotransmitter
in excitatory neurons, glutamate supplied to
45
inhibitory neurons may act as a significant pre-
cursor for GABA synthesis. Several systems are
present within inhibitory synaptic terminals
that provide GABA used for vesicular packag-
ing by VIAAT. These include proteins that facili-
tate reuptake of GABA and glutamate into the
inhibitory terminal following release (GABA
transporters such as GAT-1 and the neuronal glu-
tamate transporter EAAC-1), other transporters
that take up glutamine (system A[slc38-family]
transporters; Chaudry et al. 2002), alanine (asc
family transporters; Palacin et al. 1998; Helboe
et al. 2003; Kanai and Hediger, 2004), and leu-
cine (system L transporters, Segawa etal. 1999),
all amino acids that are released from astrocytes.
Once in the inhibitory presynaptic terminal,
these amino acids can all serve as precursors for
local glutamate synthesis by the enzymatic activ-
ity of glutaminase, alanine aminotransferase,
and branched-chain aminotransferase, respec-
tively (Yudkoff etal. 1996; Yudkoff, 1997; Sweatt
etal. 2004). In addition, other substrates may be
provided by the tricarboxylic acid (TCA) cycle,
localized in mitochondria within the synaptic
terminal. Since neurons lack pyruvate carboxy-
lase, an enzyme necessary to synthesize gluta-
mate directly from CO2 (Shank et al. 1985; Yu
et al. 1983), and these cells constantly release
glutamate and/or GABA as neurotransmitters,
they rely exclusively on astrocytes to provide
substrates for continued glutamate synthesis.
Otherwise, provision of TCA intermediates to
subserve this function will result in their deple-
tion and the rapid failure of intraterminal mito-
chondrial metabolism and energy production.
Evidence shows that in a normal brain the major-
ity of neurotransmitter recycling is accomplished
through the astrocytic glutamate-glutamine
cycle (Laake et al. 1995; Battiglioli and Martin,
1996; Rae etal.2003).
THE ASTROCYTIC
G L U TA M AT E - G L U TA M I N E
CYCLE ASA GABA
R ECYCLING MECHANISM
While interneurons do recycle GABA from
the synaptic cleft through GABA transporters
(including GAT-1), the principal source of syn-
thesized GABA packed into synaptic vesicles is
derived from decarboxylation of glutamate by
glutamic acid decarboxylase (Martin etal. 2000).
There are two primary sources for this gluta-
mate in interneurons. One is glutamate uptake
via excitatory amino acid carrier-1 transporters
(EAAC-1) located in the presynaptic terminals of
46 Part I: Homeostatic Regulators
GABAergic neurons (Arriza et al., 1994; Bjoras
etal., 1996; Nakayama etal., 1996; Eskandari etal.,
2000). A second source of glutamate is derived
from the astrocytic glutamate-glutaminecycle.
The glutamate-glutamine cycle begins with
the uptake of synaptically released glutamate by
glutamate transporters in ensheathing astrocytic
processes. In the forebrain, glutamate is taken
up by astrocytes through the astrocytic specific
glutamate transporter-1 (GLT-1) and converted
into glutamine by astrocyte-specific enzyme,
glutamine synthetase (Pines et al., 1992; Arriza
etal., 1994). Glutamine is then shuttled back to
presynaptic terminals of neurons through spe-
cific glutamine transporters localized in astro-
cytes and neurons. Glutamine is released from
astrocytes by system N transporters and trans-
ferred into neurons by system A transporters
(both members of the slc38 family; Chaudhry
et al., 2002). Once in the presynaptic terminal,
glutamine is then reconverted to glutamate by
the mitochondrial enzyme, phosphate-activated
glutaminase. Following its resynthesis from glu-
tamine, glutamate is available directly as a neu-
rotransmitter for packaging in synaptic vesicles
in excitatory terminals or is converted to GABA
by decarboxylation in inhibitory terminals (the
glutamate-glutamine-GABA cycle). Release of
synaptic glutamate or GABA then completes
the cycle (reviewed in Bak etal., 2006; Chaudry
etal.,2002).
Given the primary role of the glutamate-
glutamine cycle in taking up and inactivating
glutamate, and the linkage between this cycle
and the local neurotransmitter economy in syn-
apses (discussed earlier), it would be expected
that pharmacological approaches interfering
with the cycle at any stage would rapidly impact
neurotransmitter supply and synaptic function.
Several studies have examined the role of the
glutamate-glutamine cycle in maintaining inhib-
itory synaptic transmission. In hippocampal and
thalamic inhibitory synapses, there was a marked
dependence of transmitter release on continued
function of the glutamate-glutamine cycle. In
functional studies, it was found that application
of a glutamine synthetase antagonist, glutamate
uptake blockers, or glutamine transporter block-
ers all rapidly depleted synaptic release of GABA,
which could be reversed by a supply of exogenous
glutamine (Fricke etal., 2007; Liang etal., 2006;
Yang and Cox, 2011). This is entirely consistent
with the astrocytic glutamine-glutamate cycle
plays a primary role in providing inhibitory syn-
apses with substrates for GABA synthesis.
In contrast, in similar experimental stud-
ies, excitatory synaptic transmission was found
to be much less sensitive to disruption of the
glutamate-glutamine cycle. Although several
studies have found that epileptiform activity
in vitro depends on glutamine supply (Bacci
et al., 2002; Tani et al., 2007, 2010), excitatory
synaptic transmission exhibited less evidence
for a similar reliance. Under conditions where
the glutamate-glutamine cycle was blocked by
application of a glutamine synthetase antago-
nist (methionine sulfoximine), no compromise
in excitatory synaptic function was evident
until prolonged periods of synaptic release were
induced, encompassing hours or thousands of
synaptic stimuli (Kam and Nicoll, 2007; Tani
et al., 2014). So, although inhibitory synaptic
function appears directly linked to astrocytic
derived glutamine, continued excitatory synap-
tic transmission appears less dependent on this
astrocytic-derived glutamate precursor. This
may be due to higher reserve pools of cytoplas-
mic glutamate being accessible in excitatory
relative to inhibitory neurons, buffering the
immediate effects of loss of glutamate-glutamine
cycle function for significant periods of activ-
ity. Comparing the distinct effects of disrupting
the glutamate-glutamine cycle in inhibitory and
excitatory synapses, it is likely that the predomi-
nant proximate effect would be to reduce GABA
release from inhibitory synapses, potentially
enhancing circuit excitability.
One notable consequence of the depend-
ence of GABA replenishment on the glutamate-
glutamine-GABA cycle is that this may serve
as an intimate link, scaling inhibitory synaptic
efficacy to ongoing levels of glutamate release.
Since appropriate inhibition is necessary to con-
strain the potential for excessive glutamater-
gic excitation, this linkage to the astrocytic
glutamate-glutamine cycle may provide GABA
on demand when excitation levels are high and
reduce GABA levels under low activity condi-
tions. As already mentioned, this glutamate
release to glutamine resupply linkage occurs
within milliseconds, enhancing the potential for
an intimate interrelationship between excitatory
activity and supply of metabolites to sustain inhi-
bition (Uwechue etal., 2012). Interestingly, many
of the key players in the glutamate-glutamine
cycle exhibit functional regulation at both the
transcriptional and the posttranslational level
(e.g., Broer etal., 2004; Nissen-Meyer etal., 2011;
Yang etal., 2009), suggesting that differing activ-
ity levels in brain circuits may alter the efficacy of
 Glutamate Homeostasis as a Regulator of Neurotransmitter
this cycle by influencing expression and function
of these key players.
T H E G L U TA M AT E - G L U TA M I N E
CYCLE IN EPILEPSY
Many central nervous system disease states,
including epilepsy, Parkinsons disease,
Alzheimers disease, stroke, and traumatic brain
injury, are associated with the development of
a series of astrocytic alterations, collectively
termed reactive astrocytosis. The disease in
which reactive astrocytosis has arguably received
the most experimental attention is temporal lobe
epilepsy (TLE). The pathological hallmark of
TLE is mesial temporal sclerosis, defined as seg-
mental neuronal loss and reactive astrocytosis in
the hippocampus and associated temporal lobe
structures.
In addition to proliferation of astrocytes
and anatomic changes in cellular shape and
size, there are numerous functional and bio-
chemical changes in astrocytes accompanying
the development of reactive astrogliosis. These
include increased expression of glial fibrillary
acidic protein (GFAP) and vimentin, downreg-
ulation in both Kv4.1 and aquaporin-4 expres-
sion, reduced gap junction coupling between
astrocytes, and decreased expression of GLT-1
and glutamine synthetase (Eid etal., 2004; van
der Hel et al., 2005; reviewed in Wetherington
et al., 2008). Associated with the presence of
astrogliosis and loss of glutamine synthetase in
the hippocampus of patients with TLE, there
are abnormally high concentrations of extracel-
lular glutamate (Cavus etal., 2005; During and
Spencer,1993).
In patients with TLE, there is a juxtaposition
between pathological hyperexcitability (seizure
predisposition), astrogliosis, and accompany-
ing loss of glutamine synthetase expression. This
raises the related questions:Is loss of glutamine
synthetase (and the concomitant damaged func-
tion of the glutamate-glutamine cycle) a cause
or a consequence of epilepsy? Do these changes
contribute to seizure susceptibility, or are they
merely a reflection of the severe damage seen
in the hippocampi of patients with TLE? To
address these questions, Eid and colleagues
(2008) continuously infused the glutamine syn-
thetase antagonist methionine sulfoximine into
the hippocampus of rats for 28 days and moni-
tored animals for epilepsy development (see also
Wang etal., 2009). They found that methionine
sulfoximinetreated animals developed epilepsy
and neuropathology similar to mesial temporal
47
sclerosis in TLE. This supports a causal/con-
tributory linkage between glutamine synthetase
downregulation and the circuit hyperexcitabil-
ity generating seizures, the hallmark symptom
ofTLE.
ASTROGLIOSIS EFFECTS ON
SY NAPTIC TR ANSMISSION
A N D C I R C U I T E X C I TA B I L I T Y
Astrogliosis is a pathological hallmark of the
hippocampus in TLE patients. As discussed
previously, the development of astrogliosis is
accompanied by a number of important changes
in astrocyte structure and function, including
a marked downregulation in expression of glu-
tamine synthetase. Recent experiments have
explored the potential role of astrogliosis in excit-
ability defects that underly seizure predisposi-
tion in TLE. The role of astroglial reactivityin
the etiology of epilepsy is poorly understood.
This is in large part because of the many poten-
tially contributory changes in diverse processes
evident following an epileptogenic injury, which
complicate the interpretation of results. To iso-
late the role that reactive astrogliosis may play
in generating hippocampal excitability defects,
Ortinski etal. (2010) induced reactive astrocyto-
sis in isolation using a cell-specific viral strategy
and then examined the effects of gliosis on circuit
excitability.
Experimental astrogliosis induced by the
viral strategy employed by Ortinski and col-
leagues (2010) had several similarities to reac-
tivity seen in vivo. Astrocytes overexpressing
Green Fluorescent Protein following a high titer
viral infection exhibited hypertrophy, increased
vimentin, and GFAP expression, as well as a
downregulation in glutamine synthetase expres-
sion, all of which were viral-titer dependent.
Importantly, neighboring pyramidal neurons
and interneurons showed no alterations in their
anatomy and intrinsic properties. In hippocam-
pal regions with reactive astrocytes, GABAergic
inhibition, but not glutamatergic excitation, was
significantly compromised. To assess effects
of gliosis on circuit excitability in area CA1 of
the hippocampus, the response to activation of
direct cortical inputs to the distal dendrites of
CA1 neurons was compared between controls
and astrogliotic hippocampal slices. This path-
way has been previously shown to be extensively
hyperactive in epileptic animals (Ang etal., 2006;
Denslow etal., 2001; Wozny etal., 2005)and to be
powerfully regulated by local circuit inhibition
(Ang etal., 2005; Empson and Heinemann, 1995;
48 Part I: Homeostatic Regulators
Soltesz, 1995). In regions of extensive gliosis, there
was significant hyperactivation of CA1 by direct
cortical inputs, and this was reversed by supply
of exogenous glutamine (Ortinski et al., 2010).
Finally, all of these astrogliosis-mediated effects
on inhibitory synaptic function could be mim-
icked by blockers of the glutamate-glutamine
cycle, and these alterations in gliotic hippocampi
were occluded by these same blockers and
reversed by supply of exogenous glutamine, all
supporting a role for gliosis-induced glutamine
synthetase downregulation in the circuit excit-
ability defects underlying epilepsy.
CONCLUSIONS
One primary mechanism contributing to regu-
lation of glutamate homeostasis in the brain
is the glutamate-glutamine cycle. This cycle
involves partitioned expression of various trans-
porters and enzymes in perisynaptic astro-
cytic processes and presynaptic terminals of
excitatory and inhibitory neurons. It is critical
in regulating pathological extracellular accu-
mulation of glutamate, termination of synaptic
responses, and in resupply of neurotransmitter
in both excitatory and inhibitory neurons. This
glutamate-glutamine cycle is not only critical
in normal functions of synapses. Dysfunction
in this cycle may contribute to the etiology of
many neurological disease states, including epi-
lepsy. Epilepsy is associated with the develop-
ment of significant gliosis, which, among other
changes, results in a significant downregula-
tion in expression of the keystone astrocytic
enzyme in the glutamate-glutamine cycle, glu-
tamine synthetase. Experimental induction of
reduced glutamine synthetase function results
in epilepsy and triggers hippocampal circuit
hyperexcitability and disinhibition. These data
not only provide mechanistic insight into cir-
cuit dysfunction underlying epilepsy and other
disorders associated with gliosis but also sug-
gest novel, metabolic strategies for the develop-
ment of therapeutic agents to better treat such
diseases.
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4
Homeostasis ofNeuronal Excitability Via
Synaptic and Intrinsic Inhibitory Mechanisms
J O C H E N M E I E R , M A R C U S S E M T N E R , A N D J A K O B W O L FA R T
T H E M U LT I P L E FA C E T S
OFINHIBITION VIA
G A B A A N D G LY C I N E
RECEPTORS
In the mid-20th century, the mysterious fac-
tor I included in brain extracts that inhibited
impulse generation in neurons was discovered
to be -aminobutyric acid (GABA), that is, the
main fast inhibitory transmitter of the brain
(Bazemore et al., 1956). The other fast central
inhibitory transmitter is glycine (Curtis et al.,
1968). The respective ionotropic receptors (i.e.,
ligand-gated ion channels) are GABA type
A receptors (GABAA Rs) and glycine receptors
(GlyRs), which are co-expressed in central neu-
rons (Jonas etal., 1998; Le-Corronc etal., 2011).
They belong to the superfamily of acetylcholine
receptor type ligand-gated ion channels (also
known as Cys-loop receptors), share common
protein architecture and are permeable for both
chloride and bicarbonate (Bormann etal., 1987;
Kaila and Voipio, 1987). GABAA R and GlyR
anion channels consist of five subunits, and each
subunit consists of an extracellular N-terminal
neurotransmitter binding domain stabilized
by disulfide bridges, four transmembrane seg-
ments, and a large cytosolic protein loop between
the third and fourth transmembrane domain
at the C-terminus (Thompson et al., 2010). The
large cytoplasmic loop provides the structural
framework for functional diversification through
posttranscriptional and posttranslational
mechanisms.
As a basic principle for all ion channels, the
direction of ion currents (I) depends on the
equilibrium potential (E) of the respective ion
across the plasma membrane, which in turn is
dependent on the ion concentration gradient.
The GABA A Rs and GlyRs are permeable for
chloride and bicarbonate, the concentrations of
which are dynamically regulated by ionic trans-
port and enzyme activity, respectively (see later
discussion for details). Generally, the opening
of ion channels has two simultaneous effects: a
decrease in membrane resistance (R m), and a
shift of membrane potential (Vm) toward E.The
R m decrease always leads to shunting (inhibition)
due to Ohms law (U=R*I). The Vm effect depends
on the difference in E versus membrane poten-
tial (Vrest, if the cell is at rest). Since E of chlo-
ride (ECl) is close to the Vrest of many cell types,
the net effect of GABA A R/GlyR channel open-
ing is difficult to predict. The easiest case is an
ECl negative of Vrest (Figure4.1a); here GABA AR/
GlyR activation is clearly inhibitory in the sense
that it prevents action potential (AP) genera-
tion. If ECl is positive of Vrest and positive of the
threshold for AP generation (e.g.45 mV), then
GABA A R/GlyR activation is initially excitatory.
However, this effect is reversed when GABA AR/
GlyR activation is slow and long-lasting, because
voltage-gated sodium (Nav) channels progres-
sively inactivate, which prevents AP generation
(Figure4.1b, depolarization block). If ECl is posi-
tive or equal to Vrest but below the AP threshold,
GABA A R/GlyR activation will depolarize but
itself not evoke APs; instead it will shunt fur-
ther incoming excitatory postsynaptic potentials
(EPSPs), thus reducing the probability of AP
generation (Figure4.1c, shunting inhibition). In
addition to direct effects, GABA A R/GlyR acti-
vation may have secondary consequences; for
example hyperpolarization can trigger rebound
excitation via deinactivation of Nav channels.
Thus the transmembrane chloride gradient
needs a thorough homeostatic regulation, which
is achieved, among other mechanisms, via chlo-
ride transporters.
52 Part I: Homeostatic Regulators
(a) (b) (c)
Vm Vm Vm
+20 +20 +20
45 45 45
ECl
Vrest Vrest Vrest
ECl
Hyper- Depol Shunt
pol block
FIGURE 4.1 Scheme depicting three forms of
inhibition via chloride channel opening (i.e.,
GABAAR/GlyR activation). (a) When the chloride
equilibrium potential (ECl) is negative of the rest-
ing membrane potential (Vrest), hyperpolarization
predominates. (b) If ECl is around the threshold for
voltage-gated sodium (Nav) channel activation (around
-45mV) and depolarization relatively slow, Nav chan-
nels inactivate, decreasing the likelihood for AP gen-
eration (depolarization block). (c) Shunting inhibition
occurs due to the resistance reduction at any potential,
but its effect becomes predominant if the chloride con-
ductance is large and ECl is close to Vrest because then the
cell is clamped to ECl and all inputs are shuntedaway.
Basically, two families of cation-chloride
cotransporters exist and work in oppo-
site directions. Transporters of the sodium-
potassium-chloride cotransporter (NKCC) type
use the energy of the electrochemical sodium
gradient to load cells with chloride. On the other
hand, potassium-chloride co-transporter (KCC)-
type transporter-mediated chloride extrusion is
driven by the electrochemical potassium gradi-
ent. Hence, both transporter types are fueled by
the sodium-potassium ATPase, which establishes
the respective ionic gradients. For the purpose of
simplicity, we focus on NKCC1 andKCC2.
During early postnatal development, the
intracellular (somatic) chloride concentration
is estimated to range from 25 mM to 40 mM
(Blaesse etal., 2009), which likely reflects prepon-
derant expression of NKCC1 at this developmen-
tal stage (Plotkin etal., 1997; Hbner etal., 2001).
It has also been established that developmental
upregulation of KCC2 expression and func-
tion correlates with the progressively decreas-
ing depolarizing effect of GABAA R or GlyR
activation, likely reflecting the KCC2-dependent
reduction of the intracellular chloride concentra-
tion to about 4 to 7 mM (Rivera etal., 1999; Tyzio
etal.,2008).
Besides the developmental regulation of intra-
cellular chloride concentration through NKCC1
and KCC2 (Ben-Ari, 2002), spatial aspects of
chloride transporter function play a crucial role
for the nature of GABA action. The intracellular
chloride concentration was found to be highly
variable between axonal, somatic, and den-
dritic compartments or even from one dendritic
ECl
branch to its neighbors (Figure4.2a, 4.2b; Duebel
et al., 2006; Waseem et al., 2010; Friedel et al.,
2013). Thus, depending on the cellular compart-
ment, activation of GABA ARs or GlyRs can have
different effects on the membrane potential of
the same neuron and be inhibitory due to hyper-
polarization or shunt inhibition (Banke and
McBain, 2006; Tyzio etal., 2008; Glickfeld etal.,
2009) or depolarizing and excitatory, the latter
eventually facilitating plasticity of glutamater-
gic synaptic transmission (Staley et al., 1995;
Turecek and Trussell, 2001; Ruiz et al., 2010).
Indeed, the intracellular chloride concentration
in the axonal compartment of glutamatergic
neurons is rather high (about 52 mM; Waseem
and Fedorovich, 2010), and, consequently, activa-
tion of chloride channels at the axon initial seg-
ment or in presynaptic axon terminals is usually
depolarizing (Khirug etal., 2008)and excitatory
as it facilitates glutamate release (Turecek and
Trussell, 2001; Ruiz etal., 2010; Figure4.2a, 4.2b).
However, it should be noted that excitation via
GABA is less common than inhibition and the
effect of GABAergic excitation is also less pro-
nounced than that of glutamatergic transmis-
sion, due to the different equilibrium potential of
chloride (~ 1020 mV) and sodium (~ 120 mV)
atVrest.
As GABA A Rs and GlyRs are also perme-
able to bicarbonate (Bormann etal., 1987), ionic
crosstalk between chloride, bicarbonate, and
potassium influences the functional versatil-
ity of these receptors. Carbonic anhydrase is an
important enzyme in this context as it catalyzes
the rapid interconversion of carbon dioxide and
water to bicarbonate and protons (or vice versa).
In pyramidal neurons, expression of carbonic
anhydrase VII is developmentally upregulated
just like KCC2 but with a delayed time course
(Rivera etal., 2005). The equilibrium potential of
bicarbonate (EHCO3) is about -10mV (Figure4.2c);
that is, bicarbonate efflux during GABA AR or
GlyR activation is rather depolarizing (Kaila
 Homeostasis of Neuronal Excitability 53

(a) (b) (c)

Soma Dendrites Axon Vm

Axon +20 +20 +20 +20
Soma AIS

10 EHCO3
Dendrites
45 45 45 ECl 45
ECl ECI
Vrest ECl Vrest Vrest Vrest

(d)

GABA/glycine

(Ecl) (Synchronization)
HCO3 Cl K+
(Depolarization)
GlyR
KCC2

GABAAR Gephyrin

H2O HCO3
CA7
CO2 H+ Cl
(Hyperpolarization, Ecl)

FIGURE 4.2 Subcellular heterogeneity of chloride and bicarbonate equilibrium potentials (ECl
and EHCO3, respectively). (a) Immunochemical staining of a hippocampal neuron in cell culture showing the
somato-dendritic compartment (Winkelmann and Meier, unpublished results). The axon with axon initial seg-
ment (AIS) is drawn schematically. (b) Scheme of ECl range between different neuronal compartments. In particu-
lar, the axonal ECl can be depolarized compared to the soma. See Figure 4.1 for functional consequences. (c) The
EHCO3 is more positive than ECl, irrespectively of the neuronal compartment. (d) Upon pronounced GABAergic
transmission (e.g., during seizure-like activity) in the presence of carbonic anhydrase (CA7) and the chloride
transporter KCC2, the ECl may increase due to loading of neurons with chloride (during hyperpolarization) if local
chloride extrusion capacity is saturated (KCC2), which facilitates HCO3-dependent depolarizing GABAergic
transmission in the adult nervous system. Furthermore, KCC2 can contribute to synchronization of neural net-
work activity by increasing the interstitial potassium concentration. For citation and details see maintext.

and Voipio, 1987). The net effect of GABA A R or
GlyR activation in the presence of carbon diox-
ide and carbonic anhydrase depends on the rela-
tive permeability of bicarbonate versus chloride
(PHCO3/chloride), which can be calculated using the
Goldman-Hodgkin-Katz equation and ranges
between 0.2 and 0.6 (Kaila, 1994). This ratio
suggests that GABA AR or GlyR activation can
trigger a biphasic change of Vm (Figure 4.2d).
Several studies have shown that tonic GABA A R
activation loads neurons with chloride due
to GABA AR-mediated chloride influx that is
facilitated by bicarbonate-dependent depo-
larization (efflux; Kaila and Voipio, 1987; Kaila
et al., 1989; Staley and Proctor, 1999). If the
GABA AR-mediated current exceeds local chlo-
ride transport capacity, a significant positive
shift of ECl will occur (Staley etal., 1995), while
EHCO3 is kept fairly constant due to rapid buffering
through carbonic anhydrase activity (Pasternack
et al., 1993; Figure 4.2d). This mechanism of
neuronal aniondependent plasticity may pref-
erentially apply to dendrites as they show a high
receptor-to-volume ratio (Qian and Sejnowski,
54 Part I: Homeostatic Regulators
1990). Thus, in response to GABA application or
high-frequency GABAergic synaptic transmis-
sion, dendritic GABA AR activation in mature
neurons gives rise to a biphasic response con-
sisting of initial chloride-dependent hyper-
polarization followed by bicarbonate-driven
depolarization; in acute slice preparations, hip-
pocampal pyramidal neurons are actually excited
by this depolarization, which accounts for fre-
quency modulation of synaptic NMDA receptor
activation and synchronous gamma-frequency
(2080 Hz) firing in response to high-frequency
stimulation (Staley etal., 1995; Ruusuvuori etal.,
2004). In addition, (pathological) neuronal net-
work synchronization can occur under these
conditions (Figure4.2d) and involve an increase
in interstitial potassium concentration through
the KCC2-dependent net efflux of potassium and
chloride (Viitanen etal., 2010), which depolarizes
the neuronal membrane potential but may also
reverse the neuronal chloride load in a homeo-
static way, provided that local chloride transport
capacity is not saturated (Voipio and Kaila, 2000;
Rivera etal., 2005). This ionic crosstalk between
chloride, bicarbonate, and potassium can locally
and transiently change the efficacy ratio between
glutamatergic and GABAergic (and/or glyciner-
gic) neurotransmission and thereby contribute to
synaptic plasticity and homeostatic regulation of
high-frequency network activity. However, such
a complex interplay of ionic mechanisms must
be tightly controlled because runaway excitation
may result.
Collectively, these considerations suggest
that homeostasis of neuronal activity involves an
array of spatiotemporally regulated inhibitory
mechanisms of ionic crosstalk.
INTRINSIC PLASTICITY
What Is Intrinsic Plasticity?
The plasticity of nonsynaptic ion channels is sum-
marized by the term intrinsic plasticity (Siegel
etal., 1994; Zhang and Linden, 2003; Marder and
Goaillard, 2006). Although in principle post-
synaptic ion channels such as AMPA receptors
could count as intrinsic, the synapse is a special-
ized structure; therefore homeostatic plasticity
affecting postsynaptic receptors is differentiated
as synaptic scaling (Davis and Goodman, 1998;
Turrigiano et al., 1998; Desai, 2003), and this
subject is separately treated in chapters7 and 10.
Synaptic plasticity is a prominent cellular mecha-
nism involved in learning and memory, and this
is also the context in which intrinsic plasticity is
most frequently discussed (Abbott and Nelson,
2000; Turrigiano and Nelson, 2000; Davis and
Bezprozvanny, 2001; Daoudal and Debanne,
2003; Zhang and Linden, 2003; Debanne and
Poo, 2010). In particular, the possibility of scaling
excitability without changing the relative differ-
ences produced by synaptic plasticity in form of a
metaplasticity is an attractive feature of intrin-
sic plasticity (Abraham and Bear, 1996; Abbott
and Nelson, 2000; Turrigiano and Nelson, 2000;
Davis and Bezprozvanny, 2001; Zhang and
Linden, 2003; Narayanan and Johnston, 2010).
Somewhat interfering, but also popular, is the
concept that intrinsic plasticity could itself be
a mechanism for learning and memory (Alkon,
1984; Marder etal., 1996; Daoudal and Debanne,
2003; Disterhoft and Oh, 2006; Mozzachiodi and
Byrne, 2010; Turrigiano, 2011). Additionally,
intrinsic plasticity has been discussed in the
context of cell type identity and variability
(Golowasch et al., 1999; Padmanabhan and
Urban, 2010; Marder and Taylor, 2011), develop-
ment (Turrigiano and Nelson, 2004; Marder and
Goaillard, 2006), and brain diseases, in particu-
lar epilepsy (Beck and Yaari, 2008; Wolfart and
Laker,2015).
Much of neuronal development depends on
intrinsic plasticity. The properties of immature
neurons are different from those of adult neurons
(Spitzer, 1999; Moody and Bosma, 2005; Seki
et al., 2012), and neurons need to develop their
functions while their morphology and synaptic
inputs are being built (Llinas, 1988; Spitzer, 1999;
Moody and Bosma, 2005; Marder and Goaillard,
2006; Overstreet-Wadiche, and Westbrook, 2006;
Pratt and Aizenman, 2007). But also, follow-
ing maturation, neurons need to maintain their
functional phenotype despite constant recycling
(turnover) of short-lived ion channel proteins
(Marder and Goaillard, 2006). Thus neurons
cannot rely solely on a genetically encoded pro-
tein expression in a feed-forward open loop man-
ner; they constantly need homeostatic feedback
mechanisms to adjust their excitability (Davis,
2006; Marder and Goaillard,2006).
Short-Term Intrinsic Plasticity
Neuronal excitability is due to positive feedback
effects of voltage-gated calcium (Cav) and Nav
channels on membrane voltage (with excitation
producing excitation). Clearly, such feedback
cannot exist without inhibitory control. Starting
from the millisecond-fast APs up to day-to-day
cellular milieu changes, evolution has connected
all excitatory signals (e.g. depolarization, calcium

influx, and energy consumption) directly to a
battery of appeasing potassium channels with
many subtypes outnumbering those of excita-
tory channels (Coetzee et al., 1999; Hille, 2001;
Catterall etal., 2005; Gutman etal., 2005). Thus
control of excitation is an important function of
potassium channels; however, their even more
fundamental purpose is maintenance of Vrest for
membrane transport of metabolites, and this
function is evolutionarily much older than the
appearance of excitable cells with Nav and Cav
channels (Anderson and Greenberg, 2001). In
other words, compared to potassium channel
homeostasis, synaptic plasticity is evolutionarily
a recent luxury (Jan and Jan, 1994; Anderson and
Greenberg, 2001; Hedrich,2012).
An important factor in intrinsic plas-
ticity is the timing of the inducing stimuli.
Depolarization and calcium influx are thought to
be the key signals involved in both synaptic and
intrinsic plasticity (Thoby-Brisson and Simmers,
1998; Narayanan and Johnston, 2010; Wiegert
and Bading, 2011). Atrain of APs directly elicits
calcium- and sodium-dependent potassium con-
ductances (KCa) mediating after hyperpolariza-
tions (AHPs) of different lengths (up to minutes)
depending on the duration of the depolarization
(Gustafsson and Wigstrom, 1983; Schwindt etal.,
1989; Kim and McCormick, 1998; Sanchez-Vives
etal., 2000; Tanner etal., 2011). Although it may
be confusing to count the AHP itself as intrin-
sic plasticity, many AHP-evoking stimuli are
certainly overlapping with those evoking some
forms of intrinsic memory (Marder etal., 1996;
Golowasch et al., 1999; Aizenman and Linden,
2000). Perhaps one may speak of short-term
intrinsic plasticity in analogy to short-term syn-
aptic plasticity (changes in synaptic strength that
last up to few minutes; Zucker and Regehr,2002).
Synaptic PlasticityRelated
Intrinsic Plasticity
It is well established that experimental stimu-
lation protocols that elicit long-term synaptic
plasticity simultaneously trigger long-lasting
intrinsic plasticity. Already in the earliest in vivo
long-term potentiation (LTP) experiments, the
potentiation of extracellularly recorded popu-
lation spikes could not be fully explained by
potentiation of the EPSP alone (Bliss and Lomo,
1973). This effect involved alterations of syn-
aptic inhibition and/or intrinsic plasticity and
was called excitation-spike (E-S) coupling (Bliss
and Lomo, 1973; Andersen etal., 1980; Daoudal
and Debanne, 2003). In the hippocampus, E-S
Homeostasis of Neuronal Excitability 55
coupling reinforces synaptic plasticity in both
directions: E-S potentiation supports LTP and
E-S depression accompanies long-term synap-
tic depression (LTD; Daoudal etal., 2002; Wang
et al., 2003). However, since E-S regulation is
synapse-specific (Daoudal and Debanne, 2003),
it lacks the advantage of cell-wide homeostatic
scaling. Also, behavioral experiments found that
associative learning paradigms permanently
reduced potassium conductances mediated by
rapidly inactivating A-type (IA) and KCa types
of potassium channels in certain types of neu-
rons (Alkon, 1984; Scholz and Byrne, 1987; de
Jonge et al., 1990). Later, LTP-inducing proto-
cols were shown to increase the intrinsic excit-
ability of cerebellar and hippocampal neurons
in slice preparations (Aizenman and Linden,
2000; Armano etal., 2000; Xu etal., 2005). Not
surprisingly, considering the inducing protocols,
the cellular mechanisms underlying this intrin-
sic plasticity appear similar to those involved
in LTP; that is, they depend on calcium influx,
NMDA receptor activation, and activation of
Ca 2+/calmodulin-dependent protein kinase II
(Aizenman and Linden, 2000; Armano et al.,
2000; Xu etal., 2005; Groth etal., 2011). However,
such intrinsic plasticity accompanying synaptic
plasticity can also be evoked via strong post-
synaptic depolarization alone (Aizenman and
Linden,2000).
In pyramidal neurons of hippocampus and
cortex, two currents prominently involved in
intrinsic plasticity are the hyperpolarization-
activated cation current (IH mediated via HCN
channels), which mediates mostly shunting inhi-
bition, and the aforementioned IA (members of
Kv1, Kv3, and Kv4 subfamilies). Both IA and IH are
downregulated during LTP induction (Wang
etal., 2003; Frick etal., 2004), possibly by NMDA/
calcium-triggered internalization of membrane
proteins (Kim etal., 2007; Hyun etal., 2013). In
fact, the decrease of IA and IH during the plasticity
induction protocol can determine whether or not
the coincidence of synaptic excitation eventu-
ally induces LTP (Hoffman etal., 1997; Markram
et al., 1997; Magee, 1998; Spruston, 2008).
Interestingly, the very same LTP protocols can
lead to a reduction of excitability via IH increase
in the cornu ammonis region 1 (CA1) pyrami-
dal dendrites (Fan etal., 2005). This discrepancy
could arise from experimental differences, for
example, in the age of animals (Fan etal., 2005),
but there may also be a window for spike timing
dependent neuronal dynamics of intracellular
signaling cascades involving NMDA receptors or
56 Part I: Homeostatic Regulators
Cav channels, where the individual status/history
of the discerned neuron decides on the direction
of plasticity (Roth-Alpermann etal., 2006). These
examples identify ion channel changes as a pow-
erful metaplasticity mechanism for the bidirec-
tional control of the neuronal network (Abraham
and Bear, 1996; Narayanan and Johnston,2010).
Chronic Intrinsic Plasticity
Homeostatic intrinsic plasticity and synaptic
scaling related to changes of spontaneous activ-
ity on the time scale of days have been best stud-
ied in cell culture models (Marder et al., 1996;
Turrigiano, 1999). For example, bathing the cell
culture in blockers of synaptic inhibition not
only leads to synaptic downscaling of AMPA and
NMDA receptors but also to a long-term reduc-
tion of intrinsic excitability (Lissin et al., 1998;
Karmarkar and Buonomano, 2006). Similarly,
chronic depolarization via elevated extracel-
lular potassium, a major excitotoxic burden for
the cells, leads to permanent downregulation of
excitatory currents and upregulation of inhibi-
tory currents, even when AP activity is blocked
(Franklin et al., 1992; Golowasch et al., 1999;
Leslie etal., 2001; van Welie etal.,2004).
Although it is doubtful that there are physi-
ological conductances that can kill a neuron
rapidly by hyperpolarization, chronic silenc-
ing can eventually kill neurons (Eichler et al.,
2008). Therefore, and to achieve functional
homeostasis, it makes sense that chronic reduc-
tion of spontaneous AP activity (e.g., by bath-
ing cell cultures in Nav channel blockers or
AMPA and NMDA receptor inhibitors) results
in a compensatory long-term downregulation
of dendrotoxin-sensitive delayed rectifier (Kv1)
channels in cornu ammonis region 3 (CA3)
pyramidal cells (OBrien etal., 1998; Turrigiano
etal., 1998; Desai etal., 1999; Turrigiano, 1999;
Cudmore and Turrigiano, 2004; Karmarkar
and Buonomano, 2006). Similarly, chronic
overexpression of inward rectifier potassium
(K ir) channels involved in the resting potential
(K ir2.1), leads to a compensatory downregu-
lation of Kv currents in CA1 pyramidal cells
(Okada and Matsuda, 2008). In addition to the
quantitative ion channel expression, the sub-
cellular location of ion channels is also under
activity-dependent homeostatic control (Grubb
and Burrone, 2010; Kuba et al., 2010). All of
these mechanisms of neuronal intrinsic plastic-
ity contribute to a constancy of neuronal firing
rates (Marder et al., 1996) and most likely are
realized via a regulation on the transcriptional
level (Kirchheim etal., 2013). In addition, com-
plex activity patterns can be homeostatically
controlled. For example, the spontaneously
bursting phenotype of lobster stomatogastric
ganglion (STG) neurons, which is lost when
cut off from the network, is autonomously rees-
tablished in cell culture via intrinsic plasticity
(Turrigiano etal., 1994; Turrigiano etal., 1995;
Thoby-Brisson and Simmers,1998).
Experimental gene or RNA modulation has
revealed the enormous flexibility of intrinsic
plasticity. For example, the deletion of GABA A R
channels responsible for a tonic leak current
(6 and subunits) resulted in upregulation of
the two-pore potassium (K2P) channels, which
exactly compensated for the lost conductance
leak (Brickley etal., 2001). Furthermore, overex-
pression of Kv4 A-type channels via RNA injec-
tion in cultured STG neurons led to a functional
compensation through IH upregulation, which
restored the STG pacemaker pattern (MacLean
etal., 2003). Astonishingly, this IH plasticity was
observed even when nonconducting Kv4 chan-
nels were employed, that is, in the absence of
any functional feedback (MacLean et al., 2003;
MacLean et al., 2005). The latter indicates that
some forms of intrinsic plasticity may actually
risk feed-forward open loop regulation. Most of
the discussed forms of intrinsic plasticity, how-
ever, are realized most likely via proper home-
ostasis, that is, via intracellular set point and
feedback mechanisms that keep the neuronal
activity constant even in a changing environ-
ment (Davis, 2006). The targeted set point of
AP firing appears in most cases to be imple-
mented via the intracellular calcium level, which
can be detected by calcium sensors such as the
calcium-calmodulin-dependent protein kinase
II; this instrument faithfully translates AP fre-
quency into kinase activity and downstream
activation of transcription factors (LeMasson
et al., 1993; De Koninck and Schulman, 1998;
Davis and Bezprozvanny, 2001; Weston and
Baines,2007).
In summary, intrinsic homeostatic plas-
ticity is the capacity of cells to adapt ion chan-
nel expression, distribution, and function. We
discussed two partially overlapping forms of
intrinsic plasticity: (a) a form that accompa-
nies synaptic LTP in a permissive manner (i.e.,
anti-homeostatic) and (b) an evolutionarily
older form that counteracts deviations from Vrest,
in particular via potassium channels.

POSTTR ANSCRIPTIONAL
A N D P O S T T R A N S L AT I O N A L
MECHANISMS FOR
SY NAPTIC AND INTRINSIC
H O M E O S TA S I S O F
INHIBITION
The aforementioned homeostatic inhibitory
mechanisms often involve intracellular signal-
ing on RNA and protein levels, which includes
RNA mechanisms (splicing and editing) and
posttranslational modification of proteins (phos-
phorylation). Here we discuss examples of such
molecular plasticity.
R NA SPLICING AND
EDITING INNEURONAL
INHIBITORY PLASTICITY
As mentioned previously, chloride homeostasis
via chloride transporters such as NKCC1 is an
important determinant of GABAergic/glyciner-
gic inhibition. More than 10years ago, two RNA
splice variants of NKCC1 (NKCC1a/NKCC1b)
were isolated from human tissue (Vibat et al.,
2001), but information regarding their functional
role is still sparse. It is known that NKCC1b is
the preponderant RNA variant in the brain; in
contrast to NKCC1a, it lacks a protein kinase
Aconsensus site but contains a casein kinase II
site (Vibat et al., 2001). Regarding KCC2, two
splice variants (KCC2a/KCC2b) with different
N-terminal parts were identified (Uvarov et al.,
2007), and KCC2b seems to be upregulated
during development, whereas KCC2a expres-
sion apparently prevails in the neonatal brain
(Uvarov et al., 2009). Here again, RNA splicing
creates additional sites for protein modifica-
tion through phosphorylation, and, according
to their subcellular distribution, the different
KCC2a and KCC2b RNA splice variants seem
to function preferentially at distal and proximal
somatodendritic compartments, respectively
(Markkanen et al., 2014). However, RNA splice
variantspecific functional roles of KCC2 remain
to be determined.
Several RNA splice variants of GlyR-coding
gene transcripts exist, whereas only GABA A R 2
and 2 subunits seem to undergo functional diver-
sification through RNA splicing. GABA AR 2L
contains a 38 amino acidlong splice insert that
adds a potential calcium-calmodulin-dependent
kinase II phosphorylation site to the large cyto-
plasmic loop domain (McKinley et al., 1995).
Use of phosphomimetic molecular constructs
revealed that this site may be important for
Homeostasis of Neuronal Excitability 57
ATP-dependent regulation of run-down of
GABA-induced currents upon successive GABA
applications (Zhao etal., 2006; Zhao etal., 2009),
indicating that this site takes part in the regula-
tion of the excitation/inhibition ratio. Moreover,
the genomic sequence environment of the exon
10 is a hotspot for single nucleotide polymor-
phisms that give rise to expression of differently
spliced GABA AR 2 variants in patients with
schizophrenia (Zhao et al., 2006; Zhao et al.,
2009), a disease associated with deregulation of
the ratio between glutamatergic and GABAergic
synaptic transmission (Eichler and Meier, 2008).
RNA splicing of the GABA AR 2 subunit also
changes the protein sequence in the large cyto-
plasmic loop domain; the long 2L GABA A R
subunit contains eight additional amino acids
that harbor a protein kinase C phosphorylation
site (Whiting etal., 1990; Moss etal., 1992). RNA
splicing of GABA AR 2 is developmentally regu-
lated (Henneberger etal., 2005), varies in different
brain regions (Gutierrez etal., 1994), and is altered
in patients with schizophrenia (Huntsman etal.,
1998). Phosphorylation of the protein kinase C
site in the GABAA R 2L splice variant was impli-
cated in interaction with gephyrin (Meier and
Grantyn, 2004b), a postsynaptic scaffold protein
that facilitates neurotransmitter receptor stabi-
lization at GABAergic and glycinergic synapses
(Kirsch et al., 1993; Essrich et al., 1998; Meier
etal., 2001; Lardi-Studler etal., 2007). RNA splic-
ing of GlyR-coding gene transcripts also leads to
sequence variation in the large cytoplasmic loop
domain of 1 and 3 subunits (Malosio et al.,
1991; Nikolic etal., 1998)or changes the protein
sequence of the ligand-binding domain of GlyR
2 (Kuhse etal., 1991). While the functional rel-
evance of GlyR 1 RNA splicing remains largely
unclear, it is well established that GlyR 2 splic-
ing changes apparent neurotransmitter affinity
(Miller et al., 2004). In contrast, GlyR 3 splic-
ing impacts on subcellular receptor distribution,
receptor clustering, and desensitization kinetics
(Nikolic etal., 1998; Eichler etal., 2009; Notelaers
et al., 2012; Winkelmann et al., 2014). In par-
ticular, expression of 2B- and 3K-GlyR RNA
splice variants were shown to be upregulated in
the sclerotic hippocampus of patients with idi-
opathic temporal lobe epilepsy (TLE), suggesting
that they may contribute to neurodegeneration
(Eichler et al., 2008). In fact, nonsynaptic tonic
GlyR activation and the resulting decrease of AP
firing due to shunting inhibition were shown to
be harmful to neurons with low KCC2 protein
58 Part I: Homeostatic Regulators
expression (Eichler et al., 2008). On the other
hand, upregulation of tonic GABA A R currents
in the dentate gyrus of the epileptic tissue may
have protective effects for some cell types (Young
etal., 2009), emphasizing the implications of cell
type- and brain region-specific mechanisms for
homeostatic inhibitory mechanisms.
Gephyrin is a central organizer of postsynap-
tic GABAergic and glycinergic domains (Tretter
etal., 2012). It consists of three domains (G, C, and
E), which evolved from exon shuffling of homolo-
gous bacterial proteins involved in molybdenum
cofactor synthesis (Schwarz etal., 2009). Due to
its central role in postsynaptic receptor stabiliza-
tion, RNA splicing of gephyrin-coding mRNA
could play a significant role in regulation of the
strength of GABAergic and glycinergic synapses.
Indeed, a considerable number of RNA splice
variants were isolated (Fritschy etal., 2008)and
basically fall into two groups. The glia cell-specific
RNA splice variant (containing the C3 peptide
insert in the C domain), for example, was shown
to be involved in molybdenum cofactor synthesis
in the brain (Smolinsky etal., 2008), while RNA
splicing in the G domaincoding part of gephy-
rin was reported to regulate synaptic receptor
clustering in neurons (Meier and Grantyn, 2004a;
Bedet etal., 2006). Sequence variations of the G
domain could be disease relevant as different
gephyrin splice variants lacking several exons in
the G domain are expressed in the hippocampus
of TLE patients and downregulate the strength
of GABAergic synaptic transmission (Frstera
etal., 2010). In fact, hippocampal regionspecific
dysfunction of the RNA splice machinery and
the resulting impairment of GABAergic synaptic
inhibition in the CA3 region may contribute to
increased excitability of CA3 pyramidal neurons
(Frstera et al., 2010). In addition to cell stress
as a reason for exon skipping during gephyrin
RNA splicing (Frstera et al., 2010), deletions
in the G domaincoding part of gephyrin can
also have a genetic origin, as was evidenced in a
wide range of patients with different neuropsy-
chiatric symptoms (Lionel et al., 2013). These
findings may define a new genetic and RNA
processingdependent hotspot for pathological
gephyrin protein sequence variation. However,
whether or not downregulation of postsynaptic
gephyrin is a pathological or beneficial mecha-
nism of neuronal plasticity depends on many
factors, including spatiotemporal regulation of
ionic equilibrium potentials and crosstalk with
intrinsic neuronal plasticity, as well as the type
of neuron where this form of molecular plasticity
occurs (Gonzalez,2013).
RNA splicing can furthermore contribute to
intrinsic potassium channel plasticity mentioned
before. For example, alternative splicing in the C
terminal domaincoding region changes subcel-
lular channel trafficking of Kv3.1 channels while
it does not affect biophysical channel proper-
ties. The differential propensities of Kv3.1a and
Kv3.1b splice variants for supporting fast spik-
ing could rely on axonal trafficking of Kv3.1b
(Gu etal., 2012). Indeed, splicing of Kv3.1-coding
RNA can act as a regulatory switch between neu-
ronal compartmentspecific forms of neuronal
plasticity because only the axonal Kv3.1b effec-
tively converted slow-spiking young neurons to
fast-spiking ones, while somatodendritic expres-
sion of Kv3.1a was far less effective at increasing
the maximal firing frequency (Gu et al., 2012).
This finding suggests different properties of som-
atodendritic and axonal compartments in the
expression of synaptic and/or intrinsic homeo-
static neuronal plasticity.
In addition to the functional diversification
through RNA splicing, neurotransmitter recep-
tors and potassium channels can undergo RNA
editing. Adenosine-to-inosine RNA editing of
GABA AR 3 is developmentally upregulated,
influences receptor surface expression (Ohlson
et al., 2007; Daniel et al., 2011), and eventually
contributes to the glutamate-dependent develop-
mental and cognitively relevant 3-to-1 subu-
nit switch of GABA AR expression (Henneberger
et al., 2005). Cytidine-to-uridine RNA edit-
ing of GlyR 2 and 3 subunits changes the
apparent neurotransmitter affinity by about
10-fold (glycine) or even 50-fold (taurine), ren-
ders GlyRs responsive to GABA (Meier et al.,
2005; Legendre et al., 2009), and even leads to
spontaneous receptor activity in the nominal
absence of a neurotransmitter (Kletke et al.,
2013; Winkelmann et al., 2014). The RNA edit-
ing of GlyR 2- and 3-coding gene transcripts
is increased in TLE patients (Eichler etal., 2008).
Depending on spatiotemporal coregulation of
GlyR RNA splicing and editing and subcellular
differences in receptor location, the resulting
GlyR protein variants can elicit neurodegenera-
tion via increased tonic inhibition of neuronal
AP firing (Eichler et al., 2008; Legendre et al.,
2009) and/or trigger neuropsychiatric symp-
toms reminiscent of the psychopathology of
epilepsy due to enhancement of presynaptic neu-
ronal function (Winkelmann et al., 2014). This

suggests that RNA processing of GlyR-coding
gene transcripts is a disease-promoting mecha-
nism of neuronal molecular plasticity. On the
other hand, adenosine-to-inosine RNA editing
of Kv1.1 channels and the resulting change in the
current-voltage relationship may serve as a com-
pensatory mechanism against epileptic seizures
(Streit etal., 2011; Kirchheim etal.,2013).
Collectively, RNA processing of gene tran-
scripts coding for anion permeable neuro-
transmitter receptors, the postsynaptic scaffold
protein gephyrin, and the plasticity of potassium
channels create considerable functional diversity
and an important level for the regulation of inter-
action of homeostatic synaptic and intrinsic plas-
ticity in the healthy and diseasedbrain.
Phosphorylation
One important switch for the regulation of
protein function that also plays a major role in
functional homeostasis is protein phospho-
rylation and dephosphorylation via kinases
and phosphatases, respectively. For exam-
ple, the functional properties of NKCC1 and
KCC2 cotransporters are reciprocally regulated
by serine/threonine phosphorylation. With
no lysine = K kinase-dependent activation
of NKCC1 and inhibition of KCC2 through
Ste20-related proline alanine-rich/oxidative
stress response-1 signaling cascades (Uvarov
et al., 2007; Kahle et al., 2010) may be consid-
ered homeostatic mechanisms because these
pathways are activated in conditions of hyper-
tonic cell stress (Xu etal., 2000)and depletion of
intracellular chloride (Ponce-Coria etal., 2008).
Activity-dependent bidirectional regulation of
KCC2 function through phosphorylation is well
established (Chamma et al., 2012; Kahle et al.,
2013), and several different serine/threonine and
tyrosine phosphorylation sites in the C-terminal
domain can contribute to up- or downregula-
tion of KCC2-mediated chloride extrusion (Lee
et al., 2007; Rinehart et al., 2009; Watanabe
et al., 2009). Oxidative stress and induction of
seizure activity change the KCC2 phosphoryla-
tion status through signaling events that involve
brain-derived neurotrophic factor, calcium, pro-
tein kinase C, TrkB tyrosine kinase, and protein
phosphatases, which triggers downregulation of
KCC2-mediated chloride extrusion in the adult
brain (Rivera et al., 2002; Rivera et al., 2004;
Wake etal., 2007; Lee etal., 2010; Lee etal., 2011).
Whether or not downregulation of KCC2 chlo-
ride transport activity contributes to homeostatic
Homeostasis of Neuronal Excitability 59
or maladaptive disease-promoting mechanisms
depends on developmental characteristics of
glycinergic and GABAergic neurotransmission.
Undoubtedly, the ratio between NKCC1 and
KCC2 activity is relevant for regulation of the
nature (excitatory or inhibitory) of glycinergic
and GABAergic signaling in the central nervous
system, and altered chloride homeostasis due to
the dysfunction of NKCC1 and/or KCC2 impacts
neuronal excitability and cell volume regulation
in disease (Kahle etal.,2008).
There is compelling evidence for
phosphory lation-dependent modification of
GABA AR function. Sixteen GABA A R subunits
are known to date, and most of them can be phos-
phorylated involving serine/threonine-directed
protein kinases or kinases with substrate speci-
ficity for tyrosine residues (Luscher etal., 2011).
Basically, most of the phosphorylation sites are
located in the large cytoplasmic loop domain,
which is a central determinant of subcellular
receptor distribution and biophysical receptor
properties.
The pioneering studies by Stephen Moss dis-
closed many different phosphorylation sites and
their impact on regulation of GABA AR surface
expression (Kittler and Moss, 2003; Vithlani
and Moss, 2009). Phosphorylation of GABAA R
2S (Krishek etal., 1994)may be relevant in the
context of homeostatic regulation of brain func-
tion as it modulates GABA AR stabilization in an
activity-dependent way, involving activation of
the NMDA receptor and the protein phosphatase
calcineurin in rapid dispersal of postsynaptic
2-containing GABA ARs (Muir etal., 2010). This
may present an antihomeostatic mechanism as it
supports glutamate-biased weighting of excita-
tory and inhibitory synapses in the context
of learning-associated synaptic plasticity. Serine
phosphorylation of the GlyR subunit through
protein kinase C may also contribute to maladap-
tive forms of synaptic plasticity as it downregu-
lates postsynaptic gephyrin clustering (Specht
etal.,2011).
Gephyrin phosphorylation plays a major role
in GABAergic synaptic plasticity. In particular,
glycogen synthase kinase GSK3-dependent and
extracellular signal-related kinasemediated
phosphorylation of two adjacent serine residues
in the C domain of gephyrin reduce the num-
ber of postsynaptic gephyrin clusters and func-
tional GABAergic synapses (Tyagarajan et al.,
2011; Tyagarajan etal., 2013), which may present
another antihomeostatic mechanism. Lithium is
60 Part I: Homeostatic Regulators
a GSK3 inhibitor that is used as mood-stabilizing
drug, which again suggests that gephyrin mal-
function contributes to neuropsychiatric disease
(Frstera etal., 2010; Lionel etal., 2013). However,
we also know that GSK3-signaling impacts on a
wide range of cellular processes in the healthy
and diseased brain, including Alzheimers dis-
ease, bipolar disorder, Fragile X syndrome, schiz-
ophrenia, and stroke (Kaidanovich-Beilin etal.,
2012). Therefore, whether or not GSK3-dependent
regulation of postsynaptic gephyrin contributes
to disease or represents a beneficial homeostatic
mechanism of GABAergic synapse plasticity
remains to be determined.
Also, potassium channels are phosphoryl-
ated, and phosphorylation influences surface
expression and function of potassium channels.
There is a large number of involved potassium
channels and an even larger number of phos-
phorylation sites (Vacher and Trimmer, 2011).
Here we limit ourselves to emphasizing that
phosphorylation-dependent internalization has
been implicated as an intracellular mechanism
in the functional downregulation of potassium
channels as it occurs during LTP protocols and
epilepsy (Bernard etal., 2004; Lugo etal., 2008;
Hyun etal.,2013).
E P I L E P S Y- R E L AT E D
INTRINSIC AND
SY NAPTIC PLASTICIT Y
Epilepsy-Related Intrinsic
Plasticity
It is not coincidental that neuronal plasticity
and epilepsy share mechanisms: LTP-evoking
protocols resemble epileptic seizures very much
(Leite et al., 2005). Those most frequently dis-
cussed are cases of pathological ion channel
changes (channelopathies), which lend them-
selves to be potentially seizure-causing ones
(Chang and Lowenstein, 2003; George, 2004;
Rogawski and Loscher, 2004; Beck and Yaari,
2008). Again, the usual suspects IA (e.g., Kv4.2)
and IH (e.g., HCN1) but also T-type Cav channels
were reported as misregulated; particularly in
TLE models without hippocampal sclerosis such
as systemic pilocarpine-induced status epilepti-
cus, CA1 pyramidal cells were found hyperex-
citable due to such ion channel changes (Tsaur
et al., 1992; Castro et al., 2001; Su et al., 2002;
Bernard et al., 2004; Bender and Baram, 2007;
Dyhrfjeld-Johnsen etal., 2009; Noam etal., 2011).
Because in TLE patients with hippocampal scle-
rosis this CA1 cell population is severely depleted,
we assume that the hypothesis of seizure-evoking
mechanisms via hyperexcitable CA1 pyramidal
cells applies for the TLE patient population with-
out hippocampal sclerosis (which also exists) or
represents a presclerosis stage of human TLE.
Many other dysfunctional potassium chan-
nels have been implicated in epileptic disor-
ders (Wolfart and Laker, 2015). Examples are
G proteincoupled inward rectifier (Girk, K ir3)
channels (Signorini et al., 1997), KCa channels
of the big conductance (Brenner etal., 2005; Du
etal., 2005)and small conductance type (Schulz
et al., 2012), as well as Kv channels (e.g., Kv7.2/3
[Biervert etal., 1998; Main etal.,2000] and Kv1.1
channels [Smart et al., 1998]). A new channel-
epsy nomenclature was even proposed for potas-
sium channelrelated epilepsies (DAdamo etal.,
2013). Less frequently reported but also impor-
tant are gain-of-function mutations in potas-
sium channels leading to enhanced excitability,
as in the case of big conductancetype potassium
channels, which allow higher firing frequencies
when upregulated (Brenner etal.,2005).
In contrast to the aforementioned procon-
vulsive channelepsies, anticonvulsive ion chan-
nel changes have received much less attention.
More recently though, several forms of such
homeostatic ion channel regulations have been
highlighted (Figure 4.3a). Dentate gyrus gran-
ule cells downregulate their excitability not only
in an animal model of TLE but also in sclerotic
hippocampi of TLE patients (Stegen etal., 2009;
Young etal., 2009; Stegen etal., 2012; Kirchheim
etal., 2013). It is perhaps related to the gate-keeper
function of the dentate (Hsu, 2007)that granule
cells appear as veritable experts in the downscal-
ing of intrinsic excitability.
A whole range of inhibitory ion channels was
found permanently upregulated in granule cells
of epileptic tissue (e.g., leak channels control-
ling Vrest and input resistance, such as Kir2.1-4
channels and K2P weak inward rectifier chan-
nels; Stegen etal., 2009; Young etal., 2009)and
axonal Kv channels (e.g., Kv1.1), which control AP
delay (Kirchheim et al., 2013). There are inter-
esting molecular differences between animal
models of TLE and the human TLE; while in the
severely sclerotic tissue of a mouse TLE model,
a tonic GABA A leak conductance is increased,
respective human granule cells of TLE patients
showed enhanced IH currents (Young etal., 2009;
Stegen etal., 2012). Both IH and tonic GABA A are
slightly depolarizing but shunting inhibitory for
dentate granule cells and thereby counterbalance
the effect of increased potassium conductances

on Vrest without reversing their decrease of excit-
ability (Young et al., 2009; Stegen et al., 2012).
The differences between human and mouse gran-
ule cells could be due to the more severe epileptic
phenotype of the animal model (Isokawa, 1996;
Haussler etal., 2012)or species differences. The
similar functional outcome of these changes is
consistent with the hypothesis that neurons have
perhaps many alternative (ion channel) routes to
achieve output homeostasis via intrinsic excit-
ability (Brickley et al., 2001; Prinz et al., 2004;
Young etal., 2009; Stegen etal., 2012; Figure4.3a).
Furthermore, it appears that the intrinsic plastic-
ity is not installed to simply switch off these gran-
ule cells in overcompensation but instead to scale
down their excitability in a function-maintaining
manner during upstream hyperexcitation. For
the dentate gyrus, the upstream driving source
likely is the entorhinal cortex, which during TLE
shows enhanced excitatory output (Kobayashi
et al., 2003). Consistent with the hypothesis of
granule cell output homeostasis, a recent mod-
eling study showed that the observed intrinsic
plasticity is in a position to restore the network
performance in a task of pattern separation
despite epileptic wiring (Yim etal.,2015).
In addition to antiexcitability strategies,
pharmacological support of neuronal intrinsic
anticonvulsive mechanisms of neurons could
be a powerful concept for antiepileptic drugs.
However, one has to carefully examine the tar-
geted cell type because, depending on the cel-
lular conductance environment, increasing a
hyperpolarizing current can eventually enhance
the network excitability, for example by deinac-
tivation of excitatory channels or by supporting
inhibitory synchronization that underlies certain
epilepsy forms (McCormick and Contreras, 2001;
Wickenden, 2002; Brenner etal., 2005). Another
important aspect is changed network connectiv-
ity during epilepsy. In particular, the imbalance of
different forms of inhibition in the CA regions and
the subiculum has to be studied carefully when
considering the potential impact of intrinsic plas-
ticity during TLE (Cohen etal., 2002; Magloczky
and Freund, 2005; Cohen et al., 2006; Wittner
et al., 2009; Frstera et al., 2010; see also previ-
ous discussion for RNA processingdependent
mechanisms of neuronal plasticity).
Epilepsy-Related Synaptic
Plasticity
Activity (calcium)-dependent proteolysis impacts
on the entire cellular proteome, but we focus
Homeostasis of Neuronal Excitability 61
here on a few selected target proteins involved
in inhibitory synaptic transmission. Calpain
belongs to the family of calcium-dependent,
nonlysosomal cysteine proteases. It cleaves a
wide range of proteins most likely because ter-
tiary protein structural determinants rather than
primary amino acid sequences define substrate
specificity.
Gephyrin is a calpain substrate (Kawasaki
et al., 1997). In fact, ERK and GSK3 cooper-
ate and phosphorylate serine residues 268 and
270, respectively, in the central C domain of
gephyrin, which may render gephyrin more
susceptible to calpain-mediated proteolysis due
to phosphorylation-dependent conformational
changes (Tyagarajan etal., 2011; Tyagarajan etal.,
2013). Furthermore, we know that gephyrin is
extensively spliced in this protein region, which
may involve RNA splicing in conformational
changes and increased susceptibility of gephyrin
to proteolysis (Herweg and Schwarz,2012).
Evidence from in vivo studies may support
a pathophysiological role for proteolytic gephy-
rin processing in TLE patients (Frstera et al.,
2010; Fang et al., 2011), and recent studies have
also demonstrated downregulation of gephyrin
and GABA ARs in an animal model of epilepsy
where the muscarinic acetylcholine receptor
agonist pilocarpine is used for induction of sta-
tus epilepticus (Fang etal., 2011; Gonzalez etal.,
2013). However, KCC2 is also a calpain target
(Puskarjov et al., 2012), and NMDA receptor
activation enhances calpain-mediated proteoly-
sis of KCC2. Here again, phosphorylation seems
to play a key role in the regulation of calpain
susceptibility because pilocarpine increased
tyrosine phosphorylation and lysosomal deg-
radation of KCC2 (Lee et al., 2010). Hence,
activity-dependent calpain action on gephy-
rin and KCC2 should restrain both GABAergic
neurotransmission-dependent chloride load and
potassium-dependent (KCC2-mediated) rebound
burst activity of neurons with expression of car-
bonic anhydrase (see Figure4.2d), suggesting that
this seizure-induced molecular plasticity may
represent a homeostatic form of neuronal synap-
tic and intrinsic plasticity. Indeed, that neonatal
seizure activity increases KCC2 function (Khirug
etal., 2010)in the absence of carbonic anhydrase
(Ruusuvuori etal., 2004)supports the view that
seizure-dependent bidirectional regulation of
KCC2 function is part of a neuronal homeostatic
adaptive mechanism that aims at protecting the
system against pathological hypersynchronous
62 Part I: Homeostatic Regulators

(a)

(b)

FIGURE 4.3 Scheme summarizing a selection of intrinsic and synaptic plasticity mechanisms
relevant to hippocampal and other epilepsies. (a) In reaction to seizures invading the hippocampus
via the perforant path (red triangle) to the dentate gyrus (DG), granule cells homeostatically downscale their
intrinsic excitability via transcriptional upregulation of leak channels such as classic inward rectifier K+ (K ir2.1-4),
two-pore K+ (K 2P) channels, voltage-gated K+ (Kv1.1) channels, as well as shunting conductances such as tonic
chloride currents via GABA type Areceptors (GABA AR, * in a mouse TLE model) or hyperpolarization-activated
cation (HCN1) channels (** in sclerotic tissue of TLE patients) (Stegen et al., 2009; Young et al., 2009; Stegen
et al., 2012; Kirchheim et al., 2013). In TLE models without hippocampal sclerosis, inhibitory synaptic influ-
ences are decreased in the cornu ammonis (CA) region of the hippocampus (right panel, green triangle), and the
intrinsic excitability of particularly CA1 pyramidal neurons is further increased due to reduced expression of
dendritic A-type (Kv4) and HCN channels, as well as increased influence of T-type voltage-gated calcium chan-
nels (Cav3) (Tsaur etal., 1992; Castro etal., 2001; Su etal., 2002; Bernard etal., 2004; Bender and Baram, 2007;
Dyhrfjeld-Johnsen et al., 2009; Noam et al., 2011). (b) Epilepsy-related changes of presynaptic molecules can
increase (red, orange) or decrease (green) network excitability when expressed in glutamatergic neurons while
the opposite holds true if the changes occur in GABAergic neurons. For a review see Meier etal. (2014). These
examples emphasize the importance of specific neuron types in the regulation of neural network homeostasis.
Further abbreviations:Nav,=voltage-gated sodium channels; GlyR=glycine receptors; GABABR=GABA type B
receptors. Panel b:modified with permission from Meier etal. (2014).

neural network activity. The fact that respiratory
CO2-regulatory regimes are able to interrupt sei-
zure activity (Schuchmann etal., 2006)further-
more accentuates the relevance of bidirectional
homeostatic adaptive regulation of KCC2 expres-
sion as a function of neuronal carbonic anhy-
drase expression.
However, the homeostatic machinery may
also fail in disease conditions. Actually, there
is increasing evidence that supports a critical
role for the axonal and/or presynaptic compart-
ment in epilepsy as it may escape homeostatic
regulation (Figure4.3b; Eichler and Meier, 2008;
Meier et al., 2014). For example, changes in the
expression of presynaptic vesicleassociated
proteins such as synapsins can elicit neural
network hyperexcitability (Fassio et al., 2011).
Knockout of the presynaptic protein syndapin
I also results in an epileptic phenotype (Koch
et al., 2011). Furthermore, presynaptic GABA B
autoreceptor-mediated interneuron type-specific
reduction of inhibition is associated with net-
work hyperexcitability in an animal model of
epilepsy (Dugladze et al., 2013; Figure 4.3b).
Also, ectopic AP generation was shown to be a
critical mechanism of network hyperexcitability,
identifying axo-axonic connecting neurons as
possible targets for new therapeutic strategies to
treat epilepsy (Dugladze etal.,2012).
The multifaceted clinical picture of patients
with epilepsy involves sudden episodes of cogni-
tive dysfunction and psychiatric comorbidities,
including depression and anxiety (Currie et al.,
1971; Beyenburg etal., 2005; Garcia-Morales etal.,

2008), which suggests common neuronal and
molecular mechanisms. Arecent study identified
a presynaptic molecule that can trigger cognitive
dysfunction and anxiety depending on whether
it is expressed in principal glutamatergic neurons
or a specific type of GABAergic interneuron (i.e.,
parvalbumin-positive interneurons), respectively
(Winkelmann et al., 2014). In agreement with
possible common molecular mechanisms of epi-
lepsy, increased presynaptic functionality may
represent a critical factor in the dysregulation of
neural network homeostasis (Figure 4.3b) and
identify specific neuron types that can trigger the
diverse neuropsychiatric symptoms of the dis-
ease (Winkelmann etal., 2014; Meier etal.,2014).
In conclusion, the homeostasis of neuronal
excitability via intrinsic and synaptic inhibitory
mechanisms accompanies neurons throughout
their lifes and becomes crucial during chronic
hyperexcitability as in epilepsy. The changes are
often very specific for cell types and even for
subcellular compartments. Therefore, further
cell typespecific investigations are needed to
better understand the impact of molecules, neu-
rons, and local networks in neuropsychiatric
diseases.
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PARTII

Homeostatic Control

Systems, Cells, and Organelles

5
Role ofAstrocytes inSleep and Epilepsy
J E R O M E C L A S A D O N T E A N D P H I L I P G . H AY D O N
INTRODUCTION
Glia, glue in Greek, was used for the first time
in 1858 by German scientist Rudolf Virchow to
describe the connective tissue that surrounded
neuronal cells in the brain. Increasing evidence
over the past 30 years suggests that astrocytes,
the most abundant glial cell type, represent much
more than a supportive tissue. Beyond the buffer-
ing of extracellular potassium and the uptake and
recycling of neurotransmitters and the neuronal
energy supply, astrocytes actively participate in
the processing of the information in the central
nervous system (CNS; for reviews see Halassa
and Haydon, 2010; Verkhratsky et al., 2011;
Parpura et al., 2012, Ben Achour and Pascual,
2012; Pirttimaki and Parri, 2013). Astrocytes dis-
play a highly ramified structure of thin processes
that contact neurons, blood vessels, and other
astrocytes. Structurally, astrocytes are remark-
ably well organized. They tile in the entire brain
in a contiguous and nonoverlapping manner
(Bushong etal., 2002; Halassa etal., 2007). Each
astrocyte disposes of its own domain in which
the most distal tips of processes interdigitate and
contact with one another through gap-junction
coupling to form a multicellular network called
the astrocytic network (Giaume etal., 2010). The
thin branching processes from an individual
astrocyte can contact 600 dendrites and envelop
100,000 synapses in rodents (Bushong et al.,
2002; Halassa et al., 2007). Diversity and struc-
ture of astrocytes become even more complex in
humans where astrocytes can contact more than
1million synapses (Oberheim etal., 2009). It is
also noteworthy that contact between astrocytes
and neurons represent highly dynamic struc-
tures, since it has been shown that astrocytic
coverage of the synapse depends on neuronal
activity (Hirrlinger et al., 2004; Haber et al.,
2006; Genoud etal.,2006).
Astrocytes express potassium and sodium
channels, but unlike neurons they do not fire or
propagate action potentials along their processes
(Sontheimer et al., 1996; Dallerac et al., 2013).
Instead, astrocytes exhibit regulated increases
in intracellular calcium concentration that
are considered a form of astrocyte excitability.
Astrocytes express numerous membrane recep-
tors for neurotransmitters whose activation leads
to increases in intracellular concentration of cal-
cium that can subsequently propagate through
the astrocytic network as intercellular calcium
waves. In turn, these calcium increases trigger
the release of neuroactive substances called gli-
otransmitters to regulate synaptic transmission
and neuronal activity. This bidirectional commu-
nication between neurons and astrocytes, termed
gliotransmision, can ultimately regulate vari-
ous brain functions such as sleep, breathing,
sensory perception, pain, parturition, lactation,
osmoregulation, reproduction, learning, and
memory (for reviews see Halassa and Haydon,
2010; Oliet and Bonfardin, 2010; Verkhratsky
etal., 2011; Clasadonte etal., 2011; Parpura etal.,
2012; Ben Achour and Pascual, 2012; Pirttimaki
and Parri, 2013; Blutstein and Haydon, 2013).
In this chapter we mainly focus on the recent
development of an astrocyte-specific transgenic
mouse that allowed the evaluation of the role of
glial signaling pathways in the control of sleep.
Besides the role of glial cells in brain physiology,
astrocytes can also contribute to the pathogen-
esis of neurological disorders and diseases (for
reviews see Sofroniew and Vinters, 2010; Parpura
etal., 2012). We then discuss the recent evidence
that supports the critical role of astrocytes in the
progressive development of one of the most com-
mon brain disorders, epilepsy.
C A L C I U M E X C I TA B I L I T Y
INASTROCYTES
Unlike neurons, astrocytes do not generate action
potentials and, thus, they were thought to be qui-
escent. However, astrocytes display spontaneous
76 Part II: Homeostatic Control
intracellular calcium concentration increases
that can reach micromolar levels and are known
to affect cell physiology. The development and use
of calcium imaging techniques has been pivotal
for investigating the physiology of astrocytes. The
growth of interest in astrocytic calcium signaling
came soon after its first discovery when it was
found that calcium increases in astrocytes could
be triggered by using the excitatory neurotrans-
mitter glutamate in cultures (Cornell-Bell etal.,
1990)or by evoking synaptic release of glutamate
in hippocampal slices (Porter and McCarthy,
1996). In some circumstances, astrocytic calcium
signals may propagate to neighboring astrocytes
as intercellular calcium waves (Cornell-Bell etal.,
1990; Bowser and Khakh, 2007). Since then,
similar observations have been made in vivo in
anesthetized or freely behaving animals (Hirase
et al., 2004; Dombeck et al., 2007; Kuga et al.,
2011) and in human cortical and hippocampal
slices prepared from acutely resected surgical tis-
sue (Oberheim etal., 2009, Navarrete etal.,2013).
The synaptic and neuronal controls of astro-
cytic calcium originate from the fact that astro-
cytes express a variety of G-protein coupled
receptors for different neurotransmitters whose
activation increases the intracellular concentra-
tion of calcium through the release of calcium
from intracellular inositol 1,4,5-trisphosphate
(IP3)-sensitive calcium stores (for a review see
Perea etal., 2009). The most well-known recep-
tors involved in calcium increases in astrocytes
are the metabotropic receptors activated by
purines such as P2Y1, and the metabotropic glu-
tamate receptors 1 (mGluR1) and 5 (mGluR5),
although for the latter, a recent study has ques-
tioned its contribution to astrocytic calcium
signaling in the adult brain (Sun etal., 2013). It
is also noteworthy that the IP3-sensitive calcium
store, although predominant, is not the only
source of calcium within the astrocyte. There are
multiple other sources of calcium that can lead
to global increases in intracellular calcium con-
centrations such as mitochondria and sodium/
calcium exchangers, nonselective cation chan-
nels, and voltage-gated calcium channels in the
plasma membrane (for a review see Parpura and
Verkhratsky,2012).
An extremely interesting finding in the glial
field was the discovery in the in vivo brain that
astrocytic signals could differ in their spatial
extent and in their duration during physiological
processes such as locomotion (Nimmerjahn etal.,
2009)and in response to a variety of physiological
stimuli such as whisker, limb, odor, and visual
stimulations (Wang et al., 2006; Winship et al.,
2007; Dombeck etal., 2007; Petzold etal., 2008;
Schummers etal., 2008), supporting a functional
role for astrocytic excitability in brain physiology.
In addition, astrocytic calcium signals have been
shown to be important for the control of cerebral
arteriole diameter (Zonta et al., 2003; Mulligan
and MacVicar, 2004; Takano etal., 2006; Metea
et al., 2006; Filosa et al., 2006; Chuquet et al.,
2007; Gordon etal., 2008; Kuga etal., 2011)and
the regulation of brain blood flow during optical
signaling (Schummers etal., 2008)and locomo-
tor behavior (Nimmerjahn etal., 2009), although
Takata etal. (2013) have a differentview.
Astrocytic calcium signals have been
originally and extensively studied only in
the somata and thick branches due to tech-
nical limitations using bulk loading of
membrane-permeable organic calcium indi-
cator dyes (for a review see Tong et al., 2013).
Loading of membrane-impermeable organic
calcium indicator dyes via glass pipettes has
also been used to measure calcium signals
in astrocytes (Di Castro et al., 2011; Panatier
etal., 2011). Using this method, Di Castro etal.
and Panatier etal. observed different patterns
of calcium signals within astrocyte processes;
they can be compartmentalized, localized,
focal, or expanded and can be dependent or
independent of neuronal activity (Di Castro
etal., 2011; Panatier etal., 2011). More recently,
the use of cytosolic- and membrane-targeted
genetically encoded calcium indicators
(GCaMPs and Lck-GCaMPs, respectively)
revealed a novel type of microdomain calcium
signal (Shigetomi et al., 2011). These calcium
signals, called spotty calcium signals, are spa-
tially restricted to the membrane within the
very thin astrocytic processes and are thought
to contribute to basal cytosolic calcium lev-
els. The mechanisms underlying spotty cal-
cium signals differ from those for the more
classical aforementioned G-protein coupled
receptorsmediated calcium signals; they
involved in fact the entry of calcium from the
extracellular space through a family of nonse-
lective cation channels, the transient receptor
potential channels ankyrin 1 (Shigetomi etal.,
2011). Thus the development of new methods
for studying the diversity of calcium signals
within single astrocytes is and will be essential
to investigate in-depth diversity of functions
for astrocytes in different parts of thebrain.

ASTROCYTES RELEASE
CHEMICAL TR ANSMITTERS
T O M O D U L AT E S Y N A P T I C
AND NEURONAL ACTIVIT Y
One of the most exciting discoveries in the past
20years in the glial field has been that astrocytes
can release chemical transmitters that are able to
modulate synaptic and neuronal activity. In anal-
ogy with neurotransmitter release from neurons,
they have been termed gliotransmitters. This
discovery came from seminal works showing that
astrocytes respond with calcium elevations to
synaptically released neurotransmitters (Porter
and McCarthy, 1996; see previous discussion)
and that astrocytes could also release transmit-
ters (Parpura et al., 1994). Thus the realization
of this bidirectional communication between
neurons and astrocytes led to the concept of the
tripartite synapse (Araque etal., 1999), which not
only consists of pre- and postsynaptic elements
but also of an astrocytic element that responds to
presynaptic transmitter release and feeds back via
gliotransmitter release to affect synaptic activity.
The most-studied gliotransmitters are glu-
tamate, D-serine, gamma-aminobutyric acid
(GABA), adenosine triphosphate (ATP), and
adenosine. Calcium-dependent exocytosis rep-
resents the better characterized pathways of gli-
otransmitter release (for reviews see Gucek etal.,
2012; Zorec et al., 2012). In culture, astrocytes
express the soluble N-ethylmaleimide-sensitive
fusion protein receptor (SNARE) complex,
which is colocalized with small vesicles posi-
tive for vesicular glutamate transporters (Bezzi
et al., 2004; Zhang et al., 2004; Montana et al.,
2004), ATP-storing vesicles (Coco et al., 2003;
Zhang et al., 2007; Sawada et al., 2008), and
D-serine-containing vesicles (Mothet et al.,
2005). In addition, ultrastructural studies have
shown that astrocytic processes contain small
synaptic-like vesicles with a diameter of 30 to
40 nM (Bezzi et al., 2004; Jourdain et al., 2007;
Martineau et al., 2013). Importantly, these vesi-
cles contain glutamate and D-serine and are
clustered in close proximity to synapses (Bezzi
et al., 2004; Jourdain et al., 2007; Martineau
et al., 2013). Convincing evidence for astro-
cytic vesicular release of ATP is provided by the
dominant-negative SNARE (dnSNARE) mouse
in which exocytosis in astrocytes is selectively
inhibited and results in a reduction in ATP release
following astrocyte stimulation (Pascual et al.,
2005; see following discussion for more details
and Figure 5.1). Other gliotransmitter release
Role of Astrocytes in Sleep and Epilepsy 77
FIGURE 5.1: Proposed model of the modula-
tion of brain functions by astrocytic purines.
Astrocytes release ATP through a SNARE-dependent
exocytosis or via the gap-junction connexin 43 hemichan-
nels (Cx43), volume-activated anion channels (VAAC), or
pore-forming P2X7 receptors. In the extracellular space
(ECS), ATP is converted to adenosine by a combination
of extracellular enzymes. Ecto-nucleoside triphosphate
diphosphohydrolases (E-NTPDases) and ecto-nucleotide
pyrophosphatase/phosphodiesterases (E-NPPs) metabo-
lize ATP and adenosine diphosphate (ADP) to adenosine
monophosphate (AMP), whereas alkaline phosphatases
(ALPs) and ecto-5-nucleotidase (CD73) metabolize AMP
to adenosine. Adenosine can be taken up into the intra-
cellular space (ICS) of the astrocyte through the bidirec-
tional ectonucleotide transporters (ENTs). This passive
transport of adenosine depends on the activity of the
intracellular enzyme adenosine kinase (ADK) that elimi-
nates adenosine via phosphorylation to AMP and drives
the flux of adenosine into the astrocyte. In the ICS of the
astrocyte, adenosine can also be converted to inosine by
the enzyme adenosine deaminase (ADA). The accumula-
tion of astrocytic adenosine in the ECS leads to the tonic
activation of the neuronal adenosine A1 receptor, result-
ing in the modulation of synaptic transmission and traf-
ficking of NMDA receptors at the cell surface. In a more
integrated system, this contributes to the modulation of
cortical network activity, sleep homeostasis, cognitive
functions, and epilepsy. These brain functions attributed
to astrocytic purines are supported by a series of experi-
mental studies in which the dominant-negative form of the
SNARE complex (dnSNARE) was genetically expressed
into the astrocytes to disrupt the SNARE-dependent exo-
cytosis of ATP. See text for additional details.
78 Part II: Homeostatic Control
mechanisms have been proposed, including
membrane transporters, volume-activated anion
channels, ligand-gated P2X7 channels, and con-
nexin/pannexin hemichannels (for a review see
Malarkey and Parpura, 2008)and more recently
the two-pore domain potassium channels and
the bestrophin anion channels (Lee et al., 2010;
Woo et al., 2012; Han et al., 2013). Although
numerous studies have demonstrated the role of
gliotransmitters in synaptic transmission, there
is still debate and controversy about the mecha-
nism by which they are released, whether or not it
is calcium-dependent, and whether it has physio-
logical significance (Fiacco etal., 2007; Petravicz
etal., 2008; Agulhon etal., 2010; Hamilton and
Attwell, 2010; Wang et al., 2013). Rather than
debating the relevance of each of these mecha-
nisms, we provide a synopsis of recent work sup-
porting the functional role of gliotransmitters in
synaptic and neuronal physiology.
Release ofGlutamate
Glutamate was one of the first gliotransmit-
ters identified in the early 1990s (Parpura etal.,
1994). Since then, numerous studies have shown
that astrocytic glutamate released through a
calcium-dependent mechanism exerts many
effects on synaptic transmission and neu-
ronal excitability. By acting on presynaptic
glutamatergic receptors, including mGluR1,
mGluR2/3, mGluR5, kainate receptors, and
N-methyl-D-aspartate (NMDA) receptors, astro-
cytic glutamate strengthens or weakens inhibi-
tory and excitatory synapses and consequently
participates in synaptic plasticity (Pasti et al.,
1997; Kang etal., 1998; Liu etal., 2004a, 2004b;
Fiacco et al., 2004; Perea and Araque, 2007;
Jourdain et al., 2007; Navarrete and Araque,
2010; Bonansco etal., 2011). Although all of these
studies were mainly performed in acute brain
slice preparations, only a few have been carried
out in vivo to elucidate the role of astrocytic
glutamate in synaptic plasticity. For example,
Navarrete etal. (2012) demonstrated that astro-
cytic glutamate was involved in the generation
of in vivo hippocampal long-term potentiation
(LTP) mediated through activation of neuronal
mGluRs following stimulation of the cholinergic
septo-hippocampal pathway. Asimilar plasticity
has been observed in the cortex in vivo involv-
ing astrocytic muscarinic acetylcholine recep-
tors and neuronal NMDA receptors (Takata
etal., 2011). Finally, Han etal. (2012) showed that
astroglial cannabinoid receptors were responsible
for the impairment of spatial working memory
and NMDA-dependent hippocampal long-term
depression (LTD) in vivo induced by an acute
exposure of exogenous cannabinoids.
A hallmark of astrocytic glutamate release
is the generation of slow inward currents (SICs)
in postsynaptic neurons from slice preparations.
SICs were first identified in the thalamic neurons
where they were correlated with spontaneous
calcium spikes in astrocytes (Parri et al., 2001).
Since then, SICs have been detected in many
regions of the CNS such as the cortex (Ding etal.,
2007), hippocampus (Fellin etal., 2004), nucleus
accumbens (D Ascenzo et al., 2007), olfactory
bulb (Kozlov etal., 2006), brainstem (Reyes-Haro
etal., 2010), spinal cord (Bardoni etal., 2010; Nie
etal., 2010), and, more recently, human cortical
and hippocampal slices prepared from acutely
resected surgical tissue (Navarrete et al., 2013).
Typically, SICs are due to a calcium-dependent
release of astrocytic glutamate that preferentially
activates extrasynaptic NMDA receptors con-
taining the NR2B subunit (Fellin et al., 2004).
SICs can occur spontaneously or be synaptically
evoked (Parri et al., 2001; Fellin et al., 2004).
Although the physiological role of SICs is still
unknown or even questioned by some (Fiacco
etal., 2007), they can induce neuronal depolari-
zation and synchronize firing of CA1 pyramidal
cells over short distances of 100m (Fellin etal.,
2004), suggesting that SICs could represent a
source of excitation during epileptic discharges
as discussed later in this chapter. Interestingly,
studies conducted in spinal cord slices have
shown that SICs could be triggered by pharma-
cological blockade of glutamate uptake with the
nontransportable competitive glutamate trans-
porter blocker D-Threo--benzyloxyaspartate
(Nie et al., 2010), suggesting that changes in
ambient glutamate concentrations could rep-
resent a key component that contributes to the
regulation of this bidirectional glutamatergic
interaction between neurons and astrocytes.
However, the prevalence of this phenomenon in
other brain areas and its (patho)physiological
implications remain to be determined.
Release ofD-Serine
Convincing ultrastructural studies indicate that
astrocytes contain all the machinery that is neces-
sary for the calcium-dependent vesicular release
of D-serine (Mothet etal., 2005; Martineau etal.,
2013). This amino acid is produced within astro-
cytes through the conversion of L- to D-serine

by the specific enzyme serine racemase (Mothet
et al., 2000; Mothet et al., 2005). Acting as the
natural substrate for glycine binding sites on
the NMDA receptor, D-serine is considered an
endogenous co-agonist of the NMDA recep-
tor and thus can potentially modulate synap-
tic plasticity (Mothet et al., 2000; Mothet et al.,
2005). Indeed, in the supraoptic nucleus of the
hypothalamus of rodents, changes in the glial
coverage of synapses dependent on physiologi-
cal signals affect extracellular levels of D-serine
and consequently can lead either to LTP or LTD
in acute brain slices (Panatier etal., 2006). This
metaplastacity is critical for the modulation of
excitability of magnocellular neurosecretory
cells during neuroendocrine processes such as
lactation and parturition (Panatier etal., 2006).
Moreover, in situ studies manipulating serine
racemase activity and using calcium clamp of
astrocytes (Henneberger et al., 2010) or attenu-
ating basal calcium levels in astrocytes through
inhibition of transient receptor potential chan-
nels ankyrin 1 channels (Shigetomi et al., 2013;
see previous discussion) have revealed that astro-
cytic D-serine regulated LTP within the hip-
pocampus by targeting preferentially synaptic
NMDA receptors (Papouin etal., 2012). Arole for
astrocyte-derived D-serine in cortical plasticity
has also been suggested (Takata etal.,2011).
Release ofGABA
Despite early reports showing that astrocytes can
accumulate, synthesize, and release GABA, the
idea that this major inhibitory transmitter of the
mammal CNS could also be a gliotransmitter has
not been widely supported (for reviews see Angulo
etal., 2008; Velez-Fort etal., 2012). Several stud-
ies mainly performed in acute brain slices have
shown that astrocytes from the olfactory bulb,
thalamus, and hippocampus inhibit neighboring
neurons by releasing GABA, which generates slow
outward currents (SOCs) due to the activation of
neuronal GABA A receptors (Kozlov et al., 2006;
Jimenez-Gonzalez etal., 2011; Le Meur etal., 2012).
Interestingly, these SOCs share some similarities
with NMDA receptor-mediated SICs caused by
the release of glutamate from astrocyte. Indeed,
SOCs can occur simultaneously in neighboring
neurons and provide a source of synchronized
inhibition by targeting extrasynaptic GABAA
receptors (Kozlov etal., 2006; Jimenez-Gonzalez
etal., 2011). Although SICs are mediated through
an exocytotic release of astrocytic glutamate, the
mechanism by which GABA is released from
Role of Astrocytes in Sleep and Epilepsy 79
astrocytes is not very well described. Some have
proposed a calcium-dependent mechanism
involving the anion bestrophin anion channels in
the cerebellum (Lee etal., 2010). However, in this
case, bestrophin anion channels mediate tonic
inhibition instead of SOCs (Lee etal., 2010). The
physiological role of SOCs remains completely
unknown, although it has been suggested that,
like SICs, they could play an important role under
pathological conditions such as epileptiform dis-
charges (Jimenez-Gonzalez etal., 2011; Le Meur
et al., 2012). Interestingly, a recent study has
shown that SOCs recorded in thalamic slices from
a genetic rat model of absence epilepsy (genetic
absence epilepsy rats from Strasbourg) displayed
a reduced amplitude compared with control ani-
mals (Pirttimaki etal., 2013). The smaller ampli-
tude of SOCs seems to result from a malfunction
of the astrocytic GABA-transporter GAT-1 in
these animals, suggesting that, like SICs, SOCs
can be influenced by the ambient GABA concen-
trations. However, it remains difficult to ascribe
a precise pathological significance to the smaller
amplitude of SOCs in these epileptic animals.
ReleaseofATP
Astrocytic ATP was initially proposed as an
extracellular messenger and a primary signal
for the propagation of calcium waves through
the astrocytic network by acting directly on
purinergic receptors (Guthrie et al., 1999; for a
review see Giaume et al., 2010). Although dif-
ferent mechanisms of ATP release from astro-
cytes have been proposed (Figure 5.1), including
volume-activated anion channels, gap-junction
connexin 43 hemichannels, and pore-forming
P2X7 receptors, convincing morphological and
biochemical evidence suggests that astrocytes
may preferentially use calcium-dependent exo-
cytosis to release ATP (for a review see Gucek
et al., 2012). Additionally, Pascual et al. (2005)
have generated an inducible transgenic mouse
in which only astrocytes express a dnSNARE
domain of vesicle-associated membrane pro-
tein 2 to suppress exocytosis of gliotransmitters.
Bioluminescence imaging demonstrated that this
molecular genetic manipulation led to reduced
extracellular ATP, providing further support that
ATP release from astrocytes is mediated through
a vesicular-dependent mechanism (Pascual
etal.,2005).
Once ATP is released from astrocytes, it can
affect synaptic and neuronal activity through
its direct action on purinergic receptors. For
80 Part II: Homeostatic Control
example, in the paraventricular nucleus of the
hypothalamus of dehydrated rats, withdrawal
of the glial coverage surrounding magnocellular
neurosecretory cells influences extracellular lev-
els of ATP released from astrocytes in response
to adrenergic input (Gordon etal., 2005). During
this process, ATP has been shown to directly
activate P2X7 receptors located on magnocellu-
lar neurosecretory cells to enhance membrane
insertion of alpha-amino-3-hydroxy-5-methyl-
4-isoxazolepropionic acid receptors and thereby
cell excitability (Gordon etal., 2005). This mech-
anism is critical for facilitating the secretion of
the two neurohormones, oxytocin and vasopres-
sin, involved in the control of osmoregulation
(Gordon etal., 2005). Interestingly, further stud-
ies indicated that this physiological process also
involved an mGluR1-mediated rise in calcium in
astrocytes (Gordon et al., 2009). Another study
from Gourine et al. (2010) using in situ and in
vivo approaches has shown that astrocytes in the
brainstem contributes to the regulation of venti-
lation through a pH-induced vesicular release of
ATP that in turn increases activity of medullary
chemo-receptor neurons.
Adenosine Derived From
Astrocyte- ReleasedATP
Besides its direct actions through purinergic
receptors, ATP can also exert indirect effects
on its rapid conversion to adenosine by a com-
bination of extracellular enzymes (Figure 5.1).
Ecto-nucleoside triphosphate diphosphohy-
drolases and ecto-nucleotide pyrophosphatase/
phosphodiesterases metabolize ATP and adeno-
sine diphosphate to adenosine monophosphate
(AMP), and alkaline phosphatases (ALPs) and
ecto-5-nucleotidase metabolize AMP to adeno-
sine (Zimmermann, 2000). Subsequently, adeno-
sine can exert numerous effects in the brain that
are mediated through the activation of four sub-
types of G-protein-coupled adenosine receptors
(A1, A2A, A2B, and A3; for a review see Fredholm
etal., 2005). It is noteworthy that the breakdown
of ATP to adenosine in the extracellular space is
not the only source of adenosine. Adenosine can
be directly released from neurons and astrocytes
through a passive mechanism involving a family
of equilibrative nucleotide transporters (Boison,
2013; Figure 5.1). This direct release of adenosine
occurring typically during hypoxic conditions
has been recently debated (Martin et al., 2007;
Parkinson etal., 2009; Lovatt etal., 2012; Zhang
etal., 2012; Wall and Dale, 2013). In this case, the
extracellular concentration of adenosine depends
on the activity of adenosine kinase (ADK), an
intracellular enzyme mainly expressed in astro-
cytes that converts adenosine to AMP (Figure
5.1). This conversion creates a concentration gra-
dient that drives the influx of adenosine into the
cell through bidirectional equilibrative nucleo-
tide transporters (Figure5.1).
In situ studies have shown that adenosine
derived from astrocyte-released ATP controls
synaptic and neuronal activity. In the retina, for
example, extracellular adenosine, which derives
from ATP released by the Mller glial cells,
hyperpolarizes neurons and inhibits their firing
through the activation of A1 receptors (Newman,
2003). In addition, using pharmacology in the
astrocyte dnSNARE mouse, seminal studies have
discovered that the SNARE-mediated release of
ATP represents an important source of extracel-
lular adenosine (Figure 5.1), which suppresses
excitatory synaptic transmission through the
activation of presynaptic A1 receptors (Pascual
et al., 2005; Halassa et al., 2009; Schmitt et al.,
2012). Although this astrocytic signaling path-
way is supported by other studies (Zhang etal.,
2003; Serrano etal., 2006), it has been suggested
that SNARE-mediated release of ATP could
instead increase excitatory synaptic transmis-
sion by acting specifically on presynaptic A2A
receptors (Panatier et al., 2011). Subsequently,
direct measurements of adenosine in hipp-
pocampal slices with adenosine biosensors have
confirmed that astrocytic dnSNARE expression
reduces extracellular levels of adenosine (Schmitt
etal., 2012), supporting the idea that one source
of extracellular adenosine is SNARE-mediated
release of ATP that is hydrolyzed in the extracel-
lular space to adenosine (Figure5.1).
In addition to exerting presynaptic inhibition
via A1 receptors, adenosine derived from astro-
cytic ATP can also exert postsynaptic actions to
regulate the trafficking of NMDA receptors in
cortical and hippocampal pyramidal neurons
(Figure 5.1; Fellin etal., 2009; Deng etal., 2011;
Clasadonte etal., 2013). Using the dnSNARE mice
along with biochemical, pharmacological, and
electrophysiological approaches, further studies
demonstrated that the tonic activation of post-
synaptic A1 receptors leads to a delayed Src fam-
ily kinase-dependent tyrosine phosphorylation of
the NMDA receptor subunits NR2A and NR2B
that is required for their endocytosis (Deng etal.,
2011). In the dnSNARE animals, the reduced
extracellular adenosine tone leads to a reduced

cell surface expression of the NMDA receptors
that thereby decreases the NMDA current (Fellin
et al., 2009; Deng et al., 2011; Clasadonte et al.,
2013). These results are furthermore consistent
with earlier studies showing that dnSNARE mice
exhibit a reduced NMDA receptor-dependent
LTP at the hippocampal synapses (Pascual etal.,
2005). It is important to note that this regulation
of postsynaptic NMDA receptor trafficking by
astrocytic adenosine is a slow process. Indeed,
pharmacological studies performed in cortical
slices by Deng etal. (2011) have shown that the
reduction of NMDA receptor trafficking was
phenocopied in wild-type littermates (WT) only
when slices were incubated with the A1 receptor
antagonist 8-cyclopentyl-1,3-dimethylxanthine
(CPT) for at least 3 hours, and rescued in
dnSNARE animals only when slices were incu-
bated with the A1 receptor agonist 2-Chloro-N6-
cyclopentyladenosine for at least 1 hour. Thus
astrocytes can regulate NMDA receptors function
over different timescales. Release of glutamate
and D-serine by astrocytes provides the poten-
tial for second-to-second changes in the NMDA
receptor current, while the adenosine-dependent
pathway that regulates NMDA receptor traffick-
ing represents a much slower process. By regulat-
ing NMDA receptor trafficking, astrocytes could
contribute to homeostatic synaptic plasticity that
works over long timescales (hours to days) such
as during sleep/wake cycles (Vyazovskiy et al.,
2008) in which astrocyte-derived adenosine is
known to play important roles (Halassa et al.,
2009), as described previously. Additionally, we
will see later in this chapter that this particular
astrocytic signaling pathway is critical for the
development of temporal lobe epilepsy(TLE).
ROLE OFASTROCYTES
INSLEEP
Because adenosine is known to be a powerful
modulator of sleep (for a review see Basheer etal.,
2004), several studies have investigated whether
astrocyte-derived adenosine could contribute to
the regulation of sleep physiology (for a review
see Blutstein and Haydon, 2013). It is known that
adenosine levels rise during wakefulness; sleep
deprivation leads to adenosine elevations, and
infusion of adenosine promotes sleep while infu-
sion of adenosine receptors antagonists such as
caffeine promotes wakefulness. Although the
functions of sleep are still unresolved, recent
progress has been made in deciphering the brain
mechanisms that control sleep and wakefulness
Role of Astrocytes in Sleep and Epilepsy 81
(for a review see Brown etal., 2012). In the follow-
ing section, we focus on recent studies that have
investigated the role of astrocyte-derived adeno-
sine in sleep-related behavior.
Astrocytes Modulate Brain Activity
atthe CircuitLevel
The brain displays distinct network dynamics in
different states of anesthesia, wakefulness, and
sleep. During non-rapid eye movement (NREM)
sleep and certain forms of anesthesia, cortical
local field potential patterns are characterized
by the large-amplitude slow oscillations (< 1 Hz),
whereas small-amplitude and rapid fluctuations
appear during the two other vigilance states
rapid eye movement (REM) sleep and wakeful-
ness (Steriade, 1997). The cortical slow oscilla-
tions detected in vivo with local field potential
reflect the activity of a vast network of pyrami-
dal neurons that oscillate between a depolarized
up-state and a hyperpolarized down-state with
a high level of synchrony (Steriade etal., 2006).
While recording cortical local field potential in
vivo in anesthetized mice, it was found that the
expression of dnSNARE in astrocytes caused
a striking reduction in the power of slow oscil-
lations compared to that from WT littermates
(Fellin etal., 2009; Schmitt etal., 2012). In addi-
tion, local disinhibition of the circuits by acute
blockade of the A1 receptors with CPT increased
the power of cortical slow oscillations in WT
mice while this effect was abolished in dnSNARE
mice, confirming a reduced extracellular level
of adenosine in these transgenic animals (Fellin
et al., 2009; Schmitt et al., 2012). Importantly,
the SNARE-dependent A1 receptor modulation
of both cortical slow oscillations and synaptic
transmission was dependent on the time of the
day and more specifically dependent on the time
spent in wakefulness (Schmitt etal., 2012). These
results provide the first in vivo evidence that
astrocyte-derived adenosine modulates brain
activity at the circuit level and is potentially
involved in the control of sleep-related behavior.
Astrocytes Modulate Sleep
Homeostasis
In line with the aforementioned study dem-
onstrating that astrocytic adenosine is critical
for the control of circuit function in the cortex,
Halassa et al. (2009) performed studies to ask
whether this astrocyte-derived adenosine con-
tributes to sleep homeostasis. Sleep is modulated
by both the circadian oscillator that controls the
82 Part II: Homeostatic Control
timing of sleep and wakefulness and the sleep
homeostat that integrates the time awake and
promotes the sleep drive. The sleep drive, also
called sleep pressure, can be monitored with cor-
tical electroencephalography (EEG) by measur-
ing the power of slow-wave activity (SWA:delta
frequency range 0.54 Hz) during NREM sleep.
It is commonly accepted that sleep pressure and
the power of SWA per se increases in proportion
to the time spent awake and decreases during
sleep (Borbly and Achermann, 2005). Thus, by
combining EEG with electromyogram (EMG)
recordings in mice to monitor the three differ-
ent vigilance states NREM, REM, and wakeful-
ness, Halassa etal. (2009) found that dnSNARE
expression in astrocytes reduced the power of
SWA during NREM sleep at the onset of the sleep
period while it was normally elevated in WT mice
after a natural period of prolonged wakefulness.
In addition, astrocytic dnSNARE expression
attenuated the increased in SWA during NREM
sleep in response to acute sleep deprivation of 6
hours compared with WT mice (Figures 5.2Ai,
5.2Aii, and 5.2B) and prevented the compensa-
tory increase in sleep time that normally occurs
after sleep deprivation (Halassa et al., 2009;
Figure 5.2C). Importantly, the dnSNARE sleep
phenotype was entirely phenocopied in WT ani-
mals by chronic intracerebroventricular deliv-
ery of the A1 receptor antagonist CPT (Halassa
etal., 2009). Although these results suggest that
the SNARE-dependent release of adenosine from
astrocytes represents a major component of the
homeostatic control of sleep (Figure 5.1), some
experimental discrepancies must be noted. The
chronic inhibition of A1 receptors that reduces
the power of SWA and thereby mimics the
dnSNARE phenotype (Halassa etal., 2009)is in
apparent contradiction with the fact that acute
inhibition of A1 receptors increases the power
of cortical slow oscillations (Fellin et al., 2009;
Schmitt et al., 2012). These differences can be
explained by the fact that the effects of acute and
chronic inhibitions of A1 receptors are mediated
by two distinct mechanisms. Acute inhibition
of A1 receptors attenuates the power of cortical
slow oscillations by causing a rapid local disinhi-
bition of the circuits (Fellin etal., 2009; Schmitt
etal., 2012). On the other hand, long-term inhi-
bition of A1 receptors have been shown to cause
a delayed reduction in NMDA receptor activity
(Deng et al., 2011) and thus could explain why
dnSNARE animals displayed a reduced power
of both cortical slow oscillations (Fellin et al.,
2009; Schmitt et al., 2012) and SWA (Halassa
et al., 2009). Accordingly, it has been demon-
strated that the reduced power of cortical slow
oscillations observed in dnSNARE animals
could be phenocopied in WT animals when both
A1 and NMDA receptors were acutely inhibited
with CPT and the NMDA receptor antagonist
D-()-2-amino-5-phosphonopentanoate (D-AP5),
respectively (Fellin et al., 2009).
The idea that astrocytic adenosine controls
sleep homeostasis is supported by other stud-
ies. For example, reduced SWA during NREM
sleep have also been previously described in con-
ditional knockout mice lacking A1 receptors in
neurons (Bjorness et al, 2009). Mice, in which
the two enzymes ecto-5-nucleotidase and ADK
known to regulate adenosine metabolism (Figure
5.1) have been genetically manipulated to lower
adenosine tone in the brain, also display sleep
phenotype (Zielinski et al., 2012; Palchykova
etal., 2010). In addition, patients bearing a genetic
mutation of the intracellular adenosine deami-
nase, an enzyme involved in the inactivation of
adenosine (Figure 5.1), display an increased sleep
pressure and present a more consolidated sleep at
night (Bachmann etal.,2012).
Astrocytes, Sleep Loss, and
Memory Consolidation
An important function of sleep is offline pro-
cessing of information encountered during the
day and consolidation of memory. Conversely,
accumulation of sleep pressure after prolonged
wakefulness is associated with substantial
impairments in cognitive functions (McCoy and
Strecker, 2011; Yoo etal., 2007). Given that astro-
cytes play a major role in the homeostatic con-
trol of sleep, Halassa etal. (2009) investigated the
contribution of these glial cells in the cognitive
deficits induced by sleep loss. While high sleep
pressure in WT mice was associated with poor
performances in memory recognition tests fol-
lowing acute sleep deprivation, the lack of accu-
mulated sleep pressure in dnSNARE animals
prevented the effects of sleep loss on the consoli-
dation of recognition memory. In addition, the
chronic infusion of the A1 receptor antagonist
CPT in WT mice mimicked the dnSNARE phe-
notype by preventing the deleterious effects of
sleep deprivation on memory recognition.
Similarly, another study performed by
Florian etal. (2011) demonstrated that dnSNARE
animals exhibited better performances in spatial
memory tests than WT animals after sleep dep-
rivation. This lack of alteration in the consolida-
tion of spatial memory was consistent with the
 Role of Astrocytes in Sleep and Epilepsy 83

(a)(i) WT
Baseline (ZT7-8) Recovery after SD (ZT7-8)

EEG
5V
EMG
1s

(a)(ii) dnSNARE
Baseline (ZT7-8) Recovery after SD (ZT7-8)

EEG
5V
EMG
1s

(b) Recovery after SD (c)

Baseline
260
WT Recovery
Normalized Power

220 * * dnSNARE
60 N.S.
(0.51.5 Hz)

**

(% Recording Time)
Total Sleep Time
180 *
40

140 20

100 0
6 8 10 12 WT dnSNARE
Time (Zeitgeber)

FIGURE 5.2: Gliotransmission is essential for sleep pressure accumulation. (Ai) Representative
EEG/EMG recordings in non-rapid eye movement (NREM) sleep from wild-type (WT) mice during baseline day
(left) and recovery day after 6 hours of sleep deprivation (SD, right). Recordings during the baseline and recovery
days are shown at the same time point between the zeitgeber times 7 and 8 (ZT78). Note that the amplitude of the
EEG slow-wave activity (SWA) in WT mice increases during recovery after SD, compared to baseline. (Aii) Same
experiments as in (Ai) but performed with dnSNARE mice. Note that the amplitude of EEG SWA in dnSNARE
mice does not increase during recovery after SD, compared to baseline. (B)Analysis of the normalized power of
SWA during NREM sleep indicated that SWA was decreased in dnSNARE mice (n=7) following SD when com-
pared with WT littermates (n=8) (ANOVA:p < 0.001, F=7.91; post hoc test, *p < 0.05). (C)SD increased the total
sleep time in WT (n=8) but not in dnSNARE animals (n=9) during an 18-hour recovery period compared with
a baseline period (Students paired t test:**p < 0.01; N.S.=nonsignificant).
(B) and (C)adapted from Halassa etal. (2009).

fact that dnSNARE mice did not show a decrease
in the late phase of hippocampal LTP after sleep
deprivation when compared with WT mice.
Further, the chronic treatment of WT mice with
CPT in this study also mimicked the dnSNARE
phenotype. Overall, these studies suggest that
astrocyte-derived adenosine contributes to the
impairment of memory consolidation following
sleeploss.
Astrocytes, Sleep Loss,
and Depression
There are several links between depression and
sleep. Both monopolar and bipolar depression are
associated with sleep disturbances. Paradoxically,
it has been observed that acute sleep deprivation
has a potent and rapid antidepressant action in
severely depressed individuals (for a review see
Hemmeter etal., 2010). Some have even suggested
that insomnia, a common symptom of depres-
sion, might represent an endogenous adaptive
mechanism to counteract underlying depres-
sive mood rather than just be part of the pathol-
ogy (Adrien, 2002). However, the mechanisms
underlying antidepressant effects of sleep dep-
rivation are still relatively unknown. Given that
astrocytes modulate sleep homeostasis through
A1 receptor signaling (Halassa et al., 2009), a
recent study has investigated the potential role
of glial cells in the beneficial effects of sleep
deprivation on depression (Hines et al., 2013).
In this study, the authors first confirmed that
84 Part II: Homeostatic Control
depressed animals that were sleep-deprived for
12 hours displayed significantly less depressive
symptoms than control depressed mice. Then,
using the dnSNARE animals to disrupt astro-
cytic adenosine signaling, they found that this
genetic manipulation prevented the antidepres-
sant effect of sleep deprivation on depressive-like
behaviors. Additionally, they demonstrated that
the antidepressant effects were potentially due to
a sleep deprivationinduced increase in extracel-
lular levels of adenosine acting specifically on A1
receptors. Indeed, intracerebroventricular infu-
sion of the A1 receptor agonist 2-Chloro-N6-
cyclopentyladenosine in WT mice mimicked
the effects of sleep deprivation on depression
phenotypes. Further, similarly to dnSNARE ani-
mals, A1 receptor knockout mice were resistant
to the antidepressant effects of sleep depriva-
tion (Hines etal., 2013). Consistent with another
study in which the role of astrocyte-derived ATP
in depressive-like behaviors has been identified
(Cao etal., 2013), these results provide a mecha-
nistic insight into how sleep deprivation pro-
duces its antidepressant effects.
Astrocytes and Sleeping Behavior
During Inflammatory Response
It is known that activation of the immune sys-
tem induces an increase in sleep pressure (SWA)
during NREM sleep and thereby an increase
in time spent in NREM sleep. This increased
sleeping behavior is part of several behavioral
changes called sickness which often occur dur-
ing systemic infections (for a review see Rohleder
etal., 2012). However, the mechanisms by which
the inflammatory response promotes sleep drive
are not well understood. Interestingly, several
studies have shown an increase of extracellular
concentrations of ATP and its derivative adeno-
sine in the brain during the development of the
systemic inflammatory response (Krueger, 2008;
Gourine et al., 2007). Because astrocytes are a
major source of the adenosine that can regulate
sleep homeostasis, a recent study has suggested
that these glial cells could contribute to the
induction of sleep behavior during inflammatory
responses (Nadjar etal., 2013). In this study, sys-
temic infections in WT mice were mimicked by
peripheral lipopolysaccharide treatment. Using
EEG/EMG recordings, the authors first con-
firmed that intraperitoneal injections of lipopol-
ysaccharide in WT mice induced an increase
in both NREM SWA power and NREM sleep
time. Then they used the dnSNARE mice to ask
whether astrocytes contribute to the increased
sleep pressure during systemic infections and
whether this is a result of changes in adenosine
signaling. Astrocytic dnSNARE expression pre-
vented the increase in both NREM SWA power
and NREM sleep time that normally occur in
WT mice during the first hours following intra-
peritoneal injections of lipopolysaccharide. In
addition, intracerebroventricular delivery of
CPT to block A1 receptors in WT mice mimicked
the dnSNARE phenotype that is consistent with
the role that astrocytic adenosine and A1 recep-
tors play in the regulation of sleep homeostasis.
This work indicates that astrocytes, in addition
to playing a pivotal role in the CNS immune
and inflammatory responses by releasing sev-
eral relevant immunologically factors (Volterra
and Meldolesi, 2005; Farina etal., 2007), are also
critical for the control of sleep pressure during
systemic infections by releasing adenosine that
subsequently acts on A1 receptors.
ROLE OFASTROCYTES
INEPILEPSY
Besides their important role in maintaining CNS
functions under physiologic conditions, growing
evidence suggest that astrocytes can also contrib-
ute to the development of brain disorders such
as epilepsy (for reviews see Wetherington etal.,
2008; Seifert etal., 2010; Clasadonte and Haydon,
2012). Epilepsy represents one of the most com-
mon neurological disorders, affecting 1% of the
population worldwide. The clinical manifesta-
tion of epilepsy is the occurrence of recurring,
unprovoked spontaneous seizures caused by the
hypersynchronous discharges of a population of
neurons mainly in the hippocampus that spread
across the brain through the recruitment of other
neuronal populations (Spencer, 2002; Avoli etal.,
2002). Numerous studies have identified a variety
of mechanisms by which astrocytes could con-
tribute to the generation of seizures (for reviews
see Wetherington etal., 2008; Seifert etal., 2010).
In the following sections, we summarize only
the recent works that have investigated the role
of reactive astrocytosis and gliotransmission in
epilepsy.
Reactive Astrocytosis and Epilepsy
Reactive astrocytosis (also known as reactive
astrogliosis) is often associated with TLE in
humans (Cohen-Gadol et al., 2004) and animal
models of epilepsy (Borges et al., 2003; Shapiro
et al., 2008). How or why this process occurs is

poorly understood. Reactive astrocytosis is gen-
erally observed under pathological conditions
and is typically characterized by morphological
changes in astrocytes, which increase in size and
number (for reviews see Sofroniew, 2009; Robel
et al., 2011). In fact, reactive astrocytes display
an increase in expression of glial cytoskeletal
proteins such as the glial fibrillary acidic protein
(GFAP) and vimentin that are therefore used as
hallmarks of the epileptic brain (Wilhelmsson
etal., 2004; Pekny and Nilsson, 2005). These reac-
tive changes in astrocytes are also accompanied
by the loss of astrocytic domain organization
(Oberheim et al., 2008) and generation of new
astrocytes from stem cells (Borges et al., 2006).
It is noteworthy that these overall morphologi-
cal changes in astrocytes during epilepsy occur
together with neuronal loss and synaptic rear-
rangements within the hippocampus in a process
called hippocampal sclerosis (Borges etal., 2003;
de Lanerolle et al., 2012). In addition, evidence
from studies in human epileptic tissue reports
that reactive astrocytes undergo striking changes
in the expression of specific enzymes, such as glu-
tamine synthetase (GS; Eid etal., 2012)and ADK
(Boison, 2012). Based on recent evidence, we next
discuss how these changes in astrocyte function
may contribute to the increased neuronal excit-
ability found in epileptic tissue.
Downregulation ofGS
During Epilepsy
GS found in astrocytes is the only enzyme known
to date that is capable of converting glutamate
and ammonia to glutamine in the mammalian
brain. This reaction is important, because it
allows ammonia detoxification in the brain and
also provides a continuous supply of glutamine,
which is necessary for the synthesis of gluta-
mate and GABA in neurons. Furthermore, rapid
metabolism of glutamate via GS is a prerequisite
for efficient glutamate clearance from the extra-
cellular space (for a review see Eid et al., 2012).
Given that, it is likely that any alterations in the
glutamine-glutamate-GABA cycle could affect
the extracellular concentrations of glutamate
and GABA and consequently neuronal network
excitability. Initial clinical studies performed in
humans with TLE have shown that the accumula-
tion of extracellular glutamate was due to a slow-
ing in the conversion of glutamate to glutamine,
leading to the hypothesis that GS deficiency
contributes to the development of epilepsy (Otis
and Jahr, 1998). This idea was then supported by
Role of Astrocytes in Sleep and Epilepsy 85
several works in human and experimental epi-
lepsy in which a relationship was found between
the downregulation of GS and hippocampal
reactive astrocytosis (Eid et al., 2004; Hammer
et al., 2008). Furthermore, it has been reported
that mutation of the GS gene in humans resulted
in seizure development (Haberle et al., 2005,
2006). Finally, studies in rats have also shown
that chronic hippocampal infusion of methio-
nine sulfoximine blocks GS-induced progressive
development of seizures along with neuropatho-
logical changes like those seen in human TLE
(Eid etal., 2008; Wang etal.,2009).
Although these experimental results suggest
that loss of GS induces seizures by causing an
increase in extracellular glutamate, other stud-
ies have tested the hypothesis that loss of GS may
contribute to epilepsy by causing a depletion of
GABA in inhibitory synaptic terminals. In sup-
port of this hypothesis, studies performed in
CA1 hippocampal slices demonstrated that selec-
tive blockade of GS with methionine sulfoximine
altered GABAergic synaptic transmission (Liang
etal., 2006)without affecting glutamatergic syn-
aptic transmission (Kam et al., 2007), suggest-
ing that the glial glutamine-glutamate-GABA
cycle represents the major contributor to synap-
tic GABA release regulating inhibitory synaptic
strength. Subsequently, an elegant study per-
formed by Ortinski et al. (2010) demonstrated
that a higher titer viral transduction of astro-
cytes with enhanced green fluorescent protein
via injections of adeno-associated virus from
serotype 2/5 (AAV2/5) into the mice hippocam-
pus led to reactive astrocytosis. The mechanisms
underlying AAV2/5-induced reactive astrocyto-
sis are unknown. However, because this reactiv-
ity in astrocytes was relatively local and occurred
in the absence of alterations in adjacent neurons
and microglia, the authors suggested that it
was likely a consequence of direct interactions
between the virus itself and the astrocyte, rather
than a consequence of inflammatory processes
involving microglial activation. Thus AAV2/5,
which has a strong astrocytic tropism, can be
used as a selective tool to generate reactive astro-
cytosis isolated from other pathologic processes.
In agreement with the idea that reactive astro-
cytosis leads to a reduced expression of GS, the
authors found a pronounced downregulation of
GS associated with increased expression in glial
fibrillary acidic protein and vimentin. Then, by
using hippocampal slice preparations, they tested
the hypothesis that GS deficits, and therefore the
86 Part II: Homeostatic Control
reduced glutamine pool, could decrease inhibi-
tory synaptic transmission. Interestingly, elec-
trophysiological recordings of CA1 pyramidal
neurons located in the vicinity of enhanced green
fluorescent proteinpositive areas indicated that
spontaneous and evoked GABAergic synaptic
transmission was significantly reduced while
glutamatergic synaptic transmission remained
unaltered. In addition, treatment of the hip-
pocampal slices with glutamine rescued the
deficit in synaptic inhibition, providing further
proof that this GABA depletion was caused by
neuronal glutamine starvation. Finally, using
voltage-sensitive dye-imaging techniques along
with electrical stimulation of temporoammonic
pathways, the authors demonstrated that reactive
astrocytosis was associated with network hyper-
excitability, which was subsequently attenuated
when hippocampal slices were treated with glu-
tamine. These results suggest that GS deficits
associated with reactive astrocytosis could con-
tribute to elevated seizure susceptibility during
epileptic conditions by causing a loss of synaptic
inhibition.
Upregulation ofADK During Epilepsy
Under physiological conditions, the ambient
level of adenosine is controlled by a metabolic
clearance through astrocytes (Boison, 2013).
Whereas adenosine can be released from neu-
rons or derived from the extracellular cleavage
of ATP, the astrocyte-based enzyme ADK elimi-
nates adenosine via phosphorylation to AMP
(Figure 5.1). Because astrocytes express equili-
brative nucleotide transporters, it is the meta-
bolic clearance through ADK that drives the flux
of adenosine into astrocytes, which thereby form
a sink for the metabolic clearance of adenosine
(Figure 5.1). Because adenosine has been found
to be elevated in patients following seizures, it
has been hypothesized that adenosine released
during a seizure mediates seizure arrest and pos-
tictal refractoriness (During and Spencer, 1992).
The A1 receptor-mediated functions are largely
responsible for the anticonvulsant and neuro-
protective activity of adenosine (Fredholm etal.,
2005). Indeed, it is known that activation of A1
receptors leads to a global reduction in neuronal
excitability via inhibition of presynaptic gluta-
mate release (Masino et al., 2002) and postsyn-
aptic membrane hyperpolarization (Arrigoni
et al., 2006). Given that, it is plausible that any
alterations in ADK activity that consequently
lead to changes in adenosine homeostasis could
be responsible for the increased neuronal excit-
ability during seizures. In support of this notion,
a series of studies from Boisons group identified
ADK as one of the major molecular link between
reactive astrocytosis and neuronal dysfunc-
tion in epilepsy (for a review see Boison, 2012).
First, ADK was found to be upregulated in reac-
tive astrocytes in human and experimental epi-
lepsy causing extracellular adenosine deficiency
(Fedele etal., 2005; Aronica etal., 2011; Li etal.,
2008, 2012). Subsequently, it has been found
that transgenic overexpression of ADK in mice
induced spontaneous recurrent seizures while
mice with a genetic forebrain-selective reduction
of ADK or receiving intrahippocampal implants
of ADK-deficient stem cellderived neuronal
precursors were resistant to seizure develop-
ment (Li etal., 2008). These findings indicate that
astroglial ADK represents a promising target for
the prevention of epileptogenesis.
GLIOTR ANSMISSION
AND EPILEPSY
Based on the aforementioned observa-
tions that astrocytes release glutamate in a
calcium-dependent manner and are able to con-
trol neuronal NMDA receptors trafficking, it is
not hard to envisage that these glial cells could
contribute to epileptogenesis. In the following
sections, we discuss the recent studies that have
investigated the potential role of gliotransmis-
sion in epilepsy in situ and invivo.
Astrocytic Calcium-Dependent
Release of Glutamate in Epilepsy
A direct role of gliotransmission in the genera-
tion of epileptiform activity in situ came from
demonstration that through calcium-dependent
release of glutamate, astrocytes in the hippocam-
pus can directly excite a group of neighboring
CA1 pyramidal neurons in a synchronized fash-
ion via extrasynaptic NMDA receptor-mediated
SICs (Fellin et al., 2004). Subsequently, several
studies in brain slices (Fellin etal., 2006)and in
anaesthetized animals (Tian et al., 2005) have
shown that calcium oscillations were increased
during chemically induced epileptiform activ-
ity. Importantly, these calcium increases in
astrocytes were dampened by several known
anticonvulsant drugs (Tian et al., 2005). The
calcium-dependent glutamate release from
astrocytes and consequently the activation of
extrasynaptic NMDA receptors were then found
to contribute to neuronal death in the cortex

and hippocampus during the days that followed
chemically induced status epilepticus in mice
(Ding et al., 2007). Finally, a recent work from
Gmez-Gonzalo et al. (2010) demonstrated a
direct role of astrocytes in focal epilepsy. The
authors found that astrocytes in cortical slices
displayed massive intracellular calcium increases
in response to a surge of neuronal activity and by
signaling back to neurons they lower the thresh-
old for the generation of focal seizures. Combined
with the observations that reactive astrocytes
exhibit increased expression of mGluRs dur-
ing epilepsy (Aronica etal., 2000; Tang and Lee,
2001; Steinhauser and Geifert, 2002), which may
potentiate calcium-dependent glutamate release,
these results suggest that a hyperexcitable neu-
ronal network may arise in the brain resulting
from enhanced glutamatergic gliotransmission
and trigger a focal site of seizure initiation.
Astrocytic Control ofSynaptic NMDA
Receptor Trafficking and Epilepsy
Although initial studies suggesting a role of
astrocytes in the generation of epileptiform activ-
ity were mainly performed in situ, a direct role of
gliotransmission in epileptogenesis in vivo was
identified only recently (Clasadonte etal., 2013).
Using dnSNARE mice along with electrophysi-
ological, behavioral, and histological approaches,
we have demonstrated that genetic impairment
of the SNARE complex in astrocytes led to a
hypofunction of postsynaptic NMDA receptors
and ameliorated the development of epilepsy
that includes progressive increase in seizure fre-
quency, hippocampal sclerosis, and behavioral
abnormalities.
By using the mouse pilocarpine model of
epilepsy along with continuous video-EEG
recordings that commenced 5 days after
pilocarpine-induced status epilepticus and con-
tinued for as long as 150 days, we found that
astrocytic dnSNARE expression delayed the
occurrence of the first spontaneous recurrent
seizure (Figure 5.3A), reduced seizure sever-
ity, and attenuated the progressive increase
in seizure frequency (Figure 5.3B) that typi-
cally occurs in human TLE (Clasadonte et al.,
2013). In accordance with our previous stud-
ies showing that astrocytic dnSNARE expres-
sion leads to a reduced expression of synaptic
NMDA receptors in cortical cells (Deng et al.,
2011), whole-cell patch-clamp recording of CA1
pyramidal neurons indicated a decrease in min-
iature and evoked postsynaptic NMDA currents
Role of Astrocytes in Sleep and Epilepsy 87
in dnSNARE animals that persisted during epi-
lepsy (Clasadonte et al., 2013) (Figures 5.3Ci,
5.3Cii, and 5.3Ciii). Given that, it is likely that
this reduced activity of synaptic NMDA recep-
tors, which is expected to reduce neuronal excit-
ability, could mediate the effects of astrocytic
dnSNARE expression on epilepsy. To test this
hypothesis, we used an in situ model of epilepsy
and found that the frequency of epileptiform dis-
charges recorded in CA1 hippocampal slices was
reduced by astrocytic dnSNARE expression sim-
ilarly to results obtained on seizures in vivo. We
then observed that blocking NMDA receptors
with D-AP5 mimicked the dnSNARE phenotype
in WT slices. These results were subsequently
replicated in WT mice in vivo by using chronic
intracerebroventricular infusion of D-AP5.
Additionally, we noticed that D-AP5 treat-
ment remained ineffective on seizure activity in
dnSNARE animals, providing further proof that
the effects of astrocytic dnSNARE expression
were mediated through reduced activity of syn-
aptic NMDA receptors (Clasadonte etal.,2013).
Because it is known that long-term behavioral
abnormalities are often associated with epilepsy
(Swinkels et al., 2005; Mller et al., 2009), we
then investigated whether astrocytic dnSNARE
expression could prevent the development of
these behavioral defects (Clasadonte etal., 2013).
Consistent with previous studies (Mller et al.,
2009), we found that exploratory habits of WT
mice with epilepsy were dramatically affected in
the open field test. Epileptic WT mice displayed
a striking increase in duration of stops between
subsequent locomotor excursions. Interestingly,
these behavioral defects were significantly atten-
uated in epileptic dnSNARE mice (Figures5.3Di,
5.3Dii5.3Diii).
In the same study (Clasadonte et al., 2013),
we subsequently compared the degree of hip-
pocampal sclerosis between WT and dnSNARE
animals 8 months after pilocarpine treatment.
Immunostaining against the neuronal epitope
NeuN indicated a significant greater neuro-
protection in the hilus of the dentate gyrus
from dnSNARE mice compared to WT mice
(Figures 5.3Ei and 5.3Eii), consistent with our
previous studies demonstrating that pharmaco-
logical blockade of gliotransmission prevented
neuronal loss in the hippocampus after induction
of status epilepticus (Ding etal., 2007). In addi-
tion, immunostaining against the glial markers
glial fibrillary acidic protein and vimentin showed
a striking attenuation in reactive astrocytosis in
 (a) (b) (c)(i) (c)(iii)

(c)(ii)

(d)(i) (d)(ii) (d)(iii)

(e)(i) (f)(i) (g)(i)

(e)(ii) (f)(ii) (g)(ii)

FIGURE5.3: Gliotransmission modulates pathophysiological consequences of temporal lobe epi-

lepsy. (A)Kaplan-Meier analysis of the time of spontaneous recurrent seizure appearance after pilocarpine-induced
status epilepticus (SE) showing a delayed seizure onset in dnSNARE mice compared to wild-type (WT) mice (n=6
per genotype; log rank test, *p < 0.05). (B)The average number of seizures per day for each 1-month block was
attenuated from 2 to 5months after SE in dnSNARE mice (n=68) compared to WT mice (n=46; ANOVA:geno-
type, F=14.893, p=0.001; time, F=5.410, p=0.002; genotype time interaction, F=2.664, p=0.048; post hoc test,
*p < 0.05). Top insets represent spontaneous recurrent seizures detected in the cortical EEG of WT and dnSNARE
mice several months after SE. (Ci) Representative whole-cell patch-clamp recordings of NMDA and alpha-amino-
3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) currents in CA1 pyramidal neurons obtained from SE WT
and SE dnSNARE mice 5months after pilocarpine treatment. Note the reduced amplitude of NMDA current in SE
dnSNARE mice compared to SE WT mice. (Cii) AMPA/NMDA ratio was increased in SE dnSNARE mice (9 cells
from 4 animals) compared to SE WT mice (13 cells from 5 animals; Students t test, ***p < 0.001) 5months after
 Role of Astrocytes in Sleep and Epilepsy 89

the dentate gyrus (Figures 5.3Fi, 5.3Fii, 5.3Gi,
and 5.3Gii) as well as in the CA1CA3 areas
(Clasadonte et al. 2013). Because changes in
expression of astroglial proteins such as GS and
ADK are often associated with reactive astrocyto-
sis (see previous discussion), it is likely that these
molecular alterations are also dampened by astro-
cytic dnSNARE expression during epilepsy, pro-
viding consequently further protection against a
dysregulation in the neuronal network excitabil-
ity, though this remains to be tested.
As mentioned, astrocytic glutamate acting
on neuronal NMDA receptors could potentially
contribute to seizure generation and brain
damage. However, the inability to measure
dnSNARE-dependent changes in glutamate
levels, measured with microelectrode biosen-
sors in hippocampal slices (Clasadonte et al.,
2013), makes it unlikely that the nonprogres-
sive development of epilepsy, including seizure
occurrence, neuronal damage, and behavioral
defects, discovered in dnSNARE animals was
a consequence of an impaired release of gluta-
mate from astrocytes. Since adenosine acting
on A1 receptors is primarily known to exert
anticonvulsant (Dunwiddie, 1999; Etherington
and Frenguelli, 2004; Boison, 2012) and neu-
roprotective effects (Gomes et al., 2011), one
would have expected that the reduction of
extracellular adenosine acting on A1 recep-
tors in dnSNARE animals (Pascual etal., 2005;
Halassa etal., 2009; Schmitt etal., 2012)would
have worsened pathophysiological conse-
quences of epilepsy. This discrepancy could
be due to the difference in the timescales in
which the processes take place. Indeed, it has
been shown that short- and long-term inacti-
vations of adenosine receptors have diametri-
cally opposite effects on pathophysiological
consequences of epilepsy (Georgiev etal., 1993;
Von Lubitz etal., 1994; Johansson etal., 1996;
Rigoulot et al., 2003). This pharmacological
phenomenon termed effect inversion has been
questioned by Jacobson etal. (1996). Consistent
with the anticonvulsant role of adenosine, acute
administration of the A1 receptor antagonist
CPT decreases the threshold to NMDA-induced
seizures and increases seizure-induced brain
damage (Von Lubitz et al., 1994). In con-
trast, chronic treatment with CPT, which
most likely mimics the dnSNARE phenotype,
decreases seizure susceptibility and provides

FIGURE5.3 Continued
pilocarpine treatment. (Ciii) Pictures showing CA1 pyramidal neurons filled with biocytin (red) in SE WT and
SE dnSNARE mice during AMPA/NMDA ratio measurements. The presence of green fluorescent protein (GFP)
confirms that the dnSNARE transgene was expressed in astrocytes (green) of dnSNARE animals (Pascual etal.,
2005). (Di) Representative 30 min of track plots of control-no SE and SE-experienced WT mice placed in an open
field 8months after pilocarpine treatment. (Dii) Same experiments as in (Di) but performed with control-no SE
and SE-experienced dnSNARE mice. (Diii) The average duration of time stopped over the 30 min test period was
increased in SE WT mice but not in SE dnSNARE mice (WT no SE, n=8; WT SE, n=7; dnSNARE no SE, n=7;
dnSNARE SE, n=8; ANOVA followed by post hoc test, *p < 0.05 when WT SE is compared with WT no SE). (Ei)
Representative hippocampal sections from control-no SE and SE-experienced WT and dnSNARE mice stained
with an antibody directed against the neuron-specific epitope NeuN 8months after pilocarpine treatment. Striking
neuronal cell loss was observed in the hilus (h)of the dentate gyrus (DG) in SE WT mice but not in SE dnSNARE
mice. (Eii) Average neuronal density in the hilus of the DG (WT no SE, n=3 animals; WT SE, n=7 animals;
dnSNARE no SE, n=5 animals; dnSNARE SE, n=4 animals; ANOVA followed by post hoc test, **p < 0.01, ***p <
0.001; ns=nonsignificant). (Fi) Same hippocampal sections as in (Ei) but stained with an antibody directed against
the astrocyte-specific epitope glial fibrillary acidic protein (GFAP). GFAP expression was increased in the hilus
(h)of the DG in SE WT mice but not in SE dnSNARE. (Fii) Average GFAP area in the hilus of the DG (WT no SE,
n=3 animals; WT SE, n=7 animals; dnSNARE no SE, n=5 animals; dnSNARE SE, n=4 animals; ANOVA fol-
lowed by post hoc test, *p < 0.05; ns). (Gi) Representative hippocampal sections stained with an antibody directed
against the astrocyte-specific epitope vimentin 8 months after pilocarpine treatment. Vimentin expression was
dramatically increased in the hilus (h) of the DG in SE WT mice but not in SE dnSNARE mice. Samples were
obtained from another group of WT and dnSNARE mice that either did (SE) or did not enter SE (control-no SE).
(Gii) Average vimentin area in the hilus of the DG (WT no SE, n=11 animals; WT SE, n=11 animals; dnSNARE
no SE, n=9 animals; dnSNARE SE, n=7 animals; ANOVA followed by post hoc test, *p < 0.05, **p < 0.01, ***p <
0.001; ns). Average scale bars for (Ciii), (Ei), (Fi) and (Gi) represent 100m.
Adapted from Clasadonte etal. (2013).
90 Part II: Homeostatic Control
neuroprotection (Von Lubitz et al., 1994). The
mechanism underlying the observed protection
of long-term inactivation of A1 receptors may
rest at the level of second messenger systems
coupled to these receptors. In agreement with
this idea, we have found that one of the molecu-
lar consequences of prolonged inactivation of
A1 receptors is the reduction of NMDA recep-
tor surface trafficking (Deng etal., 2011). Thus
we suggest that this long-term process mediates
the beneficial effects of astrocytic dnSNARE
expression on pathophysiological consequences
of epilepsy.
By combining a model of epilepsy that more
closely mimics the complex feature of TLE
in patients with astrocyte-specific molecular
genetics, these findings revealed that astro-
cytes, by controlling neuronal NMDA receptor
activity, contribute to the progressive devel-
opment of TLE (Figure 5.1), including seizure
occurrence, brain damage, and behavioral
deficits. These results provide further evidence
to support the idea that astrocytes represent a
potential therapeutic target for the treatment of
epilepsy.
CONCLUDING REMARKS
In this chapter we summarized the efforts that
have been made over past decades to under-
stand the mechanisms underlying neuron-
to-astrocyte communication in the brain.
Although there remain some disparities
between areas mainly due to the lack of tools
that allow the selective manipulation of astro-
cytic signals, recent work employing the trans-
genic dnSNARE mouse line has been beneficial
to discern the role of astrocytes in more inte-
grated systems during physiological and patho-
logical conditions. As examples, we discussed
the role of astrocytes in sleep-related behavior
as well as in the etiology of epilepsy. The rela-
tionship between sleep and epilepsy has been
recognized since antiquity and demonstrates
a delicate association of brain physiology and
dysfunction. For example, sleep and sleep dep-
rivation affect the distribution and frequency
of seizures in humans. Consequently, it would
be very interesting in the future to investi-
gate whether glia represents the cellular link
between sleep and epilepsy. Much more work
still needs to be done to understand how gli-
otransmission contributes to brain physiology
and pathology. In a common effort, this will
ultimately lead to the development of new ther-
apeutics for treating brain disorders.
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6
AstrocyteNeuron Interactions
A NGUS M.BROW N A ND BRUCE R .R A NSOM
INTRODUCTION
The diversity and complexity of brain function
in part derives from the close association of het-
erogeneous cell types separated by a narrow gap
filled with interstitial fluid. Such an arrangement
optimizes rapid cell-to-cell communication and
facilitates homeostatic control of the compart-
ments present within the central nervous system
(CNS). There are two main types of cell present in
the brain: neurons, the classic electrically excit-
able cell, and glia, of which we focus on astro-
cytes. Broadly speaking, the function of neurons
is communication with other neurons, and the
function of astrocyte cells is to maintain an opti-
mal environment for neuronal activity. Thus the
dogmatic view of brain function is of neuronal
communication mediated by electrical signal-
ing. Such signaling relies on energy-dependent
maintenance of transmembrane concentrations
of sodium (Na+) and potassium (K+; Bear et al.,
2007). The transmembrane separation of ions is
essential for the action potentials that underlie
neuronal activity, but such activity results in dis-
ruption of these gradients by (a) disturbing the
equilibrium of ion distribution both in cells and
in the interstitium, which (b) requires homeo-
static mechanisms to restore ionic equilibrium
for optimal neural function (Dienel, 2009). Given
the close proximity of cells to each other, it is easy
to appreciate how activity in one cell type can
affect neighboring cells via the interstitial fluid.
This interdependent relationship lies at the heart
of astrocyte neuron interactions. In the past
decade or so, the diversity of roles assumed by
astrocytes has burgeoned to the extent that full
coverage is beyond the scope of this chapter, thus
we focus on a few key functions of astrocytes.
Neuronal activity in the form of action poten-
tials and synaptic activity disturbs the ionic
composition of interstitial space. Given the con-
strictive dimensions of the interstitial space, rela-
tively minor changes in ion movements across
neuronal membrane can result in large devia-
tions from baseline levels in the interstitial fluid
(Ransom, 2009). The most recognized of these is
the effect of neuronal activity on [K+]o, which can
increase from 3 mM to 12 mM under physiologi-
cal conditions (Ransom etal., 1992). pHo can also
be affected, the resulting interstitial acidification
being dependent on the degree of neuronal activ-
ity (Ransom et al., 1992). Synaptic activity can
result in elevated [glutamate]o at synapses, which,
given the potential excitotoxic function of gluta-
mate, can have grave consequences if not rapidly
sequestered (Choi, 1992). Such alterations in ion
concentrations also have effects on osmolarity
of the various compartments, thus intercellular
water transport is controlled by astrocytes (see
later discussion). The interstitial space is also the
site of energy substrate transfer between astro-
cytes and neurons, with astrocyte-to-neuron
metabolic interactions being increasingly real-
ized as a vital component of CNS function.
I O N H O M E O S TA S I S
[K+]o
The transmembrane ion gradients that are pre-
sent across neuronal and glial cell membranes
are vital for reliable electrical communication
throughout the CNS. In general, cell membranes
are more permeable to K+ than to Na+ at rest, thus
the resting membrane potential approaches the K+
reversal potential (Hille, 2001). Indeed, astrocytes
are exclusively permeable to K+, thus the resting
membrane potential equals the K+ reversal poten-
tial, resulting in the astrocyte membrane potential
responding in a manner predicted by the Nernst
equation to elevations in [K+]o (Kuffler & Nicholls,
1966). Excitation of neurons and axons in the
form of action potentials are a result of transient
increases in Na+ permeability followed by K+ per-
meability. The increased Na+ permeability occurs
via voltage-dependent opening of Na+ channels

leading to a Na+ influx into the cells, followed by
opening of K+ channels leading to an efflux of K+
from the cell (Hille, 2001). Given the large extra-
cellular [Na+] and the limited movements of Na+
into cells required to produce an action potential
(see Hille, 2001, for detailed calculations), there is
a negligible effect on [Na+]o resulting from action
potentials. However, given the longer duration of
opening of K+ channels, the low [K+]o, and the small
interstitial space, which acts to concentrate ions,
there is a significant increase in [K+]o as a result
of action potential conduction (Ransom et al.,
2000). Although the increase in [K+]o is apparent
in response to a single action potential, it is repeti-
tive activity that results in significant, long-lasting
elevations in [K+]o. An interesting feature of physi-
ologically evoked [K+]o elevations is that there
is a ceiling level above which the [K+]o will not
increase due to the buffering capacity of the astro-
cytes (Ransom et al., 1985). Large elevations of
[K+]o would lead to neuronal depolarization, thus
the transmembrane ion gradients must be rap-
idly restored for optimal neuronal function. One
of the first roles of astrocyte to be described was
in buffering increases in [K+]o (Kofuji & Newman,
2004). Astrocytes take up K+ via three separate
mechanisms:the Na+K+ATPase, which dominates
at modest elevations of K+; the NKCCl cotrans-
porter, which dominates at higher concentrations
(Hertz et al., 2013); and the inwardly rectifying
K+ channel (Kofuji & Newman, 2004). The astro-
cytic Na+K+ATPase has a Km value of up to 15 mM,
significantly higher that its neuronal counterpart
and a value that makes it ideally suited to buff-
ering the elevations of K+ routinely encountered
under physiological conditions (Verkhratsky &
Butt, 2007). Since astrocytes are interconnected
via gap junctions into syncitia, the K+ accumulated
during buffering activity can be redistributed via
the syncitium and released at distant sites in order
to maintain low [K+]o, a process known as spatial
buffering (Newman, 1995). Within individual
astrocytes, such as Muller cells in the retina, a
specialized form of buffering occurs known as
siphoning. Muller cells span the width of the ret-
ina but express K+ channels in a polarized man-
ner. The Kir2.1 inwardly rectifying channels are
expressed at the inner and outer plexifrom layers
where there is significant synaptic activity and
thus release of K+ from neural elements. As such,
the Kir2.1 channels are ideally located to take up
K+. The Muller cells express Kir4.1 channels at the
endfoot and vitreous borders where they release K+
into the vitreous humor and the blood (Kofuji &
Newman, 2004; Figure 6.1). In mice where the
AstrocyteNeuron Interactions 99
AQP4 aquaporin water channel was knocked out,
activity-evoked elevations in interstitial K+ were
attenuated compared to wild type, indicative of
decreased water uptake into astrocytes. In addi-
tion, there was enhanced gap-junction coupling
of astrocytes and enhanced K+ redistribution
(Strohschein etal.,2011).
pHo
Astrocytes are an important site for regulation of
pH. Such regulation is important as alterations in
pH of astrocytes can lead to changes in the pH
of the interstitium, which in turn can affect the
pH of neurons given the constricted interstitial
space separating astrocytes and neurons. pH
is of course a measurement of hydrogen, [H+],
expressed on a logarithmic scale to counter the
nanomolar concentrations of H+ in living tissue
(pH=log10[H+]). The expression of pH and the
limited range encountered under physiological
conditions can suggest that [H+] does not vary a
great deal, but relevant physiological concentra-
tions are from 10 to 300 nM (Rose & Ransom,
1998). Such a range of [H+] has very important
consequences as proteins and enzymes are
exquisitely sensitive to pH with changes of as lit-
tle as 0.05 units having large effects on function
(Stryer, 1995). From data acquired in multiple
mammalian astrocytes, a broad generalization
can be made that pHi of astrocytes contains up
to 0.2 pH units more acid than the interstitial pH
(Rose & Ransom, 1998). This implies an obvious
difference in transmembrane [H+] and a reversal
potential for H+ of about 10 mV. Given a rest-
ing membrane potential of 85 mV or so, there
Subretinal space
K+ IPL
Endfoot
Vitreous
FIGURE 6.1: Schematic illustration of K+
siphoning in a retinal Muller cells. Inwardly
rectifying Kir2.1 channels (filled circle) are expressed
in the inner plexiform layer (IPL, grey area) to absorb
K+ released during synaptic activity. K+ is distributed
within the Muller cell and is ejected via Kir4.1 channels
(open circles) located on the endfoot into the vitreous
humor and the subretinalspace.
100 Part II: Homeostatic Control
is a clear outward movement of H+ creating an
inward electrochemical gradient for H+ across the
astrocyte membrane. There are a variety of mech-
anisms in which pHi of astrocytes is regulated,
and these can be divided into Na+-dependent
and Na+-independent mechanisms. Into the
former category are the Na+/H+ exchanger,
Na+/HCO3 contransport, and Na+-dependent
Cl/HCO3 exchanger, and into the latter lie
Cl/HCO3 exchange and the H+ pump (Rose &
Ransom, 1998). The differences in astrocyte
transmembrane Na+ concentrations provide the
electrochemical gradient for select ions to couple
their movements relative to the Na+ gradient. The
Na+/H+ exchanger is present in all mammalian
cells. It is electroneutral with a 1:1 stochiometry
in which influx of 1 Na+ is countered by the efflux
of a H+, resulting in intracellular alkalization.
The Na+/H+ exchanger plays an important role
in setting the baseline pHi, a role it shares with
the HCO3 exchangers and transporters. Na+/
HCO3 cotransport is exclusive to glial cells and
absent from neurons (Verkhratsky & Butt, 2007).
The cotransporter is electrogenic due to the sto-
chiometry of transport of 1 Na+ per 2 HCO3 in
astrocytes. The resulting reversal potential of70
to 80 mV is close to the resting membrane
potential and dictates that membrane depolariza-
tion would lead to inward HCO3 transport and
alkalization, whereas membrane hyperpolariza-
tion would lead to HCO3 efflux and intracellular
acidification (Pappas & Ransom, 1994). The elec-
trogenic nature of the cotransporter is important
as it implies that membrane potential will influ-
ence transport and, perhaps more important
with regard to function, that activation of the
cotransporter will alter membrane potential. The
Na+-dependent Cl/HCO3 exchanger uses the
Na+ gradient to transport Cl into the cell and
HCO3 out, whereas the Cl/HCO3 exchanger is membrane Na+ gradient to drive glutamate into
independent of the Na+ gradient. Since the acid
extrusion mechanisms described earlier employ
the Na+ gradient, these ultimately require adeno-
sine triphosphate (ATP) to fuel the Na+K+ATPase
that maintains the uneven distribution of trans-
membrane Na+. The H+ pump (H+ ATPase) also
requires ATP to fuel pumping of H+ out of thecell.
Activity-Induced pH Changes
and Regulation
Depolarization of astrocytes, as would occur as a
consequence of elevated interstitial K+ resulting
from increased neuronal activity, causes alkali-
zation in astrocytes (Pappas & Ransom, 1994).
This may result from astrocyte-to-neuron lactate
shuttling, as lactate is cotransported with H+,
or as a result of depolarization causing inward
transport of the Na+/HCO3 cotransporter. It is
hypothesized that neuronal-induced elevation
in [K+]o ultimately leads to a negative feedback
loop that limits neuronal activity. The elevated
[K+]o causes depolarization of the astrocyte mem-
brane, which in turns leads to inward transport
of HCO3 via the Na+/HCO3 cotransporter. The
resulting astrocyte alkalization causes an acidi-
fication of the interstitium, which in turn leads
to depression of neural activity by an as yet
unknown mechanism (Rose & Ransom,1998).
NEUROTRANSMITTER
H O M E O S TA S I S
A key function of astrocytes is the recovery and
cycling of the excitatory neurotransmitter glu-
tamate at synapses. Glutamate, a nonessential
amino acid, is the dominant excitatory neuro-
transmitter in the CNS and is estimated to be
present at 80% of excitatory CNS synapses (Bear
et al., 2007). Given the potential excitotoxic
effects of uncontrolled glutamate levels in the
brain, the blood-brain barrier excludes glutamate
present in the systemic circulation from the brain
parenchyma, thus glutamate must be manufac-
tured within the brain. The source of glutamate is
ultimately glucose, with glutamate derived from
the tricarboxylic acid substrate -ketoglutyrate
via actions of the enzyme glutamate dehydro-
genase, a reaction that occurs in the presynaptic
terminal (McKenna et al., 2006). Once gluta-
mate is secreted from the presynaptic terminal
and subsequently released from the postsynaptic
receptors, it is taken up into astrocytes, rather
than back into the presynaptic terminal. This is
facilitated by glutamate transporters present on
the astrocyte membrane, which use the trans-
the astrocyte (i.e., from a compartment of low
glutamate concentration, the synaptic cleft, to
a compartment of high glutamate concentra-
tion, the astrocyte). Once inside the astrocyte
the glutamate is converted to glutamine by the
ATP-dependent enzyme glutamine synthase and
released back into the extracellular space, from
which it is taken up by the presynaptic termi-
nal. The glutamine, once inside the presynaptic
terminal, is converted to glutamate by glutami-
nase and is subsequently repackaged for vesicu-
lar release (McKenna etal., 2006). This sequence
constitutes the glutamateglutamine cycle, and it
confers several advantages on the neuron. First,
it shifts the metabolic burden from the neuron to

the astrocyte, thus sparing neuronal ATP, which
could be a vital neuroprotective mechanism dur-
ing periods of energy deprivation. Second, the
glutamine shuttled from the astrocyte to the
neuron is inert as a neurotransmitter; that is,
it does not bind to glutamate receptors and is
thus incapable of contributing to excitotoxicity
(Figure6.2).
A consequence of energy deprivation, as can
occur during stroke or anoxia or aglycemia as
separate insults, is a decrease in ATP production,
such that the tissue demand for ATP exceeds sup-
ply. The ATP requirements fall under two main
categories:signaling, involving action potentials
and synaptic potentials, and maintenance of
the hyperpolarized resting membrane potential
(Attwell & Laughlin, 2001). It is the latter that has
the most far-reaching consequences under condi-
tions of energy deprivation, as there is insufficient
ATP to fuel the Na+K+ATPase that maintains the
transmembrane ion gradients (i.e., low [K+]i and
high [Na+]o.) Thus, as a result, the ion concentra-
tions tend to equilibrate across the cell membrane
such that the resting membrane potential depo-
larizes. The equaling of the Na+ concentration
across the cell membrane has grave consequences
for cell survival as the Na+-coupled glutamate
transporters can result in an efflux of glutamate
from the astrocyte into the interstitial space,
gln gln
ATP
GL
pre GS
glu
Na+
ADP
glu glu
post
astrocyte
FIGURE 6.2: The glutamateglutamine cycle.
Glutamate (glu) released from the presynaptic termi-
nal is cleared from the synaptic cleft via Na+-coupled
glutamate transporters present on the astrocyte mem-
brane (open circle). The glutamate is converted to glu-
tamine (gln) in the astrocyte by glutamine synthase
(GS), a reaction that requires adenosine triphosphate
(ATP). The glutamine is then shuttled back to the
presynaptic terminal for conversion to glutamine by
glutaminase(GL).
AstrocyteNeuron Interactions 101
given that the direction of glutamate movements
is determined by the Na+ concentration (Attwell,
2000). The resulting extracellular glutamate has
direct access to Ca2+-permeable receptors, result-
ing in toxic Ca2+ influx into neurons triggering
apoptosis and necrosis (Choi,1992).
E N E R G Y M E TA B O L I S M
Interest was reignited in the dormant field of
brain glycogen research by several studies. These
were predominantly tissue culture studies, the
conclusions of which could be extrapolated to
more intact systems. The first study to suggest a
link between astrocytic glycogen and function
resulted from experiments in which hypotha-
lamic neurons were cultured with glial cells either
present or absent from the culture. Neurons that
were seeded in the company of glial cells survived
for longer periods of time than neurons seeded
in the absence of glial cells. The clear conclusion
was that coculturing neurons with glia confers a
benefit of an unknown type, possibly metabolic,
on the neurons, prolonging their survival in the
artificial culture environment (Whatley et al.,
1981). A subsequent study asked a more direct
question: What is the manner of protection
offered by the glial cells in cocultures of corti-
cal neurons? The conclusion from that study was
that it is not the presence per se of the glial cells
(astrocytes) that protects the neurons but rather
the glycogen contained within the astrocytes.
In cocultures in which the glycogen content of
the astrocytes was decreased, neurons did not
survive as long as when the astrocytes displayed
robust glycogen content (Swanson & Choi, 1993).
That glycogen can protect neurons is not surpris-
ing, given its role in the liver and skeletal muscle
as an energydepot.
In the more intact central model of white
matter, the rodent optic nerve, studies have high-
lighted the basic role of glycogen as a central
energy store/buffer. In an in vitro model of the rat
optic nerve, where the tissue is maintained in a
superfusion chamber held at 37C, bubbled with
95% oxygen (O2) 5% CO2, and superfused with
artificial cerebrospinal fluid containing 10 mM
glucose, the stimulus-evoked compound action
potential (CAP) can be maintained for many
hours. If the preparation is exposed to anoxia
(withdrawal of O2), the CAP falls to zero within
5 minutes (Stys etal., 1991). However, in the pres-
ence of O2, but in the absence of glucose, the CAP
fails after 20 to 30 minutes (Ransom & Fern,
1997). This delay in CAP failure in the absence
of exogenously applied glucose strongly suggests
102 Part II: Homeostatic Control
that an endogenous energy reserve is present in
the tissue that is capable of supporting axon con-
duction, albeit for a limited period of time. The
most likely candidate is glycogen. The presence
of glycogen in the CNS has been known for dec-
ades, and it has the intriguing feature of being
present only in astrocytes in the adult (Cataldo &
Broadwell, 1986). To investigate whether gly-
cogen was the energy reserve that supported
function, parallel experiments were carried out
in which the CAP was recorded in the absence
of glucose and glycogen content measured. The
results indicated that after switching to zero glu-
cose, while the CAP is maintained, the glycogen
content starts to fall, and once the glycogen level
in the tissue reaches its nadir, the CAP starts to
fail and from that point falls rapidly to zero. This
temporal correlation between glycogen content
and CAP conduction closely associates the gly-
cogen present in the tissue with the ability of the
nerve to conduct action potentials (Figure 6.3)
and leads to the prediction that the larger the gly-
cogen content of the tissue, the longer the CAP is
maintained. This was tested by altering the gly-
cogen content of the tissue prior to the period of
aglycemia. Incubating the nerves in 1 mM nor-
epinephrine decreased glycogen content, whereas
incubating the tissue in artificial cerebrospinal
fluid containing 25 mM glucose elevated gly-
cogen content. Exposing nerves to aglycemia
after increasing or decreasing the glycogen con-
tent led to an increase or decrease, respectively,
in the latency to CAP failure (Wender et al.,
2000). Similar experiments were carried out in
a mouse model, and qualitatively similar results
were found. Glycogen content decreased from
the onset of aglycemia and took 20 minutes to
reach its nadir, at which point the CAP failed.
Augmentation of glycogen content increased
CAP latency during aglycemia, whereas attenu-
ation of glycogen content increased CAP latency
(Brown etal.,2003).
In addition to glycogens ability to support
axonal conduction during periods of glucose
withdrawal, the ability of glycogen to support
physiological function was studied. Although
glucose withdrawal is an extremely useful
experimental maneuver with which to study
a substrates ability to support function in the
absence of glucose, it is artificial in that in vivo
blood glucose levels would be hypoglycemic
rather than aglycemic, although it has been cal-
culated that blood glucose levels in the brain can
approach zero during severe bouts of hypoglyce-
mia (Frier & Fisher, 2007). Indeed, aglycemia can
FIGURE 6.3: Glycogen in the astrocyte fuels axon
function. Glucose is transported from the blood vessel
to astrocytes via glucose transporters (closed circles).
In astrocytes glycogen is formed from glucose via gly-
cogen synthase (GS) and is converted, ultimately, to
lactate by glycogen phosphorylase (GP). The lactate is
shuttled from the astrocyte to the neuron via monocar-
boxylate transporters (MCTs, open circles), where it is
oxidatively metabolized.
reasonably be viewed as a consequence of iatro-
genic insulin therapy, and thus its occurrence has
been a significant clinical emergency only since
the introduction of exogenously applied insulin
to treat type 1 diabetics (Frier & Fisher, 2007).
However, it should be noted that the condition of
insulinomas would also produce such effects.
We studied the ability of glycogen to support
axon conduction in our optic nerve preparation
by imposing on the tissue a high-frequency train
of stimuli of 100 Hz. What we found was that 100
Hz stimulus for 4 minutes caused a significant
decrease in glycogen content, even in the pres-
ence of normoglycemic concentrations (10 mM)
of glucose. These results imply that, under con-
ditions where tissue energy demand exceeds glu-
cose supply, glycogen is metabolized to provide
supplemental energy substrate to support axon
conduction, a clear example of glycogen support-
ing physiological function (Brown etal.,2003).
L A C TAT E T R A N S F E R F R O M
ASTROCYTE TOAXON
The means by which glycogen supports function
has also been investigated. Clearly, glycogen is too
large a molecule to be transported intercellularly,
thus it must be metabolized in the astrocytes to
a conduit that is transported from the astrocyte
to the neuron/axon (Dringen et al., 1993). The
most likely candidate for this conduit is lactate.
In tissue culture studies, astrocyte cultures were
shown to release lactate but not glucose (Dringen
etal., 1995). This is an important point as astro-
cytes lack the enzyme glucose-6-phosphatase
and thus cannot convert glucose-6-phosphate to

glucose, as occurs in the liver. Thus once glucose
is taken up by astrocytes and is phosphorylated
to glucose-6-phosphate, the glucose is com-
mitted down a metabolic pathway that will not
result in glucose formation. Lactate is produced
by the action of the enzyme lactate dehydro-
genase on the substrate pyruvate, a reversible
reaction. The equilibrium between lactate and
pyruvate depends on the energy status of the
cell and the presence of lactate dehydrogenase
isoforms (Stryer, 1995). In astrocytes, the lactate
dehydrogenase isoform that converts pyruvate to
lactate is present. In mouse optic nerve, we have
shown that lactate can support the CAP in the
absence of exogenously applied glucose (Brown
et al., 2001), supporting the notion that lactate
delivered to neurons from astrocytes can support
axon function. The lactate movement across cell
membranes is accomplished via the actions of
membrane-bound monocarboxylate transport-
ers (MCT), which cotransport H+ with the lac-
tate. The MCT that preferentially moves lactate
out of cells is the MCT1 subtype, and it is located
on astrocytes, whereas the MCT2, which takes up
lactate into cells, is located on neurons. If lactate
does support axon conduction as a consequence
of glycogen metabolism, then interrupting lactate
transfer from astrocytes to axons should result in
a loss of axon conduction as seen by a decrease in
the CAP area. This was tested in a rodent prepa-
ration by introducing compounds that inhibit
lactate transport at the MCT (d-lactate, CIN),
resulting in reduced CAParea.
E L E VAT E D [ K + ] O S I G N A L S
A S T R O C Y T E T O M E TA B O L I Z E
G LY C O G E N
It is timely to consider the signaling mechanism
by which increased neuronal activity is sensed by
astrocytes. In the mid-1990s, the astrocyte neu-
ron lactate shuttle hypothesis was proposed, in
which uptake of glutamate into astrocytes was
coupled to glucose uptake into astrocytes. This
hypothesis has proved to be controversial and
clearly does not apply to areas of the brain devoid
of glutamatergic synapses, or white matter, which
accounts for 50% of the adult human brain vol-
ume. Amore recent hypothesis is more attractive
as it applies to the entire CNS (and possibly the
peripheral nervous system). In a study predomi-
nantly using astrocyte cultures, a key role for
elevated [K+]o was identified as the primary sign-
aling mechanism between neurons and astro-
cytes. It is universally accepted that increased
neuronal activity results in localized elevations
AstrocyteNeuron Interactions 103
in [K+]o (see previous discussion), thus K+ can
be proposed as a universal signaling molecule
indicative of increased neuronal activity. The
[K+]o activates a Na+-coupled bicarbonate trans-
porter on astrocyte membranes whose activa-
tion results in increased transport of bicarbonate
and intracellular alkalization of the astrocyte.
This in turn activates the enzyme soluble adenyl
cyclase, which converts ATP to cyclic AMP. The
cyclic AMP then promotes glycogenolysis via
glycogen phosphorylase and results in glycogen
metabolism to lactate, which is then transported
out of the astrocyte to the neuron (Pellerin &
Magistretti,1994).
G LY C O G E N A N D M E M O R Y
In a recent study on the rodent brain, a role
for glycogen in supporting the acquisition of
memories was described (Choi etal., 2012). The
experiments entailed introducing rats into a box,
from which another box was readily accessible
through a door. If the rats entered the second
box, they were given a shock through the floor
of the box, which acted as an aversive condition-
ing response. The rats were removed from the
box and then reintroduced into the box from
periods of between 1 hour and 7days later. The
latency for the rats to enter the second box was
used as an index of their memory: the longer
the latency, the better the rat had retained that
memory. Injecting 1,4-dideoxy-1,4-imino-D-
arabinitol (DAB), an inhibitor of glycogen phos-
phorylase, into the hippocampi of the rat resulted
in a dose-dependent decrease in the latency,
indicative of decreased memory function. The
DAB had to be injected before the conditioning
stimuli in order to be effective. The lactate con-
centration ([lac]o) in the extracellular space in
the hippocampi was elevated in the hour or so
after the conditioning stimuli, whereas in rats
injected with DAB no elevation in [lac]o was seen.
Injecting the rats with lactate at the same time
that they were injected with DAB attenuated the
effects of DAB, indicating that circumventing the
contribution of glycogen-derived lactate by direct
introduction of lactate in the extracellular space
restored the animals memory. Finally, using
molecular biology techniques to downregulate
expression of the monocarboxylate transporter 1
(MCT1), which takes up glycogen-derived lactate
into the neurons, decreased memory acquisition
(Suzuki et al., 2011). These data strongly sug-
gest that glycogen is required for acquisition of
memories, where it is metabolized in astrocytes
and then the resulting glycogen-derived lactate is
104 Part II: Homeostatic Control
transported out of the astrocyte and into astro-
cytes via the MCT1. This is the first description of
glycogen playing a vital role in a key physiologi-
cal brain function.
A S T R O C Y T E S FA C I L I TAT E
C S F C I R C U L AT I O N A N D
CLEANSING OFBR AINECS
In addition to comprising different cell types
(neurons and glia, of which there are subdivi-
sions of each), the CNS is compartmentalized
as follows. The cellular compartments com-
prising the cell bodies of the neurons and glia
are surrounded by the fluid-filled interstitium.
The brain parenchyma itself is enclosed within
three meninges: the pia mater, arachnoid, and
dura. The cerebrospinal fluid (CSF) is contained
within the subarachnoid space (between the
dura and arachnoid) and the ventricular system
(Suzuki etal., 2011). The CSF interacts with the
interstitial fluid across two borders, the pial glial
membrane and ependymal cells, which line the
ventricles. The pial glial membrane is present
at the loosely fenestrated epithelial cells lining
blood vessels supplying the brain via the Virchow
Robin space. The CSF is produced by the choroid
plexus cells that line the ventricles, and the posi-
tive pressure resulting from continual produc-
tion is assumed to be the main force that drives
CSF flow. The water content of the interstitium
and the CSF varies, with the CSF being more
hypotonic than the interstitium due to lack of
proteins and cholesterol (Ransom, 2009). From
the point of view of water distribution, it should
be appreciated that there is continual change in
compartmental osmolarity due to neural activ-
ity. For example, the elevated [K+] and glutamate
in the interstitium that accompany neuronal
activity will cause increases in osmolarity that
must be compensated for. Thus water move-
ment between compartments is a very important
process to ensure optimal brain function. The
AQP4 member of the aquaporin water channel
family is expressed on astrocyte membranes. Its
expression is polarized with primary expression
on endfeet surrounding blood vessels, astrocyte
processes abutting the glia limitans, and ependy-
mal cells (Ransom, 2009). The function of aqua-
porins is to facilitate water movement across
cell membranes commensurate with alterations
in water content. The role of AQP4 has derived
mainly from knockout experiments where func-
tion in genetically engineered mice lacking the
AQP4 was measured. From these experiments
several primary conclusions can be drawn. First,
AQP4 is essential for water movement across
astrocyte cell membranes; in knockout animals
the water transport is one-seventh that of control
conditions. Similarly, AQP4 plays a vital role in
water transport across the blood-brain barrier
(Papadopoulos & Verkman, 2013). The aqua-
porin member AQP1 is expressed on the apical
membrane of chorioid plexus cells and facili-
tates water movement from the chorioid plexus
blood supply into the ventricles (Papadopoulos &
Verkman, 2013). A Na+K+ATPase on the apical
membrane creates the Na+ gradient that pro-
motes water movement (Nielsen etal., 1993). CSF
absorption occurs via AQP4 channels expressed
on ependymal cells, which facilitates move-
ment of water into the venous drainage system
(Ransom, 2009). The AQP4 channels present on
astrocyte endfeet that abut the blood vessels are
a low-resistance pathway for water movement
between the blood and the interstitial fluid and
additionally provide a route for clearance of sol-
utes from the interstitium to the venous drain-
age. Experiments using fluorescent tracers and
AQP4 knockout mice confirmed the presence
of the pathway and emphasized its importance
in the drainage of clearance of large solutes,
including beta-amyloid, from the parenchyma
(Papadopoulos & Verkman, 2013). AQP4 chan-
nels and voltage-dependent Kir4.1 ion channels
are colocalized to the perivascular and subpial
endfeet at synapses. Increased synaptic activity
results in elevated interstitial [K+], which is trans-
ported into astrocytes via the Kir4.1 ion channel.
This creates osmotic pressure across the astrocyte
membrane and drives water accumulation in
astrocytes, which is subsequently redistributed
via the astrocytic syncitium (Strohschein et al.,
2011; Iliff etal.,2012).
BLOOD VESSEL DIAMETER
Functional hyperemia, first proposed over a
century ago (Verkhratsky & Butt, 2007), is the
process by which neuronal actively is coupled
to localized blood flow, controlled by blood ves-
sel diameter. The mechanism(s) of this process,
which has recently been elucidated, displays a
degree of metabolic flexibility such that the alter-
ations in blood vessel diameter are determined
by factors that are indicative of the energy status
of the tissue. One of the first studies to investi-
gate in detail the role of astrocytes in controlling
localized arteriole diameter demonstrated that
elevated [Ca2+]i in astrocytes evoked arteriole

dilation via the actions of prostaglandin E2 (Roy
& Sherrington, 1890). However, the same mecha-
nism, namely elevated astrocytic [Ca 2+]i, was also
shown to result in arteriole constriction (Zonta
et al., 2003). The apparent contradictory effects
can be explained by the degree of oxygena-
tion of the tissue (Mulligan & MacVicar, 2004).
Under conditions of low O2, there is an increase
in interstitial lactate, which blocks the uptake of
prostaglandin into astrocytes. The prostaglan-
din then acts to dilate blood vessels to deliver
increased blood (and O2) to the tissue. However,
in well-oxygenated tissue, interstitial lactate lev-
els are low and the effects of arachindoic acid
derived 20-HETE dominate, and result in arte-
riole constriction.
HUMAN AND RODENT
A S T R O C Y T E S M AY B E
Q U A L I TAT I V E LY D I F F E R E N T
The dogmatic view of astrocytes as relatively
small cells that oversee an individual nonover-
lapping domain was shattered by a recent report
that described both morphological and func-
tional features of human astrocytes (Gordon
et al., 2008). Cortical tissue was obtained dur-
ing surgery to remove focal lesions sites in the
hippocampus of patients suffering from intrac-
table epilepsy. Immunohistological analysis of
the tissue using glial fibrillary acidic protein
and diolistic staining techniques revealed that
human astrocytes vary dramatically from their
rodent cousins. The most immediate difference
was in the size of the astrocytes. Whereas rodent
astrocytes cover a field extending about 56 m in
diameter, the human astrocytes covered a field
over twice (142 m) as wide. It is calculated that
the human astrocyte domain encompasses up
to 2million synapses. More important from the
point of view of cell-to-cell communication, the
domains of the human astrocytes overlapped,
whereas those of the rodent did not, strongly sug-
gesting astrocyte-to-astrocyte communication
in the human. In addition, the dendrites of the
human astrocyte were much finer, and thus the
dendritic field of the human astrocytes had
many orders of magnitude more surface area
than that of the rodent astrocyte. However, the
most startling discovery was the presence of long
processes that emerged from the somas of the
human astrocytes. These processes could be up
to 1mm long and traversed cortical layers, hence
their description as interlaminar astrocytes. The
process expressed regularly spaced varicosities
AstrocyteNeuron Interactions 105
that contained mitochondria, clearly signal-
ing an energy-dependent role for these objects.
Functionally the human astrocytes resembled
rodent astrocytes in that they supported intra-
cellular Ca 2+ waves and responded to the pres-
ence of neurotransmitters by generating Ca 2+, but
the waves travelled four times faster than their
rodent counterparts (Oberheim et al., 2009).
The implications of these findings will almost
certainly be far reaching, with the most obvious
conclusion that astrocytes can communicate via
the extended processes over long distances.
A recent study has investigated the function
of human astrocytes further by creating chimeric
mice that possess both native murine astro-
cytes as well as transplanted human astrocytes,
that maintain their human morphology, and by
extension that express their unique human func-
tions. The chimeric mice displayed improvements
in the key psychological parameters of learning
and memory. Avariety of tests were carried out,
and in all cases the chimeric mice performed bet-
ter than wild-type mice (Oberheim etal., 2009).
Such studies, while in their infancy, are intrigu-
ing and prompt the key questions regarding what
function(s) or property(s) of the astrocytes confer
the human performance on thesemice.
CONCLUSION
Astrocytes are essential partners in the optimal
functioning of neurons. Akey aspect of this rela-
tionship is the astrocyteneuron interactions
via the interstitial fluid. The constricted inter-
stitial space leads to close proximity of the cells
such that any deviation from equilibrium of ions
affects neighboring cells. Thus the interstitial
fluid is an ideal means by which cells can commu-
nicate. The ionic disruptions caused by increased
neuronal activity (e.g., increased [K+]o, decreased
pH, and increased [glutamate]o) are all buffered
by astrocytes, as are the subsequent changes in
osmolarity resulting from these perturbations.
Of increasing importance are the metabolic
interactions between neurons and astrocytes in
the form of how neurons signal increased activity
to astrocytes and how the astrocytes respond to
such activity.
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7
Homeostatic Synaptic Scaling
atCentral Synapses
N I R A J S . D E S A I A N D E L I S A B E T H C . WA L C O T T
DEVELOPMENT AND
LEARNING
Nearly every physiological system requires
some method of homeostatic regulationsome
means of regulating its internal environment
and maintaining stable performance despite
external perturbations (Cannon, 1932). This is
perhaps especially true of the nervous system,
which not only faces the same challenges to sta-
bility as other systems (e.g., protein degradation
and turnover, environmental perturbations) but
also must, while maintaining stability, change
many of its fundamental properties over time
and in response to experience so as to allow
individuals to learn and remember (Marder and
Goaillard, 2006). Stability and change are not
strictly opposing qualities, but neither are they
complementary, and the need to balance the two
and keep them from interfering with each other
makes homeostatic regulation of the nervous sys-
tem an extraordinarily complicated problem. To
make the problem somewhat more tractable, it is
common practice to break it up into two distinct
but related (and still difficult) problems:develop-
ment and learning.
The developmental problem arises to a con-
siderable extent because of the rapidity and
complexity of synapse development (Waites
etal., 2005; Collin and van den Heurval, 2013).
In mammalian neocortex, the number of syn-
apses a typical neuron receives changes by sev-
eral orders of magnitude in the weeks just before
and just after birth. Some of these synapses are
excitatory, others inhibitory, and still others
neuromodulatory; some arise from neighboring
neurons, others from neurons millimeters away;
some are located close to the cell body, while oth-
ers are far along the dendritic arbor. The synaptic
drive coming through these synapses fluctuates
dramatically over the course of development,
because the presynaptic neurons are themselves
developing, synaptic properties change substan-
tially over developmental time, and landmark
developmental events, like eye opening, abruptly
shift input. In the absence of implausibly exten-
sive genetic instructions, it is difficult to imagine
how developing neural circuits can function, or
even avoid destructive epileptic events, without
tight feedback control. Experiments, both in
vitro and in vivo, have confirmed this intuition,
demonstrating that development of the neocor-
tex and other brain structures is guided by sen-
sory and motor experience and by neural activity
(Berardi et al., 2000; Hensch, 2004; Knudsen,
2004; Hengen etal., 2013; Keck etal.,2013).
The homeostatic requirements imposed by
learning processes are less obvious, and much
of what we understand about them comes from
theoretical and computational studies rather
than experimental ones (Miller and Mackay,
1994; Abbott and Nelson, 2000; Turrigiano and
Nelson, 2000; Nelson and Turrigiano, 2008). The
starting point for most of these studies is Hebbs
rule, which holds that a synapse connecting one
neuron to another should be strengthened if the
firing of the first neuron repeatedly and persis-
tently leads to firing of the second (Dayan and
Abbott, 2005). Many variations on this rule have
been proposed over the years, and Hebbian mod-
els of synaptic plasticity have come to dominate
biologically based theories of learning. One com-
mon feature of this work is the understanding
that Hebbian plasticity is best complemented by
homeostatic control (Abbott and Nelson, 2000).
The intuition here is simple and appealing:con-
sider two neurons (A and B) connected by a syn-
apse. If the firing of Adrives B to fire, the synapse
between them will be strengthened; this will
make it more likely that Awill drive B to fire in
the future; this will lead to more strengthening;
 Homeostatic Synaptic Scaling at Central Synapses
this will lead to more driving; and so on. Absent
some kind of constraint or negative feedback,
Hebbian plasticity can lead to situations of
runaway synaptic potentiation because of such
positive feedback loops. Moreover, even without
positive feedback, synaptic modifications pro-
duced by learning processes still likely necessi-
tate homeostatic control. Computational studies
of neural networks with biologically constrained
parameters indicate that, without stringent (and
unlikely) parameter fine tuning, such networks
are highly unstable and prone to extreme regimes
of high or low activity (Lazar et al., 2009; Watt
and Desai, 2010; Tetzlaff etal., 2011, 2012). Only
including mechanisms of homeostatic control
can produce reasonable network behavior.
What form should those mechanisms take?
This is a question that has been actively and
explicitly investigated for the better part of three
decades, in the context of both development
and learning. Several answers have been offered
(Bienenstock etal., 1982; Turrigiano, 1999; Desai,
2003; Abraham, 2008; Nelson and Turrigiano,
2008; Watt and Desai, 2010; Turrigiano, 2011;
Vitureira et al., 2012; Chen et al., 2013; Davis,
2013; Chistiakova etal., 2014). One is that a neu-
rons intrinsic excitability, as determined by its
complement of voltage- and calcium-gated ion
channels, should be modulated so as to coun-
terbalance shifts in synaptic input. Another is
that synapse number should vary as a function
of overall activity. A third is that the rules that
govern Hebbian plasticity should themselves
incorporate homeostatic features, an idea called
metaplasticity. But the idea that has perhaps
received the most attention is synaptic scaling.
In its simplest form, synaptic scaling holds that
the strengths of all of a neurons excitatory syn-
apses should be scaled up (or down) as the neu-
rons average firing rate goes down (or up; Lissin
etal., 1998; OBrien etal., 1998; Turrigiano etal.,
1998). Since its introduction more than 15years
ago, synaptic scaling has been extensively stud-
ied experimentallyin cell cultures, slice cul-
tures, acute brain slices, and in vivoand its
simplest form has been elaborated to include
contributions from inhibitory synapses and to
allow it to act on different spatial scales and time-
scales (Turrigiano, 2008; Pozo and Goda, 2010;
Davis, 2013). Many of these findings have been
further examined using computational mod-
els (van Rossum etal., 2000; Renart etal., 2003;
Savin et al., 2010; Tetzlaff et al., 2013). In this
chapter we provide a succinct overview of much
of the work to date, focusing on evidence from
109
cortical systems, though it must be noted that
closely related phenomena have been examined
using invertebrate and spinal systems (Davis
and Bezprozvanny, 2001; Davis, 2006; Pratt
and Aizenman, 2007; Rich and Wenner, 2007;
Wenner, 2011; Frank, 2014; Wenner,2014).
PHENOMENA
The field of homeostatic synaptic scaling has
been expanding rapidly, and recently numer-
ous detailed and excellent reviews of many of its
aspects have appeared (Davis, 2006; Turrigiano,
2008; Pozo and Goda, 2010; Wenner, 2011;
Turrigiano, 2012; Vitureira et al., 2012; Wang,
2012; Davis, 2013; Lazarevic etal., 2013; Vitureira
and Goda, 2013; Chen etal., 2014; Lee etal., 2014;
Pribiag and Stellwagen, 2014; Siddoway et al.,
2014; Thalhammer and Cingolani, 2014; Whitt
etal., 2014). In this section we limit ourselves to
a concise description of the basic phenomena,
including some of what is known about its molec-
ular underpinnings.
Synaptic scaling has been studied in many
ways:in the neocortex, hippocampus, and spinal
cord; in cell cultures, slice cultures, brain slices,
and the intact brain; from young animals and
mature animals; in infragranular, granular, and
supragranular cortical layers; and after various
types of pharmacological manipulation and sen-
sory deprivation. As one might imagine, these
experiments have yielded complex and (occa-
sionally) contradictory results.
Excitatory Synapses
Pyramidal neurons. By far the majority of exper-
imental studies of synaptic scaling have focused
on plasticity of excitatory synapses onto excitatory
neurons. As of this writing, there are dozens of
examples of strengthening (or weakening) of glu-
tamatergic synapses following prolonged reduc-
tions (or increases) in activity levels in networks
of neurons. One of the earliest efforts (Turrigiano
etal., 1998)still serves as an exemplar. This study
used dissociated cultures of neocortical neu-
rons. Such cultures are useful for several rea-
sons:individual neurons can be easily visualized
and identified morphologically; the environment
in which neurons develop is under experimen-
tal control; and, perhaps most important, cul-
tures exhibit pronounced spontaneous spiking
and synaptic activity, with firing rates of several
spikes per second. Pharmacological manipula-
tions of spontaneous activity can consequently
be used to explore activity-dependent plasticity.
Adopting this strategy, Turrigiano etal. reduced
110 Part II: Homeostatic Control
activity for one or more days using the sodium
channel blocker tetrodotoxin or the -amino-
3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) receptor antagonist CNQX, or increased
activity for one or more days using the GABA A
antagonist bicuculline, and then characterized
excitatory synaptic strengths by measuring the
amplitude and frequency of miniature excitatory
postsynaptic currents (mEPSCs), which were car-
ried by AMPA receptors. They found that mEPSC
amplitude (quantal amplitude) was bidirection-
ally regulated by average activity: low activity
regimes pushed mEPSC amplitudes up over a
time scale of hours, whereas high activity regimes
pushed them down. These changes in amplitudes
were not accompanied by changes in mEPSC fre-
quency. Moreover, the changes in quantal ampli-
tude appeared to be multiplicative:the data were
consistent with the synaptic plasticity process at
work scaling all excitatory synapses up (or down)
after activity deprivation (or enhancement) by a
single multiplicative factor.
Experimental studies like this, in dissoci-
ated and slice cultures, gave rise to what might
be called the canonical version of synaptic scal-
ing (Figure 7.1): prolonged changes in average
neuronal activity trigger opposing (compensa-
tory) changes in excitatory synaptic strength. The
canonical story has several parts. Scaling is slow,
with a natural timescale of hours (Turrigiano
et al., 1998; Thiagarajan et al., 2005; Stellwagen
and Malenka, 2006). Scaling is global and multi-
plicative; it depends on somatic firing and affects
all of a neurons excitatory synapses (Burrone
etal., 2002; Harms and Craig, 2005; Ibata etal.,
2008; Goold and Nicoll, 2010). Scaling is expressed
postsynaptically, as an increase or decrease in
the accumulation of glutamate receptorsboth
AMPA and N-methyl-D-aspartateat postsyn-
aptic sites (Watt et al., 2000; Wierenga et al.,
2005; Cingolani etal., 2008; Gainey etal., 2009;
Anggono et al., 2011). AMPA receptor scaling
requires the GluA2 subunit of the AMPA recep-
tor and results in a coordinated increase in the
number of GluA1/GluA2-containing receptors
(Wierenga et al., 2005; Cingolani et al., 2008;
Gainey etal., 2009; Anggono etal., 2011). Scaling
is a cell-autonomous process; it can be triggered
by manipulating the activity of a single neuron,
regardless of the behavior of the network in which
it is embedded (Burrone etal., 2002; Ibata etal.,
2008; Goold and Nicoll, 2010). Scaling depends
on intracellular calcium and requires transcrip-
tion (Ibata et al., 2008; Goold and Nicoll, 2010;
Qiu etal.,2012).
But the canonical story is incomplete, and
portions may well be wrong. Indeed, every part
has been challenged to some degree. The biggest
challenge has been the discovery of a form of syn-
aptic scaling that acts rapidly and locally, possibly
even at individual synapses (Ju etal., 2004; Sutton
et al., 2004, 2006; Aoto et al., 2008; Hou et al.,
2008; Soden and Chen, 2010; Chen etal., 2014).
This form depends not only on action potential
firing but on synaptic activation; in cultures, this
means treatment with both tetrodotoxin and glu-
tamate antagonists. Rather than transcription,
the scaling is accomplished by local protein syn-
thesis, dependent on retinoic acid, which inserts
homomeric GluA1 receptors at postsynaptic sites
(Chen et al., 2014). A couple of features of this
type of scaling are surprising. One is how local
its actions are. Amajor idea about synaptic scal-
ing has been that its global, multiplicative nature
allows it to regulate overall synaptic strength
without affecting the relative strengths of differ-
ent synapses (Nelson and Turrigiano, 2008). This
is thought to be important because, if information
is stored in relative synaptic strengths, scaling
would be able to perform a homeostatic function
while preserving stored information. Put another
way, homeostatic plasticity and Hebbian plastic-
ity might coexist without interfering with each
other. Local synaptic scaling complicates the sit-
uation considerably. One intriguing idea is that
dendritic branches, rather than the neuron as a
whole, should be considered the basic computa-
tional unit (Branco and Hausser, 2010); local scal-
ing, as long as it is not too local, could then work
together Hebbian mechanisms (Rabinowitch and
Segev, 2008). Asecond surprise is that local scal-
ing requires manipulations of both spiking and
receptor activation. Moreover, the receptor acti-
vation in question is sensitive to spontaneous
rather than spike-evoked vesicle release, and the
spiking requirement may be on presynaptic neu-
rons rather than postsynaptic ones (Sutton and
Schuman, 2009). These features raise the ques-
tion of under what conditions local scaling acts.
Possibilities include very early in development,
when synapses are just forming, or after some
kind of pathological insult.
Although postsynaptic changes are widely
seen following activity manipulation, many
studies indicate that under some conditions
chronic activity manipulation results in coor-
dinated presynaptic and postsynaptic modi-
fications (Burrone et al., 2002; De Gois et al.,
2005; Thiagarajan et al., 2005; Erickson et al.,
2006; Wierenga et al., 2006; Gong et al., 2007;
 Homeostatic Synaptic Scaling at Central Synapses 111

SC
AL
ING
P
LT
High Activity

LT NG
D ALI
Target Activity SC Target Activity

Low Activity

FIGURE7.1: Excitatory synaptic scaling. In its simplest form, scaling involves increasing or decreasing the num-
ber of excitatory receptors (red markers) at dendritic spines (blue protrusions) in order to keep spiking activity
near a target level. The number of receptors at all synapses are scaled up or down by the same multiplicative
factor, thus maintaining relative differences (ratios) between synapses. So, for example, synaptic scaling can
restore target activity while preserving changes in synapses induced by long-term potentiation (LTP) or long-term
depression(LTD).

Jakawich et al., 2010; Lindskog et al., 2010;
Lazarevic et al., 2011; Henry et al., 2012). The
presynaptic modifications can include changes
in both release probability and glutamate trans-
porter expression, which might in turn affect
how much neurotransmitter is packaged in
each vesicle. Whether presynaptic modifications
accompany postsynaptic ones may depend on
developmental age. Wierenga etal. (2006) found
that, in young cultures (<2 weeks in vitro), scal-
ing was exclusively postsynaptic in origin but
that, in older cultures, scaling included both
presynaptic and postsynaptic elements. Adiffer-
ent set of studies, using hippocampal cultures,
suggested that presynaptic/postsynaptic scaling
may involve different physical mechanisms than
postsynaptic-only scaling (Thiagarajan et al.
2005; Lindskog et al., 2010; Groth et al., 2011).
These studies demonstrated that when presynap-
tic scaling is present, the new postsynaptic recep-
tors inserted in the membrane are homomeric
GluA1 receptors and that this process involves
a pathway including the kinase CaMKII.
Both of these features are different than what
has been reported for postsynaptic-only scal-
ing, in which GluA2-containing receptors and
a CaMKK/CaMKIV pathway are implicated
(Ibata et al., 2008; Gainey et al., 2009; but see
Goel etal.,2011).
The question of whether or not scaling is
cell autonomous has received a lot of attention
(Turrigiano, 2012). Most studies suggest that it
is. For example, manipulations of spiking firing
and/or depolarization in individual neurons can
elicit scaling, even when the network as a whole is
unchanged (Burrone etal., 2002; Ibata etal., 2008;
Goold and Nicoll, 2010). However, there is evi-
dence that presynaptic activity and network-level
activity are also important (Rutherford et al.,
1998; Stellwagen and Malenka, 2006; Jakawich
et al., 2010). The network argument rests espe-
cially on the fact that two soluble, diffusible fac-
tors, brain-derived neurotrophic factor (BDNF)
and the cytokine tumor necrosis factor alpha
(TNF), play important signaling roles for syn-
aptic scaling. BDNF and TNF are released into
the extracellular space by pyramidal neurons and
glial cells, respectively. How widely these factors
spread is uncertain, but their places in the scaling
process suggest that a neuron may respond not
only to its own average activity but to that of its
near neighbors.
112 Part II: Homeostatic Control
It is likely that part of the reason so many
different answers have been given to the ques-
tions surrounding synaptic scaling is that
the questions have mainly been examined in
culture systems, which are, perhaps, in some
respects too artificial. Over the past dozen
years there have been a number of studies
examining the effects of chronic sensory dep-
rivation in vivo on synaptic properties in sen-
sory cortex (Desai et al., 2002; Maffei et al.,
2004; Goel et al., 2006; Goel and Lee, 2007;
Maffei and Turrigiano, 2008; Goel etal., 2011;
Hengen etal., 2013; Keck etal., 2013). The gen-
eral approach has been to subject animals to
sensory deprivation and then to prepare brain
slices in order to probe synaptic transmission.
These studies have clearly demonstrated that
a process very much like the synaptic scaling
previously observed in culture operates in the
intact brain, but they have also shown that
scaling is even more complicated than the cul-
ture experiments suggest. In particular, scal-
ing is sensitive to cell type, developmental age,
and the precise nature of the sensory depriva-
tion (e.g., lid suture versus intraocular tetrodo-
toxin injection versus dark rearing). Moreover,
sensory deprivation in vivo engages not only
synaptic scaling but Hebbian plasticity, mak-
ing interpretation of the experimental results
somewhat challenging.
Interneurons. The data on how prolonged
activity manipulation affects excitatory synapses
onto inhibitory interneurons are limited and
somewhat contradictory. Using culture systems
and pharmacological activity manipulation,
three different groups have reported that
days-long activity blockade increases excitatory
currents onto interneurons (Bartley etal., 2008),
decreases excitatory currents onto interneurons
(Chang etal., 2010), or has no effect (Rutherford
etal., 1998). Two of these studies also examined
the effects of activity enhancement by incubation
in bicuculline; both reported an increase in
excitatory mEPSC amplitude (Rutherford et al.,
1998; Chang et al., 2010). It is not clear how to
reconcile these results. The differences may stem
from differences between the neocortex and
hippocampus, duration of activity manipulation,
or type of interneuron examined. However, two
of the groups found opposite results for a single
class of interneuron, parvalbumin-expressing
fast-spiking interneurons (Bartley et al.,
2008; Chang et al., 2010). Certainly this is a
subject would benefit from a more varied and
systematicstudy.
Scaling up and scaling down. As should be
clear from the previous discussion, the molecular
underpinnings of excitatory synaptic scaling are
varied and a matter of considerable uncertainty.
For example, consider what is known about
global, postsynaptic-only scaling. The triggering
event for both scaling up and scaling down is an
activity-dependent change in somatic calcium
influx that then affects signaling through a
CaMKIV pathway (Ibata etal., 2008; Goold and
Nicoll, 2010). CaMKIV is a protein kinase that
is part of the larger Ca2+/calmodulin-dependent
family, which includes CaMKII and which
is closely associated with forms of synaptic
plasticity that regulate receptor synthesis
and trafficking (Wayman, 2008). It becomes
active with phosphorylation by CaMKK and
autophosphorylation following Ca2+/calmodulin
binding. But downstream of CaMKIV, scaling
up and scaling down seem to diverge. Scaling
up depends on the actions of BDNF, TNF,
the immediate early gene Arc, and the AMPA
receptor binding protein PICK1, whereas scaling
down does not (Rutherford et al., 1998; Leslie
et al. 2001; Shepherd et al., 2006; Stellwagen
and Malenka, 2006). Rather scaling down has
roles for polo-like kinase 2, the immediate
early gene Homer1a, and a ubiquitin pathway
regulated by Eph4A (Seeburg et al., 2008; Hu
etal., 2010; Fu etal., 2011). Furthermore, scaling
up is completely dependent on the scaffold
postsynaptic density protein 95, whereas scaling
down can be supported by either postsynaptic
density protein 95 or postsynaptic density protein
93 (Sun and Turrigiano, 2011). It seems then that
the signaling pathways for scaling up and scaling
down are independent, so that activation of the
one might be accompanied by deactivation of the
other. The implications of such a scenario have
yet to be elucidated.
Inhibitory Synapses
If the main purpose of synaptic scaling was to
implement firing rate homeostasis, then one
would expect it should act on inhibitory synapses
in the opposite way it acts on excitatory ones.
Considerable evidence suggests that, broadly
speaking, this is the case, though the picture
is complicated by the considerable diversity of
interneuronal types (Rutherford et al., 1997;
Kilman etal., 2002; Maffei etal., 2004; Hartman
etal., 2006; Bartley etal., 2008; Kim and Alger,
2010; Peng etal., 2010; Rannals and Kapur, 2011;
Saliba et al., 2007; Sarti et al., 2013). Inhibitory
scaling has been studied in more or less the same
 Homeostatic Synaptic Scaling at Central Synapses
way as excitatory scaling:through pharmacologi-
cal manipulations in neocortical and hippocam-
pal cultures or in vivo sensory deprivation in
sensory cortex. Together these studies indicate
that inhibitory synaptic strengths onto pyrami-
dal neurons are bidirectionally regulated by pro-
longed changes in activity levels:downregulated
after blockade, upregulated after enhancement.
The regulation appears to involve both postsyn-
aptic and presynaptic modifications, including
changes in receptor density, vesicle release, and
neurotransmitter packaging. However, there
have been some deviations from this simple
story, with increases in some classes of inhibi-
tory synapse reported after manipulations that
should reduce or least decorrelate network activ-
ity (Maffei etal., 2004; Kim and Alger,2010).
As with excitatory scaling, there are also open
questions about whether inhibitory scaling is cell
autonomous and global. Hartman et al. (2006)
demonstrated that chronic activity blockade in
hippocampal cultures downregulates inhibition
onto principal cells but that this only happens
when the activity of multiple neurons is blocked;
silencing individual presynaptic or postsynaptic
neurons was not sufficient to induce inhibitory
scaling. This suggests that inhibitory scaling
is not cell autonomous but is instead a network
property. On the other hand, Peng etal. (2010),
also using hippocampal cultures, found that
enhancing activity in multiple ways (treatment
with bicuculline or kainic acid, or overexpression
of a cation channel) upregulated inhibitory cur-
rents and that only the postsynaptic pyramidal
neurons activity mattered for this effectthat
is, scaling here was cell autonomous. Moreover,
as is true of excitatory scaling, alongside global
inhibitory scaling, there appears to be a rapid,
local, protein- and retinoic aciddependent
form of inhibitory scaling (Sarti etal., 2013). The
relationship between the two forms of scaling
remains ambiguous.
F U N C T I O N A L I M P L I C AT I O N S
Synaptic Scaling asPart
ofControlTheory
Homeostatic regulation is, of course, not
restricted to biological systems. Rather it is a
recurring feature of complex systemsbiological,
chemical, and physicalin which inputs are
transformed into outputs in the presence of noise
and external perturbations. The general theory
of how such systems behave and are regulated
is called control theory (strm and Murray,
113
2008). Efforts to understand the functional con-
sequences of synaptic scaling and to incorporate
it into computational models of neural function
have been informed by the fundamental ideas of
control theory and have, increasingly, begun to
utilize its formalism (Davis, 2006; OLeary and
Wyllie, 2011; Queenan etal., 2012; Davis,2013).
The basic problem control theory addresses
is illustrated in Figure 7.2A. A controlled sys-
tem (sometimes called a process or a plant)
receives inputs, which may include feedforward,
feedback, and noise/perturbation components.
It transforms these, through some unspecified
means, into an output. The goal of control theory
is to maintain the output as near to a target (or
set-point) value as possible, responding quickly to
external perturbations, noise, and changes in the
target value. It does this through both feedfor-
ward and feedback control. Feedforward inputs
may depend on the target and may even monitor
noise/perturbations, but they are independent of
the actual output, whereas feedback inputs are
adjusted based on an error signal correspond-
ing to the difference between the measured out-
put and the target value. To illustrate how these
two types of controls work, consider a simple
case: say that we want to use a heater to main-
tain the temperature of a room at some fixed level
throughout the course of a day. We know that the
outside temperature will be colder in the morn-
ing than in the afternoon, and so one strategy we
might employ is simply to keep the heater on high
early in the day and then turn it down low later
on. This is an example of feedforward (or open
loop) control since we used only our prior knowl-
edge of how the system behaves and did not actu-
ally measure the rooms temperature, much less
adjust the heater setting based on this measure-
ment. Feedforward control, while simple, is quite
limited and fragile; it requires a good knowledge
of system properties and may not fully compen-
sate for perturbations (e.g., the unexpected open-
ing of a window). Amuch more robust strategy
would be to monitor room temperature (output),
compare it to our desired temperature (set point),
and continually adjust the heater setting based on
the difference (error signal). This is an example of
what is called feedback (or closed loop) control.
Synaptic scaling, which is after all activity
dependent, is generally thought to be an example
of a feedback control mechanism. To understand
it in this way, we ask three questions: (a) What
is the process (system under control)? (b) How
is the error signal formed? (c) How is the error
signal used to determine a control signal that
114 Part II: Homeostatic Control

(a) Noise/
(b)
Perturbations
Feedforward
model
Proportional
(Present)
Target Controlled System
(Set Point) (Process)
Target Integral + Controlled System
+ +
(Set Point) (Past) + (Process)
Feedback
Controller
Derivative
(Future)

FIGURE7.2: Synaptic scaling as part of control theory. (A)Control theory concerns how a system that receives
inputs and transforms these into an output maintains the output near a target or set point value in the presence of
noise and other perturbations. Control mechanisms can be divided into two generic types:feedforward and feed-
back. Feedforward control signals depend on the target value and in some cases the noise and perturbation inputs;
they do not explicitly depend on the systems output. Feedback control signals depend on an error signal formed
by the difference between the system output and the desired target output. To make the distinction concrete,
consider early development of the cortex. One expects the number of excitatory synapses each neuron receives
to grow dramatically over time. One strategy for maintaining stable output is to gradually reduce all synaptic
strengths over developmental time. Such a strategy requires prior knowledge (a model) of how synapse forma-
tion and maturation proceeds across development and constitutes an example of feedforward control. Adifferent
strategy is to monitor firing rates directly and to adjust synaptic strengths continually in order to keep firing rates
within appropriate limits; this is feedback control. Synaptic scaling, as a process that depends on neural activity,
is thought to be a feedback process. (B)Feedback control can take many forms, but the most generic description
is the proportional-integral-derivative controller. This description divides feedback controls dependence on the
error (difference between target output and actual output) into three parts, which are sensitive to the present error
(proportional), the accumulation of past errors (integral), and an estimate of future errors (derivative).

appropriately adjusts the feedback input in order
to achieve control?
The process for synaptic scaling depends on
the spatial scale at which one imagines it oper-
ates. Most work, both experimental and compu-
tational, has taken the process to be the single
neuron (Turrigiano, 2008). Its inputs are the syn-
aptic currents, and its output is the firing rate or
its pattern of action potentials. This is a natural
choice because of the experiments in culture that
indicate individual neurons can respond to per-
turbations independent of the network in which
they are embedded and that identify somatic
firing as the important variable (Burrone et al.,
2002; Ibata etal. 2008; Goold and Nicoll, 2010). It
is also a choice that is straightforward to imple-
ment in a computational model (van Rossum
et al., 2000; Renart et al., 2003). However, it is
important to be aware that this is not the only
choice possible. One attractive alternative is to
assume that the process is an individual den-
dritic branch (or an even smaller structure); its
inputs are again synaptic currents but its output
now is some measure of local (restricted to the
branch) synaptic activity (Sutton et al., 2006;
Branco etal., 2008; Hou etal., 2008; Bque etal.,
2011). This idea is attractive because it prevents
any one branch from dominating over the others,
thus preserving the computational power inher-
ent in complex dendritic arbors, and it allows
for the selection of spatial patterns of input that
are particularly efficacious in controlling neu-
ronal output (Rabinowitch and Segev, 2008).
A differentand equally attractivealternative
is to consider the neural circuit as a whole to
be the process (Maffei and Fontanini, 2009). In
this case the output is some measure of network
activity, such as the average firing rate of all the
individual neurons that make up the network.
This idea is plausible because of evidence that dif-
fusible factors, such as BDNF and TNF, might
serve as signaling molecules for synaptic scaling
(Stellwagen and Malenka, 2006; Lindskog etal.,
2010); the spatial range over which they diffuse
would then set the scale for the circuit considered
to make up the process.
To form an error signal one needs two
things:an observable signal related to the output
and a target value against which to compare it. As
is often true in biology, the obvious candidate for
 Homeostatic Synaptic Scaling at Central Synapses
an observable signal is internal calcium, an ion
that closely tracks action potential and synaptic
activity and that is known to play a fundamental
role in many cellular processes. Other possibili-
ties exist, such as active monitoring of receptor
accumulation, but most of the focus in stud-
ies of this question has been on calcium and its
downstream processes (Thiagarajan etal., 2005;
Ibata etal., 2008; Goold and Nicoll, 2010; Zhao
et al., 2011). Much more uncertain is how the
target value or set point is established. It would
seem likely that the set point must (somehow) be
encoded genetically, with variations between dif-
ferent cell types and developmental ages (Davis,
2013). But, despite considerable progress in
unraveling the molecular machinery of synaptic
scaling and some intriguing ideas, the question
of set point remains almost entirelyopen.
The central problem of control theory is how to
use an error signal to generate an appropriate con-
trol signal. The most generic algorithm for doing
this is called the proportional-integral-derivative
controller (strm and Murray, 2008). This algo-
rithm is used widely in engineering and can be
captured by a simple equation (Figure7.2B):
f (t ) = K p * e(t ) + K i * e(t )dt+K d * de(t ) / dt
where f(t) is the control signal; KP, Ki, and
Kd are constants; and e(t) is the error signal
(= target output). The control signal might
directly represent a feedback input (e.g., current)
or an intermediate quantity (e.g., rate of change
of synaptic weights) that indirectly modulates
feedback input. The three terms at the right of
the equation represent the present error (propor-
tional), the accumulation of past errors (integral),
and an estimate of future errors (derivative). The
first term is the easiest to understand. It is posi-
tive when output is below the target value and
negative when it is above, with a magnitude that
scales with the size of the discrepancy. It is also
fairly easy to understand how a proportional
error might be implemented biologically, espe-
cially if the error signal was formed by internal
calcium; many biological processes relevant to
synaptic strength depend on calcium concentra-
tion. However, proportional control alone may
not capture quite how synaptic scaling works.
Proportional control will always leave the out-
put short of the target by some finite amount,
and it is prone to instability if the proportional
gain K P is set too high (strm and Murray,
2008). Moreover, most of the experimental
115
evidence indicates that synaptic scaling is a
slow process, acting over hours to days, which
proportional control does not readily accom-
modate. Accordingly, computational modeling
of synaptic scaling sometimes includes an inte-
gral term, which accumulates errors over time,
giving scaling a long history dependence and
eliminating offset (van Rossum et al., 2000;
Rowan and Neymotin, 2013; Yger and Harris,
2013). However, it is somewhat more difficult to
understand how a real neuron or neural circuit
implements integral control. Although calcium is
itself a temporally filtered measure of spiking and
synaptic activity, the timescale of the filtering is
short (no more than seconds). Instead, attention
has been focused on calcium-dependent pro-
cesses that may act on timescales much slower
than calcium itself (OLeary and Wyllie, 2011).
The derivative control term has mostly not been
used in modeling synaptic scaling because it
tends to make models very sensitive to noise and
it is difficult to justify biologically.
ComputationalIdeas
Synaptic scaling has been incorporated into
numerous computational models of corti-
cal function, both at the single-cell level and
at the network level (van Rossum etal., 2000;
Renart et al., 2003; Sullivan and de Sa, 2006;
Lazar et al., 2009; Liu and Buonomano, 2009;
Rossi Pool and Mato, 2010; Tetzlaff et al.,
2011; Keck etal., 2012; Rowan and Neymotin,
2013; Yger and Harris, 2013). It has been
implemented in various ways, including the
proportional-integral-derivative formulation
given previously, schemes in which the rate of
synaptic weight change depends on some func-
tion of current synaptic weights, and schemes
in which synaptic weights are directly normal-
ized (Dayan and Abbott, 2005; Lazar et al.,
2009; Tetzlaff etal., 2013). The modeling stud-
ies have identified several distinct functions
synaptic scaling might serve. In this subsection
we highlight five ofthem.
Firing rate homeostasis. The original
motivation for studying synaptic scaling was
to identify a biophysical mechanism that might
mediate firing rate homeostasis in cortical
networks (Turrigiano et al., 1998). Subsequent
computational studies of small networks of
spiking neurons confirmed that this is indeed a
role scaling might play (Sullivan and de Sa, 2006;
Tetzlaff et al., 2011, 2012; Rowan and Neymotin,
2013). However, these same studies indicated that
the details of how scaling is implemented can
116 Part II: Homeostatic Control
matter a great deal as to whether stabilization of
firing rates is actually achieved. In particular, to
ensure stability in the most general case, in the
presence of Hebbian plasticity and with generic
neuronal dynamics, synaptic scaling must not
only be weight-dependent but that dependence
must be strongly nonlinear (e.g., quadratic; Tetzlaff
etal., 2011, 2012). The linear multiplicative rule for
synaptic scaling proposed originally (Turrigiano
etal., 1998)does not meet this requirement, but it
may be that experiments to date have simply not
been sensitive enough to determine the exact form
scaling takes or that scaling works in combination
with some other form of homeostatic plasticity
(i.e., intrinsic plasticity) to stabilize neural circuits
(Lazar etal., 2009; Savin etal.,2010).
A different issue is that of timescale. Most
studies of synaptic scalingbut not allindicate
that scaling happens slowly, over the course of
hours (Turrigiano etal., 1998; Sutton etal., 2004,
2006; Thiagarajan et al., 2005; Stellwagen and
Malenka, 2006; Hou et al., 2008). This may be
very different than the timescale of Hebbian plas-
ticity; experimental studies of long-term poten-
tiation and depression indicate that they can be
induced in just minutes (Sjstrm et al., 2008).
The temporal mismatch might make it difficult
for the former to counter the latter in some cases.
In particular, a recent study by Zenke etal. (2013)
suggests that unless scaling can act within min-
utes, it cannot effectively stabilize the activity of
a generic, balanced network; the authors came
to this conclusion using both numerical simula-
tions and a mean field model. Afew caveats are in
orderthe nature of the network (connectivity,
excitation-inhibition balance, external inputs)
and the form plasticity takes can matter a great
deal (as is true for all models)but these results
do suggest that to counter the positive feedback
instability introduced by Hebbian plasticity,
some homeostatic mechanism other than scal-
ing might be required. Examples of fast homeo-
static mechanisms are heterosynaptic plasticity
(Chistiakova et al., 2014) and weight-dependent
Hebbian plasticity (Van Rossum etal.,2000).
Synaptic competition. Global synaptic scaling
makes plasticity at any one synapse depend on the
activity environment created by all of the other
synapses. In particular it weakens quiet synapses
relative to active ones. As such, scaling might
be a way of implementing competition between
synapses, which is thought to be important
for development of neural circuits (e.g., ocular
dominance plasticity; Bienenstock et al., 1982).
This idea was explored by van Rossum et al.
(2000) who found that combining synaptic
scaling with spike timing-dependent plasticity
resulted in neural circuits that were stable and
had synaptic weight distributions that were
approximately log-normal (Tetzlaff et al., 2011),
which compares well with quantal amplitude
distributions measured experimentally (Song
etal.,2005).
Working memory. Persistent activity in neural
circuits, particularly of the prefrontal cortex,
is thought to be the physical basis of working
memory (Fuster and Alexander, 1971; Miller etal.,
1996; Wang, 2001; Baeg et al., 2003). A number
of mechanisms for generating and maintaining
persistent activity have been proposed, including
slow, recurrent excitatory connections mediated
by N-methyl-D-aspartate receptors (Wang, 2001;
Egorov etal., 2002). Aweakness of some of these
proposals is that, in the absence of exquisite
parameter fine-tuning, they require unrealistic
assumptions about the uniformity of neuronal
and synaptic properties (Wang, 2001). Real
neural networks lack such homogeneity. Using
a biologically realistic network model, including
plausible heterogeneities, Renart et al. (2003)
demonstrated that synaptic scaling, operating
on each neuron independently, can effectively
compensate for heterogeneities in cellular
excitability and synaptic properties, and it can
allow for robust persistent activity.
Short-versus long-term memory storage.
Memory storage depends on biological
mechanisms, like long-term potentiation and
depression, that act on a time scale of minutes; and
yet memory consolidation is a process that can take
days. How to reconcile these different timescales is
a difficult problem because it requires that neural
circuits have some means of separating synapses
relevant for long-term storage, which should be
consolidated, from synapses relevant for short-term
storage, which should fade over time. Tetzlaff etal.
(2013) show that synaptic scaling, working on
an intermediate timescale (hours), might bridge
the time gap between long-term potentiation/
long-term depression and memory consolidation.
In particular, they argued that neural circuits
that combine synaptic scaling with Hebbian
plasticity intrinsically exhibit a bifurcation in their
dynamics that provides a natural way of separating
potentiated synapses bound for long-term storage
from those consigned to short-term storage.
Sleep. The purpose of sleep is a matter of
debate, but an idea that has attracted much
recent attention is that sleep promotes an overall
synaptic weakening that offsets the synaptic
 Homeostatic Synaptic Scaling at Central Synapses
strengthening that occurs during wakefulness
(Tononi and Cirelli, 2014). Called the synaptic
homeostasis hypothesis, the mechanism of this
weakening is not clear. Synaptic scaling has
been proposed as a candidate, though two lines
of evidence argue against this view: changes
during sleep in the expression of some molecular
factors tied to synaptic scaling, including BDNF,
TNF, and Arc, are in the wrong direction
to promote downscaling (Frank, 2012), and
firing patterns during sleep are complicated
and may not be consistent with downscaling
(Frank, 2012; Hengen et al., 2013; Tononi and
Cirelli,2014).
SY NAPTIC SCALING
AND DISORDERS
Although synaptic scaling has mainly been stud-
ied in the context of normal brain function,
much recent work has explored ideas about how
scaling might act under pathological conditions,
both in the young and in theold.
Neurodevelopment
Neurodevelopmental disorders are complex and
have a great variety of molecular components,
so it is remarkable that synaptic scaling has
been directly linked with two of the best-studied
experimental models:Rett syndrome and fragile
X syndrome (Henry, 2011; Na etal.,2013)
Rett syndrome. Rett syndrome is the leading
cause of mental retardation in females, with some
girls also exhibiting autistic-like features such as
a loss of social interaction and communication
(Chahrour and Zoghbi, 2007). It is produced
by loss of function mutations in a single gene,
which codes for the transcriptional regulator
methyl-CpG binding protein 2 (MeCP2). Loss of
MeCP2, an X-linked protein, is devastating:there
are few live male births in humans, and the
survivors die of encephalopathy soon after birth,
while females develop debilitating and eventually
fatal symptoms within the first year after birth.
The severity of the effects of MeCP2 loss and its
delayed onset suggest that it plays a critical role
in postnatal brain development. Even so, most of
the reported neural and synaptic abnormalities
in MeCP2-knockout mice are surprisingly mild
(Na et al., 2013). One exception is synaptic
scaling.
Two groups have independently demon-
strated that MeCP2 plays a critical role in bidi-
rectional synaptic scaling (Blackman et al.,
2012; Qiu etal, 2012). Their results are distinct
117
but complementary. Using hippocampal dis-
sociated cultures, Qui and colleagues found
that chronic (days) activity enhancement, by
application of the GABA A antagonist bicucul-
line, increased expression of MeCP2 mRNA
and decreased average mEPSC amplitude onto
pyramidal neurons. When MeCP2 expres-
sion was acutely knocked down using an RNA
interference method, the synaptic downscaling
was blocked in those neurons underexpress-
ing MeCP2. Conversely, using neocortical dis-
sociated cultures, Blackman et al. showed that
chronic activity blockade, by application of the
AMPA receptor antagonist DNQX, led to an
increase in mEPSC amplitude onto pyramidal
neurons that was absent when an RNA interfer-
ence method was used to acutely knock down
MeCP2. Together these studies indicate that
normal MeCP2 function is necessary for bidi-
rectional synaptic scaling acting in a cell auton-
omous fashion and suggest that loss of scaling
is part of pathological postnatal development in
this disorder.
Fragile X syndrome. Fragile X syndrome is the
most common inherited form of mental
retardation and the most common known
cause of autism spectrum disorder (Bagni
and Greenough, 2005). Like Rett syndrome, it
is produced by the absence of single protein,
fragile X mental retardation protein (FMRP).
Numerous synaptic and neuronal abnormalities
have been reported to be associated with loss
of FMRP, including those affecting long-term
and short-term synaptic plasticity, synaptic
morphology, and the distribution of dendritic
ion channels (Bassell and Warren, 2008; Brager
et al., 2012; Routh et al., 2013). How FMRP,
which is located near synaptic sites and acts as a
translational regulator, affects synaptic scaling
is complicated. Using mutant mice in which
Fmr1, the gene that codes for FMRP, had been
knocked out, Soden and Chen (2010) found that
FMRP was necessary for the form of synaptic
scaling that is mediated by retinoic acid and acts
locally through local protein translation (Aoto
et al., 2008). However, FMRP was apparently
not necessary for the transcription-dependent,
global form of synaptic scaling (Turrigiano
etal., 1998). The relationship between these two
types of scaling is not clear, as they may well
be mediated by different physical mechanisms,
with the local form sensitive to spontaneous
vesicle release and the global form to
spike-evoked release (Ibata et al., 2008; Sutton
and Schuman,2009).
118 Part II: Homeostatic Control
Stroke
Stroke is caused by interruptions in the blood
supply to the brain, which result in struc-
tural and functional damage to oxygen- and
energy-deprived neurons. Recovery after stroke
is a complex process, including behavioral com-
pensation, rewiring, and synaptic plasticity
(Murphy and Corbett, 2009). The first steps in
recovery appear to involve restoration of neural
activity in the peri-infarct region (i.e., the tis-
sue surrounding the area of greatest damage),
which might create a permissive environment
for axonal sprouting and dendritic spine for-
mation. The activity appears shortly (days) after
the initial stroke. This time course suggests that
synaptic scaling may be the underlying physi-
cal mechanism (Murphy and Corbett, 2009).
Evidence from rodent models that this is true
includes the poststroke hyperexcitability in the
weeks immediately following injury and the tran-
sient appearance of low-frequency spontaneous
activity (0.11.0 Hz; Carmichael and Chesselet,
2002; Schiene etal., 1996; Winship and Murphy,
2008). Also, local inflammation is common after
stroke, which might lead to release of TNF. This
cytokine is of course necessary for global synap-
tic scaling (Stellwagen and Malenka,2006).
Alzheimers Disease
A key event in the neurodegeneration associ-
ated with Alzheimers disease is the accumula-
tion of -amyloid protein in the brain (Walsh
and Selkoe, 2007). -amyloid accumulation is
toxic to neurons because it affects a number of
signal-transduction pathways. In particular, it
disrupts calcium homeostasis, with attendant
effects on synaptic function and cell death path-
ways. An intriguing, if speculative, idea is that
synaptic scaling creates a positive feedback loop
that exacerbates the damage done by -amyloid
(Small, 2008). The idea is simple: increased
-amyloid increases cytoplasmic calcium by
disrupting calcium homeostasis, leading to cell
death; this neurotoxicity reduces synaptic and
spiking activity in spared but still vulnerable
neurons; the reduced activity triggers homeo-
static synaptic scaling; scaling boosts network
activity and thus increases cytoplasmic calcium
even further, leading to more neuronal death.
Two molecular pieces of evidence support this
idea:oligomeric -amyloid decreases BDNF lev-
els in cultured neurons (Tong etal., 2004; Garzon
and Fahnestock, 2007), whereas the cytokine
TNF has been found at elevated levels in post-
mortem Alzheimers tissue (Jia et al., 2005; Ho
et al., 2005). Both of these effectsdecreased
BDNF and increased TNFwould tend to
upregulate AMPA receptor levels through synap-
tic scaling.
CONCLUDING REMARKS
Even before there was any experimental evi-
dence for synaptic scaling, it was anticipated on
general theoretical grounds by the need of neu-
ral networks for some mechanism of firing rate
homeostasis and limiting synaptic strengths
(Miller and MacKay, 1994). It was not the only
candidate homeostatic mechanism (see, e.g.,
Bienenstock et al., 1982), but it was among the
simplest and most direct. Experiments over the
past 15 or more years have demonstrated that
synaptic scalinglike processes exist in a wide
variety of neurobiological systems, including in
the neocortex, hippocampus, and spinal cord;
in mammal, Xenopus, and Drosophila; and in
young animals and mature animals. Ongoing
experiments have elucidated the properties of
synaptic scaling in considerable detail and dem-
onstrated that its phenomenology is rich and
varies depending on activity manipulation, neu-
robiological system, and age. But perhaps what
is most needed now is a better idea of its func-
tional consequences. Firing rate homeostasis was
the starting pointand remains a focus of cur-
rent workbut, as we have indicated, there are
several other possible uses for scaling, including
synaptic competition and working memory, and
it may play important roles in several neurologi-
cal disorders. For technical reasons, experiments
to date have not addressed these issues directly,
and that is the most important challenge for
futurework.
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8
Homeostatic Role ofHeterosynaptic Plasticity
N I C H O L A S M . B A N N O N , M A R I N A C H I S T I A K O VA ,
AND MA XIM VOLGUSHEV
INTRODUCTION:LEV ELS
O F H O M E O S TA S I S
Homeostatic regulation is one of the basic princi-
ples of the functioning of living organisms. In the
brain, a great variety of homeostatic mechanisms
is employed to keep a wealth of biophysical pro-
cesses within their operating range (Davis and
Bezprozvanny, 2001). These mechanisms oper-
ate at multiple nested levels, such as regulation
of metabolic activity, transmembrane gradients,
and intracellular concentration of ions; homeo-
stasis of synaptic weights; the balance of excita-
tion and inhibition; the firing rate of neurons;
and other, more global processes at systems and
network levels.
We propose that the most effective homeo-
static mechanisms are intrinsic to a specific level,
such that the sensor that measures the state
of the variable, the error detector that deter-
mines its deviation from a given set point, and
the effector that provides the negative feedback
should all be built within the same level and
utilize similar mechanisms, thus operating on a
similar timescale as the processes they are regu-
lating. Consider input-specific (or homosynap-
tic) plasticity governed by Hebbian rules that
mediates developmental plasticity, learning, and
memory. Associative synaptic plasticity intro-
duces a perturbation to the strength of neurons
inputs. This perturbation is amplified by a posi-
tive feedback on synaptic weight changes, which
is intrinsic to Hebbian-type learning rules, thus
pushing the system out of balance. We argue that
to achieve a high-fidelity homeostatic response
to a change in synaptic weights, the underlying
mechanism should have the same-level trigger
and operate on the same timescale as Hebbian
plasticity. Heterosynaptic plasticity, which
accompanies induction of homosynaptic changes
but occurs at synapses that were not active during
the induction, is well suited for this role. We con-
clude that heterosynaptic plasticity with experi-
mentally observed properties can serve as a
mechanism of homeostatic regulation of synaptic
weight changes induced by associative learning.
A S S O C I AT I V E H E B B I A N
PLASTICITY
Plasticity is a universal property of synapses,
vital for fundamental operations of the nervous
system. Synaptic plasticity mediates the forma-
tion and refinement of connectivity patterns in
the nervous system during development and also
mediates effective adaptive behavior in changing
environments throughout life. Hebbs rule (Hebb,
1949)formulates the basic principle according to
which synaptic weights change:synaptic connec-
tions that repeatedly or persistently take part in
firing of the postsynaptic neuron increase their
strength. The original rule, suggested only for
potentiation of synaptic connections, was later
extended to include depression: synapses that
consistently do not take part in firing the post-
synaptic neuron decrease their strength. In 1973,
Bliss and Lomo discovered the cellular basis of
associative learning, long-term potentiation
(LTP) in the hippocampal formation (Bliss and
Lomo, 1973). The authors found that after a brief
period of strong activity (high-frequency stimu-
lation, or afferent tetanization) the amplitude of
test responses to electric stimuli was increased,
and the increase persisted for hours. Further
research complemented the associativity rule
with the rules of cooperativity and input speci-
ficity (Bliss and Collingridge, 1993). The rule of
cooperativity states that activation should be
strong, exceeding a certain threshold, to induce
plastic changes. The rule of input specificity
maintains that changes take place only at syn-
apses that were activated during the induction
 Homeostatic Role of Heterosynaptic Plasticity
but not at other synapses. Altogether, these
Hebbian-type learning rules capture the asso-
ciative nature of synaptic changes and explain a
multitude of plastic phenomena in the nervous
system, ranging from refinement of connectivity
during development (neurons that fire together
wire together) to extraction of casual relations
between events in the environment in Pavlovian
conditioning and other types of associative learn-
ing, motor learning, and acquisition of sequences
of behavioral actions.
Spike-timing dependent plasticity (STDP)
explicitly implements relative timing between
firing of the neuron and activity at its inputs (pre-
synaptic spikes) as a determinant of the direction
and the magnitude of synaptic weight changes. In
the typical STDP learning rule, synaptic inputs
that were active shortly before a postsynaptic
action potential (pre before post) are potentiated,
while synaptic inputs active shortly after the
postsynaptic spike (post before pre) are depressed
(Markram etal., 1997; Magee and Johnston, 1997;
Abbott and Nelson, 2000; Caporale and Dan,
2008). STDP, and Hebbian-type learning rules in
general, extract casual relations between events
by using the temporal order of their occurrence.
This dictates the necessity of activity of both
presynaptic and postsynaptic neurons for plas-
ticity induction. The necessity for concomitant
pre- and postsynaptic activation for plasticity
induction and the rule of input specificity explic-
itly restrict plasticity to synaptic inputs that were
active during the induction. Thus plasticity gov-
erned by Hebbian-type learning is input specific
and homosynaptic.
Synaptic changes underlying Hebbian plas-
ticity are triggered by rises of intracellular [Ca 2+],
which under natural conditions result from fir-
ing of pre- and postsynaptic neurons at high
frequency during afferent tetanization, or with
repeatedly occurring coincidence with pre-
cise temporal relation in STDP (Golding et al.,
2002; Nevian and Sakmann, 2006; Sjstrm
and Hausser, 2006). Importantly, such activ-
ity patterns are not uncommon and may occur
naturally in the living brain. The final outcome
of the induction (whether or not long-term plas-
ticity will be induced, the direction of change,
and the magnitude of potentiation or depression)
depends on the temporal dynamics and ampli-
tude of intracellular [Ca2+] rise, the source of
Ca2+ influx, and specifics of the synapse (Lisman,
1989; Artola and Singer, 1993; Cummings etal.,
1996; Yang et al., 1999). Accordingly diverse
are the biochemical cascades triggered by the
125
calcium signal and the mechanisms of plasticity
expression (Abbott and Nelson, 2000; Malenka
and Bear, 2004; Feldman, 2009; Manninen etal.,
2010). Expression of plasticity may involve both
presynaptic changes, such as an increase of the
probability of transmitter release in LTP, and
postsynaptic changes, such as insertion or inter-
nalization of -amino-3-hydroxy-5-methyl-
4-isoxazolepropionic acid (AMPA) receptors.
Associative synaptic changes, at least at their
early phase, are induced within tens of seconds
to minutes.
H E B B I A N P L A S T I C I T Y: W H Y
IT CANNOT WORKALONE
Synaptic changes induced according to
Hebbian-type rules provide a cellular mechanism
for encoding new memories. However, learning
systems in which Hebbian-type plasticity is the
only rule that governs synaptic modifications has
two major problems:they are prone to runaway
dynamics of synaptic weights and activity, and
they introduce only a weak degree of competition
between synapses.
Propensity for runaway dynamics is imposed
by the positive feedback on synaptic weight
changes that is intrinsic to Hebbian-type learning
rules. Potentiation, by making synapses stronger,
increases their chances to take part in firing a
neuron and thus increases the probability of these
synapses to be potentiated further. Similarly,
synapses weakened by depression have a lower
chance to lead to spikes and thus an increased
probability for being further depressed. Because
of this positive feedback on synaptic weight
changes, synapses would tend to be either poten-
tiated to the maximal value or depressed to zero.
The runaway of synaptic weights leads to runa-
way dynamics of activity. Runaway potentiation
of synaptic weights to maximal values results in
overexcitability of neurons and excessive activity,
while runaway depression would lead to silencing
of neurons. Both scenarios would lead to distur-
bance of the inputoutput relations of neurons
(Chen et al., 2013), thus compromising compu-
tational abilities of neuronal networks (Skorheim
et al., 2014). Moreover, runaway potentiation of
synaptic weights and associated runaway activity
are energetically unsustainable and may lead to
pathology. Thus homosynaptic changes governed
by associative Hebbian plasticity must be con-
strained by additional plasticity rules that keep
plastic synapses and learning networks within
their operation range, maintaining a balance of
learning and network homeostasis.
126 Part II: Homeostatic Control
Hebbian-type learning rules introduce only a
weak, if any, degree of competition between syn-
apses. The idea of competition, which maintains
that synapses should strengthen at the expense
of other synapses, is well grounded from a bio-
logical perspective: while undergoing changes,
synapses might compete for shared limited
resources, such as available energy, molecules,
or plasticity factors. The need for competition
is best illustrated by activity-dependent shap-
ing of sensory representations during develop-
ment (Wiesel and Hubel, 1963; Aitkin et al.,
1970; Merzenich et al., 1975; Thompson et al.,
1983; Feldman, 2009). The canonical example
here is the development of projections of specific
thalamic afferents from the lateral geniculate
nucleus to the visual cortex from a highly over-
lapping projection of afferents representing two
eyes in early life to segregated projections into
ocular dominance columns in adults. Synaptic
changes during everyday learning might be more
subtle, but the ability of synaptic competition
to accentuate the plasticity-induced contrast
between synaptic weights might enhance learn-
ing, especially in paradigms that involve dis-
crimination (Skorheim et al., 2014). Although
specific homosynaptic induction protocols can
induce either potentiation or depression, and
thus change synaptic weights in both directions,
these bidirectional changes can hardly support
synaptic competition because they require repe-
tition of specific and independent patterns of the
input activity. For two groups of inputs to change
in opposite directions, both groups should be
repeatedly activated with specific but different
and independent patterns, such as low frequency
for induction of long-term depression (LTD) but
high frequency for induction of LTP, or be sys-
tematically active at different timings relative
to postsynaptic activity to change via an STDP
mechanism. Although possible in theory, such a
scenario imposes strict requirements on the rela-
tions between specifics of plasticity rules and pat-
terns of input activity. Thus, to support intrinsic
competition between synapses, a mechanism for
plastic changes outside of the Hebbian learning
rule is required.
MODELING
P E R S P E C T I V E : W H AT I S
NEEDED INADDITION
TOHEBBIANRULES?
The need for maintaining stability of the total
synaptic weights, the amount of synaptic drive
a neuron receives and neuronal activity, and for
strong synaptic competition in learning systems
with Hebbian-type rules has been appreciated
since early theoretical studies (von der Malsburg,
1973; Miller and MacKay, 1994; Miller, 1996). To
some extent, this can be achieved by carefully
balancing STDP rules, whereby an appropriate
negative bias balances potentiation and depres-
sion (Song etal., 2000; Van Rossum etal., 2000;
Gtig etal., 2003; Babadi and Abbott, 2010). One
problem with this solution is that it imposes strict
requirements on the relative strength (amplitude
and duration) of potentiation and depression
windows. STDP can indeed lead to stabilization
of the neurons mean firing rate if the integral
of the learning windows for potentiation and
depression is negative (Kempter et al., 2001).
This requirement is, however, not consistent with
experimental evidence that has demonstrated
an enormous heterogeneity in the parameters
of STDP windows at different synapses, cells,
developmental stages, and conditions of neuro-
modulation (Nishiyama et al., 2000; Sjstrm
etal., 2001; Zhou etal., 2005; Haas etal., 2006;
Feldman, 2009). Arelated problem is that STDP
rules adjusted to maintain stability in a neuron
subject to a certain pattern of external drive may
still lead to runaway dynamics when activity
changes, such as increase of the input correlation.
Finally, an STDP rule provides only weak com-
petition between synapses: only synapses from
presynaptic neurons that are repeatedly active
shortly after the postsynaptic spikes, and thus
repeatedly fall into the depression window, will
become depressed. This weak competition does
not mimic biologically-plausible forms, such as
competition for limited resources.
One further mechanism that was suggested to
reduce the positive feedback on synaptic weight
changes and counteract their runaway dynam-
ics is weight dependence of plastic changes (Oja,
1982). Weight dependence dictates that weaker
synapses can potentiate more, while stronger
synapses potentiate less, and in the limit do not
change (Bi and Poo, 1998; van Rossum et al.,
2000; Hardingham et al., 2007). In this limit,
weight dependence imposes bounds on synaptic
weights, though it does not prevent saturation of
synaptic weights at extreme values. Combination
of weight dependence of plastic changes with
depression-biased STDP rules improves the
stability of the activity level of model neurons
with plastic synapses (van Rossum et al., 2000;
Kempter etal., 2001; Gtig etal., 2003; Morrison
etal., 2007; Babadi and Abbot, 2010; Gilson and
Fukai,2011).
 Homeostatic Role of Heterosynaptic Plasticity
Achieving Stability Through
Normalization
A simple and robust method of stabilization com-
monly used in modeling to constrain runaway
dynamics of neuronal activity is normalization
(von der Malsburg, 1973; Oja, 1982). The concept
of normalization is that following an episode of
plasticity inducing weight changes at some syn-
apses, all synaptic weights on the cell are read-
justed so that their sum remains constant. This
normalization has typically been implemented via
multiplicative and subtractive methods. In multi-
plicative normalization (von der Malsburg, 1973;
Oja, 1982), each weight is multiplied by an amount
necessary to maintain the overall net weight (Van
Ooyen, 2001). In the subtractive method, a fixed
amount of change (decay to compensate for the
effect of homosynaptic potentiation or increase
to compensate for the effect of depression) is
added to all synapses regardless of their weight
(Miller and MacKay, 1994). While both meth-
ods of normalization, multiplicative and sub-
tractive, robustly prevent runaway dynamics of
total synaptic drive and activity, neither prevents
runaway potentiation or depression of individual
synapses. Moreover, multiplicative and subtrac-
tive normalization introduce a different degree
of competition between synapses and thus may
lead to different final distributions of synaptic
weights and functional connectivity (Miller and
MacKay, 1994; Miller, 1996; see later discussion).
The use of normalization for achieving stability
of the activity level and synaptic competition was
further elaborated in later studies (e.g., Kempter
et al., 2001; Elliott and Shadbolt, 2002; Wu and
Yamaguchi, 2006; Finelli etal.,2008).
Note that because normalization affects all
synapses of the neuron, irrespective of their
recent activity, it postulates the existence of het-
erosynaptic plasticitychanges at synapses that
were not activated during plasticity induction.
The Need forCompetition
Theoretical analyses have demonstrated that
strong competition between projecting fibers, such
that the weight of synapses formed by one fiber
should grow at the expense of synapses of other
fibers which must decrease their weights, is neces-
sary for processes such as the establishment of sen-
sory representations during development (Wiesel
and Hubel, 1963; Aitkin et al., 1970; Merzenich
et al., 1975; Thompson et al., 1983; Feldman,
2009). In the mature brain, synaptic competition
is instrumental in learning tasks that involve dis-
crimination (Skorheim etal., 2014). Competition
127
between plastic synapses, for example for limited
available resources, can also be one of the natural
ways in which the magnitude of possible change
is restricted, thus preventing excessive plastic-
ity. In a pioneering computational study, von der
Malsburg (1973), citing the limited evidence that
was available 40 years ago, suggested that the
biological constraint could be limited dendritic
space for which synapses are competing. In a later
study, Oja (1982) speculated that the physiological
basis would be competition for shared resources,
such as receptor molecules, surface area, energy
resources, or plasticity factors.
In the simplest sense, competition is pro-
vided by normalization, with different meth-
ods of normalization introducing different
degrees of competition (Miller and MacKay,
1994; Miller, 1996; Gerstner and Kistler, 2002).
Multiplicative normalization introduces a weak
competition, which permits a gradated separa-
tion of synaptic weights but cannot explain the
competitive exclusion of thalamic afferents in
ocular dominance plasticity, when neurons in
the visual cortex become predominately inner-
vated by afferents carrying information from a
single eye (Miller and MacKay, 1994). Amodel
with multiplicative normalization was able to
segregate correlated inputs from noncorrelated
inputs; however, it failed to segregate inputs
expressing stronger correlation out of a larger
pool of weaker-correlated inputs. This latter set-
ting is more realistic:inputs from different eyes
express a certain degree of correlation imposed
by the visual scene, although these correlations
are weaker than correlations between activity
of inputs from the same eye. Subtractive nor-
malization introduces strong competition suffi-
cient to solve this problem and segregate inputs
from the two eyes despite their correlation, but
it leads to emergence of synapses with extreme
weights (Miller and MacKay, 1994; Elliott and
Shadbolt, 2002). From a biological perspective,
subtractive normalization is less plausible than
multiplicative, though neither of the two has a
clear relation to biological processes that may
underlie competition in real neurons. It has
been suggested that the explanatory power of
models is limited if normalization is imposed
as a mathematical convenience and does not
represent dynamics underlying a physiological
mechanism (Van Ooyen, 2001). Ideally, com-
petition should be a consequence of the learn-
ing rule and not require explicit additional
rules (Gerstner and Kistler, 2002). By extension
it could also be proposed that normalization
128 Part II: Homeostatic Control
should be a consequence (or part of) the learn-
ing rule. Indeed, in a model that implemented
activity-dependent competition over a resource
necessary for plasticity, neurotrophic factors,
the normalization emerged naturally, with-
out being explicitly implemented (Elliott and
Shadbolt, 2002). In a stable substate of this bio-
logically inspired model, the emerged Hebbian
learning dynamics were accompanied by a mul-
tiplicative normalization that, unlike in mod-
els with imposed normalization, permitted the
segregation of correlated inputs. Thus modeling
competition for resources, which approximates
the biological reality more closely, can manifest
the normalization necessary to prevent runaway
and provide the competition to segregate inputs,
moving these features from an ad hoc rectifica-
tion to emergent properties of learningrules.
The Trigger and theTimescale
ofthe Homeostatic Mechanism
In most modeling studies, normalization is
implemented directly into the learning rules,
which update synaptic weights in each itera-
tion. As a result, first, the trigger for plasticity
also inevitably triggers the normalization, and
second, the normalization operates on the same
timescale as the plasticity rule. While these two
features are an automatic consequence of the
model design, they are suggestive of additional
characteristics required from a homeostatic
mechanism in systems with Hebbian-type
learning rules. Indeed, a few studies in which
normalization was implemented separately
converge at the conclusion that its time con-
stant should be short. In a model expressing
stable dynamics with STDP learning, the sepa-
rate timescale for homeostatic scaling regulated
by postsynaptic firing rate was relatively short,
with a time constant of 100s, which is compara-
ble to the timescale of synaptic changes intro-
duced by learning (van Rossum et al., 2000).
In a theoretical analysis of one-trial sequence
learning of place-fields in the hippocampus,
Wu and Yamaguchi (2006) concluded that for
learning processes that occur within minutes,
the physiological mechanism that constrains
synaptic weights must also operate rapidly. The
question of timescale of homeostatic mecha-
nism that is necessary for maintaining stabil-
ity of a system with plastic synapses has been
directly addressed in a recent study by Zenke
et al. (2013). The time constant of the homeo-
static mechanism in this study was separate
from the time constant of the plasticity rule.
The authors found that, to achieve robust stabil-
ity of the system, the homeostatic mechanism
must operate on a timescale comparable to the
timescale of plasticity itself (Zenke etal.,2013).
The question of the trigger for the homeo-
static mechanism that protects synapses from
runaway in the face of Hebbian plasticity is more
complicated because possibilities do not form a
one-dimensional, easily quantifiable continuum
that can be conveniently studied in simulations
(such as time constant) but rather consist of sev-
eral discrete factors, such as total synaptic weight
limited by resource and energy constraints, or
level of activity-dependent calcium influx, or fir-
ing rate of a cell or a population. These possibili-
ties are not mutually exclusive but may operate on
several different levels of homeostatic regulation.
Thus a successful physiological mechanism
that maintains stability of the dynamics of learn-
ing systems with plastic synapses must, in addi-
tion to the aforementioned requirements, operate
in tandem with homosynaptic plastic changes.
In sum, theoretical analysis clearly shows
the necessity of homeostatic mechanism(s)
that prevent runaway dynamics imposed by
Hebbian-type rules on synaptic weights and neu-
ronal activity. It also identifies certain required
features of cellular-level candidate mechanisms.
First, such mechanisms should be robust to both
the variability in the details of plasticity rules
observed in experiments and a wide range of
activity patterns. Second, they should provide
normalization of synaptic weights and support
synaptic competition. It is not clear if the nor-
malization brings about competition or arises
from competition, or if normalization and com-
petition are mediated by diverse mechanisms.
Third, they should operate on a timescale com-
parable to the timescale on which Hebbian-type
synaptic changes take place. This latter point
would be automatically fulfilled if homeostatic
and Hebbian-type changes of synaptic weights
share the trigger and are mediated by the same or
overlapping intracellular processes.
BIOLOGICAL
C A N D I D AT E : H O M E O S TAT I C
SY NAPTIC SCALING
Experimental Phenomena
A form of homeostatic plasticity that is often
considered as a mechanism that keeps overall
balance of synaptic weights and prevents runa-
way dynamics of activity induced by Hebbian
learning rules is the phenomenon of synaptic
 Homeostatic Role of Heterosynaptic Plasticity
scaling:compensatory scaling of synaptic weights
triggered by prolonged dramatic changes of
global activity. Scaling modifies synaptic weights
to counteract changes of activity. Synaptic
weights scale up after hours and days of activ-
ity blockade by tetrodotoxin (Turrigiano et al.,
1998), or hyperpolarization of neurons caused
by expression of inwardly rectifying potassium
channels (Burrone etal., 2002), and scale down
after prolonged increases of activity by block-
ade of inhibition (Turrigiano et al., 1998). This
scaling is multiplicative and thus maintains the
relative balance of synaptic weights. Originally
discovered in dissociated neuron cultures, scal-
ing has been also demonstrated in the whole brain
after hours of deafferentation (Keck etal., 2013;
Vlachos etal., 2013; Becker etal., 2013). Scaling
in the neocortex is developmentally regulated,
such that layer-4 neurons express a transient
ability to scale, and subsequently neurons from
layers 2 and 3 demonstrate the phenomena that
persist through adulthood (Turrigiano,2012).
Homeostatic synaptic scaling in cortical
neurons is dependent on somatic calcium (Ca 2+)
influx, gene transcription, and multiple intracel-
lular pathways that may operate in parallel or
interact with each other (Rich and Wenner 2007;
Ibata etal., 2008; Watt and Desai 2010; Turrigiano,
2012; Chen et al., 2014). Network-wide factors
such as activity-dependent brain-derived neu-
rotrophic factor release or tumor necrosis fac-
tor alpha from glia may also have a role in some
types of synaptic scaling (Kaneko et al., 2008;
Turrigiano, 2012). Evidence for both presynaptic
and postsynaptic changes has been found in vari-
ous experimental paradigms (Turrigiano et al.,
1998; Murthy et al., 2001; Burrone et al., 2002;
Ibata etal., 2008). However, typically, scaling is
attributed to postsynaptic mechanisms:changes
in AMPA trafficking, resulting in changes of
the amplitude but not frequency of miniature
events, and changes of the amplitude but not
short-term plasticity of evoked responses (Ibata
et al., 2008; Turrigiano, 2012; Tatavarty et al.,
2013). Significantly, the biochemical cascades
and mechanisms underlying AMPA traffick-
ing engaged by scaling are distinct from those
engaged during Hebbian plasticity (either LTD
or LTP; Turrigiano, 2012; Tatavarty etal.,2013).
Although originally described as a global,
cell-wide process, later studies raised the possibil-
ity that scaling could be quasi-local (at the spatial
resolution of dendritic branches), which could
serve for localized normalization of weights in
a computationally useful way (Burrone et al.,
129
2002; Branco etal., 2008; Rabinowitch and Segev,
2008). Realistically, scaling is not a unitary phe-
nomenon but a multitude of mechanisms aimed
to counteract dramatic, long-lasting alterations
of overall neural activity and drive the firing rate
back to a set point (Rich and Wenner 2007; Watt
and Desai 2010; Turrigiano, 2012). A common
feature of these mechanisms is that their activa-
tion requires prolonged (hours/days) deviation of
the triggering variable from its setpoint.
Homeostatic synaptic scaling, operating
alongside other plasticity mechanisms, might
play a role in the dramatic reorganizations of
connectivity observed during visual cortex devel-
opment, such as ocular dominance plasticity and
experimental paradigms of monocular depri-
vation (Desai et al., 2002; Vitureira et al., 2012;
Lambo and Turrigiano, 2013; Keck etal., 2013).
However, while scaling is well suited to adjust
long-term and dramatic alterations of activ-
ity, and probably supplements and shapes other
homeostatic mechanisms during development,
two features make it a poor candidate for serving
the acute constraining role necessary to combat
runaway dynamics imposed by Hebbian-type
plasticity rules:the timescale and the trigger for
induction.
Timescale, Competition, and
Runaway Dynamics
Homeostatic synaptic scaling operates on the
timescale of hours and days. The rapid scal-
ing takes place after 4 hours of complete silenc-
ing of cultured neurons with tetrodotoxin (Ibata
et al., 2008). This timescale is compatible with
that of developmental processes, such as for-
mation of sensory representations in norm and
pathology (Wiesel and Hubel, 1963; Aitkin
et al., 1970; Merzenich et al., 1975; Thompson
etal., 1983; Feldman, 2009), but it is at least two
orders of magnitude slower than the timescale
of associative plasticity, which changes synap-
tic weights within minutes or even tens of sec-
onds. Because of this dramatic difference of the
timescales, homeostatic synaptic scaling cannot
mediate synaptic competition or normalize syn-
aptic weights during ongoing associative synap-
tic plasticity, nor is it suitable for counteracting
runaway dynamics that can be induced within
seconds and minutes by Hebbian-type learning
rules. This inconsistency between timescales
of slow homeostatic scaling and fast associa-
tive learning has been pointed out by Wu and
Yamaguchi (2006), who concluded that synaptic
scaling does not seem to work for fast learning.
130 Part II: Homeostatic Control
Computational analysis of simple recurrent
schemes built of neurons with Hebbian-type
synapses showed that the maximal amount of
input that these simple circuits can tolerate with-
out drifting into runaway dynamics decreases
sharply with the increasing ratio of the time-
scales of the homeostatic and Hebbian plasticity.
For the ratio of 100, which is just approaching
the lower limit of experimentally measured val-
ues, the level of tolerated activity is close to zero
(Tetzlaff etal., 2012). The range of the timescales
of a homeostatic mechanism compatible with
stable operation of a neuron with Hebbian-type
plasticity has been systematically investigated
in a recent theoretical study (Zenke etal., 2013).
The results demonstrated that, for achieving
robust stability of a system with Hebbian-type
plastic synapses, the mechanism that maintains
homeostasis and prevents runaway dynamics
must operate on a timescale comparable to the
plasticity itself (Zenke etal.,2013).
This conclusion is compatible with results
of other computational studies of which we
are aware. To the best of our knowledge, in all
computer models in which synaptic scaling was
used to prevent runaway dynamics imposed by
Hebbian-type plasticity or to introduce synap-
tic competition, scaling was implemented on a
short timescale, the same or comparable to the
timescale of the Hebbian plasticity. We are not
aware of a published computational study in
which homeostatic synaptic scaling with experi-
mentally observed features (in particular the
requirement of at least 4 hours of altered activity
level to trigger synaptic changes) was shown to
be effective in preventing runaway dynamics or
supporting synaptic competition during ongoing
learning.
Realism ofExperimental Paradigm
One further concern regarding possible involve-
ment of homeostatic synaptic scaling in balanc-
ing synaptic changes induced by Hebbian-type
plasticity is the severity of changes that are
required to trigger the scaling. Typically, scaling
up is induced by a complete silencing of activ-
ity for many hours by tetrodotoxin application
(Turrigiano etal., 1998; Turrigiano, 2012). Such
dramatic and global changes of activity are nei-
ther likely nor compatible with normal operation
of the brain. A recent study demonstrated that
6 hours after complete bilateral retinal lesions,
activity in the visual cortex was reduced to ~60%
of prelesion level (Keck etal., 2013). The authors
did see evidence for homeostatic scaling after
the lesions but noted that homeostatic scaling
alone could not explain the observed recovery of
activity in the deprived cortex (Keck etal., 2013).
Note that even this more modest interven-
tion represents extreme pathology. In contrast,
Hebbian-type synaptic plasticity can be induced
by far more subtle events, and activity changes
that may result from associative synaptic plastic-
ity might be also far less dramatic.
In sum, homeostatic synaptic scaling repre-
sents a homeostatic mechanism engaged slowly
in response to extreme and long-lasting chal-
lenges, and presumably these mechanisms would
not be engaged until runaway dynamics had
created a dramatically unstable and saturated
network. Experimentally observed properties
of homeostatic synaptic scaling do not fit the
requirements delineated in theoretical studies
for a hypothetical mechanism needed in addi-
tion to Hebbian-type learning rules. Both the
trigger and the timescale of synaptic scaling
are fundamentally different from those of the
Hebbian-type plasticity.
B I O L O G I C A L C A N D I D AT E :
HETEROSY NAPTIC
PLASTICITY
Experimental Evidence
Heterosynaptic plasticity refers to changes at syn-
apses that were not presynaptically active dur-
ing the induction of plasticity. Heterosynaptic
LTD accompanying induction of LTP was first
described in the hippocampus shortly after the
phenomenon of LTP was discovered (Lynch etal.,
1977). In CA1 pyramidal neurons, induction of
LTP of Schaffer collateral-commissural synapses
at apical dendrites was accompanied by an LTD
at inputs to basal dendrites made by commissural
fibers that were not stimulated during the induc-
tion. Vice versa, induction of LTP at the basal
dendrites was accompanied by LTD at the apical
dendrites. Heterosynaptic LTD accompanying
the induction of homosynaptic LTP clearly has
potential for both balancing plastic changes and
supporting synaptic competition.
Heterosynaptic LTP was first discovered at
synapses adjacent to potentiated inputs. After
pairing one input to a CA1 neuron, synapses
formed by nearby fibers on that cell and even on
nearby neurons were potentiated too (Bonhoeffer
et al., 1989; Kossel et al., 1990; Schuman and
Madison, 1994a; Engert and Bonhoeffer, 1997).
 Homeostatic Role of Heterosynaptic Plasticity
These results demonstrated that input specificity
breaks down at short distances: LTP-protocols
induce plasticity not only at the activated syn-
apses but also at those not active during the
induction.
Studies of the spatial distribution of plastic
changes in structures with regular organization
of the inputs, such as the hippocampus or amyg-
dala, revealed a Mexican hattype profile of plas-
ticity (White etal., 1990; Royer and Par, 2003).
Induction of LTP at a set of synapses was accom-
panied by a biphasic profile of heterosynaptic
changes: a weaker LTP at nearby inputs, LTD
at more distant inputs, and finally no changes
at yet more distantly located synapses. A sym-
metrical profile was observed around the site of
LTD induction: weaker LTD at close distances
and LTP at more distant inputs (Royer and Par,
2003). Importantly, the amount of potentiation
and depression in these profiles was balanced,
so that neither LTP nor LTD induction at acti-
vated synapses resulted in net changes of the total
synaptic input. Induction of balanced profiles
of plastic changes can provide a powerful local
mechanism of both normalization of synaptic
weights and synaptic competition.
Trigger forHeterosynaptic
Plasticity:Bursts ofSpikes
and CalciumRise
Heterosynaptic changes are triggered by rises
of intracellular Ca 2+ concentration, thus shar-
ing the trigger with Hebbian-type plastic-
ity (Lisman, 1989; Artola and Singer, 1993;
Cummings etal., 1996; Yang etal., 1999). Bursts
of backpropagating action potentials induce
massive rises of [Ca 2+] throughout the dendritic
tree, which might be sufficient to reach plastic-
ity threshold at heterosynaptic sites (Spruston
etal., 1995; Staubli and Ji, 1996; Larkum etal.,
1999; Golding et al., 2002; Waters et al., 2003;
Lisman and Spruston, 2005; Sjstrm and
Hausser, 2006; Remy and Spruston, 2007).
Experimental protocols that deliver purely
postsynaptic challenges to postsynaptic neu-
rons can induce long-term plasticity. In the hip-
pocampus and neocortex, either photolysis of
caged Ca 2+ (Neveu and Zucker, 1996; Yang etal.,
1999) or postsynaptic spiking is sufficient to
induce plasticity (Kuhnt etal., 1994; Volgushev
et al., 1994; Volgushev et al., 2000; Cummings
et al., 1996, Chistiakova and Volgushev, 2009;
Lee et al., 2012). Chelating intracellular Ca 2+
impairs induction of heterosynaptic plasticity
131
(Lee etal., 2012). Mechanisms of heterosynaptic
plasticity overlap with those mediating homo-
synaptic plasticity, as indicated by a partial
occlusion between homo- and heterosynaptic
plastic changes (Kuhnt etal., 1994; Cummings
etal., 1996; Neveu and Zucker, 1996; Yang etal.,
1999; Volgushev etal., 1999). This partial occlu-
sion of expression mechanisms of homosynap-
tic and heterosynaptic plasticity may be due to
the shared calcium dependence and activation
of common intracellular cascades by the Ca 2+
rises that lead to plastic changes. One further
possibility involves shared pathways of retro-
grade signaling. Specifically the role of nitric
oxide and cannabinoid signaling pathways have
been demonstrated in both homosynaptic and
heterosynaptic plasticity. Nitric oxide has an
established role as a retrograde signal during
plasticity induced by pairing or afferent teta-
nization (Gally etal., 1990; Bhme etal., 1991;
ODell etal., 1991; Schuman and Madison 1991).
Because nitric oxide spread is not limited to
the activated synapses, it also can affect non-
activated synapses. Indeed, retrograde signal-
ing via nitric oxidedependent pathways has
been explicitly demonstrated to be involved in
induction of heterosynaptic changes (Schuman
and Madison 1994a,1994b; Volgushev et al.,
2000; Nugent et al., 2007; Bright and Brickley,
2008; Phillips et al., 2008; Lange et al., 2012;
Lee etal., 2012; reviewed in Hardingham etal.,
2013). LTD mediated by retrograde signaling
via endocannabinoids has been demonstrated
at both excitatory and inhibitory connections at
both homo- and heterosynaptic sites (Sjstrm
et al., 2004; Chevaleyre et al., 2003, 2006; Pan
etal., 2008; Chiu etal., 2010; Huang etal., 2008;
Yasuda et al., 2008; reviewed in Heifets and
Castillo, 2009). Because activation of mGluRs
can trigger the production and release of endo-
cannabinoids in a calcium-independent manner
(Maejima et al., 2001), this form of heterosyn-
aptic LTD can be Ca 2 independent but mGluR
dependent (Chevaleyre etal., 2003; Chevaleyre
etal., 2006; Pan etal., 2008; Huang etal., 2008;
Yasuda etal., 2008; Chiu etal.,2010).
Thus heterosynaptic plasticity can be induced
by the same protocols, occurs at the same time-
scale, and shares mechanisms with Hebbian-type
plasticity (for further discussion of induction of
heterosynaptic changes by the protocols that
are typically used to induce homosynaptic plas-
ticity, see Fig. 8 and related text in Chistiakova
etal.,2014).
132 Part II: Homeostatic Control

Properties ofHeterosynaptic active during the induction, will experience post-

Plasticity
Heterosynaptic, long-term plastic changes can be
induced in hippocampal and neocortical neurons
by intracellular tetanizationbursts of spikes
evoked by short, depolarizing pulses applied
through the recording electrode (Figure 8.1A;
Kuhnt et al., 1994; Volgushev et al., 1994, 1997;
Volgushev et al., 1999; Volgushev et al., 2000;
Chistiakova and Volgushev, 2009; Lee et al.,
2012). The rationale behind the intracellular teta-
nization protocol as a tool to study heterosyn-
aptic plasticity is as follows:Each neuron in the
neocortex receives thousands of synaptic inputs.
However, spiking of a neuron can be induced by
activation of only a fraction of these inputs, from
a few dozens to hundreds. Repetitive activation
of a specific group of synapses leading to repeti-
tive firing of a cell can, under certain conditions,
induce synaptic plasticity. Other synapses, not
synaptic activity that is not associated with acti-
vation of their presynaptic fibers. Because spikes
can be evoked by activation of just a small frac-
tion of all synapses, this situationpostsynaptic
activity without presynaptic activationwill
be experienced by the majority of synapses at a
neuron. Thus intracellular tetanization mimics
the conditions experienced by the majority of
neurons synapses during the induction of asso-
ciative plasticity. Moreover, because during the
intracellular tetanization no synaptic inputs were
stimulated, any synaptic changes can be consid-
ered heterosynaptic.
How much postsynaptic activity is required
to induce heterosynaptic plasticity? Our typi-
cal protocol of intracellular tetanization con-
sists of 10 bursts of three to seven spikes at 50
to 100 Hz, presented as one train or repeated
three times with 1-minute intervals (Kuhnt etal.,

(a) (b) 4 (c)

Intracellular 3
Tetanization
EPSP amplitude, mV

2 1 mV
r = 0.52
? 1 300
***
? 0
EPSP amplitude change, %

?
2 200
?
1.5 1 mV
? 1 100
0.1 s
? 0.5
EPSP amplitude, mV

0
1.2

0.8 1 mV
100
40 mV 0 1 2 3
50 ms 0.4
Initial Paired-Pulse Ratio
0
x3 10 0 10 20 30 40 50
1s Time from Intracellular Tetanization, min

FIGURE 8.1: Long-term synaptic plasticity induced by intracellular tetanization. A:Scheme of an

intracellular tetanization experiment. Bursts of short depolarizing pulses (5 pulses at 100Hz; 10 bursts at 1Hz, 3
trains of 10 bursts) were applied through the recording electrode without presynaptic stimulation to induce bursts
of action potentials. Synaptic responses were recorded before and after the intracellular tetanization. Because
no inputs were stimulated during the induction, plasticity at all synapses can be considered heterosynaptic.
B:Examples of inputs that underwent potentiation (top), depression (middle), or did not change after intracellular
tetanization. Time courses of amplitudes of excitatory postsynaptic potentials (EPSPs) evoked by the first pulse in
a paired-pulse paradigm. The timing of intracellular tetanization is indicated by the arrows above each plot. Insets
show averaged responses to paired pulse stimuli before and after intracellular tetanization, from color-coded time
intervals. In this example, LTP and LTD were induced simultaneously at two inputs to the same neuron. Input
resistance of neurons measured by responses to small hyperpolarizing pulses applied before synaptic stimuli (not
shown) remained unchanged. C:Correlation between changes of EPSP amplitude after intracellular tetanization
and initial paired-pulse ratio. Data for N=136 inputs to pyramidal neurons in slices of visual cortex (N=60
inputs) and auditory cortex (N=76 inputs). Green symbols (star, square, and triangle) refer to the example inputs
fromB.
Modified, with permission, from Chen etal.,2013.
 Homeostatic Role of Heterosynaptic Plasticity
1994; Volgushev et al., 1994, 1997; Volgushev
et al., 1999; Volgushev et al., 2000; Chistiakova
and Volgushev, 2009; Lee etal., 2012; Chen etal.,
2013). Altogether, 50 to 200 action potentials are
generated during the intracellular tetanization.
This is well within the range of spiking in vivo,
for example, during optimal stimulation of cat
visual cortex neurons (e.g., Volgushev etal., 2002,
2003) or even during slow oscillations in some
neurons (e.g., Volgushev etal., 2006). Moreover,
it is also within the range of postsynaptic activity
during typical pairing protocols used for plastic-
ity induction. For example, the number of action
potentials varied between 50 and 600 in pairing
protocols used in seven representative publica-
tions (Markram etal., 1997; Hardingham etal.,
2007; Ismailov etal., 2004; Froemke etal., 2005;
Zhou et al., 2005; Nevian and Sakmann 2006;
Sjstrm and Hausser 2006; Sjstrm et al.,
2008; see Chistiakova and Volgushev 2009, Fig.3
and Fig. 4 and accompanying text for details).
This latter point raises an intriguing question of
whether heterosynaptic plasticity can be induced
by regular pairing protocols used, for exam-
ple, to induce STDP. Our recent analysis of the
published data (Feldman 2009; Sjstrm et al.,
2001; Watt etal., 2004; Birtoli and Ulrich 2004;
Nevian and Sakmann 2006; Letzkus etal., 2006;
Hardingham et al., 2007) indicates that this is
indeed the case. The variance of response ampli-
tude changes at unpaired inputs (i.e., synaptic
inputs that were not stimulated during the pairing
procedure and thus experienced only postsynap-
tic activity) is high, suggesting that although on
average the amplitude at unpaired inputs did not
change, both potentiation and depression must
have been observed in individual cases. However,
because potentiation and depression at unpaired
inputs were roughly balanced, averaged changes
over the whole population were not significant
(see Chistiakova et al., 2014, Fig. 8 and related
text for details).
Following intracellular tetanization in
pyramidal neurons from slices of rat visual
cortex, amplitudes of synaptic responses could
increase, decrease, or remain unchanged
(Figure 8.1B). In two independent inputs onto
one cell, intracellular tetanization could simul-
taneously induce LTP in one input and LTD in
the other (Figure 8.1B). The direction of plastic
changes was correlated with the inputs initial
paired-pulse ratio, which is inversely related to
the release probability (Figure 8.1C; Volgushev
etal., 1997; Volgushev etal., 2000; Lee etal., 2012;
Chen et al., 2013). Inputs that expressed high
133
initial paired-pulse ratio, and thus had low release
probability, were typically potentiated. Inputs
that expressed low initial paired-pulse ratio, and
thus had high release probability, were typically
depressed or did not change. Thus heterosynaptic
plasticity is weight dependent, and the direction
of weight change at each synapse is determined
individually, depending on its initial properties.
Weight dependence is one further similar fea-
ture of heterosynaptic and homosynaptic plas-
ticity: it has also been reported for tetanization
or pairing-induced LTP in the hippocampus and
neocortex (van Rossum et al., 2000; Sjstrm
etal., 2001; Hardingham etal.,2007).
It has been suggested that the weight
dependence of heterosynaptic plasticity reflects
history-dependent predispositions of synaptic
inputs to undergo potentiation or depression
(Volgushev et al., 1997; Volgushev et al., 2000;
Chistiakova and Volgushev, 2009, Chistiakova
et al., 2014). In this scenario, synapses that
become weaker after a depression (and have lower
release probability) have a lower predisposition
for further depression but a stronger predispo-
sition for potentiation. Synapses that became
stronger and have higher release probability
after a potentiation will have a higher predis-
position for depression. The idea of predisposi-
tions for plastic changes is closely related to the
notion of a sliding threshold between depression
and potentiation in the Bienenstock, Cooper,
and Munro (BCM) rule (Bienenstock etal., 1982;
Yeung etal., 2004), and to a more general con-
cept of metaplasticity, according to which the
abilities of synapses to undergo potentiation or
depression depend, among other factors, on the
recent history of their plastic changes (Abraham
and Bear,1996).
In sum, heterosynaptic plasticity induced by
intracellular tetanization expresses properties
that are well suited for serving as a robust mecha-
nism of normalization of synaptic weights:(a)it
depresses strong and potentiates weak synapses,
thus preventing runaway dynamics of synaptic
weights; (b) it is induced at nonactive synapses
by the same protocols that induce homosynaptic
plasticity, providing for explicit competition; and
(c)it operates on the same timescale as homosyn-
aptic plasticity.
Modeling:Heterosynaptic Plasticity
Robustly Prevents Runaway
Dynamics
The hypothesis that heterosynaptic plasticity with
experimentally observed properties can prevent
134 Part II: Homeostatic Control
runaway dynamics of synaptic weights and activ-
ity has been tested in computer simulations
(Chen et al., 2013). The model neuron received
synaptic inputs from 100 simulated presynaptic
neurons, each firing action potentials at an aver-
age frequency of 1 Hz, with Poisson distributed
interspike intervals (Figure 8.2A). Activity of
presynaptic neurons was mildly correlated, with
averaged cross-correlation between pairs of spike
trains 0.35 + 0.05. This input led to the firing
of the postsynaptic model neuron at ~1.8 Hz. In
the example illustrated in Figure 8.2B, a STDP
rule with symmetrical windows for potentiation
and depression was implemented (Figure 8.2B,
inset). In this model with an STDP-only learning
rule, synaptic weights showed runaway dynam-
ics. By the end of the 100 s long simulation, all
synaptic weights were saturated at the maximal
value (Figure 8.2B, red). This led to a dramatic
increase of the postsynaptic firing rate from ~1.8
Hz during the first 10 s of simulation to ~6.3 Hz
during the last 10 s of simulation. In the model
with STDP windows strongly biased toward
depression (Figure8.2C, inset), synaptic weights
also expressed clear runaway dynamics, but
toward the minimum value (Figure 8.2C, red).
In this case, the postsynaptic neuron became
silent despite continuing activity of presynaptic
neurons.
Implementing in the model (in addition to
STDP) heterosynaptic plasticity with experi-
mentally observed properties effectively pre-
vented runaway dynamics of synaptic weights
and activity. In the model with symmetrical
STDP and heterosynaptic plasticity, synaptic
weights slightly increased, but did not saturate,
and formed a normal distribution around the
new mean value, within the operation range of
synapses (Figure 8.2D, red). Firing rate of the
postsynaptic neuron slightly increased from
~1.8 Hz to ~2.6 Hz. Heterosynaptic plasticity
also prevented runaway dynamics of synaptic
weights and activity toward zero produced by
STDP windows with a negative bias (Figure8.2E,
red). These results demonstrate that heterosyn-
aptic plasticity can prevent runaway of synaptic
weights to either extreme.
Further simulations demonstrated that the
stabilizing effect of heterosynaptic plasticity on
synaptic weights is long-lasting and robust to
variations in the patterns of presynaptic activ-
ity, changes of calcium threshold for plasticity
induction, and changes of parameters of STDP
learning rules over a broad range of values. This
latter point is illustrated in Figure8.3. To explore
how changing STDP parameters affects stability
of operation of the model neuron, the depression
window of STDP was fixed, while the amplitude
and the time constant of the potentiation window
was systematically varied. The range of tested
STDP rules included those strongly dominated
by depression, as well as those dominated by
potentiation (see insets in Figure8.3).
The STDP-only model expressed nonsaturat-
ing behavior only with a very limited set of STDP
rules, specifically those dominated by depression
(Figure 8.3A, blue area of the matrix). As soon
as the potentiation window of the STDP rule
was ~75% of the depression window or stronger,
synaptic weights and postsynaptic firing invari-
ably expressed runaway dynamics (Figure8.3A,
orange/red area of the matrix). Addition of het-
erosynaptic plasticity to the model robustly pre-
vented runaway dynamics and led to the stable
distribution of synaptic weights within the oper-
ation range. The model equipped with heterosyn-
aptic plasticity expressed stable behavior with all
STDP windows testedfrom almost exclusively
depressing STDP rules to those strongly domi-
nated by potentiation (Figure 8.3B). Notably,
heterosynaptic plasticity, by preventing runaway
dynamics of synaptic weight, also kept averaged
firing rate around the operating point (Chen
etal.,2013).
Thus heterosynaptic plasticity makes a broad
range of STDP parameters compatible with sta-
ble operation of neurons and neuronal networks.
This is an important feature because experimen-
tal evidence indeed shows wide variations of
STDP windows for potentiation and depression
and of their relative strength in neurons and syn-
aptic connections of different types (Abbott and
Nelson, 2000; Nishiyama et al., 2000; Sjstrm
et al., 2001; Froemke et al., 2005; Zhou et al.,
2005; Haas etal., 2006; Caporale and Dan 2008;
Feldman2009).
Modeling:Heterosynaptic Plasticity
Permits Competition
Despite its strong stabilizing effect on synaptic
weights, heterosynaptic plasticity does not pre-
vent synaptic competition and segregation of syn-
aptic weights. This has been tested in simulations
in which inputs to the model neuron were segre-
gated in two groups. The two groups of synapses
differed either by the correlation of presynaptic
firing (high in one group, low in the other) or by
the average frequency of presynaptic firing. In
the STDP-only model, inputs that were strongly
correlated were rapidly potentiated and saturated
 Homeostatic Role of Heterosynaptic Plasticity 135

(a) (b) (c)

(d) (e)

FIGURE 8.2: Heterosynaptic plasticity prevents STDP-induced runaway dynamics in a model.

A: Amodel neuron receiving synaptic inputs from 100 simulated presynaptic neurons. Presynaptic neurons fired
action potentials at an average frequency of 1Hz; their activity was mildly correlated (averaged cross-correlation
0.35 +0.05). In addition to synaptic activation, current could be injected into the postsynaptic neuron to evoke
spikes. BE: Distributions of synaptic weights at the beginning (initial synaptic weights, blue) and at the end of
simulation (final synaptic weights, red) in four neuron models. In models with only the STDP rule implemented
at each synapse, activity leads to runaway dynamics of synaptic weights (B, C). Implementing heterosynaptic
plasticity in addition to STDP rule prevents runaway dynamics of synaptic weights (D,E). B: In the model with
symmetrical windows for potentiation and depression (inset), synaptic weights express runaway potentiation,
and by the end of simulation all weights are saturated at the maximal value (0.03 mS/cm 2; note truncation of the
Y axis for the final weights). C: In the model with strong negative bias of STDP (inset), synaptic weights express
runaway depression leading to saturation of the weights of ~40percent of synapses at zero and silencing of the
postsynaptic neuron. D, E: The same models as in B and C are shown, but with the mechanism for heterosynaptic
plasticity implemented in addition to STDP. Note that synaptic weights do not express runaway dynamics and are
not saturated but remain normally distributed within the operationrange.
(Modified, with permission, from Chen etal.,2013).

at the maximum value, while distribution of the
weights of weakly correlated inputs changed lit-
tle. In the model with both STDP and heterosyn-
aptic plasticity, no inputs were saturated, but the
groups of weakly and strongly correlated inputs
formed two clearly separate distributions, both
within the operation range of synaptic weights
(Chen et al., 2013). Similarly, segregation was
observed when the two groups of synaptic inputs
differed by their average firing frequency rather
than by the level of correlation. Note that com-
petition imposed by heterosynaptic plasticity is
able to support subtle differentiation of synaptic
weight changes. For example, if an initially large
number of synapses are potentiated but later only
a portion of them remains consistently active,
heterosynaptic plasticity will be able to suppress
the remaining synapses, thus allowing for selec-
tion of only the relevant groupa process that
may mediate the differentiation stage of learning.
Thus heterosynaptic plasticity does not pre-
vent segregation of groups of synaptic inputs
exhibiting activity patterns that differ by the
degree of correlation or by firing frequency.
Moreover, it helps to preserve the ability of a
neuron with plastic synapses for further learn-
ing: unsaturated synapses have higher potential
for further changes than those potentiated to the
maximum or depressed to zero by STDP-only
learningrules.
In sum, heterosynaptic plasticity with experi-
mentally observed properties is a strong can-
didate mechanism for counteracting runaway
dynamics imposed on synaptic weights and
activity by positive feedback of Hebbian-type
learning rules. Prevention of runaway dynam-
ics by heterosynaptic plasticity is robust over
a broad range of activity patterns and details
of Hebbian-plasticity rules, such as balance of
STDP windows for potentiation and depression.
136 Part II: Homeostatic Control

(a) (b) Nonnormal

STDP only STDP + Heterosynaptic plasticity
Time constant + (ms) distribution
40 40 log(K2)
30 30 3
20 20 2
1
10 10
0
5 5
Normal
0.0002 0.001 0.0018 0.0025 0.0002 0.001 0.0018 0.0025
distribution
Potentiation window a+ amplitude, mS/cm2 Potentiation window a+ amplitude, mS/cm2

FIGURE 8.3: Heterosynaptic plasticity makes a broad range of STDP parameters compatible
with stable operation of neurons. A, B:Each box in the grids shows the DAgostino-Pearsons K 2 test for
normality of synaptic weight distribution after 100 seconds of simulations with different STDP potentiation win-
dows, with a+ and + as indicated on the X and Y axes. Insets show STDP windows for example boxes. The white
square indicates the symmetrical STDP learning rule. Synaptic weight distributions with high K 2 test values (>50),
indicating deviation from normality, typically contain most of the weights saturated at maximal or minimal val-
ues and thus expressed runaway dynamics. Note that in simulations with the STDP-only model (A), only a few
STDP rules, with strong bias toward depression, did not lead to runaway dynamics. Most STDP rules, including
examples shown in the bottom, led to runaway dynamics of synaptic weights. In contrast, the model with STDP
and heterosynaptic plasticity (B) did not express runaway dynamics over the whole range of tested STDP rules,
including those extremely biased toward potentiation or depression.
Modified, with permission, from Chen etal.,2013.

Heterosynaptic plasticity has a normalizing effect magnitude of potentiation is smaller at strong

on synaptic weight changes and can support syn-
aptic competition. Moreover, it shares the trigger,
has partially overlapping mechanisms, and oper-
ates on the same timescale as Hebbian-type plas-
ticity. Importantly, it operates at the same level
(synapses and regulation of their weights) as the
Hebbian-type plasticity. These features make het-
erosynaptic plasticity an ideal candidate mecha-
nism for homeostatic control of synaptic weight
changes.
B I O L O G I C A L C A N D I D AT E S :
OTHER MECHANISMS
C O U N T E R A C T I N G R U N AWAY
Extensive studies of Hebbian-type synap-
tic plasticity have suggested several further
mechanisms that may counteract or reduce the
tendency for runaway of synaptic weights and
activity. One is saturation of plasticity, which
is the reduction of the magnitude of potentia-
tion during a series of potentiation-inducing
episodes, with eventual complete loss of the
ability for further potentiation (Colino et al.,
1992; Huang et al., 1992). Another one is
weight dependence of plasticity, whereby the
synapses, which probably were already poten-
tiated, than at weak synapses, which did not
experience prior potentiation or were previ-
ously depressed (van Rossum et al., 2000;
Sjstrm etal., 2001; Hardingham etal., 2007).
One further mechanism is a sliding calcium
threshold for potentiation and depression,
whereby, depending on the history of recent
activity and synaptic changes, the thresholds
for potentiation and depression or intracellu-
lar calcium homeostasis change (Bienenstock
et al., 1982; Yeung et al., 2004). These notions
and mechanisms contribute to the concept of
metaplasticityhistory-dependent changes of
the abilities of synapses to undergo potentiation
or depression (Abraham and Bear, 1996). These
mechanisms are inherent to Hebbian-type plas-
ticity rules and thus are ideally suited to shape
the ability of synapses to change. By impos-
ing negative feedback on homosynaptic plas-
tic changes, they clearly can limit the runaway
tendency and thus decrease the instability of
a system with plastic synapses. A drawback of
these mechanisms, as of any mechanisms gov-
erning homosynaptic plasticity, is that they
 Homeostatic Role of Heterosynaptic Plasticity 137

(a) (b)
?
?

+
?
50 ms
?
?
Homosynaptic Homosynaptic Heterosynaptic
LTP LTD plasticity

(c) (d)
Homosynaptic LTP/LTD Heterosynaptic plasticity
Hom
Potentiation

Potentiation

Potentiation
He osy
ter nap
osy tic
n LTP
LT apt
Weight change

Weight change

Weight change
P ic

0 0 0 He
ter
os
LT ynap
Depression

Depression

Depression
Hom
osy D tic
nap
tic
LTD

Initial synaptic weight Initial synaptic weight Initial synaptic weight

FIGURE8.4: Scheme of induction of homosynaptic and heterosynaptic plasticity and the result-
ing changes of synaptic weights. A: In a typical plasticity experiment, a set of afferents to a neuron (red
inputs) is paired with bursts of postsynaptic spikes (red current step applied through the recording electrode).
All other inputs to this neuron (green ? marks) are not stimulated and thus represent heterosynaptic sites.
B: Scheme of STDP protocols for induction of homosynaptic potentiation (red, presynaptic stimulation before
postsynaptic firing), for homosynaptic depression (blue, presynaptic stimulation after postsynaptic spikes) and
purely postsynaptic challenge (green, postsynaptic spikes without presynaptic stimulation, as experienced dur-
ing plasticity induction by heterosynaptic sites, which are marked by the green ? symbolsinA). C: Scheme of
weight-dependent plastic changes in response to induction protocols from B.Left:expected outcome of homosyn-
aptic, associative Hebbian plasticity after induction of potentiation (red area) or depression (blue area). Changes at
few synapses, which were activated during the induction, are shown as circle symbols. Note the weight dependence
of both homosynaptic LTP and LTD, whereby very weak synapses exhibit less depression and very strong synapses
exhibit less potentiation. Right:expected outcome of heterosynaptic plasticity. Many more synapses may express
heterosynaptic changes (circle symbols), because the majority of synapses onto a neuron were not activated dur-
ing the induction but experienced strong postsynaptic spiking. Note that both the magnitude and the direction
of heterosynaptic changes depends on initial synaptic weight, so that initially strong synapses tend to depress,
while initially weak synapses tend to potentiate. D: Driving forces on weight changes imposed by homosynaptic
and heterosynaptic plasticity. Homosynaptic plasticity, LTP or LTD, is induced only at synapses active during the
induction and requires specific-activity patterns. Homosynaptic LTP leads to weight-dependent increase of syn-
aptic weights:potentiation is stronger at initially weak than at initially strong synapses. Homosynaptic LTD leads
to weight-dependent decrease of synaptic weights:depression of strong synapses is stronger than depression of the
weak synapses. Heterosynaptic plasticity can be induced at any synapse during episodes of strong postsynaptic
activity. Initially weak synapses are subject to weight-dependent heterosynaptic LTP. Initially strong synapses are
subject to weight-dependent heterosynapticLTD.

require presynaptic activation and cannot affect all respective synapses (Bliss and Lomo, 1973;
affect inactive synapses. This requirement lim- Daoudal et al., 2002; Zhang and Linden, 2003;
its the ability of homosynaptic mechanisms to Frick et al., 2004; Fink and ODell, 2009; Sehgal
serve as regulators of global, cell-wide synaptic et al., 2013). Excitability changes may counteract
homeostasis. synaptic changes, thus having a homeostatic effect
A family of nonsynaptic mechanisms regulat- (Zhang and Linden, 2003), or enhance and amplify
ing intrinsic excitability does not have this limita- synaptic changes, thus having an antihomeostatic
tion. These mechanisms can change excitability effect (Frick etal., 2004; Fink and ODell, 2009; see
of a dendritic branch or a whole neuron and thus Sehgal etal., 2013 for a recent review).
138 Part II: Homeostatic Control
CONCLUSIONS
The interacting mechanisms of homosynaptic
and heterosynaptic plasticity impose different
forces on synaptic weights, which drive synaptic
weights in opposite directions. Homosynaptic
plasticity induced by selective and specific activ-
ity patterns drives synaptic weights to the limits
of their dynamic range. Heterosynaptic plasticity
simultaneously introduces a normalizing driving
force that pulls synaptic weight values away from
the extremes. As a consequence, every spike, or
burst of spikes, becomes a homeostatic signal to
the cell. Because homosynaptic and heterosyn-
aptic changes are induced by the same activity
patterns and take place on the same timescale,
the weight of a synapse is determined by the bal-
ance of homosynaptic LTP, homosynaptic LTD,
and the normalizing force of heterosynaptic
plasticity (Figure 8.4B). At strongly activated
homosynaptic sites, the associative driving force
may be dominant, leading to net potentiation or
depression of these subpopulations of synapses
(Figure8.4C, left). Meanwhile the vast number of
synapses that are at that moment inactive will be
subject to the stabilizing effect of heterosynaptic
changes (Figure 8.4C, right). This allows neu-
rons to update relative strength of inputs while
keeping synaptic weights within operation range
and preserving abilities of synapses for further
modifications. Importantly, heterosynaptic
plasticity allows robust homeostasis of synaptic
weights and activity over a wide range of STDP
rules, Ca 2+ thresholds, and presynaptic activity
levels. Moreover, differential driving forces for
the weight changes at active and inactive syn-
apses introduce competition between synapses
(Figure 8.4D). This competitive force imposed
by heterosynaptic plasticity emerges simultane-
ously with Hebbian plasticity, fighting not just
over shared mechanisms or resources but push-
ing synapses with opposing driving forces, the
result of which is a balance between the forma-
tion of new patterns of synaptic weights and
biological stability. Therefore heterosynaptic
plasticity expresses all of the desired features of
an intrinsic homeostatic mechanism for stabiliz-
ing synaptic weight dynamics after learning.
ACK NOWLEDGMENTS
We are grateful for the support from the National
Institutes of Health (Grant R01MH087631)
and the Alexander von Humboldt-Foundation
(Humboldt Research Award toMV).
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9
The Blood-Brain Barrier
Y O N ATA N S E R L I N , A L O N F R I E D M A N , A N D U W E H E I N E M A N N
THE BLOOD-BR AIN BARRIER
I N T H E H E A LT H Y B R A I N
Anatomy
To maintain normal brain function, the neural
extracellular environment must be maintained
within a narrow homeostatic range; this requires
a tight regulation of transportation of cells, mol-
ecules, and ions between the blood and the brain.
Such tight regulation is maintained by a unique
anatomical and physiological barrier formed col-
lectively in the central nervous system (CNS).
Awareness of existence of a physical interface
between the CNS and the peripheral circulation
and the vascular capacity was first described
by Paul Ehrlich (1885). Ehrlich described how
dye injection into the blood circulation stained
peripheral organs but not the spinal cord and the
brain. Later, Ehrlichs student Edwin Goldmann
showed that direct injection of trypan blue into
the cerebrospinal fluid (CSF)-stained cells within
the CNS and not in the periphery (Goldmann,
1913). An additional limiting element was later
demonstrated by Reese and Karnovsky (1967)
who presented a solute exchange barrier between
the blood and the brain by means of endothelial
tight junction complexes.
Three barrier layers contribute to the separa-
tion of the blood and neural tissues:(a)a highly
specialized endothelial cells (EC) layer compris-
ing the blood-brain barrier (BBB) and partition-
ing the blood and brain interstitial fluid; (b)the
blood-CSF barrier with the choroid plexus epi-
thelium, which secretes CSF into the cerebral
ventricles and separates the blood from the
ventricular CSF; and (c) the arachnoid epithe-
lium between the blood and subarachnoid CSF
(Abbott etal.,2006).
The BBB components include the EC layer
and its basement membrane, adjoined by tight
cell-to-cell junction proteins with specific trans-
port mechanisms and pinocytic vesicles. The
endothelium is surrounded by cellular elements
such as pericytes and astroglial foot processes,
forming an additional continuous stratum that
separates blood vessels from brain tissue. Around
penetrating vessels and venules there is often some
distance between EC and brain tissue, forming
the Virchow-Robin space in which perivascular
macrophages are found, which execute some of
the immune functions of the CNS. The intimate
contact between neurons, astrocytes, microglia,
pericytes, and blood vessels, and the functional
interactions and signaling between these cells,
form a dynamic functional unit known as the
neurovascular unit. Understanding the function
of the neurovascular unit is key to our under-
standing of brain functions in health and disease,
including neuronal firing, synaptic plasticity,
regulation of blood flow, and response to injury
(thoroughly reviewed by Abbott,2014).
The neurovascular unit is illustrated in
Figure 9.1. The innermost luminal constituent
is comprised of a single specialized EC layer lin-
ing brain capillaries, exhibiting a greater num-
ber and volume of mitochondria, augmenting
the selective molecular permeability of the BBB
(Oldendorf et al., 1977). The basement mem-
brane, a 30- to 40-nm thick lamina composed
of collagen type IV, heparin sulfate proteogly-
cans, laminin, fibronectin, and other extracel-
lular matrix proteins, encompasses pericytes
and EC and is closely adjacent to the plasma
membranes of astrocyte endfeet, enclosing the
cerebral capillaries (Hawkins and Davis, 2005).
Transmembrane proteins (junctional adhesion
molecule-1, occludin, and claudins1, 3, 5, and
possibly 12)and cytoplasmic accessory proteins
(zonula occludens-1 and -2, cingulin, AF-6, and
7H6) establish the tight junctions between adja-
cent EC. Molecular and structural studies of
tight junctions reveal a complex, dynamic, and
highly regulated molecular structure. Junctional
adhesion molecules maintain tight junction
144 Part II:Homeostatic Control
FIGURE9.1: The neurovascular unit. A schematic
drawing of the neurovascular unit showing the close
spatial relationships and interactions between endothe-
lial cells, pericytes, astrocytes, microglia, and neurons.
Adopted from Abbott and Friedman,2012.
properties, claudins facilitate tight barrier capa-
bilities, and occludins and zonula occludens-1
regulate targeted signaling (Abbott et al., 2010;
Bazzoni, 2006; Chen and Liu,2012).
Pericytes are enveloping brain microvessels
and capillaries and are found in close proxim-
ity to astrocytes and neurons. The ratio of peri-
cytes to EC is assessed to be 1:3 (Shepro and
Morel, 1993). Using multiple signaling path-
ways, pericytes play a critical role in the forma-
tion and maturation of the BBB during tissue
development and regulation of tissue survival
(Daneman et al., 2010). Pericytes control over
cerebral blood flow is potentially due to a regu-
latory effect on the capillary diameter through
actin fibers in the pericytic cell body (Hamilton
etal., 2010). Pericyte dysfunction through aging
was noted in animal models (Bell et al., 2010),
and their absence results in loss of BBB integrity
and reduction in regional cerebral blood flow
(Armulik etal.,2010).
Astrocytes interact with pericytes and micro-
vascular EC by endfeet protrusions ensheathing
the capillaries. Interactions may also exist with
smooth muscle cells at arterioles. Astrocytes play
important roles in maintenance of the BBB and
in homeostasis of extracellular concentration of
transmitters, metabolites, ions, and water but
also serve as stem cells early during development
and provide for templates for migratory neuronal
streams. Interaction between astrocytes and
neurons determine synaptic transmission, clear-
ance of neurotransmitters, plasticity, and control
blood flow (reviewed by Wong etal.,2013).
Development
A key stage in the development of the BBB lies
in the early communications of the evolving
endothelium with neural cells (Stewart and
Wiley, 1981). The BBB matures during fetal life
and is well formed by birth (Keep et al., 1995;
Moos and Mllgrd, 1993; Olsson et al., 1968;
Saunders, 1992; Saunders et al., 2000; Tauc
et al., 1984). Transport mechanisms may con-
tinue to develop in mammals born in a relatively
immature state (such as the rat and mouse) and
become fully functional only in the peri- or post-
natal period (Jones et al., 1992). The develop-
ment of the vascular endothelium is now known
to be provoked by neuroepithelial signaling
through the Wnt/-catenin pathway to induce a
CNS-specific vascular system and BBB speciali-
zation (Daneman etal., 2009; Liebner and Plate,
2010; Stenman etal.,2008).
An early feature of BBB development is the
formation of tight junctions. In humans, the
brain of a 14-week fetus expresses occludin and
claudin-5 in the capillary endothelium with the
same distribution at cell margins as seen in an
adult (Virgintino etal., 2004). Human postmor-
tem studies of perinatal deaths and stillborn
fetuses from approximately 12 weeks gestation
have demonstrated that a barrier to trypan blue
exist from at least the beginning of the second
trimester, equivalently to that of the adult human
(Grontoft, 1954). Culture studies suggest that
astrocytes have a key role in regulating the tight-
ness of the BBB (Hartmann etal.,2007).
Physiology
Each of the three main CNS interface layersthe
BBB, the choroid plexus epithelium, and the epi-
thelium of the arachnoid materfunctions as a
physical, transport, metabolic, and immunologic
barrier. The barrier functions are dynamic and
respond to a wide range of regulatory signals
from the blood and the brain. Tight junctions
between adjacent cells restrict diffusion of polar
solutes through the intercellular cleft (paracel-
lular pathway). The barriers are permeable to O2
and CO2 and other gaseous molecules such as

helium, xenon, N2, and many gaseous anesthet-
ics. The permeability to xenon may provide a
high-resolution magnetic resonance imaging tool
by which small morphological alteration may be
detectable within the living tissue and also per-
mit the analysis of binding sites using molecu-
lar probing techniques. Lipid soluble substances
can pass the BBB by diffusion. Principally, the
BBB is also permeable to water; however, sol-
ute carriers on the apical and basal membranes
together with ectoenzymes and endoenzymes
regulate small solute entry and efflux. Transfer
of drugs and other agents is regulated by multi-
drug transporters that can limit their concentra-
tions within the CNS. Multidrug transporters are
ubiquitous transport proteins that exploit adeno-
sine triphosphate hydrolysis to funnel molecules
across lipid membranes; they facilitate transport
of molecules into cells but may also prevent accu-
mulation of molecules within the brain inter-
stitial space. Multidrug resistance proteins and
P-glycoprotein-like proteins are expressed at the
BBB and limit access of drugs to the brain but
also of other lipophilic molecules, including the
neurotoxic bilirubin, the degradation product of
hemoglobin, whichif entering the CNScan
cause major damage to the CNS (Abbott and
Friedman, 2012). Recent studies indicate that
transporter molecules are upregulated in some
pathological conditions and thereby reduce drug
levels within the brain and thus may explain
treatment failures in some neurological and psy-
chiatric disorders (Potschka,2010).
Large molecules (e.g., peptides and pro-
teins) with particular growth and signal-
ing roles within the CNS enter the brain in a
restricted and regulated manner by adsorptive
and receptor-mediated transcytosis. Smaller
peptides may cross the BBB by either nonspecific
fluid-phase endocytosis or receptor-mediated
transcytosis mechanisms. Similarly, 98% of
all small molecules are not freely transported
across the BBB (Pardridge, 2005). The barriers
also regulate the recruitment and entry of leu-
kocytes and innate immune elements and are
involved in both the reactive and the surveil-
lance functions of CNS immunity. Leukocyte
migration involves a complex set of adhesion
molecules at the surface of leukocytes and vas-
cular EC. Tethering and rolling of leukocytes is
achieved via integrins VLA-4 (41) and 47 cellular barrier capacity of tight junctions, the
(Laschinger and Engelhardt, 2000), and adhe-
sion molecules such as ICAM-1, VCAM-1, and
PECAM-1 contribute to the adhesion and/or
migration of distinct subsets of leukocytes to the
The Blood-Brain Barrier 145
CNS through cytokine-activated brain endothe-
lium (Greenwood etal., 2003). Transport systems
across the BBB are illustrated in Figure9.2.
TR ANSPORT OFGLUCOSE
AND AMINOACIDS
Carrier-mediated influx, which may be passive
or secondarily active, provide transport into
the CNS of essential polar molecules that can-
not diffuse through the cell membrane, such as
glucose, amino acids, and nucleosides. EC of the
brain microvasculature, astrocytes, and the cho-
roid plexus express the insulin-independent glu-
cose transporter GLUT1, a membrane-spanning
glycoprotein containing 12 transmembrane
domains with a single N-glycosylation site
(Carruthers etal., 2009). GLUT1 plays a vital role
in brain glucose uptake and is highly expressed
in cells forming the blood-tissue barriers and in
astrocytes (Mann et al., 2003). Glucose concen-
trations play a role in the regulation of GLUT1
protein concentrations: hypoglycemia induces
upregulation of GLUT1, while hyperglycemia
does not seem to exhibit an effect (Devaskar etal.,
1991; Simpson et al., 1999). Active mechanisms
for controlling sugar transport across the BBB
and in astrocytes might be influenced by acute
regulation of cell surface GLUT1 levels (Simpson
et al., 2001) and are potentially related to the
energetic condition of brain tissue. Importantly,
GLUT1 is not the sole glucose transporter at the
BBB; the GLUT4 transporter for glucose seems
to be expressed as well. GLUT3 is expressed in
neurons and is likely providing glucose uptake
into neurons, thus bypassing the glucose lactate
shuffle through astrocytes by which lactate is
provided as an energy-rich substrate.
Another important aspect is brain protection
against neuroactive substances such as aspartate
and glutamate. The BBB is largely impermeable
to these amino acids. Glutamate metabolism by
the liver provides for prompt transformation into
glutamine, and, consequently, consumption of
food containing high levels of glutamate such as
tomatoes or food additives such as soy sauce does
not affect brain function. Aspartate consumed
by food is rapidly secreted through the kidney
(Beyreuther etal.,2007).
The limited transport of circulating mono-
amines through EC is attributed to the para-
diffusional characteristics imposed by the lipid
bilayer, and specific transport proteins of the cell
membrane. Intracellular levels of amino acids
in the brain are correlated to the rates of influx
146 Part II:Homeostatic Control

Cell migration Passive Carrier- Carrier- Receptor- Adsorptive- Tight-

diffusion mediated mediated mediated mediated junction
efflux influx transocytosis transocytosis modulation

Lipid soluble drugs

Lipid soluble Amines Cytokines Cationised Polar solutes

Non-polar solutes Amino acids Insulin albumin
Glucose Transferrin Histones
Neucleosides
Small peptides

FIGURE 9.2: Potential routes for infiltration and transport across the endothelial cells forming
the blood-brain barrier (BBB). Cells may cross the BBB through or adjacent to the tight junctions. Solutes may
passively diffuse through the cell membrane. Active efflux carriers may pump some of these passively pen-
etrating solutes out of the endothelial cell. Carrier-mediated influx (passive or secondarily active) can trans-
port essential polar molecules, such as amino acids, glucose, and nucleosides, into the central nervous system.
Receptor-mediated transcytosis can transport macromolecules such as peptides and proteins across the endothe-
lium. Adsorptive-mediated transcytosis is induced nonspecifically by positively charged macromolecules and can
result in passage across the BBB. Tight junction modulation may occur, affecting the permeability of the paracel-
lular aqueous diffusional pathway.
Modified from Begley and Brightman,2003.

across the BBB, and synthesis of neurotrans-
mitters such as serotonin, dopamine, and hista-
mine are substrate limited (Smith and Takasato,
1986). For a sustainable supply of essential polar
nutrients such as glucose and amino acids to the
brain, there is a crucial role to specific solute car-
riers (SLC) across the BBB endothelium, and all
cells express a large number of SLCs in the cell
membrane (Zhang et al., 2002). A wide variety
of SLCs mediate the movement of nutrients and
solutes in and out of the brain and are found on
either the luminal or the abluminal membrane
only or inserted into both membranes of the EC.
Adetailed list of BBB SLCs are listed elsewhere
(see Table2 in Abbott etal.,2010).
TR ANSPORT OFIONS
Preservation of an optimal environment for syn-
aptic and neural function is achieved by specific
ion channels and transporters. Water molecules
can also cross the BBB through ion channels.
Due to regulated ionic movement, potassium
concentration in the CSF and brain interstitial
fluid are maintained at 2.5 to 2.9 mM despite
the higher concentration of potassium in the
plasma (approximately 4.5 mM). In fact, potas-
sium concentration can vary strongly during
body exercise, nutrition, or pathological con-
ditions (Bradbury et al., 1963; Hansen, 1985;
Medb and Sejersted, 1990)and may increase to
levels as high as 10 mM in venous blood. If such
an increase in potassium concentrations would
occur in the brain, a significant change in neu-
ronal activity, often epileptic discharges, would
be triggered. The BBB thereby protects the nerve
cells from such variations. The BBB is similarly
largely impermeable to most ions such as calcium
(Ca2+) and magnesium (Mg2+).
Within the CNS, increases in [K+]o per action
potential have been estimated to be around 0.01
to 0.02 mM (Heinemann and Lux, 1977; Lux
etal., 1986). During intense sensory stimulation
(e.g., painful stimuli), [K+]o rises in the dorsal
horn by more than 2.5 mM (Heinemann et al.,
1990; Lothman and Somjen, 1975; Sykov etal.,
1980). A baseline elevation in [K+]o by about 1
mM has also been observed during slow-wave
sleep (Amzica etal., 2002)accompanied by a fall
in [Ca2+]o by 0.2 mM (Massimini and Amzica,
2001). As information is transmitted to the

thalamus and cortex, increases in [K+]o during
sensory stimulation become smaller, probably
reflecting sparsification of action potential firing.
In the cortex, during sensory stimulation, typi-
cally [K+]o rises to about 0.4 mM (Lux and Singer,
1973). Changes in [Ca2+]o are indeed small during
sensory stimulation (in the cortex less than 0.2
mM), while during seizures [Ca 2+]o can fall from
the baseline concentration of 1.2 mM to less than
0.1 mM (Pumain et al., 1985). During seizures,
potassium concentration can increase to 12 mM
in limbic structures and to 10 mM in the cortex
(Lux et al., 1986). Unless the BBB is damaged,
this increase in [K+]o will not affect serum potas-
sium concentration. The rise in serum [K+] dur-
ing convulsions is in fact mostly due to striated
muscle activity. Based on observation during
epileptic activity, it was suggested that neurovas-
cular coupling depends on changes in potassium
and calcium concentration (Kuschinsky et al.,
1972). Indeed, elevations in potassium concentra-
tion can hyperpolarize smooth muscle cells and
thereby cause vessel dilation (Nelson and Quayle,
1995), while reductions in Ca 2+ concentration
would reduce Ca2+ entry during electrogenesis
in these cells and therefore weaken muscle con-
traction. Thus neurovascular coupling during
seizures and other pathological activities differs
from that observed during physiological acti-
vation, where other signals are used to regulate
blood flow (Attwell etal., 2010). Accordingly, the
small changes in [K+]o and [Ca2+]o may have sign-
aling functions onto other nerve cells and glia
but probably are not a major factor in physiologi-
cal neurovascular coupling. However, alterations
in the electrolyte composition may contribute to
the steady potentials observed, for example, dur-
ing slow-wavesleep.
pH also is actively regulated at the BBB
and the blood-CSF barrier (Jeong et al., 2006;
Michalke and Nischwitz, 2010). The neurovas-
cular unit and the BBB are also important in
the spatial buffering of electrolytes on neuronal
activation. Astrocytes and their position between
capillaries and neurons are connected with gap
junctions, allowing them to communicate with
each other and with capillary EC in contact with
astrocytic processes. Neuronal firing and synap-
tic transmission are associated with the influx of
Na+ and Ca++ and the extracellular increase in the
concentrations of K+ and neurotransmitters. In
addition, glucose metabolism during neuronal
activity generates water at the rate of ~28 nl/g
min-1 (Rapoport, 1976). While neurotransmitters
are recycled directly or via astrocytes, potassium
The Blood-Brain Barrier 147
is distributed spatially via astrocytes and water
is excreted from the brain. Astrocytes have a key
role in the homeostatic mechanisms maintaining
brain extracellular environment within a nar-
row limit, despite continuous neuronal activity,
and the perivascular endfeet at the BBB have a
particular role in these processes (Simard and
Nedergaard, 2004). For example, extracellular
K+ ions accumulating during neuronal activ-
ity are expected to enter astrocytes according to
the electrochemical gradient and distributed to
neighboring astrocytes (via gap junctions) and
astrocytic endfeet. The high density of inwardly
rectifying Kir4.1 on perivascular astrocytic end-
feet makes them well suited for spatial buffering,
depositing the K+ in the perivascular space. The
high density of AQP4 water channels in perivas-
cular astrocytic endfeet facilitates a similar
redistribution of water. Excess metabolic water
may join the interstitial fluid in perivascular
spaces and cleared through the CSF. Similarly,
the uptake of glutamate to astrocytes via specific
transporters (mainly EAAT1 and EAAT2) is Na+
dependent and accompanied by net uptake of
ions and water, which will similarly clear at the
perivascular space and theBBB.
MACROMOLECULES:
PEPTIDES AND PROTEINS
Endocytic vesicles account for the main deliv-
ery of large molecular weight substances such as
proteins and peptides through the BBB. Protein
synthesis in the brain is dependent on the sup-
ply of essential amino acids; most are neutral
and large and thus incapable of passive diffu-
sion to the brain. The typical concentrations of
plasma proteins are higher than the CSF protein
content, apparently due to the ability of the BBB
to preclude the penetration of such macromol-
ecules into the brain. Vesicular mechanisms
involve either receptor-mediated transcytosis or
adsorptive-mediated transcytosis enabling the
transport of diverse large molecules and com-
plexes. Asummary of a number of known tran-
scytotic mechanisms is presented elsewhere (see
Table 4 in Abbott et al., 2010). Internalization
into the endothelial cell cytoplasm and exocyto-
sis to the opposite pole of the cell occurs follow-
ing interaction between ligands and cell-surface
receptors, which leads to caveolae and vesicle
formation.
A possible mechanism for a peptide-specific
transporter protein may facilitate its entrance
through the membrane (Dogrukol-Ak et al.,
2009; Kastin and Pan, 2003). Agrowing body of
148 Part II:Homeostatic Control
evidence indicates that large molecular weight
serum proteins infiltration though a dysfunc-
tional BBB carries a potential risk for patho-
logical outcomes within the CNS. Thrombin,
plasmin, and albumin were reported to induce
local effects such as cellular activation, apopto-
sis, and epileptogenesis. The presence of some
proteins in brain interstitial fluid can initiate
signaling cascades resulting in seizures, acti-
vation of glia, synaptic plasticity, and synap-
togenesis and cell damage (David et al., 2009;
Lapilover etal., 2012; Maggio etal., 2008). The
wide presence and expression of factor Xa (con-
verting prothrombin to thrombin), tissue plas-
minogen activator (converting plasminogen
to plasmin), and the thrombin receptor PAR1
likely play a role in these pathological path-
ways (Gingrich and Traynelis, 2000; Gingrich
etal., 2000). Albumin extravasations from the
plasma into the brain milieu has been shown to
be associated with astrocytic activation and the
development of network modifications leading
to epilepsy (David etal., 2009; Ivens etal., 2007;
Nadal etal., 1995; also see later discussion).
DRUG DELIVERY
As mentioned, the fact that penetration of large
molecules from the blood into the brain is pre-
vented by the BBB evokes essential research and
translational efforts aimed at development of
novel treatments for many CNS pathologies and
new radiopharmaceuticals for radio-labeled
brain imaging techniques. A recent, thor-
ough review (Pardridge, 2012) provides an
updated outline of classical modes of drug
delivery to the brain, including transcranial
delivery of drugs or small molecules, endog-
enous carrier-mediated and receptor-mediated
transport systems within the BBB and current
approaches for reengineering of drugs to enable
BBB transport.
N E U R O N A L A N D VA S C U L A R
FUNCTIONS
It is well known that despite the fact that the
human brain constitutes only ~2% of total body
mass, over 20% of total body oxygen and energy
consumption is dedicated to proper brain func-
tion (Shulman etal., 2003). The term neurovas-
cular coupling designates an integrated system
of neuronal and vascular cells and their milieu
working in concert to maintain brain homeosta-
sis by providing the energy demands of neuronal
activity via a tight, activity-dependent regulation
of local blood flow. This complex process also
involves pericytes, microglia, and specialized cel-
lular compartments such as endothelial glycoca-
lyx (Stanimirovic and Friedman, 2012). While the
role of the intact BBB in controlling the normal
neurovascular coupling is not completely under-
stood, recent studies in injured patients hint that
under conditions in which the BBB is severely
impaired, vessels show impaired response to
neuronal activation (Winkler etal.,2012).
The BBB plays a crucial role in the main-
tenance of a strict extracellular environment
around synapses and axons. Neuron interfaces
within the CNS depend on chemical and elec-
trical signals, and thus a steady neural function
is dependent on the barrier capacity. Following
BBB dysfunction the extracellular microenviron-
ment is disturbed, thus resulting in abnormal
neuronal activity and under some conditions lead
to seizures (Marchi etal., 2007). Sensory-motor
neurological dysfunction developing after patho-
logical vascular response and BBB opening may
be attributed to reduced metabolic efficacy, cel-
lular damage, and interference of homeostatic
mechanisms such as active transporters and elec-
trolyte buffering, required for neuronal activity
(Friedman, 2011). As mentioned, the importance
of intact BBB in maintaining the orchestrated
relationship between brain activity and changes
in blood flow is considered a key determinant
in the pathophysiology of brain injuries. While
neurovascular coupling may reflect a physiologi-
cal homeostatic response to increased metabolic
demand, recent animal and human data suggest
that reduced energy supply and worsening of the
tissue metabolic state will promote cellular dam-
age and slow energy-demanding homeostatic
mechanisms as active transporters required
for neuronal repolarization. Under pathologi-
cal conditions, the physiological neurovascular
coupling may fail, and neuronal depolarization
during seizures or spreading depolarization may
be associated with no or inverse coupling (i.e.,
vasoconstriction; Dreier,2011).
BBB INDISEASE
There is no dispute that increase in the perme-
ability of the BBB is associated with brain influx
of circulating proteins, water, and cells, which
further lead to neurological manifestations.
Growing attention is currently being paid to
the potential role of more subtle and transient
changes in BBB integrity as triggers for abnormal
brain signalling, leading to a neurological disor-
der. Indeed, in recent years, remarkable human

and animal data have accumulated, revealing
that BBB failure is a major determinant of neuro-
logical disorders and in some cases may actually
be the cause of disease.
BBB Dysfunction Is Common
in Neurological Diseases
BBB dysfunction has been reported in patients
diagnosed with the most common neurologi-
cal disorders. Leaky capillaries in primary brain
tumors has become a major imaging hallmark,
seen as contrast enhancement in brain comput-
erized tomography or magnetic resonance imag-
ing. Similarly, ring enhancement representing
BBB breakdown around traumatic, ischemic, and
infectious brain injuries are common. A more
subtle increase in BBB permeability has been
reported in patients with subarachnoid hemor-
rhage and Alzheimers disease (AD). In animal
models of AD, the accumulation of amyloid is
often first seen in close proximity to blood ves-
sels, with increased vessel permeability before
significant neuronal loss. The data from ani-
mal models of AD showing subclinical seizures
(Noebels, 2011)is consistent with the hypothesis
that a leaky BBB increases the network tendency
to seize. Recent meta-analysis based on imaging
and biochemical CSF studies for assessment of
BBB leak in patients suffering from AD showed
a greater increase in BBB permeability and asso-
ciation with significant reductions in the peri-
cyte populations in the cortex and hippocampus
among AD patients compared with neurologi-
cally healthy human controls. These results were
also confirmed by postmortem brain tissue
studies (reviewed by Zlokovic, 2013). A human
apolipoprotein E isoform is particularly shown
to mediate neuronal injury in AD by leading to
damage of the cerebrovascular integrity and BBB
breakdown via activation of a CypAnuclear
factor-Bmatrix-metalloproteinase-9 pathway
in pericytes (Bell etal.,2012).
Some chronic neuropathologies such as mul-
tiple sclerosis may involve an early phase of BBB
dysfunction (involving the downregulation of
claudin 1, 3 (Wolburg and Lippoldt, 2002) that
precedes neuronal damage, suggesting that vas-
cular damage can lead to secondary neuronal
disorder (see later discussion). In epilepsy, BBB
breakdown was suggested in animal studies to
be a direct cause of epileptogenesis. Pathological
analysis in resected epileptic tissue indeed con-
firms the presence of IgG and serum albumin
within the brain, indicating BBB failure (Raabe
etal., 2012; van Vliet etal., 2007). Normal pattern
The Blood-Brain Barrier 149
of brain adenosine triphosphate-binding cas-
sette transporter expression may also change,
with upregulation of Pgp on astrocytes and
brain endothelium (Abbott etal., 2002; Marroni
etal.,2003).
BBB Dysfunction Leads to
Reorganization and Dysfunction
of the Neuronal Network
Accumulating data suggest that increased syn-
chronicity and excitability of the neuronal
network is promoted by an increase in BBB per-
meability. Acute and transient dysfunction of the
BBB may be associated with changes in the brain
extracellular ionic environment (e.g., increase in
[K+]o and decrease in [Ca 2+]o and [Mg2+]o; Ivens
et al., 2007; Marchi et al., 2007)reducing the
threshold for seizures. Clinical reports have
implied a role for BBB breakdown in familial
hemiplegic migraineassociated spreading depo-
larization (Dreier etal., 2005)and in the patho-
genesis of seizures in the cerebral hyperperfusion
syndrome (Ivens etal., 2010). It seems that while
water influx and edema may contribute to patho-
logical mechanism following BBB disruption
and may reduce perfusion and disturb neuro-
vascular coupling, additional mechanisms are
involved. Macromolecules, specifically serum
proteins, penetrate the brain after loss of BBB
integrity and were shown to accumulate within
the brain and trigger specific signaling pathways
affecting neurovascular functions. For exam-
ple, serum albumin has been shown to induce
calcium signaling and DNA synthesis in astro-
cytes in culture (Nadal etal., 1995)and in brain
slices (Nadal etal., 1998). In vivo animal studies
showed that brain exposure to serum albumin
lead to activation of transforming growth fac-
tor beta (TGF-) signalling in astrocytes. The
transcriptional response in astrocytes is asso-
ciated with increased expression of the glial
acidic fibrillary protein, a hallmark of activated
astrocytes in brain injury. Activated astrocytes
downregulate Kir4.1 and connexins (thus lead-
ing to accumulation of extracellular K+ upon
neuronal activation), downregulate EAAT1 and
EAAI12 glutamate transporters (thus leading to
accumulation of extracellular glutamate), and
upregulate inflammatory cytokines, including
TNF-, IL-6, IL-1, which were shown indepen-
dently to increase BBB permeability (Heinemann
et al., 2012). Activated astrocytes are thus in a
position to underlie neuronal hyperexcitabil-
ity via disturbance in the normal homeostasis
of K+, glutamate, and cytokinesall known to
150 Part II:Homeostatic Control
promote neuronal depolarization and plastic-
ity in excitatory synapsesat least partly due
to increased activation of N-methyl-d-aspartate
receptors (Bezzi et al., 2001; David et al., 2009;
Lapilover et al., 2012). It is thus not surprising
that when BBB breakdown persists, network
reorganization occurs, which might eventually
result in epilepsy with recurrent seizures. Apro-
longed increase in neuronal excitability has been
strongly connected to neuronal toxicity and may
point to an important mechanism linking BBB
breakdown to neurodegeneration (Tomkins
et al., 2007). Other serum proteins (in addition
to albumin), most notably the blood coagula-
tion serine protease thrombin, were similarly
shown to increase neuronal network excitability
(Maggio etal., 2008)and to facilitate hippocam-
pal long-term potentiation (Han et al., 2011;
Maggio etal.,2008).
Accumulated evidence from clinical and
animal studies supports the notion that BBB
dysfunction may serve as a primary signaling
event underlying neurovascular dysfunction.
While imaging approaches are being devel-
oped to quantitatively measure BBB breakdown
in patients (Chassidim et al., 2013), it is yet
necessary to confirm that vascular pathology
predicts specific neurological syndromes. In
stroke, BBB disturbance was shown to be a sole
parameter in magnetic resonance imaging risk
assessment for hemorrhagic transformation,
subsequent to thrombolysis in stroke patients
(Neumann-Haefelin et al., 2002). Other poten-
tial studies include those involving traumatic
and ischemic injuries and their complications,
including epilepsy and cognitive decline patients.
If such clinical studies confirm a potential role for
BBB breakdown in neurological complications,
novel treatments directly targeting BBB stability
and integrity should be developed and tested.
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10
Inflammation and Immunomodulation
inEpilepsy and Its Comorbidities
A N D R E Y M A Z A R AT I A N D A N N A M A R I A V E Z Z A N I
P R E FA C E
Inflammation is a prototypical response to injury
or infection. This homeostatic mechanism is acti-
vated in the challenged tissue in order to reestab-
lish homeostasis and initiate repair; however,
inflammation can also promote tissue injury if
prompt resolution of the inflammatory cascade
is not attained in a timely and efficient manner.
Increasing evidence provides support to a patho-
genic role of unabated brain inflammation in
various central nervous system (CNS) diseases,
including epilepsy. The induction of inflamma-
tory mediators after epileptogenic brain injuries
or seizures chiefly involves brain resident micro-
glia and astrocytes but also extends to neurons
and microvessels and promotes later leukocytes
recruitment. The involvement of glial cells in
the generation of the inflammatory cascade
in epilepsy highlights their role as key players
in innate immunity in the brain. Notably, glial
cells also contribute to normal brain develop-
ment and function via their physical and func-
tional interactions with surrounding neurons,
and the release of inflammatory mediators plays
a crucial role in many of their physiological
properties. These cells are, therefore, placed at
the intersection between health and disease, thus
representing a notable example of how runaway
homeostatic and physiological mechanisms may
lead to pathologic and harmful sequelae. In this
chapter we describe clinical and experimental
evidence supporting the role of brain inflam-
mation in pathogenic mechanisms of seizures,
cell loss, and comorbidities of epilepsy. We also
describe the novel and increasingly recognized
neuromodulatory role of inflammatory mol-
ecules and mechanisms underlying their effects
on neuronal (hyper)excitability. The identifi-
cation of the pathogenic role of inflammatory
mediators in epilepsy and its comorbidities, and
the identification of glia as main source of these
molecules, highlights putative novel targets for
therapeutic interventions beyond classical anti-
convulsive drugs that modulate neuronal chan-
nels and classical neurotransmitters.
INTRODUCTION
Inflammation is a prototypical response to infec-
tion or tissue injury, and, as such, it represents
a key homeostatic endogenous mechanism
activated for pathogen killing and removal,
thereafter promoting tissue healing and repair.
It is now well established that the evolution
of inflammation programs in tissue requires
strict control and fast resolution by endogenous
anti-inflammatory molecules in order to avoid
detrimental effects of inflammation on tissue
physiology. Indeed, there are ample examples
in the literature showing that if endogenous
anti-inflammatory control fails or is inefficient,
then inflammation results in the cell dysfunc-
tion or death. Uncontrolled inflammation has
been suggested to play a pathogenic role in sev-
eral brain diseases, as well as in acute and chronic
neurodegeneration (Craft etal., 2005; Minghetti,
2005; Glass etal., 2010). In the frame of the sug-
gested role of inflammation in CNS pathology,
increasing evidence in past 15 years has proven
a reciprocal causal link between brain inflamma-
tion and epilepsy (Vezzani etal., 2011a). In this
setting, inflammation represents a phenomenon
associated with the innate immune response of
the brain exposed to epileptogenic injuries or sei-
zures. Inflammation chiefly involves microglia
and astrocytes and is best defined as a process
comprising of rapid injury-mediated release of
cytokines and chemokines, which in turn acti-
vate downstream inflammatory mediators and
related molecular processes in glia (i.e., autocrine
action), as well as in neurons and endothelial
156 Part II:Homeostatic Control
cells of the blood-brain barrier (BBB; i.e., parac-
rine action). The induction of innate immunity
may subsequently activate adaptive immunity
mechanisms and the consequent leukocytes
extravasation in the brain parenchyma, a pro-
cess requiring an active role of the BBB (Vezzani
etal., 2011a; Vezzani and Friedman,2011).
Although the presence of inflammatory
molecules in the epileptic brain has been estab-
lished since the first characterization in 1958
of Rasmussens encephalitis (RE) as a chronic
inflammatory disease (reviewed by Varadkar
et al., 2014) the relevance of inflammation in
epilepsy has been further expanded due to novel
discoveries that (a)brain inflammation, as previ-
ously defined, is a common substrate of various
drug-resistant forms of epilepsy with differing
etiologies not necessarily linked to autoimmune
dysfunctions or active infections, and (b)inflam-
matory mediators are indeed neuromodulators
endowed of CNS-specific roles (Viviani et al.,
2007; Vezzani etal., 2011b); in particular, they can
directly affect neuronal activity independently of
their classical immune role in response to infec-
tion or systemic inflammatory challenges.
EVIDENCE LINKING BR AIN
I N F L A M M AT I O N T O E P I L E P S Y
INTHE CLINICAL SETTING
There are three main sets of evidence establish-
ing a link between brain inflammation and such
hallmarks of epilepsy as the onset and the recur-
rence of seizures, tissue neuropathology, and
comorbidities (Vezzani et al., 2011a; Aronica
etal., 2012; Vezzani etal.,2013):
Various inflammatory mediators (e.g.,
cytokines, chemokines, prostaglandins,
complement factors) are overexpressed in
surgically resected epileptogenic foci in
different forms of pharmacoresistant epi-
lepsy (e.g., tuberous sclerosis, glioneuronal
tumors, focal cortical dysplasia [FCD],
mesial temporal lobe epilesy [mTLE],RE).
Activation of innate (i.e., microglia and
astrocytes) and adaptive (i.e., extravasation of
leukocytes, autoantibodies) immune mecha-
nisms in differing forms of human epilepsy.
Drugs and treatments with
anti-inflammatory properties (e.g.,
adrenocorticotropic hormone, steroids,
intravenous immunoglobulins, plasma
exchange) show therapeutic effects in oth-
erwise drug-resistant pediatric syndromes
(infantile spasms, Lennox-Gastaut,
Landau-Kleffner, Dravet, fever-induced
refractory epileptic encephalopathy in
school age children).
Immunohistochemical evidence has shown
that activated microglia and astrocytes provide
a common source for the synthesis and release of
inflammatory mediators across epilepsies with dif-
ferent etiologies, whereas evidence for the activa-
tion of adaptive immunity appears to depend on a
particular etiology. For example, limited or scarse
infiltration of T cells has been reported in mTLE
and FCD type 1 as compared to both FCD type 2
(Iyer etal., 2010)and RE (Bien etal., 2002; Pardo
et al., 2004), and circulating autoantibodies have
been detected only in certain forms of systemic
or neurological autoimmune disorders, and only
rarely in idiopathic epilepsies (Bien and Scheffer,
2011; Vincent and Crino, 2011; Vincent etal.,2011).
Neurons, and baloon cells in FCD, also stain
for inflammatory molecules, as well as endothe-
lial cells of the BBB (Ravizza et al., 2006a;
Aronica and Crino, 2011). Notably, BBB damage
is often observed in perivascular areas in associa-
tion with the increased presence of inflammatory
mediators in perivascular astrocytes and their
endfeet and in the perivascular activated micro-
glia. Finally, the extent of microglia activation
and the level of expression of inflammatory mol-
ecules in brain cells positively correlate with sei-
zure frequency and epilepsy duration at the time
of surgery (Boer etal., 2006; Ravizza etal., 2006a;
Pernhorst etal., 2013). These observations dem-
onstrate the activation of inflammatory signaling
in human epileptic tissue; moreover, therapeu-
tic effects of anti-inflammatory treatments in
drug-resistant epilepsies highlight a possibility
of a causal relationship between brain inflam-
mation and the pathogenesis of epilepsy. Such
a causal relationship has been established using
experimental models of human disease.
EVIDENCE OFA CAUSAL
R E L AT I O N S H I P B E T W E E N
B R A I N I N F L A M M AT I O N
A N D T H E PAT H O G E N E S I S
OFSEIZURES
Innate Immunity and
Inflammation:Focus on
IL-1R1/TLR4 Signaling
Activation of innate immune mechanisms in
microglia and astrocytes has a pivotal role in the
 Inflammation and Immunomodulation in Epilepsy and Its Comorbidities 157

generation of the inflammatory cascade follow-
ing brain injuries or seizures via transcriptional
activation of NFkB-dependent genes. Activation
of the same type of signaling in neurons has dif-
ferent physiopathologic outcomes that are medi-
ated by rapid posttraslational modifications of
voltage-gated or receptor-coupled ion channels
and the consequent reduction of seizure thresh-
old (Table 10.1 and reviewed in Vezzani et al.,
2011b).
Recent data have demonstrated that epilepsy
is associated with the induction of the IL-1 recep-
tor type 1 (IL-1R1) and of Toll-like receptor 4
(TLR4) signaling in glia and neurons (Ravizza
et al., 2006a; Boer et al., 2008; Ravizza et al.,
2008a; Maroso etal., 2010; Zurolo etal., 2011). The
respective endogenous cytokines, IL-1 and high
mobility group box 1 protein (HMGB1) that acti-
vate their cognate receptors are also upregulated
in epilepsy brain tissue. Evidence of signaling
activation has been reported both in astrocytes
and in neurons in human epilepsy (e.g., malfor-
mations of cortical development, mTLE, and RE)
and in animal epilepsy models, for example those
of mTLE, absence seizures in genetic absence epi-
lepsy rats from Strasbourg (GAERS) (serving as
a model of absence epilepsy in humans), as well
as in kindling (i.e., occurence and progression
of motor seizures in response to repetitive sub-
threshold electical stimulation of limbic system;
Vezzani etal., 2011b; Aronica etal.,2012).
The role played by these signaling molecules
in seizure mechanisms (Table 10.1) has been
highlighted by pharmacological and genetic
studies. For example, blockade of the inflamma-
some (i.e., the macromolecular complex respon-
sible for the IL-1 and HMGB1 release) using
antagonists of P2X7 receptors that are activated

TABLE10.1 E X A M PL E S OFI N FL A M M ATORY SIGNA LI NG CASCA DE S AC T I VATED

I NTH E BR A I N DU R I NG SEI Z U R E S OR BYEPIL EP TOGEN IC I N J U R I E S
I NE X PER I M EN TA LMODEL S
Inflammatory Endogenous Intracellular Target Outcomes
pathway activators signaling (cell types)

IL-R1/TLR4 IL-1,HMGB1 Srckinase NR2B Seizures, cellloss

n.d. (neurons) Depression, Cognitive
NFkB HPA, 5-HT (neurons) deficits
Genes Inflammation,
(glia,BBB) BBB damage
RAGE HMGB1,S100 NFkB Glia Seizures, Inflammation
n.d. n.d. Cognitive deficits
P2X7 R ATP Inflammasome Glia Seizures,cell loss
COX-2/PGE2 Glutamate, cAMP/PKA/CREB EP2 Neuronal survival
Cytokines n.d. (neurons) Cognitive deficits
cAMP/Epac n.d. Inflammation, cell loss
EP2
(glia)

TNF-type1 TNF- PI3KAkt GluR2,AMPA GABA-A Seizures, cellloss

TNF- type 2 NFkB (neurons) Inflammation
n.d. n.d. Neuroprotection,
(glia) reduced seizures
n.d.a

Note: This table reports selected examples of inflammatory mechanisms activated in epilepsy that have been shown to play a role in
physiopathological outcomes, as demonstrated in experimental models. For additional information, see the reference list in the main
text and the latter sections of the article.
n.d.=not determined; NFkB, nuclear factor KB; NR2B=NMDA receptor subtype 2B; NMDA=N-methyl-D-aspartate;
BBB=blood-brain barrier; RAGE=receptor for advanced glycation end products; Epac=exchange protein directly activated by cAMP;
PKA, protein kinase A; CREB= cAMP response element-binding protein; PI3K=phosphoinositide 3-kinase; Akt=protein kinase B;
TNF=tumor necrosis factor; AMPA=-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; gamma-aminobutyric acid=GABA.
a
indirect data in receptor knockout mice suggest that NMDA, AMPA, and kainate receptor subtypes are downregulated by receptor
activation (Balosso etal.,2009).
158 Part II:Homeostatic Control
by ATP released by damaged cells (Engel et al.,
2012), or caspase-1 antagonists that inhibit IL-1
biosynthesis, both drastically reduce the fre-
quency of seizures in rodent models of temporal
lobe epilepsy (TLE), decrease absence seizures in
GAERS (Ravizza etal., 2006b; Akin etal., 2011;
Maroso et al., 2011) and inhibit seizure propa-
gation during kindling (Ravizza et al., 2008b).
Injection of IL-1R1 or TLR4 antagonists also
alleviates experimental seizures in a variety of
models (Vezzani etal., 1999; Vezzani etal., 2000;
Vezzani et al., 2002; Marchi et al., 2009; Auvin
etal., 2010; Maroso etal., 2010; Kwon etal., 2013).
The described anticonvulsive effects are congru-
ent with the proconvulsive activity of either
IL-1 or HMGB1 upon their intracerebral injec-
tion before the convulsive challenge (Vezzani
et al., 1999; Vezzani et al., 2000; Balosso et al.,
2008; Maroso etal., 2010; Iori etal., 2013; Balosso
et al., 2014). Accordingly, transgenic mice with
perturbed IL-1 or HMGB1 signaling show sig-
nificant alterations in their intrinsic seizure sus-
ceptibility (Vezzani et al., 2008; Maroso et al.,
2010; Iori etal.,2013).
Interestingly, recent data have shown that
proconvulsive activity of HMGB1 is specifically
linked to the redox state of its cysteine residues
after the molecule is released extracellularly.
Thus only the disulfide (i.e., oxidized) form of
this molecule activates TLR4 and promotes sei-
zures but not the all-thiol (i.e., reduced) form,
which has instead chemoattractant properties.
This finding highlights the importance of the
redox state of the extracellular milieu for medi-
ating the physiopathologic effects of HMGB1 on
neurons (Balosso etal.,2014).
One key question concerns mechanisms
by which the activation of the IL-1R1/TLR4
signaling promotes seizures. Importantly, this
signaling induces rapid (i.e., within minutes)
Src kinasemediated phosphorylation of the
NR2B subunit of the N-methyl-D-aspartate
(NMDA) receptor complex thus leading to the
increased neuronal Ca2+ influx (Viviani et al.,
2003; Balosso et al., 2008). This molecular event
underlies the proconvulsive activity of both IL-1
and HMGB1, as well as their effect in enhanc-
ing glutamate-mediated neurotoxicity. Another
mechanism of hyperexcitability mediated by
IL-1R1/TLR4 signaling is the downregulation
of the HCN1 channel and the associated Ih cur-
rent on dendrites of hippocampal pyramidal
neurons, as assessed in rats upon intraventricu-
lar lipopolysaccharide (LPS) injection (unpub-
lished data), a component of Gram-negative
bacteria, which induces inflammation by acti-
vating TLR4. Acquired channelopathies have
been reported to be induced also by tumor
necrosis-alpha (TNF-) factor affecting the
assembly and synaptic clustering of -amino-
3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) receptors and the membrane expression
of gamma-aminobutyric acid (GABA)-A recep-
tors. In particular, TNF- induces the membrane
shift toward the extrasynaptic compartment of
GLUR2-lacking AMPA receptors, a mechanism
involved in excitotoxicity and possibly in synaptic
scaling (Beattie etal., 2002; Stellwagen etal., 2005;
Stellwagen and Malenka, 2006). Furthermore,
cytokine-induced activation of intracellular
kinases changes the phosphorylation and func-
tion of voltage-gated Na+, K+, and Ca2+ channels in
neurons (Vezzani and Viviani,2015).
Additional mechanisms possibly underlying
the proictogenic properties of cytokines include
their ability to inhibit glutamate reuptake by astro-
cytes, to promote glutamate release from glia, and
to enhance NMDA-mediated glutamate release
from synaptic terminals, since all these effects pro-
mote neuronal excitability (Devinsky etal.,2013).
I N F L A M M AT O R Y M O L E C U L E S
AND EPILEPTOGENESIS
Mounting evidence suggests pathogenic role of
specific inflammatory signaling in the develop-
ment of epilepsy after a primary brain injury.
This information is emerging from studies
using epilepsy models, where pharmacologi-
cal blockade of a specific target is attained at
various times after the epileptogenic injury
(usually after status epilepticus onset) but
before epilepsy develops (Ravizza et al., 2011).
For example, individual targeting of IL-1 and
cyclooxygenase 2 (COX-2) systems, or their
combination, affords neuroprotection (Kwon
etal., 2013; No etal., 2013)and in some com-
binatorial experiments also reduces the fre-
quency of spontaneous seizures (Kwon et al.,
2013). Disease-modifying effects have been
reported also by pharmacological interference
with microglia or astrocyte activation (Ravizza
et al., 2011; Wang et al., 2015). One study
reported antiepileptogenic effects achieved
by pharmacological blockade or genetic inac-
tivation of adhesion molecules on brain ves-
sels, thus impairing leukocyte extravasation
(Fabene et al., 2008). This study, however, has
been challenged by subsequent evidence show-
ing worsening effects on seizure onset and neu-
ropathology when leukocyte extravasation was
 Inflammation and Immunomodulation in Epilepsy and Its Comorbidities
prevented (Zattoni et al., 2011). Accordingly,
adoptive transfer of T lymphocytes in immu-
nodeficient mice delayed seizures onset and
mediated neuroprotection (Deprez etal.,2013).
The absence of either TLR4 or receptor for
advance glycation end products (RAGE, a recep-
tor for the HMGB1 and S100 protein family) in
knockout mice did not interfere with the acute
phase of status epilepticus but mediated a signifi-
cant reduction in spontaneous seizure frequency
in epileptic mice (Iori etal.,2013).
This set of evidence highlights the need for
better understanding master regulators of the
inflammatory cascade that dynamically evolves
during epileptogenesis and of discerning mech-
anisms with an homeostatic role from those
contributing to the disease progression. This
information is instrumental for developing effec-
tive anti-inflammatory treatments, which can be
timely applied for arresting epileptogenesis after
a primary injury without interfering with the
beneficial role of inflammation.
T H E Y I N A N D YA N G O F T H E
I N F L A M M AT O R Y C A S C A D E
The inflammatory cascade activated by IL-1R1/
TLR4 signaling leads to synthesis and release of
various inflammatory mediators, which in turn
may contribute either to pathology or to home-
ostasis and repair. This yin and yang feature of
inflammation is well known, and many exam-
ples are reported in the literature. In general,
the physiopathologic outcomes of inflamma-
tion are strictly context dependent and as such
are chiefly determined by the extent of changes
in the levels of specific inflammatory molecules
and time of tissue exposure to them, as well as by
the type and cellular pattern of their respective
receptor expression. The microenvironment in
which tissue inflammation develops, therefore,
is instrumental for defining the outcomes. The
time of onset and development of the inflamma-
tory response after the primary injury or tissue
challenge is also crucial. A typical example is
represented by LPS-induced tolerance (Biswas
and Lopez-Collazo, 2009), a phenomenon that
protects brain tissue from subsequent ischemic
damage or seizures (Dmowska etal., 2010), con-
trasted by the LPS-mediated increase in neu-
ronal cell loss (Auvin etal., 2007)and reduction
of seizure threshold (Sayyah et al., 2003; Riazi
et al., 2010). Such opposite outcomes strictly
depend on precise timing before the induction
of tissue inflammation and the onset of brain
insult.
159
One notable example of the dual role of inflam-
matory molecules in the brain is represented by
the COX-2 induction (Table 10.1). This enzyme
is activated in epilepsy models, first in neurons
and subsequently in glial cells with a prominent
induction in astrocytes (Vezzani et al., 2012).
Genetic inactivation of COX-2 in pyramidal neu-
rons rescues cell loss when mice are exposed to
status epilepticus; accordingly neuroprotection is
attained by administering antagonists of prosta-
glandin (PG) E2 EP2 receptor from 1 hour up to 3
hours from status epilepticus onset. Interstingly,
activation of EP2 receptors at the time of status
epilepticus induction is indeed neuroprotective,
thus indicating the dual role of PGE2/EP2 sig-
nalig in determining seizure-induced cell loss
or survival. Activation of cAMP/PKA/CREB in
neurons or cAMP/Epac proinflammatory signal-
ing in glia appear to be the key determinant for
mediating cell survival or cell loss, respectively
(Jiang et al., 2012). Different effects on seizures
have also been reported depending on the type of
PG induced in a particular experimental model
and the timing of anti-COX-2 intervention
(Kulkarni and Dhir, 2009; Vezzani etal.,2012).
TNF- is another inflammatory media-
tor that plays a dual beneficial or detrimental
role. Depending on the type of receptors pref-
erentially activated in the specific pathologic
contex, this cytokine mediates anticonvulsive
effects by activating TNF- type 2 receptors or
proconvulsive effects by activating TNF- type
1 receptor (Balosso et al., 2005; Balosso et al.,
2013; Weinberg etal., 2013). The extent of TNF-
increase in the brain also matters, whereby mod-
erate increases appear to mediate inhibitory
effects on seizures while massive increases exac-
erbate seizures (Akassoglou etal., 1997; Balosso
etal.,2005).
THE ROLE OF
A DAPTIV E IM MU NIT Y
Human Evidence
The adaptive immune system can play a role in
epilepsy by activating autoantibody-mediated
mechanisms or by T cellmediated cytotoxicity.
Invasion of immunoglobulins (Ig) and T lym-
phocytes in the brain can be found in a variety
of encephalitis including RE, viral encephalitis
and a group of antibody-associated encepha-
litis defined by the presence of specific neural
autoantibodies in serum and/or CSF. In RE and
other epileptic disorders, a wide spectrum of
anti-neural antibodies has been detected which
160 Part II:Homeostatic Control
may result from a secondary immune response
against antigens released during neuronal cell
degeneration. A role of cytotoxic CD8+T lym-
phocytes has been described in RE; in particu-
lar, granzyme B positive cytotoxic T cells can be
found in close apposition to neurons and astro-
cytes with polarization of the cytotoxic gran-
ules facing the targeted cell membrane (Bien
et al., 2002; Bauer et al., 2007). MRI studies
have found that infiltrating T cells in RE occur
also outside of the epileptic network, suggesting
that epileptic activity may not be directly associ-
ated with the presence of infiltrating cells (Hauf
et al., 2009). Accordingly, an immunosuppres-
sive drug tacrolimus had ameliorating effects on
motor and cognitive dysfunctions and neuronal
cell degeneration in RE patients, but showed no
effect on seizure frequency (Bien etal., 2004; Bien
etal.,2013).
The antibody-associated encephalitides are
characterized by a prominent seizure pheno-
type often involving limbic system (i.e., limbic
encephalitis). Clinical evidence and emerging
in vitro data give support to the pathogenicity of
some of these antibodies. They can be directed
against either intracellular (e.g. GAD65, amphi-
physin, Hu, Yo and Ma2) or membrane (e.g.
the voltage-gated potassium channels (VGKC),
NMDA, AMPA, or metabotropic glutamate
receptor subunits and the GABAB receptor)
antigens. Negative passive transfer experiments
suggest that antibodies targeting intracellular
antigens are not pathogenic since they cannot
access the intracellular antigens in intact neurons
(Vincent et al., 1999). Differently, antibody tar-
geting proteins associated to the VGKC-complex
(i.e. Caspr2, LGI1, contactin-2) (Irani etal., 2010;
Lai etal., 2010; Vincent and Irani, 2010)are sug-
gested to mediate memory loss, movement disor-
ders and seizures (Thieben etal., 2004; Vincent
etal., 2004; Chan etal., 2007). Importantly, ster-
oids, intravenous immunoglobulin G (IgG), and
plasmapheresis improve the described neuro-
logical deficits, thus suggesting that these anti-
bodies are directly pathogenic (Vincent et al.,
2004). The presence of circulating and brain
tissue VGKC-complex antibodies is associated
with neuronal degeneration in the hippocam-
pus in the presence of C9neo, the end complex
of complement activation (Bien etal., 2012). This
suggests that an antibody-mediated complement
activation may mediate neuronal celldeath.
Another form of encephalitis is associated
with antibodies directed against the NR1 subunit
of the NMDAR (Dalmau etal., 2008). Clinically,
a prodromal stage with neurovegetative symp-
toms precedes the development of seizures,
memory loss and psychiatric symptoms followed
by a dramatic movement disorder (Dalmau etal.,
2011). Prolonged courses of immunotherapy
may reverse these clinical signs. Brains of these
patients show few inflammatory T cells and in
most cases neuronal loss is mild (Dalmau etal.,
2007; Bien etal., 2012). IgG binding to hippocam-
pal neurons may lead to a state of NMDAR hypo-
function (Hughes etal.,2010).
Although autoantibodies are unlikely to be a
major cause of seizures in idiopathic epilepsies,
an increasing number of studies have detected
serum autoantibodies in 2% to 7% of these
patients (McKnight et al., 2005; Majoie et al.,
2006). Immunotherapies offered occasional ther-
apeutic benefit (Giometto etal., 1998), although
their overall efficacy is often disappointing.
E X P E R I M E N TA L S T U D I E S
Pathological relevance of autoantibodies or
leuckocytes in epilepsy has been addressed in
experimental in vitro and in vivo studies. Notable
examples are reportedhere.
Antibodies Against
Membrane Antigens
Infusion of NMDAR antibodies into the rodent
brain caused a downregulation of NMDAR;
accordingly, incubation of hippocampal neuron
cultures with purified NR1-IgG from patients
with NMDAR encephalitis caused a significant
reduction in NMDAR density and in both sur-
face and total NR2A/B subunits (Hughes et al.,
2010), resulting in a reduced synaptic NMDAR
localization, which was reversed upon removal
of the antibodies. Applying patients serum anti-
bodies against AMPA receptors in hippocampal
neurons produced similar results, along with the
significant reduction in the density of membrane
AMPA receptors (Lai etal., 2009). NMDA recep-
tor antibodies also increased the concentration of
glutamate in the extracellular space (Manto etal.,
2010), and whole-cell patch-clamp recordings of
miniature excitatory post-synaptic currents in
cultured rat hippocampal neurons showed that
patients antibodies specifically decreased synap-
tic NMDAR-mediated currents, without affect-
ing AMPA receptor-mediated currents (Hughes
et al., 2010). Lalic et al. (2010) using whole-cell
patch-clamp recordings showed synaptic stimu-
lation of CA3 neurons in hippocampal slices
 Inflammation and Immunomodulation in Epilepsy and Its Comorbidities
incubated with IgG from limbic encephalitis
patients, as well as epileptiform activity and
increased mossy fiber-evoked synaptic responses.
These results were mimicked by -dendrotoxin,
an antagonist of the Shaker VGKC channel, sug-
gesting that the antibodies increase CA3 neurons
excitability by downregulating the activity of the
VGKC complex.
Antibodies Against Intracellular
Antigens
Although the cytoplasmic location of GAD
(i.e. the enzyme responsible for the synthesis of
GABA) suggests that the related antibodies are
unlikely to be pathogenic, the application of
GAD-positive sera from epilepsy patients onto
cultured hippocampal neurons increases the fre-
quency of the postsynaptic inhibitory potentials.
This evidence supports the hypothesis of patho-
logically active components in the patients sera
(Vianello etal.,2008).
Role ofLeukocytes inSeizureModels
There is evidence for the involvement of the
peripheral immune cells in some forms of epi-
lepsy. It appears that their contribution to mech-
anisms of the disease differs depending on the
nature of the epileptogenic trigger. In particular,
perivascular extravasation of CD4+ and CD8+
T lymphocytes has been reported in the mouse
hippocampus after prolonged seizures induced in
mice by either systemic injection of convulsant
doses of the muscaric receptor agonist pilocar-
pine (Fabene etal., 2008)or by intrahippocam-
pal administration of kainic acid (Zattoni etal.,
2011). This phenomenon may reflect cell extrava-
sation due to injury or seizure-induced leakage of
BBB. Alternatively, lymphocytes may be primed
to pass into the perivascular space by vessel
inflammation following cell injury or seizures.
Upregulation of adhesion molecules on endothe-
lial cells of brain microvessels and chemoattrac-
tion is induced by cytokines and chemokines
released from perivascular astrocytes and
microglia (Librizzi et al., 2007; Librizzi et al.,
2012), thus favoring luminal interactions and
extravasation of circulating leukocytes (Fabene
et al., 2008). Although the presence of circulat-
ing antigens activating T cells has not been dem-
onstrated, one might envisage that brain-born
molecules released by damaged or activated
neurons and/or glial cells may drop out of the
CSF into the systemic circulation. If these mol-
ecules express molecular mimicry of pathogens
161
(e.g., formylated peptides released from damaged
mitochondria), then they may activate adaptive
immunity. One crucial, still open question is
whether this phenomenon has some relevance
for tissue hyperexcitability or neuropathology.
Two divergent findings emerged from mouse
models of TLE. In the pilocarpine model in mice,
neurotrophils, macrophages, and T cells may
play a detrimental role in the postinjury phase
(i.e., epileptogenesis) since knockout mice lack-
ing key adhesion molecules or wild-type mice
treated with anti-integrins antibodies (i.e., two
procedures that impair leukocytes extravasa-
tion) develop less frequent spontaneous seizures
and show less cell loss as compared to wild-type
epileptic mice (Fabene et al., 2008). Conversely,
when epilepsy is triggered by intracerebral kainic
acid in mice, macrophages and T cells play a pro-
tective role by delaying the onset and reducing
the recurrence of spontaneous seizures (Zattoni
et al., 2011); prevention of neurotrophils entry
into the brain tissue appears to be one mecha-
nism of neuroprotection. For a correct interpre-
tation of these results, it is important to consider
that pilocarpine uses a peculiar mechanism of
action to induce epilepsy: it directly activates
muscarinic receptors on circulating leukocytes,
which in turn promote BBB leakage, thus allow-
ing enough pilocarpine to enter the brain to
induce seizures (Vezzani and Janigro,2009).
I N F L A M M AT I O N A N D
COMORBIDITIES
Many epilepsy patients present with inter-
ictal neurobehavioral disorders at a signifi-
cantly higher rate than the general population.
Neurobehavioral comorbidities of epilepsy have
been receiving growing attention. Indeed, with
significant progress made in the management
of seizures proper, it is becoming increasingly
obvious that effective seizure control is not nec-
essarily accompanied by the alleviation of con-
current neuropsychiatric disorders. As a result,
comorbidities may have higher negative impact
on patients quality of life than even frequency
of seizures (Gilliam etal., 1997; Garcia-Morales
et al., 2008). The importance of addressing
comorbidities of epilepsy has been recognized by
the National Institute of Neurological Disorders
and Stroke, which included them in its Epilepsy
Research Benchmarks in 2007 and again in
2013 (National Institutes of Health, National
Institute of Neurological Disorders and Stroke,
2007,2013).
162 Part II:Homeostatic Control
A dominating school of thought regarding
causes of a high rate of neurobehavioral per-
turbations among epilepsy patients is that epi-
lepsy and concurrent nonconvulsive conditions
share certain pathophysiological mechanisms.
Considering that brain inflammation has been
implicated in mechanisms of several neuropsy-
chiatric disorders, it is conceivable that inflam-
matory processes that are triggered in the brain
by an epileptogenic insult may, concurrently
with seizures, lead to the development of neu-
robehavioral abnormalities. We discuss the role
of inflammation in the pathophysiology of three
common comorbidities of epilepsy: depression,
autism, and cognitive impairments.
Depression
Between 10% and 60% of epilepsy patients pre-
sent with symptoms of depression (Mendez
et al., 1986; Kanner et al., 2012). The comor-
bidity has been examined along the lines
of pathophysiological mechanisms shared
by epilepsy and major depressive disorder
(MDD). For example, the dysfunction of cen-
tral monoamine neurotransmitters serotonin
and norepinephrine, and the hyperactivity of
hypothalamo-pituitary-adrenocortical (HPA)
axis have been regarded as key mechanisms of
MDD (Baumann etal., 1999; Ordway etal., 2003;
Carroll etal., 2007; Lowry etal., 2008; Michelsen
etal., 2008)and at the same time have been docu-
mented in patients with various forms of epilepsy
(Kondziella etal., 2007; Kanner etal.,2012).
The connection between chronic inflammation
and MDD is well established. Thus biomarkers of
inflammation have been consistently found in the
blood of depression patients (Bremmer etal., 2008;
Muller et al., 2011; Krishnadas and Cavanagh,
2012). Furthermore, patients with primarily
chronic inflammatory diseases (e.g., rheumatoid
arthritis) develop depression at a significantly
higher rate than general population (40% vs. 5%
to 15%; Bruce, 2008). Chemokine immunother-
apy is known to induce symptoms of depression
(Dantzer and Kelley, 1989). In an experimental
setting, Gram-negative infection mimicked in
rodents by LPS produces sickness behavior, which
includes such symptoms of depression as anhedo-
nia and failure to cope with stress (Dantzer, 2006).
Moreover, even depressive impairments not pri-
marily associated with inflammatory stimuli (e.g.,
depression induced by stress) are amenable to the
treatment with anti-inflammatory drugs, such as
minocycline and celecoxib (Molina-Hernandez
etal., 2008; Guo etal.,2009).
Brain IL-1 has been regarded as a key fac-
tor in the inflammation-associated depression
(Dunn et al., 2005; Goshen and Yirmiya, 2009;
Lang and Borgwardt, 2013). For example, cen-
trally administered IL-1 induced behavioral
(Dunn and Swiergiel, 2005)and neuroendocrine
(Parsadaniantz etal., 1997)symptoms of depres-
sion in rodents. Therefore, the established chronic
overexpression of IL-1 and its receptor in the
epileptic hippocampus (Ravizza et al., 2008a),
aside from contributing to mechanisms of epi-
lepsy proper, may lead to depression. Outside the
hippocampus, IL-1 is suppresses the firing of
raphe serotonin neurons (Brambilla etal., 2007),
thus conceivably leading to depression via sub-
sequent reduced activity in serotonergic afferents
(Figure10.1).
Animals with chronic epilepsy, which is pre-
cipitated by either status epilepticus or kindling,
exhibit behavioral deficits indicative of depres-
sion, particularly anhedonia and failure to cope
with stress (Koh et al., 2007; Mazarati et al.,
2007; Mazarati et al., 2008). These impairments
develop as a result of consecutive hyperactiv-
ity of the HPA axis, upregulation of 5-HT1A
autoreceptors, and subsequently diminished
raphe-hippocampal serotonergic tone (Mazarati
et al., 2008; Mazarati et al., 2009; Pineda et al.,
2011). These perturbations are not amenable to
fluoxetine therapy (Pineda etal., 2012), thus rep-
resenting a case of selective serotonin reuptake
inhibitor (SSRI)-resistant depression (discussed
further next). At the same time, direct intra-
hippocampal administration of interleukin-1
receptor antagonist (IL-1ra), an endogenous com-
petitive inhibitor peptide of IL-1 receptor type 1,
alleviated (albeit not fully reversed) depression
symptoms, including behavioral, neuroendo-
crine, biochemical, and receptor impairments
(Mazarati etal., 2010). This suggests that depres-
sion in epileptic subjects may, at least in part,
stem from the overexpression of hippocampal
IL-1 and/or its receptor, with the subsequent
involvement of the HPA axis and brain seroton-
ergic transmission (Figure10.1).
In addition to precipitating symptoms of
depression, the enhanced IL-1 signaling in the
epileptic hippocampus may also play role in the
SSRI resistance of epilepsy-associated depres-
sion. SSRI resistance represents significant chal-
lenge in MDD management, as between 30%
and 50% of depression patients do not respond
or poorly respond to this class of antidepressants
(Barbui etal., 2002; Rush etal., 2006). It has been
suggested that SSRI resistance is present in MDD
 Inflammation and Immunomodulation in Epilepsy and Its Comorbidities
Normal
Proinflammatory event
Depression
IL-1 (a) IL-1
Epilepsy (b)
HPA HPA
axis 5-HT axis 5-HT
5-HT1A
5-HT1A
FIGURE 10.1: Perturbations of inflammatory
signaling: Role in seizures and in epilepsy-
associated depression. Under normal conditions,
the expression of IL-1 in the hippocampus is relatively
low; therefore, its modulation of the hypothalamo-
pituitary-adrenocortical (HPA) axis is negligible.
Within the physiological parameters, the HPA axis
exerts little or no effects on somatodendritic 5-HT1A
receptors in raphe nuclei. Raphe 5-HT1A receptors
regulate 5-HT release from the raphe into the hip-
pocampus via equilibrated autoinhibitoryloop.
We can envisage two main, nonmutually exclusive sce-
narios:(a)a primary inflammatory event may concur
to trigger the onset and recurrence of epileptic seizures
via the activation of IL-1 signaling, and in parallel the
same mechanism may produce symptoms of depres-
sion by altering the HPA axis; (b)epilepsy may lead to
or perpetuate the overexpression of IL-1 in the hip-
pocampus, which in turn hyperactivates the HPAaxis.
A pathological hyperactive HPA axis upregulates
raphe 5-HT1A receptors. This shifts regulation of
5-HT release in favor of autoinhibition and results in
the diminished serotonergic tone, the latter producing
depression.
patients with polymorphism of 5-HT1A recep-
tor gene, which translates into the upregulation
of 5-HT1A autoreceptors (Lemonde etal., 2003;
Richardson-Jones etal., 2010). At the same time,
5-HT1A upregulation may be acquired and not
necessarily inherited. For example, an excessively
hyperactive HPA axis leads to the upregulation
of 5-HT1A autoreceptors (Bellido et al., 2004;
Judge et al., 2004). In epilepsy, the HPA hyper-
activity is triggered and sustained by chronically
enhanced IL-1 signaling (Mazarati etal., 2010).
Hence, the disruption of the latter may not only
alleviate depression but restore the effectiveness
of SSRIs. Indeed, the combined administration
of IL-1ra and fluoxetine fully reversed depres-
sion in chronic epileptic rats (Pineda et al.,
2012). This finding may be applicable not only to
epilepsy-associated depression but also to other
163
cases of SSRI-resistant MDD that do not stem
from the 5-HT1A receptor gene polymorphism
(Levin etal.,2007).
Autism
Epidemiologically, reciprocal connection
between epilepsy and autism has been well
established:approximately 30% of patients with
autism develop epilepsy at some point of their
lives, and at the same time around 30% of patients
with epilepsy as a primary diagnosis fit the cri-
teria of being diagnosed with autism (Tuchman
and Rapin, 2002; Clarke etal., 2005; Seidenberg
etal.,2009).
Causes of the comorbidity remain unclear.
Several scenarios have been suggested, ranging
from an incidental coappearance to compris-
ing two manifestations of the same pathology
(e.g., fragile X chromosome; Levisohn, 2007).
However, since both epilepsy and autism are
multifactorial and multicausative diseases, the
nature of the association between the two con-
ditions is very likely to vary among different
patients.
Causes of autism proper are being exam-
ined along three lines. Genetic mechanisms
have been extensively studied; a growing
number of genes has been identified and their
role has been supported by experimental evi-
dence (Moy and Nadler, 2008; Abrahams and
Geschwind, 2010; Eapen, 2011). At the same
time, autism concordance among monozygotic
twins is between 50% and 70%, and, despite a
growing number of candidate genes, cases of
autism traceable to a genetic trait remain at 15%
(Hallmayer etal., 2011). Further, spontaneous
resolution of autism symptoms in a cohort of
patients (or optimal-outcome individuals) sug-
gests the transient nature of etiological fac-
tors in some cases (Fein et al., 2013). These
observations strongly implicate environmental
risks. Indeed, exposure of pregnant women to
infections, valproic acid, ethanol, and stress
have been suggested as risk factors for the
development of autism in offspring (Patterson,
2009; Atladottir et al., 2010; Landrigan, 2010;
Dufour-Rainfray etal., 2011). The third school
of thought combines genetic and environmen-
tal causes under the concept of a double-hit
umbrella.
Among environmental factors, infec-
tions during pregnancy have been receiving
growing clinical and experimental support.
Epidemiological studies provide a compelling
association between maternal infection and
164 Part II:Homeostatic Control
the increased chance of development of autism
in children (Ciaranello and Ciaranello, 1995;
Jonakait, 2007; Moy and Nadler, 2008; Atladottir
etal., 2010; Atladottir etal., 2012). Atladottir etal.
found that viral infection in the first trimester
and bacterial infection in the second trimester
of pregnancy were associated with autism in the
offspring, with hazard ratios of 2.98 and 1.42,
respectively (Atladottir et al., 2010); another
study by this group found that maternal influenza
was associated with a twofoldand prolonged
episodes of fever with a threefoldincreased
risk of autism in the offspring (Atladottir et al.,
2012). Experimental studies have established
that injection of pregnant rodents with either
LPS or polyinosinicpolycytidylic acid (Poly I:C),
which mimic Gram-negative and viral infections,
respectively, lead to the development in offspring
of autism-like impairments (Patterson, 2002;
Shi etal., 2003; Fatemi etal., 2005; Boksa, 2010;
Patterson, 2011a, 2011b; Schwartzer et al., 2013).
Poly I:C-induced maternal immune activation
(MIA) in rhesus monkeys produced offspring
with impaired sociability and restricted behavior
(Bauman etal., 2013). Electrophysiological studies
in the MIA offspring revealed impaired commu-
nication between the hippocampus and prefron-
tal cortex, which represents a hallmark of autism
spectrum disorder (Baharnoori etal.,2009).
Detrimental effects of maternal infection
occur through MIA (Figure 10.2). The latter,
frequently described as the cytokine storm,
involves massive activation of inflammatory
pathways that, when acting on the embryonic
brain, induce perturbations in neuronal develop-
ment, connectivity, metabolism, and function-
ing, ultimately resulting in autism (Jonakait,
2007; Ponzio et al., 2007; Buehler, 2011; Deng
etal., 2011; Patterson, 2011b; Oskvig etal.,2012).
Concurrently with autism, MIA primes off-
spring to epilepsy (Figure 10.2). Thus the offspring
of rats treated with LPS during pregnancy exhib-
ited increased severity of pilocarpine-induced
status epilepticus, as well as exacerbated neurode-
generation in the hippocampus (Yin etal., 2013).
MIA induced by Poly I:C in genetically normal
mice increased the severity of status epilepticus
induced by intrahippocampal administration of
kainic acid and subsequent neurodegeneration
in the hippocampus of the offspring (Kirschman
etal., 2011). Prenatal exposure to Poly I:C accel-
erated the rate of hippocampal kindling epilep-
togenesis during adolescence and prolonged the
retention of a kindling-induced epileptic state
(Pineda etal.,2013).
Maternal immune
Offspring
activation
IL-6 Autism
Maternal
infection
IL-1 Epilepsy
Other
FIGURE10.2: Maternal immune activation (MIA)
as a risk factor for the development of autism
and epilepsy in the offspring. Among various
components of the MIA triggered by infection, IL-6
is necessary and sufficient for producing autism in
the offspring in the adulthood. Neither IL-6 nor IL-1
alone produces epileptic phenotype. However, com-
bined induction of IL-6 and IL-1 is both sufficient and
necessary for predisposing the offspring to epilepsy.
Hence, the autismepilepsy comorbidity following an
infection during pregnancy depends on the extent of
the exposure of embryonic brain to IL-6 andIL-1.
Among components of MIA, interleukin-6
(IL-6) has been established as a factor solely
responsible for the development of autism in off-
spring. Thus anti-IL-6 antibody coadministered
with Poly I:C in pregnant mice prevented the
occurrence of autism phenotype in the offspring,
while mice treated with recombinant IL-6 pro-
duced the offspring with autism-like behavioral
impairments similar to those induced by Poly I:C
(Smith etal., 2007). In a separate study (Pineda
et al., 2013), prenatal exposure to IL-6, but not
to IL-1, led to autism-like behavior in the off-
spring of mice, while none of the two cytokines
administered alone produced epileptic pheno-
type. However, when IL-6 and IL-1 were admin-
istered together, the offspring presented with the
accelerated hippocampal kindling rate and pro-
longed kindling retention (i.e., the effects were
similar to those induced by PolyI:C).
Conversely, immune blockade of IL-1 pre-
vented facilitatory effects of prenatal Poly I:C on
kindling epilepsy but not on autism behavior,
while the blockade of IL-6 prevented both autism
and epileptic phenotypes induced by MIA. These
observations led to a hypothesis that the develop-
ment of autism without concurrent epilepsy and
autism accompanied by epilepsy in the offspring
following MIA depends on the extent of the
exposure of embryonic brain to IL-6 and IL-1
 Inflammation and Immunomodulation in Epilepsy and Its Comorbidities
(Pineda etal., 2013; Figure 10.2). If corroborated
in patients, these findings may translate both into
early diagnostic (through measuring plasma lev-
els of the cytokines) and preventive (e.g., through
using cytokine antibodies) measures that would
respectively predict and significantly alleviate
the risk of autism and epilepsy in patients atrisk.
Postnatal inflammation has been associ-
ated with autism as well. For example, increased
blood levels of RAGE ligands such as HMGB1
and S100A9 (Boso et al., 2006; Emanuele et al.,
2010), as well as the enhanced reactivity of TLR2
and TLR4 expressing monocytes (Enstrom etal.,
2010), have been documented in people with
autism. Increased levels of monocyte chemoat-
tractant protein 1 and TNF1 were documented
in the cortex, white matter, and cerebellum of
patients with autism (Vargas etal., 2005). Acute
exposure of neonatal rat pups to a combination
of LPS to mimic Gram-negative bacterial infec-
tion, doxorubicin to produce diffuse brain dam-
age involving the forebrain and brainstem, and
p-chlorophenylalanine to deplete central seroto-
nin resulted in complex subacute perturbations
comprised of infantile spasms, social disen-
gagement, and restricted behavior (Scantlebury
etal.,2010).
Cognitive Impairments
Memory and cognitive impairments are com-
mon among epilepsy patients (Giovagnoli
and Avanzini, 1999) and have been consist-
ently reported in animal epilepsy models
(Lenck-Santini and Holmes, 2008; Muller
et al., 2009). Causes of these impairments have
been attributed to neuronal cell loss (Holmes
et al., 2002) or dysfunction (Lenck-Santini and
Holmes, 2008; Karnam etal., 2009), as well as to
adverse effects of antiepileptic drugs (Sankar and
Holmes,2004).
Several mediators of inflammation exert
detrimental effects on cognition. For example,
COX-2, an enzyme that is ubiquitously involved
in inflammatory responses and is constitu-
tively expressed in the hippocampus (Yamagata
et al., 1993), negatively regulates learning and
memory (Teather et al., 2002). The increase of
COX-2 expression has been reported in the hip-
pocampus of patients with temporal TLE (Das
et al., 2012; Desjardins et al., 2003; Rojas et al.,
2014), as well as in an animal model of TLE
(Kawaguchi etal., 2005). Deletion of COX-2 from
forebrain neurons improved memory deficits
in mice with pilocarpine-induced TLE (Levin
et al., 2012). Accordingly, a COX-2 inhibitor
165
celecoxib alleviated learning deficits in rats in
which chronic epilepsy had been induced by sys-
temically administered kainic acid (Gobbo and
OMara,2004).
Memory impairments represent one of hall-
marks of the LPS sickness (Tarr etal., 2011), thus
implicating TLR4. In addition, recent studies
have been pointing toward the RAGE-mediated
inflammatory pathway as a negative regulator
of cognitive functions. In particular, increased
RAGE signaling has been implicated in mecha-
nisms of memory impairments in Alzheimers
disease (Arancio etal.,2004).
As it has been discussed earlier, HMGB1 is
overexpressed in the epileptic tissue and may
precipitate ictogenesis via activating both TLR4
(Maroso etal., 2010)and RAGE (Iori etal., 2013).
It is therefore conceivable that stimulation of
both TLR4 and RAGE by HMGB1 may, along
with exacerbating seizures, lead to memory dys-
function (Figure 10.3). Indeed, central adminis-
tration of HMGB1 in wild type as well as TLR4
and RAGE knockout mice impaired animals
performance in the novel object recognition
test, while pharmacological blockade of TLR4 in
RAGE knockout mice abolished amnestic effects
of HMGB1 (Mazarati etal., 2011). Thus excessive
HMGB1 signaling, which has been established
in the epileptic brain, may lead to concurrent
Seizures
(b)
(a)
Proinflammatory
event
(c)
HMGB1
TLR4 RAGE
Cognitive impairments
FIGURE10.3: High mobility group box 1 protein
(HMGB1)-dependent pathways, seizures and
cognitive impairments in epilepsy. (a)Recurring
seizures amplify HMGB1 expression and its extracel-
lular release in the hippocampus. HMGB1 then may
lead to cognitive impairments via the activation of both
a Toll-like 4 receptor (TLR4) and receptor for advance
glycation end products (RAGE). Alternatively a primary
inflammatory event may lead (b) to epilepsy through
discussed mechanisms (e.g., activation of IL-1R1/TLR4
signaling) and (c)to concurrent cognitive impairments
via the activation of HMGB1 signaling.
166 Part II:Homeostatic Control
selective memory deficits via activating both
TLR4 and RAGE (Figure 10.3). In addition, in
light of a possible role of RAGE in Alzheimers
dementia, it is tempting to speculate that the
HMGB1-induced overstimulation of RAGE may
represent a mechanistic link explaining the high
incidence of comorbidity between epilepsy and
Alzheimers disease (Noebels,2011).
In conclusion, brain inflammation may
constitute if not a primary cause then at least a
confounding factor in mechanisms of neurobe-
havioral comorbidities of epilepsy. Inflammatory
processes triggered in the epileptic brain may
translate into interictal behavioral abnormalities.
Hence, inflammatory factors may represent tar-
gets for evidence-based therapeutic interventions,
as well as biomarkers that may effectively assess
the risk of the development of comorbidities of
epilepsy.
CONCLUDING REMARKS AND
T H E R A P E U T I C I M P L I C AT I O N S
There has been an ongoing interest in nonneu-
ronal mechanisms of epilepsy, epileptogenesis,
and ictogenesis. This interest is explained by a
widely acknowledged mismatch between the
advancements in understanding the role of clas-
sical neurotransmitters (particularly GABA and
glutamate) in epilepsy on the one hand and the
lack of progress in pharmacotherapy based on
targeting these neurotransmitter systems on
the other hand. Indeed, a significant propor-
tion of epilepsy patients remains nonrespond-
ent or poorly respondent to antiepileptic drugs
(even the ones of newer generations), which are
designed to effectively normalize the function
of classical transmitter systems. This mismatch
has prompted a search for alternative mecha-
nisms and novel therapeutic targets. Discovery
of active anticonvulsant roles of regulatory pep-
tides (such as somatostatin, neuropeptide Y, and
galanin), adenosine, and endocannabinoids rep-
resent examples of such efforts. The ubiquitous
nature of inflammation makes it reasonable to
assume that inflammatory responses are trig-
gered by seizures when epilepsy is a primary
condition, or reciprocally inflammation stem-
ming from other causes may prime brain to epi-
lepsy and concurrent neurobehavioral disorders
(Figures 10.1 and 10.3). The discussed data cor-
roborate these assumptions in both clinical and
experimental settings. Certainly, it remains to
be seen whether the promise of targeting inflam-
mation for the effective treatment of epilepsy
and its comorbidities will be fulfilled; however,
the accumulated evidence makes a strong case
for anti-inflammatory interventions in epilepsy
patients.
Many drugs are clinically available for the
management of various inflammatory diseases;
therefore their application in epilepsy may be
streamlined, provided that they would show effi-
cacy in clinical trials. Examples include COX-2
inhibitors, IL-1ra (Kineret; used for the treat-
ment of rheumatoid arthritis), monoclonal anti-
bodies against TNF- (Adalimumab, Infliximab,
used in rheumatoid arthritis and Crohns dis-
ease) or IL-1 (Canakinumab, approved for the
treatment of cryopyrin-associated periodic syn-
drome, clinical trials for chronic obstructive pul-
monary disease), and IL-6 (Siltuximab, clinical
trials for non-Hodgkins lymphoma and multiple
myeloma) or treatments targeting hyperactivated
glial cells (e.g., fingolimod, used in multiple scle-
rosis, minocycline). The fact that some of these
drugs do not readily permeate BBB in the normal
brain makes their use challenging, although this
may potentially limit their CNS effects to condi-
tions when BBB is damaged (as discussed earlier).
The clinical development of VX-765, a compound
that blocks IL-1 biosynthesis by inhibiting
interleukin converting enzyme, represents an
example of a more advanced approach, whereby
an anti-inflammatory drug has been evaluated
in a phase 2 clinical study for the treatment of
pharmacoresistant epilepsy (http://clinicaltrials.
gov/ct2/show/NCT01048255;www.epilepsy.com/
files/Pipeline2012/67).
ACK NOWLEDGMENTS
AV is grateful to Fondazione Monzino (Grant
20032005) and EPITARGET (FP7/20072013,
grant agreement n602102) for their support given
to the laboratory of Experimental Neurology at
the Istituto di Ricerche Farmacologiche Mario
Negri, which allowed to obtain some of the find-
ings reported in this review article.
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11
Neuroplasticity
HIROYUKIOKUNO
OVERVIEW
The neural networks of the brain are not static
but rather change dynamically throughout an
individuals lifetime. Neuroplasticity is the abil-
ity of a neural network to change in response to
the environment. At each synapse, the efficiency
of synaptic transmission is adaptively modulated
based on the historic pattern of synaptic activity.
The overall synaptic weight of individual neurons
is homeostatically regulated by compensatory
mechanisms to keep cellular excitability at stable
levels. Such synaptic plasticity serves to maintain
the excitation-inhibition balance within a neu-
ral network and provides pivotal mechanisms
underlying higher cognitive functions includ-
ing learning and memory. At the network level,
neuroplasticity is critically involved in functional
recovery from brain damage or loss of sensory
information. Malfunction of neuroplasticity may
lead to various types of brain disorders, includ-
ing addiction, psychiatric abnormalities, and
cognitive impairment.
INTRODUCTION
The brain shows a huge capacity to coordinate
complex body movements during exercise, to
store personal experiences and knowledge dur-
ing life, and to manage functional recovery from
brain damage caused by injury or stroke. The
mechanisms underlying such plastic and homeo-
static abilities of the brain are of interest not only
to researchers but also to the general public, and
understanding the mechanisms underlying this
plasticity has been one of the central themes in
neuroscience. Neuroplasticity is a comprehensive
term that encompasses changes in synaptic func-
tion, cellular excitability, and network connectiv-
ity in response to information about changes in
the internal and external environment (Cooke
and Bliss, 2006). Neuroplasticity occurs in
various areas and at various levels of the brain
(Buonomano and Merzenich, 1998; Chklovskii
et al., 2004; Kandel, 2001). At the most micro-
scopic level, neuroplasticity is achieved at the
synapse and is referred to as synaptic plasticity.
Synaptic plasticity, which modifies the efficiency
of synaptic transmission (synaptic weight) accord-
ing to the history of synaptic input, can be clas-
sified into several types depending on the mode
of action (Bliss and Collingridge, 2013; Huganir
and Nicoll, 2014). Long-term potentiation (LTP)
and long-term depression (LTD) are the two
most-studied types of synaptic plasticity (see fol-
lowing discussion). Whereas LTP and LTD act on
specific synapses that receive plasticity-inducing
synaptic inputs, other types of synaptic plastic-
ity affect all synapses of the cell. One type of
cell-wide plasticity is homeostatic synaptic scal-
ing. It is now widely thought that at each synapse,
the net synaptic weight is determined by the
summation of input-specific synaptic plasticity
and homeostatic synaptic scaling (Davis, 2006;
Turrigiano, 2008). At more macroscopic levels,
neuroplasticity involves changes in cellular and
network excitability, as well as neural network
connectivity, that lead to cortical reorganization
and remapping (Buonomano and Merzenich,
1998; Hensch, 2004; Shah etal.,2010).
In this chapter I first describe several cat-
egories of synaptic plasticity and its molecular
mechanisms. Ithen present an overview of neu-
roplasticity at the cellular/circuit levels. Finally,
I discuss malfunctions of neuroplasticity as a
possible cause of brain disorders and neuropa-
thology. Although neuroplasticity is fundamen-
tal and observed in a wide variety of species, here
I focus on vertebrate/mammalian nervous sys-
tems, unless otherwise mentioned.
NEUROPLASTICITY
AT S Y N A P T I C L E V E L S
The efficiency of synaptic transmission can be
either strengthened or weakened in response to
particular patterns of synaptic inputs, and such
176 Part II:Homeostatic Control

alternations can be sustained for a short time (A) Synaptic

(second to minutes) or can be long-lasting (hours Long-term potentiation vesicles
HFS (LTP) AMPA-R
to days, weeks, or months). Synaptic plasticity
can also be classified by local (input-specific) Pre
or global (cell-wide) phenomena. Although EPSP
each type of synaptic plasticity involves distinct
molecular and cellular mechanisms, it is gener- Post
ally accepted that the efficiency of synaptic trans- (B)
Long-term depression
mission is primarily determined by the number LFS (LTD)
of receptors for neurotransmitters that are pre-
EPSP
sent on the postsynaptic membrane (Huganir
and Nicoll, 2014). Here Isummarize several types
of input-specific and cell-wide synaptic plasticity.
(C)
Long-Term Potentiation HFS
Heterosynaptic LTD
LTP is one of the most-studied input-specific
synaptic modifications (Bliss and Collingridge, EPSP
1993, 2013). LTP is an enhancement of synap-
tic transmission efficiency that endures for a
long time (Bliss and Gardner-Medwin, 1973;
Bliss and Lomo, 1973; Douglas and Goddard, EPSP
1975; Figure 11.1A). As Hebb (1949) proposed,
when a presynaptic cell Arepeatedly or persis-
FIGURE 11.1: Several types of input-specific
tently takes part in firing a postsynaptic cell B,
long-term synaptic plasticity. (A) Long-term
the synaptic efficiency of cell B from cell A is
potentiation (LTP): Strong high-frequency synaptic
increased. This coincidence of pre- and post-
stimulation (HFS) (e.g., 100 Hz, 100 pulses) to presyn-
synaptic activation ensures input specificity of
apses evokes long-lasting (> hours) enhancement of
LTP because the synaptic efficiencies of all other
excitatory postsynaptic potentials (EPSP) at the postsyn-
postsynapses connected with nonstimulated
apses. The coincidence of presynaptic and postsynaptic
cells are unchanged. The temporal order and
activation is required for induction of LTP (Hebbs rule)
timing of presynaptic and postsynaptic activa-
and the synaptic changes are spatially restricted to the
tion are critical determinants for the synaptic
site of activation (input-specificity, i.e., homosynaptic
efficiency; the sign and amplitude of changes in
expression). (B)Long-term depression (LTD):In contrast
synaptic transmission are precisely controlled
to LTP, low-frequency synaptic stimulation (LFS) (e.g.,
with the range of tens of milliseconds, a mecha-
1 Hz, 900 pulses) to presynapses induces long-lasting
nism known as spike-timing dependent plastic-
(> hours) reduction of EPSP at the postsynapses. This
ity (Magee and Johnston, 1997; Markram etal.,
type of LTD also follows Hebbs rule and is homosynap-
1997). Interestingly, while LTP is primarily
tic. (C)Heterosynaptic LTD:In some cases, homosynap-
expressed at the site that receives strong repeti-
tic synaptic plasticity also involves changes in synaptic
tive synaptic inputs, it can also be established
efficiency at the synapses that are not directly stimulated.
at other synaptic sites if the synapses receive
This is called heterosynaptic plasticity. Heterosynaptic
weak synaptic stimulation that per se is insuf-
LTD is observed in association with homosynaptic LTP
ficient to induce LTP concurrently with the
in several brain areas and neural networks. AMPA-R;
LTP-inducing strong stimuli. This property is
AMPA-type glutamate receptor.
called associativity of LTP. LTP also is coop-
erative in the sense that, even though a single
presynaptic fiber with weak stimulation fails to focal high-frequency stimulation with a micro-
elicit LTP, many of such weakly activated fibers electrode to presynaptic fibers (Bliss and Lomo,
can cooperatively induce LTP if the presynaptic 1973; Gustafsson etal., 1987; Kauer etal., 1988),
inputs converge on a postsynaptic region to pro- spike-timing dependent plasticity protocols
vide sufficient postsynaptic depolarization. The (Magee and Johnston, 1997; Markram et al.,
associativity and cooperativity together govern 1997; Zhang etal., 1998)or postsynaptic depo-
input specificity of LTP. Selective establishment larization with optically controlled repeated
of LTP at a subset of synapses, but not at other glutamate application (Harvey and Svoboda,
sets of synapses, can be induced by applying 2007; Matsuzaki etal.,2004).

There are various types of LTP: these oper-
ate by distinct molecular mechanisms, depend-
ing on the cell type, inputs, and brain areas.
Likely the most-studied LTP is N-Methyl-
d-aspartate (NMDA)-type glutamate recep-
tor (NMDA-R)-dependent LTP, which occurs
at excitatory synapses in the CA1 hippocam-
pal region (Shaffer collateral-CA1 pyramidal
cell connections; Bliss and Collingridge, 2013).
This Hebbian-type synaptic plasticity is now
understood as follows: high-frequency stimu-
lation, such as 100 pulses at 100 Hz, induces
glutamate release from presynaptic sites. The neu-
rotransmitter activates postsynaptic -amino-
3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA)-type glutamate receptors (AMPA-Rs),
as well as NMDA-Rs. The activated NMDA-Rs
permit Ca 2+ influx, which subsequently activates
chemical reaction cascades at the postsynaptic
site to enhance the number of AMPA-Rs on the
postsynaptic surface. Anumber of pathways and
molecules have been suggested to contribute to
the NMDA-dependent LTP process (Bliss and
Collingridge, 2013; Huganir and Nicoll, 2014).
Among them, Ca2+/Calmodulin-dependent pro-
tein kinase II (CaMKII) is one of the most prom-
ising key molecules. Pharmacological inhibition
or genetic disruption of CaMKII impairs hip-
pocampal LTP (Malinow etal., 1988; Otmakhov
et al., 1997; Otmakhov et al., 2004; Silva et al.,
1992b). While many works dealing with LTP
focus on excitatory synapses between excita-
tory neurons (glutamatergic neurons), others
have investigated LTP at excitatory synapses of
inhibitory neurons (gamma-aminobutyric acid
(GABA)-ergic neurons). Interestingly, in contrast
to previously described NMDA-R-dependent
Hebbian LTP, which requires postsynaptic depo-
larization for its induction, some forms of LTP
in inhibitory neurons critically depend on post-
synaptic hyperpolarization, instead of depolari-
zation. Because this type of LTP is blocked by
conventional postsynaptic activation during its
induction, it is called anti-Hebbian LTP (Lamsa
etal., 2007; Kullmann and Lamsa,2007).
Theoretical studies have proposed that LTP
is the cellular substrate of memory (Morris
et al., 2003). Several lines of experimental evi-
dence have also demonstrated the involvement
of LTP in memory-formation processes. First,
learning-induced LTP has been observed in
the CA1 region during a passive avoidance task
(Whitlock et al., 2006) and the amygdala after
a fear-conditioning paradigm (Nabavi et al.,
2014) in free-moving rats. Saturation of LTP in
Neuroplasticity 177
the hippocampus interferes with spatial mem-
ory formation in the Morris water-maze task
(Barnes etal., 1994; Moser etal., 1998). Conversely,
learning-induced LTP occludes subsequent induc-
tion of LTP in the hippocampus (Whitlock
et al., 2006) and in the primary motor cortex
(Rioult-Pedotti et al., 2000). Furthermore, many
genetically modified mice that are deficient in LTP
show reduced/impaired memory formation (Abel
etal., 1997; Bourtchuladze etal., 1994; Grant etal.,
1992; Lee etal., 2003; Plath etal., 2006; Silva etal.,
1992a). These observations, together with theoreti-
cal considerations and modeling, strongly support
the idea that LTP is a cellular mechanism that is cru-
cial for learning and memory (Neves etal.,2008).
Long-Term Depression
LTD is a synaptic plasticity mechanism that func-
tions in the opposite fashion to LTP. The syn-
apses that receive a particular frequency range
of stimulation show long-lasting decreases in
synaptic transmission efficiency (Dudek and
Bear, 1992; Luscher and Huber, 2010; Malinow
and Malenka, 2002; Mulkey and Malenka,
1992). For example, low-frequency stimulation,
such as 1 Hz for 15 min, induces LTD in Shaffer
collateral-CA1 pyramidal cell synapses (Dudek
and Bear, 1993) (Figure 11.1B). This synaptic
modification also requires coincident activa-
tion of pre- and postsynapses (i.e., Hebbian-type
plasticity). As in the case of LTP, several types
of LTD mechanisms have been identified. Two
major types of LTD mechanisms are NMDA-R
dependent and metabotropic glutamate recep-
tor (mGluR) dependent (Bear and Abraham,
1996). In both cases, a reduction in AMPA-Rs at
the postsynaptic membrane is thought to be the
principal molecular mechanism of LTD expres-
sion at excitatory synapses in the central nervous
system. Although the mechanism of the AMPA-R
reduction in LTD is not fully understood, several
possible mechanisms, including the regulation
of endocytosis and diffusion of AMPA-Rs on the
plasma membrane, have been proposed (Man
etal., 2000; Wang and Linden, 2000; Waung etal.,
2008; Xia etal.,2000).
In the cerebellum, a center of motor coordi-
nation and postural control, LTD occurs at excit-
atory synapses between parallel fibers (PFs) and
Purkinje cells (Ito, 1989). Purkinje cells receive
two types of inputs:one from PFs from cerebel-
lar granule cells and the other from the climb-
ing fibers from the inferior olive nucleus. When
Purkinje cells receive simultaneous inputs from
PFs and climbing fibers, PF-mediated synaptic
178 Part II:Homeostatic Control
responses decrease in an mGluR1-dependent
manner (Aiba et al., 1994; Kano et al., 1997).
Because mGluR1 is a G-protein-coupled metabo-
tropic receptor and is coupled with phospholipase
C, the mGluR1 activation results in an intracel-
lular Ca2+ increase as well as in protein kinase
C (PKC) activation via IP3/diacyl-glycerol second
messenger systems. Cerebellar LTD is thought to
play critical roles in the vestibulo-ocular reflex
and in motor learning (Ito, 2000; Lee etal., 2009;
Shibuki etal., 1996; Shutoh etal.,2006).
Heterosynaptic LTP and
HeterosynapticLTD
To date, the majority of studies that have
addressed long-term synaptic plasticity have
focused on LTP and LTD at the synaptic site
where the plasticity-inducing stimuli were origi-
nally given. Because the site of change corre-
sponds to the site of stimulation, this mode of
synaptic plasticity is called homosynaptic plastic-
ity (homosynaptic LTP and homosynaptic LTD).
However, neurons also have the ability to induce
synaptic changes at sites other than the site of
homosynaptic plasticity, in order to balance the
initial input-specific synaptic change. For exam-
ple, when strong synaptic inputs induce LTP in
a subset of synapses, another subset of synapses
can undergo LTD accordingly (Abraham and
Goddard, 1983; Barry etal., 1996). Because this
type of LTD occurs at synapses that have not
received plasticity-inducing inputs, it is called het-
erosynaptic LTD (Figure 11.1C). Heterosynaptic
LTD can be envisioned as a homeostatic process
to maintain local synaptic inputs at a constant
level after a set of synapses are potentiated by
LTP-inducing stimuli (Abraham, 1996). This het-
erosynaptic mechanism could also play a role in
accentuating the contrast of synapses undergoing
LTP with surrounding unpotentiated synapses.
Heterosynaptic LTP after induction of homosyn-
aptic LTD has also been observed in some brain
areas (Nguyen, 2001; Royer and Pare,2002).
It is known that glial cells release diffusible
factors, including D-serine, adenosine triphos-
phate (ATP), and tumor necrosis factor (TNF)-
for cellcell communication (Ben Achour and
Pascual, 2010; Fields and Burnstock, 2006; Perea
et al., 2009; Stellwagen and Malenka, 2006).
Those factors, called gliotransmitters, act not only
on glia themselves but also on neighboring neu-
rons to modulate synaptic functions (Perea etal.,
2009). Because individual astrocytes contact
with hundreds of synapses of neighboring neu-
rons, gliotransmitters released from an astrocyte
likely have impacts on areas beyond single syn-
apses. Indeed, D-serine and ATP released from
glial cells have been implicated in LTP on multi-
ple neurons (Henneberger etal., 2010)and heter-
osynaptic LTD (Pascual etal., 2005), respectively.
Synaptic Tagging and
Inverse Tagging
The maintenance of long-term synaptic plastic-
ity requires new synthesis of transcripts and pro-
teins (Costa-Mattioli et al., 2009; Kandel, 2001;
Kelleher et al., 2004; Okuno, 2011). This is espe-
cially true during a particular time window after
synapses receive plasticity-inducing stimuli, when
newly synthesized plasticity-related proteins may
functionally interact with local synaptic tags. The
synaptic tagging and capture theory adopts a con-
ceptual framework in which activity-triggered
local changes at synaptic sites (i.e., synaptic tagging)
permit the use of activity-induced plasticity-related
proteins at the cell body and dendrites (i.e.,
plasticity-related protein capture) to stabilize and
maintain changes in synaptic efficacy (Frey and
Morris, 1997; Martin et al., 2000; Figure 11.2A).
Several expanded versions of this hypothesis have
been proposed and are experimentally supported
(Fonseca etal., 2004; Sajikumar etal., 2007). The
synaptic tagging and capture hypothesis explains
how protein-synthesis-dependent LTP recon-
ciles input-specific synaptic modifications with
the broad cellular distribution of newly synthe-
sized proteins that are required for LTP mainte-
nance (Govindarajan et al., 2006; Redondo and
Morris,2011).
An opposite, but nonmutually exclusive,
hypothesis is the inverse synaptic tagging model,
which assumes the existence of synaptic tags that
mark nonpotentiated synapses and play a role in
a process to prevent these synapses from being
strengthened (Figure 11.2B). A recent study has
indicated that an activity-dependent protein,
called Arc, may be involved in such an inverse
synaptic tagging process (Okuno et al., 2012).
When Arc is induced by LTP-eliciting stimuli in
neurons, the newly synthesized Arc mainly accu-
mulates at nonpotentiated synapses rather than
at potentiated synapses. The levels of synaptic
Arc accumulation were found to be negatively
correlated with the surface expression levels of
AMPA-Rs (Chowdhury etal., 2006; Okuno etal.,
2012; Shepherd etal., 2006). Because Arc induc-
tion has been shown to correlate with ongoing
cognitive activity in the hippocampus and the
cortex (Guzowski et al., 1999), and because Arc
absence causes severe memory disorders (Plath

(A) Synaptic tagging
Potentiated
synapse EPSP
Un-potentiated Un-potentiated
synapses synapses
(B) Inverse synaptic tagging
EPSP
Un-potentiated
or
synapses
Potentiated
synapse
FIGURE 11.2: Synaptic tagging and capture
model. (A) Synaptic tagging: During the induction
phase of long-term potentiation (LTP), plasticity-
inducing stimuli develop a putative signature (tag)
within the activated synapses (a red flag). This synap-
tic tag serves as a mark to distinguish the synapses to
be potentiated from other nonstimulated synapses and
stimulus-induced plasticity-related proteins (PRPs; red
circles) are then captured based on the presence of the
synaptic tags. The PRPs are required to maintain the
EPSP enhancement during the late phase of LTP. (B)
Inverse synaptic tagging: In contrast to the synaptic
tagging, the inverse synaptic tag (blue flags) is created
in synapses other than the synapse to be potentiated.
The stimulus-induced negative PRPs (blue circles) are
captured in the nonpotentiated synapses and play a role
in preventing undesired synaptic augmentation during
the late phase ofLTP.
etal., 2006), the inverse synaptic tagging of Arc
may subserve memory consolidation by prevent-
ing undesired synaptic enhancement at weak
synapses while sparing potentiated synapses
(Kim et al., 2012; Okuno et al., 2012). Such an
inverse synaptic tagging mechanism may also
participate in some forms of heterosynaptic LTD
(see previous discussion).
Synaptic Scaling
The types of synaptic plasticity described pre-
viously are expressed in spatially limited areas
Neuroplasticity 179
where synapses receive plasticity-inducing stim-
uli. In contrast, other types of synaptic plastic-
ity involve synaptic modification in a cell-wide
manner (Burrone etal., 2002; Thiagarajan etal.,
2005). Synaptic scaling (also see later discussion)
is one such cell-wide mechanism that homeo-
statically modifies the strength of all synapses
in response to global changes in synaptic inputs
(Desai et al., 2002; Turrigiano et al., 1998). For
example, when the synaptic activity of cultured
neurons is chronically suppressed by the sodium
channel blocker tetrodotoxin, all synapses in the
neurons gain synaptic strength. In the opposite
scenario, chronic enhancement of synaptic activ-
ity with GABA receptor blockers in neuronal cul-
ture results in the reduction of synaptic efficiency
at all synapses. This bidirectional change in each
synapse occurs in proportion to its initial strength
(i.e., a multiplicative function); thus this phe-
nomenon is called synaptic scaling (Figure 11.3 ;
Turrigiano, 2008). Synaptic scaling involves
several cell-wide mechanisms associated with
transcription and translation in the cell body,
but it also includes synapse-specific components
EPSP
EPSP
Chronically
low activity
Normal neuronal
activity
Chronically
high activity
FIGURE 11.3: Homeostatic synaptic scaling.
Contrary to homosynaptic plasticity, synaptic scaling
is a mechanism that influences all synapses in the cell.
Through this homeostatic mechanism, the surface
expression levels of AMPA receptors are upregulated
at all synapses under conditions of chronically low
neuronal activity (left), while surface AMPA receptor
levels are downregulated with persistent high neuronal
activity (right).
180 Part II:Homeostatic Control
(Beique etal., 2011; Sutton etal., 2006). In gen-
eral, synaptic scaling is thought to be advanta-
geous for keeping neuronal excitability within a
certain dynamic range without affecting the rela-
tive balance between strong and weak synapses
(Turrigiano,2008).
Because activity-dependent gene expres-
sion is critical for synaptic scaling and other
forms of synaptic homeostasis (Ibata et al.,
2008; Sutton et al., 2006), a number of labo-
ratories have attempted to identify the genes
involved. Several neuronal immediate-early
genes, a class of genes that is rapidly and
transiently induced by synaptic activity, have
been shown to critically contribute to syn-
aptic homeostatic changes. The immediate
early gene Arc is rapidly induced by synaptic
activation in an NMDA-R-dependent man-
ner (Link etal., 1995; Lyford etal., 1995). This
activity-regulated Arc expression and the role
of Arc in promoting AMPA-R endocytosis con-
stitutes a cell-autonomous loop that accounts
for the homeostatic control of AMPA-R sur-
face expression levels (Rial Verde et al., 2006;
Shepherd etal., 2006). When the levels of excit-
atory inputs increase, neurons express more
Arc. The induced Arc then promotes AMPA-R
endocytosis at postsynaptic sites, resulting
in a reduction in responsiveness to excitatory
inputs (Shepherd etal., 2006). By contrast, the
cells with suppressed synaptic inputs cease
to express Arc; thus Arc-induced AMPA-R
endocytosis is reduced, resulting in increased
cellular responsiveness. In this way, cellular
responsiveness is homeostatically controlled
by activity-regulated Arc expression. Indeed,
Arc knockout neurons do not show homeostatic
synaptic scaling both in vitro and in vivo (Gao
et al., 2010; Shepherd et al., 2006). Consistent
with this, a recent work has demonstrated that
Arc expression during synaptic scaling is reg-
ulated by a protein kinase called MSK1, and,
importantly, knockout of MSK1 results in loss
of synaptic scaling (Correa etal., 2012). Asimi-
lar but distinct activity-dependent mechanism
with another immediate-early gene product,
Homer1a, has been reported (Hu etal.,2010).
Neuronal activity-regulated pentraxin (Narp)
is also a major neuronal immediate-early gene
product. Unlike Arc and Homer1a, which are
postsynaptic proteins, Narp is a secreted protein
that localizes to excitatory synapses (OBrien
etal., 1999). Interestingly, activity-induced Narp
predominantly accumulates, and facilitates the
recruitment of AMPA-Rs, at excitatory synapses
on parvalbumin-expressing inhibitory neurons
(Chang et al., 2010). The Narp-mediated effect
on parvalbumin neurons results in a homeostatic
increase in inhibitory inputs within the neuronal
network, suggesting that Narp mediates cell
nonautonomous homeostatic regulation of the
excitatory-inhibitory balance (Chang etal.,2010).
NEUROPLASTICITY
AT T H E N E T W O R K L E V E L
In addition to modulations in synaptic weight and
cellular excitability, there are several additional
mechanisms for changing network connectivity,
by generating new synapses and/or by pruning
existing synapses. Such changes in connectivity
can be most dynamically observed in the brain
after a drastic perturbation of sensoryinput.
Cortical Reorganization
and Remapping
The somatosensory area of the cerebral cor-
tex receives tactile information from each part
of the body in a topologically organized man-
ner. When one part of the body is severely
damagedfor example, finger amputationthe
area representing the finger suddenly loses sen-
sory input. However, as time goes by, the area
recovers neural activity by receiving sensory
input from the remaining fingers (Merzenich
et al., 1984). This phenomenon is called corti-
cal reorganization or cortical remapping and is
thought to be a compensatory adaptive response
to the loss of a bodypart.
Similarly, cortical reorganization is observed
in other sensory cortices. One of the most-studied
areas is the primary visual cortex. In primates,
including humans, as well as many carnivorous
mammals, visual inputs from two eyes are sepa-
rately transmitted to and segregated within the
primary visual cortex (Hubel etal., 1976). When
one eye is occluded for days to weeks in a par-
ticular time window during development (i.e., a
critical period), neural activity in the visual areas
corresponding to each of the two eyes becomes
unbalanced. The area responding to the intact
eye expands, while the cortical area correspond-
ing to the occluded eye shrinks (Figure 11.4). This
phenomenon is known as ocular dominance plas-
ticity and was originally demonstrated in ani-
mal models, such as with cats and monkeys, but
has now been observed in many other species,
including mice (Gordon and Stryker, 1996; LeVay
etal., 1980; Shatz and Stryker, 1978). The molec-
ular and cellular mechanisms underlying ocu-
lar dominance plasticity have been extensively

Right
Left Monocular
deprivation
Right
Left
FIGURE 11.4: Cortical plasticity after sensory-
input deprivation. Ocular dominance plasticity in
the visual cortex. In many mammals such as primates
and cats, the primary visual cortex is organized to
create the alternating columnar structures (blue and
red stripes) corresponding to each eye during normal
development (top). When visual information from one
eye is deprived by eye-lid suture or injury, especially
in a particular time window during development (criti-
cal period), the cortical areas of the nondeprived eye
become larger than those of the deprived eye (bottom).
investigated using a combination of electrophysi-
ology, optical imaging, genetics, and molecular
biology in recent decades (Hensch,2004).
Homeostatic Response Recovery
Recent in vivo live imaging and electrophysi-
ological studies have demonstrated the dynam-
ics of homeostatic responses after sensory input
manipulation in the visual cortex. Keck et al
(2013), using in vivo 2-photon Ca 2+ imaging,
reported that neuronal activity in the visual cor-
tex of mice transiently decreased after retinal
lesions deprived the cortex of visual input, but
the cortical activity gradually recovered over one
to two days after the lesioning (Keck etal., 2013).
This homeostatic response was accompanied by
synaptic scaling (increases in mEPSC ampli-
tude) and spine-head enlargement in acute brain
preparations. Furthermore, although synaptic
scaling was observed during the early phase,
the authors also observed a reduction in inhibi-
tory inputs during the late phase of homeostatic
Neuroplasticity 181
plasticity (Keck etal., 2013). Hengen etal. (2013)
performed chronic multiunit recording from
the rat visual cortex and revealed that cellular
activity transiently dropped after eye-lid suture
(i.e., monocular deprivation) but that normal
activity resumed by six days after the depriva-
tion (Hengen etal., 2013). The authors also dem-
onstrated that mEPSC amplitude was enhanced
at six days after deprivation in ex vivo prepa-
rations, indicating that synaptic scaling con-
trols the homeostatic process in this phase. It is
worth noting that such homeostatic responses
in the visual cortex after manipulation of vision
are mediated by Arc. Arc knockout mice fail
to exhibit visual experience-induced homeo-
static plasticity of mEPSC amplitude in the
visual cortex (Gao etal., 2010), as well as ocular
dominance plasticity that normally takes place
after monocular deprivation in wild-type mice
(McCurry etal.,2010).
NEUROPLASTICITY AND
N E U R O L O G I C A L / P S Y C H I AT R I C
DISORDERS
The cognitive and executive functions of the
brain largely rely on the proper control of syn-
apse efficiency and connectivity. Such synaptic
properties are controlled by the balance between
cell-wide (e.g., homeostatic synaptic scaling)
and local (e.g., LTP/LTD) synaptic plasticity. So
what impact does synaptic plasticity impairment
have on brain functions? A number of studies
have demonstrated that gene-knockout animals
that show LTP deficits in the hippocampus also
exhibit impaired long-term memory forma-
tion (Abel et al., 1997; Bourtchuladze et al.,
1994; Grant et al., 1992; Lee et al., 2003; Plath
et al., 2006; Silva et al., 1992a). Hippocampal
LTP is also impaired in many animal models of
Alzheimers disease (Auffret et al., 2009; Gong
et al., 2004; Oddo et al., 2003). In the striatum,
exaggerated LTP in dopaminergic neurons is
associated with drug addiction (Luscher and
Malenka, 2011; Nestler et al., 1993). Moreover,
dysregulation or malfunction of homeostatic
synaptic scaling is often associated with neuro-
pathological processes, including drug addiction
(Boudreau and Wolf, 2005), epilepsy(Chang etal.,
2010; Savin etal., 2009), and neurodevelopmen-
tal disorders such as fragile X (Soden and Chen,
2010)and Rett syndromes (Blackman etal., 2012;
Qiu etal., 2012). These facts suggest that normal
brain functions require the appropriate balance
of cell-wide homeostatic and input-specific syn-
aptic plasticity.
182 Part II:Homeostatic Control
CONCLUDING REMARKS
Brain flexibility is involved in numerous pro
cesses, such as learning and memory, motor
coordination, and recovery from injury, and this
flexibility is based mainly on neuroplastic mecha-
nisms. In this chapter I described several repre-
sentative forms of synaptic plasticity and cortical
reorganization. Neuroplasticity is a broad term
that covers a variety of synaptic and nonsynaptic
plasticity mechanisms. Other neuroplastic mecha-
nisms include structural plasticity, which modifies
the shape and size of synapses (Kasai etal., 2003;
Luo, 2002), intrinsic plasticity, which controls the
biophysical properties of the plasma membrane
(Beck and Yaari, 2008), and metaplasticity, by
which the rules governing synaptic plasticity per se
are dynamically modulated by prior synaptic activ-
ity (Abraham and Tate, 1997; Hulme etal., 2013).
Currently, however, the relationship between the
different types of plasticity remains unclear. This
relationship should be clarified experimentally
and theoretically in the near future. Furthermore,
accumulating evidence is now shedding light on
the fundamental connections between neuroplas-
ticity and other research fields, such as epigenet-
ics (Guzman-Karlsson et al., 2014), glia (Barres,
2008), and metabolism (Liu et al., 2013). In par-
ticular, adult neurogenesis recently emerged as
one of the central topics in neuroscience (Gage
and Temple, 2013). Several reports have indicated
the involvement of adult neurogenesis in memory
(Imayoshi etal., 2008; Kitamura etal., 2009; Sahay
et al., 2011). Further studies of neuroplasticity
might lead to a comprehensive understanding of
our brains from the perspective of basic research
and provide clues to novel therapeutic approaches
to the many clinically unmet needs, including
memory impairment, addiction, psychiatric dis-
orders, and rehabilitation after stroke.
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12
Epigenetics
K AT J A K O B O W A N D I N G M A R B L M C K E
INTRODUCTION
Epigenetic mechanisms are self-perpetuating,
functionally relevant modifications to the
genome not involving changes in nucleotide
sequence. They include covalent modifications
of histone proteins and DNA, chromatin remod-
eling, as well as the action of noncoding RNAs.
The number and variability of epigenetic marks,
their flexibility over time, their interdependence,
and potential synergistic effects may provide the
molecular basis for any phenotypic variation in
physiological and pathological conditions. This
is particularly interesting with respect to post-
mitotic neurons in the brain. Experimental data
showed evidence for epigenetic mechanisms as
fundamental regulatory processes in neurogen-
esis and central nervous system (CNS) develop-
ment, cellular plasticity, and higher order brain
function. There is further compelling evidence
that dysfunctional epigenetic processes are
involved in the pathobiology of neurologic dis-
eases and may serve as the molecular arbiter for
integrating the effects of inherited and acquired
etiological factors and thus for modulating clini-
cal manifestations of a specific disease.
BUILDING BLOCKS
OFTHE EPIGENOME
DNA Methylation
DNA methylation is a covalent chromatin modifi-
cation, characterized by the biochemical addition
of a methyl group (CH3) to cytosine nucleo-
tides. S-adenosyl methionine (SAM) serves as
the methyl donor that is converted to S-adenosyl
homocysteine following methyl group trans-
fer (Figure 12.1). DNA methylation patterns are
directed and preserved by the action of the DNA
methyltransferase (DNMT) family, whereas the
effects of DNA methylation are mediated by
recruitment of the reader methyl-CpG-binding
domain (MBD) family, containing proteins such
as MBD1, MBD2, MBD3, MBD4, MeCP2, and
KAISO (Buck-Koehntop and Defossez, 2013;
Fournier etal., 2012; Lunyak etal.,2002).
Present in all higher eukaryotes including
humans, DNA methylation is mainly confined to
CpG dinucleotides (Dulac, 2010). However, meth-
ylation of non-CpG sites has also been reported
for embryonic stem cells, induced pluripotent
stem cells, oocytes, and the brain (Barres etal.,
2009; Clark et al., 1995; Grandjean et al., 2007;
Ichiyanagi etal., 2013; Lister etal., 2013; Varley
etal., 2013; Ziller etal., 2011). While CpG meth-
ylation is mainly thought to lock the genome and
provide a basal repressive mark, the biological
impact of non-CpG methylation remains poorly
understood. Given that this mark is highly pre-
sent in the adult mouse and human brain but
rare or absent in other differentiated cell types,
a unique role in mammalian brain development
and function can be assumed (Lister etal.,2013).
DNA methylation is usually high within
sequences of low CpG content, for example at
noncoding regions (e.g., centromeric hetero-
chromatin) and interspersed highly repetitive
elements (endogenous retroviruses and transpo-
sons). Therefore it has been suggested to prevent
genomic instability phenomena, making genome
stabilization a primary function of DNA meth-
ylation (Bestor, 2000). In contrast, regions of high
CpG density, referred to as CpG islands (CGIs),
are frequently found in the 5 end of genes and
have been identified as sites of transcription ini-
tiation (Eckhardt etal., 2006; Weber etal., 2007).
CGIs are very frequently located within pro-
moters, often in the direct vicinity of transcrip-
tion start sites. Some CGIs are not associated
with known genes, but we think there are still
unknown genes hidden in the genome. In favor
of this hypothesis respective CGIs show features
of standard gene promoters and active genes
including the binding of specific proteins (PolII),
chromatin modifications that support active
188 Part II:Homeostatic Control
FIGURE 12.1: DNA methylation and demeth-
ylation dynamics in the mammalian brain.
Potential mechanisms of DNA demethylation. Passive
DNA demethylation is thought to occur by a reduc-
tion in activity or absence of DNA methyltransferases
(DNMT) during DNA replication, whereas active
DNA demethylation involves DNA repair mecha-
nisms: 5-methylcytosine (5-mC) is hydroxylated by
ten-eleven translocation (TET) to form the key inter-
mediate 5-hydroxymethylcytosine (5-hmC). 5-mC or
5-hmC are deaminated by activation-induced cyti-
dine deaminase (AID) to form thymine (not shown)
or 5-hydroxymethyluracil (5-hmU), which are then
removed by thymine DNA glycosylases (TDG) and
exchanged by unmethylated C through the BER path-
way. Alternatively, 5-hmC may be further oxidized by
TET to form other intermediates, which are converted
to cytosine or 5-hmC may be lost during replication.
Double arrowheads indicate chemical chain reactions,
where intermediates were not shown for simplicity.
Other abbreviations: SAM = S-adenosyl methionine; Zhang, 2010). Genomic or locus-dependent DNA
SAH =S-adenosyl homocysteine; Gadd45 = growth
arrest and DNA-damage-inducible.
gene expression and RNA that is obviously tran-
scribed from these gene loci. All of these findings
point to orphan CGIs as sites of transcription
initiation (Illingworth etal.,2010).
The great majority of CGIs are unmethyl-
ated at all stages of development in all normal,
nondiseased tissue types (Bird, 2002; Edwards
etal., 2010), thereby retaining an open chromatin
structure for potential accession of transcription
factors and dynamic regulation of gene expres-
sion (Bergman and Cedar, 2013). However, when
present, promoter methylation is generally cor-
related with gene repression. In contrast to pro-
moter regions, gene body methylation shows less
stringent associations with gene silencing. There
are reports that intragenic methylation is indica-
tive for active transcription, including the regu-
lation of alternative splicing (Sati et al., 2012).
Other studies suggested a role of intragenic DNA
methylation in controlling alternative promoter
usage, particularly in the brain (Maunakea etal.,
2010). It is generally anticipated that downstream
effects of DNA methylation may result from
(a)interference with transcription factor binding,
(b) recruitment of methyl-binding proteins and
their associated regulatory complexes, and/or
(c)induced chromatin remodeling. Interestingly,
mammalian transcription factor binding sites
are more CG rich than the bulk genome, and
many contain CpG within their recognition
sequence (Deaton and Bird, 2011), suggesting a
strong interdependence.
DNA methylation is present in all higher
eukaryotes, including humans (Dulac,
2010) and, besides general stabilization of the
genome, is associated with parent-of-origin
imprinting (Edwards and Ferguson-Smith,
2007), X-chromosome inactivation in females
(Minkovsky et al., 2012), during the process of
aging (Madrigano et al., 2012; Numata et al.,
2012), in lineage commitment (e.g., during neu-
rogenesis; Ma etal., 2010), and in neural plastic-
ity (Feng etal., 2010; Guo etal., 2011a; Levenson
etal., 2006; Miller and Sweatt, 2007; Nelson etal.,
2008). Despite previous hypotheses, DNA meth-
ylation has proven to be highly dynamic not only
during development and cellular differentiation
(Jaenisch and Bird, 2003; Reik etal., 2001; Reik
and Walter, 2001)but also in adult and postmi-
totic cells (Guo etal., 2011a; Reik, 2007; Wu and
methylation loss were observed in both physi-
ologic and pathologic conditions, but no DNA
demethylase has been identified so far (Ooi and
Bestor, 2008). DNA methylation may be pas-
sively lost or diluted during the cell cycle, when
DNA methylation enzymes and/or their com-
plexes are denied access to the newly replicated
DNA (Figure 12.1, left panel). In addition, active
DNA demethylation may be facilitated in non-
dividing cells through the selective recruitment
of cytidine deaminases and DNA glycosylases
(Figure 12.1, right panel; Gong and Zhu,
2011). A common intermediate in the pro-
cess of DNA demethylation seems to be
5-hydroxymethyl-Cytosine (5-hmC). The func-
tion of 5-hmC is still matter of debate, but there
is some evidence for 5-hmC-mediated epige-
netic dynamics during postnatal development
and aging in the rodent as well as the human
brain (Hahn et al., 2013; Szulwach et al., 2011;
Wang et al., 2012). Oxidative deamination by
the activation-induced cytidine deaminase/
 Epigenetics 189

apolipoprotein B messanger RNA (mRNA)- phosphorylation, methylation, ubiquitination,

editing catalytic component family of deaminases
converts 5-hmC to 5-hmU or other interme-
diates, which may all be further processed by
base excision repair enzymes (Guo etal., 2011b).
Another enzyme considered to facilitate active
DNA demethylation is the growth arrest and
DNA damage 45 (Gadd45) family of genes (Engel
etal., 2009). Gadd45 has been suggested to act as
an adapter protein for nucleotide excision repair
and/or base excision repair enzymes promot-
ing removal of 5-methylcytosine from DNA at
selected gene-specific loci (Schafer,2013).
Posttranslational Histone
Modification
DNA is wrapped around histone octamers con-
taining two H2A, H2B, H3, and H4 proteins,
each forming the basic unit of chromatinthe
nucleosome. Each histone protein is composed
of a central globular domain and an N-terminal
tail that contains multiple sites for posttrans-
lational modification, including acetylation,
and ADP-ribosylation (Khorasanizadeh, 2004).
Intriguingly, multiple posttranslational histone
modifications may function combinatorially and/
or sequentially to regulate downstream function
(Fischle, 2008; Wang et al., 2008). Through its
complexity, the so-called histone code ensures
nuclear processes (transcription, replication,
DNA damage response) to be directed to the
required region of the genome at the appropriate
time, mediating unique cellular responses and
biological outcomes (Figure12.2).
In histone acetyltransferase, a negatively
charged acetyl group is added to lysine resi-
dues on histone proteins. An acetylated lysine
has no longer a basic side chain and interferes
with the usual electrostatic affinity between
histone proteins and the DNA backbone. It is a
hypothesis structure more permissive to gene
expression (Graff and Tsai, 2013). Histone meth-
ylation can target lysine or arginine residues
(Di Lorenzo and Bedford, 2011; Lachner and
Jenuwein, 2002). Of note, different methylation

NH2
NUCLEOSOME

C K119

Ub NH2
H2A H2B
Ub
Ub

H4 H3

P A
NH2 K36
K20 S10
K16 K27 K9
M

M M M M M M
M M
M

A
M

M M NH2
K4
M

FIGURE 12.2: Nucleosome structure and histonecode. Simplified scheme of nucleosome structure and
histone code. All histones are subject to posttranscriptional modifications, which mainly occur in N-terminal
histone tails. Posttranscriptional histone modifications include acetylation (Ac), methylation (M), phosphoryl-
ation (P), and ubiquitination (Ub), among others. Each modification has specific functions in gene regulation
(green=activation, red=inhibition), whereby interdependence of signals and crosstalk between different signals
allows very fine-tuning of gene expression. The sum of all activating and inhibiting signals define the activity state
of a specific gene. Histone acetylation has long been associated with increased gene expression. More recently,
histone lysine methylation has been linked to both gene activation and gene repression, depending on the site
and extent of methylation. The number associated with each amino acid represents its position in the sequence.
K=lysine; S=serine; NH2=N-terminal histone tail; C=C-terminaltail.
190 Part II:Homeostatic Control
marks are established by specific enzymes with
relatively little overlap in substrate and function
(Kouzarides, 2007; Wu et al., 2010). They may
further have opposing effects on gene expres-
sion, dependent on the targeted amino acid.
Methylation of H3K9, H3K27, H4K20 has been
associated with condensed chromatin states
and gene repression (Martin and Zhang, 2005),
although different effects could be observed
with mono-, di-, or trimethylation of the same
amino acid (Barski et al., 2007). H3K9 meth-
ylation has been particularly implicated in the
induction and propagation of heterochroma-
tin formation, whereas H4K20 methylation is a
key regulator of biological processes that ensure
genome integrity, such as DNA damage repair,
DNA replication, and chromatin compaction
during cell cycles (Jrgensen etal., 2013; Tardat
et al., 2007). H3K27 methylation contributes
to the inactivation of gene expression. Given
the critical role of H3K27 methylation in the
balance of gene activity, it is not surprising to
find anomalies of this system in different types
of cancer (Martinez-Garcia and Licht, 2010).
Both H3K9- and H3K27-trimethylation have
been further described as playing a crucial role
in regulating DNA methylation, thereby link-
ing different epigenetic repression systems
(Lehnertz et al., 2003; Vire et al., 2006). This
interdependence seems to hold true for most of
the genome, except CpG islands in postmitotic
neurons, where more recent evidence showed
that H3K27-trimethylation and DNA methyla-
tion are mutually exclusive and work antagonis-
tically to silence genes (Brinkman etal.,2012).
Other methylation marks seem to be restricted
to active gene promoters (e.g., H3K4 methylation
associated with transcription initiation), while
H3K36 methylation may support transcription
elongation (Rando, 2012). Monomethylation of
H3K4 distinguishes active enhancers, whereas
trimethylation of H3K4 is highly enriched at
promoters and around transcriptional start sites
(Heintzman etal., 2007). Binding of H3K4-specific
methyltransferases protects promoters of devel-
opmental genes from DNA methylation (Smith
and Meissner, 2013), further underlining its role
in gene activation. Intriguingly, developmen-
tally critical genes frequently contain a bivalent
domain within their promoters, where function-
ally opposing H3K4- and H3K27-trimethylation
marks are present at the same time. This pattern
allows lineage-specific genes to be either silenced
or activated as differentiation proceeds (Bernstein
etal.,2006).
In addition to the well-known acetylation or
methylation, histones can also be ubiquitinated
(Zhang, 2003). This modification was long con-
sidered to be associated only with the protein
degradation system, whereas its contribution to
transcriptional regulation remained uncertain.
Proteasome-dependent degradation of polyubiq-
uitinated transcription factors or linker protein
H1 are important regulatory mechanisms, but
now proteasome-independent mechanisms have
also come into focus. The carefully orchestrated
addition and removal of single ubiquitin moieties
to histones has been associated with gene regu-
lation (Davie and Murphy, 1990). There is fur-
ther evidence for an interdependence of histone
ubiquitination and other epigenetic mechanisms.
Intriguingly, H2B monoubiquitination appears
to be implicated in the recruitment of some
methyltransferases mediating di- and trimeth-
ylation of H3K4 and H3K79 commonly associ-
ated with transcriptionally active chromatin (Lee
etal., 2007). Moreover, ubiquitination of histones
may regulate histone acetylation and facilitate
histone eviction, as the sequential acetylation
and ubiquitination of H2AX has been shown to
promote histone dynamics (Ikura et al., 2007).
An interdependence of H2A ubiquitination, his-
tone, and DNA methylation has been established
and linked to X-chromosome inactivation as well
as polycomb silencing (de Napoles et al., 2004;
Sarcinella et al., 2007; Wang et al., 2004; Wu
et al., 2008). These findings promote an impor-
tant role for histone ubiquitination in transcrip-
tional regulation. However, most findings were
obtained in yeast and flies and await experimen-
tal confirmation in higher organisms. An explicit
role in the homeostasis of brain function has not
been shownyet.
NoncodingRNAs
Noncoding RNAs are small, functional RNA
molecules that are not translated into protein but
are implicated in transcriptional and posttran-
scriptional regulation of gene expression. They
include microRNAs (miRNAs; He and Hannon,
2004), long noncoding RNAs (Mercer et al.,
2009), and PIWI-interacting RNAs (P-element
induced wimpy testis in Drosophilas; Luteijn and
Ketting, 2013), all characterized by individual
biogenesis, mode of target recognition, regula-
tory properties, and transgenerational epigenetic
states. Here we focus on miRNAs, a group of
highly conserved small regulatory RNAs playing
essential roles in CNS development and neuronal
function, including proliferation of neural stem
 Epigenetics 191

and progenitor cells, neuronal differentiation
and maturation (De Pietri Tonelli et al., 2008;
Sun etal., 2013a), neurite outgrowth, and synap-
togenesis (Jovicic etal., 2013; Olde Loohuis etal.,
2012; Yoo etal., 2011). Despite their critical role
in neurodevelopment, miRNAs are by definition
not epigenetic players per se but can regulate
other epigenetic layers (Zhou et al., 2010) or be
epigenetically regulated themselves (Lopez-Serra
and Esteller,2012).
Biogenesis of miRNAs initially starts with
transcription from noncoding regions within
the genome followed by sequential cleavage
of the primary stem loop structure transcripts
(pri-microRNA) to form mature miRNAs (Kim
et al., 2009). Processing of miRNA precursors
is mediated sequentially by RNase-III enzymes
Drosha and Dicer. Finally, mature miRNAs
guide the RNA-induced silencing complex to 3
untranslated regions of complementary target
mRNAs and repress gene expression by either
destabilization of mRNA transcripts (i.e., decap-
ping, deadenylation, degradation) or repression
of mRNA translation. There is also evidence for
transcriptional and translational activation by
miRNAs, introducing a new level of complexity
(Figure 12.3; Vasudevan etal.,2007).
Studies on region-specific genetic ablation of
Dicer and the subsequent blocking of miR bio-
genesis at different developing stages are com-
patible with global effects of miRNAs on CNS
development (Davis et al., 2008; Kawase-Koga
et al., 2009). Cortex-specific Dicer conditional
knockout mice displayed a reduced neural pro-
genitor pool and abnormal neuronal differen-
tiation (Kawase-Koga et al., 2009). Similarly,
knockout of specific miR genes (e.g. miR-9,
miR-124) proved their implication in embry-
onic and adult neurogenesis (Sun et al., 2013a).
Despite the presented global relevance of miR
signaling in brain development, activity of miR-
NAs may also be locally restricted to specific cell
types or even subcellular compartments, where
they, for example, modulate synaptic activity
and neuronal connectivity (Hengst etal., 2006;
Schratt etal.,2006).

CYTOPLASM

Dicer Exportin
double stranded
mature microRNA

microRNA Drosha
RISC precursor
primary microRNA
ORF AAAAAAA transcript

NUCLEUS

epigenetic chromatin
mRNA degradation translational inhibition modifications
(perfect match) (imperfect match)

FIGURE 12.3: MicroRNA biogenesis and function. Simplified schematic on microRNA biogenesis and
RNA-induced gene silencing. Transcription of primary microRNA from miRNA genes is followed by cleavage
to precursor microRNA by the Drosha nuclear RNase III. The microRNA precursor is then exported to the cyto-
plasm by exportin via nuclear pore. In the cytoplasm, the precursor microRNA is further processed by RNase
activity of Dicer to the mature microRNA duplex. The duplex loads onto the RNA-induced silencing complex
(RISC) complex, but only one of the mature microRNA strands (red strand) mediates RNA silencing by degrading
the target mRNA or interfering with translation, while the second microRNA strand (grey strand) is degraded
itself by the RNase activity of RISC. The outcome of RISC formation varies with the degree of complementarity
of the seed sequence of microRNA with the 3 untranslated region of the target mRNA. MicroRNAs can regulate
different epigenetic layers including DNA methylation, histone modification, and chromatin remodeling or be
epigenetically regulated themselves (miR expression, miR function).
Modified from Cuellar and McManus,2005
192 Part II:Homeostatic Control
Chromatin Remodelers
Alterations in gene expression are closely related
to changes in higher-order and/or locus-specific
chromatin structure, and chromatin-remodeling
enzymes play key roles in differentiation and devel-
opment. ATP-dependent chromatin-remodeling
enzymes can be divided into three subfamilies
according to structure and function:the SWItch/
Sucrose Nonfermentable (SWI/SNF), chromodo-
main and helicase-like domain (CHD), and imi-
tation SWI families (Flaus and Owen-Hughes,
2011). Each subfamily harbors a unique domain
(Bromo, Chromo, SANT) that helps to recruit
remodeling complexes to regions of specifically
modified chromatin. Importantly, remodeling
enzymes use energy provided by ATP hydrolysis
to locally disrupt histone-DNA associations and
shift nucleosomes to alternate positions. They are
further organized into protein complexes and
may be regulated by subunit composition as sug-
gested by the isolation of SWI/SNF complexes
containing a neuron-specific subunit from the
brain of mice (Kim etal., 2001). There is evidence
that chromatin remodeling complexes are key
regulators of neural development (Potts et al.,
2011; Ronan et al., 2013; Seo et al., 2005), with
some of their components being abundantly,
sometimes exclusively, expressed within the
mammalian brain, spinal cord, or retina.
EPIGENETICS
INNEUROGENESIS AND
BR AIN DEVELOPMENT
Neurogenesis is defined as the generation of new
functional neurons and can be divided into two
phases: embryonic/developmental neurogenesis,
which basically forms the CNS, and adult neuro-
genesis, which continues at low levels in postna-
tal and adult brains (Jobe etal., 2012). Epigenetic
mechanisms carry out diverse roles in regulating
specific aspects of embryonic and adult neuro-
genesis, including stem cell renewal, neuronal
fate specification, as well as maturation and inte-
gration (Ma etal.,2010).
During development, multipotent neural
stem cells give rise to a wide range of cell types
within the CNS along the body axis. Homeobox
(Hox) genes are key regulators of neural stem cell
renewal and lineage commitment and determine
not only the identity of individual cells within
the anterior-posterior and dorso-ventral axes
of the brain, but also direct cellular migration,
axonal direction and the process of somatogene-
sis during embryogenesis and development (Park
etal.,2007).
Hox gene expression is spatially controlled
by Polycomb group (PcG) and trithorax group
(trxG) proteins, which comprise protein com-
plexes with epigenetic function implicated in
lineage commitment and cellular memory. The
specific relevance of the PcG system in neuro-
genesis and CNS development is exemplified by
neuronal defects in various PcG mouse mutants
(Fasano etal., 2009). In mice, genetic ablation of
components of the polycomb repressor complex
1 (e.g., Bmi1 polycomb ring finger oncogene,
Bmi1; Ring finger protein 2, Rnf2) interferes
with cerebral neural stem cell renewal, result-
ing in progressive postnatal growth retardation
and neurological abnormalities manifested, for
example, by an ataxic gait and sporadic seizures
(Molofsky etal., 2003; van der Lugt etal.,1994).
In the course of neocortical development,
neural progenitors produce neurons first and
astrocytes later. Evidence that the PcG system
also regulates the temporal regulation of neural
progenitor cell fate comes from studies show-
ing that ablation of several Polycomb-repressive
complex 2 components (e.g., enhancer of zeste
homolog 2, Ezh2; embryonic ectoderm devel-
opment, Eed) prolonged the neurogenic and
delayed the gliogenic phase in vivo (Hirabayashi
etal., 2009; Pereira etal., 2010), thereby severely
affecting timing of cortical development. Histone
modification dynamics, also independent from
the PcG/trxG system, are relevant for proper
regulation of neuronal differentiation, matura-
tion, and migration, as well as maintenance of
neuronal function as deduced from the specific
spatiotemporal expression of (e.g., SET domain,
bifurcated 1, Setdb1, a H3K9 specific methyl-
transferase). It is highly expressed at early stages
of mouse brain development and is particularly
located in the ventricular zone, where neural
progenitor cells reside. Setdb1 expression could
be associated with cell fate specification. Not
surprisingly, Setdb1 knockout in vivo resulted in
severe brain defects and early postnatal lethality
(Tan etal., 2012). Chromatin remodelers are also
implicated in early neurogenesis and CNS devel-
opment. Mice depleted of major components of
the SWI/SNF chromatin remodeling complex die
at peri-implantation stages. Furthermore, many
heterozygotes display neural tube abnormali-
ties (Bultman et al., 2000) and are predisposed
to tumors of neural origin (Bultman etal., 2000;
Kim etal.,2001).
Prenatal and early postnatal periods in mam-
malian development are characterized by rapid
changes in neuronal organization, providing a

critical time window during which environmen-
tal factors (i.e., intrinsic and extrinsic stimuli)
can lead to long-term influences on brain devel-
opment and adapting brain function. Epigenetic
mechanisms seem to play a critical role in
mediating the effects of (early) sensory experi-
ence on gene expression in the brain. Prenatal
stress is associated with an increased vulner-
ability to neurodevelopmental and psychiat-
ric disorders, including autism, schizophrenia,
and depression (Champagne and Curley, 2009).
Experimental studies in humans and mice have
demonstrated that prenatal exposure to mater-
nal stress as well as early life stress in the infant
induces localized and global changes in DNA
methylation associated with stable changes in
gene expression, which emerge in infancy and
are sustained into adulthood, proving a lasting
effect on infant development (Champagne and
Curley, 2009; Fagiolini et al., 2009; Suderman
et al., 2014). Nutrition is another environmen-
tal factor, which exerts a strong impact on CNS
development and function. One critical meta-
bolic pathway is the methionine cycle, in which
folate and B12 convert homocysteine to methio-
nine, which is in turn converted to SAM the
methyl group donor in all transmethylation reac-
tions (Kobow and Blumcke, 2012). Intriguingly,
the folate-dependent methionine cycle plays a
key role in neuronal homeostasis. Dietary folate
deficiency or genetic defects in folate metabolism
inhibit the methionine cycle, thereby increas-
ing developmental abnormalities of the fetal and
neonatal nervous system (Chen etal., 2001), and
have been associated with autism and epilepsy in
youth, as well as depression, schizophrenia, and
neurodegeneration in the adult (Goyette et al.,
1995; Iskandar et al., 2010; Popp et al., 2009;
Shea and Rogers, 2014; Tonetti etal., 2000). SAM
is required for maintaining DNA methylation
levels during critical periods of high neuronal
proliferation. Furthermore, homocysteine is a
neurotoxic agent that, if accumulating in the
methionine cycle, can induce aberrant activation
of NMDA receptors, calcium influx, and oxida-
tive damage (Shea and Rogers,2014).
Taken together, there is converging evidence
for a role of multiple epigenetic mechanisms dur-
ing all stages of brain development and neuronal
homeostasis. Perturbations in epigenetic gene
regulation, whether driven by genetic or environ-
mental factors, may have lasting effects on syn-
aptic function, neuronal network activity, and
proliferation and can be deleterious to the nerv-
ous system (Ramocki and Zoghbi,2008).
Epigenetics 193
S Y N A P T I C P L A S T I C I T Y,
LEARNING, ANDMEMORY
Epigenetic modifications on chromosomal DNA
do not only comprise the molecular memory of
a single cell but assist in learning and memory
as higher order brain functions. Histone acety-
lation is regarded as a promoter of memory for-
mation, whereas lack of this modification has
been causally related to cognitive impairment in
neurodevelopmental disorders, neurodegenera-
tion, and aging (Graff and Tsai, 2013; Peleg etal.,
2010). Specific forms of learning in rodents have
been correlated with increased HAT activity and
global histone acetylation, and more recently
the role of specific histone modifications (e.g.,
H4K12 and H4K5 acetylation) has been investi-
gated (Fischer, 2014; Park etal., 2013; Peleg etal.,
2010). Given that neurodegenerative diseases are
frequently associated with impaired learning
and memory, the question arises regarding how
learning ability and access to long-term memo-
ries can be improved. Studies in mice provided
evidence that both environmental enrichment
as well as pharmacological inhibition of histone
deacetylases (HDACs) re-established histone-tail
acetylation and correlated well with increased
sprouting of dendrites, increased number of
synapses, as well as reinstated learning behav-
ior and access to long-term memories (Fischer
et al., 2007; Peleg et al., 2010). Interestingly,
Hdac2 as well as Hdac3 knockout mice showed
enhanced learning behavior, whereas mice
lacking Hdac4 in the adult forebrain exhibited
impaired hippocampus-dependent memory for-
mation. Likewise, haploinsufficiency of HDAC4
in humans is linked to mental retardation
(Fischer,2014).
Rubinstein-Taybi syndrome, a congenital
anomaly syndrome characterized by intellectual
disability, postnatal growth deficiency, micro-
cephaly, and dysmorphic facial features, is caused
by heterozygous mutations in the gene encoding
the transcriptional co-activator CREB-binding
protein (CREBBP; Petrij et al., 1995). CREBBP
is known to possess intrinsic HAT activity and
plays a critical role in embryonic development,
growth control, and neuronal homeostasis by
coupling chromatin remodeling to transcrip-
tion factor recognition (Roelfsema et al., 2005).
Although CREBBP is the most thoroughly stud-
ied HAT with regard to learning and memory,
recent studies have shown a role for E1A-binding
protein (P300) and P300/CREBBP-associated
factor (PCAF) in memory processes (Barrett and
Wood, 2008). Genetic mouse models targeting
194 Part II:Homeostatic Control
either one of the three factors display impaired
memory consolidation, although mechanisms
underlying this effect seem to be poorly under-
stood. So far CREBBP function has been linked
to increased calcium (Ca2+) signaling and tran-
scriptional activation as well as synaptic plas-
ticity. Beside its ability to acetylate histone
and nonhistone proteins, CREBBP selectively
recruits transcriptional co-activators during the
formation of the transcription preinitiation com-
plex and has therefore been suggested to act as
scaffold to stabilize protein interactions with the
transcription machinery.
Histone methylation is another key regula-
tor for hippocampal and cortical gene activation
and silencing during memory consolidation.
Conditional knockout of histone methylating
and demethylating enzymes (e.g., euchromatic
histone-lysine n-methyltransferase 2, Ehmt2;
lysine-specific methyltransferase 2a and 2b,
Kmt2a/2b) in adult mice forebrain or hippocam-
pus resulted in altered histone methylation and
corresponding gene expression changes, as well
as complex behavioral abnormalities and learn-
ing impairment. Brain morphology remained
unaffected, however (Gupta-Agarwal et al.,
2012; Gupta etal., 2010; Kerimoglu etal., 2013).
Evidence for DNA methylation in learning and
memory comes from studies showing that Dnmt
inhibition blocked hippocampal memory forma-
tion associated with deregulated expression of
genes known to contribute to synaptic plasticity
(Feng etal., 2010; Miller and Sweatt, 2007). Fear
conditioning in mice increased the expression
of Dnmt3a and Dnmt3b and, moreover, induced
localized changes in DNA methylation patterns
and concomitant changes in gene expression
(Miller and Sweatt, 2007). Ten-eleven transloca-
tion family enzymes, which promote active DNA
demethylation in the nervous system, have also
been shown to regulate the expression of CNS
activity-dependent genes and memory formation
(Kaas etal., 2013). Genetic deletion of Gadd45b,
another regulator of active DNA demethyla-
tion, enhances long-term memory and synaptic
plasticity (Sultan et al., 2012). Finally, noncod-
ing RNAs are also implicated in cognitive brain
function (e.g., PIWI-interacting RNAs were
recently identified to be abundantly expressed
in the CNS and mediate activity-induced CpG
methylation and transcriptional silencing of key
synaptic plasticity-related genes).
In conclusion, epigenetic mechanisms includ-
ing histone modifications and DNA methylation
are dynamically regulated in the adult nervous
system and play a significant role in learning and
memory. However, these phenomena are mecha-
nistically not yet well understood.
NEUROLOGIC DISEASE
Epigenetic changes are fundamental in normal
development, and disruption of the epigenetic
program can unchain a variety of pathogenic
processes. Cancer was the first disease described
for altered epigenetic marks. Epigenetic silencing
of tumor suppressor genes by promoter methyla-
tion is a common event also during brain tumor
development (Hegi et al., 2009). Epigenetic
alterations in tumors frequently target essen-
tial components of DNA repair pathways (e.g.
O6-methylguanine-DNA methyltransferase
(MGMT). Most interestingly, these alterations
sensitize affected tumors to treatment with cer-
tain classes of anticancer agents (e.g., alkylat-
ing agents). Consequently, MGMT promoter
methylation in glioblastoma patients has been
associated with overall longer survival and
better response to chemotherapy (Hegi et al.,
2005). More recently, recurrent somatic muta-
tions in isocitrate dehydrogenase (IDH) genes
have been associated with glioblastoma but also
low-grade gliomas (Sun et al., 2013b; Yan et al.,
2009). Cytoplasmic IDH1 and mitochondrial
IDH2 isoforms normally catalyse the intercon-
version of isocitrate and -ketoglutarate but also
play a role in lipid synthesis, glucose sensing, or
energy metabolism. Moreover, IDH enzymes
regulate redox homeostasis and protect the cell
against oxidative stress by generating the reduc-
ing equivalent NAPDH. NADPH is essential for
the regeneration of glutathione that neutralizes
free radicals and reactive oxygen species and
for the activity of the thioredoxin system.
Although the pentose phosphate pathway is the
main source of NADPH, IDH1/2 are similarly
important (Mellai et al., 2013). Redox homeo-
stasis is linked to transmethylation and recy-
cling of SAM as universal methyl group donor.
Under oxidative conditions, SAM synthetase
can become inactivated, thus stopping the flow
of metabolites through the methionine cycle and
inhibiting, for example, DNA or histone meth-
ylation (Hitchler and Domann, 2012). In addi-
tion, -ketoglutarate is an essential cofactor of
ten-eleven translocation family enzymes and of
Jumonji C domain containing histone demethy-
lases. Mutations targeting IDH1/2 as much as
changes in the expression or activity of IDH1/2
enzymes may therefore globally alter 5-mC and
5-hmC levels (Chia etal.,2011).

Other than brain tumors, many other neu-
rological diseases primarily or secondarily
associate with epigenetic alterations, including
autism, bipolar disorder, schizophrenia, neu-
rodegeneration, stroke, and epilepsy (Kobow
and Blumcke, 2012; Kobow etal., 2013; Mehler,
2008; Moon et al., 2013; Qureshi and Mehler,
2010). As mentioned, epigenetic mechanisms
affect cell fate determination and maturation,
migration, and lamination. More recent stud-
ies suggest neuronal membrane excitation,
memory formation, and cognition are linked
to epigenetic chromatin modifications, thereby
adapting gene expression levels to functional
network activity (Dulac, 2010; Fischer et al.,
2007; Peleg et al., 2010; Qureshi and Mehler,
2010). In human and experimental epilepsy,
localized and global changes in DNA methyla-
tion have been identified and correlated with
gene expression changes. Prominent examples
of genes sensitive to DNA methylation-based
silencing in experimental and human epi-
lepsy include Reelin (Reln), a master regulator
of neuronal migration (Kobow et al., 2009),
and carboxypeptidase A6 (CPA6), an enzyme
involved in the selective biosynthesis of neu-
roendocrine peptides (Belhedi etal., 2014; Sapio
etal., 2012). Genome-wide methylation analysis
in rodent models of epilepsy identified meth-
ylation signatures distinguishing animals that
experienced status epilepticus as an initial pre-
cipitating injury from chronic epileptic animals
and/or healthy controls (Kobow et al., 2013;
Miller-Delaney et al., 2012). Increased DNA
methylation in human and experimental epi-
lepsy could be associated with either increased
DNA methyltransferase expression or increased
enzymatic activity (Williams-Karnesky et al.,
2013; Zhu etal.,2011).
Ca2+ is a major second messenger that helps
to transmit depolarization status and synap-
tic activity to the biochemical machinery of a
given neuron. Neuronal hyperactivity, as dur-
ing seizures, is intricately linked to altered
Ca2+ and redox homeostasis, neuronal energy
metabolism, and second messenger signal-
ing to the nucleus. All of these processes feed
each other and impact epigenetic processes,
and some examples have been more closely dis-
cussed in this chapter. It has previously been
suggested that initial precipitating injuries
(e.g., traumatic brain injury, inflammation,
prolonged febrile seizures, or status epilepti-
cus), which are frequently observed in acquired
epilepsies, as much as seizures by themselves
Epigenetics 195
can induce epigenetic alterations, resulting in
sustained changes of gene expression, thereby
promoting and enhancing the epileptogenic
condition (Kobow and Blumcke, 2011, 2012).
In genetically determined human epilepsies,
it can further be noted that all different types
of epigenetic players have been identified to
be affected by mutations including chromatin
remodelers (e.g., chromodomain helicase DNA
binding protein 2, CHD2; SWI/SNF-related,
matrix associated, actin dependent regulator of
chromatin, subfamily A, member 4, SMARCA4;
Alpha-thalassemia/mental retardation,
ATRX), histone modifiers (e.g. euchromatic
histone-lysine N-methyltransferase 1, EHMT1;
lysine-specific demethylase 5C, KDM5C; his-
tone deacetylase 4, HDAC4) as well as readers
of DNA methylation (e.g. methyl CpG binding
protein 2, MeCP2; Kobow and Blumcke,2012).
Taken together, a two-tailed interdepend-
ence between epigenetics and brain function
can be assumed that allows alterations in epi-
genetics to be a cause as well as a consequence
of brain malfunction and disease. In complex
diseases, knowledge about epigenetic altera-
tions can probably elucidate the origin of
some non-Mendelian inheritance and etiology
beyond genetic mutations. Phenotypic vari-
ation and disease susceptibility, response to
treatment, and the complex outcome of com-
mon and rare diseases may be compounded by
both genetic and epigenetic anomalies as well
as the interaction between them (Huidobro
etal.,2013).
CONCLUSIONS
Epigenetic mechanisms comprise a large spec-
trum of DNA-modifying molecular mecha-
nisms and play a major role in adapting brain
function to environmental stimuli during
development and adolescence but also aging.
Epigenetic alterations have been consistently
identified in many neurological diseases, open-
ing new avenues to drug treatment, as the key
molecular players represent promising targets
for pharmacological intervention. Currently,
global effects of epigenetic inhibitors impede
normal cellular functions, which is a clear limi-
tation, and severe side effects must be envis-
aged for unselective systemic epigenetic drug
treatment (Frisch et al., 2009; Harden et al.,
2009). Therefore, future studies need to address
how the enzymology can be specifically tar-
geted in the affected brain region or even at
selectedgenes.
196 Part II:Homeostatic Control
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13
Adult Neural Stem Cells
and Brain Homeostasis
ASHOK K.SHET T Y
INTRODUCTION
One of the foremost dogmas in early neurosci-
ence research was that neurons in the mamma-
lian brain are generated in their entirety during
the early developmental periods and hence are
not reinstated when they die due to either neuro-
degenerative diseases or brain injury. Although
the concept of adult neurogenesis (i.e., the forma-
tion of new neurons in the adult brain) emerged
in the 1960s through a finding that new cells are
generated in the olfactory system and dentate
gyrus (DG) of the adult brain by Altman and Das
(1965), lack of definitive markers to detect neu-
rons among tritiated-thymidine labeled newly
born cells made this discovery go mostly unno-
ticed for nearly two decades. Findings in the
1980s that neurogenesis occurs in 3-month-old
rat visual cortex (Kaplan, 1981)and adult canar-
ies learn new songs through the addition of new
neurons (Goldman and Notteboum, 1983) reig-
nited the perception of neurogenesis in the adult
brain but did not attract many researchers to
work in this area. However, a seminal finding
in the early 1990s that both neurons and astro-
cytes can be generated in culture from cells iso-
lated from the adult mammalian brain (Reynolds
and Weiss, 1992)triggered a massive interest in
examining neural stem/progenitor cells (NSCs)
and neurogenesis in the adultbrain.
Cells that produce new neurons in different
regions of the adult brain are broadly classified
as NSCs (Ihrie and Alvarez-Buylla, 2011; Ming
and Song, 2011; Yao et al., 2012). Neural stem
cells have the ability of extensive proliferation,
self-renewal, and production of all three major
phenotypes (neurons, astrocytes, oligodendro-
cytes) in the central nervous system. Progenitor
cells, on the other hand, seem to have limited
proliferation potential and lack self-renewal
ability. Multiple studies have now confirmed
the persistence of NSCs and the occurrence of
neurogenesis all through life in two regions of
the adult mammalian brain: the subventricu-
lar zone (SVZ) lining the lateral ventricles of
the forebrain and the subgranular zone (SGZ)
of the DG in the hippocampal formation (Ihrie
and Alvarez-Buylla, 2011; Ming and Song, 2011;
Yao et al., 2012; Figure 13.1). Typically, prolif-
eration of NSCs in the SVZ produces a large
pool of immature neurons (neuroblasts), which
migrate into the olfactory bulb through a path-
way called the rostral migratory stream (Ihrie
and Alvarez-Buylla, 2011, Figure 13.1). Once
they reach the olfactory bulb, they differentiate
into different types of olfactory interneurons.
Proliferation of NSCs in the SGZ, on the other
hand, results in production of new neurons and
glia. Newly born neurons then migrate into the
granule cell layer (GCL) of the DG, where they
mature, emanate dendrites into the molecular
layer to establish connectivity with axons com-
ing from the entorhinal cortex, and send axons
into the stratum lucidum of the CA3 to make
synaptic contacts with primary dendrites of CA3
pyramidal neurons (Ming and Song, 2011; Yao
etal., 2012, Figure13.1).
Furthermore, many studies support the idea
that some amount of neurogenesis also occurs
in the so-called nonneurogenic regions of the
brain (Gould, 2007). This is initially revealed
through in vitro observations that proliferat-
ing multipotent NSC-like cells can be isolated
from many nonneurogenic regions of the adult
brain, which include the cerebral cortex, stria-
tum, spinal cord, retina, ventral mesencephalon,
and substantia nigra (Palmer et al., 1999; Lie
et al., 2002; Bonfanti and Peretto, 2011). Many
in vivo studies also suggest limited neurogenesis
in several regions of the adult brain such as the
hippocampus CA1 subfield (Rietze etal., 2000),
 Adult Neural Stem Cells and Brain Homeostasis 203

A
OB
CBM
CTX

DG
SVZ
RMS
C GFAP
B &
Sox-2

ML
D GFAP & Sox-2

E Sox-2 & Ki-67

Mature
Granule Cell
GCL NeuN, Prox1
SGZ

F DCX & Ki-67

Type 3 Cells
(Neuroblasts)
Type 2 Cells
DCX
(TAPs)
sox-2, Mash1
Type 1 Cells (NSCs)
GFAP, Sox-2, Nestin

FIGURE13.1: Neurogenic regions and steps of neurogenesis. Figure Adepicts the two active neurogenic
regions in the adult brain, the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) lining
the lateral ventricle (red zones). RMS=rostral migratory stream; OB=olfactory bulb; CTX=cortex; CBM=cer-
ebellum. Figure B illustrates the various steps involved in DG neurogenesis within the hippocampus. Type 1 cells
(both radial and horizontal cells shown in green) express markers such as the glial fibrillary acidic protein, Sox-2,
and nestin. These cells occasionally divide to self-renew and to produce type 2 cells. Type 2 cells (or transit ampli-
fying cells [TAPs], shown in blue]) express Sox-2 and mammalian achaete-scute homolog 1 (Mash1) and divide
more actively to produce type 3 cells (neuroblasts). Type 3 cells express doublecortin (DCX) and exhibit limited
proliferation before maturing into dentate granule cells expressing markers such as NeuN and Prox-1. Figures C
and D show examples of radial (C)and horizontal (D)type 1 cells expressing glial fibrillary acidic protein (GFAP)
and Sox-2 in the subgranular zone of the DG. Figures E and F show proliferation of type 2 cells (i.e., cells express-
ing Sox-2 and Ki-67 in E) and type 3 cells (i.e., cells expressing DCX and Ki- in F) in the subgranular zone of the
DG. GCL=granule cell layer; ML=molecular layer; SGZ=subgranularzone.

substantia nigra of the mesencephalon (Lie etal.,
2002; Zhao et al., 2003), neocortex (Cameron
and Dayer, 2008), striatum (Bedard etal., 2006),
amygdala (Fowler et al., 2008), piriform cortex
(Shapiro et al., 2007), and hypothalamus (Yuan
and Arias-Carrin et al., 2011). While there is
still some skepticism pertaining to the functional
integration of new neurons born in nonneuro-
genic regions in normal conditions (Koketsu
et al., 2003; Bonfanti and Peretto, 2011), the
existence of multipotent NSC-like cells in these
adult brain regions displaying the ability for pro-
liferation in the presence of apt neurotrophic
factors is promising. This is because, with the
advent of new neurogenic compounds and drugs
(e.g., aminopropyl carbazole; Pieper etal., 2010;
Walker etal., 2014), these cells may be amenable
for extensive proliferation to produce large num-
bers of site-specific new neurons in neurodegen-
erative disease conditions.
204 Part II:Homeostatic Control
Thus, both adult NSC and neurogenesis stud-
ies in multiple species during the past two decades
have clearly refuted the longstanding doctrine
that adult mammalian brain does not generate
new neurons. Furthermore, Eriksson and col-
leagues (1998) offered the first proof of neurogen-
esis occurring in the hippocampus of the adult
human brain. Since then, there has been immense
attention paid to ascertaining the behavior of
NSCs and the pattern and extent of neurogenesis
in different regions of the adult brain in normal
and disease conditions. Nonetheless, because
neurogenesis was observed at lower levels in
nonhuman primates than rodents, skepticism
prevailed regarding the extent of neurogenesis in
the adult human brain such as the hippocampus
and whether a small number of newly generated
neurons can impact human behavior (Kheirbek
and Hen, 2013). A recent study, by providing
strong evidence for widespread neurogenesis
in the DG of humans throughout adulthood,
has elegantly answered the query regarding the
amount of neurogenesis that occurs in the adult
human hippocampus (Spalding etal., 2013). An
exciting finding of this study is that neurogen-
esis does not considerably decline with aging
in the human hippocampus, in contrast to a
dramatic age-related decline in neurogenesis
observed in rodents (Kuhn etal., 1996; Rao etal.,
2005; Rao et al., 2006; Hattiangady and Shetty,
2008a). Although the functional significance of
adult-born neurons in the human hippocampus
remains to be elucidated, studies in animal mod-
els have provided substantial insights regard-
ing their role in cognitive and mood function
(Kheirbek and Hen, 2013). Therefore, this chap-
ter is focused on the present knowledge concern-
ing the activity of NSCs and neurogenesis in the
adult mammalian hippocampus under standard
and certain disease environments and on how
aberrant or declined NSC activity and neurogen-
esis in neurological diseases can weaken brain
homeostasis principally with reference to cogni-
tive and mood function. The way increased hip-
pocampal neurogenesis in depressive disorders
or improved forebrain neurogenesis after stroke
may aid in functional recovery is also discussed.
NSC NICHE INTHE
HIPPOCAMPUS
Neurogenesis in the DG of hippocampus occurs
in a wide variety of species (Kuhn et al., 1996;
Gould et al., 1997, 1999; Kornack and Rakic,
1999; Eriksson et al., 1998). Interestingly, a
vast majority of proliferating cells in the SGZ
of the hippocampus differentiate into neurons
(Cameron etal., 1993; Rao etal., 2005; Rao etal,
2006), though astrocytes, oligodendrocytes, and
endothelial cells also emerge from these prolif-
erating cells (Kornack and Rakic, 1999). It is
widely believed that a subset of slowly dividing
cells expressing the glial fibrillary acidic pro-
tein (GFAP) and exhibiting a single radial pro-
cess that extends through the GCL are NSCs or
radial glial cells (type 1 cells) in this region (Seri
etal., 2001; Kempermann etal., 2004; Ihrie and
Alvarez-Buylla, 2008, Figure 13.1). These cells
also express other markers of NSCs such as the
transcription factor sex determining region Y
[SRY]-box 2 (Sox-2; Hattiangady and Shetty,
2008a), the primitive neurofilament protein nes-
tin (Filippov etal., 2003; Lagace etal., 2007), the
radial glial marker vimentin (Hattiangady and
Shetty, 2008a), and the RNA binding protein
musashi-1 (Okano etal., 2005). However, another
class of cells having short, horizontal processes
has also been suggested as type 1 cells in the SGZ
(Lugert etal., 2010, Figure13.1). Both radial and
nonradial type 1 cells seem to shuttle between
active and quiescent states (Rolando and Taylor,
2014). The quiescent nature of NSCs between
their proliferative states is widely believed to not
only prevent their exhaustion in normal con-
ditions but also to reduce chromosomal aber-
rations resulting from frequent cell division
(Rolando and Taylor, 2014). In normal condi-
tions, slow or infrequent proliferation of these
NSCs produces a pool of transit amplifying cells
(TAPs, or type 2 cells; Figure 13.1). However, the
response of radial and nonradial type 1 NSCs in
the adult DG to pathophysiological signals seems
to be dependent on the type of cues. For instance,
a study has shown that physical exercise induces
proliferation of radial NSCs whereas seizures
activate only nonradial NSCs (Lugert etal., 2010).
Type 2 cells display smaller size, show short tan-
gential processes, and typically occur in clusters
(Hsieh, 2012, Figure13.1). These cells expressing
Sox-2 and mammalian achaete-scute homolog 1
proliferate rapidly and give rise to doublecortin
expressing neuroblasts (type 3 cells; Figure 13.1)
and glia (Encinas etal., 2011). Neuroblasts later
evolve into full-fledged granule cells express-
ing the mature neuronal marker neuron specific
nuclear antigen (NeuN) and the granule cell spe-
cific transcription factor prospero-related home-
obox gene (Prox-1; Figure 13.1). These mature
granule cells also establish afferent connectivity
 Adult Neural Stem Cells and Brain Homeostasis
with the perforant path fibers coming from the
entorhinal cortex and efferent connectivity with
the CA3 subfield pyramidal neurons, which takes
about 6 to 8 weeks in the rodent hippocampus
(Zhao etal., 2006; Hsieh,2012).
Activity of NSCs is determined by multiple
signals arising from the milieu. First, a variety
of cellular components existing in and around
NSC niches such as astrocytes, oligodendrocyte
progenitors, endothelial cells, pericytes, micro-
glia, mature granule cells, and GABA-ergic
interneurons can influence NSC activity
(Palmer etal., 2000; Goldman and Chen, 2011;
Gemma and Bachstetter, 2013). Second, noncel-
lular components such as secreted molecules
and the extracellular matrix proteins play roles
(Palmer etal., 2000; Morrens etal., 2012; Hsieh,
2012). Third, studies have shown that both neu-
rons and astrocytes in NSC niches can influence
self-renewal or differentiation of NSCs (Song
et al., 2002; Tozuka et al., 2005). For example,
astrocytes have been shown to be a source of
several important paracrine factors having vital
roles in neurogenesis, which include Notch,
Sonic hedgehog, bone morphogenetic proteins,
and Wnt proteins (Ahn and Joyner, 2005; Lie
etal., 2005; Ables etal., 2010; Hsieh, 2012). Thus
adult neurogenesis is orchestrated through a
multicellular niche, where NSCs respond to
instructive signals from other cell types, result-
ing in their proliferation, self-renewal, and/
or differentiation of their progeny into mature
neurons orglia.
R E G U L AT O R S O F N S C
ACTIVIT Y AND NEUROGENESIS
I N T H E A D U LT H I P P O C A M P U S
Maintenance of NSCs and cell fate specification
of NSC-derived cells in the SGZ are mediated
through several transcription factors and sign-
aling pathways. Multiple studies have shown
that preservation of type 1 cells with expres-
sion of transcription factors Sox-2 and hairy and
enhancer of split 5 (which encodes a member of
a family of basic helixloophelix [bHLH] tran-
scription repressors) occurs through Notch sign-
aling (Ables et al., 2010; Imayoshi et al., 2010;
Lugert etal., 2010; Hsieh, 2012). Both radial and
nonradial type 1 cells have been shown to exhibit
canonical Notch activity (Lugert et al., 2010;
Lugert et al., 2012). It has been suggested that
Notch signaling blocks neuron generation from
NSCs through suppression of the expression of
proneural transcription factors (Rolando and
205
Taylor, 2014). The role of several other transcrip-
tion factors in the maintenance of NSCs have
also been noted, which include paired domain
and homeodomain-containing transcription fac-
tor, Ascl1 (another bHLH transcription factor),
forkhead domain transcription factor, repres-
sor element1 silencing transcription factor, and
nuclear hormone transcription factor (Jessberger
etal., 2008; Renault etal., 2009; Gao etal., 2011;
Hsieh,2012).
The formation of type 2 cells (TAPs) and
type 3 cells (neuroblasts) from types 1 and 2 cells
involves a sequential upregulation of transcrip-
tion factors Neurogenin 2 (a bHLH transcription
factor involved in neural fate-choice decision)
and Tbr2 (T-brain gene 2, involved in conversion
of radial glia into intermediate progenitors during
cortex development). Differentiation of neuro-
blasts into mature neurons involves the expres-
sion of neuronal differentiation 1 (Gao et al.,
2009) regulated through Wnt/-catenin signal-
ing (Kuwabara etal., 2009; Seib etal., 2013)and
expression of Sox-3, Sox-11, and Forkhead box
G1, a winged helix transcriptional repressor
(Shen et al., 2006; Wang et al., 2006; Haslinger
et al., 2009). Inhibition of Wnt activity results
in reduced neurogenesis whereas its activation
through Wnt3a protein expression enhances
neurogenesis (Lie etal., 2005). Furthermore, dele-
tion of a negative regulator of the Wnt/-catenin
pathway such as Dickkopf1 enhances neurogen-
esis (Seib etal., 2013). The survival and matura-
tion of newly generated neurons is influenced
by Prox-1 (Jessberger etal., 2008; Karalay etal.,
2011)and cyclic AMP response element-binding
protein (CREB; Jagasia etal.,2009).
Additionally, several neurotransmitters can
influence the extent of adult hippocampal neu-
rogenesis. These include the inhibitory neuro-
transmitter gamma-amino butyric acid (GABA),
serotonin (5-hyroxytryptamine [5-HT]), and
acetylcholine. The role of GABA has received
the most attention because the GABA released
by a subpopulation of interneurons expressing
the calcium binding protein parvalbumin (PV)
in the DG has been found to regulate both stem
cell quiescence and neuronal cell fate decision
by newly born cells. Specifically, PV+ interneu-
rons repress the activation of quiescent NSCs by
releasing nonsynaptic GABA under normal con-
ditions (Song etal., 2012). Such regulation medi-
ated through 2-containing GABAA Rs expressed
on NSCs is thought to be important, as it averts
the exhaustion of primary NSCs through rapid
206 Part II:Homeostatic Control
proliferation (Song et al., 2013). On the other
hand, specific activation of PV+ interneurons pro-
motes the survival of proliferating DCX+ imma-
ture neurons through establishment of immature
synapses between axons of PV+ interneurons
and newly born neurons. Pertaining to 5-HT,
multiple studies using pharmacological enhance-
ment of 5-HT levels, reduction of serotonergic
neurons, or stimulation of 5-HT receptors have
shown that 5-HT can enhance the production of
new neurons in the DG (Doze and Perez, 2012).
Likewise, acetylcholine levels also seem to influ-
ence neurogenesis because ablation of cholinergic
neurons in the medial septum decreased neuro-
genesis and administration of cholinergic ago-
nists enhanced neurogenesis in the hippocampus
(Mohapel etal.,2005).
FUNCTIONAL SIGNIFICANCE
OFNSC ACTIVIT Y AND
NEUROGENESIS INTHE
A D U LT B R A I N
Newly generated neurons (granule cells) in the
DG integrate into the existing hippocampal cir-
cuitry and become functional (van Praag et al.,
2002). They display special electrophysiological
features such as an increased amenability for
long-term potentiation (a substrate underlying
learning and memory) for about a month after
their birth, after which they exhibit similar prop-
erties as their older counterparts (Schmidt-Heber
et al., 2004; Wang et al., 2000; Ming and Song,
2011). Thus, continuous production of new den-
tate granule cells would serve to maintain a pool
of neurons in the DG with special properties.
Studies suggest that such continuous turnover of
DG granule cells is highly efficient for meeting
some of the unique computational needs of the
hippocampus (Appleby etal., 2011). It is believed
that the newly born granule cells in the adult DG
not only promote plasticity but also provide sta-
bility through their integration into the circuitry
for longer periods (Kempermann,2013).
At the behavioral level, adult DG neuro-
genesis has been suggested to be important for
hippocampus-dependent learning and memory
(Braun and Jessberger, 2014). Initially, a mul-
titude of studies correlated the extent of DG
neurogenesis with spatial learning and memory
performance using tasks such as water maze test.
Many of these investigations have suggested a link
between spatial memory function and the num-
ber of newly generated neurons, which has been
substantiated through multiple observations.
First, positive environmental conditions (physi-
cal exercise or exposure to enriched environ-
ments) that enhanced hippocampus-dependent
memory function also increased DG neurogen-
esis (Kempermann etal., 1997; Van Praag etal.,
1999). Second, aged rats with unimpaired spatial
memory retrieval ability displayed greater levels
of neurogenesis than aged rats exhibiting mem-
ory impairment (Drapeau et al., 2003). Third,
impaired hippocampus-dependent memory
function in animals that underwent stress was
associated with greatly reduced DG neurogenesis
(Gould et al., 1999). Fourth, neurogenesis abla-
tion studies performed using approaches such
as whole brain radiation or administration of
drugs that target dividing NSCs and their prog-
eny also supported a role for DG neurogenesis
in certain forms of hippocampus-dependent
memory (Shors etal., 2001; Snyder etal., 2005).
Nevertheless, conflicting results in other studies
and questions regarding the efficiency of tech-
niques used to selectively ablate DG neurogen-
esis in its entirety without altering the existing
hippocampal cytoarchitecture or circuitry raised
some doubts on the purported role of DG neu-
rogenesis in spatial learning and memory func-
tion for some time (Deng etal., 2010; Braun and
Jessberger,2014).
Follow-up studies, however, provided
stronger evidence that spatial learning astutely
inserts or eliminates newly born granule cells
depending on their stage of the development
and the functional importance, and that popula-
tions of new granule cells rescued are likely those
that are successfully integrated into the learning
and memory circuitry (Tronel etal., 2010, Deng
et al., 2010). Furthermore, a memory-retrieval
task after the water maze training preferentially
activated new granule cells that were ~4 to 6
weeks old at the time of learning the task and ~10
weeks old at the time of memory-retrieval test in
mice (Kee etal., 2007). Moreover, computational
models suggested a role for newly born granule
cells in the encoding of temporal information as
well as in the maintenance of old memories dur-
ing the encoding of new information (Aimone
etal., 2010; Deng etal., 2010; Aimone etal., 2011).
Studies utilizing sophisticated genetic ablation
techniques also showed spatial learning defi-
cits with the loss of hippocampal neurogenesis
(Dupret etal., 2008; Zhang etal., 2008; Jessberger
etal., 2009). Another study, by utilizing a combi-
nation of retroviral and optogenetic approaches
to birth date and reversibly controlling a group
 Adult Neural Stem Cells and Brain Homeostasis
of adult-born neurons in adult mice, showed that
silencing a cohort of ~4-week-old newly born
granule cells after water-maze learning substan-
tially disrupts retrieval of spatial memory (Gu
etal.,2012).
Moreover, several recent studies through
selective testing of DG circuitry point out that
newly generated neurons are critical for pattern
separation function (Clelland etal., 2009; Sahay
et al., 2011a,b; Kheirbek et al., 2012; Nakashiba
etal., 2012). Pattern separation refers to the abil-
ity to discriminate similar experiences through
the storage of similar representations in a nono-
verlapping manner (Leutgeb et al., 2007; Yassa
and Stark, 2011). The precise mechanisms by
which newly born neurons promote pattern
separation function are still being investigated.
Computational, anatomical, and electrophysio-
logical studies imply that sparseness of activation
in the granule cell layer of DG (resulting in sparse
functional connectivity between DG and CA3) is
conducive for pattern separation by enhancing
global remapping, a process by which similar rep-
resentations are encoded by nonoverlapping neu-
ronal ensembles (OReilly and McClelland, 1994).
Consistent with this idea, a recent study suggests
that modulation of DG excitability by adult-born
neurons (i.e., new granule cells) enhances sparse
coding in the granule cell layer to influence pat-
tern separation (Ikrar et al., 2013). Addition of
increased numbers of newly born granule cells
into the DG altered the excitability of mature or
existing dentate granule cells, which appeared to
be mediated through local circuit mechanisms
comprising increased excitatory drive from
newly generated granule cells onto GABA-ergic
interneurons in the dentatehilus.
Nonetheless, one of the issues being dis-
cussed in the field is whether the extent of neu-
rogenesis occurring in adulthood is adequate for
influencing hippocampus function. The amount
of neurogenesis occurring after the initial devel-
opment of the DG is not trivial, because ~9,000
new neurons (granule cells) are generated every
day in the DG of a young adult rat, and the num-
ber of new granule cells generated each month
amounts to roughly 6% of the total size of the
granule cell population (Cameron and McKay,
2001). In the human brain, that figure is ~700
new neurons per day for an annual turnover
rate of 1.75% (Kempermann, 2013). However,
the volume of DG or the hippocampus does not
increase as a function of age because a substan-
tial fraction of newly generated neurons appear
207
to die over a period of time (Gould etal., 1999;
Dayer et al., 2003; Gould et al., 2007). Thus,
despite continuous neurogenesis, the total num-
ber of granule cells in the DG remains nearly
static throughout life under normal conditions.
While newly born granule cells have been shown
to replace some granule cells that were born
during early development, they mostly seem
to replace granule cells that were born during
adulthood (Dayer etal., 2003). While the precise
lifespan of adult-born granule cells is unknown,
it is believed to be dependent on their ability to
become integrated into the hippocampus cir-
cuitry, as learning and complex experience can
promote the survival of newly born granule cells
(Kempermann, 2012). Moreover, in their matu-
ration period, newly born granule cells display
enhanced synaptic plasticity, which is evidenced
through a lower threshold for eliciting long-term
potentiation in these cells (Schmidt-Hieber
et al., 2004; Snyder et al., 2001). Furthermore,
newly born granule cells form a major compo-
nent of granule cells that contribute to long-term
potentiation in the DG because the preexisting
network is inhibited from being activated (Saxe
etal., 2006; Garthe etal., 2009). Taken together,
it emerges that newly born granule cells are more
plastic than older granule cells and are more
likely to generate action potentials in response to
an incoming stimulus. Therefore, young granule
cells respond to a broad variety of synaptic input
in comparison to old (preexisting) granule cells
that seem to respond to a more specific input.
This differential response between young and
old granule cells has been suggested to be benefi-
cial because young granule cells being tuned to a
broad variety of inputs can be good integrators,
whereas older cells displaying high input speci-
ficity can be better separators (Marn-Burgin
et al., 2012; Kempermann, 2012). These attrib-
utes are considered to be critical for the DG
to perform its functional roles, which include
facilitating pattern separation and preventing
catastrophic interference, providing contextual
and affective annotations, and contributing to
cognitive flexibility in situations where novel
information has to be integrated into previously
formed representations (Marn-Burgin et al.,
2012; Kempermann, 2012). Therefore, continu-
ous production of new granule cells seem to be
necessary for maintaining normal hippocampus
function, as it provides a pool of highly plastic
immature cells to facilitate complex computa-
tional needs (Kempermann,2012).
208 Part II:Homeostatic Control
Reduced DG neurogenesis has also been
implicated in psychiatric disorders (Bergmann
and Frisen, 2013). It has been suggested that
fewer newly born neurons impairing pattern
separation function may impair the ability to
distinguish threats from similar, but safe, situ-
ations and contribute to a generalized percep-
tion of fear and anxiety in posttraumatic stress
disorder (Kheirbek et al., 2012; Bergmann and
Frisen, 2013). Furthermore, most antidepres-
sant treatments enhance DG neurogenesis, and
some of the effects of antidepressants in animal
models are dependent on increased neurogenesis
(Santarelli etal., 2003). Thus DG neurogenesis in
the adult hippocampus is important for main-
taining homeostasis in the hippocampus, partic-
ularly its ability for promoting normal memory
and mood function.
N E U R O G E N E S I S I N T H E A D U LT
HUMAN HIPPOCAMPUS
Coherent with the results in other mammalian
species, the DG of the human hippocampus has
been validated as a neurogenic region. Eriksson
and colleagues (1998) specifically examined
putative NSCs and newly born neurons within
the DG of the adult human hippocampus using
postmortem hippocampal tissues from five can-
cer patients receiving one intravenous infusion
of 5-bromodeoxyuridine (BrdU) for diagnostic
purposes (Eriksson et al., 1998). Examination
of BrdU+ cells in the DG using BrdU-NeuN and
BrdU-GFAP dual immunofluorescence and con-
focal microscopy revealed ~22% neurons and
~18% astrocytes among newly born cells. The
remaining BrdU+ cells were smaller in size and
appeared to be quiescent undifferentiated NSCs.
Cells that double-labeled for BrdU and calbindin
were also observed, which provided further sup-
port for the occurrence of newly born granule
cells in the adult human DG. While this study
provided the first evidence for the occurrence
of NSCs and neurogenesis in the adult human
hippocampus, skepticism prevailed in the field
because some studies have suggested that neuro-
genesis is less abundant in nonhuman primates
in comparison to rodents (Amrein et al., 2011;
Kheirbek and Hen,2013).
However, a recent study provides strong evi-
dence for widespread neurogenesis in the DG of
humans throughout adulthood (Spalding et al.,
2013). The authors employed a unique birth-dating
strategy based on the peak of carbon isotope
14 (14C) that was released into the atmosphere
during the above-ground nuclear bomb tests
between 1945 and 1963 (Kempermann, 2013).
This approach relies on the principle that 14C in
the atmosphere becomes integrated into the DNA
of dividing human cells because of eating plants
that have absorbed 14C from the atmosphere or
eating animals that have consumed plants absorb-
ing atmospheric 14C (Welberg, 2013). Because 14C
is incorporated in the DNA during cell division,
the 14C content of a cell is thought to reflect 14C
levels in the atmosphere at the time of the birth
of the cell. As atomic bomb testing in the 1950s
and 1960s caused a spike in atmospheric 14C lev-
els and levels declined after 1963 (because of the
limited test ban treaty), the level of 14C in cellu-
lar DNA facilitated determination of a cells birth
date (Welberg, 2013). Spalding and colleagues,
using hippocampus from postmortem brains of
individuals who were born in different years in
the 20th century, sorted neurons from glial cells,
purified neuronal DNA, and measured 14C levels.
As the amount of incorporated 14C correlates with
the atmospheric 14C at the time of cell division,
this method allowed determination of the age of
cells. This study provided three major novel find-
ings. First, adult neurogenesis in the human brain
is restricted to the hippocampus. Second, based
on computer modeling, it is estimated that nearly
80% of DG granule cells undergo renewal in adult-
hood and ~700 new neurons are added per day for
an annual turnover rate of 1.75% (Kempermann,
2013). Third, the turnover rate was quite similar
between men and women and decreased only
modestly with aging, unlike in rodents where a
dramatic age-related decline in neurogenesis is
observed (Kuhn etal., 1996; Rao etal., 2005; Rao
etal., 2006; Hattiangady and Shetty, 2008a). Thus
the occurrence of NSCs and neurogenesis in the
adult human hippocampus has now been proved
beyonddoubt.
NSC ACTIVIT Y AND
NEUROGENESIS IN
NEUROLOGICAL DISORDERS
Altered Homeostasis of NSCs
and Neurogenesis in Temporal
Lobe Epilepsy
Epilepsy, a medical condition typified by sei-
zures and interfering with a variety of mental
functions, affects over 2million Americans and
~65 million people worldwide. Temporal lobe
epilepsy (TLE) is among the most common
types of drug-resistant epilepsy (Engel, 2001;
 Adult Neural Stem Cells and Brain Homeostasis
Strine etal., 2005). While the etiology of TLE is
unidentified in most cases, an initial precipitat-
ing injury such as status epilepticus (SE), head
trauma, brain infections, or childhood febrile
seizures leads to TLE in others (Pitkanen and
Sutula, 2002; Lewis, 2005). Classically, a dor-
mant period of several years is seen between an
initial precipitating injury and the appearance of
chronic TLE exemplified by spontaneous recur-
rent seizures (SRS) commencing from temporal
lobe foci, learning and memory difficulties, and
depression (Devinsky, 2004; Butler and Zeman,
2008; Bell etal., 2011). In addition, TLE is often
associated with hippocampal sclerosis owing
to a considerable loss of neurons in the dentate
hilus and CA1 and CA3 subfields. Over the past
decade, altered activity of NSCs and abnormal
hippocampal neurogenesis has emerged as one
of the most conspicuous pathological altera-
tions in TLE (Parent etal., 1997; Scharfman and
Gray, 2007; Kuruba and Shetty, 2009; Hester and
Danzer,2014).
Homeostasis ofNSCs and
Neurogenesis Is Distraught inthe
Early Phase ofTLE via Increased
Proliferation ofNSCs and Aberrant
Connectivity ofNewly Born Neurons
Studies in animal models highlight considerably
increased NSC proliferation and DG neurogen-
esis in the early phase of TLE (for 23 weeks fol-
lowing acute seizures or SE; Bengzon etal., 1997;
Parent et al., 1997; Hattiangady et al., 2004).
Interestingly, acute seizures do not alter the pro-
liferation of type 1 NSCs in the SGZ but instead
promote the proliferation of TAPs and immature
neurons (Jessberger et al., 2005). In agreement
with these results, the hippocampus from young
TLE patients (<24years old) displayed increased
proliferation of NSC-like cells (Blumcke et al.,
2001), signifying that the onset of TLE in pedi-
atric patients is likely also accompanied by
increased DG neurogenesis. Even though the spe-
cific mechanisms triggering the seizure-induced
surge in DG neurogenesis are still unclear, vari-
ous possibilities have been advocated. Multiple
neurotrophic factors, believed to be released from
dying neurons, deafferented neurons, and reac-
tive glia after seizures, are thought to increase the
proliferation of types 2 and 3 cells as well as pro-
mote the neuronal differentiation of their prog-
eny. The neurotrophic factors include the nerve
growth factor, brain-derived neurotrophic fac-
tor (BDNF), fibroblast growth factor 2 (FGF-2),
209
vascular endothelial growth factor, and sonic
hedgehog, all of which are known to rise after
acute seizures and to positively influence NSC
activity in the hippocampus (Lowenstein et al.,
1993; Bugra etal., 1994; Shetty etal., 2003; Croll
et al., 2004; Shetty et al., 2004; Banerjee et al.,
2005). Furthermore, increased levels of GABA
and neuropeptide Y (NPY) in the DG during the
early postseizure period likely play roles, as both
GABA and NPY have been shown to promote
DG neurogenesis (Scharfman and Gray, 2006; Ge
etal., 2007; Masiulis etal., 2011). Moreover, there
is some indirect evidence that repressor element
1-silencing transcription factor (a transcriptional
repressor that functions to limit transcription
of target genes) plays a role in seizure-induced
increased neurogenesis (Jessberger et al., 2007).
Besides these, augmented neuronal activity
during and after seizures may be promoting an
increased proliferation of NSCs, as increased
excitatory stimuli can act directly on NSCs
and influence the production of new neurons
(Deisseroth etal.,2004).
Abnormal migration of a significant frac-
tion of newly born granule cells into the hilus
and the molecular layer is one of the major
features of increased DG neurogenesis in the
early phase of TLE (Figure 13.2; Bengzon etal.,
1997; Parent etal., 1997; Scharfman etal., 2000;
Hattiangady et al., 2004; Scharfman and Gray,
2007; Shetty and Hattiangady, 2007; Hester and
Danzer, 2014). Loss or reduced levels of reelin
(a migration guidance cue released from a sub-
class of GABA-ergic interneurons in the dentate
hilus) after acute seizures is believed to be one
of the causes of aberrant migration of newly
born dentate granule cells (Gong et al., 2007).
Additional analyses has revealed that these
ectopic newly born granule cells likely become
seizure-inducing hub cells as they (i)incorpo-
rate aberrantly into the CA3 network, promote
the development of proepileptogenic circuitry,
undergo activation when epileptic rats are expe-
riencing SRS, and react with a longer latency
to onset of evoked responses following per-
forant path stimulation (Scharfman etal., 2002;
Scharfman etal., 2003a, 2003b); (ii) exhibit basal
dendrites and establish afferent connectivity
with mossy fiber terminals (axons of other gran-
ule cells; Pierce etal., 2005; Shapiro etal., 2005;
Walter etal., 2007); (iii) sprout axons (mossy fib-
ers) abnormally into the dentate inner molecu-
lar layer (Hester and Danzer, 2014); and (iv)
exhibit spontaneous bursts of action potentials
210 Part II:Homeostatic Control

Newly Born Neurons in the DG of a Naive Rat

A1 A2

SGZ
GCL
DH

GCL

SGZ

Newly Born Neurons in the DG of a Rat that

Underwent Status Epilepticus
B1 B2 Survival of Newly
GCL
Born Neurons in the
Epileptic Hippocampus
(~5 months after birth)
DH

D1 ML

GCL
GCL

SGZ SGZ
Aberrant Migration of Newly Born Neurons into the
DH in a Rat that Underwent Status Epilepticus DH
C1
GCL
D2
SGZ
GCL

SGZ
DH

DH

FIGURE 13.2: Changes in hippocampal neurogenesis following kainic acid induced status epilepticus. Newly
born neurons in the dentate gyrus (DG) of a nave adult rat (A1) and an adult rat that underwent status epilepticus
12days prior to euthanasia (B1) were visualized with immunostaining for doublecortin (a marker of newly born
neurons). A2 and B2 show magnified views of regions of dentate gyrus from A1 and B1, respectively. Note that, in
comparison to the dentate gyrus of a control rat (A1, A2), a rat that underwent status epilepticus (B1, B2) exhibits
considerably increased density of doublecortin+ new neurons and abnormal migration of newly born neurons
into the dentate hilus (indicated by arrowheads in B1). C1 is magnified view of a region from B1 showing aber-
rantly migrated newly born neurons in the dentate hilus. Figures D1 and D2 show that neurons born in the first
two weeks after status epilepticus survive for prolonged periods (~5months) and incorporate into both granule
cell layer (arrows in D1) and the dentate hilus (arrows in D2) of the epileptic hippocampus. DH=dentate hilus;
GCL=granule cell layer; ML=molecular layer; SGZ=subgranularzone.
Figure reproduced with modifications from Shetty, A.K., and Hattiangady, B.[2007], Prospects of stem cell therapy for temporal
lobe epilepsy. Stem Cells 25, 21042117

and contribute to SRS in chronically epilep- TLE also comprise similarly displaced granule
tic animals (Scharfman etal., 2000; Jung etal., cells (Houser etal.,1990).
2004; McCloskey etal., 2006). Interestingly, hip- Thus altered homeostasis of NSCs and DG
pocampal tissues obtained from patients with neurogenesis after acute seizures clearly facilitates
 Adult Neural Stem Cells and Brain Homeostasis
aberrant circuitry development. The role of this
aberrant circuitry in the development of chronic
TLE has been a subject of intense scrutiny in a vari-
ety of TLE models. While studies in some models of
TLE have raised doubts (Raedt etal., 2007; Pekcec
et al., 2007; Pekcec et al., 2011), findings in other
models of TLE are supportive of the involvement of
this circuitry as one of the pathophysiology under-
lying SRS. These include findings that reduction of
seizure-induced neurogenesis using an antimitotic
agent can decrease the frequency and duration
of SRS (Jung etal., 2004)and animals with large
numbers of abnormal cells display greater numbers
of seizures than animals with only a few abnor-
mal cells (McCloskey etal., 2006). Additionally, a
recent study demonstrates that seizure frequency
in chronic TLE is determined by multiple factors,
which include the severity of mossy fiber sprout-
ing and frequencies of hilar ectopic granule cells
and basal dendrites (Hester and Danzer, 2013), all
of which result from altered homeostasis of neuro-
genesis after seizures. Suppression of this aberrant
circuitry may also minimize comorbidities of TLE
such as cognitive and mood dysfunction because
animal studies have shown that blockage of abnor-
mal DG neurogenesis after acute seizures can
prevent seizure-induced cognitive impairments
(Jessberger etal., 2007; Pekcec etal., 2008). From
these perspectives, it appears that development
of clinically applicable therapeutic strategies that
are capable of stabilizing NSC and neurogenesis
homeostasis after acute seizures will be beneficial
for preventing chronic epilepsy and behavioral
abnormalities resulting from SE or hippocampus
injury.
Homeostasis ofNeurogenesis Is
Altered inthe Chronic Phase ofTLE
Through Altered Potential ofNSCs
and Decreased Generation ofNew
GranuleCells
Studies in animal models clearly demonstrate
greatly weakened neurogenesis in the chronic
phase of TLE (Figure 13.3; Hattiangady et al.,
2004, Hattiangady and Shetty, 2010). Moreover,
an inverse relationship was seen between the
frequency of SRS and the extent of neurogen-
esis (Hattiangady et al., 2004). Interestingly, a
study in the kainate model demonstrated that
chronic epilepsy did not affect the overall addi-
tion or long-term survival of newly born cells
in the GCL. However, only 45% of newly born
cells (i.e., BrdU+ cells) differentiated into neu-
rons, in comparison to 7380% of such cells
211
exhibiting neuronal differentiation in the intact
hippocampus (Hattiangady and Shetty, 2010).
Furthermore, differentiation of newly born cells
into S-100 + mature astrocytes or NG2+ oligo-
dendrocyte progenitors enhanced to ~79% in the
chronically epileptic hippocampus from ~25%
seen in the intact hippocampus. Thus a dramatic
decline in the neuronal fate-choice decision of
newly born cells underlies decreased neurogen-
esis in chronic epilepsy. Moreover, chronic TLE
maintains abnormal features of neurogenesis
seen in the early phase, which include an aber-
rant migration of some newly born neurons
into the hilus and occurrences of basal den-
drites from newly born neurons that incorporate
into the GCL (Hattiangady and Shetty, 2008b).
The DG from children exhibiting frequent SRS
and adult TLE patients also showed decreased
density of newly born neurons positive for the
polysialylated neuronal cell adhesion molecule
(PSA-NCAM; Mathern etal., 2002; Pirttila etal.,
2005; Crespel et al., 2005; Fahrner et al., 2007).
The potential reasons underlying the waned neu-
rogenesis in chronic epilepsy include an altered
milieu of NSC niches in the SGZ, including
decreased concentrations of neurotrophic factors
(such as BDNF, FGF-2, and IGF-1) important
for NSC activity and neuronal differentiation of
their progeny (Shetty et al., 2003; Hattiangady
etal., 2004), depletion of multipotent NSCs that
can generate neurons (Hattiangady and Shetty,
2008b), or modifications in signaling pathways
causing reduced neuronal differentiation of the
progeny of NSCs such as increased concentra-
tion of Dickkopf-1 (an inhibitor of neurogenesis
promoting protein Wnt; Busceti etal., 2007; Lie
etal.,2005).
Thus studies on hippocampi from both
animal models and TLE patients authenti-
cate that chronic epilepsy is associated with
greatly declined hippocampal neurogenesis.
Considering the functions of hippocampal neu-
rogenesis discussed earlier, it is likely that altered
homeostasis of hippocampal neurogenesis is one
of the major causes of memory dysfunction and
depression seen in TLE. These links are sup-
ported by observations that (i)the overall granule
cell density is a significant predictor accounting
for the total memory capacity in an individual
TLE patient (Pauli etal., 2006; Siebzehnrubl and
Blumcke, 2008), and (ii) increased production
of new neurons in the hippocampus is essential
for effective action of antidepressants (Santarelli
et al., 2003; Sahay and Hen, 2007; Perera et al.,
 DCX-Positive Newly Born Neurons in an Age-Matched Control Rat
A1 A2

SGZ GCL ML

DH

GCL

SGZ

DCX-Positive Newly Born Neurons at months after ICV KA administration

B1 B2 ML
GCL
SGZ

DH GCL

SGZ

DCX-Positive Newly Born Neurons at 5 months after IP KA-induced SE

C1 GCL C2
GCL
SGZ
SGZ

DH

DH

ML

D
p < 0.001
15000
Number per Dentate Gyrus

= Age-Matched Control Hippocampus

10000
= Hippocampus at 5-months post-ICV KA
= Hippocampus at 5-months post-SE
5000 p < 0.01

0
Doublecortin Positive Neurons

FIGURE 13.3: Status of dentate neurogenesis in chronic epilepsy as revealed by doublecortin (DCX) immu-
nostaining. A1 through C1 illustrate the distribution of DCX positive newly born neurons in the dentate gyrus
of an age-matched intact hippocampus (A1), a chronically injured hippocampus at 5months after an intracere-
broventricular (ICV) kainic acid (KA) administration (B1), and a hippocampus displaying robust chronic epilepsy
at 5months after the KA-induced status epilepticus (SE) (C1). Note that, in comparison to the age-matched intact
 Adult Neural Stem Cells and Brain Homeostasis 213

2007). From these viewpoints, it appears that
strategies capable of normalizing neurogenesis
levels in chronic epilepsy (such as administra-
tion of distinct neurotrophic factors, a physical
exercise regimen, exposure to an enriched envi-
ronment, antidepressant therapy, or grafting of
fresh NSCs) would be efficacious for easing these
impairments (Shetty, 2011; Barkas et al., 2012).
Positive outcomes of normalizing DG neuro-
genesis levels in chronic epilepsy will, however,
largely depend on whether or not newly born
granule cells establish normal connectivity with
the surviving target CA3 pyramidal neurons.
ROLE OF NSC AND
N E U R O G E N E S I S H O M E O S TA S I S
F O R T R E AT I N G D E P R E S S I V E
DISORDERS
Depression affects nearly 15% of the population
and a major depressive disorder (MDD) is asso-
ciated with considerable morbidity and disabil-
ity (Pandya etal., 2012). One of the hallmarks of
MDD is a reduction in the size of hippocampus,
which may be related to altered homeostasis of
NSCs and declined DG neurogenesis for pro-
longed periods (Masi and Brovedani, 2011; Lee
etal., 2013). However, other factors such as neu-
ron loss, impairments of synaptic plasticity in
the form of reduced axon branching, dendritic
regression, and reduced numbers of synapses
may also contribute to the reduced size of the hip-
pocampus (Sapolsky, 2001; Masi and Brovedani,
2011; Duman and Aghajanian, 2012). Altered
homeostasis of NSCs and neurogenesis in depres-
sion likely emerges from multiple changes in the
hippocampus. This may include decreased sero-
tonergic neurotransmission, increased levels of
stress and cortisol, and reduced levels of p-CREB
and BDNF (DSa and Duman, 2002). This is
because selective serotonin reuptake inhibitors
such as fluoxetine enhance neurogenesis through
activation of the 5HT1A receptors (Santarelli
etal., 2003), stress can increase cortisol levels and
both together can alter NSC activity and decrease
neurogenesis (Gould etal., 1997; Sapolsky, 2004),
and reduced levels of p-CREB and BDNF can
reduce neurogenesis (Lee etal., 2002; Nakagawa
etal.,2002).
While precise mechanisms by which neuro-
genesis influences mood are unknown, multiple
studies in animal models provide support for
an association between mood function and hip-
pocampal neurogenesis. For example, factors
that induce depressive-like behavior (e.g., mater-
nal separation or social hierarchy related stress)
also diminish neurogenesis (Mirescu etal., 2004;
Lajud etal., 2012). Moreover, animals exposed to
antidepressant activities (such as physical exer-
cise and exposure to enriched environments)
or treated with antidepressant drugs display
increased levels of neurogenesis with improved
cognitive and mood function (van Praag et al.,
1999; Santarelli etal., 2003; Sahay and Hen, 2007;
Lee etal.,2013).
Although doubts have been raised that an
increased level of neurogenesis after antidepres-
sant treatments may be just an epiphenomenon,
some clinical and animal model studies support
the notion that improved DG neurogenesis is a
prerequisite for the beneficial effects of antide-
pressants. First, beneficial effects from antide-
pressants happen only after a chronic treatment
regimen, which rules out an acute effect on sero-
tonergic neurotransmission and instead points
to alternative mechanisms such as the require-
ment for antidepressant-induced improvements
in neurogenesis, which would take several
weeks before drug-induced neurons become

FIGURE13.3: Continued
hippocampus, chronically epileptic hippocampi exhibit dramatically reduced density of DCX positive newly gen-
erated neurons. Arrowheads point to regions in the subgranular zone (SGZ) where neurogenesis is active. The
arrow in C1 denotes a neuron that has migrated into the dentate hilus. A2, B2, and C2 are magnified views of
regions from A1, B1, and C1 demonstrating the morphology of newly generated neurons in three groups. In the
dentate gyrus of the age-matched intact rat (A2), DCX positive newly born neurons exhibit long apical dendrites
that extend into the molecular layer (ML) through the granule cell layer (GCL). Contrastingly, in the hippocampi
from epileptic animals (B2, C2), a vast majority of DCX positive neurons display basal dendrites (indicated by
short arrows). Figure D shows that the extent of neurogenesis declines greatly in the epileptic hippocampus in
comparison to the age-matched intact hippocampus. DH=dentatehilus.
Figure reproduced from Hattiangady, B., and Shetty, A.K. (2008), Implications of decreased hippocampal neurogenesis in chronic
temporal lobe epilepsy. Epilepsia, 49 Suppl 5,2641
214 Part II:Homeostatic Control
functionally integrated (Braun and Jessberger,
2014). Second, the beneficial effect of antidepres-
sant treatment correlates with the time of onset
of increased neurogenesis, as ablation of prolif-
erating NSCs in the hippocampus blocked the
antidepressant actions of drugs fluoxetine and
imipramine (Santarelli etal., 2003). Third, norep-
inephrine and antidepressants that block its reup-
take enhance neurogenesis through increased
proliferation of quiescent NSCs in the hippocam-
pus (Jhaveri etal., 2010). Fourth, an animal study
has demonstrated that newly generated neurons
serve as a buffer for stress response through their
effects on the hypothalamicpituitaryadrenal
axis (Snyder etal., 2011). However, several other
studies contradict the positive link between
neurogenesis and antidepressants. For example,
not all drugs with antidepressant effects require
adequate neurogenesis to ease depression (David
et al., 2009). Furthermore, an increased level of
neurogenesis obtained through genetic altera-
tions in normal animals enhances pattern separa-
tion function but has no effect on mood function
(Sahay etal., 2011a). Thus altered homeostasis of
NSCs and neurogenesis is one of the features of
MDD, but it remains to be examined whether
it is one of the causes or consequences of MDD.
Nonetheless, drug therapies that enhance NSC
activity and neurogenesis seem to ease cognitive
and mood impairments seen in MDD (Sahay and
Hen,2007).
A LT E R E D N S C A C T I V I T Y A N D
N E U R O G E N E S I S H O M E O S TA S I S
INALZHEIMERS DISEASE
Alzheimers disease (AD), an age-related neuro-
degenerative disease typified by a progressive loss
of memory, affects ~5 million Americans and
~45million people worldwide. Individuals with
AD display difficulties in learning, reasoning,
judgment, and communication; depression; and
personality changes (Goedert and Spillantini,
2006; Mu and Gage, 2011). Although the risk
for developing AD doubles every five years after
age 65, younger people may also get AD. While
the precise causes of AD are still unknown, it is
widely believed that AD occurs as a result of mul-
tiple factors, which include genetics (e.g., pres-
ence of a risk gene apolipoprotein E 4 [APOE 4])
and environmental and lifestyle factors such as
heart disease, stroke, high blood pressure, dia-
betes, and obesity (Reitz and Mayeux, 2014).
There are two alterations in the brain that are
believed to contribute to the evolution of AD
symptoms. First, the accumulation of a protein
called amyloid-beta (A) in the extracellular
space leads to the formation of amyloid plaques.
Amyloid-beta proteins are liberated from a larger
integral membrane protein called the amyloid
precursor protein (APP) through sequential
cleavage by - and -secretases (Mu and Gage,
2011). Second, the accumulation of another pro-
tein, tau, inside neurons leads to the formation of
neurofibrillary tangles (Reitz and Mayeux, 2014).
As these changes progress, other alterations
become evident, which include impaired transfer
of information at synapses, loss of synapses, and
neurodegeneration (Selkoe,2001).
While pathological changes affect multiple
brain areas in AD, the hippocampus is con-
sidered one of the earliest brain regions to be
affected (Scheff and Price, 2006; Ohm, 2007; Mu
and Gage, 2011). It is generally believed that an
alteration in hippocampal neurogenesis may
underlie cognitive and mood dysfunction seen in
aging and the early stages of AD (Lazarov etal.,
2010). Characterization of senile AD brains using
markers of newly born neurons such as DCX.
PSA-NCAM surprisingly suggested increased
hippocampal neurogenesis in AD, and these
increases were correlated with the severity of the
disease (Jin et al., 2004a). There was also some
indication of increased proliferative activity
of NSC-like cells in senile AD brains, based on
increased levels of proliferating cell nuclear anti-
gen (PCNA) positive cells in the hippocampus
(Jin etal., 2004a). In contrast, a study examining
younger (pre-senile) AD brains reported no alter-
ations in the DG neurogenesis, based on analyses
of cells expressing the proliferative marker Ki-67
(Boekhoorn etal., 2006). Another study reported
reduced markers of neurogenesis in association
with increased bone morphogenetic protein-6
levels in the hippocampus of AD patients (Crews
etal.,2010).
Studies in animal models of AD have
also provided divergent findings regarding
AD-induced changes in NSC activity and neuro-
genesis. Astudy using a transgenic mouse model
of AD with the Swedish and Indiana mutations
under the platelet-derived growth factor pro-
moter reported increased neurogenesis in the
hippocampus (Jin etal., 2004b; Lopez-Toledano
and Shelanski, 2007), consistent with the obser-
vations in senile AD brains discussed earlier
(Jin et al., 2004a). However, studies in multiple
other models of AD mostly revealed decreases in
hippocampal neurogenesis. These comprise the
 Adult Neural Stem Cells and Brain Homeostasis
presinilin1 (PS1) conditional knockout mouse
(Feng et al., 2001), the mouse overexpressing
a mutant form of APP (the Swedish mutation
APP695; Haughey et al., 2002), the mutant PS1
knock-in mouse (Wang etal., 2004a), the mutant
P117L transgenic mouse (Wen et al., 2004), the
homozygous PDAPP mouse (Donovan et al.,
2006), the double knock-in mouse carrying
targeted mutations in both APP and PS-1 (Zhang
et al., 2007), the mouse overexpressing the
Swedish variant of APP and the exon 9-deleted
variant of human PS1 (Verret et al., 2007), the
triple transgenic mouse 3xTg-AD that harbors
three mutant genes (APPswe, PS1M146V, and
tauP301L; Rodriguez etal., 2008), and the Tg2576
transgenic mouse overexpressing the human APP
isoform harboring the Swedish double mutation
(Krezymon etal.,2013).
When taken together, most studies imply
changes in the homeostasis of hippocampal NSCs
and DG neurogenesis in AD, although there is
disagreement regarding upregulation versus
downregulation of neurogenesis in AD brains.
Increased neurogenesis observed in some senile
AD brains could be a compensatory response
to deafferentation and neuronal loss. This may
also be due to the effects of AD drug therapy, as
administration of AD drugs can enhance DG
neurogenesis in mouse models (Jin etal., 2006a).
Thus discrepancies in findings between studies
likely reflect differences in the stage of the disease
at the time of characterization, drug treatment
provided to patients, and methods employed for
analyzing proliferating cells. This necessitates
systematic investigation of larger cohorts of AD
brains at different stages of the disease. It will
be also necessary to consider other issues such
as the age of patient at the time of AD onset, the
type and doses of drugs taken, and the duration
of drug therapy in different AD patient popula-
tions. Decreased neurogenesis seen in several
mouse models of AD may reflect the advanced
stage of the disease at the time of analyses. This is
because in some mouse models of AD, cell prolif-
eration was significantly enhanced at early stages
of neurodegeneration, whereas the survival of
newly generated neurons was impaired at later
stages, implying increased adverse changes in the
microenvironment with time after the onset of
disease (Chen etal., 2008; Perry etal.,2012).
Nevertheless, it remains to be determined
whether therapies that normalize the homeosta-
sis of NSCs and neurogenesis would slow down
the progression of AD if such interventions were
215
applied at the early stage of disease. Animal stud-
ies performed so far are mostly supportive of
this possibility. Multiple strategies that enhance
neurogenesis have been shown to improve cog-
nitive performance in several transgenic mouse
models of AD. The strategies include physical
exercise or exposure to enriched environments
(Costa et al., 2007; Nichol et al., 2007, 2009),
inhibition of microglial activation (Biscaro
etal., 2012), choline supplementation (Velazquez
et al., 2013), neural precursor cell grafting (Ben
Menachem-Zidon, et al et al., 2014), curcumin
treatment (Tiwari et al., 2014), and electroacu-
puncture (Li et al., 2014). Thus normalizing the
homeostasis of NSC activity and neurogenesis in
the early stages of AD may considerably delay the
severity of the disease and promote better quality
of life to AD patients.
Furthermore, because impaired neurogen-
esis likely precedes the deposition of A plaques
(Chuang, 2010) and normal aging is not associ-
ated with significant decreases in neurogenesis
in humans (Spalding et al., 2013), examining
neurogenesis in patients with mild cognitive
impairment may aid in early diagnosis of AD.
Although some techniques that can indirectly
measure neurogenesis in living human brains,
such as the measurement of blood flow in the DG
or the detection of a spectroscopic biomarker by
magnetic resonance spectroscopy, are available
(Pereira etal., 2007; Manganas etal., 2007), more
advanced techniques need to be developed for
accurately quantifying neurogenesis in vivo in
healthy and disease conditions (Ho etal.,2013).
SIGNIFICANCE OF
NSC ACTIVIT Y AND
NEUROGENESIS FOR BR AIN
R E PA I R A F T E R S T R O K E
Stroke affects nearly 15 million people world-
wide each year. Of these, 5 million die due to
stroke-related complications. Among patients
who survive stroke, ~90% endure neurological
deficits, requiring rehabilitation therapy and/or
prolonged clinical care (Jorgensen et al., 1997;
Wang et al., 2012). Currently there is no effec-
tive drug or alternative therapy that adequately
reverses neurological deficits caused by stroke,
and hence nearly 50% of stroke survivors expe-
rience lifelong disability. However, the observa-
tion in animal models that NSCs in the SVZ of
the forebrain and the SGZ of the hippocampus
respond to stroke or ischemia with increased
proliferation and enhanced production of new
216 Part II:Homeostatic Control
neurons has provided a glimpse of self-repair
capability of the adult brain after insults. This
has resulted in a new direction of stroke research
focused on restoration of function through mobi-
lization of forebrain NSCs (Wang etal.,2012).
Multiple animal studies have shown that focal
cerebral ischemia stimulates NSC proliferation
in the SVZ, resulting in the production of a large
number of neuroblasts (Jin etal., 2001), which take
a detour (from their normal path of entering the
rostral migratory stream and reaching olfactory
bulb) and arrive in areas of stroke-induced brain
damage (Parent etal., 2002; Ohab etal., 2006). In
these regions, neuroblasts undergo maturation
and express markers, mature neurons such as
the microtubule associated protein-2 and NeuN.
An elegant study by Arvidsson and colleagues
(2002) has further shown that stroke-generated
new neurons, following their migration into the
severely damaged area of the striatum, differen-
tiate into mature, striatal medium-sized spiny
neurons, implying that stroke induces differen-
tiation of NSC-derived new neurons into specific
phenotypes that are destroyed by the ischemic
lesion. Studies have suggested that up-regulation
of erythropoitin after stroke promotes NSC pro-
liferation in the SVZ, the migration of newly
born neuroblasts into the peri-infarct cortex, and
angiogenesis in the damaged brain area (Wang
et al., 2004b; Tsai et al., 2006). Poststroke neu-
roblast migration has also been attributed to the
vascular production of stromal-derived factor-1
and angiopoietin-1 (Ohab etal., 2006). Follow-up
studies have also demonstrated the formation
of synapses between stroke-generated new neu-
rons and the host striatal neurons (Yamashita
etal., 2006), as well as electrophysiological prop-
erties of synaptic integration (Lai et al., 2008).
Investigations employing the global cerebral
ischemia models have also revealed the ability
of NSCs for functional neuronal replacement
after injury (Nakatomi etal., 2002). In this pro-
totype, activation of NSCs in the SVZ bordering
the hippocampus led to a massive regeneration of
hippocampal pyramidal neurons after ischemic
brain injury. Neural progenitor cells generated
from NSCs appeared to spontaneously migrate
into the hippocampus to regenerate new neu-
rons following ischemia in the CA1 subfield.
Interestingly, infusion of growth factors into the
lateral ventricle enhanced this regeneration and
promoted integration of newly added neurons
into the existing brain circuitry and ameliorated
neurological deficits (Nakatomi etal.,2002).
Importantly, NSC and neurogenesis abla-
tion approaches after ischemia have demon-
strated an exacerbation of ischemia-induced
deficits, implying that targeted addition of new
neurons by NSCs after stroke/ischemia indeed
contributes to functional recovery (Zhang etal.,
2004; Wang et al., 2012). Hence, therapies hav-
ing promise to increase the activity of NSCs after
stroke have attracted considerable attention.
Studies in animal models have uncovered multi-
ple strategies to increase forebrain neurogenesis
as well as functional recovery through stimula-
tion of NSCs after stroke. These include findings
that (i) intrastriatal infusion of glial cell-line
derived neurotrophic factor in the postischemic
period promotes several steps of striatal neuro-
genesis after stroke (Kobayashi et al., 2006); (ii)
the vascular endothelial growth factor improves
stroke-induced striatal neurogenesis (Sun et al.,
2003; Wang etal., 2007); (iii) intrathecal admin-
istration of sonic hedgehog stimulates NSC pro-
liferation and improves neurological recovery
after stroke in rats (Bambakidis etal., 2012); (iv)
overexpression of Wnt signaling enhances neuro-
genesis and improves neurological function after
focal ischemic injury in mice (Shruster et al.,
2012); (v)grafting of NSCs augments endogenous
neurogenesis and improves behavioral recov-
ery after stroke in rats (Mine et al., 2013); (vi)
physical exercise boosts neurogenesis and eases
functional deficits after stroke in rats (Zhang
et al., 2013); (vii) intravenous administration of
bone marrow derived mesenchymal stem cells or
umbilical cord derived mesenchymal stem cells
improves neurogenesis and promotes functional
recovery in neonatal and adult rats undergo-
ing ischemia (Yang et al., 2013; Iskander et al.,
2013; Tsai etal., 2014; Tsuji etal., 2014); and (viii)
improved social environment (housing a mouse
having stroke with a healthy cage mate) enhances
both BDNF levels and neurogenesis (Venna
etal., 2014). Despite these findings, causeeffect
relationship has not been established between
increased neurogenesis and functional recov-
ery after stroke. As neurogenesis enhanced by
strategies described here is often coupled with
angiogenesis, it is plausible that the improved
neurological function observed may be a result
of a combination of effects, including angiogen-
esis, neurogenesis, and axonal as well as dendritic
plasticity (Zhang etal.,2014).
Ischemic injury also enhances NSC pro-
liferation and neurogenesis in the SGZ of the
hippocampus (Liu et al., 1998; Jin et al., 2001).
 Adult Neural Stem Cells and Brain Homeostasis
Furthermore, poststroke-generated neurons are
recruited into the hippocampal networks (Geibig
etal, 2012). Studies imply that stroke stimulates
DG neurogenesis through activation of the cAMP
response element binding protein (Zhu et al.,
2004), Ca 2+ influx through L-type voltage-gated
Ca2+ channels (Luo et al., 2005), reduced neu-
ronal nitric oxide synthase and upregulation
inducible nitric oxide synthase expression (Luo
etal., 2007). The role of hippocampal neurogen-
esis in functional recovery following stroke is
unclear, however. Some studies imply that it helps
in the recovery from spatial memory deficits
(Luo etal., 2007), but direct evidence is lacking.
In contrast, a recent study shows that stimula-
tion of DG neurogenesis after stroke coincides
with an increased rate of aberrantly integrated
neurons into the hippocampus circuitry. This
abnormal circuitry development may contribute
to functional impairments, cognitive deficits, or
epilepsy often seen in stroke patients (Niv etal.,
2012). From this perspective, it may be neces-
sary to target therapies to specifically increase
SVZ neurogenesis to avoid an increased aberrant
recruitment of newly born dentate granulecells.
Studies on poststroke neurogenesis in
humans are rare. Jin and colleagues (2006b) pro-
vided evidence for stroke-induced neurogenesis
in the human brain. The status of neurogenesis
was analyzed in brain sections obtained from
human brain biopsies performed for the diagno-
sis of cerebral lesions that proved to be ischemic
strokes. Control specimens obtained from the
cerebral cortex of autopsied patients who died
without brain pathology exhibited either negligi-
ble or no proliferating cells. However, in speci-
mens from stroke patients, the cortical region
adjacent to the infarct core (ischemic penumbra)
contained considerable numbers of proliferating
Ki-67+ cells, a fraction of which co-expressed the
immature neuronal marker DCX. Examination
of MCM2 and PCNA expression in Ki-67+ cells
confirmed that Ki-67 labeling reflected recent
cell proliferation. Furthermore, the ischemic
penumbra exhibited considerable numbers of
cells positive for neuron-specific markers such as
DCX and Tuj-1. Some of the DCX+ or TuJ-1+ cells
also exhibited Ki-67 expression, suggesting that
these are newly born neurons. Although analy-
ses of biopsy specimens in this study precluded
precise identification of the source of newly born
neurons, their perivascular location suggested
that newly born neurons likely originated in
the SVZ and migrated along blood vessels into
217
the ischemic region. However, it remains to be
evaluated whether newly born neurons exhibit
long-term survival or contribute to brain repair
after stroke in humans.
OVER ALL CONCLUSIONS
It is now validated that NSCs persist in numer-
ous regions of the adult mammalian brain in a
state of quiescence but become activated in con-
ditions such as disease or injury. Channeling
these NSCs by facilitating their proliferation
and the neuronal differentiation of their prog-
eny would likely enhance neural repair after
injury or disease and help in maintaining brain
homeostasis. Neural stem cells in the adult hip-
pocampus in particular have received the most
attention because their presence has been vali-
dated in the human brain at all ages and multi-
ple animal studies signify that they are vital for
functions such as learning, memory, and mood.
The extent of NSC proliferation and neurogenesis
in the adult brain is regulated by multiple physi-
ological stimuli and signaling pathways. Neural
stem cells and hippocampal neurogenesis are
also highly sensitive to pathological conditions,
and their altered homeostasis likely contributes
to functional deficits in several neurological
disorders. First, in the early phase of TLE devel-
opment after SE or hippocampus injury, homeo-
stasis of NSCs and hippocampal neurogenesis is
severely disrupted in the form of increased pro-
liferation of NSCs and aberrant connectivity of
neurons recruited from their progeny. Clinically
applicable therapeutic strategies that can stabi-
lize NSC and neurogenesis homeostasis after SE
or hippocampus injury are therefore believed to
restrain the development of chronic spontane-
ous seizures as well as cognitive and mood dys-
function. Second, in the chronic phase of TLE,
homeostasis of hippocampal neurogenesis is
altered in the form of modified potential of NSCs
and greatly decreased generation of new neurons
(dentate granule cells). Application of approaches
capable of normalizing the potential of NSCs and
increasing the extent of normal pattern of neuro-
genesis is hence considered important for easing
cognitive and mood impairments seen in chronic
TLE. Third, impaired homeostasis of NSCs and
neurogenesis in the hippocampus is one of the
features of depressive disorders, and drug thera-
pies that improve NSC activity and neurogenesis
seem to ease cognitive and mood impairments
seen in depression. Fourth, altered NSC activ-
ity and hippocampal neurogenesis homeostasis
218 Part II:Homeostatic Control
has also been observed in AD though the extent
of changes (increase, no change, or decrease)
seemed to depend on the stage of the disease.
Hence, therapies that maintain or increase the
activity of NSCs and neurogenesis may slow
down the progression of AD, if such therapies are
applied early after the disease onset. Fifth, stroke
increases neurogenesis not only in the hippocam-
pus but also from the SVZ of the forebrain, which
has prompted the development of several strate-
gies to increase the activity of NSCs and neuro-
genesis after stroke. Such approaches have shown
better functional recovery after stroke in animal
models and hence have promise for ameliorating
functional deficits in stroke patients.
ACK NOWLEDGMENTS
This work was supported by grants from the
Texas A&M Health Science Center (Emerging
Technology Funds from the State of Texas),
National Institute of Neurological Disorders and
Stroke (R01 NS054780 & R01 NS043507), and
the Department of Veterans Affairs (VA Merit
Review Award and Research Career Scientist
Award). The author thanks Drs. B.Hattiangady,
M.S. Rao, R.Kuruba, B.Shuai, and B.Waldau for
their excellent contributions to Shetty Lab neural
stem cell and neurogenesis studies discussed in
this chapter.
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PARTIII

Homeostatic Manipulators

Preventative and Restorative Opportunities

14
Systems (Network) Pharmacology forBrain
Functionality Restoration
DORU GEORG M ARGINE A NU
OVERVIEW
The Neurologic and psychiatric diseases etiolo-
gies are as yet poorly understood; medications
merely treat the symptoms, and the prevail-
ing reductionist focus fails to grasp the brains
complexity and the intricacy of its dysfunctions.
Reductionism has led to some chief successes
in molecular biology, but it fails to account for
integrative biological functions that are emer-
gent properties occurring only at the organis-
mic level. Also, the reductionist goal of single
target-selective drugs, instead of substantiat-
ing an efficient drug design, caused a persistent
decrease in new drug discoveries, particularly
for brain diseases. In contrast to target-selective
pharmacology, emergent systems (or network)
pharmacology holds that drug action results
from multiple interactions with manifold targets
in a complex network underlying therapeutic
and collateral effects. This chapter exposes the
necessity of holistic-type systems biologybased
approaches that embrace the intrinsic complexity
of brain functional disturbances.
Brain functionality disorders are complex dis-
eases whose multifactorial and polygenic nature
do suggest a disturbance in networks of interac-
tions, and all major neuropsychiatric drugs do
in fact interact with multiple molecular targets.
The multimechanistic feature derives from the
development of those drugs based on phenotypic
observations in integrated in vivo systems, rather
than target-oriented rational drug discovery. But
systems pharmacology ultimately aims to design
ab initio drugs that would appropriately act on
the ensemble of cell components disturbed in a
given pathology, normalizing their network.
Pharmacologic support of cognitive functions
impaired in dementia-type degenerative disor-
ders is by now hardly capable of any cognitive
restoration. But normal cognition can be actually
doped by various psychostimulant drugs, whose
use became common for many healthy people,
though it raises serious safety concerns. On the
other hand, nootropic drugs were originally
meant to activate/restore deficient higher brain
activity while not inducing toxic phenomena.
The nootropic concept, tailored on the unusual
pharmacology of piracetam, remained largely
overlooked due to the lack of a straightforward
drug-receptor mechanism, while a multitude
of weak interactions have been documented for
piracetam-like nootropics. Though at odds with
the analytical quest for a definite molecular
interaction accounting for the effect, the original
nootropic concept does fit the holistic-oriented
framework of network pharmacology.
Network-oriented drug discovery requires
optimization of multiple positive and negative
influences on several targets while balancing
drug-like properties and controlling unwanted
effectsan endeavor by far more complex
than the much simpler single-target strategy.
Nonetheless, significant bioinformatics and
chemoinformatics resources and computational
methods, briefly outlined in this chapter, have
already been put forward and are progressing
rapidly.
Introduction:Brain Functionality
and Its Complexity
Brain function to ensure an organisms inte-
gration into its milieu and to exert centralized
control over all body organs is an exquisite and
demanding communicational activity that led
to emergence of the most complex system in the
universethe human brainwhose complexity
is unanimously acknowledged and increasingly
approached with concepts from mathematics,
physics, and computer science (Sporns, 2003;
Singer, 2007). Brain functionality ultimately
232 Part III:Homeostatic Manipulators
relies on the cellular property of excitability,
which is truly prototypal for neurons and sub-
tle but present also in some glia (Volterra and
Meldolesi, 2005), and on highly structured inter-
cellular (interneuronal, glia-neuronal, and inter-
glial) communication. The astounding structural
intricacy of the human brain is accompanied by
an exceptionally intense metabolism, represent-
ing a drain of over 20% of resting consumption
on an organisms energy budget (Laughlin and
Sejnowski, 2003). These very features render
the brain subject to various types of dysfunc-
tion: degeneration, physicochemical damage,
excitability troubles, and intercellular com-
munication alterations, largely deriving from
disruption of the homeostatic environment
needed for proper neuronal function (Boison
etal., 2013). They also entail the complexity and
heterogeneity of neurological (e.g., Tejada etal.,
2013) and psychiatric (Yang and Tsai, 2013;
Takahashi, 2013)diseases, as yet poorly mastered.
Consequently, the etiology of brain-specific
pathologies is but insufficiently understood, with
the corollary that brain disease medications are
mostly symptomatic and hardly curative.
This chapter highlights the idea that, for
advancing the means to restore disturbed brain
functionality, neuropsychopharmacology should
transcend the currently prevailing reduction-
ist focus and develop integrative, holistic-type
approaches aimed at embracing the intrinsic
complexity of brain functional disturbances.
Such a conceptual move is brought about by the
fairly recent ascent of systems biology (SB; Kitano,
2002; Sauer et al., 2007) that goes beyond the
mere analytic (reductionist) focus on the molecu-
lar components, taking into account the complex
and dynamic interactions within the organisms,
considered in their environment. Consequently,
the pathology is seen to reflect alterations of the
network of interactions that underlie organismic
functions, and the corresponding systems (or net-
work) pharmacology is aimed at renormalizing
that network. This chapter explores the coming
out, the current status, and the prospects of neu-
ropsychopharmacology in this conceptualshift.
The Rise ofSystems Biology
and Integrative Approaches
inNeuroscience
The advent of modern science essentially relies on
Francis Bacons Novum Organon (1620) and Ren
Descartes Discours de la mthode (1637) that laid
the bases of Western scientific inquiry. Bacons
suggestion that principles derived from particu-
lar cases might be applied to make general pre-
dictions later evolved into the inductive method.
Descartes proposal that each difficult problem
should be divided into as many simpler parts
necessary for its resolution (the analysis rule) and
that the effort to understand should start with the
simplest and easiest to know objects, gradually
rising to the knowledge of those composed (the
synthesis rule), had a foremost and long-lasting
impact on all Western science. This methodologi-
cal reductionism, which posits reducing explana-
tions to the smallest possible entities as the best
scientific strategy, entails epistemological reduc-
tionism, which admits that the knowledge of a
scientific domain about more complex objects
of study can be reduced to knowledge in scien-
tific domains about simpler objects, and derives
from ontological reductionism, which consid-
ers reality as composed of a minimum number
of kinds of simplest entities (Brigandt and Love,
2012; Fang and Casadevall, 2011). Descartes
reductionist thinking, relying on his belief that
God made the world as a clockwork mechanism,
triumphed in the classical mechanics of Newton
and culminated in the mathematically elegant
rational mechanics developed in the late eight-
eenth and early nineteenth centuries. Its full suc-
cess to explain the movement of all bodies, from
an apple falling from the tree to manmade vehi-
cles to celestial bodies, was so impressive that it
initiated the search for the mechanism of every
phenomenon as the ultimate goal of all sciences.
Descartes was so fascinated by the exquisite
clock-like automata of his time that he believed
that animals function as machines, albeit more
advanced, since being made by the hands of God
(Discours de la mthode, part5). The accumula-
tion of discoveries on cellular organization of
animals and plants culminated in the middle of
the 19th century in Theodor Schwanns cellular
theory, so that it is aptly said that biology gradu-
ated from natural history by holding the hand
of reductionism (Bose, 2013). Furthermore,
as every organism is made up of molecules, for
understanding phenomena at the organism level
biochemistry focused on biomolecules and on
biochemical reactions. The climax of the reduc-
tionist approach was reached in the third quarter
of the 20th century by the brilliant achievements
of molecular biology. These seemed to substanti-
ate the belief that all biology might be explained
in terms of chemistry and physics, as claimed
by Francis Crick (1966) in a provocative book.
 Systems (Network) Pharmacology for Brain Functionality Restoration
Along those lines, an extreme reductionist view
is the belief that consciousness and mental states
can be reduced to the chemical reactions occur-
ring in the brain (Van Regenmortel,2004).
Notwithstanding the wealth of knowledge
about individual cellular components and their
roles generated by the reductionist (analytic)
approach, it is obvious that no biological func-
tion can be attributed to an individual mole-
cule, because all the functions of organisms are
emergent properties that occur only at a given
level of complexity, while not existing at lower
levels (OConnor and Wong, 2012). This sim-
ple conclusion was imposed, among others, by
the unexpected results or just the lack of effect
of many knockout experiments performed to
reveal the functional role of a single gene (for
discussion, see Hopkins, 2008; Mazzocchi,
2012). It was further reinforced when, following
the complete mapping of the human genome,
it appeared that the number of protein-coding
genes is not larger than 21,000 (Pennisi, 2012),
which is less than four times the number of genes
of the unicellular eukaryote Saccharomyces cere-
visiae (Goffeau et al., 1996), though the human
genome has to encode for ~100 trillion cells of
many different types in the human body. This
evidently suggests that the observed complex-
ity is not encoded as such in the relatively few
genes, but it arises from the large set of mutual
interactions that they are capable of generating.
Thus the richness of information derives not
only from genes themselvesas it was the ear-
lier tenet of molecular biologybut also from
the interaction between genes and between their
respective products (see discussion in Ahn etal.,
2006; Sinha et al., 2009). Beyond the informa-
tion stored in the nucleotide sequence, epigenetic
mechanismschiefly DNA methylation and his-
tone modifications but also dynamic reorganiza-
tion of nuclear architecture and RNA and DNA
editinglargely influence the way genes are
expressed without altering the underlying DNA
sequence. Most notably, epigenetic mechanisms
account for cellular differentiation during mor-
phogenesis by selective inhibition/activation of
various genes (Reik,2007).
In neuroscience, while the Cartesian ana-
lytic approach could ensure an in-depth current
knowledge of how individual brain cells function,
it was much less successful in deciphering their
cooperation in large ensembles and could not
further our understanding of higher brain activi-
ties that integrate a huge number of neurons. The
233
detrimental effect of de facto forgetting the syn-
thesis rule, equally stated in Descartes Method,
while largely relying only on his analysis rule, is
particularly obvious in neuroscience. The insuf-
ficiency of the current understanding is evident
not only for the most challenging brain activity of
giving rise to consciousness and thought (Singer,
2007)but even for apparently much simpler and
long-studied subjects, such as the mechanism of
general anesthesia (see later discussion).
The necessity that neuroscience transcends
reductionism was strongly enhanced by the
awareness of the complexity of brain pathologies
causality. Apart from the fairly rare channelo-
pathies associated with a single mutated gene,
genetic studies have indicated that the etiologies
of common neurological (e.g. epilepsy) and psy-
chiatric (e.g. bipolar disorder and schizophrenia)
diseases involve many genes interacting mutu-
ally and with environmental risk factors, each
of which contributes only a small risk on its
own (Berkovic et al., 2006; Huang and Mucke,
2012; Szczepankiewicz, 2013; Jiang et al., 2013).
A recent example is the study of the genetic
architecture of Tourette syndrome and obses-
sive compulsive disorder (Davis etal., 2013)that
provides evidence that the two pathologies are
highly heritable and polygenic, and that a signifi-
cant majority of the heritability of both disorders
is associated with single nucleotide polymor-
phism variants. Previous attempts to identify
the individual genetic risk factors involved in
these two neuropsychiatric disorders have been
mostly unsuccessful, likely because there are
many genetic variations scattered throughout the
genome, each contributing only a small amount
to the overall risk. The complexity of the etiology
of brain dysfunction is illustrated in Figure 14.1
for the case of epileptic syndromes.
Epigenetic mechanisms and gene-
environmental interactions mediated by epige-
netic mechanisms have been recognized as fun-
damental causes of different neurodevelopmental
disorders, including autism spectrum disorders.
Likewise, there is strong evidence that neurode-
generative diseases are largely mediated by aber-
rant epigenetic mechanisms, which have been
also identified in diverse neuropsychiatric condi-
tions (for a review see Mehler, 2008). Moreover,
the complexity of brain pathologies causality
is enhanced by the confirmed participation in
various neurodevelopmental diseases associated
with epilepsy, autism disorders, and intellectual
disability of somatic mutations not present in
234 Part III:Homeostatic Manipulators
Gene 1
Epileptogenic mutations and variants
Gene 2
Gene 3
Gene N1
Gene N
FIGURE14.1: Convergence of multiple genetic and environmental factors in the etiology of epilepsy. Single gene
mutations, mostly in genes coding channel proteins, underlie only rare epileptic syndromes, while multiple gene
variants contribute to frequent polygenic epilepsies. The neurobiological spectrum of epilepsies appears fairly
continuous from a predominantly genetic to a predominantly acquired causation.
Slightly modified from Margineanu, 2012; reproduced with permission
all body cells, as they arise during embryonic
development and are undetectable in the parents
(Poduri etal.,2013).
Integrative approaches in biology date from
19th-century embryology, but the origins of a
holistic approach of animal description can be
traced back to Aristotle (384322 bc). He stated
in his treatise Metaphysics that the whole is
more than the sum of its parts, thus affirming
that a whole has emergent properties that can be
neither reduced nor deduced from the qualities
of its individual parts (Bothwell, 2006; Brigandt
and Love, 2012). This view, now held in a vari-
ety of scientific fields (Mazzocchi, 2008; Brigandt
and Love, 2012), is at the core of the SB that origi-
nates in the general systems theory that L. von
Bertalanffy developed (1950, 1968)in the 1950s.
The awareness of the limits of reductionism in
biology became so overt in the 1990s that it led to
symposia and monographs specifically devoted
to that subject (e.g., Bock and Goode, 1998).
Holistic approaches to biological data received
mainstream attention (Ideker etal., 2001; Kitano,
2002; Barabsi and Oltvai, 2004) after the pro-
gress of omics technologies permitted the simul-
taneous probing of thousands of genes and their
products, thus allowing SB to reach the molecular
level of description. SB deals with the networks
of interactions that underlie each biological
GeneticEnvironmental Head trauma
risk interaction
Perinatal
injury
Epileptogenic acquired factors
Monogenic
(chanelopathies) Stroke
Structural/
metabolic
Developmental
Genetic
lesions
Epilepsy
Tumor
Polygenic (complex
inheritance) Infectious
lesions
Previous
seizures
function and, consequently, considers that a dis-
ease expresses a disturbed network of interac-
tions rather than alteration of a critical molecular
component. Specifically, multi-omics and power-
ful bioinformatics computational tools make the
systemic approach to a better understanding/
treatment of nervous system diseases promising
(Noorbakhsh etal., 2009; Villoslada etal., 2009;
Hu etal.,2013).
An extra-scientific factor that forcefully
pushed amendment of the reductionist
approaches is the lowered yield of drug discovery
in the past two decades (Pammolli et al., 2011),
especially obvious with respect to the discovery
of novel drugs for brain diseases (Miller, 2010;
Arrowsmith, 2011). The productivity decrease in
pharmaceutical research and development is sig-
nificantly concomitant with the replacement of
the traditional phenotypic-based drug discovery
with a target-based one (Sams-Dodd, 2005). This
latter relies on the simplifying idea that a single
mutated gene, giving rise to one altered protein
causes a disease that should be treated by a selec-
tive drug targeted at that protein. In phenotypic
screening in vivo, the compounds are selected on
the basis of their ability to produce desired meas-
urable changes in the physiopathological states
of animals. In target-based screening, however,
the whole physiological context is replaced by
 Systems (Network) Pharmacology for Brain Functionality Restoration 235

16.0 14.3

% of Sys. Pharmacol. entries

14.0
12.0
10.0
7.3
8.0
6.0
3.7
4.0 2.7
1.3 1.9
2.0 0.7 0.7 0.7 0.2 0.2
0.0

e
se

e d in

ke

ia

r d nt

er

r
as

ps

as

e
io

en
Pa

rd
a

nc
ro

ss

m
ise
ise

se
ile

iso
r
St

Ca
r
e

h
di

Bi pai
Ep
ld

pr

p
zo
ric

De
a

im
iv

la
hi
ic

at
at

po
g

Sc

ve
hi
r
lo

ne

iti
yc
o
ur

ge

gn
Ps
Ne

de

Co
u ro
Ne

FIGURE 14.2: The percentage of the total number of papers indexed in the PubMed databank under the med-
ical subject heading (MeSH) systems pharmacology (Sys. Pharmacol.) that also include as MeSH as various
brain-specific pathologies. The percentage of systems pharmacology papers referring to cancer is given for com-
parison. The respective percentage value is given at the top of each column. Search completed on November 10,
2013.

the molecular interaction with only one target
(Pujol etal., 2010), the compounds being chosen
to optimize the affinity and selectivity toward a
single protein, whose involvement in a pathologi-
cal process is deemed critical.
The reductive philosophy of target-based
drug discovery was expected to substantiate an
efficient rational drug design, but it turned bit-
terly illusory (Sams-Dodd, 2005, 2013). The poor
productivity in the past decades of the pharma-
ceutical industry lured by the target-centered
approach favors a shift toward the SB-relying
systems pharmacology (Hopkins, 2008; Berger
and Iyengar, 2009; Boran and Iyengar, 2010)that
replaces drug discovery on realistic biological
grounds (Butcher, 2005). Systems pharmacol-
ogy has a view of drug action basically different
from traditional pharmacology. Traditionally,
a drug is meant to interact specifically with a
given therapeutic target that mediates the ther-
apeutic effect and only accessorily some side
effects. Any other putative interaction is con-
sidered off-target and mainly carrying the risk
of unwanted effects. On the other hand, systems
pharmacology conceives drug action as result-
ing from multiple interactions, possibly of weak
strength, with manifold targets pertaining to a
complex network that underlies both therapeu-
tic and collateraloccasionally adverseeffects.
Systems pharmacology approaches aimed to dis-
cover novel drugs for treating brain pathologies
are too recent for already-launched medicines,
but the scientific interestassessed by the num-
ber of papers devoted to these topicsis already
noteworthy (Figure 14.2), justifying optimistic
expectations.
The Necessary Track ofMultitargets
inNeuropsychopharmacology
Pharmacologys truly great idea of drugreceptor
interaction (Rang, 2006) suggested the meta-
phoric concept of a magic bullet drug that might
treat a disease upon selectively hitting a decisive
molecular target. Put forward by Paul Ehrlich as
a logical objective of antimicrobial therapy, that
metaphor largely seduced the ensemble of drug
discovery practitioners by the hope of a concep-
tually simple rational drug discovery. However,
for complex systemic diseasesas is the case
with all disturbances of brain functionalitythe
intrinsic multifactorial and polygenic nature of
the pathology turned the magic bullet into a lure.
In contrast, the SB view of diseases as arising
from the disturbance of networks of interactions
voids of sense the search for clean drugs that
would specifically interact with unique molecu-
lar targets.
The realistic alternative appears to be a
multitarget pharmacology (or polypharmacol-
ogy) by compounds that interact in a concerted
manner with multiple targets (Keith etal., 2005;
Hopkins, 2008). Drugs nonselective for a sin-
gle target but that selectively interact with sev-
eral targets involved in a given pathology have
236 Part III:Homeostatic Manipulators
been pertinently dubbed by Roth etal. (2004) as
magic shotguns. An overview of the antipsy-
chotic drugs used in the treatment of schizophre-
nia and similar severe mental disorders, recorded
in DrugBank, revealed for most of them multi-
ple targets, peaking at no less than 26 targets for
clozapine and quetiapine and 25 for olanzapine
and aripiprazole (Sun et al., 2012). Remarkably,
it was, on the other hand, highlighted that the
enriched pharmacology imparted on clozap-
ine by the multiplicity of its molecular targets,
rather than the lack of extra-pyramidal side
effects, is the origin of the clinical superiority of
this second-generation atypical antipsychotic
(Grnder etal.,2009). recent rise of the theoretical underpinning of SB,
The label magic shotgun, coined by Roth
etal. (2004) upon evaluating the mechanisms of
the drugs for mood disorders and schizophrenia,
is actually fit for most (if not all) of the drugs
effective in treating psychiatric and neurologi-
cal troubles. Thus, the antipsychotic drugs have a
wide pharmacological promiscuity, as they mod-
ulate (at nMM concentrations) the function of
quite numerous ion channels, both ligand-gated
and voltage-gated. Also the antidementia drugs
have been reported to interact with multiple ion
channels (Bianchi, 2010). Likewise, the fact that
all the reference antiepileptic drugs (AEDs) and
the more clinically useful new generation AEDs
exert their hypoexcitatory effects by interacting
with multiple ion channels was repeatedly high-
lighted (Bianchi etal., 2009; Margineanu, 2012).
Interestingly, functional modulation of multi-
ple ion channels has been clearly attested even
for the AED levetiracetam (Margineanu and
Klitgaard, 2002), which has a well-defined bind-
ing site, the synaptic vesicle protein SV2A (Lynch
etal., 2004), which is not a channel protein.
The pharmacological promiscuity of psychi-
atric, anticonvulsant, and anesthetic drugs that
modulate multiple molecular targets, particu-
larly the ligand-gated ion channels GABA A, Gly,
nACh, and 5-HT3 of the cys-loop family of recep-
tors, appears so marked that it was hypothesized
that it effectively contributes to clinical efficacy,
so that the promiscuity maps of current neurop-
harmaceuticals might provide templates against
which to screen candidate compounds (Bianchi
and Botzolakis,2010).
Systems pharmacology provides the ration-
ale to using multimechanistic drugs, but the
idea that absolute selectivity should not be a
sought-after objective for drugs aimed to treat
central nervous system (CNS) pathologies that
involve complex changes has been put forward
earlier. In a meeting in 1990 addressing the
question of whether high selectivity is always
beneficial (Fredholm and Abbott, 1990), Arvid
Carlsson highlighted the merit of correcting the
imbalance between receptors rather than a single
deficit. Likewise, in the circumscribed domain
of AEDs, at the same time that the fascination
with the rational drug design was generalized,
Lscher and Schmidt (1994) noted that, for AED
development, the target-centered rational
strategies did not show any superiority over the
older screening methods in animal models, and
they emphasized that the most clinically useful
AEDs are multimechanistic. However, before the
such lucid observations and opinions, though
expressed by high-ranking scientists, could not
deter the reductionist trend in neuropharmacol-
ogy, largely because a target-centered drug dis-
covery comforts the marketing-driven leaning of
pharmaceutical industry toward clean drugs,
deemed to be advantageous (Margineanu,2014).
Most clinically effective drugs currently
used for treating neurological and psychiatric
troubles interact with multiple targets, because
these drugs have been developed starting from
phenotypic observations, largely serendipitous,
in integrated in vivo systems, rather than by
target-oriented rational drug discovery. The vast
ensemble of neuropsychiatric drugs serendipi-
tously discovered that are highly multimecha-
nistic is illustrated in Figure 14.3 by the case of
valproate, which is truly emblematical, though
not singular, for the multiplicity of biochemi-
cal and cellular effects exerted by a fairly sim-
ple (MW=144.2) molecule. The systemic-type
phenotypic screening in CNS drug discovery
remarkably resisted the reductionist thinking
that underlies target pharmacology, as proven
by an analysis of the discovery approaches that
generated the new molecular entities approved
by the US Food and Drug Administration (FDA)
in the decade from 1999 to 2008 (Swinney and
Anthony, 2011). Surprisingly, given the main
focus on target-based approaches, phenotypic
screening led to the discovery of more first-in-
class small-molecule drugs than target-based
approaches (28 vs. 17 drugs, respectively).
Moreover, an assessment of the drug targets of
the new molecular entities approved by FDA
in the decade 2000 to 2009 revealed a trend
toward multitarget drugs, with the average
number of targets per approved drug rising to
2.5 from a previously estimated value of 1.8
(Lu etal.,2012).
 Systems (Network) Pharmacology for Brain Functionality Restoration
/NaF; P /T
Brain
cCMP
GABA
turnover
O
GHB
HO
VPA
formation
& release
... ...
!!!
Brain
Asp
HDAC1
Brain
Gly; Taurine
Brain
5-HT; DA
FIGURE14.3: Apart of the dozens of biochemical and
cellular effects of valproic acid (or valproate) that likely
contribute to the large-spectrum antiepileptic activity
(Lscher, 2002), mood-stabilizing and antimigraine
activity (Rosenberg, 2007; Chiu etal., 2013), potential
anticancer activity, and toxicity (Chateauvieux et al.,
2010)of this drug. The actions are designed by either:
(activation), | (inhibition), or (modulation). Red letters
on yellow background indicate relevance for anticon-
vulsant activity and brown letters on green background
indicate potential anticancer relevance. Asp = aspar-
tic acid, Cgmp= cyclic guanosine monophosphate,
DA=dopamine, GABA=gamma-aminobutyric acid,
GHB = gamma-hydroxybutyric acid, Gly =glycine,
HDAC1 = histone deacetylase 1, 5-HT = serotonin,
INa F = fast sodium current, INa P = lasting (permanent)
sodium current, IT = low-voltage activated (transient)
calcium current.
Apart from the multitargeted drugs, poly-
pharmacology is also performed by multidrug
therapies. This is in no way a novelty in the treat-
ment of brain diseases, since polypharmacy is a
long-standing and widespread clinical practice
both in psychiatry (Correll and Gallego, 2012;
Kukreja etal., 2013)and neurology (e.g., French
and Faught, 2009; Brodie and Sills, 2011). The aim
of any rational polytherapy is to find the com-
bination of drugs with complementary simple
mechanisms that will obtain additive, preferably
synergistic effects with infra-additive toxicity.
But the sobering reality is that by now the choice
of drug combinations remains largely empirical,
until a consistent systemic characterization of the
addressed pathology can substantiate a proper
mechanistic complementarity and an integrated
view of the multitude of potential molecular
interactions can allow mastering the toxicity.
Regardless, combining several drug molecules
raises many risks due to the potentially different
degrees of bioavailability, pharmacokinetics, and
237
metabolism (Keith et al., 2005), to unforeseen
drugdrug interactions, as well as to possible
multiplied side effects. Accordingly, the focus is
on the design by knowledge-based modification
of drug structure to achieve multifunctionality of
single-drug molecules that target multiple etio-
logical targets specific to a particular neuropsy-
chiatric disorder (Van der Schyf etal., 2006; see
later discussion).
Concerning the reality that the most effi-
cient CNS drugs are multimechanistic, one
should notice that those drug molecules pos-
sess that clinically beneficial feature simply by
chance, with the merit of the drug discovery
process being only of having duly selected them
by informed phenotypic screening and serendip-
ity and occasionally having improved them by
ADME (absorption, distribution, metabolism,
and excretion)-guided medicinal chemistry and
efficacy criteria. On the other hand, the ultimate
objective of systems pharmacology is, as men-
tioned, the ab initio design of drugs that will
appropriately act on the ensemble of cell compo-
nents disturbed in a given pathology, normaliz-
ing their network.
Might Systems Pharmacology
Rescue theControversial Nootropic
Drug Concept?
Cognitive enhancement is an unquestionable
therapeutic goal in all pathology-related treat-
ment, whether or not it involves age-linked cogni-
tive impairment. At the same time, an enhanced
and longer lasting high cognitive activity is that
typical human aspiration that begets education
and culture and whose appeal derives from both
the subjective self-confidence and the competitive
social advantage imparted by advanced mental
capability. The marked increase in life expectancy
of the population in developed countries is par-
alleled by the increased prevalence of cognitive
decline and dementia, causing cognitive troubles
to emerge as a major health threat of this cen-
tury (Bishop etal., 2010). Cognitive impairment
is emblematic for dementia-associated neurode-
generative conditions, but it is also prominent
in psychiatric disorders (Millan et al., 2012),
with cognitive enhancement standing a major
challenge to their treatment (Insel et al., 2013).
Pharmacological therapeutic approaches aimed
to support cognitive functions pathologically
affected are quite numerous (Froestl etal., 2012;
Froestl et al., 2013a, 2013b), but their success
notoriously lags behind the desire. Apart from
the restoration of impaired cognition, normal
238 Part III:Homeostatic Manipulators
neurocognitive function too can be doped by
various psychostimulant drugs (Lanni et al.,
2008). The use of psychostimulants such as meth-
ylphenidate and modafinil actually became a
common fact of life for many healthy people who
have to cope with demanding mental activities
(Sahakian and Morein-Zamir, 2007), including
professional scientists (Maher, 2008). This reality
raises serious ethical concerns (Farah etal., 2004;
Cakic, 2009; Hyman etal., 2013)related not only
to social justice but chiefly to the safety of using
strong promoters of attention and wakefulness.
On the other hand, 40 years ago, Corneliu
Giurgea, from the Belgian pharmaceutical firm
UCB, put forward the concept of nootropic
drugs (Giurgea, 1972), tailored to account for
the unusual pharmacology of piracetam, a pyr-
rolidine derivative synthesized at UCB in 1964
and marketed in 1971. That Pavlovian neuro-
physiologist coined the term nootropic (join-
ing the Greek words for mind and toward) ous, as this small molecule (MW = 142.16) has
to designate drugs that would directly activate
the integrative activities of the brain, restoring
deficient higher brain activity and opposing
brain impairment by agents such as barbiturates
and scopolamine while not inducing any toxic
phenomenon. Since his definition was simply
phenomenological, did not make any mechanis-
tic reference, and appeared to neglect the core
idea of drugreceptor interaction, the nootropic
concept hardly found any place in the major trea-
tises of pharmacology and remained controver-
sial. This apparent neglect is strongly contrasted
by the numerous early attempts to synthesize
other piracetam-like nootropics, as well as by the
steadily rising scientific interest and widespread
popularity in the lay public. Thus, even 20years
ago more than 1,650 piracetam-like compounds
had been synthesized worldwide in view of the
development of nootropics, many of them con-
taining the same 2-oxo-1-pyrrolidine acetic acid
(racetam) substructure (Gouliaev and Senning,
1994). Also, currently PubMed indicates 27,754
entries under the term nootropic (as of October 17,
2013), with 910 entries in2012.
Presently, the term nootropic is taken
as equivalent to cognitive enhancer (see, e.g.,
Lanni etal., 2008; Froestl etal., 2012)and to the
mass-medias catchy term smart drug. However,
the equivalence is but loose (Margineanu, 2011),
as it leaves aside the requirement of lack of toxic-
ity that was explicit in the original definition of
nootropic. Piracetam, which is unanimously
acknowledged as the forerunner of nootropic
drugs (Shorvon, 2001; Froestl et al., 2012), has
indeed such a low acute toxicity in all animal
species tested in preclinical studies that no LD50
could be determined. Likewise, it appeared very
well tolerated at therapeutic doses of the order
of several grams per day in several thousands of
patients included in controlled clinical studies
(Winblad, 2005). It is noteworthy that both the
lack of toxicity and the unusually high dosages
necessary to achieve therapeutic effects imply
very weak affinity of piracetam for the molecular
components in the cells of the receiving organ-
ism. Illustrative for this is the fact that piracetam
almost completely lacks affinity (pK=4.1) for the
SV2A proteinthe binding site of the pyrroli-
dine AEDs levetiracetam (pK=6.1)and brivar-
acetam (pK=7.1)in spite of the close chemical
kinship of the three compounds.
The mechanisms by which piracetam exerts
its actions remained obscure for a long time
(Giurgea, 1982) and they still appear multifari-
been reported to influence cholinergic, sero-
toninergic, noradrenergic, and glutamatergic
systems (for a review see Winblad, 2005). But
none of its effects results from a direct agonism
or antagonism, with piracetam showing no
noticeable affinity for the receptors of those neu-
rotransmitters, in contrast to the usual psycho-
tropic drugs. More recently, Ahmed and Oswald
(2010) revealed a weak positive allosteric modu-
lation by piracetam of the AMPA receptors. Such
a weak modulation might conceivably relate to
the repeatedly reported piracetam-induced
changes in physical properties of cellular mem-
branes (Mller et al., 1997; Eckert et al., 1999;
Mingeot-Leclercq et al., 2003). A recent analy-
sis of piracetam modulation of dopaminergic,
glutamatergic, or cholinergic brain systems
pinpointed the interaction with membrane
phospholipids as the most plausible mechanism
explaining piracetam action on the activities
of neurotransmitter systems (Slais et al., 2012).
Arecent neurochemical study of the prevention
by piracetam of scopolamine-induced memory
impairment (Marisco et al., 2013) suggests that
piracetam-induced improvement of memory
is associated with protection against oxidative
stress. Indeed, piracetam was shown to improve
mitochondrial dysfunction associated with oxi-
dative stress and/or aging (Keil et al., 2006),
with mitochondrial stabilization and protection
providing a likely justification of the putative
beneficial effect of piracetam on the cognition
of elderly patients. Moreover, a restoration by
piracetam of age-altered or pathology-altered
 Systems (Network) Pharmacology for Brain Functionality Restoration
fluidity of plasma membranes of various cells,
not only of neurons, via a nonspecific interac-
tion with cell membrane phospholipids could
account for the reported effects of piracetam
beyond the cognitive disorders, in a variety of
pathologies from vertigo to myoclonus to stroke
and even sickle cell anemia (see Winblad, 2005).
Furthermore, the restoring effect of piracetam
on the drug-impaired glucose uptake in eryth-
rocytes (Naftalin etal., 2004)might also derive
from its effects on membrane fluidity.
Piracetams lack of specificity and its putative
fluidizing effect on membranes remind us of the
classical case of inhalational general anesthetics,
for which a membrane lipid-located action, as sug-
gested by the Meyer-Overton linear correlation
of anesthetic potency with lipophilicity, was long
accepted as the basic mechanism. More recently,
deviations from that rule (Cantor, 2001) led to
a referral to the lateral pressure of lipids on ion
channels, after which the lipid theory was largely
superseded in the past decade by the alternative
view that anesthetics act by binding directly to
target proteins (Franks, 2006). However, not a
single but rather several targets have been identi-
fied as the most likely candidates, and currently
a multiple-receptor interaction is favored (Chau,
2010). Apart from the inhalational general anes-
thetics, another prototypal example of molecules
exerting a multitude of effects on the organism,
in particular on brain functions, while not hav-
ing any single ligandreceptor interaction that
would account all its effects, is alcoholone of
the oldest and most widely (ab)used of all psycho-
active drugs. Similarly to the case of general anes-
thetics, it has long been held that the effects on
the brain of the amphiphilic molecule of ethanol
are produced by acting primarily on membrane
lipids. However, within the past two decades it
appears that the primary sites of alcohol action
are membrane proteins (Peoples et al., 1996), a
view that has gained widespread acceptance (e.g.,
Spanagel, 2009; Komrekov and Jank, 2012). initial definition and uses the term nootropic
The primary targets of ethanol in the brain are
multiple: NMDA, GABA A, glycine, 5-HT3, and
nACh receptors, as well as L-type Ca 2+ channels
and G protein-activated inwardly rectifying K+
channels, with the initial effect on these receptors
and ion channels being followed by a second wave
of indirect effects of ethanol on monoamines,
neurosteroids, opioids, and endocannabinoids
(see Spanagel, 2009, for a review). Consequently,
it was highlighted that the fact that a molecule
as simple as the ethanol (MW=46.07) has such
239
pleiotropic effects and that, for its action, high
clinically relevant tissue concentrations (~10 mM)
are required makes it unlikely that any single
molecular mechanism, or its corresponding tar-
get, might explain all the pharmacology of that
drug (Paul,2006).
The explanatory shift in the case of inha-
lational anesthetics and alcohol from a purely
nonspecific interaction with membrane lipids
to a primary interaction with various types of
membrane proteins makes such an evolution
of the mechanistic understanding of the effects
of piracetam and chemically related nootrop-
ics conceivable as well. This is indeed suggested
by the already-mentioned allosteric interaction
with AMPA receptors and by a recent report that
piracetam induces in rat brain synaptosomes
a plasma membrane depolarization sensitive
to GABA receptors inhibitors and to AMPA/
kainate receptor inhibitors (Fedorovich, 2013).
Whether or not such an explanatory shift will
proceed with identifying other weak interactions
with proteins, it cannot question the rescue of the
original nootropic concept within the systemic
frame of network pharmacology. That holis-
tic type of concept, referring to the integrative
activity of the brain, is at odds with the analyti-
cal quest of a straightforward molecular interac-
tion that would account for all the effects. But
within the holistic-oriented conceptual frame-
work of network pharmacology, the multiplic-
ity of weak interactions is no longer a drawback,
and the weakly interacting and poorly specific
compoundsas in the case of most natural
products and many brain pharmaceuticalsare
no longer disregarded as merely dirty drugs but
instead are considered multimechanistic agents
potentially efficient to restore disturbed func-
tional networks. The future might show whether
and to what extent this possible conceptual res-
cue will have a practical outcome. On the other
hand, if one leaves aside the piracetam-inspired
simply as equivalent to cognitive enhancer,
irrespective of the requirement of lack of toxic-
ity, the term becomes a huge umbrella covering
a plethora of compounds (Froestl et al., 2012;
Froestl etal., 2013a, 2013b) actually or supposedly
enhancing one or another of the multiple mental
functionsmemory, attention, executive func-
tions, perception, language, psychomotor, and/
or executive functions (Figure 14.4)involved in
the complex concept of cognition (Nehlig, 2010;
Millan etal.,2012).
240 Part III:Homeostatic Manipulators
Cognition
Nootropic
E a certain pathology-disturbed functionality,
ns r
fu xecu
tio oto
nc ti
nc m
fu cho
tio ve
ns
y
Ps
n
Language
ptio
Me
enhancer
mo
ce
Per
ry
Attention
Neurotransmission
promoting wake/enhancing mood
Brain metabolism & homeostasis
FIGURE 14.4: Cognition as a highly complex con-
struct that can be impacted at several levels. Cognition
emerges from multiple levels of integration of con-
tributing factors, whose pharmacological modulation
might result in a cognitive-enhancing effect. While the
dietary supplements, nutraceuticals, and functional
foods claiming a procognitive effect (purportedly) act
at the basic level (e.g., via free-radicals scavenging) and
the current most popular smart drugs are stimulants,
the original definition of a nootropic drug envisioned
a direct action at the uppermost level. Such an action
remained hardly conceivable as a straightforward inter-
action with a given molecular target but becomes plau-
sible in terms of influencing a network of interactions.
Practical Matters ofSystems
Pharmacology ofCNSDrugs
A core concept of neuropsychopharmacology is
that molecules influencing behavior and those that
improve the functional state of patients afflicted
by psychopathologies chiefly act by increasing
or reducing the effect of specific combinations of
actions exerted by synaptic mediators. This princi-
ple is also held to largely underlie the pharmacology
of some neuropathologies, though less gener-
ally than in psychopharmacology. The principle
is fully compatible with systems pharmacology,
provided it is not reduced to aiming just one neu-
rotransmitter, as is customary in drug-discovery
projects. But optimizing multiple positive and
negative influences on several targets, while try-
ing to balance drug-like properties and control
unwanted effects, as implied by network-oriented
drug discovery, appears to be a fairly intimidating
endeavor when compared with the much simpler
single-target strategy. Nonetheless, significant
bioinformatics and chemoinformatics resources
and computational methods have already been put
forward and are progressing rapidly.
For systems pharmacology, attaining the
final goal of finding the drug(s) able to restore
without entailing other dysfunctions, imposes
gaining a global view of the network of molecu-
lar interactions underlying that disease state as
Memory
the first landmark. This implies a large-scale
integration, via bioinformatics, of diverse data
acquired from multiple levels of biological
Stimulant
complexity, from molecular-cellular (geno-
type, gene expression, DNAprotein binding,
proteinprotein interaction) to physiological
Neuroprotec- and clinical levels. The data, warehoused and
tive/recon-
structive
annotated within a comprehensive database,
have to be further transferred to visualization
programs to construct the network of interac-
tions involved in the syndrome tackled and the
molecular networks that define toxicity path-
ways and subtypes of disease (Schadt et al.,
2009; Arrell and Terzic, 2010; Iskar etal., 2011).
Large networks can be visualized with programs
that implement algorithms for drawing nodes
and performing basic analysis of data from
any field, be it social or biological, such as, for
example, the freely downloadable and evolving
programs NetDraw (https://sites.google.com/
site/netdrawsoftware/home) and Pajek (http://
pajek.imfm.si/doku.php?id=pajek). The charac-
terization of the molecular network undergo-
ing a disease is an ongoing process of iterative
modelexperimentation cycling, with biological
measurements leading to models amenable to
computational simulation, prediction, and then
experimentation for validation that potentially
substantiates model refinement.
In addition, to devise a disease-associated
molecular network, compounds must be char-
acterized by their action on that molecular net-
work. This characterization and annotation of
compounds should also be iteratively refined
on identification of networks associated with
off-target effects. Large-scale open-access data-
bases of chemicals and associated bioactivity
data, as well as the bioinformatics tools for the
analysis of small molecules properties in the con-
text of cellular networks, would become increas-
ingly popular and rapidly grow and diversify. The
National Health Institutes databank PubChem
(http://pubchem.ncbi.nlm.nih.gov/), launched
in 2004 as three linked databases (Substance,
Compound, and BioAssay) with a search tool of
structure similarity, quickly became a resource
for data on several hundreds of thousands chemi-
cals and their bioactivities, made readily acces-
sible (Wang et al., 2009). ChemBank (http://
 Systems (Network) Pharmacology for Brain Functionality Restoration
chembank.broadinstitute.org/) is a knowledge
environment created by the Chemical Biology
Program of Broad Institute (http://www.broa-
dinstitute.org/) to guide chemists synthesizing
novel compounds and biologists searching for
small molecules that perturb specific biologi-
cal pathways. It currently stores information
on hundreds of thousands of small molecules
and hundreds of biomedically relevant assays.
STITCH (short for Search Tool for Interactions
of Chemicals; http://stitch.embl.de/) is a data-
base of interactions between some 300,000 small
molecules and about 2.6 million proteins from
1,133 organisms (Kuhn etal., 2012). ChEMBL is
a very large open-access repository of annotated
compounds activity data taken from medici-
nal chemistry sources (https://www.ebi.ac.uk/
chembldb/), which currently houses more than
5.4 million bioactivity measurements for more
than 1million small molecules and 5,200 protein
targets (Gaulton etal., 2012). These are but a few
representative examples from a larger set of data-
bases of interest to systems pharmacology (Iskar
et al., 2011; Taboureau et al., 2012; Panagiotou
and Taboureau, 2012). Their growth largely relies
on the fact that data from in vitro binding assays
and chemical perturbation experiments are rou-
tinely deposited in public repositories, a trend
expected to extend in the future to multipara-
metric readouts and clinical and histopathologi-
cal parameters describing response to drugs at
tissue and organismic levels (Iskar etal.,2011).
Once the network of molecular interactions
involved in a given pathology is assembled with
reasonable reliability, the next step toward a sys-
tems pharmacology-derived drug is to identify
the chemical structure(s) likely to duly impact
that network. In principle, a network can be
impacted either by hitting a hub node of it or
by influencing the whole network via multiple
milder influences (Csermely et al., 2013). While
a central hit strategy seems indicated when aim-
ing to selectively damage the integrity of an
infectious agent or malignant cells, the search of
medicines for complex neurological and psychi-
atric syndromes seems likely to require a network
influence strategy, aiming to renormalize a faulty
network by mildly impacting multiple targets. At
this point, however, the understanding of net-
work dynamics in healthy versus diseased states
has not yet achieved the required reliability, and,
moreover, the methods of the network influence
strategy are as yet less developed than those of
the central hit strategy (Csermely etal.,2013).
241
Nevertheless, the chemoinformatics compu-
tational tools involved in hit identification and
lead selection by machine-learning methods that
can make reliable in silico predictions of drug
polypharmacology are evolving rapidly. Keiser
et al (2009) elaborated a similarity ensemble
approach that predicts numerous unanticipated
drug-target associations on the basis of chemical
similarities between a drug and the set of ligands
of a target. Some of the more likely predictions,
such as the antagonism of the 1 adrenergic
receptor by the antidepressives fluoxetine and
paroxetine (selective serotonin recapture inhibi-
tors), have been actually confirmed experimen-
tally. Likewise, a computational implementation
based on the similarity of drugs against sets of
molecules for which binding data is available
was able to predict with high-level precision the
entire affinity profile of a representative collec-
tion of antipsychotic drugs (Vidal and Mestres,
2010), confirming the efficiency of in silico recep-
torome screening. On the other hand, Campillos
etal. (2008) have shown that targets for drugs can
be predicted not only by exploiting similarity in
chemical structure or in activity across cell lines
but also by using phenotypic side-effect similari-
ties as an indication that two drugs might share
a target. Overall, it can be taken as proven that
chemoinformatics computational approaches are
able to predict the interaction with multiple tar-
gets so as to potentially suggest putative indica-
tions and side effects (Hopkins,2009).
The repeatedly revealed reality that modula-
tion of single-protein targets is not sufficient for
obtaining the desired restoration, and that partial
modulation of several targets can be more effec-
tive than the complete inhibition of a single tar-
get (Csermely etal., 2005; Morphy and Rankovic,
2007), has received widespread acceptance in the
case of neuropsychiatric dysfunctions that always
reflect complex molecular alterations. But the
design of multitarget drugs that have balanced
activity at each target of interest while simulta-
neously achieving a suitable pharmacokinetic
profile is a medicinal chemistry task by far more
challenging than designing a best-binding ligand
on the basis of the three-dimensional structure
of a single-target. The successful in silico recep-
torome screening, as in the examples cited ear-
lier, enhances the prospect of multitarget drug
design, but it does not actually represent drug
designing. However, the design of ligands against
profiles of multiple drug targets is clearly emerg-
ing. For example, Besnard etal. (2012) developed
242 Part III:Homeostatic Manipulators
an automated algorithm for ligand design that
adapts by multi-objective evolution, progressing
toward novel compounds with predefined mul-
titarget profiles. That algorithm was validated
by starting from the structure of the acetylcho-
linesterase inhibitor donepezil (approved for
cognitive enhancement in Alzheimers disease),
which has only minimal activity at D2 receptors,
and evolving with a dual objective of improving
D2 activity while achieving blood-brain barrier
penetration. A large proportion of the affinity
predictions of prospectively designed ligands
have been confirmed experimentally, therefore
attesting to the feasibility of designing drug leads
with multitarget profiles to achieve a desired
polypharmacology.
Concluding Remarks and Outlook
All organismic functions are emergent proper-
ties that cannot be understood outside of their
inherent complexity, and this reality is particu-
larly obvious when dealing with human brain
functions and their alteration. The current insuf-
ficient understanding of the multifactorial etiol-
ogy of neuropsychiatric diseases largely derives
from the reductionist failure to grasp the intrin-
sic complexity and heterogeneity of brain dys-
functions and has as a corollary that the existing
drugs to treat them are merely symptomatic, not
basically restorative.
However, the progress of omics technologies
and of the bioinformatics computational tools
have rendered possible holistic approaches of
the networks of interactions that underlie each
biological function, entailing a fairly recent but
rapidly mounting conceptual move from reduc-
tionism toward SB. Within the systemic concep-
tual frame, a disease is seen to reflect alteration
of a whole network of interactions, rather than
of a critical molecular component that might
be selectively targeted by a magic bullettype of
drug. Consequently, the systems (or network)
pharmacology aims to renormalize the disturbed
network by polypharmacology via multimecha-
nistic drugs that would interact in a concerted
manner with multiple targets or via suitable drug
combinations.
In fact, most clinically effective drugs that
treat neurological and psychiatric troubles actu-
ally interact with multiple targets, being promis-
cuous magic shotguns rather than magic bullets,
even in cases when the drug might have a defined
binding site. Interestingly and in some ways par-
adoxically, promiscuous compounds are typical
of smaller molecular size than target-selective
compounds (Morphy and Rankovic, 2007), as
illustrated by the highly multimechanistic val-
proate or alcohol. This might be also the case
of the old prototype nootropic drug piracetam,
a fairly small molecule showing no noticeable
specific binding and almost no acute toxicity
but reported to exert multiple weak effects on
brain neurotransmitters. Incidentally, the very
concept of a nootropic drug, deemed fuzzy in
conventional molecular pharmacology, appears
uncontroversial within the frame of systems
pharmacology.
The beneficial feature of most efficient cur-
rent CNS drugs as multimechanistic is not as yet
the result of a purposeful drug design but of the
fact that those drug molecules have been duly
selected by serendipity and phenotypic screening
in integrated in vivo systems, rather than emerg-
ing from target-oriented drug discovery. Due to
the unequaled complexity of brain functional-
ity, in CNS drug discovery the systemic-type
phenotypic screening remarkably resisted the
lure of reductionist thinking that underlies tar-
get-centered pharmacology. Beyond the domain
of CNS drugs, the striking concomitancy of
a marked decline in pharmaceutical develop-
ment of new drugs with the replacement of tra-
ditional phenotypic-based drug discovery by a
target-based one led to an increasing awareness
that phenotypic drug discovery is a viable strat-
egy with the potential to enhance innovation
(Lee etal.,2012).
The ultimate objective of systems pharmacol-
ogy is the ab initio design of drugs that would
appropriately act on the pathology-disturbed
network of molecular interactions, normaliz-
ing it. This task requires elaborating and ana-
lyzing the network of molecular interactions
underlying that disease state, which is a complex
activity, implying a large-scale computational
integration of diverse biological data and a pro-
cess of iterative model-experimentation cycling.
Subsequently, compounds must be characterized
by their action on that molecular network, which
also involves iterative refinement. The design of
multitarget drugs with due activity at several
targets and suitable pharmacokinetic profiles
involves in silico receptorome screening and
adaptive chemoinformaticstools.
Obviously, all of ideas constitute an endeavor
so highly challenging that it is not reasonable to
expect quick outcomes. However, the progress
so far has been quite rapid. Also, the awareness
 Systems (Network) Pharmacology for Brain Functionality Restoration
that systems pharmacology is the only way to
restore altered brain functionality (not just alle-
viate symptoms) with drugs is already manifest
in connection with such diverse pathologies as
schizophrenia, dementia, depression, stroke, and
epilepsy. Thus it does not seem a gratuitous state-
ment to expect that, in the not-too-distant future
system thinking might replace mere serendipity
as the main, driving force of drug discovery in
neuropsychopharmacology.
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15
Ketogenic Diets forNeurological Disorders
L I N D S E Y B . G A N O , M A N I S H A PAT E L , A N D J O N G M . R H O
INTRODUCTION
The ketogenic diet (KD) is a high-fat, low-
carbohydrate therapy for drug-resistant epilepsy
(Neal, 2008; Vining, 1998) and is increasingly
being studied for therapeutic efficacy in a num-
ber of neurological disorders, including epilepsy,
headache, neurotrauma, Alzheimers disease
(AD), Parkinsons disease (PD), sleep disorders,
brain cancer, autism, pain, and amyotrophic lat-
eral sclerosis (ALS) (Baranano & Hartman, 2008;
Stafstrom & Rho, 2012). This is a result of growing
experimental evidence for the broad neuroprotec-
tive properties of the KD and mechanistic linkages
to key cellular signaling pathways and fundamen-
tal bioenergetics processes, notably within mito-
chondria (Milder & Patel, 2012; Waldbaum &
Patel, 2010). In recent years, the field of neurome-
tabolism has been greatly amplified by interest in
dietary treatments such as the KD (Milder & Patel,
2012)and by the recognition that bioenergetic dys-
regulation may be a critical pathophysiological fac-
tor in diseases of the nervous system (Lin & Beal,
2006; Pathak, Berthet, & Nakamura, 2013). Indeed,
there is increasing appreciation for the concept of
energy failureprincipally from mitochondrial
dysfunctionas a key mechanism resulting in
neuronal death seen in neurodegenerative diseases
(Pathak, Berthet, & Nakamura,2013).
That diet and nutrition influence brain func-
tion should not be altogether surprising, and there
exist much clinical and laboratory data linking
disturbances in energy metabolism to a vari-
ety of clinical disorders (Roth, Szulc, & Danoff,
2011; Schiff et al., 2011; Waldbaum & Patel,
2010). Fundamentally, any disease in which the
pathogenesis is affected by disturbances in cel-
lular energy utilizationand this could apply to
almost every known medical conditionwould
This research was originally published in the Journal of Lipid Research. Gano LB, Patel M, Rho JM. Ketogenic
diets, mitochondria, and neurological diseases. Journal of Lipid Research. 2014 Nov; 55(11):221128. the
American Society for Biochemistry and Molecular Biology.
potentially be amenable to treatments that
restore normal metabolism. A common thread
of such diet-based therapies for brain diseases
is that metabolic substrates and nutrients can
exert profound effects on neuronal plasticity,
modifying neural circuits and cellular proper-
ties to enhance and normalize function. Further,
as there is increasing evidence for diet-induced
epigenetic mechanisms contributing causally to
the development of common chronic diseases
(McKay & Mathers, 2011; Roopra, Dingledine, &
Hsieh, 2012), greater knowledge of processes and
players such as DNA methylation, histone modi-
fications, and noncoding microRNAs will be
needed to understand the relationships between
energy dysregulation and therapeutic strategies
to counter such impairment (McKay & Mathers,
2011; Urdinguio, Sanchez-Mut, & Esteller,2009).
This chapter explores the rationale and evidence
for using the KD and related dietary treatments in
a broad range of neurological disorders and high-
lights novel mechanisms that have been implicated
in their actions. However, it is important to recog-
nize that much of the data discussed here remain
preliminary in nature. Nevertheless, the therapeu-
tic potential for dietary therapies for neurological
disorders remains almost limitless when viewed
from the perspective of salvaging neuronal bioen-
ergetic dysfunction (Masino & Rho, 2012; Milder
& Patel, 2012; Pathak, Berthet, & Nakamura,2013).
KETOGENIC DIET AND
E P I L E P S Y: H I S T O R I C A L
ASPECTS
The use of dietary manipulations to treat
epilepsyin particular, controlling seizures
through sustained fastingdates back to the
time of Hippocrates (Bailey, Pfeifer, & Thiele,
 Ketogenic Diets and Mitochondria and Neurological Diseases
2005; Wheless, 2008). In modern times, reports
of modifying diets to treat seizures emerged in
the early 20th century both in France and in the
United States (Wheless, 2008). Importantly, in
the 1920s, Drs. Stanley Cobb and W.G. Lennox
at Harvard Medical School conducted research
on the effects of starvation as a treatment for epi-
lepsy (Wheless, 2008). The effects of fasting were
seen within 2 to 3days, and hence they concluded
that a lack of carbohydrates in the diet prompted
a change in metabolism to force the utilization
of fats to produce energy (Wheless, 2008). It was
also recognized that in the fasted state, the body
produced ketone bodies (-hydroxybutyrate
[BHB], acetoacetate [ACA], and acetone) through
the liver and that a diet high in fats but low in car-
bohydrates could mimic fasting (Wheless,2008).
Dr.Russell Wilder at Mayo Clinic first referred
to this special high-fat diet as the ketogenic diet
(Wheless, 2008). During an era when antiseizure
drugs (ASDs) were scarce, the KD became quickly
popularized in large medical centers. However,
with the advent of diphenylhydantoin in 1938, the
KD quickly fell out of favor due to the simplicity
of prescribing an oral medication as opposed to a
strict and exacting dietary regimen. Nevertheless,
a variation of the KDthat is, the medium-chain
triglyceride (MCT) dietemerged later as yet
another dietary option for medically intractable
epilepsy (Huttenlocher, Wilbourn, & Signore,
1971)and remains today as an alternative to the
classic KD (Neal etal.,2009).
In recent years, there has been an explosion
in clinical use of the KD and its variants (Kossoff
etal., 2013; Muzykewicz etal., 2009), as well as
in scientific interest regarding the mechanisms
underlying their action (Lutas & Yellen, 2013;
Masino & Rho, 2012). Even beyond this resur-
gence in popularity for epilepsy, the diet has been
increasingly found to exert protective effects in
a variety of neurological diseases (Baranano &
Hartman, 2008; Stafstrom & Rho, 2012), and new
mechanistic insights have steadily emerged.
Efficacy inEpilepsy:Clinical Studies
The classic KD utilizes a fat-to-carbohydrate
plus protein ratio of 4:1 by weight, with approxi-
mately 90% of daily caloric intake coming from
fat, and the inclusion of a small amount of protein
(1 g/kg BW/day) to ensure adequate growth in
pediatric patients (Kossoff & Rho, 2009; Kossoff,
Zupec-Kania, & Rho, 2009; Kossoff et al., 2009).
A fat-to-carbohydrate ratio of 3:1 may be uti-
lized based on a patients needs and efficacy,
which underscores the importance of the need
249
for a dietician to implement and monitor the
patient while he or she is on the diet (Kossoff &
Rho, 2009; Kossoff, Zupec-Kania, & Rho, 2009;
Kossoff et al., 2009). The classic KD is primarily
based on ingestion of saturated long-chain fatty
acids (FA) (Kossoff & Rho, 2009). Upon restric-
tion of carbohydrates, ketogenesis occurs in the
liver and ketone bodies are exported to the circu-
lation (Figure 15.1). Circulating concentrations of
the major ketone bodies BHB, ACA, and acetone
have been shown to significantly increase within 1
to 3days after initiation of theKD.
A key aspect of the KD includes partial caloric
restriction (CR). Prior to starting the diet, a fast-
ing period of 24 to 48 hours is typical (Freeman,
Kossoff, & Hartman, 2007), but the need for this
requirement has been debated (Bergqvist et al.,
2005). An initial period of fasting may accelerate
seizure control and is therefore recommended for
patients with a greater need for immediate seizure
reduction (Freeman & Vining, 1999; Kossoff etal.,
2008). Adecrease in daily caloric intake of 10% to
25% is typical with the diet. Gluconeogenesis may
result from consumption of excess calories, and
therefore this slight reduction in calories is thought
to increase the efficacy of the diet by maintaining
ketosis (Bough & Rho, 2007; Masino & Rho,2012).
After initiation of the diet, seizure control
slowly increases within the first few days to weeks
(Freeman & Vining, 1999; Kossoff et al., 2008).
This is believed to be due to the gradual elevation
of circulating ketone bodies. However, since serum
levels of ketone bodies do not correlate tightly
with seizure control, it is unknown whether these
substrates are directly responsible for the clinical
effects observed (Kossoff & Rho, 2009). Despite
this uncertainty, it is known that a break from the
diet by ingestion of carbohydrates rapidly reverses
the antiseizure effects of the diet. In fact, the
onset of seizures can occur less than an hour after
administration of glucose (Huttenlocher,1976).
Historically, the KD has been primarily used
to treat epilepsy in pediatric patients. Efficacy
in seizure control with the KD is assumed to
be enhanced in younger patients and is thought
to reflect age-dependent changes in the expres-
sion of monocarboxylate transporters, which
transfer ketone bodies across the blood-brain
barrier from the systemic circulation (Pierre
& Pellerin, 2005; Prins, 2008) (Figure 15.1).
However, despite challenges in maintaining the
diet in older patientsmostly due to compli-
ance issuesimproved seizure control has been
reported as well in adolescents and adults (Klein
etal., 2010; Mady etal., 2003; Payne etal.,2011).
250 Part III:Homeostatic Manipulators

Ketogenic Diet

ATP
Fatty acids (FA)

UCP-2
Krebs cycle

FA ?
CAT CPT-1
FAO 1
Acetone Acetone BHB ACA

MRC
Acetyl-CoA ACA ACA 2
BHB BHB MCT-1 Acetoacetyl-CoA
3
BHB Acetyl-CoA
Krebs cycle Glial cell
Pyruvate Pyruvate
Glucose
Oxaloacetate Mitochondrion
Glucose Glycolysis
GLUT-1
Mitochondrion
Glucose Neuron
Hepatocyte Capillary BBB

Figure15.1 Metabolic pathways involved in ketogenic diet treatment. CAT=carnitine-acylcarnitine translo-

case; FAO=fatty acid oxidation; ACA=acetoacetate; BHB=-hydroxybutyrate; MCT-1=monocarboxylate transporter-1;
GLUT-1=glucose transporter-1; BBB=blood-brain barrier; CPT-1=carnitine palmitoyl transferase; UCP=uncoupling
protein; ATP=adenosine triphosphate; =3-hydroxybutyrate dehydrogenase, =succinyl-CoA3-oxoacid CoA trans-
ferase; = mitochondrial acetoacetyl-CoA thiolase; MRC=mitochondrial respiratory complex.
Reprinted with permission:Kim do Y, Rho JM. The ketogenic diet and epilepsy. Curr Opin Clin Nutr Metab Care 2008
Mar;11(2):11320. Lippincott, Williams & Wilkins.

Despite numerous clinical reports document-
ing efficacy of the KD against intractable epilepsy
(Kossoff & Rho, 2009), very few class 1 and 2 stud-
ies exist. After nearly a century of use, the strong-
est evidence became available only as recently as
2008. In this randomized controlled trial of 145
children, ages 2 to 16 years with daily seizures
and who did not respond to at least two ASDs, it
was shown that those who maintained the KD for
greater than 3months had a significant reduction
in the mean percentage of baseline seizures (Neal
etal., 2008). Seven percent of children on the KD
demonstrated a greater than 90% reduction in sei-
zures, compared with 0% on the control diet, and
there was a more than 50% reduction in seizures
in 38% of the patients on the KD versus 6% on the
control diet (Neal etal.,2008).
Efficacy inEpilepsy:
Preclinical Studies
Efficacy of the KD has also been shown in multi-
ple animal models of seizures and epilepsy. Rats
fed the KD for nearly 2 weeks exhibited increased
seizure thresholds after repeated intravenous infu-
sion of pentylenetetrazol (PTZ), a chemoconvul-
sant that blocks -aminobutyric acid (GABA) type
Areceptors, whereas a 3-week treatment increased
thresholds in response to a single exposure to both
PTZ and flurothyl, a volatile convulsant (Bough
etal., 2006). The KD also increased resistance in
the 6Hz model of epilepsy in mice, but the effects
required 5days on the diet and protection was lost
after 3 weeks (Hartman etal., 2008). Differences
in age-dependent protection and latency to seizure
control were shown in flurothyl-induced seizures
in juvenile and adult mice (Rho etal., 1999). The
KD increased time to the first generalized (clonic)
seizure in juvenile mice after 7 and 12days but not
after 3days in young mice or after 15days in adult
mice. However, 15days on the diet protected adult
mice against second generalized (tonic extension)
seizures, whereas juvenile mice showed no differ-
ence after 3, 7, and 12days on the diet. These and
other studies emphasize the inherent variability of
the effects of the KD as a function of the different
rodent models used, the heterogeneous methods
used for inducing seizures, as well as the dietary
protocols adopted.
Variations onthe KetogenicDiet
Despite documented efficacy of the KD against
seizure disorders, implementation remains chal-
lenging (e.g., poor compliance due to lack of pal-
atability and concerns regarding long-term health
consequences such as increases in cholesterol and
triglycerides, slow growth in pediatric patients,
 Ketogenic Diets and Mitochondria and Neurological Diseases
etc.) (Kossoff & Dorward, 2008; Kossoff, Laux,
et al., 2008; Kossoff et al., 2008). Hence clinical
researchers have sought alternative therapies that
retain antiseizure effects that are devoid of these
limitations. In addition to the MCT diet, two dis-
tinct variations on the KD theme include the mod-
ified Atkins diet (MAD) and the low-glycemic
index treatment (LGIT) (Cross & Neal, 2008)
(Figure 15.2). Finally, there is CR, a dietary prac-
tice known to reduce the progression of many
age-related diseases. Interestingly, this particu-
lar dietary strategy, which the KD was originally
designed to mimic, has been an intense focus for
researchers interested in the mechanisms under-
lying aging/longevity, neurological diseases, and a
host of other conditions (Bishop & Guarente, 2007;
Fusco & Pani, 2013; Libert & Guarente,2013).
The Medium-Chain TriglyerideDiet
The MCT diet, whichas the name implies
utilizes medium-chain triglycerides as the fat com-
ponent, was first described in 1971 and was reported
to induce antiseizure effects similar to the classic
KD (Huttenlocher, 1976; Huttenlocher, Wilbourn,
& Signore, 1971). In an initial comparison of
Classic ketrogenic Medium-Chain Triglyceride
(4:1)
3%
7%
20%
10%
70%
90%
5%
27%
25%
45%
70%
28%
Modified Atkins Low-Glycemic Index
Fat Protein Carbohydrates
Figure 15.2 Comparison of four major keto-
genic diets. Pie charts depict relative proportion of
calories provided by fat, protein, and carbohydrates for
the classic ketogenic diet (4:1 ratio by weight of fats to
carbohydrate plus protein), the medium-chain triglyc-
eride (MCT) diet, the modified Atkins diet (MAD),
and the low-glycemic index therapy (LGIT).
251
children under 10years of age on a 60% MCT diet
with the classic 3:1 KD, circulating levels of BHB
were found to be similar (Huttenlocher, 1976;
Huttenlocher, Wilbourn, & Signore,1971).
Medium-chain trigylerides produce a higher
level of ketosis compared with long-chain trigly-
erides, and this allows for a decrease in the intake
of these fats and a greater consumption of carbo-
hydrates and protein on this modified diet (Neal
et al., 2009).
The first long-term randomized trial com-
pared the effects of the classic KD and the MCT
diet in children with intractable epilepsy after
3, 6, and 12 months on these regimens (Neal
etal., 2009). The 4:1 classic KD was used for the
majority of the children, although some chil-
dren were on a 3:1 ratio. The MCT diet was com-
posed of ~15% carbohydrates, ~10% protein, 30%
long-chain FA, and 40% to 45% MCT fat. There
were no differences between dietary groups in
percentage of reduction in baseline seizures after
3, 6, and 12months. Additionally, no differences
were found between the two diets in those achiev-
ing seizure reductions greater than 50% or 90%.
Serum levels of ACA and BHB were greater in
children on the classic KD after 3 and 6months,
but only levels of ACA remained elevated in those
on the KD after 12months (Neal etal.,2009).
The Modified AtkinsDiet
The MAD was designed to mimic the high-fat
KD while allowing more liberalized intake of
protein, fluids, and calories to increase compli-
ance, especially in adults (Kossoff & Dorward,
2008)(Figure 15.2). However, the MAD still relies
on carbohydrate restriction, initially 10 g/day
in children and 15 g/day in adults; this can
be increased to 20 to 30 g/day after a couple of
months on the diet depending on seizure control
(Kossoff & Dorward, 2008). The MAD was origi-
nally developed at the Johns Hopkins Hospital
and is considered to be a modified version of
the original Atkins diet because weight loss is not
the principal goal and protein intake is liberal-
ized (Kossoff & Dorward,2008).
Seizure control with the MAD was originally
shown in 6 children and adults with intractable
epilepsy (Kossoff et al., 2003). In the 5 patients
able to maintain ketosis for 6 to 24 months, 3
demonstrated a significant reduction in seizures
and the opportunity to reduce concomitant ASDs
(Kossoff etal., 2003). Following this initial report,
the Dr.Robert C.Atkins Foundation sponsored
the first prospective study on the MAD in 20
children who experienced at least three seizures
252 Part III:Homeostatic Manipulators
per week and had prior use of at least two ASDs
(Kossoff etal., 2006). In those that maintained the
diet for at least 6months, 4 became seizure-free,
13 (65%) had a greater than 50% reduction in sei-
zures, and 7 (35%) demonstrated a greater than
90% decrease (Kossoff etal., 2006). Efficacy of the
MAD on seizure control in adults was also shown
in a prospective study of 30 adults with at least
weekly seizures and previous use of at least two
ASDs (Kossoff etal., 2008). Of those that main-
tained the diet, a greater than 50% reduction
in seizures was found in 14 (47%) patients after
3months and in 10 (33%) adults after 6months
(Kossoff etal.,2008).
The Low-Glycemic Index Treatment
The glycemic index (GI) is a measure of the rise in
circulating levels of glucose in response to inges-
tion of a specific food. The index estimates how
each gram of available carbohydrate in a food
source will elevate levels of blood glucose com-
pared to the consumption of pure glucose, which
is assigned a GI of 100. The rationale for the LGIT
was based on the clinical observation that the KD
led to a sustained drop in blood glucose levels,
and on laboratory studies showing decreased sei-
zure susceptibility in epileptic (EL) mice induced
by CR (Greene et al., 2001). The LGIT prevents
large postprandial increases in blood glucose,
resulting in more stable circulating levels of glu-
cose, and in early studies has been shown to pro-
vide seizure control in a broad range of patients
(Pfeifer, Lyczkowski, & Thiele, 2008; Pfeifer &
Thiele, 2005). This diet also allows for a greater
intake of carbohydrates (~4060 g/day), but this is
restricted to those foods with a GI of <50 (Pfeifer,
Lyczkowski, & Thiele, 2008; Pfeifer & Thiele,
2005). Total caloric intake is determined based on
patients needs, with 20% to 30% of calories com-
ing from protein and the remaining 60% from fats
(Pfeifer, Lyczkowski, & Thiele, 2008)(Figure15.2).
In an initial study of 20 epileptic patients
(534 years old), 10 out of 20 patients demon-
strated a >90% seizure reduction while on the
LGIT, despite achieving lower levels of circulating
ketone bodies (Pfeifer & Thiele, 2005). In a larger
cohort of children on the LGIT, 89% of whom
failed to respond to at least three ASDs, seizure
frequencies were reduced by greater than 50% in
42%, 50%, 54%, 64%, and 66% in patients after 1,
3, 6, 9, and 12months of treatment, respectively
(Muzykewicz etal., 2009). This study also showed
an inverse relation between efficacy of the diet
and serum glucose at certain time-points but did
not find a correlation between seizure reduction
and circulating levels of BHB during follow-ups
(Muzykewicz etal.,2009).
Caloric Restriction
As the KD was originally designed to mimic the
effects of fasting, it was postulated that CR (or
intermittent fasting) may provide similar results
without the need for a large regular intake of fats.
CR involves a reduction in total daily caloric intake
below the ad libitum level without the risk of mal-
nutrition (Bishop & Guarente, 2007; Guarente,
2013). CR is well known to ameliorate many
age-related and metabolic diseases and is the only
natural method known to increase lifespan across
multiple species (Libert & Guarente, 2013). As
such, comparisons of the KD and CR on seizure
control have been performed in animal models of
epilepsy. However, clinical studies involving CR
or intermittent fasting for epilepsy have yet to be
conducted (Hartman & Stafstrom,2013).
A study in rats of varying ages treated with mild
calorically restricted standard chow diet (90% of
recommended daily calories) and an isocaloric
KD showed protection against PTZ-induced sei-
zures (Bough etal., 1999). Rats fed the isocaloric
KD exhibited a greater threshold to PTZ-induced
seizures compared to CR-standard chow animals,
which in turn demonstrated greater protection
than those fed the same standard chow ad libitum
(Bough etal.,1999).
In the EL genetic mouse model of epilepsy,
CR (15% or 30%) delayed the development and
frequency of seizures in both juvenile and adult
animals (Greene et al., 2001). Additionally,
when compared with a previous study by the
same group that utilized the typical KD in EL
mice (Todorova et al., 2000), the authors found
that the mild form of CR (15%) resulted in
greater protection than the KD in juvenile mice.
Afollow-up study in adult EL mice showed that
CR in the context of both a ketogenic and a
high-carbohydrate/low-fat diet reduced seizure
susceptibility (Mantis et al., 2004), leading the
authors to conclude that restriction of calories,
not the composition of the diet per se, is the key
determinant of seizure control.
M EC H A N I ST IC OV E RV I E W:
KETOGENESIS
Sustained intake of a high-fat, low-carbohydrate
diet increases the rates of FA oxidation (FAO)
and gluconeogenesis. The end product of FAO is
acetyl-Coenzyme A(acetyl-CoA), which can enter
the tricarboxylic acid (TCA) cycle and reacts with
oxaloacetate to form citrate. However, under these
 Ketogenic Diets and Mitochondria and Neurological Diseases
metabolic conditions, oxaloacetate is also diverted
to gluconeogenesis and exported out of the mito-
chondria as malate. In the liver, increased produc-
tion of acetyl-CoA results in levels that exceed the
amount of oxaloacetate available for entry into the
TCA cycle, and ketogenesis is then initiated when
two acetyl-CoA molecules are combined to form
acetoacetyl-CoA (Figure 15.1). Acetoacetyl-CoA
is then condensed with another molecule of
acetyl-CoA to form 3-hydroxy-3-methylglutaryl
CoA (HMG-CoA), in a nonreversible step cata-
lyzed by the rate-limiting enzyme HMG-CoA syn-
thase 2 (HMG-CoAS2). The ketone body ACA is
then produced via the breakdown of HMG-CoA,
releasing a molecule of acetyl-CoA. ACA can then
be exported from the liver into the circulation for
uptake by tissues with high metabolic demands,
such as the heart, skeletal muscle, and the brain.
Alternatively, ACA can be further reduced to the
ketone body BHB by BHB dehydrogenase (BDH)
in a reaction that is coupled to the ratio of the oxi-
dized to reduced forms of nicotinamide adenine
dinucleotide (NAD+; i.e., NAD+/NADH) or the
spontaneous decarboxylation of ACA that yields
acetone, another ketone body. BHB and acetone
are also excreted from the liver and taken up from
the circulation into other tissues. In extrahepatic
tissues, BDH catalyzes the first reaction in ketone
body oxidation from BHB to ACA, which makes it
an important regulator of mitochondrial NAD+/
NADH status (Cotter, Schugar, & Crawford, 2013).
ACA is then further oxidized to acetyl-CoA for
entry into the TCAcycle.
KetoneBodies
The earliest demonstration of ketone bodies
inducing antiseizure effects was made by Keith
in the 1930s. Acetoacetate was shown to pro-
tect against thujone-induced seizures in rabbits
(Keith, 1933). This was followed decades later
by two additional studies demonstrating in vivo
antiseizure effects of ketone bodies (Likhodii
etal., 2003; Rho etal., 2002). In the Frings audio-
genic seizure-susceptible mouse (a model of
sensory-evoked reflex seizures), acute adminis-
tration of acetone and ACA led to an elevation
in seizure threshold, whereas the more prevalent
ketone body BHB had no effect on sound-induced
seizures. In a separate study, acetone displayed
dose-dependent antiseizure effects in four diverse
rodent models of epilepsy, including maximal
electroshock seizures, subcutaneous PTZ, amyg-
dala kindling, and the AY-9944 (an inhibitor
of cholesterol biosynthesis) model of atypical
absence seizures (Likhodii etal., 2003). Curiously,
253
studies showing antiseizure properties of the
major ketone BHB have not yet been forthcoming.
Neurotransmitters and Ion
Channel Regulation
A long-standing theory for KD action involves
changes in the levels of certain neurotransmitters
(NT), as a result of altered synthesis and/or clear-
ance from the synaptic cleft. The production of the
major excitatory NT glutamate is paradoxically
linked to the synthesis of the main inhibitory NT
GABA via the action of the biosynthetic enzyme
for GABA, glutamate decarboxylase (GAD). The
KD has been proposed to alter the metabolism of
glutamatein response to ketosisresulting in
increased levels of GABA and enhanced inhibi-
tory neurotransmission (Yudkoff etal.,2005).
Glutamate is cleared from the synaptic
space by astrocytes, which convert glutamate
to glutamine through the action of the glial
enzyme glutamine synthetase. Glutamine is
then exported to neurons where it is hydrolyzed
to glutamate and can then either be converted
to GABA or transaminated to aspartate, in a
reaction that also requires oxaloacetate. Since
the KD induces metabolic changes that require
available oxaloacetate for either incorporation of
acetyl-CoA into the TCA cycle or for gluconeo-
genesis, the production of aspartate from gluta-
mate is reduced. This may result in enhanced flux
through GAD to increase the synthesis of GABA
(Yudkoff etal.,2005).
In children fed the KD, cerebrospinal fluid
levels of GABA were increased but without a
change in glutamate concentrations (Dahlin
et al., 2005). However, rats fed the KD for 3
weeks showed a reduction in brain glutamate
levels, with no change in GABA (Melo, Nehlig,
& Sonnewald, 2006). Another study in rats fed
the KD for 3 weeks found increased levels of glu-
tamate and glutamine in the hippocampus, but
this was also associated with an overall decrease
in the transcripts of genes involved in synaptic
transmission (Bough etal., 2006). Further, using
both mild CR (i.e., 90% of daily energy require-
ments, or 10% CR) and an isocaloric KD, inves-
tigators found significant increases in the mRNA
expression of both isoforms of GAD (GAD65
and GAD67) in several brain regions and inde-
pendent of ketogenic effects (Cheng etal., 2004).
Additionally, reduced levels of aspartate were
found in a mouse model of ketosis, along with
the expected increases in acetyl-CoA, with no
change in the levels of GABA and glutamate
(Yudkoff etal., 2005). However, this same study
254 Part III:Homeostatic Manipulators
also showed increases in glutamine and GABA
upon infusion of the nitrogen donors alanine or
leucine (Yudkoff etal.,2005).
As another component of the NT theory, it
was hypothesized that the KD might also enhance
reuptake of glutamate from the synaptic cleft by
astrocytes (Bough etal., 2007). However, in rats fed
a KD for 4 to 5 weeks, there were no differences in
the levels of brain glutamate transporters and no
change in the reuptake activity of glutamate (Bough
etal., 2007). With regard to glutamatergic neuro-
transmission, a recent study demonstrated a possi-
ble alternative mechanism through which the KD
could suppress neuronal excitability. The ketone
bodies BHB and ACA were shown to directly influ-
ence presynaptic glutamate release by directly com-
peting with Cl for allosteric activation of vesicular
glutamate transporters, resulting in diminished
release of glutamate (Juge etal.,2010).
In the same study, direct application of the
potassium channel blocker 4-aminopyridine to
rat brain in vivo evoked seizures with concurrent
secretion of glutamate, and these effects were
blocked by ACA (Juge etal.,2010).
Despite these observations, whether
ketone bodies exert direct effects on excita-
tory or inhibitory neurotransmission remains
controversial. In the hippocampus, BHB and
ACA did not acutely affect GABAA, -amino-
3-hydroxy-5-methylisoxazole-4-propionate,
N-methyl-D-aspartate, kainate, or glycine recep-
tors (Thio, Wong, & Yamada, 2000). However,
ACA and BHB were later shown in vitro to reduce
the spontaneous firing rate of GABAergic neu-
rons in the substantia nigra pars reticulata, a
putative subcortical seizure gate, and this action
was dependent on opening of ATP-sensitive
potassium (K ATP) channels and GABAB receptor
activation (Ma, Berg, & Yellen, 2007). The same
group then showed that the open probability of
K ATP channels in the hippocampus in vitro was
enhanced in the presence of BHB (Tanner etal.,
2011). Further support for ketone body effects on
neuronal excitability was recently demonstrated
in sympathetic neurons in vitro. Here, BHB was
shown to be an agonist of the free FA receptor 3,
through which inhibition of N-type Ca2+ channels
was documented (Won etal.,2013). Interestingly,
the short-chain FA acetate, proprionate, and
butyrate are known agonists of free FA receptor 3.
Another link between the KD and K ATP chan-
nels was recently revealed. BCL-2-associated
agonist of cell death (BAD), a member of the
Bcl-2 proteins that govern apoptotic cell death,
was found to mediate a switch in the oxidative
metabolism of glucose versus ketone bodies, as
neurons and astrocytes from Bad -/- or BadS155A
mice exhibited reduced mitochondrial oxida-
tive metabolism of glucose but enhanced mito-
chondrial respiration in the presence of BHB
(Gimenez-Cassina etal.,2012).
Interestingly, this change in metabolism was
implicated in neuronal excitability, as Bad -/- or
BadS155A mice also demonstrated increased
resistance to KA and PTZ-induced seizures,
effects that required the opening of K ATP chan-
nels (Gimenez-Cassina et al., 2012). While K ATP
channel opening induced by low adenosine
triphosphate (ATP) levels is indeed an intrigu-
ing mechanism that couples the metabolic state
of the cell to neuronal excitability, the fact that
the KD and ketone bodies actually increase ATP
production needs to be reconciled with the K ATP
channel hypothesis (DeVivo etal., 1978; Kim do,
Vallejo, & Rho, 2010; Ma, Berg, & Yellen,2007).
Increases in the levels of the purine nucleo-
tide adenosine may also potently modulate neu-
ronal activity. Adenosine is produced from ATP
and itself produces antiseizure effects through
activation of inhibitory adenosine A1 receptors
(A1R) (Masino etal., 2012). As the KD increases
cellular levels of ATP (DeVivo et al., 1978; Kim
do, Vallejo, & Rho, 2010), this may result in
elevated levels of adenosine and subsequent
enhanced activation of A1R. In mice, targeted
deletion of A1R receptors (i.e., A1R+/- and A1R
-/-) or increased expression of adenosine kinase
(Adk-Tg), an enzyme that enhances clearance of
adenosine, causes spontaneous electrographic
seizures (Masino etal., 2011). A3-week treatment
with a KD led to decreased electrographic sei-
zures in Adk-Tg and A1R+/- mice but not in ani-
mals missing A1R receptors (A1R-/-), indicating
that adenosine may be an important mediator of
the KDs antiseizure effects (Masino etal.,2011).
Bioenergetic and Mitochondrial
Changes
Pathological changes in mitochondrial energy
metabolism and reactive oxygen species (ROS)
production are known to occur with epilep-
togenesis, and intriguingly the KD has been
found to profoundly affect these processes
(Rowley & Patel, 2013). In addition to enhancing
energy reserves, ATP levels, and the expression
of many enzymes involved in multiple metabolic
pathways in the mitochondria, the KD has also
been shown to increase mitochondrial biogen-
esis in the hippocampus (Bough et al., 2006).
Additionally, multiple studies have demonstrated
 Ketogenic Diets and Mitochondria and Neurological Diseases 255

elevated antioxidant activity, diminished produc-
tion of ROS, and decreased ROS-induced dam-
age with the KD (Masino & Rho, 2012; Milder &
Patel,2012).
Figure 15.3 summarizes the substrates and
pathways linking mitochondrial redox changes
to the TCA cycle and respiratory chain func-
tion. Impairment of mitochondrial bioenergetics
capacity can critically affect apoptosis, neuronal
excitability, and seizure susceptibility.
Antioxidant Activity, Reactive
Oxygen Species, and theRedoxState
Several studies indicate that the KD decreases
the production of ROS and limits ROS-mediated
damage, possibly by enhancing antioxidant

Figure 15.3 Mitochondrial function and neuronal excitability. Various aspects of the mitochondria
can lead to impairment of its bioenergetic capacity affecting neuronal excitability, apoptosis, and an increase
in seizure susceptibility. O2 production by complex Iand III of the electron transport chain (ETC) leads to the
production of ONOO in a reaction with NO, and H 2O2 through dismutation by the antioxidant MnSOD. H2O2 is
membrane-permeable and able to diffuse out of the mitochondria causing widespread oxidative damage. Excessive
O2 production also damages Fe-S containing enzymes involved in the tricarboxylic acid cycle such as aconitase.
OH can be formed from H2O2 through Fenton chemistry and lead to further oxidative damage of macromol-
ecules such as ETC complexes and mtDNA. Oxidative damage to mtDNA can lead to increased mutation rates
and a decrease in ETC subunit expression encoded by the mitochondrial genome. Alterations in the redox status of
GSH/GSSG and CoASH/CoASSG can cause an inability to protect against the deleterious effects of reactive oxy-
gen species. Modification of neurotransmitter biosynthesis within the mitochondria can affect levels of neuronal
excitability/inhibition. Oxidative damage to these targets can result in increased neuronal excitability resulting
from decreased mitochondrial membrane potential and adenosine triphosphate (ATP) levels affecting the Na+/
K+-ATPase and the release of cytochrome C, leading to apoptosis. mNa+C2+E=mitochondrial sodium calcium
exchanger; mCU = mitochondrial calcium uniporter; mNICE = mitochondrial sodium independent calcium
exchanger; MPT=mitochondrial permeability transition pore; GSH=glutathione; GSSG=glutathione disulfide;
CoASH=coenzyme A; CoASSG=coenzyme Aglutathione disulfide; GR=glutathione reductase; GPx=glu-
tathione peroxidase; cyto C=cytochrome C; m=mitochondrial membrane potential.
Reprinted with permission:Waldbaum S, Patel M.Mitochondria, oxidative stress, and temporal lobe epilepsy. Epilepsy Res 2010
Jan;88(1):2345. Elsevier.
256 Part III:Homeostatic Manipulators
activity. Rats fed the KD for 8 weeks showed
tissue-specific alterations in lipid peroxida-
tion, total antioxidant capacity, and antioxi-
dant enzyme activities (Ziegler et al., 2003).
Specifically, total antioxidant capacity was ele-
vated in the hippocampus, lipid peroxidation was
increased in the cerebellum, and glutathione per-
oxidase activity was enhanced in the hippocam-
pus (Ziegler etal.,2003).
The KD has also been shown to change the
mitochondrial production of hydrogen perox-
ide (H2O2) as a function of treatment duration.
A decrease in substrate-driven mitochondrial
H2O2 production was shown in rats after 3
weeks on the diet, and this was associated with
increased mitochondrial glutathione (GSH) lev-
els, depletion of which is known to occur with
seizures (Jarrett et al., 2008). The increase in
GSH was associated with elevated activity of
the rate-limiting enzyme in GSH biosynthesis,
glutamate cysteine ligase (GCL), and enhanced
expression of the GCL catalytic subunit, GCLC,
and modulatory subunit, GCLM, in rats fed the
KD (Jarrett etal.,2008).
The increase in GSH and levels of the GCL
subunits GCLC and GCLM observed by Jarrett
etal. (2008) prompted an investigation to exam-
ine the role of NF E2-related factor 2 (Nrf2), as
activation of this redox-sensitive transcription
factor is the primary mechanism that induces
this antioxidant pathway (Milder, Liang, &
Patel, 2010). Nrf2 is activated by cellular stress
and initiates transcription of a diverse set of
genes, such as antioxidant defense, drug trans-
porters, metabolic enzymes, and transcription
factors, by binding to the antioxidant or electro-
phile response elements (Suzuki, Motohashi, &
Yamamoto, 2013). This study demonstrated that
production of mitochondrial-derived H2O2 was
initially enhanced after 1day on the diet but was
significantly decreased at the 3-week time-point
(Milder, Liang, & Patel,2010).
This initial increase in H2O2 was accom-
panied by an elevation in the lipid peroxida-
tion product 4-hydroxy-2-nonenal (4-HNE),
both of which stimulate Nrf2 activity through
oxidation of the inhibitory binding partner
Kelch-like ECH-associated protein 1, result-
ing in the release and nuclear translocation of
Nrf2 (Sekhar, Rachakonda, & Freeman, 2010).
The acute rise in H2O2 and 4-HNE with the KD
coincided with increased hippocampal nuclear
expression of Nrf2 after 1 week on the KD,
indicative of enhanced Nrf2 activation. Levels of
Nrf2 remained elevated after 3 weeks on the KD,
and this was associated with increased activity
of NAD(P)H:quinone oxidoreductase (NQO), a
prototypical Nrf2 target. Despite a depletion of
GSH in liver homogenates at various time-points
(3days, 1 week, 3 weeks), levels of reduced coen-
zyme A, a measure of mitochondrial antioxidant
capacity, was decreased at 3days but elevated after
3 weeks. Likewise, in the liver, nuclear extracts
demonstrated increased Nrf2 expression after 1
and 3 weeks on the diet, accompanied by eleva-
tions in both NQO activity and the expression
of Nrf2 target heme oxygenase-1 (HO-1) after 3
weeks on the diet (Milder, Liang, & Patel, 2010).
Interestingly, a recent study found that increas-
ing Nrf2 expression in a rat model of temporal
lobe epilepsy decreased spontaneous seizures
(Mazzuferi etal.,2013).
Acute application of the ketone bodies BHB
and ACA in hippocampal slices enhanced cata-
lase activity in response H2O2 (Kim do, Vallejo, &
Rho, 2010) and decreased oxidation of carboxy-
H2DCFDA, a dye often used as an indicator of
intracellular ROS (Maalouf & Rho, 2008). In iso-
lated mitochondria, ACA and BHB have been
shown to decrease ROS levels in response to gluta-
mate by enhancing oxidation of NADH (Maalouf
etal., 2007). Additionally, ACA and BHB reduced
mitochondrial ROS both basally and in response
to the ATP synthase inhibitor oligomycin (Kim do
etal.,2007).
A possible mechanism mediating the decrease
in mitochondrial ROS production with the KD and
ketone bodies is enhanced expression of uncou-
pling proteins (UCP). Increased activity of UCP
can diminish the mitochondrial membrane poten-
tial (), resulting in a decrease in ROS production,
and this has been associated with increased resist-
ance to KA-induced seizures (Sullivan etal.,2003).
Additionally, FA can induce increases in UCP
expression possibly through enhanced activity
of transcription factors, such as the peroxisome
proliferator-activated receptor (PPAR) and the
forkhead box (FOXO) families of transcription
factors (Azzu & Brand, 2010). In mice fed the KD,
UCP activity was enhanced, and this was associ-
ated with increased levels of UCP2, UCP4, and
UCP5 in the hippocampus (Sullivan etal., 2004).
Additionally, ROS productionassessed in the
presence of oligomycin to maximize was
reduced in mice fed the KD (Sullivan etal.,2004).
Recently, BHB was shown to be an inhibitor
of class I histone deacetylases (HDAC) in vitro
and in vivo (Shimazu etal., 2013), and this activity
was associated with increased resistance to oxida-
tive stress. Specifically, BHB increased acetylation
 Ketogenic Diets and Mitochondria and Neurological Diseases
of histone H3 lysine 9 and histone H3 lysine 14
and enhanced transcription of genes regulated by
FOXO3A, including the antioxidant enzymes man-
ganese superoxide dismutase and catalase. Further,
BHB (administered in vivo for 24 hours via an
osmotic pump) decreased protein carbonylation,
4-HNE, and lipid peroxides in the kidney. Although
the authors did not report such effects in neuronal
tissue or cells, it is possible that direct inhibition of
HDACs and the ensuing transcriptional changes
may mediate some of the antioxidant effects known
to occur in the brain with theKD.
Mitochondrial Permeability
Transition
ACA and BHB both blocked neuronal death in
response to diamide, a mitochondrial permeabil-
ity transition (mPT) activator, in a mechanism
independent of oxidative stress. Additionally, ACA
and BHB mimicked the effects of the mPT blocker,
cyclosporin A, as all three increased the threshold
for calcium-induced mPT opening (Kim do etal.,
2007). More recently, it was shown that prolonged
in vitro seizure-like activity induced with
low-Mg2+ in rat glio-neuronal cocultures resulted
in depolarization of and mPT opening, with
subsequent cell death (Kovac etal.,2012).
These effects were reversed by CsA, a known
inhibitor of mPT. Despite these observations, the
causal relationships between mPT, the KD, and
epilepsy have yet to be clearly delineated.
Glycolytic Restriction/Diversion
A key feature of the KD is a relative reduction in
glycolysis and an increase in nonglucose sources
of fuel through the oxidation of FA and ketone
bodies, which ultimately feed the TCA cycle
through a process known as anaplerosis (i.e., the
replenishing of depleted metabolic cycle inter-
mediates). Glycolytic restriction is thought to be
an important mechanism mediating the antisei-
zure properties of the KD. As mentioned, CR has
been shown in a mouse model of epilepsy to ren-
der antiseizure, and possibly antiepileptogenic,
effects (Mantis etal.,2004).
The earliest clinical observation supporting
this notion is the rapid reversal of seizure con-
trol upon ingestion of carbohydrates or glucose
in patients on the KD (Huttenlocher, 1976).
Additionally, studies utilizing labeled metabolic
precursors have shown reduced oxidative metab-
olism of glucose (Melo, Nehlig, & Sonnewald,
2006; Yudkoff etal.,2005).
Use of glycolytic inhibitors has enabled fur-
ther insights into this possible mechanism for
257
seizure control. In vitro application of 2-deoxy-D-
glucose (2-DG), an inhibitor of phosphoglu-
cose isomerase, resulted in antiseizure effects
against the GABA A receptor antagonist bicucul-
line, 4-aminopyridine, and increased extracel-
lular K+ (Stafstrom etal., 2009). Additionally, in
vivo administration of 2-DG reduced seizures
induced by audiogenic and 6Hz stimulation in
mice (Stafstrom et al., 2009) and chronically in
a rat kindling model (Garriga-Canut etal., 2006;
Stafstrom etal., 2009). The antiseizure effects of
2-DG may be partially mediated by changes in
the expression of genes encoding brain-derived
neurotrophic factor and its receptor TrkB, both
of which are regulated by the activity of the tran-
scription factor neural restrictive silencing factor
(NRSF), which represses transcription by bind-
ing to the neuron restrictive silencing element
(NRSE) in promoter regions (Garriga-Canut
et al., 2006). NRSF transcriptional repression
was enhanced by 2-DG, and this was associ-
ated with a reduction in acetylation and an
increase in methylation of histone H3 lysine 9
at the NRSE promoter, epigenetic modifications
associated with suppression of gene transcrip-
tion. NRSF-mediated repression required an
interaction with the transcriptional corepressor
carboxyl-terminal binding protein that was dis-
rupted with increasing concentrations of NADH,
a cofactor that is elevated upon increased gly-
colytic flux. This suggests that the antiseizure
actions of 2-DG may be mediated by a decrease
in cytosolic and nuclear levels of NADH and
subsequent influences on histone modifications
(Garriga-Canut etal.,2006).
Interestingly, changes in histone acetylation
have also shown to be mediated by the activ-
ity of ATP-citrate lyase (ACL), which converts
glucose-derived citrate to acetyl-CoA (Wellen
etal.,2009).
Enhanced flux of acetyl-CoA from ACL
increased acetylation of H3, and this resulted in
increased expression of glycolytic enzymes. This
effect was found to be specific for acetyl-CoA
produced from glycolysis, as changes in histone
acetylation did not occur in cells supplemented
with FA under glucose-replete conditions
(Wellen etal.,2009).
Acetyl-CoA has yet another role as the
cofactor for acetylation of the -lysine residues
on histones and nonhistone enzymes by lysine
acetyltransferases (KAT) (Albaugh, Arnold, &
Denu, 2011). Three families of KATs have been
found to alter the acetylation state of a diverse
group of substrates with varying influences
258 Part III:Homeostatic Manipulators
on cellular functions (Albaugh, Arnold, &
Denu, 2011). However, these three families
of KATs have primarily been localized to the
nucleo-cytoplasmic compartments, and a mito-
chondrial KAT has yet to be confirmed despite
estimates that 65% of mitochondrial proteins are
acetylated, especially those involved in energy
metabolism and antioxidant defenses (Albaugh,
Arnold, & Denu, 2011; Hebert et al., 2013).
Recently, it was shown that the high alkaline pH
of the mitochondria in the presence of elevated
levels of acetyl-CoA allows for nonenzymatic
acetylation to occur in vitro (Wagner & Payne,
2013). This has led to speculation that compart-
mental differences in acetyl-CoA concentra-
tion can direct changes in acetylation (Albaugh,
Arnold, & Denu,2011).
Fructose-1,6-bisphosphate (FBP) also inhibits
glycolysis by diverting the metabolism of glucose
to the pentose phosphate pathway and also affords
broad antiseizure effects in vivo (Lian, Khan, &
Stringer, 2007). FBP was shown to provide the
greatest protection when compared to the KD,
2-DG, and the commonly prescribed ASD valproic
acid in the pilocarpine-, KA-, and PTZ-induced
seizure models in rats (Lian, Khan, & Stringer,
2007). Surprisingly, the KD did not demonstrate
antiseizure activity in this study, and 2-DG was
only protective in the pilocarpine model, whereas
valproic acid demonstrated limited protection in
all three models. Co-administration of lactate, to
relieve the glycolytic inhibition induced by either
2-DG or FBP, abolished 2-DG-mediated protec-
tion but only slightly reduced the antiseizure
activity of FBP (Lian, Khan, & Stringer, 2007).
This indicates that the effects of FBP are not solely
due to inhibition of glycolysis but may possibly
be a result of enhanced flux through the pentose
phosphate pathway.
Fatty Acid Oxidation
Intake of a high-fat diet, such as the KD, inherently
increases the rate of FAO, and this also changes
the levels and types of polyunstaturated fatty acids
(PUFA) in the circulation, liver, and brain. These
alterations in PUFA are a result of endogenous
production and export of these FA into the circu-
lation since large quantities of these lipids are not
a typical component of the KD (Fraser etal., 2003).
PUFA are known to possess neuroprotective prop-
erties (Michael-Titus & Priestley, 2013); therefore
it has been speculated that this lipid species may
mediate the antiseizure effects of theKD.
In pediatric patients with epilepsy, 3 to 4 weeks
of KD treatment led to elevations in circulating
levels of BHB, cortisol, and free fatty acids (Fraser
etal., 2003). Most PUFA were also increased in the
serum, including the triglyceride and phospholipid
forms of linoleic acid, arachidonic acid (AA), and
docosahexaenoic acid (DHA); triglyceride levels
of stearic acid; and palmitic acid in phospholipids
(Fraser etal., 2003). Additionally, these alterations
in FA were associated with seizure reduction in
78% of the children. Interestingly, seizure control
was correlated with circulating AA levels but not
EEG changes (Fraser etal.,2003).
Whether PUFA supplementation can ren-
der antiseizure effects remains controversial.
Supplementation with 5 g of omega-3 PUFA for
6months appeared to reduce the frequency and
severity of seizures in a small observational study
(Schlanger, Shinitzky, & Yam, 2002). However, in
a 12-week randomized, placebo-controlled paral-
lel group study in adults with epilepsy, adminis-
tration of eicosapentaenoic (EPA) and DHA (1 g
EPA, 0.7 g DHA daily) reduced seizure frequency
for the first 6 weeks of treatment, but this effect
did not persist despite sustained increases in
DHA and EPA and decreases in AA and linoleic
acid in the plasma (Yuen etal., 2005). In a further
randomized, blinded trial of PUFA supplementa-
tion (EPA plus DHA, 2.2 mg/day in a 3:2 ratio over
12 weeks) in adults with uncontrolled epilepsy,
there was a lack of clear efficacy when compared
to placebo (Bromfield et al., 2008). However, it
is unclear whether different doses, duration, or
ratios of PUFA might have been effective.
In rats fed the KD, there were marked reduc-
tions of PUFA levels in plasma and adipose tis-
sue but enhanced mobilization of the PUFA AA
and DHA to the liver and brain (Taha, Ryan, &
Cunnane, 2005). This was accompanied by an ini-
tial increase in the plasma levels of BHB, followed
by a reduction by day 10 of the diet. Following a
calorie-restricted KD, mRNA expression of the
rate-limiting enzyme for ketone body production,
HMG-CoAS2, was found to increase in both the
liver and the brain, in contrast to isocaloric stand-
ard chow, which showed enhanced expression only
in the liver (Cullingford, Eagles, & Sato,2002).
Collectively, these and other animal studies
suggest that diet-induced changes in brain con-
tent and metabolism of PUFA may be impor-
tant contributors to antiseizure effects of the KD
(Taha, Burnham, & Auvin,2010).
Bioenergetics Reserve and
Mitochondrial Respiration
Alterations in bioenergetic metabolites by the
KD have been shown in several animal studies.
 Ketogenic Diets and Mitochondria and Neurological Diseases
DeVivo and colleagues first demonstrated sig-
nificant increases in ATP and the ATP/ADP ratio
(and other parameters of bioenergetic reserve
capacity) in the brain of rats fed the KD for 3
weeks (DeVivo etal., 1978). Further, decreases in
creatine with no change in phosphocreatine were
noted in this study. However, while a later study
failed to confirm elevations in ATP and ATP/ADP
ratios, the KD was shown to increase the ratio
of phosphocreatine to creatine (Bough et al.,
2006). In vitro studies have further supported
the concept that KD metabolites can enhance
bioenergetic function. For example, BHB and
ACA prevented depletion of ATP in hippocam-
pal slices in response to H2O2 and inhibitors of
complex I (e.g., rotenone) and complex II (e.g.,
3-nitropropionic acid) of the electron transport
chain (ETC) (Kim do, Vallejo, & Rho, 2010). In a
mouse model of global cerebral ischemia, admin-
istration of BHB immediately following bilateral
common carotid artery ligation ameliorated the
decline in tissue levels of ATP (Suzuki et al.,
2001). Thus, collectively, there is both in vivo and
in vitro evidence for the KD and ketone bodies
enhancing ATP production inbrain.
Consistent with the KDs favorable effects on
energy production, changes in the expression of
enzymes involved in metabolic pathways have
also been reported. Two studies revealed changes
in gene expression in the hippocampus of mice
and rats fed the KD (Bough et al., 2006; Noh
et al., 2004), and both demonstrated enhanced
expression of numerous enzymes involved in
mitochondrial metabolism (Noh et al., 2004),
such as the TCA cycle and oxidative phosphoryl-
ation (Bough etal., 2006). However, there was not
complete concordance with respect to the gene
expression changes for all metabolic enzymes
(Bough etal.,2006).
Mitochondrial respiration and ETC activity
have been shown to be elevated in animals fed the
KD or with in vitro application of ketone bodies.
BHB increased oxygen consumption and ATP
production in purified mitochondria in the pres-
ence of two inhibitors of complex I, 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) and
rotenone (Tieu etal., 2003). Further, in a model of
glutamate excitotoxicity, ACA and BHB increased
complex I-driven oxygen consumption through
increased NADH oxidation (Maalouf etal.,2007).
TCA Cycle Effects and Anaplerosis
As numerous studies have shown that the KD can
increase ATP levels, bioenergetic capacity, and
transcription of enzymes in energy-producing
259
pathways, it is also likely that levels of meta-
bolic intermediates may be altered. Specifically,
the KD may enhance neuronal ATP production
through refilling of TCA cycle intermediates
from the increase in acetyl-CoA levels from the
oxidation of ketone bodies. These actions may
contribute to antiseizure effects. In this regard,
the anaplerotic substrate triheptanoin has been
shown to raise seizure thresholds in mouse sei-
zure models and to help restore levels of TCA
intermediates (Hadera et al., 2014; Willis et al.,
2010). Further, replenishment of the TCA cycle
may counter seizure-induced energy failure
and augment inhibitory neurotransmission
(Kovac, Abramov, & Walker, 2013). For example,
-ketoglutarate, which serves a dual function as
a TCA cycle intermediate and as the immediate
precursor to glutamate, which can be converted
to GABA, directly links anaplerosis to neuronal
excitability (Kovac, Abramov, & Walker,2013).
A C T I VAT I O N O F
E N E R G Y- S E N S I N G
S I G N A L I N G PAT H WAY S
It is well established that multiple signaling path-
ways have evolved to sense states of diminished
energy stores, such as seen during exercise and
CR, and that activation of these pathways results
in restoration of cellular homeostasis and integ-
rity (Libert & Guarente, 2013; Mattson, 2012).
Data regarding the association of the KD with
these energy-sensing pathways are limited, but
the similarities between the protective mecha-
nisms evoked by the KD and those known to be
influenced by these pathways indicates substan-
tial overlap in their mechanisms and downstream
effects.
Peroxisome Proliferator-Activated
Receptors
The PPAR family of nuclear receptors represents
one signaling pathway that may mediate some
of the beneficial effects of the KD (Sampath &
Ntambi, 2005). PPARs are transcription factors
that bind to the PPAR response element (PPRE)
of promoter regions. They include three iso-
forms: PPAR, PPAR, and PPAR/ (Sampath
& Ntambi, 2005). FA are endogenous ligands
for PPARs, and PPAR agonists are used in the
treatment of lipid disorders such as type II dia-
betes mellitus and high cholesterol (Sampath &
Ntambi, 2005). The induction of FAO is a known
metabolic effect of PPAR activation, which ren-
ders these transcription factors an attractive
mediator for the clinical effects of theKD.
260 Part III:Homeostatic Manipulators
Fenofibrate, a PPAR agonist, was shown to
exhibit antiseizure properties similar to the KD
(Porta etal., 2009). Rats fed the KD and a diet con-
taining fenofibrate showed increased threshold to
PTZ-induced seizures and an increased latency
to spike-and-wave discharges lasting longer than
5 minutes in the lithium-pilocarpine model,
compared with control-fed animals (Porta etal.,
2009). In a separate study, acute injection of the
PPAR agonist FMOC-L-leucine protected adult
magnesium-deficient mice against audiogenic
seizures, and this was reversed by administra-
tion of the PPAR antagonist GW9662 (Maurois
et al., 2008). In contrast, FMOC-L-leucine did
not exhibit antiseizure activity in another sei-
zure model, the 6 Hz test, and another PPAR
ligand, rosiglitazone, did not reduce audiogenic
seizures (Maurois et al., 2008). Rosiglitazone
has been shown to reduce oxidative stress in the
lithium-pilocarpine model of epilepsy, and this
was associated with an amelioration of neuronal
death in the hippocampus and an enhancement
of SOD activity and levels of GSH, as well as a
reduction in the Nrf2 target HO-1 (Yu et al.,
2008). Further, the PPAR-selective agonist
ciprofibrate was shown to selectively increase
mRNA levels of HMG-CoAS2, the rate-limiting
enzyme in the production of ketone bodies, and
that of two other known PPAR targets, acyl CoA
oxidase and medium chain acyl-CoA dehydroge-
nase (Cullingford, Dolphin, & Sato,2002).
AMP-ActivatedKinase
AMP-activated kinase (AMPK) is induced by
low levels of AMP and inhibited by high con-
centrations of ATP; therefore, AMPK functions
as a direct sensor of the energy state of the cell.
Activation of AMPK leads to the phosphorylation
of multiple substrates, resulting in concurrent
inhibition of anabolic pathways and increases
in catabolic metabolism to enhance the produc-
tion of ATP (Hardie & Sakamoto, 2006). These
effects are known to include an upregulation of
FAO and increased mitochondrial biogenesis
(Hardie & Sakamoto, 2006). Similar to PPARs,
agonists of AMPK are used to treat type II diabe-
tes mellitus and metabolic syndrome (Hardie &
Sakamoto,2006).
The KD increased AMPK activity in liver
and skeletal muscle of mice, and this was associ-
ated with increased gene expression of enzymes
involved in FAO and a reduction in transcript lev-
els of enzymes in lipid synthesis pathways in the
liver (Kennedy etal., 2007). Increased activation
of AMPK was also found in the liver of rats on the
KD, but this was not associated with enhanced
activation in the brain (McDaniel etal.,2011).
Mammalian Target ofRapamycin
The mammalian target of rapamycin (mTOR) is
another protein kinase that exerts multiple effects
on energy metabolism through the actions of two
distinct complexes, mTOR complex 1 (mTORC1)
and mTOR complex 2 (mTORC2) (Johnson,
Rabinovitch, & Kaeberlein, 2013). Unlike the PPARs
and AMPK, however, mTOR is activated during
high-energy states, and this results in an induction
of protein synthesis and mRNA translation, among
other actions that promote growth and cellular
proliferation (Johnson, Rabinovitch, & Kaeberlein,
2013). Importantly, AMPK inhibits mTORC1
through direct phosphorylation of both a subunit
of this complex and an upstream regulatory protein
(Johnson, Rabinovitch, & Kaeberlein, 2013). In rats,
the KD inhibited activation of the mTOR path-
way in the liver and brain (McDaniel etal., 2011).
Additionally, in the KA model, enhanced activation
of mTOR was found in the hippocampus of rats fed
a standard diet, and this effect was blocked after
7days on the KD (McDaniel etal., 2011). Given that
mTOR inhibition is believed to retard the processes
of epileptogenesis (Wong, 2013) and that the KD
can decrease mTOR signaling, it is conceivable that
metabolism-based treatments could render antiepi-
leptogenic effects.
Sirtuins
The sirtuins (SIRT17) are a family of NAD-
dependent enzymes known to influence mul-
tiple aspects of cellular homeostasis, including
metabolism and antioxidant activity. They were
originally classified as Class III HDACs; how-
ever, it is now known that individual isoforms
also demonstrate desuccinylase, demalonylase,
and ADP-ribosyltransferase activities (Libert &
Guarente, 2013). The sirtuins are direct sensors of
the energetic and redox state of the cell, principally
through NAD+, which is required for their cata-
lytic activity. Additionally, the beneficial effects
of CR may be mediated by the sirtuins, as well
as the AMPK and mTOR pathways (Guarente,
2013; Houtkooper, Williams, & Auwerx, 2010).
The diversity and abundance of their histone and
nonhistone substrates, as well as their specific
localization within different cellular compart-
ments, reflect the extent of their broad influence on
energy metabolism (Guarente, 2013; Houtkooper,
Williams, & Auwerx, 2010). Collectively, the sir-
tuins are known to enhance oxidative phosphoryla-
tion and reduce glycolysis, in addition to providing
 Ketogenic Diets and Mitochondria and Neurological Diseases
increased resistance to oxidative stress (Libert &
Guarente, 2013). The substantial overlap in the
activities of these regulators of cellular homeosta-
sis with the known mechanisms of the KD suggests
possible involvement of the sirtuins in the effects of
this dietary modification. Certainly, the observa-
tion the BHB is a ClassIHDAC inhibitor (Shimazu
etal., 2013)is compelling in this regard.
One study examined levels of SIRT1 and SIRT3
in interscapular brown adipose tissue from mice
on the KD for 1 month (Srivastava et al., 2013).
Protein expression of SIRT1 was elevated, but lev-
els of SIRT3 were reduced compared to mice on
a standard diet (Srivastava et al., 2013). A sepa-
rate study examining gene expression changes in
the hippocampus of rats fed the KD for 3 weeks
found increased levels of Sirt5, although this was
reported in the supplemental results section and
not discussed by the authors (Bough etal.,2006).
Further, SIRT3 and SIRT5, major regulators of
mitochondrial energy metabolism, exhibit extensive
regulation of the acetylation and succinylation state
of numerous enzymes in the TCA cycle, FAO, and
ketogenesis (Rardin, Newman, etal., 2013; Rardin,
He, et al., 2013). Interestingly, activity of HMG-
CoAS2, the rate-limiting enzyme in ketogenesis, is
regulated by both deacetylation through SIRT3 and
desuccinylation by SIRT5, two posttranslational
modifications thought to inhibit enzymatic activ-
ity (Rardin, Newman, etal., 2013; Rardin, He, etal.,
2013; Shimazu etal., 2010). Finally, separate studies
in Sirt3-/- and Sirt5-/- mice demonstrated reduc-
tions in the circulating levels of BHB under fasting
but not basal conditions (Rardin, He, et al., 2013;
Shimazu etal.,2010).
E V I D E N C E A N D I M P L I C AT I O N S
FOROTHER NEUROLOGICAL
DISORDERS
Mitochondrial Dysfunction
inNeurological Diseases
Mitochondrial dysfunction has recently been
recognized as a common mechanism under-
lying many neurological disorders (Cali,
Ottolini, & Brini, 2012; de Castro, Martins, &
Tufi, 2010; Lin & Beal, 2006; Pathak, Berthet,
& Nakamura, 2013). The role for mitochondrial
energetics and signaling as important media-
tors of neuronal death, a common feature of
neurodegenerative diseases (Johri & Beal,
2012), is increasingly being understood. Specific
pathological changes include a reduction in
ATP production via oxidative phosphoryla-
tion or direct inhibition of specific complexes
261
of the ETC and elevations in the production of
mitochondrial-derived ROS, which can alter
cellular signaling. Additionally, given the simi-
lar mechanisms of these processes in neurode-
generative diseases, and the beneficial effects of
the KD and ketone bodies on multiple aspects
of mitochondrial function, there has been an
increase in the use of metabolism-based treat-
ments for neurological diseases (Baranano &
Hartman, 2008; Stafstrom & Rho,2012).
Alzheimers Disease
AD is characterized by an accumulation of
neurofibrillary tangles and amyloid plaques
comprised of misfolded aggregates of tau and
amyloid- (A) proteins, respectively, result-
ing in neuronal death (Mattson, 2012). AD is
an age-related neurodegenerative disease and is
known to arise from both genetic and environ-
mental influences. Older adults with AD are at
increased risk for the development of epilepsy,
and similar mechanisms, such as deficits in
mitochondrial energy metabolism and eleva-
tions in oxidative stress, are thought to contrib-
ute to both pathological states (Mattson, 2012;
Palop & Mucke, 2009). Therefore, there is grow-
ing interest in the use of the KD to delay the
progressionofAD.
In fasted patients with AD or mild cogni-
tive impairment (MCI), acute ingestion of a
MCT drink increased BHB levels and cogni-
tive function compared to placebo (Reger etal.,
2004). Further, in a separate study of older
adults with MCI, a low-carbohydrate (5%10%
of calories) diet for 6 weeks enhanced a meas-
ure of verbal memory versus preintervention
scores, whereas there was no change in those
assigned to the high-carbohydrate (50% of calo-
ries) diet (Krikorian et al., 2012). Additionally,
the improvement in memory in those on the
low-carbohydrate diet was positively correlated
with levels of urinary ketones (Krikorian et al.,
2012). Finally, Henderson and colleagues treated
152 patients having mild to moderate AD daily
with 20 g of MCT for 3months and found that
MCT-treated patients (lacking the APOE4 allele)
achieved significantly higher scores on the
cognitive subscale of the Alzheimers Disease
Assessment Scale at two different time-points
versus placebo-treated controls. Interestingly,
postdose serum BHB levels correlated posi-
tively with improvement in scores (Henderson
etal.,2009).
However, in a mouse model of AD, consump-
tion of a KD high in saturated FA reduced total
262 Part III:Homeostatic Manipulators
levels of A but did not affect cognitive func-
tion (Van der Auwera et al., 2005). In vitro,
BHB reduced hippocampal neuronal cell death
from exposure to the proteolytic fragment of the
-chain of the amyloid precursor protein, A1-42
(Kashiwaya etal., 2000). Additionally, it has been
shown that a ketone ester diet lessens amyloid
and tau pathologies and improves learning and
memory performance in a mouse model of AD
(Kashiwaya etal., 2013). Taken together, there are
an increasing number of studies pointing to the
neuroprotective benefits of the KD and its meta-
bolic substrates.
Parkinsons Disease
The hallmark neuropathological finding in PD is
the degeneration of dopaminergic neurons in the
substantia nigra. Amain mechanism thought to
contribute to this excitotoxic cell death is defects
in complex I of the ETC (Johri & Beal, 2012).
Hence it follows that metabolism of ketone bod-
ies may circumvent this deficit and allow for oxi-
dative phosphorylation to occur (Maalouf etal.,
2007). In PD patients treated with the KD for 4
weeks, scores on Unified Parkinsons Disease
Rating Scale improved, although this study had
a very small sample size and did not include con-
trols (Vanitallie etal., 2005). Moreover, this study
showed that switching monounsaturated FA and
PUFA for saturated fats prevented the increase in
cholesterol expected from intake of a high-fat diet
(Vanitallie etal., 2005). In a prospective study of
older adults, increased consumption of total fats,
monounsaturated FA, and PUFA were associated
with a lower risk of PD (de Lau etal.,2005).
Following treatment with MPTP, an inhibitor
of complex Icommonly used to model PD, BHB
reduced dopaminergic neuronal cell death in
substantia nigra pars compacta in vivo, and this
was associated with increased levels of dopamine
and improved rotarod performance, demonstrat-
ing an attenuation of motor deficits (Tieu etal.,
2003). Additionally, BHB reduced cell death
in vitro in mesencephalic neurons exposed to
1-methyl-4-phenylpyridinium, the active metab-
olite of MPTP (Kashiwaya etal.,2000).
BrainCancer
Cancer cells are known to undergo dramatic
metabolic alterations, including a preference for
ATP production via glycolysis and enhanced lac-
tic acid production, despite the presence of oxy-
gen for oxidative phosphorylation (Seyfried &
Shelton, 2010). This shift in metabolism was first
noted by Otto Warburg and was subsequently
coined the Warburg effect (Warburg, 1956).
Defects in mitochondrial function resulting
in diminished oxidative phosphorylation are
thought to be main contributors to cancer cell
metabolism. Since cancer cells preferentially use
glucose for energy, and the KD reduces glycolytic
flux and enhances oxidative metabolism, high-fat
KDs may represent potentially viable treat-
ments to limit oncogenesis (Seyfried etal., 2011).
Indeed, this conceptual approach was demon-
strated in a compelling manner in a mouse astro-
cytoma model, indicating that plasma glucose is
an accurate predictor of tumor growth more than
the specific origin of dietary calories (Seyfried
etal.,2003).
In a later study employing a mouse model of
malignant glioma, the KD reduced tumor growth
and increased survival, and this was associated
with a decrease in tissue levels of ROS (Stafford
et al., 2010). Additionally, the KD induced gene
expression changes in the tumor tissue to more
closely resemble the pattern found in the normal
brain. Further, the KD also caused an upregu-
lation of enzymes involved in oxidative stress
resistance, such as glutathione peroxidase 7 and
peroxiredoxin 4, in tumor tissue but not normal
specimens (Stafford etal.,2010).
The same research group later showed in a
similar model that administration of KetoCal, a
commercially available 4:1 KD, greatly increased
survival (Abdelwahab et al., 2012). When
KetoCal was given in conjunction with radiation
therapy, a supra-additive effect was found as vis-
ualization of tumor cells diminished below the
levels of detection in 9 out of 11 animals. Further,
when these animals were placed back on a stand-
ard diet, no regrowth of tumors was found after
100days (Abdelwahab etal.,2012).
In a more recent study, expression of cytosolic
glycolytic enzymes and mitochondrial ketolytic
enzymes were altered in sections of malignant
gliomas compared with adjacent normal brain
tissue from adults (Chang, Olson, & Schwartz,
2013). The most common pattern observed was
upregulation of the cytosolic glycolytic enzymes
with a reduction in the mitochondrial ketolytic
enzymes in the brain tumor tissue.
Neurotrauma
Acute neurotrauma results in metabolic changes
in the brain, including diminished glycolysis, and
also activates excitotoxic and neuroinflamma-
tory cascades (Algattas & Huang, 2014; Babikian
et al., 2010). Despite the glycolytic restriction
observed, the KDthrough a multiplicity of
 Ketogenic Diets and Mitochondria and Neurological Diseases 263

other neuroprotective mechanismsmay coun-
ter the pathophysiological changes seen after
traumatic brain injury (TBI). In an animal model
of TBI, cortical contusion volume was reduced in
rats fed the KD (Prins, Fujima, & Hovda,2005).
Further, infusion of BHB 3 hours after injury
increased cerebral uptake of BHB and amelio-
rated the injury-induced reduction in cortical
ATP levels (Prins et al., 2004). A recent study
in a rat model of TBI demonstrated that the KD
decreased mRNA levels of Bax, a Bcl-2 protein
mediating apoptosis, and reduced cerebral edema
and apoptosis (Hu etal., 2009). Additionally, ini-
tiation of the KD 4days before insulin-induced
hypoglycemia reduced neuronal death in rats
(Yamada, Rensing, & Thio,2005).
Further support for a metabolic approach
toward TBI treatment is provided by Davis and
colleagues, who showed that fasting for 24 hours
following controlled cortical impact in rats
resulted in increased tissue sparing and improve-
ments in mitochondrial function and that these
effects were a result of ketones and not hypogly-
cemia (Davis etal.,2008).
Amyotrophic Lateral Sclerosis
Mitochondrial dysfunction is also thought to
contribute to the progression of ALS, a disease
characterized by degeneration of motor neurons
in the cortex and spinal cord (Rothstein, 2009).
In ALS mice, the KD improved motor func-
tion, as evidenced by increased time to failure
on rotorod performance, and this was associated
with preservation of motor neurons in the ven-
tral horn of the spinal cord (Zhao et al., 2006).
In mitochondria isolated from the spinal cord of

Ketogenic Diet
LGIT MAD MCT CR

Glycolysis/ Fatty Acid Oxidation

Ketogenesis/
Ketone Bodies

mTOR AMPK PPARs Sirtuins HDACs

TAC Cycle Antioxidant Mitochondrial Histone

Ion Channels
Anaplerosis Capacity Function Acetylation

Synaptic Integrity Bioenergetics ROS

Neuroprotection

Figure15.4 Proposed mechanisms for the neuroprotective effects of the ketogenic diet (KD) and
its variants. The dietary interventions are shown in orange; the metabolic effects of the diets are shown in blue;
the energy-sensing pathways that may mediate the effects of the dietary alterations are shown in red; the cellular
effects resulting from the diets and/or the energy-sensing pathways are shown in green; the broad protective effects
of the diets and the resulting cellular effects are in cyan. LGIT=low-glycemic index treatment; MAD=modified
Atkins diet; MCT=medium-chain triglyeride diet; CR=caloric restriction; mTOR=mammalian target of rapa-
mycin; AMPK=AMP-activated kinase; PPARs=peroxisome proliferator-activated receptors; HDACs=histone
deacetylases; TCA=tricarboxylic; ROS=reactive oxygen species. Solid black lines indicate links proven in the
literature; dashed black lines represent possible but as yet unproven links. See text for further details.
264 Part III:Homeostatic Manipulators
a transgenic mouse model of ALS (SOD1-G93A),
addition of BHB in vitro enhanced ATP produc-
tion, and this effect was maintained in the pres-
ence of the complex Iinhibitor rotenone but not
the complex II inhibitor malonate (Zhao et al.,
2006). Additionally, the preservation of ATP lev-
els by BHB was associated with increased neu-
ronal survival in the presence of rotenone but not
malonate (Zhao etal.,2006).
Pain and Inflammation
Multiple lines of experimental evidence suggest
shared fundamental mechanisms responsible for
chronic pain syndromes and epilepsy, particularly
the involvement of cellular membrane-bound ion
channels (Bialer, 2012). Specifically, both chronic
pain and epilepsy are characterized by enhanced
neuronal excitability, and, whatever the relevant
mechanisms may be, metabolic approaches
toward treatment (e.g., the KD, inhibition of gly-
colysis through fasting or 2-DG, etc.) can alleviate
neuropathic pain (Masino & Rho, 2012; Ruskin,
Kawamura, & Masino, 2009). Additionally,
ASDs are often prescribed for chronic pain,
further suggesting similarities in the patho-
physiology of these two disorders (Masino &
Ruskin, 2013). In juvenile and adult rats, the KD
reduced pain, as assessed by increased latency to
hind-paw withdrawal in a test of thermal nocio-
ception, and inflammation, as measured by
hind-paw swelling in response to injection of the
immune-potentiator Freunds complete adjuvant
(Ruskin, Kawamura, & Masino, 2009). Despite
attaining similar levels of circulating ketone
bodies, the anti-inflammatory and hypo-algesic
effects of the KD were more prominent in juvenile
mice (Ruskin, Kawamura, & Masino,2009).
Other Disorders
There are a number of other reports suggesting
that the KD (or other dietary manipulations) can
effectively treat diverse neurological disorders
such as autism and migraine. For example, in the
BTBR mouse, an inbred model that recapitulates
the core behavioral features of autism spectrum
disorder and that does not exhibit altered suscep-
tibility to seizures, the KD increased sociability
and communication while reducing repetitive
behaviors (Ruskin et al., 2013). A small study
examined the use of the MAD in treating chronic
headaches in adolescents and demonstrated
reduced headache severity in the three patients
that completed the study (Kossoff et al., 2010).
However, the MAD did not reduce headache
frequency; unfortunately, the small study size
and limited number of patients completing the
3-month study prevent any definitive conclusions
(Kossoff etal.,2010).
Summary
The KD is a broad-spectrum therapy for multiple
forms of epilepsy in both children and adults. The
utility of the KD and variations of this diet for
the treatment of a variety of neurodegenerative
disorders suggests common central mechanisms
that restore imbalances in energy metabolism
(Figure 15.4). The numerous mechanisms known
to partially mediate the effects of the KD (such
as increases in FAO, reductions in glycolysis,
enhancement of the cellular responses to oxida-
tive stress, etc.), indicate that manipulation of
these specific pathways may represent an attrac-
tive paradigm for experimental therapeutics.
Specifically, bioenergetic substrates and enzymes
may be desirable drug targets for the treatment
of many neurological diseases. Additionally, the
signaling pathways that evolved to sense the cel-
lular energetic state and provide resistance to
metabolic stress may provide the best means to
mimic the KD. Future research in this burgeon-
ing area may lead to the elucidation of additional
novel mechanisms that mediate the pleiotropic
neuroprotective effects of theKD.
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L. Ho, J. Suh, N. Humala, M. Thiyagarajan, J.
Wang, and G. M.Pasinetti. 2006. A ketogenic diet
as a potential novel therapeutic intervention in
amyotrophic lateral sclerosis. BMC Neurosci7:29.
Ziegler, D.R., L. C.Ribeiro, M. Hagenn, I. R.Siqueira,
E. Araujo, I. L.Torres, C. Gottfried, C. A.Netto,
and C. A. Goncalves. 2003. Ketogenic diet
increases glutathione peroxidase activity in rat
hippocampus. Neurochem Res 28:17931797.
16
Dietary Manipulations
AT H A N A S I O S E VA N G E L I O U A N D M A R T H A S P I L I O T I
INTRODUCTION
The brain is the most active organ in the body
in terms of energy consumption and therefore
particularly vulnerable to disruptions of energy
resources. Energy is available in the form of
adenosine triphosphate(ATP), the major source
of biological free energy in higher organisms.
Consequently, impaired ATP production threat-
ens brain homeostasis and integrity. Many
normal and pathological situations are associ-
ated with either increased energy demands or
impaired brain energy production. Some of these
situations are depicted in Table 16.1. In all catego-
ries in Table 16.1, one question that has occupied
scientists for many years is how to restore energy
balance when and where deficits exists and how
to improve existing normal energy balance in the
brain. Accordingly, dietary manipulations that
improve metabolic function or prop up ATP pro-
duction may have a vast number of implications
in health and disease. In the past years there has
been accumulating evidence that various nutri-
ents that can be ingested in food or by themselves
may prove to be effective in improving brain
energy balance. In this chapter we provide an
overview of the nutrients that can impact and
improve brain energy homeostasis.
L-CARNITINE AND
CARNITINEESTERS
Carnitine (-hydroxy--trimethylaminobutyric
acid) is an essential cofactor for the transport of
long-chain fatty acids across the inner mitochon-
drial membrane into the mitochondrial matrix,
where they are broken down via -oxidation
(Evangeliou and Vlassopoulos, 2003; Foster,
2004; Pekala etal., 2011; Virmani and Binienda,
2004; Figure 16.1). In addition to its principal
function, carnitine also buffers potentially toxic
acyl-CoA metabolites and modulates the ratio of
acyl-CoA/CoA (Di Lisa etal., 1997; Stumpf etal.,
1985; Figure 16.2). The later regulates the activity
of many mitochondrial enzymes involved in the
citric acid cycle, gluconeogenesis, the urea cycle,
and fatty acid oxidation (Di Lisa et al., 1997;
Evangeliou,1992).
Carnitine is a nonessential nutrient, first
described in the early beginnings of the 20th cen-
tury. It was initially considered to be an essen-
tial vitamin; it was later discovered that it could
be produced in the liver, kidney, and brain from
amino acid precursors (lysine and methionine).
Human skeletal muscle, heart, liver, kidney, and
brain are able to synthesize -butyrobetaine,
an immediate precursor of carnitine, but only
the liver, kidney, and brain have the ability to
convert this substrate to carnitine (Vaz and
Wanders, 2002). The reaction is catalyzed by
-butyrobetaine hydroxylase. Due to the very
low activity of -butyrobetaine hydroxylase in
fetal and neonatal age, carnitine is considered
a vitamin for newborn babies (Ling etal., 2012;
Rebouche and Seim,1998).
In humans, 75% of carnitine intake comes
from dietary sources (Li etal., 1992; Pekala etal.,
2011). These include most animal foods; however,
due to losses in food cooking and preparation,
there are few data on the total content ingested
(Rospond and Chopicka, 2013). Carnitine is not
metabolized and is excreted, mostly as free carni-
tine, in theurine.
Homeostatic mechanisms conserve the
amount of L-carnitine in the body and under
normal circumstances regulate blood and tis-
sue levels within relatively narrow limits. The
uptake of L-carnitine into most tissues of the
body, including muscles and neurons, involves
carrier-mediated transport systems, which
maintain high tissue-to-plasma concentration
ratios. Especially in the brain, carnitine is trans-
ported from the plasma into neurons by Na+- and
energy-dependent processes and may involve the
cation transporter OCTN2 (Berezowski et al.,
2004; Naecz etal.,2004).
272 Part III:Homeostatic Manipulators

TABLE16.1 SIT UAT IONS W IT H I M PA IR ED OR I NCR E ASED

EN ERGY DEM A N DS
Normal Situations Pathological Situations

Impaired Energy Increased Energy Impaired/Increased Energy

Demands Demands Demands
Aging Pregnancy Inborn errors of metabolism
Infancy Autism
Childhood Parkinsons disease
Puberty Huntingtons disease
Other stress situations Alzheimers disease
Amyotrophic lateral sclerosis
Depression
Schizophrenia
Bipolar disorder
Cancer
Epilepsy

In addition to effects on lipid metabolism, pyruvate dehydrogenase with subsequent reduc-

carnitine influences carbohydrate and protein
metabolism. This is achieved different ways. As
already mentioned, carnitine affects the ratio of
acyl-CoA/CoA. This relation is crucial in mito-
chondrial metabolism, modulating the activity of
many mitochondrial enzymes, including, among
others, pyruvate dehydrogenase, a very impor-
tant enzyme in glucose metabolism (Evangeliou
and Vlassopoulos, 2003; Ramsay, 2000). Ahigh
ratio of acyl-CoA to CoA reduces the activity of
tion of carbohydrate breakdown (Holness and
Sugden, 2003). L-Carnitine supplementation
reduces the acyl-CoA/CoA ratio, thus increas-
ing the amount of nonesterified CoA essential
for lipid and carbohydrate utilization (Chapela
etal., 2009; Ramsay, 2000; Ramsay and Arduini,
1993). Moreover L-carnitine is an energy car-
rier through the mitochondrial membrane and
so modulates the availability of acetyl-CoA
and consequently controls the activity of

CYTOSOL
Carnitine
Acyl-CoA Acylcarnitine

CPT I Outer Mitochondrial Membrane

Acylcarnitine CoA
Carnitine Acylcarnitine

Carnitine

CT CT Inner Mitochondrial Membrane

CPT II CAT
Acylcarnitine
CoA
Carnitine Carnitine
Acyl-CoA Carnitine
Acylcarnitine
CoA
MITOCHONDRIAL MATRIX
-oxidation Acetyl-CoA

FIGURE16.1: Transport of long-chain fatty acids across the inner mitochondrial membrane into the mitochon-
drial matrix. CAT=carnitine acetyl translocase; CPT I=carnitine palmitoyl transferase I; CPT II=carnitine
palmitoyl transferase II; CAT=carnitine acetyl translocase.

Carnitine Acylcarnitine Urine
AcylCoA CoA
Toxic organic acids
Valproic acid
FIGURE 16.2: Carnitine buffering of toxic acyl-CoAs
by conversion to acylcarnitine and modulation of the
ratio of acyl-CoA/CoA. Valproic acid, used broadly
as an antiepileptic, causes an iatrogenic organic aci-
demia and is also buffering through carnitine. This
reaction is a two-way chemical reaction modulating
the acyl-CoA/CoA ratio. The acyl-CoA/CoA ratio is
very crucial in mitochondrial metabolism by control-
ling enzymes of the Krebs cycle: pyruvate dehydoge-
nase complex, pyruvate carboxylase, citrate synthase,
acetyl-CoA carboxylase, adenine nucleotide translo-
case, glutamate dehydrogenase, malate dehydrogenase,
and succinyl-CoA ligase.
the respiratory chain complex (Virmani and
Binienda, 2004). Indeed, the majority of carni-
tine deficiency syndromes are associated with
mitochondrial dysfunction and vice versa (Geier
and Geier, 2013; Kaido et al., 1997; Weinberg
and Baughman, 2006). In addition, experimen-
tal and clinical data provide evidence for ben-
eficial effects of L-carnitine in situations with
mitochondrial dysfunction (Binienda, 2003;
Binienda etal., 2005; La Guardia etal., 2013). In
particular, carnitine increases function of the
respiratory chain and antioxidation and lipid
peroxidation tolerance capacity in skeletal mus-
cle mitochondria. L-carnitine supplementation
also has protein sparing effects; the activation of
the -oxidation pathway, mediated through car-
nitine, reduces the breakdown of branched-chain
amino acids (BCAAs) via an internal feedback
mechanism (inhibition of the branched chain
keto-dehydrogenase) and promotes protein
synthesis resulting in a leaner body (Feller and
Rudman, 1988; Owen etal.,1994).
Several primary carnitine deficiency syn-
dromes have been described, and the major-
ity of them are characterized by hepatopathy,
cardiomyopathy, myopathy, myoglobinuria,
and nonketotic hypoglycemia (Magoulas and
El-Hattab, 2012; Pons etal., 1997; Pons and De
Dietary Manipulations 273
Vivo, 1995). These symptoms often are accom-
panied by central nervous system (CNS) signs
such as hepatic encephalopathy in which the
major pathologic finding is swelling of astro-
cyte cytoplasm, while less often expanded
mitochondria and myelin sheath splitting in
the white matter are observed (Kimura and
Amemiya, 1990; Malaguarnera, 2013; Virmani
and Binienda, 2004). Similar symptoms to
primary carnitine deficiency are observed in
secondary carnitine deficiencies occurring
in some inborn errors of metabolism and in
some acquired conditions such as in patients
under valproate therapy (Morand et al., 2012;
Okumura et al., 2011; Stumpf et al., 1985;
Vreken etal.,1999).
Beyond the changes in carnitine metabo-
lism observed in inborn errors of metabolism
and the role of carnitine in the prevention of
clinical symptoms related to these disorders,
carnitine seems to play an important role as
adjunctive treatment in some neurodegen-
erative disorders and also in the prevention of
consequences associated with aging (Calabrese
etal.,2010).
Carnitine andAging
Clinical and experimental data in recent
decades has shown the relation of carnitine
metabolism to aging and the beneficial effect of
carnitine on the amelioration of aging conse-
quences. Animal experiments have shown that
the aging process is accompanied by carnitine
deficiency in brain, myocardium, and skeletal
muscle (Costell et al., 1989; Flanagan et al.,
2010; Maebashi et al., 1982; Rebouche, 1992).
Carnitine deficiency in these tissues is accompa-
nied by disturbances in mitochondrial genesis,
morphology, and function. These mitochon-
drial alterations are increasingly recognized as
components of the aging process, and experi-
mental work provides evidence that L-carnitine
raises mitochondrial energy-producing capa-
bilities by reversing the age-associated decline
in mitochondrial enzyme activities and thereby
protecting mitochondria from aging (Arockia
Rani and Panneerselvam, 2001; Noland et al.,
2009; Savitha etal., 2005). Consistent with this,
clinical work done in centenarians indicates
that oral administration of carnitine produces
a reduction of total fat mass, increases total
muscular mass, and facilitates an increased
capacity for physical and cognitive activity by
reducing fatigue and improving cognitive func-
tions (Malaguarnera etal.,2007).
274 Part III:Homeostatic Manipulators
Carnitine andAutism
Autism spectrum disorder (ASD) is highly
genetic and multifactorial, with many interact-
ing risk factors. Recent studies have implicated
abnormalities in mitochondrial metabolism
(Guevara-Campos et al., 2013; Legido et al.,
2013; Rossignol and Frye, 2012). Interestingly,
most individuals with ASD and mitochondrial
dysfunction do not have a specific genetic muta-
tion, raising the possibility that mitochondrial
dysfunction may be acquired, at least in a sub-
group of children with ASD (Dhillon etal., 2011;
Hadjixenofontos etal., 2013). Consequently, one
suggested approach to treating patients diag-
nosed with an ASD entails the administration
of mitochondrial respiratory chain cofactors to
enhance mitochondrial function. Such a strat-
egy would employ stimulation of enzyme activ-
ity by supplying precursors or co-enzyme and
alternative substrates (Frye etal., 2013a, 2013b).
L-carnitine is among the suggested options for
treatment, and a recent prospective, double-blind,
placebo-controlled trial demonstrated that
L-carnitine therapy for 3months among subjects
diagnosed with an ASD significantly improved
on clinical measurements recorded by trained
professionals and parents of study subjects (Geier
etal.,2011).
Carnitine and Valproate
Valproate treatment is known to reduce blood
and muscle carnitine levels (Anil et al., 2009;
Morand et al., 2012). Reduced carnitine lev-
els in patients under valproate therapy may
be associated with severe hepatotoxicity and
hyperammonemia (Hamed and Abdella, 2009).
In addition, in some rare cases treatment with
valproate is associated with the development of
coma and life-threatening cerebral edema, even
in the absence of concomitant hepatic failure
(Triggs etal.,1997).
Numerous studies have demonstrated the
beneficial effects of carnitine supplementation
in the treatment of valproate-induced hepa-
totoxicity (Bhmer et al., 2010; Perrott et al.,
2010; Sonik et al., 2011). In November 1996, a
panel of pediatric neurologists met to update
the consensus statement issued in 1989 by a
panel of neurologists and metabolic experts
on L-carnitine supplementation in childhood
epilepsy. The panelists agreed that intravenous
L-carnitine supplementation is clearly indicated
for valproate-induced hepatotoxicity and over-
dose (De Vivo etal.,1998).
Acetyl-L-Carnitine
Acetyl-L-carnitine is an endogenous molecule
synthesized in mitochondria by the enzyme
acetyl-L-carnitine transferase and is the predom-
inant acylcarnitine in normal tissues (Hudson
and Tabet, 2003; Pettegrew etal., 2000; Rebouche,
2004). Acetyl-L-carnitine facilitates the influx
and efflux of acetyl groups across the mitochon-
drial inner membrane (Reuter and Evans, 2012;
Virmani and Binienda, 2004). Some of acetyl-L-
carnitines proposed neuroprotective benefits
involve improved mitochondrial energetics and
function, antioxidant activity, stabilization of
membranes, protein and gene expression modu-
lation, and enhancement of cholinergic neuro-
transmission (Ando etal., 2001; Assaf etal., 2012;
Haorah et al., 2011; Kathirvel et al., 2013; Patel
etal., 2010; Pillich etal., 2005; Traina etal., 2008;
Virmani etal.,2001).
Multiple clinical studies have shown that
acetyl-L-carnitine may be of benefit in treating
aging-related consequences, neurodegenerative
disorders, peripheral neuropathies, and cognitive
impairment related to various conditions (Beghi
et al., 2013; Bianchetti et al., 2003; De Grandis,
2007; Hudson and Tabet, 2003; Malaguarnera
et al., 2008; Montgomery et al., 2003; Zanelli
etal.,2005).
C R E AT I N E
Creatine (Cr; -methyl-guanidinoacetic acid)
was discovered and isolated from meat extract
in 1835 by Chevreul. Although studies regard-
ing its physiology have been published, until
recently research for its clinical application was
lacking. Since Roger Harris and his colleagues
have demonstrated that Cr loading is able to
enhance muscle Cr and phospho-Cr (PCr) con-
tent, Cr supplementation has been largely used
to improve exercise capacity in healthy individu-
als and athletes (Francaux and Poortmans, 1999;
Harris etal., 1992; Hoffman etal.,2006).
Work by Stockler et al. (1994, 1996) and
Salomons et al. (2001) identified the first disor-
ders of Cr metabolism and their clinical presenta-
tions that involve CNS and respond positively to
Cr therapy. Accordingly, researchers are focused
on the possible role of Cr supplementation on
brain metabolism. It is well known that Cr treat-
ment provides neuroprotection against various
toxic insults in fetal rat ventral mesencephalic
and striatal cultures (Andres etal., 2005). There
is some evidence that Cr treatment can be
applied in neurodegenerative disorders such as

Huntingtons and Parkinsons diseases (Bender
etal., 2008; Hass etal., 2007; Hersch etal., 2006;
Tabrizi et al., 2005). In the past few years there
has been growing evidence about Crs neuropro-
tective effect on acquired brain disorders such as
brain trauma, perinatal brain damage, and epi-
lepsy, as well as a variety of pathologies in which
energy metabolism and oxidative stress play an
etiological role (Adcock et al., 2002; Evangeliou
et al., 2009b; Iqbal et al., 2013; Sakellaris
etal.,2006).
Cr is distributed throughout the body with
95% found in skeletal muscle. The remaining
5% is located in the brain, liver, kidney, and
testes (Walker, 1979). Cr content in the body is
maintained by nutritional intake and endog-
enous biosynthesis roughly equally. The dietary
source is restricted to animal sources, such as
meat. In vegetarians it is likely that the synthe-
sis route is upregulated. Chronic supplemen-
tation of Cr results in downregulation of the
Cr transporter (CRT; Guerrero-Ontiveros and
Wallimann,1998).
The first step of Cr biosynthesis probably occurs
mainly in the kidney with L-arginine:glycine
amidinotransferase (AGAT), an enzyme that
catalyzes the conversion of arginine and glycine
to form guanidinoacetate and ornithine, whereas
the liver is likely to be the principal organ accom-
plishing the subsequent methylation of guanidi-
noacetate to Cr (Bera etal., 2008; Lage etal., 2013;
Walker, 1979). Cr phospokinase (CPK) catalyses
the phosphorylation and dephosphorylation of Cr
and PCr, respectively (Hespel etal., 2001). Cr and
PCr play an essential role in the storage and trans-
mission of phosphate-bound energy (Balestrino
etal.,2002).
Endogenous synthesis of Cr occurs in the
brain as AGAT and S-adenosyl-L-methionine:g
uanidinoacetate N-methyltransferase (GAMT)
are expressed in most cell types, particularly
neurons, oligodendrocytes, and astrocytes, it
is generally agreed that the majority of Cr syn-
thesis occurs peripherally in the kidney (via
AGAT), liver, and pancreas (via GAMT; Bard
and Braissant, 2010; Braissant et al., 2011;
Brosnan etal., 2011; Brosnan etal., 2007; Edison
et al., 2007). Following biosynthesis or inges-
tion, Cr is transferred from blood plasma by
specific sodiumand chloride-dependent CRTs
located in skeletal muscle, kidney, heart, brain,
and liver (Allen, 2012; Snow and Murphy, 2001).
Importantly, Cr enters the brain via these CRTs at
the blood-brain barrier (BBB), which emphasizes
Dietary Manipulations 275
the potential benefits of oral Cr supplementa-
tion to treat brain-related disorders (Allen, 2012;
Nash etal., 1994; Ohtsuki etal., 2002). Questions
arise about the rate of Cr crossing the BBB.
Recent research shows that the CRT SLC6A8 is
expressed by microcapillary endothelial cells at
the BBB, but it is absent from their surrounding
astrocytes. This raised the concept that the BBB
has a limited permeability for peripheral Cr and
that the brain supplies a part of its Cr by endog-
enous synthesis (Braissant, 2012). Recent data
on Cr metabolism shows that in physiological
conditions, Cr is taken up by the CNS from the
periphery through SLC6A8 at BBB but in limited
amounts, and that the CNS also needs its own Cr
synthesis (Braissant,2012).
Cr is taken up by neurons and oligoden-
drocytes through CRTs where, together with
endogenous Cr, it participates in the regulation
of energy balance. The effect of Cr in energy
metabolism is depicted in Figure 16.3. As noted,
recent research on Cr has demonstrated positive
therapeutic results in various acquired clinical
disorders, especially for those with disturbed
energy balance.
Creatine and BrainTrauma
The most common cause of death and disability
after severe trauma in childhood is traumatic
brain injury (TBI). Of all trauma deaths, 25% are
caused by head injury (Tepas et al., 1990). TBI
leads to functional deficits caused in primary
and secondary mechanisms. While for primary
mechanisms the damage occurs at the time of
impact and results from the mechanical insult
itself, for secondary mechanisms the cellular
damage occurs not immediately after the trauma
but develops within minutes, hours, or even days,
and during this time medical intervention, phar-
maceutical or mechanical, is usually applied.
One of the first events caused by the secondary
mechanisms is mitochondrial dysfunction asso-
ciated with the disruption in cellular calcium
homeostasis. Maintenance of cellular calcium
homeostasis is intimately related to ATP use and
synthesis, which are key to proper brain function-
ing. At this point enhanced neuron survival may
be improved by providing an adequate supply of
ATP immediately after trauma, and at this point
Cr could play an important role; recent accu-
mulative literature support this idea (Sullivan
etal., 2000). Specifically, it is well known that Cr
supplementation increases intramuscular and
cerebral stores with both Cr and PCr (Dechent
276 Part III:Homeostatic Manipulators

Glycolysis b-oxidation ATP dependent processes

at cell membrane, place1
in cytosol, at cytoskeleton
BRAIN at organelles
Acetyl-CoA
ATP
Energy deficit
Endogenous Krebs situations
creatine synthesis cycle ATP
CK Neurodegenerative
CK disorders
NADH place2
PCr pool
Mitochondrial
respiratory chain

Creatine + ATP CK ADP + PCr

creatinine

CRT
SLC6A8 BBB BBB

Dietary creatine
intake
Exogenously
administered
creatine

FIGURE 16.3: Simplified schema of creatine (Cr) metabolism and its relationship to ATP. Cr taken up into the
brain via Cr transporters (CRT SLC6A8) is transformed to the high-energy compound PCr by Cr kinase (CK).
ATP synthesis mainly from carbohydrate (glucose) and less from fat occurs via three series of metabolic path-
ways:glycolysis and -oxidation, the Krebs cycle, and the mitochondrial respiratory chain. When ATP is rapidly
depleted, CK catalyzes the donation of a phosphate group from phosphocreatine (PCr) to ADP, producing more
ATP to buffer energy needs. Conversely, when energy is released, an individual phosphate group is cleaved from
ATP and bound to Cr to rejoin the PCr pool. PCr is used to buffer cytosolic ATP/ADP ratios and for local ATP
consumption, e.g., by cytosolic metabolic enzymes, ATP-requiring contractile processes or cell motility, organelle
transport or ATP-dependent cell signaling or at the cell membrane by ATP-requiring ion pumps, ATP-gated
ion-channels or ATP-regulated receptors (place 1). In addition, PCr plays an important role in managing energy
deficits in neurodegenerative diseases such as Parkinsons and Alzheimers (place2).

etal., 1999). The increase of these stores may offer
therapeutic benefits by stimulating protein syn-
thesis or reducing protein degradation, stabiliz-
ing biological membranes, and preventing ATP
depletion, which occur in patients with TBI. The
experimental work by Lifshitz et al. (2003) and
Hausmann et al. (2002) demonstrated that Cr
reduced cortical damage and protected mito-
chondrial energetics in the brain while reducing
the spread of secondary spinal cord injury.
Although there is plethora of experimental
work dealing with the neuroprotective role of Cr
in TBI, few clinical studies have been published
on the same subject. In clinical work done by our
team in 39 children and adolescents 1 to18years
old with TBI, Cr supplementation (given for
6 months at a dose of 0.4 g/kg/day orally)
improved results in several parameters, including
duration of posttraumatic amnesia, duration of
intubation and intensive care unit stay, disability,
good recovery, self-care, communication, loco-
motion, sociability, personality/behavior, and
neurophysical and cognitive function (Sakellaris
etal., 2006; Sakellaris etal.,2008).
Creatine and Epilepsies
It is well known that Cr is involved in the patho-
physiology of epilepsy and that epileptic seizures
are accompanied by increased CPK concentra-
tion levels. Thus in patients with a recent loss
of consciousness and abnormal movements,
serum CPK concentration is a useful, practical,

and relatively accurate parameter to assist in the
differentiation of epileptic seizures from either
vasovagal syncope or psychogenic nonepileptic
seizures (Neufeld et al., 1997; Petramfar et al.,
2009). Early experimental work done from
Glatzner et al. (1979) suggested that sources
other than skeletal muscle alone contribute to
the increased CPK activity after grand mal sei-
zures. A later magnetic resonance spectroscopy
study in humans suggested that an abnormal
N-acetylaspartate/Cr (or choline) ratio will poten-
tially provide a useful indicator of poor seizure
control condition in patients with temporal lobe
epilepsy (Mendes-Ribeiro et al., 1998). Several
relevant publications suggest that an increase in
Cr may reflect reactive astrocytosis (mesial tem-
poral sclerosis), even when MRI volumetric stud-
ies show normal hipoccampi (Cendes etal., 1997;
Kuzniecki and Jackson, 1995). Crs antiepileptic
action on immature brains was first introduced
by Holzman et al. (1998). Specifically, rat pups
were injected with Cr for 3days before exposing
them to hypoxia on postnatal day 10 or 20. Before
and during hypoxia, the electrocortical activ-
ity or 31P nuclear magnetic resonance spectra
were measured. At 10 but not 20days, Cr injec-
tions increased brain Cr/nucleotide triphosphate
ratios, decreased hypoxia-induced seizures and
deaths, and enhanced brain PCr and ATP recov-
eries after hypoxia. Thus Cr protected the meta-
bolically immature brain from hypoxia-induced
seizures and, perhaps, from cellular injury.
In another study (Royes et al., 2006), Cr
supplementation protected not only against
methyl-malonic acid-induced convulsions but
also against methyl-malonic acidinduced oxi-
dative damage. Furthermore, experimental work
from the same group demonstrated that physi-
cal training, Cr supplementation, or their com-
bination attenuated pentylenetetrazol-induced
seizures and oxidative damage in vivo, provid-
ing evidence that combined Cr supplementation
and physical exercise may be a useful strategy in
the treatment of convulsive disorders (Rambo
etal.,2009).
Another interesting finding is the fact that
the ketogenic diet, a metabolic therapy suc-
cessful in the treatment of refractory epilep-
sies, also impacts Cr metabolism. Experimental
work done by Bough etal. (2006) demonstrated
that the ketogenic diet induced a striking 46%
increase in the density of mitochondrial profiles
in tissue taken from ketogenic dietfed animals
compared to controls. Furthermore, in the same
Dietary Manipulations 277
experimental work and in light of the afore-
mentioned findings, the investigation of energy
metabolites within the hippocampal tissue dem-
onstrated that the ketogenic diet induced an
increase in the PCr/Cr energy-store ratio and tis-
sue levels of -hydroxybutyrate, although ATP,
ADP, and AMP levels did not change appreciably.
Based on these observations, we gave a Cr
supplement to a group of patients for whom
the ketogenic diet was partially successful
(Evangeliou A, Asprangathou D, Karanika E,
Spilioti M., unpublished data). Our rationale
was to precipitate a further rise in the PCr/Cr
energy-store ratio in order to induce an increase
in energy production in the brain, thus enhanc-
ing the efficacy of the diet. Preliminary data gives
us some hope that the ketogenic diet in combi-
nation with Cr might be more effective in the
treatment of refractory epilepsies. Specifically, in
10 children with therapy-resistant epilepsy
receiving the ketogenic diet, Cr supplementation
was added in doses up to 5 g daily. The ketogenic
diet had a partial effect in all of these children,
and when Cr was added, 3 of the children experi-
enced a further seizure reduction of 90% to 100%
from baseline.
M E L AT O N I N
Melatonin (N-acetyl-5-methoxytryptamine) is
a signaling molecule released as a hormone of
the pineal gland predominantly during night.
It regulates the sleepwake cycle by chemically
causing drowsiness and lowering body tempera-
ture (Al-Omary, 2013; Hardeland, 2012; Rios
et al., 2010). Therefore, interventions that target
sleep may improve child health and child and
family distress but may also ameliorate core and
related symptoms of autism (Gunol etal., 2011;
Rossignol and Frye, 2011). Most of its biological
effects are mediated by G-protein-coupled recep-
tors MT1 and MT2, with its main action within
the CNS. In mammals, these receptors are
expressed in various tissues and organs, includ-
ing the brain (Dubocovich et al., 2010; Levoye
etal.,2006).
Melatonin secretion decreases during aging.
Decreased levels of melatonin, with concomi-
tant upregulation of MT1 and MT2 receptors
exceeding that observed during normal aging,
have been repeatedly described in neurodegen-
erative disorders, especially Alzheimers disease
and other types of senile dementia, in some
mood disorders, and after prolonged drug treat-
ment (e.g., antidepressants; Boyce and Hopwood,
278 Part III:Homeostatic Manipulators
2013; Bubenik and Konturek, 2011; Carvalho
etal., 2009; Mishima etal., 1999; Rosales-Corral
etal.,2012).
Melatonin also acts as a direct free-radical
scavenger and indirect antioxidant by stimulat-
ing gene expression for antioxidant enzymes
(superoxide dismutase, catalase, glutathione
peroxidase, glutathione reductase) or to increase
their activity (Fischer et al., 2008; Reiter et al.,
2010). Unlike other antioxidants, melatonin can
easily cross all morphophysiological barriers,
such as the BBB, and enter cells and subcellu-
lar compartments. Based on all these properties
there is accumulating evidence suggesting the
potential of melatonin in neurodegenerative con-
ditions such as Parkinsons disease, Alzheimers
disease, Huntingtons disease, amyotrophic
lateral sclerosis, epileptic seizures, and stroke
(Cecon and Markus, 2011; Pandi-Perumal etal.,
2013; Polimeni etal.,2014).
Melatonin and Insomnia
Insomnia is a common sleep disorder that can
make it hard to fall asleep, hard to stay asleep, or
both, despite the opportunity for adequate sleep.
Endogenous melatonin levels decrease with age,
probably due to an age-related decline in circa-
dian rhythmic functions or the calcification of
the pineal gland (Dijk et al., 1999; Ortiz et al.,
2008). Since endogenous melatonin has benefi-
cial effects on sleep in humans and helps stabi-
lize physiological circadian rhythms, including
the sleepwake cycle, declining melatonin levels
may contribute to the common complaint of poor
sleep quality seen among the elderly (Touitou
etal., 1984; Wade etal., 2011; Wade etal., 2010).
This has raised the possibility of improving sleep
in elderly patients with insomnia by appropri-
ately timed treatment with melatonin (Al-Aama
et al., 2011; Lyseng-Williamson, 2012; Simn
Padilla et al., 2009). The prevalence of chronic
sleep onset insomnia is common not only in
elderly people but in all ages (Blader etal., 1997;
Cunnington etal., 2013; Luthringer etal.,2009).
A recent meta-analysis involving 1,683 sub-
jects of all ages demonstrated that melatonin
significantly improved sleep in subjects with
primary sleep disorders compared to placebo.
In this study, melatonin reduced sleep-onset
latency, increased total sleep time, and improved
overall sleep quality compared to placebo to a sta-
tistically significant degree (Monterrosa-Castro
et al., 2013). According to the same study, the
effects of melatonin on sleep are modest but do
not appear to dissipate with continued mela-
tonin use. Although the absolute benefit of
melatonin compared to placebo is smaller than
other pharmacological treatments for insomnia,
melatonin has a role in the treatment of insom-
nia given its relatively benign side-effect profile
compared to these other agents (Ellis etal., 2012;
Ferracioli-Oda etal.,2013).
Melatonin andAutism
Sleep difficulties, particularly insomnia, occur in
more than 50% of children with autism spectrum
disorders (Bain, 2006; Couturier etal., 2005). In
addition, there is increasing evidence that sleep
disorders in children with autism are associated
with disrupted melatonin secretion (Doyen etal.,
2011; Goldman etal., 2012). Accordingly, numer-
ous studies have suggested the use of melatonin
for sleep problems of children and adults with
autism (Cortesi et al., 2012; Malow et al., 2012;
Tordjman etal.,2005).
Melatonin and Neurodegenerative
Disorders
Numerous studies suggest melatonins neuro-
protective role in neurodegenerative diseases
(Galli-Carminati etal., 2009; Huang etal., 2009;
Srinivasan etal., 2011). Intensive research in the
past few years has indicated melatonins benefi-
cial effects in neurodegenerative disorders. Brain
oxidative damage has been implicated as a com-
mon link in the pathogenesis of such diseases.
Melatonin crosses the BBB easily and acts as a
free-radical scavenger, and its broad spectrum of
antioxidant activities in many CNS neurodegen-
erative diseases has been well documented and
reviewed (Galli-Carminati etal., 2009; Gehrman
et al., 2009; Sliwinski et al., 2007; Wang, 2009;
Weishaupt etal.,2006).
BR ANCHED CHAIN
AMINOACIDS
Metabolism
The BCAAs (leucine, isoleucine, and valine) con-
stitute an important subgroup of the indispensa-
ble amino acids. BCAAs are the most hydrophobic
of the amino acids and play crucial roles in deter-
mining the structures of globular proteins as well
as the interaction of the transmembrane domains
of membranous proteins with phospholipid bilay-
ers (Brosnan and Brosnan, 2006). Despite their
similarity, the three BCAAs play subtly different
roles in proteins. Their side chains differ in size,
 Dietary Manipulations 279

shape, and hydrophobicity. Therefore, they have
different predilections for different secondary
structure motifs. Most proteins have a relatively
high proportion of these amino acids. Indeed,
most dietary proteins consist of ~20% BCAAs.
They comprise some 35% of the indispensable
amino acid requirements of mammals (Harper
etal.,1984).
The three BCAAs support numerous meta-
bolic processes ranging from the fundamental
role as substrates for protein synthesis to meta-
bolic roles as energy substrates, precursors for
synthesis of alanine and glutamine and the neuro-
transmitter glutamate, and modulators of muscle
protein synthesis via the insulin-signaling path-
way (Ahlborg et al., 1974; Anthony et al., 2001;
Hutson etal., 2001; Li etal., 2003; Patti etal., 1998;
Ruderman, 1975; Xu etal., 1998). Degradation of
BCAAs in skeletal muscle is linked to production
of alanine and glutamine and maintenance of
glucose homeostasis (Layman, 2003). The catabo-
lism of the BCAAs is shown in Figure16.4.
Not all tissues can oxidize BCAAs. Whereas
muscle has BCAA aminotransferase activity, the
liver lacks this enzyme. However, the liver has
branched chain -ketoacid-dehydrogenase and
can oxidize the branched-chain ketoacids. As
shown in Figure 16.4 all three BCAAs are con-
verted to branched-chain ketoacids while their
further metabolism employs distinct pathways
to end products of glucose and/or ketone bod-
ies. Ketone bodies and glucose can then be oxi-
dized as a respiratory fuel by extrahepatic tissues,
helping the organism in energy-stress situations
such as diabetes, epilepsy, neurodegenerative dis-
ease, and psychiatric disorders (Cole etal., 2010;
Doisaki et al., 2010; Iwasa et al., 2010; Piscopo
etal., 2011; Richardson etal., 2004; Scarna etal.,
2003; Sevy etal.,2006).
BCAA and Their Role intheCNS
Beyond direct metabolic effects, the BCAAs par-
ticipate directly and indirectly in a variety of
important biochemical functions in the brain.
BCAAs are crucial for homeostasis of gluta-
mate, an essential excitatory neurotransmitter.
Specifically, BCAAs (especially leucine) provide
neurons with a constant supply of glutamate,
which both neurons and glia oxidize robustly
(Bixel et al., 2001; Sweatt et al., 2004). he

Isoleucine Leucine Valine

-KG Branched chain -KG Branched chain -KG Branched chain
aminoacid aminoacid aminoacid
aminotransferase aminotransferase aminotransferase
Glutamate Glutamate Glutamate

-keto--methylvalerate -ketoisovalerate -ketoisocaproate

glucose
CoA Branched chain CoA Branched chain CoA Branched chain
ketoacid- ketoacid- ketoacid-
pyruvate dehydrogenase dehydrogenase dehydrogenase
CO2 CO2 CO2
a-methylbutyryl-CoA Isovaleryl-CoA Isobutyryl-CoA
malate
acetyl-CoA acetyl-CoA
acetoacetate

acetyl-CoA

malate
succinyl-CoA
Krebs
Cycle
fumarate

succinate
succinate

FIGURE 16.4: BCAA metabolism: isoleucine, leucine, and valine and their conversion to ketone bodies and
glucose.
280 Part III:Homeostatic Manipulators
A
BBB
Brain
SERUM
BCAA Aromatic aminoacids
B 1982; Gijsman et al., 2002; Islam et al., 2010;
BBB
Brain
SERUM
Decrease of
Aromatic aminoacids
(TRP, PHE, TYR)
Decreased levels of
neurotransmitters
5HT, DE, NE
FIGURE 16.5: BCAAs compete with aromatic amino
acids (phenylalanine, tyrosine and tryptophan)
for brain entry. a. Under normal conditions, some
amount of BCAAs and aromatic amino acids cross
the blood brain barrier and enter the brain. b. When
plasma BCAA concentrations rise, which can occur in
response to food ingestion or BCAA administration,
brain BCAA concentrations rise, and aromatic amino
acids decline.
BCAAs readily cross the BBB, with the influx of
leucine exceeding that of isoleucine and valine
(Oldendorf, 1971; Smith et al., 1987; Yudkoff,
1997). Astrocytes, which are in close approxima-
tion to brain capillaries, are probably the initial
site of metabolism of leucine (Hannuniemi and
Oja, 1981; Yudkoff etal., 1994). Amitochondrial
BCAA aminotransferase is very active in these
cells. Indeed, from 30 to 50% of all -amino
groups of brain glutamate are derived from leu-
cine alone (Gijsman et al., 2002; Yudkoff et al.,
2005b). However, homeostatic mechanisms
maintain a very low intrasynaptic concentration
of glutamate, partially accomplished by uptake
and conversion of glutamate to glutamine inside
astrocytes (Yudkoff etal., 2005b).
BCAAs influence brain function not only
by participating in glutamate homeostasis but
also in other aspects. Thus they influence brain
metabolism by modifying transport of aromatic
amino acids (tryptophan, phenylalanine, tyros-
ine) at the BBB. BCAAs compete with phenylala-
nine and tyrosine for brain entry, and studies in
healthy volunteers indicate that BCAAs produce
cognitive and endocrine effects consistent with
attenuation of central dopamine neurotransmis-
sion. When plasma BCAA concentrations rise,
which can occur in response to food ingestion
or BCAA administration, brain BCAA concen-
trations rise, and aromatic amino acids concen-
trations decline. Such effects occur acutely and
chronically (Bastone et al., 1995; Berry et al.,
Oldendorf, 1971; Sevy etal.,2006).
BCAAs enter the brain via a transporter,
located at the BBB on CNS capillary endothe-
lial cells, that is almost fully saturated at normal
plasma amino acid concentrations and com-
petitively shared by a number of large neutral
amino acids, including the aromatic amino acids
(Oldendorf, 1971; Pardridge, 1983; Pardridge and
Choi, 1986; Smith etal., 1987). As a consequence,
the ingestion of BCAAs causes rapid elevation
of their plasma concentrations, increases their
uptake into the brain, and decreases the brain
uptakes and levels of the aromatic amino acids
(Figure16.5).
BCAA Applications inCNS Diseases
BCAAs have been administered under a num-
ber of circumstances to both healthy humans
and individuals with certain neurological dis-
eases. A study done in patients with mania
demonstrated that acute administration of
BCAA decreased ratings of mania. The authors
of this study suggested that the beneficial effect
of BCAA to these patients could be explained
through the decrease of tyrosine availability to
the brain (Scarna etal., 2003). Elevated tyrosine
availability to the brain may contribute to the
development of central/mental fatigue during
and after sustained exercise. Supplementation
with BCAAs should prevent the exercise-induced
increase in the plasma concentration ratio of
free tryptophan to other large neutral amino
acids (including BCAAs) and thereby prevent
an elevation in the level of serotonin in the brain
(Hassmn etal.,1994).
BCAA supplementation enhances the cog-
nitive recovery of patients with severe TBI by
restoring hippocampal function after a TBI
(Aquilani etal., 2005; Cole etal., 2010). Aseries
of studies demonstrated that deficient clear-
ance of the large neutral amino acid phenyla-
lanine was associated with tardive dyskinesia,

that the administration of BCAAs significantly
decreased disease symptoms over placebo, and
that the observed symptom reduction was sig-
nificantly correlated with a diminished avail-
ability of phenylalanine to the brain of adult men
with psychosis (Mosnik etal., 1997; Richardson
et al., 2006; Richardson et al., 1999; Schultz
etal.,2001).
Older studies suggest a significant benefit
in terms of maintenance of extremity muscle
strength and continued ability to walk in patients
with amyotrophic lateral sclerosis. The posi-
tive effect for these patients could be explained
through the BCAAs, glutamate dehydrogenase
activation, and, consequently, glutamate decrease
(Bastone et al., 1995; Plaitakis et al., 1988).
Evidence is quite mixed however, with some evi-
dence of toxicity, increased mortality, and lack of
efficacy (Anonymous, 1993; Tandan etal.,1996).
BCAA and Epilepsy
As the major excitatory neurotransmitter in the
CNS of mammals, glutamate is essential for all
of our behaviors (Meldrum, 2000; Shepherd and
Huganir, 2007; Usherwood et al., 1968). Proper
glutamate metabolism is necessary to ensure syn-
aptogenesis, neurogenesis, neurite outgrowth,
and programmed cell death (Mattson, 2008).
Glutamate may have detrimental effects on neu-
rons when present in excessive amounts and has
been implicated in the pathogenesis of a number
of neurological disorders. Excessive sustained
activation of glutamate receptors can kill neu-
rons, particularly under conditions of reduced
energy availability and increased oxidative stress,
and is one of the major mechanisms of epilep-
togenesis (Hampson and Manalo, 1998; Hazell,
2007; Mattson, 2003; Zeng etal., 2007). Thus glu-
tamate homeostasis is very important for proper
neurotransmission. The brain must provide neu-
rons with a constant supply of glutamate while at
the same time the intrasynaptic glutamate level
must be kept low to maximize the signal-to-noise
ratio upon the release of glutamate from nerve
terminals to minimize the risk of excitotoxicity
consequent to excessive glutamatergic stimula-
tion of susceptible neurons (Sakai et al., 2004;
Yudkoff etal., 2005b).
Glutamate metabolism is accomplished
through complicated biochemical pathways.
BCAAs influence glutamate metabolism in
multiple ways. First, BCAAs (especially leu-
cine) provide neurons with a constant sup-
ply of glutamate: 30% to 50% of all -amino
groups of brain glutamine are derived from
Dietary Manipulations 281
leucine (Kanamori et al., 1998; Sakai et al.,
2004; Yudkoff et al., 2005b). BCAAs also serve
as ammonia donors to glutamate, participating
in the conversion of glutamate to glutamine in
the glutamate-glutamine cycle (Evangeliou etal.,
2009a; Yudkoff etal., 2005b). Proper function of
the glutamate-glutamine cycle is important not
only in glutamate homeostasis but also in the
prevention of epileptic seizures. An increasing
number of studies have suggested that an abnor-
mal amplification of glutamatergic activity is
involved in the pathophysiology of seizures and
certain types of medically refractory epilepsies
(Bernard etal., 1997; Eid etal., 2008; Guo etal.,
2010; Scimemi etal.,2006).
However, it appears that for buffering
of brain glutamateif levels of this excitatory
and potentially toxic neurotransmitter become
elevatedother mechanisms are recruited, such
as the BCAA effect on glutamate dehydrogenase
(GDH). GDH is a mitochondrial enzyme link-
ing the Krebs cycle to the multifunctional amino
acid glutamate. GDH catalyzes the reversible
interconversion of glutamate to -ketoglutarate
and ammonia using NADP(H) and NAD(H) as
cofactors, thus interconnecting amino acid and
carbohydrate metabolism. Thereby GDH plays
a pivotal role between carbohydrate and protein
metabolisms, controlling production and con-
sumption of the messenger molecule glutamate
in neuroendocrine cells (Karaca et al., 2011;
Spanaki etal.,2010).
In the past few years there has been increased
evidence that GDH dysfunction participates in
the pathophysiology of epileptogenesis. Clinical
and experimental data demonstrated that GDH
activity was significantly decreased in the tempo-
ral cortex in patients with temporal lobe epilepsy,
and it is proposed that significant alterations in
the enzyme activities may contribute to decreased
metabolism of glutamate, leading to its accumu-
lation (Kang et al., 2006; Malthankar-Phatak
et al., 2006). In this point of view, BCAA may
help in glutamate buffering by activating gluta-
mate dehydrogenase and in this way converting
excessive glutamate amounts to -ketoglutarate
(Plaitakis, 1989; Plaitakis etal., 1988). Our inte-
grated proposal of BCAA action on glutamate
metabolism is illustrated in Figure16.6.
Experimental evidence also shows that
BCAAs alone have antiepileptic properties. An
infusion of a BCAA solution (300 mg/kg) has been
found to increase the seizure threshold in rats to
the proconvulsant drug picrotoxin, an antagonist
on the GABA-benzodiazepine receptor complex.
282 Part III:Homeostatic Manipulators

Postsynaptic-Neuron
Presynaptic-Neuron
-KG

GDH
glutamate glutamate

glutamine

BCAA
pool
NH3 glutamate Astrocyte

GDH
glutamine -KG

FIGURE 16.6: BCAAs effects on glutamate homeostasis: BCAAs may affect glutamate homeostasis in three
ways:1. BCAAs - especially leucine - provide neurons with a constant supply of glutamate; 2.The intrasynaptic
glutamate level must be kept low to maximize the signal-to-noise ratio upon the release of glutamate from nerve
terminals to minimize the risk of excitotoxicity consequent to excessive glutamatergic stimulation of susceptible
neurons. This is partially achieved by converting glutamate to glutamine inside the astrocytes; 3. BCAA may
help in glutamate buffering by activating glutamate dehydrogenase and thus convert excessive glutamate to
-ketoglutarate.

The effect was similar regardless of whether all
three BCAAs were given together or separately
(Skeie etal., 1992; Skeie etal.,1994).
BCAA and KetogenicDiet
Over the past few decades, the ketogenic diet
has become one of our valuable weapons in the
management of intractable seizures. However,
not all patients have truly benefited from this
therapy. In addition, the application of this diet
is limited by the fact that many patients cannot
tolerate it while others present with side effects
(Freeman etal., 2007; Kang etal., 2004). Recent
efforts have been made to improve its tolerabil-
ity such as the application of the low glycemic
index treatment or the modified Atkins diet
(Kossoff et al., 2003; Pfeifer and Thiele, 2005).
The addition of BCAA to the classic ketogenic
diet appears to be a new promising advance-
ment toward this effort. Work done in our
clinic demonstrated that the use of BCAA could
increase the effectiveness of the ketogenic diet
(Evangeliou etal., 2009a). From the same study,
none of our patients had a remarkable reduc-
tion in the level of urine ketosis after the sup-
plementation of BCAAs. Moreover, in all of the
patients, no reduction in ketosis was recorded
despite the change in the fat-to-protein ratio
from 4:1 to2.5:1.
Experimental and clinical data indicate
that BCAAs and the ketogenic diet share some
common properties. First, both could favor
-aminobutyric acid (GABAergic) neurotrans-
mission. It is well known that the ketogenic
diet may limit the availability of oxaloacetate
to the aspartate aminotransferase reaction, an
important route of brain glutamate handling.
As a result, more glutamate becomes accessible
to the glutamate decarboxylase reaction to yield
GABA, the major inhibitory neurotransmitter
and an important antiseizure (antiepileptic)
agent (Yudkoff etal., 2004). These experimental
data are supported by a clinical trial in 26 chil-
dren suffering from refractory epilepsy where
the ketogenic diet increased the GABA lev-
els in cerebrospinal fluid (Dahlin et al., 2005).
However, experimental data from another
research group demonstrated that BCAAs
decreased the concentration of glutamate in
the thalamus and cortex without affecting
GABA concentrations. A logical explanation
of this phenomenon is the glutamate decrease
through the BCAA cycle as proposed by Hutson
et al. (2001). BCAAs enter astrocytes where

transamination catalyzed by the mitochon-
drial branched-chain aminotransferase pro-
duces glutamate and branched-chain ketoacids.
Because branched-chain ketoacids are poorly
oxidized in astrocytes, they exit astrocytes
and can be taken up by neurons (Hutson etal.,
1998). Inside neurons, they are transaminated
back to the BCAAs by the neuronal cytosolic
branched-chain aminotransferase, in a pro-
cess consuming glutamate. These BCAAs exit
neurons and return to astrocyte. Another pos-
sibility for decreasing glutamate is through
BCAA glutamate dehydrogenase activation
(Plaitakis, 1989; Plaitakis et al., 1988). This
aspect is supported by work in which glutamate
dehydrogenase was significantly decreased in
temporal, neocortical, and hippocampal tissues
obtained from medically intractable epilepsy
patients (Malthankar-Phatak etal., 2006). Thus
BCAAs, by decreasing the effects of glutamater-
gic neurotransmission, could facilitate those
of GABAergic neurotransmission (Dufour
et al., 2001b). In addition, the ketogenic diet
appears to favor the synthesis of glutamine,
an essential precursor to GABA. This occurs
because ketone body carbon is metabolized to
glutamine and also because there is increased
consumption of acetate in ketosis, which is con-
verted quickly into glutamine in the brain by
astrocytes. Similarly leucine, one of the main
BCAAs, facilitates the production of glutamine.
In particular, it serves as an ammoniac donor
in the formation of glutamine from glutamate.
Moreover, leucine is related to many more bio-
chemical reactions that take place in ketosis.
Specifically, ketosis may favor the release of
glutamine from the brain, which is exchanged
for blood leucine at the BBB by specific trans-
porters. Because brain glutamine is formed in
astrocytes from glutamate, the overall effect
would be to favor the release of glutamate
from the nervous system (Yudkoff etal., 2005a;
Yudkoff et al., 2004). These properties of leu-
cine are of paramount importance because a
delayed removal of glutamate in the brain leads
to increased levels of glutamate in neuronal
synapses, which is one of the pathophysiologic
mechanisms implicated in epileptogenesis
(Wong etal.,2002).
Although encouraging, clinical data on
an antiepileptic action of BCAA are still lim-
ited. Many questions need to be answered.
How high can we go with the BCAA dose?
Could we provide leucine alone as the most
ketotic of BCAAs? Providing exclusively
Dietary Manipulations 283
leucine as an adjunctive treatment to the
ketogenic diet is impossible because it is toxic
when consumed out of proportion to valine
and isoleucine. Downregulation of branched
chain kinase and inhibition of branched
chain kinase by -ketoisocaproate activates
branched-chain -ketoacid dehydrogenase.
Activated branched-chain -ketoacid dehy-
drogenase depletes -ketoisovalerate and
-keto-L-methylvalerate and therefore also
depletes valine and isoleucine. Lack of valine
and isoleucine inhibits protein synthesis. The
consequence is that leucine should not be
consumed in large amounts without valine
and isoleucine, even though only leucine pro-
motes protein synthesis (Scarna et al., 2005).
High dietary BCAAs should be well toler-
ated because of the reserve of branched chain
-ketoacid dehydrogenase activity present
in tissues of the body. However, here also,
theoretically the safety margin of maximally
activated branched-chain -ketoacid dehydro-
genase may be exceeded at high BCAA intake.
There is a need, therefore, to know the maxi-
mal capacity for BCAA disposal. Not enough
information is currently available to make such
calculations (Harris etal.,2005).
Another reason to be concerned with BCAA
supplementation is the fact that our study is the
only clinical study on epileptic patients, and we
must be very cautious with their administra-
tion. Our concerns are supported by the fact that
although the majority of publications discuss
the antiepileptic action of BCAAs, there is some
experimental research addressing the epilepto-
genic action of BCAAs in genetic absence epi-
leptic rats of Strasbourg (GAERS; Dufour etal.,
2001a). Interestingly, in the same experimental
model seizures are connected with increased
GDH expression in the thalamus of both young
and adult GAERS and in the cortex of young
GAERS (Dutuit etal., 2000). This is in contrast
to what happens in patients with temporal lobe
epilepsy, where the convulsions are connected to
low GDH levels and the BCAA could help by acti-
vating GDH (Malthankar-Phatak etal.,2006).
Another question that needs to be answered
is related to the use of BCAAs as monotherapy
in patients with epilepsy. There is no doubt that
the antiepileptic action of BCAAs is based only
on experimental data. Further studies, espe-
cially those on patients with intractable epilepsy
receiving two to three antiepileptic regimens and
not on the ketogenic diet, are required for more
concrete conclusions.
284 Part III:Homeostatic Manipulators
PROBIOTICS
Complex organisms, including humans, devel-
oped symbiosis as a common way to overcome and
survive with genetic and metabolic deficiencies
(Resta, 2009; Tylianakis, 2009). Evolutionarily,
symbiosis has proven beneficial for all partners
considered and has become our way of life. As
a result, Homo sapiens largest organ is the
gut microbiota, comprising two major divi-
sions of bacteria, Bacteroidetes and Firmicutes
(Neish, 2009; Resta, 2009). The recognition that
the gut microbiota influences several signaling
pathways led to the suggestion of the concept
of a microbiotagutbrain axis (Montiel-Castro
etal., 2013). The gutbrain interation is the bidi-
rectional communication between the gut and
the brain that occurs through multiple pathways
that include hormonal, neural, and immune
mediators (De Palma etal.,2014).
Gut microbiota have a very important role
in stress situations. Exposure to stress results in
alterations of the braingut axis ultimately lead-
ing to the development of a broad array of gas-
trointestinal disorders, including negative effects
on intestinal microbiota (Konturek et al., 2011).
Accordingly, one area of flourishing research
involves the relationship between the intesti-
nal microbiota (as well as the related functional
integrity of the gastrointestinal tract) and mental
health (Bested etal., 2013a, 2013b, 2013c).Recent
clinical studies demonstrate that the adminis-
tration of beneficial microbes, through supple-
mentation and/or fecal microbial transplant, can
affect end-points related to mood state (glycemic
control, oxidative status, uremic toxins), brain
function (functional MRI), and mental outlook
(depression, anxiety, autism; Bested etal., 2013c;
Chen etal.,2013).
Probiotics andAutism
Children with autism often exhibit symptoms of
the gastrointestinal system (Adams et al., 2011;
McElhanon etal., 2014). These symptoms include
diarrhea, constipation, vomiting/reflux, abdomi-
nal pain/discomfort, gaseousness, and unusually
foul-smelling stools (Chaidez etal., 2014; Hsiao,
2014; Kang et al., 2014). In the past few years,
increasing numbers of studies have implicated
correlation between not only gastrointestinal
but also other autistic symptoms and abnor-
mal intestinal probiota (Kang etal., 2013; Wang
et al., 2013). In a very recent study, the partici-
pants demonstrated strong positive correlation of
autism severity with the severity of gastrointesti-
nal dysfunction (Tomova etal.,2015).
Furthermore, many studies have demon-
strated the restoration of microbiome intestinal
mucosa after administration of probiotics, and
in this sense probiotics can be useful to restore
the microbial balance in the intestine, to relieve
gastrointestinal problems, and to attenuate
immunological abnormalities (Fond etal., 2015;
Rook etal., 2014). It is well known that much of
the immune system is located in the intestine
and that children with autism disorders suf-
fer from immune system dysfunction (Samsam
etal.,2014).
Probiotics can effect microbiota composition
and intestinal barrier function and alter intes-
tinal immune responses, thus improving the
gutbrainmicrobiota axis (Butel, 2014; Smith
etal., 2014). The administration of probiotic bac-
teria to address changes in the microbiota might,
therefore, be a useful novel therapeutic tool with
which to restore normal gut microbiota, reduce
inflammation, restore epithelial barrier func-
tion, and potentially ameliorate behavioral
symptoms associated with some children with
autism (Critchfield et al., 2011; Hemarajata and
Versalovic, 2013; Szajewska, 2013). Although
there are many experimental studies that dem-
onstrate the beneficial effect of probiotics on
animal models, there are no randomized clini-
cal trials to prove this in humans (Gilbert etal.,
2013; Hsiao etal., 2013). In an ongoing study at
our clinic, probiotics were administered to 30
children, ages 4 to 10years, with autism. Sixteen
of these children had gastrointestinal symptoms
and 14 did not. Probiotics had beneficial effects in
both groups of patients. Significant improvement
(>12 units on the Childhood Autism Rating Scale)
was recorded in 5 patients (pre-scale: 36.00
1.39 [mean SD]) from the group with intestinal
symptoms and in 4 from the group with no intes-
tinal symptoms (pre-scale 34.1 1.21; Evangeliou
A, Asprangathou D, Karanika E, Spilioti M,
unpublished data). Although these data are very
preliminary, there is a body of emerging evidence
that probiotics may be used in autistic behavior
as an additional or alternative therapy.
O M E G A - 3 FAT T Y A C I D S
Omega-3 fatty acids are polyunsaturated fatty
acids, and three main types are found in the
human diet:-linolenic acid (ALA), docosahex-
aenoic acid (DHA), and eicosapentaenoic acid
(EPA). DHA and EPA are found in seafood,
while ALA is found in nut and plant oils (Bent
et al., 2009; Zhang et al., 2011). Interestingly,
fish do not produce EPA and DHA, but the oils

are synthesized by single-cell marine organ-
isms that are eaten by fish (Harris, 2004). While
the human body can synthesize both DHA and
EPA from ALA, it cannot synthesize any of these
three types of fatty acids ex novo, and, accord-
ingly, these substances are dietary essentials
(Bent etal., 2009; Freeman etal., 2006). Omega-3
fatty acids are critical to brain development and
function (Denis et al., 2013; Innis, 2008; Zhang
et al., 2011). Lack of these fatty acids has been
implicated in a number of mental health condi-
tions over the lifespan, from developmental dis-
orders and mental retardation in childhood to
depression, bipolar disorder, schizophrenia, and
borderline personality disorder; stress, hostil-
ity, and aggression in adulthood; and cognitive
decline, dementia, and Alzheimers disease in
late adulthood (Andreeva etal., 2011; Bent etal.,
2011; Fotuhi et al., 2009; Marano et al., 2013;
Rondanelli etal., 2010, 2011). Agrowing body of
clinical research trials has been conducted with
variable outcomes to all these situations. Thus in
the past 30years a substantial number of obser-
vational and epidemiological studies have sug-
gested that mental illness, in particular mood
disorders, is associated with reduced dietary
intake and/or cellular abundance of omega-3
polyunsaturated fatty acids (Ross etal.,2007).
Despite the many studies that have been done,
the results are questionable. A meta-analysis
of randomized, placebo-controlled trials of
omega-3 fatty acid treatment of major depres-
sive disorder to determine efficacy and examine
sources of heterogeneity between trials suggested
a small, nonsignificant benefit of omega-3 fatty
acids for major depression. The authors of the
same study suggested that nearly all of the treat-
ment efficacy observed in the published literature
may be attributable to publication bias (Bloch
and Hannestad,2012).
Moreover, in another review of the published
literature on the effects of omega-3 fatty acids on
measures of cognitive function in normal aging,
incidence and treatment of dementia suggested
that the available data are insufficient to draw
strong conclusions about the effects of omega-3
fatty acids on cognitive function in normal aging
or on the incidence or treatment of dementia;
however, also from the same study, the authors
proposed that limited evidence suggests a possi-
ble association between omega-3 fatty acids and
reduced risk of dementia (Issa etal.,2006).
Despite the aforementioned concerns and
the lack of sufficient randomized double-blind
studies, the health benefits of omega-3 essential
Dietary Manipulations 285
fatty acids may be especially important in
patients with psychiatric disorders, due to
high prevalence rates of smoking and obesity
and the metabolic side effects of some psycho-
tropic medications (Freeman etal., 2006). Thus
while it is not currently possible to recommend
omega-3 polyunsaturated fatty acids as either
a mono- or adjunctive therapy in any mental
illness, the available evidence is strong enough
to justify continued study, especially with
regard to attentional, anxiety, and mood disor-
ders (Ross etal., 2007). Moreover, a number of
clinical studies have shown omega-3 fatty acids
are essential for normal infant vision devel-
opment, and adequate amounts of DHA and
other omega-3 fatty acids in the diet of preg-
nant women also appear to be important in
normal infant vision development (Dennehy,
2011; Hoffman et al., 2009; SanGiovanni
etal.,2000).
CONCLUSION
Here we have outlined a diverse array of dietary
manipulatons that can impact brain homeo-
stasis directly or indirectly. We hope and
expect much new research is on the horizon
due to increased patient interest and along-
side the potential for limited toxicity and the
broad implications for practical and affordable
treatment.
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17
Exercise
M ARKUSDWOR AK
INTRODUCTION
Participation in regular physical activity has
been associated with positive effects on physi-
cal and mental performance. Physical exercise
affects the cardiovascular system, the pulmo-
nary system, and whole-body metabolism and is
an important factor in the prevention of cardio-
vascular diseases and metabolic diseases such
as type 2 diabetes and obesity. Other than the
positive effects on the peripheral systems, there
is accumulating evidence that physical activ-
ity can also significantly affect brain structures
and brain functioning. The exercise-induced
effects on the brain are partially mediated by
alterations in endogenous peptides, amino
acid transport through the blood-brain barrier
(BBB), and neurotransmitter alterations as well
as changes in energy metabolism. Since physical
activity is linked to neuronal activity and neu-
ronal firing itself consumes much of the energy
in the brain, physical exercise is closely associ-
ated with changes in brain energy metabolism.
With ongoing physical activity, the consump-
tion of oxygen (O2) and glucose increases in
brain regions involved in motor movement.
However, with increasing exercise intensity,
brain activation increases and changes in brain
energy homeostasis occur, since neurons prefer
lactate to glucose during high energy conditions
as their primary energy substrate, which raises
the production of the high-energy substrate
adenosine triphosphate (ATP). Furthermore,
high-intensity exercise increases the ratio of
metabolic demand to metabolite availability
with an accompanied change in brain energy
status. This chapter gives an overview of the
effects of physical activity on homeostatic pro-
cesses in the brain with a special consideration
of brain energy metabolism and its potential
implications on physical and mental health and
performance.
NEUR AL CONTROL
OFMOVEMENT
Exercise involves the contraction of muscle tis-
sue through an increase in spatial and temporal
recruitment of motor units. The neural organiza-
tion of motor control is organized hierarchically,
with smaller and simpler elements of movement
integrated into more complex elements at higher
levels of the nervous system. Simple motor com-
mands such as reflexes are directly controlled by
the spinal cord. Special types of neurons within
the central nervous system (CNS) play a key
role in motor movement. These efferent nerves
are called motoneurons, since they project their
axons outside the CNS to directly or indirectly
control muscles. Each motoneuron sends its axon
to one muscle and innervates a limited number
of muscle fibers, a complex called the motor unit.
Motoneurons are activated by interneurons of
different motor programs or reflex centers as well
as by descending tracts from the brainstem and
the forebrain. The average motoneuron receives
many thousands of synaptic inputs (Brnnstrm,
1993) that relay instructions from higher neu-
ronal centers and provide signals from sensory
modalities in the periphery. Motoneurons that
control different muscles are located in the spinal
cord and in the brainstem. Motoneurons supply-
ing different muscles can be activated with great
precision by these different neuronal sources,
which determine the degree of activation and
the exact timing of the motoneurons of a given
muscle. The recruitment and fine control of
motoneurons to produce voluntary coordinated
movements therefore requires that multiple
influences interact in the appropriate sequence
and with the right balance.
The region of the cerebral cortex most
involved in the planning, control, and execu-
tion of voluntary movements is the motor cortex.
The motor cortex is located in the rear portion

of the frontal lobe and is divided into two main
areas:Area 4 (primary motor cortex) and Area 6.
The primary motor cortex forms a thin band
along the central sulcus. Area 6 is wider and is
further subdivided into two distinct subareas.
The lateral portion of Area 6 (premotor area)
guides body movements by integrating sensory
information and controls muscles that are clos-
est to the bodys main axis, while the medial
part (supplementary motor area) is involved in
planning more complex movements. However,
besides the motor cortex, other cortical areas
also play important roles in generating voluntary
movements, such as the prefrontal cortex and the
posterior parietal cortex.
The basal ganglia, found below the motor
cortex, further help to organize motor programs
for complex movements. The main basal ganglia
are the striatum (caudate nucleus and the puta-
men), the globus pallidus, the substantia nigra,
the nucleus accumbens, and the subthalamic
nucleus, which are tightly interconnected since
they also receive information from several dif-
ferent regions of the cerebral cortex. The impor-
tance of these subcortical nuclei for normal
brain function and behavior is emphasized by
the numerous and diverse neurological condi-
tions associated with basal ganglia dysfunction,
which include disorders of behavior control such
as Tourette syndrome and obsessivecompulsive
disorder; dystonia; psychostimulant addiction;
and movement disorders, the most notable of
which are Huntingtons disease, which primarily
involves damage to the striatum, and Parkinsons
disease, which is caused by the degeneration of
the dopamine-producing cells in the substantia
nigra (Stocco etal., 2010; Fix,2008).
The timing and coordination of motor tasks
and the integration of sensory and motor infor-
mation is coordinated in the cerebellum, and its
damage causes loss of coordination. The cerebel-
lum and the basal ganglia receive information
from various parts of the brain and send this
information to the motor cortex via neurons in
the thalamus, forming the cortico-basal ganglia
loop and the cortico-cerebellar loop. The neu-
rons from the cortex send their axons via the cor-
ticospinal tract to the opposite side of the body
and connect either to the appropriate cranial
nerves or to the appropriate spinal nerve in the
spinal cord, where the spinal nerves connect to
particular muscles or muscle groups, thereby giv-
ing them the potential to directly control move-
ments. Therefore, the conductance of voluntary
movements is generated by changes in neuronal
Exercise 299
signaling across several cortical and subcortical
areas associated with changes in brain neuro-
transmission and metabolism (Ide and Secher,
2000; Figure17.1).
Activity in the frontal regions of the brain
has been related to affective and perceptual
responses to acute bouts of exercise (Nybo and
Nielsen, 2001; Petruzzello etal., 2006). Dynamic
movements are associated with cortical activa-
tion and increases in blood flow to the primary
sensorimotor area and supplementary motor
area (Orgogozo and Larsen, 1979). Changes in
neuronal activity and the cerebral metabolic rate
for glucose determined after running indicate
an involvement of the hypothalamus, the poste-
rior parietal cortex, the temporoparietal cortex,
the prefrontal cortex, as well as the premotor
and the primary motor cortex (Orgogozo and
Larsen,1979).
Neuroimaging studies of physical activity in
humans have shown differences in brain activ-
ity that are closely related to physical activity.
Examination of baseline spectral frequency
distributions of electroencephalograms (EEGs)
has revealed increased activation in the theta
Cortex
Central Command
Basal ganglia
Cerebellum
Brainstem
Motoric pattern
coordination Spinal Cord
Motoneurons
Sensory feedback
Effektor organs
Muscle
Movement
Sensory receptors
FIGURE 17.1: Neural regulation of movement.
Motor movements are planned and refined in the motor
cortex, basal ganglia, and cerebellum. Interneurons
within the spinal cord and brainstem send its axon to
the muscles and innervate the muscle fibers to gener-
ate movement. Sensory feedback initiates, informs, and
modulates the output at any stage of motor control.
300 Part III:Homeostatic Manipulators
(48 Hz), alpha (813 Hz), and beta (1320 Hz)
spectral bands and higher mean frequency in the
delta (0.254 Hz), theta, and beta bands in more
active or aerobically fit individuals (Hillman
etal., 2008; Bashore, 1989; Dustman etal., 1990;
Lardon etal., 1996; Mecklinger etal., 1992). These
findings suggest that physical activity influences
baseline electrocortical function and thus has the
potential to affect cognitive operations.
In addition to the effects of exercise on base-
line cortical functioning, motoric movements
are directly associated with changes in neuronal
activity. During and following aerobic and anaer-
obic exercise, there is an increase in EEG activ-
ity in the theta, alpha, and/or beta frequencies
(Crabbe and Dishman, 2004; Kamijo etal., 2004;
Nielsen etal., 2001; Nybo and Nielsen, 2001). The
increase in EEG activity is associated with the
workload of exercise. Studies show that brain
activity increases above resting levels at higher
workloads especially during anaerobic activities
and during muscle fatigue during graded exer-
cise (Bailey et al., 2008; Brmmer et al., 2011).
The increases in EEG activity were seen across
EEG frequencies (theta, alpha 1, alpha 2, beta 1,
beta 2) and electrode sites between the frontal
(F3, F4, F7, F8), central (C3, C4), and pariental
(P3, P4) lobes. Furthermore, EEG changes were
not localized to either hemisphere. While older
studies showed an increase of around 20% in
activity after exercise close to exhaustion with
a mean maximum heart rate of 200 beats per
minute (Beaussart et al., 1959), further stud-
ies confirmed that the increase in alpha activity
was strongest within the first 10 min after exer-
cise and returned to pre-exercise baseline after
30 min of recovery (Mechau etal., 1998; Boutcher
etal.,1988).
The neuronal activity pattern within the
brain depends further on the kind of exercise
and participants physical exercise preferences
(Schneider et al., 2009). Studies with stand-
ardized low resolution brain electromagnetic
tomography to assess EEG activity showed an
increase of alpha and beta activities immedi-
ately post-exercise widely across the brain. While
immediately after exercising all three kinds of
movement led to a significant increase of beta
activity in the Brodmann area 7, an area that
plays an important role in motor coordination,
15- and 30-min post-exercise a specific activation
pattern in the beta-frequency range character-
ized by an decrease in frontal brain activity and
an increase in occipital regions was recognized
after bike and treadmill exercises but not after
arm crank exercise, indicating that specific brain
activation patterns are linked to different kinds
of exercise and participants physical exercise
preferences (Schneider et al. 2009; Hanakawa
etal.,2003).
Therefore neuronal activity within differ-
ent brain regions is directly affected by exer-
cise, where the magnitude of brain activation
increases with the intensity and type of exercise
(Williamson etal., 1999; Schneider etal., 2009),
and the brain may become maximally stimu-
lated when exercise is performed at a level near
exhaustion (Kayser,2003).
B R A I N E N E R G Y M E TA B O L I S M
DURING EXERCISE
Cerebral Blood Flow and O2
Consumption
In addition to the changes in peripheral circu-
lation during in physical exercise also cerebral
blood flow (CBF) increases. One major factor
responsible for the increase in CBF during exer-
cise is the increase in neuronal activity needed
to co-ordinate motor control (also described as
the central command), including ventilatory
control and control of circulation (Goodwin
etal., 1972; Ide and Secher 2000). Furthermore,
CBF is influenced by the vast sensory input from
mechanically and chemically sensitive nerves
within the muscles (Sato et al., 2009 a, 2009b).
Central command and integration of informa-
tion from activation especially of the mechani-
cally sensitive nerve fibers work in concert to
elicit the increase in CBF. The relative contri-
bution of these influences varies depending on
the exercise model, and arm muscles appear to
require more neural integration from the part
of the brain interrogated by the middle cerebral
artery than is the case for the leg muscles.
An increase in CBF during brain activation
elicited by exercise has also been shown with
near-infrared spectroscopy (Ide et al., 1999).
Besides the changes in whole CBF, regional
changes in CBF (rCBF) and metabolism have been
found during cerebral activation. In humans, CBF
is transiently increased in the thalamus, ante-
rior cingulate, insula, and sensorimotor cortex
during static handgrip (Rogers et al., 1990) and
in the thalamus and insula during dynamic
cycling exercise (Romer et al., 2006). A distinct
increase in both rCBF and regional metabolism
has become synonymous with cerebral activa-
tion as visualized by single-photon emission
tomography, positron emission tomography, and

functional magnetic resonance imaging. The
motor tasks involved in exercise have been asso-
ciated with an increase in rCBF and confirmed
in numerous follow-up studies (Gonzalez-Alonso
etal., 2004; Madsen etal., 1993; Ide and Secher,
2000; Sato etal.,2009).
Early studies showed that cerebral oxygenation
increases during activation by exercise (Astrand
etal., 1964). This is in contrast to the progressive
reduction in muscle oxygenation with increas-
ing workload. Also, brain function deteriorates
when its average oxygenation becomes reduced
more than 10% (Gonzalez-Alonso et al., 2004;
Madsen et al., 1999; Van Lieshout et al., 2001;
Van Lieshout etal., 2003), whereas skeletal mus-
cles maintain their activity despite O2 saturation
below 10% (Astrand etal., 1964). The capillaries
within skeletal muscle are positioned in direct
contact to the muscle cells. However, within the
brain the capillaries are protected by extension of
the astrocytes covering the entire capillary net-
work, thereby creating the BBB, which directly
builds an obstacle between capillaries and neu-
rons. During brain activation, O2 consumption
increases and the diffusion distance for O2 can
become critical. To satisfy the enhanced neuronal
metabolism, an increase in rCBF is required to
generate an elevated O2 gradient. Maximal exer-
cise is associated with a reduced mitochondrial
O2 tension as CBF is influenced by the reduction
in partial pressure of carbon dioxide in the blood
in addition to a decline in arterial O2 content
(Nielsen et al., 1998; Nielsen et al., 2002). The
reduced cerebral oxygenation in turn affects the
recruitment of motor units, and supplemental O2
enhances cerebral oxygenation and work capac-
ity without effects on muscle oxygenation (Secher
etal.,2008).
The exercise-related changes in brain activ-
ity are closely connected to changes in CBF and
oxygen consumption and may occur primary or
even secondary to the metabolic changes during
prolonged exercise (Dalsgaard and Secher, 2007;
Davis and Bailey,1997).
Physical activity leads to an increased meta-
bolic rate in the active tissues, foremost in the
active limb locomotor muscles but obviously
also in the heart and the respiratory muscles and
in activated brain structures associated with a
given physical activity. Exercise-related brain
activation is associated with an increase in brain
energy usage, since the brain is one of the most
energy-demanding organs in the body. Although
constituting only 2% of body mass, brain energy
utilization account for ~20% of those of the whole
Exercise 301
organism, as defined by changes in brain oxygen
and glucose metabolism and the associated pro-
duction on ATP, the energy currency of brain cells
(Magistretti, Pellerin, Rothman, and Shulman,
1999). This association is based on the direct rela-
tionship between neuronal activity and energy
consumption as suggested as early as 1890 by Roy
and Sherrington. Energy metabolism is highly
organized within cells and tightly coupled to neu-
ronal activity, where postsynaptic potentials and
associated ATP-consuming ion pumping domi-
nate the energy requirements for neuronal sign-
aling (Ames, 2000; Dhar and Wong-Riley, 2009;
Magistretti 1999; Alle et al., 2009; Magistretti
et al., 1999; Buzski et al., 2007). Eighty-seven
percent of brain energy consumption is propor-
tional to neuronal firing rates, most of which is
consumed in the restoration of membrane poten-
tial after postsynaptic potentials, whereas only
13% of the energy is used to maintain the rest-
ing potential (Attwell and Gibb, 2005, 179; Alle
etal., 2009, 165; Magistretti, 2009). This postsyn-
aptic potentialslinked increase in brain energy
consumption is a function of elevated activity of
the sodium-potassium pump (Na+K+-ATPase),
which is triggered by the influx of sodium ions
and efflux of potassium ions accompanying post-
synaptic potentials or action potential depolari-
zations. Therefore, increases in neuronal activity
are tightly correlated with elevated concentra-
tions of intracellular Na+ and extracellular K+.
These changes lead to increased Na+K+-ATPase
activity to restore ionic gradients across the mem-
brane to their resting levels. Supporting evidence
was provided by a recent study that demonstrated
a coupling between transcriptional regulation of
Na+K+-ATPase and that of the glutamate trans-
porters involved in glutamate reuptake as an
outcome of increased neuronal activity (Rose
et al., 2009). Therefore, the increase in excita-
tory neurotransmission and associated neuronal
activity are closely linked with increases in brain
energy usage. At high exercise intensity and
hence intense regional neuronal activity, energy
demand may exceed energy supply and an imbal-
ance may occur in brain regions activated during
exhaustive exercise associated with changes in
brain energetics (Dworak etal.,2007).
Glucose and Lactate Metabolism
Glucose represents the main fuel for the human
brain and can support its energy demands.
Glucose can produce metabolic intermediates,
such as lactate and pyruvate, which do not neces-
sarily enter the tricarboxylic acid cycle but rather
302 Part III:Homeostatic Manipulators
can be released via circulation. Glucose can also
be incorporated into lipids, proteins, and glyco-
gen or serve as the substrate for neurotransmit-
ters such as -aminobutyric acid, glutamate, and
acetylcholine (Hoyer, 1990). Moreover, glucose
is not the only source of ATP at a given time in
the brain: other molecules can also contribute
to ATP production, although in smaller quanti-
ties. For example, glycogen, located primarily
in astrocytes, is an important reserve of glucose
equivalents and contributes ~1% of the cerebral
metabolic rate of glucose in humans and rats
(Gruetter, 2003). During exercise, the brains
energy demands increase significantly. Transient
increases in local cerebral glucose use in the
somatosensory, auditory, and visual cortices; the
dorsal raphe; and specific areas of the hypothala-
mus, amygdala, and hippocampus have been
reported in response to acute strenuous treadmill
running in rats (Vissing etal.,1996).
Besides glucose, other substrates can also be
used to provide sufficient energy to the cells (Ide
et al. 2000; Schurr, 2006). For example, during
long-term fasting the concentrations of ketone
bodies are increased, and they replace glucose as
the primary energy source for the brain (Owen
etal., 1967). Energy metabolism is highly organ-
ized to provide sufficient energy, mostly in the
form of ATP, during periods of high energy
demand as well. From rest to high-intensity
exercise, skeletal muscles increase their energy
requirements more than 50-fold, which is asso-
ciated with ATP consumption and significant
increases in lactate production. Under resting
conditions, the skeletal muscle and the brain
release only small quantities of lactate. However,
both tissues continuously take up lactate,
although in lesser quantities than simultaneously
released, resulting in the small net lactate release
(van Hall etal.,2009).
The monocarboxylic acids such as lactate
and pyruvate are taken up by the brain when the
blood level increases. Such transport across the
BBB is facilitated by the monocarboxylate trans-
porter. This transporter is expressed widely
throughout the rodent brain, with the high-
est concentrations in areas of high neuronal
activity and metabolism such as the cortex, the
hippocampus and the cerebellum (Hertz and
Dienel, 2005; Pierre and Pellerin, 2005; Pellerin
etal., 1998; Pierre and Pellerin, 2005). Pyruvate,
another monocarboxylic acid and the end prod-
uct of glycolysis, is converted to acetylcoenzyme
A, a substrate for the tricarboxylic acid cycle and
subsequent respiration, albeit only indirectly
through nicotinamide adenine dinucleotide
(NADH) and flavin adenine dinucleotide for-
mation. Alternatively, pyruvate is reduced to
lactate while reoxidizing cytosolic NADH to
NAD+ as a prerequisite for substrate flow in
anaerobic glycolysis. The near-equilibrium state
of the responsible enzyme, lactate dehydroge-
nase, implies that the ratio of lactate to pyruvate
in cytoplasm reflects that of NAD+ to NADH.
Thus a high ratio of lactate/pyruvate in cerebral
arterial-venous differences or cerebrospinal fluid
is taken to indicate insufficient oxygen supply.
Nevertheless, pyruvate seems to be less effective
than lactate as substrate, at least during reperfu-
sion/ischemia (Phillis etal.,2001).
During light exercise, a small increase in
brain lactate release, relative to lactate uptake,
is observed that might be related to increased
neural activity. With increasing exercise inten-
sity, more lactate is produced in skeletal muscles
and released to the blood. The increase in arte-
rial lactate concentration during intense exer-
cise makes lactate available to the brain, and the
brain switches from a net producer of lactate to
an importer while glucose uptake is reduced (Ide
etal., 2000, Kemppainen etal., 2005). In fact, the
arterial lactate concentration is highly correlated
with the unidirectional brain lactate uptake. The
lactate taken up from skeletal muscle can act as
an intercellular energy shuttle within the brain
during high-intensity exercise (Dalsgaard et al.,
2006). Under situations of a high energy demand,
neurons prefer lactate to glucose as their primary
energy source, which raises the production of
ATP (Schurr, 2006; Magistretti etal., 1999), and,
under exercise conditions, lactate oxidation is
responsible for approximately 8% of the brain
energy requirements (van Hall etal., 2009). The
lactate taken up by the brain is almost completely
oxidized and does not accumulate in the brain
under the various conditions, which is remark-
ably different from skeletal muscle in which near
complete oxidation was observed only during
exercise (van Hall et al., 2009). The changes in
blood biochemistry during exercise can directly
affect brain activity and cortical functioning,
since exercise at high intensities, with blood lac-
tate accumulation, decreases significantly the
EEG beta-2, beta-1, and alpha-1 frequency bands
(Bailey etal.,2008).
The metabolic response to brain activation in
exercise can be defined as the cerebral metabolic
ratio (MR; uptake O2/glucose + 1/2 lactate). At
rest, brain energy is provided by a balanced oxi-
dation of glucose and the MR is close to 6, but

further activation provokes an excessive uptake
of glucose relative to O2. While the MR remains
stable during light exercise, it is decreased dur-
ing prolonged exhaustive exercise where blood
lactate remains low, but when vigorous exercise
raises blood lactate, the brain takes up lactate
in an amount similar to that of glucose, result-
ing in a reduced MR by 30% to 40% (Ide and
Secher, 2000; Sato et al., 2009). Interestingly,
studies showed that the MR depends also on the
central command, since a decrease in the MR
is also observed under partial neuromuscular
blockade, when the will to exercise is intense
(Sato et al., 2009). Intense regional neuronal
activity in cerebral regions during high exer-
cise intensities cause energy demand to exceed
production, in turn draining energy reserves,
which is hypothesized to result in local energy
depletion such as brain glycogen and might play
a role in central fatigue (Dalsgaard etal., 2002;
Dworak etal., 2007; see also Central Fatigue as glycolysis and oxidative phosphorylation,
section).
Nucleotide and Nucleoside
Metabolism
At high intensities exhaustion times are short,
whereas at low intensities exercise can be main-
tained longer. This relationship is determined by
the maximal use of energy sources available to
the organism:alactic anaerobic (Phosphocreatine
and ATP), lactic anaerobic (anaerobic glyco-
lysis), and aerobic (sugar and fat oxidation). For
short-duration exercises, alactic anaerobic and
lactic anaerobic metabolism provide sufficient
energy to the organism, whereas for lower-power
outputs aerobic metabolism becomes the main
energy source and maximum glucose and fat oxi-
dation rates set the limits (Monod and Scherrer,
1965; Wilkie, 1981). One vital function of the
metabolic network is to maintain stable concen-
trations of cellular ATP by adjusting the reac-
tion rates to ensure a continuous energy supply
for sustaining electrophysiological activity and
maintaining normal function in the brain. ATP
is the currency of cellular energy and besides its
role as major energy molecule is also an impor-
tant molecule in cell-to-cell signaling (Burnstock
et al., 2011). Thus anabolic and catabolic pro-
cesses must be well balanced to maintain con-
stant levels ofATP.
ATP acts as an activity-dependent metabo-
lite and signaling molecule between neurons and
glia and modifies membrane potentials through
ATP-modulated potassium channels (Fields
and Stevens, 2000; Peters et al., 2004; Pascual
Exercise 303
et al., 2005), thus strongly influencing neuronal
information processing, synaptic strength, gene
expression, and protein, fatty acid, and glycogen
synthesis (Fields and Stevens, 2000; Peters etal.,
2004; Pascual etal.,2005).
Intracellular ATP levels drop from 4 to 8 mM
to 2 to 3 mM during muscle fatigue (Harris,
1996). The energy charge of the adenylate pool
([ATP]+1/2[ADP]/ ([ATP]+[ADP]+[AMP]) has
been proposed as a control parameter in the reg-
ulatory interactions by which biological homeo-
stasis is maintained (Atkinson and Walton,
1967). Decrease in ATP concentration results
in increases in ADP and more so in adenosine
monophosphate (AMP) and adenosine ADO,
but without drastic changes in the overall energy
charge. In normal brain tissue, the energy charge
is closer to 0.85 (Derr and Zieve, 1972) and is
regulated by direct participation of adenine
nucleotides in energy-converting processes, such
and many energy-expending biosynthetic path-
ways, such as those of amino acids, nucleic acids,
protein, fatty acids, and cholesterol synthesis
(Thompson and Atkinson,1971).
During intense neuronal activation such
as exhausting physical activity, when the cen-
tral command and sensory input to the brain is
intense, metabolic demand exceeds metabolic
availability and results in an increased break-
down of ATP associated with an increase in the
nucleotide ADO (Basheer etal.,2004).
Previous studies showed significantly
higher ADO concentrations in the rat neostria-
tum and in the hippocampus during the active
period of rats compared to the sleeping period,
a finding that was interpreted as an associa-
tion of ADO concentrations with motor activ-
ity (Huston etal., 1996). The increase in brain
ADO concentrations seems to depend on exer-
cise intensity. While moderate exercise did
not affect brain ADO levels, intense exercise
increased the ratio of metabolite demand to
metabolite availability with an accompanied
production of ADO from ATP breakdown
(Dworak etal.,2007).
These observations were supported by show-
ing that minimal exercise did not affect ADO
concentrations in the basal forebrain if meas-
ured via microdialysis (McKenna et al., 2007).
The observed findings of increased ADO con-
centrations during exhausting exercise might
reflect a state of bioenergetic stress, possibly as a
result of an increased breakdown of high-energy
phosphates.
304 Part III:Homeostatic Manipulators

FIGURE17.2: Homeostatic neurometabolic regulation during exercise. The increased excitatory neu-
rotransmission during intense exercise results in a higher activity of Na+-K+-ATPase to restore the concentration
gradient. Na+-K+-ATPase activity is a high energy consuming process, resulting in the breakdown of adenosine
triphosphate (ATP) and the generation of adenosine. Adenosine acts via A1 receptors to inhibit neurotransmit-
ter release and provoke a hyperpolarization, thereby inhibiting neuronal activity. 5-HT=serotonin, LC=locus
coreuleus, NA=noradrenalin.

The changes in brain ATP and ADO concentra-
tions during exercise can directly affect behavior.
ADO acts as a neuromodulator, and its effects are
mediated via specific G-protein coupled recep-
tors, namely A1, A2a, A2b, and A3 (Haas etal.,
2000). The net effect is a presynaptic reduction
in transmitter release in wakefulness-promoting
networks, including the cholinergic and mono-
aminergic systems, as well as a postsynaptical
hyperpolarization and accompanied EEG desyn-
chronization (Rainnie et al., 1994; Latini et al.,
2001). The basal forebrain and the mesopontine
cholinergic neurons, whose discharge activity
plays an integral role in EEG arousal and mainte-
nance of wakefulness, are especially postsynapti-
cally inhibited by endogenous ADO (Latini and
Pedata, 2001; Arrigoni etal., 2006; Figure17.2).
Neurotransmission
During Exercise
Associated with the changes in neuronal signaling
throughout the brain regions involved in motor
control, physical activity directly produces alter-
ations in endogenous peptides, increased amino
acid transport through the BBB, and changes
in the release of various neurotransmitters (Ide
etal., 1999, Herholz etal., 1987; Meeusen etal.,
1995). The release of most neurotransmitters is
influenced by exercise, since it was shown that
physical activity directly affects the central dopa-
minergic, glutamatergic, noradrenergic, and ser-
otonergic systems (Meeusen etal.,1995).
Dopamine
In the brain, dopaminergic systems are primar-
ily involved in reward-motivated behavior, motor
control, and control of the release of several impor-
tant hormones. Exercise increases dopamine lev-
els in the brain through a calcium-dependent
process that regulates numerous brain functions
(Sutoo etal., 2003). Dopamine levels were regu-
lated by exercise in epileptic and spontaneously
hypertensive rats, demonstrating the possibility
that exercise can be used to improve symptoms
of Parkinsons disease or dementia (which are
associated with low dopamine levels; Sutoo etal.,
2003). Regular aerobic exercise has a protective
effect on D2 dopamine receptor levels and inhib-
its modifications in brain dopamine metabolism
due to the aging process (Uysal etal., 2005). The
release of dopamine by neurons is necessary for
sustaining neural activity and working memory
(Surmeier etal., 2007). Dopamine is also a pre-
cursor to norepinephrine and epinephrine, and
levels of dopamine are responsive to levels in
serotonin.

Synthesized from dopamine, norepinephrine
acts in the central and sympathetic nervous sys-
tems by binding to adrenergic receptors. Brain
norepinephrine levels are affected after acute
bouts of exercise (running or swimming). Most
studies found a decrease or no affect in brain nor-
epinephrine levels. It seems that acute exercise
results in a depletion of brain norepinephrine
levels probably due to an acceleration in nor-
epinephrine turnover by activating hydroxylase
activity (Chaouloff, 1989). After chronic exercise
training, brain norepinephrine levels increased
significantly. The effect of norepinephrine neu-
rotransmission also shows a region-specific pat-
tern. While norepinephrine levels decreased due
to acute exercise in the brainstem, hippocampus,
midbrain, and hypothalamus, increased norepi-
nephrine levels were observed in the striatum,
cortex, and preoptic area (Meeusen et al., 1995;
Dishman et al., 2000). Therefore, brain norepi-
nephrine levels can be directly affected by exer-
cise, but the alterations differ between specific
brain regions and depend on the type of exercise
(aerobic vs. anaerobic).
Glutamate
Glutamate is the most common neurotransmit-
ter in the brain, as well as the major excitatory
neurotransmitter involved in many aspects of
brain function, including learning and memory
(Chaddock et al., 2011). Glutamatergic activity
changes as a function of behavioral states, since
cortical glutamate levels increase progressively
during periods of increased neuronal activity
such as waking and rapid eye movement (REM)
sleep and decrease progressively in non-rapid
eye movement (NREM) sleep, when neuronal
activity is decreased significantly (Dash et al.,
2009). Animal studies have shown that tread-
mill running significantly increases glutamate
levels during and for a short while after exercise
in rats (Leung etal., 2006)and may influence the
levels of glutamate receptors such as NR2B and
mGluR5. (Zhang etal., 2010). Moreover, most of
the brain energy consumption is directly associ-
ated with glutamatergic (Attwell and Laughlin,
2001). These observations are in accordance
with the increase in neuronal activity within
different brain regions during and shortly after
exercise.
5-Hydroxytryptamine (Serotonin)
Serotonin is closely regulated by physical activity.
It has been found that at least 30 min of daily aer-
obic activity such as running, biking, or walking
Exercise 305
is needed to elevate serotonin (5-HT) synthesis
in the brain, whereas anaerobic activities such as
weight lifting and stretching exercises have not
been associated with increased brain serotonin
levels. Hence, it seems that brain 5-HT levels
depend on exercise quality (aerobic vs. anaerobic,
local vs. whole-body) and quantity (duration).
Chronic activity wheel running or treadmill
running results in small increases in basal levels
of 5-HT in the dorsal raphe nucleus (Dishman
et al., 1997) and increased turnover of 5-HT in
the brain cortex (Yoo etal; 2000). Chronic wheel
running also attenuates stress-induced c-Fos
induction in serotonergic neurons of the dor-
sal raphe nucleus and increases levels of 5-HT
1A inhibitory autoreceptor mRNA (Greenwood
etal., 2003; Greenwood etal., 2005). The antide-
pressant and anxiolytic effects of exercise have
been clearly demonstrated in different studies,
and the positive effects might be partially due to
the exercise-induced change in 5-HT neurotrans-
mission (Salmon etal., 2001; Young etal.,2007).
The interactions between brain neurotrans-
mitters, metabolites, and their specific receptors
could also play a central role in the onset of cen-
tral fatigue during prolonged exercise.
Central Fatigue
Physical fatigue can originate within the muscle,
which is known as peripheral fatigue, or within
the CNS, which is known as central fatigue.
Several biological mechanisms have been pro-
posed to explain CNS fatigue (Davis etal., 1997;
Meeusen etal., 2006), mainly focused on altera-
tions in several neurotransmitters and metabo-
lites including 5-HT, dopamine, acetylcholine,
andADO.
The original central fatigue hypothesis indi-
cates that an exercise-induced increase in extra-
cellular 5-HT concentrations in several brain
regions contribute to the development of fatigue
during prolonged exercise. 5-HT has been linked
to central fatigue because of its established
effects on sleep, drowsiness, and loss of motiva-
tion (Meeusen et al., 2006). Several nutritional
and pharmacological studies have attempted to
manipulate central serotonergic activity during
exercise, but this work has yet to provide robust
evidence for a significant role of 5-HT in the
central fatigue process (Davis and Bailey 1997;
Meeusen etal., 2006). Under normal physiologi-
cal conditions, 5-HT is unable to cross the BBB,
therefore cerebral neurons are required to syn-
thesize it by themselves and overall brain 5-HT
levels arelow.
306 Part III:Homeostatic Manipulators
The precursor of 5-HT is tryptophan, and
the synthesis as well as the transport through
the BBB of 5-HT is driven by the blood supply
of free tryptophan. Tryptophan is transported
via the L-system, the amino acid transporter sys-
tem, which also transports the other large neutral
amino acids, including the three branched-chain
amino acids (BCAA) and that compete with
tryptophan for entry into the brain. Therefore,
the amount of tryptophan transported into the
brain depends not only on the concentration of
free tryptophan in the bloodstream but also on
the concentrations of the BCAA, since these
make up approximately 75% of the total large
neutral aminoacids.
The concentration of free tryptophan in the
plasma is mainly influenced by the change in
plasma free fatty acids, as these are also trans-
ported bound to albumin in the plasma. An
increased release of fatty acids from the adipose
tissue during exercise results in an increase in
their concentration in plasma and displaces
some of the tryptophan from albumin, thereby
elevating the plasma level of free tryptophan
(Bloomstrand,2001).
Also, the plasma concentrations of amino
acids vary during exercise and are dependent
on the type of exercise and the duration and
intensity and of the exercise period (Henriksson,
1991). During short-term exercise, there is an
increase in most amino acids, whereas prolonged
exercise causes a decrease in the concentration of
most amino acids, such as BCAA (Bloomstrand
et al., 2000). The change in the plasma ratio of
free tryptophan/BCAA during prolonged exer-
cise depends in part on exercise intensity; an
increased ratio was found during the last hours
of exercise of a 5-hour ergometer exercise at 75%
of the maximal oxygen uptake, whereas no sig-
nificant change was found during exercise at 50%
of the maximal oxygen uptake (Strder et al.,
1997). Thus a marked increase in the plasma ratio
of free tryptophan/BCAA is found after exercise
with longer duration and at higher intensities,
which favors the transport of tryptophan into the
brain and also the synthesis and release of 5-HT
from some neurons that could be responsible for
fatigue during and after sustained heavy exercise
(Meeusen etal., 2006; Bloomstrand etal., 2000;
Figure17.3).
Another potential neurotransmitter in cen-
tral fatigue is dopamine. Dopamine is an excita-
tory neurotransmitter that regulates arousal,
motivation, endurance performance, and mus-
cular coordination. Animal studies showed
that during prolonged physical activity, brain
dopamine activity increased (Bailey etal., 1993;
Gerald etal., 1978), which may influence exercise
performance (Freed etal., 1985). Brain serotonin
and dopamine content progressively increased
during exercise, but at the point of exhaustion
a marked fall in tissue dopamine content was
apparent. Animal studies show that at the point
of fatigue dopamine extracellular concentrations
are low, possibly due to the interaction with brain
serotonin (Davis et al., 1997) or a depletion of
central catecholamines (Bailey etal.,1992).
Acetylcholine modulates arousal and temper-
ature regulation and might play a role in fatigue.
During exercise, the levels of acetylcholine
decrease due to a drop in plasma choline levels.
However, the results in studies about the effect of
acetylcholine on central fatigue are conflicting.
Conlay et al. (1992) found that plasma choline
levels decrease by 40% after a marathon, whereas
some studies have found that supplementation
with choline citrate improved performance in
long-distance runners and other studies did not
find any improvement in time to exhaustion after
choline supplementation (Spector et al., 1995).
The same study also found that plasma choline
levels had not changed in either the placebo or
the choline-supplemented groups. Therefore
more research is needed to investigate acetylcho-
lines effects on fatigue.
Neuromodulators such as cytokines, ammo-
nia, and ADO can also influence central fatigue
during exercise. Increases in several cytokines
have been associated with reduced exercise toler-
ance associated with acute viral or bacterial infec-
tion. Accumulation of ammonia in the blood and
brain during exercise could also negatively affect
the CNS function and fatigue (Meeusen et al.,
1995; Davis etal.,1997).
Recent studies suggest that ADO might play
a central role in central fatigue (Dworak et al.,
2007). ADO is derived from the metabolism of
ATP and is a well-known homeostatic sleep fac-
tor that links energy metabolism with neuronal
activity (Brown etal., 2012). Systemic and central
administrations of ADO or ADO A1 receptor ago-
nists induced sleepiness and impaired vigilance
(Brown etal., 2012). Stimulation of neuronal ADO
receptors mediates presynaptic inhibition in the
transmitter release, including cholinergic, adren-
ergic, and serotonergic neurons (Rainnie et al.,
1994; Arrigoni etal., 2006)as well as postsynaptic
hyperpolarization that regulate alertness, sleep,
and wakefulness (Basheer et al., 2004; Latini
and Pedata, 2001). Intense exercise increases
 Exercise 307

Blood BBB Brain

intracellular extracellular

Lactate Lactate ATP ATP

Protein
AD AD

FFA LNNA Dopamin

Albumin Acetylcholine
Norephedrine
Tryptophan BCAA
Albumin Tryptophan 5-HT

FATIGUE

FIGURE 17.3: Neuronal mechanisms of central fatigue. Prolonged and intense exercise causes an
increased release of fatty acids from the adipose tissue and elevated concentrations in plasma free fatty acids
(FFA). FFA displace some of the tryptophan from albumin, thereby elevating the plasma level of free tryptophan.
Also, the concentrations of most amino acids, such as BCAA, drop during prolonged exercise thereby increas-
ing the plasma ratio of free tryptophan/BCAA. Free tryptophan is transported via large neutral amino acids
(LNNA) transporter through the blood-brain barrier (BBB) into the brain, thereby increasing the level of brain
tryptophan and 5-HT. Exercise also increases the blood concentrations of lactate. Peripheral lactate can enter the
brain via specific transporters and provide additional energy under intense physiological conditions. Increased
neuronal activity during intense and prolonged exercise is associated with changes in dopamingergic, cholinergic,
and adrenalinergic neurotransmission and an increased cotransmission of adenosine triphosphate (ATP) to the
extracellular space. ATP is metabolized to adenosine ADO. ADO acts via neuronal ADO receptors by mediating
presynaptic inhibition in the transmitter release, including cholinergic, adrenergic, and serotonergic neurons, and
results in a postsynaptic hyperpolarization that can directly affect behavior, performance, and fatigue.

brain ADO concentrations due to an increased
energy demand versus limited energy availabil-
ity (Dworak etal., 2007; Brown etal., 2012). The
increase in brain ADO concentrations and release
to the extracellular space during exercise is a
potential mediator of fatigue due to the inhibitory
action of ADO on excitatory neurotransmission
(Rainnie etal., 1994; Arrigoni etal., 2006). This
relationship is also supported by studies show-
ing that exercise improves sleep quality (Driver
etal., 2000; OConnor, Youngstedt, 1995). Intense
(anaerobic) bicycle exercise increases restorative
NREM sleep (also known as stage 4 sleep), which
correlates closely with brain ADO levels (Dworak
etal., 2008). During NREM sleep, in turn, resto-
ration of energy stores occurs particularly of ATP
and glycogen in muscles and in the brain, which
is needed for anabolism, such as protein and gly-
cogen synthesis to support recovery and regenera-
tion processes (Kong et al., 2002; Dworak et al.,
2010; Brown etal.,2012).
Other evidence for an ADO role in central
fatigue is the exercise performance supporting
effects of caffeine. Caffeine is a potent ADO
antagonist and CNS stimulant that easily crosses
the BBB due to its lipophilic properties (Graham
et al., 2001). It has been shown to counteract
most of the inhibitory effects of ADO on neuro-
excitability (Fredholm etal., 1999; Gervitz etal.,
2001), neurotransmitter release (Okada et al.,
1997), and arousal (Brown etal., 2012). In addi-
tion, blocking CNS ADO receptors may help to
explain the fatigue-delaying properties of caf-
feine. Ingestion of caffeine has been shown to
delay fatigue during prolonged intense exercise
in both human and animal models (Cole et al.,
1996; Costill et al., 1978; Jackman et al., 1996;
Kovacs et al., 1998). Caffeine does not improve
maximal oxygen capacity directly but could per-
mit the athlete to train at a greater power output
and/or to train longer. It has also been shown to
increase speed and/or power output in simulated
race conditions (Graham etal.,2001).
Thus the capabilities of ADO in modulating
neuronal activity and alertness by linking energy
metabolism with neuronal activity provide a
308 Part III:Homeostatic Manipulators
central role for the nucleotide ADO in the reg-
ulation of central fatigue during intense and/or
long-lasting exercise.
B E H AV I O R A L E F F E C T S
OFEXERCISE
Regular participation in physical exercise has
positive outcomes on the cardiovascular system,
the pulmonary system, and whole-body metabo-
lism and is an important factor in the prevention
of cardiovascular diseases and metabolic diseases
such as type 2 diabetes and obesity. Besides the
positive effects on the cardiometabolic systems,
physical activity significantly affects brain struc-
tures and brain functioning, and exercise-related
changes in brain metabolism, neurotransmis-
sion, and neuronal firing have direct implications
on behavioral factors.
Emerging evidence suggests that physi-
cal activity may confer health-protective ben-
efits for several neurological diseases, including
Parkinsons disease, Alzheimers dementia, and
ischemic stroke, as well as injuries from falls
attributable to neuromuscular declines associ-
ated with physical inactivity among the elderly
(Karp etal., 2006; Wilson etal., 2002; Colcombe
etal.,2003).
Physical training has been observed to selec-
tively enhance brain health and cognition on
the molecular level, including angiogenesis,
synaptogenesis, and neurogenesis, as well as to
upregulate a number of neurotrophic factors
such as brain-derived neurotrophic factor and
insulin-like growth factor 1, which are necessary
for neuronal differentiation and synaptic plas-
ticity (Uysal et al., 2005; Vaynman et al., 2006;
Cotman et al., 2007; Cotman and Berchtold,
2002; Cotman and Engesser-Cesar, 2002). Also,
in animals, voluntary exercise leads to increased
axon regeneration and neuronal growth com-
pared to being sedentary (Molteni et al., 2004).
Exercise increases the expression of genes that
are required for rapid neural growth in the
brain and the growth directly correlated with
the amount of exercise the animal participated
in, specifically the total distance the animal had
run (Molteni et al., 2004). The exercise-induced
alterations in neurogenesis and brain plasticity
occurred in different brain regions such as the
hippocampus, hypothalamus, and dentate gyrus
(Farmer etal., 2004). Neurogenesis in the hypo-
thalamus is linked to improvements in learn-
ing as well as in memory (Cotman et al., 2007;
Ruscheweyh etal., 2011). These and other studies
show clearly that voluntary exercise can impact
neurotrophin levels and neurogenesis, induc-
ing activity-dependent plasticity (reviewed in
Cotman etal.,2007).
The homeostatic control of brain energy
metabolism and the associated changes in neu-
ronal activity can influence sleep-wake behavior
directly. Epidemiological studies have consist-
ently supported the view that acute and chronic
exercise promotes sleep (Vuori et al., 1988;
Youngstedt etal., 1997). Exercise at higher inten-
sities in particular reduces sleep onset latency
and increases total sleep time and the amount of
homeostatic regulated NREM slow-wave sleep
with accompanied reductions in REM sleep
(Driver etal., 2000; Shapiro etal., 1981; Dworak
etal.,2008).
The exercise-induced alterations in sleep
quality may be related to changes in brain
energy metabolism. High-intensity exercise
results in an increased brain energy demand,
increased ATP consumption, and coincident
increases in brain ADO concentrations (Dworak
etal., 2007), reflecting an energy deficit, which
is also observed during prolonged wakeful-
ness (Basheer et al., 2004). Due to its inhibi-
tory actions, ADO serves as an sleep-promoting
factor in the CNS and might be a key player
in mediating the positive effects of exercise on
sleep quality (Dworak etal.,2007).
CONCLUSION AND OUTLOOK
Besides the positive effects of exercise on the
peripheral system, there is accumulating evi-
dence that physical activity influences brain
functioning. The exercise-induced effects on
the brain are related to alterations in neuronal
signaling, neurotransmission, endogenous pep-
tides, amino acid transport through the BBB,
and metabolism. The exercise-induced changes
in these systems depend on the quality and
quantity of exercise and underlie homeostatic
factors within the brain. Therefore exercise
has the potential to affect brain homeostasis at
the cellular, molecular, and behavioral levels,
implying that physical activity participation is
beneficial to brain health, cognition, and mood.
Such evidence highlights the importance of
promoting physical activity across the lifespan
to reverse recent obesity and cardiovascular
disease trends, as well as to prevent or reverse
cognitive and neural decline. Accordingly,
physical activity can serve to promote health
and performance in individuals while also less-
ening the health and economic burden placed
on our society.

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18
Sleep
K R IST INA SIMEONE, CHA Z JOHNSON, K AELI SA MSON,
H A R R I S O N R O U N D T R E E , T I M S I M E O N E , A N D L E I L A TA R O K H
INTRODUCTION
Proper sleep hygiene can exert beneficial effects
on several physiological systems. Here we focus
on how sleep influences the central processing
of cognition, stress, behavior, body weight, brain
energy (adenosine triphosphate [ATP]) and waste
removal, and immune responses. Research sug-
gests that the neurobiology associated with suf-
ficient sleep interacts with these processes in a
homeostatic manner to promote optimal day-
time functioning.
Sufficient sleep can be achieved with 7 to 9
hours for adults, 8.5 to 9.5 hours for teenagers,
10 to 11 hours for school-aged children, and
12 to 14 hours for preschool children (Bonnet and
Arand, 2003; Institute of Medicine Committee
on Sleep Medicine and Research, 2006; National
Sleep Foundation, 2013). Decades of research
have taught us that when the brain is deprived
of sleep, neurobiological instabilities can result.
Depending on the length of deprivation, the
behavioral readouts of these instabilities can
be quantified during the deprivation itself and
subsequent days. These behavioral instabili-
ties include impairments of cognitive process-
ing, reaction time, and vigilance; reduction of
stress thresholds; promotion of negative affec-
tive behaviors (including the inability to focus or
concentrate, irritability, anxiety, aggression, and
depressive-like behaviors); changes in appetite;
and suboptimal immune responses.
Unfortunately, insufficient sleep is a grow-
ing public health concern for multiple reasons
(Institute of Medicine Committee on Sleep
Medicine and Research, 2006). First, the increase
in societal pace, accessibility of artificial light,
and repeated prolonged periods of wakefulness
can result in chronic insufficient sleep for adults
and children. Second, the neuropeptides and
neurotransmitters involved in sleep-wake cycles
can be dysregulated in neurological disorders
and affective disorders. Indeed, insufficient sleep
and sleep disorders can manifest as comorbid
conditions of several neurological disorders and
affective disorders, including anxiety disorders,
depression, epilepsy, attention deficit hyperactiv-
ity disorder (ADHD), autism, Alzheimers dis-
ease, and Parkinsons disease. Third, considering
the effects of insufficient sleep, sleep disorders
can simultaneously be comorbid with cognitive
impairments and/or affective disorders. Finally,
sleep neurobiology can be targets of pharmaco-
logical treatments. Tiredness, drowsiness or trou-
ble sleeping are side effects of many drugs used to
treat anxiety and depression (including aripipra-
zole, serotonin reuptake inhibitors [SSRIs], and
serotonin norepinephrine reuptake inhibitors
[SNRIs]), epilepsy (ethosuximide, gabapentin,
phenytoin, levetiracetam, zonisamide), autism
(aripiprazole, risperidal), Alzheimers disease
(donepezil, galantamine, rivastigmine), and
Parkinsons disease (carbidopa with levodopa).
In this chapter we first describe the neuro-
biology of sleep architecture. We discuss the
intersections of neurobiology, sleep, and phar-
macology in high-incidence disorders, including
anxiety disorders (1:6), depression (1:10), epi-
lepsy (1:26), ADHD (1:9 children), autism (1:68
children), Alzheimers disease (1:8 over the age of
65 and 1:2 over the age of 85), and Parkinsons
disease (1:313) (Alzheimers Association, 2012;
Institute of Medicine Committee on the Public
Health Dimensions of the Epilepsies, Board on
Health Sciences Policy, 2012; National Institute of
Mental Health; Parkinsons Disease Foundation,
2014). Next we discuss how intentionally struc-
turing sleep as a preventative measure or as an
adjuvant restorative therapy may improve cogni-
tion and/or behavior in normal individuals and
lessen the degree of cognitive/behavioral comor-
bid afflictions in individuals with neurological or
affective disorders. We conclude by highlighting
 Sleep 315

how sleep may provide restorative influence deprivation); thus results are discussed in the
on brain regulators of body weight, brain ATP context of the protocol employed.
levels and damage/waste removal systems, and
neuro-immune responses. SLEEP ARCHITECTURE
This chapter is structured to highlight a wide AND NEUROBIOLOGY
array of studies and is not intended to be com- In this section, we discuss the electroencepha-
prehensive. It is important to note that care was lography (EEG), neurobiology and pharmacol-
taken to determine the order in which topics are ogy of non-rapid eye movement sleep (NREMS),
presented based on their interrelationships and rapid eye movement sleep (REMS), wake, and
sections are not concluded with causal specu- wake-sleep transition.
lation. In addition, many sleep studies employ Sleep architecture describes the timing and
sleep deprivation protocols, which vary in terms the predominance of different brain rhythms
of duration (full or partial deprivation), timing during NREMS (stages 1, 2, and 3/4) and REMS
(early or late), and recurrence (acute or chronic (Figure 18.1, left panel). Each NREMSREMS

Adults Sleep Architecture Neurobiology

REM

NREM (80% of sleep) BF VLPO MLPO LH TMN

ACH/A/GABA GABA GABA OX/ACH/A/GABA HA
- S1: Theta, less alpha NERM
- S2: Spindles, k- ViPAG/ SN/VTA/VPAG R LC LDT PPT
complexes SLD
SLEEP (7 hr)

LPT DA 5-HT NE ACH(R)

- S3: Delta, SWA
Motor
neurons
Thalamus
REM (20% of sleep)
- Alpha BF VLPO MLPO LH TMN
- Theta ACH/A/GABA GABA GABA OX/MCH/A/GABA HA
REM
24 hr period

LDT PPT
S1
S2
S3

ViPAG/ SN/VTA/VPAG R LC
SLD ACh(R)
LPT DA 5-HT NE
VM Spinal Motor
GABA/glycine interneurons neurons

WAKE Thalamus
- Beta
BF VLPO MLPO LH TMN
- Alpha ACH/A/GABA GABA GABA OX/MCH/GABA HA
WAKE (17 hr)

- Theta Wake
- Gamma ViPAG/ SN/VTA/VPAG R LC LDT PPT
STD ACh (R)
LPT DA 5-HT NE

Motor
neurons
Frequencies of brain rhythms
- Very slow - Beta (914 Hz)
oscillation (0.11 Hz) - Sigma (1116 Hz)
- Delta SWA (1.24.5 Hz) - Beta (1630 Hz)
- Theta (58 Hz) - Gamma (40100 Hz)

FIGURE 18.1: Brain rhythms and the corresponding neurobiology during each state of vigi-
lance. Left: The schematic depicts a 24-hour period divided into time spent awake and asleep. During sleep,
the non-rapid eye movement sleep (NREM) stages (S) 13 (blue) and the rapid eye movement (REM; purple)
transitions and cycles are depicted. Each state is magnified, and the specific brain rhythms are identified. The
corresponding frequencies that comprise each rhythm are below (grey). Right:Aligned with each state of vigi-
lance is a simplified schematic of some of the known underlying neurobiology during NREM sleep (blue), REM
sleep (purple), and awake stages (green). The boldness of the font indicates the relative changes in different neu-
ronal populations:bold indicates strong signaling; normal font indicates mild/moderate signaling; lighter font in
NREM sleep indicates reduced activity when compared to awake; lighter font in REM indicates reduced activity
when compared to NREM sleep; and light grey indicates minimal or no signaling. We highlighted the activity of
the orexin (OX), adenosine (A), histamine (HA), serotonin (5-HT), dopamine (DA), norepinephrine (NE), ace-
tylcholine (ACh), and GABA systems. BF=basal forebrain, LC=locus coeruleus, LDT=lateral dorsal tegmental
nucleus, LH = lateral hypothalamus, MLPO = medial lateral preoptic area, PPT = pedunculopontine nucleus,
R=raphe nuclei, SLD=sublateral dorsal nucleus, SN=substantia nigra, TMN=tuberomammillary nucleus,
VLPO=ventral lateral preoptic area, VPAG=ventral periaqueductal grey, VTA=ventral tegmentalarea.
316 Part III:Homeostatic Manipulators
cycle transitions from NREMS stages 1, 2, 3/4,
2, 1 to REMS and is approximately 90 to 120
minutes in duration in adults. NREMSREMS
cycles occur several times during sleep, and often
waking in the morning occurs following REMS.
REMS durations range from approximately 10
minutes to 1 hour and increase with subsequent
episodes. Thus, overall there is a higher amount
of NREMS during early sleep, and a higher inci-
dence of REMS during later sleep. Analyses of
sleep architecture can be used as a tool to identify
problems with specific types or phases ofsleep.
The neurobiological landscape responsible
for promoting sleep and wake states employ the
same peptides and transmitters that (i)are dys-
regulated in neurological disorders and affective
disorders and (ii) are targets of pharmacothera-
pies. These include gamma-aminobutyric acid
(GABA), serotonin, norepinephrine, acetyl-
choline, adenosine, orexin, histamine, and
dopamine. Figure 18.1 (right panel) depicts the
relative changes in activity of some of these sys-
tems during NREMS and REMS and waking. In
this section we briefly describe the sleep archi-
tecture and how relative levels of these molecules
play a critical role in the homeostatic balance of
NREMS and REMS and wake states (see Figure
18.1 throughout this section).
NREMS
EEG and Neurobiology
During stage 1 sleep, there is a reduction in alpha
waves and an increase in theta power (Figure
18.1). During stage 2 sleep, which occupies 45%
to 55% of our total sleep time, our body tempera-
ture decreases, our heart rate slows, our muscle
tone relaxes, and sleep spindles (sigma 1116 Hz)
and K-complexes become apparent on the EEG.
During stage 3, which accounts for 15% to 25% of
our total sleep, we are in deep sleep and our brain
rhythms oscillate at high amplitude (75 mV)
and low frequency (i.e., delta frequency range
from 0.5 to 5 Hz also known as slow-wave activity
(SWA)). Delta power is associated with a reduc-
tion in heart rate and an increase in parasympa-
thetic activity. The amount of delta power and
stage 3/4 sleep declines across the course of the
night and is thus a marker of sleep homeostasis.
During NREMS, GABAergic neuro-
transmission is responsible for inhibiting the
wake-promoting regions (discussed later) and
inducing SWA (Chou etal., 2002; Gallopin etal.,
2000). GABA projections from the mediolateral
preoptic nucleus are necessary for inducing sleep,
and additional projections from the ventrolateral
preoptic nucleus (VLPO) are necessary for main-
taining sleep (Chou et al., 2002; Gallopin et al.,
2000; Gaus etal., 2002; Gong etal., 2004; Hassani
etal., 2009; Hassani etal., 2010; Lu etal., 2000;
Manns etal., 2000; McGinty and Sterman, 1968;
Saper etal., 2005a; Saper etal., 2010a; Sherin etal.,
1996; Sherin et al., 1998; Suntsova et al., 2002;
Szymusiak et al., 1998; Takahashi et al., 2009).
During NREMS, additional GABAergic neuro-
transmission from the basal forebrain and lateral
hypothalamus (LH) and melanin-concentrating
hormone from the LH contribute to the inhibi-
tion of cortical and subcortical activity (Alam
etal., 2002; Bittencourt etal., 1992; Hassani etal.,
2010; Kilduff and de Lecea, 2001; Koyama etal.,
2003; Saito etal., 2001; Verret etal.,2003).
Pharmacology
GABA. Drugs that enhance GABAergic signal-
ing, such as benzodiazepines (e.g., clonazepam),
are used to treat anxiety, insomnia, agitation, and
seizures in epilepsy and are used as sedatives and
hypnotics. Benzodiazepines are associated with
increasing NREMS activity and sleep duration.
However, with sleep-promoting effects on neuro-
biology, a potential side effect of benzodiazepines
is prolonged daytime drowsiness. Other seda-
tives/hypnotics that also increase GABA inhibi-
tion to promote sleep include zolpidem, zaleplon,
and eszopiclone.
REMS
EEG and Neurobiology
During REMS, there is a reduction of the over-
all GABAergic tone, spindles and slow waves.
Increased activation of acetylcholine and glu-
tamate projections to the thalamus and cortex
induce phasic activation of the visual and limbic
circuits, neuronal network desychronization, and
low-amplitude theta and alpha rhythms (510
Hz) associated with REMS (Brown et al., 2012;
Dang-Vu etal., 2010). Electromyogram is used to
verify muscle atonia during REMS to distinguish
this state from wakefulness.
Pharmacology
Acetylcholine. Acetylcholine is a key neurotrans-
mitter during REMS. Increasing acetylcholine
levels at the synapse is associated with reducing
the latency to REMS onset and increasing REMS
duration. One treatment strategy for Alzheimers
disease is to increase levels of acetylcholine at the
synapse by inhibiting the enzyme responsible for

degrading acetylcholine (acetylcholine esterase)
with a reversible acetylcholine esterase (AChE)
inhibitor, such as donepezil, galantamine, or
rivastigmine. Indeed, treatment with donepezil
increases REMS in people with Alzheimers dis-
ease (Moraes Wdos etal.,2006).
GABA, serotonin, norepinephrine-enhancing
drugs. REMS can be reduced by drugs that
enhance GABA, serotonin, or norepineph-
rine (Boissard et al., 2002; Curtis et al., 1968;
Fedirchuk and Dai, 2004; Fenik et al., 2004;
Jelev et al., 2001; Kayama et al., 1992; Lai
et al., 2001; Leonard and Llinas, 1994; Morales
et al., 1987; Morrison et al., 2003; Perrier and
Delgado-Lezama, 2005; Steriade et al., 1990).
As described, GABA-activating drugs promote
NREMS and thus reduce time spent in REMS.
Serotonin and norepinephrine are active neu-
rotransmitters during wake and NREMS and
are virtually absent during REMS. Drugs that
increase levels of serotonin and/or norepineph-
rine, specifically at the synapse, include SSRIs,
SNRIs, and monoamine oxidase inhibitors. These
drug classes are used to treat depression and
ADHD and can result in an increase in NREMS
rhythms, a reduction in REMS incidence, and
a delay in the overall drive to induce REMS
(Wilson and Argyropoulos, 2005). Due to the
fact that serotonin is also involved in promoting
wakefulness, insomnia is a side effect ofSSRIs.
Wake
Sleep-disorder symptoms, such as fragmenta-
tion or inappropriate awakenings during sleep,
involve the neurobiology underlying wakeful-
ness. Symptoms such as longer sleep latency
involve the neurobiology underlying the drive to
fallsleep.
EEG and Neurobiology
Projections from the master circadian pace-
maker, the suprachiasmatic nucleus, activate
orexin (also referred to as hypocretin) neurons
in the LH to promote wakefulness. Orexin pro-
jections to the ascending reticular activating
system excite several nuclei to increase arousal,
attention, and muscle tone (Sakurai, 2007;
Szymusiak and McGinty, 2008). These efferent
targets include dopamine neurons (in the sub-
stantia nigra), serotonin neurons (in the dorsal
raphe nuclei), norepinephrine neurons (in the
locus coeruleus), histamine neurons (in the tub-
eromammillary nucleus), and acetylcholine neu-
rons (in the pedunculopontine and laterodorsal
Sleep 317
tegmental nuclei). These regions then send pro-
jections to glutamatergic thalamic and cortical
structures to increase the cortical EEG desyn-
chronization and higher frequency rhythms (in
the alpha, beta, theta, and gama power), muscle
tone, attention, and alertness.
Pharmacology
Alterations of each of the following wake-
promoting systems, either by disorders or phar-
macology, can negatively influencesleep.
Orexin receptor blockers.An increase in sleep
fragmentation, or the number of awakenings dur-
ing sleep, is a common comorbid sleep-disorder
symptom in mental retardation, anxiety and
stress disorders, depression, Alzheimers,
Parkinsons, and epilepsy (Lee-Chiong, 2008).
Orexin-expressing neurons in the LH are a wake
trigger, and dysfunction of the orexin system is
associated with sleep fragmentation, increased
awakenings, narcolepsy, restless leg disorder,
and epilepsy (Sakurai etal., 2010). Novel strate-
gies that suppress orexin activity via dual orexin
receptor antagonism improved sleep quality in
normal mice in clinical trials and in a mouse
model of epilepsy with a comorbid sleep disorder
(Roundtree etal., submitted). This class of drugs
has a faster half-life than other sedatives, and
thus there is minimal daytime drowsiness.
Dopamine, serotonin and norepinephrine
inhibiting agents. Atypical antipsychotics, such
as risperidone, are used to treat schizophrenia
and bipolar disorder and to reduce self-harming,
aggressive, and tantrum behaviors in children
with autism. These compounds antagonize
wake-promoting neurotransmission by blocking
dopamine receptors and exerting antiserotonin-
ergic, antinorepinephrinergic, and antihistamin-
ergic activity. Risperidone has been reported to
improve sleep in autistic children with irritability
and related behaviors (Kent etal.,2013).
In people with depression, treatment with
olanzapine, another atypical antispyschotic,
improved sleep efficiency, total sleep time, and
sleep latency (Lazowski etal., 2014). However, by
blocking wake-promoting neurotransmission,
these compounds have fatigue and sedative side
effects. Reduced levels of dopamine and seroto-
nin are associated also with Parkinsons disease
and depression, respectively, both disorders with
comorbid daytime drowsiness.
Dopamine, serotonin and norepineph-
rine enhancing agents. Stimulants including
amphetamines and cocaine increase synaptic
318 Part III:Homeostatic Manipulators
concentrations of monoamines, primarily dopa-
mine and norepinephrine (but also serotonin and
histamine), via multiple mechanisms of action.
Modafinil, used for the treatment of narcolepsy,
promotes wakefulness primarily by increasing
dopamine and histamine levels.
Antihistamines. Histamine promotes
wakefulness and the ability to focus. First gen-
eration antihistamines, such as diphenhy-
dramine (an active ingredient in more than 40
over-the-counter allergy and sleep-aid prod-
ucts), are able to cross the blood-brain barrier
(BBB). These antihistamines can promote sleep
at night but can also cause daytime drowsiness.
Second-generation antihistamines, such as lorat-
adine, have minimal BBB permeability and thus
have minimal effects on sleep architecture.
Wake-Sleep Transition
Two-Process Model ofSleep
Regulation
The factors that determine when we fall asleep
and establish our sleep homeostasis are typically
described with a two-process model of sleep regu-
lation (Daan etal., 1984; Tarokh and Achermann,
2013). The first is the internal circadian process
(process C). Process C determines the ideal
timing of a sleep episode based on internal fac-
tors and has a 24-hour rhythm governed by the
suprachiasmatic nucleus. The second is the sleep
homeostatic process (process S). Process S is
dependent on the amount of time elapsed since
the last sleep episode and is an internal driver to
ensure sleep is attained to re-establish our sleep
homeostasis. The actual timing and consolida-
tion of wake and sleep (i.e., when we fall asleep)
are regulated by the intersection of process S and
processC.
Pharmacology
Recent studies suggest that adenosine (which
increases with sleep deprivation) is a key neu-
robiological somnogen that drives process
S and can be used as a marker of sleep pres-
sure (Porkka-Heiskanen et al., 1997a;
Porkka-Heiskanen and Kalinchuk, 2011). Other
somnogens that may participate in process S
include melatonin and immune factors (dis-
cussed later).
Adenosine and adenosine receptor block-
ers. Adenosine is a purine nucleoside somnogen
that acts on its adenosine type 1 receptor (A1R) to
inhibit wake-promoting basal forebrain and LH
activity (Liu and Gao, 2007). Adenosine also acts
in the tuberomammillary nucleus and basal fore-
brain to increase VLPO GABAergic signals to
enhance process S or the drive to sleep (Basheer
etal., 2004; Benington etal., 1995; Huang etal.,
2007; Huston et al., 1996; Porkka-Heiskanen
et al., 1997a; Radulovacki et al., 1984). Caffeine
and theophylline, found in tea, are stimulants
that antagonize A1R and thus interfere with
adenosines ability to promotesleep.
Melatonin and a receptor agonist. The drug
ramelteon is a selective melatonin receptor ago-
nist that decreases the latency to NREMS (or sleep
onset) and increases NREMS duration (Fisher
et al., 2008; Spadoni et al., 2011). Activation
of melatonin receptors in the suprachiasmatic
nucleus is thought to shift the timing of the cir-
cadian system promoting sleep onset directly
and reinforcing regular evening sleepiness and
sleep onset (Neubauer, 2008). In preclinical stud-
ies, exogenous melatonin had similar effects as
ramelteon; however, unfavorable pharmacoki-
netics has led to variable results in human clini-
cal trials. It does appear that prolonged-release
formulations of melatonin do decrease sleep
onset and increase sleep duration (Fisher et al.,
2008; Spadoni etal., 2011; Srinivasan etal., 2011).
In addition, melatonin has potent antioxidant
activities that may contribute to additional effects
during sleep (discussed later).
Pathology
Reduction of adenosine tone is associated with
brain pathology that expresses reactive astro-
cytes or astrogliosis (Boison, 2006, 2008).
Astrogliosis is associated with an increase of
adenosine kinase, the enzyme responsible for
converting adenosine into adenosine monophos-
phate. Astrogliosis can occur in neurological
disorders that are associated with sleep comor-
bidities, including epilepsy, Alzheimers disease,
and Parkinsons disease, however, reported loca-
tions are typically in non-sleep regulating brain
regions. One recent study identified astroglio-
sis in the sleep-regulating LH of epileptic mice
(Roundtree etal., submitted).
P R E V E N TAT I V E
A N D R E S T O R AT I V E
OPPORTUNITIES
Proper hygiene to ensure sufficient sleep can
exert preventative and restorative effects on
many physiological processes. Here we highlight
the effects of sleep on the central processing of
(i)cognitive processing, (ii) stress responses and

behavior, (iii) brain regulators of body weight,
(iv) ATP production and damage/waste removal,
and (v)neuro-immune responses.
How Does Sleep Influence
Cognition?
Cognition andSleep
Successful processing and consolidation of
different types of memories and next-day
decision-making are strengthened when suf-
ficient sleep is attained. Neuroimaging signals
indicate strong activation of the hippocampus
and striatum during NREMS SWA. This activity
is hypothesized to reflect a replay of coordinated
hippocampal-striatal activity and contrib-
ute to the selective strengthening of memories
(Dang-Vu etal., 2010). When early sleep remains
intact, recall of previously learned declarative
tasks is enhanced, suggesting that NREMS SWA
facilitates declarative memory consolidation pro-
cesses (Marshall et al., 2006; Plihal and Born,
1997). When later sleep stages rich in REMS
remain intact, recall of tasks requiring proce-
dural skills is enhanced (Walker etal.,2002).
The cognitive processes that are impaired fol-
lowing different severities of sleep deprivation
depend on the extent of regional or global decline
in alertness and attention and whether these pro-
cesses are able to rely on compensatory support
(Killgore, 2010). While execution of basic logical,
planning, and rule-based tasks is resilient to one
night of sleep deprivation, the ability to inno-
vate, revise, or effectively handle distractions and
unexpected situations can be impaired (Harrison
and Horne, 2000). According to the Centers for
Disease Control and Prevention and the National
Sleep Foundation, there is a positive correlation
between the increased chronicity (or number of
consecutive days) of partial sleep deprivation and
impaired cognitive capacities as measured by
the number of automobile accidents and medi-
cal mistakes made by first responders (Institute
of Medicine Committee on Sleep Medicine and
Research, 2006; Machi et al., 2012; Waggoner
etal., 2012). Further, the effect of chronic partial
sleep deprivation (i.e., 14days of less than 6 hours
per night) on cognitive function is dose depend-
ent (Van Dongen etal.,2003).
Implications forChildren
and Adolescents
During childhood and adolescence, brain growth
and the generation and pruning of new syn-
apses occur simultaneously with the cognitive
Sleep 319
processing of extensive abstract concepts, school
curricula, relationships, emotions and behav-
iors. During development, the brain experiences
approximately 2 to 5 more hours of sleep each
night (dependent on age), when compared to
adults. Chronic partial sleep deprivation in chil-
dren and young adults is associated with poorer
cognitive performance. In young adults, reduced
SWA is associated with reduced sensory motor
and cognitive procedural memories (Limoges
et al., 2013). Further, child-reported sleep prob-
lems are a predictor of child-and teacher-reported
academic problems and teacher-reported inat-
tentiveness (Becker,2014).
Disorders, Cognition, andSleep
Cognitive impairments occur with Alzheimers
dementia and can be a comorbid condition of
other disorders and diseases, including autism,
ADHD, depression, epilepsy, and Parkinsons dis-
ease. Sleep disturbances among these populations
correlate with the degree of cognitive impair-
ment. Poor sleep is an indicator of impaired
performance on selective attention, declarative
memory, sensory-motor abilitiy, and procedural
memories in autistic young adults (Limoges etal.,
2013). Likewise, there is a positive correlation
between sleep disturbances and impaired cogni-
tive functioning in Parkinsons and Alzheimers
patients, epilepsy patients, and children and
adults with ADHD (Fisher etal., 2014; Kim etal.,
2014; Piperidou etal., 2008; Pistacchi etal.,2014).
Sleep Therapy
Restoration of sleep quality is associated with
improvements in cognitive performance in peo-
ple with autism, epilepsy, ADHD, Parkinsons dis-
ease, and Alzheimers disease (Fisher etal., 2014;
Kim etal., 2014; Limoges etal., 2013; Piperidou
et al., 2008; Pistacchi et al., 2014). Interestingly,
increased theta power (of 48 Hz), which is asso-
ciated with normal cognition during wakeful-
ness, and delta power during NREMS SWA is
associated with improved cognitive performance
and better recall in people with Alzheimers
disease (Hot etal., 2011). Increased theta power
during stage 1 NREMS sleep is associated with
improved recall in patients with Parkinsons and
Alzheimers diseases (Kempf etal.,2009).
Pharmacology
Acetylcholine and histamine are wake-promoting
molecules that are involved in the network pro-
cessing of different types of learning, memory
320 Part III:Homeostatic Manipulators
consolidation, recall, and next-day thinking and
are also targets of neurological conditions and/or
pharmacotherapies.
Acetylcholine. Increasing synaptic acetylcho-
line levels with AChE inhibitors enhance cogni-
tive abilities in people with Alzheimers disease.
Histamine. The daytime drowsiness side
effect of first-generation antihistamines may
impair cognitive function and induce confusion.
These side effects can be avoided by utilizing
second-generation antihistamines, which have
minimal BBB permeability.
How Does Sleep Influence
Stress and Behavior?
As depicted in Figure 18.2, sleep efficiency, stress,
and affective behaviors are cyclically interde-
pendent, thus there are many subsections within
this section. Maintaining the categorical inde-
pendence of each subsection was designed to
easily visualize their interrelationships while
avoiding causality. We begin by discussing the
interactions between stress responses and sleep
efficiency followed by affective behaviors and
pharmacology.
Stress Responses
Responses to psychological and physiologi-
cal stressors are mediated by the limbic-
Insufficient
sleep
D C A D
B efficient response or a more severe and prolonged
Negative
Stress
behaviors
FIGURE18.2: Cyclic interdependencies of insufficient
sleep, stress, and negative behaviors. (A) Insufficient
sleep can elevate stress molecules. (B) Elevated stress
responses are associated with negative behaviors
including anxiety, reduced threshold for aggression,
irritability, and impulsiveness. (C) Insufficient sleep
can promote negative behaviors including anxiety,
hyperactivity, distractibility, aggression, disinhibition,
oppositional behavior, risk taking, irritability, and
impulsiveness. (D)Both stress and negative behaviors
are associated with insufficientsleep.
hypothalamic-pituitary-adrenal (LHPA) axis
and sympathetic nervous system. Key LHPA
molecules are corticotropin releasing hormone
(CRH) and glucocorticoids (cortisol in humans
and corticosterone in rodents; Arborelius et al.,
1999; McEwen and Stellar, 1993). Stress sig-
nals from cortical, hippocampal, and amygdala
regions converge onto the hypothalamic para-
ventricular nucleus to elicit the secretion of CRH.
CRH acts on the anterior pituitary and ultimately
causes the release of glucocorticoids from the
adrenal glands. The response is terminated by
glucocorticoid negative feedback. Each transient
stress response is superimposed on its circadian
rhythm. The circadian rhythm of CRH and corti-
sol expression peaks in mid-morning and reaches
a nadir in the evening.
Activation of the sympathetic nervous sys-
tem increases release of norepinephrine and
epinephrine. Stressor-induced increases in heart
rate and/or blood pressure are mediated in part
by the enhanced sympathetic activation and
the subsequent actions of norepinephrine and
epinephrine. In terms of circadian rhythms,
norepinephrine and epinephrine reach a nadir
approximately 1 hour after sleep onset (Irwin
etal.,1999).
Implications for children. It is well known
that the duration and severity of the LHPA
response is initially programmed early in life.
The expression ontogeny of CRH and its recep-
tors differ in a brain region specific manner
(Korosi and Baram, 2008), and the epigenetic
programming of the LHPA is exquisitely sen-
sitive to early life experiences (Fenoglio et al.,
2004; Fenoglio etal., 2005; Fenoglio etal., 2006a;
Fenoglio etal., 2006b; McEwen and Stellar, 1993).
Enriched or stressful early life experiences can
shift the programming pendulum to either an
response, respectively (Biggs etal., 2011; Fenoglio
etal., 2006b; Ivy etal., 2008; Spaeth etal., 2013;
Watanabe etal.,2010).
Insufficient Sleep and
Stress Responses
The glucocorticoid response is resilient to
acute and chronic sleep deprivation protocols
in rodents and humans (see Figure 18.2; Fadda
and Fratta, 1997; Kant etal., 1984; Novati etal.,
2008; Redwine et al., 2000). However, follow-
ing acute partial sleep deprivation (sleeping for
approximately 4 hours), increased sympathetic
activity contributes to increased heart rate and
blood pressure variability and levels of serum

norepinephrine (Dettoni etal., 2012; Irwin etal.,
1999). In addition, hypothalamic CRH levels
increase following chronic partial sleep dep-
rivation (5 days of REMS deprivation; Koban
etal.,2006).
LHPA Axis Tone and Behavior
Increased CRH activity and LHPA axis tone is asso-
ciated with behaviors such as anxiety, irritability,
aggression, and impulsiveness (see Figure 18.2).
Increased LHPA activity is also a predispos-
ing factor for depression-like psychopathologies
(Roman etal.,2006).
Insufficient Sleep and Behavior
Insufficient sleep increases the manifestation
of behaviors including anxiety, hyperactivity,
distractibility, aggression, disinhibition, oppo-
sitional behavior, risk taking, irritability, and
impulsiveness (see Figure 18.2). Children who
experience insufficient sleep may express day-
time sleepiness by being overactive and hav-
ing a difficult time focusing. In children with
autism, increased sleep disturbances are a pre-
dictor of anxiety (May et al., 2013). A recent
study reported that inconsistent sleep schedules
and short sleep durations are associated with
behavioral difficulties in children (Biggs et al.,
2011). Further, self-reported sleep problems (by
children) predicted child- and teacher-reported
behavioral problems, including reactive aggres-
sion (Becker,2014).
Insufficient sleep is also a predisposing factor
for depression-like psychopathologies (Roman
et al., 2006). In animal models, chronic partial
sleep deprivation (4 hours/day for 1 wk) leads to
a blunted adrenocorticotropic hormone response
(but normal glucocorticoid response as described
previously), which resembles depression (Novati
et al., 2008). Self-reports from bipolar patients
indicate that periods of sleep deprivation can
trigger depressive episodes (Proudfoot et al.,
2012). Child-reported sleep problems is a predic-
tor of child- and teacher-reported social func-
tioning, feelings of loneliness, lower friendship
satisfaction, and lower self-worth (Becker,2014).
Behavior and InsufficientSleep
Insufficient sleep can be a comorbid condi-
tion experienced by people with anxiety and
stress-related disorders (see Figure 18.2).
Comorbid symptoms in adults with general-
ized anxiety disorder and ADHD can have
increased latency to sleep onset and reduced
Sleep 321
sleep efficiency. People with posttraumatic stress
disorder (PTSD) report increased sleep latency,
less duration, and increased awakenings (Brown
et al., 2011; (Cohen et al., 2009). Patients with
insomnia report more severe symptoms of anxi-
ety and depression (Mason and Harvey, 2014).
Even in young preschoolers, insufficient sleep
is related to psychiatric symptoms, particularly
anxiety (Steinsbekk etal.,2013).
Major Depressive Disorder
In contrast to the effects in bipolar disorder, anxi-
ety, stress, and PTSD, there is a well-documented
antidepressant effect of sleep deprivation in peo-
ple with major depressive disorder (Gillin, 1983;
Landsness etal.,2011).
Pharmacology
Norepinephrine. Currently, the most effective
treatment strategy for PTSD includes cognitive
behavioral therapy with either prazosin or an
SSRI. Prazosin blocks central and peripheral nor-
epinephrine (adrenergic) alpha 1 receptors. This
reduces sympathetic responses associated with
anxiety (elevated heart rate and/or increased
blood pressure) and can enhance the ability to
focus while awake and increase the probability of
REMSsleep.
Serotonin. Reduced serotonin levels are asso-
ciated with increased depression, aggression,
irritability, and hostility in humans and animals
(Fuller, 1996; Kamphuis et al., 2012). Reduced
sleep efficiency (24-hour deprivation and partial
deprivation) is associated with reducing sero-
tonin tone (Figure 18.2; Asikainen et al., 1997;
Elmenhorst etal., 2012). Two advantages of SSRIs
(which specifically inhibit serotonin uptake to
selectively increase extracellular serotonin lev-
els), such as fluoxetine, are the associated reduc-
tion in aggression (Fuller, 1996) and increase
ofNREMS.
Alcohol. Alcohol consumption reduces REMS.
Imaging analyses during REMS indicates a sig-
nificant increase in amygdala and mesolimbic
dopaminergic activation. Future studies are
required to test the hypothesis that these signals
reflect the processing of events that have emo-
tional/motivational value (Perogamvros et al.,
2013).
Caffeine. Excessive caffeine intake is asso-
ciated with insomnia, lower stress thresholds,
increased aggressive behaviors, and increased
anxiety (see Fredholm etal., 1999, for extensive
review). In youth with major depressive disorder,
322 Part III:Homeostatic Manipulators
there is a higher incidence of caffeine intake
and sleep problems, and caffeine use is associ-
ated increased symptoms of anxiety (Whalen
etal.,2008).
Sleep Therapy
Ensuring sufficient sleep may be an effective
therapeutic adjuvant to attenuate stress, anxiety,
and/or depressive symptoms. Indeed, improve-
ment of sleep quality increases clinical outcomes,
lessens depression, improves quality of life, and
increases physical health in individuals with
PTSD (Germain et al., 2008). Sufficient sleep is
associated with significantly fewer behavioral dif-
ficulties in children (Biggs etal., 2011). Further,
improved sleep is associated with improved social
ability of autistic children (May etal.,2013).
How Does Sleep Influence
Ghrelin and Leptin, Brain
Regulators of Body Weight?
Attainment of sufficient sleep is one mechanism
to promote healthy weight and reduce the risk
for metabolic disorders. Insufficient sleep is a
significant contributing factor of metabolic dis-
orders for many reasons; here we focus on two.
Ghrelin and leptin are two appetite-regulating
neuropeptides. Ghrelin is secreted from the
stomach and acts at the hypothalamus to pro-
mote hunger. In contrast, leptin is secreted
from fat cells and acts at the hypothalamus to
promote satiety. Partial chronic sleep depriva-
tion (less than 6 hours per night or late sleep
onset) is associated with increased levels of
ghrelin and reduced levels of the leptin in the
blood. Studies indicate that adults who report
having less than 5 hours of sleep per night are
at higher risk of developing diabetes than those
who achieve 7 hours. In multiple, large-cohort
human studies, partial chronic sleep depriva-
tion (less than 6 hours per night or late sleep
onset) is associated with increased daily caloric
intake, self-reported increases in hunger,
increased BMI, weight gain, and the develop-
ment of obesity (Spaeth et al., 2013; Spiegel
etal., 2004; Srivastava etal., 2013; Taheri etal.,
2004; Watanabe et al., 2010). Further, glucose
tolerance is impaired following restriction of
either total sleep time or NREMS SWA (Spiegel
et al., 1999; Tasali et al., 2008). Collectively,
these data suggest that attainment of sufficient
sleep is one preventative/restorative therapy to
promote healthy weight and reduce the risk for
metabolic disorders.
How Does Sleep Influence
Brain ATP Levels and Damage/
Waste Removal Processes?
Recent studies have examined the role of sleep in
the homeostatic regulation of brain ATP levels
and the removal of damage and waste products
within the brain milieu. These processes have
dynamic interactions among synaptic transmis-
sion, cognitive processes, adenosine levels, sleep,
reactive species production, and the pathology,
which can occur in neurological disorders asso-
ciated with sleep and cognitive comorbidities
(Figure18.3).
ATP
ATP, the brains energy currency, is critically
important for brain homeostasis. Mitochondria
produce approximately 97% of ATP, and, at the
cost of 25,000 molecules of ATP to transmit one
bit of information, it is estimated that 41% is
expended on synaptic transmission (Hall et al.,
2012; Harris et al., 2012; Ligon and Steward,
2000). Recent studies suggest that sleep can pro-
mote restoration of brain ATP levels. This notion
is supported by data indicating that enzymes that
promote ATP production (including pyruvate
kinase, adenylate kinase, and malate dehydro-
genase) peak at the beginning of the rest phase,
both glucose and ATP levels increase during
early NREMS, and deprivation of sleep reduces
levels of ATP precursors, glycogen, and glucose
(Dworak etal., 2010; North etal.,1981).
Damage, Pathology, and
Neurological Disorders
In addition to producing ATP, mitochondria can
induce cell damage/injury and trigger cell death.
ATP is produced via mitochondrial complexes
Ithrough V.Impairment of electron transport at
complex I results in (i) reduced ATP-producing
respiratory rates and (ii) increased production
of reactive species by the binding of misshuttled
electrons to oxygen and nitrogen (species include
superoxide, nitric oxide, hydroxyl radical, hydro-
gen peroxide, nitric dioxide, and peroxynitrite)
(Fariss etal., 2005; Simeone etal., 2014; Sullivan
etal., 2003; Sullivan etal., 2004). High levels of
these reactive species cause oxidative damage to
DNA, RNA, proteins, lipids, and cell membranes.
The cascades of events that can result from mito-
chondrial impairment can induce a range of
pathology from minor cell injury to celldeath.
Normal aging and neurological disor-
ders, including epilepsy, Alzheimers disease,
 Sleep 323

Mitochondria Impaired mitochondria

Disrupted
Synaptic Low levels High levels
ATP Less ATP synaptic
transmission of RS of RS
transmission
?
Cognitive Less Cognitive Oxidative
Adenosine damage to
Processes adenosine impairments
lipids,
proteins,
nucleic
Disrupted
Sleep acids
sleep

Pathology:
Pathology: Astrogliosis
Cell injury/death

FIGURE 18.3: Dynamic multidirectional crosstalk among mitochondria, neurotransmission, pathology, cogni-
tion, and sleep. Left:Mitochondria play a critical role in regulating adenosine triphosphate (ATP) and reactive
species (RS) homeostasis, both factors upstream of synaptic transmission and cognitive processes. ATP is readily
converted into the somnogen adenosine. Right: Impaired mitochondria, specifically a complex I reduction of
function, is associated with reduced ATP and elevated RS, both implicated in impairing synaptic transmission
and cognitive processing. Impaired mitochondria are upstream of pathological features and are associated with
neurological disorders that have cognitive and sleep disorder comorbidities. The precise nature of the impairment
and the location within the brain will determine which circuitry and behavioral outputs are influenced.

and Parkinsons disease are associated with
a higher incidence of complex I impairment,
increased levels of reactive species and subse-
quent oxidative damage (Brown et al., 2004;
DiMauro etal., 1999; Fariss etal., 2005; Head
etal., 2009; Joseph etal., 2000; Simeone etal.,
2014; Wallace, 1992). Deficient pools of ATP
and elevated reactive species negatively impact
synaptic transmission and are associated with
cognitive impairments (Chouhan et al., 2012;
Guo etal., 2005; Hall etal., 2012; Harris etal.,
2012; Hongpaisan et al., 2003; Hongpaisan
etal., 2004; Hu etal., 2006; Kamsler and Segal,
2003; Kann et al., 2011; Knapp and Klann,
2002; Lee etal., 2012; Ligon and Steward, 2000;
Simeone et al., 2014; Thiels et al., 2000), a
hallmark feature of Alzheimers disease and a
potential comorbid condition of epilepsy and
Parkinsons disease.
Astrogliosis is a pathology that can occur in
neurological disorders, including epilepsy and
Alzheimers disease. Astrogliosis and the con-
comitant increase in adenosine kinase imply a
reduction of adenosine tone. Studies are needed
to determine whether reduced adenosine tone
occurs in the brain regions, such as the basal fore-
brain or lateral hypothalamus, that are known to
regulatesleep.
Damage and Waste Removal
The homeostatic mechanisms responsible for
reducing oxidative damage and neurotoxic waste
products is critical to prevent overaccumulation.
Although these processes are not entirely under-
stood, recent studies suggest that they may occur
more during sleep. Two waste removal mecha-
nisms include the antioxidant system and the
glia-lymphatic system.
Antioxidants. Reactive species are reduced
by many endogenous antioxidants during both
the day and the night. The antioxidant and som-
nogen melatonin is elevated during the night,
peaking after sleep onset (Redwine etal., 2000).
This reactive species clearance mechanism is not
sensitive to sleep deprivation, as melatonin levels
do not change following acute, partial sleep dep-
rivation (Redwine et al., 2000). However, levels
of the more potent antioxidant glutathione are
significantly reduced following sleep deprivation
(DAlmeida etal., 2000; Silva etal.,2004).
Glia-lymphatic system. Recent reports indi-
cate that oxidative damage and neurotoxic waste
products (i.e., metabolites, soluble proteins
including -amyloid, excess fluid, etc.) are cleared
via a glia-lymphatic system. The exchange rate of
cerebral spinal fluid with fresh cerebral spinal
fluid appears to be higher during periods of sleep
324 Part III:Homeostatic Manipulators
(Iliff and Nedergaard, 2013; Nedergaard, 2013;
Xie etal.,2013).
Insufficient sleep is comorbid to neurologi-
cal disorders that can express pathology, includ-
ing astrogliosis, oxidative damage, cell injury,
and cell death. Future studies are required to
determine whether insufficient sleep influences
the glia-lymphatic system and whether reduced
damage/waste clearance during the night pro-
motes the development of pathology.
Nutritional Pharmacology
Supplements and foods that target mitochondria
and are rich in antioxidants, including ascorbic
acid and alpha tocopherol (vitamins C and E), can
be used to minimize accumulation of oxidative
damage. Additional compounds of mono- or pol-
ytherapies (including pyruvate and melatonin)
have also been used in preclinical treatments
for sleep disorders in epilepsy (Simeone et al.,
2014).The ketogenic diet (KD), a diet high in fat
and low in carbohydrates and proteins, is highly
effective in reducing medically refractory sei-
zures. As a disease-modifying treatment, the KD
is now being explored for its efficacy in autism,
Alzheimers disease, and Parkinsons disease. The
KD improves mitochondrial function, restores
complex IATP-producing rates, reduces reactive
species, increases endogenous antioxidants, and
enhances adenosine tone. Further, recent studies
have found that the KD improves sleep in chil-
dren and animal models of epilepsy (Fenoglio
et al., 2009; Hallbook et al., 2007; Masino and
Geiger, 2008; Roundtree et al., submitted;
Sullivan etal.,2003).
How Does Sleep Influence
Neuro-Immune Response Factors?
The relationship between sleep and the immune
system is bidirectional and complex. Here we high-
light two examples:(i)immune factors can act as
somnogens in specific brain areas to promote sleep,
and (ii) sleep can enhance immune responses.
Immune Factors Are Somnogenic
During infection, increased levels of proin-
flammatory cytokines, such as TNF-alpha and
IL-1beta, can act in the brain to promote sleep by
inducing NREMS (Figure 18.4). Studies suggest
that this is in part mediated via signal convergence
onto the prostaglandin2 (PGD2)-adenosine path-
way (Cao et al., 1996; Terao et al., 1998). PGD2
can increase NREMS or REMS when injected
into different brain regions (i.e., the preoptic
area, the lateral ventricles, or the subarachnoid
IL-1, IL-6, TNF, NF-
AA
COX
Protaglandin H2
PGI PGE2
PGF2a
PGD2
- Acts on DP1R
- Increases adenosine
FIGURE18.4: Immune factors, prostaglandins, aden-
osine and sleep. One mechanism by which immune
factors such as IL-1 beta, IL-6, TNF alpha, or NF kappa
beta may promote sleep is by activating the cyclooxy-
genase (COX)-prostaglandin (PGD) pathway. COX
induces the conversion of arachidonic acid to prosta-
glandin (PG) H2, which can be converted into PGD2.
PGD2 acts on its receptor (DP1R) to increase adenosine,
an endogenous somnogen, in sleep-promoting regions.
space of the ventral forebrain; Huang etal., 2007;
Pandey etal., 1995; Ram etal., 1997; Terao etal.,
1998). PGD2 acts on its D prostanoid receptor 1
in the basal forebrain, which increases levels of
the somnogen adenosine and activation of the
VLPO (Huang et al., 2007; Kristensson et al.,
2010; Mizoguchi etal., 2001; Saper etal., 2005b;
Saper etal., 2010; Sherin etal.,1996).
Sleep Can Enhance
Immune Responses
While anti-inflammatory cytokines peak dur-
ing the day, activity of many proinflammatory
factors peak following sleep onset, including lev-
els of cytokines, activation and proliferation of
proinflammatory APC and T cells, lymphocyte
accumulation in lymph nodes, and phagocyto-
sis (Besedovsky et al., 2012; Lange et al., 2003;
Lange et al., 2010). Recent studies suggest that
sleep not only increases the acute number of
antigen-specific T helper cells following vacci-
nations (in these studies for hepatitis A) but also
promotes immunological memory as evidenced
by effects persisting one-year postvaccination
(Bollinger et al., 2011; Lange et al., 2003; Lange
etal., 2011; Phillips etal.,2008).
PROPER SLEEP HYGIENE
Routine and proper sleep hygiene is essential.
Based on the recommended amount of time adults
and children spend in sleep, approximately 30% of

brain activity in adults and 44% in children occurs
during sleep. According to the National Sleep
Foundation and the National Institute of Health
Center for Sleep Disorders Research, the following
guidelines can be used to improve sleep hygiene.
During the day, spend time outside in natural
light. Avoid exercising, eating large meals, and
drinking immediately before bed. Avoid caffein-
ated and alcoholic beverages.
Maintain a consistent and positive bedtime
routine that ensures a sufficient number of hours
of sleep is achieved. Develop a bedtime routine
that begins 30 min before falling asleep, prefer-
ably at the same time each night. This routine
should include relaxing in a cool, quiet room,
which has reduced lighting and lacks electronic
sources of light. Commonly used artificial light
has more blue frequencies, which is associated
with reduced levels of melatonin, an important
somnogenic neuropeptide (Lockley etal.,2003).
Ensuring sufficient sleep offers a preventa-
tive measure or an adjuvant restorative therapy
that may improve cognition and/or behavior in
normal individuals and lessen the degree of cog-
nitive/behavioral comorbid afflictions in indi-
viduals with neurological or affective disorders.
ACK NOWLEDGMENTS
The authors would like to thank the National
Institutes for Healths National Institute of
Neurological Disorders and Stroke (NS072179)
for support, E. Staneck for his technical assis-
tance, and A. Sachs for his editorial assistance.
Please note the first author has published under
the names K.A. Dorenbos, K.A. Fenoglio, and
K.A. Simeone.
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19
Botanicals
DA NA EK ST EIN A ND ST E V EN C .SCH ACH T ER
INTRODUCTION
Herbal therapy had been the main type of medi-
cal treatment worldwide up until the last cen-
tury. With the development of modern Western
medicine, in parallel with the pharmaceutical
industry and regulatory agencies, the wide use
of botanicals was replaced by refined pharma-
ceutical compounds, specifically targeted at
disease processes. This change has been enabled
by each great advance in the understanding of
pathophysiology, extending from the genomic
and through the proteomic and system levels.
Novel pharmaceutical medications were ration-
ally designed to act at very specific biologic sites
and reverse the pathologic state. These new treat-
ments widely replaced the traditional herbal rem-
edies, and their introduction coincided with the
development of an entire system aimed at prov-
ing their safety and efficacy, thereby establishing
the gold standard of evidence-based medicine.
Botanicals are now considered part of com-
plementary and alternative medicine or comple-
mentary health approaches (CHA)the latter is
the preferred term most recently introduced for
these medical practices by the National Center
for Complementary and Alternative Medicine
(2013). CHA are defined as medical remedies
that are not part of the mainstream Western
type of medicine. They include natural prod-
ucts, mind and body practices, and practices of
whole health systems, such as homeopathy or
traditional Chinese medicine (TCM). Herbs (or
botanicals), minerals, vitamins, and probiotics
are all considered natural products. Traditional
herbal treatments were never completely aban-
doned in the developing parts of the world, such
as China, Korea, India, and South America,
and the use of CHA has become increasingly
prevalent in Western countries over the past
few decades, especially in attempts to enhance
wellness or treat the symptoms of chronic dis-
eases, reflecting disappointment with modern
Western treatments. For example, according to
the most recent national survey conducted in
2007 (Barnes etal., 2008), nonvitamin/nonmin-
eral natural products were the most widely used
type of CHA, with 17.7% of Americans reporting
their consumption.
Interestingly, this return to traditional rem-
edies by consumers coincidentally occurred in
parallel with the development of systems biology,
focusing on the complex physical and functional
relations between cellular molecules, organs,
and whole organisms (Ideker etal., 2001; Kitano,
2002), and the emergence of network pharmacol-
ogy. Network pharmacology, discussed in detail
in chapter14 - Systems (network) pharmacology
for brain functionality restoration, refers to treat-
ment comprising combinations of compounds to
maximize the required therapeutic effect and
minimize undesired adverse effects of the active
components (Hopkins, 2007; Keith etal., 2005).
Conceptually, this type of combination poly-
therapy is the basis of herbalism, where various
active compounds are contained in every plant
(herb) derived extract and mixtures of herbs are
used for treatment of medical conditions. Indeed,
several publications have recently attributed net-
work pharmacology characteristics to herbal
treatment as practiced in TCM (Li and Zhang,
2013; Zhang etal.,2013).
The introduction of system biology and net-
work pharmacology is particularly relevant to
the treatment of epilepsy, one of the most com-
mon neurological disorders. Whereas epilepsy
(a chronic neurologic condition characterized
by recurrent epileptic seizures; Blume et al.,
2001)used to be viewed only as a seizure disor-
der, the current widely used definition acknowl-
edges that epilepsy is a disorder of the brain
characterized by an enduring predisposition
to generate epileptic seizures and by the neu-
robiologic, cognitive, psychological, and social
consequences of this condition (Table1, p.471)
334 Part III:Homeostatic Manipulators
(Fisher et al., 2005). This definition reflects the
broader, network-like character of epilepsy,
implies a complex pathophysiology, and suggests
the need for treatments aimed at alleviating both
the occurrence of epileptic seizures as well as the
related consequences or comorbidities.
In addition, a great emphasis has been put
on mitigating the adverse effects of antiepileptic
drugs (AEDs). Accordingly, one of the four 2014
National Institute of Neurological Disorders
and Strokes benchmarks for epilepsy research is
Improve treatment options for controlling sei-
zures and epilepsy-related conditions without
side effects.
Epilepsy, like many other central nervous
system (CNS) disorders, can be viewed as dis-
equilibrium of normal brain homeostasis, and
therefore the goal of treatment should be to
restore healthy homeostasis. While using net-
work pharmacology as a strategy to discover new
AEDs has already been proposed (Margineanu,
2013), we present the case for considering botani-
cals as potential therapies for epilepsy, consistent
with the principles of network pharmacology.
T R A D I T I O N A L T R E AT M E N T
W I T H B O TA N I C A L S U S E S
MIXTURES OFHERBS
According to TCM, health is perceived as a bal-
anced, homeostatic state. Disease is caused by
unbalance of various defined functional and
energetic systems, as diagnosed by a complex
of symptoms derived from observation, auscul-
tation and olfaction, interrogation, and pulse
analysis. Therefore, treatment is aimed at restor-
ing the homeostasis of the organism. Chinese
herbal medicine is the most widely used among
all TCM practices. More than 8,800 different
medicinal compounds, extracted mainly from
plants but also from animals and minerals, are
known. These products are grouped into three
classes. The lowest class has a low therapeutic
index (ratio of toxic dose to effective dose), with
herbs meant to be taken in small doses to treat
specific disorders for short periods of time. The
medicinal herbs in the middle class are safer but
are also not recommended for prolonged use. The
top class includes products that are the safest and
are recommended for daily consumption to pro-
mote general health and longevity (Sucher,2013).
Combination herbal therapy is a fundamen-
tal principle of TCM, and the herbal combination
formulas have been developed over thousands
of years by taking into consideration both
theoretical and empirical aspects of the proper-
ties of single herbs and combinations. The con-
temporary traditional Chinese physician usually
chooses from about 500 classical formulas, each
combining 4 to 10 medicinal herbs on average.
The prescription formulas are chosen to con-
form to the jun (ruler, monarch) chen (minis-
ter, official) zuo (assistant) shi (enabler) principle
that guides specific combinations. The mon-
arch is the ingredient directed to act against
the main cause and symptoms of the disease.
The ministers attack the underlying causes of
the disease and the accompanying symptoms and
complications. The assistant compounds help
the two leading types of drugs to achieve their
effects by counteracting their potential adverse
effects and by treating secondary symptoms of
the disease. The enablers direct the actions of
the other drugs to ensure that together the other
ingredients do not exceed the patients capacity
to cope with their actions (Sucher, 2013). These
prescription formulations, based on combina-
tions of herbs with various actions, are designed
to achieve homeostasis in systemic conditions, as
well as in brain disorders, and whether they treat
disease with a lower risk of undesirable adverse
effects compared to Western medicines requires
furtherstudy.
Dose-related adverse effects are particularly
problematic consequences of Western treatments
for chronic CNS disorders. A systematic review
and network meta-analysis of the tolerability of
AEDs for refractory focal epilepsy included 8,546
patients in 43 different trials taking a total of 11
AEDs (Bodalia et al., 2013). Overall, the odds
ratio for premature drug withdrawal due to devel-
opment of adverse effects for all AEDs versus pla-
cebo was 3.27. The lack of high-quality trials of
herbal treatments precludes comparison between
these treatments and modern AEDs, whether for
efficacy, safety, or tolerability, but recent trials
of botanicals have suggested better tolerability
compared to phenobarbital (Li etal.,2009).
B O TA N I C A L S C O N TA I N
MIXTURES OFACTIVE
COMPOUNDS
Individual plants frequently exert numerous
beneficial effects on disease states. Curcumin,
for example, has been shown to ameliorate vari-
ous neurological conditions (Kulkarni and Dhir,
2010). The homeostatic effect of curcumin on
the brain was nicely demonstrated in an animal
model of chronic epilepsy induced by chemical

kindling. This plant extract not only alleviated
epileptic seizures and improved depression,
learning, and memory but was shown to nor-
malize epilepsy-induced changes in brain nor-
epinephrine, serotonin, and nitrite level and
acetylcholinesterase activity as well (Choudhary
etal.,2013).
The complexity of botanical treatments can
be at least partially explained by the plethora
of compounds with various effects that can
be extracted from each individual plant. For
example, about 80 different phytocannabinoid
compounds can be extracted from the cannabis
sativa plant. The most psychogenic compound,
9-tetrahydrocannabinol (9-THC), has received
the most attention. However, numerous phyto-
cannabinoids with very weak or no psychotropic
effects can be extracted from the cannabis plant.
These include, among others, cannabidiol (CBD),
cannabigerol (CBG), cannabichromene (CBC),
9-tetrahydrocannabivarin (9-THCV), canna-
bidivarin (CBDV), 9-tetrahydrocannabinolic
acid (9-THCA), and cannabidiolic acid (CBDA)
(Izzo et al., 2009). There is evidence of ancient
medical use of cannabis in China (named
ma-fen), Assyria (azallu or gan-zi-gun-nu),
and India (Indian hemp). Cannabis was pre-
scribed for the treatment of a variety of systemic
and neurological conditions, including fever,
arthritis, weight loss, injuries, depression, impo-
tence, pain, inflammation, epilepsy, migraines,
neuralgia, cramps, and tetanus (Mechoulam and
Hanu, 2001). The purported ability to ameliorate
a large diversity of pathologic conditions suggests
the possibility of a general, homeostatic effect of
this plants constituent compounds. However,
this effect ceases to exist beyond a certain dose
of the remedy, when adverse effects are fre-
quently encountered. This limitation of cannabis
has been well known, and its psychotropic and
gait-disturbing properties when taken in excess
were mentioned even in ancient Chinese records
(Mechoulam and Hanu,2001).
Modern science has managed to decipher
many of the balancing effects of cannabis by
identifying different mechanisms of action of
many of its compounds. For example, 9-THC
was found to increase appetite and stabilize
weight in patients with AIDS (Beal etal., 1995),
and CBD showed anxiolytic activity and increase
in serotonergic activity in animal models (Izzo
etal., 2009), properties that may explain its use
for the treatment of depression. The potential
of cannabis to help in the treatment of epilepsy
Botanicals 335
may be explained on the basis of the comple-
mentary activity of different compounds on sev-
eral mechanisms, since 9-THC and CBD were
found to inhibit T-type calcium channels (Ross
etal., 2008), CBD regulates intracellular calcium
concentration (Ryan etal., 2009), and 9-THCV
modulates inhibitory activity (Ma etal.,2008).
HER BAL EXTR ACT ED
C O M P O U N D S E X E R T A VA R I E T Y
O F C O M P L E M E N TA R Y E F F E C T S
The therapeutic effects and restoration of homeo-
stasis exerted by botanicals may potentially be
explained by the combined properties of numer-
ous single compounds extracted from plants.
Each one of these compounds is usually not a
magic bullet, as desired for pharmaceutical
drugs, but rather a molecule with various sites of
action that mildly modulates physiological pro-
cesses. For example, CBD, the major nonpsycho-
tropic compound of cannabis, has been reported
to exert its effects on signaling pathways by bind-
ing to at least 22 different sites (Izzo etal., 2009).
Its influence on intracellular calcium concen-
trations (a major modulator of physiologic and
pathologic processes in the CNS) is of particu-
lar interest, since CBD was shown to increase or
decrease those concentrations, depending on the
excitatory status of the cell, therefore exerting
its action by promoting intracellular homeosta-
sis of this important element (Ryan etal., 2009).
Though many of the clinical consequences of
CBD have been associated with its mechanisms of
action, other associations remain to be revealed.
Another example of a homeostatic herbal
compound is the alkaloid Huperzine A, extracted
from the plant Huperzia serrata. This plant has
been traditionally used in China for the treat-
ment of many conditions, including contusions,
strains, swelling, and schizophrenia (Ma et al.,
2007). Huperzine A is approved in China for
treatment of dementia. Arandomized controlled
trial performed in the United States showed only
classIII evidence of its efficacy in mild to mod-
erate Alzheimers disease, demonstrating cogni-
tive benefit after 16 weeks of treatment with 0.8
mg Huperzine Aa day compared to placebo, and
failed to show efficacy at the primary end point of
improvement in scores on the cognitive subscale
of the Alzheimer's Disease Assessment Scale with
a daily dose of 0.4 mg (Rafii etal., 2011). However,
many preclinical studies reported cognitive
improvement after treatment with Huperzine A,
and numerous mechanisms of action support the
336 Part III:Homeostatic Manipulators
same line of evidence (Zhang et al., 2008). The
main mechanisms of action of Huperzine Aare
inhibition of acetylcholinesterase (Tang et al.,
1989) and antagonism at the NMDA receptor
(Zhang and Hu, 2001). Additionally, it was found
to exert other influences in laboratory studies rel-
evant to cognitive dysfunction, as well as other
neurological disorders, such as multiple sclero-
sis, amyotrophic lateral sclerosis, pain, epilepsy,
and stroke, by protecting against oxidative stress,
inhibiting apoptosis, promoting neurogenesis,
decreasing inflammation, and modulating ion
currents (Bialer etal., 2009; Hemendinger etal.,
2008; Li and Hu, 2002a, 2002b; Ma et al., 2013;
Pohanka etal., 2011; Tian etal., 2013; Wang etal.,
2008; Yu etal., 2013). The homeostatic properties
of Huperzine A, similarly to those of other herbal
products, are dose-dependent, implying the need
for physician-guided administration, despite
the common notion that natural means safe
and thus that the consumption of herbal reme-
dies does not have to be as rigorously monitored
as do pharmaceuticals. This dose-dependent
homeostasis is reflected by dual opposite actions
of the compound: it supports the proliferation
of cultured neuronal stem cells when used in
low concentrations as opposed to inhibition of
their proliferation in high concentrations (Ma
etal.,2013).
I M P R O V I N G T R E AT M E N T
OF CNS DISORDERS BASED
ON LESSONS FROM USE OF
B O TA N I C A L S
The main protocol for drug development in
the modern pharmaceutical industry has been
target-based. This approach implies in-depth
acquaintance with disease processes and with the
mechanisms of action of candidate compounds.
However, since the productivity of this approach
has waned, for example in epilepsy, the need for
a paradigm shift emphasizing the disease to be
treated and its manifestations has been stressed
(Schmidt and Schachter, 2014). Our knowledge
of mechanisms involved in pathophysiological
processes of diseases and exerted by drugs is at
best only partial (Sams-Dodd, 2013), and tradi-
tional herbal remedies represent a rich potential
source for a pipeline of novel, systems-based,
disease-centered medications (Sucher, 2013).
In this way, botanicals can be used according
to their traditional indications after acquiring
stringent evidence for their safety and efficacy,
using modern testing protocols and manufac-
turing methods. Constituent herbal compounds
can be used in combination, based on their
mechanisms of action, to achieve best therapeu-
tic results; and botanicals may lead to discov-
ery of new target sites for an enhanced rational
approach to treatment combining the best of
traditional and modern approaches. However,
these great opportunities are limited by numer-
ous challenges encountered when performing
clinical studies and by the incomplete informa-
tion available to date on the biological actions of
botanicals in various CNS disorders (Head and
Kelly, 2009; Izzo et al., 2009; Namjooyan et al.,
2014; Sucher,2006).
I N T E G R AT I N G H E R B S A N D
HERBAL COMPOUNDS IN
WESTERN MEDICINE AFTER
THEIR RIGOROUS TESTING
BY THE PRINCIPLES OF
EVIDENCE-BASED MEDICINE
Despite their thousands of years of use, most
herbal remedies have never been rigorously
tested using modern preclinical animal mod-
els or in human randomized controlled trials
for safety, efficacy, and tolerability, as required
by regulatory agencies such as the US Food and
Drug Administration, before they can be pre-
scribed for specific diseases. The most promising
botanicals should be studied using a bench-to-
bedside paradigm with full incorporation of
regulatory oversight. Such an approach for the
treatment of epilepsy is the Harvard Epilepsy
Botanical Program (Schachter, 2009) launched
by one of the authors (SS). This program was
established to support preclinical evaluation of
herbal therapies for epilepsy. Its goals are (a) to
identify herbal therapies and compounds iso-
lated from them that have promising activity
in animal epilepsy models and relevant in vitro
assays, (b)to conduct the preclinical studies nec-
essary to proceed with early-stage clinical stud-
ies, (c)to plan and initiate these clinical studies.
As part of the program, numerous mixtures and
individual herbal extracts and isolated com-
pounds have been tested through the National
Institute of Neurological Disorders and Strokes
Anticonvulsant Screening Project and in in vitro
assays of neuronal receptor or ion channel func-
tion. More than half of these products (70%) have
shown promise in preclinical testing. The lead
product of this pipeline is Huperzine A, men-
tioned earlier. This compound was found to be
extremely potent in suppressing seizures in ani-
mal models of seizures (Bialer etal., 2009)and is
now advancing to clinical trials.

The main challenges limiting the design of
clinical studies on the use of botanicals and the
interpretation of their results are the consequence
of the large variability in plants contents. The pre-
cise chemical composition of plants extracts are
not only influenced by the exact strain, place of
growth, and weather but also by even minor dif-
ferences in the extraction method. Additionally,
the interpretation of results of studies performed
in regions of the world where herbal medicine
has been traditionally implemented is problem-
atic, since the practice of diagnostics is differ-
ent in these regions than in Western medicine,
using a more holistic approach. Furthermore,
the prescription of natural remedies usually
includes individualized mixtures of botani-
cals, aimed not only to be directly ingested but
sometimes prepared as infusions or inhaled. The
relative ease of purchasing herbal remedies as
compared to prescription medications and the
perception of botanicals as safe lead in many
cases to their worldwide empirical use in practice
well before they have been stringently studied.
However, these obstacles should not prevent the
medical community from aiming to implement
FIGURE19.1: Traditional medical systems make use of mixtures of botanicals containing combinations of herbs,
and, in turn, numerous single chemical compounds.
Botanicals 337
the highest research standards when studying
botanicals.
M A N U FA C T U R I N G P R O D U C T S
T O A F F E C T N E W LY
D I S C O V E R E D TA R G E T S
Botanicals may lead to discovery of new physi-
ologic pathways that will help us better under-
stand disease processes and develop therapies.
For example, research on the mechanisms of
action of cannabis compounds revealed their
receptors (Matsuda et al., 1990; Munro et al.,
1993), which had been previously unknown, and
consequently the endocannabinoid (Mechoulam
and Ben-Shabat, 1999) system was revealed.
The elements of this complex system were then
promptly proposed as targets for the pharmaceu-
tical industry (Piomelli etal.,2000).
Traditionally used herbal medications may
also lead to discovery of new pathways and
possible pharmaceutical targets involved in
the pathophysiology of various medical condi-
tions. For example, as mentioned earlier, cur-
cumin has been shown to ameliorate various
brain disorders, including epilepsy, depression,
338 Part III:Homeostatic Manipulators
and Alzheimers disease (Kulkarni and Dhir,
2010). Treatment with curcumin in an animal
model of epilepsy was superior to phenytoin
in alleviating memory deficits in addition to
seizures and depressive symptoms. Since, in
contrast to phenytoin, curcumin also normal-
ized brain levels of neuroepinephrine and ace-
tylcholinesterase activity (Choudhary et al.,
2013), these mechanisms could be targeted
by the pharmaceutical industry in the devel-
opment of novel antiepileptic drugs. Other
possible targets revealed by the bioactivity
of curcumol, a volatile essential oil extracted
from the curcumin plant, are the 2 and sub-
units of the glycine receptor, expressed mainly
in the hippocampus (Wang et al., 2012). The
hippocampal glycine receptors, which induce
tonic inhibition, have been implied in the
pathophysiology of learning and memory defi-
cits (Wang etal., 2012)and of epilepsy (Eichler
etal., 2008). Current modern methods for stud-
ying the biological actions of small molecules
should be adapted to facilitate the study of the
mechanisms of action of herbal medicines and
to find new therapeutic targets.
SUMMARY
Treatment of CNS disorders with botanicals may
re-establish homeostasis by their complementary
multitargeted effects at the levels of mixtures,
extracts, and compounds (Figure 19.1). These
effects may be exploited for therapeutic benefit,
either by use of the herbal remedies themselves
after modern testing or by targeting new disease
pathways revealed by the botanicals with indi-
vidual compounds.
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20
The Role ofAcupuncture
inNociception Homeostasis
N A N N A G O L D M A N , TA K A H I R O TA K A N O , B E N J A M I N T. K R E S S ,
AND MAIKEN NEDERGA ARD
CHALLENGES AND PROGRESS
I N U N D E R S TA N D I N G
ACUPUNCTURE
Acupuncture is a form of traditional Chinese
medicine that has been practiced for more than
4,000 years. Despite widespread use, however,
there is a lack of understanding of how acu-
puncture derives its therapeutic efficacy. This
is true in part because the indications for acu-
puncture treatment are exceedingly diverse
and may include wide-ranging applications for
gastrointestinal (e.g., heartburn) dermatologic
(e.g., acne), or psychiatric disorders (e.g., anxi-
ety, insomnia, or depression), among many oth-
ers. The most common use of acupuncture is for
the relief from pain. Although the exact protocol
often varies depending on the practitioner, the
treatment for pain typically utilizes a fine needle
that is inserted a depth of ~1 to 2inches into spe-
cific points (acupoints) on the patients body. The
needle is then intermittently rotated to initiate
acupunctures analgesic properties, or, in certain
variations, heat or a low-grade electric current
are applied to the needles in order to amplify or
prolong the treatments therapeutic efficacy.
Many historical events contribute to why so
little is known about the pathways involved in
acupuncture-mediated analgesia. Acupuncture is
based on the philosophical principles of Eastern
medicine that traditionally do not search for bio-
logical mechanisms as underlying causes of dis-
ease. For example, the anatomical and functional
distinctions of acupoints are based on the ancient
theory of communication channels, called merid-
ians, that are said to facilitate transmission of the
bodys energy source, or Qi. According to this
theory, it is critical to have a balance of energy
streaming through the major 12 meridians in the
body. Blockade of such energy flow is thought
to result in diseases (Yang etal., 2011), however
the metaphysical nature of this hypothesis has
hindered acupuncture from being the subject of
empiricalstudy.
The evolving political climate in China also
contributed to the lack of information about
the biological mechanisms of acupuncture.
Due to widespread epidemics of infectious dis-
eases and the comparative success of Western
medicine, particularly at promoting notions of
public hygiene, the Kuomintang government
in China outlawed many traditional Chinese
medical practices in the early 20th century and,
in 1929, closed schools devoted to the teaching
of traditional Chinese medicine (Veith, 1973;
Yang et al., 2011). This decision resulted in at
least two decades of official abandoning of acu-
puncture. It was not until Mao Tse-Tung and the
Communist Party rose to power in 1949 that the
use of traditional Chinese medicine regained
widespread popularity. However, in order to
limit the growing influence of Western powers
over Chinese culture, the Communist Party did
not encourage objective studies of acupuncture,
which in turn created a distrust in the Western
world toward acupunctures therapeutic effi-
cacy. Several clinical studies have addition-
ally reported a lack of benefit of acupuncture
over placebo (Moore and Berk, 1976;Petrie and
Hazleman, 1986), which has further hindered
the integration of acupuncture into Western
medical practices.
Other studies, however, support the clini-
cal benefits of acupuncture, including those
conducted in experimental chronic pain
animal models, which have the advantage
of lacking potentially confounding placebo
effects (Cantwell, 2010; Koski, 2011). A recent
meta-analysis of 35 eligible randomized
342 Part III:Homeostatic Manipulators
controlled trials indicates that acupuncture
provides a significant benefit over placebo when
used for treatment of chronic neck, back, or
osteoarthritis pain (Vickers et al., 2012). Such
data have contributed to the growing popular-
ity of acupuncture and the growing recognition
of its therapeutic value by Western agencies
such as the World Health Organization and
the US National Institutes of Health (NIH)
(World Health Organization, 2003). For
example, acupuncture is currently practiced
in more than 160 countries around the world
(Zhao, 2008), and in a consensus statement in
1997 the NIH recognized that acupuncture
may serve as an adjunct treatment or accept-
able alternative therapy for postoperative den-
tal pain, headache, menstrual cramps, tennis
elbow, fibromyalgia, myofascial pain, osteoar-
thritis, carpal tunnel syndrome, and low back
pain (Acupuncture, 1997). According to the
NIH National Center for Complementary and
Alternative Medicine, the 2007 National Health
Interview Survey revealed that an estimated
3.1million US adults and 150,000 children used
acupuncture in 2006. Nevertheless, the lack of
a defined mechanistic basis of acupuncture
has led to skepticism by physicians who do not
subscribe to its underlying philosophical prin-
ciples. In particular, the absence of verifiable,
anatomical acupoints or meridians represents
a major conceptual barrier to Western medi-
cine (Ahn et al., 2008). As a result, until very
recently, acupuncture has not been the focus of
rigorous empirical study, and thus is not com-
monly recommended as a first-line treatment.
Consequently, the general public is forced to
seek treatment from potentially underquali-
fied practitioners, in many cases under uncon-
trolled conditions. Defining the mechanistic
basis for acupuncture-mediated analgesia is
thus a critical step for its acceptance to the
health-care system and also an essential step
for improving its safety and clinical benefits.
Although skepticism remains, California and
Maryland recently mandated acupuncture cov-
erage in all health insurance plans for individu-
als and small groups.
ANALGESIC ACTIONS
OFADENOSINE
Adenosine, a purine nucleoside, has potent
antinociceptive properties in both animals
and humans. Adenosine signals through
four distinct receptors: A1, A 2A, A 2B, and A 3.
Of the adenosine receptors, the A1 receptor
has received the greatest attention in the field
of pain research due to its widely distributed
expression pattern in the dorsal spinal cord,
especially in lamina II (substantia gelatinosa;
Choca et al., 1988; Horiuchi et al., 2010). In
this region, most afferent sensory nerves
make connections with postsynaptic neurons.
Similarly important for antinociception is the
localized expression pattern of A1 receptors in
the descending projection of the dorsal horn
(Choca etal.,1988).
In both the peripheral and central nervous
system, the endogenous release of adenosine
inhibits pain transmission by inhibiting the
activity of nociceptive pathways (Guo et al.,
2013; Taylor, 2009), and many groups have
identified A1 receptors as the main adenosine
receptor involved in the analgesic actions of
adenosine (Burnstock, 2007; Sawynok, 1998;
Sawynok and Liu, 2003). Interestingly, manipu-
lations such as depolarization (high K+) or the
application of agonists, including capsaicin,
substance P, N-methyl-D-aspartate (NMDA),
morphine, or serotonin (5-HT), have all been
shown to trigger elevations in extracellular
adenosine concentrations (Latini and Pedata,
2001), suggesting that adenosine release may
constitute a physiological regulator of pain
signaling. In support of this concept, acti-
vation of peripheral and central vanillinoid
receptors (TRPV1) among primary afferent
C-fibers has been shown to trigger adenosine
release that attenuated inflammatory and ther-
mal pain transmission directly by inhibiting
TRPV1-mediated Ca 2+ entry into dorsal root
ganglion neurons (Puntambekar et al., 2004).
The results of that study also reported that
enhanced capsaicin concentrations overcame
the adenosine-induced inhibition of nocicep-
tive signaling, suggesting that adenosine and
capsaicin exert competitive inhibition of the
TRPV1 receptor. The response profile to cap-
saicin is particularly interesting because it sug-
gests that agonists of TRPV1 and adenosine
exert homeostatic control over nociceptive
signaling, presumably to facilitate rapid acti-
vation (detection and propagation of a noxious
thermal or inflammatory stimulus) followed
by rapid reset, respectively, of the nociceptive
circuit (to maintain the functional sensory unit
and prepare for detection of future threats).
In further support of this concept, systemic
administration of various A1 receptor agonists
has been shown to produce analgesic effects
in a variety of acute pain models in animals
 The Role of Acupuncture in Nociception Homeostasis
(Gong etal., 2010). For example, several studies
show that intrathecal injection of A1 receptor
agonists induced analgesia in various animal
models of acute pain. These tests include tail
flick, tail immersion, hot-plate, formalin, acetic
acid, capsaicin models, and others (Nascimento
et al., 2010; Song, 2011; Zahn et al., 2007).
Similarly, mice lacking the A1 receptor (knock-
out animals) exhibit a lower pain threshold in
hyperalgesia tests (Wu et al., 2005). Further,
the analgesic effect of intrathecal adenosine
administration was abolished in A1 receptor
knockout mice (Johansson etal.,2001).
In addition to suppressing acute pain trans-
mission, agonists of A1 receptors have also been
shown to reduce chronic pain (Figure 20.1).
The agonist R-PIA inhibits mechanical allo-
dynia induced by spinal nerve ligation in
rats (Hwang et al., 2005; Song, 2011). R-PIA
also increases the heat pain threshold in rats
that underwent an injury in the spinal cord
(Horiuchi et al., 2010). Another selective A1
receptor agonist, CPA, inhibits pain induced by
arthritis or neuropathy in rats (Curros-Criado
and Herrero,2005).
Spinal cord
Systemic
Acupoint
Site of pain
FIGURE 20.1: Site in which injection of A1 receptor
agonists have been documented to have an analgesic
effect in chronicpain.
Intratecal administration (Curros-Criado and Herrero,
2005; Horiuchi etal., 2010; Hwang etal, 2005; Johansson
etal., 2001; Song etal., 2011; Zahn etal.,2007)
Systemic administration (Gong etal., 2010; Nascimento
etal.,2010)
Acupoints (Goldman etal., 2010; Hurt and Zylka,2010)
Local administration of adenosine receptor agonist at the
locus of pain. A1 receptor agonists have analgesic effects
while A2 receptors mediate proalgesic effects (Karlsten
etal., 1992; Taiwo and Levine,1990).
The agonists in all studies were injected locally, except
for the systemic administration in which the agents
were delivered intravascular.
343
ADENOSINE SIGNALING
I S A K E Y M E D I AT O R
OFTHE ANALGESIC ACTIONS
OFACUPUNCTURE
While most investigations of the biological mech-
anisms of acupuncture have focused on central
endorphin signaling (Han, 2004;Neumann etal.,
1996; Zhao, 2008), other lines of work indicate
that acupuncture produces analgesia by act-
ing locally (Melzack et al., 1977). For example,
acupuncture-induced analgesia is most potent
when applied close to rather than distant to
the locus of pain and normally restricted to the
ipsilateral side (Lao et al., 2004; WM Li, 2005),
pointing to local mechanisms. In addition, many
acupoints reside along the major nerves (Gunn
et al., 1976). In fact, a systematic analysis con-
cluded that 70% of acupoints are found either
along the pathways of major peripheral nerves,
superficial branches of peripheral nerves, or at
sites where cutaneous nerves emerge from the
deep fascia (Bossy, 1979). For example, Zusanli
(ST36), a widely used and studied acupoint, is
located above the deep peroneal nerve, and, upon
stimulation, the needle tip lands in close proxim-
ity to the nerve fiber (Yang etal.,2007).
Multiple lines of work have documented
that adenosine triphosphate (ATP) is released
in response to either mechanical and electrical
stimulation or heat (Sauer et al., 2000; White
etal., 1982). Once released, ATP acts as a trans-
mitter that binds to purinergic receptors, includ-
ing P2X and P2Y receptors (Abbracchio et al.,
2009; Burnstock, 2007). ATP is not transported
back into the cell but rather is rapidly degraded to
adenosine, inosine, or IMP by ectonucleotidases
or deaminases before reuptake (Burnstock, 2007).
Based the literature discussed previously, it was
hypothesized that the mechanical stimulation
over the unique anatomical sites in which acu-
puncture is commonly applied leads to increased
levels of extracellular adenosine, which in turn
mediates the analgesic actions of acupuncture.
The first evidence in support of this hypothesis
was documented in a series of experiments con-
ducted by the Nedergaard lab group (Goldman
et al., 2010). To determine whether adenosine
was involved in the antinociceptive effects of acu-
puncture, Goldman and colleagues first asked
whether the extracellular concentration of aden-
osine increases during the needle insertion and
gentle manipulation associated with a typical
acupuncture treatment. The results showed that,
in mice, acupuncture triggered a localized release
of tri-, di-, and mono-phosphorylated adenosine
344 Part III:Homeostatic Manipulators

nucleotides (ATP, ADP, and AMP) in addition to
adenosine. This was consistent with the obser-
vation that tissue damage is associated with an
increase in extracellular nucleotides and adeno-
sine (Fredholm, 2007). The authors acquired
direct confirmation of their hypothesis by collect-
ing samples of interstitial fluid by a microdialysis
probe implanted in the tibialis anterior muscle/
subcutis of adult mice at a distance of ~0.5mm
from the Zusanli point, which is located below
and slightly lateral for the midline of the knee
(Zhao, 2008). Adenine nucleotides and adeno-
sine were quantified using high-performance
liquid chromatography before, during, and after
acupuncture (Cui etal., 2010;Volonte etal., 2004).
At baseline, the concentrations of ATP, ADP,
AMP, and adenosine were in the low nanomolar
range, as previously reported (J Li etal., 2003; J
Li etal., 2005). Acupuncture applied by manual
rotation of the acupuncture needle every 5 min
for a total of 30 min sharply increased the extra-
cellular concentrations of all purines detected.
Adenosine concentration increased 24-fold dur-
ing the 30-min acupuncture session.
The critical role of adenosine in mediating
the analgesic effects of acupuncture was further
tested by locally injecting the selective A1 recep-
tor agonist, 2-chloro-N(6)-cyclopentyladenosine
(CCPA; Lohse etal., 1998)in two murine mod-
els of either inflammatory pain or neuropathic
pain (Goldman etal., 2010). Inflammatory pain
was assessed by quantification of the withdrawal
threshold from a thermal or tactile stimulus
following the injection of complete Freunds
adjuvant (CFA) into the right paw of both A1
knockout mice and wild-type mice. As a result
of the CFA injection, both groups of mice devel-
oped mechanical allydonia (Von Frey filament
touch test) and thermal hyperalgesia (thermal
test). When the adenosine A1 receptor ago-
nist CCPA was locally injected into the Zusanli
acupoint, it produced transient antinociceptive
effects reflected by a reduction of both mechani-
cal and thermal hypersensitivity. However, in A1
receptor knockout mice, CCPA failed to reduce
pain. These observations indicated that A1 recep-
tor expression was necessary for CCPA-mediated
pain suppression in inflammatory pain.
Neuropathic pain was modeled using the ligation
injury of the sciatic nerve (Vadakkan etal., 2005),
in which pain peaked 5 to 7days after nerve liga-
tion. CCPA injected in the Zusanli point of the
ipsilateral leg reduced neuropathic pain with an
efficacy that was comparable to its suppression
of inflammatory pain (Figure 20.2c, d). In each
model, the antinociceptive effect of CCPA was
transient and consistent with a local mechanism
of pain suppression, as it did not alter sensitivity
to painful stimulation in the contralateral leg. In
addition, injection of CCPA into the contralat-
eral leg did not alter the pain threshold of the
ipsilateral leg. These experiments revealed that
localized stimulation of A1, whether through an
agonist or via acupuncture, alleviated symptoms
associated with inflammatory pain and neuro-
pathic pain (Figure20.2).
Goldman and colleagues observations indi-
cated that the antinociceptive action of acupunc-
ture is mediated by activation of A1 receptors
located on ascending nerves. Thus medications

Acupuncture

Autocrine action to fibroblasts

ATP

ATP AMP Adenosine A1R

ADP
IMP Inosine
Fibroblasts Nociceptive
Skeletal muscle nerve

FIGURE 20.2: Proposed mechanism of acupuncture-induced analgesia. Insertion and rotation of acupuncture
needle triggers release of adenosine triphosphate (ATP) from tissue fibroblasts and skeletal muscle induces release
of ATP. Extracellular ATP is in part converted to adenosine and activates A1 receptors. ADP=adenosine diphph-
sphate, AMP=adenosine monophosphate, IMP=inosine monophosphate.
 The Role of Acupuncture in Nociception Homeostasis 345

that interfere with A1 receptors or adenosine
metabolism may improve the clinical benefit of
acupuncture. For example, it has been previously
shown that inhibition of adenosine metabo-
lism prolongs the biological effects of adenosine
(Sawynok, 2007). In accordance with this find-
ing, the Nedergaard group hypothesized that
the inhibition of adenosine deaminase would
prolong the beneficial effects of acupuncture.
The concept was tested using an Food and Drug
Administrationapproved adenosine deaminase
inhibitor, deoxycoformycin. Combined injections
of deoxycoformycin with acupuncture resulted
in antinociceptive effects that lasted 2.0 to 2.5 hrs
longer than acupuncture alone (Goldman etal.,
2010). In addition to further proving that acu-
puncture acts via adenosine receptors, the results
of these studies imply that it is possible to extend
the clinical utility of acupuncture as a safe and
effective treatment for pain. The observations
may be particularly important for the manage-
ment of patients with chronic pain who require
long-term treatment, as expanding the duration
of acupunctures analgesic effects could require
fewer sessions to achieve continued therapeutic
benefits.
ACUPUNCTURE TRIGGERS
RELEASE OFADENOSINE
INHUMAN SUBJECTS
An open question is whether acupuncture per-
formed by an experienced practitioner, and
according to standard technique, similarly trig-
gers the release of adenosine in human subjects.
The possibility existed that adenosine release
in mice exposed to acupuncture was an experi-
mental artifact, since the acupuncture needles
utilized were disproportionally large compared
to the small body weight of mice. Takano and
colleagues, also from the Nedergaard lab group,
addressed this important question directly
by collecting microdialysis samples of inter-
stitial fluid before, during, and after deliver-
ing 30 min of conventional acupuncture in the
Zusanli point in human volunteers (Takano
et al., 2012). Similar to the findings from mice,
the analysis showed that the interstitial adeno-
sine concentration increased significantly dur-
ing acupuncture and remained elevated for
30 min after the acupuncture (Figure 20.3;
Takano et al., 2012). Interestingly, the analy-
sis showed that acupuncture not only had to be
delivered at the Zusanli point in order to achieve
analgesia but that the needle had to be rotated to
evoke significant adensoine release (Figure 20.3).
Acupuncture-mediated adenosine release was
not observed if acupuncture was delivered in tis-
sue outside the Zusanli point (Figure20.3).
Other studies of acupuncture have sug-
gested that the analgesic properties may depend
on the proximity not only to peripheral nerves
but also to regions of enhanced vascularization.
Consistent with this concept, a recent study of
acupoints suggested that the ST36 (Zusanli) and
ST37 (Shangjuxu) acupuncture points exhibit
distinct structural characteristics with a higher
complexity of the local vasculature (C Liu etal.,
2014). X-ray phase contrast imaging in this study
revealed that at the acupuncture points, the
microvascular bed included multiple larger size
vessels (~50100 m in diameter) with bifurca-
tions and smaller vessels (1550m in diameter),
whereas nonacupuncture point areas exhib-
ited few larger vessels and essentially none of
the smaller vessels. Combined with our finding
that robust adenosine release and subsequent

Adenosine
Percent change from baseline

A B AMP
C # Adenosine
250 # AMP
I. Acupoint with/ * ADP 250
* ATP ADP
without rotation 200 **
Percent change

* 200 ATP
II. Non-acupoint *
150 150
with rotation
100 100
Microdialysis
probe 50 50
0 0
Baseline Recovery 1 Acupuncture Without Non-acupoint
Acupuncture Recovery 2 rotation

FIGURE20.3: Acupuncture induces release of adenosine in human subjects. (A)Extracellular purines were col-
lected by a microdialysis probe while acupuncture session was given. (B)Transient increase of tissue adenosine by
acupuncture in human. (C)Adenosine increase was observed only when a needle was inserted in an acupoint and
rotated (Takano etal., 2012). AMP=adenosine monophosphate, ADP=adenosine diphphsphate, ATP=adeno-
sine triphosphate.
346 Part III:Homeostatic Manipulators
analgesia were detected if the acupuncture nee-
dle was inserted and rotated in the Zusanli
point, whereas no analgesia was detected when
the needle was simply inserted without rota-
tion, these observations suggest that the proxim-
ity of acupuncture placement to the peripheral
nerves and to regions of dense vascularization
are important factors that influence the analgesic
properties of acupuncture. They further suggest
that an important determinant of acupunctures
analgesic benefits may be the mode of the applied
stimulus, such as the tissue stretching that results
from needle rotation, which may offer further
insights into the analgesic properties of other
alternative therapies. These potentially include
certain forms of therapeutic massage, such as the
practice known as acupressure, which involves
mechanical manipulation of the acupoints with-
out the need for needle insertion.
O T H E R P U R I N E R G I C - M E D I AT E D
SIGNALING MECHANISMS
A C T I VAT E D B Y A C U P U N C T U R E
Goldman and Takanos studies of experimental
acupuncture in mice and traditional acupunc-
ture in humans strengthened the role of adeno-
sine in acupuncture-mediated antinociception.
However, neither study established the source or
mechanism of adenosine release. Work done by
Helene Langevin has made major strides in this
area and has greatly expanded what we know
today about the cellular response to acupuncture
needle insertion. Langevin et al. first observed
that acupuncture points seem to be mainly located
along connective-tissue planes between muscles,
or between muscle and bone. Her analysis showed
that more than 80% of acupuncture points in the
arm are located along connective-tissue planes
(Langevin and Yandow, 2002). Langevins team
next observed that the fibroblasts residing in the
connective tissue, as far as several centimeters
away from the needle, reorganized their internal
cytoskeleton and changed shape upon insertion
of the acupuncture needle, becoming large and
flat (Langevin etal., 2007). The analysis showed
that fibroblasts in connective tissue respond to
static stretching of the tissue by expanding and
remodeling their cytoskeleton within minutes
both ex vivo and in vivo. In search of a mecha-
notransduction mechanism, Langevin et al.
tested the hypothesis that the mechanism of
fibroblast expansion in response to tissue stretch
involves extracellular ATP signaling. In support
of this concept, fibroblasts express high levels of
connexin 43, which mediates purine release in
multiple cell types (Kang etal., 2008;Wilgenbus
et al., 1992). Subsequent studies showed that
the acupuncture-induced shape change is also
associated with a sustained release of ATP from
fibroblasts and is dependent of P2Y purinergic
receptors (Goldman etal., 2013; Langevin etal.,
2013). However, it appears that the changes in
fibroblast shape are not mediated by adenosine,
since A1 receptor antagonists failed to inhibit
cytoskeletal reorganization induced by ATP
(Goldman etal.,2013).
The same cellular cascade can be observed
by simply stretching the tissue for approximately
30 min, about the same time duration of needle
retention during a typical acupuncture treat-
ment. Interestingly, and perhaps holding impor-
tant implications for enhancing the therapeutic
benefit of acupuncture, the work by Langavin
etal. also found that letting go of the needle after
needle rotation does not cause the tissue to imme-
diately unwind from the needle. The whorl of
connective tissue remains intact as long as the
needle remains under the skin, causing the tissue
to be stretched for a prolonged period. Langevins
team found that the tissue changes with acu-
puncture are associated with a large-scale relaxa-
tion of the connective tissue wherein fibroblasts
initiated a specific Rho-dependent cytoskeletal
reorganization that was required for full tissue
relaxation. Rho is an intracellular signaling mol-
ecule known to play a role in cell motility and the
remodeling of cell-surface proteins that connect
the fibroblast to its surrounding matrix.
Altogether, multiple lines of work have doc-
umented that the extracellular concentration
of ATP, ADP, AMP, and adenosine increases
locally in tissue exposed to traditional mechan-
ical acupuncture. While adenosine and A1
receptor activation play a key role in the anal-
gesic action of acupuncture (Goldman et al.,
2010), indirect evidence points to the impor-
tance of P2Y receptor mediated cytoskeletal
remodeling of fibroblasts (Goldman etal., 2013;
Langevin etal., 2013). Similarly, P2X receptors
are regarded as pain receptors, and both P2X4
and P2X3 receptor antagonists have shown
promising effects in preclinical and clinical
studies (Burnstock, 2013; Tsuda et al., 2013).
Acupuncture is usually not linked to pain sen-
sation, likely because the manipulation of the
needles is slow and gentle, such that ATP con-
centration does not rise fast enough to over-
come desensitization and reach the threshold
 The Role of Acupuncture in Nociception Homeostasis 347

A B 100 Control
Deoxycoformycin
ATP Adenosine 80

Touch test (%)

release release
60 ** ** **
** ** ** **
PAP **
A1R 40
CD39 CD73 cAMP
ATP AMP Adenosine PKA 20
Deaminases
ADP 0
IMP
T 0.5h 1h 1.5h 2h 2.5h 3h 3.5h 4h
Inosine
DCF Before acupuncture
Before CFA

FIGURE 20.4: Ectonucleotidase activity. (A) Extracellular adenosine can be produced from the breakdown
of adenosine triphosphate (ATP) by CD39 and subsequently by prostatic acid phosphatase (PAP) or CD73.
Deoxycoformycin (DCF) blocks AMP/adenosine deaminases. (B)Administration of DCF prolonged the analgesic
effect of acupuncture (Goldman etal., 2010). ADP=adenosine diphosphate, AMP=adenosine monophosphate,
IMP=inosine monophaosphate, PKA=protein kinase, ACFA=complete Freunds adjuvant.

for activation of P2X receptors. Excessive nee-
dle handling, however, will trigger pain, which
may reflect a consequence of higher amplitude
(suprathreshold) increases in interstitial ATP
that then exceed the capacity for degradation to
ATPs antinociceptive purine derivatives. Thus
acupuncture appears to exert therapeutic prop-
erties by capitalizing on innate homeostatic
mechanisms of purine-mediated nociceptive
activation and inhibition, such as evoking the
controlled release of ATP and its inhibitory
degradation products.
LOCAL INJECTION OF
ECTONUCLEOTIDASES:
A POTENTIAL NOVEL
A N T I N O C I C E P T I V E S T R AT E G Y
Activation of A1 receptors by endogenous adeno-
sine has been implicated as key step in mediating
actupunctures analgesic properties. In light of
this, molecular targets important for purinergic
metabolism may be highly relevant for the treat-
ment of pain. Adenosine is primarily metabo-
lized by adenosine deaminase, which removes an
amine group from adenosine to form inosine in
the subcutis and muscles, (Figure 20.4a; Cunha
and Sebastiao, 1991). AMP is readily converted
to IMP by AMP deaminase, while the kinetics
of the dephosphorylation of AMP to adenosine
are slow (Goldman et al., 2010). IMP, inosine,
and AMP are not ligands for purine receptors,
as these degradation pathways quickly eliminate
adenosine. Of the purine ligands, only adenosine
can activate the A1 receptor, albeit a recent report
suggested that AMP also binds to A1 receptors
(Rittiner et al., 2012). When deoxycoformy-
cin was locally administered to inhibit deami-
nase activity and adenosine metabolism, the
analgesic effect of acupuncture was prolonged
(Figure 20.4B; Goldman et al., 2010). In an ele-
gant series of experiments, Mark Zylkas group
recently showed that local injection of prostatic
acid phosphatase, which hydrolyses a phosphate
group from a wide variety of proteins and other
biomolecules including AMP, attenuated chronic
pain for 3 days without acupuncture treatment,
suggesting that endogenous extracellular AMP
is a sufficient source for production of analgesic
levels of adenosine (Hurt and Zylka,2012).
Acupuncture treatment for chronic pain
is typically given at multiple sessions to pro-
duce longer lasting reductions in pain (Guerra
de Hoyos et al., 2004;Thomas et al., 2005).
Acupuncture used in this manner is interest-
ing because long-lasting activation of A1 recep-
tors may activate downstream processes that
counteract inflammation and neural plasticity
that is associated with chronic pain. Adenosine
has in multiple lines of work been shown to
have potent anti-inflammatory roles (Flogel
etal., 2012; Hasko and Pacher, 2008; Ohta and
Sitkovsky, 2001; Sawynok, 1998). Adenosine
interacts with specific G protein-coupled recep-
tors on inflammatory and immune cells to
regulate their function, and injurious stimuli,
ischemia, and inflammation all result in release
of adenosine in tissue (Fredholm, 2007). The
released adenosine then acts as an immunosup-
pressant. For example, mice that were deficient
in A2 receptors showed increased inflamma-
tion tissue damage (Ohta and Sitkovsky, 2001).
Novel insights into how acupuncture achieves
therapeutic benefits open up new strategies for
treating painsuch as manipulation of local
adenosine signalingthat do not necessarily
involve acupuncture.
348 Part III:Homeostatic Manipulators
POTENTIAL CONFOUNDING
EFFECT OFCAFFEINE
Caffeine is a nonspecific antagonist of adenosine
receptors that is present in a wide variety of food
and beverages (Marchand etal., 1995). It is esti-
mated that 90% of the population worldwide con-
sumes caffeine daily, making it the most widely
consumed psychoactive drug (Frary etal., 2005).
The popularity of caffeine can be attributed to its
stimulatory effects, including wakefulness and
enhanced mood (Haskell etal., 2005; Lieberman
et al., 2002). Caffeines high blood-brain bar-
rier permeability allows it to readily enter the
central nervous system, where it then produces
psychoactive effects largely through downstream
consequences of interfering with noradre-
negic, dopaminergic, or serotonergic signaling
via A1 receptor activation (Nehlig et al., 1992).
Neurons in these nuclei are highly sensitive to
caffeine and their metabolic activity activated
after intake of just 1 mg/kg caffeine (Nehlig etal.,
1992). Recently, positron emission tomography
was used to visualize binding sites of caffeine in
the human brain, and the analysis established
that there was 50% occupancy of the brains
A1 adenosine receptors when individuals con-
sumed caffeine throughout the day (Elmenhorst
etal.,2012).
The relationship between caffeine and acu-
puncture is intriguing because it could explain
why a number of clinical trials have found that
acupuncture is not always effective. Caffeines
competition with adenosine binding at the A1
receptor would interfere with the antinociceptive
mechanism of acupuncture. However, the bio-
logical effects of caffeine are complex, making it
difficult to predict its effect on pain. For example,
caffeine has analgesic effects at nonphysiologi-
cal high concentrations and may, at lower doses,
enhance efficacy of other analgesics by actions
not related to the A1 receptors. For example, caf-
feine can improve drug absorption, reduce drug
metabolic clearance, activate norepinephrine
signaling, and alter mood and alertness (Derry
et al., 2012;Sawynok and Yaksh, 1993). Caffeine
may also indirectly interfere with the antinoci-
ceptive action of several agents such as amitrip-
tyline and oxcarbazepine by blocking the A1
receptor (Sawynok,2011).
A recent study revealed very interesting
dose-dependent interactions when caffeine
is combined with both manual and electro-
acupuncture. Mor and colleges took into
account that the average daily caffeine consump-
tion in China is 20 times lower than Western
countries. Mice received plantar incision surgery
and were then treated with acupuncture nee-
dling after administration of acute and chronic
caffeine. For the chronic preadministration of
caffeine, two different doses of caffeine were
administered, one that mimicked the average
daily consumption in Western countries and one
that mimicked the average daily consumption in
China. The analysis showed that acute preadmin-
istration of caffeine (10mg/kg, i.p.) completely
reversed acupuncture analgesia in both mechan-
ical and electro-acupuncture. Interestingly, the
Western dose of caffeine (70mg/kg/day) admin-
istered during 8days in the drinking water also
reversed acupuncture analgesia. However, the
Chinese dose (4mg/kg/day) administered during
the same period did not reverse the effects of acu-
puncture (Mor et al., 2013). It is interesting to
note that the number of studies in China favoring
acupuncture treatment is much higher than in
Western countries. Daily caffeine consumption
is a plausible explanation for this discrepancy.
CONCLUSION
Chronic pain, often defined as pain persist-
ing longer than six months, is a heterogeneous
group of medical conditions that have significant
impact on an individuals physical, emotional,
psychological, and financial distress. According
to an NIH estimate in 2001, more than 50million
Americans suffer from chronic pain, and approx-
imately 45% of the population visit clinical offices
for pain at some point in their lives (National
Institutes of Health, 2001). Although the anal-
gesic effect of acupuncture for the treatment of
chronic pain is well documented (Kelly 2009;
Weidenhammer etal., 2007; Zhao, 2008), its bio-
logical basis is not well understood, and it has
only recently been the subject of extensive study.
Clear benefits of acupuncture set the procedure
apart from traditional analgesic treatments,
which include nonsteroidal anti-inflammatory
agents (such as ibuprofen and naproxen), and
opioids (such as morphine and codeine). Aspirin
and other nonsteroidal anti-inflammatory agents
are generally ineffective in treating neuropathic
pain (Portenoy, 2000). While they are effective
in inflammatory pain states, there is a ceiling
effect to the pain relief they provide, and adverse
effects to the gastric system may become a serious
health concern with high doses or with long-term
use (Ashburn and Staats, 1999; Mukherjee etal.,
2001; Ong etal., 2007; Portenoy, 2007). Opioids
are frequently used for cancer pain manage-
ment, but due to the risks of increased tolerance,
 The Role of Acupuncture in Nociception Homeostasis
dependency, and psychological and hormonal
adverse effects, many providers are now ques-
tioning the expanded role of long-term opioid
therapy (Ballantyne and Mao, 2003;Noble etal.,
2010; Rowbotham etal., 2003). Thus the lack of
alternative approaches to treat chronic pain is a
large societal problem.
Acupuncture is an effective and relatively
cheap procedure that in controlled clinical stud-
ies has been shown to effectively lessen the burden
of chronic pain. Acupuncture is also a procedure
that is rarely associated with side effects when ster-
ile needles are utilized (Lao, 1996). Another key
advantage is that acupuncture does not adversely
affect cognitive functions and presents no risk for
dependence (Yamashita etal., 2000). Unraveling
the mechanistic foundation of acupuncture has
identified adenosine and A1 receptors as key
effectors of acupunctures analgesic properties.
Manipulations of adenosine metabolism which
prolong A1 receptors activation (CFA and pros-
tatic acid phosphatase) have already been shown
to enhance the beneficial effects of acupunc-
ture, whereas A1 receptor antagonists potently
inhibit acupunctures analgesic actions. These
observations indicate that acupunctures analge-
sic properties have a clear molecular basisone
that is susceptible to other forms of manipula-
tion and may form the basis of novel treatments
for chronic pain. Future work may even design
local approaches to reduce the burden of chronic
pain by manipulation of purine signaling in the
absence of acupuncture itself.
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21
Meditation
GALLE DESBORDES
INTRODUCTION
Meditation has been practiced for centuries by
people across the world to improve well-being
and reduce suffering. Scientific research from
the past several decades suggests that medita-
tion practice can yield objective, measurable
health benefits. In particular, evidence is grow-
ing that meditation practice may offer protection
against the deleterious effects of chronic stress,
which engenders a dysregulation of homeostatic
and allostatic processes and can lead to seri-
ous health disorders. The brain is both affected
by and centrally involved in the regulation of
chronic stress (Maier, 2003). It has been pro-
posed that meditation practice is a form of mind
training that can enhance health by improving
emotional regulation and increasing resiliency
to stress (Benson, 1975; Kabat-Zinn, 1990), pos-
sibly via the generation of enduring, beneficial
changes in the brain through better regulation
of inflammation and neuroplasticity mecha-
nisms (Davidson and McEwen, 2012; Lutz
et al., 2007; Ornish et al., 2008, 2013; Slagter
etal.,2011).
Allostasis (stability through change) is a
model of physiological regulation that is a gener-
alization of the well-known concept of homeosta-
sis and accounts for cases in which physiological
set points are not constant but vary as a func-
tion of bodily needs and competing motivation.
Allostatic regulation necessitates the extensive
involvement of the central nervous system as the
main coordinator of regulatory responsesboth
behavioral and physiological (Schulkin, 2004;
Sterling and Eyer,1988).
While the stress response is adaptive in the
short term, chronic stress exacts a cost on the
organism known as allostatic load, the cumu-
lative physiological burden enacted on the body
through attempts to adapt to lifes demands
that can accelerate disease processes (McEwen,
1998, 2004; McEwen and Stellar, 1993; Schulkin,
2004). Allostatic load has been proposed as
a marker of cumulative biological risk and a
predictor of mortality and decline in physi-
cal functioning status (Beckie, 2012; McEwen
and Seeman, 1999; Seeman et al., 2001, 2010).
Allostatic load adversely affects multiple physi-
ological systems in the organism (including the
nervous, endocrine, and immune systems) and
can manifest in the form of chronic inflamma-
tion, overactivation of the sympathetic nervous
system, underactivation of the parasympathetic
nervous system, and premature cellular aging
(e.g., shorter telomeres). Allostatic load has been
proposed as a major contributor to the increased
occurrence of a broad range of modern-day ill-
nesses, including brain disorders such as major
depression, posttraumatic stress disorder, and
other chronic anxiety disorders (McEwen,
2003). Recent studies support the intriguing
possibility that these deleterious effects may
be counteracted by specific brain training
programs (Bryck and Fisher, 2012), includ-
ing meditation-based interventions designed
to promote well-being and prosocial behavior
(Davidson and McEwen,2012).
While the field of meditation science has
not yet directly addressed how meditation may
affect homeostatic control of the brain, a grow-
ing number of studies suggest that meditation
affects physiological mechanisms that can dis-
rupt or restore homeostasis in the brain. In this
chapter we review the current body of literature
relating to the short- and long-term effects of
meditation practice on the central nervous sys-
tem and on peripheral systems that are directly
or indirectly involved in homeostaticor
allostaticregulation of the brain. Specifically,
the topics covered include the effects of medita-
tion training on (i)blood pressure, (ii) immune
function, (iii) telomeres, (iv) autonomic regula-
tion of the heart, (v)HPA axis regulation, and (vi)
neuroplasticity.
354 Part III:Homeostatic Manipulators
W H AT A R E M E D I TAT I O N
PR ACTICES?
Meditation practices have existed since prehis-
toric times (Johnson, 1982) and are currently
practiced globally by many people, both in reli-
gious and secular (including clinical) contexts.
Any attempt to define meditation is fraught
with debate, and this review is not the place to
enter into these academic considerations. As a
starting point, most meditative practices dis-
cussed in this chapter are more or less adequately
described by the following working definition,
offered by Shapiro (1982): Meditation refers to
a family of techniques which have in common a
conscious attempt to focus attention in a nonana-
lytical way and an attempt not to dwell on discur-
sive, ruminating thought. However, other types
of meditation not covered by this definition exist,
such as the loving-kindness and compassion
meditation practices described later. Different
traditions and schools of thought offer a wide
variety of meditation practices, and interested
readers are referred to the vast literature dedi-
cated to these topics. Overall, the full spectrum
of meditation practiceswhich vary in terms of
their purpose, specific instructions, and expected
resultsis therefore extremely broad. The generic
term meditation (often used without further
elaboration in the scientific literature) can refer
to widely diverse practices, likely with varying
effects on physiology. Recent attempts to organ-
ize these practices into a few categories for the
purpose of scientific research resulted in active
debate (Awasthi, 2012; Josipovic, 2010; Lutz etal.,
2008; Travis and Shear, 2010a, 2010b). In sum-
mary, experts agree that there is no such thing
as meditation as a single, monolithic practice.
This important distinction can help explain the
variety of physiological effects reported in the
scientific literature todate.
Some of the first meditation practices investi-
gated scientifically, in nonexpert as well as expert
practitioners, were Transcendental Meditation,
also known as TM (Rosenthal, 2011; Wallace
et al., 1971), and the Relaxation Response
(Benson, 1975; Benson et al., 1974b). Since the
1980s, another increasingly popular type of med-
itation practice offered in the clinical context is
mindfulness, first introduced by Kabat-Zinn
(1982). Nowadays, numerous mindfulness-based
interventions exist that are centered on mind-
fulness meditation instruction and practice,
typically in the form of a six- to eight-week
group intervention. These programs include
mindfulness-based stress reduction (MBSR),
which is the original format (Kabat-Zinn, 1990,
2013); mindfulness-based cognitive therapy
(MBCT), which is a hybrid between MBSR and
cognitive behavioral therapy originally designed
to prevent depression relapse in patients with a
history of major depression but that has been
increasingly applied to other clinical and non-
clinical populations (Segal et al., 2013; Teasdale
etal., 1995); and other mindfulness-based inter-
ventions with varying duration and components
(Kabat-Zinn, 2003). Other practices derived
from mindfulness include third-wave forms
of psychotherapy such as acceptance and com-
mitment therapy (Hayes, 2004; Hayes et al.,
1999) and dialectical behavioral therapy (Lau
and McMain, 2005; Linehan, 1993; Robins etal.,
2004). A growing scientific literature points to
wide-ranging benefits from mindfulness-based
programs, from decreases in anxiety, depression,
and chronic pain to improvements in immune
function and more (Kabat-Zinn, 2013). However,
the methodological quality of the scientific litera-
ture on meditation for health has been criticized
(Ospina etal., 2007). Arecent review concludes
that mindfulness-based programs show moder-
ate evidence of improved anxiety, depression,
and pain but low evidence of improved stress/
distress, mental health related quality of life, and
positive mood (Goyal et al., 2014), even though
participants in mindfulness-based interventions
often report an increase in well-being (reviewed
in Chambers etal., 2009; Grossman, 2004; Rubia,
2009). Clearly, more high-quality research is
needed, but the preliminary evidence to date is
very encouraging, as we reviewlater.
Other types of meditative or contempla-
tive practices are receiving increasing interest
from the scientific community. Practices col-
lectively referred to as loving-kindness or
compassion meditation (which encompass
several distinct forms of practices) are aimed at
cultivating loving-kindnessa form of uncondi-
tional love toward all beings (Salzberg, 1995)or
compassionthe feeling that arises in witnessing
anothers suffering and that motivates a subse-
quent desire to help (Goetz et al., 2010). Unlike
mere empathic resonance, which can lead to
burnout, secondary traumatic stress, or compas-
sion fatigue (a deterioration of ones resiliency,
coping, and empathic abilities), true compas-
sion is considered beneficial to the individual
who experiences it (Klimecki etal., 2014; Singer
and Bolz, 2013). In recent years, a number of

programs have been proposed to train indi-
viduals in cultivating loving-kindness and com-
passion via meditative practices presented in
a secular format adapted to a modern lifestyle
(Germer, 2009; Gilbert, 2005; Hofmann et al.,
2011; Jazaieri et al., 2013; Makransky, 2007;
Ozawa-de Silva and Negi, 2013; Salzberg, 1995;
Wallmark et al., 2012). Recent scientific studies
indicate that loving-kindness and compassion
practices may have multiple beneficial effects,
such as improvements in chronic pain and psy-
chological distress (Carson et al., 2005); reduc-
tions in depression, anxiety, rumination, and
self-criticism (Gilbert and Procter, 2006; Kemeny
et al., 2012); reduced inflammation response
(Pace etal., 2009, 2010, 2013); and increased vagal
tone as assessed by heart rate variability (HRV;
Kok etal., 2013). Afew studies have also begun
to investigate the neural correlates of these prac-
tices, although much more work is needed in
this area (Desbordes etal., 2012b; Garrison etal.,
2014; Klimecki etal., 2013, 2014; Lee etal., 2012;
Leung et al., 2013; Mascaro et al., 2013; Weng
etal.,2013).
Other types of meditative practices have
been scientifically studied, for instance visu-
alization practices (Kozhevnikov et al., 2009);
gTummo or inner heat yoga (Benson et al.,
1982; Kozhevnikov etal., 2013); and body aware-
ness practices such as yoga, qigong, tai-chi, the
Alexander Technique, the Feldenkrais Method,
and so on (Mehling etal., 2011; Schmalzl etal.,
2014), which are beyond the scope of this review.
E F F E C T S O F M E D I TAT I O N
O N A L L O S TAT I C M A R K E R S
Early scientific experiments on meditators
seemed to suggest that meditative states were
associated with a hypometabolic state and
had calming, relaxing, stress-reducing effects
(Benson etal., 1974a; Wallace etal., 1971; Young
and Taylor, 1998), even though it was recognized
early on that some types of meditation could
also increase arousal (Amihai and Kozhevnikov,
2014; Corby et al., 1978; Shapiro, 1982; Shapiro
and Walsh,1984).
The benefits of meditation practice for stress
reduction purposes have been widely reported.
In this section we review some of the most nota-
ble studies to date showing the effects of medi-
tation practices on some allostatic biomarkers.
Allostatic load can be estimated from markers of
inflammation, metabolism, blood pressure, and
telomeres, as well as by measuring the integrity of
Meditation 355
several intercorrelated physiological systems such
as the sympathetic and parasympathetic nervous
systems and the hypothalamicpituitaryadrenal
(HPA) axis (McEwen and Seeman, 1999; Seeman
etal., 2001,2010).
Blood Pressure
Early studies suggested that Transcendental
Meditation and the related Relaxation Response
practice could reduce blood pressure and nor-
malize hypertension (Benson et al., 1974b;
Wallace etal., 1971). Later studies seem to con-
firm this finding, albeit amid some controversy
(Canter and Ernst, 2004; Orme-Johnson et al.,
2005; Parati and Steptoe, 2004). A more recent
meta-analysis of nine randomized controlled
trials concluded that TM could decrease blood
pressure, with an effect size of 4.7mm Hg (95%
confidence interval:7.4 to 1.9mm Hg) for sys-
tolic blood pressure and 3.2mm Hg (95% confi-
dence interval:5.4 to 1.3mm Hg) for diastolic
blood pressure, respectivelyeffect sizes that
are considered clinically meaningful (Anderson
et al., 2008). There is also preliminary evidence
suggesting that the MBSR intervention can
reduce blood pressure as well. Asingle-arm study
of breast and prostate cancer patients undergo-
ing MBSR found a significant pre- to postinter-
vention 2.1 mm Hg decrease in systolic blood
pressure but no effect on diastolic blood pressure
(Carlson et al., 2007). A randomized controlled
trial of MBSR in prehypertensive patients, with
an active control condition consisting of progres-
sive muscle relaxation training, found that MBSR
could significantly reduce blood pressure in this
population compared to the control intervention,
with a 4.8-mm Hg reduction in systolic blood
pressure and a 1.9-mm Hg reduction in diastolic
blood pressure (Hughes etal.,2013).
Immune Function
Some evidence suggests that meditation training
may improve immune function in healthy indi-
viduals. Based on previous studies indicating that
chronic stress can diminish antibody response to
vaccines (Glaser etal., 1998; Kiecolt-Glaser etal.,
1996; Yang and Glaser, 2002), Davidson et al.
(2003) conducted a randomized controlled trial
of MBSR in a workforce population in which
subjects in both the intervention group and the
waitlist control group received the influenza vac-
cine at the end of the eight-week period. Subjects
who had received MBSR (N=25) showed small
but statistically significant increases in antibody
356 Part III:Homeostatic Manipulators
titers to influenza vaccine compared with control
subjects (N=16).
On a broader level, it has been proposed that
meditation practice might help regulate chronic
inflammation (e.g., Oke and Tracey, 2009).
Chronic inflammation is especially relevant
to homeostatic regulation of the brain, as the
immune system and the central nervous system
form a bidirectional communication network
(Maier, 2003; McEwen, 2006). Chronic inflam-
mation has been associated with a number of
diseases that can affect the brain (Anthony and
Pitossi, 2013), including inflammatory diseases
of the blood vessel wall (with increased risk of
thrombotic stroke) and other cardiovascular
diseases, Alzheimers disease (Ferreira et al.,
2014; Maccioni et al., 2009; Rubio-Perez and
Morillas-Ruiz, 2012), Parkinsons disease (Hirsch
et al., 2012; More et al., 2013), brain metastasis
(Hamilton and Sibson, 2013), and even type II
diabetes (Donath and Shoelson, 2011)which
can be associated with brain dysfunction (Biessels
etal., 2014). Chronic inflammation may also be
involved in the process of age-related cognitive
decline (Simen et al., 2011) and normal aging
(Pizza etal., 2011; Sparkman and Johnson, 2008).
Chronic inflammation is believed to be caused by
a combination of genetic predisposition, lifestyle,
and psychosocial factors; it is, therefore, suscep-
tible to sociobehavioral changes such as those
promoted by meditation-based interventions and
perhaps by other meditation-specific neuroplas-
ticity mechanisms (Coe and Laudenslager, 2007;
Davidson and McEwen,2012).
Some recent studies indicate that meditation
interventions may indeed reduce inflammation.
In a randomized controlled trial in a popula-
tion of foster-care adolescents (N=71) exposed
to early life adversity (a known risk factor for
increased inflammation), a six-week compassion
meditation intervention had some effects on sali-
vary concentration in C-reactive protein (CRP),
an inflammatory marker. While there was no
significant difference between the intervention
group and the control group in CRP levels over-
all, the number of practice sessions attended by
the subjects in the meditation group was corre-
lated with a pre- to postintervention reduction
in CRP, suggesting a possible dose-response rela-
tionship (Pace etal.,2013).
Inflammation can also be assessed in
response to an acute stressor, such as the Trier
Social Stress Test (TSST), a commonly used
standardized protocol for reproducibly inducing
psychological stress in the laboratory. The TSST
consists of a public speaking and mental arith-
metic task performed in front of a panel of emo-
tionally nonresponsive confederates presented as
behavioral experts. Even though the stressor
itself lasts only a few minutes, the TSST reliably
activates the autonomic nervous system, HPA
axis, and innate immune inflammatory response
over a time course of several hours (Dickerson
and Kemeny, 2004; Kirschbaum etal.,1993).
Several studies of meditation have used the
TSST to assess changes in inflammatory response
to stress after several weeks of meditation train-
ing. In a randomized controlled trial of compas-
sion meditation compared to an active control
intervention (based on health discussion groups)
conducted in healthy college students, Pace etal.
(2009) found a post hoc association between com-
passion meditation practice time and decreased
plasma levels of interleukin-6 (a proinflammatory
cytokine), although there was no group differ-
ence between the two interventions. In a rand-
omized controlled trial of MBSR compared to the
Health Enhancement Program (an ad hoc active
control intervention), Rosenkranz et al. (2013)
also found comparable cortisol responses to the
TSST after both interventions but significantly
smaller flare in the MBSR group in response to
topical application of capsaicin cream to forearm
skin, indicating reduced inflammation response.
In another randomized controlled trial of MBSR
with an active control group, levels of adrenocor-
ticotrophin hormone (the hormone that stimu-
lates cortisol release) during the TSST were lower
in the MBSR group than in the control group
(Hoge etal.,2012).
Several recent studies also suggest that medi-
tative states may have immediate effects on
inflammatory gene expression. In a recent study
conducted on experienced meditators taking
part in a meditation retreat, Kaliman etal. (2014)
found a downregulation of the proinflammatory
genes RIPK2 and COX2 and of several histone
deacetylase (HDAC) genes after engaging in
mindfulness meditation for eight hours. In addi-
tion, the extent to which some of those genes were
downregulated was associated with faster corti-
sol recovery to a TSST. In a study of both experi-
enced and novice practitioners of the Relaxation
Response, Bhasin etal. (2013) found that imme-
diately after engaging in the Relaxation Response
practice, the expression of genes associated with
energy metabolism, mitochondrial function,
insulin secretion, and telomere maintenance was

enhanced, whereas the expression of genes linked
to inflammatory response and stress-related
pathways was reduced, with stronger effects in
experienced practitioners than in novices. While
these recent findings are very promising, more
research is needed to determine whether medi-
tation affects inflammatory gene expression over
the longterm.
Telomeres
Telomeres are protective caps at the ends of
chromosomes. Their maintenance requires the
action of the ribonucleoprotein enzyme known
as telomerase. The degradation, or shortening, of
telomeres threatens chromosome integrity and is
implicated in the process of aging as well as cancer
(Aubert and Lansdorp, 2008; Corey, 2009; Eitan
etal., 2014; Falandry etal., 2014; Kong etal., 2013;
Oeseburg etal., 2010; Zhu etal., 2011). Measures
of telomere length and telomerase activity have
been used to assess cellular aging (Aubert and
Lansdorp, 2008; Mather et al., 2011; Njajou
et al., 2009). Importantly, stress can accelerate
telomere shortening (reviewed in Starkweather
etal., 2014). Epel etal. (2004) found that women
with the highest levels of perceived stress had
telomeres shorter on average by the equivalent
of at least one decade of additional aging com-
pared to low-stress women. In addition, telom-
erase activity in peripheral-blood mononuclear
cells, although not telomere length, was inversely
associated with six major cardiovascular disease
risk factors in healthy women, suggesting that
telomerase activity may be a more direct and
potentially earlier predictor than telomere length
of long-term cellular viability, genomic stability,
and disease processes (Epel etal.,2006).
In the field of meditation science, telomeres
and telomerase activity are gaining interest as
possible objective markers of health improve-
ments associated with meditation practice. For
example, in a study of long-term meditation prac-
titioners engaged in a three-month-long, intense
meditation retreat (known as the Shamatha pro-
ject), telomerase activity was significantly greater
in retreat participants at the end of the retreat
than in matched control subjects, and mediation
analyses based on longitudinal psychological
assessments suggested that increases in perceived
control and decreases in negative affectivity con-
tributed to increased telomerase activity (Jacobs
etal.,2011).
In a recent pilot study of mindfulness train-
ing for healthy eating in overweight and obese
Meditation 357
women, both the mindfulness group and the
waitlist control group showed an increase in tel-
omerase activity over four months, with nega-
tive correlations between changes in telomerase
activity and changes in chronic stress, anxiety,
dietary restraint, dietary fat intake, cortisol,
and glucose (Daubenmier etal., 2012). This pre-
liminary finding is in line with a previous study
of intensive lifestyle changes (including daily
meditation practice) in prostate cancer patients
who showed significantly increased telomerase
activity after three months, where the increases
in telomerase activity were significantly associ-
ated with decreases in low-density lipoprotein
(LDL) cholesterol and decreases in psychological
distress (Ornish etal., 2008)and with increased
relative telomere length after five years of follow
up (Ornish etal.,2013).
Finally, a recent pilot study on a small num-
ber (N = 15) of experienced practitioners of
loving-kindness meditation and matched control
subjects suggested that, in women, the amount
of loving-kindness meditation practice over the
lifetime was associated with longer telomeres
(Hoge etal.,2013).
Overall, these preliminary findings support
the possibility that meditation training may have
positive impacts on telomere length and telomer-
ase activity.
Autonomic Regulation oftheHeart
One of the main markers of allostatic load is HRV,
the healthy beat-to-beat fluctuations in heart
rate that reflect autonomic (sympathetic and
parasympathetic) influences on cardiac activity
(Malik etal., 1996). Diminished HRV is an inde-
pendent risk factor for mortality in patients with
heart disease (Berntson et al., 1997; Freeman,
2006; Friedman and Thayer, 1998; Malik et al.,
1996; Malliani et al., 1991; Mujica-Parodi et al.,
2009; Stein etal., 1994)and has been associated
with a variety of pathological states and disposi-
tions, including anxiety and depression (Gorman
and Sloan, 2000). For instance, low HRV is asso-
ciated with both acute (state) anxiety (Fuller,
1992; Jnsson, 2007; Watkins et al., 1998) and
chronic (trait) anxiety and clinical anxiety dis-
orders (Cohen and Benjamin, 2006; Cohen etal.,
2000; Friedman, 2007; Klein et al., 1995; Licht
etal., 2009; McCraty etal., 2001). HRV decreases
in relation to increased depression severity, even
in the absence of cardiovascular disease (Brown
et al., 2009; Kemp et al., 2010). Consequently,
high HRV has been proposed as an indicator of
358 Part III:Homeostatic Manipulators
good physical and psychological health (Porges,
2011; Thayer etal., 2009,2012).
Meditative states are usually associated with
higher vagal parasympathetic activity, as indi-
cated by higher HRV either during or imme-
diately after engaging in a meditation practice
session (Cysarz and Bssing, 2005; Ditto et al.,
2006; Kubota et al., 2001; Nesvold et al., 2012;
Peng etal., 1999, 2004; Phongsuphap etal., 2008;
Takahashi etal., 2005; Tang etal., 2009; Wolever
etal., 2012; Wu and Lo, 2008). However, it should
be noted that certain advanced forms of medi-
tation practices seem to increase sympathetic
activation in expert practitioners (Amihai and
Kozhevnikov, 2014; Corby etal., 1978; Kox etal.,
2012). Areview of the literature on the complex
autonomic changes associated with meditative
states is beyond the scope of this chapter. The
question of interest here is whether the prac-
tice of meditation has lasting, beneficial influ-
ences on autonomic functioning that may affect
homeostatic regulation of the brain. In other
words, is autonomic function altered in medita-
tion practitioners, even outside periods of for-
mal meditation practice, such as during normal
resting conditions or during tasks that challenge
the autonomic nervous system? Recent studies
have linked even brief training in meditation
(from five days to several weeks) to a longitu-
dinal increase in resting HRV. In a single-arm
pilot study of patients presenting with depres-
sion and anxiety six months after surgery for
spontaneous subarachnoid hemorrhage, rest-
ing HRV was found to significantly increase
pre- to post-MBSR training, while depression
scores significantly decreased (Joo et al., 2010).
Longitudinal increases in resting HRV have also
been found with meditation-based interventions
other than MBSR. In a waitlist-controlled field
experiment of university workers taking part in
a six-week program for learning loving-kindness
meditation (with a one-hour class per week that
included guided meditation practice and group
discussion), the experimental condition (medi-
tation versus waitlist control) significantly pre-
dicted an increase in resting HRV (Kok et al.,
2013). In our own recent study, healthy adults
were randomized to either mindful-attention
meditation training, compassion meditation
training, or an active control intervention based
on health education classes (see Desbordes etal.,
2012b). One of our assessments measured resting
HRV before and after each of the three eight-week
interventions. Preliminary analyses indicate that
participants who underwent either type of medi-
tation training (mindful-attention or compas-
sion) showed an increase in resting HRV pre- to
postintervention, with a significant correlation
between increased HRV and decreased depres-
sion score (Desbordes etal., 2012a).
In summary, while specific meditative states
are associated with a variety of changes in auto-
nomic activation, recent evidence suggests that
the regular practice of meditation may have
lasting, beneficial influences on autonomic
functioning.
It should be noted that the autonomic nerv-
ous system and the HPA axis can be indepen-
dently affected by a psychosocial stressor such
as the TSST (Schommer etal., 2003). Therefore,
it does not necessarily follow from the effects of
meditation on autonomic function that medita-
tion will have comparable effects on the HPA
axis (e.g., as assessed by cortisol levels). Next we
review how meditation may affect regulation of
the HPAaxis.
HPA Axis Regulation
One of the main systems involved in allostatic
regulation is the HPA axis, a major neuroen-
docrine subsystem that controls physiological
responses to stress, such as increased levels of cor-
tisol (Dickerson and Kemeny, 2004; Hellhammer
et al., 2009; Kirschbaum et al., 1993; Marques
et al., 2010; Steptoe et al., 2007). Cortisol levels
have been proposed as an objective measure of
HPA axis activation in chronic stress (Herbert
and Cohen, 1993) and as an outcome measure
for meditation interventions (Matousek et al.,
2010). Salivary cortisol is considered the method
of choice for measuring free cortisol levels in the
context of stress research (Hellhammer et al.,
2009). However, obtaining meaningful measures
of cortisol can be challenging. Cortisol levels
exhibit wide variations throughout a 24-hour
cycle, following a circadian rhythm controlled
by the hypothalamic suprachiasmatic nucleus
(part of the HPA axis). While the daily cortisol
curve normally follows a stereotypical profile,
with a sharp morning rise and a slow descending
slope in the afternoon and evening, reaching its
minimum level during the night (Krieger etal.,
1971), many factors can perturb this rhythm,
and erratic cortisol patterns have been associated
with metabolic abnormalities, fatigue, depres-
sion, psychosocial factors, and poor quality of
life in general (Chida and Steptoe, 2009; Debono
et al., 2009). In addition, flattened or abnormal

diurnal cortisol rhythms are a predictor of early
mortality in some types of cancer (Sephton etal.,
2000,2013).
To obtain meaningful cortisol measurements
as part of a research study, it is therefore neces-
sary to measure cortisol at very specific times
relative to the subjects waking time, if possible
at multiple time points during a 24-hour cycle,
or at short intervals immediately after waking
to assess the cortisol awakening response (Clow
etal., 2004; Fries etal., 2009; Hellhammer etal., tions of cortisol may have deleterious effects on
2009; Pruessner et al., 1997). Alternatively, one
can measure cortisol response to acute stress-
ors such as the TSST (Kirschbaum et al., 1993).
Finally, it should be noted that cortisol levels vary
with age, gender, and certain pharmacological
treatments, including oral contraceptives.
Previous reports of the effects of medita-
tion training on cortisol levels showed mixed
results (for a review see Matousek et al., 2010).
Some studies in cancer patients found that
participation in MBSR was associated with a
global decrease in cortisol levels (Carlson etal.,
2007; Witek-Janusek etal., 2008), while another
study reported an increase in cortisol awaken-
ing response (Matousek et al., 2011)arguably
an improvement in this patient population since
stressed individuals tend to exhibit a blunted
(i.e., abnormally flat) cortisol daily profile (Chida
and Steptoe, 2009). Other studies found no sig-
nificant changes in cortisol after MBSR, although
their methodological design has been criticized
on the basis of inadequate cortisol sampling (e.g.,
insufficient number of time points), small sample
sizes, or lack of control over confounding varia-
bles such as diet, physical exercise, or sleepwake
cycle (Matousek et al., 2010). However, even
carefully designed randomized controlled trials
have also yielded unclear cortisol outcomes. For
example, a longitudinal study of MBCT in remit-
ted depression patients with six-month follow-up
showed no significant changes in any cortisol
measure (cortisol awakening response, diurnal
slope, or area under the curve; Gex-Fabry etal.,
2012), although it may be due to the fact that cor-
tisol patterns are erratic and difficult to detect
in depressed patients (Peeters et al., 2004). In a
nonclinical community sample, a randomized
controlled trial of MBSR compared to an active
control intervention found no difference between
the groups in posttraining cortisol response to
a TSST, although the amount of mindfulness
practice (i.e., dose) predicted a steeper (arguably
more salubrious) diurnal cortisol slope following
Meditation 359
training (Rosenkranz etal., 2013). More longitu-
dinal studies with careful methodology for corti-
sol assessments are needed to elucidate the effects
of meditation training on HPA axis activation in
various populations.
The hypothesis that meditation practice may
contribute to lower abnormally elevated corti-
sol levels, at least in some clinical populations,
should be of interest in the context of homeo-
static control of the brain since high concentra-
the brain, especially in the hippocampus. It is
well known that the hippocampus becomes atro-
phied and presents functional abnormalities in
humans and animals exposed to severe stress or
depression (Bora etal., 2012; Bremner etal., 2000;
Colla etal., 2007; Davidson etal., 2002; Malykhin
etal., 2010; Shah etal., 1998; Sheline etal., 1996,
1999). It has been suggested that this hippocam-
pal atrophy results from prolonged exposure to
high concentrations of cortisol (Lupien et al.,
1998). While normal levels of cortisol activate
mineralocorticoid receptors and facilitate hip-
pocampal long-term potentiation (and mem-
ory functions), high concentrations of cortisol
additionally activate glucocorticoid receptors
that have debilitating effects on hippocampal
function, including a dampening of long-term
potentiation (Kerr et al., 1989). Therefore, cor-
tisol exerts a concentration-dependent biphasic
(inverted U-shape) influence on the expression
of hippocampal plasticity (Diamond etal., 1992).
While these mechanisms have been investigated
mostly in animal models, studies in humans
also indicate that elevated basal cortisol levels
predict reduced hippocampal volume and defi-
cits in hippocampus-dependent memory tasks
in the aging population (Lupien et al., 1998;
Sapolsky, 1992). These complex interactions
between cortisol levels and hippocampal struc-
ture and function may underlie the connection
between stressful experiences and dampening of
hippocampal neurogenesis (Gould and Tanapat,
1999; Gould etal., 1998; Rao etal., 2010). The link
between cortisol levels and hippocampus size in
chronic stress may take the form of a positive
feedback loop between pathologically high lev-
els of cortisol and neuronal damage in the hip-
pocampus, which may in turn further reduce
HPA downregulation and promote hypercorti-
solemia (reviewed in Davidson etal.,2002).
Most interestingly, hippocampal atrophy
seems to be reversible to some extent (Gould etal.,
2000; Jacobs etal., 2000; Malykhin etal., 2010).
360 Part III:Homeostatic Manipulators
For example, chronic treatment with antidepres-
sants increases neurogenesis in the hippocampus
(Santarelli et al., 2003; Vermetten et al., 2003).
Since hippocampal atrophy can be reverted when
stress is reduced, the question naturally arises
whether similar effects can be achieved by reduc-
ing perceived stress, via the practice of medita-
tion for example. Recent studies suggest that
meditation practice may indeed promote growth
(or prevent shrinkage) in the hippocampus.
Long-term meditation practitioners have larger
hippocampi and greater grey matter density in
the hippocampal complex than matched control
subjects (Hlzel etal., 2008; Luders etal., 2009,
2013a, 2013b). Remarkably, hippocampus growth
was also observed longitudinally after only eight
weeks of meditation practice in meditation-nave
participants, in healthy subjects undergoing
MBSR (Hlzel et al., 2011) and in healthy sub-
jects undergoing mindful-attention or compas-
sion meditation training (Desbordes etal., 2014).
In addition, neuroplastic effects of meditation
have been reported not only in the hippocam-
pal complex but also in other brain regions, as
reviewednext.
Neuroplasticity
In a seminal study of the effects on brain
structure of lifetime meditation experience,
Lazar etal. (2005) found greater cortical thick-
ness in the right anterior insula and dorsolat-
eral prefrontal cortex regions in Vipassana
(or Insight) meditation practitioners than in
matched control subjects, with a significant cor-
relation between cortical thickness and years
of meditation experience. This study was the
first to suggest that meditation practices might
offset age-related cortical thinning and grey
matterloss.
Other studies (reviewed in Fox et al., 2014;
Tang et al., 2015) have since extended these
findings. In another cross-sectional study of
Vipassana meditators and matched controls,
Hlzel et al. (2008) used voxel-based morpho-
metry (Ashburner and Friston, 2000) to assess
grey matter concentration. This study confirmed
the earlier finding in Vipassana meditators of
increased grey matter in the right anterior insula,
which is involved in interoceptive awareness,
presumably reflecting the cultivation of bodily
awareness during this type of training. This study
also reported greater grey matter concentration
in the left inferior temporal gyrus and right hip-
pocampus in meditators compared to matched
controls. Pagnoni and Cekic (2007) investigated
grey matter volume in relation to attentional
performance in Zen meditators compared with
matched controls. They found that, contrary to
the control subjects, meditators did not display
the expected effects of age on grey matter volume
and on performance in a computerized sustained
attention task. Vestergaard-Poulsen etal. (2009)
found higher grey matter density in the brain
stem of experienced practitioners of Tibetan
Dzogchen meditation compared to matched
controls, particularly in the medulla oblongata
region of the dorsal brain stem, which contains
autonomic nuclei involved in cardiac and res-
piratory functions. Grant etal. (2010) found that
compared to matched controls, Zen meditators
had both lower pain sensitivity and greater cor-
tical thickness in pain-related areas (the dorsal
anterior cingulate and secondary somatosensory
cortex), with a positive correlation between corti-
cal thickness in the anterior cingulate and years
of meditation practice. A series of studies by
Luders and colleagues conducted on long-term
meditators from various traditions demonstrated
widespread differences between meditators and
matched controls in several brain structural
measures. These measures included larger grey
matter volume in the right orbito-frontal cortex
(Luders et al., 2009), larger hippocampus and
greater grey matter in the hippocampal complex
(Luders et al., 2013a, 2013b); greater structural
connectivity within major white matter path-
ways (Luders etal., 2011); local (voxel-wise) dif-
ferences in grey matter asymmetry between left
and right hemispheres (Kurth et al., 2015); and
greater cortical gyrification (a measure of corti-
cal folding curvature) in the fusiform gyrus, pre-
central gyrus, cuneus, and especially the right
anterior dorsal insula (where gyrification was
positively correlated with years of meditation
practice; Luders et al., 2012), again pointing to
an important role of the right anterior insula. In
a study of Theravadan Buddhist practitioners of
loving-kindness meditation, Leung et al. (2013)
found significantly greater grey matter volume
in meditators than in matched controls in the
right angular gyrus and right posterior parahip-
pocampal gyrus. While neuroplastic effects of
meditation in the hippocampus had been found
in several other studies (reviewed previously),
this study was the first to highlight differences
in the right angular gyrus, a region known to be
activated by cognitive empathy and perspective
taking and thereby potentially involved in com-
passion and care for others (Adolphs, 2009; Van
Overwalle, 2009; Saxe and Kanwisher,2003).

While these studies suggest that the brains
of meditators show some structural differ-
ences from the brains of nonmeditators, the
cross-sectional nature of these studies does not
allow testing of whether meditation practice
caused these differences or whether individuals
with particular brain features were more drawn
to becoming long-term meditation practitioners.
However, recent longitudinal studies of subjects
without prior meditation experience indicate
that changes in brain structure can be detected
after only a few weeks of meditation practice.
After participating in the eight-week MBSR
program, healthy subjects showed significantly
higher longitudinal increases in grey matter
concentration in the amygdala, hippocampus,
posterior cingulate cortex, temporo-parietal
junction, and cerebellum compared with waitlist
control subjects (Hlzel etal., 2010, 2011). After
one month of integrative body-mind training
(a meditation training program based on tra-
ditional Chinese medicine in the form of daily
30-minute sessions totaling only 11 hours of
practice), a college-student population exhibited
increases in fractional anisotropy (a measure of
white matter integrity) in the corona radiata, a
major white-matter tract connecting multiple
brain structures to the anterior cingulate cortex,
a region previously implicated in self-regulation
(Tang etal., 2010, 2012). In our longitudinal med-
itation study mentioned previously (Desbordes
etal., 2012b), preliminary analyses of structural
brain data suggest changes in several subcorti-
cal regions (including increased hippocampal
volume) after eight weeks of training in either
mindful-attention meditation or compassion
meditation (Desbordes etal., 2014). In the com-
passion meditation participants, we also found
a significant increase in cortical thickness in the
dorsomedial prefrontal cortex (Singleton et al.,
2014), a region activated during empathy tasks
that, in another study, showed increased activa-
tion correlated with greater empathic accuracy
after eight weeks of training in compassion medi-
tation (Mascaro etal.,2013).
Taken together, these findings support the
view that meditation practices, as a form of
brain training, may promote neuroplasticity
and impact multiple brain functions, including
homeostatic and allostatic processes.
CONCLUSION
The scientific investigation of meditative prac-
tices is still at an early stage. Larger longitudi-
nal studies, ideally in the form of randomized
Meditation 361
controlled trials with active control interven-
tions, are warranted to rigorously test the effects
of meditation-based interventions on different
physiological (and psychological) mechanisms
that may improve healthin particular regarding
homeostatic and allostatic regulation processes
in the brain and other physiological systems. The
field is rapidly progressing and maturing into a
full-fledged multidisciplinary research domain,
with a growing number of investigators world-
wide collectively building a rigorous body of
evidence supporting the view that meditation
practices may offer wide-ranging health benefits.
The effects of meditation are especially remark-
able in the context of allostatic loadthe cumu-
lative, deleterious effects of chronic stress on our
bodies that not only diminish our quality of life
but can also promote chronic inflammation and
accelerate aging and disease processes. We expect
that future research will further establish the
legitimacy of meditation practices as low-cost,
accessible therapeutic interventions for a variety
of illnesses and preclinical conditions.
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22
Neurotrophic Regulation
inNeurorestoration oftheBrain
C A I X I N S U , M I C H E L R AT H B O N E , A N D S H U C U I J I A N G
HISTORY
O F N E U R O R E S T O R AT I O N
As far back as 1700 bc, ancient Egyptians docu-
mented their observations that injuries to the
head and spinal cord resulted in permanent
paralysis, loss of speech, and other functions that
were not recoverable (Cunha, 1949). Over time,
clinicians began to focus on neuroprotective
measures that limit further injury after central
nervous system (CNS) insults and surgeries to
ablate, remove, or reduce the circumstances that
impact the functionality.
About 100years ago, with the development of
microscopy techniques, scientists began to study
the CNS in more detail at the cellular level. The
work from Santiago Ramon y Cajal, the father
of modern neuroscience, laid the foundation for
future studies on CNS function restoration. He
pointed out that the restoration of nerve paths
in the CNS is frustrated by two negative condi-
tions:the lack of substances able to sustain and
invigorate the indolent growth of the sprouts and
the absence of catalytic agents capable of attract-
ing and directing the axonic current to its desti-
nation (Lobato,2008).
Neurorestoration, as a new concept, emerged
in the 1990s. With the escalating development
in the basic science field, in particular molecu-
lar biology, electrophysiology, and computa-
tional neuroscience, scientists became able to
examine the CNS in more detail, from molecu-
lar and cellular levels to circuitry and system
levels. Neuromodulation, gene therapy, and
cellular grafting techniques were fervently dis-
cussed in scientific research. While there is no
general agreement on the standard definition
of neurorestoration, Bednar defined it as the
structural and/or functional improvement in the
pathophysiology of the CNS following an insult
(Bednar and Perry,2012).
N E U R O R E S T O R AT I O N ,
TOBRING BACK
T H E H O M E O S TA S I S
OFTHECNS
Equipped with the knowledge from scientific
studies of the CNS, we are now beginning to
understand how the CNS integrates the regula-
tory and restorative forces of the body to foster
health and cope with environmental changes
after CNS disorders. Under normal physiologi-
cal conditions, the CNS manages a multitude of
highly complex interactions to maintain its bal-
ance and return systems to functioning within a
normal range. Homeostasis is achieved through
a delicate balance of electrical, chemical, molecu-
lar, cellular, hormonal, immunological, spatial,
and behavioral factors. To function properly,
neurons require a fairly strict electrical potential
gradient across the neural membranes, and this
gradient is determined by the acid-base balance
and the concentration of ions, both intra- and
extracellularly, the availability of neurotrans-
mitters and receptor sites, and the blood levels
of a variety of metals (Leisman, 1990). However,
neurons do not function independently; they are
affected by other cellular or even bodily activi-
ties. For example, astrocytes, the guardians of
neurons, constantly modulate neuronal activity
and support neuron survival and synaptic trans-
mission by regulating neurotransmitters and
neurotrophic factors (Benarroch, 2005; Seifert
and Steinhauser, 2013). Even respiration and
heart rate (which change blood pH, oxygen, and
glucose availability in the CNS) can contribute
to maintaining the complex homeostasis of the
CNS. The interconnection of the homeostasis
web is virtually endless.
Homeostatic regulatory mechanisms only
work within certain limits to buffer the CNS
from changes in the internal milieu. In 1939,
372 Part III:Homeostatic Manipulators
Walter Cannon postulated the theory that there
is a safety threshold in homeostatic regulation
that ensures that variations from basal physi-
ological levels (set points) do not reach danger-
ous extremes that might impair the functions of
the cells or threaten the existence of the organ-
ism because adaptive reactions are automatically
triggered to return the affected physiological
system to a basal state (Cacioppo and Berntson,
2011). Beyond that threshold, under a pathologi-
cal condition such as a CNS disorder, the system
will react by utilizing any or all compensatory
bodily mechanisms to re-establish the dynamic
homeostasis necessary for proper function-
ing. Unfortunately, CNS disorders such as head
trauma, spinal cord injury, stroke, epilepsy, and
Alzheimers disease (AD) do not all affect the
same area or portion of neural tissue. Rather,
they result in a disturbance in the delicate and
precisely tuned homeostatic environment.
N E U R O R E S T O R AT I O N ,
INCLUDING BUTNOT LIMITED
T O N E U R O R E G E N E R AT I O N
The adult CNS normally has a very limited capac-
ity to recover after the loss of function due to the
difficulty of neuronal regeneration after axon
injury. For a very long time, the prevailing dogma
held that there is no regeneration in the adult
CNS. However, some optimistic scientists contin-
ued conducting research on CNS repair. In a 1974
study, researchers observed widespread collateral
sprouting of nociceptive dorsal root axons in the
dorsal horn of the spinal cord following rhizot-
omy (Goldberger and Murray, 1974). Similarly,
others demonstrated that lesioned noradren-
ergic and cholinergic neurons were capable of
extending axons over long distances within the
brain and spinal cord (Bjorklund et al., 1971;
1975; Katzman etal., 1971). Later, using the new
horseradish peroxidase tracing technique, David
and Aguayo (1981) examined the origin, termi-
nation, and length of axonal growth after focal
CNS injury in the adult rat. When peripheral
nerve segments were used as bridges between
the medulla and spinal cord, they demonstrated
that axons from neurons at both these levels
grew approximately 30 mm. The turning point
for CNS regeneration, however, was the publica-
tion of the landmark paper from Eriksson etal.
(1998). For the first time, they showed that new
neurons, as defined by bromodeoxyuridine labe-
ling and neuronal markers, were generated from
dividing progenitor cells in the dentate gyrus of
adult humans. These findings not only confirmed
that CNS axons were able to regenerate but also
reinforced a concept that has driven virtually all
subsequent research in this field: that the CNS
nonpermissive environment inhibits axonal
regeneration following injury.
Neurorestoration is both a process and a goal
and includes, but is not limited to, neuroregener-
ation. The goal of neurorestoration is to improve
the structure of the tissue or organ involved
(Bednar, 2008). Ideally, neuroprotection at the
early stage is mandatory to limit further injury.
However, effective neuroprotectants have largely
failed over a range of CNS injury types and mod-
els, and the search for an effective therapy is
ongoing. Angiogenesis, as an antecedent require-
ment for neuroplasticity, stimulates endogenous
recovery mechanisms, including neurogen-
esis, synaptogenesis, and neuronal and synaptic
plasticity. These events are all involved in the
long-term repair and restoration process of the
brain after an ischemic event (Ergul etal.,2012).
AN APPROACH TOREDUCE
THENONPERMISSIVE AND
S T I M U L AT E T H E P E R M I S S I V E
ENVIRONMENTCUES
CNS disorders are caused by neurodevelop-
mental insults or occur when neurodegenera-
tive processes disrupt the homeostatic balance.
A detailed discussion of the pathological fea-
tures of CNS disorders is beyond the scope of
this chapter and has been discussed elsewhere
(Dumont etal., 2001; Freire, 2012; Menon, 1999).
However, only when the pathophysiological
changes and principles governing recovery of
function are more adequately understood will it
be possible to formulate strategies to restore the
function ofCNS.
As Cajal opined more than a century ago,
and as many researchers (Hou etal., 2008; Yang
and Yang, 2012) have echoed more recently,
the fundamental inhibition to CNS functional
recovery is the nonpermissive environment.
Under extreme conditions, such as spinal cord or
brain trauma, the tissue goes through mechani-
cal injury:compression, impact, distraction, lac-
eration/transection, and so on. Neurons that do
survive the initial injury are further bombarded
by secondary injuries such as neurogenic shock,
hemorrhage, ischemia, excitotoxicity, calcium
and fluid-electrolyte disturbances; immunologic
injury; apoptosis; and disturbances in mito-
chondrial function (Dumont etal., 2001). These
 Neurotrophic Regulation in Neurorestoration of theBrain
secondary injury mechanisms can exacerbate
damage, limit restorative processes, and contrib-
ute to overall morbidity and mortality.
Regeneration follows degeneration. A bur-
geoning body of evidence has shown that the
failure of axon regeneration after a CNS insult is
due to an inadequate or inappropriate regenera-
tive response from damaged CNS axons and to
a CNS environment that inhibits regeneration.
This inhibitory environment actually contains
many molecules that promote axon growth as
well as molecules that inhibit it, but the balance
of activities in the damaged CNS does not favor
the regeneration of adult CNS axons, with the
inhibitory factors usually upregulated after the
disorder.
CNS neuronal inhibitors fall into three classes.
The first class is secreted by glial scar, which is the
main physical and biochemical barrier to axonal
regeneration after CNS injury (Bovolenta et al.,
1991; Sandvig et al., 2004). Numerous studies
have identified extracellular matrix component
chondroitin sulphate proteoglycan (Fitch and
Silver, 1997; Mironova and Giger, 2013)and kera-
tan sulfate proteoglycan (Imagama etal., 2011)as
key molecules contributing to glial scar inhibi-
tion. An abundance of individual chondroitin
sulphate proteoglycans have been found, includ-
ing neurocan (Asher etal., 2000; McKeon etal.,
1999), neural/glial antigen (Levine, 1994), versi-
can (Asher et al., 2002), and brevican (Yamada
etal.,1997).
Compelling evidence has also revealed that
CNS myelin formed by mature oligodendrocytes
profoundly inhibits neurite outgrowth. A num-
ber of myelination-related inhibitory molecules
have been identified, including myelin-associated
glycoprotein (Chiquet, 1989; Domeniconi et al.,
2002; Filbin, 1995), NogoA (Chen et al., 2000;
Schwab, 2004), and oligodendrocyte myelin gly-
coprotein (Guo et al., 2007; Raiker et al., 2010).
In addition to glial scar and myelination-related
inhibitors, several axon guidance molecules have
been found to cause growth cone collapse after
CNS injury, including semaphorins, ephrins,
netrins, and slits (Fan et al., 2013; Pasterkamp
and Verhaagen, 2006). This list of inhibitory mol-
ecules is by no means exhaustive; many more
have yet to be identified. It is generally believed
that these inhibitors collectively contribute to the
regenerative failure of injured CNS axons invivo.
At the other end of the spectrum are mecha-
nisms that promote neuronal outgrowth and
regeneration after CNS injury. These include
373
neurotrophic factors, growth-promoting extra-
cellular matrix molecules, cell adhesion mol-
ecules, and other small molecules. While they
are present in a healthy and intact CNS, stimula-
tion by the endogenous regulatory mechanisms
after CNS injury has not yielded a positive result
as the molecules are not sustained long enough
to promote regeneration. If their outgrowth and
regenerative abilities could be extended, the end
result might change.
As has been widely demonstrated, neuro-
trophic factors play a pivotal role in neurores-
toration and neuroprotection in CNS disorders
such as Alzheimers disease (AD), Parkinsons
disease (PD), amyotrophic lateral sclerosis (ALS),
Huntingtons disease, spinal cord and brain inju-
ries, and other neurodegenerative and neuro-
traumatic pathologies (Aloe etal., 2012; Connor
and Dragunow, 1998; Dreyfus, 1989). While
our understanding of their roles in CNS disor-
ders is not complete, nerve growth factor (NGF),
brain-derived neurotrophic factor (BDNF), and
fibroblast growth factor 2 (FGF2) have been
examined in numerous studies, which investi-
gated both the preventive and therapeutic effects
on neuronal survival as well as the negative
impact of neurotrophin stimulation. Another
emerging area of interest is the therapeutic
potential of purinergic signaling in the patho-
physiology of CNS disorders. Geoffrey Burnstock
(1972, 2013)introduced the concept of purinergic
signaling when he presented the first evidence of
the action of adenosine triphosphate (ATP) as a
neurotransmitter. Broadly speaking, the puriner-
gic system includes purines in many different
formats: purine bases such as adenine and gua-
nine; their corresponding nucleosides (adenosine
and guanosine and their metabolic products), as
well as purine nucleotides. With the cloning of
various purinergic receptor subtypes, such as P1
and P2, the roles of adenosine and ATP as neu-
rotransmitters and neuromodulators in the CNS
have become well accepted (Burnstock, 2013).
Chapter 2 of this book discusses adenosine and
ATP in the homeostatic control of CNS disorders
in detail.
In contrast to the enthusiasm shown for
adenine-based purines, little attention has
been given to the potential extracellular roles
of nonadenosine-based purine nucleosides or
nucleotides. However, there is some evidence
that these purines, particularly guanosine, may
also act as neurotransmitters or neuromodula-
tors in the CNS. Our lab, together with others,
374 Part III:Homeostatic Manipulators
has shown evidence that guanosine may play an
important role in CNS signaling and may exert
neuroprotective and neurorestorative effects in
response to physiological and pathological con-
ditions (Rathbone etal., 1999; 2008), discussed in
detaillater.
In summary, after a CNS disorder, there are
both inhibitory and stimulating environmental
factors at work; unfortunately, the stimulating
effects are overwhelmed by the inhibitory effects.
Reducing the nonpermissive environmental cues
while stimulating a more permissive environ-
ment is the primary goal of neurorestoration.
S T R AT E G I E S
Neurorestoration is interpreted differently by
various professions. For example, a surgeon may
employ a surgical intervention to effect neurores-
toration. Rehabilitation, physical and occupa-
tional therapists employ exercise, acupuncture, or
noninvasive brain stimulation in the treatment of
many CNS disorders (Demirtas-Tatlidede et al.,
2013; Schroer and Adamson, 2011). Exercise
and acupuncture are discussed in c hapter20 in
thisbook.
Cell-based replacement therapy is one of
the newest and most discussed neurorestora-
tive strategies both in the literature and in
clinical studies for CNS disorder treatment.
Stem cell related transplant is the prevailing
approach: embryonic stem cell studies have
gradually been replaced by research employ-
ing adult stem cells such as neural stem cells,
induced pluripotent stem cells, and mesenchy-
mal stromal stem cells (Dutta etal., 2013; Farin
etal., 2009). In addition to stem cell transplanta-
tion, Schwann cells (Duncan etal., 1981; Li and
Lepski, 2013; Wiliams and Bunge, 2012), olfac-
tory ensheathing cells (Bretzner etal., 2010; Xu
etal., 1995), and enteric glia (Jiang etal., 2003b,
2003c) have also been extensively studied to
determine their potential benefits.
In non-cell-based strategies, a large body of
molecules stimulate the permissive microenvi-
ronment through neurotrophic effects, including
neurotrophic factors (including but not limited
to NGF, BDNF, and FGF2), purinergic molecules,
and so on; other strategies aim at reducing inhi-
bition, either by blocking the existing inhibitors
or modulating receptors, using antibodies and/
or receptor antagonists, or reducing apopto-
sis, inflammation, or immunity effects. Among
these molecules, neurotrophic factors have been
shown to play fundamental roles in neuroresto-
ration. Caution should be taken in the utilization
of neurotrophic factors as they can play dual roles
and can elicit opposing signals in different dis-
eases (Hefti etal., 2006; Mantyh etal.,2011).
One of the central themes for neurorestora-
tion after CNS disorder is to understand which
factors are required to stimulate recovery in
surviving nerves so that the nervous system
can rewire, leading to recovery of function.
In addition to disease-specific allopathic treat-
ment, global holistic approaches that focus on
maintaining, regulating, and restoring CNS
homeostasis will offer a long-lasting and system-
atic improvement. Many neurotrophic factors
share common signal transduction pathways
that influence numerous aspects of neuronal
function and survival. The convergent factors in
these signaling transduction cascades provide
new target sites for the development of novel
agents that could be used to treat a variety of
CNS disorders.
In this chapter we focus on several neuro-
trophic factors and small molecules including
guanosine, especially on their ability to stimulate
endogenous neurogenesis. The common signal-
ing pathways are also examined to explore the
mechanisms behind their therapeutic effects.
Our intention is not to focus in detail on any one
regulatory factor but to examine from a global
perspective how the CNS integrates the regu-
latory and restorative processes of the body to
foster health and cope with environmental chal-
lenges, highlighting what our group has added to
the current knowledge of neurorestoration.
NEUROTROPHIC EFFECT
T O S T I M U L AT E P E R M I S S I V E
ENVIRONMENT
For many years, NGF was the only known neu-
rotrophic factor. Recently, however, the list of
the substances that may be classified as potential
neurotrophic factors has expanded considerably.
Purinergic molecules like adenine-based purines
(adenosine and ATP) and nonadenine-based
purines (guanosine) have garnered wide interest
due to their neurotrophic effects (Burnstock, 2013;
Rathbone etal., 1999). Some hormones and neu-
rotransmitters such as insulin, thyroid hormones,
and sex steroids have been shown to affect neurite
extension and survival and can also be categorized
as neurotrophic factors (Arnold and Gorski, 1984;
Recio-Pinto etal., 1986). In addition, there is evi-
dence that interleukins and other lymphokines
(IL-1beta, IL-2, IL-6) may also function as growth
factors for certain neuronal and glia-like cells in
the brain (Rothwell and Strijbos,1995).
 Neurotrophic Regulation in Neurorestoration of theBrain
NGF
In the neurotrophin family, NGF was the first
identified and is the best characterized mem-
ber. Studies have shown that NGF is distributed
in high levels in regions of the CNS innervated
by the magnocellular cholinergic neurons of
the basal forebrain (hippocampus, olfactory
bulb, and neocortex) and in regions containing
cell bodies of these neurons (septum, nucleus
of the diagonal band of Broca, nucleus basalis
of Meynert). The highest level of NGF mRNA
expression occurs within the hippocampus and
cortex (Connor and Dragunow, 1998; Goedert
etal.,1986).
There has been widespread interest in NGF
as a neurotrophic factor in the CNS since its dis-
covery. In advanced AD, cholinergic basal fore-
brain neurons, which provide the major source
of cholinergic innervation to the cerebral cor-
tex and hippocampus, undergo selective and
severe degeneration. With evidence from both
tissue and animal studies, and given the lack of
an effective treatment for AD, the application of
NGF in AD patients seems justified (Everall and
Kerwin, 1990; Harbaugh, 1989; Mobley, 1989). In
1993 the first case of intracranial infusion of NGF
to an Alzheimers patient was reported. After one
month of NGF infusion, tests of verbal episodic
memory were improved, but other cognitive tests
were not. No adverse effects of the NGF infu-
sion were found. The results of this single case
indicated that NGF may counteract cholinergic
deficits in AD and suggested that further clini-
cal trials of NGF infusion in AD were warranted
(Olson etal., 1992; Seiger etal., 1993). However, in
a three-patient case study, researchers concluded
that while long-term intracerebro-ventricular
NGF administration may cause certain poten-
tially beneficial effects, the intraventricular route
of administration is also associated with nega-
tive side effects, including constant back pain
and rapid weight loss, and appear to outweigh
the positive effects of the protocol. Alternative
routes of administration, and/or lower doses of
NGF, perhaps combined with low doses of other
neurotrophic factors, may shift the balance more
positively (Eriksdotter-Jonhagen etal.,1998).
In addition to extensive study in AD, NGF
has also been examined in other CNS disorders.
After focal ischemia or stroke, numerous studies
have demonstrated that NGF, alone or combined
with other treatments, can improve functional
recovery (Cheng et al., 2009; Luk et al., 2004;
Zhu etal., 2011). In a PD model, NGF exhibited a
potential neuroprotective and neurorescue effect
375
on nigrostriatal dopaminergic neurons and may
lead to better functional restoration (Chaturvedi
etal., 2006; Li etal., 2000; Olson etal., 1991). In
Huntingtons disease, mitochondrial dysfunc-
tion plays an important role, and one study has
shown that NGF signaling can activate mito-
chondrial regulator PGC-1 expression, improve
behaviour, and restore sensorimotor ability and
reduce neuronal loss (Chen etal.,2012).
Studies have shown that NGF-dependent
functional improvement is due in part to
increased neurogenesis. In a dementia rat model,
NGF, which was at least partially mediated
through increased cholinergic tone, promoted
neurogenesis in the adult hippocampus, which
may be due to the nootropic action of NGF
(Frielingsdorf etal., 2007). In cerebral ischemia,
NGF administration improved functional recov-
ery by stimulating neurogenesis in the ipsilat-
eral striatum and subventricular zones (SVZ;
Tonchev, 2011; Zhu et al., 2011). In an in vitro
model, the extracellular signals of NGF and bFGF
induced cholinergic neuron differentiation from
endometrial stem cells (Noureddini etal.,2012).
Unfortunately, since NGF cannot cross the
blood-brain barrier, it has to be administered
directly into the brain, either intraventricularly
by surgery or biologically through gene ther-
apy. Intraventricular delivery is usually asso-
ciated with negative side effects such as pain.
Gene therapy, however, is promising. Three
recent Phase I clinical trials for AD are explor-
ing this prospect. In the first study, human NGF
genetically engineered into autologous grafted
fibroblasts was delivered into eight individuals
with mild AD. The results suggested diminu-
ation in the rate of cognitive decline, and serial
PET scans showed significant increases in cor-
tical 18-fluorodeoxyglucose after treatment
(Tuszynski et al., 2005). Another study used an
adeno-associated virusbased gene delivery tech-
nique to administer NGF to the brain to treat AD
symptoms and progression (Mandel, 2010). This
study has passed Phase I clinical safety testing,
and a multicenter Phase II clinical trial is cur-
rently ongoing. Another recent Phase I clinical
trial using encapsulated cell biodelivery of NGF
to the basal forebrain of AD patients showed pos-
itive neurological outcomes in two of six patients,
and no NGF-related adverse events were found
(Eriksdotter-Jonhagen etal.,2012).
Caution should be taken in the utilization of
NGF in different diseases. While NGF plays a sig-
nificant role in the survival of sensory and sympa-
thetic neurons after injury and inflammation, it is
376 Part III:Homeostatic Manipulators
also a major factor in pain promotion and hyper-
algesia (Hefti et al., 2006; Mantyh et al., 2011).
By binding to its receptor TrkA on nociceptors,
several different pathways are activated, including
ion channels and the transient receptor potential
vanilloid receptor, and secondary neurotransmit-
ters, including substance P and BDNF (Cattaneo,
2010; Hefti etal., 2006; Mantyh etal., 2011), which
cause immediate and long-term excitability.
Based on this information, in an example such as
back pain, it might be more beneficial to employ
NGF neutralizing antibodies or antagonists that
have been designed to fight different kinds of pain
rather than NGF itself (Cattaneo, 2010; Hefti etal.,
2006; Kivitz etal., 2013; Mantyh etal.,2011).
BDNF
BDNF was the second neurotrophic factor to be
discovered. It is a protein with a molecular mass
of 27 kD that was originally purified from pig
brain and was shown to support the survival of,
and fiber outgrowth from, cultured embryonic
chick sensory neurons in 1982 (Barde etal.,1982).
BDNF has been observed to be widely distrib-
uted within the CNS, including the hippocam-
pus, amygdala, thalamus, projection areas of
the olfactory system, inner and outer pyramidal
layers of the neocortex, claustrum, septum, cer-
ebellum, and superior colliculus (Connor and
Dragunow, 1998; Gall etal., 1992; Phillips etal.,
1990). In neurodegenerative diseases, many
studies have demonstrated reduced levels of
BDNF (Murer et al., 2001). For example, BDNF
mRNA expression was found to be reduced in
AD hippocampus specimens (Murray et al.,
1994; Phillips et al., 1991). A significant reduc-
tion in BDNF protein within the hippocampus
and temporal cortex of the postmortem human
AD brain was also found in a later study (Connor
et al., 1997). Decreased BDNF protein has also
been demonstrated in the substantia nigra in
PD (Howells et al., 2000). Furthermore, stud-
ies have shown that in Huntingtons disease,
loss of huntingtin-mediated BDNF transcrip-
tion leads to loss of trophic support to striatal
neurons, which subsequently degenerate in the
hallmark pathology of the disorder (Binder and
Scharfman, 2004; Zuccato etal., 2001). In anxi-
ety disorders, some studies showed reduced
BDNF levels in individuals; however, this was not
consistent across the various anxiety disorders
(Suliman etal., 2013). In CNS pain, electrophysi-
ological and behavioral data demonstrate that
inhibition of BDNF signal transduction inhibits
central pain sensitization (Croll etal., 1999; Pezet
etal., 2002). In epilepsy, however, studies showed
that BDNF mRNA and protein are markedly
upregulated in the hippocampus by seizure activ-
ity in both animal models and a patient sample
(Kokaia etal., 1995; Takahashi etal., 1999). Also,
overexpression of BDNF in transgenic mice led to
spontaneous seizures (Croll et al., 1999). BDNF
circulates systemically outside the CNS via the
cardiovascular system. Golden et al. (2010)
showed that elevated levels of circulating BDNF
in middle-aged and elderly adults increased the
risk of high blood pressure and cardiometabolic
dysfunction. BDNF preferentially binds to p75
neurotrophin receptor (p75NTR) rather than
TrkB on sympathetic neurons. Recent studies
show that selective activation of the p75NTR
in sympathetic neurons causes axon degenera-
tion. Therefore, elevated BDNF in the left ven-
tricle after ischemia-reperfusion may stimulate
p75NTR-dependent denervation of peri-infarct
myocardium (Lorentz,2013).
Since the purification of BDNF in 1982,
extensive literature has been published regard-
ing its central role in brain development, physi-
ology, and pathology. BDNF was demonstrated
to have survival and growth-promoting actions
on both central and peripheral neurons (Acheson
etal., 1995). BDNF exerts its growth-promoting
effect mostly by encouraging synapse growth
and facilitating synaptic plasticity, stimulat-
ing synaptogenesis. Studies have shown that, in
postnatal rat hippocampal slice cultures, BDNF
increased spine density in apical dendrites of
CA1 pyramidal neurons (Tyler and Pozzo-Miller,
2003) and enhanced the expression of synaptic
proteins (Tartaglia etal., 2001). Transgenic mice
overexpressing BDNF had an increased number
of synapses (63%) and increased synaptic vesi-
cle docking in area CA1 (Aguado et al., 2003).
A blockade of BDNFTrkB signaling prevented
spine head enlargement, whereas synaptic stim-
ulation plus the addition of exogenous BDNF
induced spine enlargement in the absence of
postsynaptic spikes (Tanaka etal.,2008).
The importance of BDNF in regulating synap-
tic transmission, plasticity, and growth suggests
that it has a crucial role in cognitive function.
Indeed, numerous studies have demonstrated
that a reduction in BDNF level not only impaired
long-term potentiation (LTP) and reduced the
number of synapses but was associated with a
deficit in learning and memory (Linnarsson
etal., 1997; Mu etal.,1999).
 Neurotrophic Regulation in Neurorestoration of theBrain
We now understand that the trophic prop-
erties of BDNF may lead to novel therapeutic
options in traumatic brain injury and stroke, in
neurodegenerative diseases such as PD and AD,
and perhaps even in neuropsychiatric disorders
(e.g., depression). In contrast, pathological levels
of BDNF-dependent synaptic plasticity may con-
tribute to conditions such as epilepsy and chronic
pain sensitization (Binder and Scharfman,2004).
Preclinical studies have shown promising
results for BDNF as a synaptic repair molecule in
neurodegenerative diseases. For example, intra-
ventricular infusion of BDNF or adeno-associated
virusinduced BDNF activity increases the
number of neurons in the adult olfactory bulb,
striatum, septum, and thalamus (Benraiss etal.,
2001; Pencea et al., 2001; Zigova et al., 1998).
Furthermore, in a neurotoxin 6-OHDA (oxido-
pamine) PD model, BDNF attenuated the loss of
dopaminergic axons and prevented rotational
asymmetry (Shults etal., 1995; Yoshimoto etal.,
1995). Moreover, BDNF has been shown to pro-
tect and/or repair hippocampal neurons and
synapses despite beta amyloid (A) build-up
and neuronal toxicity in a mouse model of AD
(Nagahara and Tuszynski,2011).
Even though the beneficial effects of BDNF on
neuronal functions have been discussed exten-
sively in the literature, few clinical trials using
BDNF have been conducted (four in ALS and
one in diabetic neuropathy; Lu etal., 2013), and
the results have been inconclusive. In a Phase I/II
open-label trial for ALS, subcutaneously admin-
istered BDNF showed a delay in the percentage
of forced vital capacity decline and an improve-
ment in walking time, but a Phase II/III trial did
not replicate these benefits (Liu et al., 2012). Lu
etal. (2013) postulated that the clinical failure of
BDNF may be due to the fact that BDNF is cleared
rapidly in vivo from where it was administered
and does not easily penetrate into the spinal cord
parenchyma.
Currently, several approaches are actively
being explored to deliver BDNF into the CNS.
Invasive procedures include using a catheter
or implantable pumps (Dittrich et al., 1996) or
implantation of a biodegradable polyethyl-
ene glycol-based hydrogel device containing
polylactic-co-glycolic acid microparticles encap-
sulated with BDNF (Bertram etal., 2010; Lampe
et al., 2011). Noninvasive approaches such as
nanoparticle molecular carriers of BDNF are also
being explored (Gabathuler, 2010; Gral et al.,
2013). In addition to BDNF itself as a potential
377
drug, other strategies to manipulate BDNFTrkB
signaling have also shown potential benefits.
Such strategies include small-molecule TrkB
agonists or modulators and TrkB agonistic anti-
bodies (Lin etal., 2008; Massa etal., 2010; Qian
et al., 2006). An even more promising strategy
is to pharmacologically enhance the expression
of endogenous BDNF in the brain using a small
molecule approach.
N E U R O T R O P H I N ( N G F/ B D N F )
S I G N A L I N G PAT H WAY
There are dozens of neurotrophic factors, and by
examining their nucleotide sequences and, there-
fore, evolutionary relatedness, they can be sepa-
rated into different families. The neurotrophin
family, including NGF, BDNF, NT-3, and NT-4,
is perhaps the best understood and most widely
expressed in the brain. Their intracellular signal-
ing pathways have also been extensively studied.
Neurotrophins regulate neuronal survival
and other aspects of cellular and biological
functions through the activation of correspond-
ing receptors. There are two distinctive classes
of neurotrophin receptors: p75NTR and the
tyrosine kinases (Trk) receptor. Trk-mediated
signaling promotes survival and growth, while
p75NTR-mediated signaling regulates celldeath.
T R K S I G N A L I N G PAT H WAY
The Trk receptors, a closely related family of
receptor tyrosine kinases, recognize neuro-
trophins with a relatively high degree of bind-
ing specificity. Biological effects of each of the
four mammalian neurotrophins are mediated
through activation of one or more of the three
members of the Trk receptor: TrkA, TrkB, and
TrkC. TrkA preferentially binds NGF, TrkB pre-
fers BDNF and NT-4/5, and TrkC interacts only
with NT-3. In addition, NT-3 can bind with the
other Trk receptors with less affinity (Chao, 2003;
Reichardt, 2006). Upon neurotrophin-binding
activation, the Trk receptor becomes autophos-
phorylated, and the autophosphorylation sites
form docking sites for the interaction of down-
stream signaling molecules. These molecules
trigger distinct downstream cascades of target
enzymes and other biological effects, includ-
ing neuronal differentiation and survival
(Chao, 2003; Huang and Reichardt, 2001). Three
well-conserved downstream signaling pathways
have been characterized, including Ras/extracel-
lular signal-regulated kinase (ERK)which is
now known as mitogen-activated protein kinase
378 Part III:Homeostatic Manipulators
(MAPK), phosphatidylinositol 3-kinase (PI3K)
and phospholipase C gamma (PLC-gamma)
pathways (Chao, 2003; Reichardt,2006).
The MAPK pathway is regulated by the activ-
ity of the Ras protein, which serves as a transducer
of signal from Trk to ERK proteins (Denhardt,
1996). The typical process starts from the activa-
tion of Ras by guanosine-5-triphosphate (GTP)
binding; following that, Ras recruits a serine
kinase of the Raf family to the membrane, where
it is activated. This initiates another cascade in
which Raf activates MEK, and then MEK phos-
phorylates and activates ERK. ERK is abundant
and multifunctional, regulating many different
cellular activities through transcription factors
such as c-myc, CREB, and c-Fos (Cobb, 1999;
Davis, 1993). By altering transcription factor
levels and activity, MAPK regulates cell cycle,
proliferation, and differentiation. Studies have
shown that MAPKs can mediate neuron survival
and neurite outgrowth (Fukuda etal., 1995)and
can regulate activity-dependent neuronal events
such as synaptic plasticity and LTP (English and
Sweatt, 1997; Grewal etal.,1999).
PI3K can be activated by three different
mechanisms, either directly by phosphoryl-
ated receptors, through docking proteins such
as the insulin receptor substrate family of pro-
teins (Myers and White, 1996), or by Ras protein
(Kodaki et al., 1994; Rodriguez-Viciana et al.,
1994). Upon activation, PI3K further stimulates
the formation of second messenger phosphati-
dylinositol (3,4,5)-trisphosphate, which activates
the downstream effector Akt (also known as pro-
tein kinase B, which is a serine/threonine-specific
protein kinase). Akt is best known for the
anti-apoptotic effects it generates by render-
ing downstream apoptogenic factors inactive in
many cell systems (Kennedy etal., 1997; Zhuang
etal., 2011). In neurons, PI3K has been shown to
mediate cell survival, induce glutamate excito-
toxicity/sensitivity, and initiate neurite process
outgrowth (Fryer etal.,2000).
The Trk receptor can also activate
PLC-gamma, which will result in the activation
of Ca2+ and protein kinaseregulated path-
ways and promote synaptic plasticity. Release
of intracellular stores of calcium can have
many effects, including activation of Ca2+/
calmodulin-dependent protein kinases and an
increase in the production of cAMP; both have
powerful effects on neurons. Studies show that
after transient exposure to NGF, the induc-
tion of the peripheral nerve type 1 sodium
channel gene in PC12 cells is mediated through
the PLC-gamma pathway (Choi etal.,2001).
P 7 5 N T R S I G N A L I N G PAT H WAY
P75NTR is a member of the tumor necrosis
factor (TNF) receptor superfamily. It consists
of an extracellular domain that includes four
cysteine-rich motifs, a single transmembrane
domain, and a cytoplasmic domain that includes
a death domain similar to those present in
other members of the TNF family. Originally,
p75NTR was identified as a low-affinity receptor
for NGF, but it was subsequently shown to bind to
each of the neurotrophins (BDNF, NT-3, NT-4/5)
with a similar affinity (Frade and Barde, 1998;
Hempstead, 2006; Johnson etal.,1986).
P75NTR is a key signaling component for
multiple ligands that regulate a wide array of
biological responses, some of which are opposite
(e.g., cell survival vs. programmed cell death),
depending on the cellular and pathological
context.
To date, three signaling pathways have
been elucidated following neurotrophin bind-
ing to p75NTR. One major pathway is the Jun
N-terminal kinase (JNK), also known as the
stress-activated protein kinase signaling cas-
cade. Signaling through this cascade results in
activation of p53 and apoptosis, causing more
neuronal cell death following injury (Coulson
etal., 2000; Reichardt, 2006). The crucial role of
JNK pathway-mediated apoptosis in the p75NTR
signaling pathway has been demonstrated in
many studies (Chen et al., 2009; Deshmukh
et al., 1996). Effectors reported as involved in
JNK pathway-induced apoptosis include CDC42,
apoptosis signal regulated kinase 1, Rac guano-
sine triphosphatase, P53, Bax, and Caspase (Chen
et al., 2009; Harrington et al., 2002; Reichardt,
2006; Troy etal., 2002). Increased expression of
p75NTR is a response to many forms of injury,
including neural axotomy, mechanical damage,
stroke ischemia, seizure, and so on. Several stud-
ies have demonstrated that p75NTR is responsi-
ble for cell death after injury in vivo. While the
mechanism of how p75 induces apoptosis is not
completely understood, some studies have shown
a link from proneurotrophin to p75-induced
apoptosis. Following injury or seizure, neuro-
trophins were released in uncleaved precursor
format-proneurotrophin. Antibodies specific to
proneurotrophins prevented cell death accord-
ingly (Harrington et al., 2004; Volosin et al.,
2008). Further studies are needed to elucidate
 Neurotrophic Regulation in Neurorestoration of theBrain
the pathways regulating proteolytic processing of
proneurotrophin in the uncleaved versus mature
form. In addition to regulating apoptosis and cell
death after injury, p75NTR has also been shown
to contribute to neurodegeneration. Studies have
demonstrated that there is a high level of p75NTR
expression in the cholinergic neurons of the adult
basal forebrain, which is severely affected in AD.
Some studies indicated that beta amyloid (142),
the major component of the hallmark plaque
found in AD patients, is a proapoptotic ligand
for p75NTR and triggers p75-mediated cell death
(Costantini et al., 2005). In addition, pro-NGF
was also found to contribute to AD pathology
via the p75NTR pathway (Pedraza et al., 2005;
Podlesniy etal.,2006).
Even though p75NTR mediates cell death
and neurodegeneration, it also can promote cell
survival through different pathways. Nuclear
factor-kappa B (NF-B) cascade is the major
signaling pathway induced by p75NTR activation
and contributes to neuronal survival (Hamanoue
etal., 1999; Middleton etal., 2000). In response
to ligand binding, p75NTR binds TNF receptor
associated factor-6 proteins through the intra-
cellular domains and activates a kinase cascade
that culminates in activation of IKK and IKK
and subsequent phosphorylation of IB subunits,
which targets them for ubiquitination and pro-
teosomal degradation. IB degradation releases
NF-B, which is a ubiquitously expressed tran-
scription factor. The released NF-B translocate
to the nucleus where they stimulate specific gene
expression and regulate cell survival.
A third pathway through the Rho family of
small guanosine triphosphatases has been iden-
tified, which activates RhoA, thereby inhibiting
neurite outgrowth (Chen etal., 2009; Reichardt,
2006; Yamashita et al., 1999). RhoA plays an
important role in transducing extracellular sig-
nals to changes in cytoskeletal proteins and
causes growth cone repulsion and collapse (Wu
etal.,2005).
Binding of each of the neurotrophins to
p75NTR also evokes activation of cellular sphin-
gomyelinase, which results in increased ceramide
production (Dobrowsky etal., 1994). Ceramide is
a versatile molecule that can promote both apop-
totic and prosurvival mechanisms by activating
many different signaling pathways, including
ERK, PI3K, JNK, and NF-B, as well as TrkA
(DeFreitas et al., 2001; MacPhee and Barker,
1997; Muller et al., 1998; Song and Posse de
Chaves,2003).
379
Crosstalk occurs inside the p75NTR pathway.
For example, while many studies have shown that
p75NTR/NF-B can enhance neuronal survival
(Chen etal., 2009; Foehr etal., 2000; Hamanoue
etal., 1999), research has also shown that the acti-
vation of the p75NTR is the underlying mecha-
nism of neuronal damage by A peptides in AD.
The p75NTR further activates p38 and JNK in a
death domain-dependent manner, followed by
NF-B translocation, p53 activation, and apopto-
sis (Costantini etal.,2005).
Even though both the p75NTR and TrkA
receptors lead to the activation of NF-B, they pro-
ceed via distinctly different proximal-signaling
intermediates and contribute to different cellu-
lar outcomes. Blocking p75-mediated activation
of NF-B in PC12 cells significantly enhanced
apoptosis. In contrast, blocking NF-B induction
via TrkA significantly inhibited the neurite for-
mation process (Foehr etal.,2000).
FGF2
Fibroblast growth factor (FGF) is a superfam-
ily with 23 members and 4 receptors identified
to date. In the mid-1980s, FGF2 (also known as
basic FGF) was the first FGF family member to
be isolated and cloned (Abraham et al., 1986a,
1986b). FGF2 is the most abundant member of
FGF in the CNS and is widely expressed dur-
ing development and postnatally, to both neu-
rons and glial cells (Eckenstein et al., 1991;
Emoto et al., 1989). FGF2 has proven to be the
most functionally versatile growth factor in the
CNS (Reuss and von Bohlen und Halbach, 2003;
Zechel etal., 2010)and is fundamentally involved
in the development of the CNS, the promotion of
axonal growth, the maintenance of plasticity in
adulthood, angiogenesis, and, most important,
the stimulation of adult neurogenesis.
Evidence has shown that FGF2 can modu-
late synaptic plasticity and axonal branching in
vitro and in vivo. Early studies demonstrated that
FGF2 is capable of promoting neurite extension
in PC12 cells (Rydel and Greene, 1987; Togari
et al., 1985) and rat cerebral cortical neurons
(Morrison et al., 1986) and axonal branching
in hippocampal neurons (Aoyagi et al., 1994).
Application of FGF2 enhanced LTP in rat hip-
pocampal slices (Terlau and Seifert, 1990).
Intracerebroventricular injection of epidermal
growth factor and FGF2 promoted the genera-
tion of LTP in synapses in the dentate gyrus of
anaesthetized rats (Ishiyama etal.,1991).
380 Part III:Homeostatic Manipulators
In addition, studies have also documented
the angiogenic properties of FGF2 in embryonic
development (Leconte etal., 1998), as well as in
the adult myocardium after ischemia (Iwai-Kanai
etal., 2002; Unger etal., 2000; Yanagisawa-Miwa
et al., 1992). FGF2 and vascular endothelial
growth factor play synergistic roles in angio-
genesis. They are released into the wound site
during haemostasis and promote the formation
of new blood vessels, thus stimulating angiogen-
esis (Przybylski, 2009). In a brain injury model
of chronic hypoxic-ischemia, the enhanced
endogenous angiogenesis and neurobehavioral
functions were demonstrated to be mediated by
upregulation of FGF2 (Seo etal.,2013).
Numerous studies have confirmed the impor-
tant role FGF2 plays in adult neurogenesis. In the
adult CNS, FGF2 is expressed in the SVZ and the
subgranular zone of the hippocampal dentate
gyrus (Ernfors et al., 1990; Zheng et al., 2004).
FGF2 has been extensively studied and found to
be a potent modulator of proliferation and dif-
ferentiation for multipotent neural progenitors.
The ability of FGF2 to induce proliferation of
adult stem cells has been clearly demonstrated
in in vitro studies using adult mouse striatum
progenitors (Gritti etal., 1996)and hippocampal
progenitors (Gage etal., 1995; Ray etal., 1993)by
maintaining these progenitors in the cell cycle
and preventing them from further differentia-
tion. In vivo studies have also provided evidence
of FGF2-induced neuronal precursor cell prolif-
eration in the neurogenic regions of the adult rat
brain (Kilpatrick and Bartlett, 1993; Rai et al.,
2007; Wagner etal.,1999).
F G F 2 S I G N A L I N G PAT H WAY
FGFs elicit their diverse effects through acti-
vation of cell surfacebound high affinity Trk
receptors FGFR1-4 (Friesel and Maciag, 1999;
Jaye etal., 1992). The binding of FGFs to FGFRs
is mediated in part by an additional interaction
with heparin sulfate proteoglycans. This inter-
action results in dimerization of the receptors,
resulting in autophosphorylation and transpho-
sphorylation of tyrosine residues, which further
activates the intracellular signaling pathways.
Among FGF receptors, FGFR1 is widely
expressed but is nevertheless confined to specific
neuronal populations in the adult CNS (Asai
et al., 1993; Yazaki et al., 1994). FGF2/FGFR1
signaling has been shown to regulate adult neu-
rogenesis in the SVZ of the adult rat (Mudo etal.,
2007). It also modulates inflammatory signaling
through the surface glycoprotein CD200, which
regulates microglial activation (Woodbury and
Ikezu, 2013). Manipulation of FGF2/FGFR1
signaling has been a focus of therapeutic devel-
opment for many neurodegenerative disorders,
such as AD, multiple sclerosis (Rottlaender etal.,
2011; Ruffini etal., 2001), PD (Baron etal., 2012),
and traumatic brain injury (Sun etal.,2009).
The intracellular signaling pathway of FGF2/
FGFR1 activation has not been fully elucidated.
From available studies, we know that both
phosphotyrosine-dependent and -independent
mechanisms are involved, and common path-
ways such as MAPK, PI3K, and PLC-gamma,
which regulate proliferation, differentiation, cell
survival, and apoptosis, are also triggered under
different conditions.
The most common pathway employed by
FGF2 is the MAPK pathway. The upstream
components of the signaling pathway, includ-
ing fibroblast growth factor receptor substrate
2 (FRS2) and the Src homology 2 domain, con-
tain transforming protein (Shc). FGFR1 can
phosphorylate FRS2 and Shc. The phosphoryl-
ated FRS2 continues to bind the adapter protein
growth factor receptor bound 2 (GRB2) and the
protein tyrosine phosphatase, nonreceptor type
11. Shc and GRB2 form a complex with the gua-
nine nucleotide exchange factor Son of sevenless
proteins (SOS). Further on, SOS activate the Ras/
Raf/MEK1/2/ERK1/2 signaling cascade, as dis-
cussed in the TrK section. This cascade leads to
phosphorylation of the target transcription fac-
tor ELK1 (Katoh, 2006; Suyama etal., 2002; Yang
etal.,2008).
The FRS2/GRB2 complex might also trigger
the PI3K pathway through a connection with
GRB2-associated binding protein 1 (GAB1). The
assembly of the FRS2/GRB2/GAB1 complex leads
to activation of the PI3K pathway and down-
stream effector proteins such as Akt (Hadari
et al., 2001; Ong et al., 2001). FGF2 is a known
inducer of epithelial-to-mesenchymal transition
(EMT). FGF2 induces EMT via PI3K (Ko and
Kay, 2005; Lee and Kay, 2003; Lee and Kay, 2006).
FGF2/FGFR1 also can trigger the PLC-
gamma signaling pathway in the process of
hydrolyzing membrane phospholipids in cells.
Upon activation, PLC-gamma in turn hydrolyzes
phosphatidylinositol 4,5 bisphosphate to diacyl-
glycerol (DAG) and inositol trisphosphate (IP3).
DAG and IP3 are second messengers. DAG acti-
vates protein kinase C delta; IP3 activates the IP3
receptor and induces the release of Ca(2+) from
 Neurotrophic Regulation in Neurorestoration of theBrain
intracellular Ca(2+) storage into the cytoplasm
(Kim etal.,2003).
NONADENINE-BASED PURINES
Since neurotrophins such as NGF and BDNF
cannot cross the blood-brain barrier, their thera-
peutic potential is limited. However, other strat-
egies that are being pursued include developing
drugs that mimic the action of growth factors on
receptors, stimulating growth factor synthesis,
and enhancing the effects of endogenous growth
factors.
Our lab made the initial discovery of the
effects of extracellular guanosine on cells (Kim
et al., 1991). Over the past two decades, we
have focused on the study of nonadenine-based
purines, especially guanosine. One of our studies
showed that guanosine can cross the blood-brain
barrier. Exogenous guanosine (8 mg/kg body
weight) entered the rat CNS within 7.5 minutes
of intraperitoneal administration and resulted
in a doubling of the guanosine concentration in
the brain (Jiang etal., 2008b). This unique char-
acteristic of guanosine makes it advantageous in
the potential future application of CNS disorder
treatment.
We discovered (Kim et al., 1991; Rathbone
et al., 1992; 1999) and many others confirmed
(Bocklinger et al., 2004; Moretto et al., 2009;
Thauerer et al., 2012) that exogenous guano-
sine exerts numerous neurotrophic effects. One
mechanism of action is by stimulating synthesis
and release of purines and trophic factors, such as
NGF. In our 1995 study, we found that cultured
neonatal mouse cortical astrocytes synthesized
NGF mRNA and released immunoreactive NGF
(ir-NGF) into the culture medium. The addition
of 10 M guanosine to the cultures increased
ir-NGF release by six-fold after 24 hours and
increased NGF mRNA six-fold after four hours
(Middlemiss et al., 1995b). Subsequent studies
showed that extracellular guanosine promotes
neurite outgrowth in PC12 cells and synergis-
tically enhances NGF-dependent neurite out-
growth (Gysbers and Rathbone, 1992, 1996a).
The enhancement of the neuritogenic effects of
NGF by GTP and guanosine may have physi-
ological implications in sprouting and functional
recovery after neuronal injury in the CNS due
to the high levels of nucleosides and nucleotides
released from dead or injured cells (Gysbers and
Rathbone, 1996a).
Extensive literature has demonstrated that
guanosine has neurotrophic effects both in vitro
381
and in vivo and can help functional recovery
in different CNS disorders such as hypoxia-
ischemia (Moretto et al., 2009; Thauerer et al.,
2012), stroke (Chang et al., 2008; Connell
et al., 2013; Rathbone et al., 2011), spinal cord
injury (Jiang etal., 2003a; 2007, 2008a), seizure
(Schmidt etal., 2000; 2005; Soares etal., 2004),
and PD (Giuliani etal., 2012; Su etal.,2009).
The purinergic system is emerging as a key
player in the endogenous control of neural stem
cells. Ulrich et al. (2012) discussed the impor-
tance of adenine-based purinergic signaling
molecules (ATP, uridine-5'-triphosphate, and
adenosine) in regulating the synchronized pro-
liferation, migration, differentiation, and death
of neural stem cells during brain and spinal
cord development. In our lab, we discovered
that systemic administration of guanosine can
stimulate the proliferation of endogenous pro-
genitor/stem cells. In a traumatic spinal cord
injury rat model, we found that daily intraperito-
neal administration of guanosine for two weeks
enhanced functional improvement in correlation
with an increase in myelination in the injured
cord. The functional improvement and remyeli-
nation observed after systemic administration
of guanosine is due to the effect of guanosine/
guanine on the proliferation of adult progenitor
cells and their maturation into myelin-forming
cells (Jiang et al., 2008a). Furthermore, in a rat
model of Parkinsonism, we showed that guano-
sine treatment increased cellular proliferation in
the substantia nigra and SVZ, and ameliorated
symptoms. Proliferating cells in the SVZ were
noted in nestin-positive adult neural progenitor/
stem cells. This finding indicated that guanosine
protected cells from apoptosis and stimulated
intrinsic adult progenitor/stem cells to become
dopaminergic neurons in rats with proteasome
inhibitor-induced PD (Su etal.,2009).
Guanosine treatment has some advantages
over autologous cell transplant and neuronal
growth factor techniques. First, as a naturally
occurring compound, it is nontoxic, and, unlike
with cell transplantation, there is no immune
reaction. Another unique characteristic of
guanosine is that it can cross the blood-brain
barrier (Jiang et al., 2008b). This evidence sug-
gests that the purinergic system may be a target
for new strategies to treat neurodevelopmental
disorders and neurodegenerative diseases.
Our studies showed that other nonadenine-
based purine derivatives can also stimulate NGF
production. Neotrofin, leteprinim potassium
382 Part III:Homeostatic Manipulators
(AIT-082) is a novel, metabolically stable deriva-
tive of the purine hypoxanthine. Addition of
AIT-082 to cultured PC12 cells significantly
enhanced NGF-mediated neurite outgrowth
from PC12 cells (Middlemiss et al., 1995a).
Exposure of mouse astrocytes to AIT-082 stimu-
lated messenger RNA production for NGF, NT-3,
and bFGF in cultured mouse astrocytes (Glasky
et al., 1995). Some preliminary experimental
evidence indicates that AIT-082 effects on neu-
rite outgrowth observed in vitro can also occur
in vivo. In the latter, AIT-082 increased NGF
mRNA synthesis in the region of injury when
administered orally to rats following basal fore-
brain lesions induced by stereotaxic injection
of ibotenic acid (Glasky et al., 1997). AIT-082
administration to rats resulted in a small but
significant advance in the onset of nerve sprout-
ing into denervated adjacent skin. This sprout-
ing process is NGF-dependent (Rathbone etal.,
1999). However, unlike treatment with exog-
enous NGF, AIT-082 treatment did not cause
hyperalgesia. These data not only indicate that
AIT-082 can stimulate neurite outgrowth in vivo
as in in vitro but also that AIT-082 may be thera-
peutically useful since, unlike NGF, it does not
cause hyperalgesia.
GUANOSINE SH AR ES
COMMON SIGNALING
TR ANSDUCTION CASCADES
W I T H N G F, B D N F, A N D F G F 2
Even though the detailed intracellular
mechanism(s) involved in the effects of guano-
sine on neuroprotection, neurogenesis, and
functional recovery of CNS disorders remains
to be fully elucidated, knowledge gained from
previous studies allows us some insight into
guanosines working network (Figure22.1).
Guanosine-activated cAMP-dependent
mechanisms are thought to activate the MAPK
cascade and potentially other protein kinases
(Rathbone et al., 1991; Tomaselli et al., 2005).
Within the MAPK pathway, guanosine has been
found to activate and stimulate phosphorylation
of ERK-1 and -2 in cultured astrocytes that may
act to promote NGF synthesis and release (Di
Iorio etal., 2001; Rathbone etal., 1998; Schwartz
and Mishler, 1990). The MAPK cascade has been
identified as the major pathway by which NGF
induces growth and differentiation in PC12 cells
(Rathbone et al., 1999) and may, therefore, rep-
resent a convergent mechanism for NGF and
guanosine.
Numerous studies have also found that the
neuroprotective effect of guanosine on ischemia
involves augmentation of glutamate uptake,
which is modulated by BK channels and the
activation of the PI3K pathway. Moreover, neu-
roprotection caused by guanosine depends on
the increased expression of phospho-Akt pro-
tein (Dal-Cim et al., 2011; Schmidt et al., 2008;
Tavares etal.,2005).
We also found that guanosine can stimu-
late endogenous stem/progenitor cell prolifera-
tion in a number of models (Jiang etal., 2008a;
Su etal., 2009). To further elucidate the mecha-
nisms underlying guanosine-triggered neuro-
genesis, we used an in vitro neural stem cell line
model and found that guanosine can stimulate
neural stem cell proliferation partially through
the cAMP-CREB pathway (Su et al., 2013).
Guanosine might also exert its neurogenesis
effect by stimulating endogenous FGF2 (Su etal.,
2009) or BDNF (unpublished data) accumula-
tion, or by working synergistically on common
signaling cascades downstream.
In an in vitro model employing cultured rat
astrocytes, we found that the antiapoptotic effect
of guanosine is mediated by PI3-kinase/Akt/
protein kinase B pathway activation (Di Iorio
etal., 2004). Utilizing an in vitro model of PD,
Giuliani etal. (2012) found that guanosine pro-
tected SH-SY5Y neuroblastoma cells when they
were exposed to neurotoxin 6-hydroxydopamine
(6-OHDA) and promoted their survival by reg-
ulating the p-38 and JNK pathways. Moreover,
guanosine also potentiated an early increase
in the phosphorylation of the anti-apoptotic
kinase Akt and increased the expression of
the anti-apoptotic Bcl-2 protein induced by
6-OHDA.
Guanosine has also been shown to contribute
to lipid homeostasis in the CNS. Cholesterol plays
a key role in the regulation of synaptic physiology
and neurotransmission as well as in those mech-
anisms underlying synaptic plasticity (Koudinov
and Koudinova, 2001). Moreover, cell choles-
terol depletion has been linked to a reduction in
amyloid beta formation. One study showed that
guanosine was able to increase cholesterol efflux
from astrocytes and C6 rat glioma cells through
PI3K/ERK-1/2 pathways (Ballerini etal.,2006).
The diverse effects of extracellular guanosine
on many cellular components of the CNS may
be related to its uptake into cells but may also be
due to its ability to release adenine-based purines
from cells; however, it has also been suggested
 Neurotrophic Regulation in Neurorestoration of theBrain 383

FIGURE 22.1: Guanosine may mediate its biological effects through growth factor pathways,
directly or indirectly, by sharing signaling transduction cascades. This schematic represents the pos-
sible mechanisms by which guanosine might mediate its biological effects. Guanosine, a small nonadenine purine
compound, may act through GPCR(s), or unknown receptor(s), to regulate signaling molecules (activating the
molecules as indicated by green arrow [], or inhibiting the molecule as indicated by green arrow end []). The
involvement of these molecules indicates that guanosine shares common signaling cascades with growth factors,
including NGF, BDNF, and FGF2. The three major signaling pathways (PLC-gamma, PI3K, MAPK) emanating
from receptor tyrosine kinases (RTKs) are shown here. RTKs are widely expressed transmembrane proteins that
act as receptors for growth factors, neurotrophic factors, and other extracellular signaling molecules. The Trk
receptor family and the FGF receptor family are two classes of RTKs. Guanosine was shown to activate the PI3K/
Akt and MAPK pathways. In the PI3K/Akt pathway, Akt has an anti-apoptotic effect through its effects on Bad and
caspases. In the MAPK pathway, activated Ras activates a cascade of kinases including Raf, MEK, and ERK (also
known as MAP kinases). ERKs stimulate many known effectors. Each of these pathways then exerts a number of
nuclear and nonnuclear actions with short- and long-term consequences for the cells. It was also demonstrated
that guanosine can induce glia cells to produce growth factors, which will further exert the biological effects.

that its mechanism of action may occur through 2011, a study using aDELFIA Eu-GTP binding
a specific G-protein coupled receptor. In 2002, assay further showed that the existing guano-
Traversa etal. showed that there is specific guano- sine binding site is a G-protein-coupled receptor
sine binding site in the rat brain membrane. In (Volpini etal.,2011).
384 Part III:Homeostatic Manipulators
TA R G E T I N G T H E C O N V E R G E N T
MOLECULES ONTHE COMMON
SIGNALING TR ANSDUCTION
CASCADE
As discussed previously, after CNS disorders, a
plethora of supportive (e.g., neurotrophic fac-
tors) and hostile molecules (such as semaphorins,
ephrins, myelin-secreted inhibitory glycopro-
teins, and repulsive axon guidance molecules)
are generated in the microenvironment (Chiquet,
1989; Fan etal., 2013; Filbin, 1995). Like the sup-
porting molecules, the nonpermissive inhibitory
cues also act through their respective receptors
to affect the collapsing of growth cones via sev-
eral pathways (Hou et al., 2008). Many of these
signaling pathways share common mechanisms
in a variety of different CNS disorders; therefore,
identifying this pathways role in the disease will
help to identify novel pharmacological targets
and design new modifiers for CNS disorders.
Many neurodegenerative disorders, such as
AD, PD, and polyglutamine diseases, share a
common pathogenic mechanism: the abnormal
accumulation of disease-causing proteins due
to either the mutant proteins resistance to deg-
radation or overexpression of the wild-type pro-
tein. In 2013, a research group at Baylor College
of Medicine in the United States published a
strategy to identify therapeutic entry points for
such neurodegenerative disorders by screening
for genetic networks that influence the levels of
disease-driving proteins (Park etal., 2013). To test
this principle, they used spinocerebellar ataxia
type 1 (SCA1), which is an inherited neurode-
generative disease characterized by a progres-
sive loss of cerebellar neurons. It is caused by the
expansion of a CAG triplet repeat located in the
N-terminal coding region of the disease-causing
gene ATXN1. Pathogenic ATXN1 contains an
abnormally elongated stretch of the amino acid
glutamine. This causes the protein to sponta-
neously misfold and form aggregates (Donato
etal.,2012).
Studies (Emamian et al., 2003; Jorgensen
et al., 2009; Park et al., 2013) have shown that
neurodegeneration in SCA1 parallels with the
levels of the mutant ATXN1 protein, and over-
expression of wild type ATXN1 results in neuro-
degeneration. Genetic network analysis revealed
that the MAPK cascade was the most enriched
where 6/10 genes belonged to the canonical
MAPK pathway (ERK1, ERK2, MED2, MEK3,
MEK6, and MSK1). ATXN1 is known to impair
motor performance; to determine the effects of
the MAPK pathway on the CNS, a motor per-
formance test was carried out in drosophila. The
use of small interfering RNA (siRNA) to decrease
the MEK, ERK-1/2, and MSK1 drosophila homo-
logues resulted in increased motor performance
and lifespan. Decreasing upstream MAPK path-
way homologues suppressed ATXN1 eye defects
and improved motor and lifespan phenotypes. It
was also found that ATXN1 levels were sensitive
to S776 phosphorylation. Further study revealed
that the MAPK kinases were implicated and that
MSK1 was able to phosphorylate the consensus
sequence associated with S776. Based on these
previous findings, in 2013 Park etal. designed a
proof-of-principle strategy to determine whether
the MAPK pathway could serve as a pharmaco-
logical target for SCA1. Human cells expressing
ATXN1 were treated with a PDI84352 (MEK1/2
inhibitor), GW5704 (RAF1 inhibitor), and a
Ro31-8220 (MSK1 inhibitor). Pharmacological
inhibition of the MAPK pathway led to decreased
ATXN1. Moreover, the addition of MAPK inhibi-
tors to cerebellar slices decreased ATXN1 levels
(Emamian et al., 2003; Jorgensen et al., 2009;
Park etal.,2013).
It has become evident that second messengers
such as cAMP, PKA, calcium, and diacylglycerol
can control ERK signaling via the small G pro-
teins Ras and Rap (Grewal etal., 1999). In intra-
cellular signal transduction, cAMP facilitates
the transfer of the effects of hormones such as
glucagon and adrenaline through the cell mem-
brane. It is involved in the activation of protein
kinases and regulates the effects of adrenaline
and glucagon. cAMP also binds to and regulates
the function of ion channels. Numerous studies
have shown the particularly beneficial effects of
cAMP to promote axonal regeneration, includ-
ing but not limited to spinal cord injury (Hannila
and Filbin, 2008; Kajana and Goshgarian, 2008;
Lau etal., 2013; Qiu etal., 2002; Whitaker etal.,
2008). Qiu et al. showed that by increasing the
intracellular levels of cAMP and protein kinase
Aactivity, neurons overcame myelin-based inhi-
bition. Many different methods have been studied
in an effort to increase intracellular cAMP:stim-
ulating adenylate cyclase with forskolin (Kilmer
and Carlsen, 1984), using cell-permeable cAMP
analogues such as dibutyryl cAMP or Sp-cAMPS
(Bhatt et al., 2004; Cai et al., 1999; Song et al.,
1998), initiating an intracellular signaling cas-
cade that prevents cAMP degradation by using
neurotrophins such as NGF and BDNF (Cai etal.,
1999; Hannila and Filbin, 2008), and inhibiting
 Neurotrophic Regulation in Neurorestoration of theBrain
PDE4 activity (the major source of phosphodi-
esterase activity in the CNS) with an antibody
(Iona etal.,1998).
As discussed in the previous section, the
p75NTR signaling pathway can induce neuronal
death, reduce axonal growth, and decrease syn-
aptic function. It has been hypothesized that the
induction of p75NTR may be part of a homeo-
static program that removes defective neurons,
axons, and synapses upon limited injury and
degeneration. Adownside of this function is the
exacerbated damage that p75NTR signaling pro-
duces following severe lesions and in pronounced
neurodegeneration. Because these circumstances
are prevalent in neural injury, stroke and neuro-
degenerative diseases in humans, there is a strong
rationale for inhibiting p75NTR cell death sign-
aling as a therapeutic approach for these condi-
tions (Ibanez and Simi, 2012). Direct application
of neurotrophin to push the balance of Trk versus
p75 signaling toward Trk cell survival signaling is
an obvious approach (Yano and Chao, 2000). In
addition, antibody or antagonist to p75NTR also
prevents apoptosis (Wang et al., 2008). Further
studies are needed to address the time window
for application. Astudy in myocardial-infarcted
mice may give us some hint. This study showed
that reduced BDNF levels modulated the early
inflammatory and neovascularization responses,
leading to improved survival and reduced cardiac
remodeling at day 28 post-myocardial infarction
(Halade etal.,2013).
CONCLUDING REMARKS
As widely demonstrated, neurotrophic factors
play a pivotal role in the neurorestoration and
neuroregeneration of damaged CNS neurons,
not only by potentiating the effects of intrinsic
growth-promoting programs but also by coun-
teracting at least some of the inhibitory signal-
ing within the CNS. Growth factors such as NGF,
BDNF, and FGF2 have been widely applied in
both preclinical and clinical studies, adminis-
tered alone or in combination with other phar-
macotherapeutic agents, which indicates their
fundamental role in neurorestoration. When
contemplating the use of growth factors in the
treatment of CNS disorders, the particular dis-
ease must be taken into consideration. For exam-
ple, using BDNF in neurodegenerative diseases
such as AD or PD will supply neurotrophic sup-
port and stimulate restoration (Lu et al., 2013;
Murer et al., 2001); however, in epilepsy and
seizures, further BDNF administration will
385
exacerbate the condition (Binder and Scharfman,
2004). Whether BDNF affects neurons positively
or negatively depends on the intracellular signal-
ing pathways it activates. For example, reduced
BDNF and TrK B receptor have been found in AD
patients, and beta amyloid (142), a pro-apoptotic
ligand for p75NTR, triggered p75-mediated cell
death in AD pathology. BDNF treatment not only
enhances the Trk/Akt cell survival signaling but
also precludes the production of A, thus sup-
pressing the p75 neurodegeneration pathway.
Despite the well-demonstrated beneficial
effects of growth factors, one practical issue with
their application is that they cannot cross the
blood-brain barrier and they rapidly degrade
in the circulation, which limits their thera-
peutic potential. Small molecules have no dif-
ficulty crossing the blood-brain barrier. If they
can mimic the action of growth factors on their
receptors, stimulate growth factor synthesis,
potentiate the effects of endogenous growth, or
even manipulate the activities of key cell signal-
ing molecules in a common mechanism in favor
of restoration, they will represent a logical and
promising strategy.
In this regard, the functions of adenine-based
purines such as adenosine and ATP as neuro-
transmitters and neuromodulators in the CNS
have garnered wide interest in the past few dec-
ades (Rathbone etal., 1991; 1999; 2008). As our
lab discovered, and as has been confirmed by
many others, nonadenosine-based purine nucle-
osides and nucleotides, particularly guanosine,
play an important role in CNS signaling and
may exert neuroprotective and neurorestorative
effects in response to physiological and patho-
logical conditions (Rathbone etal., 1999; 2008).
Most important, we recently discovered that
guanosine stimulates proliferation of endogenous
progenitor/stem cells in the adult mammalian
nervous system following chronic disorders. The
intracellular mechanisms involved in the effects
of guanosine have yet to be fully elucidated.
We have suggested that guanosine might exert
its effect through a G-protein coupled receptor.
Both the cAMP-independent and -dependent
mechanisms have been involved (Gysbers and
Rathbone, 1996b). Several in vitro studies have
shown that the cAMP-dependent mechanism, the
MAPK pathway, including ERK-1 and ERK-2 (Di
Iorio etal., 2001; Rathbone etal., 1998; Schwartz
and Mishler, 1990), and the cAMP-CREB path-
way (Su etal., 2013)are involved. Guanosine may
either directly stimulate endogenous growth
386 Part III:Homeostatic Manipulators
factor generation (including NGF, BDNF) or
crosstalk with the TrK pathway signaling cas-
cade through ERK and PI3K, thereby inhibiting
p75 NTR signaling to reduce apoptosis. Other
mechanisms underlying the biological effects of
guanosine are still to be discovered.
Despite the few but impressive clinical suc-
cesses, multiple breakthroughs in animal models,
and speedy progress in the development of basic
science research, there is still a gap from bench
to bedside, and few successful therapies or drugs
are available to treat CNS disease. Guanosine
is an example of a molecule that is able to exert
biological effects by regulating common intracel-
lular signaling cascades. By doing so, it is able to
promote cell proliferation, differentiation, sur-
vival, and neurogenesis. Identifying the common
pathways in CNS disorders will provide novel
pharmacological targets and, more important,
will open new avenues for combination thera-
pies. The combination therapies that have tar-
geted multiple barriers to neuronal functional
recovery have provided encouraging progress
and represent better therapeutic interventions.
Neurotrophic factors play a fundamental role
in building a permissive microenvironment for
restoration. Intracellular signaling pathways of a
variety of CNS disorders share a common sign-
aling cascade. Research targeting the converging
molecules on the common signaling transduc-
tion cascade may lead to the development of a
new generation of therapeutics with better selec-
tivity and potency.
ACK NOWLEDGMENTS
We thank Ms. Ann Kolkin for her helpful sugges-
tions and for her critical editorial review.
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PARTIV

Homeostatic Therapies
for Disease and Dysfunction
23
Epilepsy
DETLEVBOISON
INTRODUCTION
Epilepsy is one of the most frequent neurological
conditions, affecting about 1% of the population
or 70 million persons worldwide. It is a het-
erogeneous syndrome characterized by spon-
taneous recurrent seizures, which can be brief
periods of altered consciousness or absences
but can also generalize and involve motor func-
tions leading to convulsions. Whereas seizures
are generated by excessive electrical discharges
in neuronal clusters, the cellular and molecular
basis of epilepsy is still largely unknown. It is
now widely accepted that epilepsy is far more
complex than merely a disruption of the balance
between neuronal excitation and inhibition,
and in particular the contribution of inflam-
matory, glial, bioenergetic, and epigenetic
changes to the pathophysiology of epilepsy has
gained much attention recently (Boison et al.,
2011; Pan et al., 2005; Qureshi and Mehler,
2010; Ravizza et al., 2008; Seifert et al., 2010;
Vezzani et al., 2008; Williams-Karnesky et al.,
2013; Williamson et al., 2005). Most forms of
epilepsy are triggered by precipitating injuries,
which lead to blood-brain barrier (BBB) disrup-
tion, inflammatory responses, and microglial
and macroglial activation. In particular, astro-
gliosis and disruption of key astrocyte depend-
ent homeostatic functions such as aquaporin
expression, ion buffering, and adenosine equi-
librium play important roles in epileptogenesis.
Those mechanisms certainly play a role in post-
traumatic epileptogenesis, where disruption of
the BBB and astrogliosis are significant factors.
Consequently, epilepsy presents as a disorder of
complex network dysfunction, which is not only
characterized by recurrent seizures but also by
comorbidities ranging from sleep dysfunction
to cognitive impairment, depression, and psychi-
atric problems (Hermann etal., 2008; LaFrance
et al., 2008; Lin et al., 2012). Intriguingly, it
has become clear that comorbidities occur
early in the disease and frequently precede the
emergence of epileptic seizures, often by years
(Kanner, 2012; Rudzinski and Meador, 2013). It
is tempting to speculate that epileptic seizures
are merely one more symptom of a complex syn-
drome in which major homeostatic functions of
the brain are disrupted.
DISRUPTION OFNET WORK
H O M E O S TA S I S I N E P I L E P S Y
Current epilepsy treatment is based on the pre-
sumption that neuronal dysfunction is the
primary cause of epilepsy. However, current
antiepileptic drugs (AEDs) that have neuronal
targets and have been designed to suppress sei-
zures as the major symptom found in epilepsy
are ineffective in about one-third of all persons
with epilepsy; importantly, those drugs (i) fail
to treat the comorbidities of epilepsy and (ii)
fail to affect the underlying pathogenetic pro-
cesses (Brooks-Kayal et al., 2013; Sorensen and
Kokaia, 2013). There is a substantial amount of
evidence implicating nonneuronal mechanisms
as a major contributing factor to the develop-
ment of epilepsy (i.e., epileptogenesis). Among
those, inflammatory and immunomodulatory
processes, as well as the integrity of the BBB,
are involved, and glial dysfunction is therefore
a major player in the disruption of homeostatic
functions in epilepsy (Figure 23.1; Boison, 2012b;
Coulter and Eid, 2012; Steinhauser et al., 2012).
The identification of nonneuronal mechanisms
contributing to the syndrome of epilepsy offers
new hopes to treat not only the hallmark seizures
of epilepsy but also the accompanying comor-
bidities and to halt those processes that lead to
the development of epilepsy. The next sections
describe key homeostatic functions that are dis-
rupted in clinical epilepsy as well as in animal
models of the disease.
FIGURE23.1: Disruption of network homeostasis in epilepsy. Astrocytes play a key role in the mainte-
nance of network homeostasis in the brain. Astrocytic endfeet couple to the vasculature, whereas distinct processes
contribute to the tripartite synapse. Through neurovascular coupling, astrocytes can directly link vascular signals
to neuronal activity, whereas neuronal activity can directly influence vascular responses. A selection of major
homeostatic regulators is shown, with pathological alterations in epilepsy shown in red. Water and ion homeosta-
sis depend largely on the water channel aquaporin 4 (AQ4, in pink), the potassium channel Kir4.1 (in green), the
sodium potassium ATPase (in orange), and the sodium potassium chloride cotransporter (in violet). In epilepsy,
delocalization of AQ4 impairs potassium uptake through Kir4.1, contributing to a rise in synaptic K+. Astrocytes
control synaptic glutamate via reuptake through the glutamate transporter GLT-1 (in blue), through intracellular
conversion of glutamate into glutamine via glutamine synthetase (GS, in blue), and through the Ca 2+-dependent
release of glutamate as gliotransmitter. In epilepsy, decreased GLT-1 dependent glutamate uptake, decreased met-
abolic clearance through GS, and increased astroglial release of glutamate all contribute to an increase in synaptic
glutamate. Astrogliosis and associated overexpression of adenosine kinase (ADK, in red) in the epileptic brain
lead to increased metabolic clearance of adenosine (ADO) and resulting adenosine deficiency. Disruption of the
blood-brain barrier leads to the extravasation of albumin and TGF, which trigger astrogliosis and compromise
the buffering capacity of astrocytes. Growth factors such as brain derived neurotrophic factor (BDNF) lead to the
activation of transcriptional programs in neurons and via transactivation of the TrkB receptor (in green) with the
adenosine A 2AR (in orange) can trigger astrogliosis and overexpression of ADK. Inflammatory mediators, such as
interleukin 1 (IL-1) or tumor necrosis factor alpha (TNF) can compromise GLT-1-dependent glutamate uptake
and can trigger the astroglial release of glutamate. Mitochondria play a major role in the regulation of ATP and
may release reactive oxygen species (ROS) in epilepsy. See the text for more details.

Ion and Water Homeostasis
In the brain, the homeostasis of water and ions
is intricately linked and controlled by channels,
which are permeable to water and/or ions, such
as K+, Na+, Ca2+, and Cl. Apart from tightly con-
trolled ion fluxes through neuronal membranes,
glial cells exert a major control over water and ion
homeostasis (Seifert etal., 2010; Steinhauser etal.,
2012). Remarkably, a major part of the energy
expenditure of the brain is devoted to the con-
trol of ion fluxes. Aquaporin 4 (AQP4) is a glial
water transport channel, which is altered as a
consequence of brain injury (Guo etal., 2006)and
directly implicated in epileptogenesis (Binder
etal., 2012). The channel is normally localized to
both the perivascular endfeet and within peri-
synaptic processes of astrocytes, where it per-
mits the bidirectional exchange of water between
the extracellular space and the blood (Nagelhus
etal., 2004; Nielsen etal., 1997; Rash etal., 1998).
However, AQP4 redistributed primarily to peri-
synaptic processes in human mesial temporal lobe
epilepsy and therefore might be a contributing
factor of hyperexcitability through dysregulation
of water and K+ homeostasis (Eid etal., 2005). In
support of this view, AQP4 knockout mice have
an increased volume of extracellular space and are
less susceptible to pentylenetetrazole-induced sei-
zures (Binder et al., 2004a; Binder et al., 2004b).
Glial water flow is tightly linked to K+ transport
via the inward rectifying K+ channel, Kir4.1, which
colocalizes with AQP4 on the astrocyte membrane
(Hsu etal., 2011). Likewise, elevation of extracel-
lular K+ to millimolar concentrations influences
neuronal excitability and exacerbates epileptiform
activity (Feng and Durand, 2006). The importance
of the maintenance of K+ clearance mechanisms is
indicated by findings that polymorphisms in the
gene for Kir4.1 are associated with human epi-
lepsy and that a glial specific deletion of Kir4.1 in
mice reduced K+ clearance from the synaptic cleft;
deficiencies in the homeostasis of K+ clearance
therefore constitute a feasible mechanism for the
seizure phenotype in human patients (Haj-Yasein
et al., 2011; Heinemann et al., 2000). Together
these findings suggest an intricate interplay
between water and potassium homeostasis and a
direct involvement of water and ion homeostasis
in the pathogenesis of epilepsy.
Glutamate and Glutamine
Homeostasis
Glutamate transporters located in astrocytes
are responsible for the reuptake and clearance
Epilepsy 403
of more than 80% of synaptic glutamate and
therefore play an essential role in terminat-
ing excitatory neurotransmission (Anderson
and Swanson, 2000). The homeostasis of gluta-
mate, but also of its metabolic product GABA,
depends on the glutamate-glutamine cycle in
which astrocytes convert the transported gluta-
mate into glutamine via glutamine synthetase.
Glutamine is transported back into neurons,
where it is transformed back into glutamate
(or into GABA following decarboxylation of
glutamate). Any disruption of astrocyte func-
tion in epilepsy is therefore expected to affect
glial glutamate homeostasis. Virus-induced
gliosis was recently used to trigger a reduc-
tion in the expression of astroglial glutamine
synthetase; as a consequence, adjacent neu-
rons displayed a specific deficit in inhibitory
synaptic function, however, in the absence of
changes in excitatory function (Ortinski et al.,
2010). Those non-cell-autonomous deficits in
inhibition were mimicked by blockade of the
glutamate-glutamine cycle and reversed by
exogenous glutamine. This study directly dem-
onstrated that astrogliosis per se could alter
neuronal excitability via disruption of astroglial
glutamate homeostasis. In line with those find-
ings, epileptogenic tissue resected from patients
with temporal lobe epilepsy (TLE) was charac-
terized by a significant reduction in glutamine
synthetase as a likely explanation for increased
extracellular glutamate in TLE (Eid etal., 2004).
Glutamine synthetase deficiency might be a pos-
sible cause for TLE since the sustained microin-
fusion of a glutamine synthetase inhibitor into
the hippocampus of rats triggered recurrent
seizures that continued for several weeks and
resulted in neuropathological features reminis-
cent of TLE (Eid et al., 2008). Thus astrocytes
directly control the homeostasis of critical neu-
rotransmitters via specific uptake and intracel-
lular metabolic mechanisms. However, glial cells
can also directly affect the homeostasis of neuro-
transmitters and neuromodulators through gli-
otransmission, as evaluated in the next section.
Glial Dysfunction and
Gliotransmitter Homeostasis
Hippocampal sclerosis is a characteristic patho-
logical hallmark of TLE (Malmgren and Thom,
2012), and surgical resections of epileptogenic
brain tissue have demonstrated that onset
zones for chronic temporal lobederived and
posttraumatic seizures correlate with gliotic
404 Part IV:Homeostatic Therapies

scarring. Astrocytes influence the pathogenesis Adenosine Homeostasis

and pathophysiology of epilepsy by the homeo-
static control of synaptic transmission via the
release of gliotransmitters such as glutamate,
adenosine 5-triphosphate (ATP), and d-serine
(Haydon and Carmignoto, 2006) in addition
to the reuptake of neurotransmitters such as
glutamate (Coulter and Eid, 2012) or neuro-
modulators such as adenosine (Boison, 2012b).
An astrocytic basis of epilepsy was proposed
based on findings suggesting that prolonged
episodes of neuronal depolarization evoked by
the astrocytic release of glutamate contributes
to epileptiform discharges (Tian et al., 2005).
Astrocytes play important upstream homeo-
static roles in controlling uptake, degradation,
and recycling of neurotransmitters. In addi-
tion, glial dysfunction in the BBB, as well as
neuroimmunological functions governed by
glia, have been implicated not only in seizure
generation (i.e., ictogenesis) but most impor-
tantly in the pathophysiological processes that
lead to the development of epilepsy (i.e., epi-
leptogenesis). Because glia communicate with
each other and assume a role that is upstream of
neuronal function, perturbations of glial home-
ostasis can affect entire neuronal networks.
Those network effects of glia might indeed be a
reason why neuronal networks in epilepsy syn-
chronize; similarly, fluctuations in homeostatic
functions of glia might explain why seizures are
sporadic.
Epileptic seizures trigger an increase in extracel-
lular adenosine, and it is this seizure-induced
increase in adenosine that normally terminates
a seizure and leads to postictal brain shutdown;
consequently, adenosine is an endogenous anti-
convulsant of the brain (Boison, 2012b). It affects
neuronal excitability via pre- and postsynaptic
adenosine receptors, which contribute to the
control of neuronal Ca2+ and K+ fluxes; in addi-
tion, it provides important homeostatic control
over mitochondrial bioenergetics and epige-
netic functions through interference with DNA
methylation (Boison, 2013; Masino et al., 2011;
Williams-Karnesky et al., 2013). Adenosine
prevents seizures via activation of adenosine A1
receptors, which couple to inhibitory G proteins.
Deletion of the A1R in mice causes lethal status
epilepticus (SE) following traumatic brain injury
or exposure to an excitotoxin (Fedele etal., 2006;
Kochanek etal., 2006), whereas therapeutic aden-
osine augmentation effectively stops seizures
(Boison, 2012a). A crucial role for A1 receptors
in seizure control are supported by clinical find-
ings of A1R variants that have been associated
with the development of posttraumatic epilepsy
(Wagner et al., 2010). Synaptic levels of adeno-
sine are largely controlled by metabolic clearance
through the astrocyte-based enzyme adenosine
kinase (ADK), which is a phosphotransferase
converting adenosine into adenosine monophos-
phate (AMP; Boison, 2013; Figure 23.2). ADK is

FIGURE 23.2: Adenosine homeostasis:functions of adenosine and therapeutic adenosine aug-

mentation. Red: ATP is the major source of adenosine, whereas the metabolic clearance of adenosine is largely
mediated by adenosine kinase, which in the adult brain is primarily located in astrocytes. Green:Adenosine exerts
neuroprotection and seizure suppression via the activation of neuronal A1Rs; in addition adenosine has procogni-
tive and antipsychotic properties via activation of neuronal A 2ARs. Blue: Adenosine plays an epigenetic role via
the regulation of DNA methylation, a mechanism that is antiepileptogenic. Violet: Several strategies have been
developed to therapeutically augment adenosine signaling in the brain. Those include systemic ADK inhibitors,
systemic A1R agonists, stem cell therapy, and gene therapy.

upregulated and causes adenosine deficiency in
epileptogenic sclerotic tissue in a variety of rodent
models of epilepsy, as well as in human specimens
resected from patients with TLE and hippocam-
pal sclerosis (Aronica etal., 2011; Li etal., 2008).
Acausal role for overexpressed ADK in epilepsy
is suggested by findings that transgenic or viral
overexpression of ADK was sufficient to trigger
recurrent electrographic seizures in mice (Li
etal., 2008; Shen etal., 2014). In addition, homeo-
static functions of the adenosine system appear to
play a crucial role in epileptogenesis. Both trans-
genic animals with forebrain-selective reduction
of ADK and recipients of adenosine-releasing
cells or devices showed a significant attenuation
of the development of epilepsy (Li et al., 2008;
Williams-Karnesky et al., 2013). These findings
indicate that astroglial ADK is a promising target
for the prediction and prevention of seizures in
epilepsy.
Blood-Brain Barrier
A breakdown of the BBB has been intricately
linked to epileptogenesis (Heinemann et al.,
2012)and plays an instrumental role in the devel-
opment of pharmacoresistance in chronic epi-
lepsy (Loscher and Potschka, 2002). Clinical data
document BBB disruption in patients with post-
traumatic epilepsy (Tomkins et al., 2008) and
suggest an epileptogenic role of BBB disruption
in patients with brain tumors (Marchi et al.,
2007). BBB disruption has been associated with
the extravasation of albumin and transforming
growth factor beta (TGF-) signaling (Cacheaux
etal., 2009; Ivens etal., 2007), which contributes
to the transformation of astrocytes into their
reactive form, a process that also involves the
initiation of a transcriptional program leading
to further astrocyte activation, inflammation,
and reduced clearance capacity for glutamate
and K+ (Cacheaux et al., 2009; David et al.,
2009). The reduced buffering capacity of trans-
formed astrocytes for glutamate and K+ appears
to be most critical during repetitive activa-
tion. Experimental blockade of TGF- signal-
ing following BBB disruption decreased those
transcriptional responses and prevented epi-
leptogenesis (Cacheaux etal., 2009; David etal.,
2009). More recently, BBB disruption following
kainic acidinduced epileptogenesis in rats was
monitored longitudinally via MRI. Those MRI
data showed BBB leakage at 1 day and 6 weeks
after SE in the hippocampus, entorhinal cortex,
amygdala, and piriform cortex (van Vliet et al.,
2014). In this case kainic acidinduced SE was
Epilepsy 405
the initial trigger for BBB dysfunction, which
became apparent as soon as one day after the
event. Thus BBB disruption as evaluated by MRI
might constitute a valuable biomarker and target
for the prediction and prevention of epileptogen-
esis following an insult to thebrain.
Growth Factors
Epileptic seizures can induce wide-ranging
changes in growth factors, neurotrophins, and
transcription factors (Grabenstatter etal., 2012).
Akey role in epileptogenesis has been ascribed to
brain-derived neurotrophic factor (BDNF) and
its receptor TrkB (Grabenstatter etal., 2012). In
neurons, BDNF activates the JAK/STAT pathway,
cAMP response element binding protein, induc-
ible cAMP early repressor, and early growth
response factors that induce a shift in the expres-
sion of specific subunits of the GABA AR, as well
as the expression levels of N-methyl-D-aspartate
receptors (Kim et al., 2012; Lund et al., 2008;
Roberts et al., 2006). In astrocytes, activation
of TrkB has been linked to the development of
astrogliosis, a mechanism that is also influenced
by transactivation of the TrkB receptor through
the adenosine A2AR (Brambilla et al., 2003).
Therefore, an injury- or seizure-induced surge
in adenosine (Clark et al., 1997) could trigger
and potentiate pathogenetic astroglial changes
through increased TrkB signaling. Growth factor
dependent mechanisms that contribute to epilep-
togenesis via the disruption of glial homeostatic
functions are a new area of research that requires
increased attention.
Immunomodulatory and
Inflammatory Responses
Immune and inflammatory processes play cru-
cial roles in the development of epilepsy, and
anti-inflammatory therapies might have antie-
pileptogenic properties (Aronica et al., 2012).
Importantly, components of the proinflamma-
tory interleukin-1/Toll-like receptor (IL-1R/
TLR) signaling pathway are overexpressed in
surgically resected specimen from a human with
TLE (Ravizza etal., 2008), whereas experimental
activation of the IL-1R/TLR pathway has been
linked to the precipitation and recurrence of
seizures (Vezzani et al., 2011a). In addition, the
activation of the IL-1R/TLR pathway by endog-
enous ligands or by viral or bacterial mimetics
can increase excitability of the brain by inducing
rapid posttranslational changes in voltage- and
ligand-gated ion channels. Among the endog-
enous ligands are proinflammatory cytokines,
406 Part IV:Homeostatic Therapies
such as interleukin-1 (IL-1), or danger signals,
such as High Mobility Group Box 1 (HMGB1),
which can be released from injured or activated
cells (Vezzani etal., 2011b). HMGB1 is the endog-
enous ligand of TLR4 and normally bound to
chromatin. However, following either cell dam-
age or neuronal hyperexcitability, this factor
can be released into the extracellular space. The
proepileptogenic role of HMGB1 is supported by
recent data showing that blockade of the TLR4
pathway significantly delays the onset of seizures
(Maroso etal., 2010). Likewise, engineered mice
with defects in IL-1R/TLR signaling are intrin-
sically resistant to seizures. Together, inflamma-
tory processes and disruption of the BBB might
support chronic hyperexcitability of the brain via
compromised homeostatic functions dependent
on glial mechanisms on multiple levels.
Mitochondrial Bioenergetics
Mitochondria are characterized by important
homeostatic functions that influence neuronal
excitability, including production of ATP, fatty
acid oxidation, control of apoptosis and necrosis,
regulation of amino acid cycling, neurotransmit-
ter biosynthesis, and regulation of cytosolic Ca2+
homeostasis. Mitochondrial oxidative phospho-
rylation is the main pathway for the generation
of ATP, which in turn is intricately related to
adenosine. Uncoupling of mitochondrial oxida-
tive phosphorylation leads to an increase in aden-
osine, which is independent of ATP (Doolette,
1997). Energy depletion in general is marked
by the accumulation of AMP, a direct precur-
sor of adenosine. At the same time, increased
AMP signals an impending energy crisis and
activates the serine/threonine AMP-activated
protein kinase (AMPK; Zong etal., 2002), which
in turn interrupts ATP-consuming reactions,
activates ATP-generating pathways (Hardie,
2004), and promotes mitochondrial biogenesis
(Reznick and Shulman, 2006). Therefore enhanc-
ers of mitochondrial biogenesis or other strate-
gies that enhance ATP increase adenosine via
increased availability of the adenosine precursor
ATP (Masino and Geiger, 2008). Based on those
mechanisms, it can be hypothesized that when-
ever mitochondrial function is altered (in both
directions), an increased production of adeno-
sine can be expected. Certain inherited epilepsies
are associated with mitochondrial dysfunction;
however, little is known about its role in acquired
epilepsies such as TLE. Recent lines of evidence
suggest that mitochondrial oxidative stress and
dysfunction are key factors not only resulting
from seizures or injuries to the brain but also
contributing to epileptogenesis (Waldbaum and
Patel, 2010). Mitochondria can produce reactive
oxygen species and therefore are uniquely vul-
nerable to oxidative stress, which can severely
affect the production of ATP, the stability of mito-
chondrial DNA, and synaptic glutamate homeo-
stasis, which can all affect neuronal excitability
and seizure susceptibility. Therefore, improving
mitochondrial bioenergetics may offer the dual
benefits of restoring metabolic dysfunction and
adenosine homeostasis and is a potential strategy
to affect not only seizures but also the pathoge-
netic processes involved in the development of
epilepsy.
Epigenetics
The methylation hypothesis of epileptogenesis
suggests that seizures by themselves can induce
epigenetic chromatin modifications and thereby
aggravate the epileptogenic condition (Kobow
and Blumcke, 2011). Epigenetic modifications,
which alter gene transcription without modify-
ing the underlying DNA sequence, are highly
plastic and can respond rapidly in response to
environmental cues, an important endogenous
mechanism for temporally and spatially control-
ling gene expression. Changes in histone acetyla-
tion and methylation, as well as changes in DNA
methylation, once thought to occur only in divid-
ing cells, have been shown to also occur in mature
cells in the central nervous system (Feng et al.,
2010; Ma et al., 2009). Tellingly, these changes
occur regularly and rapidly. Methylation of
DNA in the central nervous system has attracted
increasing attention recently, with new research
showing activity-induced proliferation of neural
precursor cells via active DNA demethylation
(Ma et al., 2009). Altered DNA methylation in
the brain has also been implicated in psychiatric
and neurological conditions, including epilepsy
(Kobow etal., 2009; Ma etal., 2009). DNA meth-
ylation requires the donation of a methyl group
from S-adenosylmethionine, a process that is
facilitated by DNA methyltransferase enzymes.
The resulting product, S-adenosylhomocysteine
(SAH) is then further converted into adenosine
and homocysteine by SAH hydrolase. Critically,
the equilibrium constant of the SAH hydrolase
enzyme lies in the direction of SAH forma-
tion (Kredich and Martin, 1977); therefore, the
reaction will proceed only when adenosine and
homocysteine are constantly removed (Boison
et al., 2002; Kredich and Martin, 1977). In
the adult brain, removal of adenosine occurs
 Epilepsy 407

Epileptogenic condition Adenosine therapy

S-adenosylmethionine
DNMT DNMT
Methylated DNA Methylated DNA

S-adenosylhomocysteine S-adenosylhomocysteine
Epileptogenesis
Homocysteine Antiepileptogenesis
Adenosine (ADO) (HCY)
Adenosine (ADO)
Adenosine kinase (ADK)
Seizures AMP Seizure suppression

FIGURE23.3: Epigenetic role of adenosine. Red:Adenosine is an obligatory end product of transmethyla-

tion reactions. If adenosine is not constantly removed by adenosine kinase (ADK), its levels increase and block
DNA methylation. In the chronic epileptic brain, ADK is increased, and this increase in the metabolic clearance of
adenosine is thought to drive DNA methylation. Correspondingly, the chronically epileptic brain is characterized
by hypermethylated DNA. DNA hypermethylation and reduced adenosine are both thought to drive epileptogen-
esis and disease progression in epilepsy. Green:Adenosine augmentation therapy restores adenosine and thereby
not only exerts seizure suppression but also blocks DNA methylation, leading to a reversal of the hypermethylated
state found in chronic epilepsy. Through this epigenetic mechanism, adenosine augmentation therapy is thought
to prevent epileptogenesis. Blue:Reactions dependent on DNA methyltransferases (DNMT).

largely via the astrocyte-based enzyme ADK,
which is overexpressed in epilepsy (Boison,
2013; Fredholm, 2012; Pignataro et al., 2008).
If metabolic clearance of adenosine through
ADK is impaired, SAH levels rise (Boison etal.,
2002). SAH in turn is known to inhibit DNA
methyltransferases through product inhibition
(James et al., 2002). Since disruption of adeno-
sine homeostasis and adenosine deficiency has
been implicated in epileptogenesis, local thera-
peutic adenosine augmentation is an effective
strategy to acutely suppress seizures in modeled
epilepsy (Boison, 2009). In addition, possible epi-
genetic effects of adenosine augmentation in the
treatment of epilepsy, including the potential to
modulate DNA methylation status, have recently
been investigated in our laboratory, making use
of adenosine-releasing silk-based brain implants,
which provided long-term suppression of epilep-
togenesis (Figure 23.3; see also next chapter and
Williams-Karnesky etal.,2013).
Reconstruction ofNetwork
Homeostasis inthe Therapy
ofEpilepsy
The mechanisms discussed in the preceding sec-
tions offer novel opportunities for the treatment
and prevention of epilepsy via the therapeutic
manipulation of homeostatic control mecha-
nisms. Those approaches differ from conventional
single-target or defined-target symptomatic
therapies by affecting entire networks on several
different levels. The synergistic modulation of
multiple known and unknown pathways may
offer new hope to treat epileptic seizures as well
as associated comorbidities comprehensively
and to directly prevent those processes that lead
to the development of epilepsy and an aggrava-
tion and progression of the epileptic phenotype.
Interestingly, older AEDs such as valproate, car-
bamazepine, and phenytoin, which are relatively
unspecific and may act on different targets and
mechanisms, are still the clinical mainstay for
epilepsy therapy, whereas newer AEDs, which are
cleaner and more specific to better character-
ized mechanisms and pathways, did not lead to a
major overall improvement in the therapy of the
epilepsies (Vajda, 2007). The following sections
report recent progress in severalareas.
Restoration ofGlutamate
Homeostasis
Although few studies have directly addressed
glutamatergic therapies in animal models of
epilepsy, four strategies might have the potential
to reconstruct glutamate homeostasis in epilepsy.
First, -lactam antibiotics increase glutamate
transporter expression on astrocytes (GLT-1)
in vivo and thereby increase the clearance rate
of glutamate from the synaptic cleft (Rothstein
et al., 2005). In an in vitro study, the -lactam
antibiotic ceftriaxone (10M for 2days) increased
glutamate uptake in primary human astrocytes
through NFB-dependent activation of the
408 Part IV:Homeostatic Therapies
major astroglial glutamate reuptake transporter
excitatory amino acid transporter 2 (EAAT2; Lee
etal., 2008). Second, daily administration of lev-
etiracetam (50 mg/kg, i.p) for 20days beginning
one day after a traumatic brain injury triggered
by controlled cortical impact in rats reversed
the TBI-induced decrease in regional glutamate
transporter expression, indicating that at least
one AED has the potential to restore glutamate
homeostasis (Zou et al., 2013). Third, epilepti-
form activity was supported by the maintenance
of glutamine transport into neurons (Tani etal.,
2010), whereas intact glutamine synthetase func-
tion appears to be crucial for seizure prevention
by driving the intracellular metabolic clearance
of extracellular glutamate (Ortinski etal., 2010).
Therapies aimed at reconstructing glutamine
synthetase function generally appear to have sci-
entific merit; however, the concept that reduced
glutamine synthetase expression and activity
are required for seizure generation has recently
been challenged based on findings suggesting
normal expression levels but different distribu-
tion of the enzyme (Papageorgiou et al., 2011).
Fourth, astrocytes affect neuronal function by
the uptake and release of glutamate; decreased
uptake or increased release of glutamate might
contribute to the initiation of focal ictal dis-
charges (Gomez-Gonzalo et al., 2010; Halassa
etal., 2010). New findings suggest that astroglial
A2ARs, through direct antagonistic interaction
with the astroglial glutamate transporter GLT-1,
play major roles in setting the tone of extracel-
lular glutamate (Matos etal., 2012a; Matos etal.,
2012b). Aprimary role of astrocytic A 2ARs might
be to sense synaptic transmission (which gener-
ates ATP adenosine signals) in order to adjust
extracellular levels of glutamate by modulation
of transporter activity (Matos et al., 2012b) or
by controlling the vesicular release of glutamate
(Li etal., 2001; Nishizaki, 2004; Nishizaki etal.,
2002), thereby controlling and adjusting neu-
ronal excitability. Thus therapeutic enhancement
of astroglial glutamate uptake or attenuation
of the astrocytic release of glutamate could be
achieved pharmacologically by blocking A 2ARs
and thereby may constitute alternative therapeu-
tic strategies for the treatment of epilepsy that
still need to be developed and tested in preclini-
cal and clinical studies.
Adenosine Augmentation
As mentioned, adenosine deficiency is a patholog-
ical hallmark of epilepsy and sufficient to trigger
electrographic seizures (Boison, 2012b); conse-
quently, adenosine augmentation therapies are a
direct rational therapeutic approach and highly
effective in suppressing and preventing seizures
in rodent models of epilepsy (Figure 23.2; Boison,
2009). To avoid widespread systemic side effects of
adenosine, local or brain-specific treatment strat-
egies are required. Four successful approaches
have been demonstrated: (i) Silk-based brain
implants engineered to release defined doses of
adenosine with known kinetics prevented kindled
seizures in the rat. Importantly, a transient dose
of adenosine delivered during kindling robustly
attenuated epilepsy development, indicating a
potential antiepileptogenic effect of therapeutic
adenosine augmentation (Szybala et al., 2009).
(ii) Stem cells engineered to release adenosine
were used as infrahippocampal cell grafts and
shown to prevent acute and induced seizures, to
attenuate development of astrogliosis, to prevent
overexpression of ADK, and to prevent develop-
ment of spontaneous recurrent epileptic seizures
in mice and rats (Li etal., 2008; Li etal., 2007).
(iii) Agene therapy designed to selectively target
ADK in astrocytes based on RNA interference
technology almost completely abrogated any
spontaneous seizure activity in epileptic Adk-tg
mice (Theofilas etal., 2011). (iv) Finally, a high-fat
low-carbohydrate ketogenic diet (see later discus-
sion) suppressed seizures in mice via increased
activation of adenosine A1 receptors (Masino
et al., 2011). Based on the potent antiictogenic
and antiepileptogenic properties, the translation
into clinical practice is likely. Possible hurdles are
the development of local adenosine augmentation
therapies, which might best be achieved by gene
therapy; however, dietary alternatives provide a
readily available alternative.
Restoration ofthe DNA Methylome
According to the notion seizures beget sei-
zures, the seizures in epilepsy often increase in
frequency and severity during the course of the
disease. Several causes for the progressive course
of epilepsy have been proposed, including epi-
genetic modifications that lead to changes in
neuronal cell activity. While hypermethylation
of DNA in the central nervous system is associ-
ated with epilepsy (Kobow and Blumcke, 2011;
Qureshi and Mehler, 2010), there has been a lack
of evidence directly demonstrating a causative
role for increased DNA methylation in epilepsy
progression. We therefore explored the role
of adenosine as a potential therapeutic agent

capable of reversing epigenetic changes associ-
ated with epilepsy (Williams-Karnesky et al.,
2013). We confirmed increased DNA methyla-
tion in the hippocampus of epileptic rats; hyper-
methylation of the epileptogenic hippocampus
was associated with increased metabolic clear-
ance of adenosine through increased expression
of astroglial ADK (Figure 23.3). Importantly,
the transient delivery of adenosine to the brain
via an engineered silk-based brain implant cor-
rected the epilepsy-associated changes in DNA
methylation and prevented disease progression
in these animals long term. These findings dem-
onstrate that changes in DNA methylation pat-
terns are a key determinant of the progression
of epilepsy and that adenosine augmentation
therapies may reverse DNA hypermethylation
and break the cycle of increasing seizure sever-
ity (Williams-Karnesky et al., 2013). Our work
suggests a biphasic response of the DNA meth-
ylome in response to an epileptogenesis trig-
gering insult: acute DNA hypomethylation as
a presumably epileptogenesis-triggering event
followed by chronic DNA hypermethylation pre-
sumably required to maintain the epileptic state
and to promote disease progression. Importantly
this biphasic response can be directly related
to biphasic expression changes of ADK dur-
ing the course of epileptogenesis (Boison, 2008;
Li et al., 2008; Williams-Karnesky et al., 2013).
Acute DNA hypomethylation has also been
found within 24 hours after a traumatic brain
injury and has been associated with microglial
activation (Zhang et al., 2007). Those epige-
netic changes might become future diagnostic
tools to predict outcome following a potential
epileptogenesis-triggering event, such as trau-
matic brain injury (Conley and Alexander, 2011).
Adenosine augmentation via ADK inhibitors
might be a therapeutically viable option to pre-
vent epileptogenesis (Boison, 2013). ADK inhibi-
tors are highly effective in raising hippocampal
adenosine (Pak etal., 1994)and have been used in
preclinical drug development for the treatment of
epilepsy and chronic pain (McGaraughty et al.,
2005). However, drug development efforts have
largely been abandoned due to major side effects
of chronic drug use leading to cardiovascular
depression and liver toxicity (Boison, 2013). If,
however, a transient therapy with an ADK inhibi-
tor has long-lasting antiepileptogenic effects, we
may already have suitable drugs that could be
repurposed for the indication antiepileptogenesis
(Boison,2013).
Epilepsy 409
Drug Transporters in
the Blood-Brain Barrier
The expression of transporters for AEDs and
enzymes such as cytochrome P450 that metabo-
lize AEDs in glial cells of the BBB determine
whether antiepileptic treatments can reach
the epileptogenic brain areas. Expression of
these multidrug transporters, which include
ATP-binding cassette proteins and the human
P-glycoprotein, is severely disturbed in the epi-
leptic brain and might contribute to pharmacore-
sistance in intractable epilepsies; overexpression
of either the transporters or of AED-metabolizing
enzymes in the BBB has therefore been linked
to pharmacoresistant epilepsy (Ghosh et al.,
2010; Loscher, 2007; Loscher and Delanty, 2009;
Loscher et al., 2011; Luna-Tortos et al., 2008).
Association studies investigating drug trans-
porter gene polymorphisms within the context
of pharmacoresistance in epilepsy have yielded
conflicting results: whereas pharmacoresist-
ance to carbamazepine has been associated
with single nucleotide polymorphisms (SNPs)
in ATP-binding cassette transporters, polymor-
phisms in the human multidrug resistance gene
could not be associated with pharmacoresistance
in epilepsy. Overexpression of P-glycoprotein in
epilepsy depends on a cyclooxygenase 2 medi-
ated signaling pathway (van Vliet et al., 2010).
It was shown that cyclooxygenase 2 blockade
with anti-inflammatory drugs normalized
P-glycoprotein expression in rats and improved
brain penetration of phenytoin (van Vliet etal.,
2010). In contrast to direct transporter inhibi-
tion, this therapeutic strategy might have transla-
tional value to preserve physiological functions,
while specifically normalizing the pathological
transporter functions (Potschka,2010).
Anti-Inflammatory and
Immunomodulatory Therapies
As outlined, brain insults, such as SE or trau-
matic brain injury, trigger inflammatory
processes thought to play a crucial role in epi-
leptogenesis (Vezzani et al., 2011a). Therefore
anti-inflammatory therapies are potentially
antiepileptogenic. In line with this notion, the
cyclooxygenase 2 inhibitor celecoxib, admin-
istered chronically for a duration of 2 weeks
beginning 24 hours following an SE prevented
hippocampal neuronal injury and reduced the
incidence and frequency of spontaneous recurrent
seizures in rats (Jung etal., 2006). IL-1 appears
to be a key player in the onset of injury-induced
410 Part IV:Homeostatic Therapies
inflammation (Vezzani and Baram, 2007).
Consequently, pretreatment of rodents with an
IL-1 receptor antagonist prior to an SE signifi-
cantly reduced SE onset and BBB damage (Marchi
etal., 2009); however, pretreatment regimens will
also affect the quality and quantity of the precipi-
tating injury level, and therefore any interpreta-
tions regarding antiepileptogenic outcome must
be treated with caution. More recently, the selec-
tive interleukin-converting enzyme/caspase-1
inhibitor VX-765 was used to block the biosyn-
thesis of IL-1; this approach led to a reduction of
chronic epileptic activity in mice (Maroso etal.,
2011). Rapamycin is an immunosuppressive drug
acting on the serine/threonine protein kinase
mammalian target of rapamycin (mTOR), which
regulates many forms of synaptic plasticity in the
adult brain and has been linked with plasticity
changes during epileptogenesis (Cao etal., 2009).
mTOR signaling is also involved in the expres-
sion of glutamate transporter 1 in astrocytes (Wu
et al., 2010). Thus dysregulation of mTOR sign-
aling in astrocytes might critically affect major
homeostatic systems of the brain thought to be of
relevance for epileptogenesis. In a rodent model
of epilepsy, the mTOR pathway was found to be
upregulated following kainic acidinduced SE
and was linked to chronic epileptogenesis (Zeng
et al., 2009). If mTOR activation plays a role in
epileptogenesis, then mTOR blockade should
have antiepileptogenic effects. Consequently,
rapamycin blocked SE-induced mossy fiber
sprouting and the frequency of spontaneous sei-
zures in two independent studies (Buckmaster
et al., 2009; Zeng et al., 2009). Since seizures
were recorded only during the treatment period
(Zeng et al., 2009) it remains to be determined
whether the observed therapeutic effects were
anti-ictogenic or antiepileptogenic. Based on the
studies discussed previously, anti-inflammatory
therapies are certainly promising candidates for
antiepileptogenesis. To be effective, those thera-
pies should be initiated as soon as possible fol-
lowing a precipitating event and maintained for
approximately 2 weeks, a time window during
which inflammatory and immunomodulatory
processpeak.
Dietary Interventions
Mounting evidence suggests that diet plays a
major role in setting the homeostatic stage in
the brain. High-fat/low-carbohydrate ketogenic
diets have been in clinical use for over 80years
to treat refractory epilepsies, particularly those
occurring during childhood (Kossoff and Rho,
2009; McQuarrie and Keith, 1927). Importantly,
ketogenic diet therapy is effective in epilepsies
that are refractory to conventional AED treat-
ment (Mackay et al., 2005; Sirven et al., 1999).
Moreover, the literature reports on patients
treated with a ketogenic diet that remained sei-
zure free after diet reversal, suggesting poten-
tial antiepileptogenic effects of the diet (Kossoff
and Rho, 2009). Last, ketogenic diet therapy not
only affects epileptic seizures but also normal-
izes behavioral symptoms thought to be comor-
bid with epilepsy (Masino et al., 2013). Given
those characteristics, ketogenic diet therapy is
a prototypic homeostatic therapy. This notion
is supported by several beneficial mechanisms
thought to be activated by ketogenic diet therapy.
Ketogenic diet therapy forces the brain to use
ketones in lieu of glucose as the primary energy
source, and it is those metabolic changes leading
to disruption of glutamatergic synaptic transmis-
sion, inhibition of glycolysis, increase in adeno-
sine, and activation of ATP-sensitive potassium
channels that are thought to underlie the thera-
peutic effects of this type of dietary intervention
(Bough, 2008; Bough etal., 2006; Kalapos, 2007;
Lutas and Yellen, 2013; Ma et al., 2007; Masino
et al., 2011; Yellen, 2008). A large body of evi-
dence supports the notion that a ketogenic diet
leads to increased adenosine signaling in the
brain (Masino and Geiger, 2008, 2009; Masino
et al., 2012; Masino et al., 2009) most likely via
reduced expression of ADK (Masino etal., 2011).
Reduction of ADK expression seems to be a gen-
eral mechanism to shift the brain into a protective
mode (Pignataro et al., 2008). As an additional
mechanism, activation of the metabolically sensi-
tive ATP-sensitive potassium channel was shown
to be instrumental in the resistance to behavioral
and electrographic seizures in vivo, an effect that
was dependent on Bcl-associated death protein
modifications that reduce glucose metabolism
(Gimenez-Cassina etal., 2012). Metabolic thera-
pies can also effect gene expression as a possible
explanation for antiepileptogenic effects of the
diet. It was shown that the glycolytic inhibitor
2-deoxy-d-glucose potently reduced the pro-
gression of kindling epileptogenesis and blocked
seizure-induced increases in BDNF and its recep-
tor, TrkB. This effect was found to depend on the
neuron-restrictive silencer factor, a transcrip-
tion factor that recruits the reduced nicotina-
mide adenine dinucleotide (NADH)-binding
C-terminal binding protein, a corepressor

generating a repressive chromatin environment
around the BDNF promoter (Garriga-Canut
etal., 2006). Together, these findings suggest that
a ketogenic diet exerts antiepileptic effects by a
combination of acute mechanisms and genomic
mechanisms, which may lead to long-term altera-
tions of neuronal circuitry. Additional dietary
components such as branched-chain amino acids
(Novarino et al., 2012), certain oils (Bandero
etal., 2013; Koutroumanidou etal., 2013; Shawki
etal., 2013), or medium-chain triglycerides (Liu
and Wang, 2013)all have potential for the meta-
bolic therapy of epilepsy and are readily translat-
able. In particular, medium-chain triglycerides
with an uneven number of carbon atoms, such
as triheptanoin, have shown significant promise
in enforcing a ketone-based metabolism in the
brain with proven antiepileptic efficacy in several
rodent models of epilepsy (Hadera et al., 2013;
Kim etal.,2013).
Lifestyle
Lifestyle choices can have a significant impact
on brain function. Physical exercise is known
to increase neurogenesis in the adult brain (van
Praag et al., 1999), a mechanism thought to
protect the brain from hippocampal degenera-
tion and that has been demonstrated to enhance
cognitive function (Miltiadous et al., 2013).
Pathological neurogenesis, as triggered by epi-
leptic seizures, in contrast, is thought to aggra-
vate the epileptogenic cascade. Several lines of
evidence implicate newly generated neurons in
structural and functional network abnormali-
ties in the epileptic brain, such as aberrant mossy
fiber reorganization, persistence of immature
dentate granular cell structure, and the abnormal
migration of newborn neurons to ectopic sites in
the dentate gyrus (Parent and Lowenstein, 2002).
Whereas the significance of exercise-induced
neurogenesis on seizure propensity is unknown,
exercise-induced neurogenesis is certainly of
value to ameliorate cognitive comorbidities of
epilepsy. While clinical studies indicate that per-
sons with epilepsy have in general reduced their
physical activity levels as a result of their condi-
tion (Jalava and Sillanpaa, 1997), clinical studies
evaluating potential benefits of exercise for epi-
lepsy are surprisingly lacking.
Several lifestyle choices influence adenosine
signaling in the brain; intense physical exercise
has been shown to increase adenosine in the
brain (Dworak etal., 2007). Adenosine levels in
the brain also depend on sleep (Huang etal., 2011;
Epilepsy 411
Porkka-Heiskanen and Kalinchuk, 2011)and are
increased by bacterial endotoxins (Ramakers
et al., 2011). Consequently, any lifestyle-related
changes in adenosine homeostasis will have a
direct impact on the susceptibility of epilep-
tic seizures. Conversely, methylxanthines such
as caffeine or theophylline, well-characterized
antagonists of adenosine receptors, have in gen-
eral acute proconvulsant activities; however, the
chronic use of methylxanthines leads to adaptive
increases in the adenosine A1 receptor and thereby
enhances the antiepileptic activity of endogenous
adenosine (Boison, 2010). Overall, it is estimated
that up to 80% of all persons with epilepsy are
habitual consumers of caffeine. While chronic
caffeine use might be beneficial to alleviate cog-
nitive side effects of AEDs and might increase the
capacity of endogenous adenosinedependent
seizure-control mechanisms, caffeine might also
play a crucial role in sudden unexpected death
in epilepsy (SUDEP; Shen et al., 2010). On one
hand, caffeine can prevent respiratory arrest and
SUDEP (Shen etal., 2010), but SUDEP in people
usually occurs at night during sleep when caf-
feine levels are lowest. It needs to be determined
in careful clinical studies whether daytime caf-
feine use may increase nighttime SUDEP risk
and whether caffeine in general is of any benefit
for epilepsy and its comorbidities.
Age andGender
The incidence of epilepsy peaks in the juvenile
(<5 years of age) and in the elderly populations
(>60years of age; Annegers etal., 1999). However,
treatment outcome may be age dependent. In gen-
eral, epilepsy with an age of onset <12years has a
lower recurrence risk following discontinuation
of medication than that of an older age of onset
(Berg and Shinnar, 1991, 1994). In contrast, the
probability of attaining and maintaining remis-
sion after medication withdrawal is a function
of age of onset and duration of the seizure disor-
der, without a special role for puberty (Shinnar
and Moshe, 1991). Children with higher meta-
bolic rates usually require higher drug dosages,
as measured in milligram drug per kilogram
body weight, than adults. Whereas ketogenic
diets traditionally have mostly been used for the
treatment of childhood epilepsies, a recent study
suggests that ketogenic diet therapy is equally
effective in adult and adolescent patients (Nei
etal.,2014).
Hormones have a marked effect on neuronal
excitability. Estrogen reduces the concentration
412 Part IV:Homeostatic Therapies
of GABA and reduces the number and chloride
flux of GABA A receptors; in addition, estrogen
activates N-methyl-D-aspartate receptors in the
hippocampus (Morrell, 2002). Thereby estrogen
exerts potent proconvulsive effects that may trig-
ger seizures, when seizure thresholds are patho-
logically lowered. Conversely, progesterone has
opposite effects on GABAergic and glutamatergic
neurotransmission, resulting in anticonvulsant
effects. For those reasons, catamenial, menstrual
cycleassociated seizure patterns, arise in women
with seizures more likely to occur in the premen-
strual and ovulatory phases (i.e., at times when
estrogen levels are high and progesterone levels
are low; Hattemer etal., 2006). AED elimination
can depend on both sex and age. Fertile women
often have faster drug metabolism rates than
age-matched men, whereas the rate of metabo-
lism decreases with age in both sexes. An efficacy
analysis of newer AEDs by gender, however, did
not show any major differences in efficacy and
tolerability with the exception of zonisamide,
which was better tolerated by women (Morrell,
1996; Yerby, 2000a, 2000b).
CONCLUSIONS AND
OUTLOOK
The excitability of the brain is determined by
neurons, which in turn are connected to net-
works, which in turn are broadly controlled by
the homeostatic environment of their surround-
ings, which to a large degree is under the control
of glial cells. It becomes clear that complex neu-
rological syndromes, such as epilepsy, which are
not only defined by a dominant symptom (i.e., a
seizure) but also by a growing number of associ-
ated comorbidities, can best be explained by the
disruption of network homeostasis. Disruption
of network homeostasis implies the simultane-
ous and concordant dysregulation of several
molecular pathways, which in turn can affect
each other and lead to a progression of the dis-
ease in the sense that seizures beget seizures.
This old concept has a lot of truth and can best
be explained by the self-reinforcing interplay of
several homeostatic systems that become pro-
gressively dysregulated during disease progres-
sion. Conventional AEDs with a target-centric
mode of action are unlikely to affect network
homeostasis or prevent the progressive maladap-
tive changes occurring during epileptogenesis,
which can be considered a lifelong process of dis-
ease progression. Novel therapeutic interventions
based on adenosine, epigenetic mechanisms, or
dietary and lifestyle interventions might hold
promise to affect network homeostasis as a novel
conceptual strategy to treat and prevent epilepsy
on the network level. Adenosine emerges as a pro-
totype homeostatic network regulator with the
proven capability to control network activity both
through receptor-dependent as well as through
receptor-independent epigenetic and bioener-
getic mechanisms (Boison, 2013). Consequently,
therapeutic adenosine augmentation has been
demonstrated to suppress epileptic seizures (Li
etal., 2008; Li etal., 2007), and to prevent disease
progression and epileptogenesis (Li et al., 2008;
Williams-Karnesky etal., 2013), but also to pre-
vent psychosis and improve cognition (Shen etal.,
2012) without any known adverse effects. The
robust antipsychotic and procognitive effect of
adenosine in mice (Shen etal., 2012)suggests that
therapeutic adenosine augmentation might com-
bine anticonvulsant with cognition-enhancing
effects. In preclinical toxicity studies of intrathecal
adenosine in dogs, no side effects were observed
with intrathecal adenosine infused chronically
for 26days (Chiari etal., 1999). Likewise, intrath-
ecal adenosine was tested in humans in escalating
doses of up to 2 mg without any adverse effects
(Eisenach etal., 2002a, 2002b). Several therapeu-
tic adenosine augmentation strategies ranging
from gene therapy to dietary intervention are
currently in preclinical development. It is pos-
sible that adenosine-augmenting therapies will
be introduced into the clinic as a novel class of
homeostatic network therapy within the next
10years. Challenges will include developing strat-
egies to confine adenosines action to identifiable
target areas and cells; the advent of cell-type spe-
cific gene therapy vectors might offer a promising
strategy to manipulate adenosine homeostasis in
a localized and cell-type selective manner.
ACK NOWLEDGMENTS
The author is indebted to outstanding experi-
mental work from his research team. The
authors work is funded through grants from the
National Institutes of Health (R01 NS065957, R01
MH083973, R01 NS061844), the US Department of
the Army (W81XWH-12-1-0283), Citizens United
for Research in Epilepsy, the Parkinsons Northwest
Group, and the Legacy Hospital Foundations.
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24
Traumatic BrainInjury
R A J G . K U M A R A N D A M Y K . WA G N E R
A C U T E PAT H O P H Y S I O L O G Y
A N D A C U T E H O M E O S TAT I C
DISRUPTION
Primary Injury Mechanisms:
Contusions, Axonal Stretch/
Disruption
Traumatic brain injury (TBI) is characterized by
both a primary and a secondary injury. A pri-
mary brain injury is classified as a focal or dif-
fuse injury. Focal injuries can be categorized as
lesions, contusions, lacerations, and other cir-
cumscribed intracranial abnormalities caused
by collision forces that result in tissue compres-
sion below the cranium at the site of impact and/
or of tissue opposite to the impact (Andriessen
et al., 2010). The rapid acceleration/decelera-
tion injury that results in diffuse axonal injury
(DAI) is a common mechanism that results
from high-speed motor-vehicle accidents and
creates damaging shear, tensile, and compres-
sive forces within the brain tissue (Gentry etal.,
1988). Naturally, the severity of DAI depends
largely on the duration, magnitude, and direc-
tion of the angular acceleration, as well as the
associated impact (Gennarelli, 1993). With severe
DAI, there can be more significant damage than
superficial axonal injuries. The deeper white
matter structures, as well as midline structures
like the corpus callosum, may be also affected
(Halliday, 1999). Secondary injury is the broad
characterization of multifactorial biochemical
cascades that lead to cellular disruption and, in
severe cases, cell death of central nervous system
(CNS) tissue. These secondary-injury manifesta-
tions of TBI can have a delayed clinical presenta-
tion and result in cerebral hypotension, hypoxia,
ischemia, excitotoxicity, hormone abnormalities,
disruption of ion homeostasis, and CNS inflam-
mation (Chesnut etal., 1993). These components
of secondary injury are complex, and some can
persist for hours to days, while others persist for
months after injury and result in chronic symp-
toms or deficits.
Excitotoxicity
Excitatory amino acids (EAA) such as glutamate
and aspartate are important in normal excita-
tory brain function, yet in excess they can result
in excitotoxicity, a phenomenon that initiates a
whole host of secondary-injury mechanisms that
can lead to cell death. After TBI, excessive EAA
concentrations have been documented in cer-
ebrospinal fluid (CSF; Wagner et al., 2005) and
pericontusional microdialysate fluid (Bullock
etal., 1998; Werner and Engelhard, 2007). EAA
levels become elevated as a result of mechanical
axon damage and subsequent influx of EAAs
from axons. Excessive glutamate levels may
over activate specific ion channels, especially
the N-methyl-D-aspartate (NMDA) channel
(Bullock et al., 1998), resulting in an uncon-
trolled influx of sodium and calcium, along with
associated potassium efflux, which leads to acute
neuronal swelling and cell death. Intracellular
calcium may activate lipid peroxidases, proteases,
and phospholipases that in turn facilitate the pro-
duction of intracellular free radicals, which then
leads to cell membrane and DNA damage. Also,
potassium efflux will result in rapid astrocytic
swelling as cells absorb excess potassium in an
attempt to maintain ionic homeostasis (Bullock
et al., 1995). It is postulated that this process
may cause rapid cytotoxic edema, a primary
factor leading to elevated intracranial pressure
following severe TBI. Furthermore, excessive
EAA levels have also been implicated with mito-
chondrial damage. Healthy mitochondria play
an integral role in regulating intracellular Ca2+
homeostasis via various mechanisms (Gunter
et al., 1994). However, excessive Ca2+ exposure
that accompanies elevated glutamate levels dis-
rupts brain mitochondrial electron transfer and
energy production due to excess mitochondrial

calcium levels and increased reactive oxygen spe-
cies (ROS) production, which leads to oxidative
damage, disturbance of synaptic homeostasis,
and apoptosis (Sullivan et al., 1999; White and
Reynolds, 1996; Xiong etal., 1997). Mitochondrial
dysfunction also leads to energy failure after TBI,
decreased adenosine triphosphate production,
and decreased oxygen consumption by 40% to
50% (Maas et al., 2008). Alterations in mito-
chondrial integrity, specifically mitochondrial
permeability transition pore translocation, are
a fundamental component of disrupted calcium
homeostasis that results in subsequent apoptosis.
The importance of this translocation with regard
to neuroprotection is evidenced by the neuropro-
tective effects of pharmaceutical agents that tar-
get the mitochondrial transition pore in animal
models (Mazzeo etal.,2009).
In addition to EAA such as glutamate, dopa-
mine (DA) is also upregulated following TBI
through increased tyrosine hydroxylase, the
rate-limiting enzyme involved with DA synthesis
(Kobori etal., 2006; Yan etal., 2001). DA facili-
tates the development of acute cellular pathway
dysfunction, including Na+/K+ ATPase, cell
metabolism, Ca 2+ release, and the NMDA recep-
tor through cAMP-regulated phosphoprotein
32 kDa and protein phosphatase-1 (Bales et al.,
2009; Greengard etal., 1999; Nishi etal., 2002).
Additionally, high synaptic DA levels can be rap-
idly oxidized to form reactive DA semiquinones
(Hastings, 1995). Oxidized DA can generate
hydrogen peroxide and superoxide, which can
lead to significant oxidative stress (Bales et al.,
2009). Interestingly, DAergic fibers can also mod-
ulate striatal glutamate excitotoxicity (Chapman
et al., 1989; Filloux and Wamsley, 1991), and
depleting DAergic projections into the striatum
prior to ischemic injury can be neuroprotective
(Globus etal.,1987).
Inflammation
The CNS historically has been considered an
immunologically privileged organ due to the
physical separation of the brain from the periph-
ery with the blood-brain barrier (BBB). In TBI,
BBB integrity is acutely compromised. TBI is
characterized by a significant inflammatory
response that includes not only CNS-mediated
inflammatory markers but also peripheral
inflammation (Schmidt etal., 2005). Among the
key mediators of the inflammatory response are
proinflammatory cytokines, chemokines, and
cell adhesion molecules (Ghirnikar et al., 1998;
Traumatic BrainInjury 421
Jeong et al., 2013). Proinflammatory cytokines
(e.g., IL-1 and IL-6) are elevated after injury
through synthesis by peripheral cells and resi-
dent glia in the CNS. These cytokines can initi-
ate inflammatory pathways and lead to increased
neurotoxicity via oxidative injury (Feuerstein
etal., 1998). Additionally, there is evidence that
cytokines can play a dual neuroprotective role
through angiogenic, neurotrophic, and other
mechanisms known to attenuate CNS damage
(Jeong et al., 2013; Morganti-Kossmann et al.,
2002). While evidence is convincing regard-
ing the existence of an elevated inflammatory
response acutely following TBI (Bell etal., 1997;
Buttram etal., 2007; Hensler etal., 2002; Maier
et al., 2001; Shiozaki et al., 2005; Singhal et al.,
2002; Wang etal., 2013), the balance and nuanced
role of the relatively detrimental versus neuropro-
tective role of various inflammatory markers is
still largely unclear. Some studies have suggested
a dose-related effect (Wang etal., 2013). That is,
there are potential benefits of cytokines and other
inflammatory markers when under the control of
physiological regulatory mechanisms; however,
extremely high levels may be harmful. There
are also time-dependent effects of inflammatory
markers following injury. A recent TBI clinical
study (Johnson et al., 2013) identified activated
microglia and phagocytic macrophages in exten-
sive regions of the corpus callosum up to 18years
following a single TBI. Work from our group
suggests that a chronically sustained elevations
in IL-1, IL-6, IL-8, IL-10, and TNF in the first
3months after injury increases the risk for worse
global outcomes at 6 and 12months post-injury
(Kumar etal., 2014). Posttraumatic inflammation
is an important clinical consideration following
injury, and clinicians and researchers should fur-
ther examine the balance between the neuropro-
tective versus the detrimental effects of elevated
inflammatory states.
BBB Disruption and Reactive
Astrocytosis
Experimental animal models of TBI demonstrate
that BBB permeability is increased following
injury (Shapira etal., 1993). The BBB breakdown
is understood to be biphasic in nature, with rapid
deterioration within the first few hours followed
by subsequent improvement in integrity start-
ing 3 to 7 days after injury (Shlosberg et al.,
2010). In human TBI, BBB permeability usually
returns to normal within days to weeks after
injury (Kirchhoff etal., 2006; Stahel etal., 2001).
422 Part IV:Homeostatic Therapies
BBB deterioration initiates soon after the trau-
matic insult through shearing of endothelial cells
located in brain microvessels (Rodrguez-Baeza
et al., 2003). This phenomenon affects normal
BBB regulation, cerebral blood flow (CBF), and
metabolic processes that can result in more
serious secondary complications (Shlosberg
et al., 2010), such as ischemia that induces tis-
sue hypoxia (Fischer etal., 2002). Other mecha-
nisms of BBB disruption and ischemia following
TBI may include cerebral vasospasm (Lee etal.,
1997), cerebral pressure autoregulatory dysfunc-
tion (Rangel-Castilla etal., 2008), and coagulop-
athy (Nekludov etal., 2007). The resultant effects
of BBB breakdown influence other downstream
secondary-injury cascades, including edema,
inflammation, and cell death. The BBB is a com-
ponent of the larger neurovascular unit that
also includes astrocytes, pericytes, microglia,
neurons, and the extracellular matrix (Shlosberg
etal., 2010). Physiologically, these units perform
many homeostatic functions that influence neu-
ronal survival. Specifically, astrocytes maintain
extracellular ion concentrations, influence glu-
tamate clearance, and produce inflammatory
cytokines and chemokines (Chen and Swanson,
2003). Reactive astrocytes can play a damaging
role after CNS injury by facilitating the develop-
ment of glial scarring (Fawcett and Asher, 1999).
However, experimental TBI work by Myer and
colleagues (2006) showed that reactive astrocyto-
sis also helps preserve neural tissue and reduces
excess inflammation following moderate injury.
Ischemia and OxidativeStress
TBI also initiates the development of oxidative
stress, which refers to the generation of ROS. It
is well established that ROS can lead to oxidative
stress and resultant damage to lipids, proteins,
and nucleic acids (Halliwell, 1991). Further, ROS
have been associated with TBI-related patho-
genesis, including tissue loss (Shao etal., 2006),
reduced synaptic integrity (Ansari et al., 2008),
and cytoskeletal and mitochondrial damage
(Singh etal., 2007). Several animal studies have
shown evidence for the neuroprotective proper-
ties of antioxidant agents, such as propofol and
erythropoietin, in both lowering the extent of
oxidative injury (Ozturk et al., 2005, 2008) and
reducing neuronal apoptosis (Liao etal.,2008).
The brain is vulnerable to the development of
cerebral ischemia as a part of secondary injury.
Soon after impact, CBF regulation and cerebral
oxygen metabolism are impaired. Imaging
studies show that ischemia is a significant prob-
lem in the first 24 hours after injury, and the
volume of ischemic brain is correlated with
worse global outcomes at 6months (Coles etal.,
2004). Among the principle causes of cerebral
ischemia are systemic arterial hypotension, ele-
vated intracranial pressure, edema, blood carbon
dioxide concentrations, focal tissue compres-
sion, and microvascular disease (Hekmatpanah
and Hekmatpanah, 1985; Pfenninger etal., 1989;
Sarabia et al., 1988; Obrist et al., 1984). Since
the brain is so susceptible to ischemic events, it
is vital to maintain the CBF and cerebral oxy-
gen metabolism during acute care manage-
ment. Correspondingly, the The Guidelines for
the Management of Severe Head Injury (Brain
Trauma Foundation, 2007) for acute TBI are
particularly focused on treating hypotension
and hypoxia, as it is estimated that nearly 90% of
those who die following TBI show some evidence
of ischemia (Greve and Zink,2009).
Potential Acute Neuroprotectants
The purine ribonucleoside adenosine can have
a beneficial influence on secondary-injury cas-
cades in TBI (Headrick et al., 1994), including
improved Ca2+ homeostasis, attenuated EAA
release, and reduced oxidative stress. Adenosine
controls many functions through coupling of
either inhibitory (A1, A3) or stimulatory (A 2a, A 2b)
G-protein receptors and the spatial distributions
of these receptors within the brain (Fredholm
etal., 2005). Adenosine binding to either A1 or A 2
receptors affect glutamate and DA neurotrans-
mission (Boison, 2008). One animal study mod-
eling status epilepticus found that A1-receptor
activation was a key mediator in reducing neu-
ronal cell loss and convulsions (Fedele et al.,
2006). Another animal study found that A 2
receptor inhibitors can have potent neuroprotec-
tive properties by preventing apoptosis and syn-
aptotoxicity (Silva etal.,2007).
Neurotrophic factors, including brain-derived
neurotrophin factor (BDNF) and nerve-growth
factor, have important roles in cell differentia-
tion, growth, proliferation, and CNS cell survival
(Hetman etal., 1999; Sofroniew etal., 2001). After
TBI, neurotrophins in both CSF (Chiaretti etal.,
2008)and serum (Kalish and Phillips, 2010)are
depressed in association with the severity of
injury. There is some evidence that mesenchymal
stem cells have the capacity to upregulate neuro-
trophic factors and have shown some ability to
enhance recovery in the acute stages following

TBI (Kim et al., 2010). However, there is some
recent biomarker evidence that neurotrophins
are not neuroprotective early after injury, par-
ticularly for older individuals with severe TBI
(Failla etal., 2015a; Failla etal., 2015b).
Furthermore, acute TBI triggers a significant
stress response that is not limited to the CNS.
Work from our lab suggests that endogenous
serum estradiol levels are highly associated with
mortality (Wagner et al., 2011a), though higher
estradiol/testosterone ratios in CSF may be neuro-
protective (Garringer etal., 2013). Accumulating
evidence in trauma and critically ill populations
suggests that, in humans, serum estradiol is asso-
ciated with a stress response that carries a higher
risk for mortality (May etal., 2008; Spratt etal.,
2006, 2008). Work from our lab also shows cortisol
is elevated in the CSF (Santarsieri etal., 2014)and
serum (Wagner etal., 2011a) acutely after injury.
Clinical data show that elevated CSF cortisol is
associated with poor outcomes (Santarsieri etal.,
2014). Interestingly, glucocorticoid and corticos-
teroid drugs, which were once commonly used
in TBI medical management, have fallen out of
favor due to convincing evidence from clinical
trials that their use leads to increased mortal-
ity rates (Alderson and Roberts, 2005; Edwards
et al., 2005; Roberts et al., 2004). Importantly,
estradiol and cortisol levels are dependent on
progesterone, and temporal elevations in serum
estradiol and serum/CSF cortisol after TBI
are likely the product of adrenal progesterone
production. Due to the well-established neu-
roprotective properties of progesterone in exper-
imental models (Stein, 2011), clinical research
efforts have targeted progesterone therapies
(Wright et al., 2007). However, recent phase III
multisite clinical trials have failed to show a ben-
eficial effect when progesterone was given early
after moderate to severe TBI (Skolnick et al.,
2014; Wright etal., 2014). Additional evaluation
of how progesterone treatment impacts homeo-
static disruption of physiological cortisol and
estradiol production after severe TBI may aid
in identifying whether some subpopulations do
derive benefit from progesterone as a neuropro-
tective treatment.
Another consideration with acute TBI neu-
roprotection is the protein beta-cell lymphoma 2
(BCL-2). This anti-apoptotic factor works to pre-
vent the mitochondrial cytochrome C release and
subsequent caspase-mediated cell death. Data
show that acute CSF levels of BCL-2 have strong
prognostic potential as a marker of cell death
Traumatic BrainInjury 423
(Wagner et al., 2011b). Specifically, individuals
with persistently higher levels in the first week
were at risk for poor outcome at 6 and 12months;
however, the group whose BCL-2 levels started
near control levels in the first few days after injury
but showed a delayed rise in BCL-2 had the best
recovery, suggesting a prosurvival upregulatory
mechanism after severe TBI. Genetic variation
in BCL-2 may also influence outcome after TBI
(Hoh etal.,2010).
Acute Pathophysiology
Integrative Summary
Acute TBI is characterized by direct disruption of
brain tissue homeostasis, including alterations in
CBF, cerebral vasospasm, massive excitotoxicity
and oxidative stress, inflammation, and edema.
Experimental studies have provided the field
with a better understanding of acute TBI and
the secondary-injury cascade. However, as Maas
and colleagues (2010) explain, clean experimen-
tal conditions do not seem to transfer into the
dirty clinical situation. Clinical TBI represents
a complex spectrum and heterogeneous range
of pathologies that make a single golden bul-
let neurotherapy impractical (Beauchamp etal.,
2008). Due to patient heterogeneity, it is difficult
to gauge whether targeted pathophysiological
processes associated with a given neurotherapy
have the same effect in all individuals. Also then,
a one-size fits all approach to clinical interven-
tions is not likely to be successful. Some com-
pounds thought to be neuroprotective may not
be beneficial for all individuals after TBI. Thus
there is a continued need for well-designed clini-
cal characterization studies to further evaluate
heterogeneity involved with the acute pathophys-
iological events associated with secondary injury
in humans. This information should be used to
guide individualized treatment approaches and
timelines as well as to understand how pleio-
tropic treatments or treatment combinations may
confer therapeutic benefit.
T B I PAT H O L O G Y A N D
BIOSUSCEPTIBILITY
TOCHRONIC CONDITIONS
Common Mechanisms ofInnate
Homeostatic Heterogeneity
To date, few TBI preclinical and clinical trials
incorporate factors such as sex, age, baseline
behavioral function, and variable standard care
therapeutics in their trial design. A few studies
424 Part IV:Homeostatic Therapies
that have specifically examined sex effects have
observed significant sex-specific differences in
treatment response. For example, both experi-
mental and clinical studies show women may not
benefit from a treatment such as hypothermia as
much as men with similar injuries (Bayir etal.,
2004; Suzuki etal., 2003; Wagner etal., 2005). TBI
populations are also heterogeneous with respect
to baseline prognostic potential for recovery and
risk for complications. Factors such as genetics,
propensity for autoimmune responses, brain
plasticity, and atrophy are highly specific to indi-
viduals and play an integral role in homeostatic
restoration and susceptibility to common post-
traumatic chronic conditions such as epilepsy,
cognitive dysfunction, depression, migraine, and
neuroendocrine dysfunction. This section exam-
ines these common TBI phenotypes in the con-
text of innate patient heterogeneity.
Genetics/Epigenetics
Increasing evidence supports the role of genetic
variation in influencing pathophysiological
mechanisms and outcome (Bonneh-Barkay etal.,
2010; Diamond et al., 2014; Failla et al., 2013;
Hoh etal., 2010; Miller etal., 2010; AK Wagner
et al., 2010). An overall understudied area to
date in brain injury, genetics, could influence
secondary-injury mediators (Garringer et al.,
2013) and processes (Conley et al., 2014), and
factors such as sex could interact with genetics
to moderate acute secondary-injury cascades,
such as with acute DA dysfunction (Wagner
etal.,2007).
The emerging field of epigenetics, which
refers to genetic control by factors other than an
individuals DNA sequence, can influence how
genes turn on and off to determine which pro-
teins are transcribed. Specifically, initial work
suggests injury-induced epigenetic changes in
the brain may affect TBI-related pathology and
contribute to heterogeneity in recovery and bio-
susceptibility to complications after TBI, (Gao
etal., 2006; Lundberg etal., 2009; Schober etal.,
2012; Wang etal., 2011; Zhang etal., 2007). Two
primary epigenetics mechanisms relevant to
TBI include histone posttranslational modifica-
tions and DNA methylation (Jaenisch and Bird,
2003); injury-induced changes in these post-
translational modifications may underlie some
of the temporally dynamic genetic associations
that have been observed after TBI. For example,
work from our lab shows a temporally dynamic
association between the serotonin transporter
gene (SLC6A4) variation, a common depression
risk allele, and the development of posttraumatic
depression (PTD; Failla etal., 2013). Specifically,
after TBI, a history of premorbid mood disor-
der and SLC6A4 genotype differentially influ-
ences PTD risk. As an additional methylation
site exists for SLC6A4 S-allele carriers compared
to L-carriers (Failla etal., 2013), we hypothesize
that injury-specific differences in SLC6A4 meth-
ylation may be one contributor to differences in
PTD risk over time. The precise role of epigenetic
modifications in injury and recovery, as well as
how injury-induced epigenetic modifications
drive homeostatic disruption and normalization,
is still largely unknown. However, rehabilitation
strategies after TBI may be potent modifiers of
the epigenetic remarking process and an impor-
tant mechanism by which rehabilitation strate-
gies impact recovery.
Autoimmunity
Another source of homeostatic heterogene-
ity is autoimmunity. Antibodies specific for
self-antigens are involved in normal physiologi-
cal conditions. These immunoglobulins facili-
tate removal of dead cells through phagocytosis
in a process known as opsonization. After TBI,
due to BBB disruption, antibodies are prevalent
in greater quantities in the brain (Baloyannis
and Gonatas, 1979) and resident brain cells are
exposed to greater quantities of autoantibod-
ies (Soares etal., 1995). Damaged CNS cells can
release antigens that enter the blood circulation
following BBB breach that can trigger autoanti-
body production. While theoretically contrib-
uting to heterogeneity with secondary injury
and recovery, the complexity and consequences
(positive or negative) of autoantibody produc-
tion following TBI is still unclear. One study,
using an experimental injury model, identified
a neuroprotective role for circulating immuno-
globulin G autoantibodies to bind to dying neu-
rons that were proximal to the injury site after
TBI (Stein et al., 2002). Also, there is evidence
that S100b can be released from brain astrocytes
and contribute to an autoimmune response,
and S100b has been shown to be a reliable bio-
marker after sports-related concussions (Marchi
et al., 2013). A recent study also found that the
antiglial fibrillary acidic protein autoantibody
can enter living astroglial cells and compromise
glial cell health, and an increase in these levels
were negatively correlated with outcome (Zhang
etal., 2014). Also, a clinical TBI study examined

antipituitary antibodies and found evidence for
the role of autoimmunity in TBI-induced pitui-
tary dysfunction (Taniguchi etal., 1999). Because
of the latent development period required for
autoantibody production (Ritter et al., 2014), it
is possible that autoimmunity could interplay
with other long-term degenerative (Ghirnikar
et al., 1998) and inflammatory (Schmidt et al.,
2005) processes thought to contribute to sus-
tained disruption of homeostasis and poor recov-
ery afterTBI.
Plasticity
Brain plasticity following TBI can take on many
forms, from synaptic strength alterations to reor-
ganization of functional brain networks. Neural
networks that were present prior to injury are
often altered in a dynamic and heterogeneous
way during recovery. In one study utilizing mag-
netoencephalography, an imaging method that
utilizes electromagnetic fields that are generated
by the net effect of the slow ionic flow of networks
of neurons, researchers showed that synchrony in
neural signals mirrored cognitive recovery fol-
lowing a brain injury (Castellanos et al., 2011).
Functional magnetic resonance imaging studies
suggest that considerable functional reorganiza-
tion occurs following TBI. Studies show evidence,
using working memory paradigms, of increased
cerebral activation at lower workloads, even when
actual performance does not differ from controls
(McAllister etal., 1999). Other studies have sug-
gested altered hemispheric recruitment patterns
(Kasahara et al., 2011; Russell et al., 2012) in
task-based brain activations following TBI that
are not observed in control subjects. In addi-
tion to reorganization, neurogenesis contributes
to plasticity following TBI. Adult neurogenesis,
especially within the hippocampus, is a tightly
controlled process from proliferation of neural
stem cells to differentiation, migration, and inte-
gration into long-range connections. Following
experimental TBI, there is a well-documented
increase in neurogenesis immediately follow-
ing both focal and diffuse TBI (Bye etal., 2011).
A loss of neuronal integrity in fronto-limbic
regions following TBI may facilitate neurogen-
esis that is highly linked to chronic BDNF levels
(Rossi etal.,2006).
Plasticity-dependent alterations that affect
recovery and homeostatic function are also man-
ifested in the context of hippocampal long-term
potentiation (LTP), which is critical for learning
and memory formation (Bliss and Collingridge,
Traumatic BrainInjury 425
1993). BDNF, a neurotrophin ubiquitous in the
hippocampus, is critical in synapse maintenance,
particularly with LTP, via activity-dependent
secretion of BDNF (Kovalchuk et al., 2002).
Despite the possibility that BDNF is not directly
neuroprotective early after TBI (Failla et al.,
2015a; Failla etal., 2015b), it may benefit recov-
ery over the long term through its role in LTP,
where BDNF may be an underlying substrate that
facilitates persistent long-term memory storage
(Bekinschtein etal., 2008a, 2008b). Hippocampal
BDNF is chronically decreased in TBI (Chen
et al., 2005), and there is evidence that thera-
pies thought to increase BDNF expression in the
brain, such as environmental enrichment (Chen
etal., 2005)and exercise (Griesbach etal., 2009),
are promising therapies for cognitive recovery
post-TBI. Hippocampal BDNF expression is
linked to spatial memory in experimental TBI
studies (Griesbach etal., 2009). Thus BDNFs role
in LTP may be important in the critical balance
of synaptic maintenance in cognitive recovery.
Importantly, there are differential expression
patterns of BDNFs target receptors with age
(Croll etal., 1998; Tapia-Arancibia etal., 2008)in
both the prefrontal cortex (Romanczyk et al.,
2002) and the hippocampus (Webster et al.,
2006). Tropomyosin-related kinase B expres-
sion peaks in young/middle adulthood and then
decreases with age (Romanczyk etal., 2002). Thus
an age-specific shift in BDNF receptor expression
could impact BDNF signaling, complicating the
application of therapies that target BDNF. Age
appears to be a major factor in how BDNF genet-
ics and levels early after TBI influence recovery
(Failla etal., 2015a; Failla etal., 2015b). The mod-
ifying effect of age on the relationship between
chronic BDNF and outcome remains in question.
Neurodegeneration and Atrophy
Cerebral atrophy is a widespread phenomenon
in TBI (MacKenzie etal., 2002). Recent advances
in diffuse tensor imaging and related tools pro-
vide valuable information regarding changes in
white matter integrity following DAI (Huisman
et al., 2004). The cingulate gyrus, uncinate fas-
ciclus, and superior longitudinal fasciculus have
all been related to cognitive deficits post-TBI
(Turken et al., 2008) and are implicated in cir-
cuits that facilitate depression (Hamani et al.,
2011; Mayberg, 2003; Mettenburg et al., 2012).
In TBI, a few studies have examined regionally
specific atrophy in fronto-limbic regions such
as the hippocampus (Tate and Bigler, 2000)and
426 Part IV:Homeostatic Therapies
the anterior cingulate (Chen etal., 2008). Recent
work has shown in moderate to severe TBI that
the degree of fronto-limbic structural atrophy in
the hippocampus and other regions was associ-
ated with PTD (Hudak etal., 2011). Interestingly,
these authors also show that regional grey mat-
ter atrophy is closely linked to damaged white
matter tracts (Warner et al., 2010), suggesting
overall reductions in fronto-limbic connectiv-
ity can occur after TBI. While BDNF may also
influence white matter tracts and functional
fronto-limbic circuits, these relationships are less
clear (Egan et al., 2003; Kennedy et al., 2009).
Fronto-limbic connectivity and regional volumes
have been linked to genetic variation in several
studies examining healthy populations, as well
as those with major depressive disorder (MDD),
and results also implicate gene variation within
monoaminergic systems (Bartrs-Faz etal., 2002;
Bertolino etal., 2009; Honea etal., 2009; Pacheco
etal., 2009; Pezawas etal., 2005)and neurotro-
phins (Egan et al., 2003; Kennedy et al., 2009;
Kim et al., 2013; Montag et al., 2010; Pezawas
etal., 2008; Radua etal.,2014).
Another consideration regarding neurode-
generation in the context of TBI is chronic trau-
matic encephalopathy (CTE), which has gained
mainstream media attention due to football
players in the National Football League. CTE is
a progressive tauopathy that results from repeti-
tive mild TBI or concussion that is traditionally
associated with athletes and military service
members that are at high risk for repetitive brain
trauma (McKee et al., 2013). This pathology is
characterized by an accumulation of hyperphos-
phorylated tau abnormalities that begin focally,
as neurofibrillary and astrocytic tangles that
spread to adjacent cortices, and eventually leads
to extensive neurodegeneration affecting many
brain regions, including the medial temporal lobe
structures, diencephalon, and brainstem (Stein
et al., 2014). Importantly, the tauopathy asso-
ciated with CTE is believed to be unique from
other tauopathies such as Alzheimers disease.
McKee and colleagues (2013) have character-
ized progressive staging of CTE pathology from
I to IV (least to most progressive) based on the
density of neurofibrillary tangles and coverage in
regions of the brain. Despite the established link
between repetitive blows to the head and CTE
incidence, the contribution of age, sex, genetics,
stress, and substance abuse to disease pathophys-
iology is currently unknown (Stein etal., 2014).
To date, CTE has been diagnosed only through
postmortem brain examination; therefore the
prevalence of the disorder is unknown.
Epilepsy
Posttraumatic epilepsy (PTE) is a common com-
plication following TBI, with a strong relation-
ship between seizure risk and severity and type
of head injury (closed or penetrating; Agrawal
etal., 2006; Annegers etal., 1998). Importantly,
one study found that over 80% of patients that
have a seizure post-TBI will have a recurrent sei-
zure within one year (Haltiner etal., 1997). The
risk factors for epileptogenesis following TBI
likely involve multiple mechanisms; those with
a subdural hematoma or brain contusion are at
an increased risk (Annegers etal., 1998; Haltiner
etal., 1997). Acommonly implicated pathology
in both TBI and epilepsy is glial scarring, which
is connected to microglial activation follow-
ing initial insult. One study found that, after
TBI, IL-1, and the granulocyto-macrophage
colony-stimulating factor work to regulate glial
scarring at the site of focal injury (Giulian etal.,
1994). In epilepsy, activated microglia, proin-
flammatory cytokines, and leukocytes from
the brain and periphery initiate inflammatory
cascades that result in neuronal hyperexcitabil-
ity, which can result in seizures (Vezzani and
Friedman, 2011). The role of inflammation in
epilepsy is well established in non-TBI popula-
tions. Evidence from our group shows that CSF/
serum ratios of IL-1 were associated with PTE
risk over time, and IL-1 gene variation moder-
ated serum IL-1 levels in this study (Diamond
et al., 2014). In addition to immunologically
based mechanisms of epileptogenesis, reduction
in bilateral hippocampal volume, specifically
in the dentate gyrus, is associated with TLE
(Golarai etal., 2001). It is also postulated that,
similar to TBI, excessive production of excita-
tory amino acid receptors during seizures can
generate ROS and nitric oxide that contrib-
ute to oxidative stress (Lancelot et al., 1998).
These mechanisms can also lead to changes in
excitatory/inhibitory neurotransmitter homeo-
stasis, including EAAs, such as aspartic acid,
that can have significant effects on epileptogen-
esis (Agrawal et al., 2006; Janjua et al., 1990).
Further, other studies are emerging suggesting
that genetic variation within neurotransmit-
ter signaling pathways can moderate seizure
susceptibility in the context of TBI (Darrah
et al., 2013; Diamond et al., 2014; AK Wagner
etal.,2010).

Cognitive Dysfunction
Monoaminergic Neurotransmission
Acute brain trauma can result in damage to the
hippocampus (Hicks etal., 1993), frontal cortex
(Dixon etal., 1987), and striatum (Dietrich etal.,
1994). These brain regions all share the common
characteristic of involvement in DA circuitry
through interactions with glutamate (Centonze
etal., 1999; Granado etal., 2008; OCarroll etal.,
2006). The loss of DAergic fibers after injury
specifically in the ventral tegmental area and
substantia nigra can influence synaptic and den-
dritic integrity in the frontal cortex and striatum
(Bales etal., 2009). Also, after brain injury there
is a rapid increase in tyrosine hydroxylase (Yan
et al., 2001; Yan et al., 2007), an enzyme neces-
sary for DA synthesis. Not surprisingly, increased
tyrosine hydroxylase expression is associated
with acute increases in DA levels in multiple
brain regions (Massucci et al., 2004; McIntosh
etal., 1994); however, over time, DA levels appear
to be reduced in the chronic period after TBI
(Massucci et al., 2004). This phenomenon may
reflect more of a functional hypoDArgic state
(Wagner et al., 2009) rather than frank DA cell
loss. Notably, low DA levels have been associated
with schizophrenia (Goldberg et al., 1991) and
Parkinsons disease (Bernheimer et al., 1973),
and elevated levels have been associated with
deficits in learning and memory (Morrow etal.,
2000). Numerous clinical studies of DAergic ago-
nists (discussed specifically in greater detail later
in this chapter) have shown efficacy for treating
cognitive deficits afterTBI.
Diaschisis
As previously discussed, secondary brain injury
is characterized by significant alterations in
brain neuroplasticity, neurotransmitter activity,
and other cellular dysfunctions. Translational
therapeutic neurorepair and rehabilitation
approaches are intended to limit destructive
secondary-injury cascades and attenuate post-
traumatic regional neurodepression that leads
to diaschisis. Diaschisis refers to functional
depression of neurons remote from, but ana-
tomically connected to, the primary site of injury
(Von Monakow, 1969). Mechanisms of recov-
ery from diaschisis, by nature, often result from
restoration of homeostatic functions after TBI,
including normalization of cerebral metabolism
and restoration of neurotransmitter systems of
brain plasticity (Duffau, 2006)and cortical reor-
ganization (Kleim et al., 2002). However, brain
Traumatic BrainInjury 427
injury recovery does not coincidentally paral-
lel resolution of diaschisis. Further, much of the
work done in clinical rehabilitation is guided by
the framework of reducing neural depression and
diaschisis (Garcia etal.,2011).
Depression
Neurotrophic Support
In healthy individuals, serum BDNF has been
implicated as a sensitive biomarker to depres-
sive symptoms associated with MDD (Sen etal.,
2008). Specifically, it is known that both stress
and TBI are associated with attenuated hip-
pocampal BDNF expression (Marmigre et al.,
2003; Murakami et al., 2005).Hippocampal vol-
ume is also reduced following TBI (Jorge et al.,
2007), suggesting that BDNF expression may be
involved in PTD pathophysiology. While there is
evidence for the effectiveness of serum BDNF as
a biomarker for MDD development (Hashimoto,
2010; Karege et al., 2002; Sen et al., 2008), it
remains unclear whether or not serum BDNF
is a viable marker for depression in TBI patients
and its potential as a biomarker in the context of
homeostatic dysfunction, plasticity, and recovery.
Further study on BDNF links to depression path-
ways may identify potential therapeutic targets to
treat PTD. There is also some suggestion that the
depression-risk polymorphism BDNF val66met
could mediate the relationship between BDNF
levels and depression (Gatt etal., 2009), with one
TBI study finding that the val66met SNP (specifi-
cally Val/Val homozygotes) influences treatment
responses to antidepressants for PTD (Lanctt
etal.,2010).
Inflammation
It is well known individuals with TBI are more
likely to have elevated inflammatory cytokines
and their receptors in both the peripheral blood
and CSF (Kumar etal., 2014; Miller etal., 2009).
One of the most commonly occurring neurobe-
havioral complications after TBI is PTD, with
TBI survivors at a nearly 10 times increased risk
compared to the general population (Bombardier
et al., 2010). However, the pathophysiological
mechanisms underlying PTD development are
poorly understood. The literature provides a
growing body of evidence demonstrating a rela-
tionship between inflammation and depression
outside the context of TBI. Interestingly, MDD is
more prevalent in patients with conditions that
lead to chronic inflammation (e.g., cardiovas-
cular disease, type II diabetes, and rheumatoid
428 Part IV:Homeostatic Therapies
arthritis) compared to the general population
(Steptoe, 2007). Due to the well characterized
role of inflammation in both TBI and depression
pathology, respectively, it is reasonable to suggest
a potential role for inflammation in PTD devel-
opment. Work from our group shows that acute
elevations in the CSF cytokine soluble surface
markers sVCAM-1, sICAM-1, and sFAS conferred
a nearly four-fold increase in odds of developing
PTD 6months post-injury (Juengst etal., 2014).
Despite this promising initial finding, there is
still uncertainty regarding the temporal rela-
tionship between inflammation and PTD, such
as the impact of chronic serum cytokine eleva-
tion (Kumar et al., 2014), the role of peripheral
inflammation, and examination of inflammatory
cells such as microglia and leukocytes. Given the
lack of unequivocal evidence of the use of selec-
tive serotonin reuptake inhibitors (SSRIs) in PTD
treatment (Fann etal., 2009), further work in this
area could have important implications for iden-
tifying therapeutic targets that restore a physi-
ological inflammatory environment afterTBI.
Serotonergic Dysfunction
In addition to neurotrophins and inflamma-
tory markers, serotonin signaling has been also
implicated with the development of depression
(Krishnan and Nestler, 2008). Serotonergic neu-
rons have widespread connections that impact
both mood and cognition. Evidence from experi-
mental TBI models suggests that there is an acute
elevation of serotonin, or 5-hydroxytryptamine
(5-HT), levels followed by a chronic hypomono-
aminergic state (Bales et al., 2009; Busto et al.,
1997). Therapeutic targets of the 5-HT (1A)
receptor confer neuroprotective effects, leading
to improvements in cognitive-related outcomes
and a reduction in hippocampal cell loss (Kline
etal., 2002). However, SSRI treatment after TBI
does not significantly improve cognitive symp-
toms or depressive symptomology (Wang et al.,
2011). The reason for this relatively counterintui-
tive finding is unclear. Experimental work with
SSRIs have also failed to yield a benefit in behav-
ioral outcomes, including cognitive performance,
despite some seemingly beneficial effects on neu-
rogenesis (Wang etal., 2011; Wilson and Hamm,
2002). As previously elaborated in this chapter, it
is possible that genetic variability within the ser-
otonergic pathway influences PTD risk, and this
source of innate heterogeneity in TBI response
could potentially influence the response to thera-
peutics (Failla etal.,2013).
Headache and Migraine
Posttraumatic headache (PTH) is the most com-
monly reported symptom following TBI, with
prevalence estimates ranging from 30% to 90%
(Lew etal., 2006; Theeler etal., 2013). When PTH
persists for over 1month after injury, migraine/
probable migraine is the most likely headache
phenotype, occurring in nearly 40% of those
who report PTH symptoms (Lucas et al., 2012).
Among those with a preexisting history of head-
aches, PTH is diagnosed when symptoms are
increased or intensified soon after the primary
injury (Lucas, 2011). Pain following posttrau-
matic migraine (PTM) likely is due to neuro-
genic inflammation (Moskowitz, 1993). Often
referred to as the trigeminovascular pathway,
this pathway involves the release of neuropep-
tides, such as substance P, neurokinin A, and
calcitonin gene-related peptide, near sensory
fibers innervating the meninges, where they can
sensitize peripheral nociceptors (Longoni and
Ferrarese, 2006; Williamson and Hargreaves,
2001). In TBI, neurogenic inflammation has been
connected to the development of edema, vaso-
dilation, and functional deficits (Nimmo et al.,
2004). Cortical spreading depression has been
observed in brain injury (Lauritzen et al., 2011;
Strong etal., 2002)and is a phenomenon closely
tied to migraine pathology, specifically migraine
aura (Hadjikhani etal., 2001). This phenomenon
involves an intense depolarization of neuronal
and glial membranes that results in acute changes
in [K+] and [Na+] that leads to neuronal excitabil-
ity (Kraio and Nicholson, 1978). Individuals with
PTM are at a greater risk for brain infarcts and
white matter lesions compared with the general
population (Kruit MC et al., 2004). Some have
proposed that alterations in cerebral autoregula-
tion can influence the ischemic lesions (Reinhard
et al., 2007). Interestingly, during migraine
attacks there is an increase in CBF in cingulate,
auditory, and visual cortices and also the brain
stem (Weiller etal., 1995). Despite the possibility
that it is likely a product of neurogenic inflam-
mation and autoregulatory dysfunction, the
mechanisms underlying PTM remain under-
studied todate.
Migraine headaches have also been linked
with 5-HT transmission (Raskin, 1991). In
fact, many drugs effective in treating migraine,
directly or indirectly, influence 5-HT transmis-
sion or receptor functioning (Raskin, 1991).
Triptans, a class of 5-HT-1B/D agonist drugs, are
commonly used for acute management of PTM.

They work by binding serotonin receptors in
meningeal trigeminal afferents and mediate neu-
rogenic inflammation. They also can constrict
vessels dilated by calcitonin gene-related peptide
(Lucas, 2011). For chronic management of PTM,
the most common therapies are tricyclic antide-
pressants, -blockers, calcium-channel block-
ers, and antiepileptic drugs (AEDs). Therapies
that are less commonly indicated are SSRIs or
serotoninnorepinephrine reuptake inhibitors
(Lucas, 2011). There is no universal treatment
protocol for individuals presenting with PTM.
Overall, management of headache disorders after
TBI requires a balance of treatment of physical
headache or migraine symptoms and comorbid
cognitive, emotional, and sleep disorders.
Neuroendocrine Dysfunction
A majority of patients with moderate to severe
TBI also have some kind of pituitary dysfunction
comorbidity that results in hypogonadism (Agha
et al., 2004; Lieberman et al., 2001; Schneider
etal., 2006; J.Wagner etal., 2010). Work from our
lab shows that acute hypogonadism affects nearly
all cases of moderate to severe TBI. Also, among
TBI cases with acute hypogonadotropic hypo-
gonadism, approximately 40% go on to develop
persistent hypogonadotropic hypogonadism
(PHH), while the remainder have early-resolving
hypogonadotropic hypogonadism (Wagner etal.,
2012). Additional work from our group shows
that in a prospective cohort of men with severe
TBI, a two-step screening of testosterone at 12 to
16 weeks post-injury can predict persistent hypo-
gonadotropic hypogonadism status over the first
year with high sensitivity and specificity (Barton
etal.,2015).
Stress-related models have shown that eleva-
tions in cortisol levels can also contribute to
hypogonadism (Kalantaridou etal., 2004), which
could be a primary reason why acute serum hor-
mone profiles are dynamically altered following
injury (Wagner et al., 2011a). TBI activates the
hypothalamicpituitaryadrenal (HPA) axis that
increases glucocorticoid production (Grundy
et al., 2001; Lucas et al., 2006). Munck and col-
leagues (1984) demonstrate the classic paradigm
that the anti-inflammatory effects of glucocorti-
coids following the stress response help to medi-
ate recovery. However, more recent work shows
that there is a bidirectional connection between
the immune and endocrine systems through
the HPA axis (Maier, 2003). Correspondingly,
it is believed that failure of glucocorticoids to
Traumatic BrainInjury 429
mediate an anti-inflammatory immune response
in the CNS could be responsible for the pro-
longed inflammatory response observed after
TBI (Besedovsky and del Rey, 2000; Sapolsky
et al., 2000). To address this point, work from
our group specifically studied the bidirectional
neuroendocrine-immune relationships in the
context of TBI. We found that cortisol medi-
ated the relationship between CSF inflammation
and global outcomes (Santarsieri et al., 2014).
Correlational analysis indicated that, among
those in a low cortisol state, individuals with
unfavorable outcomes display a negative correla-
tion between CNS inflammatory load and corti-
sol. However, those with a high cortisol state and
unfavorable outcomes have a significant positive
correlation between cortisol and CNS inflamma-
tion. These findings suggest that poor outcomes
after TBI may be the result of a dysfunction in
neuroendocrine-immune relationships where
too much, and also too little inflammation can
have a negative effect on outcome.
PL E IO T ROPIC , E N V I RON M E N TA L ,
A N D I N DI V I DUA L I Z E D
I N T E RV E N T ION ST R AT EG I E S:
HOM EO STAT IC- V E R SUS
PL A ST IC I T Y-SU PP OR T E D
M EC H A N I SM S
Common Considerations
forTreatment
To date, pharmacological drugs to treat TBI have
failed to show efficacy due to the high degree
of heterogeneity and complicated pathophysi-
ology that accompanies the injury. Therapies
that target only one physiological mechanism
largely have not conferred an overall benefit
(Maas etal., 2010). As such, the reality is that a
single golden bullet treatment does not exist
for TBI. Health-care providers face the difficult
task of individualizing treatment to optimize
recovery. Correspondingly, progress in reha-
bilitation research toward that end is being pur-
sued through an integrative omics approach.
Derived from our work in TBI, rehabilomics
is a novel framework to address the needs of
this population that incorporates a transdisci-
plinary approach to understanding the biology,
function, prognosis, complications, treatments,
adaptation, and recovery of patients (Wagner,
2010). The reason individuals with similar mech-
anisms of injury and clinical management have
vastly different recovery trajectories could be
430 Part IV:Homeostatic Therapies
multifaceted. For example, intrinsic factors, such
as age and sex, which play a large role in most
conditions, must be considered. Additionally,
genetic variation as well as gene by environ-
ment interactions and epigenetic considerations
may affect an individuals response to treatment
as well as present a risk factor for particular
TBI-related complications. Biomarkers can serve
as a valuable tool to study the individuality and
neurobiology of TBI as a complex disease, and
information from preclinical and clinical bio-
marker studies have moved the field forward in
better understanding the heterogeneity involved
with TBI-specific pathophysiology. For clinical
providers, biomarkers may represent a permis-
sive plastic versus a dysfunctional homeostatic
state, either of which may have prognostic value
with regard to outcome. Perhaps more impor-
tant, they may also aid as dynamic markers of
treatment response. Correspondingly, both tra-
ditional and nontraditional therapies discussed
here encompass pharmacotherapies, physio-
therapies, and biologics commonly used to treat
TBI, and, where possible, biomarkers associated
with treatment mechanisms and response are
discussed.
Pharmacotherapies
Stimulants
Psychostimulants, including D-amphetamine
(AMPH) and methylphenidate (MPH), have been
prescribed recently to patients with TBI. MPH
acts as a DA transporter blocker, while AMPH
works as a transporter substrate that promotes
DA release into the extracellular space and also
release through reverse transport (Fleckenstein
et al., 2007). Despite varying mechanisms of
action, both AMPH and MPH have similar
effects and are commonly prescribed for atten-
tion deficit hyperactivity disorder (ADHD;
Scahill etal., 2004). Importantly, typical dosing
for MPH in a setting of ADHD is considerably
higher than for TBI (Whyte et al., 1997). MPH
use is more common in TBI compared with
AMPH, with multiple clinical studies showing its
efficacy in improving cognitive performance and
function, especially in the domains of attention
and memory (Gualtieri and Evans, 1988; Kaelin
etal., 1996; Kim etal., 2006; Whyte etal., 1997,
2004). Also, experimental TBI models show that
prolonged MPH treatment after controlled corti-
cal impact normalizes many components of pre-
synaptic striatal DA neurotransmission (Wagner
etal.,2009).
DA-Agonist
There is evidence to suggest that the concentra-
tion of DA and its receptors can lead to frontal lobe
deficits that can result in a wide variety of cognitive
issues (Jokinen etal., 2013; Narayanan etal., 2013).
Patients with Parkinsons disease have DA depletion
from neurodegeneration of the nigrostriatal path-
way that results in prefrontal dysfunction. After
TBI, there is DA system disruption that is intimately
involved with cognitive deficits (Bales etal., 2009).
However, systemic DA-agonists could be benefi-
cial or detrimental based on the time of adminis-
tration after injury. In addition to stimulants, two
DA-agonist drugs that have been studied and used
clinically in TBI are bromocriptine and amanta-
dine hydrochloride (AMH). Bromocriptine is a
mixed D2 agonist that has a dose-specific agonist/
antagonist effect. At low doses (2.55 mg/kg), bro-
mocriptine has been associated with DA neuronal
firing and evidence that it acts as a partial D2 auto-
receptor antagonist (Jackson etal., 1990; Lieberman
and Goldstein, 1985). At higher doses (>10 mg/kg),
bromocriptine acts as more of a D2 autorecep-
tor agonist, causing inhibition of DA release and
metabolism (Maruya etal., 2003). In clinical TBI,
there are reports that low-dose bromocriptine can
result in improvements in prefrontal and attention
deficits (McDowell et al., 1998); however, another
study that administered 10 mg/day of bromocrip-
tine to a population with moderate to severe TBI
did not see benefits in cognitive recovery (Whyte
etal.,2008).
AMH, which has also been used as an anti-
viral therapy for influenza type-A (Atkinson
et al., 1986) as well in Parkinsons disease
(Godwin-Austen et al., 1970; Metman et al.,
1998) and multiple sclerosis (Cohen and Fisher,
1989), has been used as a DA enhancing agent
for TBI. AMH may increase extracellular DA
concentrations through NMDA antagonism and
reuptake inhibition through the DA transporter;
however, chronic AMH use could increase DA
transporter activity (Harun and Wagner, 2014).
AMH can be efficacious in increasing arousal
and cognition, especially for agitated, restless,
or apathetic patients (Van Reekum et al., 1995;
Sawyer et al., 2008). Also, a recent multicenter
randomized control trial showed that 4-week
administration of AMH accelerated recovery
and led to benefits in patients in a minimally con-
scious state (Giacino etal., 2012). This work pro-
vides promising evidence that AMH may have
significant utility as a therapeutic to improve
functional recovery afterTBI.

Cholinergics
After a brain injury, a rapid elevation in ace-
tylcholine (ACh) is observed (Saija etal., 1988).
The cholinergic system is integral to cogni-
tive function, including attention, arousal, and
memory. Altered cholinergic states could be a
factor leading to cognitive deficits following TBI
(Dixon etal., 1997), even in the absence of spe-
cific damage to the hippocampus (Lyeth et al.,
1990). Importantly, acute elevations in ACh are
followed by a chronically depressed cholinergic
state that may contribute to prolonged mem-
ory deficits (Dixon et al., 1995). The reason for
decreased ACh in the chronic stages post-TBI
could be altered activity of choline acetyltrans-
ferase, the enzyme responsible for synthesiz-
ing ACh. Specifically, choline acetyltransferase
activity is responsible for attenuating the acute
hypercholinergic state, but this enzyme may
also contribute to a chronic hypofunctioning
state (Levin et al., 2014). Within the context of
a hypocholinergic state, individuals with TBI
have more sensitivity to anticholinergic phar-
macotherapies compared with healthy individu-
als (Griffin et al., 2003). Therefore, drugs with
anticholinergic effects should be used with cau-
tion in patients with TBI (Arciniegas etal., 2000;
Levin etal., 2014; Stanislav,1997).
A systematic review examined the efficacy of
various cholinergic agents following TBI (Poole
and Agrawal, 2008). Studies involving acetylcho-
linesterase inhibitors showed modest improve-
ments in cognitive benefits. The authors cite
issues with timing of administration of the drug,
with early administration during the hypercho-
linergic state being neurotoxic and later admin-
istration being more effective. Also, studies that
used a combined therapy of physostigmine and
lecithin, a choline precursor, showed no major
additive effects from this combined therapy to
improve memory (Levin et al., 1986; Walton,
1982), and these cholinesterase inhibitors are not
recommended for clinical administration (Poole
and Agrawal, 2008). However, other inhibi-
tors, including donepezil, galantamine, and
rivastigmine, have shown therapeutic potential
in chronic TBI (Tenovuo, 2005). Additionally,
CDP-choline, an ACh precursor and key interme-
diate in the biosynthesis of phosphatidylcholine,
has also been shown to improve global outcome
and improve motor and cognitive deficits fol-
lowing TBI (Calatayud Maldonado et al., 1991).
However, a large multisite clinical trial featuring
CDP-choline did not show benefit (Zafonte etal.,
Traumatic BrainInjury 431
2012), leaving questions as to how innate het-
erogeneity and differences in standard care may
have influenced treatment response.
Antidepressants
Three of the most common classes of antidepres-
sants include SSRIs, tricyclic antidepressants,
and monoamine oxidase inhibitors (MAOIs).
These three drug classes have been studied and
are used to treat posttraumatic behavioral and
mood disorders, including PTD. The most com-
prehensive review of treatment for PTD was
done by Fann and colleagues (2009), who iden-
tified 13 studies that examine pharmacothera-
pies, including 7 studies with SSRIs. The only
study with class I evidence was a randomized
clinical trial examining the efficacy of sertraline
compared to placebo (Ashman etal., 2009). The
study found no statistically significant difference
between the two treatment arms. Another study
with classII evidence compared treatment with
MPH or sertraline versus placebo (Lee et al.,
2005). This study found that both experimen-
tal groups showed improvements in depressive
symptoms compared to placebo; however, MPH
showed added benefit beyond sertraline for
cognitive-related symptoms. Other studies have
shown sertraline was well tolerated and provided
benefits for patients with PTD (Fann etal., 2009;
Rapoport etal., 2008; Turner-Stokes etal., 2002).
Studies involving SSRIs have examined citalo-
pram and fluoxetine; however, evidence for their
efficacy is less established (Horsfield etal., 2002;
Perino et al., 2001). There is limited evidence
for TCA use for treating PTD. One small study
examined desipramine in a severe TBI popula-
tion, but results are hard to interpret due to study
limitations (Wroblewski etal., 1996). Two other
studies examined amitriptyline for PTD and
found no treatment effects (Dinan and Mobayed,
1992; Saran, 1985). Intervention with MAOIs
have not been well studied (Newburn etal., 1999;
Saran, 1985). With this evidence in mind, current
knowledge about efficacy of antidepressants in
PTD treatment is unclear. Due to the heterogene-
ity of TBI, many researchers posit that PTD has
varying pathophysiology compared with MDD,
thus it is possible that antidepressants efficacious
for MDD may not provide the same benefits in
TBI (Failla etal., 2013; Saran, 1985). Due to the
high prevalence of PTD, there is a pronounced
need for more high-quality studies assessing
antidepressant use and other treatments in this
population.
432 Part IV:Homeostatic Therapies
Anticonvulsants
As previously discussed in detail elsewhere in
this chapter, seizures are a common complication
following TBI; however, treatment studies to date
and prophylaxis guidelines shed little light on
how AEDs might influence outcome. Acompre-
hensive systematic review examining the efficacy
of AEDs following acute TBI (Schierhout and
Roberts, 1996) included six published studies,
and the prophylactics studied included: pheny-
toin (McQueen etal., 1983; Pechadre etal., 1991;
Temkin et al., 1990; Young et al., 1983), carba-
mazepine (Gltzner et al., 1983), and pentobar-
bital (Manaka, 1992). The authors of the review
calculated a pooled relative risk for early seizure
prevention of 0.34 but, due to the heterogeneity of
the included studies, could not make any claims
regarding late seizure risk or development of
PTE following therapy. Agrawal and colleagues
(2006) posit that there is no current evidence that
one drug is better than the other. It is noted
that a recent randomized controlled trial found
that levetiracetam shows modest improvement
over phenytoin (Szaflarski et al., 2010) for criti-
cally ill patients with TBI. Additionally, experi-
mental work in the controlled cortical impact
model shows that daily levetiracetam treatment
can improve neurological recovery and neuro-
plasticity through modulation of inflammation
and excitatory pathways (Zou etal., 2013). Despite
this, there still remains a lack of consistent evi-
dence that acute therapies for posttraumatic sei-
zures affect the eventual development of PTE.
Researchers still have significant work ahead to
determine new therapeutic targets, examine how
genetic heterogeneity might influence treatment
response, and better understand how acute pro-
phylactic therapies after TBI reduce early seizure
risk andPTE.
Anti-Inflammatory Strategies
As discussed earlier, TBI is characterized by a
significant inflammatory response. The rapid
activation of microglia and upregulation of pro-
inflammatory cytokines leads to a significant
inflammatory load in the acute and subacute stages
of brain injury (Kumar et al., 2014). Among the
relatively new considerations for TBI therapeutic
interventions are anti-inflammatory treatments.
The goal of these therapies is not to halt the inflam-
matory process in its entirety, as there is a fairly
well-established neuroprotective role of inflam-
mation in TBI (Morganti-Kossmann etal., 2002),
but to ameliorate the proinflammatory cascade
to lessen the likelihood of a sustained chronic
inflammatory state. To date, anti-inflammatory
therapies for TBI have provided inconclusive
results. Some studies have examined the use of
corticosteroids; however, this did not prove to
be efficacious in a TBI population and in fact
resulted in an elevated mortality risk (Edwards
et al., 2005). Others have examined the use of
nonsteroidal anti-inflammatory therapies such as
minocycline, which have been efficacious in other
CNS injury models (Alano et al., 2006; Maier
et al., 2005; Stirling et al., 2004). Additionally,
there is some evidence that the interleukin-1
receptor antagonist (IL-1ra) can attenuate neu-
ronal damage after injury due to its effects on
IL-1-initiated damage (Toulmond and Rothwell,
1995)and effects on downstream neurotrophins,
such as nerve-growth factor (Dekosky et al.,
1996). IL-1ra use has been well studied in other
disease models, such as rheumatological diseases
(Bresnihan etal., 1998; Cohen etal., 2002; Horai
etal., 2000)and type II diabetes mellitus (Larsen
et al., 2007), but it is understudied in TBI as an
anti-inflammatory therapy.
Ziebell and Morganti-Kossmann (2010)
explain that the challenge with anti-
inflammatory therapies in TBI is achieving mild
immunosuppression that modifies but does not
entirely eliminate the inflammatory response.
With this in mind, there is a definite need for
continued research in experimental and clinical
models to understand the temporal dynamics
of both pro- and anti-inflammatory cascades in
TBI to better differentiate the neuroprotective
and detrimental properties of posttraumatic
inflammation. As inflammation is implicated
in several chronic complications after TBI,
this knowledge may be useful in driving tar-
geted therapeutics for patients with multiple
TBI-related conditions.
Physiotherapies
Exercise
There is a wealth of evidence that physical activ-
ity can improve and enhance mood, quality of
life, and cognitive function in both healthy and
neurologically impaired populations (Erickson
etal., 2011; Taylor etal., 1985; Wolin etal., 2007).
Exercise used alone or in conjunction with other
cognitive and pharmaceutical interventions
(Archer et al., 2012) can endogenously increase
neurotrophins (Griesbach et al., 2004) that (at
least chronically) are important to neuronal
plasticity and repair after TBI. One randomized

controlled trial in TBI showed a significant
improvement in depressive symptoms and an
overall higher quality of life after aerobic exercise
(Hoffman et al., 2010). Interestingly, multiple
studies have found positive effects with Tai Chi
Chuan Qigong exercise on mood and self-esteem
(Blake and Batson, 2009; Gemmell and Leathem,
2006). Animal models of TBI have identified
voluntary exercise as a robust intervention that
leads to cell proliferation and cell survival in
the dentate gyrus of the hippocampus, which
may accelerate learning and memory deficits
that are characteristic in neurological disorders
when introduced at an appropriate timeframe
after TBI (Griesbach etal., 2004; Hillman etal.,
2008). Exercise also leads to a corresponding up
regulation of BDNF protein, which in turn can
have positive effects on learning, memory, and
mood (Erickson et al., 2011). In fact, research-
ers posit that the positive effects on cognition
that result from exercise are mediated through
BDNF (Griesbach etal., 2009). Exercise confers
other neuroprotective effects by reducing oxi-
dative stress and free radical production (Lima
etal., 2009)and reducing cytokine overproduc-
tion (Ding etal.,2006).
Despite the postulated neuroprotective
effects, the timing of exercise intervention fol-
lowing TBI is an important consideration. There
is considerable controversy in the mild TBI/con-
cussion literature regarding when athletes can
return to play after injury, as early return prior
to resolution of associated pathophysiological
cascades can exacerbate symptoms and increase
the risk for a repeat, more serious head injury
(Lovell et al., 2004). Clinical and experimen-
tal TBI studies have shown that exercise in the
acute stages following injury can negatively affect
recovery (Kozlowski etal., 1996; Majerske etal.,
2008). These findings suggest that later exercise
is better to achieve neuroprotection/restoration,
and acute exercise can have negative effects on
outcomes. One hypothesis for these findings
may be the reduction in BDNF protein upregula-
tion that occurs with acute versus more delayed
exercise (Griesbach etal., 2004), which may be a
neuroprotective response in the acute phase since
BDNF target receptors for apoptosis are elevated
during that time (Rostami et al., 2014). In sum,
evidence exists that exercise has potentially
potent neurorestorative properties following
TBI, particularly when considerations are taken
regarding timing, intensity, and individualized
limitations to maximize benefits.
Traumatic BrainInjury 433
Mindfulness Meditation
During rehabilitation of individuals with TBI
or cognitive deficits, it is imperative to imple-
ment learning and cognitive strategies to opti-
mally engage patients during therapy. Also,
results from neuropsychological tests can be
used to guide personalized strategies for learning
(Cicerone etal., 2011). Aquiet and low-stimulant
environment is critical to engage a patient in
a single task. In addition, learning should be
conducted in a procedural, step-by-step man-
ner with repetition at each stage (Haslam etal.,
2011; McCulloch, 2007; Vakil, 2005). One highly
developed behavioral strategy that could be
adapted for a cognitively impaired population
is mindfulness meditation, which is designed to
target attention and moment-to-moment aware-
ness (Kabat-Zinn etal., 1985). This technique has
been well established as a treatment for chronic
pain (Kabat-Zinn, 1982, 1994). However, others
have found evidence for its efficacy in a clinical
acquired brain injury population for reducing
mental fatigue, a common symptom following
TBI (Johansson et al., 2012; Olver et al., 1996).
Another study found that mindfulness medita-
tion can improve cognitive deficits, specifically
stimulus overselectivity, a behavioral phenom-
enon showing characteristic hyperattentiveness
to certain stimuli and lack of attention to others
(McHugh and Wood, 2013). Another study found
preliminary evidence that mindfulness-based
therapy reduced depressive symptoms after TBI
(Bdard et al., 2013). Mindfulness-based thera-
pies may be promising as a nonpharmaceutically
based intervention during periods of stress and
depression after TBI. However, further replica-
tion studies, strategies for optimizing interven-
tion in populations with cognitive impairment
and behavioral problems, and examination of
who can benefit the most from these strategies
should be explored.
Cognitive Rehabilitation
and Strategy Training
Cognitive deficits are commonly observed
after TBI and can be a significant burden on
caregivers as well as limit potential benefit
from other aspects of rehabilitation therapy.
Rehabilitation strategies can improve the cogni-
tive issues that result from TBI through various
strategy-training approaches; these approaches
largely target implicit elements of learning and
cognition (Cicerone etal., 2011), and applicabil-
ity can be tailored to individualized needs across
434 Part IV:Homeostatic Therapies
a wide range of deficits and homeostatic dys-
function (Sohlberg and Turkstra, 2011). These
issues include attention, visuospatial function-
ing, language and communication, memory, and
executive functioning. Cognitive rehabilitation
approaches in practice are complex and multi-
factorial but can be broadly characterized into
two categories: (a) cognitive function retrain-
ing and (b) functional compensation (Ponsford
et al., 2012). The former strategy focuses on
attenuating specific cognitive functions that are
impaired after injury through specific, organ-
ized neuropsychological exercises (Malec et al.,
1984; Novack et al., 1996; Ben-Yishay et al.,
1987). It is important to note that cognitive
function retraining is built on the foundation
of a bottom-up approach, which makes the
assumption that recovery from injury is based
on amelioration of specific cognitive impair-
ments (Ponsford etal., 2012). The second broad
category of cognitive rehabilitation interven-
tion is functional compensation, which empha-
sizes improvements in various domains of life,
including mobility, self-care, domestic life, and
community. This approach could be considered
a top-down approach as it focuses on building
on a persons strengths to compensate for cog-
nitive impairments and personalize therapy for
individual personal goals (Ponsford etal., 2012).
Improved task performance can be achieved
through internal strategies (e.g., mnemonics;
Kaschel etal., 2002; Ryan and Ruff, 1988; Thoene
and Glisky, 1995), external mechanisms, and
environmental restructuring (e.g., cueing or
memory aids; Evans etal., 2003; Wilson, 1991). It
is important to note that the cognitive rehabilita-
tion literature has certain limitations. For exam-
ple, many studies incorporate training that uses
artificial tasks that are not likely to be encoun-
tered by patients with TBI. Also, few studies have
been able to incorporate robust ways to quantify
the ability of an individual to use a strategy to
improve performance in real-world settings.
Importantly, there is a lack of incorporation of
preclinical animal work that examines cognitive
rehabilitation and strategy training. Work from
our lab (Wagner et al., 2013) discusses a clini-
cally relevant animal model using nonspatial
pretraining to discriminate implicit and explicit
learning through the evaluation of spatial and
nonspatial learning and memory constructs
within the Morris water maze. Overall, there is a
need for further study to refine our understand-
ing in the biological foundations of cognitive
rehabilitation and how to normalize the homeo-
static underpinnings of cognition to optimize
recovery.
Acupuncture
The biological mechanisms underlying the effi-
cacy of acupuncture, an alternative medicine that
dates back over 2,000years in eastern Asia (Wu,
1996), is controversial, with nearly one-third
of studies providing no biological rationale
(Moffet, 2006). Studies that have discussed the
physiological mechanisms attributed the ben-
efits of acupuncture to neurochemical, segmen-
tal (gate theory), autonomic regulation, local
effects, and effects on brain function (Moffet,
2006). The National Center for Complementary
and Alternative Medicine (2014) of the National
Institutes of Health cites the major benefits of
acupuncture as regulating the nervous system
by dynamically affecting the release of pain bio-
chemicals (e.g., endorphins), as well as altering
the release of neurotransmitters and neurohor-
mones that affect parts of the CNS related to sen-
sation, such as immunology and blood flow. Due
to the overlap of the proposed mechanisms of
action of acupuncture and the pathophysiology
of CNS disorders, like TBI, it is reasonable to sug-
gest a potential therapeutic use for different neu-
rological complications (Ernst etal., 2001). Wong
and colleagues (2011) conducted a review of the
literature and found four randomized controlled
trials that studied traditional Chinese acupunc-
ture in TBI populations. The authors noted that
although some studies supported the efficacy of
acupuncture, the overall methodological qual-
ity of the studies were low, making it difficult
to draw conclusions. Patients with specific TBI
complications, such as PTM and PTD, which do
not have well-established pharmacological inter-
ventions to date, may be promising candidates
for acupuncture targets. However, the evidence
for efficacy of acupuncture for migraine (Linde
et al., 1996, 2005; Loh et al., 1984) and MDD
(Mukaino etal., 2005; Wang etal., 2008)is mixed
and needs further study. In sum, the physiologi-
cal mechanisms of acupuncture, although not
entirely established in itself, are likely wide rang-
ing and have systemic effects on the CNS, spe-
cifically in the treatment of chronic pain (Vickers
et al., 2012). As previously emphasized, many
TBI pharmacological drugs have failed to show
efficacy due to the characteristic high degree of
heterogeneity in the secondary-injury cascade.
It is possible that acupuncture may be a logical

alternative form of therapy due to its more global,
pleiotropic effects; however, at the present time
there is not enough evidence to make a defini-
tive claim. Thus there is a need for well-designed
experimental and clinical studies that specifically
address the physiological process and homeo-
static mechanisms that acupuncture therapy may
have to offer individuals withTBI.
Biologics
Progesterone/Estradiol
Numerous experimental studies have suggested
a neuroprotective role for steroids, including
progesterone and estradiol (Roof et al., 1994;
Schumacher et al., 1996, 2004; Stein, 2001;
Wright et al., 2001). As noted earlier, clinical
TBI studies have shown significant alterations
in endogenous serum and CSF hormonal pro-
files after injury (Garringer et al., 2013; Rogers
and Wagner, 2006; Wagner et al., 2011a).
Experimental studies show that progesterone
has strong neuroprotective effects that target
cerebral edema, as well as reduce neuroinflam-
mation, BBB deterioration, and cellular necrosis
(Stein etal., 2008). Estradiol treatment has also
been shown to be neuroprotective in animal
models by improving synaptic transmission and
ameliorating excitotoxicity and oxidative injury
(Stein, 2001). There is limited research on the
use of estradiol as a therapy in clinical TBI. Two
published studies have shown safety and initial
clinical efficacy of progesterone therapy for acute
TBI (Wright et al., 2007; Xiao et al., 2008), but
phase III trials did not support a beneficial effect
(Skolnick etal., 2014; Wright etal., 2014). Given
the complexity of endogenous hormone physi-
ology after TBI, future research should aim to
understand how hormone treatments influence
peripheral and CNS hormone profiles and their
downstream effects on other mechanisms of sec-
ondary injury.
VitaminD
Recent evidence implicates vitamin D deficiency
in chronic fatigue after TBI (Schnieders et al.,
2012). Vitamin D deficiency is highly prevalent
in the elderly (Gloth and Tobin, 1995; Visser
et al., 2003), pregnant women (Andran et al.,
2002), the obese (Buffington et al., 1993), and
individuals with osteoporosis (Lips etal., 2006).
Furthermore, the heterogeneity of TBI pathology
makes vitamin D use in combination with other
therapies logical. Specifically, vitamin D hormone
(VDH) has begun to be evaluated as a cotherapy
Traumatic BrainInjury 435
with progesterone to enhance its neuroprotective
effects following TBI by targeting inflammatory,
apoptotic, and excitotoxicity pathways (Cekic
and Stein, 2010). One animal study in TBI shows
that the combined use of a pharmacological-dose
of VDH (5g/kg) and progesterone was not sig-
nificantly different with respect to memory tasks
compared to progesterone alone; however, com-
bined therapy did reduce neuronal loss and reac-
tive astrocyte reduction (Tang etal., 2013). Given
the complexity of VDH and progesterone, the
exact mechanisms of actions are still unclear, but
some posit that the systemic anti-inflammatory
properties of both agents may complement
each other both in the brain and in the periph-
ery (Stein and Cekic, 2011). With this in mind,
progesterone and vitamin D may be a logical
approach for combined therapy for TBI patients
with vitamin D deficiencies; to date there is still
sparse evidence for the efficacy of progesterone
and VDH therapy in humans.
Omega-3 FattyAcids
A healthy diet rich in omega-3 fatty acids
has powerful neuroprotective effects and can
increase molecules important for normal brain
function, including BDNF, which can lead to
improvements in synaptic and cognitive plastic-
ity in the injured brain (Wu etal., 2004; Zuccato
and Cattaneo, 2009). Several studies have shown
that omega-3 supplementation in animal mod-
els of TBI lead to BDNF elevations, reduced
oxidative stress, and improved cell signaling
(Salem et al., 2001). Interestingly, a recent ani-
mal study found evidence that progesterone may
play a large neuroprotective role in specifically
omega-3 deficient rats through the maintenance
of neuronal signaling by supporting membrane
stability and axonal growth (Tyagi etal., 2014).
Dietary and supplementation of omega-3 has also
been linked to improvements in MDD (Levant,
2011; Lotrich etal., 2013). Also, one experimen-
tal study showed decreased depressive symp-
toms as well as increased plasma 5-HT levels
and lower IL-6 levels following dietary intake of
omega-3 (Park etal., 2012). Omega-3 supplemen-
tation reportedly also has efficacy in migraine
treatment (Harel etal., 2002; Simopoulos, 2002).
Due to positive links between omega-3 and
TBI-related pathologies, there is promise for the
clinical utility of omega-3 supplementation for
homeostatic restoration in patients with TBI.
However, well-designed randomized trials are
needed.
436 Part IV:Homeostatic Therapies
Dietary Polyphenols
Polyphenols are a broad group of organic
substances that contain an excess of phenol
groups. Two of the most common groups that
are believed to have CNS-protective properties
include curcumin and flavonoids. Among the
benefits of polyphenols are powerful antioxidant
and anti-inflammatory properties. Flavonoids
can interact with neuronal signaling, specifi-
cally protein kinase and lipid kinase pathways,
which can inhibit apoptosis and promote neu-
ronal survival (Spencer, 2009). Foods rich in fla-
vonoids include vegetables and fruits, especially
berries, green tea, and red wine. Curcumin is a
common dietary and medicinal product used in
India that is found in turmeric and curries. One
study found that dietary curry consumption has
positive effects on cognition in a neurologically
intact, elderly population (Ng et al., 2006). In
addition to curcumin and flavonoids, resveratrol
is another powerful polyphenol that may have
protective effects in the heart, brain, and kidney,
in addition to its antiaging properties (Kalantari
and Das, 2010). Thus dietary phenols appear to
have promising neuroprotective properties that
may be logical therapeutic approaches to target
in reducing secondary-injury cascades that result
from TBI. However, like other biologic therapies
discussed, more treatment studies in TBI are nec-
essary to move this area of research forward.
CONCLUSIONS
As the literature here suggest, TBI is a complex
disease process whereby injury and innate het-
erogeneity play a prominent role in acute and
chronic recovery. In response to primary injury,
inflammation, excitotoxicity, and BBB disruption
lead to reactive astrocytosis, ischemia, and oxida-
tive stress. These acute processes lead to chronic
inflammation, impaired neurotransmission, and
other types of CNS dysfunction. Further, the lit-
erature is mixed on if, how, and when potential
neuroprotectants such as neurotrophins and sex
and stress steroids confer benefits in the context
of TBI. Persistent aberrations in humoral mark-
ers (inflammation, neurotrophins, sex/stress
steroids) are likely contributors to chronic condi-
tions and complications after TBI. Though sev-
eral agents have been tested, there are currently
no known effective neuroprotectants for treat-
ing TBI. However, a better understanding of the
likely chronic disease patho-mechanisms, as well
as the mechanistic impact of innate heterogene-
ity, may help minimize risk for complications
after TBI and aid in the design of effective per-
sonalized therapies. In addition to currently
used pharmacotherapies for TBI management,
emerging physiotherapies and biologics may
have relevance in managing the wide range of
conditions and complications associated with
TBI. Newer approaches, including comparative
effectiveness studies and randomized controlled
trials that incorporate effective biological indica-
tors for stratification, and monitoring of treat-
ment effects may further aid in the identification
of effective homeostatic treatments that restore
function for individuals dealing with the chronic
effects ofTBI.
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25
Adenosine and Alzheimers Disease:
A Possible EpigeneticLink
D AV I D B L U M , U R S U L A S . S A N D A U , O L I V I E R B O U S I G E S ,
L U I S A V. L O P E S , VA N E S S A F L AT E N , E M I L I E FA I V R E ,
LUC BUE , A NNE-L AUR ENCE BOU T ILLIER, A ND DE T LE VBOISON
ALZHEIMERS DISEASE
Generalities, Symptoms, and Lesions
Alzheimers disease (AD) was first described
more than 100 years ago. Corresponding to the
rise in life expectancy, its incidence has increased
dramatically, and current forecasts speak in
terms of a doubling of the number of persons
affected every 20 years (Wittchen and Jacobi,
2005). AD is the most frequently encountered
form of dementia (about 70% of cases of demen-
tia). The earliest and most frequent manifesta-
tions are benign memory disorders relating to
recent facts (Amieva et al., 2005; Ritchie et al.,
2001). This is followed by a slow evolution of
symptoms, which gradually spread to organiza-
tional and programming disorders, such asapha-
sia (Martin and Fedio, 1983; Murdoch et al.,
1987), apraxia (Foundas etal., 1999; Mozaz etal.,
2006), and agnosia. In most cases, AD appears as
a sporadic multifactorial disease resulting from
the interaction of different environmental, epige-
netic, and genetic factors that might facilitate its
onset. Various epidemiologic studies have identi-
fied risk factors and protective factors (Reitz C-terminal fragment is released by -secretase.
etal., 2011). Not only aging but also cardiovascu-
lar factors such as high blood pressure, diabetes,
and obesity are examples of the former (Ballard
et al., 2011; Pasinetti and Eberstein, 2008).
Conversely, a healthy lifestyle (physical and intel-
lectual activities, fish consumption, among other
factors) seems to have protective effects (Belarbi
etal., 2011; La Rue, 2010; and references herein).
Recently, genome-wide association studies have
allowed for the identification of genes associated
with the disease (Lambert and Amouyel, 2011;
Bettens etal.,2013).
The definitive diagnosis of AD is based on
the observation of characteristic brain lesions,
usually found during a postmortem examina-
tion: senile plaques and neurofibrillary tangles.
Neurofibrillary degeneration is due to the patho-
logical accumulation in the neuron of a naturally
present protein, the Tau protein, while the amy-
loid pathology is characterized by the extracel-
lular accumulation of the amyloid-beta peptide
(A) that is normally present in low concentra-
tions. Soluble oligomeric forms of amyloid pep-
tides are thought to promote a significant part of
their toxic effects at synapses, thereby contribut-
ing to plasticity deficits (Brouillette et al., 2012;
Benilova etal., 2012; and references herein). A
peptides either are derived from the -amyloid
precursor protein (APP) or result from the
combined action of the two distinct proteo-
lytic enzymatic activities of - and -secretase.
(Checler, 1995; De Strooper et al., 2010). The
activity of -secretase is now well characterized
as an acid protease (-site-APP cleaving enzyme
1 [BACE1]), which releases the N-terminal frag-
ment of the amyloid peptides. In comparison, the
Numerous studies suggest that presenilins
1 (PS1) and 2 (PS2) are themselves carriers of the
-secretase activity. Depending on the preseni-
lins involved, the -secretase activity appears
to be associated with a multiproteoic complex
of high molecular weight implicating at least
three other proteins:nicastrin, anterior pharynx
defective 1 homolog, and presenilin enhancer 2
homolog (De Strooper,2010).
Neurofibrillary degeneration consists of
the intraneuronal accumulation of proteina-
ceous fibrils forming flame-shaped neurons
454 Part IV:Homeostatic Therapies
into paired helical filaments (for reviews see
Buee et al., 2000; Sergeant et al., 2008). The
major antigen of paired helical filaments corre-
sponds to Tau protein, which is phosphorylated.
Tau is a neuronal protein essentially located
within the axonal compartment. Its structure
makes it essential for the organization, stabi-
lization, and dynamics of microtubules (Buee
et al., 2000; Sergeant et al., 2008), but recent
data also emphasize that Tau has other impor-
tant neuronal functions at the dendritic and
nuclear levels (Ittner et al., 2010; Morris et al.,
2011 for a review; Sultan etal., 2011). The physi-
ologic and pathologic functions of Tau are also
regulated by posttranslational modifications
such as phosphorylation. In AD and related
disorders, aggregated Tau proteins are always
found hyperphosphorylated. These changes in
phosphorylation may affect a number of Tau
functions and facilitate Tau aggregation (Buee
etal., 2000; Sergeant etal., 2008). During nor-
mal aging, Tau hyperphosphorylation occurs
in the hippocampal formation. In AD, once
the hippocampus is involved, amyloid plaques
may be present, and the Tau pathology spreads
to the basal forebrain and several cortical areas
in an anatomically defined pattern (Dujardin
et al., 2014) along neuronal projections defin-
ing the Braak stages of Tau pathology (Braak
and Braak, 1991). These stages are well corre-
lated to the severity of dementia (Duyckaerts
et al., 1997; Grober et al., 1999). Although Tau
is normally considered an intracellular protein,
even in its abnormal conformational state, it is
also found in the cerebrospinal fluid (CSF) of
patients as well as in the extracellular space of
diseased brains, suggesting a spreading mecha-
nism in a prion-like process (Jucker and Walker,
2013). These findings support the instrumental
role of Tau pathology in cognitive alterations,
as evidenced by reductions in several forms of
long-term potentiation (LTP) and long-term
depression, two well-known manifestations of
hippocampal synaptic plasticity in transgenic
models mimicking AD-like Tau pathology
(Hoover etal., 2010; Polydoro etal., 2009; Van
der Jeugd etal., 2011; Burnouf etal.,2013).
InflammationandAD
Besides A load and Tau pathology, both inflam-
matory processes and astrogliosis appear to be
integral components of AD onset and progres-
sion (Heneka and OBanion,2007).
Affirmation of the role that reactive astro-
cytes play in the pathophysiology of AD stems
from several observations. Postmortem charac-
terization of brain specimens from human AD
patients reveal that the marker for glial activa-
tion, S100, is elevated (Sheng et al., 1994; Van
Eldik and Griffin, 1994)and that activated astro-
cytes are found juxtaposed to amyloid plaques
(Wisniewski and Wegiel, 1991). Furthermore,
there is an increase in all isoforms of the astro-
cyte selective marker glial fibrillary acidic pro-
tein during disease progression (Kamphuis etal.,
2014). Importantly, there is a tight association
between astrogliosis and either aberrant A or
Tau function. Exogenous application of the A1-42
peptide to the rodent cortex causes rapid activa-
tion of astrocytes, as well as increased expression
of the astroglial adenosine A 2A receptor, lead-
ing to a decrease in astroglial glutamate uptake,
which may underlie glutamatergic dysfunction
and excitotoxicity in AD (Matos et al., 2012).
Importantly, the astroglial response occurs in
the absence of senile plaques, indicative of a
direct A-mediated effect on astrocyte func-
tion (Perez et al., 2010). Considering that A,
acting as an acute toxin is a potent glial activa-
tor, it remains possible that astrocyte activation
could be an early event in disease onset, occur-
ring even in the absence of amyloid deposition
(Moreira etal., 2008; Nunomura etal., 2000). In
line with this, glial activation has been detected
at very early stages in the AD brain (Cagnin etal.,
2001). Furthermore, in a population-based study,
increased gliosis has been found before the devel-
opment of AD lesions (Wharton et al., 2009).
The presence of reactive astrocytes in early AD
is of great importance considering that cogni-
tive deficits and neurodegeneration precede the
formation of senile plaques in multiple trans-
genic mouse models of AD (Hsia et al., 1999;
Westerman etal., 2002). Not only are astrocytes
activated by A1-42, but they may also contrib-
ute to disease progression and plaque formation
(Nagele etal., 2004; Nagele etal., 2003). Within
the AD brain there is evidence that A accu-
mulates within astrocytes (Kurt et al., 1999).
Furthermore, there is a positive correlation with
the amount of astrocytic A42 and AD pathol-
ogy. Single and double immunohistochemistry
to label plaques and markers for either astrocytes
or neurons within human AD brain specimens
suggests that astrocytes, which accumulate A42,
may lyse and be the source for small plaques that
form within the subpial portion of the molecular
layer of the cerebrocortex (Nagele etal.,2003).
Alterations in astrocyte function are not lim-
ited to A toxicity but are also associated with

Tau pathology (Belarbi etal., 2011; Schindowski
etal., 2006). Hippocampal sclerosis is a neuropa-
thology that has been described in brains from
patients with dementia, AD, and, most com-
monly, mesial temporal lobe epilepsy. The pathol-
ogy of hippocampal sclerosis is characterized by
astrogliosis and severe loss and redistribution of
neurons (Attems and Jellinger, 2006). Correlative
analysis of case histories and postmortem brain
specimens from patients with hippocampal scle-
rosis reveal a strong positive correlation between
AD and the presence of both glial and neuronal
tauopathy within the sclerotic tissue (Beach etal.,
2003). The putative role for astrocytes in tauopa-
thies has further been investigated in THY-Tau22
transgenic mice. As a tauopathy model, these
mice express a double-mutated 4-repeat human
tau, which causes hyperphosphorylated Tau
and aggregation throughout the brain. The hip-
pocampal pathology of THY-Tau22 mice includes
astrogliosis, as evidenced by increased glial fibril-
lary acidic protein expression, without neuronal
cell loss. THY-Tau 22 mice have altered synap-
tic function and plasticity and impairments of
learning and memory (Van der Jeugd etal., 2011;
Schindowski etal., 2006). Considering the asso-
ciation of aberrant astrocyte function in both
Tau and amyloid-mediated dementia and AD,
glial cells may play a pivotal role in regulating
disease progression and behavioral outcomes.
Thus restoring normal astrocyte function could
be of importance for managing symptoms or dis-
ease progression.
Inflammatory processes are an important
hallmark of AD (for reviews see Heneka and
OBanion, 2007; Wyss-Coray, 2006). This is sub-
stantiated by recent genetic data indicating that
variants located in genes notably involved in
innate immunity regulation such as CR1, TREM2
or CD33 modulate AD risk in genome-wide asso-
ciation studies (Lambert et al., 2009; Guerreiro
et al. 2013; Jonsson et al., 2013; Naj et al., 2011;
Bradshaw et al., 2013). Further, a recent study
points out that both acute and chronic systemic
inflammation, characterized with increases in
serum tumor necrosis factor-alpha (TNF), is
associated with an increase in cognitive decline
in AD (Holmes etal., 2009). Microglial cells seem
to play an important role in AD-related central
inflammatory processes. Whether microglial
activation is protective or deleterious is still a
matter of debate in AD research and has been dis-
cussed elsewhere (e.g., see Wyss-Coray, 2006, for
a review). Some data indicate that microglia may
promote A clearance. For instance, removing
Adenosine and Alzheimers Disease 455
CX3CR1-mediated signaling between neurons
and microglia results in reduction of -amyloid
levels and deposition in APP mouse models,
while microglial reaction is increased (Lee etal.,
2010b). In accordance, intrahippocampal injec-
tion of lipopolysaccharide (LPS), a well-known
promoter of inflammation, can reduce A load
in vivo (Herber et al., 2004; Wyss-Coray et al.,
2001). In line with these findings, recent data
indicate that favoring the M2 microglial pheno-
type by reducing Nlrp3 inflammasomes leads
to increased amyloid clearance and improved
memory performance in APP mice (Heneka
etal., 2013). Besides resident microglia, it has also
been shown that peripheral monocytes could
infiltrate the brain and contribute to amyloid
deposit reduction (Simard etal., 2006). Depletion
of the chemokine receptor CCR2, known to be
involved in the accumulation of the mononuclear
phagocyte in the brain, results in an increase of
A burden and memory defects in APP mouse
models (El Khoury etal., 2007; Naert and Rivest,
2011). However, in opposition to the aforemen-
tioned results, some studies have concluded that
microglia are unable to remove A deposits but
instead produce cytotoxic damages (Fang et al.,
2010; Wegiel et al., 2003). Puzzlingly, a recent
study suggests that microglia ablation does not
modulate plaque formation in an APP transgenic
model (Grathwohl etal.,2009).
Therefore, the precise role of innate immu-
nity toward the amyloid side of AD remains
unclear. At early stages, microglia and astro-
cytes would help to clear amyloid but, with
time, chronic microglial activation and change
in astrocyte functionality would contribute to
the detrimental outcome. Further adding to
this complexity, little is known about the rela-
tionship between inflammation and the Tau
side of AD. Previous work readily described
neuroinflammatory processes in several models
of AD-like Tau pathology (Bellucci etal., 2004;
Lourent et al., 2011; Sasaki et al., 2008; Zilka
et al., 2009). It has been stressed in particular
that microglial activation would even precede
tangles formation (Yoshiyama et al., 2007).
Interestingly, microglial activation would be
detrimental toward Tau pathology. Indeed, LPS
administration in rTg4510 mice exacerbated
hippocampal and cortical Tau phosphoryla-
tion (Lee etal., 2010a). This is in line with other
data showing that LPS significantly induced
Tau hyperphosphorylation through activation
of cyclin-dependent kinase 5 in the 3xTg trans-
genic model, without affecting APP processing
456 Part IV:Homeostatic Therapies
(Kitazawa et al., 2005). Involvement of micro-
glia in processes underlying Tau pathology has
been further emphasized by demonstrating
that removal of the microglial CX3CR1 recep-
tor leads to enhanced Tau pathology (Bhaskar
etal., 2010). These observations fit well with the
ability of proinflammatory mediators, known
to be released by microglial cells, such as IL1
or TNF, to promote Tau phosphorylation and
even its neuritic aggregation (Gorlovoy et al.,
2009; Li et al., 2003). Together, these observa-
tions fit with recent data demonstrating a good
correlation between innate immunity and
phosphorylated-Tau levels in the brain of AD
patients (Zotova etal.,2013).
Overall, the aforementioned data stress that
astrogliosis and neuroinflammatory events,
especially those mediated by microglial cells,
may have an instrumental role in AD, while
their respective contribution to the amyloid and
Tau sides remain unclear so far. It is also indi-
cated, however, that modulation of brain glial
cells may affect mechanisms underlying AD
pathogenesis.
Lesions, Seizures, and
Memory LossinAD
As already outlined, inflammatory processes
appear to be crucial in AD pathophysiology
(Heneka and OBanion, 2007). Several lines of
evidence now indicate that astrocyte and micro-
glial activation could be an early event in the
disease, occurring even in the absence of A
deposition (Heneka and OBanion, 2007). In
support of this notion, AD patients have glial
activation, which is evident at very early stages
of disease progression (Cagnin et al., 2001) and
appears to contribute to the development of amy-
loid plaques (Nagele et al., 2004). Importantly,
AD is now considered as a complex syndrome
comprised not only of cognitive impairment but
also of comorbid conditions that include seizures
and changes in sleep homeostasis. Those comor-
bid changes cannot readily be explained by amy-
loid or Tau deposits, or by neuronal cell loss, and
indicate that a more global disruption of network
homeostasis might contribute to the complex
symptomatologyofAD.
AD research and therapy development have
traditionally been dominated by the perception
that cognitive impairment in AD is due to neu-
ronal cell loss. Therefore, therapy development
has focused largely on neuroprotective strate-
gies, which fail to reverse cognitive impairment
in AD. In contrast, the role of glia and of home-
ostatic control mechanisms in the pathogen-
esis of AD have remained understudied. Thus
therapies aimed at reconstructing homeostatic
network function are of interest to improve
cognitive function in AD. Importantly, cogni-
tive dysfunction in AD occurs prior to neu-
rodegenerative changes in several models of
AD. This implies that neuroprotective strate-
gies might not be sufficient to improve cogni-
tive function in AD and that strategies aimed
at reinstating normal network activity deserve
investigation. If cognitive impairment in AD
is not primarily due to the loss of neurons but
can be linked to a deficiency in homeostatic
control mechanisms, then cognitive decline in
AD might be a therapeutically reversible pro-
cess. In support of this hypothesis the antie-
pileptic drug levetiracetam effectively reduced
abnormal epileptiform activity in hAPP mice.
Importantly, chronic treatment with this drug
also reversed hippocampal remodeling, behav-
ioral abnormalities, synaptic dysfunction,
and deficits in learning and memory in those
animals (Sanchez et al., 2012). These findings
suggest that aberrant network activity might
causally be linked to synaptic and cognitive
deficitsinAD.
EPIGENETIC CHANGES
AND MEMORY LOSS
I N A D : F R O M M E T H Y L AT I O N
T O A C E T Y L AT I O N
Epigenetic mechanisms are able to influence gene
expression without altering the DNA sequence,
and the coordinated action of epigenetic programs
on thousands of genes leads to diverse phenotypes.
In dividing cells, such changes are heritable, but
in postmitotic neurons, they participate in the
dynamic regulatory mechanisms maintaining
their functional status. As the epigenetic profile
of young identical twins are essentially indistin-
guishable, older ones show substantial differ-
ences in their epigenetic marks (Fraga etal., 2005;
Martin, 2005; Mastroeni etal., 2011). Remarkably,
the onset of AD in identical twins can differ by
more than 20 years (Cook et al., 1981; Nee and
Lippa, 1999). Thus epigenetic mechanisms are
thought to mediate the interaction between genetic
and environmental factors (e.g., diet, heavy metal
exposure, stress), and they could represent a link
between life events, risk factors, and pathophysi-
ological processes underlying the emergence of
neurodegenerative disorders suchasAD.

Epigenetic Marks ofthe Chromatin
Epigenetic modifications are found either on the
DNA or the histone proteins. DNA methylation
is involved in key cellular processes, includ-
ing X-chromosome inactivation, imprinting,
and transcriptional silencing of specific genes.
DNA methylation is a covalent biochemical
modification occurring predominantly on the
cytosines (5mC) of so-called CpG islands, cata-
lyzed by the family of DNA methyltransferases
(DNMT1, DNMT3A and DNMT3B; Goll and
Bestor, 2005). So far, no direct DNA demethyl-
ase has been identified, but it was recently found
that ten-eleven translocation enzymes (TET1,
TET2, and TET3) could convert aberrant DNA
methylation of cytosines (5mC) into hydroxym-
ethylation (5hmC), thereby providing a means
for active demethylation (Tahiliani etal., 2009).
Such a mechanism has also been found in neu-
rons (Guo et al., 2011). Methylation of CpG
island promoters correlates with transcrip-
tional silencing, either by a direct interference
of transcription-factor binding or through the
recruitment of repressive methyl-binding pro-
teins such as MeCP2 (Bogdanovic and Veenstra,
2009). Of note, Gadd45b, involved in DNA
repairlike mechanisms, has been proposed to
drive activity-dependent demethylation at spe-
cific promoters (Bdnf and Fgf) in neurons (Ma
etal.,2009).
In contrast to DNA, histones can undergo
multiple posttranslational modifications such
as methylation, acetylation, phosphorylation,
sumoylation, ubiquitination, ADP-ribosylation,
and others, among which acetylation is the most
well studied. Histone acetylation is tightly regu-
lated by opposing enzymes, the histone acetyl-
transferases (HATs) add an acetyl group on lysine
residues in the N-terminal histone tails, while
the histone deacetylases (HDACs) hydrolyze
the acetyl group. It is believed that such modi-
fications remove positive charges from histone
proteins and lower their interactions with the
negatively charged DNA structure, thereby con-
ferring a more relaxed conformation to the chro-
matin. This has an impact on transcription factor
and chromatin regulator accessibility, ultimately
modulating gene transcription (Kouzarides,
2007; Li etal., 2007). Recent data suggest that the
co-occurrence of histone marks may act as allos-
teric regulators of chromatin complexes (Rando,
2012). It is noteworthy that histone- and DNA
methylation modifications work tightly together
in cells, maintaining, for instance, active genes
Adenosine and Alzheimers Disease 457
undermethylated and repressed genes undera-
cetylated (Cervoni and Szyf, 2001), thereby main-
taining cellular homeostasis.
DNA Methylation and Susceptibility
GenesinAD
Basically, DNA methylation perturbations have
been shown to affect some susceptibility genes
involved in AD and hence can drive changes on
the pathophysiology of the disease. For example,
the high CpG content (72%) found in the APP
promoter makes it a valuable target for control
by methylation (Yoshikai et al., 1990). This was
further confirmed as complete demethylation of
the APP gene was reported in an AD postmortem
cortical sample but not in a normal control or a
Picks disease patient (West etal., 1995). Specific
demethylation of the APP promoter was also
reported to occur with age, a major risk factor for
AD, in the parietal cortex of 10 neurologically nor-
mal individuals (Tohgi etal., 1999). Several stud-
ies showed a general decrease in 5mC amounts
with age (Cooney, 1993; Wilson and Jones, 1983;
Wilson etal., 1987). Regarding the mechanisms,
immunoreactivity for DNMT1 and methylation
corepressor complex components were mark-
edly decreased together with 5mC in some AD
cases (Mastroeni et al., 2010). In addition, in a
primate model of AD, DNMT1 was decreased in
the cortex and associated with hypomethylation
of the APP promoter and increased APP mRNA
expression (Wu et al., 2008). The presenilin-1
(PSEN1) promoter was also hypomethylated
in late-onset AD brains compared with con-
trols (Wang et al., 2008). These results failed to
be reproduced in another study investigating
the methylation state of selected loci (including
microtubule-associated protein Tau MAPT, APP,
and PSEN1) in AD disease cases when compared
with controls, which failed to detect any signifi-
cant modifications, despite a high preservation
of CpG methylation of gene promoters with
postmortem delay. However, small changes in
the methylation of DNA promoters in vulner-
able cells might have been missed using total
homogenates of frontal cortex and hippocampus
(Barrachina and Ferrer,2009).
By contrast, higher A concentration results
in promoter hypermethylation, particularly that
of the neprilysin gene, a major A-degrading
enzyme in the brain, thereby leading to decreased
neprilysin levels in the hippocampus of AD
patients (Chen etal., 2009; Gunzburg etal., 2007;
Iwata et al., 2000; Yasojima et al., 2001). This
458 Part IV:Homeostatic Therapies
suggests that A-induced methylation could
exert positive feedback on its own production
through such methylation processes.
In summary, some genes involved in AD
(APP, PSEN1) are found hypomethylated in
patients and animal models of AD. Interestingly,
in AD patients, global DNA hypomethylation
was observed in the entorhinal cortex, one of
the first structures to be affected by the disease
(Mastroeni et al., 2010). Global DNA hypo-
methylation was also found in an AD monozy-
gotic twin compared with his normal sibling
(Mastroeni et al., 2009). Of note, such global
hypomethylation is also involved in the deregu-
lation of other genes, including those contrib-
uting to neurodegeneration (e.g., aberrant cell
cycle, apoptosis, and inflammatory processes) in
AD (Mastroeni etal.,2011).
DNA Methylation and
theMethionine CycleinAD
Both homocysteine (Hcy) and folate are criti-
cal components of a series of biosynthetic path-
ways essential for DNA methylation. S-adenosyl
methionine (SAM), generated by methionine, is
a methyl donor for methylation reactions. The
resulting product, S-adenosylhomocysteine
(SAH), can be further processed in Hcy and
adenosine (ADO) by SAH hydrolase. Then rem-
ethylation of Hcy to methionine (the methionine
cycle) predominates over the catabolic degra-
dation of Hcy (Finkelstein, 2000). Folate and
vitamin B12 are essential cofactors required to
regenerate methionine from Hcy. Chronic eleva-
tion in Hcy levels results in parallel increases in
SAH, whose intracellular accumulation can be
pathologic as it is a potent product inhibitor of
DNMTs (Hoffman et al., 1980). Thus sustained
hydrolysis of SAH to Hcy and ADO is essential to
maintain normal methylation of DNA, as well as
a proper methionine regeneration.
Vitamin B deficiency in humans and in
animal models results by itself in global hypo-
methylation. In AD patients the levels of Hcy
are elevated, and the pathology is associated
with folate and vitamin B12 deficiency (Morris,
2003; Seshadri et al., 2002). Folate is signifi-
cantly decreased in AD CSF (Serot etal., 2001),
as well as SAM and one of its synthesizing
enzymes, methionine S-adenosyltransferase, in
the CSF and brain of AD patients (Bottiglieri
et al., 1990; Morrison et al., 1996). DNA meth-
ylation inhibition has been observed together
with increased SAH levels in the brain (Kennedy
etal., 2004)and of Hcy in plasma (Clarke etal.,
1998; Smith, 2008). Deficits in folates and ele-
vated Hcy levels in APP mutant mice impaired
DNA repair in hippocampal neurons, sensitiz-
ing them to oxidative damage induced by A
(Kruman etal., 2002). The resulting elevated Hcy
levels and corresponding decrease in SAM in
AD (Tchantchou etal., 2006)may decrease APP
promoter methylation and increase its expres-
sion, thereby leading to increased A load. In
vitro, expression of the BACE and PSEN 1 genes
is enhanced after folate deprivationinduced
hypomethylation and restored to normal when
folate deprivation is accompanied by SAM sup-
plementation (Fuso et al., 2005). In vivo, expo-
sure of APP-overexpressing transgenic mice
to a folate/B12/B6deficient diet is associated
with enhanced SAH relative to SAM, as well as
PS1 hypomethylation leading to PS1 upregu-
lation but also BACE upregulation, enhanced
A deposition, and an accelerated appearance
of intraneuronal A in line with the develop-
ment of cognitive deficits (although the latter
was quite modest; Fuso et al., 2008; Fuso et al.,
2011). Furthermore, inhibiting methylation via
vitamin B deficiency is responsible for glycogen
synthase kinase 3 beta (GSK3; one of the most
important protein kinases regulating Tau phos-
phorylation) upregulation, promoting GSK3beta
protein increase and leading to abnormal
hyperphosphorylated Tau (Nicolia et al., 2010).
SAM treatment reversed PS1 hypomethylation
and consequently inhibited the upregulation
of vitamin B deficiencyinduced PSEN1 and
also BACE1 expression. Those changes reduced
amyloid production and plaque spreading and
increased Tau phosphorylation and spatial
memory in TgCRND8 mice (expressing human
APP695 with the Swedish [KM670/671/NL] and
Indiana [V717F] mutations) and wild-type mice
(Fuso etal., 2011; Fuso etal., 2012). Several stud-
ies have tested the benefits of high-dose supple-
ments of folate, vitamin B12, and vitamin B6 in
humans (Aisen etal., 2003; Aisen etal., 2008)and
demonstrated that vitamin B treatment reduced
the elevated Hcy levels in AD patients, without
any significant benefits in cognitive function,
however. It was further reported that a cocktail of
folate, vitamin B6, -tocopherol, SAM, N-acetyl
cysteine, and acetyl-L-carnitine in early-stage
AD patients led to significant improvements in
all scoring systems (dementia rating scale, neu-
ropsychiatric inventory, and activities of daily
living) for all patients (Chan etal.,2008).

Histone ModificationsinAD
Histone acetylation is the major histone modifi-
cation under the scope of Alzheimers research.
Other histone modifications, such as methyla-
tion, phosphorylation, and ADP-ribosylation
have been linked to memory functions but, so
far, not to AD (Grff etal., 2011; Fischer 2014).
The exact outcome of the role and the fate of
acetylation in AD is not clear, as many conflict-
ing data have been reported at all levels stud-
ied (cellular, animal, human pathology), likely
due to the great heterogeneity of models and
approaches that have been used. Unfortunately,
even if acetyltransferase activities seem to be
relatively preserved with postmortem and stor-
age duration, acetylated histones are mostly
unstable in postmortem tissues and only a
few results are available in the human brain
(Barrachina etal., 2012; Monoranu etal., 2011).
In addition, brain tissues reflect a mixture of
neuronal and glial populations, the ratio of
which is exacerbated by the pathology, so it is
difficult to conclude histone acetylation levels
in neurons through global analyses. A recent
study reported that HDAC2 levels were signifi-
cantly elevated in the hippocampal area CA1
and the entorhinal cortex in AD patients, as
early as in Braak and Braak stage I/II, suggest-
ing HDAC2 increase could be an early event in
the progression of AD (Grff et al., 2012). As
HDAC2 was recently described to negatively
regulate memory and synaptic plasticity in the
healthy rodent brain (Guan etal., 2009; Hanson
et al., 2013), elevated levels of HDAC2 in AD
may also accompany the cognitive decline of
the human neurodegenerating brain. This result
nicely fits the current literature promoting the
use of HDAC inhibitors in memory-related dis-
eases (Fischer etal., 2010; Grff and Tsai, 2013b),
because histone acetylation has been associated
with facilitation of learning and memory (Grff
and Tsai, 2013a). Indeed, many studies have
now tested the potential of HDAC inhibitors in
AD models for their ability to restore memory
functions. Injection of trichostatin Ain the hip-
pocampus of APP/PS1 mice increased the levels
of acetyl H4 and contextual freezing perfor-
mance upto wild-type values and restored hip-
pocampus synaptic dysfunction (Francis etal.,
2009). Administration of various pan-HDACs
inhibitors (HDACi; sodium valproate, sodium
butyrate, suberanilohydroxamic acid) was able
to reinstate associative memory in APP/PS19
mice (Kilgore etal., 2010). Treatment with the
Adenosine and Alzheimers Disease 459
broad HDACi sodium butyrate of p25/Cdk5,
which present neurodegeneration, neurofibril-
lary pathology (Cruz etal., 2003), and intracel-
lular A accumulation (Cruz et al., 2006), not
only increased H3 and H4 acetylation levels in
mice but also induced sprouting of dendrites,
increased the number of synapses, and resulted
in the reestablishment of learning abilities, as
well as access to long-term memories that had
been ablated by prior hyperactivation of p25/
Cdk5 (Fischer et al., 2007). Administration of
the pan-HDACi phenylbutyrate (PBA), a model
of amyloid pathology, in the tg2576 mice was
able to reverse spatial memory loss (Ricobaraza
et al., 2009) and fear conditioningrelated
memory (Ricobaraza etal., 2011). Interestingly,
PBA also successfully impacted AD physi-
opathology, as it reduced the hyperphosphoryl-
ated form of Tau via an increase of the GSK3
inactive form (Ricobaraza et al., 2009) and
decreased amyloid beta pathology as well as
inflammation (Ricobaraza etal., 2011). Chronic
PBA treatment also restored the loss of den-
dritic spines and synaptic plasticity in the
hippocampus in 6- and 16-month-old tg2576
mice (Ricobaraza et al., 2011; Ricobaraza
et al., 2012)., Importantly, such treatments
were efficient against memory dysfunctions
even in a very severe stage, as described in the
APPPS1-21 mouse strain following sodium
butyrate treatment (Govindarajan etal., 2011).
Recent reports performed with more selective
inhibitors also showed an interesting thera-
peutic potential obtained with an HDAC1
inhibitor MS-275 (Entinostat) in APPPS1-21
mice (behavioral impairment improvement
and significant reduction of amyloid plaque
deposition in the hippocampus and corti-
cal regions; Zhang and Schluesener 2013) or a
mercaptoacetamide-based class II inhibitor in
3xTG AD mice (decrease of A40, A42, and
Thr181 phospho-Tau levels and spatial memory
performance improvement; Sung etal., 2013). It
is noteworthy that HDACi might affect not only
histone acetylation but other cellular proteins
as well. For example, Tau was shown to be acet-
ylated by the HAT p300 and deacetylated by the
classIII HDAC SiRT1 (Min etal., 2010). ATau
intrinsic acetyltransferase activity was also
reported (Cohen etal., 2013). The impact of Tau
acetylation in the pathology seems to lead to an
aggravation of the tauopathy (Min etal., 2010;
Cohen et al., 2011; Irwin et al., 2012), but this
has been recently debated (Cook et al., 2014).
460 Part IV:Homeostatic Therapies
Ultimately HDACi treatment might lower the
phosphorylation status of Tau as reported in
response to phenylbutyrate (Ricobaraza et al.,
2009)or mercaptoacetamide (Sung etal., 2013).
However, such links remain to be further tested.
In contrast to these in vivo beneficial effects
of increasing acetylation with HDACis, early evi-
dence obtained in cellular models rather linked
AD-related genes with upregulation of histone
acetylation. For instance, the APP C-terminal
peptide (AICD), a product of APP cleavage by
the -secretase, was shown to interact with the
acetyltransferase TIP60 through the nuclear
adaptor Fe65, and the resulting complex was able
to enhance gene transcription (Cao and Sudhof,
2001). The AICD fragment induced histone H3
and H4 hyperacetylation and the expression
of cytotoxic genes, an effect further enhanced
by high-dose HDAC inhibitor administration
(Kim et al., 2004). Another study reported that
familial-AD mutations on PS genes inhibited the
production of an intracellular peptide N-Cad/
CTF2 originally cleaved through WT PS1,
thereby preventing the degradation of the acetyl-
transferase CBP and resulting in upregulated
CBP/CREB-mediated transcription (Marambaud
et al., 2003). However, whether these mecha-
nisms exist in vivo remains unknown. Of note,
epidemiologic studies show that AD risk is sig-
nificantly reduced in a population that consumes
large quantities of curcumin (Chandra et al.,
1998; Chandra etal., 2001), a molecule that has
been shown to display acetyltransferase inhibi-
tor on CBP/P300 proteins (Morimoto et al.,
2008) and to interfere with the formation of
amyloid plaques in AD rodent models (Ishrat
et al., 2009; Lim et al., 2001; Ono et al., 2004;
Yang et al., 2005). Overexpression of SIRT1,
which mainly deacetylates nonhistone proteins,
rescued neurons against neurotoxicity in p25/
Cdk5 mice (Kim et al., 2007), as well as in the
APPswe,PSENdE9 AD mouse strain (Donmez
etal.,2010).
In contrast, modulation of the APP sig-
nalling pathway (with a specific APP-targeted
antibody) in primary neurons induced the dea-
cetylation of H3 and H4, together with caspase
6-dependent degradation of the acetyltrans-
ferases CBP and p300 (Rouaux et al., 2003). It
is noteworthy that caspase-6 activity has been
observed in neurofibrillary tangles in the brain
of AD patients (Albrecht etal., 2007), suggesting
that such loss of acetyltransferases could occur
in diseased neurons and produce a net decrease
in histone acetylation. Deletion of the genes
encoding presenilins PSEN1 and PSEN2 lead to
a downregulation of genes regulated through the
CREB/CBP complex, which are associated with
decreased LTP, long-term memory deficits, and
hyperphosphorylated Tau (Saura et al., 2004).
In cellular systems, overexpression of wild-type
PSEN1, but not PSEN1 variants containing
mutations associated with familial AD, stimu-
lated the transcriptional activity of CBP and
p300 (Francis et al., 2007; Francis et al., 2006).
Moreover, CRE/CBP-dependent gene expression
was altered in PSEN1 and APP mice (Soreghan
et al., 2005). Interestingly, an in vivo study
showed that increasing CBP levels with admin-
istration of viral particles in triple transgenic
AD mouse hippocampus restored learning and
memory deficits, likely through BDNF upregula-
tion (Caccamo etal., 2010). Therefore, CBP level
preservation seems to be important in patholo-
gies such as AD. In addition, a recent study in
humans established that AD patients presented
increased nuclear translocation of an endog-
enous p300/CBP inhibitor (EP300-interacting
inhibitor of differentiation 1 [EID1]) in their hip-
pocampal neurons (Liu etal., 2012). EID1 over-
expression in mouse brain reduces hippocampal
LTP and impairs spatial memory as well as his-
tone H3 acetylation (Liu etal., 2012). Altogether,
this series of data supports strategies that could
target CBP HAT activity as an interesting thera-
peutic pathway to target treatment development
in AD (Schneider etal., 2013; Valor etal., 2013).
Such drugs are currently being developed, and a
HAT activator treatment in wild-type mice was
recently shown to enhance maturation of newly
generated neurons in the adult and sustain spa-
tial long-term memory (Chatterjee et al., 2013),
but it remains to be tested in AD models.
In conclusion, multiple epigenetic mecha-
nisms are dysregulated in AD, and it is difficult to
clearly link specific epigenetic changes to patho-
logical effects, as many molecular players and
mechanisms are involved. However, the thera-
peutic modulation of histone acetylation stands
out as a very promising strategy with proven effi-
cacy on plasticity and memory-related symptoms
and to a certain degree also on the pathophysiol-
ogy in animal models (Grff etal., 2011; Fischer
2014). Manipulating the global DNA hypometh-
ylation observed in AD brains, especially as it
is also possible with specific diets in the elderly,
may be of great interest in AD pathology as well
(Narayan and Dragunow,2010).

ADENOSINE AS
A H O M E O S TAT I C
R E G U L AT O R I N A D
Adenosine:Origin and Receptors
ADO is an endogenous neuromodulator control-
ling brain function via activation of high-affinity
A1 or A2A, low-affinity A2B, or low-abundance
A3 adenosine receptors that feed into a multi-
tude of different neuronal and glial downstream
pathways (Blum etal., 2003; Boison etal., 2009;
Sebastiao and Ribeiro, 2000, 2009). Synaptic lev-
els of ADO are regulated by an astrocyte-based
ADO cycle and metabolic clearance through
phosphorylation into adenosine monophosphate
(AMP) via adenosine kinase (ADK; Boison,
2008). Importantly, the ADK/ADO system is an
upstream regulatory component of complex neu-
ronal interactions. Changes in this system affect
brain function on the network level by simulta-
neous modulation of several downstream path-
ways (Boison, 2009; Boison etal., 2009; Sebastiao
and Ribeiro, 2000). Evidence that ADO dysregu-
lation is associated with AD pathology is evident
from the observed decrease in hippocampal A1
receptors in AD patients (Stone etal., 2009). The
postulated deficiency in ADO signaling in AD is
consistent with cognitive impairment, seizures,
and sleep disorders, which are all characteristic
comorbiditiesofAD.
Caffeine asa Cognitive
NormalizerinAD
Caffeine is one of the most consumed psychoac-
tive drugs. At high millimolar concentrations,
irrelevant for normal consumption, caffeine
can act at the level of ryanodine receptors and
cyclic nucleotide phosphodiesterases, but it is
now well established that under normal physi-
ological conditions, the effects exerted in the
brain by caffeine depend on its ability to block
adenosine A1 and A 2A receptors (Fredholm
et al., 1999; Fredholm et al., 2005). In humans,
caffeine is largely demethylated to its dimethyl
metabolic intermediates, with over 80% of orally
delivered caffeine metabolized to paraxanthine
(1,7-dimethyl-xanthine), and about 16% is con-
verted to theophylline (3,7-dimethylxanthine)
and theobromine (1,3-dimethylxanthine), both
potent antagonists of adenosine receptors.
Adenosine receptors have a crucial neu-
romodulatory role and regulate both synaptic
transmission and plasticity (Linden, 2001)either
by directly modulating synaptic responses or
Adenosine and Alzheimers Disease 461
by interfering with other receptors (Ribeiro
and Sebastiao, 2010). Adenosine receptors are
G-protein coupled receptors that mostly regu-
late, in opposite directions, the second messen-
ger cAMP. While A1Rs are coupled to inhibitory
Gi/o proteins, thus decreasing cAMP levels, A2A
receptors are coupled to excitatory Gs proteins,
increasing cAMP levels. Activation of these
receptors also modulates Ca 2+ channels and
the phospholipase C pathway. Through these
actions and by modulating both the release and
the uptake of different neurotransmitters, the
balance between adenosine A1 and A2A recep-
tors activation allows the fine-tuning of synaptic
transmission and plasticity in the hippocampus
(Sebastiao and Ribeiro, 2009). Upon aging, as
well as under chronic noxious brain conditions,
increased ADO levels are associated with an
increase in A2A receptor density and effects and
a decrease in A1 receptors (Cunha et al., 2001;
Lopes et al., 1999; Rebola et al., 2003). Thus, by
blocking adenosine receptors, caffeine emerges
as a potential drug to modulate brain function
and cognition (de Mendonca and Cunha,2010).
The widespread consumption of caffeine
underlies the importance of studying the conse-
quences of its chronic intake to aging-associated
cognitive decline, particularly in pathologi-
cal conditions. Caffeine consumption increases
alertness and improves cognition in physiologi-
cal conditions (Fisone etal., 2004; Haskell etal.,
2005; Sawyer etal., 1982; Smit and Rogers, 2000).
Caffeine also improves attention and information
processing (Lorist and Tops, 2003). In rodents,
evidence from the past few years supports the
cognitive-enhancing properties of caffeine in a
variety of behavioral tasks that evaluate learning
and memory (Takahashi et al., 2008; Marques
etal., 2011). More recently, and in line with such
experimental evaluations, a recent study points
out that caffeine improves long-term memory in
humans (Borota etal.,2014).
However, only recently has the relationship
between the consumption of caffeine and the
pathophysiology of many neurodegenerative dis-
eases been firmly established (Rahman, 2009).
Even if some controversy may still exist (Eskelinen
and Kivipelto, 2010), caffeine consumption has
beneficial effects not only on aging, reducing the
associated cognitive decline in healthy subjects
(Johnson-Kozlow etal., 2002; Ritchie etal., 2007;
van Gelder etal., 2007), but also in AD patients.
Indeed, different epidemiological reports high-
light the inverse correlation between caffeine
462 Part IV:Homeostatic Therapies
consumption and the incidence or severity of
AD. In the Canadian Study of Health and Aging,
daily coffee drinking decreased the risk of AD
by 31% during a 5-year follow-up (Lindsay etal.,
2002). In line with this, another study from the
same population reports that coffee drinkers at
midlife had a lower risk of dementia and AD later
in life compared with those who drank little or
no little coffee. The lowest risk (65% decreased)
was found in people who drank three to five cups
per day (Eskelinen etal., 2009). Finally, a retro-
spective study has shown an inverse correlation
between coffee intake and the occurrence of
AD later on in life. Indeed, while age-matched
controls had an average daily caffeine intake
of 198.7 135.7 mg during the corresponding
20years of their lifetimes, patients with AD had
a lower average daily caffeine intake of 73.9 97.9
mg during the 20years that preceded diagnosis
of AD (Maia and de Mendonca, 2002). Other ret-
rospective data proposed that high plasma caf-
feine levels were associated with a reduced risk
of dementia or a delayed onset in patients with
mild cognitive impairment (Cao etal., 2012). It is
also noteworthy that the recent Honolulu-Asian
Aging Study did not find a significant associa-
tion between caffeine intake and dementia risk
(Gelber etal., 2011). However, the authors inter-
estingly reported that, at autopsy, patients in the
highest quartile of caffeine intake (>277.5 mg/d)
were less likely to have any of the neuropathol-
gical lesions, such as AD-related lesions, micro-
vascular ischemic lesions, cortical Lewy bodies,
hippocampal sclerosis, or generalized atrophy.
Therefore, available epidemiological data support
that caffeine consumption is able to slow down
cognitive decline in the elderly and reduces the
risk of developing AD. Of note, while this looks
also to be the case in Parkinsons disease (Ross
etal., 2001), caffeine has been recently suggested
to exhibit detrimental effects in Huntingtons
disease (Simonin et al., 2013), suggesting that
caffeine is not protective in all neurodegenerative
conditions and that its effects depends on under-
lying instrumental mechanisms.
Caffeine, Adenosine Receptor
Targeting, and AD:From Lesions
toInflammation
Caffeine mitigates cognitive decline induced by
A and reduces amyloid burden in transgenic
mice overexpressing mutated APP (APPsw)
in preventive, but also therapeutic paradigms.
Indeed, APPsw mice chronically treated from 4
to 9.5 months of age with caffeine (300mg/L by
drinking water corresponding to 500mg/d in
humans) were cognitively improved in several
behavioral tasks that evaluated working and
spatial memories and exhibited reduction of
hippocampal A1-40 and A1-42 (Arendash et al.,
2006). Importantly, a similar treatment of APPsw
mice at late pathological stages (1819 months)
for 4 to 5 weeks reversed memory deficits and
reduced amyloid deposition as well as soluble
A levels in both entorhinal cortex and hip-
pocampus (Arendash et al., 2009). Such benefi-
cial effects of caffeine against A production has
recently been confirmed by another group in an
experimental model of sporadic AD based on
feeding rabbits a cholesterol-enriched diet that
elevates both A levels and Tau phosphoryla-
tion in the brain (Prasanthi etal., 2010). In this
study, rabbits fed a cholesterol-enriched diet were
treated with low doses of caffeine (0.5 to 30 mg/d)
through drinking water, corresponding to a max-
imal 60mg/d consumption in humans. In this
paradigm, caffeine significantly decreased A
production in accordance with Arendash etal.s
results (Prasanthi et al., 2010). Interestingly,
reduced production of A140 and A142 was also
observed in a neuroblastoma model overexpress-
ing mutated APP following treatment with con-
centrations of caffeine below 10 M (Arendash
etal., 2006), further supporting the idea that caf-
feine impacts on mechanisms underlying amy-
loid peptide production. In accordance, chronic
caffeine treatment of APPsw mice has been asso-
ciated with decreased PS1 and BACE1 protein
expression as well as increased insulin-degrading
enzyme levels, the latter presumably contribut-
ing to enhanced A degradation (Arendash etal.,
2006; Prasanthi et al., 2010). Effects of caffeine
on BACE1 expression could relate to its ability
to reduce c-Raf1 activity, possibly through PKA
activation (Arendash et al., 2009). In addition,
caffeine could reduce GSK3 expression and/or
activity and thereby influence A production
(Arendash et al., 2009). However, a direct effect
of caffeine on -secretase activity remains elu-
sive, and mechanisms linking caffeine and A
production/clearance deserve further evaluation.
Finally, it is noteworthy that although the ben-
eficial effects of coffee on cognitive decline and
decreased AD risk in humans has been mostly
ascribed to caffeine, other coffee constituents may
play an important role. Indeed, two recent stud-
ies support that noncaffeine components of cof-
fee display neuroprotective effects in drosophila

and C.elegans amyloid models though activation
of the Nrf2 detoxification pathway (Dostal etal.,
2010; Trinh etal.,2010).
Doses used in experimental studies describ-
ing beneficial effects of caffeine achieve plasma
concentrations of caffeine and metabolites
below 10M (Georgiev etal., 1993). Taking into
account the brain-to-plasma ratio (Kaplan etal.,
1989), and even if the involvement of other tar-
gets cannot be excluded (Fredholm et al., 1999;
Guerreiro etal., 2008), such levels are essentially
compatible with adenosine receptor inhibition
(Fredholm et al., 1999; Muller and Jacobson,
2011). Therefore, beneficial effects of caffeine
could then be mostly ascribed to its effects on
adenosine receptors. Previous studies indicate
that A1 receptor activation increases soluble APP
production, a nonamyloidogenic form of APP
(Angulo etal., 2003). Thus A1 receptor blockade
by caffeine could reduce the nonamyloidogenic
pathway, an effect considered deleterious in the
AD context. However, the involvement of the A1
receptor blockade by caffeine in the modulation
of amyloid production and toxicity remains to be
thoroughly evaluated, as does the potential role
of adaptive changes of A1 receptors promoted by
chronic caffeine consumption (Jacobson et al.,
1996; Johansson et al., 1997). Indeed, adaptive
increases in A1 receptor expression are expected
to promote the neuroprotective potential of
endogenous ADO (Cunha, 2008) and have been
suggested to be involved in protection afforded
by caffeine in ischemia or traumatic brain injury
(Jacobson et al., 1996; Johansson et al., 1997; Li
etal.,2008).
Caffeine could also mediate its long-term
effects through A2A receptor blockade. During
aging, we and others have found compelling evi-
dence of cortical and hippocampal increases of
A2A receptor expression/function. Specifically,
in the hippocampus of aged rats, A2A receptor
expression is nearly two-fold higher compared to
young rats (Cunha etal., 1995; Lopes etal., 1999).
More important, the A2A receptor-dependent
activation of glutamate release becomes more
pronounced as aging progresses and shifts
from protein kinase C-mediated signaling to
cAMP-dependent effects (Lopes et al., 1999;
Rebola etal., 2005). This is accompanied by clear
behavioral deficits in hippocampus-dependent
tasks, such as spatial memory in rats (Diogenes
etal., 2011). Accordingly, rats overexpressing hip-
pocampal A2A receptors also exhibit behavioral
deficits, including spatial memory defects as well
Adenosine and Alzheimers Disease 463
as LTP impairments (Lopes et al., unpublished
data). Interestingly, other detrimental condi-
tions associated with cognitive impairment, such
as hypoxia, diabetes, or epilepsy, share similar
A 2A receptor overactivation (Lopes et al., 1999;
Batalha etal., 2013; Lopes etal., 2011, for review).
Recently, we demonstrated decreased adult hip-
pocampal LTP and cognitive/memory impair-
ment in a chronic stress induced aging-like
model, generated by maternal separation during
the early postnatal period, in association with
increased hippocampal A 2A receptor expression
(Bathala etal., 2013). Strikingly, we observed, in
adults, a normalization of synaptic and cognitive
dysfunctions following A2A receptor blockade
with the selective antagonist KW6002, support-
ing an instrumental role of A2A receptor dys-
regulation in the genesis of synaptic dysfunction
underlying cognitive impairment within the
context ofaging.
As A2A receptors are dysregulated in AD
(Albasanz et al., 2008), and given the impact
of caffeine toward both cognitive and patho-
logical processes, the blockade of A 2A receptors
appears to be a rational strategy to mitigate sev-
eral detrimental components in AD. Besides
A-production, convergent data indicate that caf-
feine protects against the synapto-neurotoxicity
induced by A through blockade of A 2A recep-
tors. Caffeine indeed protected against the death
of rat primary hippocampal neurons induced
by an adenovirus carrying a mutated APP gene
(Stoppelkamp et al., 2011). These data are in
accordance with former work showing that, in
primary cultures of cerebellar granule cells, low
doses of caffeine (125M) are able to counteract
A25-35 toxicity, an effect mimicked by ZM241385,
an A2A receptor antagonist but not CPT, a selec-
tive A1 receptor antagonist (DallIgna et al.,
2003). These protective effects were confirmed
in vivo. Subchronic treatment with caffeine at a
dose of 30mg/kg protected against aversive and
working- memory deficits induced by intrac-
erebroventricular (ICV) injection of A2535 in
mice (DallIgna et al., 2007) and mimicked by
administration of SCH58261, a selective A2A
receptor antagonist. A2A receptor blockade,
through intraperitoneal injection of SCH58261
and KW6002 or genetic knockout, also pre-
vented working-memory impairment as well as
synaptic loss induced by ICV injection of A142
(Canas etal., 2009; Cunha etal., 2008). Improved
working-memory following A 2A receptor block-
ade was thought to be related to prevention of
464 Part IV:Homeostatic Therapies
synaptotoxicity promoted by A-through modu-
lation of p38 MAPK and mitochondrial function
(Canas etal.,2009).
Interestingly, memory improvement pro-
moted by A2A receptor blockade following ICV
injection of A was not observed in amnestic
conditions induced by MK801 or scopolamine
(Cunha etal., 2008). This reinforces the idea that
A2A receptor blockade targets specific synaptic
mechanisms rather than general mechanisms
controlling memory (Cunha et al., 2008; and
Gomes et al., 2011, for discussion). However,
this possibility should be tempered since, physi-
ologically, the A2A receptor blockade is procog-
nitive and particularly favors working memory,
but apparently not reference memory that needs
memory consolidation (Wei et al., 2011; Zhou
et al., 2009). This raises the possibility that the
overall cognitive effect observed in the para-
digms discussed previously may not be entirely
related to normalization of synaptic defects
induced by A, but rather involve more com-
plex regulation. Further, the extent to which A2A
receptor modulation impacts on amyloidogenesis
remains totally unknown sofar.
Contrasting with the available encouraging
data supporting that caffeine and A 2A receptor
blockade impacts on the amyloid side of AD,
much less is known about the impact of adeno-
sine receptor modulation on Tau pathology.
So far, few data are available, and most studies
have focused on the effects of caffeine. Caffeine
protected against the death of rat primary hip-
pocampal neurons expressing a mutated Tau
protein (Stoppelkamp etal., 2011). Furthermore,
caffeine mitigated Tau hyperphosphorylation
induced by a cholesterol-rich diet in rabbits
(Prasanthi et al., 2010). In line with this, caf-
feine, at doses irrelevant of human consump-
tion, reduced Tau phoshorylation in rat primary
cortical neurons (Currais et al., 2011). Caffeine
effects would rely at least on GSK3 modula-
tion, but the underlying mechanisms remain
largely unknown. However, we recently obtained
results supporting the notion that caffeine miti-
gates memory defects and Tau pathology in a
transgenic mouse model of AD- Tau pathology
(Laurent etal., 2014a). Whether these effects are
mediated through adenosine receptors remains
unclear. Several years ago, A1 receptor activation
was found to promote Tau phosphoryation in a
neuroblastoma model (Angulo etal., 2003), sup-
porting that the effect of caffeine toward Tau may
be mediated through this receptor subtype. More
recently, we established that reducing A2A recep-
tor function improves memory and Tau pathol-
ogy in a Tau transgenic mouse model (Laurent
etal., 2014b). This would suggest that A 2A receptor
modulation impacts on both pathological sides
of AD, supporting interest toward A 2AR-based
strategiesinAD.
Data from DallIgna et al., (2003) and Canas
et al. (2009) suggest that protection afforded by
A2A receptor blockade against A is ascribed to
neuron-autonomous mechanisms. As discussed
earlier in this chapter, astroglial and microglial
reactions are AD hallmarks, and both type of
cells express A2A receptors. Therefore, nonneu-
ronal autonomous mechanisms are also expected.
The role of A2A receptors expressed by astro- and
microglial cells is far from understood (for a
review see Hasko etal., 2005). A2A receptors may
promote activation and proliferation of astroglial
cells (Hindley etal., 1994; Ke etal., 2009)as well as
their ability to release glutamate (Nishizaki, 2004),
while A2A receptor blockade counteracts the astro-
cyte induction driven by basic fibroblast growth
factor (Brambilla etal., 2003). However, other stud-
ies indicate that A2A receptor stimulation inhibits
nitric oxide and TNF release by astrocytes (Brodie
et al., 1998). Concerning microglial cells, it has
been shown that A2A receptor stimulation causes
microglial activation (Orr etal., 2009)and potenti-
ates the release of nitric oxide as well as prostaglan-
dine E2 release from these cells (Fiebich etal., 1996;
Saura etal., 2005). Also, different experimental evi-
dence support the anti-inflammatory effect of A2A
receptor blockade in different neuropathological
situations (Dai etal., 2010a; Rebola etal., 2011; Yu
et al., 2008). In particular, A2A receptor blockade
mitigates LTP defects as well as microglial activa-
tion and IL1 release following LPS administra-
tion (Rebola etal., 2011). Of interest, the role of A2A
receptors may vary depending on the pathophysio-
logical context and in particular with regard to the
local environment. Indeed, although A2A receptors
may have anti-inflammatory functions under low
glutamate conditions, their function shifts toward
a proinflammatory function under high glutamate
concentrations following a transduction shift (Dai
etal., 2010b).
A 2AR upregulation has also been observed
in the microglia from AD patients (Angulo
etal., 2003). Further, experimental studies point
out that amyloid peptides can promote an A2A
receptor upsurge in both micro- and astroglial
cells (Orr etal., 2009; Matos etal., 2012). Recent
observations indicate that inhibitory effects of

A on astrocytic glutamate uptake is prevented
by A2A receptor blockade (Matos et al., 2012).
Together, this would suggest a tight relationship
between glial A2A receptors and pathological pro-
cesses underlying AD, which needs to be deeply
investigated.
Adenosine, Adenosine Receptors,
and Seizures
Seizures and epilepsy are a common comorbid-
ity of AD (Menendez, 2005; Palop and Mucke,
2009). Within the sporadic AD population there
is an 87% increase in the incidence of unpro-
voked clinical seizures compared to the aged
matched reference population (Amatniek et al.,
2006; Menendez, 2005). In addition to these
behavioral seizures, it is postulated that the
numerous episodes of severe confusion reported
by AD patients is the occurrence of silent (i.e.,
subclinical, electrographic) seizures (Thomas,
1997). So far, seizure occurrence in AD has
been ascribed to the amyloid side through the
use of several APP transgenic mouse models.
Transgenic mice that carry the human APP with
the Swedish and Indiana mutations (hAPP J20)
exhibited spontaneous nonconvulsive seizures in
cortical and hippocampal networks associated
with synaptic plasticity deficits (Minkeviciene
et al., 2009; Palop, 2007). In addition, seizure
thresholds in the hAPP transgenic mice were
reduced in response to pentylentetrazol (Del
Vecchio etal., 2004; Palop, 2007). The spontane-
ous and chemically induced seizure phenotypes
in the APP mutant were attenuated by genetically
removing the endogenous Tau protein, thereby
restoring synaptic function and reducing hyper-
excitability (Roberson et al., 2007; Roberson
etal., 2011). These data raise the possibility that
amyloid may be instrumental in seizure occur-
rence observed in AD patients but also that these
seizures relate to A-Tau interaction. To this end,
targeted gene therapy with a Tau antisense oligo-
nucleotide designed to decrease endogenous Tau
throughout the brain reduced seizure severity
in two independent chemically induced seizure
models (DeVos etal., 2013). Interestingly, epilep-
tic activity and reduced seizure thresholds have
been observed in different tauopathies, includ-
ing fronto-temporal dementia linked to chro-
mosome 17 and progressive supranuclear palsy
(Garcia-Cabero et al., 2013; Beach et al., 2003;
Nygaard etal., 1989). However, whether AD-like
Tau pathology is also able to promote seizure
occurrence has yet to be established.
Adenosine and Alzheimers Disease 465
Astrogliosis and the associated increase
in ADK leading to focal adenosine deficiency
is sufficient to trigger seizures (Boison, 2008).
Given the tight association between astrogliosis
and neuropathological lesions found in AD, it is
tempting to hypothesize that ADO dysfunction
may be instrumental for AD-related seizures. In
line with this idea, ADO deficiency as a result
of transgenic overexpression of ADK is suffi-
cient to promote severe learning deficits in the
Morris water maze and in Pavlovian condition-
ing (Yee etal., 2007). The role of ADO deficiency
and ADK on the generation of AD-related
seizures and memory deficits remains to be
established in reliable models and human
brain tissue. Importantly, this possibility sug-
gests that reduction of the adenosinergic tone
by increasing its metabolic clearance through
overexpressed ADK is deleterious. Although
this notion apparently contrasts with the pos-
sible protective effect expected from adenosine
receptor blockade, especially of the A 2A subtype,
it needs to be stressed that the net effects of dis-
rupted ADO homeostasis affect bioenergetic
network regulation and encompass adenosine
receptor dependent, as well as adenosine recep-
tor independent, aspects of homeostatic net-
work control by ADO. Future work needs to
address adenosine receptor independent and
epigenetic functions of ADO within the context
of AD, integrate those functions with the bet-
ter characterized adenosine receptor depend-
ent functions of adenosine, and ascribe specific
roles of adenosine-dependent homeostatic net-
work disruption to cognitive and seizure phe-
notypes of AD. Eventually, it might be possible
to treat or possibly cure AD synergistically by
reconstructing homeostatic bioenergetic net-
work functions that depend on correct ADO
homeostasis. Therefore, carefully tailored focal
ADO augmentation strategies, perhaps in com-
bination with A 2A receptor blockade, might hold
promise for the future therapyofAD.
Adenosine asa Potential Epigenetic
Modulator forMemory Improvement
As already described in detail, DNA methylation
status is dependent on a biochemical transmeth-
ylation reaction whereby SAM donates a methyl
group to unmethylated cytosines in DNA and is
converted to SAH. SAH is further hydrolyzed to
ADO and Hcy. ADO is cleared by ADK-mediated
phosphorylation to AMP and Hcy is converted
to methionine in a folate-dependent manner.
466 Part IV:Homeostatic Therapies
Importantly, DNA methylation is dependent on
the continuous removal and subsequent equi-
librium constants of SAH, ADO, and Hcy (Lu,
2000; Boison etal., 2002). Thus an accumulation
of ADO or Hcy prevents the biochemical conver-
sion of SAM to SAH and consequentially inhibits
DNA methylation.
The effects of ADO tone on the DNA meth-
ylation status are evident from humans with
a missense mutation in the Adk gene. Loss of
ADK function prevents ADO clearance, which
consequently blocks the methionine cycle lead-
ing to an increase in plasma SAH and SAM lev-
els, while Hcy levels remain normal or mildly
elevated (Bjursell etal., 2011). These findings are
recapitulated in two rodent models of hyper-
methioninemia involving either global or focal,
forebrain restricted, ADK knockout. Global
ADK knockout mice have a pathological shift
in the transmethylation pathway as evidenced
by decreased blood adenine levels and increased
Hcy levels (Boison et al., 2002). Similarly, focal
ADK knockout increases hippocampal ADO
3.3-fold (Shen et al., 2011) and decreases DNA
methylation by 30% (Williams-Karnesky et al.,
2013). Independent of permanent genetic muta-
tions that influence the methionine cycle, both
systemic and focal administration of pharmaco-
logical agents also influence the DNA methyla-
tion status. Systemic administration of the ADK
inhibitor 5-iodotubercidin (3.1 mg/kg ip once
every day for 5 days) decreases the global DNA
methylation status by 35% in the hippocam-
pus of wild-type mice. Likewise, focal deliv-
ery of ADO and Hcy to nave rats (ICV, single
bolus) decreases hippocampal DNA methyla-
tion (Williams-Karnesky etal., 2013). Thus DNA
methylation is responsive to de novo changes in
ADO tissuetone.
While low ADK and high ADO can decrease
DNA methylation, the transmethylation path-
way can also be shifted to increased meth-
ylation as a result of high ADK and low ADO
tone. DNA hypermethylation is observed with
both a pharmacological, focal delivery of SAM
to nave rats (ICV, single bolus) and a patho-
logical, injury-induced increase in ADK (kainic
acidinduced status epilepticus). Importantly,
using cultured ADK knockout cells transiently
transfected with either the short or long isoform of
ADK, the nuclear (long) isoform has the greatest
effect on increasing the DNA methylation status
(Williams-Karnesky et al., 2013). As an obliga-
tory end product of transmethylation, the ADO
tone nonspecifically drives the transmethylation
pathway by regulating substrate availability. Any
site-specific DNA methylation changes would be
mediated by DNMT1, 3a or 3b complexes (Goll
and Bestor, 2005; Caiafa etal., 2009; Feng etal.,
2010; Zampieri etal., 2012). Thus the ADO tone
does not regulate site-specific DNA methylation
but instead the homeostasis of the methylome.
Therefore, any modification of this tone is likely
to impact on gene expression. Ultimately, this
tone may have a significant impact on the expres-
sion of AD susceptibility genes or their regulators
or of those implicated in memory and plasticity
(see previous discussion).
Albeit a global biochemical shift in the trans-
methylation pathway has profound physiological
effects, humans without ADK have abnormal
liver function, develop encephalopathy, and have
severe progressive neurological deficits includ-
ing learning and memory impairments and sei-
zures (Bjursell etal., 2011). Similarly, mice with
a global ADK knockout develop the fatal liver
disease neonatal hepatic steatosis (Boison et al.,
2002). Transgenic mice with ADK overexpres-
sion throughout the brain have working and ref-
erence memory impairments in the Morris water
maze (Yee et al., 2007). Furthermore, patients
with mesial temporal lobe epilepsy including hip-
pocampal sclerosis frequently have dementia as a
comorbidity and are characterized by increased
astrogliosis and ADK expression.
The association between pathological changes
in ADK expression and memory deficits may be
through the inability to mount de novo changes
in the DNA methylation status. It is now read-
ily accepted that the epigenome of mature cells
within the central nervous system normally
undergoes modifications that involve both his-
tones and DNA methylation (Feng et al., 2010;
Ma et al., 2010). These changes occur regularly
and rapidly and can include both active meth-
ylation and demethylation. For example, an
episode of neural synchronization induces hip-
pocampal DNA methylationdependent changes
in the transcription of immediate early genes
and initiates a cascade of transcription factors
contributing to long-term neuronal and circuit
plasticity (Nelson et al., 2008). Furthermore,
activity-induced adult neurogenesis is depend-
ent on GADD45b-mediated active DNA
demethylation of the Bdnf and Fgf1 genes within
dentate granular neurons (Ma et al., 2009).
Aproper regulation of DNA methylation may be
of prime importance for fear-memory formation,

particularly hypermethylation of the PP1 gene
and demethylation of the reelin gene (Miller and
Sweatt, 2007). Altered PP1 levels (through ADO
tone-induced methylation deregulation) might
then subsequently impact histone modifications
(phosphorylation but also acetylation) related to
long-term memory (Koshibu etal., 2009). Thus by
manipulating the endogenous ADO tone to shift
the transmethylation pathway to either increase
or decrease the DNA methylation status, it may
be feasible to restore learning and memory defi-
cits by promoting neurogenesis, neuronal plastic-
ity, and memory signaling.
CONCLUSION
Network homeostasis is strongly compromised in
AD, and there is an interest to reinstate network
function as well as neuroglial communication
to improve cognition as well as mitigate lesions.
The adenosinergic system is a regulatory system
impaired in AD that deserves interest as a drug-
gable entity. On one hand, ADO itself is a critical
modulator of both network function and epige-
netic regulation. Reconstitution of ADO-based
homeostatic network functions would be of ther-
apeutic interest and thus deserves preclinical and
clinical evaluations. On the other hand, ADO
A2A receptor dysregulation may be instrumental
in AD. In line with this notion, several lines of
evidence indicate that caffeine administration
and A2A receptor blockade constitute promising
therapeutic approaches for AD. So far, no clini-
cal trials have been performed to date to evalu-
ate the extent by which caffeine can slow down
disease progression in AD patients. Further,
although A2A antagonists appear safe and toler-
able in Parkinsons disease patients (for reviews
see Morelli et al., 2009; Pinna, 2009; Simola
et al., 2008), no study has evaluated effects in
AD patients, and the major drawback for the use
of A2A receptor antagonists in AD is the lack of
experimental studies and pilot clinical studies
assessing their effects on cognitive impairment
during aging or in AD patients. Overall, modu-
lation of the adenosinergic system is certainly
important to consider for the treatment of AD,
but precise evaluations of its importance for
pathological and memory processes await fur-
ther experimental work in the future.
ACK NOWLEDGMENTS
David Blum and Luc Bue are supported by
France Alzheimer, LECMA/Alzheimer Forschung
Initiative, LabEx (Excellence Laboratory)
Adenosine and Alzheimers Disease 467
DISTALZ (Development of Innovative Strategies
for a Transdisciplinary approach to Alzheimers
disease), Inserm, CNRS, Universit Lille 2, Rgion
Nord/Pas-de-Calais, ANR (ADORATAU),
ERA-Net (ABeta-ID) and FUI MEDIALZ. Olivier
Bousiges and Anne-Laurence Boutillier are sup-
ported by CNRS, Universit de Strasbourg,
Hopital Universitaire Strasbourg, ANR (ANR-12-
MALZ-0002-01 ATACTAD), the Indo-French
Centre for the Promotion of Advanced Research
(IFCPAR No.4803-3) and Alsace Alzheimer 67
association. Vanessa Flaten holds a grant from
Inserm/Region Nord pas de Calais. Detlev Boison
is supported through grants from the National
Institutes of Health (NS065957, MH083973,
NS061844), the US Department of the Army
(W81XWH-12-1-0283), and the Legacy Good
Samaritan Foundation. L.V. Lopes is an iFCT
investigator. David Blum is an Inserm investigator.
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26
Brain Homeostasis and Parkinsons Disease
DEEP TILALL
PA R K I N S O N S D I S E A S E :
AN OVERVIEW
Parkinsons disease (PD) is a chronic, progres-
sive, neurodegenerative disorder of the basal
ganglia. In its worldwide prevalence it is second
only to Alzheimers disease, affecting 5 million
people globally, and its occurrence is expected to
markedly increase in the coming decades due to
an increase in lifespan of the population (Olanow
and Schapira, 2013). As with Alzheimers, the
principal risk factor for acquiring PD increases
with age with incidence rates rising exponen-
tially after 50 years of age (Bower et al., 2000;
Driver etal., 2009). The disorder does not show
any geographical and/or gender-specific perva-
siveness; it is a common disabling disorder and is
often seen in people from all races and geograph-
ical locations with clinical signs emerging also in
the young (Lees etal., 2009; Massano and Bhatia,
2012; Tolosa etal.,2006).
PAT H O P H Y S I O L O G Y
AND NEUROCHEMICAL
F E AT U R E S O F P D
PD results primarily from the loss of the nigros-
triatal dopaminergic (DAergic) neurons in the
substantia nigra pars compacta (SNpc), although
additional neuronal systems such as catechola-
minergic, serotonergic, and brainstem nuclei are
also affected, albeit less severely. Atime-course
analysis of the pathological process in PD
showed that the neurodegeneration reaches
the dorsal motor nuclei of the hypoglossus and
vagus nerves and raphe nuclei, as well as the
anterior olfactory nucleus and olfactory bulb,
before it extends to the mesencephalic areas,
which includes the substantia nigra (SN) where
a majority of the cell damage takes place (Braak
etal., 2003). The cell bodies of the nigrostriatal
neurons reside in the SNpc and project primar-
ily to the putamen. These neurons contain the
pigment neuromelanin (Marsden, 1983), which
gives these neurons their distinct color. Loss of
these neurons produces the classic gross neu-
ropathological finding of SNpc depigmenta-
tion, highly characteristic of PD. The pattern
of the SNpc neuronal cell loss also parallels the
expression of dopamine transporter (Uhl etal.,
1994)and is in line with the findings that deple-
tion of dopamine (DA) is most pronounced in
the dorsolateral putamen (Bernheimer et al.,
1973), which is the main site for the projections
of these neurons. At the onset of PD symptoms,
approximately 80% of the putamental DA is
depleted and approximately 60% of the SNpc
DAergic neurons arelost.
The PD-associated loss of DAergic neu-
rons displays a characteristic pattern that is
highly distinct and different from neuronal
loss in normal aging. Fearnley and Lees (1991)
reported that while in normal aging the dor-
somedial part of the SNpc is affected, in PD
the cell loss is more concentrated in the ven-
trolateral and caudal regions. Also, the stria-
tum terminal loss is more pronounced than
the magnitude of SNpc DAergic neuron loss
(Bernheimer etal.,1973).
Apart from neuronal cell loss, PD patients
also show formation of proteinaceous intraneu-
ronal or intraglial inclusions, known as Lewy
bodies (LBs) composed of a dense core of filamen-
tous material enshrouded by filaments of 10 to
20 nm in diameter (Goedert, 2001a, 2001b). LBs
are found in all affected regions, have an organ-
ized structure of a dense hyaline core surrounded
by a clear halo, and contain cytoplasmic aggre-
gates composed of numerous proteins including
-synuclein, parkin, ubiquitin, and neurofila-
ments (Forno, 1996; Spillantini etal., 1998). The
influences that lead to this neuronal cell loss and
the formation of LBs is still unknown. However,
a common perception points toward complex
interactions between genetic (nonsporadic) and
environmental (sporadic)cues.
482 Part IV:Homeostatic Therapies
CLINICAL SYMPTOMS OF PD
PD is characterized by four cardinal motor fea-
tures:bradykinesia (slowness of movements with
a progressive loss of amplitude or speed during
alternating rapid body movements; Jankovic,
2008; Marsden, 1982; Rodriguez-Oroz et al.,
2009), rest tremor, rigidity, and postural and
gait impairment. Numerous non-motor symp-
toms are also associated with PD. These include
neuropsychiatric features such as anxiety, panic
attacks, depression, apathy, dysautonomia (i.e.,
constipation, urinary dysfunction, excessive
sweating), sleep disorders (e.g., insomnia, rest-
less leg syndrome, excessive daytime sleepiness),
sensory dysfunction (e.g., loss of smell, decreased
color and contrast discrimination), pain, and
fatigue. Since PD is a progressive neurological
disease, these non-motor features may be present
before presentation of any classical motor symp-
tom phenotype and therefore present a poten-
tial utility for diagnosis and determination of
disease-progressionstage.
ETIOLOGYOFPD
PD is a multifactorial disorder caused by a
combination of genes and environmental fac-
tors. Until 1997, the notion that heritability has
a role in PD was contentious; indeed, PD was
considered to be the archetypal nongenetic
disorder (Farrer, 2006). However, in the past
decade, multiple mutations in genes have been
described in families with a Mendelian pattern
of PD inheritance (Bonifati et al., 2003; Kitada
etal., 1998; Leroy etal., 1998a; Leroy etal., 1998b;
Paisan-Ruiz et al., 2004; Polymeropoulos et al.,
1997; Singleton etal., 2003; Valente etal., 2004a;
Zimprich etal., 2004). Genome-wide association
as well as linkage mapping studies have identified
many causal mutations and candidate genes as
well as multiple susceptibility variants that might
trigger PD (Maraganore etal.,2005).
GENETIC FORMSOFPD
PD was long considered a sporadic disorder, and
the hypothesis that heredity underlies the etiol-
ogy of PD traditionally found little support from
epidemiological data. The majority of PD cases
are still sporadic: only about 10% (Thomas and
Beal, 2007) of patients report a positive family
history. Aseminal study by Leroux in 1880 first
recognized familial aggregation of parkinson-
ism and suggested that heritable factors might
increase disease susceptibility. The past decade
of research in PD has led to the discovery of
several monogenic (a form of disease for which
a mutation in a single gene is sufficient to cause
the disease) forms of the disorder and several risk
factors that increase susceptibility to PD. They
collectively account for about 30% of the famil-
ial and 3% to 5% of the sporadic cases (Klein and
Westenberger, 2012). Today we know of about 18
distinct chromosomal regions that are more or
less convincingly related to PD. Only six of these
specific regions contain genes with mutations
that conclusively cause monogenicPD.
Chromosomal locations associated with PD
are designated as PARK, and they are numbered
in chronological order of their identification
(PARK1, PARK2, PARK3, and so on; Klein and
Westenberger, 2012). All of the currently known
monogenic PD forms are autosomal, either dom-
inant or recessive. In autosomal-dominant disor-
ders, one mutated allele of the gene is enough to
cause the disease, whereas in autosomal-recessive
disorders two mutations (the samehomozygous
or differentcompound heterozygous), one on
each gene copy (allele), are necessary to cause the
phenotype. Mutations in SNCA (PARK1/4) and
LRRK2 (leucine-rich repeat kinase 2 or darda-
rin,/PARK8), VPS35 (vacuolar protein sorting
35), EIF4G1 (eukaryotic translation initiation
factor 4 gamma 1), and GBA (glucocerebrosi-
dase) are responsible for autosomal-dominant
PD forms, and mutations in parkin (PARK2),
PTEN-induced putative kinase 1 (PINK1/PARK6),
DJ-1 (PARK7), ATP13A2 (PARK9), FBXO7
(F-box only protein 7 gene), and PLA2G6 (phos-
pholipase A2, group VI) are accountable for PD
that displays an autosomal recessive mode of
inheritance.
AUTOSOM AL DOMINANT
FORMSOFPD
SNCA (PARK1/PARK4)
SNCA was the first pathogenic PD gene dis-
covered that had a missense mutation. SNCA
codes for a protein called -synuclein, which is
expressed throughout the brain and is involved
in learning, synaptic plasticity, vesicle dynam-
ics, and DA synthesis (Lotharius and Brundin,
2002; Sidhu et al., 2004). The SNCA gene has
six exons encoding for -synuclein. Natively
unfolded -synuclein has no secondary structure
and is nontoxic. Once bound to the phospholipid
membrane, -synuclein forms the helical struc-
ture (Giasson et al., 2001). The point mutants
of SNCA tend to form stable sheets and thus
 Brain Homeostasis and Parkinsons Disease
exacerbate the formation of toxic oligomers,
protofibrils, and fibrils. Therefore, it is believed
that the missense SNCA mutations cause PD
through a toxic gain of function (Bertoncini
etal., 2005), and LBs may represent the attempt
to purge the cell of toxic damaged -synuclein
(Chen and Feany, 2005). Transgenic overexpres-
sion of -synuclein causes neurotoxicity with
aggregation and accumulation of -synuclein.
In vivo, mice demonstrate various neuropatho-
logical changes, including neuronal atrophy,
dystrophic neurites, and astrogliosis, accompa-
nied by -synuclein-positive LB-like inclusions
(Fernagut and Chesselet, 2004). Similarly, viral
delivery of -synuclein within the SN of adult
rats results in marked loss of DA neurons, dys-
trophic changes, and aggregated -synuclein
pathology (Yamada et al., 2004). In human PD
cases, -synuclein can also be phosphorylated
and found within LB inclusions (Fujiwara etal.,
2002; Nonaka etal., 2005). However, it is a mat-
ter of debate whether the phosphorylated mon-
omer disrupts the amphipathic association of
-synuclein with lipids or is especially toxic if not
sequestered and degraded.
PARK1/PARK4 contributes to SNCA muta-
tions (missense) and duplications/triplications,
respectively. The SNCA 209G>A (Ala53Thr)
mutation was identified through genome-wide
linkage studies in several families with autoso-
mal dominant parkinsonism (Athanassiadou
et al., 1999; Bostantjopoulou et al., 2001; Choi
etal., 2008; Ki etal., 2007; Polymeropoulos etal.,
1997; Puschmann etal., 2009; Spira etal., 2001),
and two further missense mutations 88G>C
(Ala30Pro; Kruger et al., 1998) and 188G>A
(Glu46Lys; Zarranz et al., 2004) were subse-
quently identified. Seventeen duplications of the
entire coding region of SNCA have been reported
to date, 13 in PD families and 4 in sporadic cases,
one in which it was shown that the mutation
arose de novo (Ahn et al., 2008; Brueggemann
et al., 2008; Chartier-Harlin et al., 2004; Fuchs
etal., 2009; Fuchs etal., 2007; Ibanez etal., 2004;
Ibanez etal., 2009; Ikeuchi etal., 2008; Nishioka
etal., 2006; Troiano etal., 2008; Uchiyama etal.,
2008). Triplications of the SNCA gene were found
in three independent families (Farrer etal., 2004;
Ibanez etal., 2009; Singleton etal.,2003).
Patients with SNCA mutations usually have
early-onset PD (age of onset 50years) with an
initially good response to levodopa treatment
to increase DA levels. However, the disease has
a rapid progression and often presents with
483
dementia and cognitive decline and sometimes
with atypical features such as central hypoven-
tilation and myoclonus. LBs are present and
spread through the SN, locus ceruleus, hypo-
thalamus, and cerebral cortex (Polymeropoulos
etal.,1996).
LRRK2 (PARK8)
Mutations in LRRK2 are the most common and
known cause of autosomal dominant PD with a
frequency ranging from 2% to 40% in different
populations with the highest occurrence among
North Africans and Jewish individuals (Brice,
2005; Lesage etal., 2006; Ozelius etal., 2006). The
LRRK2 gene encodes for a protein called darda-
rin with two enzymatic domains (GTPase and
kinase) and multiple proteinprotein interaction
domains. One segment of the dardarin protein is
called a leucine-rich region because it contains a
large amount of a amino acid known as leucine.
Proteins with leucine-rich regions appear to
play a role in activities that require interactions
with other proteins, such as transmitting signals
or helping to assemble the cytoskeleton. Other
parts of the dardarin protein are also thought to
be involved in proteinprotein interactions. The
LRRK2 gene is active in the brain and other tis-
sues throughout thebody.
Seven mutations (Asn1437His, Arg1441Cys,
Arg1441Gly, Arg1441His, Tyr1699Cys, Gly
2019Ser, and Ile2020Thr) are known to cause
PD. Patients respond favorably to levo-
dopa therapy, and dementia is not common.
Neuropathological findings are mostly inconsist-
ent, showing both LB (and sometimes tau- and
ubiquitin-containing inclusions) pathology and
pure nigral degeneration without LBs, with or
without neurofibrillary tangles. The pathogenic
mechanism leading to PD caused by LRRK2
mutations is still uncertain. Since LRRK2 is a
big protein interacting with so many proteins,
any changes in these interactions due to muta-
tions can have profound effects on its functions
and thereby can change the susceptibility to the
disease.
VPS35
VPS35 is a protein involved in retrograde
transport of proteins from endosomes to the
trans-Golgi network. Recently the p.D620N
mutation in VPS35 was discovered as a new cause
of PD in two independent exome sequencing
studies on Swiss (Vilarino-Guell etal., 2011)and
Austrian families (Zimprich et al., 2011).
484 Part IV:Homeostatic Therapies
Frequency of this mutation carrier is low and has
been estimated to represent about only 0.1% of
the PD population (Kumar etal., 2012). Patients
afflicted with this mutation develop classical
late-onset, levodopa-responsive Parkinsonism
reminiscent of sporadic PD, albeit with a slightly
earlier age ofonset.
EIF4G1
EIF4G1 is a eukaryotic translation initiator fac-
tor. The p.R1205H mutation in the EIF4G1 was
discovered in a French family inheriting famil-
ial parkinsonism (Chartier-Harlin etal., 2011). It
is dominantly inherited and has a very low fre-
quency of 0.02% to 0.2% in the PD population
(Tucci etal.,2012).
GBA
GBA is an enzyme with glucosylceramidase
activity that is needed to cleave, by hydroly-
sis, the beta-glucosidic linkage of the chemical
glucocerebroside, an intermediate in glycolipid
metabolism. It is localized in the lysosome.
Heterozygous mutations in this gene are consid-
ered to be a major risk factor for PD (Sidransky
etal., 2009). The mutations in the GBA gene deter-
mined to date are Asn370Ser and Leu444Pro.
The Asn370Ser mutation is commonly found
in Ashkenazi Jews with a carrier frequency of
5% (Lwin et al., 2004). However, the mutations
have a much lower penetrance than the classical
Mendelian mutations. Patients with GBA patho-
genic mutations have typical PD with possibly a
slightly earlier onsetage.
AUTOSOM AL R ECESSIV E
FORMSOFPD
Homozygous or compound heterozygous muta-
tions in each of five genesparkin (PRKN/
PARK2), PTEN induced putative kinase 1
(PINK1/PARK6), Parkinson protein 7 (DJ-1/
PARK7), F box only protein 7 gene (FBX07/
PARK15), and Phospholipase A2 (PLA2G6)can
cause autosomal recessive forms of early-onset
parkinsonism, usually without atypical clinical
signs (Bonifati,2014).
Parkin (PRKN/PARK2)
PARK2 gene encodes for the protein parkin,
which is involved in ubiquitin-mediated deg-
radation of proteins. Mutation in parkin causes
the most common autosomal recessive form of
PD, which accounts for most PD cases in people
under 30years of age (juvenile PD, age of onset
21years). Pathology underlying PARK2-related
PD does not generally show LB pathology.
However, thepatients do have substantia nigral
cell loss and gliosis. Marked cognitive or vegeta-
tive disturbances are rare. PD patients suffering
from this mutation show excellent and sustained
response to levodopa (Lohmann etal.,2003).
PINK1 (PARK6)
PINK1 is a mitochondrial serine/threonine-
protein kinase encoded by the PINK1 gene and
is believed to protect cells from stress-induced
mitochondrial dysfunction. Mutation in the
PINK1 protein is responsible for causing the
second most common form of autosomal reces-
sive PD. Frequency of PINK1 mutations is in the
range of 1% to 9% with considerable variation
across different ethnic groups (Bonifati et al.,
2005; Healy et al., 2004; Klein et al., 2005; Li
et al., 2005; Rogaeva et al., 2004; Valente et al.,
2004b). It is characterized by early onset in most
patients (mean age of onset = 30 years) with
symptoms ranging from dystonia to hyperre-
flexia. Psychiatric disturbances such as depres-
sion, anxiety, and psychosis are also found
frequently. However, patients respond excellently
to Levopoda treatment.
DJ-1 (PARK7)
The third gene associated with autosomal reces-
sive PD is mutated in about 1% to 2% of patients
(Pankratz etal., 2006). This seems to be a rare dis-
order with a clinical phenotype similar to PARK6.
PARK7 gene encodes for the protein DJ-1, which
functions as a redox-sensitive chaperone and as a
sensor for oxidative stress, and protects neurons
against oxidative stress and cell death. About 10
different point mutations and exonic deletions
have been described for this gene, mostly in the
homozygous or compound-heterozygous state.
Given that the mutations are so uncommon,
there is not enough data to draw any conclusion
about the possible role of heterozygous mutations
in thisgene.
ATP13A2 (PARK9)
ATP13A2 encodes a large lysosomal P-type
ATPase with 1180 amino acids and 10 trans-
membrane domains. Since the lysosomal degra-
dation pathway can clear -synuclein aggregates
by macroautophagy, lysosomal dysfunction,
caused by mutations in ATP13A2, might con-
tribute to the pathogenesis of parkinsonism (Pan
etal., 2008). Malfunctioning of this gene causes a
 Brain Homeostasis and Parkinsons Disease
juvenile atypical parkinsonism characterized by
pyramidal degeneration, dementia, and supra-
nuclear gaze palsy, which is levodopa-responsive.
FBXO7 (PARK15)
The FBXO7 gene encodes the F-box only protein
7 protein, which plays a role in phosphorylation-
dependent ubiquitination. Mutations in this
gene cause a recessive form of juvenile parkin-
sonism with pyramidal disturbances. The brain
pathology in patients with PARK15 remains
unknown.
PLA2G6
The PLA2G6 gene encodes for the
calcium-independent phospholipase A2 enzyme
that plays a role in phospholipid remodeling and
apoptosis. Recessive mutations in this gene results
in levodopa-responsive dystonia-parkinsonism,
pyramidal signs, and cognitive/psychiatric fea-
tures with onset in early adulthood (Khateeb
et al., 2006; Morgan et al., 2006; Paisan-Ruiz
etal., 2009). Magnetic resonance imaging in PD
patients showed brain atrophy with or without
iron accumulation. Brain pathology in patients
with PLA2G6 mutations also shows widespread
accumulation of LBs in different rain regions,
suggesting links with typical PD (Paisan-Ruiz
etal.,2012).
Despite all the evidence available confirming
the hereditary basis of PD, the field remains highly
controversial. While studies cited here and data
from monozygotic and dizygotic disease in longi-
tudinal twin studies have indicated that PD has a
genetic contribution, larger cross-sectional twin
studies have argued against heritability (Piccini
etal., 1999; Tanner etal., 1999; Wirdefeldt etal.,
2004). As PD is not a homogeneous, penetrant
trait (Simon et al., 2002), even within families
that are now known to be affected by monogenic
PD, the age of onset, symptoms, and end-stage
pathology can be quite variable.
INFLUENCE
O F E N V I R O N M E N TA L FA C T O R S
Studies analyzing the effects of environmental
toxins on PD are in a nascent stage, although
examples are available in history that points
toward a possible link. Epidemiological research
indicates that rural living, pesticide use,
well-water consumption, and certain occupa-
tions, including mining and welding, are associ-
ated with an increased risk of PD (Firestone etal.,
2005; Jankovic, 2005; Priyadarshi et al., 2001).
485
More males are affected than females (in a ratio
of 1.5:1.0), although whether this reflects work-
place exposure, sex-linked genetic variability,
or the protective effect of estrogen is unknown
(Farrer, 2006). In principle, the progressive
neurodegeneration of PD can be produced by
chronic neurotoxic exposure or limited expo-
sure, which then perpetuates a cascade of del-
eterious events. Examples include exposure to
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP; Langston et al., 1983), which produces
symptoms identical to PD. Rotenonea mito-
chondrial poison in the environmenthas also
been indicted in human epidemiological stud-
ies with an elevated risk of PD (Tanner, 1992).
By contrast, protective effects have been noted
for cigarette smoking and alcohol and caffeine
intake (Hernan et al., 2002), but it is not clear
how these agents influence disease risk. These
examples reinforce the concept that environmen-
tal factors do modify PD susceptibility.
EFFECTS
OFENDOGENOUSTOXINS
Apart from genetic and environmental factors
that are implicated in the etiology of PD, there
is also evidence that endogenous toxins such
as distortion of metabolic pathways may be
responsible for PD neurodegeneration. Changes
in normal metabolism might create toxic sub-
stances due to environmental factors or inher-
ited differences in metabolic pathways (Dauer
and Przedborski, 2003). Cohen (1984) showed
that endogenous toxins, which may be the nor-
mal metabolism products of DA pathway, can
generate harmful reactive oxygen species that
can prove detrimental to the cells (Cohen, 1984).
Also consistent with this hypothesis is a report
that patients harboring specific polymorphisms
in the gene encoding xenobiotic detoxifying
enzyme cytochrome P450 may be at a greater risk
of developing early-onset PD (Sandy etal., 1996).
Furthermore, isoquinoline-derivative neurotox-
ins that act similarly like MPTP and are toxic to
DAergic neurons have been recovered from PD
brains (Nagatsu,1997).
Apart from these examples, other causes of
PD include heterogenerative disorders, in which
parkinsonism is a manifestation of the disease
rather than a direct effect itself, such as demen-
tia with LBs, fragile-X-associated tremor/ataxia
syndrome, and so on; structural brain lesions
(cerebrovascular disease, infectious brain lesions,
postencephalitic, etc.) drug use (antipsychotics
486 Part IV:Homeostatic Therapies
such as haloperidol, olanzapince, etc.), and vari-
ous psychogenicforms.
PAT H O G E N E S I S O F P D
The pathogenesis of PD remains a mystery in
itself. Different hypotheses have been put for-
ward to explain the pathogenesis of the disease,
the most common being misfolding and aggrega-
tion of proteins, mitochondrial dysfunction, and
consequent oxidative stress, impairment of the
ubiquitin-proteasome system, and neuroinflam-
mation. However, these factors are not mutually
exclusive, and one of the major aims of current
PD research is to find a common connection
between them. The finding that oxidative dam-
age to -synuclein enhance its ability to mis-
fold and aggregate (Giasson et al., 2000) is one
example of such an interaction. Another highly
uncertain issue in the field is whether the multi-
ple cell-death related pathways activated during
PD neurodegeneration lead to a common cell fate
such as apoptosis or remain highly divergent.
Protein Aggregation and Misfolding
Several age-related neurodegenerative disor-
ders share a common pathology of abnormal
deposition of misfolded and aggregated proteins,
although the composition and localization of
these aggregates varies in different disorders.
Aggregated or soluble misfolded proteins can
become neurotoxic through a variety of mecha-
nisms. They can directly cause cell damage by
deforming the cell or interfering with the intra-
cellular trafficking in neurons. Protein inclusions
might also sequester proteins that are essential
for cell survival. Strict quality-control mecha-
nisms determine the rate of protein synthesis as
well as its degradation (Balch etal., 2008; Powers
etal., 2009)and normally prevent such aggregates
from forming. However, prolonged exposure to
various stressors place an incredible burden on
these mechanisms. When these mechanisms fail,
aggregation-prone proteins abnormally accumu-
late, as observed in neurodegenerative diseases
such as PD (Ross and Poirier, 2004). The common
observation of protein aggregation in different
diseases suggests that the protein deposition per
se is toxic to the neurons and that there should be
a correlation between inclusion body formation
and neurodegeneration. This relation, however,
is not convincingly shown in postmortem tis-
sue samples from sporadic PD patients. Instead,
the formation of aggregates may reflect a state of
cellular distress and represent a way to sequester
harmful, soluble misfolded proteins from the cel-
lular milieu (Kopito, 2000). Several lines of study
now indicate that protein aggregation, sequestra-
tion, and inclusion body formation is perhaps
a defensive measure aimed at removing toxic
soluble misfolded proteins (Auluck et al., 2002;
Cummings et al., 1999; Cummings et al., 2001;
Warrick et al., 1999). The question therefore
remains as to what causes these aggregated pro-
teins and inclusion bodies to become toxic. One
answer could be protein-damaging modifications
as well as dysfunction of chaperones and other
proteosomal machinery that may indirectly con-
tribute to the toxicity. The cause of PD in patients
with either -synuclein mutations or DAergic
neurodegeneration is thought to be related to
(i) mutant -synuclein, (ii) excess levels of nor-
mal -synuclein adopting toxic conformations
(Bussell and Eliezer, 2001), or (iii) interference
with the cellular handling of misfolded proteins.
In sporadic PD, there is a similar focus on both
protein-damaging modifications and dysfunc-
tion of protein-degradation systems. However,
irrefutable evidence that misfolded proteins and
related aggregates are toxic to DAergic neurons
in PD is still lacking.
Ubiquitination and RelatedStress
The ubiquitin-proteasome system (UPS) is one of
the major protein-degradation systems believed
to go awry in PD. UPS also degrades proteins that
are misfolded and/or damaged, possibly caused
by mutation, environmental stress, or intrin-
sic folding inefficiency apart from degradation
of key regulatory proteins that control signal
transduction, cell cycle progression, apoptosis, as
well as cellular differentiation (Ciechanover and
Brundin, 2003; Ciechanover etal., 2000; Hershko
and Ciechanover, 1998). Ubiquitinated proteins,
heat shock proteins/chaperones, and components
of the UPS have all been shown to accumulate
within LBs, and both protein misfolding and UPS
dysfunction are implicated in neurodegenerative
processes in sporadic PD (Ii etal., 1997; Lennox
et al., 1989; Lowe et al., 1990; Schlossmacher
etal., 2002). The presence of nondegraded, stable
ubiquitin conjugates in the brains of PD patients,
as well as in genetic and toxic models of PD, sug-
gests that misfolded proteins in sporadic PD are
properly recognized and ubiquitinated but not
efficiently degraded. Such inefficient degradation
may be the result of intrinsic resistance to pro-
teasomal degradation of -synuclein-containing
aggregates (Lee etal., 2004)or a reflection of the
 Brain Homeostasis and Parkinsons Disease
difficulty in unfolding stable protein aggregates
by the 19S subunits of the proteasome complex
(Voges etal.,1999).
The tissue content of abnormally oxidized/
aggregated proteins (which may misfold)
increases with age (Beckman and Ames, 1998),
and neurons are particularly susceptible because
they are postmitotic. Cells respond to the mis-
folded proteins by increasing the production
of chaperones. However, with aging the abil-
ity of cells to produce a variety of chaperones is
impaired, as is the activity of the proteosomal
system. This provokes a vicious cycle, with the
excess misfolded proteins further inhibiting an
already compromised proteasome (Dauer and
Przedborski, 2003). All of these factors converge
and generate a proteotoxic insult to thecells.
Mitochondrial Dysfunction
Another factor involved in the pathogenesis of
PD is mitochondrial dysfunction (Mandemakers
et al., 2007; Parker and Swerdlow, 1998).
Mitochondrial dysfunction due to oxidative
stress, mitochondrial DNA deletions, or altered
mitochondrial morphology and the interaction
of pathogenic proteins with mitochondria all
result in DAergic neurodegeneration. However,
the most definitive evidence of mitochondrial
dysfunction in PD has come from studies using
MPTP, a toxicant that causes parkinsonian
symptoms in humans, rodents, and primates by
inhibiting the mitochondrial complex-I of the
electron transport chain (Sandy et al., 1993).
Similar to MPTP, other complex-I inhibitors
such as rotenone, maneb, paraquat, fenzaquin,
and trichloroethylene result in the loss of nigral
DAergic neurons in the mouse model of PD,
implicating mitochondrial dysfunction in its
pathogenesis (Betarbet et al., 2000; Gash et al.,
2008). Also, it has been shown that PD patients
have significantly decreased complex-I activity
within the SN (Heo et al., 2012; Schapira et al.,
1990). Recently, more convincing evidence of
mitochondrial dysfunction in PD has been
reported in conditional knockout MitoPark
mice, which have a disrupted mitochondrial
transcription factor A (Tfam) gene in DAergic
neurons (Ekstrand et al., 2007). These mice
exhibited reduced mitochondrial DNA expres-
sion and attenuated expression of respiratory
chain function in DAergic neurons in SN, along
with behavioral impairments and striatal DA
depletion, mimicking progressive PD pheno-
types, starting from 18 weeks ofage.
487
OxidativeStress
Oxidative stress has been long implicated in the
pathogenesis of PD. Mitochondrial dysfunction
and oxidative stresses are not mutually exclusive
events, and one plays a role in the formation of
the other (Cardoso et al., 1999; Cecarini et al.,
2007; Turrens, 2003). Reactive oxygen species
(ROS), which form a major component of oxida-
tive stress, are generated within mitochondria
in several sites of mitochondrial electron trans-
port chains. Excessive production of ROS and/or
defective ROS removal can potentially damage
cellular lipids, proteins, and DNA. Mitochondrial
dysfunction is closely related to the increased
ROS formation in PD. Postmortem studies have
consistently shown high levels of oxidation of
lipids, proteins, and nucleic acids in the SNpc
of sporadic PD brains (Alam etal., 1997; Dexter
etal., 1989; Floor and Wetzel, 1998; Jenner, 2003;
Tsang and Chung, 2009; Yoritaka et al., 1996).
The deterioration of DAergic neurons is triggered
by the pathogenic mitochondrial mechanism
enabling ROS production. Coincidentally, the
postmortem brain tissue of the SN in PD patients
indicates stimulated levels of lipid peroxida-
tion, high oxidation of proteins and DNA, and
reduction of glutathione, which strengthens the
notion that this type of stress is involved (Sian
et al., 1994). Also, significant alterations of the
antioxidant defense system, in particular reduc-
tion in glutathione, were found in the SNpc of PD
patients (Sian etal.,1994).
B R A I N H O M E O S TAT I C
S Y S T E M S I N V O LV E D I N P D
Symptoms in PD can be broadly classified into
two groups:(i)motor symptoms and (ii) nonmo-
tor symptoms. Motor symptoms are those that
relate to how one moves and include tremor,
bradykinesia, rigidity, and postural instabil-
ity. Non-motor symptoms are those that do
not involve movement, coordination, physical
tasks, or mobility. Many studies have shown
that nonmotor symptoms may precede motor
symptomsand a Parkinsons diagnosisby
years. The most recognizable early nonmotor
symptoms include loss of sense of smell, con-
stipation, sleep disturbances, mood disorders,
depression, fear, and anxiety. Conceivably, the
smooth operation of routine motor and nonmo-
tor functions requires a structurally complex
neuronal circuitry with a high degree of inter-
connectivity that utilizes a number of different
neurotransmitters, including DA, glutamate,
488 Part IV:Homeostatic Therapies
acetylcholine, norepinephrine, serotonin, and
-aminobutyric acid (GABA). Traditionally
research in PD has focused mainly on neurode-
generation of DAergic nigrostriatal cells as the
key cause of motor dysfunctions. On the other
hand, nonmotor symptoms associated with PD
are not fully associated with respect to specific
neurotransmitter dysfunctions.
The basal ganglia is not only a group of func-
tionally related and strongly interconnected nuclei
that form the key components of the complicated
circuitry that mediates motor and cognitive func-
tion and behavior; it is also connected with differ-
ent parts of the cerebral cortex and the thalamus,
as well as with various structures leading to the
red nucleus, brainstem reticular formation, and
spinal cord (Pollack, 2001), all of which play a
role in both normal motor and nonmotor func-
tions of the body. The role of the basal ganglia in
motor function has been extensively investigated,
and the pathological changes to these structures
in PD are well known (Galvan and Wichmann,
2008; Halliday et al., 2005). However, its role in
nonmotor functions is not as widely understood.
Due to the interconnectivity of different neu-
rotransmitter systems, disruption in any one of
them will have consequences in the symptoma-
tology of PD. For example, the motor function
of the striatum is dependent on the equilibrium
reached between DA and acetylcholine (Zhou
et al., 2003), while DAergic activity within the
SNpc is itself modulated by glutamatergic and
GABAergic innervations (Fink and Gothert,
2007). Disturbances in central noradrenergic sys-
tems may be causative in the onset of depression
and anxiety by interactions with serotonin and
DA (Richard, 2005). The observation that selective
serotonin reuptake inhibitors (SSRIs), adminis-
tered to treat the depressive symptoms associated
with PD, can affect the motor symptoms suggests
that there may be an interaction between sero-
tonin and dopamine in PD (Vajda and Solinas,
2005). Thus, although loss of DA neurons in the
basal ganglia is most often associated with the
disease and its characteristic motor symptoms, it
is clear that the changes in DA interactions with
other neurotransmitters and subsequent effects
both within and outside the basal ganglia need to
be considered when looking at the wider (nonmo-
tor) symptoms reported in patients withPD.
Dopaminergic Neurotransmission
The pathological hallmark of PD is the preferen-
tial loss of DAergic neurons in SN resulting in
a loss of DA in the striatum. This DA loss also
extends to other parts of the basal ganglia, mani-
festing the motor symptoms observed in PD. In
support of this hypothesis, postmortem DA lev-
els are extensively reduced in the globus pallidus,
with a possible correlation between the pattern of
DA depletion and the presence of tremor in PD
(Rajput etal., 2008). Different kinds of dopamine
receptors are widely present in the brain, which
are classified as D1 to D5 receptors. D1 mRNA
expression levels are highest in the dorsal stria-
tum and nucleus accumbens but are also found
in the cortex and at lower levels in the hippocam-
pus, thalamus, and hypothalamus. Similar to
D1, the D2 receptor is highly expressed in spiny
projection neurons (SPNs) in the dorsal and ven-
tral striatum. D2 mRNA is also expressed in the
cortex, hippocampus, and hypothalamus. D3 is
present in particular in the nucleus accumbens,
with only very low levels in the dorsal striatum,
hippocampus, and cortex. Low levels of D3 auto-
receptors have been detected in SN and ventral
tegmental area neurons. D4 shows the lowest
expression levels among all DA receptors in the
basal ganglia. The D5 receptor has been detected
in the cortex, dorsal and ventral striatum, hypo-
thalamus, and hippocampus. In the dorsal stria-
tum, D5 receptors are localized in SPNs and in
cholinergic interneurons.
There are two distinct pathways that process
signals through the basal ganglia:the direct path-
way and the indirect pathway. These two pathways
have opposite net effects on thalamic target struc-
tures. Excitation of the direct pathway has the net
effect of exciting thalamic neurons (which in turn
make excitatory connections onto cortical neu-
rons). Excitation of the indirect pathway has the
net effect of inhibiting thalamic neurons (render-
ing them unable to excite motor cortex neurons).
The normal functioning of the basal ganglia appar-
ently involves a proper balance between the activi-
ties of these two pathways. One hypothesis is that
the direct pathway selectively facilitates certain
motor (or cognitive) programs in the cerebral cor-
tex that are adaptive for the present task, whereas
the indirect pathway simultaneously inhibits the
execution of competing motor programs. An
upset of the balance between the direct and indi-
rect pathways results in motor dysfunctions.
Therapeutic strategies to relieve PD
symptoms are mostly focused on increas-
ing the DA levels in the brain. DA receptor
stimulation in the dorsal striatum is the pri-
mary mechanism by which these drugs help
 Brain Homeostasis and Parkinsons Disease
alleviate the motor features associated with PD.
L-3,4-dihydroxyphenylalanine (L-DOPA) ther-
apy is most commonly used to treat motor symp-
toms of PD. L-DOPA is a precursor to DA, and
once it crosses the blood-brain barrier (which DA
cannot), it nonspecifically increases the concen-
tration of DA at dopamine receptors in the brain,
thereby relieving some of the cardinal signs
of PD. In some cases, dopamine receptor ago-
nists are used for symptomatic treatment of PD.
Recently monoamine oxidase-B (MAO-B) inhib-
itors have been used to treat some symptoms of
PD. MAO-B inhibitors increase the level of DA
in the basal ganglia by blocking its metabolism.
They inhibit MAO-B, which breaks down DA
secreted by the DAergic neurons. The reduction
in MAO-B activity results in increased L-DOPA
in the striatum.
However, DA replacement therapies often
have little or no effect on nonmotor manifesta-
tions of PD. Since the DAergic system is spared in
the early stages of PD, diagnosis and treatment of
nonmotor symptoms are of clinical importance
at early stages of the disease, even before motor
symptoms have become apparent.
Glutamatergic Neurotransmission
Glutamate is the most abundant excitatory neuro-
transmitter in the central nervous system. It exerts
its effects through ionotropic glutamate receptors
(iGluRs, which include N-methyl-D-aspartate
[NMDA], -amino-3-hydroxy-5-methylisoxazole
propionic acid [AMPA], and kainate receptors),
which are involved in fast synaptic transmission
and metabotropic glutmate receptors (mGluRs,
subtypes 18) that mediate slow synaptic trans-
mission. All of these receptors subtypes are
expressed throughout the brain, including the
basal ganglia, and in peripheral organs, suggest-
ing that glutamate is also involved in biological
processes other than neuronal transmission.
The striatum is the primary input nucleus
in the basal ganglia. Besides dense innervation
from DAergic SN neurons, the striatum receives
excitatory afferents from the glutamatergic neu-
rons in the cerebral cortex, the thalamus, and the
amygdala. The striatum then modulates signal-
ing to other brain regions through the basal gan-
glia output nuclei, the internal part of the globus
pallidus (GPi) and the SN pars reticulata. The loss
of DA neurons is believed to cause an increase
in glutamatergic activity in the basal ganglia.
Preclinical studies using NMDA and AMPA
antagonists have demonstrated improvements
489
in various motor symptoms of PD (Lange etal.,
1997), particularly in those patients that do not
respond to L-DOPA therapy (Rodriguez et al.,
1998). Binding studies in human postmor-
tem tissue revealed that the levels of the NR1/
NR2B NMDA receptor, the AMPA receptor,
and mGluR5 are increased in the putamen of
patients with levodopa-induced dyskinesia (LID)
compared to those without (Calon et al., 2003;
Ouattara etal., 2011). These findings support the
hypothesis of enhanced glutamatergic neuro-
transmission in LID and that the selective inhi-
bition of glutamatergic hyperactivity may be an
effective strategy for the treatment of some PD
symptoms. Other studies have shown that mod-
ulation of glutamatergic neural tone in patients
with PD may also have a beneficial effect on cog-
nitive impairment (ONeill and Dix,2007).
Apart from iGluRs, mGluRs, which are
expressed abundantly in basal ganglia, are also
involved in some PD symptoms, although their
direct effects are not properly understood. It is
hypothesized that antagonism of subtypes 1 and
5 can protect against nigrostriatal degenera-
tion, whereas enhancing the other receptor sub-
types may help relieve parkinsonian symptoms
by inhibiting glutamate release and/or reduc-
ing inhibition of lateral globus pallidus neurons
(Conn etal.,2005).
Overall, it is safe to postulate that activation
of glutamatergic receptors may be effective in pre-
clinical models of anxiety, pain, depression (via
AMPA), and sleep dysfunction, whereas inhibi-
tion of glutamatergic neurotransmission may
result in increased anxiety and executive dys-
function but also lessen depression (via NMDA)
and improve cognitive function. However, stud-
ies are needed to confirm this hypothesis and to
design novel strategies based on this to treatPD.
GABAergic Neurotransmission
GABA is the major inhibitory neurotransmitter
in the brain. GABA signaling occurs via iono-
tropic GABA A and metabotropic GABA B recep-
tors, which are expressed in all human basal
ganglia structures. In the striatum, the two dif-
ferent types of GABAergic SPNs express DA
receptors and give rise to the direct and indirect
signaling pathways (Gerfen and Surmeier, 2011).
Therefore, SPNs are affected by postsynaptic
changes, resulting in an altered activity pat-
tern for both pathways. This signaling alteration
changes the output of the basal ganglia, causing
some of the typical motor features seen in PD
490 Part IV:Homeostatic Therapies
patients, including stiffness and bradykinesia.
GABAergic neurotransmission itself is altered in
PD. Binding studies in postmortem tissue indi-
cate that LID is associated with increased levels
of GABA A receptors in the internal globus palli-
dus and decreased density of GABAB receptors in
the putamen and in the external globus pallidus
in PD patients compared to controls. (Day etal.,
2006). The description of these region-specific
receptor alterations is interesting. However,
follow-up studies are required to extend these
findings to the development of therapeutic
strategies.
Advanced PD is also associated with
GABAergic SPNs characterized by truncated
dendrites and a lower number of spines (McNeill
et al., 1988; Stephens et al., 2005) due to deple-
tion of DA. Although several of the nonmotor
symptoms associated with PD have been shown
to involve GABAergic neurotransmission, such
as pain, sleep disorders, and depression, there is
a lack of evidence as to how these symptoms are
modulated by changes in endogenous or circulat-
ing GABA concentrations in PD. Studies in ani-
mals and in individuals without PD suggest that
decreases in GABAergic neurotransmission gen-
erally result in the reduction of these symptoms
(Barone,2010).
Adrenergic Neurotransmission
Alpha 2-adrenergic (-2a and 2c) receptors are
widely distributed throughout the basal ganglia.
Studies in mice have demonstrated that these
receptors modulate the sensitivity of DAergic
receptors, and activation of -2 receptors facili-
tates movements produced by activation of the
direct pathway of the basal ganglia motor circuit
(Colosimo et al., 2006). Noradrenergic systems,
in conjunction with serotonergic and DAergic
mechanisms, appear to play an important role
in the etiology of depression in PD. A recent
positron emission tomography study has shown
that noradrenergic innervations in depressed PD
patients are much lower than those in healthy
controls (Remy etal., 2005). Keeping in mind this
observation, a number of selective noradrenergic
reuptake inhibitors are currently undergoing
clinical trials for the treatment of depression and
anxiety in PD (Fox etal.,2008).
Reduced levels of noradrenaline have been
observed in patients with PD compared to normal
subjects (Barbic etal., 2007). Loss of noradrena-
line, which occurs at early stages of PD, has been
reported to worsen disease progression, either by
increasing the susceptibility of DAergic neurons
to degeneration or by inhibiting the repair of
neurons that are already damaged (Fornai etal.,
2007). In addition, it has been reported that loss
of noradrenergic innervation facilitates the onset
of dyskinesia during DA replacement therapy
(Colosimo and Craus,2003).
Among other nonmotor symptoms associ-
ated with PD, some studies have shown that sig-
nificantly more patients with PD reported pain
as compared to healthy controls. Like serotoner-
gic and DAergic systems, noradrenaline is a key
neurotransmitter of the endogenous pain system.
As sustained pain induces noradrenergic feed-
back inhibition (Pertovaara, 2006), lower levels
of noradrenaline would most likely be associated
with increased symptoms of pain as observed in
PD patients. However, since other neurotrans-
mitter systems are also involved in the pathology
of pain, it would be safer to suggest that there may
be some interplay between these various neuro-
transmitter systems in the propagation ofpain.
Cholinergic Neurotransmission
Cholinergic neurons are found in the basal gan-
glia (i.e., pedunculopontine nucleus [PPN]) and
brainstem, as well as in the striatum (a major-
ity of interneurons). These neurons are a major
source of acetylcholine, which mediates its
effects via nicotinic and muscarinic receptors.
Whereas neurons in the basal ganglia project-
ing to the SNpc, subthalamic nucleus, globus
pallidus, and striatum contain only muscarinic
receptors, both subtypes of cholinergic recep-
tors are found on neurons projecting to the
substantia nigra pars reticulata (Zhou et al.,
2003). In accordance with their expression,
deficits in the cholinergic system are believed
to play a role in the etiology of cognitive dys-
function and dementia in PD (Williams-Gray
etal., 2006). Reduction in cholinergic activity in
the cortex and degeneration of cholinergic neu-
rons in the nucleus of Meynert is closely asso-
ciated with declining cognitive functions in PD
(Perry et al., 1985). Recent studies also impli-
cate the role of PPN neurons in the etiology of
gait disturbances in PD (Karachi et al., 2010).
This study revealed that PD patients with gait
and balance abnormalities had a lower number
of cholinergic neurons in their PPN than PD
patients without these signs or unaffected con-
trols. Cholinergic PPN neurons are also shown
to undergo degeneration in patients with PD
(Rinne etal.,2008).
 Brain Homeostasis and Parkinsons Disease
Autonomic nervous system dysfunction (e.g.,
sleep disruption, sexual dysfunction, etc.) is
common in PD, affecting up to 80% of patients
(Zesiewicz etal., 2003). It has been suggested that
cholinergic dysfunction might be responsible
for, or contribute to, autonomic dysfunctions in
PD (Vernino etal., 2009). It is being speculated
that inhibition of or diminished cholinergic
neurotransmission in clinical and preclinical
paradigms of PD results in attention dysfunc-
tion, cognitive impairments, and executive dys-
function. Conversely, decreased stimulation
or enhanced cholinergic neurotransmission is
associated with lessening of several nonmotor
symptoms, including anxiety, apathy, attention
dysfunction, orthostatic hypotension, cognitive
impairment, and sleep disorders, which are more
autonomous in function.
Serotonergic Neurotransmission
The basal ganglia, including the globus pallidus
and SN, receive numerous innervations from
the serotonergic (5-HT) neurons in the brain-
stem raphe nuclei. 5-HT neurons innervate both
DAergic neuronal cell bodies of the SN and the
region of their terminal projections in the stria-
tum. The anatomical interaction of the 5-HT
system with DAergic components of the basal
ganglia facilitates functional modulation of DA
neurotransmission by serotonin in the normal,
non-parkinsonian brain. This mainly occurs via
5-HT1A and 5-HT1B receptors, which facilitate DA
release, while stimulation of the 5-HT2C receptor
inhibits DA release (Alex and Pehek,2007).
Levels of serotonin, its metabolite
5-hydroxyindoleacetic acid, serotonin trans-
porter, and tryptophan hydroxylase are reduced
in striatal tissue from PD patients compared to
controls (Kish etal., 2008). Low levels of seroto-
nin have been linked to alterations in mood, such
as depression and changes in sleep architecture
in PD patients, with a reduction of serotonin
levels of up to 50% in cortex and basal ganglia
(Scatton et al., 1983). Selective SSRIs, which
inhibit presynaptic reuptake of serotonin via the
serotonin transporter, have been used in several
studies to improve depression symptoms in PD
patients (Richard et al., 2012). However, not all
patients respond to SSRI treatment, suggesting
that PD-related depression is not fully attribut-
able to perturbations in serotonin signaling.
As mentioned in the discussion on adren-
ergic neurotransmission, one of the other com-
mon symptoms associated with PD is pain.
491
Approximately 80% of people diagnosed with PD
experience pain (Beiske etal., 2009). Serotonergic
pathways apart from adrenergic pathways have
been implicated in modulating pain. It has been
shown that treatment of PD patients with seroto-
nin and adrenaline reuptake inhibitors resulted
in varying degrees of pain relief for 65% of
patients with PD (Djaldetti etal.,2007).
Thus stimulation of the serotonergic system
in PD appears to result in decreases in several
nonmotor symptoms, including anxiety, depres-
sion, and possibly pain. Dampening the trans-
mission of serotonin leads to deficits in cognition,
executive function, and possibly fatigue, thereby
making serotonergic therapy as a treatment for
PD highly complicated.
Norepinephrine-Mediated
Neurotransmission
Along with acetylcholine receptors, norepineph-
rine has been postulated to be involved in pos-
tural instability and gait abnormalities observed
in PD (Grimbergen etal., 2009). These manifes-
tations are unresponsive to L-DOPA therapy.
However, treatment with a DA and norepineph-
rine reuptake inhibitor improved freezing of gait
in patients with advanced PD who also received
deep brain stimulation (Moreau et al., 2012).
These data suggest that modulation of norepi-
nephrine signaling might have some beneficial
effects on certain symptomsofPD.
Adenosine-Mediated
Neurotransmission
Adenosine is a ubiquitous endogenous purine
nucleoside that acts as a synaptic modulator, and
its effects are transmitted by at least four receptor
subtypes:A1, A2A, A2B, and A3 receptors. The A1,
A2B, and A3 adenosine receptors are widely dis-
tributed throughout the brain. In contrast, A2ARs
are localized within the basal ganglia, with the
highest concentration in the caudate-putamen
(Rosin etal., 1998; Schiffmann etal., 1991). A2ARs
are able to interact antagonistically with postsyn-
aptic dopamine D2 receptors and are also thought
to interact with presynaptic glutamatergic recep-
tors. Apart from this, A2ARs also colocalize with
dendritic spines of enkephalin-rich striatopallidal
GABAergic neurons of the indirect pathway. Thus
A2ARs are functionally relevant to the operation
of the indirect pathway of the basal ganglia sys-
tem (Morelli etal., 2007; Schiffmann etal.,2007).
Patients with PD have altered adenosine
A 2AR expression in the basal ganglia, suggesting
492 Part IV:Homeostatic Therapies
a pathogenic role for these receptors. In a post-
mortem study of patients with PD, a 2.95-fold
increase was observed in A 2AR density in the
putamen compared with healthy subjects (Varani
et al., 2010). Another autopsy study demon-
strated that patients with PD and dyskinesia who
were treated with levodopa had increased lev-
els of A2AR and corresponding messenger RNA
compared with levodopa-treated patients with-
out dyskinesia (Calon etal., 2004), suggesting a
tightly liked relation between A2ARs, dyskinesia,
and PD symptoms.
In animal models of PD, adenosine
A2AR-specific antagonists have been shown to
consistently reverse motor deficits such as
tremors and rigidity and/or enhance the effect
of DA-based pharmaceuticals (Hauser and
Schwarzschild, 2005). This effect may result from
action on the indirect pathway while allowing
DAergic action by the D1-mediated direct path-
way. Moreover, A 2AR antagonists are also neuro-
protective in animal models of PD (Morelli etal.,
2007). Thus A 2AR antagonists can be considered
prodopaminergic agents and could potentially
reduce the effects associated with DA depletion
in PD. Despite all the data available showing the
protective effects of A2AR antagonists, the exact
mechanism responsible for the neuroprotective
potential of adenosine antagonists is not clear,
although modulation of brain glutamate and
aspartate release are one potential hypotheses.
As described previously, disorders of sleep
and wakefulness are extremely common in
PD, with an estimated prevalence of 98%.
Adenosine has long been accepted as a media-
tor of sleep, although the contributions of the
adenosine receptor subtypes are still a matter
of intense debate. There is increasing evidence
that adenosine A 2AR play a critical role in the
sleep-promoting effects of adenosine and most
likely mediates the sleep-inhibiting effects of
the nonselective adenosine receptor antagonist
caffeine. Accordingly, it has been suggested that
insomnia be considered as a possible adverse side
effect of A2ARs antagonists used in the treatment
of PD (Ferre etal.,2007).
Epidemiological and preclinical data suggest
that caffeine may confer neuroprotection against
the underlying DAergic neuron degeneration and
may influence the onset and progression of PD. It
has been demonstrated that among the various
pharmacological effects attributed to caffeine,
this agent exerts A2AR antagonistic properties.
Thus it is possible that caffeine and its derivatives
via the modulation of A2AR might reduce PD
symptoms and possibly exert neuroprotective
effects (Prediger,2010).
Altogether, alterations in adenosine-mediated
neurotransmission impact upon DAergic
neuronal loss, cognition, pain, and sleep dis-
turbances. However, the effect of adenosine
modulation has not yet been determined in PD
models.
I N F L A M M AT O R Y
RESPONSEINPD
Inflammation in the central nervous system is a
prominent and common feature of neurodegener-
ative diseases, including PD. Neurodegeneration
of substantia nigral neurons leads to a plethora
of inflammatory responses induced by soluble
factors secreted from injured neurons, which is
also termed neuroinflammation (Tansey and
Goldberg, 2010). However, whether neuroinflam-
mation is a causal/trigger factor or a secondary
consequence in PD remains unknown. It appears
there is complex interplay between neuroinflam-
mation and other proposed pathogenic mecha-
nisms of PD such as mitochondrial dysfunction
and oxidative stress (Witte etal.,2010).
Inflammation, which is generated by an
activated immune system, is a strictly regulated
self-defensive mechanism against pathogenic
stimuli or injury that results in the protection
of the host organism by clearance of pathogenic
stimuli or debris to promote the healing pro-
cess (Khandelwal etal., 2011; Stone etal., 2009).
Both innate and adaptive immune responses
have been implicated in the pathophysiology of
PD. While innate immunity does not require the
presence of a specific antigen to develop, adap-
tive immunity occurs when specific antigens
are presented and recognized by lymphocytes.
Immunogenic factors released by injured dopa-
minergic neurons have the potential to trig-
ger a detrimental innate and adaptive immune
response, thereby amplifying the pathological
process. Neuroinflammatory responses in PD
consist of phenotypic and morphological activa-
tion of microglia cells, astrogliosis, and lymphatic
infiltration of T-cells (Brochard et al., 2009;
Hirsch and Hunot, 2009). It also leads to release
of proinflammatory cytokines, chemokines, ele-
ments of the complement cascade, and increased
expression of several enzymes that are involved
in the excess production of reactive oxygen and
nitrogen species (Lull and Block, 2010; Tansey
and Goldberg,2010).
 Brain Homeostasis and Parkinsons Disease
E V I D E N C E O F I N F L A M M AT I O N
INPD:POSTMORTEM STUDIES
Initial evidence of the involvement of inflam-
mation in the progression of PD stems from
a postmortem study over 20 years ago, which
demonstrated the presence of activated micro-
glia in the SNpc of a PD patient (McGeer etal.,
1988). This initial finding was later confirmed
by other postmortem studies (Desai Bradaric
et al., 2012; Imamura et al., 2003; Mirza et al.,
2000). Enzymes associated with inflamma-
tion, such as inducible nitric oxide synthase and
cyclooxygenase-2, have also been identified post-
mortem in PD brains (Hunot etal., 1996; Knott
et al., 2000). Several cytokine levels, including
tumor necrosis factor- (TNF-), TNF- recep-
tor, interleukin (IL)-1, IL-6, interferon (IFN)-,
and the inflammation-related transcription fac-
tor NFk, were elevated in the SN of PD patients
(Boka etal., 1994; Mogi etal., 1994a; Mogi etal.,
1994b; Mogi etal., 2007). Inflammatory markers
analyzed in the cerebrospinal fluid of PD patients
also showed elevated levels of TNF-, IL-1, and
IL-6 (Blum-Degen etal., 1995; Mogi etal., 1994a;
Mogi etal., 1994b; Zhang etal.,2008).
CELLS AND
N E U R O I N F L A M M AT O R Y
PAT H WAY S I N V O LV E D I N P D
A glial reaction involving astrocytes and micro-
glial cells and lymphocytic infiltration has been
described in several animal models of PD includ-
ing MPTP, 6-hydroxydopamine, rotenone, and
genetic mouse models. Microglia are active sen-
sors of the body that mediate innate immune
responses in the brain via antigen-presenting
and effector functions such as phagocytosis. They
also possess various beneficial functions, such
as neurotrophic factor release, removal of toxic
substances, neuronal repair, synaptic remod-
eling, synaptic pruning, and guidance to neu-
ral stem cells during synaptogenesis. Microglial
activation can be stimulated by aggregation of
mutant protein such as -synuclein and other
cellular mediators that are released by degener-
ating neurons (Lull and Block, 2010). Following
activation in the early phases, microglia migrate
through the site of damage and secretes pro- and
anti-inflammatory cytokines. Microglial activa-
tion can also be acute or chronic depending on
the type and duration of the external stimuli or
activating factor. Whereas short-term activation
of microglia is generally believed to be neuropro-
tective, chronic activation has been implicated
493
as a potential mechanism in neurodegenera-
tive disorders. Thus increased time of inflam-
mation flares the amount of proinflammatory
molecules and subsequently the neurons are
eventually affected by neurodegenerative envi-
ronment (Czeh et al., 2011; Smith et al., 2012).
The role of microglia in neurodegenerative dis-
eases is unequivocal. However, the mechanism
that underlies the switch from neuroprotective to
autoaggressive effector microglia, which causes
neurodegeneration, is as yet unknown (Hanisch
and Kettenmann, 2007; Khandelwal etal.,2011).
Astrocytes are the most abundant cells in
the brain, heavily outnumbering neurons. They
are dispersed throughout the central nervous
system, where one astrocyte occupies its own
territorial region without any overlap with the
territory of another. Astrocytes perform regula-
tory and supportive roles, such as biochemical
and nutritional support for neurons, extracel-
lular ion balance, neuronal cellular activity and
blood flow, and repair of scarring of brain and
spinal cord tissue (Sofroniew and Vinters, 2010).
Apart from this, astrocytes also have immune
functions that limit the spreading of inflamma-
tory cells and infectious agents from the dam-
aged area to healthy parenchyma by secreting
pro- and anti-inflammatory molecules (Hamby
and Sofroniew,2010).
Glial reaction in pathological situations of the
central nervous system such as PD can play either
a beneficial or a detrimental role. The former
properties are thought to be possibly mediated by
the production of a variety of trophic factors, the
uptake of any excess of glutamate, or the removal
of cell debris; the latter can result from the pro-
duction of a host of cytotoxic molecules rang-
ing from ROS and nitrogen oxygen species to
cytokines. The astrocytic reaction is a well-known
neuropathological characteristic of the SN in PD.
Glial fibrilliary acidic protein-specific staining in
PD patients showed that astrocytes are heteroge-
neously distributed within the mesencephalon
in healthy individuals (Damier etal., 1993). The
density of astrocytes is low in the SNpc, which
is severely affected in PD, whereas the density is
high in the ventral tegmental area and the cat-
echolaminergic cell group A8, areas that are less
affected in PD. Therefore, vulnerable neurons in
patients with PD might have few surrounding
astroglial cells, which detoxify oxygen free radi-
cals by glutathione peroxidase in healthy indi-
viduals, and this limited astroglial environment
might be a susceptibility factor for the disorder.
494 Part IV:Homeostatic Therapies
A30% increase in the density of astroglial cells
in the SN of patients at postmortem was detected
by quantitative analysis (Damier et al., 1993).
Looking at this evidence, it might be safe to pos-
tulate that in a disease like PD, in which not all
neurons die at the same time, the very first cells
to succumb to the pathological process activate
the neighboring glial cells. Once activated, glial
cells, and especially microglia, can engage in
the production of toxic molecules that can pro-
mote the demise of surrounding compromised
neurons.
Lymphocytes might also participate in the
inflammatory reaction in the brains of patients
with PD. Different studies have reported higher
densities of cytotoxic T lymphocytes (CD8+ and
CD4+) in the SN of PD patients as compared to
healthy controls (Brochard etal., 2009; McGeer
etal., 1988). These cells were in close contact with
blood vessels (suggesting migration from the
bloodstream) and near to melanised dopamin-
ergic neurons (suggesting an interaction between
the lymphocytes and the dopaminergic neurons).
The CD8+ and CD4+ T cells were not detected in
the red nucleus, an area not affected in PD, sug-
gesting that this infiltration is highly selective
for the lesioned brain areas (Hirsch and Hunot,
2009). These data indicate that some peripheral
cells can enter the brain parenchyma during the
neurodegenerative process; therefore, changes
in blood-brain barrier function might occur in
the brains of patients with PD. The presence of
neuroinflammatory processes in portmortem
tissues has also been confirmed on a molecu-
lar basis. Increased concentrations of TNF-,
2-microglobulin, epidermal growth factor,
transforming growth factor (TGF), TGF1,
and interleukins 1, 6, and 2 has been found in
the striatum and SN of patients with PD, indicat-
ing that there are neuroinflammatory processes
in the affected brain regions of patients with PD.
However, these studies do not help to determine
whether such changes are involved in the patho-
logical process or are merely a consequence of
neuronal degeneration.
THER APEUTIC
INTERVENTIONS
F O R N E U R O I N F L A M M AT I O N
An increasing amount of evidence, such as
that discussed previously as well as continuing
research, has shown that neuroinflammation is
involved in pathogenesis of PD. However, the ori-
gin and role of these neuroinflammatory changes
need to be established. Neuroinflammation
might be a simple consequence of neuronal
changes or degeneration. Alternatively, neuro-
inflammatory processes might be a main cause
of the disease. Whatever the origin of the neu-
roinflammatory processes in PD, therapeutic
intervention aimed at prevention or downregu-
lation of these immune-associated mechanisms
could be of great use to stop disease progression
or even halt the pathological process. Different
commonly used analgesics and antipyretics such
as nonsteroidal anti-inflammatory drugs (e.g.,
ibuprofen) and cyclooxygenase inhibitors can
have protective effects on the symptomatology
of PD (Rees et al., 2011; Reksidler et al., 2007;
Teismann and Ferger, 2001; Teismann et al.,
2003a; Teismann etal., 2003b).
Strategies aimed at suppressing the activa-
tion of glial cells and their inflammatory prop-
erties by use of various drugs is another way
medications are being used to study their effects
on PD. Administration of drugs with a broad
spectrum of action on inflammation would
be more likely to protect dopaminergic neu-
rons efficiently than more selectively targeted
drugs. Some of these drugs include agonists of
PPAR, a receptor that regulates fatty acid stor-
age and glucose metabolism (Dehmer et al.,
2004; Quinn et al., 2008; Swanson et al., 2011;
Whitehead, 2011), adenosine receptor antago-
nists (Bibbiani et al., 2003; Hauser et al., 2011;
Hodgson etal., 2010; Kanda etal., 1998; LeWitt
etal., 2008; Pourcher etal., 2012), 7 nicotinic
acetylcholine receptor agonists (Acker et al.,
2008; Mazurov et al., 2005), as well as immu-
notherapy (Laurie et al., 2007; Reynolds et al.,
2010; Reynolds etal.,2009).
NEUROTROPHIC
FA C T O R S I N P D
Neurotrophic factors are neuroprotective
secreted peptides that promote development,
influence neuronal survival and axonal growth,
and modulate neuronal functions during devel-
opment. Neurons that fail to obtain a sufficient
quantity of the necessary neurotrophic factors
die by a process called programmed cell death.
In response to injury, many trophic factors and
their receptors have been shown to increase in
concentration, suggesting an endogenous regen-
erative response by these molecules (Hughes
etal., 1999). Furthermore, these factors serve as
neuroprotectant molecules against cytotoxic cell
damage.
 Brain Homeostasis and Parkinsons Disease
It has been proposed that the loss of endoge-
nous target-derived trophic support for selective
neuronal populations may lead to the neuronal
degeneration characteristic of PD and other
neurodegenerative diseases, but direct support
for this hypothesis is currently lacking (Connor
and Dragunow, 1998). Initial in vitro studies for
determining the effects of different neurotrophic
factors on dopaminergic neuronal populations
included assays of: brain-derived neurotrophic
factor (BDNF; Hyman et al., 1991; Hyman
etal., 1994)and glial-derived neurotrophic fac-
tor (GDNF; Engele etal., 1996; Lin etal., 1993).
In MPTP- and 6-hydroxydopamine-lesioned
animal models with PD, BDNF (Frim et al.,
1994) and GDNF (Gash et al., 1996; Tomac
et al., 1995) have been shown to promote the
survival of neurons and ameliorate PD symp-
toms. Supranigral implants of fibroblasts engi-
neered to secrete human BDNF are shown to
protect dopaminergic neurons from MPTP
toxicity (Frim et al., 1994). These neuroprotec-
tive effects of BDNF may be mediated by its
antioxidant-promoting capacity. BDNF also
stimulates DA activity and turnover and is
hypothesized to play a role in the compensatory
actions of surviving DA neurons in early-stage
PD (Blochl and Sirrenberg, 1996; Murer et al.,
2001). Although BDNF has been found to pro-
tect DA neurons in cell culture and animal mod-
els of PD, its large molecular size may prevent
its delivery to DA neurons. It has been suggested
that alternative strategies for BDNF delivery
may be more effective, such as using endogenous
factors to induce BDNF expression or transplan-
tation of BDNF-releasingcells.
GDNF supports the survival of several differ-
ent neuronal populations in both the central and
the peripheral nervous system. GDNFs potential
therapeutic value for PD was first recognized in
1993 when it was purified and shown to promote
the growth and survival of midbrain embryonic
dopaminergic neurons (Lin et al., 1993). Later
studies showed that GDNF encouraged neuronal
fiber outgrowth and improved motor function
when delivered into the cerebral ventricles or
directly into the striatum or SN in both rodent
and primate models of PD (Bjorklund et al.,
2000; Chiocco et al., 2007; Peterson and Nutt,
2008; Siegel and Chauhan, 2000). Similar to
rodent studies, the positive effects of GDNF have
also been demonstrated in nonhuman primate
PD models (Gash etal., 1995; Gash etal., 1996;
Zhang etal.,1997).
495
Despite its high efficacy in rodents and non-
human primate models, neurotrophic factor
therapy has not provided expected beneficial
effects in PD patients. The lack of symptomatic
relief seen in these patients may be due to the
inadequate penetration of neurotrophic factors
from the cerebrospinal fluid into the striatum
upon intracerebroventricular delivery, suggest-
ing that the factors did not diffuse sufficiently to
reach the degenerating nigrostriatal fibers and
neurons (Kordower, 2003). Later studies involv-
ing intraputamental injections in PD patients
(Patel et al., 2005), GDNF delivery via intrapu-
tamental pumps in monkeys (Maswood et al.,
2002), transplantation of GDNF-secreting cells
in rodents (Akerud etal., 2001; Cunningham and
Su, 2002), and in-vivo gene therapy using viral
vectors (Bilang-Bleuel et al., 1997; Zheng et al.,
2005)have all shown beneficial effects of modu-
lating GDNF levels on PD symptoms.
CONCLUDING REMARKS
AND FUTURE DIRECTIONS
PD is predominantly a motor disorder; how-
ever, other variable indicators (such as nonmo-
tor symptoms, depression, sleep disturbances,
cognitive dysfunction, etc.) cannot be ignored
in its pathophysiology. Cumulatively, all of these
symptoms are enervating and can have a major
impact on the quality of life of patients. Since
there is currently no cure for PD, current thera-
peutic strategies are focused on (i) delaying the
progression of the disease, (ii) providing symp-
tomatic relief from the symptoms, (iii) causing as
few adverse effects as possible via different medi-
cations and therapeutics, and (iv) developing new
therapeutics to treat PD. No single agent cur-
rently treats both the motor and the nonmotor
manifestations of the disorder. Indeed, effective
treatment of the nonmotor symptoms remains a
major unmet needinPD.
Genes implicated in Mendelian forms of PD as
well as various risk factors associated with them
have provided new insights into the pathogen-
esis of the disease. However, known PD-causing
genes account for only a small fraction of mono-
genic forms. Robust high-density single nucleo-
tide polymorphism genotyping technologies
and data analysis programs, combined with the
analysis of copy-number variations and large
pathogenic genomic rearrangements, will help
in identifying novel loci associated with PD. It is
also imperative to more closely study the cumu-
lative effect of different geneenvironment and/
496 Part IV:Homeostatic Therapies
or genegene interactions to evaluate the overall
picture for the various factors contributing to the
PD phenotype.
It is now well accepted that abnormal protein
degradation and accumulation is a critical fac-
tor mediating the degeneration of dopaminergic
neurons in PD. As the oxidative environment in
neurons is also believed to occupy a central role in
neurodegeneration, of particular relevance to PD
is the increased detection of dopamine quinones
under conditions of oxidative stress (Bisaglia
etal., 2007). These quinones are a highly impor-
tant species since they are known to interact with
proteosomal and mitochondrial machineries to
impair their functions (Berman and Hastings,
1999). Thus, despite the active debate over the
sole cause of PD, it is likely that genegene and
geneenvironment interactions combined with
oxidative and proteolytic stress, as well as mito-
chondrial dysfunction, are all intricately linked
in a pathogenic feedforward cycle responsible for
the progression of PD. This gives rise to a multi-
factorial situation where all the factors are con-
volutedly linked, making the development of a
single therapeutic agent for treatment of PD even
more complex.
Deficits in dopaminergic neurotransmit-
ters are largely responsible for the characteris-
tic motor symptoms observed in patients with
PD. However, numerous nonmotor disorders
are prevalent in many patients, even those with
mild or early-stage disease. Study of the neu-
ropathology of these nonmotor symptoms has
shown that modulation of cholinergic, seroton-
ergic, adenosinergic, glutamatergic, GABAergic,
and noradrenergic neurotransmitter systems are
involved in the etiology of these symptoms in
addition to also having a role in maintenance of
smooth motor function via interactions with the
dopaminergic system. The development of PD
symptoms arises from a comprehensive disrup-
tion of normal signal transmission throughout
the basal ganglia because of modulation of each
of these types of neurotransmitters.
Our most significant hindrance in under-
standing the complexity of these neurotrans-
mitter systems arises from the fact that our
knowledge regarding the effect of various param-
eters on the expression levels of receptors and
transporters in the human brain is very scarce.
Greater cellular, subcellular, and molecular
resolution of the expression profiles, linked to
disease-specific changes, would further facilitate
the identification of therapeutic approaches for
PD. It should also be kept in mind that rodent and
nonhuman primate models can only partially
recapitulate the pathological changes that occur
in patients with PD. The expression patterns of
neurotransmitter receptors and transporters in
normal and disease-like states, their conforma-
tion and subunit composition, and the interact-
ing partners of receptors, as well as the binding
affinity between a specific drug target and a test
compound are only some of the parameters that
may vary significantly between species. These
factors present a challenge for the translation
of therapeutic strategies from disease models to
patients and highlight the importance of validat-
ing preclinical data in human tissue or subjects
whenever feasible.
A major scientific breakthrough will occur if
we one day we develop agents that alleviate both
the motor and the nonmotor symptoms of PD
through interaction with several of the affected
homeostatic neurotransmission systems in the
brain. Due to the plethora of dynamic interac-
tions that occur between different neurotrans-
mitter systems in PD, it is likely that the future
of PD treatment will depend on type (and tim-
ing) of symptoms displayed by each patient, and
these factors will help us provide more effective
treatment of both the motor and the nonmotor
symptoms.
With a mounting body of evidence suggest-
ing that neuroinflammation, and microglial
activation in particular, is harmful to DA neu-
rons of the SNpc, it is logical to assume that an
anti-inflammatory regimen may protect against
PD-related DA neuron degeneration. However,
anti-inflammatory drugs may not provide magi-
cal answers for treatment of PD. Although pre-
clinical trials of anti-inflammatory drugs have
largely been successful, the time of administra-
tion is a caveat that cannot be ignored. Indeed,
most preclinical trials administer drugs prior
to or concomitantly with neurotoxin insult,
an option that, for now, given the absence of
presymptomatic biomarkers, is not available to
PD patients. With the unfortunate fact that PD
symptoms do not arise until a majority of DA
neurons have already degenerated, treatment
prior to disease onset creates a clinical treatment
barrier, an important aspect that needs to be con-
sidered when developing future drug therapies
forPD.
Various neurotrophic factors are being stud-
ied for therapeutic intervention of DA neuron
loss in PD in both animal models and clinical
 Brain Homeostasis and Parkinsons Disease
trials. To date, the most promising of these appear
to augment the cell loss associated with PD.
Additionally, findings from neurotrophic factor
studies suggest that a combination of therapeutic
approaches using different delivery methods or
endogenous induction may offer the most effec-
tive therapy against the devastating neurodegen-
erative effectsofPD.
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27
Brain Homeostasis and Addiction
L E T I S H A R . W YAT T
ADDICTION OVERVIEW
Drug addiction, or substance dependence, is a
national epidemic. In the United States, costs
related to illicit drug use reaches nearly $200 bil-
lion annually (National Drug Intelligence Center,
2011). As reported by the US National Drug
Intelligence Center, these direct and indirect costs
cover crime, health, and a decline in workforce
productivity and ultimately affect individuals
from many cultural, racial, and socioeconomic
backgrounds. By diagnostic standards, addiction
is characterized as the transition from casual
to compulsive drug use whereby an individual
spends more of his or her time in drug-related
versus non-drug-related activities (Zernig etal.,
2007). Within the past three decades, clinicians
have modified the definition of substance use in
the Diagnostic and Statistical Manual of Mental
Disorders, distinguishing between those that
abuse and those who are dependent (American
Psychiatric Association, 2000). Many physi-
cians and behavioral researchers in the field
agree about the stages of the drug addiction
cycle: acquisition, tolerance, dependence, with-
drawal (abstinence), and relapse (Le Moal and
Koob, 2007). These distinct stages are thought to
evolve from initial sensitivity and then tolerance
acquired during drug use (Vetulani,2001).
All drugs fall into one of six classifica-
tions: (a) narcotics, (b) stimulants, (c) depres- tive learning by encoding predictions for future
sants, (d) cannabinoids, (e) hallucinogens, and
(f) inhalants (Feltenstein and See, 2008). Each
class comes with its own behavioral characteri-
zation, for example narcotics such as heroin are
classically known to produce feelings of eupho-
ria (National Drug Intelligence Center, 2011).
Stimulants such as nicotine and cocaine, on the
other hand, are known for eliciting feelings of
exhilaration (National Institute on Drug Abuse,
2012). For sensations of relaxation, drug users
typically turn to depressants and cannabinoids
(e.g., ethanol and marijuana, respectively). It is
common to have an array of reactions to hallu-
cinogenic drugs such as ecstasy and lysergic acid
diethylamide (LSD), including rapid shifts in
emotion, enhanced tactile sensitivity, and impul-
sivity. Finally, inhalants typically cause stimula-
tion and the loss of inhibition (National Institute
on Drug Abuse,2012).
MESOCORTICOLIMBIC
R E WA R D C I R C U I T R Y
INADDICTION
Several anatomical structures have been identi-
fied as neural substrates of drugs of abuse, includ-
ing the ventral tegmental area (VTA), prefrontal
cortex (PFC), nucleus accumbens (NAc), amyg-
dala, and hippocampus. These areas of the brain
have distinct features and interconnected nuclei
where cellular modulation by drugs may elicit
obvious or subtle changes in behavior. Studies in
this area have shown that the VTA and NAc are
likely mediators of addictive behaviors. The VTA,
a midbrain structure distinct from the substantia
nigra, is one of the most commonly studied brain
areas with respect to drug reward. The VTA is
largely comprised of dopamine (DA)-releasing
cells. As theorized, the normal physiological
function of these dopaminergic neurons include
(a) the release of DA as a function of pleasure
or liking, (b) a mechanism to aid in associa-
rewards that are necessary for survival through
firing rates and the release of DA, and (c)incen-
tive salience or wanting (Ikemoto, 2007). Drugs
of abuse are capable of corrupting the system
by stimulating the release or slowing down the
reuptake of DA in the VTA, thereby eliciting a
high or extreme pleasure response that becomes
reinforcing for drug use (Ikemoto, 2007). The
NAc is designated as having a specific role in
directly mediating behavioral output by control-
ling information flow to the thalamus, striatum,
and PFC via the ventral pallidum (Pierce and
510 Part IV:Homeostatic Therapies
Kumaresan, 2006). Some investigations differen-
tiate the role of the accumbens shell versus the
core for the rewarding effect of drugs, with the
shell as the most involved in limbic processes and
the core the most involved in motor functions
(Ikemoto, 2007; Pierce and Kumaresan,2006).
Historically, research has centered on the
involvement of the mesocorticolimbic reward
pathways, which specifically describes afferent
connections between the VTA and regions such
as the NAc, amygdala, hippocampus, and medial
PFC (Pierce and Kumaresan, 2006). This path-
way is involved in the emotional, motivational,
and contextual influence on behaviors (Pierce
and Kumaresan, 2006). Drugs of abuse can affect
both pathways, thereby altering behavior and
creating a vicious cycle that results in addic-
tion. Some studies argue that local drug admin-
istration into the NAc does not increase DA but
rather may enhance the upstream firing rate of
dopaminergic neurons in the VTA (Pierce and
Kumaresan,2006).
The mesocorticolimbic areas are enriched
with receptors for DA, -amino butyric acid
(GABA), glutamate, serotonin, and endoge-
nous opioids (Volkow et al., 2012), all of which
have been identified as key regulators of drug
responses and addictive behavior. Notably, DA,
GABA, and glutamate have been linked to the
actions of depressants, stimulants, and narcot-
ics. Central opioid receptors (i.e., mu, kappa, and
delta)typically those found in the NAcare
responsible for behavioral effects of opioid drugs
such as morphine or heroin (Lutz and Kieffer,
2013). Although mechanistically the differ-
ent classes of drugs have associations with dif-
ferent receptors in the central nervous system
(CNS), they share some common neurobiological
underpinnings. For instance, as mentioned ear-
lier, all drugs of abuse alter the firing of neuronal
dopaminergic cells and elevate basal DA levels.
Moreover, genetic analyses have also shown that
individual differences in gene expression and
function of various neurotransmitters correlate
with vulnerability to drug self-administration
(for a review, see Goldman etal.,2005).
DRUG ADDICTION MODELING
INANIMALS
Animal modeling of drug addiction is a not an
easy task, though it is critical for the understand-
ing of the molecular basis of the disease and the
development of new therapies. Due to the com-
plexity of the progressive stages in addictive
behavior, no one model can encompass all aspects
of the disease. Nonetheless, proper selection of
the animal model to be used in preclinical testing
is essential to ensure validity of the interpreta-
tion of data with respect to the human condition.
Based on the hypotheses of the investigator and
the drug to be studied, procedures utilized for
modeling addiction in animals willassess
1. Acute drug effect and sensitivity via
experimenter administration
2. Acquisition (intoxication/binging),
often through self-administration using
intracranial self-stimulation or operant
chambers
3. Motivation using operant chambers to
illuminate the reinforcing effects of the
drug and investigate how hard the animal
will work for thedrug
4. Acute withdrawal following abrupt
sobriety after several sessions of drug
exposure by monitoring for shaking,
ptosis, chewing, and heightened anxiety.
Alternatively, withdrawal can be studied
using the conditioned place avoidance
test, which is achieved by providing
the drug in a defined environment over
several exposures and then allowing the
animal to experience drug withdrawal in
the same environment. The animal will
display conditioned place avoidance and
thus will spend less time in the environ-
ment associated with drug withdrawal
(Heidbreder,2011).
5. Reinstatement/craving and relapse, using
a drug/cue reinstatement procedure
whereby animals are drug exposed over
many sessions to a conditioned stimu-
lus, then are given an extinction period
during which the drug and conditioned
stimulus are absent until drug-seeking
behavior is gone, followed by re-exposure
to either the drug or a conditioned stimu-
lus; behavior is then compared to initial
drug responses (Heidbreder,2011).
The subjective effect of drugs is comparatively
more challenging to test in animals without their
verbal feedback. Thus behavioral tests have been
designed to assess the rewarding effects of drugs.
The conditioned place preference (CPP) para-
digm measures the amount of time spent in a
specific compartment where an animal has been
trained to associate the effects of a particular

test substance. When the amount of time spent
in the conditioned compartment of the appa-
ratus increases, then it can be inferred that the
test substance was rewarding (Becker etal., 2012;
Heidbreder, 2011). Tests for drug discrimina-
tion also measure reward. For this, animals are
trained to associate the feelings provided from an
injected drug or non-drug (typically saline) with
food delivered through a lever press. Subsequent
test substances administered to the animal will
cause the animal to press either the drug-food
lever or the saline-food lever, indicating the sub-
jective effect of the test substance compared to
the drug (Heidbreder,2011).
From a psychological perspective, drug
addiction is a disorder of compulsivity and/or
impulsivity. Not to be confused with impulsivity
(or the tendency toward rapid, unplanned reac-
tions to internal or external stimuli), compulsiv-
ity is defined as the performance of repetitive
behaviors to reduce or prevent anxiety or dis-
tress (Grant and Potenza, 2006). Both are related
to the craving and intoxication/binge stages of
addiction (Koob and Le Moal, 2008). To model
these endophenotypes in animals, the delayed
discounting and the 5-choice serial reaction
time tasks are often utilized. These tests are able
to evaluate impulsive choice and determine any
impairments in inhibitory control in animals
and can be used as a measure of risk for increased
drug-seeking and binge-like patterns of addic-
tion (Heidbreder,2011).
This chapter provides a review of homeostatic
mechanisms involved in drug addiction and
offers current examples in the literature where
investigators have used the aforementioned ani-
mal models to describe the involvement of vari-
ous homeostatic systems in drug addiction. The
mechanisms underlying the progression from
substance use to abuse and dependence are dis-
cussed in light of brain homeostasis. The chap-
ter concludes with a brief description of current
pharmacological strategies and also nonpharma-
cological approaches involving the modulation
of brain homeostasis as an alternative treatment
strategy for addiction.
O V E R V I E W O F H O M E O S TAT I C
CONTROL SYSTEMS
R E L E VA N T T O A D D I C T I O N
As outlined in the introductory portion of this
book, brain homeostatic mechanisms are impor-
tant for normal and optimal function of human
physiology. Regulation of brain homeostasis
Brain Homeostasis and Addiction 511
is critical for mechanisms underlying addic-
tive behavior, and altered homeostasis can have
serious consequences regarding substance use.
Several systems exist and work together to main-
tain the ideal brain environment for neuronal
functions. The release of excitatory or inhibi-
tory neurotransmitters plays an integral role
in maintaining the localized synaptic milieu.
Neuromodulation by molecules that are neither
directly excitatory nor inhibitory, but rather alter
the response of groups of neurons over a long dis-
tance, is also important in brain homeostasis (Ito
and Schuman, 2008). Additionally, inflammatory
immune responses, endocrine functions, energy
balance, and neurotrophic factors contribute to
maintaining brain homeostasis. Currently, there
is some debate as to whether inherent altered
brain homeostasis proceeds drug addiction or,
alternatively, drug exposure begets dysfunctional
homeostatic responses. Using recent neurophar-
macological and behavioral studies, here we dis-
cuss the various systems and their involvement
in the compulsive intake and addictive patterns
of the different classes ofdrugs.
Neuromodulators
Dopamine Homeostasis
We begin with DA homeostatic control of
addictive behavior, as the majority of addiction
research emphasizes a key role for DA in these
processes. However, because DA has been so
extensively covered in addiction research (see
Blum etal., 2012), a brief overview of the involve-
ment of DA in drug reward and addiction is
introduced in this section. DA has a long history
of being considered a molecular target, since all
drugs of abuse enhance brain levels of this mol-
ecule. DA is a neuromodulator responsible for
both motor and cognitive functions. Produced by
neurons in the midbrain, namely the substantia
nigra and VTA, firing patterns of DA-releasing
neurons and increases in extracellular DA in the
NAc is thought to be a way in which the brain
encodes natural reward (Ikemoto, 2007). In both
humans and rodents, DA receptors (D1-like and
D2-like) and the DA transporter are responsible
for regulating DA homeostasis, and each is sus-
ceptible to expression changes following drug
exposure (Becker etal.,2012).
Adenosine Homoeostasis
Adenosine is a potent homeostatic regulator of
network function that activates A1, A2A, A 2B, and
A3 adenosine receptors, which are expressed
512 Part IV:Homeostatic Therapies
throughout the brain (Diogenes etal., 2014). Intra-
and extracellular adenosine levels are rapidly
regulated by the astrocyte-based enzyme adeno-
sine kinase, responsible for adenosine metabo-
lism to adenosine 5-monophosphate (Boison,
2013). In both humans and animals, A1 receptor
activation has been linked to alterations in glu-
tamate and DA activity in the brain. Adenosine
agonists reduce striatal DA levels and inhibit cor-
tical glutamate function (Dohrman etal., 1997).
Glutamate, DA, and other neurotransmitters
modulated by adenosine are known mediators
of addictive behaviors (Lingford-Hughes et al.,
2010; Sebastiao and Ribeiro,2009).
In support of a glial basis of addiction, rodent
studies demonstrate an upregulation of reactive
astrocytes following drug administration, with-
drawal, and abstinence (Baydas and Tuzcu, 2005;
Fattore et al., 2002; Kelso et al., 2011). Several
lines of evidence support the notion that astro-
gliosis increases adenosine kinase, resulting in
adenosine hypofunction (Boison, 2011; Li et al.,
2008). Notably, addictive behavior is a recog-
nized comorbidity in epilepsy, a condition char-
acterized by pathological hypofunction of the
endogenous anticonvulsant adenosine (Boison,
2011). In addition, seizures are a common side
effect for some recreational drugs and a symptom
of drug withdrawal.
Recent studies have more directly addressed
a role for adenosine in mechanisms underly-
ing drug addiction and/or responses to drugs of
abuse. Wu etal. (2013) assessed changes in brain
adenosine following the administration of mor-
phine. The findings from this study indicate that
adenosine levels are significantly reduced during
opiate dependency and withdrawal in C57BL/6J
mice compared to 129Sv1 mice, which do not
develop opiate physical dependence to morphine
under the same conditions. Additionally, opiate
withdrawal behavior was modified after genetic
alteration of adenosine kinase expression in
mice (Wu et al., 2013). Based on their findings,
and in conjunction with previous studies in the
literature, the authors suggest that adenosines
involvement in opiate addiction and withdrawal
is in part linked to its modulation of glutamate
release and the action of other excitatory neuro-
transmitters in the brainstem (Wu etal.,2013).
Such reports suggest that disruption of aden-
osine homeostasis, possibly induced by glial acti-
vation, might be implicated in the development
of addictive behavior and that therapeutic adeno-
sine augmentation might be a novel strategy to
break addictive behavior. Interestingly, in 2004
and 2009 the antiepileptic drugs Gabapentin
and Vigabatrin were highlighted for their poten-
tial to treat addiction (Brodie et al., 2009; Raby
and Coomaraswamy, 2004). Although both
drugs are known to increase the level of circu-
lating GABA neurotransmitter, the underlying
anti-addictive mechanism has not been fully
identified. Nonetheless, evidence from the litera-
ture support the idea that adenosine homeostasis
plays a critical role in the regulation of addictive
behavior.
Serotonin Homoeostasis
Serotonin (5-hydroxytryptamine; 5-HT) func-
tions as both a neurotransmitter and a neuro-
modulator with important regulatory roles in the
CNS for mood, sleep, and some cognitive pro-
cesses such as learning and memory, as well as
intestinal motility (Berger etal., 2009; Gonzalez
et al., 2013). 5-HT is produced by serotonergic
neurons originating in the raphe nucleus and
released via vesicles into the synaptic cleft, target-
ing most areas of the brain. The action of seroto-
nin is dependent on 5-HT receptors, and the level
of this molecule is modulated by reuptake via the
monoamine transporter (5-HTT). 5-HT recep-
tors modulate both excitatory and inhibitory neu-
rotransmission and may alter the release of other
neurotransmitters (e.g., GABA, glutamate, and
DA) and various hormones (Nichols and Nichols,
2008). This system has been exploited by clini-
cians for its antidepressant effects since the dis-
covery of selective serotonin reuptake inhibitors
(SSRIs). Also, because inhibition of serotonergic
activity enhances mood and reduces consumma-
tory behaviors, researchers have focused on the
role of serotonin in psychostimulant, depressant,
and hallucinogenicdrugs.
The 5-HTT has received the most atten-
tion in investigations related to drugs of abuse.
However, recent studies on the G-protein cou-
pled receptors 5-HT1B, -HT2A, -HT2C, and the
ligand-gated ion channel 5-HT3, have also impli-
cated these receptor subtypes in mediating some
of the behavioral effects of drugs like ecstasy
(or 3,4-methylenedioxymethamphetamine;
MDMA), alcohol, cocaine, and methampheta-
mine (for a review, see Higgins and Fletcher,
2003). Studies by Bengel etal. (1998) have shown
that administration of ecstasy and ampheta-
mine causes an increase in serotonin levels in
mice. Additionally, by making use of 5-HTT
homozygous and heterozygous knockout mice,

the authors provided support that acute behav-
ioral responses such as increased locomotor
activity are likely mediated through the 5-HTT,
an MDMA-induced behavioral effect that was
eliminated in the 5-HTT null animals. These
data implicate a regulatory role for serotonin in
the behavioral response to ecstasy that may serve
as a new therapeutic target.
SSRIs such as fluoxetine elevate the level of
serotonin in the brain and are capable of modify-
ing behavioral reactions to cocaine and alcohol.
For instance, Burmeister and colleagues (2003)
report that fluoxetine can dose-dependently
attenuate cocaine reinstatement after extin-
guishing cocaine-seeking behavior in rats. More
recently, Sawyer et al. (2012) evaluated these
findings in rhesus macaques after clinical tri-
als using SSRIs failed to consistently reduce
cocaine abuse. The authors found that after 4
weeks of daily cocaine self-administration fol-
lowed by a 2-week extinction period, 10 mg/kg
fluoxetine attenuated cocaine reinstatement. This
was also the first study to demonstrate a reduc-
tion in drug-induced DA overflow by fluoxetine,
an effect that Sawyer and colleagues attribute to
desensitization of 5-HT2A receptors. Similarly, a
low although behaviorally relevant dose of eth-
anol (2.5 g/kg) injected directly into the CA3
region of the hippocampus in mice produces a
significant reduction in local serotonin clearance
(Daws etal., 2006). These authors also found that
inactivation of 5-HTT, as investigated via 5-HTT
knockout mice, results in a potentiation of the
sedative/hypnotic effects of ethanol. Similar
findings were not achieved when administering
fluoxetine to C57BL/6J mice, demonstrating that
the genetic loss of 5-HTT provides a more robust
alteration in ethanol-induced behavior than non-
selective pharmacological inactivation of 5-HTT
(Daws etal.,2006).
Although the psychological effects of
serotonin-altering drugs are relatively short,
the spike in serotonergic activity is thought to
be neurotoxic, and these acute neurobiological
changes can be very detrimental, as evidenced
in long-term ecstasy users who reportedly suffer
from cognitive and memory impairments and
psychiatric problems (Parrott, 2002). Much of
the problem regarding alterations in serotonin
homeostasis, particularly excess, has to do with
an increase in impulsivity. Users of drugs that
enhance serotonergic function are often found
to have comorbid psychiatric disorders such as
increased general anxiety and impulsiveness
Brain Homeostasis and Addiction 513
(Parrott, 2002). This is not surprising considering
the involvement of serotonin in biological pro-
cesses, including impulsivity, anxiety, depression,
and schizophrenia (Parrott, 2002). The working
hypothesis is that serotonin overexcitability leads
to a greater release of serotonin from neurons
in the raphe nucleus projecting to mesocorti-
colimbic areas that mediate impulsive behavior
and decision-making (Dalley and Roiser, 2012).
While several studies provide evidence that sero-
tonin and certain receptor subtypes are capable
of mediating genetic risk for addiction (Herman
and Balogh, 2012), increases in impulsivity are
proposed to be particularly problematic for drug
users due to impulsive behavior during relapse
after drug withdrawal. However, self-reports of
impulsivity from drug users have inconclusively
linked this cognitive deficit to addiction, where
some studies report no differences in impulsivity
scores between ecstasy users and nave controls
and others suggest that ecstasy use induces neu-
rocognitive deficits in impulsivity (Rogers etal.,
2009; Schilt etal., 2010). Notably, these types of
cognitive hypotheses are difficult to test and are
limited by not knowing the inherent responses in
participants and whether higher basal impulsiv-
ity is a risk factor for drug use or, rather, if ecstasy
use either directly or indirectly enhances impul-
sivity (Halpern etal.,2011).
Endocannabinoid Homeostasis
Endocannabinoids are a family of lipids capa-
ble of modulating neuronal synapses (Pertwee,
2006). Such endogenous cannabinoids as ara-
chidonoylethanolamide (or anandamide) and
2-arachidonoylglycerol (2-AG) interact with
the central G-protein coupled CB1 cannabinoid
receptors to mediate several neurobiological pro-
cesses (Pacher et al., 2006). The CB1 receptor is
thought to largely influence chemical messengers
such as GABA and glutamate, thereby affect-
ing learning and memory (Wang and Lupica,
2014). Notably, these receptors have been recog-
nized to be sensitive to the exogenous cannabi-
noid 9-tetrahydrocannabinol (9-THC) and
are involved with the drug effects of marijuana
(Clapper etal.,2009).
As with several other abused drugs, THC is
capable of enhancing DA neurotransmission in
the mesolimbic reward pathway. Intense inter-
est has been placed on investigating the mecha-
nism for these actions. With regard to marijuana,
THC-induced activation of CB1 receptors elicits
GABA inhibition in the NAc, allowing for an
514 Part IV:Homeostatic Therapies
increase in circulating DA levels and eventu-
ally leading to drug tolerance and addiction
(Clapper etal., 2009; Rashidy-Pour etal., 2013).
Rashidy-Pour et al. tested various synthetic
cannabinoids in rats and found that intracra-
nial injection of CB1 receptor agonists into the
VTA could potentiate the rewarding properties
of morphine in a CPP test. Interestingly, these
agonists independently produced rewarding
effects in the animals. It has been well docu-
mented that endocannabinoids released from
the postsynaptic neuron activate CB1 receptors
located presynaptically and so are thought to act
in a retrograde fashion (Pertwee, 2006). In this
way modulation of the endocannabinoid sys-
tem typically participates synergistically with
drugs of abuse to cause an increased reward
response and reinforcement. For example, sev-
eral studies report elevated endocannabinoids
following administration of alcohol, nicotine,
and morphine. Collectively, these studies show
reductions in drug self-administration, reward,
and withdrawal in CB1 receptor knockout mice
and following administration of CB1 receptor
antagonists (see Pacher etal., 2006, for a detailed
review).
Studies that investigate the changes in anan-
damide and 2-AG homeostasis in the various
stages of drug addiction target enzymatic deg-
radation of these proteins via fatty acid amid
hydrolysis (FAAH) and monoacylglycerol lipase
(MAGL), respectively (Maldonado et al., 2013).
Consensus from a review of the literature sug-
gests that in FAAH-deficient mice, the endo-
cannabinoid system is differentially involved in
consumption, reward, and withdrawal of etha-
nol and nicotine. Pharmacological inhibition of
FAAH activity by URB597 shows a more com-
plex effect, whereby cue-induced reinstatement
of nicotine seeking was reduced in rats but not
self-administration (Forget etal., 2009). Because
of the critical central role of 2-AG in energy
homeostasis, adverse effects are associated with
genetic manipulation of MAGL (Jung et al.,
2012), however, blockade with an MAGL inhibi-
tor (JZL184) in morphine-dependent mice was
able to reduce precipitated withdrawal induced
by naloxone (Ramesh etal., 2011). Selective inhi-
bition of MAGL over 2-AG completely alleviated
all measured signs of morphine withdrawal in
these mice (Ramesh et al., 2011). Moreover, the
inhibitors did not substitute for THC or other
cannabinoid receptor agonists, and, while pro-
viding great face validity as a model for morphine
withdrawal, they represent a reasonable new
target for the treatment of opiate dependence
(Ramesh etal.,2011).
Neurotransmitters and Receptors
Glutamate Homoeostasis
Glutamate is the main excitatory neurotransmit-
ter in the CNS. Ionotropic glutamate receptors,
located throughout the brain at synapses, bind
glutamate and allow for the influx of calcium
(Dingledine et al., 1999). The balance between
release and elimination of glutamate between
ionotropic receptors (-amino-3-hydroxy-5
-methyl-4-isoxazolepropionic acid [AMPA] and
N-methyl-D-aspartic acid [NMDA]), metabo-
tropic glutamate receptors (mGluR), and glial
cells is essential for the regulation of extracel-
lular glutamate. Glutamate neurotransmission is
of importance in addiction, largely with regard
to synaptic plasticity associated with drug rein-
forcement and reinstatement processes (Koob
and Le Moal, 2008). Pre- and postsynaptic glu-
tamate receptors located in the NAc are involved
in communication to the PFC, an area that is
heavily involved in generating learned behaviors
and behavioral adaptations to the environment
(Kalivas, 2009). Such corticostriatal connections
are altered in the presence of abused drugs. In the
literature, studies involving glutamate predomi-
nately investigate narcotic (opiates, e.g., heroin
and morphine) and stimulant (cocaine)drugs.
Reinforcement studies have demonstrated
that alterations in glutamate tone are part of the
process of the escalation of drug-taking. While
cocaine facilitates glutamate release, morphine
reduces the amount of extracellular glutamate
in some mesocorticolimbic areas of the brain
(i.e., VTA, NAc, dorsal striatum). Analyses of
AMPA and NMDA receptor expression in rats
after cocaine self-administration revealed upreg-
ulation of AMPA receptors in the VTA as an
effect of drug reinforcement (Choi et al., 2011).
Mechanistically, one theory involves changes
at dendritic spines. Morphological changes in
spine diameter and density via expression of
proteins for spine formation as well as expres-
sion of AMPA receptors and synaptic glutamate
transporters lead to increased excitability of
the brain regions involved, triggering cravings
and eventually drug tolerance (Choi etal., 2011;
Kalivas, 2009). Glutamate neurotransmission
has also been shown to modulate reinstatement
after drug withdrawal. Circulating glutamate lev-
els and AMPA receptor expression is reportedly

elevated in the PFC and NAc during cue-induced
drug-seeking following cocaine, heroin, and
methamphetamine self-administration, increas-
ing the reactivity of this area thereby inten-
sifying cravings and susceptibility to relapse
(Conrad et al., 2008; LaLumiere and Kalivas,
2008; Parsegian and See, 2014). Recent stud-
ies have also pointed to an adenosine and DA
receptor component for regulating the func-
tion and expression of AMPA receptors for drug
reinstatement. Specifically, Hobson et al. (2013)
suggest that stimulating adenosine A1 receptors
reduces AMPA receptor function via an antago-
nistic interaction with D1 receptors. The authors
results support the idea of the importance of
AMPA receptor signaling in the NAc for cocaine
reinstatement.
Glutamate homeostasis is also highly regu-
lated by transporters and exchangers on neurons
and nearby glial cells that modulate its release
and elimination. The excitatory amino acid trans-
porters (EAAT1-5) are responsible for glutamate
transport in many different brain regions, includ-
ing the striatum, hippocampus, and cerebellum.
Astrocytic EAAT2, also known as GLT-1 (glu-
tamate transporter-1), accounts for the major-
ity of glutamate transporter activity in the CNS
(Robinson, 1998). Studies have shown that GLT-1
mRNA is decreased in morphine-dependent rats
and that activation of the glutamate transporter
reduces morphine tolerance and dependence
(Wu etal., 2008). Yan and colleagues (2013a) also
investigated the mechanism for this and hypoth-
esized that aquaporin-4 (AQP4), which coexists
with GLT-1, is deficient in rodents, leading to
reduced opiate dependence. Interestingly, GLT-1
expression and glutamate reuptake was reduced
in wild-type mice treated with morphine, an
effect that was not observed in AQP4-deficient
mice (Yan et al., 2013a). The authors conclude
that regulation of glutamate homeostasis through
transporter (i.e., GLT-1 and AQP4) expression
may be a novel strategy for treating addiction.
GABA Homoeostasis
GABA homeostasis has been studied extensively in
the context of sedative drugs such as barbiturates,
benzodiazepines, and alcohol. Both the ionotropic
GABAA and metabotropic GABAB receptors
respond to GABA neurotransmitter and are capa-
ble of eliciting inhibitory responses from neurons
by either preventing firing of action potentials or
neurotransmitter release, respectively. Neurons in
the VTA are primarily DAergic and GABAergic
Brain Homeostasis and Addiction 515
in nature, and accordingly GABA has a major
role in modulating DA neurotransmission in this
part of the brain. Some drugs specifically target
the GABAergic neurons in the VTA and cause a
reduction in their activity. Consequently, GABA
inhibition of DA release is attenuated, thereby
enhancing the rewarding properties of such drugs
(Addolorato etal.,2012).
GABA A receptor physiology is quite complex.
The various subunits (i.e., , , , and ) come
together in many different combinations to form
a pentameric receptor (Uusi-Oukari and Korpi,
2010). Each subunit, with its subtypes, has been
implicated in drug addiction in various ways,
and at this time a specific molecular target, even
within this family of receptors, has not be iden-
tified. However, as evidenced by the number of
publications in databases of scientific journals,
it is clear that the GABAA 1 subunit receives
the most attention in addiction research, likely
due to the ubiquitous expression of this subtype.
Most studies of the GABA A receptor have investi-
gated alcohol addiction, primarily due to a more
comprehensive understanding of GABA receptor
physiology as well as behavioral similarities in
the sedative effects of ethanol and CNS depres-
sive effects after GABA receptor activation.
The role of GABA A receptors is complex and
differs depending on the stage of addiction and
the specific brain region that is targeted by the
drug. For instance, the expression of accumbal
GABA A receptors has been demonstrated to be
important for ethanol intake and reinforcement
in rodents where greater expression results in
more intake (Nie et al., 2011). In addition, this
receptor subtype is downregulated with chronic
ethanol exposure and also has been shown to be
endocytosed in the hippocampus during with-
drawal (Sarviharju etal., 2006; Shen etal., 2011).
Similarly, benzodiazepines elicit disinhibition of
GABA to increase the amount of circulating DA.
Several groups have proposed this mechanism, as
benzodiazepine drugs are allosteric modulators
of GABAA receptors and have been extensively
studied because of their high addictive liability
(e.g., alprazolam or Xanax and diazepam or
Valium). Benzodiazepines cause sleepiness and
have been used to treat anxiety, convulsions,
and sleep disorders. Tolerance and dependence
quickly develops, and this is likely due in part to
the drugs strong interaction with GABAA recep-
tors (Tan etal.,2010).
Drug responses and addictive behavior medi-
ated by GABAB receptors seemingly function
516 Part IV:Homeostatic Therapies
a little differently. Findings in the literature
point toward a potential for GABAB receptors to
reduce drug self-administration. The use of the
GABAB receptor agonist baclofen has shed some
light on the potential mechanism for its sup-
pression of drug-related behaviors. Presumably,
GABAB receptors located on DAergic cell bod-
ies in the VTA cause an inhibition of DA release
to the NAc (Filip and Frankowska, 2008). Using
rats and an intracranial self-stimulation para-
digm, Slattery etal. (2005) showed that baclofen
dose-dependently elevated the intracranial
self-stimulation reward threshold for cocaine.
The authors suggest that activating GABAB recep-
tors may represent a strategy for reducing the
rewarding effects of cocaine. However, a recent
ethanol self-administration study in baboons
showed that baclofen could reduce ethanol intake
but also consumption of vehicle, in this case the
orange-flavored drink Tang, suggesting that
the GABAB receptor agonist may just generally
increase consummatory behavior (Duke et al.,
2014). Tests of baclofen on alcohol-dependent
individuals are underway, and so far small clini-
cal trials have shown that the drug does not
produce any untoward side effects and can mod-
erately reduce alcohol intake (Leggio etal., 2010;
Leggio et al., 2013). Currently a phase 2 clinical
trial is ongoing to evaluate the safety and efficacy
of baclofen in abstinent individuals (clinicaltri-
als.gov, NCT01604330). However, more tests
are needed to determine the efficacy of GABAB
agonists for other drugs of abuse and stages of
addiction.
Cholinergic Homoeostasis
In the CNS, acetylcholine is a principle inhibi-
tory neurotransmitter with functions also as
a neuromodulator (Itier and Bertrand, 2001).
Acetylcholine is synthesized primarily in cholin-
ergic neurons of the basal forebrain from choline
and acetyl-CoA. The ligand-gated nicotinic and
G-protein coupled muscarinic (M1M5) acetyl-
choline receptors are responsible for the action
of this neurotransmitter (Hurst et al., 2013).
Nicotinic acetylcholine (nAch) receptors are
broadly classified as muscle type and neuronal
type and share structural homology with with
others in the cys-loop receptor family (i.e., GABA
and glycine receptors). Seventeen subtypes make
pentameric ligand-gated ion channels, resulting
in much variation in how the receptors can come
together and thus allowing very diverse actions.
While muscle nicotinic receptors always function
postsynaptically, neuronal forms are found both
presynaptically where they can influence the
release of multiple neurotransmitters and post-
synaptically (for classical neurotransmission;
Hurst etal., 2013). In the case of nicotinic recep-
tors, the high permeability to calcium allows for
great influence over intracellular signaling cas-
cades and thus gene activation and neurotrans-
mitter release (Hurst etal.,2013).
The cholinergic system has been shown to
be targeted by several different drugs of abuse.
The majority of the literature suggests that the
role of the cholinergic system is dependent on
the receptors involved within the drug response.
Accordingly, disruption in acetylcholine homeo-
stasis largely affects the level of reinforcement,
self-administration, and behaviors related to
withdrawal and relapse. For example, genetic
deletion of the muscarinic M5 receptor attenu-
ates cocaine self- and operant administration in
rats, and ethanol intake is significantly reduced
in mice after repeated administration of partial
nAch receptor agonists varenicline and cytisine
(Sotomayor-Zarate et al., 2013; Thomsen et al.,
2005). The authors report that the mechanism
behind these findings relates to muscarinic ace-
tylcholine receptor-dependent increase in DA
release in the VTA, a process that is lost with
deletion of the M5 receptor (Sotomayor-Zarate
etal., 2013; Thomsen etal., 2005). Alternatively,
muscarinic M4 receptor knockout mice have
increased cocaine operant self-administration
(Schmidt etal., 2011). In this case, Schmidt etal.
(2011) suggests that the deletion of M4 acetyl-
choline receptors, which colocalize with D1 DA
receptors on GABAergic interneurons within the
midbrain, may cause an indirect activation of DA
receptors.
With regard to drug behavioral effects, alter-
ations to acetylcholine homeostasis may affect
drug responses that lead to addiction as well as
withdrawal and relapse. Ethanol is of particular
interest as individuals often co-abuse alcohol and
cigarettes or nicotine (Dawson etal., 2013). In a
study where mice lacking the 2 subunit of nAch
receptors underwent a battery of ethanol-related
behavioral tests and self-administration, the
authors found that mice lacking this particular
subunit had reduced time to recovery from etha-
nol hypnosis, enhanced anxiety, and no change in
ethanol intake (Dawson etal., 2013). In another
study, the rewarding effect of morphine (meas-
ured via the CPP paradigm) was reduced in mus-
carinic M5 knockout mice (Basile et al., 2002).

In rats, pretreatment with the nAch receptor
antagonist mecamylamine caused a significant
reduction in the delayed discounting impulsivity
test when paired with cocaine (Xie etal., 2012).
The authors state that the data indicate that nAch
receptor populations, likely in central regions
such as the frontal cortex and NAc, are responsi-
ble for some impulsive decision-making and that
these processes in turn may influence relapse in
individuals addicted to cocaine (Xie etal., 2012).
Overall, these associations between drug behav-
iors and the discrete molecular targets often
identified by investigators from human genetic
studies, serve as a platform for further study and
provide promising targets for the development of
novel treatments for various stages of addiction.
Inflammatory Immune Responses
Many investigators have taken several approaches
to study the role of immune inflammatory
responses in addiction. These include looking
at changes in immune genes of drug addicts,
delivery of such genes to the brain of rodents
to assess alterations in behavior using animal
models of addiction, and investigating the effect
of pharmaceutical drug therapies on immunity
and inflammation (Crews et al., 2011). Recent
studies have shown that immune gene expres-
sion is altered in the presence of drug stimuli.
The transcription factor NF-kB (nuclear factor
kappa-light-chain-enhancer of activated B cells)
has been studied in this context due to its role
in activating the production of immune signal-
ing molecules such as chemokines and cytokines
(Crews et al., 2011). Highly expressed in mono-
cytes and microglia, NF-kB is necessary for
proper immune development, and dysregulation
of this transcription factor can induce inflamma-
tory responses (Crews and Vetreno, 2011; Garg
and Aggarwal,2002).
Microglia
Microglial cell signaling is essential to the brains
immune system. Microglia make up about 12% of
brain cells and have a very specific role in regulat-
ing inflammatory immune responses to infection
and injury. Microglia recognize threats, travel to
the site of injury, and in some instances act as
macrophages to phagocytose foreign materials.
These scavenger cells are activated for several rea-
sons, including altered extracellular potassium
levels and in the presence of proinflammatory
cytokines (Czirr and Wyss-Coray, 2012). In such
cases, their activated status calls for signaling
Brain Homeostasis and Addiction 517
and amplification of cell surface Toll-like recep-
tor proteins (Crews et al., 2011). Beyond this
stage, activated microglia secrete chemokines
leading to protease release (Crews et al., 2011).
These regulatory functions serve to return the
brain to a normal homeostatic environment.
Of interest to the topic of addiction, these cells
have been shown to be sensitive to and activated
by substance abuse. For example, a recent study
tested immune-suppressing drugs ibudilast and
minocycline for their ability to reduce metham-
phetamine self-administration in rats (Snider
etal., 2013). The rationale for these experiments
were based on previous work demonstrating that
drug use causes microglia-induced neuroinflam-
mation and that methamphetamine specifically
stimulates the production of proinflammatory
cytokines. Trained rats were systemically treated
with ibudilast and minocycline, resulting in
a significant reduction in methamphetamine
consumption (Snider etal., 2013). Although the
reduction of methamphetamine-induced glial
activation was not measured in these animals,
the findings suggest that modulating microglial
activity may be a new avenue for treating drug
addiction.
Cytokines
Cytokines are considered by some to be hor-
mones of the immune system that are responsible
for modulating responses to inflammation and
injury (Dinarello, 2000; Kronfol and Remick,
2000). Cytokines do not readily cross the
blood-brain barrier (BBB); however, most of these
small molecules can be produced and secreted in
the brain by astrocytes and microglia targeting
their respective receptors to modulate brain activ-
ity (Kronfol and Remick, 2000). Classification of
cytokines is dependent on their biological activ-
ity, and they are typically grouped into three
categories:proinflammatory, anti-inflammatory,
and hematopoietic (referring to the ability to
alter the responses of blood cells; Kronfol and
Remick, 2000). The proinflammatory cytokines,
interleukins (IL)-1 and -6, and tumor necrosis
factor-alpha (TNF-), have been implicated in
addiction with studies showing that several of the
ILs can increase sensitivity to amphetamines and
modulate the DA system (Kronfol and Remick,
2000). Methamphetamine increases IL-1 and
TNF- expression in the hypothalamus and NAc,
respectively (Yamada and Nabeshima, 2004). The
authors propose that TNF- acts as a neuropro-
tectant against drug addiction and neurotoxicity
518 Part IV:Homeostatic Therapies
by enhancing vesicular reuptake of DA (Yamada
and Nabeshima, 2004). A similar finding was
reported by Tien et al. (2011), demonstrating
that neonatal exposure to lipopolysaccharide
(LPS; an endotoxin responsible for the inflam-
matory effects of infection by Gram-negative
bacteria) causes increased behavioral sensitiza-
tion and reinstatement for methamphetamine.
Lipopolysaccharide exposure has been formerly
linked to persistent increases in inflammatory
cytokines IL-1 and IL-6 and dysregulation of
the DAergic system in the brains of rats (Tien
etal., 2011; Tien etal., 2013). The findings from
this study were interpreted to mean that inflam-
mation early in life may affect the susceptibility
to drug addiction later inlife.
Histamine
Some endocrine functions, particularly the
histaminergic system of the hypothalamus,
have been demonstrated to be able to modulate
immune inflammatory responses. Histamine
does not cross the BBB but instead is produced
locally and may also be found in the gut via
production in white blood cells (Brabant et al.,
2010; Smolinska etal., 2014). Typically the hypo-
thalamus has endocrine and metabolic functions
and, through the pituitary, links the nervous
system with several endocrine systems (e.g.,
hypothalamic-pituitary-adrenal axis [HPA] and
hypothalamic-pituitary-gonadal). The normal
physiological function of histamine is to elicit
local immune responses and to act as a neuro-
transmitter for the regulation of sleep patterns
(Smolinska etal., 2014). Because this molecule is
largely considered a proinflammatory signaling
molecule, it has been studied in the context of var-
ious neurological disorders such as schizophre-
nia and multiple sclerosis (Panula and Nuutinen,
2013). Behavioral neuroscientists have honed in
on histamine and its potential role in drug addic-
tion due to influences on the DAergic system and
brain reward (Brabant et al., 2010) and, more
recently, the abuse of over-the-counter antihis-
tamine drugs such as Benadryl (Forest, 2008).
Notably, in several species histamine innerva-
tion and the presence of histamine receptors
(i.e., H1, H2, and H3) has been found to be high in
brain areas that are deemed important for drug
addiction, including the NAc and VTA (Brabant
et al., 2010). Naturally these mechanisms could
translate to substance abuse and addiction. Early
studies into the relevance of histamine recep-
tor presence in these brain regions have led to
groundbreaking findings suggesting that antago-
nism or ablation of the histaminergic system can
promote the rewarding effects of drugs (Brabant
etal.,2010).
Administration of histaminergic drugs
causes changes in responses to amphetamines,
opioids, and ethanol. The behavioral effects
after administration of various abused drugs are
thought to be differentially mediated by the three
histamine receptors. Dimenhydrinate, an H1
receptor antagonist was shown to be a rewarding
drug for rats tested in the CPP paradigm (Halpert
et al., 2003). Stimulation of H3 receptors has an
opposite effect on drug behavior. For instance,
methamphetamine-induced increases in his-
tamine are significantly decreased by pretreat-
ment with histamine H3 agonists. Consequently,
the reduction of hypothalamic histamine levels
resulted in an increase in stereotypical behav-
iors induced by the administration of metham-
phetamine (Kitanaka et al., 2011). Additionally,
in investigating the effects of methamphetamine
administration on histamine release in mice,
Morisset et al. (2002) observed an increase in
histamine neuron activity in synaptosomes
of mouse cerebral cortex and also increased
tele-methylhistamine (a histamine metabolite)
levels in several brain regions. Studies by these
laboratories and others attribute such effects to
the direct or indirect modulation of midbrain DA
levels by histaminergic drugs. The caveat to some
of this work is the fact that histaminergic drugs
are not entirely specific to the three histamine
receptors and are capable of modulating other
neurotransmitter systems. Thus no clinical trials
are yet underway and future studies are needed
to establish the neural mechanisms underlying
alterations in drug reward by changes in hista-
mine homeostasis.
Endocrine Function
Sex Steroids
Although the majority of basic laboratory and
preclinical research utilize male subjects, those
that have studied addiction neural circuitry,
behavior, and underlying mechanisms in male
and female animals often report sex differences.
Such differences are likely due, in part, to differ-
ential patterns of brain organization and func-
tion between males and females. Considering
physiological differences that exist in circulating
sex hormones, it is reasonable to expect sexu-
ally dimorphic responses to drugs in males and
females. Even though sex differences cannot be

exclusively explained by differences in circulat-
ing levels of sex steroids, mounting evidence
support the notion that sex hormones do indeed
influence many of the previously discussed
homeostatic systems. Testosterone and estrogen
(i.e., estradiol) are well known for their func-
tions in vertebrate development and reproduc-
tion (for a comprehensive review, see Viveros
et al., 2012). The mechanism of action for the
former involves direct activation of androgen
receptors located throughout mammalian tissues
and subsequent genomic influence via transloca-
tion to the nucleus, or a conversion to estradiol
and binding to estrogen receptors (Hiipakka
and Liao, 1998). Similarly, cytosolic estrogens
bind to their respective receptors, which enter
the nucleus, bind to DNA, and alter gene expres-
sion (McEwen, 2002). Of interest to this discus-
sion, and keeping with the theme of homeostatic
systems, this section focuses on the role that sex
steroids play in mediating activation of the brain
in the presence of abused drugs, leading to the
progression of addiction. Surprisingly, in this
regard, fewer studies have investigated the direct
influence of testosterone on addictive behaviors
than estrogen (Carroll etal., 2004). Both testos-
terone and estrogen are derived from cholesterol
and produced in the testes of males and ovaries
of females, respectively; however, testosterone
function is not exclusive to males nor is estro-
gen to females (Neil and Kulkarni, 2011). Despite
being synthesized in areas outside of the brain
and their importance for reproductive functions,
these steroid hormones are permissible across the
BBB, and testosterone and estrogen receptors are
dispersed throughout thebrain.
Testosterone fluctuations in males are largely
related to social interactions and reproductive
status (Becker etal., 2012). As with hormone var-
iations during estrous in females, the pattern of
testosterone release in males may influence drug
behaviors. Due to the difficulty and potential
stressful confound of monitoring estrous status
in addiction animal models, most studies utilize
surgical alternatives such as gonadectomies and
hormone replacement to study the effect of ster-
oid hormones on drug behaviors. Vetter-OHagen
and colleagues (2011) found that ethanol prefer-
ence in gonadectomized male rats was higher
compared to gonadectomized males receiving
testosterone hormone replacement. In addi-
tion, although the amount of ethanol consumed
did not differ between the groups, testosterone
replacement in castrated males was sufficient to
Brain Homeostasis and Addiction 519
return ethanol intake to levels of intact males.
In a study of 10- to 12-year-old boys, regression
analysis of testosterone levels was able to pre-
dict illicit drug use and substance abuse prob-
lems during young adulthood (Reynolds et al.,
2007). Moreover, recent studies report reduced
testosterone during withdrawal in opiate- and
cocaine-dependent individuals (Wisniewski
etal., 2007; Yee etal., 2014). Undoubtedly, testos-
terone activity in the brain contributes to drink-
ing behavior, some suggest as an influence over
psychological factors; however, the mechanism(s)
have yet to be elucidated.
In humans, the prevalence of addiction to
alcohol and marijuana is high in males while,
conversely, women have been found to have
dependence problems with cocaine and psycho-
stimulant drugs (Becker et al., 2012). Females
also appear to be different than males in many
other drug-related measures, including earlier
age of first use as well as the rate of drug use esca-
lation and quantity (Becker et al., 2012). Many
researchers have focused on the involvement of
estrogen and its modulation during the female
menstrual cycle. Estrogen receptors alpha (ER)
and beta (ER) reside in the cytoplasm of cells
throughout many tissues; however, notably, the
ER subtype is localized in the brain (Matthews
and Gustafsson, 2003). Ovarian hormones appear
to primarily be involved with drug acquisition
and sensitization, though mounting evidence
points toward a role for such hormones in the
reinforcing effects of drugs. Studies of estradiol
in rodents have shown that the administration of
estradiol to gonadectomized females facilitates
the acquisition of cocaine self-administration
(Hu et al., 2004). This is supported by previ-
ous evidence that cocaine use varies over the
course of the estrous cycle (Hu et al., 2004).
Investigations of DA levels in the dialysate of ova-
riectomized females, castrated males, and intact
females and males revealed that after treatment
with estradiol, cocaine-induced DA release was
greater in the dorsolateral striatum (and not the
NAc) in females compared to males. In addition,
locomotor sensitization after systemic cocaine
administration was higher in females, indicat-
ing sex differences in behavioral sensitization
(Cummings et al., 2014). A recent review high-
lights the developing role of ovarian hormones
in mediating drug reward within the mesolimbic
area and HPA-axis, suggesting that processes that
are altered in dueling brain systems contribute to
a net change in brain reward, which eventually
520 Part IV:Homeostatic Therapies
leads to attenuation in positive drug reinforce-
ment (e.g., tolerance; Bobzean etal., 2014). Once
this occurs, the drug user then consumes increas-
ing amounts of drug to re-experience the initial
rewarding sensation (Bobzean etal., 2014). Taken
together, the authors results suggest that estra-
diol possibly mediates long-term neural changes
related to the progression from voluntary to com-
pulsive use (Cummings etal.,2014).
Neurosteroids
Unlike sex steroids, neurosteroids are hormones
produced de novo, directly in the brain (Helms
et al., 2012). Neurosteroids are derived from
local cholesterol or the metabolism of steroid
precursors that are synthesized in the adrenals
and gonads (Mellon and Griffin, 2002) and are
capable of modulating both excitatory and inhib-
itory (i.e., NMDA receptor and GABA recep-
tor, respectively) activity and neurotransmitter
release (Helms etal., 2012). While their normal
function is to provide rapid homeostatic modu-
lation of neurotransmission, pregnenolone sul-
fate, dehydroepiandrosterone sulfate (DHEAS),
and allopregnanolone (among others) are a few
naturally occurring neurosteroids that have
been recognized as regulatory molecules in drug
addiction. The pharmacological action of preg-
nenolone in the brain involves enhancement of
GABA A receptor activity (Besheer et al., 2010).
Allopregnanolone is also a GABAergic neuro-
steroid with hypnotic and anxiolytic effects in the
brain (Reddy, 2010). DHEAS, the sulfated metab-
olite produced from the conversion of DHEA to
testosterone, antagonizes GABA A receptor activ-
ity and has been implicated in age-related bone
and skin pathologies as well as neuropsychiatric
and mood disorders (Mo etal.,2006).
Collectively, investigations on neurosteroids
and abuse point toward the idea that neuroster-
oids may be a viable avenue for preventing addic-
tion to some substances. Studies have shown
that pregnenolone can reduce operant ethanol
intake in alcohol-preferring rats, a genetic model
of excessive alcohol intake (Besheer etal., 2010).
The mechanism for this is still under investi-
gation, but the authors suggest that the key to
pregnenolone-induced reduction of ethanol
intake is related to the alcohol-dependent pheno-
type and potentially the idea that this neuroster-
oid can improve cognitive deficitsa common
comorbidity linked to alcohol addiction (Besheer
et al., 2010). With regard to relapse to cocaine,
allopregnanolone has been shown to reduce
cue-induced reinstatement in rats (Schmoutz
et al., 2014). Conversely, chronic morphine
administration reduces the level of neurosteroids
(e.g., progesterone and pregnenolone sulfate) in
male rats (Yan and Hou, 2004). The authors report
that the reduction of neurosteroid concentration
was related to a morphine-induced inhibition of
the HPA axis resulting in lower levels of precur-
sors such as testosterone and corticosterone (Yan
and Hou, 2004). Using repeated administration
of morphine and naloxone-induced withdrawal
to test mice for the development and expression
of morphine dependence and tolerance, Ren and
colleagues (2004) found that DHEAS prevented
tolerance to morphine-induced analgesia and a
significant reduction in morphine dependence
measured via withdrawal symptoms.
Surprisingly, a recent extensive study of
drugs from all five classifications reported that
pregnenolone reduced the firing of VTA DA
neurons, increased the level of DA in the NAc,
and blunted behavioral disturbances only to
THC or Cannibis sativa (Vallee etal., 2014). It is
clear from the literature that the effects of neu-
rosteroid on addiction is complex and subject to
confounding factors, including species differ-
ences, endogenous levels of circulating steroids,
and experimental conditions (e.g., time of day;
Helms etal., 2012). However, neurosteroids still
warrant continued investigation as underlying
mechanisms may provide additional insight into
the diverse neuropathology of different abused
drugs and the advantage of allosteric modulation
of neurotransmitter systems.
Energy Balance and Metabolic
Disturbance
Energy balance is one of the basic processes
necessary to survival. The body is intricately
designed to maintain energy homeostasis, and
disturbances in metabolism can have highly dele-
terious effects throughout the peripheral nervous
system and CNS. The main signaling molecules
responsible for proper energy balance are peptide
hormones leptin, ghrelin, and insulin. Leptin and
ghrelin are two appetite-regulating hormones.
Leptin is produced primarily in white adipose
tissue and has also been found in the placenta,
stomach, and the pituitary (Ahima and Osei,
2004). The traditional role of leptin is to activate
receptors in the hypothalamus, thereby promot-
ing appetite suppression by counteracting the
feeding stimulants neuropeptide Y (Ahima and
Osei, 2004). Ghrelin acts in opposition to leptin

in that it stimulates hunger (Klok et al., 2007).
Human P/D1 (or rodent X/A-like) cells in the
stomach are central to the production and release
of ghrelin, which increases just before hunger and
diminishes after eating (Kirchner et al., 2012).
The main function of insulin, coming from beta
cells of the pancreas, is for carbohydrate and fat
metabolism. This is achieved by removing excess
glucose from the blood stream.
Feeding and other activities important for
survival (e.g., mating) are considered to be nat-
ural rewards. As such, when performing any
of these activities, the reward brain circuitry is
activated as a means to motivate toward these
behaviors. This is the same reward circuitry that
is hijacked by many drugs of abuse, and similar
DA-mediated mechanisms are at play. Notably, a
deficiency in energy status increases the salience
and hedonic value of food and drinks. Both lep-
tin and insulin are known to reduce food- and
drug-seeking in animals (Cummings etal., 2007).
Similarly, these energy homeostatic neuropep-
tides have influence over the mesocorticolimbic
DA reward system (Fulton et al., 2000; Jerlhag
et al., 2007). Preclinical studies have explored
the potential for leptin to modulate drug-related
behavior. Interestingly, leptin-deficient ob/ob
mice have been shown to have reductions in
their locomotor responses and sensitization to
amphetamine. These findings were substantiated
in other models, including a reversal in behav-
iors after a leptin infusion as well as reductions
in DA released from the NAc in slice prepara-
tions (Fulton et al., 2006). With regard to drug
reinstatement, Shalev etal. (2001) demonstrated
that intracerebral ventricular infusion of leptin
in rats can attenuate heroin-seeking induced by
food deprivation. The authors theorize that lep-
tin is involved in modulating motivational states
linked to food restriction or deprivation (Shalev
etal., 2001). In contrast, ghrelin, when adminis-
tered to the VTA and ventricles of mice, causes
a significant increase in ethanol consumption.
Additionally, accumbal DA release and alcohol
CPP was abolished in ghrelin receptor knock-
out mice as well as wild-type mice treated with
receptor antagonists (Jerlhag etal.,2009).
Fewer studies can be found in the literature
relating energy homeostasis and drug addiction
in humans. A meta-analysis by Aguiar-Nemer
and colleagues (2013) of publications on the topic
revealed that leptin levels may be considered a
biomarker for the risk of relapse in alcoholics
and smokers. The 12 studies identified during
Brain Homeostasis and Addiction 521
the analysis indicated that leptin increases dur-
ing abstinence is potentially related to a reduc-
tion in DAergic activity in the reward system
(Aguiar-Nemer et al., 2013). Finally, a study of
the effects of ghrelin on alcohol craving and use
in heavy drinkers is being evaluated in a clini-
cal trial that is currently recruiting participants
and is slated to complete by the fall of 2020.
Participants will be tested for alcohol craving
and use after receiving an infusion of ghrelin or
placebo, and investigators will use fMRI to assess
function of brain regions associated with incen-
tive salience and reward (clinicaltrials.gov, NCT
NCT01779024).
Neurotrophic Factors
Neurotrophic factors are a family of signaling
molecules with many integral roles in the brain,
including neuronal growth and maturation,
maintenance of mature neurons, and synap-
tic plasticity (Autry and Monteggia, 2012; Poon
et al., 2013). Within the family are three clas-
sifications (a) neurotrophins including neuro-
trophin -3 and -4, nerve growth factor (NGF),
and brain-derived neurotrophic factor (BDNF);
(b) glial cell line-derived neurotrophic factor
family ligands (e.g., glial cell line-derived neu-
rotrophic factor [GDNF]); and (c) neuropoietic
cytokines (Deister and Schmidt, 2006). Since the
discovery of these proteins more than several
decades ago, they continue to be intensely stud-
ied in order to fully understand the diversity of
their biological roles (Bolanos and Nestler, 2004).
While a few studies have looked at the contri-
bution of NGF and neurotrophin-3 in addictive
behavior, BDNF and GDNF have received the
most attention to date with regard to synaptic
plasticity and various neuropsychiatric disorders
(Autry and Monteggia, 2012). Thus this section
focuses specifically on the links between drug
addiction and brain homeostasis involving these
two neurotrophic factors.
BDNF
Within the past couple of decades, a potential
link between neurotrophic factors and addic-
tion has been made via chronic drug studies
in animals while investigating the underlying
mechanisms for observed plasticity in mesocor-
ticolimbic regions (Bolanos and Nestler, 2004).
Mounting evidence supports the notion that
BDNF is an important modulator of drug
reward. Protein and mRNA of this neurotrophic
factor is expressed in mesocorticolimbic areas,
522 Part IV:Homeostatic Therapies
and several groups have reported that cocaine,
ethanol, and amphetamine alter the levels of
BDNF in several brain regions key to reward pro-
cesses, including the striatum, frontal cortex, and
the amygdala (Meredith etal., 2002; Raivio etal.,
2012). Alcohol abuse appears to be more complex
regarding BDNF. Logrip etal. (2009) found that
acute ethanol self-administration increases cor-
tical BDNF expression in male C57Bl6/J mice,
whereas long-term exposure (6 weeks) to ethanol
reduces BDNF in the cortex. The authors con-
sider such findings to be representative of how
BDNF is involved in the escalation of drug use
to addiction. In this study BDNF appeared to be
protective early on but did not recover 2 weeks
after ethanol deprivation, indicating that this
neurotrophic factor may be involved with the
inflexibility of drug addiction (Grimm et al.,
2003; Logrip etal.,2009).
To directly assess the role of BDNF signaling
on addictive behaviors, some groups have investi-
gated drug-seeking and relapse after BDNF infu-
sion into reward areas. While a study by Berglind
etal. (2007) reports that infusing BDNF into the
medial PFC suppresses cocaine-seeking, another
group has shown that cocaine-seeking behavior
is enhanced for up to 30 days after withdrawal
in rats receiving intra-VTA infusions of BDNF
(Lu etal., 2004). Notably, there was no effect of
NGF infusion on cocaine-seeking or BDNF infu-
sion when delivered to the substantia nigra (Lu
et al., 2004). Although differential roles of vari-
ous brain regions are evident, these data support
the notion of BDNF-mediated development of
drug addiction.
GDNF
GDNF is expressed throughout the CNS and at its
highest levels in the striatum, thalamus, cortex,
and hippocampus. GDNF activity in the adult
brain is primarily located in the midbrain where
its expression is kept low under normal condi-
tions and then supplied to regions of injury via
retrograde transport by the striatum (Carnicella
and Ron, 2009). Interestingly, studies show that
this neurotrophic factor has a mostly universal
effect on drug self-administration, reward, and
relapse in that activating GDNF signaling attenu-
ates drug (i.e., psychostimulant, morphine, and
ethanol) behaviors. This has been demonstrated
by intra-VTA infusions of GDNF in rats as well as
infusion of anti-GDNF antibodies in mice, result-
ing in significant reductions in cocaine reward
or place preference and locomotor sensitization
responses following chronic drug administration
(Messer etal., 2000). Similarly, GDNF attenuates
ethanol self-administration and relapse when
infused into the VTA but not the substantia nigra
of rats (Carnicella etal., 2008)and significantly
reduces methamphetamine self-administration
and relapse when delivered to the striatum in
mice (Yan etal., 2013b).
Since neurotrophic factor signaling path-
ways are so vast and critical to the mainte-
nance and survival of neurons, one idea for the
way they facilitate various stages of addiction,
mainly reward and relapse, is based on findings
that these proteins can alter neuronal plasticity.
Although the mechanisms are still under inves-
tigation, several groups report that increased
BDNF expression enhances dendritic formation
(Bolanos and Nestler, 2004). As a consequence
of increased synaptic connections at dendrites,
synaptic strength and neuronal signaling is also
altered (Bolanos and Nestler, 2004). Furthermore,
neurotrophic factors activate several complex
signaling pathways involving phospholipase C,
phosphatidylinositol-3-kinase, and extracellu-
lar signal-regulated protein kinase. In doing so,
and depending on which pathway is activated, a
rise in intracellular calcium or cellular structural
changes is initiated and thus long-term poten-
tiation is increased, causing an elevation in drug
sensitivity and reward (Bolanos and Nestler,
2004). Surely these mechanisms come into play
during chronic drug exposure, and so it is plausi-
ble to attribute some aspects of addiction to plas-
ticity changes mediated by neurotrophic factors.
A LT E R N AT I V E A D D I C T I O N
THERAPIES
Most currently approved treatments for drug
addiction utilize pharmacotherapies that mod-
ulate the various neurotransmitter systems or
act as replacement substances. Interestingly, in
the United States the only treatments approved
by the Federal Drug Administration are use-
ful for alcohol, nicotine, and opioid addiction.
Those suffering with alcohol use disorders have
the most options. Aside from the moderate suc-
cess of self-help programs such as Alcoholics
Anonymous, which describes necessary actions
and spirituality leading toward recovery (Detar,
2011), pharmacological interventions are availa-
ble, including the alcohol dehydrogenase blocker
disulfiram (Antabuse), the opioid antagonizing
drug naltrexone (Vivitrol ), and NMDA recep-
tor modulator acamprosate (Campral ). Of late,

clinical trials have tested the anti-alcohol efficacy
of nalmefene, an opioid antagonist with a longer
half-life, better bioavailability, and reduced tox-
icity compared to naltrexone (Hoofnagle, 2012).
Two large studies show that pharmacological
treatment with nalmefene effectively reduces
alcohol intake in dependent individuals who
are unable to reduce consumption on their own
(Gual et al., 2013; van den Brink et al., 2013).
Opioid addiction is often treated with the syn-
thetic opioids methadone or buprenorphine.
Similarly, nicotine-dependent individuals have
the option of pharmacological treatment with
nicotine replacements or varenicline, which is
a partial nicotinic acetylcholine receptor ago-
nist (Tomek et al., 2013). Although these treat-
ments are available, they have limited efficacy,
and addiction remains a pervasive problem, par-
tially due to patient noncompliance and also due
to a poor understanding and the complexity of
underlying mechanisms of action of these drugs.
Accordingly, scientists are seeking alternative
strategies for blocking the progression to addic-
tion and preventing relapse.
Researchers are beginning to investigate the
ability of biologic compounds to act as thera-
pies for addiction to certain drugs. Several com-
pelling recent preclinical studies suggest that
N-acetylcysteine (NAC) may be a novel thera-
peutic for cocaine addiction. NAC is a cysteine
prodrug that purportedly acts at mGluR2/3
receptors to facilitate the exchange of extracel-
lular cysteine for intracellular glutamate, thereby
normalizing glutamate levels (Moran et al.,
2005). This process is important for cocaine
addicts since chronic use of this psychostimulant
drug results in reduced levels of glutamate out-
side of the cells within the NAc and PFC regions
(Moran et al., 2005). Taking this work further,
Murray and colleagues (2012) tested the utility
of NAC as an antidrug therapy in rats using an
operant self-administration paradigm, finding
that animals reduced cocaine-seeking in both
early- and late-stage addiction without affecting
the amount of cocaine consumed. The authors
concluded that this work supports the potential
for NAC to act as a therapy to prevent relapse,
but not reinforcement, for cocaine via regulation
of glutamate homeostasis. Since then, NAC has
also been tested clinically in cocaine-dependent
patients and has resulted in similar effects as
seen in the preclinical studies (Schmaal et al.,
2012). In humans, the link between NAC and
regulation of glutamate homeostasis seems to
Brain Homeostasis and Addiction 523
be centered on impulsivity, which is a predic-
tor of relapse in drug addiction. Schmaal et al.
(2012) was able to show that impulsivity scores in
cocaine-addicted patients were predictive of the
amount of NAC-induced changes in glutamate.
Although this study had limitations related to
relatively moderate sample sizes and confound-
ing comorbid addictions in the patient popu-
lation (e.g., alcohol and nicotine), others have
provided evidence that NAC may work therapeu-
tically in patients with dependence on marijuana,
pathological gambling, and nicotine (Schmaal
etal.,2012).
Promising new therapies have come to frui-
tion for those with cannabis (or marijuana)
substance abuse and dependence problems.
These largely exploit the notion that those with
cannabis addiction are also more likely to have
concomitant mental health problems. Such
pharmacotherapies as lithium and divalproex,
two mood-stabilizing drugs, have been shown
to improve cannabis withdrawal symptoms
(Danovitch and Gorelick, 2012). Nonetheless, the
continued development of alternative treatments
for addiction is slow, costly, and largely limited
by the complexity of drug interactions with dif-
ferent parts of the brain. Accordingly, research-
ers are turning to natural interventions that are
capable of modulating several brain homeostatic
mechanisms.
Exercise
Some say that there are similarities between the
act of using drugs and the act of exercise. This
commonality is thought to exist based on the
fact that both produce positive affective states.
Preclinical studies support the idea that exercise
may have many benefits for substance-dependent
people. Particularly with regard to cocaine and
methamphetamine, exercise has been shown to
reduce self-administration at several stages of
addition throughout the progression from ini-
tial drug use to abuse and dependence (Smith
and Lynch, 2011). Several drugs have been stud-
ied for the effect of exercise on reducing intake
and progression to addiction, with much of the
work focused on drug withdrawal and reinstate-
ment after abstinence. Devaud and colleagues
(2012) report that rats with access to running
wheels that voluntarily exercised (measured
as wheel turns) had a significant increase in
pentylenetetrazol-induced seizure threshold after
removal from an ethanol liquid diet. In an oper-
ant responding paradigm, rats that had access to
524 Part IV:Homeostatic Therapies
a running wheel displayed a reduction in rein-
statement after extinction and abstinence from
cocaine self-administration compared to seden-
tary rats (Smith etal., 2012). In recent years the
Solinas group has demonstrated in a number of
studies that environmental enrichment (EE; i.e.,
access to ramps, running wheels, and toys) causes
a significant reduction in craving and the pro-
pensity for relapse. Cocaine-seeking in rats was
reduced after 1-week exposure to EE compared
to standard environment, and this effect was
lost upon discontinuation of the enriched hous-
ing conditions (Chauvet etal., 2012). In addition,
studies show that exercise attenuates the positive
reinforcing effect in rodents when challenged
with other drugs of abuse such as heroin (Smith
and Pitts, 2012). The ability for exercise to slow
the escalation from drug use to abuse has been
demonstrated to be more effective in adolescent
rodents than adult rodents (Zlebnik etal., 2012).
Moreover, humans undergoing treatment for
substance-use disorders have been more success-
ful with the incorporation of exercise (Weinstock
etal.,2008).
Behavioral and psychological mechanisms
involved in the ability of exercise to reduce drug
intake are likely related to (a) reductions of the
drug as a positive reinforcer when both the
drug and exercise are available at the same time;
(b) a reduction in potential comorbid disorders
(e.g., depression or anxiety), which are risk fac-
tors of substance abuse and reduced by exercise
in humans; and (c) a general improvement in
affective state since in most conditions exercise
increases feelings of well-being and self-esteem
in humans (Smith and Lynch, 2011). Nader etal.
(2012) substantiated this theory in a study of the
influence of EE versus standard environment
on cocaine addiction in mice. In this study, the
authors found that in young mice, removal of EE
after exposure prevented the protective effect of
the enriched conditions and thus these animals
reward for cocaine was higher and they were more
vulnerable to addiction. This effect was attrib-
uted to emotional distress related to the loss of
EE measured via levels of corticotropin-releasing
factor (Nader etal., 2012). Related to the neuro-
biology behind exercise-induced reductions in
drug-taking, many lines of evidence suggest the
involvement of neurotransmitters also relevant
to drug addiction. For example, studies show that
exercise is capable of increasing DA in the rodent
basal ganglia (Meeusen etal., 1997). In addition,
increased glutamate levels caused by ischemia are
normalized after exercise, a mechanism that may
similarly be related to exercise-induced attenua-
tion of drug-seeking behavior and relapse (Smith
and Lynch, 2011). Neurotrophins have also been
implicated, as several studies support the idea
that exercise promotes neurogenesis in the hip-
pocampus, a process that may be involved in the
prevention of drug- and cue-induced cravings
and relapse (Smith and Lynch,2011).
Although the effect of exercise-mediated
alterations in brain homeostasis on addictive
behavior seems to be an interesting avenue, sev-
eral questions remain unanswered. Studies inves-
tigating how much exercise and when exercise
needs to be performed in relation to drug use and
the progression to addiction is one. Furthermore,
of the different types of exercise, which type is
the most therapeutic? Another area for more
research is whether sex hormones, which defi-
nitely influence addictive behavior, have differ-
ential effects of exercise in males and females.
And last, gaining a better understanding of the
underlying biological mechanisms involved and
how this relates to alterations of brain homeosta-
sis after drug use will help inform clinicians on
the best strategies for exercise as an alternative or
conjunctive therapy for addiction.
Mindfulness Training and Meditation
Mindfulness training is a technique that theo-
retically involves shifting ones attention to the
immediate experience in a nonjudgmental and
accepting kind of way. This skill is at the core of
meditation, is often learned through repetition
or practice, and has been shown to be particu-
larly beneficial in individuals with concomi-
tant mental health and substance use disorders
(Brewer etal., 2010). Meditation is rooted in reli-
gious practices (i.e., Hindu-based transcendental
meditation or Buddhist meditation); however,
although mindfulness relies on Buddhist ideas,
when used therapeutically the spiritual associa-
tion is not made (Dakwar and Levin, 2009). Two
major aspects of mindfulness training involve
the development of improved attention and
acceptance skill sets in those with addiction. This
allows the individual to pay attention to present
internal and external stimuli and become accept-
ing of these observations in a nonjudgmental
way. Mindfulness-training techniques have been
applied as ways to manage stress, change behav-
ior, aid cognition, and prevent relapse. Relapse
prevention therapy is not mindfulness treatment
per se but rather uses mindfulness as a strategy to

help manage cravings. For example, addicts are
trained to expect that cravings will occur but will
eventually pass and to accept and learn to deal
with them (Dakwar and Levin,2009).
A growing number of clinical studies have
investigated the effectiveness of mindful-
ness training for substance misuse and abuse.
Currently, the literature provides evidence that a
mindfulness approach, sometimes coupled with
more contemporary treatments, can be useful for
preventing anxiety, depression, stress, cravings,
and impulsive thinking (Bowen and Enkema,
2014). Such studies utilize various mindfulness
techniques and individuals with differing racial
backgrounds and addictions to a variety of drugs
(i.e., alcohol, cocaine, methamphetamine, and
marijuana), and all report positive relationships
between psychological states after this therapy
and craving and drug use during the subsequent
months (de Dios et al., 2012; Witkiewitz and
Bowen, 2010). Furthermore, when polysubstance
abusers were tested in measures of working mem-
ory, behavioral inhibition, and decision-making,
those who received mindfulness training per-
formed better than those who did not (Alfonso
etal., 2011). The authors believe that these tech-
niques warrant continued examination to deter-
mine whether mindfulness training represents a
novel therapy for reducing neuropsychological
deficits and relapse in drug addicts.
The proposed theory for the efficacy of mind-
fulness training and meditation is related to a
potential role in attenuating cravings, facilitating
cue extinction, and reducing compulsive behav-
iors (Dakwar and Levin, 2009). Neurobiological
information on this hypothesis is difficult to
attain since all studies are done on humans.
Clinicians have been strategic in their experi-
mental design to investigate mechanisms behind
the efficacy of mindfulness training. Subject
plasma analysis and also brain imaging can be
applied pre-, post-, and/or during the interven-
tion. The release of the stress-induced hormone
cortisol is a common pathological trait in addicts
that has been linked to craving and drug use
(Brewer etal., 2010). Thus it is logical to believe
that attention and acceptance of the present with
a nonjudgmental attitude instead of rumination
would elicit alterations in circulating cortisol in
those with substance abuse problems; however,
this has not been explicitly studied. In addition,
neural mechanisms to support reported changes
in memory, decision-making, and impulsivity
resultant from mindfulness training have been
Brain Homeostasis and Addiction 525
empirically tested using functional and struc-
tural neuroimaging. Findings from several stud-
ies show that neuroplastic changes occur in the
fronto-limbic areas, and others after mindfulness
mediation (for a review, see Hlzel et al., 2011),
and that these are likely to be beneficial for recov-
ery from addiction.
A major limitation is that much of the work
in the field of mindfulness training and addic-
tion utilizes small sample sizes. This raises the
question as to whether studies with nonsignifi-
cant effects actually lack statistical power. In
addition, as mentioned earlier, investigators may
employ different styles of mindfulness training
and often cast a wide net for study participants.
Although more controlled studies are needed on
the different types of mindfulness techniques
and their utility for treating substance abuse,
mindfulness-based meditation has great poten-
tial for restoring brain homeostasis in addicts,
especially in light of success with various appli-
cations such as stress reduction and behavior
modification.
Acupuncture
Acupuncture, a form of traditional Chinese med-
icine, is a technique involving stimulation of var-
ious points along the skin using needles, heat, or
pressure (Ernst, 2006). This practice is thought to
restore the imbalance of flow (or qi) along merid-
ians in the body (Ernst, 2006). The prevalence
of the use of acupuncture to treat drug-related
disorders in the United States was climbing until
2002 when two large-scale clinical trials reported
negative results (White, 2013). These two studies
showed no difference between the acupuncture
and sham treatment in maintaining abstinence
in individuals dependent on alcohol and cocaine
(White, 2013). Despite the lack of research evi-
dence to support the ability of acupuncture to
reduce drug dependence, it is still used in clinical
practice for this problem. White (2013) believes
that this is due to the large variability in the many
parameters involved with acupuncture, including
bilateral versus unilateral application of needles
and the use of electrical stimulation. In addition,
acupuncture methods appear to be successful
on discrete components of the drug-addiction
stages (i.e., craving and withdrawal; White,
2013). Arecent study that examined tobacco ces-
sation within the Chinese population at a treat-
ment center found that individuals who elected
to receive acupuncture in addition to nicotine
replacement therapy had higher cessation rates
526 Part IV:Homeostatic Therapies
after 6 months (Chang et al., 2013). Although
there were several limitations to the study, the
results suggest that acupuncture could be a bene-
ficial and attractive supplementary treatment for
addictions to a wide range of substances. Further
evaluation is needed, but acupuncture appears to
be a safe, potential alternative therapy for drug
addiction.
Herbal Remedies
In China, herbal remedies have long been a
way to treat ailments. However, only within the
past couple of decades has this approach come
into acceptance elsewhere. Clinical trials have
recently brought to light the notion that herbal
medicine can be an alternative strategy to tra-
ditional pharmacological treatments for addic-
tion. In addition, discoveries providing possible
mechanisms of action for various herbal com-
pounds support their utility and aid in the design
of new therapies. To date, several traditional
herbs have been tested for their ability to reduce
behaviors associated with addiction, and some
have even gained popularity in Western culture.
Plant extracts such as ginseng, Kudzu, kava, and
St. Johns Wort are pharmacologically active and
have been described as beneficial for treating a
variety of ailments.
One of the most popular and most stud-
ied herbs is ginseng (Panax quinquefolium in
America). The major compound responsible for
most action(s) of ginseng in the CNS is ginse-
nosides. More than 20 ginsensoides have been
identified and are thought to have an inhibitory
and excitatory influence on neurotransmission,
effecting learning and memory and nociception
(Attele et al., 1999). Because of the diversity of
ginsengs structural constituents and effects on
the brain, it has gained attention for its potential
as an anti-addiction remedy. This herb report-
edly reduces methamphetamine and morphine
behaviors in rodents, an effect resultant from
the antagonist action of psuedoginsenoside-F11
(PF11) on opioid signaling and DA release (Wu
et al., 2003). Ginseng is known to have both
excitatory and inhibitory actions in the CNS, but
in a recent study by Hao and colleagues (2007),
mice that were pretreated with PF11 followed by a
systemic injection of morphine showed no devel-
opment of behavioral sensitization in locomotor
responses, thereby suggesting an inhibition of
morphine-induced DAergic neurotransmission.
Furthermore, in vivo microdialysis revealed that
animals pretreated chronically or with higher
doses of PF11 had a reduction in the level of
morphine-induced reduction of extracellular
glutamate in the medial PFC (Hao etal., 2007).
Taken together, PF11 may represent a novel ther-
apy for addiction, particularly to morphine and
methamphetamine relapse.
Daidzin and daidzein, two extracts from the
root of the East Asian Puerariae plant (or Kudzu),
have been used to treat hypertension and reduce
fever and nausea (Lu et al., 2009). These herbal
extracts reportedly reduce metabolism of liver
monoamines and serotonin. Additionally, with
regard to alcohol, daidzin is capable of inhib-
iting mitochondrial aldehyde dehydrogenase
in the liver (Overstreet et al., 2003). Kudzu has
been studied for the ability to reduce free-choice,
continuous-access ethanol intake in rodents.
Although at this time few preclinical studies
exist, this natural medicine has been tested in
human clinical trials where alcohol consump-
tion in alcohol-dependent individuals was exam-
ined following treatment with Kudzu root. The
results have been mixed. In a recent trial, Lukas
etal. (2005) reported that subjects who received
and took the herbal remedy for a period of 7 con-
secutive days drank significantly fewer alcoholic
beverages during laboratory testing compared
to the number of beverages consumed prior to
treatment. The authors provide evidence that
Kudzu may work by enhancing the effects of the
first beverage, since pretest craving scores were
not significantly altered by Kudzu treatment.
Because those treated with Kudzu drank smaller
volumes and took more sips of alcohol for subse-
quent drinks, also reporting feeling more intoxi-
cated after the first drink, Kudzu appears to be a
way to curb binge-drinking (Lukas etal., 2005).
Compared to other trials, the one by Lukas and
colleagues differs in that a natural setting (i.e.,
the lab testing area resembled a small studio con-
taining an entertainment center, kitchen, and
couches) was utilized, a more concentrated dose
of Kudzu was tested similar to preclinical testing,
and the amount of active ingredient and com-
pliance was verified by urine analysis. Differing
from the aforementioned study, a recently listed
clinical trial will test the efficacy of Kudzu in
heavy drinkers predetermined to be alcohol
dependent (clinicaltrials.gov, NCT01853293).
The interaction of Kudzu with the metabolism
of ethanol has been well documented. Acting
as an inhibitor of acetaldehyde dehydrogenase,
much like disulfiram, this natural medicine has
the potential to be a strong anti-alcohol therapy.

Additionally, Kudzu binds to benzodiazepine
receptors on GABA receptors in brain regions
that are neural substrates of alcohol (e.g., cortex,
cerebellum, and hippocampus; Lu etal.,2009).
Lesser-studied plant extracts such as kava and
St. Johns Wort have been clinically used for the
treatment of psychological disorders such as anx-
iety and depression, respectively (Lu etal., 2009;
Overstreet et al., 2003). Notably, lipid-soluble
kava lactones and St. Johns Wort modulate brain
neurotransmitter concentrations (e.g., DA, sero-
tonin, and GABA), allowing for a role in promot-
ing abstinence and reducing cravings in addicts
(Lu etal., 2009; Overstreet etal., 2003). Thorough
preclinical testing and human clinical trials are
still needed to better understand efficacy, mech-
anisms, and the interaction of various drugs of
abuse with these herbal medicines. In addition,
herbs such as Kudzu and kava are heavily regu-
lated as they can be very toxic, particularly to the
liver. Some of these herbs have been utilized for
centuries in other parts of the world; however,
their safety and toxicity for the masses have not
been thoroughly investigated and documented.
CONCLUSIONS
In summary, brain homeostasis is critical for
proper physiological function and also appears
to be very much involved in the progression of
drug use to addiction. Drug interactions with
many of the homeostatic systems eventually
lead to the modulation of DA-related neuronal
activity in the mesocorticolimbic regions as well
as influence excitatory and inhibitory neural
transmission. Despite the need for much more
testing, the likelihood that brain homeosta-
sis manipulation of various stages of addiction
seems plausible. Current investigations along the
lines of alternative treatment options (e.g., diet,
exercise, meditation, acupuncture, and herbal
medicines) for addiction are promising as ways
to overcome some of the challenges related to the
time and funds required to develop new phar-
macotherapies. Natural treatments as alternative
ways to restore normal brain homeostasis should
also prove beneficial in continuing to inform
researchers on the complex neurological disorder
of addiction.
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28
Anxiety and Stress Disorders
E VA M A R A M A R C O A N D M A R A - PA Z V I V E R O S
INTRODUCTION
Neurobiological Basis
ofEmotional Homeostasis
All living organisms try to achieve equilibrium
with the changing environment, which is called
homeostasis. Emotional homeostasis attempts to
achieve a balance in an individuals emotional
state; however, maintaining stability against
physical and psychological events that may hap-
pen in everyday life frequently requires a change
in homeostasis, known as allostasis. The brain,
through a great variety of complementary and
often redundant mechanisms, exerts a principal
role not only in maintaining emotional homeo-
stasis but also in adapting to situations that
may threaten survival. Notably, if these adap-
tive mechanisms do not cease when the threat
ends, or are overused by excessive exposure to
challenges, then the body and brain acquire
cumulative changes, called allostatic load, that
may increase the risk for psychopathologies,
including anxiety and stress-related disorders
(McEwen,2003).
Emotional processing comprises the
evaluation of the emotional salience of the
stimulus perceived (e.g., appetitive vs. aversive),
its relationship with previous experiences, and
the context in which it arises. This process also
includes the behavioral, endocrine, and auto-
nomic manifestations of the emotional response,
as well as the subjective feelings that accompany
this response. Abnormal responses to threat or
stress may lead to fear and anxiety, the emotional
processes that have been the most extensively
studied. In general, the valuation of external
or internal stimuli is formed by limbic brain
structures, which include the hippocampus, the
amygdala, and prefrontal cortex (for a review
see de Kloet et al., 2005; Jankord and Herman,
2008; Joels and Baram, 2009). Here we focus
on these brain areas that are key central nodes
in the control of emotional homeostasis and
stress response. The adaptive response to physi-
cal and/or psychological challenges comprises a
peripheral hormonal response through activation
of the hypothalamic-pituitary-adrenal (HPA)
axis, together with an autonomic response medi-
ated by the sympathetic and parasympathetic
nervous systems. These hormonal and autonomic
responses mediate important adaptive functions
in preparing the organism for responding to
threat or stress, by increasing vigilance, modu-
lating memory, mobilizing energy stores, and ele-
vating cardiovascular function. On the one hand,
glucocorticoid hormones (cortisol in humans
and corticosterone in rodents), the principle
endproducts of the HPA axis, act on the brain
to maintain homeostatic balance. First, stress
stimulates the release of corticotropin-releasing
hormone (CRH) from the paraventricular
nucleus (PVN) of the hypothalamus and amyg-
dala. The CRH secretion from the PVN, in turn,
increases peripheral adrenocorticotropic levels,
which stimulate the adrenal glands to secrete glu-
cocorticoid hormones. Glucocorticoids activate
mineralocorticoid and glucocorticoid receptors
that, once coupled to the hormone, translocate to
the cell nucleus where they act as regulators of
gene transcription. On the other hand, stimula-
tion of the lateral nucleus of the hypothalamus
activates the sympathetic system, provoking
an increase in blood pressure and heart rate,
sweating, piloerection, and pupillary dilatation,
whereas the innervation of the parasympathetic
nervous systems, mainly the vagus and splanch-
nic nerves, from limbic regions is thought to
mediate visceral symptoms affecting the gastro-
intestinal and genitourinary systems, also altered
in anxiety disorders (see de Kloet et al., 2005;
Joels and Baram, 2009; Ulrich-Lai and Herman,
2009, for extended reviews on the subject).
536 Part IV:Homeostatic Therapies
Neural Circuitries
The amygdala is the integration site within the
limbic network where the generation of fear
and anxiety occurs in response to environmen-
tal stimuli. The amygdala is critically involved
in emotion processing; this brain structure
receives perceptual informationsimple and
rapidlythrough the sensory thalamus but also
highly processed informationmore veridi-
cal but slowervia the sensory cortex. Both the
thalamic and the cortical pathway project to the
lateral nucleus of the amygdala, classically con-
sidered the sensory interface of the amygdala
and a key site of plasticity. In contrast, the lateral
nucleus of the amygdala projects, both directly
and indirectly, to the central nucleus, the out-
put region of the amygdala. The central nucleus
and the medial amygdaloid nucleus have lim-
ited projections to the PVN, and also through
-aminobutyric acid (GABA) neurons to the bed
nucleus of the stria terminalis; thus it has been
suggested that the activation of the HPA axis may
result from a disinhibitory process (Jankord and
Herman, 2008). Taken together, the amygdala
participates in implicit learning, emotion pro-
cessing, and memory storage; it is not only lim-
ited to fear. In addition, the amygdala is involved
in the development of emotional responses to
the environmental context; in this regard, inputs
from the hippocampus have been related to con-
text conditioning. The amygdala also plays a key
role in the emotional modulation of memory,
emotions influence on attention and percep-
tion, social responding, and emotion inhibition
and regulation (see LeDoux, 2000; Phelps and
LeDoux, 2005, for review). Human studies have
further confirmed the implication of the amyg-
dala in emotional processing. Bursts of electro-
encephalographic activity have been recorded in
the amygdala during recollection of specific emo-
tional events (Halgren, 1981), and the electrical
stimulation of the amygdala has been reported
to evoke emotional experiences, mostly fear and
anxiety (Gloor etal., 1982), as well as the recol-
lection of emotionally charged remote memories
(Brothers,1995).
The hippocampus is involved in the circadian
glucocorticoid rhythm and inhibition of the HPA
axis response to stress. Indeed, the hippocampus
can detect a wide range of circulating glucocor-
ticoid concentration given the high expression
of both glucocorticoid and mineralocorticoid
receptors in this brain area (Reul and de Kloet,
1986; Herman et al., 1989). Projections to the
hypothalamus are largely emitted by the ventral
hippocampus/ventral subiculum, thus control-
ling the activity of the HPA axis in response to
stressors. The ventral hippocampus/ventral sub-
iculum contact PVN projecting neurons in the
bed nucleus of the stria terminalis, the medial
preoptic area, the dorsomedial hypothalamus,
and other hypothalamic nuclei. These relay areas
are densely populated with GABAergic neu-
rons, allowing for a putative two-neuron relay
between glutamatergic hippocampal outflow
and GABA neurons controlling the HPA stress
response (Herman et al., 2003; Jankord and
Herman,2008).
Last, the prefrontal cortex has been shown to
play a key role in the regulation of mood, emo-
tion, and stress (Myers-Schulz and Koenigs, 2012;
Shansky and Lipps, 2013). The medial prefrontal
cortex (mPFC) has long been implicated in the
modulation of anxiety and other emotional behav-
iors. Actually, it is strategically located to modulate
the output of limbic and monoaminergic neuronal
networks and seems to participate in the interpre-
tation and valuation of the external stimuli, as well
as in the prediction of the social outcomes of the
behavioral response (Nishijo etal., 1988; Damasio,
1998; Baxter et al., 2000). The mPFC modulates
visceral control centers in the brainstem, includ-
ing cholinergic neurotransmission originating in
the basal forebrain (Gaykema etal., 1991), noradr-
energic (NE) neurons from the locus coeruleus
(Jodo etal., 1998), dopaminergic neurons project-
ing from the ventral tegmental area and substantia
nigra (Carr and Sesack, 2000), and serotonergic
(serotonin: 5-hydroxytryptamine [5-HT]) neu-
rons emerging from the dorsal and median raphe
nucleus (Hajos etal., 1998). In addition, the mPFC
is a vital component of a distributed extrahypo-
thalamic network that modulates activation and
feedback inhibition of the HPA axis. Different
subregions of the mPFC differentially modulate
the behavioral and systemic response to psycho-
logical stress; the prelimbic region of the mPFC
suppresses the HPA axis response to acute stress,
while the infralimbic cortex activates autonomic
PVN outputs to promote stress-induced activation
of the HPAaxis.
However, neither the prelimbic nor the
infralimbic cortexes directly innervate the PVN;
their connections to modulate the HPA axis relay
through diverse subcortical nodes (Vertes, 2004;
Holmes and Wellman, 2009). In general, the
amygdala activates the HPA axis in response to
acute stress whereas the hippocampus and PFC
inhibit the HPA response (Herman et al., 2003;
Jankord and Herman, 2008; see Figure28.1).

External
STIMULUS Environmental
AFFERENT
SYSTEMS
PREFRONTAL
CORTEX
EMOTIONAL
PROCESSING
EFFERENT
SYSTEMS PARASYMPATHETIC
& SYMPATHETIC
NERVOUS SYSTEM
EMOTIONAL VISCERAL
RESPONSES SYMPTOMS
FIGURE 28.1: Scheme of the main neural circuitries involved in emotional processing. PVN, paraventricular
nucleus.
In the past few decades, neuroimaging studies
have advanced considerably our understanding of
the specific brain regions and circuitries that are
affected in anxiety disorders. Indeed, changes in
resting brain metabolism have been described in
the scans of patients diagnosed with anxiety dis-
orders when compared with healthy volunteers.
In addition, many studies have been conducted
in healthy volunteers after exposure to a fear con-
ditioning paradigm or during an experimentally
induced panic attack. One of the most consistent
findings is a hyperactivity of the amygdala dur-
ing symptom provocation that is related to the
experienced symptoms of fear. The anterior cin-
gulated cortex and the insula are also involved in
the development and maintenance of anxiety dis-
orders (Rauch etal., 2003; Bremner, 2004; Damsa
etal., 2009). The amygdala is consistently involved
in studies of social anxiety disorders (SAD), and
increased activity in limbic and paralimbic regions
have also been described in SAD, although much
remains to be investigated within the complexity
of these repetitive disorders (Freitas-Ferrari etal.,
2010). Functional neuroimaging studies have
recurrently demonstrated amygdalar hyperacti-
vation in posttraumatic stress disorder (PTSD;
Brohawn et al., 2010; Vermetten et al., 2007). In
contrast, hypoactivity of the mPFC, the anterior
cingulated cortex, and sometimes the hippocam-
pus, have been identified in a variety of anxi-
ety disorders, particularly PTSD and SAD (Shin
et al., 2006; Freitas-Ferrari et al., 2010; Hayes
etal., 2011). Altogether, symptoms of anxiety are
Anxiety and Stress Disorders 537
Internal
mileau
HIPPOCAMPUS
AMYGDALA
HYPOTHALAMUS
PVN
HORMONAL BEHAVIOR
RESPONSE RESPONSE
considered to be due to a pathologically hyperac-
tivated and insufficient top-down regulation by
frontal brain regions. Worth mentioning, neuro-
imaging techniques may be considered not only
as a promising tool to reveal the neural circuit-
ries underlying anxiety disorders but also of great
value for the discovery and development of novel
therapeutic tools, as functional neuroimaging
may predict the efficacy and therapeutic value of
drugs (Holzschneider and Mulert,2011).
Neurochemical Basis
ofEmotional Homeostasis
A wide variety of neurotransmitter systems are
involved in the complex circuitry that controls
emotional homeostasis. These include monoamin-
ergic transmitters, NE, 5-HT, and dopamine and
the amino acid transmitters, GABA and glutamate.
The neurotransmitter systems that have been best
studied in relation to stress responsiveness involve
the HPA axis and the central NE system. Additional
neurochemical systems participate in the control of
stress and emotional responses, including neuro-
peptide Y (NPY), substance P, and opioid and can-
nabinoid (CB) systems, among others. Preclinical
and clinical studies supporting a role for some of
these neurochemical agents in emotional homeo-
stasis are reviewed in the next section, as well as
their potential application as pharmacological
therapies in the management of anxiety disor-
ders. Overall, the fine-tuning of these circuitries
under basal conditions, together with their coor-
dinated response under adverse or stressful
538 Part IV:Homeostatic Therapies
stimuli, guarantees an adequate emotional adap-
tation to the changing environment. However,
abnormalities in the neuroanatomic circuits that
support emotional regulation and stress respon-
siveness and/or in the diversity of chemical neu-
rotransmitter systems that control the emotional
homeostasis may be associated with the appear-
ance of anxiety disorders. Disproportional anxi-
ety responses, both in intensity or duration, or
the arousal of anxiety responses in the absence of
relevant risksthat is, a dysfunction of emotional
homeostasisconstitutes a maladaptive response
to the environment and possibly signal the appear-
ance of a psychiatric disorder (i.e., anxiety and/or
stress-related disorders).
DYSFUNCTIONS OF
E M O T I O N A L H O M E O S TA S I S :
ANXIETY AND STRESS
DISORDERS
Recent surveys demonstrate that anxiety and
stress disorders are the most prevalent of
psychiatric diseases and have high personal and
societal costs (Wittchen et al., 2011). Despite
being common, the severity of these disorders can
vary considerably, from mild worries to incapaci-
tating symptoms that interfere with functioning
(Bernstein and Shaw, 1997). Actually, anxiety
disorders categorize a large number of disorders
in which the primary feature is abnormal or inap-
propriate anxiety. As previously stated, anxiety is
an adaptive component of the reactive response to
stress and fear that integrates the flight or fight
phenomenon, thus preparing the body to either
fight and protect itself or to flee from a dangerous
situation. However, in the absence of any recog-
nizable stimulus or when the stimulus does not
warrant such a reaction, these anxiety symptoms
become pathological, and an anxiety disorder
might be diagnosed. Indeed, anxiety disorders are
marked by excessive fear (and avoidance), often
in response to specific objects or situations and
in the absence of true danger. Anxiety disorders,
such as panic disorder, social and specific phobia,
generalized anxiety disorder (GAD), and PTSD,
represent a highly prevalent and strongly disa-
bling class of psychiatric disorders (Bekker and
van Mens-Verhulst, 2007; Shin and Liberzon,
2010). Notably, cognitive impairments are fre-
quently observed in patients with anxiety disor-
ders (Gualtieri and Morgan, 2008). More precisely,
PTSD that develops after prolonged inescapable
stress experience of exceptional severity (Rubin
et al., 2008) has been associated with a number
of cognitive impairments, including basic defi-
cits in attention, extinction processes, concen-
tration, and memory (Isaac etal., 2006; Parsons
and Ressler, 2013). In Europe, a recent report has
indicated that the most frequent mental disor-
der is anxiety disorders (14%), with an estimated
number of 61.5million of persons affected across
all ages (Wittchen etal.,2011).
The fourth edition of the Diagnostic
and Statistical Manual of Mental Disorders
(American Psychiatric Association, 1994)groups
disorders into diagnostic classes on the basis
of the subjective criterion of shared phe-
nomenological features and includes GAD,
obsessive-compulsive disorder (OCD), panic
disorder, phobias (including social phobia),
and PTSD under the category of anxiety disor-
ders. More recently, the American Psychiatric
Association (2013) approved a new edition of
the Diagnostic and Statistical Manual of Mental
Disorders, which incorporates significant scien-
tific advances in more precisely identifying and
diagnosing mental disorders. Regarding the area
of anxiety, refinements of criteria and symp-
toms across the lifespan constitute the most
relevant change. Some disorders included in the
broad category of anxiety disorders are now in
three sequential chapters: Anxiety Disorders
(including specific phobia, social phobia, panic
disorder and panic attack, GAD, substance/
medication-induced anxiety disorder, anxiety
disorder due to another medical condition, etc.),
Obsessive-Compulsive and Related Disorders,
and Trauma- and Stressor-Related Disorders.
R E L E VA N C E O F
T R A N S L AT I O N A L S T U D I E S
FROM ANIMAL MODELS
Animal models are of great relevance for under-
standing anxiety and stress disorders (see Hohoff,
2009; Cryan and Sweeney, 2011; Neumann etal.,
2011, for more focused reviews on the subject).
Most animal models evaluate unconditioned or
spontaneous behaviors, involve simple or com-
plex classical and/or operant conditioning pro-
cedures, or use brain-stimulation paradigms.
General locomotion and exploration in a novel
environmental context is commonly used to
evaluate unconditioned or spontaneous behav-
ior in animals. Exploratory activity appears to
be a function of general activity level; impulsiv-
ity or the need to become familiar with a strange
territory opposes with anxiety or the desire to
escape from the unknown and unpredictable.

As examples of this class, the open field and
hole-board tests are common, as is the social
interaction test (File and Hyde, 1979; File and
Seth, 2003)or the hyponeophagia (Shephard and
Broadhurst, 1983). Animal models based on con-
ditioned behaviors typically consist of an operant
response cued by different light signals. In this
class we can use the Geller-Seifter conflict test
(Sepinwall et al., 1978) or the Vogel water-lick
water test (Vogel et al., 1971). Conditioned pro-
cedures are often subjected to the criticism of
including food or water deprivation, and, in turn,
motivation and reward cannot be excluded of the
discussion of results (see File etal., 2005; Griebel
and Holmes, 2013, for review). The delineation of
specific neural pathways mediating conditioned
and unconditioned fear can logically guide the
design and conduct of clinical studies investigat-
ing the neurobiological mechanisms of anxiety
disorders such as GAD (unconditional fear) and
phobic disorders (conditionedfear).
Most paradigms claim at least face validity,
as they are fear inducing, and/or a predictive
validity in that they are relatively selective for
clinically used anxiolytics. Notwithstanding, the
validity of animal models of anxiety has usually
been restricted, implicitly or explicitly, to the
success or failure of a given model in predicting
the clinical anxiolytic potency of pharmacologi-
cal agents (predictive validity); however, screen-
ing may not be the only function of these animal
models. Animal models also provide important
contributions to revealing the neurobiologi-
cal mechanisms of anxiety disorders, as well as
important information on vulnerability factors,
age-dependent differences, and sexual dimor-
phisms, among others. Indeed, animal models
are of great value for the investigation of genetic
and/or environmental factors that may predis-
pose for the development of anxiety disorders. In
particular, rodent and nonhuman primate stud-
ies of motherinfant interactions are particularly
compelling, given the important clinical impli-
cations of the early life environment. Sex dif-
ferences have also been extensively studied, and
literature on animal studies has largely docu-
mented male versus female differences in behav-
ior, neurobiological parameters, and response to
stress (Toufexis etal., 2006; Simpson and Kelly,
2012; Simpson etal., 2012). In general, female ani-
mals appear to be more vulnerable than males to
both acute and chronic stress exposure. Actually,
the elevation in plasma corticosterone levels in
response to stress is higher and more persistent in
Anxiety and Stress Disorders 539
female rats and the HPA axis negative feedback
greater (e.g., Louvart et al., 2006; Mazor et al.,
2009). Notwithstanding, gonadal steroid hor-
mones may play a critical role in mediating the
sex differences in the response to stress as well as
in anxiety disorders, since sex differences involve
differences in gonadal steroid hormone secretion
(i.e., estrogen and androgen) and causes differ-
ent effects on neuronal functioning (e.g., Patchev
and Almeida, 1998; McCormick etal., 2002; Ter
Horst etal.,2009).
Targeted mutations leading to anxiety-like
responses in transgenic mice have suggested
roles for serotonin receptor subtype 1A (5-HT1A),
CRH, GABA, neuropeptide Y, cholecystokinin
(CCK), and substance P neural systems in the
control of emotional states. A huge volume of
potential anxiolytic drugs has been generated in
animal models; however, the clinical outcome
of these investigations has been disappointing,
as promising results with novel agents in rodent
studies have very rarely translated into effec-
tiveness in humans (Griebel and Holmes, 2013).
We briefly describe the participation of some of
these neurochemical systems in emotionality and
anxiety disorders (see, e.g., Nutt, 2001; Charney and
Drevets, 2002; Cryan and Sweeney, 2011; Griebel
and Holmes, 2013, for more extensive reviews), and
their potential as anxiolytic drugs in the clinic is
discussed in the corresponding section.
GABA and Benzodiazepine Systems
Clinical and preclinical data have established
that benzodiazepine (BDZ)-receptor agonists
exert anxiolytic effects, and the functionality
of BDZ receptors may be altered in anxiety dis-
orders (Nutt and Malizia, 2001). The BDZ and
GABA A receptors are part of the same macromo-
lecular complex; these receptors present different
binding sites that are functionally coupled and
regulated in an allosteric manner. Central BDZ
receptors are extensively expressed throughout
the brain but are most densely concentrated in the
cortical grey matter (Choi etal., 1981). Responses
similar to those seen in anxiety (i.e., increases in
heart rate, blood pressure, plasma cortisol, and
chatecolamines) were observed in rodents after
the administration of BDZ-receptor inverse ago-
nists, such as -carboline-3-carboxylic acid ethy-
lester (Braestrup etal., 1982; Dorow etal., 1983).
Actually, classical BDZs have long been reported
to exert anxiolytic-like effects by increasing the
affinity of GABA A receptors for the neurotrans-
mitter GABA. Along the same lines, transgenic
540 Part IV:Homeostatic Therapies
mice studies further supported the role of BDZ
and GABA A receptors in emotional homeosta-
sis and enabled the understanding of the spe-
cific role of each GABA A-receptor subunit. The
GABA A-receptor 2 subunit seem to mediate, at
least partially, the anxiolytic effect of BDZ ago-
nists, since the anxiolytic action of diazepam
disappeared in mice lacking the 2 subunit but
remained in mice lacking 1 and 3 subunits
(Low etal., 2000; McKernan etal., 2000). In con-
trast, GABA A receptor 1 may mediate the seda-
tive, amnesic, and anticonvulsant effects of BDZs
(Rudolph et al., 1999; McKernan et al., 2000).
These findings have important implications
for the investigation of the pathophysiology of
anxiety and also for the development of
BDZ-receptor agonists selective for their anxio-
lytic properties and devoid of adverse side effects
such as sedation or amnesia (for a review see
Rudolph and Knoflach,2011).
Serotonin NeurotransmissionSystem
The 5-HT system has also been shown to play
a role in fear and anxiety disorders in animal
models and in humans (Ressler and Nemeroff,
2000; Garvey et al., 1995; Iny et al., 1994).
Multiple studies in animals indicate that stress
exposure increases 5-HT turnover in the mPFC,
nucleus accumbens, amygdala, and lateral hypo-
thalamus (Inoue etal., 1994). The effect of stress
in activating 5-HT turnover may stimulate both
anxiogenic and anxiolytic pathways within the
forebrain, depending on the region involved and
the 5-HT receptor subtype that is predominantly
stimulated. Graeff etal. (1993) proposed that the
serotoninergic innervation of the amygdala and
the hippocampus mediate anxiogenic responses
by 5-HT2A receptor stimulation, whereas sero-
toninergic innervation of hippocampal 5-HT1A
receptors may mediate responses to aversive
events. In accordance, 5-HT1A receptor knockout
mice show behaviors consistent with heightened
anxiety (Ramboz etal.,1998).
CRH and Glucocorticoid
In rodents, CRF1 antagonists generally do not
show anxiolytic effects under basal conditions,
but rather they block the heightened anxiety
produced by such factors as stress or selective
breeding for high anxiety (Rotzinger etal., 2010).
In humans, by examining a population-based
cohort of individuals exposed to the World Trade
Center collapse, genetic markers related to gluco-
corticoid signaling were identified in individuals
who developed PTSD. In particular, polymor-
phisms at FKBP5, a modulator of glucocorticoid
receptor sensitivity, may influence circulating
cortisol levels and PTSD severity (Yehuda, 2006;
Sarapas etal.,2011).
Norepinephrine
NeurotransmissionSystem
Abnormalities in the NE system have long been
implicated in the pathophysiology of anxiety dis-
orders (Ressler and Nemeroff, 2000). Exposure to
stressful stimuli increases central NE function;
stress exposure increases NE turnover in the
locus coeruleus, hypothalamus, hippocampus,
amygdala, and cerebral cortex (Cassens et al.,
1981). The firing rate of locus coeruleus neurons
also increases during exposure to stress and other
fear-conditioning stimuli (Rasmussen et al.,
1986; Abercrombie and Jacobs, 1987). Actually,
NE release seems to play a critical role in fear
learning since the acquisition of fear-conditioned
responses requires an intact central NE system
(Rasmussen etal., 1986; Abercrombie and Jacobs,
1987; Bremner etal., 1996; Charney and Deutch,
1996). Responsiveness of locus coeruleus neu-
rons to future novel stressors can be enhanced by
chronic exposure to some stressful experiences.
Therefore, the NE system seems to participate in
the process by which single or repeated exposure
to aversive stimuli or pharmacological agents can
increase the behavioral sensitivity to subsequent
stressors, the so-called behavioral sensitization.
This effect may result from a stress-mediated
alteration in the sensitivity of presynaptic 2-NE
synthesis and release.
Cholecystokinin
CCK was originally isolated in the gastrointesti-
nal system but is found extensively throughout
the nervous system, with particularly high con-
centrations distributed throughout the limbic
system. CCK is synthesized as a 115 amino acid
preprohormone and is converted into multi-
ple isoforms: the sulphated CCK-8 is predomi-
nant in the central nervous system, and CCK-5
(pentagastrin), and CCK-4 are also relevant. In
general, CCK binds to two receptors, CCK1 and
CCK2CCK is one of the neuropeptides impli-
cated in anxiety both in animal models and
humans (Woodruff etal., 1991; Harro etal., 1993;
Rotzinger et al., 2010; Bowers et al., 2012). CCK
agonists show anxiogenic effects in a number of
animal models; indeed, CCK administration is
frequently employed to induce panic attacks in

animal models (Rotzinger and Vaccarino, 2003).
However, the effects of CCK antagonists often
are not seen unless the system is potentiated.
The CCK2 receptor appears to play a key role in
anxiety, but it remains to be seen whether effica-
cious CCK antagonists can treat anxiety disorders
or depression. Clinical trials in anxiety disorders
and CCK antagonists have not been promising.
The CCK2 antagonist CI-988 had no effect in
patients with GAD (Adams et al., 1995; Pande
etal., 1999), nor did it block the panic-inducing
effects of CCK-4 (van Megen et al., 1997). The
CCK2 antagonist L-365,260 had no effect in
patients with panic disorder (Kramer etal.,1995).
NeuropeptideY
NPY is a 36-amino acid peptide that is widely
distributed in the central nervous system. There
are currently five identified NPY receptors,
which are also widely distributed in the central
nervous system, particularly throughout the
frontal cortex and limbic regions (Redrobe etal.,
2002). NPY has been implicated in anxiety dis-
orders (see Rotzinger et al., 2010, for a review).
In animal models, NPY administration has been
reported to induce anxiolytic-like effects in vari-
ety of behavioral models. In contrast, the admin-
istration of NPY-receptor antagonists enhances
anxiety, thus providing strong evidence for
NPYs role in modulating anxiety responses. In
humans, plasma NPY levels have been proposed
to represent a biologic correlate of resilience to or
recovery from the adverse effects of stress; a bio-
marker of great relevance for the clinical study
of PTSD (Cohen etal., 2012; Yehuda etal., 2006;
Sajdyk etal.,2008).
The EndocannabinoidSystem
The endocannabinoid (eCB) system consists of
two CB receptors, one mainly central (CB1 recep-
tor) and a second one (CB2 receptor) tradition-
ally considered as peripheral although it has been
recently described in several brain regions. Two
principle endogenous ligands have been discov-
ered: N-arachidonoylethanolamine (AEA) and
2-arachidonoylglyceril (2-AG), together with
the enzymes involved in their metabolism (see
Maccarrone, 2010). Over the past few decades, an
extent literature on the eCB system has emerged to
give support to the central role played by the eCB
system in emotional control (Viveros etal., 2005;
Moreira and Lutz, 2008; Moreira and Wotjak,
2010; Marco and Laviola, 2012). The eCB system
is known to constrain activation of the stress
Anxiety and Stress Disorders 541
response through distributed actions in limbic
and hypothalamic circuits in the brain (Riebe
and Wotjak, 2011; Hill and Tasker, 2012) and is
involved in the extinction of emotionally aver-
sive memories (Marsicano etal., 2002; Plendl and
Wotjak, 2010; Gunduz-Cinar et al., 2013) and
habituation and adaptation to stress (Patel et al.,
2005; Hill etal., 2010), processes that are dysregu-
lated in PTSD. Taken together, it may seem that
reduced AEA signaling could be associated with
impaired extinction of aversive memories and an
increase in intrusive symptoms. Regarding the
contribution of eCB ligands on emotion, a differ-
ential, and maybe opposite, role has been proposed
for AEA and 2-AG. However, much research is
needed to clarify the complex role of each com-
ponent of the eCB in emotional homeostasis and
anxiety disorders.
PRESENT AND FUTURE
H O M E O S TAT I C T H E R A P I E S
FORANXIETY AND STRESS
DISORDERS
Benzodiazepines and agents acting on the sero-
tonergic system are currently the main drugs
employed in the management of anxiety dis-
orders (Sandford et al., 2000; Millan, 2003).
However, in recent years a search for new drugs
beyond classical treatments have focused most
intensively on the 5-HT, neuropeptide, gluta-
mate, and eeCB systems (Griebel and Holmes,
2013). Selective 5-HT reuptake inhibitors (SSRIs;
e.g., citalopram, paroxetine, fluvoxavine, fluox-
etine, etc.) and serotonin-norepinephrine reup-
take inhibitors (e.g., venlafaxine, duloxetine,
etc.) are useful first-line agents for most anxiety
disorders, particularly given the frequent comor-
bidity with mood disorders. They both present
an extremely broad spectrum of action in anxi-
ety disorders. However, their use is limited by a
delayed onset and incomplete response in many
patients. Research on the neurobiological under-
pinnings of anxiety disorders has continued for
several decades, and the search for novel phar-
macological treatments is driven by the growing
medical need to improve on the effectiveness
and the side-effect profile of existing drugs (see
Ravindran and Stein, 2010; Reinhold etal., 2011;
Griebel and Holmes, 2013, for a more complete
update on anxiety disorders pharmacotherapy).
In general, benzodiazepines are still use-
ful in the management of panic disorder and
GAD. Serotonergic agents are preferred for the
treatment of OCD, while other antidepressants,
542 Part IV:Homeostatic Therapies
such as tricyclic antidepressants or monoam-
ine oxidase inhibitors, are generally reserved as
second- and third-line strategies due to toler-
ability issues. Antidepressant drugs, primarily
acting on monoamine reuptake, are effective for
the treatment of a spectrum of anxiety disorders
including SAD, GAD, panic disorder, and PTSD.
Notwithstanding, the anxiolytic effects of antide-
pressant drugs may result from interactions with
the GABAergic system. As an example, phenelzine
induces an anxiolytic-like effect in the rat elevated
plus maze, possibly through the elevation of GABA
concentration (Paslawski etal., 1996). Azapirones,
including buspirone, are partial 5-HT agonists
with low abuse potential that have been effectively
used for GAD. In case of treatment resistance,
anticonvulsants and atypical antipsychotics are
employed as an adjuvant in the treatment with
antidepressants (Ravindran and Stein,2010).
As mentioned, GABA has been recently
involved in extinction; thus augmenting
extinction-based psychotherapy with GABA-
agonist drug treatment may be a potential
strategy to investigate. In regard to benzodiaz-
epine drug development, research has focused
on the development of selective agonists with
increased responsiveness at GABA A-receptor
2-subunit but limited actions to the 1 and 3
subunits (see Rudolph and Knoflach, 2011). The
development of the CRH antagonist has also
significantly influenced the pharmacotherapy
of anxiety disorders. Indeed, there is potential
for developing targets for the CRF-2 receptor
and other peptides, such as vasoactive intesti-
nal peptide, involved in the regulation of stress
(Ravindran and Stein,2010).
Furthermore, enhancement of eCB signal-
ing has achieved promising anxiolytic effects in
a variety of animal studies, thus emerging as an
attractive strategy for potential therapeutic appli-
cation (Pacher etal., 2006; Piomelli, 2006; Marco
et al., 2011). In recent years, promising results
have been achieved in the field of the eCB sys-
tem as a potential clinical target in the treatment
of the PTSD. Actually, drugs acting on the eCB
system have been proposed to enhance the effec-
tiveness of exposure therapy (see Passie et al.,
2012; de Bitencourt et al., 2013; Rabinak and
Phan, 2014; Trezza and Campolongo, 2013, for
reviews). Trials examining the effects of agents
that potentiate eCB signaling in PTSD, either
alone or in conjunction with treatments that
modulate glucocorticoids signaling, will provide
proof of principal for the contribution of eCB
signaling to PTSD or will help to elucidate more
precisely the respective functional roles of 2-AG
and AEA in PTSD development and expression.
Despite further studies being needed; prelimi-
nary data indicate that CB agonists may be effec-
tive in the clinical treatment of PTSD. At present,
a first clinical trial (phase IV) on the efficacy of
delta-9-tetrahydrocannabinol treatment for the
management of PTSD is being conducted; adult
subjects of both genders are being recruited,
and first results will be available soon (http://
ClinicalTrials.gov/show/NCT00965809).
In spite of the fact that cannabidiol CBD
may not be considered a component of the eCB
system, notable anti-anxiety properties have
been extensively reported for this non-psycho-
active phytocannabinoid present in the plant
of Cannabis sativa (Guimares et al., 1990;
Moreira etal., 2006). CBD not only prevents the
anxiogenic effects of high doses of tetrahy-
drocannabinol (Zuardi et al., 1982) but also
decreases anxiety symptoms in healthy subjects
submitted to a simulated public-speaking para-
digm (Zuardi etal., 1993). More recently, using
a similar paradigm, Bergamaschi et al. (2011)
showed that CBD reduced public-speaking
anxiety in treatment-nave social phobic
patients and successfully decreased subjective
anxiety in SAD patients (Crippa et al., 2011).
Accordingly, neuroimaging studies have shown
that CBD impairs connectivity between the
prefrontal and subcortical regions (Fusar-Poli
et al., 2010), attenuates blood oxygenation
leveldependent responses to fearful faces in
the amygdala and cingulate cortex (Fusar-Poli
et al., 2009), and decreases activation in the
left amygdala-hippocampal complex and left
posterior cingulate gyrus (Crippa et al., 2004).
Although the pharmacological mechanisms
involved in CBD effects are still poorly under-
stood (Campos etal., 2012), one of the primary
mechanisms may involve fatty acid amide
hydrolase inhibition and thus facilitation of
AEA signaling (Bitencourt etal.,2008).
At present, pharmacological therapies
are usually combined with concomitant psy-
chotherapeutic interventions. Indeed, phar-
macotherapy should be used as an adjunct to
behavioral or psychotherapeutic interventions.
Among psychotherapeutic interventions, cogni-
tive behavioral therapy, particularly exposure
therapy, has been shown to be highly effective
in the treatment of anxiety disorders (Goossens
et al.,2007). Exposure therapy consists of the

systematic and repeated exposure of patients
to the anxiety-provoking stimulus or situation
until their fear subsides. In this regard, changes
in brain structure and function have been dem-
onstrated following successful treatment with
exposure therapy. Actually, altered patterns in
neural functioning were observed after success-
ful exposure therapy in a sample of individuals
with spider phobia (an example of specific pho-
bia); a decrease in the activation of the amygdala
was observed, together with a normalization of
insular and anterior cingulated cortex activity
(Goossens et al., 2007). Similarly, recovery from
PTSD was correlated with the activity in the hip-
pocampus and subgenual anterior cingulated cor-
tex (Dickie etal.,2011).
Furthermore, advances in deep brain stimu-
lation have opened new avenues for the applica-
tion of this novel strategy in the management
of anxiety disorders. Depression and OCD have
benefited from deep brain stimulation since 2005,
when the US Food and Drug Administration
approved vagal nerve stimulation for the man-
agement of treatment-resistant depression;
in 2009 it was approved for refractory OCD
(Heeramun-Aubeeluck and Lu, 2013). More
recently, a double-blind study demonstrated a sig-
nificant improvement of PTSD patients resistant
to standard treatment following repeated medial
prefrontal cortex deep transcranial magnetic
stimulation (a wide multicenter study is suggested
to substantiate these findings; trial registra-
tion:ClinicalTrials.gov identifier:NCT00517400;
Isserles et al., 2013), while striatal deep brain
stimulation seems to reduce conditioned fear
and enhance extinction memory in rodents
(Rodriguez-Romaguera etal.,2012).
SEX DIFFERENCES IN
PHYSIOLOGICAL AND
PAT H O L O G I C A L E M O T I O N A L
H O M E O S TA S I S
In general, females are affected approximately
two to three times more frequently than males
by anxiety disorders (Wittchen et al., 2011).
Accordingly, epidemiological studies report
higher rates of PTSD among women compared
to men (Breslau and Anthony, 2007); in addi-
tion, symptoms appear to be more severe and
quality-of-life outcomes are significantly worse
in women (Holbrook etal., 2002; Tolin and Foa,
2006). Sexual differences in PTSD vulnerability
may rely on the intimate, interpersonal nature
of potentially traumatic events that women are
Anxiety and Stress Disorders 543
more likely to experience (i.e., higher rates of
severe intimate-partner violence and sexual
assault across the lifespan); the profoundly cor-
rosive and damaging meanings inherent in these
kinds of violations; the possibility that women
may be prone to heightened levels of emotional
reactivity, which could confer additional risk for
PTSD; and the potential that gender differences in
behavioral and psychological sequelae to trauma
influence diagnostic presentation (e.g., internal-
izing vs. externalizing symptoms; Pratchett etal.,
2010). Neuroanatomical sex differences have been
reported in children with maltreatment-related
PTSD; boys exhibited larger prefrontal lobe
cerebrospinal fluid volumes and smaller sple-
nium, smaller cerebral volumes and corpus cal-
losum, and greater lateral ventricular volume
increases than girls. An association between
enhanced brainstem activity and PTSD diagnosis
has been described for men but not for women,
and the hippocampal fear response was greater
in men compared to their female counterparts
(De Bellis and Keshavan, 2003). Epinephrine and
norepinephrine has been associated with PTSD
symptoms following trauma exposure in men
but not in women (Hawk etal., 2000). Moreover,
sex differences in corticotropin-releasing fac-
tor (CRF) receptor functionality, which is more
sensitive to low CRF levels but less adaptable
to high levels in women, may possibly increase
the risk for PTSD development among women
(Bangasser et al., 2010). In addition, sex dif-
ferences in cortisol and catecholamines levels
among subjects with PTSD may also be of criti-
cal relevance. Among PTSD subjects, womens
salivary cortisol levels decreased during the day
while the opposite occurred in men (i.e., a daily
increase in cortisol levels; Freidenberg et al.,
2010; Viveros etal.,2012).
ANXIETY DISORDERS
DURING THELIFETIME
Anxiety, as already described, presents as auto-
nomic hyperactivity, with raised blood pres-
sure and heart frequency, increased sweating,
and dryness of mouth and throat, along with
cognitive and affective states of increased vigi-
lance, distractibility, and apprehension. Motor
fatigue and insomnia are common. In children,
anxiety disorders include separation anxiety,
avoidance disorders (of strangers), and over-
anxious disorderin sum, a general apprehen-
sion of the world. Anxiety disorders appear as
early as in childhood, critically affecting further
544 Part IV:Homeostatic Therapies
neurocognitive development and thus interfering
with school and academic achievement, social
functioning, and social integration (Drysdale
et al., 2014; Kessler et al., 2005). Anxiety disor-
ders might persist throughout the lifespan or at
least have an enduring impact. Recent data indi-
cate that GAD and PTSD incidence significantly
differ by age group; for GAD, evidence suggests
highest rates (3.4%) among the elderly (65+) and
considerably lower rates (1.7%) in the 14 to 65 age
group, while PTSD rates seem to decline by age
(1434: 2.9%, 3565: 1.3%, 65+:1.1%; Wittchen
etal.,2011).
Anxiety Disorders inChildren
and Adolescents
Anxiety disorders are among the most com-
mon pediatric psychopathologies. The diagnosis
of anxiety disorders peaks during adolescence
(Drysdale et al., 2014; Kessler et al., 2005), and
the presence of an anxiety disorder in childhood
or adolescence seems to predict its persistence
into adulthood, as well as the appearance of other
psychiatric disorders, particularly depression
(Stein and Lang,2002).
The pharmacological intervention of anxiety
disorders in children and adolescents was initially
based on successful strategies applied in adults.
However, increasing concern about the efficacy
and adequacy of these therapies rapidly rose, and
clinical trials in youth have been performed in
the past few years also considering the appear-
ance of adverse side effects and complications
(see Kratochvil etal., 2002, for a review). Some of
the most relevant studies in the youth are briefly
described here, despite the fact that the psychop-
harmacological management of pediatric anxiety
disorders is out of the scope of this chapter and
can be investigated elsewhere (e.g., Peters and
Connolly, 2012; Riddle etal.,2013).
When pharmacotherapy is indicated, the
SSRIs appear to be particularly suitable for
the treatment of anxiety disorders in children
and adolescents given their safety and effi-
cacy. Fluvoxamine has arisen as an effective
therapy for children and adolescents diag-
nosed with social phobia, separation anxiety
disorder, or GAD (Research Unit on Pediatric
Psychopharmacology, 2001) as well as for
the management of OCD in youth (Riddle
et al., 2001). Similarly, fluoxetine successfully
reduced anxiety scores in pediatric patients.
Sertraline was also effective in the management
of GAD (Rynn etal., 2001)and SAD (Compton
etal., 2001)in children and adolescents. In all
of these clinical studies, adverse side effects
were minimal. In addition, the efficacy of the
SSRI treatment in a pediatric population of
OCD patients was long-lasting with only mild
side effects that tended to disappear with time,
mainly nausea, headache, insomnia, abdominal
pain, hyperkinesia, nervousness, and vomiting
(Cook etal., 2001; Thomsen etal., 2001). Taken
together, the treatment of anxiety disorders
in children and adolescents should generally
involve a multimodal approach that is a com-
bination of pharmacological intervention with
cognitive behavioral therapy. However, the
clinician must always weight whether the con-
dition of the child is sufficiently serious to war-
rant pharmacological intervention (Velosa and
Riddle, 2000; Kratochvil et al., 2002; Riddle
etal.,2013).
Anxiety Disorders inthe Elderly
An increasing body of evidence suggests that
medication and psychotherapy for anxiety dis-
orders in older adults is not as effective as in
younger people. Indeed, only a minority of
old-age patients receives appropriate medica-
tion. In spite of the fact that anxiety disorders
are common in later life, the number of clini-
cal trials in the old population is scarce (see
Voshaar, 2013; Wetherell et al., 2013, for recent
updates). Actually, little is known about the
natural course, trajectories, and determinants
of anxiety disorders in old age, or regarding the
specific needs for interventions in this age group
(Riedel-Heller etal., 2006). Therefore, the design
of appropriate clinical trials in the elderly is of
critical urgency as well as the development of
age-specific psychological and pharmacological
therapies.
Recently, a European study on mental
health (Wittchen et al., 2011) highlighted
the urgency for incidence longitudinal stud-
ies especially across defined age cohorts (i.e.,
in children and adolescents, adults, and older
adults) in order to better understand the onset
and natural course of anxiety disorders as well
as the identification of critical trajectories.
Such data would provide better guidance for
preventive trials and targeted early interven-
tions, and they may suggest important cues for
preventing the development of co- and multi-
morbidity of anxiety disorders.

COMORBIDITIES OFANXIETY
DISORDERS
It has long been known that anxiety symptoms
and comorbid anxiety are common in depres-
sive syndromes (Fawcett, 1997; Merikangas etal.,
2003; Kessler et al., 2005). Comorbid anxiety
disorders have also been associated with a more
frequent history of substance abuse and higher
ratings for suicidal ideation, and high rates of
anxiety disorders have been reported in bipolar
disorders (Lee and Dunner, 2008). In both cases,
the presence/severity of anxiety symptoms nega-
tively impact the clinical management of the psy-
chiatric disorders of major depression (Fawcett
and Kravitz, 1983)and bipolar disorder (Lee and
Dunner, 2008). Given the high prevalence and
risk rate of comorbid anxiety disorders in depres-
sive and bipolar patients, it is of great relevance
to evaluate such symptoms in clinical practice,
since the presence of comorbidity may eventu-
ally serve as a guide to choosing the appropriate
pharmacological therapy.
CONCLUSIONS AND
FUTURE REMARKS
At present, despite that several drugs, mainly
benzodiazepines and serotonergic agents, are
available and effective in the management of
anxiety disorders, the understanding of the
neural and chemical basis of these disorders
has enabled the discovery of novel drugs for
potential use in the clinic. In a recent European
study on mental health (Wittchen et al., 2011),
the authors highlight that early intervention
is critical for an improvement in the primary
diagnosed disorder but may also be particularly
important for preventing the development of co-
and multimorbidity since the early treatment
of a temporally primary disorder may prevent
secondary comorbidity. Moreover, clini-
cians may control for the presence of poten-
tial comorbidities in the selection of a therapy.
Longitudinal studies along the lifespan are
critical, since age-specific studies should be
accomplished for the accuracy in the selection
of appropriate therapies.
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29
Malignant Brain Cancer Management
withMetabolic Therapy
T HOM AS N.SE Y FR IED A ND PUR NA MUK HER JEE
G L I O B L A S T O M A M U LT I F O R M E
Glioblastoma multiforme (GBM) is the most
malignant of the primary brain cancers, with
only about 12% of patients surviving beyond
36 months (long-term survivors) (Fisher and
Buffler, 2005; Krex etal., 2007; Patil etal., 2012;
Stupp et al., 2009). Most GBMs are heterogene-
ous in cellular composition, consisting of tumor
stem cells, mesenchymal cells, and host stromal
cells; hence the name glioblastoma multiforme
(Chen et al., 2010; Ohgaki and Kleihues, 2009;
Prestegarden et al., 2010; Rubinstein, 1972; Tso
et al., 2006). Primary GBM appears to arise de
novo, while secondary GBM is thought to arise
from less malignant glial tumors (Lopes et al.,
1993; Ohgaki and Kleihues, 2009). In addition to
the neoplastic cell populations, tumor-associated
macrophages/monocytes (TAM) also comprise a
significant cell population in GBM, sometimes
equaling the number of tumor cells (Morantz
etal., 1979; Nishie etal., 1999; Phillips etal., 1982;
Seyfried, 2001; Seyfried et al., 2010; Shinonaga
et al., 1988). TAM can indirectly contribute to
tumor progression through release of proinflam-
matory and pro-angiogenic factors (Lewis and
Murdoch, 2005; Nishie et al., 1999; Seyfried,
2001; Seyfried etal., 2010). Neoplastic cells with
macrophage characteristics might also contribute
to the sarcomatous characteristics of many GBM
(Huysentruyt etal., 2011; Lopes etal., 1993). The
neoplastic cells in GBM invade through the neural
parenchyma well beyond the main tumor mass,
making complete surgical resections exceed-
ingly rare (Fisher and Buffler, 2005; Kallenberg
etal., 2009; Talacchi etal., 2010). Moreover, GBM
exhibits the highest incidence of systemic metas-
tasis among glial neoplasms, while many cells
appearing as TAM within GBM can actually be
neoplastic macrophages/microglia (Huysentruyt
etal., 2011; Lopes etal., 1993). Despite extensive
analysis from the cancer genome project, no
mutation is known that is unique to the GBM and
no genetic alterations are seen in major signaling
pathways in about 15% of GBM (Parsons et al.,
2008). Moreover, few of the personalized molec-
ular markers available are considered important
for GBM analysis or therapy (Holdhoff et al.,
2012). Recent evidence suggests that the somatic
mutations seen in cancer cells can arise as down-
stream secondary effects of disturbed energy
metabolism and are unlikely to provide useful
information for therapeutic treatment strategies
for the majority of GBM patients (Johnson etal.,
2014; Poff etal., 2014; Seyfried, 2012a). Reactive
oxygen species (ROS), generated from protracted
disruption of oxidative phosphorylation, cause
the nuclear genomic instability and mutations
seen in tumor cells (Cooke et al., 2003; Iglesias
et al., 2012; Seoane et al., 2011). Therefore, the
genomic approach to brain cancer management
is unlikely to produce effective therapies in light
of the underlying metabolic origin of the disease.
T H E C U R R E N T S TA N D A R D
OFCARE FORGLIOBLASTOMA
The current standard of care for GBM and many
malignant brain cancers includes maximum sur-
gical resection, radiation therapy, and chemo-
therapy (Fisher and Buffler, 2005; Mason et al.,
2007; Mrugala, 2013; Stupp etal., 2009). The toxic
alkylating agent temozolomide (Temodar) is the
most common chemotherapy used for treating
GBM. Most GBM patients also receive periop-
erative corticosteroids (dexamethasone) as part
of the standard of care, which is often extended
throughout the course of the disease (Chang
etal., 2005; Koehler, 1995; Seyfried etal., 2010).
The anti-angiogenic drug bevacizumab (Avastin)
is also given often to GBM patients despite the
Food and Drug Administrations removal of
554 Part IV:Homeostatic Therapies
100
90
80
70
Survival (%)
60
50
40
30
20
10
0
0 1 2 3 4 5
Time (years)
Number at risk
Combined 254 175 76 39 23
Radiotherapy 278 144 31 11 6 3
FIGURE 29.1: Kaplan-Meier estimates of over-
all survival of patients with glioblastoma
multiforme by treatment group. The two patient
groups included radiotherapy alone (n=278) and radi-
otherapy with temolozomide (n=254). Overall patient
survival has remained largely unchanged from the
study published in 2005 (Stupp etal.,2005).
Reprinted with permission from Stupp etal. (2009). Readers
are referred to the original papers for details.
bevacizumab for breast cancer due to toxicity
and lack of efficacy (Burton and Dooren, 2011;
Seyfried etal., 2012). There have been no major
advances in GBM management for over 50years,
though use of temozolomide has produced
marginal improvement in patient survival over
radiation therapy alone (Souhami et al., 2004;
Stupp et al., 2009). Despite conventional treat-
ments, prognosis remains poor for most patients
with high-grade brain tumors (Davis et al.,
1998; Fisher and Buffler, 2005; Krex etal., 2007;
Mrugala, 2013; Souhami etal., 2004; Figure 29.1).
It is our view that the therapies used as part of the
standard of care are responsible in part for the
poor prognosis for patients withGBM.
MITOCHONDRIAL
ABNORMALITIES
INMALIGNANT
BR AINTUMORS
Substantial evidence collected from numer-
ous investigators over many years indicates that
mitochondrial abnormalities are the hallmark of
all cancers, including brain cancer (Ordys etal.,
2010; Seyfried et al., 2014a). These mitochon-
drial abnormalities reduce energy production
through oxidative phosphorylation (OxPhos)
(Arismendi-Morillo, 2009; Arismendi-Morillo
and Castellano-Ramirez, 2008; Bayley and
Devilee, 2010; Carew and Huang, 2002; Cuezva
et al., 2002; John, 2001; Kiebish et al., 2008;
Combined
Radiotherapy
Ordys et al., 2010; Pedersen, 1978; Ramanathan
etal., 2005; Roskelley etal., 1943; Villalobo and
p < 0.0001 Lehninger, 1979). The ultra structure of mito-
chondria in malignant brain tumors differs mark-
edly from the ultra structure of normal tissue
mitochondria (Arismendi-Morillo, 2009, 2011;
Arismendi-Morillo and Castellano-Ramirez,
n = 287
n = 286
2008). In contrast to normal mitochondria,
6 7
which contain numerous cristae, mitochondria
from GBM tissue samples show swelling with
partial or total cristolysis (Figure 29.2). Cristae
14 6
contain the proteins of the respiratory com-
0
plexes and play an essential structural role in
facilitating energy production through OxPhos
(Cogliati etal., 2013; Galluzzi etal., 2010; Ordys
et al., 2010; Stroud and Ryan, 2013). The struc-
tural defects in human glioma mitochondria are
also consistent with lipid biochemical defects in
murine gliomas. We showed that cardiolipin,
the signature phospholipid of the inner mito-
chondrial membrane, was abnormal in five inde-
pendently derived mouse brain tumors (Kiebish
etal., 2008; Kiebish etal., 2009). Cardiolipin con-
trols the efficiency of OxPhos, and any alterations
in the content or fatty acid composition of car-
diolipin will reduce cellular respiration (Chicco
and Sparagna, 2007; Claypool and Koehler, 2012;
Fry and Green, 1981). In addition to these find-
ings, Oudard and etal (1997) also indicated that
the high glycolytic activity seen in malignant gli-
omas could arise from mitochondrial structural
abnormalities. Hence substantial morphological
and biochemical evidence exists showing that
respiratory capacity is defective in most tumors,
including gliomas.
Based on numerous findings in human gli-
oma cell lines and tissues, it is not likely that the
majority of malignant brain tumors are capa-
ble of producing adequate amounts of energy
through OxPhos (Arismendi-Morillo, 2009, 2011;
Arismendi-Morillo and Castellano-Ramirez,
2008; Oudard et al., 1996; Oudard et al., 1997;
Oudard etal., 1995). Besides these ultrastructure
findings, Renner and colleagues (2010) showed
that tumor cells isolated from human GBM could
produce adenosine triphosphate (ATP) in the
presence of potassium cyanide. Cyanide blocks
cytochrome c oxidase and kills normal control
cells, which obtain energy through OxPhos.
Mitochondrial energy production in the pres-
ence of cyanide suggests that OxPhos is not likely
the origin of the energy produced in these GBM
cells. Similar findings were reported recently
for mitochondria in pancreatic tumor cells
 Malignant Brain Cancer Management with Metabolic Therapy 555

Normal Mitochondria GBM Mitochondria

inter membrane
matrix cristac inner space outer
membrane membrane

FIGURE29.2: Typical ultrastructure of a normal mitochondrion and a mitochondrion from GBM

tissue. Normal mitochondria contain elaborate cristae, which are extensions of the inner membrane and con-
tain the protein complexes of the electron transport chain necessary for producing ATP through OxPhos. The
mitochondrion from the GBM (m)is enlarged and shows a near total breakdown of cristae (cristolysis) and an
electron-lucent matrix. The absence of cristae in GBM mitochondria indicates that OxPhos would be deficient.
The arrow indicates an inner membrane fold. Bar:0.33m. Method of staining:uranyl acetate/lead citrate. The
GBM mitochondria was reprinted with permission from Arismendi-Morillo and Castellano-Ramirez (2008). The
normal mitochondria and diagram are from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/mito.htm.

(James etal., 2013). These and other studies sug-
gest that OxPhos is deficient in malignant glio-
mas and other malignant cancers and that energy
through oxidative metabolism alone would be
incapable of maintaining viability in gliomacells.
T H E WA R B U R G E F F E C T
INMALIGNANT
BR AINTUMORS
Otto Warburg (1931, 1956a) first proposed that
all cancers arise from irreversible damage to cel-
lular respiration. As a result, cancer cells increase
their capacity to ferment lactate even in the pres-
ence of oxygen in order to compensate for their
insufficient respiration. Although confusion has
surrounded Warburgs hypothesis on the ori-
gin of tumor cells (Koppenol etal., 2011; Zu and
Guppy, 2004), his hypothesis has never been for-
mally disproved and remains a credible explana-
tion for the origin of cancer (Cuezva etal., 2004;
Ferreira, 2010; Seyfried, 2012h; Seyfried and
Shelton, 2010). Consequently, Warburgs expla-
nation for the origin of cancer can no longer be
viewed as a hypothesis but can now be viewed as
a theory (Seyfried, 2012a).
The key points of Warburgs theory are
(a) insufficient respiration initiates tumorigen-
esis and ultimately cancer, (b) energy through
glycolysis gradually compensates for insufficient
energy through respiration, (c)cancer cells con-
tinue to ferment lactate in the presence of oxy-
gen, and (d)respiratory insufficiency eventually
becomes irreversible (Pedersen, 2007; Warburg,
1931, 1956a, 1956b, 1969). Warburg referred
to the phenomenon of enhanced glycolysis in
cancer cells as aerobic fermentation to high-
light the abnormal production of lactate in the
presence of oxygen (Pedersen, 2007; Warburg,
1931, 1956a, 1956b, 1969). The Warburg effect
refers to the aerobic fermentation of cancer cells
(Ferreira, 2010; Seyfried, 2012h). Substantial evi-
dence exists showing that gliomas produce lactate
(Lichtor and Dohrmann, 1986; Seyfried, 2012g;
Seyfried and Mukherjee, 2005). Lactate is the end
product of pyruvate fermentation. This would
be expected for any tumor cell with quantitative
or qualitative or abnormalities in mitochondria.
The large number of mutations found in tumor
cells is considered downstream epiphenomena
of damaged or insufficient respiration (Seyfried
etal., 2014b).
As the result of insufficient respiration,
cancer cells must rely on nonoxidative energy
metabolism to maintain energy balance and via-
bility. Consequently, aerobic fermentation plays a
role in producing energy through substrate-level
phosphorylation in the cytoplasm (glycolysis;
Seyfried et al., 2014a; Warburg, 1956b). Besides
556 Part IV:Homeostatic Therapies
aerobic fermentation in the cytoplasm, the tricar-
boxylic acid cycle substrate level phosphorylation
might also produce ATP through nonoxidative
metabolism. It can be difficult to determine,
however, the degree to which mitochondrial
ATP production arises from coupled respira-
tion or from tricarboxylic acid cycle substrate
level phosphorylation (Chinopoulos etal., 2010;
Phillips et al., 2009; Schwimmer et al., 2005;
Seyfried, 2012d). A protracted reliance on non-
oxidative energy metabolism involving glucose
and amino acid fermentation with substrate
level phosphorylation can cause genomic insta-
bility and other recognized hallmarks of cancer
(Seyfried etal., 2014a). Emerging evidence indi-
cates that the function of DNA repair enzymes
and the integrity of the nuclear genome are
dependent to a large extent on the energy derived
from normal respiration (Chandra and Singh,
2011; Delsite et al., 2003; Kulawiec et al., 2008;
Lu etal., 2009; Rasmussen etal., 2003; Smiraglia
etal., 2008; Veatch etal., 2009; Yang etal., 2010).
In other words, the Warburg effect and genomic
instability ultimately arise from a protracted
insufficiency of OxPhos (Seyfried et al., 2014a).
It remains speculative whether respiratory insuf-
ficiency is irreversible, as Warburg suggested, or
might be reversed through metabolic therapy.
It is difficult to imagine, however, how respira-
tion or the mitochondria cristolysis seen in GBM
could be easily reversed (Figure29.2).
ROLE OFGLUCOSE AND
G L U TA M I N E I N B R A I N
TUMOR PROGRESSION
Glucose is the predominant fuel of the brain,
but it also fuels tumor cell glycolysis as well as
serving as a precursor for glutamate synthesis
(McKenna etal., 2006; Seyfried and Mukherjee,
2005; Seyfried et al., 2003; Warburg, 1956a).
Using linear regression analysis, we showed
that the growth rate of the CT-2A experimen-
tal mouse astrocytoma was directly dependent
on blood glucose levels (Seyfried et al., 2003).
The higher the blood glucose levels, the faster
the tumors grew. As glucose levels fall, tumor
size and growth rate fall. Hyperglycemia not
only contributes to rapid tumor cell growth but
also enhances white matter damage in patients
receiving radiation therapy (Szerlip etal., 2011).
Hyperglycemia is also linked to poor prognosis
in humans with malignant brain cancer (Champ
and Klement, 2015; Derr et al., 2009; McGirt
et al., 2008). In other words, our findings in
mice were corroborated with similar findings in
humans.
Moreover, we found that the expression of
insulin-like growth factor 1 (IGF-1) was also
dependent on circulating glucose levels (Marsh
etal., 2008a; Seyfried etal., 2003). IGF-1 is a cell
surface receptor linked to rapid tumor growth
through the PI3K/Akt signaling pathway (Marsh
et al., 2008a). The association of plasma IGF-1
levels with tumor growth rate is due in part to
elevated levels of blood glucose. These findings
in animal models and in brain cancer patients
indicate that tumor growth rate and prognosis is
dependent to a significant extent on circulating
glucose levels. Glucose is the prime fuel for gly-
colysis, which drives growth of most brain cancer
(Oudard etal., 1996; Oudard etal., 1997; Seyfried
and Mukherjee, 2005). As long as circulating glu-
cose levels remain elevated, brain tumor growth
will be difficult to manage.
In addition to glucose, glutamine is also
suggested to play an important role in tumor
energy metabolism (Alberghina et al., 2014;
DeBerardinis and Cheng, 2010; Gao etal., 2009;
Wise et al., 2008; Yuneva, 2008). Recent stud-
ies suggest that inhibition of the mitochondrial
OxPhos, and more specifically of Complex I, is
able to promote glutamine utilization (Fendt
et al., 2013). Glutamine is also a major energy
fuel for immune cells (Newsholme et al., 2003).
As neoplastic myeloid cells can contribute to the
GBM tumor cell population, glutamine becomes
an important fuel for GBM growth and inva-
sion (Huysentruyt et al., 2011). In contrast to
extracranial tissues where glutamine is the most
available amino acid, glutamine is tightly regu-
lated in the brain through its involvement in the
glutamate-glutamine cycle of neurotransmis-
sion (Hawkins, 2009; McKenna et al., 2006).
Glutamate is a major excitatory neurotransmitter
that must be cleared rapidly following synaptic
release in order to prevent excitotoxic damage
to neurons (Hawkins, 2009; Takano etal., 2001).
Glial cells possess transporters for the clear-
ance of extracellular glutamate, which is then
metabolized to glutamine for delivery back to
neurons. Neurons metabolize the glutamine to
glutamate, which is then repackaged into synap-
tic vesicles for future release (Hawkins, 2009). The
glutamate-glutamine cycle maintains low extra-
cellular levels of both glutamate and glutamine
in normal neural parenchyma. Disruption of the
glutamate-glutamine cycle can cause neurotox-
icity and provide neoplastic GBM cells access
 Malignant Brain Cancer Management with Metabolic Therapy
to glutamine, as we recently described (Seyfried
etal., 2014b).
CYTOMEGALOVIRUS:AN
O N C O M O D U L AT O R O F B R A I N
T U M O R E N E R G Y M E TA B O L I S M
Many cancers including GBM are infected with
human cytomegalovirus (HCMV), which acts
as an oncomodulator of tumor progression
(Dziurzynski et al., 2012; Hawkins and Croul,
2011; Michaelis etal., 2009). Products of the virus
can damage mitochondria in the infected tumor
cells, thus contributing to a further dependence
on glucose and glutamine for energy metabo-
lism (Bozidis etal., 2010; Cobbs, 2013; Seyfried,
2012c; Williamson and Colberg-Poley, 2009; Yu
etal., 2011). The virus often infects cells of mono-
cyte/macrophage origin, which are considered
the origin of many metastatic cancers, includ-
ing GBM (Dziurzynski etal., 2011; Huysentruyt
etal., 2011; Munzarova etal., 1991; Pawelek and
Chakraborty, 2008; Seyfried and Huysentruyt,
2013). Indeed, we proposed that neoplastic
microglia/macrophages were the most invasive
cells within GBM (Huysentruyt etal., 2011). GBM
malignancy is correlated with the titer of HCMV
infection: the higher the titer, the greater the
malignancy (Dziurzynski et al., 2012). HCMV
infection increases glucose and glutamine metab-
olism seen in tumor cells (Chambers etal., 2010;
Yu etal., 2011). Human fibroblasts infected with
HCMV require glutamine for ATP production.
Infected cells starved for glutamine beginning 24
hours postinfection failed to produce infectious
virions (Chambers et al., 2010). Thus HCMV
infection activates the mechanisms needed to
switch the anapleurotic substrate from glucose
to glutamine (Chambers et al., 2010). We sug-
gest that HCMV infection will contribute to the
progression of GBM through an oncomodulatory
effect on tumor cell energy metabolism.
D OE S T H E C U R R E N T STA N DA R D
OF C A R E ACC EL ER AT E G BM
R EC U R R E NC E A N D PRO G R E SSION
T H ROUG H EF F EC T S ON E N ERGY
M E TA BOL ISM?
It is our view that the current standard of care
for GBM and other malignant brain cancers con-
tributes to tumor recurrence and progression.
This prediction comes from new information
describing how the standard of care can enhance
the availability of glucose and glutamine within
the tumor microenvironment (Seyfried et al.,
557
2014b). It is well documented that neurotoxicity
from mechanical trauma (surgery), radiotherapy,
and chemotherapy will increase tissue inflamma-
tion and glutamate levels (Di Chiro etal., 1988;
Kallenberg et al., 2009; Lee et al., 2010; Monje
etal., 2007). Local astrocytes rapidly clear extra-
cellular glutamate, metabolizing it to glutamine
for release to neurons. In the presence of dead
or dying neurons, however, surviving tumor
cells and the TAMs will use astrocyte-derived
glutamine for their energy and growth. TAMs
also release pro-angiogenic and growth fac-
tors, which further stimulate tumor progres-
sion (Seyfried, 2001, 2012b; Seyfried etal., 2010).
Radiation damage to tumor cell mitochondria
will hasten a dependence on glucose and glu-
tamine for growth and survival (Seyfried and
Shelton, 2010; Warburg, 1956a). The HCMV
infection in the neoplastic GBM cells will further
accelerate tumor cell growth through increased
metabolism of glucose and glutamine (Yu etal.,
2011). Tumor radiation will also upregulate
the PI3K/Akt signaling pathway, which drives
glioma glycolysis and chemotherapeutic drug
resistance (Elstrom et al., 2004; Kargiotis et al.,
2010; Seyfried and Shelton, 2010; Xu etal., 2005;
Zhuang et al., 2009). In contrast to normal glia
that metabolize glutamate to glutamine, neoplas-
tic glioma cells secrete glutamate. It is not clear if
the secreted glutamate is derived from glutamine
in the necrotic microenvironment or is synthe-
sized from glucose. Glioma glutamate secre-
tion is thought to contribute in part to neuronal
excitotoxicity and tumor expansion (Takano
et al., 2001). Any treatment that increases neu-
rotoxicity will reduce patient survival (Lawrence
etal., 2011). The standard of care increases cen-
tral nervous system toxicity. These observations
indicate that the current standard of care creates
a metabolic environment that would facilitate
GBM progression.
In addition to the enhancing effects of radia-
tion and HCMV on GBM energy metabolism,
most GBM patients are also given high-dose
glucocorticoids (dexamethasone) (Chang etal.,
2005). Although dexamethasone is given to
reduce radiation-associated brain swelling and
tumor edema, dexamethasone significantly
elevates blood glucose levels (Hans etal., 2006;
Harris etal., 2013; Kargiotis etal., 2010; Lukins
and Manninen, 2005; Noch and Khalili, 2009).
Glucose fuels tumor cell glycolysis as well as
serving as a precursor for glutamate synthesis
(McKenna etal., 2006; Seyfried and Mukherjee,
558 Part IV:Homeostatic Therapies

2005; Seyfried et al., 2003; Warburg, 1956a).
Any therapy that elevates blood glucose will
not be in the best interest of the GBM patient.
Many GBM patients are also treated with the
anti-angiogenic drug bevacizumab (Avastin).
Bevacizumab targets leaky blood vessels, thus
enhancing hypoxia and radiation-induced
necrosis in the tumor microenvironment.
Increased hypoxia will further enhance tumor
cell glycolysis and select for those tumor cells
with the greatest invasive properties (de Groot
et al., 2010; Iwamoto et al., 2009; Reardon
et al., 2011). Bevacizumab also exacerbates
radiation-induced necrosis, which will create a
more favorable environment for tumor recur-
rence (Jeyaretna et al., 2011). Viewed collec-
tively, these findings further illustrate how the
current standard of care will create a microen-
vironment that facilitates the energy needs of
tumor cells and the inevitable recurrence of the
tumor. The process is illustrated in Figure29.3.
Although the existing standard of care for
malignant brain cancer will increase patient sur-
vival over the short term (months) compared to
the no therapy option (Lawrence et al., 2012),
it is clear how this therapeutic strategy can accel-
erate the energy metabolism of surviving tumor
cells. Moreover, the malignant phenotype of
brain tumor cells that survive radiotherapy is
often greater than that of the cells from the origi-
nal tumor (Kargiotis etal., 2010). Treatments that
increase tumor energy metabolism will facilitate
tumor cell growth and survival, thus decreas-
ing overall patient survival. The data show that
only about 1.1% of GBM patients (6/532) become
long-term survivors following radiation therapy
(Figure 29.1). While GBM is certainly a deadly
disease, it remains to be determined if the current

Dexamethasone-induced
Radiation-induced
necrosis Hyperglycaemia

Dead and
dying neurons
Gln
Glu
Gln
RAC
Glu Tumour
Proinflammatory
cytokines
Glu Glu Proangiogenic factors
Gln: Glu cycle
Glu

Dying Gln TAM

Gln
neurons

Glu

Monocytes
from circulation

FIGURE 29.3: How the standard of care can accelerate brain tumor growth and recurrence.
GBM and other high-grade brain tumors consist of multiple neoplastic cell types as well as tumor-associated mac-
rophages (TAMs), which release proinflammatory and pro-angiogenic factors. All these cells will use glucose and
glutamine (Gln) as major metabolic fuels for their growth and survival. Recent evidence suggests that nearly all
GBM are infected with human cytomegalovirus, which enhances glucose and glutamine metabolism in the tumor
cells. Increased glutamate (Glu) concentrations will arise after radiation/drug-induced necrosis. Reactive astro-
cytes (RA) take up and metabolize glutamate to glutamine, whereas hyperglycemia will arise after corticosteroid
(dexamethasone) therapy. Together, these standard treatments will provide a microenvironment that facilitates
tumor cell growth, survival, and the likelihood of tumor recurrence (Seyfried etal.,2010).
With permission from Lancet Oncology.
 Malignant Brain Cancer Management with Metabolic Therapy
standard of care makes the disease even worse
(Fisher and Buffler, 2005; Stupp etal., 2009). This
situation is even more disturbing, as the standard
of care is not considered curative but only pallia-
tive (Preusser etal.,2011).
EXPLOITING MITOCHONDRIAL
DYSFUNCTION FOR THE
M E TA B O L I C M A N A G E M E N T
OF GBM
GBM, like most cancers, is primarily a disease
of energy metabolism (Seyfried, 2012a; Seyfried
etal., 2012; Seyfried etal., 2011). Rational strate-
gies for GBM management should therefore be
found in therapies that specifically target tumor
cell energy metabolism. As glucose is the major
fuel for tumor energy metabolism through lac-
tate fermentation, the restriction of glucose
8 5
Diet
Plasma [Glucose] (mM)
Initiation
4
Managed Growth
Unmanaged
Growth
2 levels. Moderate restriction of intake will reduce
[Glucose]
[Ketones]
0
0 3 10 17 24
Days of Treatment
FIGURE29.4: Relationship of circulating levels
of glucose and ketones (-hydroxybutyrate,
-OHB) to tumor management. The glucose and
ketone values are within normal physiological ranges
under fasting conditions in humans and will produce
anti-angiogenic, anti-inflammatory, and pro-apoptotic
effects. We refer to this state as the zone of metabolic
management. Metabolic stress will be greater in tumor
cells than in normal cells when the whole body enters
the metabolic zone. The values for blood glucose in mg/
dl can be estimated by multiplying the mM values by
18. The glucose and ketone levels predicted for tumor
management in human cancer patients are 3.1 to 3.8
mM (5565 mg/dl) and 2.5 to 7.0 mM, respectively.
These ketone levels are well below the levels associ-
ated with ketoacidosis (blood ketone values greater
than 15 mmol). Elevated ketones will sustain meta-
bolic pressure on tumor cells, buffer daily fluctuations
in blood glucose levels, and protect the brain from
hypoglycemia.
Modified from a previous version (Seyfried etal.,2008).
559
becomes a prime target for GBM management.
However, most normal cells of the brain also
need glycolytic pathway products, such as pyru-
vate, for energy production through OxPhos. It
therefore becomes important to protect normal
brain cells from drugs or therapies that disrupt
glycolytic pathways or cause systemic reduc-
tion of glucose. It is well known that ketones
can replace glucose as an energy metabolite and
can protect the brain from severe hypoglyce-
mia (Drenick etal., 1972; VanItallie and Nufert,
2003; Veech, 2004). Hence the shift in energy
metabolism associated with a low carbohydrate,
high-fat ketogenic diet (KD) administered in
restricted amounts (KD-R) can protect normal
brain cells from glycolytic inhibition and the
brain from hypoglycemia.
It is important to mention that the therapeu-
tic benefit for the KD is best when the diet is con-
sumed in moderately restricted amounts rather
than in unrestricted amounts. Blood glucose lev-
els are lower while ketone levels are higher when
4 the diet is consumed in restricted amounts than
Plasma [Ketones] (mM)
in unrestricted amounts (Seyfried et al., 2003;
3 Zhou etal., 2007). Moreover, a prolonged unre-
stricted consumption of the KD can cause dyslip-
2
idemia, insulin resistance, and elevated glucose
these conditions (Meidenbauer etal., 2014). The
1
mild body weight loss associated with the dietary
restriction also creates a healthier homeostatic
30
0.0 state. This contrasts with the weight loss associ-
ated with radiation and chemotherapies, which
are toxic to the body. Hence, the KD-R can
enhance general physiological health while plac-
ing metabolic stress on tumorcells.
When systemic glucose availability becomes
limiting, most normal cells of the body will tran-
sition their energy metabolism to fats and ketone
bodies. Ketone bodies are generated almost exclu-
sively in liver hepatocytes largely from fatty acids
of triglyceride origin during periods of fasting
(Krebs etal., 1971; VanItallie and Nufert, 2003).
Arestriction of total caloric intake will facilitate a
reduction in blood glucose and insulin levels and
an elevation in ketone bodies (-hydroxybutyrate
and acetoacetate). Most tumor cells are unable to
use ketone bodies for energy due to abnormalities
in mitochondria structure or function (Seyfried,
2012e; Seyfried et al., 2012). Ketone bodies can
also be toxic to some cancer cells (Maurer etal.,
2011; Skinner et al., 2009). Nutritional ketosis
induces metabolic stress on tumor tissue that
is selectively vulnerable to glucose deprivation
(Seyfried et al., 2012). Hence, metabolic stress
560 Part IV:Homeostatic Therapies
will be greater in tumor cells than in normal cells
when the whole body is transitioned away from
glucose and to ketone bodies for energy.
The metabolic shift from glucose metabo-
lism to ketone body metabolism creates an anti-
angiogenic, anti-inflammatory, and pro-apoptotic
environment within the tumor microenviron-
ment (Jiang and Wang, 2013; Mukherjee et al.,
2004; Mukherjee et al., 2002; Mulrooney et al.,
2011; Seyfried, 2012e; Seyfried et al., 2003). The
general concept of a survival advantage for tumor
cells over normal cells occurs when fermentable
fuels are abundant but not when they become
limiting (Seyfried, 2012f). Figure 29.4 illustrates
the changes in whole-body levels of blood glu-
cose and ketone bodies (hydroxybutyrate)
that are predicted to metabolically stress tumor
cells while enhancing the metabolic efficiency
of normal cells. This therapeutic strategy was
illustrated previously in cancer patients and in
preclinical models (Champ et al., 2014; Chang
etal., 2013; Fine etal., 2012; Nebeling etal., 1995;
Simone et al., 2013; Stafford et al., 2010; Zhou
etal., 2007; Zuccoli etal.,2010).
THE CALORIE-RESTRICTED
KETOGENIC DIET AS A
N O N T O X I C M E TA B O L I C
THER APY FORBR AIN CANCER
Emerging evidence suggests that metabolic
therapies using KDs that lower glucose levels
can help retard GBM growth in younger and
older patients (Nebeling et al., 1995; Rieger
et al., 2014; Woolf and Scheck, 2015; Zuccoli
et al., 2010). We showed that reduced intake of
either a high-carbohydrate diet or a high-fat,
low-carbohydrate KD had similar effects in
reducing aggressive brain tumor growth in mice
(Zhou etal., 2007). The macronutrient composi-
tion of commercial KDs for tumor treatment has
been described in detail (Abdelwahab etal., 2012;
Zhou etal., 2007). Restricted diets are those that
deliver fewer total calories in order to lower circu-
lating glucose and insulin levels (Seyfried etal.,
2003). Moreover, the KD-R has some advantages
over regular diet restriction as it delivers the
optimum metabolic stress to the cancer cells and
protects normal cells by reducing the glucose
and elevating the ketone level further (Seyfried
etal., 2005). Nebeling and colleagues (1995) first
showed that the KD was an effective nontoxic
management for advanced stage astrocytoma
in children. Ketone bodies (-hydroxybutyrate
and acetoacetate) become an alternative fuel for
brain energy metabolism when glucose levels
are reduced (Cahill and Veech, 2003; Morris,
2005; VanItallie and Nufert, 2003; Veech et al.,
2001). Ketone bodies have neuroprotective and
anti-inflammatory action against a number of
neurological and neurodegenerative diseases
(Maalouf et al., 2009; Seyfried, 2014). Ketone
body metabolism reduces oxygen free radicals
while enhancing metabolic efficiency of normal
cells (Milder and Patel, 2012; Stafford etal., 2010;
Veech et al., 2001). It is also important to rec-
ognize that circulating ketone levels will rarely
exceed 7 to 9mmol in most nondiabetic patients
since excess ketones will be excreted in the urine
(Veech etal., 2001). Hence, ketones are consid-
ered good medicine for several neurological
and neurodegenerative diseases in patients with
normal physiology (Cahill and Veech, 2003;
Freeman and Kossoff, 2010; Seyfried, 2014;
VanItallie and Nufert,2003).
The KD, in addition to lowering glucose
availability to the tumor microenvironment,
could potentially lower brain glutamine lev-
els, thus restricting availability of this energy
metabolite for tumor growth (Bergman et al.,
2010; Yudkoff et al., 2007). Previous studies
with refractory pediatric astrocytoma and adult
GBM suggest that KDs are therapeutically effec-
tive against malignant brain cancer (Nebeling
etal., 1995; Zuccoli etal., 2010). The KD-R could
be even more therapeutic if combined with
nontoxic drugs that also target glycolysis (e.g.,
2-deoxyglucose or 3-bromopyruvate; Ko et al.,
2004; Marsh et al., 2008b). The KD-R could
also further enhance GBM survival when com-
bined with the anti-HCMV drug, valganciclovir
(Valcyte). HCMV infection of tumor cells can
contribute to elevated metabolism of glucose
and glutamine (Yu et al., 2011). Recent evi-
dence indicates that valganciclovir can enhance
survival of GBM patients (Soderberg-Naucler
et al., 2013). However, this finding is contro-
versial, and further studies will be needed to
verify these initial observations (Cobbs, 2014;
Solomon et al., 2014). On the other hand, we
think the KD-R could possibly reduce HCMV
load and activation state through an effect of
viral energy metabolites.
Poff et al. (2014) also recently showed a
synergistic interaction between the KD and
hyperbaric oxygen therapy (HBO2T). The KD
reduces glucose for glycolytic energy, while also
reducing nicotinamide adenine dinucleotide
phosphate-oxidase levels for anti-oxidant poten-
tial through the pentose-phosphate-pathway.
HBO2T will increase ROS in the tumor cells
 Malignant Brain Cancer Management with Metabolic Therapy
while the ketones will protect normal cells
against ROS damage and from the potential for
central nervous system oxygen toxicity (Veech,
2004; Veech et al., 2001). Glucose deprivation
will enhance oxidative stress in tumor cells,
while increased oxygen can reduce tumor cell
proliferation (Chen etal., 2009; Spitz etal., 2000).
In contrast to radiation therapy, which also kills
tumor cells through ROS production (Harrison
and Blackwell, 2004), the KD + HBO2T will
kill tumor cells without causing toxic collateral
damage to normal cells. Some KDs might also
enhance the therapeutic action of radiation ther-
apy against brain and lung tumors (Abdelwahab
etal., 2012; Allen etal., 2013). It will be impor-
tant to compare and contrast the therapeutic
efficacy of conventional radiation therapy with
HBO2T when used with the KD-R. It would also
be important to determine if HBO2T can even-
tually replace radiation therapy for managing
malignant brain cancer.
According to the evidence presented here,
the KD-R can represent a viable nontoxic option
to the current standard of care for managing
malignant brain cancer. The KD-R can tar-
get tumor cells globally without harming nor-
mal neurons and glia. The blood-brain barrier
is less of an issue with the KD-R therapy than
with conventional therapies. Although KD-R
therapy could be a more rational approach to
malignant brain cancer management than is
the current standard of cancer, the KD-R is not
without some shortcomings. Compliance can
be a major obstacle in attempting to implement
the KD-R (Seyfried et al., 2012). Some people
can have difficulty in maintaining blood glu-
cose and ketones in the ranges needed to target
angiogenesis and to control tumor growth and
inflammation. Considerable patient discipline
and motivation is required for implementing
the KD-R as a therapy. Many neurooncolo-
gists are also unfamiliar with the principles
and concepts about how the therapy controls
tumor growth or how metabolic therapy can be
used as an alternative to the standard of care.
Consequently, some patients might be discour-
aged from using the KD-R. Nevertheless, we
remain hopeful that the metabolic approach
to brain cancer management using the KD-R
together with synergistic drugs, valganciclovir,
and possibly HBO2T will offer the best chance
for quality of life and longer term survival for
GBM patients. Neurooncologists should test
this hypothesis.
561
CONCLUSIONS AND HOPE
FORTHEFUTURE
As long as brain cancer is viewed as something
other than a metabolic disease, it is unlikely
that there will be major progress in improv-
ing progression-free survival for the major-
ity of those with the disease (Seyfried, 2012b).
The current standard of care for GBM offers lit-
tle hope of improved quality of life or long-term
patient survival (Lawrence et al., 2012). If GBM
becomes viewed as a metabolic disease, however,
we might anticipate major advances in treatment
and substantial enhancement of progression-free
survival. Metabolic therapy targets the abnor-
malities in tumor cell energy metabolism without
causing toxicity. Unfortunately, there is a criti-
cal lack of clinical trials using metabolic therapy
as an alternative to the current standard of care.
Nevertheless, we remain hopeful that the meta-
bolic approach to brain cancer management using
the KD-R together with synergistic drugs and pos-
sibly HBO2T will offer the best chance for quality
of life and longer term survival for GBM patients.
Metabolic therapy should become the new stand-
ard of care if the goal is to improve the overall sur-
vival and quality of life of GBM patients.
ACK NOWLEDGMENTS
This work was supported in part from National
Institutes of Health grants (HD-39722, NS-55195,
and CA-102135), a grant from the American
Institute of Cancer, and the Boston College
ExpenseFund.
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30
Obesityand Diabetes
Nature, Nurture, andBeyond
CHARLESMOBBS
OBESITY AND
DIABETESEPIDEMIOLOGY
A N D H E A LT H C O N S E Q U E N C E S
Biological evolution is largely the history of opti-
mizing extraction of bioavailable energy from
the environment, since energy availability (or
the lethal extraction of those resources by preda-
tors) has been a rate-limiting factor for survival.
Akey characteristic of the bioenergenetic econ-
omy is the flexibility to adapt to the availability
of nutritional resources. Notably, this includes
the capacity to use alternate substrates to pro-
duce adenosine triphosphate (ATP), depending
on availability. Since glucose produced from
photosynthesis is the ultimate source of all bio-
energetics potential, most cells and organisms
use glucose preferentially to make ATP when it
is available. When it is less available than other
substrates, cells and organisms quickly adapt
to metabolize those substrates. A classic exam-
ple of this flexibility is the lac operon, which is
activated to metabolize lactose when it is present
in the absence of glucose, and which, however,
is tellingly inhibited when glucose is present
(Lewis, 2005). As described in this chapter, such
metabolic flexibility may be relevant to obesity
on low-carbohydrate diets (Brehm et al., 2003;
Kennedy etal.,2007).
In addition to adapting to the presence of
alternate sources of bioenergetic potential, cells
and organisms must also be able to survive in
the absence of a continuous intake of nutritional
resources. Of particular relevance is the daily
cycle of sunlight that drives the daily cycle of met-
abolic economy. Aclassic example is the adapta-
tion of plants to the dark, since the production of
bioenergetics potential via photosynthesis ceases
without sunlight. However, diurnal animals gen-
erally follow the same metabolic logic, acquiring
nutritional resources during the day and using
stored resources at night (nocturnal animals, of
course, do the inverse but the logic is the same).
Thus almost all organisms store bioenergetic
potential when nutritional resources are in excess
of current need (usually during the day), then use
the stored form when environmental resources
are inadequate for current need (usually at night).
Although this minimal rhythm of storage and use
of stored resources is dictated by the daily cycle of
sunlight, organisms must also adapt to the possi-
bility of longer periods of nutritional deficit (e.g.,
during winter or drought). Thus energy resources
must be stored in excess to those needed during
the nonacquisition phase of theday.
On the other hand, other constraints limit
the optimum degree of nutritional stores. For
animals, excess storage of nutrients in prey spe-
cies would reduce mobility to escape predation,
whereas excess nutrients in predators would
reduce mobility for predation (though in the
latter case this might be self-limiting). Mobility
is also required for many other functions in
animals, including migration for reproduction
(especially in birds and insects). Thus for animals
homeostatic mechanisms have evolved to main-
tain an optimum balance between storing nutri-
tional resources for use during nutritional deficit
and maintaining mobility.
Several lines of evidence suggest that adipos-
ity is highly regulated. One such line of evidence
is the relative stability of body weight:although
most people can lose weight by limiting caloric
intake, almost all of this weight is gained back
over the next year (Davis et al., 2009). Of note
is that the weight gained back is almost exactly
the weight lost, indicating that total adiposity is
highly regulated (Davis et al., 2009). Similarly,
after removal of adipose tissue by lipectomy, adi-
posity is eventually restored to the original level
with remarkable precision (Lopez et al., 2007).
 Malignant Brain Cancer Management with Metabolic Therapy
In fact these and similar observations moti-
vated the lipostatic hypothesis, that adiposity
is defended, such that perturbation by either
overfeeding or underfeeding is quickly followed
by homeostatic responses to return to the initial
weight (Lopez etal.,2007).
Although it has been argued that, for humans
at least, homeostatic mechanisms to maintain
nutritional resources are more important than
mechanisms to prevent excess storage, this is
unconvincing, since human survival has his-
torically also required a high degree of mobil-
ity, possibly more than most species (Pijl, 2011).
Nevertheless, nutritional deficiency has arguably
been the major cause of human mortality and
morbidity throughout history (given the evidence
that malnutrition impairs immune responses to
infectious diseases). Certainly the specter of mal-
nutrition has framed the distinctly pessimistic
Malthusean view of human progress, a view that
continues to exert robust traction eventoday.
Thus from a historical perspective it is shock-
ing that in the waning of the 20th century, which
saw so many triumphs of public health, includ-
ing antibiotics, public hygiene, and the green
revolution, the major threat to global health was
the phenomenon of excess storage of nutritional
resources, apparently overriding homeostatic
mechanisms evolved to prevent precisely this
phenomenon. Nevertheless, it is now clear that
the worldwide epidemic in obesity and associ-
ated disorders is a major threat to global health
(James, 2008) and plausibly will decrease life
expectancy in the US population for the first
time in history (Olshansky etal., 2005). Indeed,
it is becoming increasingly clear that obesity is
a major risk factor for a remarkably broad range
of diseases and thus will constitute an increasing
burden on public finances (Daviglus etal., 2003;
Fontaine et al., 2003; Oster et al., 1999; Peeters
etal., 2003; Thompson etal., 1999). The main dis-
ease for which obesity is a major risk factor is type
2 diabetes (Overweight, obesity, and health risk,
2000), although this perhaps begs the question of
in what sense type 2 diabetes is a disease. Thus,
in themselves, elevated adiposity and elevated
blood glucose would plausibly be protective, in
the sense that their opposites are lethal. Certainly
epidemiological studies have overwhelmingly
confirmed that elevated blood glucose (and its
marker hemogloblin A1c) are major risk fac-
tors for a wide variety of pathologies, especially
kidney failure, retinopathy leading to blindness,
and peripheral neuropathy leading to a wide
variety of pathological consequences including
571
amputations. More important, interventional
studies have clearly demonstrated that interven-
tions leading to weight loss substantially reduce
the risk of a variety of pathologies, notably diabe-
tes, and interventions that reduce blood glucose
similarly substantially reduce the risk of dia-
betic complications. In view of the major health
threat represented by the dramatic increase in
the prevalence of obesity in the past 20years, it
is naturally of great interest to understand the
physiological mechanisms regulating adipos-
ity in humans. Of particular interest, in view of
the observation that adipose levels are typically
defended, it is of great interest to understand
why average adiposity has robustly increased in
humans in the past 20years.
ROLE OFTHE VENTROMEDIAL
HYPOTHALAMUS INENERGY
BALANCE AND GLUCOSE
H O M E O S TA S I S
The first clue to the mechanisms regulating adi-
posity actually came from clinical observations.
In these cases, published around 1900 (Bray,
1993), it was reported that patients with tumors
in the hypothalamic/pituitary area exhibited a
syndrome of obesity and sterility. These obser-
vations remained anecdotal until a classic study
published in 1940 using the relatively recently
invented stereotactic instrument (Hetherington
& Ranson, 1940)]. This study, which used a stere-
otaxic instrument to guide electrode placement,
allowing electrolytic lesion of a specific and veri-
fiable lesion in the brain, was among the first to
demonstrate the function of a specific part of
the in the control of a physiological function.
Specifically, lesions targeted to the ventromedial
nucleus of the hypothalamus in rats (but prob-
ably always lesioning adjacent arcuate nuclear
areas as well) produced profoundly increased
adiposity relative to sham-lesioned rats. The
increased adiposity was associated with increased
food intake. These studies were repeated in many
species many times over many years with reli-
ably the same results. A particularly relevant
variation on these studies was to pair-feed the
lesioned rats so that their food intake was the
same as (nonobese) sham-lesioned rats. These
pair-fed rats still became obese, only more slowly
(Hetherington & Ranson, 1940). Thus it became
clear that neurons in the area of the ventrome-
dial hypothalamus (VHM) control not only food
intake but also, apparently, metabolic rate as well.
Subsequent studies demonstrated that lesions in
this area of the hypothalamus, especially if they
572 Part IV:Homeostatic Therapies
included the area ventral to the ventromedial
nucleus (i.e., the arcuate nucleus) produced not
only sterility (as was observed in humans) but
also loss of function of all neuroendocrine sys-
tems, including glucocorticoid secretion (the
hypothalamus-pituitary-adrenal axis), growth
hormone secretion, and thyroid secretion, simi-
lar to effects of fasting (Ahima etal.,1996).
An even more informative variation on the
VMH lesion studies were the remarkable stud-
ies by Hervey, who connected rats given VMH
lesions to normal rats using parabiosis (Hervey,
1959). Remarkably, the rats with the VMH lesions
gained weight, as expected, but the control rats
lost almost all fat and frequently starved to death
(Hervey, 1959). These studies were the first to
clearly suggest that adiposity is controlled by a
circulating factor acting on hypothalamic neu-
rons. As described later, this hypothesis has been
largely confirmed by genetic analysis, although
many questions remain.
Sadly, the original clinical observations that
hypothalamic tumors in humans are often asso-
ciated with obesity have been robustly replicated.
Aparticularly common lesion of this type occurs
in children, generally in the form of hypotha-
lamic craniopharyngioma (Brabant et al., 1996;
Roth etal., 1998), a nonmalignant, slow-growing
tumor that nevertheless can cause a range of
debilitating symptoms, including hyperphagia
and obesity. In some cases, the hyperphagia may
resolve after resection of the tumor, but in oth-
ers hyperphagia and obesity may be caused by
the treatment to remove the craniopharyngioma.
In any case, these fortunately rare cases substan-
tially corroborate the unique role of the hypo-
thalamus in regulating human energy balance.
A pharmacological method to produce obe-
sity by hypothalamic lesion was discovered ser-
endipitously in the course of toxicity screening
of the compound gold-thioglucose (GTG), which
was being successfully developed as a treat-
ment for rheumatoid arthritis. During toxicity
screening it was observed that after i.p. injec-
tion of GTG at doses near but not quite LD50,
many mice became obese after treatment (Owen,
Parson, & Crispell, 1953). When this observation
was made, it was clear that hypothalamic lesions
cause obesity in a range of species, so investiga-
tors assessed if the obesity produced by GTG was
associated with hypothalamic lesions. In fact i.p.
injection of GTG reliably lesions the ventrome-
dial nucleus of the hypothalamus (Mori & Perry,
1967), although careful analysis demonstrated
that the center of the lesion was in the cell-poor
area between the ventromedial nucleus and the
arcuate nucleus (Bergen et al., 1998). Of par-
ticular interest, in some mouse strains the GTG
lesion produces not only obesity but diabetes as
well (Matsuo & Shino, 1972). It is not clear why
GTG specifically targets neurons in the ventro-
medial nucleus (although it was subsequently
shown to also target neurons in the nucleus
of the solitary tract/area postrema [Powley &
Prechtl, 1986]). However, GTG did not produce
lesions in diabetic mice (Debons etal., 1974), sug-
gesting that GTG may target neurons specifically
adapted to sense glucose as part of their function
to sense and regulate energy balance and glucose
homeostasis. As described later, these observa-
tions supported and motivated the glucostat
hypothesis (Mayer, 1953) and subsequent dis-
covery of glucose-regulated hypothalamic neu-
rons (Oomura etal., 1969). Because of these and
later results described later, the VMH has been a
major focus of studies investigating the causes of
obesity (Yi, Scherer, & Tschop, 2011). Specifically,
a major hypothesis animating the field has been
that impairments of neurons in the VMH (with
later emphasis specifically, including the arcuate
nucleus) to properly sense (and thus to underes-
timate) signals of nutritional sufficiency may be a
major contributor to obesity and diabetes (King,
2006; Myers & Olson, 2012). As described later,
this hypothesis has been in many ways supported
and still animates much research in the field,
although other brain areas are increasingly of
interest (Myers & Olson,2012).
A similar protocol, neonatal injection with
monosodium glutamate, produces a lesion
somewhat similar to the that produced by GTG,
although even more ventral, completely destroy-
ing the arcuate nucleus while damaging only a part
of the ventromedial nucleus in mice (Bergen etal.,
1998). Interestingly, however, in Chinese hamsters
this protocol produces robust damage to both the
arcuate nucleus and the ventromedial nucleus and
causes diabetes in the absence of obesity (Komeda,
Yokote, & Oki, 1980). This was an early indication
that this area of the brain controls glucose metab-
olism as well as energy balance, a concept that was
subsequently amply corroborated.
Following on these and related observa-
tions, particularly observations supporting the
glucostat hypothesis, several studies from the
Sherwin group demonstrated that inhibiting
glucose metabolism in the ventromedial nucleus
rapidly stimulated counterregulatory responses
that increase blood glucose whereas local infu-
sion of glucose or lactate in the presence of
 Malignant Brain Cancer Management with Metabolic Therapy
systemic hypoglycemia prevented counter-
regulatory responses (Borg et al., 1994; 1997;
1999; 2003). These studies clearly demonstrated
glucose-sensing neurons in the VMH play a
major role in peripheral glucose homeostasis.
GENETIC BASIS OFOBESITY
AND DIABETES INMICE
Throughout the 20th century and even more so
in the 21st century, researchers have sought the
causes of disease by genetic analysis. They have
also used genetics to clarify mechanisms causing
obesity (although, as described earlier, hypotha-
lamic lesions caused by environmental pertur-
bations have guided much research). Anecdotal
observation certainly supports that obesity runs
in families, and more rigorous research compar-
ing identical to fraternal twins has consistently
suggested that body mass index is roughly 50%
heritable (Hjelmborg etal., 2008; Maes, Neale, &
Eaves, 1997). Until recently, however, tools were
not available to isolate the specific genes involved
in human obesity.
However, tools for the genetic analysis of
complex traits, and the availability of single gene
polymorphisms determining these traits, were
developed much earlier for mice than humans.
Mice have long been the species of choice for
genetic studies in mammals because for hun-
dreds, if not thousands, of years, mice have been
the subject of recreational breeding by mouse
fanciers, who generally bred for esthetically
pleasing (cute) traits, including hair color.
One such mouse variant was the yellow or agouti
mouse, selected for its esthetically pleasing coat
color. This coat color was determined by a single
gene that segregated as a single Mendelian domi-
nant gene, suspected as early at 1927, although
rigorous analysis of one such prominent gene,
called yellow lethal, did not begin until about
1950 (Morgan, 1950). This simple heredity pat-
tern, coupled with the simple phenotype, made
this gene of great interest to mammalian geneti-
cists. Eventually this gene was bred onto the
standard C57Bl/6J background, which provided
a stark contrast:in the presence of the gene, mice
were pale yellow, but in the absence of the gene,
mice were black. Also of increasing interest,
though at the time possibly underappreciated,
the yellow color segregated with a variety of other
phenotypes, including increased susceptibility to
various cancers and obese phenotypes (Morgan,
1950). This obese phenotype had been noted pre-
viously but was not robust on other genetic back-
grounds. The complexity of the phenotypes and
573
their idiosyncratic dependence on genetic back-
ground led to much confusion in the course of
these early studies. On the C57Bl/6J background,
however, the obesity associated with the agouti
gene was quite robust and entailed hyperphagia.
As with VMH lesions, however, even when agouti
mice were pair-fed with wild-type mice, they
became obese, although at a slower rate. These
observations clearly suggested a potentially
hypothalamic locus for the obesity-producing
effects of the agouti gene, but the link between
coat color and energy balance was completely
obscure for manyyears.
Two other single genes causing obesity, though
segregating as a Mendelian single recessive gene,
were discovered through a completely differ-
ent method. Jackson Laboratories has been the
largest breeder of mice for scientific research for
much of the 20th century. As part of its program
it has instituted a systematic screening program
of each mouse for visible phenotypes, the purpose
of which was to discover spontaneous mutations.
The program was inspired by the highly produc-
tive studies of Morgan and colleagues study-
ing induced mutations in Drosophila (Morgan,
1911). This program has produced some of the
most informative mutations (leading ultimately
to the discovery of the relevant genes) in biomed-
ical research. One of the genes, initially called the
obese or ob gene, was originally discovered in a
complex cross, but eventually the gene was bred
to the standard C57Bl/6J background (Coleman
& Hummel, 1973). Homozygous ob/ob mice on
the C57Bl/6J were robustly obese and hyper-
phagic; as with VMH lesions and with the agouti
mutation, pair-fed ob/ob mice still became obese
but at a slower rate (Coleman & Hummel, 1973).
Furthermore, ob/ob mice exhibited a wide range
of phenotypes, including sterility, suggesting that
the gene involved was involved in a wide range
of physiological functions (Coleman & Hummel,
1973). On the other hand, hypothalamic lesions
also produced a similar wide range of functions,
but the relation of the ob gene to hypothalamic
function was completely obscure for decades.
A mutation in another gene produced pheno-
types similar to mutations in the ob gene but
was designated the diabetes (db) gene because
on the earliest backgrounds studied, the obe-
sity was associated with robust hyperglycemia
(Hummel, Dickie, & Coleman, 1966), compared
to the relatively mild hyperglycemia produced by
the ob gene. However, further breeding demon-
strated that the ob gene and the db gene produced
essentially identical phenotypes when placed
574 Part IV:Homeostatic Therapies
on the same genetic backgrounds (Coleman &
Hummel, 1973; 1975). Nevertheless, the same
studies clearly demonstrated that the obese and
the diabetes genes were physically distinct. The
similarity of the phenotypes produced by muta-
tions in these distinct genes suggested that their
functions might be part of the same system.
Following the pioneering studies of Hervey
(1959), Coleman developed the hypothesis that
the ob gene and the db gene code for a hormone
and its receptor (without specifying which was
which). As indicated earlier, Herveys studies
had shown that when obese rats with lesions in
the VMH were joined by parabiosis with normal
rats, the normal rats rapidly lost weight whereas
the obese rats retained their obesity. These stud-
ies suggested that a circulating satiety factor
normally acts on the hypothalamus, but when
the hypothalamus is damaged it can no longer
respond to this factor, leading to increased levels
of the factor in proportion to increased adipos-
ity. To address this hypothesis, this group car-
ried out a parabiotic analysis, first linking the
circulatory systems of db/db mice to wild-type
mice (Coleman & Hummel, 1969). In Colemans
now-classic studies, whereas db/db mice contin-
ued to gain weight, the wild-type mice connected
to db/db mice starved to death (Coleman &
Hummel, 1969), just as in Herveys VMH-lesioned
rats (Hervey, 1959), leading Coleman to conclude
that db/db mice fail to sense a peripheral signal of
satiety or adiposity, thus leading to an overabun-
dance of this signal, which when reaching the
circulation system of wild-type mice led to star-
vation. In a subsequent study, Coleman linked
ob/ob mice to db/db mice, leading to death by
starvation in the ob/ob mice, whereas the db/db
mice continued to gain weight (Coleman, 1973).
Furthermore, when ob/ob mice were linked to
wild-type mice, both the wild-type and ob/ob
mice continued to gain weight, although ob/ob
mice gained weight somewhat more slowly than
when not linked to wild-type mice (Coleman,
1973). From these studies Coleman concluded
that the obese mouse is unable to produce suf-
ficient satiety factor to regulate its food con-
sumption, whereas the diabetes mouse produces
satiety factor, but cannot respond to it because
of a defective satiety center (Coleman, 1973).
These predictions were borne out by subsequent
molecular genetic studies, but. as described later,
the actual situation is probably more complicated
thanthat.
By the 1990s genetic techniques, particu-
larly the present of restriction enzyme length
polymorphisms, had developed substantially to
the point that single-gene mutations causing a
specific phenotypes could be determined by posi-
tional cloning. The first mutation known to cause
obesity to be isolated using this approach was the
Agouti (viable Yellow) gene (Bultman, Michaud,
& Woychik, 1992; Miller et al., 1993). Isolation
of this gene surprisingly demonstrated that the
mutation was not in the coding region of the gene
but in the promoter, leading to ectopic expression
of the peptide coding for the gene (Miller etal.,
1993). The wild-type version of the Agouti gene
is normally expressed almost exclusively in hair
follicles and the epidermis (Miller et al., 1993).
Since mechanisms regulating coat color have
been extensively elucidated, it seemed plausible
that enhanced expression of the peptide coded
by the Agouti gene would likely interfere with
these mechanisms. Since the product appeared to
be a secreted peptide, the most plausible hypoth-
esis was that the peptide served to antagonize
the action of alpha-melanin-stimulating hor-
mone (-MSH), whose action at the melanocor-
tin receptor is known to promote the formation
of eumelanin, the main compound producing
dark coat color. Thus it was of great interest that
shortly after the discovery of the agouti peptide,
it was shown to block the activity of -MSH at the
melanocortin receptor, reducing the production
of cAMP and eumelanin (Lu etal.,1994).
While these studies did not directly demon-
strate the cause of the obesity, the authors also
reported that the agouti protein antagonized the
binding of -MSH to the homologous melanocor-
tin receptor, the MCR4 receptor (Lu etal., 1994).
The authors suggested that obesity might there-
fore be caused by the action of the agouti gene
to antagonize VMH -MSH acting outside the
skin. The obvious hypothesis was that -MSH was
acting on the MC4R receptor in the hypothala-
mus. However, at that point there had been lit-
tle evidence that -MSH played an important role
in regulating energy balance. There were reports
of modest effects to reduce food intake by -MSH
(Poggioli, Vergoni, & Bertolini, 1986; Tsujii &
Bray, 1989), but these effects were not substan-
tially distinguishable from the modestly anorec-
tic effects of many other peptides.
Shortly after the cloning of the Agouti gene,
the obese gene was also cloned by positional clon-
ing (Zhang etal., 1994). The cloning of the obese
gene was somewhat more technically challeng-
ing because the mutation in this gene that causes
obesity is recessive, in contrast to the dominant
character of the Agouti gene. In any case the
 Malignant Brain Cancer Management with Metabolic Therapy
likely function of the protein encoded by the
obese gene was immediately clear from the cod-
ing sequence, which indicated a secreted poly-
peptide made predominantly in adipose tissue
(Zhang etal., 1994). Thus the cloning of the obese
gene immediately suggested that this gene coded
for the circulating product originally suggested
by the parabiosis studies of Hervey and especially
Coleman. The studies of Hervey suggested that
the product of the obese gene would act in the
VMH, and the studies of Coleman suggested that
the product would act via a receptor.
These hypotheses were quickly corroborated.
First, the protein product encoded by the obese
gene was demonstrated to reduce food intake
and body weight when injected into either ob/ob
mice or wild-type mice but had no effect in db/db
mice (Halaas etal., 1995); thus the coded protein
was christened leptin, based on the Greek word
for light or, as the authors indicated, thin.
Based on the published sequence, Tartaglia etal.
labeled leptin and screened various tissues and
cell types for binding activity, eventually clon-
ing a gene from choroid plexus that bound the
peptide (Tartaglia etal., 1995). Of particular note
was that the gene mapped near the diabetes locus,
coded for a single membrane receptor similar to
the IL-6 cytokine receptor and a variant of which
was expressed in the hypothalamus (Tartaglia
etal., 1995). Shortly thereafter the diabetes gene
was cloned by positional cloning and shown to
code for the same protein as reported by Tartaglia
etal. (1995); Lee etal. (1996); Chen etal. (1996).
Once leptin was available for study, it was
quickly demonstrated that, consistent with the
hypothalamic localization of the leptin recep-
tor, the hormone was particularly effective when
infused directly into the third ventricle of the
hypothalamus, which largely targets the ven-
tromedial/arcuate hypothalamic area (Schwartz
et al., 1996). In particular, infusion of leptin
into the ventromedial nucleus produced a rapid
reduction in blood glucose (Minokoshi, Haque,
& Shimazu,1999).
While the 1990s were a high point in the field
of obesity, the hopes that leptin might be the
equivalent of insulin, and that obesity is caused
by a deficiency that could be treated by hormone
replacement, were quickly disappointed. In fact,
it was clear almost at once that obesity is almost
always associated with increased, rather than
decreased, leptin secretion, suggesting that obe-
sity, if anything, is characterized by leptin resist-
ance, analogous to insulin resistance (Frederich
et al., 1995). Subsequent clinical trials failed to
575
observe a clinically meaningful weight loss after
treatment with leptin (Heymsfield et al., 1999).
Of particular interest was that Agouti mice were
characterized by relatively elevated expressions
of leptin in adipose tissue, raising the possibil-
ity that leptin acts through the melanocortin
system and that these mice are relatively resist-
ant to leptin (Mizuno etal., 1996). The parabiotic
studies of Coleman suggested that the product of
the obese gene product (now referred to as leptin)
might be the hypothetical feedback humoral
factor(s) (presumably a hormone or hormones)
that accounts for the starvation that occurs in
the control animal linked to the obese animal.
However mice expressing transgenic leptin,
whose levels are on the order of those observed
in db/db mice, do not starve to death and indeed
eventually increase adiposity to almost normal
levels (Qiu et al., 2001). This suggests that rats
with hypothalamic lesions, and (leptin recep-
tor deficient) db/db mice, express other factors
in addition to leptin that account for the starva-
tion phenotype. In view of the inefficacy of leptin
treatment in human obesity (Heymsfield et al.,
1999), the search for such factors would appear to
be particularly compelling.
A major breakthrough in the field came from
the demonstration that targeted disruption of the
MC4R receptor, which is not associated with skin
color, causes obesity in mice (Huszar etal., 1997).
Although this genetic study was somewhat at
odds with the pharmacological studies suggest-
ing that -MSH (the ligand for the MCR4 recep-
tor) produces relatively modest reductions in
food intake, subsequent studies amply corrobo-
rated the importance of the melanocortin system
in the regulation of energy balance and glucose
homeostasis. A key set of observations were
based on the regulation of the gene that codes for
-MSH, proopiomelanocortin (POMC). This gene
codes for the precursor protein that is proteolyti-
cally processed into several active neuropeptides,
including beta-endorphin, -MSH, and cortico-
tropin (also known as ACTH), depending on the
tissue (hence the rather complex name). The gene
for this precursor peptide was originally isolated
from pituitary tumor cells on the basis of its cod-
ing for ACTH but was then shown to also code
for beta-endorphin (Roberts & Herbert,1977).
The POMC precursor in the pituitary, from
which it was originally cloned, is processed to
produced ACTH, which is then released into
systemic circulation to stimulate glucocorti-
coid production when acting on the adrenal
glands through the MCR2 receptor. Interestingly
576 Part IV:Homeostatic Therapies
Cushings disease, a condition characterized by
hyperactivity of the adrenal gland and thus of
elevated glucocorticoid secretion, can be caused
by pituitary tumors that hypersecrete ACTH.
Cushings disease is characterized most notably
by insulin resistance (sometimes diabetes) asso-
ciated with syndromic deposition of adipose tis-
sue in unusual areas such as the upper back. Thus
this product of the POMC gene can, under unu-
sual circumstances, drive a metabolic syndrome
including obesity. While beta-endorphin is also
produced by processing POMC in the pitui-
tary, the function of pituitary beta-endorphin is
notknown.
POMC is also expressed in the VMH as well
as brainstem, where it is differentially processed
to produce -MSH rather than ACTH (-MSH
is derived from a further cleavage of ACTH,
releasing the N-terminal peptide). The func-
tion of hypothalamic POMC had been obscure
(a popular hypothesis was that the key pep-
tide was beta-endorphin, which might regulate
reproductive function), but after it became unex-
pectedly clear that the receptor for -MSH (the
MC4R receptor) plays a key role in regulating
energy balance, a role for hypothalamic POMC
in energy balance was assessed. The key obser-
vations were that expression of hypothalamic
POMC was reduced in ob/ob (leptin-deficient)
and db/db (leptin receptor deficient) mice, as well
as by fasting (Mizuno etal., 1998; Schwartz etal.,
1997; Thornton etal., 1997), supporting previous
observations suggesting that leptin acts through
the hypothalamic melanocortin system (Mizuno
et al., 1996). Subsequent studies demonstrated
that ablation of neuronal POMC, leaving pitui-
tary POMC intact, produces massive obese phe-
notypes (Smart, Tolle, & Low,2006).
A key observation linking POMC to the leptin
system was that transgenic restoration of POMC
in ob/ob mice reversed obese phenotypes by
about 50% and completely reversed impairments
in glucose homeostasis in ob/ob mice independ-
ent of improvements in body weight and food
intake (Mizuno et al., 2003). Subsequent stud-
ies using alternative approaches generally cor-
roborated these studies (Balthasar et al., 2004;
Berglund etal.,2012).
Another neuropeptide expressed in the VMH
(more precisely in the medial arcuate nucleus) and
implicated in the regulation of energy balance is
neuropeptide Y (NPY). In contrast to the lep-
tin system and the melanocortin system, whose
role in energy balance was largely discovered by
genetic analysis, the role of the NPY system was
discovered by more conventional pharmacologi-
cal methods. The first evidence was that NPY,
which was originally discovered as a fairly abun-
dant peptide in the whole brain with no clear
function, is expressed in particularly high levels
in the hypothalamus, leading to assessment of its
effect on food intake (Clark et al., 1984). These
studies demonstrated that infusing NPY into
the third ventricle stimulated food intake (Clark
etal., 1984). This observation was not particularly
remarkable since the infusion of many neuro-
peptides into the third ventricle either stimulate
or inhibit food intake, and the significance of
these pharmacological effects are rarely clear.
However, in a key follow-up study, Sanacora etal.
demonstrated that NPY expressed specifically in
the arcuate nucleus was relatively induced both
in genetically obese Zucker rats (which were sub-
sequently shown to be characterized by a muta-
tion in the leptin receptor [Takaya etal.,1996])
and by fasting (Sanacora et al., 1990). Thus the
regulation of NPY is precisely inverted relative
to POMC (Mizuno et al., 1998; Schwartz et al.,
1997; Thornton etal., 1997). However, in contrast
to POMC, complete genetic ablation of NPY pro-
duces little if any metabolic phenotype (e.g., on
food intake, body weight, hyperphagic response
to fasting, and weight loss in response to leptin)
although such mice exhibit an enhanced suscep-
tibility to seizures (Erickson, Clegg, & Palmiter,
1996). These studies raise major concerns about
the physiological importance of NPY for the
regulation of energy balance. Nevertheless, abla-
tion of NPY does reduce obese phenotypes in the
complete absence of leptin (Erickson, Hollopeter,
& Palmiter, 1996), similar to effects of transgenic
restoration of POMC (Mizuno etal.,2003).
Similar to NPY in hypothalamic location,
regulation, and function is the neuropeptide
Agouti-related peptide (AgRP) (Ollmann et al.,
1997). The gene for this neuropeptide was dis-
covered in a search of a plausible hypothalamic
homolog to the wild-type Agouti gene, which in
wild-type mice is expressed principally in the
skin, not the brain (Miller et al., 1993). In con-
trast, AgRP is expressed in the hypothalamus;
of particular interest, AgRP also antagonizes
the MC4R receptor, and, even more impres-
sively, transgenic overexpression causes obesity
without changing coat color (Ollmann et al.,
1997). In contrast to NPY, which is expressed
in many, if not most, areas of the brain, AgRP is
expressed almost exclusively in the medial aspect
of the arcuate nucleus and in fact co-expresses
in NPY neurons in that area (Hahn etal., 1998).
 Malignant Brain Cancer Management with Metabolic Therapy
Furthermore, both NPY and AgRP are induced
by fasting and in genetically obese mice (Mizuno
& Mobbs, 1999). It was therefore greatly disap-
pointing that genetic ablation of both NPY and
AgRP had no evident effect on energy balance
(Qian et al., 2002). Nevertheless, ablation of
AgRP neurons in adult, though not neonatal,
neurons leads to dramatic reduction in food
intake (Luquet et al., 2005), suggesting that at
least in adults something produced by these neu-
rons (though apparently neither AgRP nor NPY)
plays an important in regulating energy balance
under normal circumstances.
Another key neuropeptide, expressed in the
lateral hypothalamus, is melanocyte concen-
trating hormone (MCH) (Qu et al., 1996). This
neuropeptide is elevated in the lateral hypothala-
mus of genetically obese ob/ob mice, and central
infusion increases food intake (Qu et al., 1996).
Furthermore, in contrast to the lack of pheno-
type by ablation of NPY and AgRP, ablation of
MCH produces a lean (though not starving) phe-
notype (Shimada et al., 1998), and overexpres-
sion produces modest obese phenotype (Ludwig
etal.,2001).
An unexpected consequence of the remarka-
ble discoveries in the 1990s was a shift away from
an emphasis on the ventromedial nucleus and
toward the arcuate nucleus, primarily because
NPY/AgRP and POMC neurons were reported
to be expressed in the arcuate nucleus. This was
puzzling because many previous studies had
demonstrated that lesions of the arcuate nucleus,
especially by neonatal exposure to i.p. monoso-
dium glutamate (MSG), produced only very mild
obesity, without hyperphagia, compared to the
far more robust obesity with hyperphagia pro-
duced by VMN lesions produced by electrolytic
destruction or GTG (Bunyan, Murrell, & Shah,
1976). Of particular pertinence, MSG lesions did
not produce hypophagia, as would be expected
if NPY/AgRP neurons play an important role in
enhancing food intake and producing pro-obese
phenotypes, consistent with the lack of metabolic
phenotypes produced by ablating both NPY and
AgRP (Bunyan, Murrell, & Shah,1976).
On the other hand, these apparently para-
doxical observations were not as simple as they
appeared. First, although it is widely assumed
that POMC neurons are confined to the arcuate
nucleus, this is not the case. NPY/AgRP neurons
are confined entirely to the medial aspect of the
arcuate nucleus and thus are almost completely
eliminated by neonatal MSG (Bergen etal., 1998).
In contrast, POMC neurons are expressed in the
577
lateral aspect of the arcuate nucleus as well as
substantially lateral to and outside of the bound-
aries of this nucleus (Bergen etal., 1998). While
MSG did reduce POMC expression (presum-
ably by destroying the medial POMC neurons),
it appears that the lateral POMC neurons were
spared (Bergen etal., 1998), and of course MSG
did not produce the robust obesity produced by
genetic ablation of POMC (Smart, Tolle, & Low,
2006) or the relevant MC4R receptor (Huszar
et al., 1997). In contrast, GTG reduced total
POMC by about the same amount (presumably
targeting mainly POMC neurons outside the
arcuate and near the boundary of the VMN) and
also of course produced extremely robust obe-
sity and hyperphagia (Bergen etal., 1998). These
data clearly suggested that a set of neurons GTG
lesions, presumably targeting the larger part of
the VMN in addition to POMC neurons, plays
a key role in regulating energy balance (Bergen
et al., 1998). However, in contrast to the NPY/
AgRP and POMC, the relevant neuropeptides
(if any) remained elusive. Nevertheless, the
observations may be explained at least in part
by evidence that VMN neurons directly regulate
(activate) POMC neurons (Sternson, Shepherd, &
Friedman,2005).
A series of molecular manipulations has led
to a better understanding of the nature of these
VMN neurons that play a key role in regulating
energy balance. Akey breakthrough was the rec-
ognition that the transcription factor steroid fac-
tor 1 is highly expressed in VMN neurons (as well
as adrenal and gonadal glands), ablation of which
(with appropriate steroid replacement to main-
tain viability) causes obesity, eventually leading
to mice with twice the weight of wild-type mice
(Majdic etal., 2002). On the other hand, the obe-
sity in these mice develops relatively late and is
not characterized by robust hyperphagia so dif-
fers from the classical robust obesity characteris-
tic of VMN lesions, for example, by GTG (Majdic
etal.,2002).
An unexpected line of investigation arose
from the observation that chronic infusion of
the neurotrophin brain-derived neurotrophic
factor (BDNF) reduces body weight (Lapchak &
Hefti, 1992), although as discussed previously
such pharmacological effects are widely reported
for many neuropeptides (e.g., NPY), so in itself
this observation generated limited interest at
the time. However, in contrast to NPY, genetic
ablation of BDNF (heterozygous only because
homozygous ablation is lethal) also led to the
expected hyperphagia and weight gain (Kernie,
578 Part IV:Homeostatic Therapies
Liebl, & Parada, 2000). Of even greater interest,
forebrain-specific postnatal ablation of BDNF
produced an even more robust hyperphagia
and obesity (Xu et al., 2003), almost as great as
observed in leptin-deficient mice (the gold stand-
ard for genetic obesity). This was of particular
interest because other than the hippocampus,
the hypothalamus expresses the highest levels
of BDNF, and in the hypothalamus, BDNF is
expressed almost exclusively in the VMN and
is induced by nutritional stimulation possibly
downstream of POMC neurons via the MC4R
(Xu etal., 2003)as well as glucose (Unger etal.,
2007). Furthermore, ablation of BDNF in VMN
recapitulates the obesity observed after forebrain
ablation of BDNF (Unger et al., 2007), whereas
infusion of BDNF into the VMH reduces food
intake and increases energy expenditure (Wang
etal., 2010). The role of BNDF became even more
difficult to resolve when it was discovered that the
form of BDNF most relevant to energy balance is
produced in the dendrites, not the cell body, of
VMN neurons, where it plays a key role in medi-
ating responses to leptin (Liao etal.,2012).
However, in contrast to neuropeptides, whose
mode of action is presumably via G-protein cou-
pled receptors, BDNF is thought to act primarily
through trophic effects via trkB, the BDNF recep-
tor (Lobo etal., 2010). This raised the hypothesis
that BDNF in dendrites from VMN neurons acts
to stabilize connections with neurons whose pro-
jections express the trkB receptor (Liao et al.,
2012). Since trkB receptors in dopamine-receptor
1 expressing neurons originating in the nucleus
accumbens appear to play a key role in mediat-
ing reward, and these neurons project to the lat-
eral hypothalamus as do dendrites from VMN
neurons, we hypothesized that BDNF in VMN
dendrites plays a key role linking food-induced
reward with food intake and obesity (Schwartz &
Mobbs, 2012). Such a hypothesis might explain
why atypical antipsychotic drugs robustly pro-
mote obesity in patients given these drugs, which
otherwise remains a major medical mystery
(Pramyothin & Khaodhiar,2010).
HYPOTHALAMIC
NUTRITIONAL SIGNALS:
S U B S T R AT E C O M P E T I T I O N
AS A CAUSE OF OBESIT Y
In addition to the robust effects of leptin, which
serves as a signal to hypothalamic neurons to
report adipose stores (Ahima & Flier, 2000),
two other hormonal signals, insulin (Woods &
Nolan, 1997) and ghrelin (Kojima et al., 1999),
also report more short-term status of nutritional
resources. However, the physiological signifi-
cance of these signals remains to be determined,
since >95% reduction of brain insulin recep-
tors produces no effect on body weight in male
mice and only an extremely marginal increase
in female mice (Bruning etal., 2000). Similarly,
ablation of ghrelin or its receptor produces lit-
tle if any effect on energy balance though there
is some effect on glucose homeostasis (Sun etal.,
2008). Therefore, among hormonal signals, leptin
is by far the most implicated in regulating energy
balance, although many lines of evidence argue
against a clear role of the leptin system in the
vast majority of human obesity (e.g., leptin sup-
plementation has no significant effect in obese
humans [Heymsfield etal.,1999]).
As indicated, the hypothesis that glucose-
sensing neurons in the VMN play an important
role in energy balance, as described by the glu-
costat hypothesis (Mayer, 1953), animated much
research in metabolism for several decades, but
this hypothesis had been largely abandoned by
the 1980s (Even & Nicolaidis, 1986) and effec-
tively abandoned after the discovery of leptin.
However, an important role for hypothalamic
nutrient sensing in regulating energy balance
arose unexpectedly from studies on the role of
fatty acid synthase in cancer (Loftus etal., 2000).
Based on the hypothesis that cancer cells must
synthesize fatty acids at a high rate to allow for
membrane and organelle replication, Kuhajda
et al. synthesized C75, a potent inhibitor of
fatty acid synthase with promising anti-tumor
properties (Kuhajda et al., 2000). In course of
characterizing the physiological effects of this
compound, Loftus etal. observed that C75 sub-
stantially reduced food intake, usually an indi-
cation of toxicity (Kuhajda etal., 2000). To their
credit however, these investigators examined in
detail the mechanism of anorexia, and found
the mechanism to require elevated hypotha-
lamic malonyl-CoA, an expected consequence
of inhibiting fatty acid synthase, which normally
uses malonyl-CoA in synthesizing fatty acids
(Kuhajda et al., 2000). Since a classic effect of
malonyl-CoA (which is normally exported to the
cytoplasm when glucose metabolism is active)
is to reduce carnitine palmitoyltransferase 1A
(Cpt1a), the rate-limiting enzyme for beta oxi-
dation of fatty acids, these investigators boldly
hypothesized that inhibiting hypothalamic beta
oxidation inhibits food intake and body weight,
probably in interaction with hypothalamic
 Malignant Brain Cancer Management with Metabolic Therapy
glucose-sensing neurons (Kuhajda et al., 2000).
This was bold because the standard view, based
on studies of cortical neurons, was (and gen-
erally still is) that neurons cannot support
beta oxidation (i.e., metabolism of fatty acids),
although astrocytes can (Edmond et al., 1987).
Nevertheless, based on studies in the periph-
ery, Ruderman et al. had hypothesized that
such substrate competition might indeed exist
in glucose-sensing neurons and thus play a role
in the physiological function of these neurons
(Ruderman etal.,1999).
The hypothesis that enhanced beta oxidation
in hypothalamic neurons promotes obese pheno-
types was supported by the report that inhibition
of hypothalamic Cpt1a (also called liver-specific
Cpt1) reduced food intake and hepatic glu-
cose production (Obici et al., 2003). Similarly,
pro-obese phenotypes of ghrelin are mediated
in part by induction of hypothalamic beta oxi-
dation (Andrews etal., 2008). Specifically, ghre-
lin induced hypothalamic expression of Cpt1(a),
and inhibition of Cpt1a activity with etomoxir
blocked the effects of ghrelin to increase food
intake (Andrews et al., 2008). Consistent with
these observations, we reported that fasting and
hypoglycemia produces changes in gene expres-
sion (including elevation of Cpt1a) indicating a
shift away from glucose utilization and toward
beta oxidation (Mobbs et al., 2004; Poplawski
etal., 2010; Poplawski, Mastaitis, & Mobbs, 2011).
We suggested that at least some of these effects are
due to activation by hypoglycemia and/or fast-
ing of PPAR- (Poplawski etal., 2010; Poplawski,
Mastaitis, & Mobbs, 2011), whose activation
during fasting (by free fatty acids) mediates the
activation of Cpt1a and inhibition of glucose
metabolism (Muoio etal., 2002). Since PPAR- in
principle activates the same genes as PPAR- (they
both target genes with PPAR response ele-
ments, this hypothesis could also accommodate
evidence that hypothalamic PPAR- promotes
obese phenotypes, especially under conditions
of excess free fatty acids (e.g., on a high-fat diet)
(Diano etal., 2011; Ryan etal.,2011).
In sum, these data support the hypoth-
esis that metabolism of glucose and free fatty
acids in specialized nutrient-sensing hypo-
thalamic neurons are in competition, with
fatty acid metabolism (beta oxidation) driving
pro-obese phenotypes and glucose oxidation
opposing obese phenotypes. Since free fatty
acids activate fatty acid oxidation (presum-
ably at the expense of glucose oxidation), this
could account for the phenomenon of obesity
579
produced by a high-fat diet, at least in rodents
(Surwit etal.,1988).
It should be noted that the effects of dietary
composition on obesity are complex, as we have
previously described (Mobbs etal., 2007). Thus,
in mice, a high-fat diet with relatively normal
carbohydrate and protein concentrations pro-
duces obesity relative to a high-carbohydrate,
low-fat diet (Surwit etal., 1988), but a ketogenic
diet even higher in fat with low carbohydrates
and protein actually reduces adiposity (Kennedy
etal., 2007). Asimilar diet in humans also facili-
tates weight loss (Brehm etal., 2003). The mecha-
nisms mediating these effects are not known
but may depend on the unique metabolic state
produced by ketogenesis (Kennedy et al., 2007;
Mobbs etal.,2007).
GENETIC OBESITY
INHUMANS
Genetic studies in mice, implicating leptin and the
melanocortin system, led to the discovery (using
the candidate gene approach) that mutations in
these systems also play a key role in the regula-
tion of human adiposity. For example, mutations
in leptin (Montague etal., 1997), the leptin recep-
tor (Clement etal., 1998), the MCR4 receptor (Yeo
etal., 1998), and POMC itself (Challis etal., 2002;
Jackson etal., 1999)can cause obesity in humans.
The most common cause of single-gene mutations
that cause obesity in humans arise in the MC4R
receptor (Yeo etal.,1998).
Noncandidate gene approaches, especially
more recent studies with very high power
(>200,000 individuals), such as genome-wide
association studies (GWAS), have corroborated
that obesity is indeed heritable but that polymor-
phisms in no single gene can account for more
than a very small amount (<1%) of the variance
in body mass index. The main candidate for
human obesity that has arisen from these stud-
ies is the fat mass and obesity associated (FTO)
gene (Hinney, Vogel, & Hebebrand, 2010). While
polymorphisms in this gene are the most reliably
associated with adiposity in humans (Hinney,
Vogel, & Hebebrand, 2010), the functional sig-
nificance of the FTO polymorphism is unclear,
including whether increased or decreased activ-
ity of FTO is associated with obesity. One line of
evidence suggests that fasting reduces hypotha-
lamic FTO and glucose induces it (i.e., regulation
similar to BDNF and POMC), so it seems likely
that decreased activity of FTO (whatever it is
doing) is associated with obesity. Nevertheless,
although FTO is presently the most promising
580 Part IV:Homeostatic Therapies
genetic basis of human obesity, the functional
basis of this risk remains to be determined.
A commonly held hypothesis is that human
obesity is actually quite simple to explain:calo-
ries consumed are simply greater than calories
expended over time, thus leading to the storage
of excess calories that accumulate over time,
due at least in part to an inherent bias toward
weight gain (Schwartz etal., 2003). Thus between
ages 50 and 70, average weight gain is about 0.8
pounds/year (Mozaffarian etal., 2011). This plau-
sibly reflects that caloric consumption each year
is very slightly greater than caloric expenditure
(indeed, it is remarkable that the difference is not
greater). While this is an appealing simple poten-
tial mechanism, it is difficult to reconcile with the
evidence that while it is relatively easy for most
people to lose substantial weight in a supervised
program, weight is regained relatively quickly.
A typical example is a recent study examin-
ing weight gain in patients who had within the
last year lost about 17 pounds due to intentional
weight loss, largely for health reasons (Sherwood
et al., 2013). Patients were then encouraged to
maintain weight loss by following a manual and
log book promoting standard weight loss strat-
egies, in addition to two follow-up phone calls
(Sherwood et al., 2013). These patients gained
about 10 pounds in the following 2years; that is,
they gained weight over five times faster than the
rate at which they likely accrued these 10 pounds
to begin with, despite intensive efforts to main-
tain the weight (likely far more effort than the
original exerted to prevent the original weight
gain). Similar rapid gain after substantial weight
loss has been widely exemplified by many celebri-
ties (e.g., Oprah Winfrey [Shugart,2011]). These
observations clearly indicate that a slow accrual
of adiposity due to a slight mismatching between
caloric intake and caloric expenditure may
account for the original increase in adiposity but
that this accrual entails regulatory adjustments
that effectively defend the new body weight.
O B E S I T Y: N E I T H E R N AT U R E
NOR NURTURE
This review has focused on the genetic basis of
energy balance, the study of which in mice has
led to profound insights on the molecular mech-
anisms involved. Nevertheless, this approach
clearly has major limitations in that, despite
extensive progress in the past 20years, we seem
hardly closer to understanding human obesity
(with the exception of vary rare individuals whose
obesity is caused by single-gene mutations) than
we were before the heroic age of discovery in the
regulation of energy balance and glucose homeo-
stasis. In particular, it is clear from GWAS stud-
ies that no single polymorphism can account for
any but a tiny fraction of the variance of adipos-
ity, and even aggregating all the genes implicated
by GWAS, no common mechanisms emerge as
particularly clear or dominant. In any case, the
best evidence for the total combined effect of all
polymorphisms on adiposity is likely to be no
more than about 50% of variance of adiposity
(Hjelmborg et al., 2008; Maes, Neale, & Eaves,
1997). Thus it is unclear that focusing on poly-
morphisms in individual genes is likely to provide
substantial insight into causes of human obesity.
It might be assumed that the increase in the
prevalence of obesity over the past 20 years,
clearly not due to changing genotypes, must be
due to changing environment, most likely in the
increased palatability and reduced cost of pro-
cessed food. While this would appear to be plau-
sible, there is at present no clear evidence that
any specific environmental factor (specifically
including sucrose or fructose) has significantly
contributed to the remarkable increase in obesity
observed in the past 20years.
We therefore suggest that an underappreci-
ated phenomenon may provide the most promis-
ing avenue to understand human obesity:a major
source of variance in adiposity that appears to
be neither genetic nor environmental. This phe-
nomenon was observed in the context of obesity
produced in rodents placed on a high-fat diet
(Surwit etal., 1988). While rodents generally gain
substantial weight when given access to a high-fat
diet, the mechanisms mediating this phenome-
non are unclear. It is plausible that at least part
of the mechanism has to do with activation of
the PPAR system, which in turn may promote
hypothalamic beta oxidation (and reduce hypo-
thalamic glucose metabolism), thus promoting
obese phenotypes as described earlier. Possibly
of greater interest is the basis of the individual
differences in response to a high-fat diet. Genetic
background can profoundly influence response
to weight gain on a high-fat diet (Levin et al.,
1997; Surwit etal., 1991). However, as in human
obesity, it appears that no single genetic poly-
morphism can account for more than a relatively
small amount of the variance in diet-induced
obesity (Fawcett etal.,2009).
However, a relatively underappreciated phe-
nomenon is the surprising degree of variability in
weight gain on a high-fat diet in genetically homo-
geneous inbred C57Bl/6J mice (Koza etal., 2006).
 Malignant Brain Cancer Management with Metabolic Therapy
Indeed, the coefficient of variation of genetically
homogeneous mice on essentially identical diets
(Koza etal., 2006)is about the same as the coeffi-
cient of variance of human body mass index. This
observation leads to the rather surprising con-
clusion that neither heredity nor environment
is chiefly responsible for individual variance in
adiposity. We have obtained preliminary evi-
dence supporting that variance of weight gain on
a high-fat in genetically similar C57Bl/6J mice, as
well as across different mouse strains, correlates
with hypothalamic expression of the same small
set of genes (unpublished data). Variation in the
expression of these genes probably arose from
stochastic processes that arose during develop-
ment or aging. Understanding the role of these
genes in determining body weight may lay the
groundwork for the finally understanding, and
manipulating, hypothalamic determinants of
energy balance.
An alternative source of variability could
be the population of microbes in the intesti-
nal tract. This hypothesis was initially most
prominently supported by the study of Backhed
etal. who reported that germ-free mice exhibit
relatively reduced adiposity and that introduc-
tion of microbiota (derived from the cecum of
conventionally raised mice) increased adiposity
by 60% (Backhed et al., 2004). This increased
adiposity was attributed to the likely effect of
bacteria to enhanced absorbance of monosac-
charides from the gut, leading to enhanced
lipogenesis (Backhed etal., 2004). Other expla-
nations of this effect have included direct
gut-brain nutrient sensing, possibly modulated
by intestinal bacteria (Moran & Shanahan,
2014). On the other hand, not all studies
have supported this hypothesis. For example,
germ-free mice were reported to weigh the
same as conventional mice and actually gain
more weight on a high-fat diet than conven-
tional mice (Fleissner et al., 2010). Similarly,
germ-free rats were reported to weigh the
same as conventional rats (Swartz etal., 2013).
Nevertheless, the gut microbiome continues to
constitute an intriguing potential mechanism
to explain variance of adiposity independent of
heredity or environment.
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31
Autism Spectrum Disorder and Homeostasis
S U S A N A . M A S I N O , J E S S I C A A . F O R T I N , M I C H E L L E I . M U R P H Y,
L I S A S A A , A N D D AV I D N . R U S K I N
OVERVIEW
Diverse Symptoms and Disrupted
Homeostasis
Autism spectrum disorder (ASD) affects chil-
dren and adults worldwide. Approximately
1 in 68 children (diagnosed at age 8)have ASD,
with a higher prevalence in males (1 in 42)than
females (1 in 189; Centers for Disease Control
and Prevention, 2014). Prevalence in adults
was recently estimated to be 1 in 100 (Brugha
et al., 2011). Current overall ASD rates repre-
sent a roughly 30-fold increase from the classic
survey in 1966 (Lotter, 1966), which reported
1 in ~2,200. This dramatic change in preva-
lence over time may be influenced by diagnosis
parameters and changes in disorder classifica-
tion, but evidence is mounting that suggests it
may partly reflect a real increase due to a variety
of environmental factors. Risk factors for ASD
are known to include genetic and environmen-
tal components; even the genetic component is
complex and extremely heterogeneous (Persico
and Napolioni, 2013), thus making the gene-by-
environment interaction contributing to ASD a
staggering matrix of unknown and uncontrolla-
ble variables in the human population.
For the majority of children with ASD, espe-
cially those diagnosed well after age 2, many of
the core symptoms are difficult to remediate.
With the cause(s) and underlying mechanism(s)
unknown, finding successful treatments can
seem like a circular problem. Commonly
used tools for the diagnosis of ASD are the
Diagnostic and Statistical Manual of Mental
Disorders (DSM) of the American Psychiatric
Association (2013) and the International
Statistical Classification of Diseases code of the
World Health Organization (2010). Two major
changes occurred in the recently adopted ver-
sion V of the DSM. Previously, autistic disorder
was one of several differential diagnoses based
on social deficits, impaired communication,
and restricted and repetitive behaviors and
interests. With the revision, autistic disorder
and the other possible diagnoses with these core
symptoms are all grouped under ASD; thus pre-
vious differential diagnoses no longer exist in
DSM V:Aspergers syndrome was removed, and
pervasive developmental disorder falls under
the umbrella ofASD.
The common core features of ASD accord-
ing to DSM V are social communication deficits
and fixated interests and repetitive behaviors.
The second major change, which has been less
controversial, is bringing together social deficits
and language problems into the single category
of social communication problems. (The other
major symptom category, restricted and repeti-
tive behavior, remains intact.) This change is
based on the opinion that language difficulties
in people with ASD are limited to the social
domain. Examples include difficulties in recip-
rocal conversation, delays in language devel-
opment, adherence to specific nonfunctional
routines, and intense preoccupations. Often
these behavioral impairments lead to decreased
social support and a lack of meaningful relation-
ships with peers (Kim and Lord, 2012). People
with ASD experience multiple behavioral and
physiological symptoms, which range from mild
to severe.
The great heterogeneity of symptoms and
severity among individuals with ASD makes it
difficult to identify specific causal mechanisms
and etiologies that apply to all or even most
patients with the disorder. Known genetic factors
account for ~10% to 20% of ASD cases, leaving
a large fraction of patients with uncertain eti-
ologies (Betancur, 2011). ASD is recognized to
be highly heritable, however, and a recent pop-
ulation study involving over 2 million people
 Autism Spectrum Disorders and Homeostasis
established that heritability provides about 50%
of the risk (Sandin et al., 2014). Most of these
identified genetic factors are common, however,
and/or common to other disorders and comor-
bidities; in most cases it appears to be the com-
plex interactions among these genes and with the
environment that produces an ASD diagnosis.
Differences and similarities in ASD due to gen-
der, environmental factors, prenatal influences,
genetics, and physiology may eventually reveal
mechanisms that lead to the disorder and thus
potential interventions and therapeutic targets.
Given this complexityunlikely to be resolved
into a common mechanism or therapeutic
targeta focus on restoring homeostasis could
promote broad-based improvement regardless of
the underlying cause(s) ofASD.
Depending on severity, an ASD diagnosis
can be devastating and result in lifelong dis-
ability. With a disorder so common and per-
sistent, and increasing in prevalence, there are
major economic implications. Expenditures
for ASD sufferers have been estimated to be at
least two-fold to six-fold over controls (Barrett
etal., in press; Peacock etal., 2012; Wang etal.,
2012). Total costs increase with severity (Lavelle
etal., 2014), as do out-of-pocket costs (Raz etal.,
2013). Annual cost per ASD patient has been
estimated at $35,000 (Horlin et al., 2014), and
total cost per patient over a lifetime as $2mil-
lion to $3million (Buescher etal., 2014; Ganz,
2007); total annual expenditure in the United
States is roughly $8 billion (Trasande and Liu,
2011). Around the world, practical, effective,
and economically viable therapies are needed.
Alongside new therapies, prevention by avoid-
ing environmental risk factors may become
an important strategy in addressing rising
incidence.
Herein we present a viewpoint that frames
ASD treatment in the context that core ASD
symptoms are often difficult to treat and are often
accompanied by neural, immune, and/or meta-
bolic dysregulationall with diverse underlying
cause(s) that may change over time even within
individuals. Targeting systemic dysregulation
with strategies that restore homeostasis may
be an effective approach. Therapies shown to
improve behavioral symptoms may be effective
because they restore homeostasis at least partially
and thus may help reveal key underlying factors
in ASD. In keeping with this perspective, we pro-
vide examples of homeostatic dysregulation in
ASD, current and potential treatment strategies,
and future research.
587
COMORBIDITIES
ASD is often accompanied by comorbid
physiological and psychological conditions,
including epilepsy, aggression, anxiety, gastro-
intestinal problems, altered sensory experiences,
motor impairments, and sleep disturbances
(Argyropoulos et al., 2012). Diverse and com-
mon comorbidities suggest that multiple aspects
of dysregulation can converge on a common
behavioral phenotype, and thus some aspects
of dysregulation may be shared widely among
patients with ASD regardless of the underlying
cause of the disorder. Recently, in an attempt
to understand the interactions between ASD
and comorbidities and among common comor-
bidities, patients with ASD were diagnosed into
several comorbid clusters based on electronic
records: (a) a seizure cluster; (b) a psychiatric
cluster; (c)a cluster that included gastrointestinal
disorders, auditory disorders, and infection, and
(d)an unresolved cluster containing most (~90%)
patients (Doshi-Velez et al., 2014). Nevertheless,
this classification scheme underscores the broad
symptomatology beyond the behavioral criteria
that define an ASD diagnosis. Here we outline
common comorbidities and focus on opportuni-
ties for neural, immune, and metabolic homeo-
stasis that may in turn address multiple aspects
ofASD.
Neural
ASD is typically thought of as a developmental
neurological disorder and is diagnosed based
on behavioralthough substantial evidence is
mounting for addressing comorbid metabolic
and immune dysfunction, discussed in more
detail later. The most common comorbidities are
in the neural realm and range from seizure dis-
orders, sensory-motor abnormalities, psychiatric
conditions, and disrupted sleep. A high comor-
bidity of epilepsy and abnormal EEG, motor
delay and dysfunction, and sensory sensitivity all
point to abnormal neural activity. Hyperactivity
and aggression are also common, and a variety of
psychoactive drugs are prescribed with the hope
that core symptoms might be alleviated along
with these comorbid symptoms.
Epilepsy is a frequent comorbidity, with
recent estimates ranging from 7% to 21% of chil-
dren diagnosed with ASD also having a diag-
nosis of epilepsy (Amiet et al., 2013; Carlsson
et al., 2013; Cuccaro et al., 2012; Doshi-Velez
et al., 2014; Kantzer et al., 2013); this range of
incidence is undoubtedly due to sampling from
different populations. An abstract presented at
588 Part IV:Homeostatic Therapies
the International Meeting for Autism Research
in 2014 (Supekar et al., 2014) presented a much
higher rate based on analyzing clinical data of
over 1 million patients at Stanford University
Medical Center: epilepsy was diagnosed in 41%
of ASD patients up to18 years old. Although
the rate remained high, there was a decrease in
comorbid seizures in adults with ASD (~24%
in ASD patients between the ages of 18 and 35
and ~15% in those over 35). Conversely, a recent
analysis screened children diagnosed with epi-
lepsy for comorbid behavioral disorders and
found that 21% of epileptic children also met
the diagnostic criteria for autism (Reilly et al.,
2014). In a cluster analysis of pediatric/adolescent
ASD aimed at comorbid epilepsy specifically,
patients fell into five clusters, one of which (5%
of patients) had a high (29%) epilepsy rate, early
ASD diagnosis, sensory abnormalities, and high
repetitive behaviors, whereas two other clusters
of high-functioning individuals (38% of patients)
had the lowest (8%) epilepsy rates (Cuccaro
etal.,2012).
Ultimately, estimates of the comorbid-
ity between epilepsy and ASD are necessarily
conservativeASD might not be diagnosed once
a child has a primary diagnosis of epilepsy, and, in
children diagnosed with ASD, seizures could be
occurring without clear convulsions or impair-
ment of consciousness. Febrile seizures and sei-
zures due to acute trauma, infection, or metabolic
illness are not diagnosed as epilepsy because they
have a known provocation (Tuchman and Rapin,
2002). However, seizures due to these events may
impact brain homeostasis at a subclinical level
and cause ongoing microseizures undetectable
behaviorally or with a scalp EEG (Stead et al.,
2010). For example, febrile seizures increase the
risk of future seizures (Berg, 2008; Mohebbi
et al., 2004), suggesting the initial seizures had
lasting effects on neural activity and/or seizure
threshold. These seizure-inducing events may
push a vulnerable central nervous system out of
homeostasis and into a ASD diagnosis.
Seizures represent dysregulated neuronal
activity, and diverse seizure types are associated
with ASD, including complex partial, atypical
absence, myoclonic, and tonic-clonic (Tuchman
and Rapin, 2002). Because the rate of epilepsy
in children in the general population is only 2%
to 3% (Amiet et al., 2013; Kohane et al., 2012;
Mouridsen et al., 2011), there is strong indica-
tion that common underlying factors influence
this comorbidity. Regarding excessive excitability,
mutations in genes encoding ion channels, such
as the sodium channel genes SCN1A and SCN2A,
have been identified in epilepsy and also in ASD
(Betancur, 2011). Additional gene mutations, such
as in protocadherin 19 and those associated with
metabolic disorders and intellectual disabilities,
have also been identified in patients with comor-
bid ASD/epilepsy (Betancur and Coleman,2012).
There is a rich array of mouse models with
varying relationships between ASD and seizures.
Some mouse ASD models are frankly epilep-
tic (Peagarikano etal., 2011; Zhao etal., 2010);
some have heightened seizure susceptibility but
no spontaneous seizures (Musumeci etal., 2000;
Pineda et al., 2013; Qiu et al., 2009; Sala et al.,
2011); some have little or no change in seizure
susceptibility (Radyushkin et al., 2009; Ruskin
et al., 2013). Conversely, some mouse models of
epilepsy also have autistic symptoms (Greco etal.,
2013; Meidenbauer et al., 2011). Interestingly,
mice in these latter models often become epi-
leptic after autistic symptoms are evident and so
are time-variant for the comorbidity. Many ASD
models remain uncharacterized or incompletely
characterized for seizures or seizure suscepti-
bility; conversely, many mice used as models of
epilepsy or with a lower seizure threshold have
never been tested for symptoms of autism. As a
whole, these animal models represent an expand-
ing research landscape in which core symptoms
of ASD can be precipitated by single-gene muta-
tions, complex gene interactions, and/or environ-
mental factors. Perhaps the best animal models to
test treatment efficacy combine a complex genetic
predisposition with epigenetic prenatal and post-
natal environmental factors.
Diverse additional comorbidities involving
the nervous system are also often associated with
ASD. Arecent review concluded that 30% to 60%
of autistic children use at least one psychotropic
medication (Siegel, 2012), and it was estimated
previously that up to 70% of autistic children
with intellectual disabilities were prescribed at
least one psychotropic medication (Tsakanikos
et al., 2006). The range of coexisting neuropsy-
chiatric diagnoses includes seizure disorders,
as noted previously, as well as attention deficit/
hyperactivity disorder (60% have poor attention
and concentration), anxiety disorder (up to 74%
have anxiety or significant fears), affective dis-
orders, obsessive compulsive disorder (37% have
obsessive phenomena), and Tourettes disorder
(Tsai, 1999). Up to 43% of people with ASD also
have a history of self-injury (Tsai,1999).
 Autism Spectrum Disorders and Homeostasis
People with ASD experience a range of
intellectual, sensory, and motor impairments.
With the recent inclusion of Aspergers syn-
drome within the diagnosis of ASD (disputed
by some:Tsai and Ghaziuddin, 2014), IQ scores
range greatly, but prior to the latest revision of
the DSM, ~50% of autistic children were char-
acterized as having an intellectual disability
(IQ<70; Charman et al., 2011). Sensory abnor-
malities include severe reactions to loud or unu-
sual noises, extreme disturbances with certain
tactile sensations, and resistance to wearing
footwear (Kim and Lord, 2012). Children with
ASD also experience motor impairments (Hilton
et al., 2014), with up to one-third experiencing
delays in reaching motor milestones (Weissman
etal.,2008).
Sleep disorders are also common in children
with ASD and contribute to a range of other
impairments. Sleep abnormalities include late
sleep onset, sleep maintenance discontinuity,
and early morning awakening (Kohane et al.,
2012; Polimeni etal., 2005; Sivertsen etal., 2012).
When children with ASD experience psychiatric,
sensorimotor, and sleep disordersin addition
to cognitive and behavioral abnormalities asso-
ciated with ASDit further decreases quality of
life and increases their need for therapy.
Metabolic
Metabolic abnormalities are prevalent in the
autistic population. Gastrointestinal problems
are also quite common (Doshi-Velez etal., 2014;
Ibrahim etal., 2009; Wang etal., 2011)and might
relate to a disordered gut microbial community
(De Angelis et al., 2013; Williams et al., 2011;
Williams et al., 2012). There are also distur-
bances in specific metabolites (El-Ansary et al.,
2010; James et al., 2006; Page and Coleman,
2000; Spilioti et al., 2013) and enzymes (Marie
etal., 2002; Muratore etal., 2013; Sivendran etal.,
2004), often indicating impaired redox homeo-
stasis and reduced methylation capacity. Some of
these may be causative for rather than comorbid
with behavioral/cognitive autistic symptoms.
Mitochondrial dysfunction is among the
most common metabolic comorbidities (Adams
et al., 2011b; El-Ansary et al., 2010; Oliveira
etal., 2005), and multiple lines of evidence from
clinical, genetic, and biochemical studiesin
humans and animal modelssupport a role for
mitochondrial dysfunction in ASD. The mito-
chondrion generates cell adenosine triphosphate
(ATP) and integrates multiple signaling cascades
589
(Horbinski and Chu, 2005), and mitochondrial
dysfunction is linked directly to a number of
neurological conditions (Garcia-Escudero et al.,
2013; Maruszak and Zekanowski, 2011; Pathak
etal., 2013; Zhu and Chu, 2010). In addition, as
mitochondrial-based energy production is criti-
cally important for neurodevelopment, mito-
chondrial dysfunction might contribute to the
developmental pathology of ASD (Haas,2010).
In a comprehensive literature review exam-
ining mitochondrial dysfunction, children
with ASD had a 5.0% prevalence rate while
the general population had a rate of approxi-
mately 0.01%, making it the most common
metabolic abnormality associated with autism
(Frye and Rossignol, 2011; Rossignol and Frye,
2012). However, this prevalence rate of mito-
chondrial disorders could even be underesti-
mated due to differences in clinical techniques
(Giulivi et al., 2010). In a study comparing 80
ASD and 73 control children, the children
with ASD had decreased antioxidant abilities
and increased oxidative stress (James et al.,
2006). Furthermore, children with ASD showed
lower serum carnitine and pyruvate levels and
increased ammonia and alanine levels, further
suggesting a mitochondrial defect (Oliveira
etal., 2005). We currently lack a detailed mech-
anistic understanding of the role of mitochon-
dria in ASD; however, in addition to regulating
cellular proliferation and differentiation, these
organelles are also involved in other aspects
of brain function such as neuroinflammation,
modulating GABA activity, and maintaining
calcium homeostasisall implicated in ASD
(Breitenkamp etal., 2014; Depino, 2013; Fatemi
etal., 2009a; Fatemi etal., 2009b).
It is now well established that metabolic
abnormalities are common with chronic neu-
rological conditions, particularly at the cellular
level, but translational research leveraging this
information into new therapies is lacking and
is becoming increasingly urgent. ASD is not
unique among neurological disorders as many
have comorbid metabolic dysfunction, yet in
ASD more widespread and systemic metabolic
changes are more prevalent; a growing number
of reviews conclude that mitochondrial dysfunc-
tion can lead to autistic symptoms (Dhillon etal.,
2011; Frye and Rossignol, 2011; Giulivi et al.,
2010; Hagberg et al., 2014; Legido et al., 2013;
Rossignol and Frye, 2012; Villafuerte, 2011). This
remains an unexplored area of research whereby
improving mitochondrial function could help
590 Part IV:Homeostatic Therapies
restore metabolic homeostasis and reduce diverse
symptoms and comorbidities.
Immunological
Many of the organ systems in ASD patients
show inflammation-related changes in immune-
related proteins and ribonucleic acids (Depino,
2013). In the brain and cerebrospinal fluid, an
inflammatory-like state was quantified by ele-
vated cytokines and activated microglia and
astrocytes in postmortem ASD brains; notably,
the age range of the subjects was 5 to 44 years
old, suggesting that this immune-activated state
starts early and can be lifelong (Vargas et al.,
2005). Elevated cytokine levels were also found
in living ASD children aged 3 to 10years (Vargas
etal., 2005). These findings were again supported
by additional studies on the same postmortem
brains, as well as new autistic brain samples (Chez
etal., 2007; Morgan etal., 2010). Microarray stud-
ies have found that in ASD brains there is a dys-
regulation of immune-related genes (cytokines
and chemokines; Lintas etal.,2012).
It has been suggested that the inflammatory-
like state in the central nervous system may
be related to abnormalities in the peripheral
immune system, but the connection between
peripheral immune abnormalities and the
altered behavior in ASD is not well understood
(Careaga et al., 2010). Compared to controls,
as well as nonautistic children with develop-
mental disabilities, autistic children exhibited
elevated cytokines and chemokines (including
interleukin-1, IL-6, IL-8, and IL-12p40) in blood
plasma. Additionally, these immune factors were
positively associated with impaired communi-
cation and behavioral abnormalities (Ashwood
et al., 2011a, 2011b). Furthermore, other stud-
ies found that in vitro blood mononuclear cells
show altered cytokine responses (Enstrom etal.,
2010; Jyonouchi et al., 2005; Patterson, 2009).
Genetic susceptibility to environmental factors
could bias the subject toward both brain and
immune-related abnormalities simultaneously;
ASD-associated gene candidates regulate both
brain and immune system development/function
(Careaga etal., 2010). Despite the need for more
research, it is well known that elevated peripheral
cytokines can cause dramatic changes in behav-
ior (Patterson,2009).
A large epidemiologic study found that
some autoimmune diseases (rheumatoid arthri-
tis, celiac disease, and type 1 diabetes) are
more common in mothers of autistic children
(Atladttir etal., 2009). Additionally, about 12%
of mothers of children with ASD have antifetal
brain antibodies in their serum, which is sig-
nificantly higher than in mothers of typical chil-
dren (Careaga etal., 2010). The role of maternal
immune and metabolic environment in utero
has received increasing attention and is one area
where there is a particularly high correlation
between clinical evidence and behavioral and
physiological outcomes in experimental models
ofASD.
ETIOLOGY OFAUTISM
SPECTRUM DISORDERS
Data about ASD etiology has increased
dramaticallyyet, paradoxically, for most
patients their particular etiology remains murky
or completely unknown. We do not attempt
a thorough review of this complex topic here,
and where possible we relate to our overarching
theme of homeostasis.
Genetic
In recent decades the known genetic basis for
ASD has improved as most known medical
conditions associated with ASD have had their
genetic underpinnings discovered. Thus the field
has progressed over two decades from known
medical conditions being associated with ~10%
of ASD cases (Rutter et al., 1994) to known
genetic conditions being associated with up to
20% of ASD cases (Betancur, 2011). This is still a
relatively low number, and genetics are undeni-
ably important:twin studies have always shown
that ASD has a very high heritability. Studies of
monozygotic twins indicate heritability rates of
>70%, and these might be underestimates as even
monozygotic twins are not necessarily geneti-
cally identical due to the possibility of different
within-pair copy number variations and different
X chromosome inactivation patterns (Posthuma
and Polderman,2013).
The genetic basis for ASD is clearly com-
plex, and disorders in over 100 specific genes
have been associated with ASD (Betancur, 2011).
These genes code for a wide array of protein
productsenzymes, ion channels, transcription
factors, transmitter and growth factor recep-
tors, cell adhesion molecules, and so onand the
genetically based ASD cases have a wide range of
incidence, down to published single case studies.
There are also over 40 genomic disorders (i.e., copy
number variations in which multigene sequences
are deleted or duplicated) that are associated with
 Autism Spectrum Disorders and Homeostasis
ASD (Betancur, 2011). In addition to all this het-
erogeneity, there is also varying penetrance of the
genetic/genomic disorder to a diagnosed autistic
phenotype, ranging from high (e.g., fragile X
syndrome, tuberous sclerosis) to low (Duchenne
muscular dystrophy, noonan syndrome). Even a
complete understanding of genetic contributions
is unlikely to lead to broadly effective therapies
for ASD in the near future.
Environmental
The role of the environment in ASD includes
many factors, including the prenatal environ-
ment. The perinatal environment and outcomes
are also factors: perinatal distress and/or low
birth weight associates with ASD diagnosis
(Froehlich-Santino et al., 2014; Gardener et al.,
2011; Losh et al., 2012). Child vaccination has
repeatedly been shown to have no association
with ASD (Taylor et al., 2014). However, mater-
nal immune activation (MIA)due to bacteria
or virusesoccurring during gestation can lead
to modified behavior in the offspring even into
adulthood. The influence of outside environmen-
tal factors is challenging to dissect but may reveal
important contributing factors and vulnerabili-
ties as discussedlater.
MIA-induced inflammation triggers an
increase of proinflammatory factors including
interleukins and activates immune cells in the
decidua, bathing the fetus in proinflammatory
compounds and antibodies and increasing the
likelihood of the child developing ASD (Patterson,
2011b). For example, an epidemiological study of
over 10,000 ASD cases found that a viral infec-
tion in the mother during the first trimester, and
any infection (viral or bacterial) during the sec-
ond trimester, was associated with an ASD diag-
nosis in the child (Atladttir etal., 2010). Similar
findings have been reported in more recent stud-
ies (Atladttir etal., 2012; Lee etal., 2015; Zerbo
et al., in press). This clinical finding has been
replicated in rodents and recently in primates
(Bauman etal., 2014; Hsiao etal., 2012; Malkova
et al., 2012; Smith et al., 2007). Rodent models
have been developed using, for instance, synthetic
agents that induce an immune response such as
poly-(I:C) (evokes antiviral-type response) and
lipopolysaccharide (evokes antibacterial-type
response). Offspring demonstrate the core symp-
toms of autism (Malkova etal., 2012; Smith etal.,
2007), some with symptoms only in males (Xuan
and Hampson, 2014), and also mitochondrial
dysfunction (Giulivi et al., 2013). In rodents,
591
MIA-induced effects on offspring behavior are
lacking in animals lacking IL6, can be prevented
by maternal injection of anti-IL6 antibodies and
can be replicated by maternal IL6 injection in lieu
of poly-(I:C) injection (Smith et al., 2007); thus
augmented IL6 levels during gestation seem suffi-
cient. These data seem to rule out a prior hypoth-
esis that detrimental effects on offspring were due
to diminished IL6 due to antipyretic treatment
during maternal viral infectioninduced fever
(Torres, 2003), as IL6 is necessary for several
aspects of nervous tissue development. It seems
very likely that, as with so many signaling agents,
there is a balanced and optimal IL6 level during
gestation (Marz et al., 1999), as well as an opti-
mal balance between IL6 and other interleukins
(Meyer etal.,2008).
Regarding factors from the outside environ-
ment, one multiyear study found an associa-
tion between incidence of ASD and proximity
of mothers (particularly in the second and third
trimesters) to agricultural pesticide use (Shelton
etal., 2014), and children with ASD have signifi-
cantly higher levels of heavy metals than con-
trols (Adams et al., 2007) with exposure level
and symptom severity correlating (Adams etal.,
2013). Many other outside factors (e.g., dietary
supplements, anticonvulsants, air pollution,
drinking-water source, electromagnetic radia-
tion, among others) have been postulated or are
being investigated. With so many factors, and a
disorder with such complex etiology, causation
from the environment will be difficult to ascer-
tain. Where possible, it would seem prudent to
limit exposure to suspicious environmental fac-
tors, particularly in individuals who may be at
higher risk such as those with a family history of
ASD, prenatal or perinatal risk factor(s), or expo-
sure to a combination of environmental risk fac-
tors that may be additive or synergistic.
Epigenetic
Epigenetic modification of DNA is a means by
which gene expression can be regulated with-
out any change in DNA sequence. This regu-
lation takes the form of DNA methylation,
histone methylation, or histone acetylation.
There is increasing interest in a possible epige-
netic component of ASD etiology, comporting
with changes in methylation capacity in autistic
patients (Adams etal., 2011b; James etal., 2006;
Muratore etal., 2013). Several studies have found
a significantly altered gene methylation pattern,
or methylome, in ASD patients compared to
592 Part IV:Homeostatic Therapies
controls (Berko etal., 2014; Nardone etal., 2014),
even when the control subjects are the monozy-
gotic twins of the patients (Wong et al., 2014).
As one example, changes in brain-derived neu-
rotrophic factor (BDNF) have been associated
with ASD (Kasarpalkar etal., 2014), and changes
in BDNF gene expression and DNA methylation
have been associated with early life adversity
in general (Jirtle and Skinner, 2007). New evi-
dence suggests altered BDNF methylation status
is similar in blood and brainthus offering the
potential for a much-needed early biomarker
(Kundakovic etal., in press).
Epigenetic dysregulation correlates with
symptom severity (Wong etal., 2014); immune-
and synapse-related genes are heavily involved
(in accord with other evidence; Nardone et al.,
2014). Methylation problems appear in much
the same set of genes implicated in ASD through
frank genetic dysfunction (Berko et al., 2014),
and epigenetics is a means by which a pre- or per-
inatal influence can cause long-term altered gene
expression because changes in DNA methylation
can be stable or very long-lasting (Tordjman etal.,
2014). Furthermore, the methylome is heritable,
such that the methylation status of a parent dur-
ing gamete generation is passed on to offspring.
This is one possible explanation for the finding
that paternal age is an ASD risk factor (Hultman
etal., 2011), even though fathers obviously can-
not contribute to the gestational environment.
Many nongenetic parental risk factors could be
similary passed on (Mouridsen et al., 2007), as
could epigenetic changes associated with mater-
nal aging, also a risk factor (Berko etal.,2014).
H O M E O S TAT I C T R E AT M E N T S
FORA SPECTRUM
OFSYMPTOMS
A perspective that most cases of ASD are related
to disrupted neural, metabolic, and/or immuno-
logical homeostasisrather than highly selective
pharmacological targetsfocuses treatments
more on broad-based strategies to alleviate multi-
ple symptoms. Certainly ongoing research efforts
need to focus simultaneously on both mechanis-
tic underpinnings and symptom relief to bet-
ter understand and treat ASD: therapies shown
to help with symptoms of ASD could also help
reveal underlying mechanisms. Here we outline
some evidence as examples, focusing on treat-
ments that are established as safeand perhaps
with multiple health benefitsand offer sugges-
tions for further research into related treatment
opportunities.
Pharmacological Approaches
Pharmacological attempts at treatment have been
used with varying degrees of success. None of
these were developed for ASD per se, and many
deal only with comorbidities. In many cases, sei-
zures can be controlled with anticonvulsant drugs
(although roughly one-third of ASD/epilepsy
patients have drug-refractory epilepsy: Sansa
etal., 2011), but even when these agents have been
prescribed also to help irritability and aggression,
meta-analysis shows they are ineffective against
comorbidities other than epilepsy (Hirota etal.,
2013). Hyperactivity, impulsiveness, and atten-
tion problems can be controlled with stimulants
acting on dopamine or norepinephrine systems
(Baribeau and Anagnostou, 2014; Cortese etal.,
2012; McCracken et al., 2014; Pearson et al.,
2013), though side effects can be worse in ASD
sufferers than in controls (Doyle and McDougle,
2012). Atypical neuroleptics can be helpful with
irritability and aggression, but again, adverse
side effects can be prominent (Baribeau and
Anagnostou, 2014; Elbe and Lalani, 2012; Moyal
etal., 2014; Politte and McDougle, 2014). Anxiety
may be alleviated by serotonergic drugs (Vasa
etal.,2014).
There has been less success in pharmacologi-
cally ameliorating the core autistic symptoms,
with most attention given to repetitive and ste-
reotyped behavior. After years of use, the con-
sensus is that serotonin reuptake inhibitors are
poorly effective against repetitive behavior, espe-
cially in children (Baribeau and Anagnostou,
2014; Doyle and McDougle, 2012; Williams etal.,
2013); however, atypical antipsychotics have
emerged as useful in limiting these symptoms
(Ching and Pringsheim, 2012; Dove etal., 2012),
and, as noted, adverse side effects of antipsychot-
ics can be dose-limiting. For social behavior,
some individual studies have noted improve-
ment possibly relating to successful alleviation
of comorbidities (e.g., Pearson et al., 2013), but
there is poor meta-analytical evidence of any
drug treatment significantly promoting prosocial
behavior (Farmer etal., 2013). However, experi-
mental clinical research is in progress with alter-
native agents such as oxytocin, D-cycloserine,
memantine, and bumetanide (Baribeau and
Anagnostou, 2014; Doyle and McDougle, 2012);
some of these may offer more broad homeo-
static effects. For pharmacological treatments
of either core symptoms or comorbidities, there
have been calls for more placebo-controlled ran-
domized trials, larger sample sizes, and inclu-
sion of quality-of-life measures (Mohiuddin and
 Autism Spectrum Disorders and Homeostasis
Ghaziuddin, 2013; Politte and McDougle, 2014;
Vasa etal.,2014).
Here we take special notice that evidence of
purinergic dysfunction in ASD: purine abnor-
malities have been associated with autism but
until recently were not linked to the core symp-
toms of the disorder. We previously published
a hypothesis, a review, and initial data relating
ASD and the purine adenosine (Masino et al.,
2011a; Masino etal., 2011c). Adenosine is an ubiq-
uitous purine nucleoside that links metabolism
to neuronal function (Dunwiddie and Masino,
2001; Fredholm, 2011). Its recognized roles have
evolved significantly from initially being termed
a retaliatory metabolite (Newby etal., 1985) FIGURE 31.1: The relationship between adenosine
i.e. a neuromodulator participating in cell energy
cycles, with a predominant function to inhibit
synaptic transmission and limit energy demand
during conditions with a net energy loss due to
dephosphorylation of ATP. Since this initial and
still valid characterization as a distress signal
(Fredholm, 2007), it has been recognized that
adenosine is a powerful endogenous antiseizure
molecule (Dunwiddie, 1999; Fedele et al., 2006;
Kawamura et al., 2014), effective in stopping all
types of seizures, and ever-present as a dynamic
regulator of neuronal activity based on small
physiological changes in the internal and external
neuronal milieu. Regarding ASD, diverse stimuli
that have been reported to ameliorate symptoms
of ASD would all be predicted to increase aden-
osine; in parallel, autistic symptoms would be
predicted to be reduced by increased adenosine
(Masino etal., 2011a; Masino etal., 2009; Masino
etal., 2011c). Figure 31.1 is a schematic illustrat-
ing some aspects of the relationship between
symptoms of autism and effects of adenosine.
Directly targeting adenosine receptors has been
perennially challenging due to cardiovascu-
lar and other side effects. However, decades of
research have yielded some new developments
(Korboukh etal., 2012; Luongo etal., 2012). For
example, Ken Jacobson has identified detailed
structural specificity for new agonists (Tosh etal.,
2012), which would have multiple clinical uses.
Previous strategies identified adenosine amine
congeners as potential clinically-useful receptor
agonists (Von Lubitz et al., 1999). A compound
extracted from an herb, N6-(4-hydroxybenzyl)
adenine riboside, designated T111, has shown
dual effects of increasing adenosine signal-
ing via actions at adenosine A2a receptors and
the ENT1 adenosine transporter (Huang et al.,
2011). In addition to these and potential other
pharmacological options, the ketogenic diet is
593
SYMPTOMS OF AUTISM
Seizures
Repetitive
behaviors
Disrupted
sleep Anxiety
Decreased
Seizure-reducer repetitive behaviors
Sleep-promoter Decreased anxiety
Adenosine
and symptoms of autism. Adenosine has multiple
impacts on physiology and behavior. Increased aden-
osine would be predicted to ameliorate multiple core
and comorbid symptoms of autism.
Adapted from Masino etal. (2011c).
a metabolic treatment that increases adenosine
based on recent in vivo and in vitro data (see later
discussion).
Another purine, the cellular fuel molecule
and adenine nucleotide ATP, is a topic of recent
rodent research and a pending clinical trial with
the ATP receptor blocker suramin. This drug has
a century-long history of use in humans against
sleeping sickness; thus it is well characterized for
side effects, may be appropriate for short-term
use, and reversed numerous behavioral, bio-
chemical, neuroanatomical, and metabolic dys-
functions in a mouse model of ASD (Naviaux
et al., 2014; Naviaux et al., 2013). These effects
were interpreted as due to reduced inflamma-
tion, which would be of major homeostatic ben-
efit in ASD. Some questions remain at this early
stage: Is the effect generalizable to other mod-
els of ASD? Is the effect truly mediated by ATP
receptors, and which subtype(s) as suramin is not
a highly selective drug (Hohenegger etal., 1998)?
How does the drug affect behavior when it does
not seem to reach portions of the brain beyond
the brainstem (Naviaux et al., 2014), and is the
mechanistic effect peripheral, possibly on the gut
microbiome or enteric nervous system activity?
Nevertheless, this is an exciting development and
a new target.
Overall, the pharmacological approaches
available and used to date would not be expected
to have a broad homeostasis-restoring benefit for
persons with ASD. Ultimately, a less selective drug
might be a better approach to autismone that
594 Part IV:Homeostatic Therapies
has multiple effects, even some not well under-
stood. Often these less selective or dirty drugs
have fewer side effects and good safety profiles,
are less likely to produce tolerance, and might
represent a homeostatic approachironically
based on poor pharmacology. Such a drug
may already be approved by the Food and Drug
Administration, and a screen that could deline-
ate efficacy in autism would be a valuable tool. To
date, the pharmacological treatment of ASD and
its associated comorbidities has identified some
effective drugs and potential therapeutic tar-
gets, but much further study and advancement is
needed to develop specific treatments with lim-
ited side effects to pharmacologically treatASD.
Metabolic and Dietary Approaches
As noted, metabolic approaches have an enor-
mous potential due to the prevalence of metabolic
dysfunction in ASD and the potential to treat
core symptoms via re-establishing metabolic
function. Such an approach could have a wide but
concerted influence in ASD and improve multi-
ple aspects of overall health.
Diet
Diets remain popular ASD treatments:one study
found 17% of children and adolescents with ASD
had taken some type of specially formulated vita-
min or supplement, and 16% were on a modi-
fied diet (Witwer and Lecavalier, 2005). Dietary
approaches for this disorder have remained
popular for many reasonssome parents favor
approaches other than medication, and there is a
high comorbidity of gastrointestinal symptoms,
some of which may be alleviated or reduced with
diet. In cases with known metabolic abnormali-
ties, multiple reports suggest removing or adding
specific dietary constituents can prevent or allevi-
ate autistic symptoms (Anonymous, 1968; Hooft
etal., 1968; James etal., 2004; Page and Moseley,
2002; Page etal., 1997; Spilioti etal.,2013).
Many diets and dietary supplements have
been tried to treat ASD. In randomized, con-
trolled trials, the gluten-free, casein-free diet,
perhaps the most well-known ASD treatment
to the public, has been split between find-
ings of effectiveness and ineffectiveness; fail-
ure in a placebo-controlled trial certainly
diminished enthusiasm in the field (Elder
etal., 2006; Knivsberg etal., 2002; Seung etal.,
2007; Whiteley et al., 2010). Other diets have
not been in randomized, controlled trials. For
dietary manipulations, studies to better link
subpopulations of ASD to their optimal target
diets and supplements would be of great use. As
the microbiome is apparently abnormal in ASD,
diets that specifically modulate the intestinal
flora could be of use. Regarding supplements, a
meta-analysis concluded that there is little evi-
dence for effectiveness of vitamin B6/magne-
sium supplementation (Nye and Brice, 2005);
randomized, placebo-controlled studies have
found no effect of omega-3 fatty acids, dimeth-
ylglycine, or digestive enzymes (James et al.,
2011; Kern etal., 2001; Munasinghe etal., 2010),
but a vitamin/mineral supplement was signifi-
cantly helpful with some symptoms (Adams
etal., 2011a). Many other supplements have been
tried in studies that were open label/not placebo
controlled, and those with suggested beneficial
effects should advance into well-controlled stud-
ies given clear positive effects of placebo (e.g.,
Kern et al., 2001). Individualized dietary treat-
ment remains a promising new area that could
yield new therapies with few side effects.
One dietary approach that has been tried
with success in ASD patients and in animal
models is the ketogenic diet (KD; Arvio et al.,
2010; Evangeliou etal., 2003; Ruskin etal., 2013;
Spilioti etal., 2013). This high-fat, very low car-
bohydrate diet is a remarkably effective nonphar-
macological treatment for patients with epilepsy,
even in cases poorly responsive to anticonvul-
sant drugs (Kinsman et al., 1992; Neal et al.,
2009; Suo et al., 2013). Furthermore, there are
many decades of clinical experience with KDs.
Strikingly, some refractory patients (consist-
ently at least 10%15% across studies) become
seizure-free, and at least 50% achieve over 50%
seizure reduction (Hallbk etal., 2015; Hassan
et al., 1999; Kang et al., 2007; Neal et al., 2009;
Suo etal., 2013). Asubset of these can be weaned
off the KD and remain seizure-free (Martinez
et al., 2007; Patel et al., 2010), suggesting that
KDs engage long-lasting, possibly epigenetic,
anticonvulsant mechanisms (Boison etal., 2013;
Kobow etal., 2013). KDs limit glucose availability
and so recruit ketone bodies (made in the liver
from -oxidation of fat) to be used as a substi-
tute cellular fuel, a major switch in body/brain
metabolism. Clearly, KDs would be useful in the
treatment of epilepsy when comorbid with ASD.
But based on a hypothesized link between autism
and adenosine (see previous discussion) and evi-
dence that KD feeding augments brain adenosine
levels (Kawamura et al., 2014; Kawamura et al.,
2010; Masino and Geiger, 2008; Masino et al.,
 Autism Spectrum Disorders and Homeostasis 595

2011b), KDs should improve autistic symptoms. three-chamber test in which test mice have the
Indeed, this is what is found in those few clini- option of spending time in a chamber alone or
cal studies that addressed this issue (Arvio etal., in a chamber with a novel mouse (and in which
2010; Evangeliou etal., 2003; Spilioti etal., 2013). normal strains of mice prefer to spend time
In the largest of these, 18 of 30 ASD patients that with a novel mouse), BTBR mice fed a control diet
maintained KD treatment had improvements in were not social, whereas BTBR mice maintained
autistic symptoms ranging from minor to sub- on a KD were social and preferentially spent time
stantial (Evangeliou et al., 2003). However, this in the chamber with a novel mouse (Figure 31.2;
study used a nonstandard KD protocol and has Ruskin etal., 2013). When sociability was meas-
not yet been replicated. Notably, given the asso- ured another way (i.e., in time spent in direct
ciation of mitochondrial defects in ASD, it is par- social contact with the novel mouse), KD-fed
ticularly relevant that KDs are known to promote BTBR mice were significantly more social than
mitochondrial biogenesis and aid energy bal- their control-fed counterparts (Figure 31.3). KD
ance (ATP production) (Bough etal., 2006; Noh feeding also improved social transmission of a
etal., 2004; Srivastava etal., 2013; Sullivan etal., food preference and significantly reduced repeti-
2004)and are used to treat patients with electron tive behavior (Ruskin etal., 2013). These findings
transfer chain defects (Kang etal.,2007). whereby the KD improved symptoms of ASD
We tested the KD/ASD interaction in a mouse have recently been confirmed in another rodent
model of ASD, the BTBR mouse, an inbred autism model, gestational valproic acid exposure
strain in which all mice show low sociability (Ahn etal.,2014).
in the home cage and in testing environments KDs are challenging for some patients
(Babineau et al., 2013; Moy et al., 2007). In the because they are necessarily quite restrictive

500 baseline sociability social novelty

(phase 1) (phase 2) (phase 3)

control diet ***

400
ketogenic diet
**
Time in chamber (s)

300

200

100

0
for stranger mouse
center
to be empty

for stranger mouse

center
to be empty

stranger mouse
center
empty

stranger mouse
center
empty

novel mouse
center
familiar mouse

novel mouse
center
familiar mouse

FIGURE 31.2: Sociability and preference for social novelty in BTBR mice and dietary effects. Data are from
three-chamber testing. In baseline (in the absence of other mice), test mice did not prefer one side over the other,
as expected. With a stranger mouse in one chamber (phase 2), BTBR mice on control diet did not significantly pre-
fer that side, but BTBR mice fed the ketogenic diet (KD) did prefer to spend time in the stranger mouse chamber.
With a novel stranger mouse placed in the opposite chamber (phase 3), BTBR mice fed the KD (but not those fed
the control diet) significantly preferred to spend time with the novel mouse over the familiar mouse. Outlier data
were identified with Grubbs test and discarded. Statistics are paired t tests of side times, corrected for multiple
comparisons (**p < 0.01, ***p < 0.001). No other paired side-by-side comparisons were significant.
Data adapted from Ruskin etal. (2013).
596 Part IV:Homeostatic Therapies
regarding food types. Some investigators
have supplemented KDs with medium-chain
triglycerides (MCTs) so that carbohydrate
restriction need not be so strict. More so than
the long-chain triglycerides in typical KDs,
MCTs are easily absorbed and metabolized
to ketone bodies. MCT-containing KDs have
been used clinically with success in epilepsy
(Ebus et al., 2014; Huttenlocher et al., 1971;
Neal et al., 2009) and were used in the larg-
est clinical ASD/KD study to date (Evangeliou
et al., 2003). Alternatively, MCTs, which are
flavorless, colorless oils, can simply be added
to a normal diet, and MCTs used in this man-
ner have recently been shown to be anticon-
vulsant in rodent models (Socaa et al., 2015;
Wla et al., 2015). A specific subtype of MCT
is one in which the fatty acids have backbones
composed of an odd number of carbons, as
opposed to the even-chain carbon fatty acids
typically found in nature. These fats, par-
ticularly triheptanoin (the triglyceride of the
odd-chain fatty acid heptanoic acid), are also
300 control diet
ketogenic diet
***
250
***
200
Contact time (s)
150
100
50
0 effect or simply because the diet may be health-
phase 2 phase 3
FIGURE 31.3: Dietary effects on sociability in BTBR
mice measured with social contact in three-chamber
testing. Social contact is defined here as physical con-
tact of the test mouse mouth, nose, or forepaws with the
stranger mouse or the small cage keeping the stranger
mouse in place. Feeding a ketogenic diet significantly
increased time in social contact with the stranger
mouse (phase 2) or the total social contact with both
stranger mice (phase 3). Outlier data were identified
with Grubbs test and discarded. Statistics are unpaired
t tests between diets, corrected for multiple compari-
sons (***p < 0.001).
Data adapted from Ruskin etal. (2013) (phase 3)and unpub-
lished data (phase2).
easily absorbed and metabolized to ketone
bodies, but at the end of -oxidative metabo-
lism a three-carbon chain compound is left
over, which can be converted to succinyl-CoA.
Succinyl-CoA is a tricarboxylic acid cycle
intermediatethus dietary supplementation
with an odd-chain MCT could boost the tricar-
boxylic acid cycle (termed anaplerosis) and so
aid in faulty energy metabolism. Patients with
epilepsy due to glucose transporter deficiency
(i.e., GLUT1 deficiency, a condition treated
successfully with the classical ketogenic
diet: Klepper and Leiendecker, 2013) showed
improvement with seizures and neuropsycho-
logical performance when triheptanoin sup-
plemented their regular diet (Pascual et al.,
2014). Notably, in a mouse model of Rett syn-
drome, an ASD-related genetic disorder that
until recently was subsumed under autism in
the DSM, a triheptanoin-supplemented normal
diet improved a large number of metabolic and
physiological dysfunctions, delayed death, and
enhanced behaviors including social behaviors
(Park et al., 2014). It is unclear if odd-chain
MCTs truly have different effects than the
normal even-chain. However, MCTs, odd- or
even-chain, would be an easily studied (and
harmless) food supplement to study clinically
in ASD patients. KDs, MCT/KDs, and MCTs
appear to be dietary treatments with signifi-
cant effects on metabolism and neural func-
tion and indeed may represent approaches that
normalize several aspects of neural and meta-
bolic function. Clearly, they deserve careful
clinicalstudy.
For existing diet-based therapies, parents
report marked improvements with various
dietary changes; some may be due to a placebo
ier overall. Given the number of variables in a
diet, combined with the etiological heteroge-
neity in ASD, there is of course the possibility
that particular diets or supplements might ben-
efit individuals with particular etiologies (e.g.,
Pietropaolo etal., 2014). As noted, this area could
benefit from significantly more research to define
and discriminate beneficial dietary approaches
within subgroups of the population with ASD.
Therefore, the search for safe and effective die-
tary treatments of ASD is ongoing, could impact
multiple aspects of homeostasis, and also has the
potential to improve overall health. More detailed
information on dietary manipulations with spe-
cific nutrients can be found in Chapter16 of this
volume.
 Autism Spectrum Disorders and Homeostasis
Exercise
Exercise has perhaps been inadequately stud-
ied and used as a targeted therapy in ASD.
Physical activity is, like diet, a holistic treat-
ment with simultaneous effects on multiple
systems. It avoids the side effects of medica-
tions and the complications of diets, is a boon
to overall health, and, depending on how and
when it is presented, may be viewed by pediatric
patientsand even adultsas fun. In children
with ASD, exercise can reduce stereotypy and
self-stimulatory behaviors (Anderson-Hanley
et al., 2011; Neely et al., 2015; Watters and
Watters, 1980), improve cognitive and executive
function (Anderson-Hanley et al., 2011), and
help with strength, agility, and running speed
(Hilton etal., 2014; Oriel etal., 2011). All of these
studies were performed after acute exercise.
Another study found that chronic (14 weeks)
exercise significantly reduced stereotypic behav-
ior, and this reduction remained for one month
after exercise cessation (Bahrami et al., 2012).
The lasting effects of chronic exercise could be
due to multiple factors, including epigenetic
effects, but the precise pathways by which exer-
cise has these acute and chronic effects is unclear.
In accord with the adenosine/ASD hypothesis
(Masino and Geiger, 2008; Masino et al., 2009;
Masino et al., 2011a), acute exercise elevates
adenosine and its major metabolite inosine in
brain (Dworak et al., 2007); chronic exercise
downregulates adenosine receptor expression in
the brain, consistent with a homeostatic receptor
response to chronically high activation by aden-
osine (Clark etal., 2014; Costa etal., 2012); and
chronic exercise reduces the painful response
to tactile sensitivity in neuropathic pain in an
adenosine receptor-dependent manner, sug-
gesting enhanced adenosinergic transmission
(Martins etal.,2013).
Microbiome
Because an aberrant microbiome is related to
cognitive and behavioral problems (see previous
discussion)and possibly causative through as
yet undetermined pathwaysit is possible that
normalizing the gut microbial community could
then normalize these problems. Normal intes-
tinal flora can be reconstituted, for instance by
fecal transplantation, which has seen extraor-
dinary success in treating Clostridium difficile
infection (Landy et al., 2011) and is in rand-
omized controlled trials for other bowel prob-
lems and metabolic syndrome (Singh etal., 2014).
In the MIA mouse model of ASD, which includes
597
a disordered microbiome, oral treatment with
Bacteroides fragilis (a prominent component of
a healthy microbiome) normalized the microbi-
ome and some bacterial metabolites and normal-
ized elevated stereotyped behavior and anxiety
but did not affect impaired sociability (all tested
several weeks after treatment; Hsiao etal., 2013).
Interestingly, when vitamin/mineral supple-
ments improved autistic symptoms in children,
those children who were most likely to benefit
had low starting levels of vitamin K and biotin,
vitamins largely produced by healthy gut flora
(Adams etal., 2011a). Thus, in some cases, dietary
supplementation could be compensating for an
abnormal microbiome. Further animal experi-
ments in the vein of Hsiao et al. (2013) could
determine the extent to which the microbiome
influences behavior and cognition. It remains
to be seen if full fecal transplantation from a
microbiomically normal donor would lead to a
more complete normalization of the ASD hosts
behavior.
Behavioral Approaches
As noted, a range of clear physiological changes
can be found in patients with ASD, and early
intervention with cognitive and behavioral
therapies is the most effective treatment to date.
There is also evidence that some children can
escape the diagnosis of autism and improve
their behavior over time such that they no longer
meet the diagnostic criteria (Fein et al., 2014).
Various cognitive and behavioral treatment
options are available, and translation of clinic
and university-based programs into the commu-
nity has produced promising results, with behav-
ior skills training focusing on childcaregiver
joint attention, reciprocal imitation training, and
symbolic play, and social skills training on prag-
matic communication, language use, and social
perception and interaction (Adams et al., 2012;
Corbett etal., 2014; Goods etal., 2013; Ingersoll
et al., 2012; Landa et al., 2011; Warreyn et al.,
2014). While these programs and treatments
have experienced some success in ameliorating
the core symptoms of autism in children, further
research is needed into the empirical techniques
that cause these improvements, early ASD detec-
tion and intervention, adolescent and adult ther-
apies, and cultural/social barriers influencing
program implementation.
Behavioral/cognitive therapy is one
approach that directly targets the core symp-
toms of autism, and perhaps because it is
a holistic approachengaging all systems
598 Part IV:Homeostatic Therapies
simultaneouslyit is an ideal example of a home-
ostatic therapy. Unfortunately it is also a domain
in which it is difficult to access why it works at a
mechanistic level to then develop a better dose.
However, because no other therapy can replace
behavioral experience with social situations and
reciprocal communication, in the absence of a
true magic pill this modality would seem to be
an enduring and perhaps permanent therapeutic
option for ASDand perhaps an underexplored
and/or underutilized therapy for other neuro-
logical disorders.
Early Intervention and Prevention
Early behavioral and anatomical markers and
manifestations of ASD have recently been
explored with the goal of providing early inter-
vention and diagnosis (Barton et al., 2012).
Similar to the findings of Courchesne etal. (2011)
in deceased adult ASD brains, studies in young
children have found increased whole-brain vol-
ume especially in the white and grey matter of
the cerebral cortex, but there remains a great deal
of heterogeneity and research needed to identify
more specific brain regions implicated in ASD
(Amaral etal., 2008; Barton etal., 2012). Parents
of children later diagnosed with ASD have also
reported early symptoms including decreased
responsiveness to parent voices, blunted recipro-
cal caregiver attention, increased irritability and
regulatory difficulties, and abnormal sensory
reactivity (Barton etal., 2012). Even after an ASD
diagnosis, intense and early behavioral interven-
tion, rather than medication, can lead to a com-
plete loss of ASD symptomology in some patients
(Fein et al., 2014), although subtle differences
remain compared to controls (Troyb etal., 2014;
Tyson etal.,2014).
Certainly preventing maternal infection
during pregnancy as much as possible is a prac-
tical and effective strategy to reduce the risk
for ASD. Paul Patterson (2011a) strongly pro-
moted this approach and a better public aware-
ness of the risks for multiple conditions in his
book Infectious Behavior. As a simple strategy
beyond prevention, could targeted treatment
with anti-inflammatory agents help reduce ASD
riskparticularly in a population with increased
genetic predisposition? This is a strategy that
could easily be tested in validated animal models.
For example, the MIA experimental model seems
quite robusteffective in primates and rodents,
with timing similar to the human conditionand
is perhaps one of our better models of ASD to
date. Combining MIA with a polygenic model
of increased risk for ASD could be a particularly
exciting and relevant animalmodel.
Awareness of the prenatal period and the
specific impact of MIA-induced increased risk
for ASD is coming to the forefront in parallel
with information about the metabolic and physi-
ological prenatal environment. For example,
mothers with gestational diabetes, high blood
pressure, obesity, and other conditions are more
likely to have a child with ASD or other neu-
rodevelopmental disorders (Polo-Kantola et al.,
2014; Sullivan et al., 2014; Xu et al., 2014). To
our knowledge there are no validated animal
models in which metabolic dysregulation during
gestationor even in generalincreases risks for
ASD; however, animal models available currently
may exhibit behavioral characteristics of ASD
and simply need to be characterized as such to
add a new cadre of research tools forASD.
CONCLUSIONS AND FUTURE
DIRECTIONS
In sum, ASD spans a wide range of symptoms
and language and cognitive abilities throughout
the lifespan. While diagnosis is based on deficits
in social communication and interactions, as
well as restricted, repetitive behaviors and inter-
ests, ASD is often accompanied by comorbidities
such as seizures, anxiety, depression, and gastro-
intestinal issues. ASD has a complex and in the
vast majority of cases poorly understood under-
lying etiology. To date, rational treatment strate-
gies for the core symptoms are lacking, and with
so little known about the cause(s) of most cases
of autism, designing effective therapies remains
extremely challenging. Clinical and experimen-
tal evidence suggests that abnormal neuronal
excitability, metabolism, and/or immune func-
tion are common features of ASD, and there-
fore a focus on restoring homeostasis may offer
the most broad-based solution for reducing core
behavioral symptoms and possibly comorbidi-
ties. An increased attention to restoring or main-
taining homeostasis should perhaps extend to the
gestational period, as adverse immune or meta-
bolic conditions during fetal development may
increase the risk of an autism diagnosis. Overall,
strategies that promote neural, metabolic, and
immune homeostasis, combined with proven
behavioral interventions, may offer the best avail-
able treatment and long-term outcomes forASD.
At this time, translational ASD research
should embrace therapeutic opportunities to
 Autism Spectrum Disorders and Homeostasis
improve or support bioenergetics and network
homeostasis in the nervous system and support
health and homeostasis of overall physiology.
This could be via explicit systemic approaches
(such as exercise or diet), increasing endog-
enous homeostatic molecules (such as adeno-
sine, BDNF), anti-inflammatory strategies
(exercise, dietary changes, supplements, or
pharmaceuticals), maternal health interven-
tion and disease prevention, and other targeted
general improvements in neural, metabolic,
or immunological function. Individual symp-
toms and practicality of a treatment approach
can be taken into consideration in the clinic.
Alongside early intervention, a synergis-
tic approach that combines supporting and
restoring normal physiology of these complex
systems along with behavioral/cognitive ther-
apy may offer the best chance for lasting and
significant improvement.
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INDEX

A1 receptor (A1R), 491. See also Adenosine receptor Acupuncture, 341349

caffeine on, 461,463 for addiction, 525526
A1 receptor (A1R) agonists, on chronic pain, 343,343f on adenosine
A 2A receptor (A 2AR), 491. See also Adenosine receptor analgesic actions of, 342343,343f
acupuncture activation of, 342347, 343f, 344f, 346f release of, in humans, 345346, 345f,346f
(See also Acupuncture) adenosine increase in,347
in Alzheimers disease, 464465 adenosine signaling in analgesia of, 343345,344f
caffeine and, 348, 461, 463465 ATP, ADP, and AMP increases from,347
in epilepsy, 404,404f biological mechanisms of,434
on glutamate,408 caffeine on,348
in Parkinsons disease, 491492 clinical benefits of, 341342
in seizure control,404 with deoxycoformycin,345
A 2A receptor (A 2AR) antagonists history of,341
in neuroprotection, 422,492 principles of,525
in Parkinsons disease,492 purinergic-mediated signaling by, other, 346347
Ab initio drugs, 231,237 for traumatic brain injury, 434435
Acamprosate,522 understanding, challenges and progress in, 341342
Acetoacetate (ACA), 250f,253 Adalimumab,166
on GABAergic neuron firing,254 Adaptive immunity, in epilepsy, 159160
on glutamate release, presynaptic,254 Addiction, 509527. See also specific substances
on reactive oxygen species levels,256 alternative therapies for, 522527
Acetylcholine acupuncture, 525526
in addiction, 516517 biologic compounds,522
in central fatigue,306 exercise, 523524
on neurogenesis, hippocampal, 205206 herbal remedies, 526527
in Parkinsons disease,490 mindfulness training and meditation, 524525
in REMS sleep, 315f, 316317 animal modeling of, 510511
on sleep and cognition, 319320 approved therapies for, 522523
after traumatic brain injury,431 compulsivity in,511
Acetylcholine receptor type ligand-gated ion channels, diagnosis of,509
51. See also specifictypes drug classification in,509
Acetylcholinesterase, after traumatic brain injury,431 endocrine function in, 518520
Acetyl-coenzyme A(acetyl-CoA) neurosteroids,520
carnitine on availability of, 272273 sex steroids, 518520
in ketogenesis, 250f, 252253 energy balance and metabolic disturbance in,
ketogenic diets on, 257258 520521
metabolites of,271 with epilepsy,512
Acetyl-coenzyme A(acetyl-CoA)/CoA, carnitine on, homeostatic control systems overview in,511
271, 272,273f inflammatory immune responsesin
Acetyl-L-carnitine,274 cytokines, 517518
Action potentials, transmembrane separation of histamines,518
ionsin,98 microglia,517
612 Index

Addiction (Cont.) TRPV1 activation on release of,342

mesocorticolimbic reward circuitry in, 509510 Adenosine 5-monophosphate (AMP), in addiction,512
N-acetylcysteine for,523 Adenosine deaminase (ADA),33
neuromodulator homeostasis in, 511514 Adenosine kinase (ADK), 32, 33, 404405,404f
adenosine, 511512 in Alzheimers seizures,465
dopamine,511 on DNA methylation,466
endocannabinoids, 513514 in epilepsy, upregulation of, 77f,86
serotonin, 512513 on memory, 466467
neurotransmitter and receptor homeostasis in, Adenosine kinase (ADK) inhibitors, therapeutic,38
514517 Adenosine receptor, 342. See also
cholinergic, 516517 A1 receptor (A1R); A 2A receptor(A 2AR)
GABA, 515516 actions of,461
glutamate, 514515 acupuncture on, 342347, 343f, 344f, 346f (See also
neurotrophic factors in,521 Acupuncture)
BDNF, 521522 in addiction, 511512
GDNF,522 caffeine on, 348,461
prevalence of,509 Alzheimers disease and, 462465
stages of,509 function of, 33,33f
Adenosine ADO, 3138,422 methylxanthines on, 318,411
acupuncture on, 343345,344f in Parkinsons disease, 491492
analgesic actions of, 342343,343f seizures and,465
increase in,347 signalingof,34
release of, in humans, 345346, 345f,346f subtypes and distribution of, 3334,33f
in addiction, 511512 Adenosine receptor blockers, on sleep-wake
in ADK/ADO system,461 transition,318
in Alzheimers disease, 452467 (See also Adenosine signaling
Alzheimers disease, adenosinein) in acupuncture analgesia, 343345,344f
astrocyte-released, 77f,8081 exercise and sleep on,411
in astrocytes Adenosine ADO therapy,3738
formation and regulation of, 33f,35 for epilepsy, 408,409
release of, 77,77f augmentation of, 407, 408,409
from ATP,3435 neuroprotective effects of, 38,422
in autism, 593,593f Adenosine triphosphate (ATP). See ATP (adenosine
on bioenergetics,3435 triphosphate)
on blood-brain barrier,35 Adiposity. See also Obesity and diabetes
brain levels of,3133 regulation of, 570571
in central fatigue, 306308,307f ADK/ADO,461
effect inversionin,38 ADO,461
in epilepsy, 404405, 404f,408 on DNA methylation,466
epigenetics of, 406407, 407f,409 ADO deficiency, in Alzheimers seizures,465
homeostasis of, 404405,404f Adrenergic neurotransmission. See also specifictypes
exercise on, 303304 in Parkinsons disease,490
formation, metabolism, and transport of, 3233,32f Adsorptive-mediated transcytosis,147
functions of,461 Aerobic fermentation, of cancer cells (Warburg effect),
on GABAergic transmission,36 262, 555556,555f
on glutamatergic transmission,3637 Age, on epilepsy, 411412
for memory improvement, 465467 Aging
neuromodulation and homeostatic regulation by, in anxiety and stress disorders, 544545
33,33f carnitine and,273
as neuromodulator,3537 melatonin in,277
neurovascular couplingof,35 Aglycemia,102
on pain transmission, 342343,343f ATP production in,101
in Parkinsons disease, 491492 Agouti mouse,573
receptors for,461 Agouti-related peptide (AgRP), 576577
on seizures, 254,465 AIT-082,381
on sleep, 81, 305, 307,308 Albumin
on sleep-wake transition,318 blood-brain barrier disruption on,405
in synaptic plasticity,3637 on brain, 148,149
synthesisof,31 Alcohol
 Index 613

on BDNF levels,522 nerve growth factor on,376

brain targets of,239 neural stem/progenitor cells in, 214215
on endocannabinoids,514 neurofibrillary tangles in, 214, 261, 452453
GDNF and,522 neurotrophic factors in,373
histaminergic drugs on responses to,518 pathogenesis of,384
on sleep,321 reactive astrocytosis in, 47,454
Alcohol addiction (alcohol use disorder), 509527. See seizures in,465
also Addiction symptoms in,452
alternative therapies for, 523527 synaptic scaling in,118
acupuncture,525 Tau protein in,454
daidzin and daidzein,526 tumor necrosis factor in,455
exercise, 523524 in twins, onset of,456
kudzu, 526527 vitamin B12 deficiency in,458
mindfulness training,525 Alzheimers disease, adenosine in, 452467
approved treatments for, 522523 amyloid plaques in, glial activation in,456
BDNF and,522 epigenetic changes and memory loss in, 456460
cholinergic homeostasis in, 516517 chromatin in,457
ghrelin on alcohol craving in,521 DNA methylation and methionine
pregnenolone sulfate and,520 cycle in,458
receptorsin DNA methylation and susceptibility genes in,
GABA, 515516 457458
muscarinic,516 epigenetic functions in,456
nicotinic acetylcholine,516 histone modifications in, 459460
serotonin homeostasis in, 512513 as homeostatic regulator, 461467
Allopregnanolone,520 ADK/ADO system in,461
Allostasis, 353,535 caffeine adenosine receptor targeting in,
Allostatic load,353 462465
Alpha 2-adrenergic (-2) receptors, in basal caffeine as cognitive normalizer in, 461462
ganglia,490 receptors for,461
-alpha-melanin-stimulating hormone (-MSH),574 seizures and,465
-linolenic acid (ALA), 284285 inflammation and, 454456
-synuclein,486 symptoms and lesions in, 453454
Alzheimers disease Amantadine hydrochloride (AMH), for traumatic
adenosine signalingin,35 brain injury,430
amyloid-beta peptide (A) in, 118, 214, 261, Amino acids. See also specifictypes
379,453 exercise and,306
p75NTR and,379 Aminos
BDNF in,376 blood-brain barrier on acid transport of,
blood-brain barrier dysfunction in,149 145146,146f
caffeine on risk of, 461462 Amitriptyline, on REMS sleep,431
clinical presentation and characteristics of, 214, Ammonia, in central fatigue,306
261,453 AMP-activated kinase (AMPK), ketogenic diets on,
DNA methylationin 260,263f
chromatin and,457 AMPA receptor,489
hypomethylation in, 458,460 cocaine, heroin, and methamphetamine on,
energy demands in,272t 514515
epidemiology of,214 scaling of,110
epilepsy in,465 Amphetamine. See also Methamphetamine
folate in, CSF,458 addiction to ( See also Addiction)
GSK3-signalingin,60 serotonin homeostasis in, 512513
on hippocampus, 214215,455 on BDNF levels,522
history of,452 histaminergic drugs on responses to,518
homeostatically controlled mechanismsin,35 on sleep, 317318
homocysteine in,458 Amygdala
inflammation in, 454456 in addiction, 509510
ketogenic diets on, 261262 in anxiety disorders,537
lesions in, 453454 in emotional processing, 536,537f
seizures, memory loss, and,456 in post-traumatic stress disorder,537
microglial cells in,455 in social anxiety disorder,537
614 Index

Amyloid-beta peptide(A) cholecystokinin in, 539540

in Alzheimers disease, 118, 214, 261, 379,453 classical treatmentof
p75NTR and,379 antidepressants,541
progression and plaque formation in,454 benzodiazepines, 540541
caffeine on,462 SSRIs and SNRIs, 540541
glial activation by,454 classification of, DSM-IV,538
p75NTR and,379 clinical presentationof
on promoter hypermethylation, 457458 in adults,543
Amyloid plaques in children, 543544
formation of,454 comorbidities of,545
glial activation in,456 CRH and glucocorticoids in,539
Amyotrophic lateral sclerosis(ALS) in elderly, 544545
branched chain amino acids for,281 emotional homeostasisin
energy demands in,272t dysfunctions of,538
ketogenic diets on, 263264 neurobiology of,535
neurotrophic factors in,373 neurochemical basis of, 535538
Anandamide,513 endocannabinoid system in,540
Anaplerosis, ketogenic diets on,259 future of,545
Anoxia, ATP production in,101 GABA and benzodiazepine systems in,539
Anterior cingulate cortex, in anxiety disorders, homeostatic therapies for, 540542
537,537f deep brain stimulation,543
exposure therapy on,543 deep transcranial magnetic stimulation,543
Antibody-associated encephalitides, adaptive immu- GABA agonist drugs,541
nity in,160 neural circuitries for, 536537,537f
Anticonvulsants. See also specificagents neuropeptide Y in,540
for seizures in autism,592 norepinephrine system in,539
for traumatic brain injury,432 prevalence of, by age,544
Antidepressants. See also specificagents serotonin system in,539
for anxiety and stress disorders,541 sex differences in,543
on neurogenesis APP C-terminal peptide (AICD),460
dentate granule cell,208 APP promoter, 457458
hippocampal, 213214 Aquaporin
for traumatic brain injury,431 in cytotoxic edema, 1314,13f
Antiepileptic drugs (AEDs), 407. See also specificagents functions of,104
adverse effects of,334 in potassium homeostasis,1011
elimination of, gender on,412 in vasogenic edema,14
failures of,401 Aquaporin 4 (AQP4)
multiple ion channel actions of,236 brain injury on,403
neuronal targets of,401 in cytotoxic edema, 1314,13f
target-centered rational strategy failure for,236 in epileptogenesis, 11,403
for traumatic brain injury,432 expression of,104
Antiglial fibrillary acidic protein autoantibody,424 functions of,104
Antihistamines, on sleep,318 in potassium homeostasis,1011
Anti-inflammatory therapy. See also specifictypes in vasogenic edema,14
for epilepsy, 409410 Arachidonoylethanolamide,513
for traumatic brain injury,432 2-Arachidonoylglycerol (2-AG), 513514
Antioxidants Arachnoid epithelium,3
ketogenic diets on, 255257 anatomy of,143
melatonin as,278 physiology of, 144145
sleep and,323 Arc, in synaptic scaling,180
Antipsychotics Arcuate hypothalamus, in obesity, 575, 576577
atypical ( See also specific agents) Aripiprazole, targets for,236
for seizures in autism,592 Arousal, adenosineon,36
on sleep,317 Aspartate
for schizophrenia, targets for,236 functions of,420
Anxiety and stress disorders, 535545 traumatic brain injury on, 420421
animal model translational studies on, 538539 Associative Hebbian plasticity, 124125
BDNF in,376 Associative learning, hippocampal long-term
in children and adolescents,544 potentiation in,124
 Index 615

Associative synaptic plasticity,124 [K+]0 elevation in, on glycogen metabolism,103

Astrocyte,7590 [K+]0 in, neuronal activityon,98
adenosinein lactate astrocyte to axon transfer in, 102103
from ATP released, 77f,8081 in neurotransmitter homeostasis, 100101,101f
formation and regulation by, 33f,35 synaptic activityin,98
setpoints for tone of, 33f,35 Astrocyte neuron lactate shuttle hypothesis,103
ATP release in,7980 Astrocytic network,75
in blood-brain barrier, 144,144f Astrocytic transporters, 12,13f
in brain development,4 Astrocytosis, reactive. See Reactive astrocytosis
calcium excitability in,7576 Astroglial cells. See Astrocyte
cell volume homeostasis microdynamics of, 1217 Astrogliosis,47
(See also Cell volume homeostasis micrody- reactive ( See Reactive astrocytosis)
namics, astroglial) seizures from,465
channels expressedby,75 sleep and, 318,323
cytoarchitecture and distribution of, 3,4,5f on synaptic transmission and circuit
discoveryof,3 excitability,4748
distribution of,493 in temporal lobe epilepsies,47
D-serine release in, 77f, 7879,404 Atkins diet, modified, 251252,251f
in epilepsy, 8486,404 ATP (adenosine triphosphate)
ADK upregulation in, 77f,86 acupuncture on,347
glutamine synthetase downregulation in,8586 adenosine in,593
reactive astrocytosis in,8485 as adenosine source,3435
in epilepsy, and sleep,8184 alternate substrates for,570
on brain activity at circuit level,81 astrocyte release of, 77, 77f,7980
in inflammatory response and sleep behavior,84 adenosine from, 77f,8081
on sleep homeostasis, 77f, 8182,83f in brain homeostasis,322
sleep loss, depression, and,8384 brain levelsof,34
sleep loss, memory consolidation, and,8283 exercise and, 302, 303304,304f
functions of, 46, 5f, 75, 98,493 ketogenic diet on,259
GABA releasein,79 mitochondrial
glutamate releasein,78 in epilepsy,405
homeostasis regulation in, 36,5f sleep and,322
of calcium and sodium,1718 neuronal functionsof,34
human vs. rodent, quality of,105 requirements for,101
in Parkinsons disease, inflammatory reaction in, sleep on,322
493494 ATP13A2 (PARK9) Parkinsons disease, 484485
potassium permeability of,7,98 ATP-binding cassette proteins, in epilepsy,409
receptors expressedby,75 ATP-citrate lyase (ACL),257
signals of, physiologic stimuli for,76 Atypical antipsychotics. See also specificagents
SNARE complex in, 77,77f for seizures in autism,592
structureof,3 on sleep,317
swelling and cell volume recovery in,13f Atypical neuroleptics. See also specificagents
on transmitters modulating synaptic and neuronal for autism,592
activity, 7778,77f Autism spectrum disorder (ASD), 586599
types of,34 adenosine in, 593,593f
Astrocyteneuron interactions,98105 carnitine and,274
action potentialsin,98 comorbidities of, 587590
blood vessel diameter in, 104105 epilepsy, 163165, 164f, 587588
in CSF circulation and brain extracellular space general,587
cleansing,104 immunologic,590
energy metabolism in, 101102,102f metabolic, 588590
glycogen and memory in, 103104 neural, 587588
human vs. rodent,105 sleep disorders,589
interdependenceof,98 core features of, DSMV,586
in ion homeostasis, 98100 (See also specificions) energy demands in,272t
[K+]0, 9899,99f epidemiology of,586
pH0,99100 etiology of, 163164, 590592
pH changes and regulation, environmental,591
activity-induced,99100 epigenetic, 591592
616 Index

Autism spectrum disorder (ASD) (Cont.) hypothalamic, in obesity,579

genetic, 586587, 590591 -amyloid protein. See Amyloid-beta peptide(A)
expenditures on,587 -hydroxybutyrate (BHB), 250f,253
future directions in, 597598 on glutamate release, presynaptic,254
homeostasis disruption in, 586587 on histone deacetylases, 256257,261
ketogenic diets on,264 on reactive oxygen species levels,256
maternal immune activation in, 164, 164f,591 -lactam antibiotics, for epilepsy, 407408
prevention of,598 Bevacizumab
melatonin and,278 on brain tumor energy metabolism,558
mitochondrial metabolism in,274 for gliobastoma multiforme, 553554
prevention of,597 BHB dehydrogenase (BDH1),253
probiotics and,284 Bicarbonate equilibrium potential, 5253,52f
purinergic dysfunction in,593 subcellular heterogeneity of, 52,53f
risk factors for,586 Bienenstock, Cooper, and Munro (BCM) rule,133
symptoms of, heterogeneity of, 586587 Bioenergetics, adenosine on,3435
treatments for, homeostatic, 592598 Bioenergetics in epilepsy, mitochondrial,406
behavioral approaches,597 ketogenic diets on, 254255,255f
diet, 594596, 595f,596f ketogenic diets on bioenergics reserve in, 258259
early interventions,597 Biologics. See also specificagents
exercise,597 for addiction,522
microbiome,597 for traumatic brain injury, 435436
pharmacological, 592594,593f Bipolar disorder
principles,592 adenosine signaling in,3738
Autoimmune disease. See also specifictypes anxiety disorders with,545
with autism,590 energy demands in,272t
in traumatic brain injury, 424425 GSK3-signalingin,60
Autonomic nervous system homeostatically controlled mechanismsin,35
meditation on, 357358 Blood-brain barrier (BBB), 3, 143150
in Parkinsons disease,491 adenosineon,35
Axonal stretch/strain disruption, from traumatic brain anatomy of, 143144,144f
injury,420 development of,144
Axon guidance molecules,373 in disease, 148150
epilepsy, 149, 405,409
Bacteroides fragilis, for autism,597 general mechanisms, 148149
Basal ganglia, 299. See also neurological,149
specific parts and neurotransmitters neuronal network, 149150
in motor function,488 drug delivery in,148
in Parkinsons disease,488 glucose and amino acid transport in, 145146,146f
signal processing pathways in,488 inflammation on,156
Basement membrane, in blood-brain barrier,143 ion transport in, 146147,146f
Basic FGF,379 leukocyte migration across,145
BCL-2-associated agonist of cell death (BAD),254 neuronal and vascular functions of,148
BDNF-TrkB signaling, 376, 377, 405, 410,578 neurovascular unit in, 3, 143144,144f
BDNF val66met,427 peptides and proteins in, 147148
Benzodiazepine physiology of, 144145,146f
addiction to ( See also Addiction) traumatic brain injury on, 47, 421422
GABA receptors in,515 Blood-CSF barrier,143
for anxiety and stress disorders, 540541 Blood pressure, meditation on,355
Benzodiazepine-receptor agonists, on anxiety, 539,540 Blood vessel diameter, astrocyteneuron interactions
Benzodiazepine receptor, in anxiety,539 in, 104105
Benzodiazepine system, in anxiety and stress Body weight
disorders,539 BDNF on, 577578
Bergmann glia,4 excess ( See Obesity and diabetes)
bestrophin-1,16 gut microbiota on,581
[Ca 2+]i on,1718 regulation of, 570571
Beta-cell lymphoma 2 (BCL-2), in traumatic brain sleep on,322
injury recovery,423 Botanicals, 333338
Beta-oxidation for addiction, 526527
carnitine palmitoyltransferase 1A on,578 Cannabis sativa,335

CNS disorder treatment improvement and,336
curcumin, 334335
evidence-based medicine for, 336337
herbal extracted compounds, 335336
history and current use of,333
Huperzine A(Huperzia serrata), 335336
mixtures of,337f
mixtures of active compounds in, 334335
mixtures of herbs in,334
newly discovered targets in, products for, 337338
Brain. See also specifictopics
function and complexity of, 231232
integrative activity of,239
metabolism of,232
after traumatic brain injury, atrophy of, 425426
Brain cancer. See also specifictypes
blood-brain barrier dysfunction in,149
epigenetics in,194
ketogenic diets on,262
malignant, 553561 (See also
Glioblastoma multiforme)
Brain-derived neurotrophic factor (BDNF), 373,
376377
in addiction, 521522
on body weight, 577578
in epilepsy,405
in neuroprotection,422
in Parkinsons disease, 376,495
therapeutic,377
after traumatic brain injury,425
in depression,427
Brain trauma. See Traumatic brain injury(TBI)
Branched chain amino acids (BCAAs), 278283
carnitine on,273
in central fatigue, 306,307f
for CNS disease, 280281
CNS functions of, 279280,280f
epilepsy and, 281282,282f
on glutamate homeostasis, 279280, 280f, 281,282f
ketogenic diet and, 282283
metabolism of, 278279,279f
Brevican,373
Buddhist meditation, 360. See also Meditation;
Mindfulness-based stress reduction(MBSR)
Buprenorphine, for opioid addiction,523
Bursts of spikes, in heterosynaptic plasticity, 131,
132,132f
[Ca 2+]
in heterosynaptic plasticity,131
in neuronal hyperactivity,195
[Ca 2+]0, blood-brain barrier on,147
Caffeine
on acupuncture effects,348
on adenosine receptors, 318, 411, 461, 463465
adenosine receptor targeting of, and Alzheimers
disease, 462465
on Alzheimers disease
as cognitive normalizer, 461462
Index 617
risk of, 461462
on A-induced decline,462
on BACE1 expression,462
cognitive effects of, 461462
complex actions of,348
on epilepsy,411
prevalence of use of,348
on sleep, 321322
Calcium excitability, in astrocytes,7576
Calcium, intracellular ([Ca 2+]i)
in astrocyte homeostasis regulation,1718
on cerebral vascular tone,17
on neuronal excitability,17
Calcium sliding threshold,136
Caloric restriction, 252, 559560,559f
Calpain,61
Canakinumab,166
Cancer. See also Brain cancer; specifictypes
energy demands in,272t
epigenetics in,194
somatic mutations in,553
Cannabinoid addiction. See
Addiction; Marijuana addiction
Cannabis sativa addiction, 335, 509527
endocannabinoid homeostasis in, 513514
mindfulness training on,525
Capsaicin, on adenosine concentrations,342
Carbamazepine. See also Antiepileptic drugs(AEDs)
targets and mechanisms of action of,407
for traumatic brain injury,432
Carbonic anhydrase, on bicarbonate in chloride chan-
nels, 5253,53f
L-Carnitine, 271274,272f
on acetyl-CoA availability, 272273
acetyl-L-carnitine and,274
on acyl-Co-A/CoA, 271, 272,273f
aging and,273
autism and,274
dietary sources of,271
functions of, 271272
homeostatic mechanisms on,271
on long-chain fatty acid transport, 271,272f
on metabolism
carbohydrate and protein, 271272
lipid, 271,272f
valproate and,274
Carnitine deficiency syndromes,273
Carnitine esters, 271274,272f
Carnitine palmitoyltransferase 1A (Cpt1a)
on beta-oxidation,578
malonyl-CoA on,578
Cation channels. See also specifictypes
in astrocyte volume regulation,1617
CB1 cannabinoid receptor, 513514,541
CB2 cannabinoid receptor,541
CBD
for anxiety and stress disorders, 541,542
effects of,335
CCR2,455
618 Index

CD4+T lymphocytes, in seizure models,161 Cholecystokinin (CKK), in anxiety and stress disor-
CD8+T lymphocytes ders, 539540
in Rasmussens encephalitis,160 Cholinergic receptors and neurotransmission. See also
in seizure models,161 specifictypes
Ceftriaxone, for epilepsy, 407408 in addiction, 516517
Celecoxib in Parkinsons disease, 490491
on epilepsy from brain insults,409 Cholinergics. See also specificagents
on major depressive disorder,162 for traumatic brain injury,431
Cell adhesion molecules,373 Chondroitin sulphate proteoglycan,373
Cell-based replacement therapy,374 Choroid plexus,3
Cell volume homeostasis microdynamics, Choroid plexus epithelium
astroglial,1217 anatomy of,143
cytotoxic edema in, 12,13f physiology of, 144145
fundamentalsof,12 Chromatin, in Alzheimers disease,457
plasma membrane channels in, 1217 (See also Chromatin remodelers,192
Cell volume regulation, astroglial) Chronic intrinsic plasticity,5657
regulatory volume decrease in, 12,13f Chronicpain
vasogenic edemain,12 A1 receptor agonists on, 343,343f
Cell volume regulation, astroglial, 12,13f acupuncture for, 343, 343f (See also Acupuncture)
cation channels in,1617 definition of,348
chloride channels in,1516 prevalence of,348
potassium channelsin,14 Chronic traumatic encephalopathy (CTE),426
principles of plasma membrane channels in, 12,13f CIC-2,15
water channels in,1314 CIC receptor family,1516
Central fatigue, exercise and, 305308,307f Circadian pacemaker,317
Ceramide,379 Citalopram. See also
Cerebellum,299 Selective serotonin reuptake inhibitors (SSRIs)
Cerebral atrophy, after traumatic brain injury, 425426 for anxiety disorders,541
Cerebral blood flow, in exercise, 300301 efficacy of,431
Cerebral hyperperfusion syndrome, blood-brain bar- for traumatic brain injury,431
rier breakdown in,149 Cl-/ HCO3- exchanger,100
Cerebral ischemia, from traumatic brain injury,422 Clozapine, targets for,236
Cerebral metabolic ratio, 302303 Cocaine
Cerebrospinal fluid(CSF) on AMPA receptors, 514515
circulation of, and extracellular space on BDNF levels,522
cleansing,104 estrous cycle on use of,519
production and flow of,104 GDNF and,522
Change, stability and,108 on glutamate,514
Channelopathies,60 on sleep, 317318
ChemBank, 240241 Cocaine addiction, 509527. See also Addiction
ChEMBL,241 acupuncture for,525
Chemokines. See also specifictypes cholinergic homeostasis in, 516517
on adhesion molecules,161 exercise for,524
astrocytes on,421 GABA receptors in,516
in autism,590 mindfulness training for,525
in immunotherapy, depression from,162 N-acetylcysteine for,523
in inflammation, 421,517 nicotinic acetylcholine receptors in,516
in epilepsy, 155, 156,157t serotonin homeostasis in, 512513
in Parkinsons disease,492 Cognition
NF-kB in,517 complexity of, 239,240f
Chloride channels epigenetic chomatin modifications in,195
in astrocyte volume regulation,1516 exercise on,308
inhibition via opening of, 51,52f nootropic drugs on, 239,240f
Chloride equilibrium potentials, subcellular heteroge- pharmacologic support of,231
neity of, 52,53f sleep on, 319320
Chloride transporters, cation. See also specifictypes Cognitive behavioral therapy
functionof,52 for anxiety and stress disorders, 542543
potassium-chloride cotransporter in, 52,53f for autism, 597598
sodium-potassium-chloride cotransporterin,52 Cognitive impairment

from epilepsy, inflammation in, 165166,165f
life expectancy on, increased,237
from traumatic brain injury,427
Cognitive rehabilitation, for traumatic brain injury,
433434
Compassion meditation, 354355. See also Meditation
Competition, 127128
in heterosynaptic plasticity, 129130, 134136
normalization in,127
synaptic
Hebbian plasticity and, 126,127
in synaptic scaling,116
Complementary health approaches (CHA), 333. See
also specifictypes
Compulsivity,511
Control theory, 113,114f
proportional-integral-derivative controller in,115
synaptic scaling in, 113115,114f
Contusions, from traumatic brain injury,420
Cooperativity, rule of,124
Cortical remapping,180
Cortical reorganization, 180181,181f
Corticotropin-releasing factor (CRF) receptor, gender
differences in,543
Corticotropin releasing hormone(CRH)
in anxiety and stress disorders,539
on sleep and stress responses, 320321
in stress response,535
Cortisol
in addiction,525
FKBP5 polymorphisms on,540
on hippocampus,359
meditation on, 358359
after traumatic brain injury,429
Cotransporters. See also specifictypes
KCC2, 5254, 53f,57,59 Cytotoxic CD8+T lymphocytes, in Rasmussens
Na+/ HCO3-,100
in neuronal excitability homeostasis,52
sodium-potassium-chloride, 6, 52, 53f,59
COX-2
in inflammatory cascade, 157t,159
on learning and memory,165
COX-2 inhibitors
on epilepsy
from brain insults,409
inflammation in,166
on major depressive disorder,162
CpG islands (CGIs), 187188,457
orphan,188
CpG methylation, 187,457
Craniopharyngioma, hypothalamic,572
C-reactive protein (CRP), meditation on,356
Creatine, 274277
biosynthesis and metabolism of, 275,276f
blood-brain barrier crossing by,275
brain trauma and, 275276
distribution of,275
epilepsy and, 276277
for Huntingtons and Parkinsons diseases, 274275
Index 619
ketogenic diets on,277
metabolism disorders of, 274275
neuroprotection by, 274275
for Parkinsons disease, 274275
CREB-binding protein (CREBBP), 193194
CRF1 antagonists, on anxiety,540
Curcumin, 334335
for traumatic brain injury,436
Cushings disease,575
proopiomelanocortin in,576
Cys-loop receptors, 51. See also specifictypes
Cytokines. See also specifictypes
in addiction, 517518
astryocyte upregulation of, 149150
in autism,590
blood-brain barrier transport of,146f
in central fatigue, after exercise,306
classification of,517
in depression after traumatic brain injury,427
exercise on,433
functions of,517
in inflammation
in epilepsy, 155159, 157t, 161, 164, 405406
in posttraumatic epilepsy,426
in traumatic brain injury, 421422,
426428,432
maternal immune activation on, 164165
meditation on,356
neurotrophic,521
in Parkinsons disease, 492,493
sleep and, 324,324f
Cytokine storm,164
Cytomegalovirus, human (HCMV)
in brain cancer,557
on tumor cell energy metabolism,557
encephalitis,160
Cytotoxic edema, 12,13f
AQP4 in, 1314,13f
after traumatic brain injury,420
Daidzein, for addiction,526
Daidzin, for addiction,526
D-amphetamine (AMPH), for traumatic brain
injury,430
DCBIP, on infarct size in cerebral artery occlusion,
15,1819
Deep brain stimulation
for anxiety and stress disorders,542
for depression and OCD,543
Deep transcranial magnetic stimulation, for
PTSD,543
Dehydroepiandrosterone sulfate (DHEAS), in
addiction,520
9-Tetrahydrocannabinol (THC), 513514
for posttraumatic stress disorder,541
Dentate granule cell neurogenesis, 202, 203f,204
antidepressants on,208
functional significance of, 206208
620 Index
Dentate granule cell neurogenesis (Cont.)
in memory and learning,206
in pattern separation,207
population and turnover of,207
in psychiatric disorders,208
Dentate gyrus (DG). See also Hippocampus
neural stem/progenitor cells in, 203f, 204205
neurogenesis in, adult, 202204,203f
Deoxycoformycin, acupuncture with,345
Depressant addiction, 509527. See also Addiction
Depression
adenosine signalingin,35
anxiety with,545
deep brain stimulation for,543
on hippocampus, size,213
homeostatically controlled mechanismsin,35
inflammationin
with epilepsy, 162163,163f
after traumatic brain injury, 427428
neural stem/progenitor cells and neurogenesis for,
213214
in Parkinsons disease, 482, 487,488
GABAergic neurotransmission in,490
glutamatergic neurotransmission in,489
noradrenergic neurotransmission in,490
serotonergic neurotransmission in,491
sleep and, 321322
sleep loss, astrocytes, and,8384
Descartes, Ren,232
Desipramine, on REMS sleep,431
Development,brain
epigenetics in, 192193
learning and, 108109
Dexamethasone
on brain tumor energy metabolism, 557558,558f
for gliobastoma multiforme,553
DHEAS, in addiction,520
Diabetes, 570581. See also Obesity and diabetes
diabetes (db) gene, 573575
Diaschisis
definition of,427
after traumatic brain injury,427
Diet, 271285. See also
Ketogenic diet (KD); specifictypes
for autism, 594596, 595f,596f
on brain function,248
branched chain amino acids in, 278283
creatine in, 274277
on energy demands, 271,272t
for epilepsy, 410411
L-carnitine and carnitine esters in, 271274,272f
melatonin in, 277278
omega-3 fatty acids in, 284285
probiotics in,284
therapies based on,248
Diffuse axonal injury (DAI),420
Disease-associated molecular networks, 240241
Disulfiram,522
DJ-1 (PARK7) Parkinsons disease,484
DNA demethylation, 188189,188f
DNA hypermethylation, in epilepsy, 406, 407f, 408409
DNA hypomethylation
in Alzheimers disease, 458,460
in traumatic brain injury,409
DNA methylation, 187188,188f
adenosine kinase on,466
ADO tone on,466
in Alzheimers disease
chromatin and,457
methionine cycle and,458
susceptibility genes and, 457458
definition of,457
in epilepsy, 406, 407f, 408409
functions and locations of,457
in gene silencing, 190,195
S-adenosylhomocysteine hydrolase in, 458,465
S-adenosyl methionine in, 188, 189f, 193,465
vitamin B on,458
DNA methylome restoration, for epilepsy, 407f,
408409
DNA methyltransferase (DNMT),187
Docosahexaenoic acid (DHA), 284285
Dopamine(DA)
in addiction, 511,512
adenosine on systems for,37
in central fatigue,306
exercise on, 304305
in Parkinsons disease, 427, 481,488
after traumatic brain injury, in cognitive
dysfunction,427
traumatic brain injury on,421
Dopamine-enhancing agents, on sleep, 317318
Dopamine-inhibiting agents, on sleep,317
Dopamine neuron loss, in Parkinsons disease, 481,
488489
in striatum,488
in substantia nigra,488
Dopamine quinones, in Parkinsons disease,496
Dopamine receptors,488
Drug discovery. See also specific drugs and disorders
magic shotguns in,236
multitarget pharmacology in, 235236
phenotypic vs. target-based screening in,236
rational,235
target-based, 234235
Drugs ofabuse
addiction to, 509527 (See also Addiction)
classification of,509
D-serine, 77f, 7879,404
Dystrophin-associated protein complex (DAPC)
AQP4in,10
AQP4Kir4.1 interactions in,1011
Kir4.1in,9
Dzogchen meditation, 360. See also Meditation
EAAC-1 (excitatory amino acid carrier-1 transporters)
in GABA reuptake,45
in glutamate uptake,4546
 Index 621

Ecstasy addiction, 509527. See also Addiction definition of, 187,424

serotonin homeostasis in, 512513 DNA demethylation in, 188189,188f
Ectonucleoside transporters (ENTs), in astrocytes,77f DNA methylation in, 187188,188f
Ectonucleotidases. See also Acupuncture in epilepsy, 195,409
local injection of,347 adenosine in, 406407, 407f,409
Edema functions of,456
cytotoxic, 12,13f histone modification in, posttranslational,
AQP4 in, 1314,13f 189190,189f
after traumatic brain injury,420 in learning, 193194
glibenclamide on,16,19 in memory, 193195
vasogenic,12,14 in neurodevelopmental disorders,233
Eicosapentaenoic acid (EPA), 284285 in neurogenesis and brain development, 192193
EIF4G1 Parkinsons disease,484 in neuronal membrane excitation,195
Emergent properties, 233, 234,242 noncoding RNAs in, 190191,191f
Emotional homeostasis in synaptic plasticity, 193194
anxiety and stress disorders on, 538 (See also in systems pharmacology, 233234,234f
Anxiety and stress disorders) in traumatic brain injury,424
dysfunctions of,538 Epilepsy, 401407. See also
neurobiology of,535 Temporal lobe epilepsy(TLE)
neurochemical basis of, 535538 A1 receptors in, 404,404f
sex differences in,543 addiction with,512
Emotional processing,535 adenosine homeostasis in, 404405,404f
Encephalitides, antibody-associated,160 adenosine signaling in,3738
Endocannabinoid receptors,513 in Alzheimers disease,465
Endocannabinoids AQP4 in, 11,403
in addiction, 513514 astrocytes in, 8486,404
endogenous,513 ADK upregulation in, 77f,86
Endocannabinoid (eCB) system glutamine synthetase downregulation in,8586
in anxiety and stress disorders,540 with autism, 587588
drugs enhancing, for anxiety and stress inflammation in, 163165,164f
disorders,541 BDNF in,376
Endocytic vesicles, on blood-brain barrier,147 blood-brain barrier breakdown in, 149,405
Energy balance. See also Mitochondrial bioenergetics brain injury in,409
in addiction, 520521 branched chain amino acids on, 281282,282f
ventromedial hypothalamus in, 571573 clinical manifestations of, 6263,84
Energy deprivation, ATP in,101 connexinsin,11
Energy metabolism creatine and, 275, 276277
astrocytes in, 101102,102f definition of, 333334
in cancer cells (Warburg effect), 262, 555556,555f DNA hypermethylation in, 406, 407f, 408409
in malignant brain tumors, standard of care on, DNA methylation in,406
557559,558f energy demands in,272t
Environmental factors. See also specifictypes epidemiology of,401
in autism,591 epigenetics in, 195, 233234, 234f,409
in epilepsy, 233234,234f adenosine and, 406407, 407f,409
in Parkinsons disease, 485, 486487 geneenvironment interactions in, 233234,234f
Ephrins,373 glial dysfunction and gliotransmitter homeostasis
Epigenetics, 187195 in, 403404
of Alzheimers disease, 456460 glial fibrillary acidic protein and vimentin in, 47, 85,
chromatin in,457 87, 88f89f
DNA methylation and methionine cycle in,458 gliotransmission in, 8690, 403404
DNA methylation and susceptibility genes in, glutamate release in,8687
457458 NMDA receptor trafficking in, synaptic, 8790,
functions of,456 88f89f
histone modifications in, 459460 glutamate and glutamine homeostasis in,403
in autism, 591592 glutamate-glutamine cyclein,47
in brain development, 192193 growth factors in,405
in brain tumors,194 homeostatically controlled mechanismsin,35
chromatin remodelers in,192 IL-1R1 signaling in, 157158,157t
in cognition,195 IL-1R/TLR signaling pathway in, 405406
622 Index

Epilepsy (Cont.) in adenosine transport,3334

immunomodulatory and inflammatory responses Equilibrium potential,51
in, 405406 ERK, 377378
intrinsic plasticity in, 6061,62f ERK signaling, second messengers on,384
ion and water homeostasis in,403 Estradiol
ketogenic diets on, 248261, 410411 (See also in addiction, 518520
Ketogenic diet (KD), on epilepsy) on dopamine release, cocaine-induced,519
Kir4.1 in,9,403 for traumatic brain injury, 423,435
mitochondrial bioenergetics in,406 Estrogen
multidrug transporter expression in,409 on GABA and GABA receptors, 411412
network homeostasis disruption in, 401403,402f on NMDA receptors,412
nonneuronal factors in,401 Ethanol. See Alcohol
pannexin 1in,12 Ethosuximide. See also Antiepileptic drugs(AEDs)
pathophysiology of,401 on sleep,314
pharmacoresistance in,409 Excitability homeostasis, neuronal,5163
potassium channelrelated,60 cotransportersin,52
prevalenceof,84 epilepsy-related
reactive astrocytosis in, 47,8485 intrinsic plasticity in, 6061,62f
synaptic plasticity in, 6163,62f synaptic plasticity in, 6163,62f
TLR4 signaling in, 157158,157t GABA and glycine receptor inhibition in, 5154,
after traumatic brain injury,426 52f,53f
water and [K+]0 homeostasisin,11 inhibitory plasticity in, neuronal,5760
Epilepsy, homeostatic therapies for, 407412 phosphorylation in,5960
adenosine augmentation in, 407, 408,409 RNA splicing and editing in,5759
age and gender on, 411412 intrinsic plasticity in,5456
anti-inflammatory and immunomodulatory thera- chronic,5657
pies in, 409410 definitionof,54
blood-brain barrier drug transporters in,409 short-term,5455
dietary interventions in, 410411 synaptic plasticityrelated,5556
DNA methylome restoration in, 407f, 408409 mitochondrial function and, 254255,255f
glutamate homeostasis restoration in, 407408 Excitation-spike (E-S) coupling,55
lifestyle choices in,411 Excitatory amino acids(EAA)
methylxanthines on,411 functions of,420
network homeostasis reconstruction in,407 traumatic brain injury on, 420421
Epilepsy, inflammation and immunomodulation in, Excitatory amino acid transporter 2 (EAAT2,
155166 GLT-1),515
adaptive immunity in, 159160 in GABA reuptake,45
comorbidities and, 161165 in glutamate transport,515
autism, 163165,164f in glutamate uptake,46
depression, 162163,163f localizationof,45
memory and cognitive impairments, Excitatory amino acid transporter (EAAT), in gluta-
165166,165f mate transport,515
neurobehavioral disorders, 161162 Excitatory synaptic scaling, 109112
evidence on, clinical,156 in interneurons,112
experimental studies on, 160162 in pyramidal neurons, 109112,111f
antibodies against intracellular antigens in,161 Excitotoxicity, from traumatic brain injury, 420421
antibodies against membrane antigens in, 160161 Exercise, 298308
leukocytes in,161 for addiction, 523524
glial cells in,155 on adenosine signaling,411
inflammatory cascade in, yin and yang of,159 amino acids and,306
inflammatory molecules and epileptogenesis in, for autism,597
158159 behavioral effects of,308
overview of,155 on brain,298
pathophysiology of, 155156 cerebral blood flow and O2 consumption in, 300301
seizure pathogenesis in, 156158,157t on epilepsy,411
therapeutic implications of,166 fatigue in, central, 305308,307f
Epistemological reductionism,232 metabolismin
Equilibrative nucleoside transporters (ENTs) glucose and lactate, 301303
in adenosine release,36 nucleotide and nucleoside, 303304,304f
 Index 623

neural control of movement and, 298300,299f in children and adolescents,544

on neurogenesis,411 on cocaine and alcohol addiction behavior,513
neurotransmission in, 304305 on depression,241
dopamine, 304305 with epilepsy,163
glutamate,305 efficacy of,431
serotonin,305 on neurogenesis,213
outlook for,308 on serotonin,513
positive effects of,298 for traumatic brain injury,431
on sleep, 305, 307,308 Fluvoxamine. See also
for traumatic brain injury, 430433 Selective serotonin reuptake inhibitors (SSRIs)
tryptophan and,306 for anxiety disorders,541
Exposure therapy, for anxiety and stress disorders,542 in children and adolescents,544
Extracellular space cleansingf, and CSF circulation, Focal cortical dysplasia (FCD), inflammation in,156
astrocytes in,104 Focal injury,420
Extracted compounds, herbal, 335336 Focal ischemia, nerve growth factor on,376
Folate
Familial hemiplegic migraine-associated spreading functions of,458
depolarization,149 in neuronal homeostasis,193
Fatigue Folate deficiency, in Alzheimers disease,458
central, exercise and, 305308,307f Fragile X mental retardation protein (FMRP), in syn-
peripheral,305 aptic scaling,117
Fat mass and obesity associated (FTO) gene, 579580 Fragile X syndrome
Fatty acid amid hydrolysis (FAAH), in endocannabi- GSK3-signalingin,60
noid addiction,514 synaptic scaling in,117
Fatty acid oxidation(FAO) Free radicals, melatonin on,278
high-fat, low-carbohydrate diet on,252 FRS2/GRB2 complex,380
ketogenic diets on,258 Fructose-1,6-bisphosphate (FBP), on glycolysis,258
FBXO7 (PARK15) Parkinsons disease,485 Functional hyperemia, 104105
Fecal transplantation, for autism,597
Feeding, reward brain circuitry in,521 GABA. See also Glutamate
FGF2/FGFR1,380 in addiction, 515516
on PLC-gamma pathway, 380381 in anxiety and stress disorders,539
Fibroblast growth factor (FGF), 379380 astrocyte release of, 77, 77f,79
Fibroblast growth factor 2 (FGF2),373 discoveryof,51
Fibroblast growth factor (FGF) receptors,380 in epilepsy,403
Fibroblast growth factor (FGF) signaling pathway, estrogen on, 411412
380381 functions of,489
Fingolimod,166 glutamate-glutamine cycle on,4547
Firing rate homeostasis, 115116,118 glutamate in synthesisof,45
5-HT1A receptor, in inflammation-associated depres- on hippocampal neurogenesis, 205206
sion, 163,163f in inhibitory synaptic terminals,44
5-HT1B receptor, in addiction,512 in ketogenic diet,253
5-HT2A receptor, in addiction,512 in Parkinsons disease, 489490
5-HT2C receptor, in addiction,512 insleep
5-HT3, in addiction,512 NREMS, 315f,316f
5-HT3 receptor, in addiction,512 REMS, 315f,317
5-Hydroxyindoleacetic acid, in Parkinsons sourcesof
disease,491 in interneurons,4546
5-Hydroxytryptamine (serotonin) presynaptic,45
exercise on,305 synaptic cleft removalof,44
on hippocampal neurogenesis, 205206 GABA agonists, for anxiety and stress
in sleep,305 disorders,541
5-Nucleotidases,33 GABA A receptors (GABA AR), 51,489
FKBP5 polymorphisms, on cortisol and PTSD,540 in addiction, 515516
Flavonoids, for traumatic brain injury,436 neurosteroids and,520
Fluoxetine. See also anion channelsof,51
Selective serotonin reuptake inhibitors (SSRIs) in anxiety, 539540
on aggression,321 in chloride channel opening, 51,52f
for anxiety,541 phosphorylationof,59
624 Index

GABA B receptors,489 discoveryof,75

in addiction, 515516 in epilepsy, 155, 403404
GABA-enhancing drugs, on REMS sleep,317 Na+/ HCO3- cotransport,100
GABAergic neurotransmission Glial-derived neurotrophic factor (GDNF)
adenosineon,36 in addiction, 521,522
progesterone on,412 in Parkinsons disease,495
Gabapentin. See also Antiepileptic drugs(AEDs) Glial fibrillary acidic protein (GFAP), in epilepsy,85
for addiction,512 Glial scar, 373, 403404
on sleep,314 Glia-lymphatic system, sleep and, 323324
GABA receptors Glibenclamide, on cerebral edema,16,19
in addiction, 515516 Glioblastoma multiforme, 553561
estrogen on, 411412 cytomegalovirus in,557
inhibition via, 5154, 52f,53f epidemiology of,553
physiology of,515 glucose and glutamine on, 556557
GABA vesicular transporter (VIAAT),44 isocitrate dehydrogenase (IDH) gene mutations
Gamma-amino butyric acid. See GABA in,194
Gap junctions management of, current standard ofcare
astrocyte,11 energy metabolism in, acceleration from,
in K+ homeostasis,11 557559,558f
GBA, in Parkinsons disease,484 survival with, 553554, 554f, 558559
GCLM,256 management of, homeostatic
Gender future hopes for,561
on antiepileptic drug elimination,412 ketogenic diet in, calorie-restricted, 559561,559f
on corticotropin-releasing factor receptor,543 mitochondrial dysfunction exploitation in,
in epilepsy, 411412 559560,559f
in post-traumatic stress disorder symptoms,543 metastasis of, systemic,553
Geneenvironment interactions, 233234,234f mitochondrial dysfunction in, 554555,555f
General systems theory,234 reactive oxygen species in,553
Gene silencing,138 tumor-associated macrophages/monocytes in,553
DNA methylation in, 190,195 glutamate use by,558
histone methylation in,194 standard of care on,557
RNA-induced, 191,191f Warburg effect in, 555556,555f
Genetics Gliomas
in Alzheimers disease, 457458 isocitrate dehydrogenase (IDH) gene mutations
in autism, 586587, 590591 in,194
in obesity and diabetes ( See also mitochondrial dysfunction in, 554555,555f
under Obesity and diabetes) Gliotransmission
human, 579580 definitionof,75
mice, 573578,574 in epilepsy, 8690, 403404
in Parkinsons disease, 482485, 495 (See also under glutamate release in,8687
Parkinsons disease) NMDA receptor trafficking in, synaptic, 8790,
in synaptic scaling,180 88f89f
of traumatic brain injury,424 functionsof,75
Gephyrin,58 in sleep pressure accumulation, 8283,83f
calpain as substrate for,61 Gliotransmitters, 7778, 77f,178
in neuropsychiatric disease,60 in epilepsy, 403404
phosphorylation of,5960 Glucocorticoids. See also specifictypes
Ghrelin in anxiety and stress disorders,539
in addiction,521 on sleep and stress responses,320
on alcohol craving,521 sleep deprivation on, 320321
in obesity, 578579 in stress response,535
sleep on,322 Glucose
synthesis and role of, 520521 blood-brain barrier on, 145146,146f
Ginseng, for addiction,526 in brain cancer progression,556
GlialCAM, in megalencephalic exercise and metabolism of, 301303
leukoencephalopathy,19 in glioblastoma multiforme management,
Glialcells 559560,559f
in addiction,512 ventromedial hypothalamus on, 571573
amyloid plaque on,456 Glucose-regulated hypothalamic neurons,572
 Index 625

Glucose transporters,145 in brain cancer progression, 556557

Glucostat hypothesis, 572,578 brain cancer therapy on, current, 557,558f
GLUT1,145 cytomegalovirus on,557
GLUT3,145 in epilepsy,403
GLUT4,145 Glutamine synthetase(GS)
Glutamate, 100, 489. See alsoGABA in epilepsy, 47,8586
in addiction, 512, 514515 for seizure prevention,408
astrocyte release of, 77, 77f, 78,408 in temporal lobe epilepsy, 47,403
astrocyte uptake of,408 Glutathione(GSH)
brain cancer treatment on, 557,558f mitochondrial, 255f,256
in epilepsy,8687 sleep deprivation on,323
in epilepsy, homeostasis of,403 Glutathione peroxidase7,262
exercise on,305 Glycemic index (GI),252
functions of,420 low-glycemic index treatment and, 251f,252
in GABA synthesis,45 Glycine receptor (GlyR),51
in ketogenic diet, 253,254 activation of, in chloride channel opening, 51,52f
release-elimination balance of,514 anion channelsof,51
in sleep,305 inhibition via, 5154, 52f,53f
synaptic cleft removalof,44 Glycogen
transport of, excitatory amino acid transporters in energy metabolism, 101102,102f
in,515 [K+]0 elevation in astrocyte metabolism of,103
traumatic brain injury on, 420421 in memory acquisition, 103104
Glutamate cysteine ligase (GCL),256 Glycolytic restriction/diversion, in ketogenic diets,
Glutamate decarboxylase (GAD),253 257258
Glutamate dehydrogenase (GDH),281 Growth arrest and DNA damage 45 (Gadd45)
Glutamate-glutaminecycle genes,189
astrocyte, 100101,101f Growth-promoting extracellular matrix molecules,373
in GABA recycling,4547 GSK3 inhibitors, on neuropsychiatric disease,60
in epilepsy,47 Guanosine
functions of,4445 in CNS signaling, 373374
in inhibitory synaptic transmission,46 on ischemia,382
Glutamate homeostasis,4448 on lipid homeostasis,382
astrocytes in, 100101,101f in neurogenesis and neurorestoration, 381,382
astrogliosis on,4748 neurotrophic effects of,381
branched chain amino acids in, 279280, 280f, signaling transduction cascade of, NGF, BDNF, and
281,282f FGF2 pathways and, 382383,383f
in epilepsy, restoration of, 407408 targeting convergent molecules in, 384385
GABA sources in, presynaptic,45 on stem/progenitor cell proliferation,382
GLT-1 and SN1in,45 Gut microbiota,284
glutamate-glutamine cycle in, astrocyte, 4445, in autism, 589,597
101102,102f on body weight,581
GABA recycling via,4547
transportersin,44 Hallucinogen addiction, 509527. See also Addiction
vesicular packagingin,44 Harvard Epilepsy Botanical Program,336
VIAATin,44 Heart rate variability (HRV), meditation on, 357358
Glutamate receptor 1 (mGluR1),76 Hebbian plasticity, 108, 124126, 136137
Glutamate receptor 5 (mGluR5),76 associative, 124125
Glutamate receptor, in addiction, 514515 runaway dynamics in,125
Glutamatergic neurotransmission spike-timing dependent plasticity rules in,126
adenosine on,3637 synaptic competition in, weak,127
in Parkinsons disease, 489490 Hebbs rule, 108,124
progesterone on,412 Herbal extracted compounds, 335336. See also
Glutamatergic therapies, for epilepsy, 407408 Botanicals
Glutamate transporter-1 (GLT-1, EAAT2),44 Herbal remedies. See Botanicals
in addiction,515 Heroin. See also Opioid addiction
in GABA reuptake,45 on AMPA receptors, 514515
in glutamate uptake,46 on mesocorticolimbic reward circuitry,510
localizationof,45 Heroin addiction, 509527. See also Addiction
Glutamine exercise on,524
626 Index
Heterosynaptic long-term depression,178
Heterosynaptic long-term potentiation,178
Heterosynaptic plasticity, 124136
definition of,130
experimental evidence for, 130131
Hebbian plasticity and, 124126, 136137 (See also
Hebbian plasticity)
induction scheme for, 137f,138
levels of homeostasis and,124
modeling of, 126128
competition in, 134136
competition in, need for, 127128
runaway dynamics prevention in, 133134,
135f,136f
stability through normalization in,127
STDP rule balancing in,126
trigger and timescale in,128
weight changes in, synaptic,126
properties of, 132133,132f
role of,124
runaway counteracting mechanisms in, other,
136137
synaptic scaling in, 128130
experimental paradigm for, 129,130
timescale, competition, and runaway dynamics
in, 129130
tetanization induction of, 132133,132f
trigger for,131
weight dependence of,131
High mobility group box 1 protein (HMGB1)
in epilepsy, cognitive impairments in, 165166,165f
in seizures, 157158, 157t,406
Hippocampal sclerosis
in Alzheimers disease,455
in temporal lobe epilepsy, 60, 62f, 85, 87, 209,403
Hippocampus. See also specificareas
in addiction, 509510
Alzheimers disease on, 214215
antidepressants on neurogenesis in, 208, 213214
in anxiety disorders,537
cortisol on,359
in depression, size of,213
in emotional processing, 536,537f
meditation on,360
neural stem/progenitor cells in, 203f, 204205
regulators of, 205206
neurogenesis in, adult, 202204, 203f,208
stress-induced atrophy of, 359360
Histamine
in addiction,518
function of,518
on sleep and cognition, 319320
Histamine receptors, in addiction,518
Histone
acetylation of, 189, 189f, 457,460
in memory formation,193
in Alzheimers disease, 459460
methylation of, 189190,189f
in memory consolidation,194
posttranslational modification of, 189190, 189f,457
ubiquitination of,190
Histone acetyltransferase,189
Histone code, 189,189f
Histone deacetylases (HDACs),457
-hydroxybutyrate on, 256257,261
in epilepsy,195
ketogenic diet on,263f
on learning and memory,193
meditation on genes for,356
valproate on,237f
Histone deacetylases (HDAC) inhibitors, for
Alzheimers disease, 459460
HMG-CoA synthase 2 (HMG-CoAS2),253
Homeobox (Hox) genes,192
Homeostasis, brain. See also specifictopics
levels of,124
regulatory mechanisms overview for, 371372
Homeostatic response recovery,181
Homeostatic synaptic scaling. See Synaptic scaling
Homer1a,180
Homocysteine, in Alzheimers disease,458
Homosynaptic long-term potentiation, 137f,138
heterosynaptic LTD with,130
Homosynaptic plasticity,178
expression mechanisms of,131
Hebbian-type learning in, 124,125
induction of, 126, 137f,138
weight dependence of,131
Hormones, on neuronal excitability, 411412
5-HT3, in addiction,512
5-HT receptors
in addiction,512
in inflammation-associated depression, 163,163f
Hub cells,209
Huntingtons disease
adenosine signaling in,3738
basal ganglia dysfunction in,299
BDNF in,376
caffeine in,462
creatine for, 274275,278
homeostatically controlled mechanismsin,35
mitochondrial dysfunction and NGF signaling
in,375
neurotrophic factors in,373
Huperzine A(Huperzia serrata), 335336
Hydrocephalus,14
obstructive,14
Hydrogen peroxide (H2O2),256
4-Hydroxy-2-nonenal (4-HNE),256
3-Hydroxy-3-methylglutaryl CoA (HMG-CoA),
250f,253
5-Hydroxyindoleacetic acid, in Parkinsons
disease,491
5-Hydroxymethyl-cytosine (5-hmC), in DNA methyla-
tion, 188189
5-Hydroxytryptamine (serotonin). See Serotonin
(5-hydroxytryptamine,5-HT)
Hyperactivity, neuronal, Ca 2+ in,195
 Index 627

Hyperbaric oxygen therapy (HBO2T), with ketogenic in Parkinsons disease ( See

diet, 560561 Neuroinflammation, in Parkinsons disease)
Hyperemia, functional, 104105 pathophysiology of, 155156
Hyperglycemia, in brain cancer progression,556 on sleep pressure,84
Hyperpolarization-activated, inward rectifier Cl- cur- from traumatic brain injury,421
rent (IClh),15 yin and yang of,159
Hypocretin,317 Infliximab,166
Hypogonadotropic hypogonadism, after traumatic Influenza type-A, amantadine hydrochloride for,430
brain injury,429 Inhalant addiction, 509527. See also Addiction
Hypothalamic craniopharyngioma,572 Inhibitory plasticity, neuronal,5760
Hypothalamic-pituitary-adrenal (HPA)axis phosphorylation in,5960
in allostatic regulation, 358,535 RNA splicing and editing in,5759
meditation on, 358360 Inhibitory synaptic scaling, 112113
in stress response, 535, 537538 Innate immunity,492
Hypothalamus, in obesity in Alzheimers disease, 455456
beta-oxidation in,579 in inflammation, 156158,157t
nutritional signals of, 578579 microglia in,493
Hypothalamus, ventromedial(VMH) Inosine,597
in energy balance and glucose homeostasis, 571573 Input specificity, rule of, 124125
on food intake and metabolic rate, 571572 In silico receptorome screening,241
in obesity, 573576 Insomnia. See also Sleep
melatonin and,278
IL-1R1 signaling, in epilepsy, 157158,157t Insula
IL-1ra, for epilepsy, from inflammation,166 in anxiety disorders,537
Immune function exposure therapy on,543
in epilepsy, 405406 meditation on,360
meditation on, 355357 Insulin
sleep on, 324,324f in addiction,521
Immunoglobulin G autoantibodies, on traumatic brain in obesity,578
injury,424 synthesis and functions of,521
Immunomodulation, in epilepsy, 155166. Insulin-like growth factor 1 (IGF-1), in brain cancer
See also Epilepsy, inflammation and progression,556
immunomodulationin Integrative approaches. See also specifictypes
Immunomodulatory therapies, for epilepsy, 409410 in biology,234
Impulsivity,511 in neuroscience, 232235,234f
in serotonergic drug addiction, 512513 Interleukin-1 receptor antagonist (IL-1ra), for trau-
Inborn errors of metabolism, energy demands in,272t matic brain injury,432
Infectious brain injuries, blood-brain barrier dysfunc- Interleukin-1/Toll-like receptor (IL-1R/TLR) signaling
tion in,149 pathway, in epilepsy, 405406
Inflammation Interleukin-1 (IL-1)
in addiction, 517518 in addiction, 517518
cytokines in, 517518 on epilepsy,410
in Alzheimers disease, 454456 in inflammation, 409410
in autism,590 depression with, 162163,163f
on blood-brain barrier,156 maternal, in autism in offspring, 163164,163f
in brain,155 sleep and, 324,324f
depressionand Interleukin-6 (IL-6)
with epilepsy, 162163,163f in addiction, 517518
after traumatic brain injury, 427428 maternal, in autism in offspring, 163,163f
in epilepsy, 155166, 405406 (See also Epilepsy, Interneurons
inflammation and immunomodulationin) excitatory synaptic scaling,112
IL-1 in, 409410 inhibitory synaptic scaling, 112113
innate immunity and, 156158,157t Interstitial space composition, ionic,98
ketogenic diets on,264 Intrinsic plasticity, 5456,182
maternal immune activation-induced chronic,5657
in autism, 164, 164f,591 definitionof,54
in autism, prevention of,598 in epilepsy, 6061,62f
meditation on,356 flexibilityof,56
in neurodegeneration,492 functionsof,54
628 Index
Intrinsic plasticity (Cont.)
short-term,5455
synaptic plasticityrelated,5556
Inverse synaptic tagging, 178179,179f
Inward rectifier Cl- current (IClh),
hyperpolarization-activated,15
Inward rectifying potassium channels (Kir),78
Ion channels. See also specificions
ion currents and equilibrium potentialin,51
ketogenic diets on, 253254
opening of, 51,52f
Ion currents (I),51
Ion homeostasis. See also specificions
astrocyteneuron interactions on,98100
[K+]0, 9899,99f
pH0,99100
in epilepsy,403
Ionotropic glutamate receptors (iGluRs), in Parkinsons
disease,489
IP3-sensitive calcium, in astrocytes,76
Isocitrate dehydrogenase (IDH) gene mutations, in
glioblastoma and gliomas,194
Isoleucine, 278283. See also Branched chain amino
acids (BCAAs)
Jun N-terminal kinase (JNK),378
K+
astrocyte uptakeof,99
in epilepsy,403
siphoning of,7,8
spatial buffering of, 67, 7f,8,147
spatial redistribution of,78
uptake of,6,7f
[K+]0
astrocyte permeabilityto,98
blood-brain barrier on, 146147
elevation of, in glycogen metabolism, astrocyte,103
homeostasis of, 9899,99f
astrocyteneuron interactions on, 9899,99f
neuronal activityon,98
Kainate receptor,489
Kava,526
KCC2 cotransporter
on chloride channels, 5254,53f
phosphorylationon,59
RNA splicingon,57
Keratan sulphate proteoglycan,373
Ketogenesis, 252259
acetyl-CoA in, 250f, 252253
antioxidant activity, reactive oxygen species, and
redox state in, 255257
bioenergetic and mitochondrial changes in,
254255,255f
bioenergetics reserve and mitochondrial respiration
in, 258259
fatty acid oxidation in,258
glycolytic restriction/diversion in, 257258
ketone bodies in,253
mitochondrial permeability transition in,257
neurotransmitters and ion channel regulation in,
253254
steps of, 250f, 252253
TCA cycle effects and anaplerosis in,259
Ketogenic diet (KD), 248264
on adiposity,579
on Alzheimers disease, 261262
on amyotrophic lateral sclerosis, 263264
on autism, 264, 594595
on brain cancer,262
branched chain amino acids and, 282283
on creatine,277
definition of,248
with hyperbaric oxygen therapy, 560561
on migraine headache,264
mitochondrial dysfunction in neurological diseases
and,261
neuroprotective effects of, 263f,264
on neurotrauma, 262263
on pain and inflammation,264
on Parkinsons disease,262
radiation therapy with,561
restricted consumption of,559
for sleep,324
variations of, 250252
caloric restriction, 252, 559560,559f
low-glycemic index treatment, 251f,252
medium-chain triglyceride diet,251
modified Atkins diet, 251252,251f
Ketogenic diet, calorie restricted (KD-R), 252,
559560
for glioblastoma multiforme, 559f, 560561
Ketogenic diet (KD), on epilepsy, 248261,
410411
efficacyof
clinical studies of, 249250,250f
preclinical studies of,250
energy-sensing signaling pathway activation in,
259261
AMP-activated kinase in,260
mammalian target of rapamycin in,260
peroxisome proliferator-activated receptors in,
259260
sirtuins in, 260261
historical aspects of, 248249
ketogenesis in, 252259 (See also Ketogenesis)
variations of, 250252,251f
Ketone bodies, 253, 559560,559f
from branched chain amino acids, 279,280f
fasting on,302
Kineret,166
Kir 2.1 inward rectifying channels, 99,99f
Kir4.1 channels,89
in epilepsy,9,403
on K+ spatial buffering,147
K siphoning,7,8
+
in Mller cells, 99,99f
Kudzu, for alcohol addiction, 526527
 Index 629

Lactate heterosynaptic, 130131,178

astrocyte to axon transfer of, 102103
astrocyte-to-neuron shuttling of,100
metabolism and, exercise and, 301303
Lactate dehydrogenase,103
Lactatepyruvate equilibrium,103
Lactate shuttle hypothesis, astrocyte neuron,103
L-DOPA, for Parkinsons disease,489
Learning
associative, hippocampal long-term potentiation
in,124
COX- 2 on,165
dentate granule neurogenesis in,206
development and, 108109
epigenetics in, 193194
Leptin
in addiction,521
discovery of,574
functions of,578
sleep on,322
synthesis and role of,520
Leptin receptor, in obesity,578
Leptin system, proopiomelanocortin in,576
Leucine, 278283. See also Branched chain amino acids
(BCAAs)
Leukocytes
blood-brain barrier migration across,145
in seizure models,161
Leukoencephalopathies, CIC-2in,15
Levetiracetam. See also Antiepileptic drugs(AEDs)
binding site of,238
on epileptiform activity in mice,456
on ion channels,236
on sleep,314
for traumatic brain injury, 408,432
Levodopa-induced dyskinesia(LID)
GABA receptors in,490
glutamatergic receptors in,489
Lewy bodies, in Parkinsons disease,481
Limbic-hypothalamic-pituitary-adrenal (LHPA)
axis tone of, on behavior,321
in sleep and stress responses, 320321
Lipostatic hypothesis,571
Lithium,5960
Long-chain fatty acid transport, across mitochondrial
membrane, 271,272f
Long-term depression (LTD), 175, 176f, 177178
adenosine on,3637
calcium sliding threshold for,136
in cerebellum, 177178
excitatory synaptic scaling on, 110,111f
heterosynaptic,178
homosynaptic,178
Long-term potentiation (LTP), 175, 176177,176f
adenosine on,3637
associativity of,176
calcium sliding threshold for,136
cooperative,176
excitatory synaptic scaling on, 110,111f
hippocampal, in associative learning,124
homosynaptic, 137f, 138,178
heterosynaptic LTD with,130
input specificity of, 176,176f
in memory, 177,181
NMDA-type glutamate receptordependent,177
types of,177
Loving-kindness meditation, 354355, 360. See also
Meditation
Low-glycemic index treatment (LGIT), 251f,252
LRRK2 (PARK8) Parkinsons disease,483
Lymphocytes
in Parkinsons disease, inflammatory reaction
in,494
T
in Rasmussens encephalitis,160
in seizure models,161
Lysergic acid diethylamide (LSD) addiction, 509527.
See also Addiction
Lysine acetyltransferases (KAT), 257258
Major depressive disorder(MDD)
anxiety disorders with,545
with epilepsy, inflammation in, 162163,163f
on hippocampus,213
neural stem/progenitor cells and neurogenesis for,
213214
sleep and, 321322
after traumatic brain injury, 427428
inflammation in, 427428
neurotrophic support in,427
serotonergic dysfunction in,428
Malonyl-CoA,578
Mammalian target of rapamycin (mTOR)
blockade of, for epilepsy,410
ketogenic diets on, 260,263f
Mania, branched chain amino acids for,280
MAPK pathway, 378,382
FGF2 in,380
guanosine-activated cAMP-dependent mechanisms
on,382
Marijuana addiction, 509527.
See also Addiction
endocannabinoid homeostasis in, 513514
mindfulness training on,525
Maternal immune activation (MIA), in autism, 164,
164f,591
prevention of,598
MC4R receptor
destruction of, in obesity,574
in obesity, 574, 575,578
-MSH on,574
MDMA addiction. See also Methamphetamine
serotonin homeostasis in, 512513
Medial prefrontal cortex (mPFC)
in anxiety and emotional
behaviors, 536,537f
in anxiety disorders,537
630 Index
Meditation, 353361. See also specifictypes
for addiction, 524525
on allostasis and allostatic load,353
on autonomic regulation of heart, 357358
on blood pressure,355
on brain homeostasis,353
on brain, research on,353
on cortisol, 358359
on C-reactive protein,356
definition of,354
on hippocampus,360
on HPA axis regulation, 358359
on immune function, 355357
on neuroplasticity, 360361
on stress response,353
on telomeres,357
for traumatic brain injury,433
types of practices of,354
Medium-chain triglyceride (MCT) diet,251
for autism,596
Megalencephalic leukoencephalopathy, astrocyte
volume controlin,19
Melanocyte concentrating hormone (MCH),577
Melatonin, 277278
aging and,277
autism and,278
on free radicals and antioxidants,278
functions of, 277278
insomnia and,278
neurodegenerative disorders and,278
sleep deprivation on,323
on sleep-wake transition,318
Membrane-bound monocarboxylate transporters
(MCT), lactate transport by, 103,302
Memory
adenosine for improvement of, 465467
adenosine kinase on, 466467
COX- 2 on,165
dentate granule neurogenesis in,206
epigenetics in, 193194
epilepsy on, inflammation in, 165166,165f
long-term potentiation in, 177,181
piracetam on,238
synaptic scalingin
in short- vs. long-term storage,116
in working memory,116
Memory consolidation
astrocytes in,8283
histone methylation in,194
Memory formation
epigenetic chomatin modifications in,195
glycogen in, 103104
histone acetylation in,193
Mesocorticolimbic reward circuitry
in addiction, 509510
in feeding,521
Metabolic disorders. See also specifictypes
in addiction, 520521
in autism, 589590
Metabolic ratio, cerebral, 302303
Metabotropic glutamate receptors (mGluRs), in
Parkinsons disease,489
Metaphysics,234
Metaplasticity, 54, 56, 109, 133, 136,182
Methadone, for opioid addiction,523
Methamphetamine
on AMPA receptors, 514515
on histamine release,518
Methamphetamine addiction. See also Addiction
ginseng on,526
mindfulness training on,525
serotonin homeostasis in, 512513
Methioninecycle
DNA methylation in Alzheimers disease and,458
in neuronal homeostasis,193
Methodological reductionism,232
1-Methyl-4-phenylpyridinium,263
Methyl-CpG-binding domain (MBD) family,187
Methyl-CpG binding protein 2 (MeCP2), in synaptic
scaling,117
3,4-Methylenedioxymethamphetamine (MDMA). See
Methamphetamine
O6-Methylguanine-DNA methyltransferase (MGMT),
epigenetic alterations in,194
Methylphenidate(MPH)
for traumatic brain injury,430
use of, by healthy people,238
Methylxanthines. See also Caffeine
on adenosine receptors, 318,411
on epilepsy,411
Microbiome treatment, for autism,597
Microbiota, gut,284
in autism, 589,597
on body weight,581
Microbiotagutbrain axis,284
Microglia
in addiction,517
in Alzheimers disease,455
cell signaling by,517
in innate immunity,493
regulatory functions of,517
in Tau pathology, 455456
MicroRNA (MiRNA), 190191,191f
Migraine headache
ketogenic diets on,264
after traumatic brain injury, 428429
Mindfulness-based cognitive therapy (MBCT), 354,
359. See also Meditation
Mindfulness-based stress reduction (MBSR), 354. See
also Meditation
on blood pressure,355
on cortisol,359
on heart rate variability,358
on hippocampus,360
on immune function, 355356
on neuroplasticity,361
Mindfulness meditation, 354. See also Meditation
for addiction, 524525
 Index 631

applications of, 524525 on adenosine concentrations, 342,512

components of,524 on endocannabinoids,514
for traumatic brain injury,433 GDNF and,522
Minocycline on glutamate, 514,515
on addiction,517 on mesocorticolimbic reward circuitry,510
for inflammatory diseases,166 on neurosteroids,520
on major depressive disorder,162 Morphine addiction. See also Addiction
for traumatic brain injury,432 acupuncture for,348
Mitochondria cholinergic homeostasis in, 516517
cell damage/injury from, sleep and, 322323,323f ginseng on,526
ketogenic diets on, 254255,255f on progesterone and pregnenolone sulfate,520
respiration in, 258259 Motoneurons,298
neuronal excitability and function of, 254255,255f Motor cortex, 298299
normal function of,555f Movement. See also specific disorders
sleep and, 322323,323f neural control of, 298300,299f
traumatic brain injury on,421 Mller cells,4
Mitochondrial bioenergetics, in epilepsy,406 K+ siphoning in, 99,99f
ketogenic diets on, 254255,255f Multidrug resistance proteins
ketogenic diets on reserve of, 258259 ATP release via,17
Mitochondrial dysfunction at blood-brain barrier,145
in autism, 589590 Multidrug therapies,237
in brain tumors, 554555,555f Multidrug transporters,145
in Huntingtons disease,375 in epilepsy,409
in neurological diseases,261 Multifunctional drugs. See also specifictypes
in Parkinsons disease,487 alcohol as,239
Mitochondrial membrane, long-chain fatty acid trans- anesthetics in,239
port across, 271,272f piracetam in, 238239
Mitochondrial permeability transition (mPT), single-drug molecules as,237
ketogenic diets on,257 Multi-omics,234
Mitogen-activated protein kinase (MAPK) pathway, Multiple sclerosis
378,382 amantadine hydrochloride for,430
FGF2 in,380 blood-brain barrier dysfunction in,149
guanosine-activated cAMP-dependent mechanisms FGF2/FGFR1 signaling manipulation for,380
on,382 fingolimod for,166
Modafinil histamine in,518
for narcolepsy,318 Huperzine Aon,336
use of, by healthy people,238 Multitarget pharmacology, 235237,237f
Modified Atkins diet, 251252,251f Muscarinic (M1-M5) acetylcholine receptors,516
Molecular plasticity, 5760. See also Inhibitory plastic- Myelin-associated glycoprotein,373
ity, neuronal Myelination-related inhibitors,373
Monoacylglycerol lipase (MAGL), in endocannabinoid
addiction,514 N6-(4-hydroxybenzyl) adenosine riboside (T1-11),593
Monoamine neurotransmission, after traumatic brain N-acetylcysteine(NAC)
injury,427 for Alzheimers disease,458
Monoamine oxidase inhibitors (MAOIs) for cocaine addiction,523
for anxiety,542 Na+ channels, voltage-gated, in cell volume,16
for Parkinsons disease,489 Na+-dependent Cl-/ HCO3- exchanger,100
on REMS sleep,317 Na+/ HCO3- cotransport,100
for traumatic brain injury,431 in glial cells,100
Monoamine transporter (5-HTT), in addiction, Na+/ H+ exchanger,100
512513 Na+-K+-2Cl cotransporter (NKCC).
Monocarboxylate transporters (MCT), lactate trans- See Sodium-potassium-chloride
port by, 103,302 cotransporter(NKCC)
Monoclonal antibodies, for epilepsy from Na+/K+-ATPase, exercise on, 301,304f
inflammation Na+/ K+-ATPase pump,6
against IL-1,166 Nalmefene, 522523
against IL-6,166 Naltrexone,522
against TNF-,166 Narcotic addiction, 509527
Morphine Narp, in synaptic scaling,180
632 Index
NCCa-ATP channel, in cell volume homeostasis,16
Neotrofin (lepteprinim, AIT-082), on nerve growth
factor production,381
Neprilysin gene,457
Nerve growth factor (NGF),373
AIT-082 on, 381382
guanosine on,381
in neuroprotection,422
in pain promotion and hyperalgesia,376
in restoration,375
Netrins,373
Network pharmacology, 231243. See also Systems
(network) pharmacology
Neural/glial antigen,373
Neural restrictive silencing factor (NRSF), 257,410
Neural stem/progenitor cells (NSCs), 202218
activity of, determining,205
in Alzheimers disease, 214215
in brain repair after stroke, 215217
definition of,202
in depressive disorders, 213214
functional significance of, 206208
in hippocampus, 203f, 204205
adult human,208
regulators of, 205206
stem vs. progenitor cells in,202
in subventricular and subgranular zones,202
in temporal lobe epilepsy, 208213
early, proliferation and connectivity in,
209211,210f
homeostasis and neurogenesis in, 208209
NSC potential and new granule cells in, 211213,
212f213f
Neurocan,373
Neurodegeneration. See also specific disorders
inflammation in,492
melatonin and,278
in traumatic brain injury, 425426
Neurofibrillary tangles, in Alzheimers disease, 214,
452453
Neurogenesis
definition of,192
epigenetics in, 192193
exercise on, 308,411
from nerve growth factor,376
pathological,411
in subventricular and subgranular zones,202
Neurogenesis, adult, 202218. See also
Stem cells, adultneural
brain areas of, 202204,203f
FGF2 in, 379380
in hippocampus, 202204,203f
human,208
regulators of, 205206
neural stem/progenitor cells in, 202 (See also
Stem cells, adult neural)
functional significance of, 206208
in nonneurogenic brain regions, 202203
stem vs. progenitor cells in,202
in subventricular and subgranular zones, 202,203f
after traumatic brain injury,425
Neurogenesis homeostasis, for depressive disorders,
213214
Neurogenin2,205
Neuroinflammation, in Parkinsons disease, 493494
inflammatory response in,492
postmortem studies of,493
therapy for, 494495
Neuromyelitis optica (NMO), AQP4 OAPsin,10
Neuronal activity-regulated pentraxin (Narp), in syn-
aptic scaling,180
Neuronal excitability homeostasis, 5163. See also
Excitability homeostasis, neuronal
Neuronal hyperactivity, Ca 2+ in,195
Neuron restrictive silencing 23 element (NRSE),257
Neurons
functionsof,98
silencing of, 56, 113, 125, 129, 130,135f
Neuropeptide Y(NPY)
in anxiety and stress disorders,540
on body weight,577
in obesity, 576577
Neuroplasticity, 175182. See also specifictypes
cortical reorganization and remapping in,
180181,181f
definition of,175
functions of,175
homeostatic response recovery in,181
homeostatic synaptic scaling in, 109, 128130, 175,
181 (See also Synaptic scaling)
meditation on, 360361
neurological/psychiatric disorders and,181
ocular dominance plasticity in, 180181,181f
synaptic plasticity in, 175180 (See also
Synaptic plasticity)
in traumatic brain injury,425
Neuropoetic cytokines,521
Neuroregeneration, 372. See also Neurorestoration
Neurorestoration, 371386, 374377
bench to bedside gap in,386
definition of,371
fibroblast growth factor in, 379380
fibroblast growth factor signaling pathway in,
380381
goal of, 371372
guanosine signaling transduction cascade of, NGF,
BDNF, and FGF2 pathways and, 382383,383f
targeting convergent molecules in, 384385
history of,371
homeostasis regulatory mechanisms and, 371372
neuroregeneration in,372
neurotrophin (NGF/BDNF) signaling pathway
in,377
nonadenine-based purinesin
AIT-082, 381382
guanosine,381
nonpermissive environment in, reducing,
372373,384

p75NTR signaling pathway in, 378379
permissive environment cues in, stimulating,373
permissive environment stimulation in, 374377
(See also specifictypes)
BDNF in, 376377
hormones and neurotransmitters in,374
interleukins and lymphokines in,374
nerve growth factor in, 375376
purinergic molecules in,374
research on, early,372
scope of,372
strategies for,374
TRK signaling pathway in, 377378
Neurosteroids,520
Neurotransmitters. See also specifictypes
in addiction, 514517
astrocyteneuron interactions in homeostasis of,
100101,101f
on hippocampal neurogenesis, 205206
ketogenic diets on, 253254
metabolism of, local synaptic,45
packagingof,44
synaptic cleft removalof,44
vesicular contentof,44
Neurotrophins (neurotrophic factors), 373, 374. See
also specifictypes
in addiction, 521522
exercise benefits for,524
in neuroprotection, 422423
in Parkinsons disease, 494495
Neurotrophin (NGF/BDNF) signaling pathway,377
Neurovascular coupling,148
Neurovascular unit, 3, 143144, 144f,422
NF E2-related factor 2 (Nrf2),256
NF-kB (nuclear factor kappa-light-chain-enhancer of
activated B cells), in addiction,517
Nicotine addiction, 509527. See also Addiction
acupuncture on, 525526
nicotine replacement for,523
varenicline for,523
Nicotine, on endocannabinoids,514
Nicotine replacement, for nicotine addiction,523
Nicotinic acetylcholine (nAch) receptors
in addiction, 516517
classification of,516
NMDAR antibodies, in epilepsy, 160161
NMDA receptor,489
astrocytic control of trafficking of, in epilepsy,
8790, 88f89f
estrogen on,412
NMDA-type glutamate receptor
(NMDA-R)-dependent LTP,177
N-methyl-D-aspartate (NMDA), on adenosine
concentrations,342
Nociception homeostasis, acupuncture in, 341349. See
also Acupuncture
NogoA,373
Nonadenine-based purines, on nerve growth factor
production
Index 633
guanosine,381
neotrofin,381
Noncoding RNAs, 190191,191f
Nootropic drugs, 231, 237239,240f
Norepinephrine (noradrenaline)
exercise and,305
in fear learning,540
in pain,490
in Parkinsons disease, 490, 491492
on sleep,321
Norepinephrine-enhancing agents, on sleep, 317318
Norepinephrine-inhibiting agents, on sleep,317
Norepinephrine neurotransmission
in anxiety and stress disorders,539
in Parkinsons disease,491
in stress response,537
Normalization,127
Notch signaling,205
NREMS sleep, 315f,316
Nuclear factor-kappa B (NF-B) cascade, p75NTR
activation of,379
Nucleosome, 189,189f
Nucleus accumbens(NAc)
in addiction, 509510
dopamine in,511
Nutrition. See also Diet
on brain function,248
prenatal, on CNS development and function,193
for sleep,324
O2 consumption, in exercise, 300301
O6-methylguanine-DNA methyltransferase (MGMT),
epigenetic alterations in,194
obese (ob) gene, 573575
Obesity and diabetes, 570581
calories myth in,580
dietary composition on,579
energy balance and glucose homeostasis in, ventro-
medial hypothalamus in, 571573
epidemiology of, 570571
fat mass and obesity associated (FTO) gene, 579580
genetic, in humans, 579580
genetic, in mice, 573578,574
Agouti-related peptide in, 576577
arcuate hypothalamus in, 575, 576577
diabetes (db) gene in, 573575
early studies on,573
leptin in,574
melanocyte concentrating hormone in,577
neuropeptide Y in, 576577
obese (ob) gene in, 573575
proopiomelanocortin in, 575577
ventromedial hypothalamus in, 573576
-melanin-stimulating hormone in,574
ghrelin in, 578579
glucostat hypothesis in, 572,578
health consequences of,571
hypothalamic beta-oxidation,579
hypothalamic nutritional signals in, 578579
634 Index
Obesity and diabetes (Cont.)
insulin in,578
lipostatic hypothesis of,571
microbiota on, gut,581
nature nor nurture in, 580581
Obsessive compulsive disorder (OCD). See also
Anxiety and stress disorders
deep brain stimulation for,543
DSM on,538
pharmacological treatment of, 541542
in youth,544
Ocular dominance plasticity, 180181,181f
Olanzapine
on sleep,317
targets for,236
Oligodendrocyte myelin glycoprotein,373
Omega-3 fatty acids, 284285
for traumatic brain injury,435
Omics technologies, 234,242
for traumatic brain injury,429
Ontological reductionism,232
Opioid addiction. See also Addiction
acupuncture for,348
buprenorphine for,523
exercise on,524
ginseng on,526
methadone for,523
Opioids. See also specifictypes
histaminergic drugs on responses to,518
on mesocorticolimbic reward circuitry,510
Orexin,317
Orexin receptor blockers, on sleep,317
Orkand model, 67,7f
Orphan CpG islands,188
Orthogonal arrays of particles (OAPs),10
Ovarian hormones, in addiction, 518520
Oxidative stress
ketogenic diets on, 255257
in Parkinsons disease,487
from traumatic brain injury,422
OxPhos
in glioblastoma multiforme mitochondria,
554555,555f
insufficiency of, in Warburg effect,556
P2X receptors, acupuncture and,347
P2Y1 receptor,76
P2Y receptor, acupuncture and,347
p75NTR
-amyloid protein and,379
nuclear factor-kappa B cascade activation by,379
p75NTR signaling pathway, 378379,385
Pain
CNS, BDNF in,376
ketogenic diets on,264
in Parkinsons disease, 482, 489492 (See also
Parkinsons disease)
Pain, chronic
A1 receptor agonists on, 343,343f
acupuncture for, 343, 343f (See also Acupuncture)
definition of,348
prevalence of,348
Pannexin 1 (panx1)
functionof,12
in potassium homeostasis,12
Parasympathetic nervous system
in anxiety and stress disorders, 535,537f
meditation on, 353, 355, 357,358
in stress response, 535,537f
PARK1/PARK4 Parkinsons disease, 482483
PARK4 Parkinsons disease, 482483
PARK6 Parkinsons disease,484
PARK7 Parkinsons disease,484
PARK8 Parkinsons disease,483
PARK15 Parkinsons disease,485
PARK72 Parkinsons disease,484
PARK genes,482
Parkin (PRKN/PARK72) Parkinsons disease,484
Parkinsons disease, 481497
adenosine signaling in, 35,3738
amantadine hydrochloride for,430
autonomic nervous system dysfunction in,491
basal ganglia dysfunction in,299
basal ganglia in,488
BDNF in,376
brain homeostatic systems in, 487492
adenosine-mediated,491
adrenergic,490
basal ganglia in,488
cholinergic, 490491
dopaminergic, 488489
glutamatergic, 489490
motor and non-motor symptoms in, 487488
norepinephrine-mediated,491
serotonergic,491
cells in, 493494
clinical symptoms of,482
motor, 487488
non-motor, 487488
creatine for, 274275,278
depression in, 482, 487,488
GABAergic neurotransmission in,490
glutamatergic neurotransmission in,489
noradrengic neurotransmission in,490
serotonergic neurotransmission in,491
dopamine in, 488489
endogenous toxins in, 485486
energy demands in,272t
environmental factors in, 485, 486487
etiology of,482
future directions in, 495497
genetic forms of, 482,495
genetic forms of, autosomal dominant, 482484
EIF4G1,484
GBA,484
LRRK2 (PARK8),483
SNCA (PARK1/PARK4), 482483
VPS35, 483484
 Index 635

genetic forms of, autosomal recessive, 484485 pH

ATP13A2 (PARK9), 484485 blood-brain barrier on,147
DJ-1 (PARK7),484 definitionof,99
FBXO7 (PARK15),485 pH0
Parkin (PRKN/PARK72),484 activity-induced changes in, and regulation,100
PINK1 (PARK6),484 astrocyte regulation of,99100
PLA2G6,485 homeostasis of,99100
homeostatically controlled mechanismsin,35 Phenytoin
ketogenic diets on,262 P-glycoprotein on brain penetration of,409
Lewy bodies in,481 on sleep,314
neuroinflammationin targets and mechanisms of action of,407
inflammatory response in,492 for traumatic brain injury,432
postmortem studies of,493 Phosphatidylinositol 3-kinase (PI3K) pathway,378
therapy for,494 Phospholipase C gamma (PLC-gamma) pathway,378
neuroinflammatory pathways in, 493494 FGF2/FGFR1 triggering of, 380381
neurotrophic factors in, 373, 494495 Phosphorylation, in inhibitory plasticity,5960
pain in,482 Physical exercise, 298308. See also Exercise
adenosine pathways in,492 Phytocannabinoids,335
GABAergic neurotransmission in,490 PI3K/Akt signaling pathway, tumor radiation on,557
glutamatergic neurotransmission in,489 Pia, glial membrane of,104
noradrenaline in,490 PINK1 (PARK6) Parkinsons disease,484
serotonergic pathways in,491 Piracetam, 231,238
pathogenesis of, 384, 486487 on cell membranes, 238239
mitochondrial dysfunction in,487 lack of specificity of, 238239
oxidative stress in,487 on memory, 231,238
protein aggregation and misfolding in,486 PLA2G6 Parkinsons disease,485
ubiquitination and related stress in, 486487 Plasticity, 308. See also Neuroplasticity; specifictypes
pathophysiology and neurochemical Hebbian, 108, 124126, 136137
features of,481 intrinsic, 5456, 182 (See also Intrinsic plasticity)
prevalence of,481 epilepsy-related, 6061,62f
reactive astrocytosisin,47 molecular (inhibitory), 5760 (See also
risk factors for,481 Inhibitory plasticity, neuronal)
sleep disorders in, 482, 487, 489491 structural,182
adenosine in,492 PLC-gamma. See Phospholipase C gamma
autonomic nervous system in,491 (PLC-gamma) pathway
GABA in,490 Polycomb (PcG) system,192
glutamatergic receptors in,489 Polyglutamine diseases,384
serotonin in,491 Polypharmacology, 235237,237f
therapeutic strategies for,495 Polyphenols, dietary, for traumatic brain injury,436
Paroxetine. See also Polytherapy, rational,237
Selective serotonin reuptake inhibitors (SSRIs) Polyunsaturated fatty acids (PUFA), ketogenic diets
for anxiety disorders,541 on,258
on depression,241 Posttraumatic epilepsy,426
Pattern separation,207 Posttraumatic headache,428
Pedunculopontine nucleus (PPN), in Parkinsons Post-traumatic stress disorder (PTSD)
disease,490 amygdala in,537
Pentobarbital, for traumatic brain injury,432 cortisol in,540
Pericytes, 144,144f deep transcranial magnetic stimulation for,543
Peripheral fatigue,305 development of,538
Peroxiredoxin4,262 gender differences in,543
Peroxisome proliferator-activated receptors sleep in,321
(PPARs),579 THC for,541
ketogenic diets on, 259260,263f Potassium channels
Persistent hypogonadotropic hypogonadism (PHH), in astrocyte-mediated potassium homeostasis,79
after traumatic brain injury,429 in astrocyte volume regulation,14
PGE2, in inflammatory cascade, 157t,159 inward rectifying (Kir),78
P-glycoprotein, in epilepsy,409 phosphorylationof,60
P-glycoprotein-like proteins, at blood-brain Potassium-chloride cotransporter (KCC), on chloride
barrier,145 channels, 5254,53f
636 Index

Potassium homeostasis microdynamics, in autism,593

extracellular,612 on neural stem cells, 380381
astrocytesin Purines. See also
aquaporins in,1011 Adenosine; ATP (adenosine triphosphate)
connexinsin,11 homeostatic regulationof,31
pannexinsin,12 salvage of,3132
potassium channels in,79 Pyramidal neurons, synaptic scalingin
astroglial,11 excitatory, 109112,111f
Kir4.1 in, 89,403 inhibitory, 112113
K+ siphoning in,7,8
K+ uptake and spatial K+ buffering in, 67,7f,8 Quetiapine, targets for,236
Orkand model of, 67,7f
potassium and sodium ion concentrationsin,6 Radiation therapy
TREK and TWIK in,9,14 on brain tumor energy metabolism, 557558,557f
Potassium ion ( K+). See also K+ with ketogenic diet,561
astrocyte uptakeof,99 on PI3K/Akt signaling pathway,557
in epilepsy, elevation of,403 Ramelteon, on sleep-wake transition,318
siphoning of,7,8 Rapamycin, in epilepsy,410
spatial buffering of, 67, 7f,8,147 Ras/extracellular signalregulated kinase (ERK),
spatial redistribution of,78 377378,384
uptake of,6,7f Rasmussens encephalitis (RE), 156, 159160
Prazosin, on sleep,321 Rational drug discovery,235
Prefrontal cortex(PFC) Rational polytherapy,237
in addiction, 509510 Reactive astrocytosis,47
in anxiety disorders,537 in Alzheimers disease, 47,454
in emotional processing, 536,537f in epilepsy, 47,8485
meditation on,360 in Parkinsons disease, 493494
Pregnenolone sulfate in stroke,47
in addiction,520 from traumatic brain injury, 47, 421422
morphine addiction on,520 Reactive astrogliosis. See Reactive astrocytosis
Presenilin-1 (PSEN1) promoter, 457458 Reactive oxygen species(ROS)
PRKN/PARK72 Parkinsons disease,484 ketogenic diets on, 255257
Probiotics,284 n tumor cells,553
Progenitor cells,202 in Parkinsons disease,487
Progesterone from traumatic brain injury,422
on GABAergic and glutamatergic Receptor for advance glycation end products (RAGE),
neurotransmission,412 in epileptogenesis, 157t, 159, 165,166
morphine addiction on,520 Receptor-mediated transcytosis,147
for traumatic brain injury, 423,435 Redox state, ketogenic diets on, 255257
Programmed cell death,494 Reductionism, 231, 232233
Proopiomelanocortin (POMC) epistemological,232
in leptin system,576 methodological,232
in obesity, 575577,579 ontological,232
Proportional-integral-derivative controller,115 Reelin,209
Prostaglandin 2 (PGD2), sleep and, 324,324f Regulatory volume decrease (RVD), 12,13f
Protein aggregation and misfolding, in Parkinsons VRAC in,13f
disease,486 Rehabilomics,429
Pseudoginsenoside-F11 (PF11),526 Relaxation Response, 354. See also Meditation
Psychostimulants. See also specifictypes on blood pressure,355
addiction to, 509527 (See also Addiction) on immune function, 356357
GDNF and,522 REMS sleep, 315f, 316317
on sleep, 317318 Repressor element 1-silencing transcription factor,
for traumatic brain injury,430 205,209
use of, by healthy people,238 Reservatrol,436
Psychotherapy, for anxiety and stress disorders, Retaliatory metabolite, 31, 38,593
542543 Rett syndrome, 117, 181,596
Purinergic signaling,373 RhoA,379
Purinergic system Risperidone, on sleep,317
acupuncture on, 346347 RNA editing, in inhibitory plasticity,5859
 Index 637

RNAs, noncoding, 190191,191f in MDMA addiction, 512513

RNA splicing, in inhibitory plasticity,5758 Serotonin-inhibiting agents, on sleep,317
Rubinstein-Taybi syndrome,193 Serotonin neurotransmission system
Runaway dynamics in anxiety and stress disorders,539
heterosynaptic plasticity in prevention of, 133134, in exercise,305
135f,136f Serotonin-norepinephrine reuptake inhibitors (SNRIs)
mechanisms counteracting, other, 136137 for anxiety disorders, 540541
normalization on,127 on REMS sleep,317
positive feedback on synaptic weight changes on,125 Serotonin receptors, in addiction, 512513
spike-timing dependent plasticity rules and, Sertraline
134,135f for anxiety in children and adolescents,544
synaptic scaling in, 129130 for traumatic brain injury,431
SeSAME syndrome,8
S100b,424 Set points,372
S-adenosylhomocysteine hydrolase (SAH), 33, Sex steroids, in addiction, 518520
406407,407f Short-term intrinsic plasticity,5455
in DNA methylation, 458,465 Silencing
S-adenosyl methionine (SAM),458 epigenetic, of tumor suppressor genes,194
in DNA methylation, 188, 189f, 193,465 gene,138
Schizophrenia DNA methylation in, 190,195
adenosine signaling in, 35,3738 histone methylation in,194
antipsychotics for, targets for,236 RNA-induced, 191,191f
energy demands in,272t neuron, 56, 113, 125, 129, 130,135f
GSK3-signalingin,60 transcriptional, 194,457
histamine in,518 Siltuximab,166
homeostatically controlled mechanismsin,35 Similarity ensemble approach,241
Secondary injury, in traumatic brain injury,420 in silico receptorome screening in,241
Seizures. See also Epilepsy Sirtuins (SIR1-7), ketogenic diets on, 260261,263f
in Alzheimers disease,465 SLC6A4 variation, after traumatic brain injury,424
from astrogliosis,465 Sleep, 314325
inflammation in pathogenesis of, 156158,157t adenosine on, 36, 81, 305,307
Selective serotonin reuptake inhibitors (SSRIs). See also on adenosine signaling,411
specificagents astrocytes in,8184
for anxiety disorders, 540541 on brain activity at circuit level,81
in children and adolescents,544 in inflammatory response and sleep behavior,84
on cocaine and alcohol addiction behavior,513 on sleep homeostasis, 77f, 8182,83f
on REMS sleep,317 sleep loss, depression, and,8384
for traumatic brain injury,431 sleep loss, memory consolidation, and,8283
Semaphorins,373 on ATP, brain,322
D-Serine, 77f, 7879,404 circadian pacemaker in,317
Serotonergic neurotransmission on cognition, 319320
in depression after traumatic brain injury,428 on damage and waste removal, 323324
in Parkinsons disease,491 on damage, pathology, and neurological disorders,
Serotonin (5-hydroxytryptamine,5-HT) 322323,323f
in addiction, 512513 dopamine-enhancing agents on, 317318
on adenosine concentrations,342 dopamine-inhibiting agents on,317
biosynthesis of,512 exercise on,308
exercise on,305 on ghrelin, leptin, and body weight,322
functions of,512 glutamate in,305
on neurogenesis, 205206 immune factors on, 324,324f
in Parkinsons disease,491 on immune responses, 324,324f
receptors on action of,512 mitochondria and, 322323,323f
on sleep, 305,321 neuro-immune response factors and, 324,324f
Serotonin-altering drugs, addictive, 512513 norepinephrine-enhancing agents on, 317318
Serotonin-enhancing agents norepinephrine-inhibiting agents on,317
on REMS sleep, 315f,317 nutritional pharmacology for,324
on sleep, 317318 in Parkinsons disease, 482,487
Serotonin homeostasis adenosine in,492
in addiction, 512513 autonomic nervous system in,491
638 Index

Sleep (Cont.) Spatial K+ buffering, 67, 7f,8,147

GABA in,490 Sphingomyelinase,379
glutamatergic receptors in,489 Spikes, bursts of, in heterosynaptic plasticity, 131,
serotonin in,491 132,132f
serotonin-enhancing agents on, 317318 Spike-timing dependent plasticity (STDP),125
serotonin in,305 Spike-timing dependent plasticity (STDP)rules
serotonin-inhibiting agents on,317 competition between synapses in,126
sleep-wake transition in,318 in Hebbian plasticity,126
on stress and behavior, 320321,320f heterogeneity in parameters of,126
sufficient,314 heterosynaptic plasticityon
synaptic scaling in, 116117 competition in, 134135
Sleep architecture, neurobiology and, 315318,315f runaway dynamics prevention and, 134,135f
definition of,315 in stable neuron operation, 134,136f
NREMS, 315f,316 learning,125
NREMS-REMS cycles in, 315316,315f neuron stabilization from,126
REMS, 315f, 316317 regular pairing protocols on,133
wakefulness, 317318 timescale in,128
Sleep disorders Spinocerebellar ataxia type 1 (SCA1),384
with autism,589 Spiny projection neurons (SPNs)
with major depressive disorder, 321322 dopaminergic,488
melatonin and,278 GABAergic, 489490
in Parkinsons disease, 482, 487, 489491 Spontaneous recurrent seizures (SRS), in temporal lobe
in post-traumatic stress disorder,321 epilepsy,209
Sleep hygiene, 324325 Stability, change and,108
Sleep, insufficient Stem cell related transplant,374
adenosine signaling in,3738 Stem cells, adult neural, 202208. See also Neural stem/
astrocytes and,8283 progenitor cells(NSCs)
astrocytes, depression, and,8384 Stimulant addiction, 509527. See also Addiction
on behavior,321 Stimulants. See also specifictypes
on brain,314 GDNF and,522
on glucocorticoids, 320321 on sleep, 317318
on glutathione,323 for traumatic brain injury,430
homeostatically controlled mechanismsin,35 use of, by healthy people,238
on melatonin,323 STITCH (Search Tool for Interactions of
on stress response, 320321 Chemicals),241
Sleep pressure St. Johns wort,526
gliotransmission in accumulation of, 8283,83f Strategy training, for traumatic brain injury, 433434
inflammationon,84 Stress-activated protein kinase signaling cascade,378
Sleep therapy,322 Stress, prenatal, on neurodevelopmental and psychiat-
for cognition,219 ric disorders,193
Sleep-wake transition,318 Stress response
Slits,373 meditation on,353
Slow inward currents (SICs) sleep on, 320,320f
from astrocyte glutamate release,78 sleep on, insufficient, 320321,320f
in epilepsy,86 Striatum,489
Slow outward currents (SOCs), from astrocyte GABA Stroke,118
release,79 on ATP production,101
Small guanosine triphosphatases,379 disability from,214
SNCA (PARK1/PARK4) Parkinsons disease, 482483 epidemiology of,214
Social anxiety disorder (SAD), amygdala in,537 GSK3-signalingon,60
Sodium, intracellular ([Na+]i), in astrocyte homeostasis nerve growth factor on,376
regulation,18 neural stem/progenitor cells for brain repair after,
Sodium-potassium-chloride cotransporter (NKCC),6 215217
on chloride channels, 52,53f reactive astrocytosisin,47
phosphorylationon,59 synaptic scaling in,118
Soluble N-ethylmaleimide-sensitive fusion protein Structural plasticity,182
receptor (SNARE) complex, in astrocytes, Subarachnoid hemorrhage, blood-brain barrier dys-
77,77f function in,149
Solute carriers (SLC),146 Substance abuse. See also specific substances
 Index 639

addiction in, 509527 (See also Addiction) weight dependence of changes in,126
anxiety disorders with,545 Synaptic plasticityrelated intrinsic plasticity,5556
classification of,509 Synaptic scaling, 54, 108118, 109, 128130, 175,
Substance P, on adenosine concentrations,342 179180,179f
Substantia nigra,299 in Alzheimers disease,118
acetoacetate and -hydroxybutyrate on,254 canonical, 110,111f
BDNF in,376 as cell autonomous, 111112
dopamine from,511 computational ideas on, 115117
dopamine neurons in, 262, 317,536 in control theory, 113115,114f
neurogenesis in, 202203 definition of, 109, 128129
in Parkinsons disease, 262, 381, 481, 484, 490,492 in development and learning, 108109
on sleep architecture,315f excitatory, 109112
in traumatic brain injury,427 in interneurons,112
Succinyl-CoA,596 in pyramidal neurons, 109112,111f
Sudden unexpected death in epilepsy (SUDEP), caf- experimental studies on, 128129
feine in,411 as feedback control mechanism, 113114
Suprachiasmatic nucleus,317 firing rate homeostasis in, 115116
Suramin,593 fragile X mental retardation protein in,117
Susceptibility genes, DNA methylation in Alzheimers in fragile X syndrome,117
disease and, 457458 genes in,180
SWItch/ Sucrose Nonfermentable (SWI/SNF) inhibitory, 112113
complex,192 local and rapid forms of,110
Symbiosis,284 MeCP2 in,117
Sympathetic nervous system in memory
in anxiety and stress disorders, 535,537f short- vs. long-term storage of,116
ketone body effects in,254 working,116
meditation on, 353, 355, 357,358 Narp in,180
norepinephrine in, 305, 320,321 in neocortex,129
sleep and,320 neurological/psychiatric disorders and,181
in stress response, 320, 535,537f realism of,130
Synapse in Rett syndrome,117
development on,108 scaling up and down in,112
Hebbs rule on,108 in sleep, 116117
neurotransmitter metabolism in, local,45 in stroke,118
Synaptic competition study methods for,109
Hebbian plasticity and, 126,127 synaptic competition in,116
in synaptic scaling,116 timescale, competition, and runaway dynamics in,
Synaptic drive,108 129130
Synaptic plasticity, 124, 175180 timescale in,116
adenosine in,3637 Synaptic tagging, 178,179f
associative,124 inverse, 178179,179f
definition of,175 Synaptic tagging and capture theory,178
epigenetics, learning, and memory in, 193194 System N glutamine transporter(SN1)
epilepsy-related, 6163,62f in glutamine release,46
inverse synaptic tagging in, 178179,179f localizationof,45
long-term depression in, 175, 176f, 177178 (See also Systems biology,232
Long-term depression(LTD)) Systems (network) pharmacology, 231243,333
heterosynaptic,178 ab initio drugs in, 231,237
homosynaptic,178 brain functionality and complexity in, 231232
long-term potentiation in, 175, 176177, 176f (See for brain functionality restoration, 231243
also Long-term potentiation(LTP)) chemoinformatics tools for, 240241
heterosynaptic,178 disease-associated molecular networks in, 240241
homosynaptic,178 on drug action,235
neurological/psychiatric disorders and,181 emergent properties in,233
saturation of,136 epigenetics and geneenvironment interactions in,
synaptic scaling in, 179180, 179f (See also Synaptic 233234,234f
scaling) fundamental tenets of,231
synaptic tagging in, 178,179f integrative neuroscience approaches in,
weight dependence of,136 232235,234f
640 Index
Systems (network) pharmacology (Cont.)
multitargets in, 235237,237f
network dynamics discovery in,241
nootropic drugs in, 231, 237239,240f
outlook for, 242243
practical matters of, for CNS drugs, 240242
PubMed papers on, 235,235f
purpose of,232
reductionism and, 232233
similarity ensemble approach in,241
systems biology in,232
T1-11,593
Tai Chi Chuan Qigong, for traumatic brain injury,433
Tamoxifen
on focal ischemia,15,18
on VRAC channels,15,18
Tardive dyskinesia, branched chain amino acids for,
280281
Target-selective pharmacology,231
drug discovery in, 234235
Tauopathies. See also specific disorders
Alzheimers disease as, 455 (See also
Alzheimers disease)
astrocytes in,455
chronic traumatic encephalopathy as,426
epileptic activity and seizure thresholds in,465
microglia in, 455456
tau acetylation on,459
Tau protein, in Alzheimers disease,454
Tbr2,205
Tele-methylhistamine, in addiction,518
Telomeres, meditation on,357
Temozolomide, for gliobastoma multiforme, 553554
Temporal lobe epilepsy (TLE). See also Epilepsy
astrogliosisin,47
clinical picture of,6263
COX-2 in,165
dentate gyrus granule cellsin,60
etiology of,209
gephyrinin,61
gephyrin splice variantsin,58
gliotransmission in, 8690, 88f89f (See also
Gliotransmission, in epilepsy)
glutamate-glutamine cyclein,47
glutamine synthetase in,403
GlyR RNA splice variantsin,57
GlyR 2- and 3-coding gene transcriptsin,58
granule cell upregulations in,6061
hippocampal sclerosis in, 60, 62f, 85, 87, 209,403
IL-1R/TLR signaling pathway in, 405406
Kir4.1 in,9,403
neural stem/progenitor cells in, 208213
homeostasis and neurogenesis in, 208209
NSC potential and new granule cells in, 211213,
212f213f
proliferation and connectivity in, 209211,210f
pathophysiology of,209
reactive astrocytosis in,8485
spontaneous recurrent seizures in,209
Testosterone
in addiction, 518519,520
on traumatic brain injury, 423,429
Tetanization, heterosynaptic plasticity induction via,
132133,132f
Tetrahydrocannabinol (THC), 513514
for posttraumatic stress disorder,541
Theophylline
on adenosine receptors, 318,411
from caffeine metabolism,461
on epilepsy,411
Theravadan Buddhist meditation, 360. See also
Meditation
3-Hydroxy-3-methylglutaryl CoA (HMG-CoA),
250f,253
Thrombin, on brain, 148,150
Tibetan Dzogchen meditation, 360. See also
Meditation
Tight junctions,144
TLR4 signaling, in epilepsy, 157158,157t
T lymphocytes
in Rasmussens encephalitis,160
in seizure models,161
Traditional Chinese medicine(TCM)
acupuncture in, 341349 (See also Acupuncture)
botanicals in, 334 (See also Botanicals)
combination therapy in,334
on health,334
Transcendental Meditation. See also Meditation
on blood pressure,355
research on, early,354
Transcranial magnetic stimulation, for PTSD,543
Transcriptional silencing, 194,457
Transcytosis
adsorptive-mediated,147
receptor-mediated,147
Transforming growth factor (TGF-B) signaling,
blood-brain barrier disruption on,405
Transient receptor potential (TRP) channel genes, in
cell volume homeostasis,1617
Transient receptor potential (TRP) channel, in
osmotransduction,13f
Traumatic brain injury (TBI), 420429
autoimmunity in, 424425
brain plasticity in,425
branched chain amino acids for,280
cognitive dysfunction in,427
creatine and, 275276
depression in, 427428
DNA hypomethylation in,409
epilepsy in,426
etiology of,422
genetics and epigenetics of,424
headache and migraine in, 428429
history of,371
innate homeostatic heterogeneity in, 423426
ketogenic diets on, 262263
neurodegeneration and atrophy in, 425426
 Index 641

neuroendocrine dysfunction in,429 standard of care on,557

neuroprotectants for, potential, 422423 Tumor necrosis factor (TNF) receptor family,378
neurotrophic factors in,373 p75NTR signaling pathway in, 378379
pathophysiology of, 420423 Tumor necrosis factor (TNF-)
blood-brain barrier and reactive astrocytosis in, in addiction, 517518
47, 421422 in Alzheimers disease,455
contusions and axonal stretch/strain in,420 in inflammatory cascade, 157t,159
excitotoxicity in, 420421 sleep and, 324,324f
inflammation in,421 TWIK,9
integrative summary on,423 2-arachidonoylglycerol (2-AG), 513514
ischemia and oxidative stress in,422
stress response from,423 Ubiquitination, in Parkinsons disease, 486487
Traumatic brain injury (TBI) intervention strategies, Ubiquitin-proteasome system (UPS),486
429436 Uncoupling proteins (UCPs),256
biologicsin
omega-3 fatty acids,435 Valine, 278283. See also Branched chain amino acids
polyphenols, dietary,436 (BCAAs)
progesterone/estradiol,435 Valproate. See also Antiepileptic drugs(AEDs)
vitaminD,435 carnitine and, 273,274
common considerations in, 429430 multimechanistic actions of, 236, 237f,242
omics and rehabilomics in, 429430 targets and mechanisms of action of,407
pharmacotherapies in, 430432 Vanillinoid receptor (TRPV1) activation, on adenosine
anticonvulsants,432 release,342
antidepressants,431 Varenicline, for nicotine addiction,523
anti-inflammatory strategies,432 Vasogenic edema,12,14
cholinergics,431 Ventral tegmental area(VTA)
stimulants,430 in addiction, 509510
physiotherapies in, 432435 dopamine in,511
acupuncture, 434435 pregnenolone sulfate on,520
cognitive rehabilitation and strategy training, Ventromedial hypothalamus(VHM)
433434 in energy balance and glucose homeostasis, 571573
exercise, 432433 on food intake and metabolic rate, 571572
mindfulness meditation,433 in obesity, 573576
TREK (TWIK-related K+ channels),9,14 VIAAT,44
Tricarboxylic acid (TCA)cycle Vigabatrin, for addiction,512
GABA from substrates of, presynaptic,45 Vimentin,204
ketogenic diets on,259 in epilepsy, 47, 85, 87, 88f89f
acetyl-CoA and glutamate in, 252253 Vipassana meditation, 360. See also Meditation
mitochondrial redox changes in, 255,255f VitaminB
via glycolytic restriction/diversion,257 deficiency of, hypomethylation from,458
Tricyclic antidepressants (TCAs) on DNA methylation,458
for anxiety,542 VitaminB12
for traumatic brain injury,431 deficiency of, in Alzheimers disease,458
Triheptanoin functions of,458
for autism,596 Vitamin D, for traumatic brain injury,435
for epilepsy, 259,411 Vitamin D hormone (VDH), for traumatic brain
Trithorax (trxG) system,192 injury,435
TrkB signaling Volume-activated anion channels (VAAC), astrocyte
in epilepsy, 59,405 release of ATP via,77f
glycolytic restriction/diversion on,257 Volume-regulated anion channel (VRAC)
Trk receptors,377 in Cl- conductance,15
Trk signaling pathway, 377378 on regulatory volume decrease, 13f,15
TRPCs,17 tamoxifen on,15,18
Tryptophan VPS35 Parkinsons disease, 483484
in central fatigue,306 VX-765, for epilepsy from inflammation,166
exercise and,306
Tumor-associated macrophages/monocytes (TAM), in Wakefulness, 317318
gliobastoma multiforme,553 in Parkinsons disease, 482, 487, 489, 490, 491,492
glutamate use by,558 Warburg effect, 262, 555556,555f
642 Index

Waste removal, sleep on, 323324 BDNF on,577

Water and ion microdynamics, 319. See also excess ( See Obesity and diabetes)
specificions gut microbiota on,581
astrocytes cells in, 36,5f regulation of, 570571
calcium and sodium dynamics in,1718 sleep on,322
cell volume homeostasis in, 1217 (See also Weight dependence, of plastic changes,126
Cell volume homeostasis microdynamics)
potassium homeostasis in, 612 (See also Yellow mouse,573
Potassium homeostasis microdynamics)
Water channels, in astrocyte volume regulation,1314 Zen meditation, 360. See also Meditation
Water homeostasis, in epilepsy,403 Zonisamide, 314, 412. See also Antiepileptic drugs
Weight,body (AEDs)