Progress in Psychobiologyan D Physiological Psychology: Elsevier

Progress in
PSYCHOBIOLOGYAN D
PHYSIOLOGICAL PSYCHOLOGY
E d i t e d b y S T E V E N J. F L U H A R T Y
Department of Animal Biology
Laboratories of Pharmacology
School of Veterinary Medicine
University of Pennsylvania
Philadelphia, Pennsylvania
H A R V E Y J. G R I L L
Graduate Groups of Psychology and Neuroscience
University of Pennsylvania
Philadelphia, Pennsylvania
Volume 18
ELSEVIER
ACADEMIC
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Contributors
Numbersin parenthesesindicatethe pageson whichthe authors' contributionsbegin.
Alan M. Rosenwasser, Department of Psychology, University of Maine,

Orono, Maine 04469 (1)
Loretta n . Flanagan-Cato, Department of Psychology and Institute
for Neurological Sciences, University of Pennsylvania, Philadelphia,
Pennsylvania 19104 (39)
Timothy J. Bartness, Departments of Biology and of Psychology,
Neurobiology and Behavior Program, and Center for Behavioral Neuro-
science, Georgia State University, Atlanta, Georgia 30303 (69)
Diane E Day, Department of Biology, Neurobiology and Behavior
Program, and Center for Behavioral Neuroscience, Geoergia State
University, Atlanta, Georgia 30303 (GA)
Alan C. Speetor, Department of Psychology, University of Florida,
Gainesville, Florida 32611 (101)
vii
Preface
In Volume 18 of Progress in Psychobiology and Physiological Psychology,

I am pleased to welcome a new editor, my colleague Harvey Grill, to this
series. Harvey replaces Adrian Morrison who has served as Co-Editor since
Volume 12. During that fifteen years, Adrian worked with the late Alan
Epstein and for two volumes with me. Adrian's editorial skills were
considerable as was his broad appreciation of behavioral neuroscience--a
vision that kept his volumes current and exciting. Harvey and I pledge to
continue to emulate the editorial energy and dedication that Adrian, and all
the past editors, have brought to this series.
Harvey Grill is an ideal person to continue the lineage of behavioral
neuroscientists whom have edited this distinguished series since its inception
in 1966. This list includes the co-founders of P4, Eliot Stellar and Jim
Sprague, the addition of Alan Epstein when Eliot retired his editorial pen to
become Provost at Penn, the recruitment of Adrian Morrison when Jim
became an emeritus Professor, and finally, me to replace Alan when he was
tragically killed in 1992. Harvey is widely recognized as a leading investi-
gator on the neurological controls of gustation, eating and body weight
regulation and, in that regard, his research expertise overlaps with both
Alan Epstein and Eliot Stellar. Harvey's work has been instrumental in
challenging the long held view that the neural controls of motivated
behavior reside exclusively in the hypothalamus by demonstrating the
impressive integrative capacity of the brainstem, an important theme that
shares much with Jim Sprague's early work on collicular control of vision
and Adrian Morrison's analysis of medullary-pontine regulation of sleep.
Harvey is past contributor to this series (in Volume 11 with Rent Berridge),
a past recipient of the APA Early Career Development Award and a past
president of the Society for the Study of Ingestive Behavior. I look forward
to working with Harvey and am certain that this series and its readership
will benefit from his vision, creativity and dedication to the field of
behavioral neuroscience.
Steve Fluharty
Volume 18 consists of four original chapters covering a broad range of

contemporary topics in behavioral neuroscience. The first of these chapters
ix
x Preface
is contributed by Alan Rosenwasser who revisits a topic he wrote about in

Volume 13. Circadian control is central to behavioral regulation (e.g.,
activity, energy, thermal) and must therefore feature in the neural mediation
of behavioral systems. Not surprisingly then, circadian neurobiology is
currently one of the most broadly embraced model systems in behavioral
neuroscience. Insights into the multifaceted nature of the circadian
contribution to biological systems come from genetic, cellular, as well as
systems levels of analysis. Alan Rosenwasser skillfully reviews the current
status of this rapidly developing field. He focuses on the mammalian
suprachiasmatic nucleus system emphasizing inputs to the "clock," their
neurochemical phenotype, and outputs from the "clock" to behavioral and
other effector systems. Another virtue of this chapter is its integration of
current data and organizing principles drawn from the analysis of non-
vertebrate species and cellular system.
Lori Flanagan-Cato's essay focuses on the neuroendocrine controls of
female reproductive behavior in the rat. Mating behavior in the female rat
consists of a prominent reflex known as lordosis in which the female exhibits
dorsiflexion of the back thereby facilitating copulation. Although this reflex
is elicited by flank stimulation provided by the mounting male, it will only
occur when estrogen levels are high. Thus, behavioral receptivity is precisely
timed to coincide with ovulation to maximize the chances of successful
pregnancy. The neural circuitry that governs the lordosis reflex has been
studied extensively and has revealed the importance of estrogen-responsive
neurons in the ventromedial nucleus of the hypothalamus (VMH). However,
until very recently, it was not possible to elucidate all of the synaptic
connections between the VMH and the epaxial muscles that subserve this
behavior. Flanagan-Cato first reviews research from her own laboratory
that utilizes pseudo-rabies viral tract tracing to identify pathways from the
VMH through the periaqueductal gray, medullary reticulospinal and
terminating on motor neurons in lumbar ventral horn that innervate the
female flank muscles. She then goes on to describe more recent experiments
suggesting that estrogen may modulate the synaptic strength of this circuit
by controlling dendritic spines on neurons intrinsic to the VMH, as well as
those that project to lordosis relevant brain circuitry. The elucidation of
these estrogen-induced changes within a defined neural circuit emphasizes
why the study of lordosis continues to be one of the best models to
investigate hormones and their effects on behavior.
The last few years have witnessed unprecedented advances in our
understanding of the neurobiological controls of feeding behavior. This
period of rapid discovery was ushered in by the identification of leptin as an
adiposity hormone that acts in the brain to control food intake and energy
expenditure commensurate with fat stores. Since its discovery by Friedman
and colleagues in 1995, progress has been swift in identifying the many
Preface xi
neurochemical systems in brain that are regulated by leptin. Almost all of

this research has focused on the final common path of ingestion, food
consumption during a meal. However, as Tim Bartness points out in his
chapter, the long term regulation of food intake and energy homeostasis is a
much richer landscape involving many adaptive changes in food searching
strategies and storage. Particularly neglected in this regard is the
contribution of hoarding to long-term physiological regulation. Bartness
begins with a comprehensive review of the literature on food hoarding in a
variety of rodent models. He then proceeds to consider the possibility that
some of the neural control systems that act with the consummatory phase of
food ingestion may also act within the context of food hoarding and he
reviews data from his own laboratory that begins to investigate this
important issue. Perhaps most significantly, Bareness' provocative essay
reminds us that the continued focus on elucidation of neural mechanisms
without corresponding detailed analysis of behavior threatens to widen the
gap between cellular and behavioral neuroscience at precisely the time so
many of us are working to close it.
The development of strategies for unraveling the taste sensory code is at
the heart of Alan Spector's contribution. There are dramatic differences in
the taste-guided behavior of omnivores, herbivores, and carnivores based on
evolutionary pressures related to their unique diet history. The neural basis
of taste-guided behaviors has, however, lagged behind the depth of
understanding in the coding of other sensory modalities. In fact, the
nature and operating characteristics of taste receptors was largely unknown
until very recently. Spector and his associates employ a research strategy
that combines psychophysical analysis of taste-guided behavior with
selective gustatory receptive field denervation to investigate the hypothesis
that taste nerves innervate functionally specialized populations of taste
receptors. Spector reviews a fascinating set of findings from his laboratory
and integrates these results with current information on taste receptors, taste
systems neuroscience, neural developments, and recovery of function.
Steve Fluharty
Harvey Grill
Contents of RecentVolumes
Volume 7 Behavioral Modulation of Visual Responses in

Evolution of the Visual System in the the Monkey: Stimulus Selection for Attention
Early Primates and Movement
John Allman Robert H. Wurtz, Michael ]3. Goldberg, and
The Role of Rhythmical Brain Activity in David Lee Robinson
Sensorimotor Integration Brain Pathways for Vocal Learning in Birds:
Barry R. Komisaruk A Review of the First 10 Years
Thermoenergetics and the Evolution of Fernando Nottebohm
Pheromone Communication Neuronal Activity of Cingulate
Delbert D. Thiessen Cortex, Anteroventral Thalamus,
Visceral Involvement in Feeding: There Is and Hippocampal Formation in
More to Regulation Than the Hypothalamus Discriminative Conditioning: Encoding
Donald Novin and Dennis A. Vander-Weele and Extraction of the Significance of
Author Index-Subject Index Conditional Stimuli
Michael Gabriel, Kent Foster, Edward Orona,
Steven E. Saltwick, and Mark Stanton
Volume 8 Neural Mechanisms in Taste Aversion
The Subdivision of Neocortex: A Proposal to Learning
Revise the Traditional View of Sensory, Motor, John H, Ashe and Marvin Nachman
and Association Areas Thirst: The Initiation, Maintenance, and
L ]i Diamond Termination of Drinking
Behavioral and Neurnphysiological Barbara J. Rolls, Roger J. Wood, and
Consequences of Visual Cortex Damage: Edmund T. Rolls
Mechanisms of Recovery The Pineal Gland: A Regulator of Regulators
Peter D. Spear Russel J. Reiter
Brainstem Regulation of Behavior during Author Index-Subject Index
Sleep and Wakefulness
Adrian R. Morrison
Stress, Arousal, and the Pituitary-Adrenal Volume 10
System: A Psychoendocrine Hypothesis Neuronal Plasticity Maintained by the Central
John W. Hennessy and Seymour Levine Norepinephrine System in the
Postprandial Satiety Cat Visual Cortex
Gerald P. Smith and James Gibbs Takuji Kasamatsu
The Ontogeny of Suckling and Ingestive Behavioral Analysis of CNS Pathways
Behaviors and Transmitter Systems Involved in Conduction
Elliott M. Blass, W. G. Hall, and and inhibition of Pain Sensations and Reactions
Martin H. Teicher in Primates
Mother Young Reunions Charles Y. Vierck, Jr., Brian Y. Cooper,
Michael Leon Ore Franzdn, Louis A. Ritz, and
Author Index Subject Index Joel D. Greenspan
The Engram Found? Initial Localization of the
Volume 9 Memory Trace for a Basic Form of Associative
Principles of Organization of Sensory-Perceptual Learning
Systems in Mammals Richard F. Thompson
Michael M. Merzenieh and ,Ion H. Kaas In collaboration with
xiii
xiv Contents of Recent Volumes
David A. McCormick, David G. Lavond, Mechanisms of Brain-Stimulation Reward

Gregory A. Clark, Ronald E. Kettner, and John Yeomans
Michael D. Mauk Author Index-Subject Index
Twenty Years of Classical Conditioning
Research with the Rabbit
L Gormezano, E. James Kehoe, and Volume 14
Beverly S. Marshall Satiety, Specifications, and Stop Rules:
Author Index-Subject Index Feeding as Voluntary Action
Douglas G. Mook
Drinking Elicited by Eating
Volume 11 F. Scott Kraly
Taste Reactivity as a Measure of the Neural Neural Snbstrates of Aggression and Rage
Control of Palatability in the Cat
Harvey J. Grill and Kent C. Berridge Allan Siegel and Martin Brutus
TrigeminaI Orosensation and Ingestive Investigating the Neural Circuitry of Brain
Behavior in the Rat Stimulation Reward
H. Philip Zeigler, Mark F. Jaequin, and James R. Stellar
Maria G. Miller Author Index-Subject Index
The Stomach: A Conception of Its Dynamic
Role in Satiety
Paul R. MeHugh and Timothy 11. Moran Volume 15
Functional Organization of the W-, X-, and Suckling Physiology and Behavior of Rats:
Y-Cell Pathways in the Cat: A Review and An Integrated Theory of Ingestion and
Hypothesis Satiety
S. Murray Sherman Dennis iV. Lorenz
Author Inde~Subject Index Brain Neuronal Unit Discharge in Freely
Moving Animals: Methods and Application
in the Study of Sleep Mechanisms
Volume12
Dennis McGinty and Jerome M. Siegel
Carbohydrates Do Not Always Produce Satiety:
Sleel~Wake States, Sucking, and Nursing
An Explanation of the Appetite- and
Patterns in Young Rats
Hunger-Stimulating Effects of Hexoses
Harry N. Shah" and Myron A. Hofer
Paula J. Geiselman
Taste, Feeding, and Pleasure
How Running Accelerates Growth
Thomas R. Scott
Katarina Tomljenovic Borer
Author Index-Subject Index
Limbic-Motor Integration
Gordon J. Mogenson
Brain Monoaminergic Unit Activity in Volume 16
Behaving Animals Ontogeny of Ingestive Behavior
Barry L. Jacobs Elliott M. Blass
Neurobiology of an Attribute Model of Memory Insulin and the Brain: A Mutual Dependency
R. P. Kesner and B. V. Dimattia Stephen C. Woods
Index Dopamine and Food Reward
Gerard P. Smith
Volume 13 Sensory Mechanisms in the Behavioral
Control of Body Fluid Balance: Thirst
Memories of Mammaries: Adaptations to
and Salt Appetite
Weaning from Milk
Alan Kim Johnson and Robert L. Thunhorst
Paul Rozin and Marcia L. Pelchat
Behavioral and Cellular Analysis of Adrenal
Tachykinins and Body Fluid Regulation
Steriod and Angiotensin Interactions Mediating
G. de Caro, M. Perfumi, and M. Massi
Salt Appetite
Brain Mechanisms of Aggression as Revealed
Steven J. Fluharty and Randall R. Sakai
by Electrical and Chemical Stimulation:
Author Inde~Subject Index
Suggestion of a Central Role for the Midbrain
Pefiaqueductal Grey Region
Richard Bandler Volume 17
Behavioral Neurobiology of Circadian Integrative Gastrointestinal Actions of the
Pacemakers: A Comparative Perspective Brain-Gut Peptide Cholecystokinin in Satiety
Alan M. Rosenwasser Gary ~L Schwartz and Timothy 1t. Moran
Contents of Recent Volumes xv
Fear and Its Neuroendocrine Basis Rita J. Valentino, Andre L. Curtis,

Jay Schulkin Michelle E. Page, Luis A. Pavovich,
Sleep Circuitry, Regulation, and Function: Sandra M. Leehner, and Elisabeth Van Bockstaele
Lessons from c-fos, Leptin, and Timeless A Model for the Control of
Priyattam J. Shiromani Ingestion--20 Years Later
The Locus Coeruleus-Noradrenergic John D. Davis
System as an Integrator of Stress Responses Author Inde~Subject Index
PROGRESS IN PSYCHOBIOLOGY AND PHYSIOLOGICAL PSYCHOLOGY, VOL. 18
Neurobiology of the Mammalian Circadian System:

Oscillators, Pacemakers,and Pathways
Alan M. Rosenwasser
Department of Psychology
University of Maine
Orono, Maine 04469
I. Introduction
The last few years have seen dramatic progress in the elucidation of the
mammalian circadian timing system at the molecular, cellular, neural
systems, and behavioral levels. These advances have led to improved
understanding of the neuroanatomy, neurochemistry and molecular
neurobiology of the circadian pacemaker, as well as the synchronization
(entrainment) of the pacemaker by both photic and nonphotic inputs.
In this chapter, the author reviews how recent research in these areas has
revealed the structure and function of the mammalian circadian timing
system, and emphasizes how advances at disparate analytical levels are
converging on an integrated view of this critical biobehavioral regulatory
system.
II. Conceptual and Methodological Foundations

In its simplest possible configuration, a circadian timing system may be
conceived as comprising three distinct components: a circadian pacemaker,
an input pathway allowing for environmental synchronization (entrain-
ment) of the pacemaker, and an output pathway transmitting the circadian
timing signal to otherwise nonrhythmic effector systems. Of course, real
circadian systems include multiple interacting (coupled) circadian oscilla-
tors, regulated by a complex array of input pathways, and modulating
downstream regulatory systems via a complex array of output pathways.
Further, real circadian systems may also involve feedback mechanisms,
allowing a pacemaker to modulate activity in its own input pathways, or
allowing downstream effector systems - for example, those regulating
behavioral state, hormone secretions or body temperature - to modulate
the pacemaker.
Copyright 2003, Elsevier Inc.

All rights reserved.
0363-0951/03 $35.00
2 Alan M. Rosenwasser
In principle, stimuli that alter the overt expression of a given circadian

rhythm may exert these effects at any level of the circadian system, or even
at the level of downstream effector systems. For example, body temperature
is increased by strenuous motor activity and decreased by rest, and if activity
and rest occur habitually at the same time each day, then such effects may
influence the waveform of the daily body temperature rhythm, even though
the mediating neural pathways may entirely bypass the circadian system.
Indeed, thermoregulatory effects of activity and rest are thought to be
responsible for about half of the normal amplitude of the daily temperature
rhythm, while circadian pacemaker-dependent processes are thought to be
responsible for the other half. While such downstream effects comprise the
primary subject matter of regulatory physiology, circadian biologists
typically refer to such effects as "masking," to emphasize that downstream
effects may obscure one's ability to deduce the behavior of the circadian
pacemaker. While this bias is reflected in the organization of the present
chapter, it should be noted that recent studies have begun to emphasize the
functional significance of masking and its important contribution to daily
activity patterns in nature (Mrosovsky, 1999b).
The most direct and effective approach to the experimental isolation
of pacemaker-mediated effects is through the study of free-running
(i.e., nonentrained) circadian rhythms. Under so-called constant conditions
(generally, either constant darkness or constant light), circadian rhythms
free-run and express the endogenous free-running period of the underlying
circadian pacemaker (Aschoff, 1981). Thus, stimuli that alter the free-
running circadian period of an expressed circadian rhythm must also alter
the period of the pacemaker, either directly, or indirectly, via action on a
pacemaker input pathway. Similarly, since the pacemaker can be assumed to
maintain a more-or-less constant phase relationship to the expressed
rhythms it controls, stimuli that evoke abrupt phase shifts of an expressed
rhythm can also be assumed to shift the phase of the underlying pacemaker
to the same degree. In general, the magnitude and direction of the phase-
shifting response to a given stimulus depends on the circadian phase of
stimulation, and the function characterizing this relationship for any specific
phase-shifting stimulus is referred to as a phase-response curve (PRC).
Analysis of PRCs provides a critical methodological tool for probing the
responsiveness and sensitivity of the circadian pacemaker to various forms
of acute perturbation (Pittendrigh, 1981).
Thus, stimuli that alter the period or phase of an expressed free-running
circadian rhythm are thought to alter the period or phase of the underlying
circadian pacemaker. Generally, tonic stimulation (for example, constant
light or chronic drug treatment) is used to alter free-running period, while
phasic stimulation (for example, a brief light pulse or an acute drug
treatment) is used to evoke phase shifts (Aschoff, 1981; Pittendrigh, 1981).
Neurobiology of the Mammalian Circadian System 3
In either case, these experimental methods usually involve relatively long

exposures to constant conditions, as well as continuous and relatively
noninvasive monitoring of some overt circadian rhythm. Since studies of
behavioral circadian rhythms - for example, of the locomotor activity
rhythm - readily permit such long-term noninvasive monitoring, beha-
vioral studies have been particularly useful in the identification of effective
stimuli.
While behavioral studies of free-running circadian rhythms provide a
convenient and powerful in vivo model for examining the underlying
circadian pacemaker, several researchers have examined the circadian
pacemaker somewhat more directly, using in vitro brain slice preparations.
In such preparations, circadian rhythmicity may be expressed in the
spontaneous neuronal activity (Gillette, 1996; Harrington, 2000; Prosser,
2000), biochemistry (Murakami et al., 1991; Tominaga et al., 1994a)
or genomic processes (Asai et al., 2001; Wilsbacher et al., 2002) within
putative circadian pacemaker tissues. Relative to behavioral studies, in vitro
studies generally allow for relatively limited duration time-series data
collection, and are thus not optimal for analysis of circadian rhythm
parameters requiring long-term measurements, such as free-running
period. On the other hand, in vitro experiments allow for the direct
application of potential chemical phase-shifting stimuli - neurotransmitters
and drugs - to the circadian pacemaker, in isolation from its normal afferent
connectivity. In this chapter, the author presents the results of both in vivo
and in vitro studies of the mammalian circadian pacemaker, including
examples of both convergent and nonconvergent evidence derived from these
two sources.
Ill. Photic Effects on the Mammalian Circadian Pacemaker

The most well studied circadian phase-shifting stimulus is light, and circadian
systems appear to be universally responsive to relatively brief (usually
seconds to minutes) light pulses interrupting otherwise constant darkness
(DD). The photic PRC characterizing the phase-shifting effects of light
pulses has been characterized in diverse species, and while the exact shape
of the function may differ in detail among species, all photic PRCs share
certain common features. Specifically, photic stimulation during late
subjective day or early subjective night (i.e., at around subjective dusk)
results in phase delays (i.e., shifts to later timing); photic stimulation during
late subjective night or early subjective day (i.e., at around subjective dawn)
results in phase advances (i.e., shifts to earlier timing); and photic stimulation
is relatively ineffective through the middle of the subjective day (Pittendrigh,
1981). The Syrian hamster has been one of the most commonly used
mammalian species in behavioral studies of circadian phase shifting, due to
the relatively precise timing of free-running activity rhythms in this species.

In hamsters, the photic PRC exhibits maximal phase delays at around
circadian time (CT) 13-15, and maximal phase advances at around CT 17-19
(Figure 1A) (Daan and Pittendrigh, 1976; Takahashi et al., 1984) (by
convention, CT 12 is defined as the beginning of subjective night, as marked
by the time of activity onset in a nocturnal species such as the hamster).
While brief light pulses evoke abrupt phase shifts of the circadian
pacemaker, free-running circadian period is affected by the intensity of
continuous illumination. Extensive behavioral experiments reveal that
increasing light intensities commonly lengthen free-running period in
nocturnal animals and generally shorten free-running period in diurnal
animals (Aschoff, 1979, 1981) (lengthening of free-running period can be
thought of as an increase in the average phase delay or as a decrease
in the average phase advance, per cycle, relative to objective time, while
shortening of free-running period can be thought of as a decrease in the
A B
2 - LightPulse 02~1 Triazo/am
N,-..
..:
03 0
7\
-1
(o
~_.-2 , I , I , , I , I ,
-2
, r , t ~ , I ~ I ,
4 8 12 16 20 24 0 4 8 12 16 20 24
C D
2i ~ Novelty
4 Dark Pulse
J~ 1
03 0
2
0
-2
-4
, I , I , , I , I , , , , , I I ,
4 8 12 16 20 24 0 4 8 12 16 20 24
Circadian Time (h) Circadian Time (h)
FIGURE 1 Selected phase-response curves (PRCs) obtained from free-running Syrian

hamsters maintained in running-wheel cages under either continuous darkness (panels A, B,
and C) or continuous light (panel D). A: 15-rain light pulses (Daan and Pittendrigh, 1976);
B: peripheral triazolam injections (Turek and Losee-Olsen, 1986); C: novelty-induced activity
(2-hr exposure to novel environment containing running wheel) (Mrosovsky et al., 1992);
D: 6-hr dark pulses (Boulos and Rusak, 1982b). Original published data (square symbols) have
been superimposed with fitted nth-order polynomial functions by the present author. Circadian
time 12 marks the beginning of the subjective night, as indicated by the onset of locomotor
activity in the nocturnal hamster.
average per-cycle delay or an increase in the average per-cycle advance).

While these generalizations are qualified by numerous exceptions, especially
among insects and diurnal primates, period lengthening as a function of
increasing illuminance has been observed rather consistently among the
nocturnal mammals, including the rodents, which have provided the most
substantial database on mammalian circadian rhythms (Pittendrigh and
Daan, 1976; Aschoff, 1981).
IV. Nonphotic Effects on the M a m m a l i a n Circadian P a c e m a k e r

The mammalian circadian pacemaker is also responsive to a variety of
nonphotic stimuli. For example, a number of behaviorally arousing stimuli -
including social interactions, cage cleaning, and exposure to novel
environments - can induce circadian phase shifting (Mrosovsky et al.,
1989; Mrosovsky, 1996). In contrast to photic phase shifting, the PRCs for
these stimuli are characterized by maximal phase advances during
midsubjective day, and by relatively smaller phase delays during late
subjective night. For example, in Syrian hamsters and mice, robust phase
advance shifts are seen in response to several hours of exposure to a novel
environment equipped with a running wheel during the midsubjective day
(Figure 1C) (Reebs and Mrosovsky, 1989; Wickland and Turek 1991;
Mrosovsky et aI., 1992; Challet, Takahashi, and Turek, 2000). Further, only
those animals showing substantial locomotor activity in the novel running
wheel show reliable phase shifting, suggesting that novelty-induced phase
shifting may be mediated by one or more physiological correlates of
locomotor activity (Reebs and Mrosovsky 1989; Mrosovsky et al., 1992).
More recently, however, it has been demonstrated that similar phase shifting
may be induced by sleep deprivation, even in the absence of substantial
locomotor activity (Antle and Mistlberger, 2000).
The recognition that activity- and sleep-related factors are effective
circadian phase-shifting stimuli has forced researchers examining putative
pharmacological phase-shifting agents to consider the possibility that
apparent drug effects on the circadian pacemaker are mediated by effects
on behavioral state (Mrosovsky, 1997). For example, acute administration of
the benzodiazepine, triazolam, yields a PRC that is essentially identical to the
PRC for novelty-induced activity (Figure 1B) (Turek and Losee-Olsen, 1986),
and prevention of locomotor activity for several hours following triazolam
treatment has been reported to block its phase shifting effect in hamsters (van
Reeth and Turek, 1989). On the other hand, other benzodiazepines can
apparently evoke circadian phase shifting in the absence of induced loco-
motor activity in hamsters (Biello and Mrosovsky, 1993; Maywood et al.,
1997), and even triazolam can induce phase shifting without concomitant
behavioral activation in both squirrel monkeys (Mistlberger, Houpt, and
Moore-Ede, 1991) and hamsters (Marchant and Morin, 1999). The effects of
benzodiazepines and other GABAergic agents on circadian pacemaker
regulation will be discussed more completely in a subsequent section.
It has been suggested that the circadian pacemaker's phase-shifting
response to diverse stimuli may be characterized by two distinct PRCs: a
photic PRC, characterized by pacemaker sensitivity primarily during
subjective night, and a nonphotic PRC, characterized by pacemaker
sensitivity primarily during subjective day (Morin, 1991; Smith, Turek, and
Takahashi, 1992; Rosenwasser and Dwyer, 2001). Nevertheless, it is clear
that all nonphotic PRCs are not identical. Thus, the data summarized by
Smith et al. (1992) reveal considerable variation among nonphotic PRCs in
the timing of both the delay-to-advance transition and the advance peak.
Indeed, examination of their summary data suggests the possibility of two
separate subfamilies of nonphotic PRCs, one characterized by maximal
phase-advances at around CT 5-6 (like that seen for novelty-induced
activity), and another with maximal phase-advances at around CT 9-10 (like
that reported for behaviorally-arousing saline injections) (Mead et al., 1992).
This distinction is also supported by in vitro data showing that direct appli-
cation of serotonin receptor agonists to the SCN evokes maximal circadian
phase advances at CT 6, while similar application of neuropeptide Y (NPY),
another neuromodulator involved in circadian pacemaker regulation, evokes
maximal phase advances at CT 10 (Prosser, 1998). Further, other nonphotic
stimuli may phase shift the circadian pacemaker following a pattern that does
not resemble any of these PRCs. For example, in Syrian hamsters, several
hours of physical restraint results in phase delays when administered near
subjective dusk, but does not produce phase advances at any phase (van
Reeth et aI., 1991; Dwyer and Rosenwasser, 2000a). While restraint-induced
phase shifts are by definition nonphotic, and are likely to be mediated in
part via restraint-induced alterations in behavioral state, such phase shifts
are obviously not dependent on evoked locomotor activity.
In addition to evoking circadian phase shifts, stimuli related to behavioral
arousal or activity can also modify the free-running circadian period. Thus,
the continuous availability of running wheels, which obviously provide a
much greater opportunity for physical activity than do standard laboratory
cages, results in shortening of free-running period in rats, mice and hamsters
(Yamada et al., 1988; Edgar, Martin, and Dement, 1991; Mrosovsky, 1999a),
and in rats, individual differences in activity levels are correlated negatively
with free-running period (Yamada et al., 1990). Additional evidence for
activity-dependent feedback effects on the circadian pacemaker include the
following: (1) in mice, bouts of regularly scheduled daily exercise, whether
"voluntary" (i.e., running wheel access) or forced (i.e., treadmill running) can
effectively entrain the otherwise free-running circadian pacemaker (Edgar
and Dement, 1991; Marchant and Mistlberger, 1996); (2) daily bouts of
daytime locomotor activity can modulate the steady-state phase of mice

entrained to light-dark cycles (Mistlberger and Holmes, 2000); and (3)
running wheel access contributes to the long-term stability and coherence of
circadian rhythm expression under both free-running (Lax, Zamora, and
Madrid, 1998) and entrained (Campuzano et al., 1999) conditions in rats.
V. The Strange Case of Dark Pulses

In several mammalian species, the circadian pacemaker is also phase shifted
by pulses of darkness interrupting otherwise constant light (LL) (Ellis,
McKlveen, and Turek, 1982; Boulos and Rusak, 1982a, b; Barbacka-
Surowiak, 2000; Dwyer and Rosenwasser, 2000a; Rosenwasser and Dwyer,
2002). Dark pulse-induced circadian phase shifting was interpreted originally
as reflecting a mirror image of photic phase shifting (Boulos and Rusak,
1982a, b). According to this hypothesis, the phase-shifting effects of dark
pulses are mediated by altered activity within photic entrainment pathways,
and the circadian pacemaker might be expected to show maximal sensitivity
to dark pulses during the same temporal window as to light pulses - that is,
during the subjective night. More recently, however, this effect has been
viewed as an example of nonphotic phase-shifting, mediated by dark-induced
alterations in behavioral state (e.g., Mrosovsky, 1996). According to this
hypothesis, dark pulse-induced phase shifting is mediated by altered activity
within nonphotic entrainment pathways, and the circadian pacemaker should
be maximally sensitive to dark pulses during subjective day, as is the case
for other behavioral state-related phase-shifting stimuli.
The dark pulse PRC is indeed similar to the PRCs for novelty-induced
activity and for triazolam, in that all three PRCs exhibit large phase
advances during midsubjective day (Figure 1D). In addition, dark pulse-
induced phase shifting during midsubjective day can be blocked by the use of
physical restraint to prevent locomotor activity (Reebs, Lavery, and
Mrosovsky, 1989; van Reeth and Turek, 1989), and may be related to
dark-induced daytime wakefulness (Mistlberger, Belcourt, and Antle, 2002).
On the other hand, the novelty-triazolam- and dark pulse-PRCs are clearly
not identical, in that dark pulses evoke reliable phase advances throughout
the first half of the subjective night, opposite to the phase delays evoked
by light pulses at this phase, while novelty and triazolam treatments fail
to evoke phase shifting during early subjective night (Rosenwasser and
Dwyer, 2001). In recent work, we also found that dark pulse-induced
phase shifting during early subjective night is not related to induced activity,
and is not blocked by prevention of activity (Dwyer and Rosenwasser,
2000a; Rosenwasser and Dwyer, 2002). Instead, the magnitude of dark
pulse-induced phase shifting is dependent on background light intensity,
consistent with a photic mediation hypothesis.
Taken together, these observations suggest that the dark pulse PRC may
comprise a complex function, reflecting different phase-shifting mechanisms
at different circadian phases. Specifically, dark pulse-induced phase shift-
ing may be mediated by induced activity during midsubjective day,
when spontaneous activity is typically low, but via a photic mirror-image
mechanism during early subjective night, when spontaneous activity is
typically maximal. In order to confirm this hypothesis, we constructed a
simple quantitative model of the dark-pulse PRC based on the summation
of two distinct component PRCs: a nonphotic (i.e., state-dependent) PRC,
based on the novelty and triazolam PRCs in the published literature, and a
photic mirror-image PRC, based on inversion of the reported photic PRC to
light pulses. We found that this model indeed predicts the unique form of
the dark pulse PRC (Rosenwasser and Dwyer, 2001).
VI. Neurobiology of the Circadian Pacemaker

Following initial reports that lesions of the hypothalamic suprachiasmatic
nucleus (SCN) abolish the expression of behavioral and neuroendocrine
circadian rhythms in the rat (Moore and Eichler, 1972; Stephan and Zucker,
1972a), these ablation findings were rapidly confirmed and extended using a
wide array of experimental approaches, including in vivo and in vitro
electrophysiology, functional metabolic mapping, and fetal tissue transplan-
tation. Together, these studies yielded strong convergent evidence that the
SCN is indeed the site of the primary circadian pacemaker in the mammalian
brain (Klein, Moore, and Reppert, 1991). In part because mathematical
modeling had suggested the possibility that a circadian pacemaker could be
constructed from an ensemble of coupled, high-frequency (i.e., noncircadian)
oscillatory units, it was important to determine whether circadian timing in
the SCN is fundamentally a cellular process, or whether multicellular
network interactions are required to generate a circadian oscillation. Studies
using a variety of in vitro models, including long-term SCN cell or tissue
culture (Tominaga, Inouye, and Okamura, 1994; Mirmiran, Koster-Van
Hoffen, and Bos, 1995), simultaneous recording of multiple single units via
multielectrode plates (Welsh et al., 1995; Herzog et al., 1997; Shirakawa et al.,
2000; Nakamura et al., 2002), and optical monitoring of calcium flux
(Colwell, 2000) or gene expression within individual SCN neurons have now
provided compelling evidence that circadian oscillation is indeed a cell-
autonomous process, expressed within many, and possibly all, individual
SCN neurons. On the other hand, this multitude of cellular circadian
oscillators normally interact to produce coherent pacemaker-like behavior
(Low-Zeddies and Takahashi, 2001), and the mechanisms underlying intra-
SCN oscillator coupling have not been identified completely. Early studies
using tetrodotoxin revealed that individual neuronal oscillators in the SCN
apparently remain synchronized even in the absence of sodium-dependent

action potentials, both in vivo and in vitro (Schwartz, Gross, and Morton,
1987; Shibata and Moore, 1993), and it has been variously suggested that gap
junctions, glial coupling, calcium-dependent action potentials, and/or local
diffusible signals may be responsible for maintaining interoscillator synch-
rony (for reviews see Miche and Colwell, 2001; Shirakawa, Honma, and
Honma, 2001).
In the last several years, analysis of the fundamental circadian oscillatory
mechanism has been extended to the molecular-genetic level. Following the
initial identification of a mammalian homologue of the identified Drosophila
"clock gene," Per, evidence has accumulated that a number of specific genes
and gene products serve similar (though not identical) functions in the core
circadian mechanism of flies, mammals, and possibly all other animals
(Dunlap, 1999; Young, 2000). While certain mammalian clock genes, inclu-
ding the mammalian Per genes, were identified in part via their homology
with Drosophila clock genes, Clock was identified via genomic analysis of an
induced circadian behavioral mutation (Vitaterna et al., 1994). At present,
putative mammalian clock genes include three Per genes (Perl, Per2, Per3),
Tim, Clock, Brnal, CKle, and two plant cryptochrome gene homologs (Cryl
and Cry2), all of which are expressed within SCN neurons.
These genes and their protein products interact to form an autoregulatory
transcription-translation feedback loop that defines the molecular core of the
circadian oscillator (Figure 2) (Dunlap, 1999; Shearman et al., 2000; Young,
2000). Thus, CLOCK and BMAL form protein heterodimers that exert
positive drive on transcription of the Per and Cry genes, while the PER and
CRY proteins form both homo- and heterodimers that negatively regulate
CLOCK and BMAL activity. This feedback loop results in rhythmic
transcription of specific clock genes within the in vivo (Shearman et al., 1997,
2000; Miyamoto and Sancar, 1999; Okano, Sasaki, and Fukada, 2001) and in
vitro SCN (Asai et al., 2001; Wislbacher et al., 2002). In addition, molecular
outputs from the core oscillator result 11 in rhythmic expression of various
clock-controlled genes (CCGs; i.e., genes that are controlled by, but not part
of, the core circadian oscillator loop), which in turn serve as the
basis for rhythmic outputs to myriad other cellular processes (Shearman
et al., 2000). Ultimately, these molecular processes are reflected in circadian
behavioral rhythmicity, as amply documented by analysis of altered
circadian pacemaker function in mice carrying null or loss-of-function
mutations of Per, Clock, and Cry genes (Vitaterna et al., 1994; Thresher et al.,
1998; van der Horst et al., 1999; Shearman et aI., 2000; Albrecht et aI., 2001;
Low-Zeddies and Takahashi, 2001), and in tau-mutant hamsters carrying a
mutation of the C K l e gene (Ralph and Menaker, 1988; Lowrey et al., 2000).
Traditionally, the SCN has been characterized anatomically as compris-
ing distinct ventrolateral and dorsomedial subdivisions. Recently, however,
\
!
f j,"'
( zi j
FIGURE2 Essential elements of the core molecular loop underlying circadian timing at the
cellular level in mammals. The transcription factors CLOCK (Clk) and BMAL1 (B) form
protein heterodimers exerting positive drive on the transcription of several clock genes,
including the Per ("period") genes Per1, Per2, and Per3, and the Cry (cryptochrome) genes
Cry1 and Cry2. The protein products of these genes dimerize in several different combinations,
including PER-CRY (as shown here) and PER-PER pairings. After nuclear translocation,
PER and CRY inhibit the transcriptional effects of CLOCK-BMAL1 through direct protein-
protein interaction, and thus exert negative autoregulation of their own transcription. This
negative feedback results in circadian expression of Per and Cry transcripts. CKIe acts posttrans-
cts posttranslationally to degrade PER and inhibit its nuclear translocation, thus regulating the
period of the rhythm. At the behavioral level, this model predicts (1) the shortening of free-
running period seen in tan-mutant hamsters carrying a mutation of the CKIe gene, (2) the
lengthening of free-running period seen in Clock-mutant mice, and (3) the loss of coherent free-
running rhythms seen in Clock mice and in Per- and Cry-knockout mice (see text for references).
this anatomical scheme has been reconceptualized as comprising distinct

S C N " c o r e " and "shell" subnuclei, a concept that m a y better a c c o m m o d a t e
species differences in the anatomical distribution o f S C N neuropeptides
(Figure 3) (Moore, 1996, 1997; M o o r e and Silver, 1998). While the vast
majority o f S C N neurons contain the inhibitory amino acid neurotrans-
mitter, G A B A , separate core and shell subdivisions are distinguished readily
by peptide phenotype. A great m a n y neuropeptides have been localized
in the SCN, but the core and shell divisions o f this nucleus have been
m o s t c o m m o n l y identified by the concentration o f arginine vasopressin
(AVP)-positive neurons in the S C N shell, and by vasoactive intestinal
peptide (VIP)- and gastrin-releasing peptide (GRP)-positive neurons in the
S C N core (Moore, 1996, 1997). B e y o n d this basic organization, several
species differences have been noted, even a m o n g nocturnal rodents. F o r
example, the hamster S C N contains a very distinct S C N core subnucleus,
consisting o f photoresponsive calbindin-positive cells, that is absent in the
rat ( M o o r e and Silver, 1998).
Hypothalarnus, Thalamus, Basal Forebrain

ons, BF (ACh)
Medulla (NE)
Post Hyp (HA)
IGL (NPY, GABA)
~phe (5HT)
Retina (GLU, SP, PACAP)
FIGURE 3 Core and shell organization of the suprachiasmatic nucleus (SCN). The vast
majority of SCN neurons release the inhibitory amino acid transmiter gamma-aminobutyric
acid (GABA). In the SCN core (light gray), GABA is commonly colocalized with one or more
neuropeptides, including vasoactive intestinal polypeptide (VIP) and gastrin-releasing peptide
(GRP), while neurons of the SCN shell (dark gray) frequently contain GABA colocalized with
arginine vasopression (VP). SCN core neurons project to other core neurons, to SCN shell
neurons, and to extra-SCN targets, most prominently in the diencephalon and basal forebrain;
SCN shell neurons project to other shell neurons, and to extra-SCN targets, but not to SCN
core neurons. This anatomical organization implies that the flow of information within the
SCN is generally from core to shell. Consistent with this suggestion, the three most well-
characterized SCN afferent systems, originating in the retina, the intergeniculate leaflet of the
thalamus (IGL), and the mesencephalic raphe nuclei, converge within the SCN core. Retinal
afferents contain the excitatory amino acid transmitter glutamate (GLU) as well as the
neuropeptides substance P (SP) and pituitary adenyl cyclase activating peptide (PACAP);
raphe afferents contain serotonin (5HT); and IGL afferents contain neuropeptide Y (NPY)
and GABA. Beyond these core afferents, several less well-characterized afferent systems
converge in the SCN shell, including acetylcholine (ACh)-containing projections from the basal
forebrain (BF) and pons, medullary norepinephrine (NE)-containing projections, and histamine
(HA)-containing projections from the posterior hypothalamus (Post Hyp). A number of other
anatomically identified but functionally uncharacterized SCN afferent systems have been
omitted from this figure, and are not discussed in the present chapter.
A l t h o u g h the specific f u n c t i o n s o f these chemically-defined S C N cell

p o p u l a t i o n s are n o t fully k n o w n , a r e a s o n a b l e heuristic is that the S C N core
serves to collect a n d collate p a c e m a k e r inputs, while the shell is p r i m a r i l y
responsible for g e n e r a t i o n of the circadian t i m i n g signal. These suggestions
are consistent with findings that (1) m a j o r S C N efferent systems converge
in the core s u b n u c l e u s (Moore, 1996, 1997); (2) s p o n t a n e o u s circadian
r h y t h m i c i t y in n e u r o n a l activity, n e u r o p e p t i d e release, cFos, a n d Per gene
expression are seen m o r e reliably in the S C N shell t h a n in the core (Inouye,
1996; S u m o v a et al., 1998; Y a n et al., 1999; H a m a d a et al., 2001; N a k a m u r a
et al., 2001), a n d t h a t (3) a d m i n i s t r a t i o n o f S C N core peptides such as V I P
and GRP can mimic both light-induced phase shifting and Per gene
expression in the SCN, in vivo and in vitro (Albers et al., 1991; Piggins, Antle,
and Rusak, 1995; McArthur et al., 2000; Nielsen, Hannibal, and Fahrenkrug,
2002). On the other hand, the view that SCN core and shell functions reflect
circadian entrainment and circadian pacemaking functions, respectively, is
probably too simplistic, since (1) light-evoked responses are seen in SCN shell
as well as in core neurons; (2) certain nonretinal SCN afferent systems
converge within the SCN shell; and (3) in vitro studies have revealed
independent circadian rhythmicity in secretion of both SCN core and SCN
shell peptides, which may free-run with different periods in the same tissue,
implicating separate core and shell oscillators (Shinohara et al., 1995;
Nakamura et al., 2001).
VII. Pacemaker Inputs: The Retinohypothalamic Tract

The circadian pacemaker is normally entrained by environmental light-dark
cycles, and indeed, the original identification of the SCN as a likely locus for
the circadian pacemaker emerged from studies designed to identify retinal
pathways underlying photic entrainment (Chase, Seiden, and Moore, 1969;
Stephan and Zucker, 1972b). Since these early studies revealed that lesions
of the primary visual pathways do not abolish photic entrainment, the
identification of a retinohypothalamic projection terminating in the SCN
(Moore and Lenn, 1972) served to focus attention on the possible
importance of this structure. While the SCN was soon determined to
house the circadian pacemaker itself, rather than an entrainment mechan-
ism, the retinohypothalamic tract (RHT) was eventually shown by selective
ablation to be both necessary and sufficient for photic entrainment of the
pacemaker (Johnson, Moore, and Morin, 1988a). The anatomy of the RHT
has now been characterized extensively in a variety of mammalian species.
The RHT originates from a distinct subset of retinal ganglion cells separate
from those giving rise to the primary visual pathways (Moore, Speh, and
Card, 1995), and terminates mainly in the SCN, as well as more sparsely in
the antero-lateral hypothalamus, subparaventricular zone (sPVZ), and
supraoptic region (Johnson, Morin, and Moore, 1988b; Levine et al., 1991).
In addition, RHT collaterals also project to specific thalamic targets,
including the intergeniculate leaflet (IGL) (Figure 4) (as discussed below, the
IGL is itself a major component of the circadian system).
Remarkably, retinally degenerate strains of mice, in which nearly all
classical photoreceptors (i.e., rods and cones) are lost by early adulthood,
exhibit normal circadian responses to light (Foster et al., 1993). More
recently, similar findings have been reported in genetically engineered mice
with a developmental absence of both rods and cones, demonstrating
conclusively that circadian light entrainment is dependent on a novel, nonrod,
~/JT"
Mediation o -photic input
Circadian outputs
FIGURE4 Overviewof functional neuroanatomical pathways in the mammalian circadian
system. Major SCN afferent systems originating in the retina and raphe nuclei also target the
IGL, which in turn projects to the SCN. Retinal projections to the SCN and IGL mediate
photic input to the circadian system, raphe projections to the SCN and IGL mediate the effects
of certain nonphotic, behavioral state-related signals, and IGL SCN projections are involvedin
mediation of both photic and nonphotic signaling to the SCN pacemaker. As described in the
text, photic and nonphotic pathways generally interact to produce mutually antagonistic effects
on the circadian pacemaker. Thus, photic signals evoke circadian phase shifting during
subjective night and antagonize nonphotic phase shifting during subjective day, while signals
related to arousal and wakefulness evoke phase shifting during subjective day and antagonize
photic phase shifting during subjective night. These antagonistic interactions are mediated in
part at the level of the SCN, but the scheme presented here suggests that the IGL is also a
probable locus for interaction between photic and nonphotic signals - this hypothesis is largely
unexplored.
noncone photoreceptor (Freedman et al., 1999). Indeed, it now appears that

the protein melanopsin, found specifically in the small subset of retinal
ganglion cells giving rise to the R H T , serves as a circadian photoreceptor
molecule within a novel population of photosensitive R H T retinal ganglion
cells (Berson, Dunn, and Takavo, 2002; Hannibal et al., 2002; H a t t a r et al.,
2002). Thus, light entrainment of the circadian pacemaker is mediated by a
dedicated and unique system of photoreceptors, retinal neurons, and central
pathways, entirely distinct from those mediating visual perception.
R H T terminals release the excitatory amino acid neurotransmitter,
glutamate, in response to photic stimulation, and extensive evidence from
both in vivo and in vitro studies indicates that glutamate acts through both
N M D A and n o n - N M D A receptors and a variety of intracellular signaling
molecules (e.g., C A + + , nitric oxide, calmodulin, PKC, P K G , CREB, and
others) (Ding et al., 1994; Gillette, 1996; Mintz et al., 1999) and immediate
early-response genes (IEGs) including c-fos (Kornhauser et al., 1996),
leading to increased expression of Per1 and Per2, and other clock genes
(Shigeyoshi et al., 1997; Moriya et al., 2000). The protein products of these
genes represent state variables of the molecular oscillator, such that
alterations in their transcription levels, when superimposed on the ongoing
circadian transcription cycle, correspond functionally to phase shifts of the
oscillator (Figure 5).
~Light pulse
GLU, SP
PACAP
t~
#.
Arousal
,~NPY
GABA
5HT
Subjective night Subjective day Subjective night Subjective day
FIGURE 5 A simple qualitative-molecular model for circadian phase shifting by photic and
nonphotic signals, and for their mutually antagonistic interaction. In this "phase-only" model,
amplitude is fixed and the underlying state variable (here, Perl transcript level) can oscillate
only within predetermined upper and lower bounds, such that Perl level represents the phase of
the molecular oscillator. During the subjective night, Perl levels are relatively low (solid line),
and light pulses (or corresponding neurotransmitters and/or intracellular messengers) induce an
abrupt increase in transcript level (arrow). Early in the night, when Perl levels are normally
decreasing, this increase in transcription essentially forces the oscillator to repeat part of its
normal trajectory, and is thus equivalent to resetting the oscillator to an earlier phase, resulting
in a permanent phase delay (dashed line). In contrast, late in the night, Perl levels are normally
increasing, such that a light-induced increase in transcription forces the oscillator to omit part
of its normal trajectory, equivalent to resetting the oscillator to a later phase, and resulting in a
permanent phase advance. Opposite to light pulses, arousal-related signals (or corresponding
neurotransmitters and/or intracellular messengers) induce abrupt decreases in Perl transcrip-
tion, resulting in phase-delays during early subjective day and phase advances during late
subjective day. Thus, the model predicts that photic and nonphotic phase-response curves
should have essentially identical shape, but should be phase-displaced by 180 (12 circadian
hours) along the horizontal axis - these predictions are at least roughly consistent with
experimental observations (cf. Rosenwasser and Dwyer, 2001). Further, this model accounts for
the general insensitivity of the circadian pacemaker to photic phase shifting during mid-
subjective day and to nonphotic phase shifting during midsubjective night: since the underlying
state variable can only vary within a predetermined range, stimuli that increase Perl
transcription are ineffective when transcript levels are already maximal, and stimuli that
decrease Perl transcription are ineffective when transcript levels are already minimal.
Nevertheless, despite these periods of insensitivity, nonphotic signals would remain capable
of counteracting light-evoked increases in transcription, and photic signals would remain
capable of counteracting arousal-evoked decreases in transcription. Finally, it should be
mentioned that the exact waveform and phasing of the photic and nonphotic PRCs would
obviously depend on the exact waveform and phasing of the underlying spontaneous
transcription cycle, here presented arbitrarily as two interlocking circular arcs centered over
midsubjective day and midsubjective night.
In addition to glutamate, RHT terminals also release two identified

peptide cotransmitters, substance P (SP) and pituitary adenyl cyclase
activating peptide (PACAP). SP appears to play an important role in RHT
transmission, since selective SP antagonists block light-induced phase
shifting and IEG expression in vivo (Abe et al., 1996; Challet et al., 1998,
2001), as well as glutamate receptor-mediated phase shifting in vitro (Kim
et al., 2001). By itself, SP can mimic at least one component of the photic
PRC (phase delays during early subjective night) both in vivo and in vitro
(Piggins and Rusak, 1997; Hamada et al., 1999). At least in vitro, the phase
shifting effects of SP appear to depend on SP-evoked GLU release, and can
be blocked by the NMDA antagonist, MK-801 (Hamada et al., 1999). In
contrast, PACAP administration has been reported to either antagonize or
mimic the effects of glutamate on circadian phase shifting and Per gene
expression in vitro, depending on dose and on circadian phase (Hannibal
et al., 1997, 2001; Chen et al., 1999; Harrington et aI., 1999; Nielsen et al.,
2001). Specifically, when administered at relatively high doses, PACAP
blocks the effects of glutamate during subjective night and evokes phase
advances during subjective day, but when administered at much lower
doses, PACAP actually mimics or potentiates the effects of glutamate on the
SCN pacemaker.
VIII. Pacemaker Inputs: The Geniculohypothalamic Tract

An additional major SCN afferent system arises from the intergeniculate
leaflet (IGL), a distinct retinorecipient region of the lateral geniculate
complex, intercalated between the dorsal and ventral LGN (Moore and
Card, 1994; Morin, 1994; Harrington, 1997). The projection from the IGL
to the SCN is referred to as the geniculohypothalamic tract (GHT), and
GHT neurons release both neuropeptide Y (NPY) and GABA (Figure 4).
Retinal signals are conveyed to the IGL in part by axon collaterals of RHT
neurons (Pickard, 1985), and GHT and RHT terminal fields are largely
coextensive within the SCN core (Moore, 1996, 1997). It is thus not
surprising that early functional studies emphasized the possible role of the
IGL/GHT system in providing a secondary, indirect pathway for photic
entrainment of the circadian pacemaker. While the IGL/GHT system is
neither necessary nor sufficient for photic entrainment, IGL lesions subtly
alter the circadian phase-shifting effects of light pulses, and modify the
effects of illuminance level on free-running circadian period (Harrington,
and Rusak, 1986, 1988; Pickard, Ralph, and Menaker, 1987; Pickard, 1989,
1994; Edelstein and Amir, 1999). However, IGL lesions can either increase
or decrease photic phase shifting at different PRC phases, and have been
reported to shorten free-running circadian in constant darkness while
lengthening period in constant light (Harrington and Rusak, 1986, 1988;
Pickard, Ralph, and Menaker, 1987). The complexity of the reported

behavioral effects of IGL lesions is also mirrored by the finding that NPY
release in the SCN can be triggered by both light pulses and dark pulses, but
at different phases (Shinohara et al., 1993).
On the other hand, more consistent effects on photic entrainment have
been obtained in studies that directly manipulate GHT-related neurotrans-
mitters in the SCN. Thus intra-SCN administration of NPY inhibits photic
phase shifting in vivo (Weber and Rea, 1997), direct coapplication of NPY to
the SCN can block glutamate-induced phase shifting in vitro (Biello,
Golombek, and Harrinton, 1997), and NPY application to the SCN in vitro
can block the phase-shifting effect of an immediately preceding light pulse
delivered in vivo (Yannielli and Harrington, 2000). Conversely, local
application of an NPY antiserum potentiates photic phase shifting in vivo
(Biello, 1995). NPY-dependent inhibition of photic-RHT-mediated phase
shifting appears to depend on the Y5-type NPY receptor (Yannielli and
Harrington, 2001a, b). The other major GHT transmitter, GABA, appears
to display similar antagonistic interactions with RHT signals, since intra-
SCN administration of GABA-A or GABA-B agonists inhibits phase shift-
ing by light pulses (Gillespie et al., 1997) and by coadministered NMDA
(Mintz et al., 2002). Further, GABA-A agonists evoke circadian phase
shifting in SCN tissue slices in vitro (Tominaga et al., 1994). However, since
many or most SCN neurons contain GABA, it is difficult to separate the
effects of GABAergic SCN afferents from those mediated by GABA release
from intrinsic SCN neurons.
In contrast to the somewhat modest effects of IGL lesions on photic
regulation of the circadian pacemaker, this structure appears to play a
preeminent role in nonphotic regulation of the circadian pacemaker. Thus,
IGL lesions abolish the phase-shifting effects of novelty-induced wheel
running (Janik and Mrosovsky, 1994; Wickland and Turek, 1994) and
benzodiazepine administration in hamsters (Johnson et al., 1988c; Biello,
Harrington, and Mason, 1991; Meyer, Harrington, and Rahmani, 1993;
Maywood et al., 1997; Schuhler et al., 1999), as well as the period-shortening
effect of running-wheel access in rats (Kuroda et al., 1997) and the
entrainment effect of scheduled daily treadmill activity in mice (Marchant,
Watson, and Mistlberger, 1997). Further, novelty-induced activity evokes
IEG expression in NPY-positive IGL neurons, especially at midsubjective
day when the circadian pacemaker is maximally sensitive to such stimuli
(Janik and Mrosovsky, 1992; Janik, Mikkelsen, and Mrosovsky, 1995).
Surprisingly, triazolam injections fail to evoke lEG expression in the IGL
(Zhang et al., 1993a) - or for that matter, in the SCN or raphe nuclei
(Cutrera, Kalsbeek, and Pevet, 1993; Zhang et al., 1993a) - even though
triazolam-induced phase shifting has been commonly attributed to induced
activity. While light-evoked IEG expression is also seen in the IGL, such
expression is not temporally gated, and does not colocalize with NPY (Janik
and Mrosovsky, 1992; Janik, Mikkelsen, and Mrosovsky, 1995). In addition,
circadian phase shifting mimicking the nonphotic PRC is evoked by daytime
electrical stimulation of the IGL (Rusak, Meijer, and Harrington, 1989),
in vivo or in vitro NPY administration (Huhman and Albers, 1994;
Harrington and Schak, 2000), and by intra-SCN administration of direct
and indirect GABA-A agonists (Smith, Inouye, and Turek, 1989). In contrast
to the photoinhibitory effects of NPY discussed above, the daytime phase-
shifting effects of this peptide are mediated by the Y2-type receptor
(Golombek et al., 1996; Huhman et al., 1996; Gribkoff et al., 1998). These
observations indicate that behavioral state-related cues evoke circadian phase
shifting at least in part by stimulating NPY and GABA release from GHT
terminals within the SCN. Finally, the interaction between photic-RHT and
nonphotic-GHT entrainment signals is apparently characterized by recipro-
cal antagonism, since the phase shifting effects of NPY (in vivo: Biello and
Mrosovsky 1995; in vitro: Biello, Golombek, and Harrington, 1997), GABA-
A agonists (in vivo: Joy and Turek, 1992; Mintz et al., 2002), and novelty-
induced activity (Mrosovsky, 1991; Biello and Mrosovsky, 1995) can all be
blocked by a concurrent light pulse or by glutamate coapplication.
The effects of RHT and GHT neutrotransmitters on circadian phase
control are likely to converge and interact at the level of the core molecular
clock mechanism (Figure 5). Per1 and Per2 expression within SCN neurons
is elevated above normally low nighttime levels by light exposure, while Per
gene expression is decreased below normally high daytime levels in
association with both novelty- and triazolam-induced phase shifting
(Maywood et al., 1999; Horikawa et al., 2000; Maywood and Mrosovsky,
2001), and by NPY application in vitro (Fukuhara et al., 2001). Thus,
changes in these putative molecular state variables of the circadian clock
may provide a common mechanism, not only for photic and nonphotic
phase shifting, but also for the reciprocal antagonism between these two
pacemaker input pathways.
IX. Pacemaker Inputs: Role of the Mesencephalic Raphe Nuclei

The third major SCN afferent system converging on the SCN core
originates from the serotonergic midbrain raphe, and specifically, the
median raphe nucleus (Meyer-Bernstein and Morin, 1996; Moga and
Moore, 1997). In addition, ascending serotonergic projections originating
in the dorsal raphe nucleus innervate the IGL, providing a second
potential route for serotonergic regulation of the SCN circadian pace-
maker (Figure 4). Extensive evidence has implicated serotonergic projec-
tions to the SCN (and IGL) in two distinct functions: (1) modulation of
photic effects on the circadian pacemaker during the subjective night,
and (2) mediation of nonphotic, behavioral state-related effects on the

pacemaker during subjective day (Morin, 1999; Mistlberger et al., 2000;
Rea and Pickard, 2000).
Photic phase shifting and light-induced IEG expression in the SCN are
attenuated by (1) electrical stimulation of the median or dorsal raphe
(Meyer-Bernstein and Morin, 1999; Rea and Pickard, 2000), (2) systemic
or intra-SCN administration of the 5HT1A/5HT7 agonist, 8-OH-DPAT
(Glass, Selim, and Rea, 1994; Rea, Glass, and Colwell, 1994; Weber,
Gannon, and Rea, 1998), or (3) intra-SCN administration of 5HT1B
agonists (Rea, Glass, and Colwell, 1994; Pickard et al., 1996; Pickard and
Rea, 1997). In addition, light-induced phase shifting is also inhibited by high
levels of behavioral activity, apparently via arousal-induced serotonin release
at the SCN (Mistlberger and Antle, 1998). Conversely, photic phase shifting
is potentiated by intracerebral or by local intra-SCN administration of the
serotonergic neurotoxin 5,7-DHT (Smale et al., 1990; Morin and Blanchard,
1991; Bradbury, Dement, and Edgar, 1997), as well as by the 5HT1A/5HT7
antagonist NAN-190 (Rea et al., 1995) or the selective 5HT1A autoreceptor
agonist, WAY 100635 (Smart and Biello, 2001). Thus, serotonin appears to
negatively regulate photic effects on the circadian pacemaker via several
mechanisms, including postsynaptic 5HT1A and 5HT7 receptors located on
SCN neurons, somatodendritic 5HT1A autoreceptors located within the
raphe, and 5HT1B receptors located presynaptically on RHT terminals
(Pickard et al., 1999; Gannon, 2001; Smith et al., 2001).
In addition to modulating photic entrainment, serotonergic mechanisms
also directly regulate the circadian pacemaker during subjective day. The
circadian pacemaker can be phase shifted by electrical stimulation of the
midbrain raphe nuclei (Meyer-Bernstein and Morin, 1999; Glass, DiNardo,
and Ehlen, 2000) as well as by in vivo (Tominaga et al., 1992; Edgar et al.,
1993; Cutrera, Saboureau, and Pevet, 1996; Ehlen, Grossman, and Glass,
2001) or in vitro (Prosser, Miller, and Heller, 1990; Shibata et al., 1992;
Prosser, 2000) administration of serotonin and/or 5HT1A/5HT7 receptor
agonists, including 8-OH-DPAT. The PRC for serotonergic stimulation is
characterized most consistently by large phase advances during midsubjec-
tive day, and closely resembles the PRCs for novelty-induced activity, sleep
deprivation, and benzodiazepine and NPY administration, as described
above. The ability of direct 5HT application to the in vitro SCN to evoke
circadian phase shifts indicates that stimulation of intra-SCN 5HT
receptors is sufficient to phase shift the pacemaker. Nevertheless, in vivo
experiments utilizing direct intracerebral 8-OH-DPAT administration have
identified several potential loci within the circadian system for serotonergic
phase shifting, including the SCN, the IGL, and the median and dorsal
raphe nuclei (Mintz et al., 1997; Challet et al., 1998; Ehlen, 2001). Further,
the phase-shifting effect of systemic 8-OH-DPAT can be blocked by
destruction of serotonin terminals within the SCN via local application of

5,7-DHT or by IGL lesions, but not by intra-IGL destruction of serotonin
terminals, suggesting that peripheral 8-OH-DPAT evokes circadian phase
shifts via stimulation of both serotonergic SCN afferents as well as
nonserotonergic IGL afferents, rather than via 5HT receptors in the SCN
itself (Schuhler et al., 1998, 1999). Finally, 8-OH-DPAT-induced phase
shifting during subjective day can be inhibited by photic stimulation in vivo
(Penev, Zee, and Turek, 1997) and by glutamate in vitro (Prosser, 2000),
indicating a reciprocal antagonism between photic-RHT and serotonergic
afferents to the circadian pacemaker. Since 8-OH-DPAT reduces Per1 and
Per2 expression in SCN neurons (Horikawa et al., 2000), it is likely that
these molecular elements underlie the reciprocal antagonism between photic
and serotonergic phase shifting, as suggested above for RHT/GHT
interactions (Figure 5).
Several studies have directly examined the potential role of serotonergic
afferents to the circadian system in mediating the effects of behavioral
state on the circadian pacemaker. Arousal, wakefulness and motor activity
are all associated with increased forebrain serotonin release (Jacobs and
Fornal, 1999), and serotonin content in the rat SCN is correlated positively
with spontaneous activity level and negatively with free-running period
(Shioiri et al., 1991). These observations suggest that increased locomotor
activity may shorten free-running period in part by increasing SCN
serotonin release. Since phase shifting by 8-OH-DPAT is not dependent
on drug-induced activity (Bobrzynska, Godfrey, and Mrosovsky, 1996a), it
appears that serotonergic activation instead mediates the effects of
behavioral state on the circadian pacemaker. Indeed, both locomotor
activity and sleep deprivation increase SCN serotonin release (Grossman
et al., 2000; Mistlberger et al., 2000), and state-dependent serotonin release
appears to partially mediate (1) the effects of activity level on free-running
period in mice (Mistlberger et aI., 1998), (2) phase-shifting by sleep depri-
vation in hamsters (Grossman et al., 2000), (3) entrainment by restricted
daily running wheel access (Edgar, Reid, and Dement, 1997) or scheduled
daily treadmill activity (Marchant, Watson, and Mistlberger, 1997) in
mice, and (4) activity-dependent inhibition of photic phase shifting in
hamsters (Mistlberger and Antle, 1998).
Triazolam-induced circadian phase shifting is also blocked by global
serotonin depletion (Penev, Turek, and Zee, 1995), destruction of intra-SCN
serotonin terminals (Cutrera, Kalsbeek, and Pevet, 1994), or neurotoxic
lesions of the median raphe (Meyer-Bernstein and Morin, 1998). In contrast,
however, neither intra-SCN serotonin lesions, nor any of several 5HT
receptor antagonists, inhibit circadian phase shifting by novelty-induced
activity (Bobrzynska, Vrang, and Mrosovsky, 1996b; Antle et al., t998;
Meyer-Bernstein and Morin, 1998). Along with findings that IEG
expression is stimulated in IGL neurons by novelty-induced but not by

triazolam-induced activity (Zhang et al., 1993a; Janik, Mikkelsen, and
Mrosovsky, 1995), these results indicate that the mechanisms underlying
triazolam- and novelty-induced phase shifting are partially distinct.
Specifically, these two phase shift stimuli exhibit differential dependence
on both serotonergic neurotransmission and IGL integrity, even though
both effects have commonly been attributed to evoked activity.
X. Other Afferent Systems: Acetylcholine,

Norepinephrine, and Histamine
Several other chemically-identified pathways provide afferent input to the
circadian system, including noradrenergic projections from the locus
coeruleus, cholinergic projections from the basal forebrain and pontine
tegmentum, and histaminergic projections from the posterior hypothalamus
(Panula et al., 1989; Bina, Rusak, and Semba, 1993; Moga and Moore,
1997). In addition, noradrenergic and cholinergic projections both innervate
the IGL, providing an alternate pathway by which these transmitter systems
could alter SCN circadian pacemaker function. Unlike the retinal,
geniculate, and raphe projections described above, which form generally
overlapping terminal fields in the SCN core, these afferents target
preferentially the SCN shell (Moore, 1996, 1997) (Figure 3). While less
studied than the SCN core afferents, sufficient data exist to suggest that
these SCN shell afferents also contribute to circadian pacemaker regulation.
Relatively early studies suggested an important role for acetylcholine
in circadian pacemaker regulation. Thus, intracerebroventricular adminis-
tration of the nonspecific cholinergic agonist, carbachol, induces circadian
phase shifts after in rats, mice, and hamsters (Zatz and Herkenham, 1981;
Earnest and Turek, 1985; Mistlberger and Rusak, 1986; Meijer, van der Zee,
and Dietz, 1988; Wee and Turek, 1989), central administration of the
cholinergic antagonist, mecamylamine, blocks light-induced phase shifting
and SCN IEG expression in hamsters (Keefe et al., 1987; Zhang et al.,
1993b), and implantation of a carbachol-secreting pellet near the SCN
shortens free-running period in rats (Furukawa et al., 1987). While the PRC
for carbachol-induced phase shifting was first described as similar to the
photic PRC, surprising variability among subsequent studies complicated
this conclusion. More recently, direct intra-SCN carbachol administration
was shown to mimic both the daytime phase advances that characterize the
nonphotic PRC as well as the early-night phase delays and late-night phase
advances that characterize the photic PRC (Bina and Rusak, 1996).
Identification of the cholinergic receptor subtype mediating these effects
has also been difficult: while the results of early studies favored a nicotinic
mechanism, Bina and Rusak (1996) found that local SCN application of
muscarinic but not nicotinic antagonists blocks carbachol-induced phase

shifting at all effective circadian phases. Further, these issues have not been
resolved by in vitro studies of cholinergic phase shifting: one research team
has reported that nicotinic stimulation of SCN brain slices produces
mecamylamine-sensitive phase advances throughout the subjective day and
night (Trachsel, Heller, and Miller, 1995; O'Hara et al., 1998), while another
has reported that muscarinic agonists produce atropine-sensitive phase
advances, but only during subjective night (Liu and Gillette, 1996; Liu et al.,
1997). Perhaps most surprisingly, both studies failed to detect phase delays
at any phase, in marked contrast to the in vivo data. These results indicate
that fundamental differences between behavioral and in vitro studies of
circadian phase shifting may result from the absence of functional SCN
afferents in the brain slice preparation. Indeed, carbachol-induced phase
shifting in vivo can be blocked by the NMDA receptor antagonist, MK-801
(Colwell, Kaufman, and Menaker, 1993), suggesting that the phase-shifting
effects of cholinergic drugs may be due in part to modulation of RHT
glutamate release, a mechanism that would be absent in in vitro studies.
In rats, chronic administration of the alpha-adrenergic agonist, clonidine,
shortens free-running period in both constant light and constant darkness
(Rosenwasser, 1996), but the magnitude of this effect increases as a function
of increasing illumination, which itself normally lengthens circadian period
(Dwyer and Rosenwasser, 2000b). Similarly, in hamsters, chronic clonidine
administration blunts the phase shifting effects of brief light pulses (Dwyer
and Rosenwasser, 2000b), and acute clonidine treatment evokes circadian
phase shifts according to the photic-type PRC (Rosenwasser, Vogt, and
Pellowski, 1995). Taken together, these results suggest that alpha-adrenergic
mechanisms affect the circadian pacemaker directly, and also modulate
photic input to the pacemaker. While the anatomical loci of these effects are
unknown, chemotoxic lesions of noradrenergic terminals using DSP-4 has
been reported to block the phase-shifting effect of clonidine, suggesting that
presynaptic alpha-2 autoreceptors may be involved (Rosenwasser, Vogt,
and Pellowski, 1995). Indeed, alpha-2 adrenergic receptors have been
identified within several components of the circadian system, including the
SCN, IGL and raphe (Rosin et al., 1996; Talley et al., 1996). In a very recent
study, our lab has found that the effects of clonidine on flee-running period
in rats are not altered by IGL lesions, suggesting that this drug acts either in
the SCN or in other afferent systems to alter circadian pacemaker function
(unpublished data).
Histamine treatment induces circadian phase shifting after intracerebral
administration in vivo (rats: Itowi et al., 1990; hamsters: Harrington, Biello,
and Panula, 2000) and in SCN slices in vitro (hamsters: Cote and
Harrington, 1993); in both preparations, the phase shifting effects of
histamine appear to follow the form of the photic PRC. In addition,
pharmacological inhibition of histamine synthesis reduces the phase shifting

effects of light pulses (Itowi et aI., 1991; Eaton, Cote, and Harrington,
1995), suggesting that histamine release normally participates in photic
entrainment. In contrast, however, antagonists directed at all known
histamine receptors failed to alter photic phase shifting (Eaton et al., 1996).
Instead, in vitro studies suggest that histamine may affect the circadian
pacemaker via direct binding to a nonglutamatergic site on the N M D A
receptor, to exert positive modulation of glutamate-evoked currents (Meyer,
Hall, and Harrington, 1998).
XI. Multiple Oscillators

To this point, the review of current circadian neurobiology presented in this
chapter has treated the SCN as the locus of the circadian pacemaker, but in
fact, the circadian system comprises a multiplicity of circadian oscillators
and possibly circadian pacemakers as well. As reviewed earlier
(Rosenwasser, 1986), circadian systems may exhibit complex dissociations
among multiple rhythmic subcomponents. For example, two or more
discrete daily activity epochs may emerge from the single normally
consolidated activity period, a phenomenon known as "splitting." Such
phenomena at the behavioral level strongly imply the existence of an
underlying multioscillatory neurobiological circadian system. Interest in
these complex phenomena appears to have been deprioritized for several
years, coincident with the ongoing maturation of molecular approaches to
the core pacemaker mechanism. However, in the last few years, molecular
approaches - and especially the finding that mammalian clock genes are
expressed not only in the SCN, but also in a variety of brain regions and in
many peripheral tissues as well - have spurred renewed interest in the
identification and functions of multiple circadian oscillators within the
circadian timing system.
At the neuronal level, the observation that individual SCN cells express
the molecular mechanisms responsible for generating a circadian time signal
demonstrates that the SCN pacemaker is itself composed of numerous,
potentially autonomous but normally coupled, circadian oscillators. Even at
the molecular level, however, the clock genes P e r l , Per2 and Per3 may
exhibit a degree of autonomy and functional specialization (Albrecht et al.,
1997, 2001; Bae et al., 2001; Zheng et al., 2001). According to one
hypothesis, P e r l and Per2 may represent state variables of Pittendigh's
"morning" and "evening" oscillators, respectively (Daan et al., 2001;
Steinlechner et al., 2002). Ultimately, it might be necessary to integrate this
molecular model with other recent findings suggesting that morning and
evening oscillators may be represented by different subpopulations of SCN
neurons (Jagota, de la Iglesia, and Schwartz, 2000; Nakamura et al., 2001).
In addition, it is not known if hypothesized intra-SCN morning and evening

oscillators are related to the separate intra-SCN oscillators driving
independent secretion of core and shell peptide rhythms in certain
preparations.
In addition to multiple oscillators within the SCN, recent evidence
suggests that certain non-SCN neural and neuroendocrine tissues are
capable of expressing at least damped autonomous oscillations. Thus,
cultured mammalian retinae display persisting circadian rhythmicity
in melatonin secretion (Tosini and Menaker, 1996), while more recent
studies have demonstrated self-sustaining oscillations of Per gene expression
in cultured endocrine tissues (pineal, pituitary), diencephalic nuclei
(e.g., hypothalamic arcuate and paraventricular nuclei, thalamic paraven-
tricular nucleus), and in the olfactory bulbs (Abe et al., 2002b). While the
relationships between these extra-SCN neural oscillators and the SCN
pacemaker have not been elucidated, it appears that at least certain types
of rhythm splitting may indeed involve dissociations between intra- and
extra-SCN clocks (Abe et al., 2001, 2002a).
Similar techniques have also been used to reveal rhythmic Per expression
in liver, lung, kidney, and other peripheral tissues (Sakamoto et al., 1998;
Zylka et al., 1998; Yamazaki et al., 2000), and it has recently been suggested
that the long-elusive mechanism of the feeding-entrainable oscillator
(cf. Mistlberger, 1994; Stephan, 2002) is based on a Per-dependent circadian
oscillator in the liver (Stokkan et al., 2001). These observations indicate
that the SCN pacemaker normally serves to entrain both central and
peripheral secondary oscillations generated by a broadly distributed
population of autonomous cellular oscillators, but that under certain
conditions (e.g., restricted food access), these downstream oscillators are
capable of adaptive disengagement from SCN control.
XII. S u m m a r y and Conclusions

The primary pacemaker for the mammalian circadian system is contained
within the SCN, and the mechanisms underlying the pacemaker function of
this structure are rapidly being elucidated at the molecular, cellular, and
neuroanatomic levels. The SCN contains a large number of normally
coupled but potentially autonomous cellular oscillators which generate a
circadian time base via the expression of a complex molecular feedback
loop. The activity of the core molecular loop results in the circadian
expression of a large number of clock-controlled genes, which in turn
regulate coordinated circadian rhythms in the metabolism, electrical
activity, and neurotransmitter and peptide release of SCN neurons. These
rhythmic processes underlie interactions among subpopulations of func-
tionally related SCN neurons, resulting in the emergence of multiple
intra-SCN oscillators, which may correspond with anatomically recognized

SCN subnuclei, or with theoretical entities such as morning and evening
oscillators. In addition, these processes result in the transmission of
circadian timing signals to both passive targets and inherently rhythmic
secondary oscillators throughout the brain and periphery.
The core molecular loop is entrained by a number of convergent SCN
afferent pathways. Photic signals are transmitted from a specialized set of
photoreceptive retinal ganglion cells via a dedicated neural pathway to the
SCN, and activity in this pathway results in the release of glutamate as well
as multiple peptidergic cotransmitters. Glutamate acts through both
NMDA and non-NMDA receptors, which in turn activate a number of
intracellular signaling pathways, resulting in the expression of IEGs
including c-fos, and in increased transcription of specific clock genes,
including P e r l and Per2. These clock genes function as state variables of the
circadian oscillator, such that alterations in the levels of their protein
products are functionally equivalent to oscillator phase shifts.
Other major SCN afferents arise from the IGL and raphe nuclei, which
form terminal fields that largely overlap the RHT terminal field in the SCN
core, and release NPY and GABA, and serotonin, respectively. These
afferents serve to regulate photic signaling in the SCN during subjective
night and to mediate the phase-shifting effects of nonphotic stimuli,
including behavioral activity and arousal, during subjective day. Retinal and
nonretinal afferents interact via both pre- and post-synaptic mechanisms,
and in general, are mutually antagonistic. The mutual antagonism between
photic and nonphotic inputs to the circadian pacemaker is mediated at least
in part by opposing effects of these pathways on Per expression.
Several other SCN afferent systems converge within the SCN shell,
including cholinergic, noradrenergic, and histaminergic projections.
In general, these inputs have been associated with photic but not with
nonphotic entrainment. While the data are somewhat variable, acute
activation of these pathways appears to result in circadian phase shifts that
more-or-less mimic the phase shifting effects of light pulses. At present,
interactions between SCN core afferents and SCN shell afferents are not
well understood at either the behavioral, cellular or molecular levels, and
this would appear to represent a fruitful area for future investigation.
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Hypothalamic Neural Circuitry: ATarget

for the Behavioral Effects of Steroids
Loretta M. Flanagan-Cato
Department of Psychology and Institute for Neurological Sciences
University of Pennsylvania, Philadelphia, Pennsylvania 1910
I. Introduction
Various behaviors and brain functions manifest sexual dimorphisms and
estrous cycle fluctuations, including ingestive behaviors, emotion, and
cognitive functions (Blaustein and Wade, 1976). A well studied example is
female rodent sexual behavior, in which striking effects of both gender and
phase of the estrous cycle are seen. During the hours before and after
ovulation, a female rodent becomes sexually receptive. Males detect this
fertile period via chemosensory cues and attempt to mount a receptive
female. A female responds to the ensuing flank and vaginal-cervical
stimulation by standing rigidly with her back dorsiflexed, thereby
facilitating copulation. This response, termed the lordosis reflex, is gated
by the hormone estrogen, which is released in large amounts by the ovary as
ovulation approaches. Ovariectomy abolishes the lordosis reflex, and this
effect is reversed by estrogen replacement. The lordosis response is not
displayed by intact males or even by adult castrated males treated with
ovarian hormones (Phoenix eta/., 1959). Therefore, this behavior has been
an illuminating model system for revealing the cellular mechanisms
underlying sex-specific, activational effects of estrogen. The brain region
targeted by estrogen to control this behavior is the ventromedial nucleus
of the hypothalamus (VMH). Therefore, a better understanding of VMH
neural circuitry would assist in explaining the neural basis of sexually
dimorphic, hormone-gated behaviors.
The neurological hierarchy that governs the motor component of
the lordosis reflex has been studied extensively (Brink, Morell, and
Pfaff, 1979; Brink and Pfaff, 1980; Kow, Zelman, and Pfaff, 1980;
Schwartz-Giblin, Halpern, and Pfaff, 1984). Briefly, the epaxial muscles
that execute the lordosis posture are innervated by motor neurons in
the lumbar ventral horn. The lordosis response is not a spinal reflex,
but requires medullary reticulospinal, and not corticospinal, projections.
39
Copyright 2003, Elsevier Inc.
0363-0951/03 $35.00
40 Loretta M. Flanagan-Cato
The medullary premotor neurons, in turn, must receive input from the
periaqueductal gray. Finally, for normal lordosis behavior, the periaque-
ductal gray requires innervation from the VMH. Behavioral studies have
shown that the VMH is a key site for estrogen to facilitate this behavior
(Mathews and Edwards, 1977; Pfaff and Sakuma, 1979a, b; Davis et al.,
1982; Pleim et al., 1989).
Although the details of the lordosis-relevant macrocircuitry have been
studied most extensively in laboratory rats, research in other species indicates
that this pathway has been conserved across evolution. In particular, the
importance of the VMH for female sexual behavior has been documented in
other mammals, ranging from guinea pigs (Goy and Phoenix, 1963),
hamsters (Malsbury, Kow, and Pfaff, 1977; Takahashi and Lisk, 1985;
Sterner, Meisel, and Diekman, 1992) and cats (Leedy and Hart, 1985), to
sheep (Blache, Fabre-Nys, and Venier, 1991). Studies in reptiles, such as
whiptail lizards (Rand and Crews, 1994; Kendrick, Rand, and Crews, 1995),
have indicated that this neural mechanism is phylogenetically old.
Furthermore, electrophysiological recordings have supported an active role
of the VMH in female sexual behavior in nonhuman primates (Aou,
Oomura, and Yoshimatsu, 1988). Thus, the mechanisms of the behavioral
effects of estrogen exerted on VMH circuitry in laboratory rats may pertain
to diverse vertebrate species.
II. Neural Connectivity of the V M H

A. AFFERENTS
Brain regions that send projections to the VMH share several features. First,
they all have estrogen receptor-containing neurons (Pfaff and Keiner, 1973;
Stumpf, Sar, and Keefer, 1975; Simerly et al., 1990; Don Carlos, Monroy,
and Morrell, 1991). Second, these forebrain regions all are activated by
female sexual behavior (Pfaus et al., 1993; Polston and Erskine, 1995),
although the hindbrain afferents have not been as well studied in this regard.
And third, the VMH maintains reciprocal projections with most of these
regions. Thus, VMH afferents form a distributed network of estrogen-
responsive neurons that participate in mating behavior.
Anterograde and retrograde studies (Luiten and Room, 1980; Kita
and Oomura, 1982; Fahrbach, Morrell, and Pfaff, 1989) are in general
agreement that the VMH receives dense projections from the ventral
subiculum, medial preoptic area, anterior hypothalamic area, the cortico-
medial amygdala, the peripeduncular nucleus, and the dorsomedial nucleus
of the hypothalamus. Substantial inputs also arrive from the bed nucleus of
the stria terminalis, lateral septum, medial parvocellular paraventricular
Hypothalamic Control of Sexual Behavior 41
nucleus (PVN), ventral premamillary nuclei, and the periaqueductal gray.

The hindbrain provides noradrenergic projections, serotonin fibers from the
pontine and mesencephalic raphe nuclei (Willoughby and Blessing, 1987),
and a cholecystokinin (CCK) projection from the superior lateral
parabrachial nucleus (Inagaki et al., 1984; Zaborszky et al., 1984; Fulwiler
and Saper, 1985; Ingrain et al., 1989). Most of the afferents to the VMH
terminate in the neuropil surrounding the VMH, although fibers from
the parvicellular PVN appear to terminate within the cell-dense core of
the VMH (Yamano et al., 1985).
In some cases, neurotransmitter systems have been identified within
specific afferent pathways. For example, vomeronasal information may be
carried by the significant projections from the medial amygdala and the
preoptic area to the VMH, with gonadotropin releasing hormone (GnRH)
serving as one of the neurotransmitters in the pathway from the preoptic
area (Merchenthaler et al., 1984; Dudley and Moss, 1988; Kow and
Pfaff, 1988; Dudley, Rajendren, and Moss, 1996; Jennes et al., 1997). At
least one source of vaginal-cervical information is the noradrenergic
projection to the neuropil surrounding the VMH (Crowley, Rodriquez-
Sierra, and Komisaruk, 1977; Hansen, Stanfield, and Everitt, 1980,
1981; Thornton et al., 1989; Vathy and Etgen, 1989; Etgen, 1990;
Vathy et al., 1991; Vincent and Etgen, 1993). Peptidergic input from the
PVN includes oxytocinergic and enkephalinergic pathways (Yamano et al.,
1985; Flanagan et al., 1993). Enkephalin immunoreactivity was found
in axons terminating on GABA-containing VMH neurons. Based on
their ultrastructure, the enkephalinergic terminals were presumably inhi-
bitory. Thus, enkephalin may inhibit these GABAergic VMH neurons,
thereby disinhibiting VMH output (Commons et al., 1999). In general, the
electrophysiological actions of various neurotransmitters in VMH correlate
with their effects on sexual behavior (Kow and Pfaff, 1988). In addition
to relaying male-originating sensory cues, afferents to the VMH may
convey environmental cues, such as circadian phase or photoperiod.
In addition, energy status cues may be integrated in the VMH in part
through the molecular antagonism of thyroid receptors and estrogen
receptors (Dellovade et al., 1996).
In summary, for a few of the afferents to the VMH, the neurotransmitter,
behavioral effect, and likely physiological signal have been well character-
ized. However, the functions and chemical mediators of many forebrain
connections, including the major inputs from the lateral septum, bed nucleus
of the stria terminalis, and the anterior hypothalamus are the least
understood. It will be interesting to explore the specific information these
pathways carry, how they might be chemically coded, and where they
synapse within the dendritic arbor of VMH neurons to allow appropriate
weighting and integration.
B. EFFERENTS
As described above, the VMH projection to the PAG has been considered
crucial for the lordosis response. The neurotransmitter phenotype of the
VMH neurons that project to the periaqueductal gray has been explored
with traditional tracers and antibodies to several peptidergic neurotrans-
mitters. Enkephalin, substance P, and prolactin, have emerged as candidates.
Each of these peptides has been localized to neurons in the ventrolateral
VMH (Harlan, Shivers, and Pfaff, 1983b; Yamano et al., 1986; Dornan,
Malsbury, and Penney, 1987; Akesson and Micevych, 1988). In addition,
central application of each of these peptides facilitates the lordosis response
(Harlan, Shivers, and Pfaff, 1983b; Dornan, Malsbury, and Penney, 1987;
Pfaus and Pfaff, 1992). It is not known whether these peptides are colocalized
with each other or with other neurotransmitters. Thus, it remains uncertain
how these peptides may interact to influence sexual behavior.
The PAG has been delineated into subdivisions, based on behavioral,
functional and anatomical studies (Beitz, 1995; Bandler and Keay, 1996).
Four longitudinal columns have been proposed, namely dorsolateral,
dorsomedial, lateral, and ventrolateral, although there are some minor
discrepancies in nomenclature. In addition to the longitudinal columns,
there is a medial zone juxtaposed to the aqueduct. Recent studies have
implicated the lateral/ventrolateral columns as being preferentially involved
in lordosis. First, lesions of the lateral/ventrolateral, but not the dorsal,
column disrupt the lordosis response, but not maternal behavior (Lonstein
and Stern, 1998). Second, anterograde tracing studies indicate that the
ventrolateral portion of the VMH preferentially innervates the caudal
ventrolateral/lateral column of the PAG (Canteras, Simerly, and Swanson,
1994). Third, retrograde transneuronal tracing from lumbar epaxial muscles
involved in lordosis yields the densest labeling in the ventrolateral/lateral
PAG (Daniels, Miselis, and Flanagan-Cato, 1999). Finally, unpublished
observations indicate that sexual behavior preferentially induces Fos
expression in the ventrolateral/lateral PAG (Calizo and Flanagan-Cato).
In addition to the periaqueductal gray, the VMH has numerous other
projection targets (Canteras, Simerly, and Swanson, 1994). Some of these
projections provide access to neural circuitry related to goal-directed
behaviors through direct and indirect projections to the nucleus accumbens
and the subpallidal region. VMH projections also contact emotionally-
relevant learning and memory pathways, including direct projections to the
central and lateral nuclei of the amygdala. VMH projections to the hind-
brain and medial amygdala may modulate sensory input received by the
VMH. The contributions of other VMH projection targets to sexual
behavior have not been well studied. Some of these projections may
contribute to other functions of the VMH (as discussed below).
C. INTRINSIC CIRCUI:FRY OF VMH
As a starting point, the VMH has been subdivided into the dorsomedial,
rostral, central, and ventrolateral zones, and the surrounding shell based on
differences in connectivity, cellular morphology, and chemoarchitecture
(Ramon y Cajal, 1911; Millhouse, 1973; Van Houten and Brawer, 1978;
Fahrbach, Morrell, and Pfaff, 1989; Canteras, Simerly, and Swanson, 1994).
For example, soma size in the ventrolateral and rostral regions is larger than
soma size in the dorsomedial and central regions (Madiera, Ferriera-Silva,
and Paula-Barbosa, 2001). The sparse neurons in the surrounding shell, also
referred to as the fiber plexus, the neuropil, or the lateral rim, are conspicuous
for their enormous size (Van Houten and Brawer, 1978). Analyses of the
subdivision-specific afferents and projection targets also indicate unique
patterns of connectivity for these subdivisions (Canteras, Simerly, and
Swanson, 1994). The dorsal and ventrolateral VMH often project to, and
receive afferents from, different compartments of the same brain regions (Ter
Horst and Luiten, 1987; Fahrbach, Morrell, and Pfaff, 1989; Canteras,
Simerly, and Swanson, 1994). For example, the ventrolateral subdivision
receives selective input from the septal area, ventral premammillary nucleus,
medial preoptic area, medial amygdala, ventral subiculum, and amygdalo-
hippocampal area (Ter Horst and Luiten, 1987; Fahrbach, Morrell, and
Pfaff, 1989). Unlike the ventrolateral subdivision, the dorsomedial region has
only weak projections to the preoptic region, lateral hypothalamus, and
ventral premammillary nucleus, but reliable input to infralimbic, prelimbic,
and anterior cingulate areas (Canteras, Simerly, and Swanson, 1994).
Furthermore, there is a specific flow of information between these
subdivisions, with the ventrolateral subdivision projecting to the rostral
and dorsomedial zones, but not to central VMH (Fahrbach, Morrell, and
Pfaff, 1989). At the same time, the ventrolateral region does not receive
projections from the other VMH subdivisions (Ter Horst and Luiten, 1987).
Thus, the ventrolateral subdivision may serve as a filter, receiving much of the
extranuclear information, then passing it onto the dorsomedial and rostral
subdivisions. The rostral region may be especially involved in lordosis-
relevant projections (Akaishi and Sakuma, 1986; Sakuma and Akaishi, 1987).
There are a host of differences in neurochemical markers between the
subdivisions. For instance, the ventrolateral VMH selectively expresses
estrogen receptor, progesterone receptor, oxytocin receptor, enkephalin,
substance P, and prolactin (Pfaff and Keiner, 1973; Harlan, Shivers, and
Pfaff, 1983b; De Kloet et al., 1986; Yamano et al., 1986; Akesson and
Micevych, 1988; Blaustein et al., 1988; Simerly et al., 1990; Don Carlos,
Monroy, and Morrell, 1991), whereas the dorsal VMH selectively expresses
androgen receptor, CRH-2 receptors, SF-1, and neurotensin fibers
(Lisciotto and Morrell, 1990; Shinoda et al., 1995; Makino et al., 1998).
Another special feature of neurons within the ventrolateral subdivision is

the presence of whorl bodies (Van Houten and Brawer, 1978). Such
subcellular structures are also found in progestin receptor-containing
neurons in the arcuate nucleus (Leranth, Shanabrough, and Naftolin,
1991). Also particular to the ventrolateral subdivision are neurons with
acristic mitochondria (Van Houten and Brawer, 1978), possibly indicative of
neurons detecting energy availability.
Several types of evidence suggest that the ventrolateral VMH is
particularly important for the lordosis reflex. First, the highest densities of
receptors for estrogen, progestin, and oxytocin are found in this region
(Brown et al., 1989; Johnson et al., 1989; Romano, Krust, and Pfaff, 1989),
each of which facilitates sexual behavior. Also, female sexual behavior
selectively induces immediate early gene expression in the ventrolateral
VMH (Flanagan et al., 1993; Pfaus et al., 1993; Tetel, Getzinger, and
Blaustein, 1993; Polston and Erskine, 1995). Transynaptic labeling from the
lordosis-producing muscles is found mainly in the ventrolateral subdivision
of the VMH (Daniels, Miselis, and Flanagan-Cato, 1999). In addition, the
effect of hormone-dependent changes in dendritic spines and synapses are
localized to the ventrolateral, not dorsomedial, VMH (Calizo and
Flanagan-Cato, 2000; Madiera, Ferriera-Silva, and Paula-Barbosa, 2001).
Finally, recent behavioral and anatomical studies have supported a role for
the caudal ventrolateral periaqueductal gray in the lordosis reflex (Lonstein
and Stern, 1998; Daniels, Miselis, and Flanagan-Cato, 1999), which receives
dense terminal fields from the ventrolateral, not dorsomedial, VMH
(Canteras, Simerly, and Swanson, 1994). Collectively, these data suggest
that the ventrolateral VMH, rather than the dorsomedial VMH, plays a
special role in the estrogen-dependent lordosis response.
The synaptic organization of the functional elements within the VMH has
remained enigmatic for several reasons, including the lack of distinguishing
morphological or topographical features of the interneurons and projection
neurons and the lack of a known topographical organization of the axons
arriving into the VMH shell. Investigations of cellular morphology have
provided rudimentary information about the synaptic organization of the
VMH. The neurons within the core have simple dendritic arbors, usually
with two to three dendrites, some of which extend into the VMH shell
(Millhouse, 1979-1981). The shell contains axonal processes from other
brain regions, containing various neurotransmitters, including norepinephr-
ine, serotonin, gonadotropin releasing hormone, and oxytocin (Swanson
and Hartman, 1975; Merchenthaler et al., 1984; Schumacher et al., 1989).
There also are a few neurons found in the shell (Millhouse, 1979 1981).
Transneuronal tracing studies have shed additional light on the synaptic
organization of the VMH (Daniels, Miselis, and Flanagan-Cato, 1999).
Such studies have used pseudorabies virus (PRV), a neurotropic virus that is
transported retrogradely and can be detected immunohistochemically. A

unique advantage of this viral tracer is that it is transported retrogradely
across synapses, thus providing labeling of multisynaptic connections to the
site of tracer injection. One can identify primary, secondary, and tertiary
afferents to a given neural target by examining the pattern of viral labeling
in the nervous system at various times after tracer injection. Because the
virus self-replicates, there is no dilution of the tracer as it is transported
across synapses. This strategy was applied to the lordosis motor pathway by
injecting PRV into the lumbar epaxial muscles. Shortly after tracer injection,
the virus only appeared in the spinal cord, particularly in the lumbar ventral
horn. PRV then was sequentially detected in the medullary reticular
formation, the periaqueductal gray, and the VMH (Daniels, Miselis, and
Flanagan-Cato, 2001).
The first wave of labeling in the VMH defines a portion of the lordosis-
relevant projection neurons, found mostly in the rostral region and
ventrolateral shell (Daniels and Flanagan-Cato, 2000). Only about 3% of
the PRV-labeled neurons expressed nuclear estrogen receptor. Although not
yet tested, it would be expected that after a longer incubation time, the
estrogen receptor containing neurons also would become labeled with PRV.
Thus, the microcircuitry seems to have at least two distinct elements,
lordosis-relevant projection neurons and estrogen receptor-containing
neurons, as illustrated in Figure 1. Additional evidence for two distinct
sets of neurons, one directly responding to estrogen, the other projecting to
the PAG is discussed below. The segregation of the PRV-labeled projection
;in
PVN?)
otordosis-relevant
brain circuitry
,i, spine s ~,,,j~.,.,~,, .~u,u,.
FIGURE 1 A schematic of neuronal elements present within the ventrolateral VMH. A cluster
of estrogen receptor-containing neurons is found at the ventrolateral pole of the V M H .
Projection neurons are found in this area and in the fiber plexus ventrolateral to the VMH.
There are also nonestrogen receptor-containing, nonprojection (undefined) neurons that display
estrogen-induced changes in spine density. It is proposed that the estrogen receptor-containing
neurons innervate the projection and/or the undefined neurons.
neurons from the estrogen receptor-containing neurons suggests that

estrogen does not simply have direct effects on the excitability of the final
common pathway for the lordosis response. But given the electrophysiolo-
gical evidence for estrogen-induced changes in the final common pathway,
estrogen must influence the projection neurons transynaptically. It is
now important to understand how communication from the estrogen
receptor-containing neurons to the projection neurons is configured,
both anatomically and neurochemically. Substance P may be one of
several neurotransmitters that provide such intercellular signaling (Daniels,
Miselis, and Flanagan-Cato, 2003).
Another approach to functionally identify VMH neurons is an
immunocytochemical assay for the induction of immediate early genes by
sexual behavior (Flanagan et al., 1993). Several studies suggest that flank
stimulation caused by a mounting male makes a minor contribution to the
induction of Fos in the VMH by sexual behavior, whereas vaginal cervix
stimulation, (VCS), caused by penile intromissions, accounts for much of
the Fos induction (Pfaus et al., 1993; Polston and Erskine, 1995). These
results are consistent with electrophysiological studies showing that VMH
neurons are responsive to flank and VCS stimulation (Bueno and Pfaff,
1976). Ejaculations may increase Fos levels in the female VMH further
(Coolen, Peters, and Veening, 1996). There has been some attempt to further
identify the type of cells activated by sexual behavior. Approximately 20-
50% of the Fos-labeled neurons in the VMH after VCS were also labeled for
estrogen or progestin receptors (Tetel, Celentano, and Blaustein, 1994;
Auger, Moffatt, and Blaustein, 1996). It is interesting that many of the
neurons activated by sexual behavior do not express estrogen receptor. This
results suggests that nonestrogen receptor containing neurons in the VMH
may be important for sexual behavior.
An important goal is to identify the major neurotransmitters and
modulators expressed by the neuron types of the lordosis relevant micro-
circuitry, especially, the estrogen receptor-containing neurons and the
projection neurons. Based on ultrastructural analysis of synapses,
both excitatory and inhibitory synaptic contacts exist in the VMH, with
approximately half of these being intrinsic to the VMH (Nishizuka and Pfaff,
1989). About one third of the synapses are axospinous and appear exclusively
excitatory. About 10-15 percent of the axodendritic synapses appear to be
inhibitory. Both glutamate and GABA, and their respective receptors, have
been found in the VMH (Gratten and Selmanoff, 1997), but these
transmitters and receptors have not been linked to a particular cell type.
Numerous studies have indicated that GABA activity in the VMH is
regulated by estrogen levels and modulates the lordosis response (reviewed in
(McCarthy, 1995)). Electrophysiological recordings of dissociated VMH
neurons found spontaneous GABAergic inhibitory postsynaptic currents
Hypothalamie Control of Sexual Behavior 47
(Jang et al., 2001). Although labeling for GAD65/67 is often used to identify
GABAergic neurons, GAD mRNA has been difficult to detect in the VMH
by situ hybridization histochemistry (Mirkes and Bethea, 2001). Never-
theless, GABA immunoreactivity has been visualized in VMH neurons using
electron microscropy (Commons et al., 1999). Immunocytochemistry studies
have detected GABAergic neurons within the primate VMH; in fact, all of
the progestin receptor-containing VMH neurons expressed GAD (Leranth,
Shanabrough, and Naftolin, 1991). Behavioral pharmacology studies have
demonstrated that GABA and glutamate participate in the lordosis response.
In particular, acute, local infusion of a glutamate N M D A receptor agonist
inhibits sexual behavior (Kow et al., 1985; McCarthy, Curran, and Feder,
1991). Conversely, a similar infusion of a GABA-A receptor agonist into the
VMH promotes sexual behavior (McCarthy, Malik, and Feder, 1990). In
summary, there is ultrastructural, electrophysiological, histochemical, and
behavioral evidence that both GABA and glutamate are important
neurotransmitters within the VMH. It will be a fundamental advance in
our understanding of the VMH to specify which cell types in the VMH
manufacture these critical neurotransmitters.
In conclusion, the VMH has several interconnected subdivisions, and the
ventrolateral region seems especially critical for the lordosis response. The
estrogen receptor-containing neurons and the PAG-projecting neurons are
largely separate populations, and both are clearly important for sexual
behavior. However, little is known about the specific input each receives or
how the estrogen receptor containing neurons influence the projection
neurons. It is also not yet clear whether these cell types are inhibitory or
excitatory for sexual behavior.
I l l . Sexual Dimorphisms of the V M H

Given the prominent role the VMH plays in a sex-specific behavior it is not
surprising that gender differences are apparent in the adult VMH. Many
such differences are established early in development. The prepattern of the
VMH can be detected at the beginning of the third trimester of gestation,
between embryonic days 13 and 17 in rats. The emergence of this nuclear
condensation depends on the activity of an orphan receptor, SF-1, which
regulates various steroidogenic P450 genes (Shinoda et aL, 1995). Neurons
immunoreactive for estrogen receptor appear as early as embryonic day 13
and migrate to the ventrolateral pole of the preVMH by embryonic day 15
(Tobet et al., 1999). An important trophic factor for defining the boundaries
of neuronal migration to form the VMH may be GABA ( Tobet et al., 1999;
Dellovade et aL, 2001). At embryonic day 22, the female VMH contains twice
the levels of the serine/threonine kinase Raf-1 compared with males (Whorf
and Tobet, 1992). Conversely, on the day of birth, the male VMH contains
twice the number of cells containing phosphorylated CREB compared with

females (Auger, Hexter, and McCarthy, 2001a). Finally, a sex difference in
GABA-A receptor activity emerges shortly after birth, with a more rapid
decay of the GABA-A-mediated current in females compared with males
(Smith et al., 1996). Thus, sexual dimorphisms in VMH biochemistry and
electrophysiology are manifested before or shortly after birth.
At later ages, numerous sex differences have been reported. For instance,
the volume of the adult VMH is approximately 25% larger in males
compared with females (Dorner and Staudt, 1969). This difference in
volume cannot be explained by differences in either total number or size of
neurons, but instead appears to derive from the amount of neuropil
(Madiera, Ferriera-Silva, and Paula-Barbosa, 2001). A gender difference in
the innervation of the ventrolateral VMH from the fornix contributes to
this, with the male VMH receiving about 25 percent more terminals than the
female VMH (Larriva-Sahd, Rondan-Zarate, and Ramirez-Degollado,
1995). In addition to dimorphisms in gross structure, there are also
differences in steroid responsiveness between the male and female VMH. In
particular, both androgen receptor and aromatase activity in the ventro-
lateral VMH are greater in males than in females (Roselli, Horton, and
Resko, 1985; Roselli et al., 1998). Conversely, there are more ERoe, ER~
(Brown et al., 1988; Scott et al., 2000), and progestin receptors (Rainbow,
Parsons, and McEwen, 1982b; Bogic, Gerlach, and McEwen, 1988; Lauber,
Romano, and Pfaff, 1991) in the female VMH.
Sex differences in the synaptic organization of ventrolateral VMH
neurons also have been examined. At the ultrastructural level, males have
a higher density of spine and shaft synapses than females (Matsumoto and
Arai, 1986; Larriva-Sahd, Rondan-Zarate, and Ramirez-Degollado, 1995).
Similarly, a stereological electron microscopy study of shaft and spine
synapses in the VMH across the life span in rats revealed a sex difference at
postnatal five days, which became very robust after puberty at 45 days, with
males having twice as many spine synapses as females (Pozzo Miller and
Aoki, 1991). Furthermore, the synaptic organization within the VMH is
differentially regulated in males versus females. Estrogen decreases dendritic
spine density in adult male rats (Frankfurt and McEwen, 1991a; Lewis,
McEwen, and Frankfurt, 1995), whereas estrogen generally increases spine
density and axospinous synapses in females (Carrer and Aoki, 1982;
Frankfurt et al., 1990; Frankfurt and McEwen, 1991b; Calizo and
Flanagan-Cato, 2000) (but see Calizo and Flanagan-Cato, 2002). Estrogen
appears to be a physiological regulator of spine density in male rats because
estrogen treatment reverses the castration-induced increase in dendritic
spine density in the male VMH (Frankfurt and McEwen, 1991a; Lewis,
McEwen, and Frankfurt, 1995).
There also are sex differences in neurotransmission in the VMH. For

example, the levels of glutamate, GABA, and the synapse-associated protein
GAP-43 in the VMH are higher in males than diestrus females (Shughrue
and Dorsa, 1993; Gratten and Selmanoff, 1997). Such differences in
neurotransmitter activity suggest that dimorphisms in afferent input to the
VMH may maintain differences in VMH behavioral output. This hypothesis
is supported by observations that manipulation of certain neurotransmitter
systems or afferents to the VMH unmasks the lordosis response in male rats
(Yamanouchi and Arai, 1985; Moreines et al., 1988). To the extent that
dendritic spines are induced and maintained by neuronal activity, the
sex difference in spine density in the VMH implies intrinsic differences in
either connectivity, basal synaptic activity, or both.
Neonatal exposure to gonadal hormones is critical for various sex
differences in the adult VMH. In particular, neonatally castrated males have
a female-like VMH, and, conversely, females treated neonatally with
testosterone or estrogen have a male-like VMH. Masculinization of the
VMH by perinatal testicular secretions has been shown for the general
volume (Matsumoto and Arai, 1983), the number of synapses (Pozzo Miller
and Aoki, 1991; Larriva-Sahd, Rondan-Zarate, and Ramirez-Degollado,
1995), and estrogen-induced electrophysiological changes (Sakuma, 1984).
Some of these neonatal effects may be mediated by nerve growth factor
(Yanase et al., 1988).
In summary, fundamental gender differences emerge in the VMH early
in its development, including sexual dimorphisms in synaptic organization.
As summarized in Table 1, the sex differences discovered to date would
TABLE 1
COMPARISON OF MALE AND FEMALE VMH
Parameters that are greater in males

Number of cells containing phosphorylated CREB on day of birth
Total volume
Amount of neuropil
Aromatase activity
Androgen receptor levels
Glutamate levels
GABA levels
GAP-43 levels
Innervation via the fornix
Spines and shaft synapse number
Parameters that are greater in females
Levels of Raf-1 at embryonic day 22
Estrogen receptor levels
Progestin receptor levels
Rate of decay of GABA-A currents
suggest that the male VMH has a more complex synaptic connectivity than
the female VMH. However, it is hard to interpret differences in the number
of synapses and spines without a better understanding of the functional
and chemical properties of presynaptic and postsynaptic neurons. Future
investigations of the components of the local circuitry of the VMH in
both sexes would potentially explain the neurological underpinnings of the
sexual dimorphism of this behavior.
IV. The Effect of Estrous Cycle on V M H Activity

A. CELLULAREFFECTS OF ESTROGEN
The VMH is the key site of ovarian steroid action to promote the lordosis
reflex (Mathews and Edwards, 1977; Pfaff and Sakuma, 1979a, b; Davis
et al., 1982; Pleim et al., 1989). A significant population of estrogen-receptor
containing neurons has been detected in the VMH using receptor
autoradiography, immunocytochemistry, and in situ hybridization histo-
chemistry (Pfaff and Keiner, 1973; Simerly et al., 1990; Don Carlos,
Monroy, and Morrell, 1991). Thus, to ascertain the mechanisms of estrogen
action that control sexual behavior, the cellular effects of estrogen in the
VMH have been investigated extensively.
Early studies inferred that the effects of estrogen were dependent on
changes in gene expression. For example, the behavioral effects of estrogen
were disrupted by drugs that blocked messenger RNA or protein synthesis
(Rainbow et al., 1982a; Yahr and Ulibarri, 1986). In addition, estrogen
treatment markedly up-regulates the cellular machinery for protein
synthesis, including increases in nuclear area and in stacked rough
endoplasmic reticulum (Jones, Pfaff, and McEwen, 1985). Both estrogen
receptors, ERa and ERI~, are members of the superfamily of steroid
receptors which function as ligand-dependent transcription factors (Beato,
1989; Carson-Jurica, Schrader, and O'Malley, 1990). Estrogen treatment is
associated with changes in the expression of several dozen proteins in the
VMH (Jones, McEwen, and Pfaff, 1987). Although the identities of all the
estrogen-regulated proteins have not been completely determined, many
examples are related to neurotransmission, including peptide neurotrans-
mitters, neurotransmitter receptors, and synapse-related proteins. Both
estrogen receptor subtypes are found in the brain, with different, although
somewhat overlapping, distributions (Shughrue, Lane, and Merchenthaler,
1997). Within the VMH, both receptor subtypes are present, but ERa is
more abundant (Shughrue et al., 2002). Like ERa, ERI~ is confined to the
ventrolateral-most pole of the caudal VMH (Shughrue and Merchenthaler,
2001). Female transgenic mice with disrupted expression of the ERa sustain
a major deficit in sexual behavior (Ogawa et al., 1998). However, such
deficits are not apparent in transgenic animals with a null mutation for ERfl
(Ogawa et al., 1999). Thus, ERo~ appears to play a more vital role in this
behavior.
In some cases it is difficult to determine whether cellular effects of estrogen
are secondary to these traditional genomic effects or instead rely on some
nongenomic mechanism. Estrogen receptor has been detected in dendrites
and axons in the VMH (Blaustein et al., 1992), suggesting extranuclear, and
perhaps more rapid, effects of estrogen. Several studies have suggested that
estrogen interacts with signal transduction pathways normally associated
with membrane receptors. For instance, the induction of oxytocin receptors
by estrogen in the VMH is mediated by protein kinase C (Bale et al., 2001).
Also, estrogen treatment increases phosphorylation of DARPP-32 in
ventrolateral VMH (Auger et al., 2001b). It is not clear if these effects are
secondary to genomic events, such as increased transcription of dopamine
receptors, or are due to a more direct interaction with second messenger
pathways. Finally, rapid (three to five minutes) electrophysiological effects of
estrogen have been detected in the VMH (Minami et al., 1990). Together
these data suggest that in addition to the well known transcriptional effects of
estrogen in the VMH, there are parallel and/or sequential effects of estrogen
on various signaling pathways.
B. EFFECTS ON NEURONAL ACTIVITY

Estrogen-induced changes in VMH neural activity have been documented
electrophysiologically with single unit recordings both in an anesthetized
in vivo preparation (Bueno and Pfaff, 1976; Akaishi and Sakuma, 1986;
Sakuma and Akaishi, 1987) and in vitro extracellular recordings in a slice
preparation (Kow and Pfaff, 1988). Estrogen treatment does not alter basal
firing rate, but it increases the excitability of the VMH units. Estrogen
treatment also decreases the antidromic activation threshold and the
absolute refractory period of the projection neurons of the VMH that
descend in the lateral, but not posterior, pathway (Akaishi and Sakuma,
1986; Sakuma and Akaishi, 1987). The selective effect of estrogen on the
laterally projecting neurons is consistent with knife cut studies that showed
that lateral, but not posterior, projecting neurons are critical for lordosis
(Manogue, Kow, and Pfaff, 1980). Although most of these recordings were
obtained after four to ten days of continuous estrogen treatment, a time
course study showed that the electrophysiological effects of estrogen do not
occur until after three days of continuous estrogen exposure
(Tashiro, Kondo, and Sakuma, 1998). These changes in VMH neuronal
firing activity were well correlated with changes in behavior. Furthermore,
blockade of action potentials with the local application of tetrodotoxin to
the VMH prevents estrogen-mediated lordosis behavior (Harlan et al.,
1983a). Thus, it is well established that estrogen increases the excitability of

VMH neurons, including projection neurons. Together with the finding,
discussed above, that excitatory neurotransmitters tend to promote sexual
behavior, these results suggested that excitation in the VMH is critical to
mediate the effect of estrogen on lordosis.
Although many effects of estrogen are not apparent for many hours after
treatment, early estrogen-induced increases in neuronal activation also are
important for the expression of behavior. In particular, the administration of
either a GABA-A receptor agonist anesthetic or a glutamate NMDA receptor
antagonist at the time of estrogen administration blocks the effect of estrogen
on sexual behavior (Roy, Lynn, and Clark, 1985; Fleischmann, Vincent, and
Etgen, 1991). Both of these results suggest that at a time before estrogen is
behaviorally effective, and perhaps before many of the genomic effects have
occurred, some neuronal excitation is present which is obligatory for the
behavior to ultimately occur. In fact, this neural activation appears to
mediate some of the genomic effects of estrogen (Quinones-Jenab et al.,
1996). Such findings hint at a cascade of effects across multiple elements
within the VMH. However, the details remain sketchy about the cascade of
cellular and intercellular events that sets the stage for sexual behavior to
occur, as well as the interrelationships between estrogen, neuronal activation,
and gene expression.
Because of the abundance of estrogen receptor-containing neurons in
the VMH and the efficacy of VMH-targeted infusions of estrogen, it has
been common to interpret various effects of estrogen as being mediated
within the neurons that express estrogen receptors. However, it is important
to consider that various afferents to the VMH also express estrogen
receptors, including input from the hindbrain, preoptic area, and amygdala.
Thus, although local infusions of estrogen are sufficient to promote the
behavior in ovariectomized rats, in intact cycling females transynaptic
influences also may be important. Furthermore, considering the evidence
discussed above for multiple elements within the local VMH network, some
of the local effects also may be transynaptic. As an example, the effects of
estrogen on sexual differentiation and GnRH secretion appear to require
intercellular signaling (Garcia-Segura et al., 1994). Therefore, it will be
important to distinguish direct intracellular effects of estrogen in the
VMH from those that are secondary to changes in local interneuron activity.
Again, a better understanding of the synaptic organization of the VMH
would aid in unraveling these likely combinatorial effects of estrogen.
C, ESTROGEN-INDUCED CHANGESIN SYNAPTIC ORGANIZATION

A driving force for ascertaining the microcircuitry of the ventrolateral VMH
is that estrogen is known to increase the number of synapses in the VMH,
and it is necessary to know the relevant connectivity within the VMH before
such changes can be appreciated. Specifically, electron microscopy studies
showed that estrogen treatment promotes the development of axodendritic
synapses in the VMH, with a trend toward increased axospinous synapses
(Nishizuka and Pfaff, 1989; Frankfurt and McEwen, 1991b). Due to the
constraints of electron microscopic analysis, however, little has been learned
about the phenotype of the neurons undergoing these estrogen-induced
changes, in terms of neurochemistry, morphology, or function within the
lordosis circuit.
Later studies, therefore, turned to Golgi impregnation techniques which
allowed the entire neuronal profile to be visualized. Although synapses
could not be resolved, dendritic spines, small protrusions which form
specialized sites of synaptic contact, could be viewed. These postsynaptic
structures have been proposed to provide connective, electrical, and/or
biochemical functions in neurophysiology. Based on their ability to
compartmentalize calcium, spines may allow for synaptic integration
(Harris and Kater, 1994; Segal, 1995; Yuste and Denk, 1995). In early
Golgi impregnation studies, spine density in the VMH increased two-fold
after treatment with estradiol (Frankfurt et al., 1990). In addition, the
density of dendritic spines in the VMH fluctuated during the estrous cycle,
with an increased density occurring on proestrus compared with diestrus.
A more recent stereological evaluation of Golgi impregnation extended
those initial findings (Madiera, Ferriera-Silva, and Paula-Barbosa, 2001). In
particular, during proestrus, spine density was increased by 30 percent in the
ventrolateral VMH compared with diestrus, with no change in the
dorsomedial VMH.
Despite the advantage of visualizing the entire neuron, various limitations
of the Golgi technique have prevented further revelations about estrogen-
induced neural plasticity. For instance, spine density analysis using camera
lucida is time intensive, and therefore researchers only analyzed an
arbitrarily chosen subset of dendrites. Also, Golgi impregnation is not
compatible with other methods, such as tract tracing or immunocytochem-
istry, which might reveal the projections, receptors, or neurotransmitters
of the impregnated neurons. To circumvent these constraints, individual
VMH neurons were iontophoretically injected with Lucifer yellow and
analyzed morphologically with confocal laser scanning microscopy (Calizo
and Flanagan-Cato, 2000). Unlike Golgi impregnation, this technique
is compatible with concomitant labeling for fluorescent tracers and
immunohistochemically stained proteins, making it possible to identify
functional characteristics of the neurons that exhibit estrogen-induced
changes in spine density. The basic morphological features of VMH cells
filled with Lucifer yellow were very consistent with those described for Golgi
impregnated VMH neurons, in terms of soma size, number of dendrites and
length of dendrites (Flanagan-Cato, Calizo, and Daniels, 2001). These

experiments confirmed and extended previous studies by showing that
estrogen increased spine density in the ventrolateral, not dorsal, VMH.
Two kinds of evidence suggested that the effect of estrogen on spine
density was transynaptic (Calizo and Flanagan-Cato, 2000). First, the
dendrites of the filled neurons could be classified into three mutually
exclusive groups: long primary dendrite, short primary dendrites, and
secondary dendrites. Comparisons of the long versus short primary
dendrites indicated that they differed markedly in both length and
orientation. Such distinguishing features suggest that the different types of
primary dendrites are positioned to receive differential innervation. This
possibility was intriguing in light of the finding that estrogen treatment
induced spines on the short, but not the long, primary dendrites. An
interesting hypothesis arising from these observations would be that
estrogen-sensitive terminals selectively innervate the short primary dendrites
and induced spines there. The identity of the putatively dendrite-selective
afferents remains uncertain. Secondly, subsequent immunostaining for
estrogen receptor indicated that none of the filled neurons expressed
detectable nuclear estrogen receptor. This result makes it doubtful that
spines were induced by a direct genomic action of estrogen, and instead
suggests a transynaptic mechanism. For lack of a better understanding of
their function, these neurons are tentatively referred to as "undefined."
A subsequent study focused on VMH neurons that project to the
periaqueductal gray (Calizo and Flanagan-Cato, 2002). Analysis of the
dendritic arbor indicated that projection neurons had a unique morphology
compared with the undefined VMH neurons. Two populations of projection
neurons were identified: those within the cluster of estrogen receptor-
containing neurons, and those lateral to the cluster. The projection neurons
within the cluster also responded very differently to estrogen, with a
decrease in spines on the long primary dendrites. Finally, the majority
(97%) of the projection neurons did not express estrogen receptors. Taken
together with the previous study, three cell types have been identified:
estrogen receptor-containing neurons, projection neurons, and undefined
neurons. The ventrolateral VMH may encompass several distinct territories,
as there were differential responses in the projection neurons depending on
their proximity to estrogen receptor-containing neurons. As with spine
induction, the mechanism of spine elimination appears to be indirect, as the
projection neurons rarely expressed estrogen receptor. Thus, the estrogen
receptor-containing neurons influence both the undefined and the projection
neurons, but perhaps in different ways, given the opposite effects on spine
density.
In summary, the cell filling technique has allowed us to assemble more
information about the effects of estrogen on the ventrolateral VMH
microcircuitry. Thus far, it has indicated functional differences within the

dendritic arbor of VMH neurons, complex effects of estrogen on the
connectivity of VMH neurons, and at least four classes of VMH neurons.
This information has contributed to a preliminary model of VMH synaptic
organization (Figure 1).
V. Other Putative Functions o f the V M H

It is important to acknowledge that other functions have been attributed to
the VMH. For example, various studies have indicated that the VMH inhibits
maternal nurturing behavior (Sheehan et al., 2000) but may promote
maternal aggression (Hansen, 1989). Electrical stimulation of the VMH
induces aggression against conspecific males (Kruk et al., 1984), although this
effect could be mediated by fibers of passage. An unexplored hypothesis is
that these changes in social behaviors may be secondary to changes in
metabolism. Hunger-induced irritability has been observed in a human case
of VMH lesion (Reeves and Plum, 1969). The VMH also may promote male
sexual behavior (McGinnis, Williams, and Lumia, 1996), which is not
surprising given the abundance of androgen receptor. In addition, the VMH
may influence gonadotropin-releasing hormone neurons in sheep (Anderson
et aI., 2001; Goubillon, Caraty, and Herbison, 2002).
In addition to these social and reproductive functions, many studies have
implicated the VMH in the control of caloric homeostasis, as alluded to
above. This suggestion was initially based on the effects of VMH lesions on
body weight, food intake (Brobeck et al., 1943), and insulin secretion
(Powley, 1977), and the responsiveness of VMH neurons to glucose (Borg
et al., 1995; Borg et al., 1997; Oomura, 1983). Obesity and hyperphagia also
have been noted in a woman with a discreet neoplasm that bilaterally
destroyed the VMH (Reeves and Plum, 1969), and after unilateral surgical
resection of the VMH (Muller, Roeder, and Orthner, 1973). The effects of
VMH lesions on food intake may be secondary to disruptions of vagal
function, particularly enhanced substrate-induced insulin secretion (Powley,
1977), and adjacent brain regions may be involved (Gold, 1973). More
recently, obesity has been observed in transgenic mice with a null mutation
of the SF-1 gene, in which neurogenesis of the VMH is disrupted (Majdic
et al., 2002). This increase in body weight could be explained by not only
increased food intake, but also by markedly decreased spontaneous motor
activity and modestly increased plasma insulin levels. Biochemical markers
also suggest a role for the VMH in calorie regulation. For instance, a specific
glucose transporter isoform, GLUT4, and the ATP-sensitive potassium
channel are expressed in the VMH (Miki et al., 2001). Also, the TrkB
receptor and its ligand brain-derived neurotrophic factor (BDNF) may
regulate food intake in the VMH (Xu et al., 2002). In particular, abnormally
low expression of either the TrkB receptor or BDNF is associated with

obesity and hyperinsulinemia, in the absence of any defect in the leptin
system. Anatomical studies have not detected direct projections from the
VMH to either parasympathetic or sympathetic preganglionic neurons,
although there are projections to visceral sensory centers, such as the
nucleus of the solitary tract and the parabrachial nucleus (Canteras et al.,
1994). Effects of the VMH on metabolism also may be mediated through its
projections to the PAG, which integrates various behaviors with autonomic
responses.
As noted in the beginning of this review, there are sexual dimorphisms
and estrous cycle effects on food intake. Given the abundance of estrogen
receptor in the VMH, as well as the possible role of the VMH in food intake,
it might seem logical to propose that the VMH also mediates the effect of
estrogen on food intake. To date, it has been difficult to pinpoint the locus
of estrogen action with respect to food intake. Although the VMH, PVN
and medial preoptic area each have been implicated (Wade and Zucker,
1970; Butera and Beikirch, 1989; Dagnault and Richard, 1997), there is also
evidence to the contrary for all of these ( King and Cox, 1973; Dagnault and
Richard, 1997; Hrupka, Smith, and Geary, 2002). Furthermore, the
predominant receptor in the PVN is ER/3 (Hrabovszky et al., 1998), and
studies in transgenic animals suggest that the inhibitory effect of estrogen on
food intake is mediated by ERa (Geary et al., 2001). Although the evidence
for the VMH mediating the effect of estrogen on food intake is weak, a
compelling alternative has yet to be found.
As more is discovered about additional functions of the VMH, it becomes
apparent that our current understanding of its topography is far from
complete. Future studies of both VMH structure and function are needed
for a unified account of VMH organization.
VI. Studies of the H u m a n V M H

Limited neuroanatomical studies have been conducted on the human VMH.
As in other species, the pear-shaped human VMH has a higher density of
cells in the distal regions compared with the central zone. Also, there is a
cell-poor zone surrounding the human VMH. The human VMH is
innervated by the central nucleus of the amygdala (Mufson, Benoit, and
Mesulam, 1988), as seen in other species. Various peptides have been
detected in the human VMH including somatostatin, thyrotropin-releasing
hormone, GnRH, substance P, preproenkephalin, and preprodynorphin
(Mai et al., 1986; Mufson, Benoit, and Mesulam, 1988; Stopa et al., 1991;
Fliers et al., 1994; Sukhov et al., 1995). As discussed above, most of these
peptides have been found in the VMH of other animals.
A few studies have supported the notion that, as in other species, the
primate VMH manifests sexual dimorphisms and mediates sexual behavior.
First, estrogen and oxytocin receptors were demonstrated in the human VMH
(Loup et al., 1991; Donahue et al., 2000), indicating that the human VMH
responds to some of the same steroid and peptide signals as the rodent
VMH. Second, a sex difference was found in the staining for Golgi apparatus,
a marker of metabolic activity, as a proportion of cell size in the human
VMH, indicating that this brain region is more active in males versus females
(Ishunina et al., 2001). As in other animals, androgen receptor is found in the
VMH of baboon, rhesus, and cynomolgus monkeys, and humans (Clancy,
Bonsall, and Michael, 1992; Wu, Nathanielsz, and McDonald, 1995), with
higher levels in male VMH versus female VMH in both cynomolgus monkeys
and humans (Michael, Clancy, and Zumpe, 1995; Fernandez-Guasti et al.,
2000). As mentioned above, neuronal activity in the VMH is increased during
sexual activity in female macaque monkeys (Aou, Oomura, and Yoshimatsu,
1988). Finally, a recent functional imaging study in humans has suggested
sex-specific activation of the ventromedial hypothalamic area after exposure
to putative pheromones (Savic et al., 2001). In particular, the male VMH was
selectively activated by estrogenic compounds, whereas the female VMH was
selectively activated by androgenic compounds. These findings suggest that
the human VMH is not only sexually dimorphic, but also that it may process
sexually relevant information.
The VMH has been the target of neurosurgical intervention for "sexual
deviance" (Muller, Roeder, and Orthner, 1973). The so-called sexual
deviants were a diverse group, including cases of pedophilia, hypersexuality,
exhibitionism, and homosexuality. The authors claim that lesions of the
VMH caused changes in both sexual orientation and drive, based on self
report. There have been concerns about the lack of data presented for the
pre- and postsurgical conditions, as well as the scientific and ethical
justifications for this intervention (Swaab, 1997). A slightly more recent
study of unilateral VMH lesions found no effects on sexual orientation,
although sexual drive was diminished (Dieckmann, Schneider-Jonietz, and
Schneider, 1988). In a case study of a neoplasm-induced discrete lesion of
the VMH, sexual behavior was not assessed, probably because the
disruptions of metabolism, food intake, pituitary hormones, and mood
were so profound (Reeves and Plum, 1969). Thus, there is scant evidence,
based on VMH damage, for evaluating the role of the VMH in human
sexual behavior at this time.
VII. Conclusions
In summary, there is a substantial database on the macrocircuitry of the
rodent VMH, including its afferent and efferent connections with the
hindbrain, hypothalamic, and limbic regions. There are well over a dozen
neurochemicals that may act within the VMH to participate in the control
of the lordosis reflex and in some cases such neurochemicals have been
colocalized with specific afferent pathways. The projection from the VMH
to the periaqueductal gray has been considered especially critical for lordo-
tic behavior, and several peptide neurotransmitters have been implicated in
this pathway. In addition to extranuclear afferents and descending
projections, anatomical and behavioral pharmacological evidence suggest
that intranuclear connections are an integral component of the lordosis
circuitry.
Recent results have been used to construct a working model of the
lordosis-relevant microcircuitry of the VMH (Figure 1). At present, this
model is necessarily simple because of uncertainty about various connec-
tions and neurotransmitters. Our ongoing studies are designed to elaborate
on this model to provide a sharper definition of how steroid hormones gate
sexual behaviors by modifying the synaptic organization within the VMH.
A long term goal is to generate a model of the synaptic organization of the
VMH in both the female and male brain to explain the sexual dimorphism
of lordosis behavior. Such models present testable hypotheses to examine in
detail how estrogen engages a behavioral switch at the level of synapses
within a confined neural circuit.
In addition to expanding our knowledge about the role of neural
plasticity in gating a motivated behavior, the VMH provides an alternative
venue for studying the regulation of dendritic spines. Spines have been
vigorously studied in higher brain structures as possible enduring physical
indicators of learning and memory processes. Such studies have revealed
some of the intracellular signaling involved in spine induction. Because spine
density in the VMH fluctuates with the estrous cycle, the VMH presents an
opportunity to study not only physiological induction of dendritic spines,
but also the physiological dismantling of spines.
Finally, the VMH presents an opportunity to better understand hypo-
thalamic functional neuroanatomy. The medial zone of the hypothalamus
has been identified with integrating motivated behaviors, and the VMH in
particular has been implicated in the control of reproductive and
ingestive behaviors. By gaining insight into the microcircuitry that controls
the lordosis response, general principles of hypothalamic networks may
emerge.
Acknowledgment
The author was supported by National Institutes of Heath Grant MH64371.

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Food Hoarding: A Quintessential Anticipatory

Appetitive Behavior
Timothy J. Bartness 1"2 and Diane E. Day t

Departments of Biologj and of Psychology:
Neurobiology and Behavior Program, and
Center for Behavioral Neuroscience
Georgia State University, Atlanta, Georgia 30303
I. Introduction
The study of ingestive behavior has a long and extensive history emerging in
its present form from the fields of animal behavior, learning/psychology,
physiology, and anatomy/neuroscience. One early influence came from
Wallace Craig, an animal behaviorist who in 1918 coined the terms
'appetitive' and 'consummatory' for the two-part sequence of eating,
drinking, and sexual behaviors (Craig, 1918). The first part of this sequence
is appetitive behaviors that are involved in seeking the goal object (food,
water, a mate) and these responses are a flexible and nonstereotyped form of
behavior that brings the animal into physical contact with its goal (Craig,
1918). The second part of this sequence is consummatory behaviors (from
consummate not consume), that are reflexive, stereotyped, and are the final
act once the goal object has been contacted (Craig, 1918).
The consummatory phase of ingestive behavior has received the most
extensive study. That is, food and water intake have been almost exclusively
investigated, most likely because the environment and the behaviors are
relatively easily arranged and measured, respectively. Thus, typically a cage
with a food and water source and gravimetric/volumetric assays are all that
are needed. As for the appetitive phase of ingestive behavior, however, there
is comparatively little known about the search for food or 'foraging,' given
its pervasive nature across animal taxa (for review see Stephens and Krebs,
1986). Perhaps this is because of the difficulty in conducting field studies
of foraging or the problem of creating a laboratory-based analog of this
behavior. In addition, 'food hoarding' - the storage of food for
later ingestion, is another appetitive ingestive behavior with widespread
expression among animals species, but has received even less attention than
foraging (for review see Vander Wall, 1990).
69
Copyright 2003, ElsevierInc.
0363-0951/03 $35.00
70 Timothy J. Bartness and Diane E. Day
Therefore, the primary purpose of this review is to focus on the appetitive

side of ingestive behavior, especially food hoarding, and attempt to integrate
what is known about the mechanisms underlying this behavior - in
particular peripheral energy-related mechanisms and central nervous
systems controls. We will focus on the results from laboratory experiments
of hoarding in small mammals where mechanisms are most easily
determined. We will not cover the interesting work on foraging, hoarding,
and memory in birds that is amenable to laboratory study nor the
laboratory-based studies of optimal foraging strategy.
II. Early Beginnings in the Study of Hoarding in the Laboratory

The curiosity with food hoarding stems from its appetitive nature - food is
acquired today for possible consumption tomorrow. Indeed, the first
laboratory study of food hoarding by Wolfe (1939) was triggered by this
puzzle; laboratory rats (Rattus norvegicus) would store (cache) food beyond
any apparent energetic need even if that required traveling considerable
distances carrying the food. Therefore, he tested whether this behavior was
amenable to 'quantitative investigation' (Wolfe, 1939). At this historical
point, and even today, the importance of hoarding in the behavioral
repertoire of energy-related responses for rats was not known or minimized.
This was most likely because rats do not possess the sine quo non of species
where food hoarding is an important behavioral response for energy
acquisition - specialized anatomical structures for holding substantial
quantities of food while traveling, such as cheek pouches in some rodents
and sublingual pouches or distensible esophagi in birds (for review see
Vander Wall, 1990). Indeed, investigations of wild rats (Rattus norvegieus)
indicate that they do not hoard in nature because no food has been found in
their burrows except for occasional observations of food buried near the
burrow of lactating rats (Pisano and Storer, 1948; Calhoun, 1962; Lore and
Flannelly, 1978; Takahhashi and Lore, 1980; Whishaw and Whishaw, 1996).
At best, wild rats will take food from its source and carry it some distance
away to a semiconcealed or concealed place to eat it and return to the food
source to get more food only after this food is completely eaten (Whishaw
and Whishaw, 1996). Some food is taken home to their burrows, however,
but it is consumed immediately perhaps so that it is not stolen by other rats
(Whishaw and Whishaw, 1996). Thus, the notion that food hoarding of
highly domesticated rat strains in laboratory resembles that of rats in the
wild is questionable at best. Nonetheless, clearly, food-carrying behavior
occurs in both the laboratory and in nature and therefore this response more
likely reflects a common behavior between laboratory rats and their wild
counterparts. Moreover, because of their extensive study in laboratories in
Food Hoarding 71
general, many of the attempts to discover the mechanisms underlying food

hoarding only have been done using laboratory rats. This leaves us with the
choice to ignore these data because this is a 'nonnatural behavior' for this
animal, or to consider it as suggestive of 'natural food hoarders' - we choose
the latter. Therefore, whenever possible, we have focused on food hoarding
studies using wild-trapped species or their offspring in laboratory
experiments. The most commonly studied of these species are Syrian
hamsters (that are quite domesticated themselves (Murphy, 1985)) and
Siberian hamsters (that are more frequently out-bred with wild-trapped
stock (e.g., Day, Mintz, and Bartness, 1999)).
Wolfe's paper (Wolfe, 1939) opened the door to the study of hoarding in
the laboratory because it demonstrated that food hoarding was quantifiable,
but given the times, much of the early work was conducted by traditional
learning theorists and the explanations for the hoarding involved 'instinct'
(Morgan, 1947), 'frustration' (Hunt and Willoughby, 1939), 'goal-lessness'
(Miller, 1945) and 'security' (Bindra, 1948). These arguments and their
empirical support are not especially insightful for our focus on the
mechanisms underlying hoarding here because of the implicit and explicit
anthropomorphism; therefore, these findings and interpretations will not be
discussed.
Morgan et al. (1943) outlined a 'deficit hypothesis' based on their findings,
those of Wolfe (1939) and of Hunt and Willoughby (1939) that continues to
govern thought and guide experiments today. Simply stated, animals hoard
because of a bodily depletion or deficit, such as energy decreases due to
fasting; moreover, as the deficit/depletion continues to grow, it eventually
reaches a threshold triggering food hoarding (Morgan, Stellar, and Johnson
1943). This type of deficit/depletion is not viewed by these and other
researchers as equivalent to the more short-term deficit induced by 'hunger;'
instead, the signal to hoard is generated across a longer time period of days
or weeks in their view (Bindra, 1948). This deficit hypothesis for food
hoarding is not that different from more modern notions about the
physiological signals that trigger food intake, such as those embodied in the
'lipostatic theory' of Kennedy (1953), where deficits in lipid stores or
utilization trigger food intake. As we shall see, just as it is the case for food
intake, some data are not a good fit for the deficit/depletion model.
III. Some Environmental Influences on Food Hoarding

Before discussing the possible peripheral and central mechanisms under-
lying the control of food hoarding, it is important to note the environ-
mental conditions under which food hoarding is stimulated or inhibited.
Although we will note some species-specific differences in the effects of an
environmental component on food hoarding, the effects of environmental

change on food hoarding are more similar than different across species.
A. PHOTOPERIOD
For many animals in nature, the ratio of the hours of daylight-to-darkness
(i.e., the photoperiod) is a principal cue signaling changes in the season
(for review see Bartness and Goldman, 1989). The photoperiod cue is
transduced into a biochemical cue by the pineal gland through its secretion of
the hormone melatonin and triggers a constellation of physiological
and behavior responses including alterations in energy balance that
are important for the discussion here (for review see Bartness and Wade,
1985). Conventional wisdom would suggest that as the days shorten,
signaling the approach of fall/winter - a time when food availability
decreases and environmental temperatures drop, food hoarding should
increase. This is exactly what was found with species of Peromyscus (white-
footed mice and deer mice) where the higher the latitudinal origin of these
wild-trapped mice, the more they hoarded when they were exposed to short
'winter-like days' (Barry, 1976). Siberian hamsters (Phodopus sungorus, the
species used in our hoarding studies discussed throughout), also increase food
hoarding in short days versus long days (Masuda and Oishi, 1988). Imposing
a fast that is a primary stimulator of food hoarding in most species however
(see below), does not further stimulate hoarding in short- versus long-days -
even though Siberian hamsters lose ~30% of their body fat in short days
(Wade and Bartness, 1984; Bartness et al., 1989) and therefore would be
expected to be more energetically challenged by a fast in this photoperiod
(Wood and Bartness, 1996b). Syrian hamsters (Mesocricetus auratus), known
for their ability to hibernate during the early years of their domestication
but no longer able to do so now readily, increase food hoarding in short
days before hibernation (Lyman, 1954). Indeed, it has been suggested
that formation of a hoard is a prerequisite for entering hibernation (Lyman,
1954). Not surprisingly, short day-induced increases in food hoarding are
blocked by pinealectomy in Syrian hamsters (Fleming, Scardicchio, and
Scardicchio, 1986), a treatment that renders hamsters unable to respond to
photoperiod cues (Hoffman and Reiter, 1965); for review see Bartness and
Goldman, 1989).
B. COLD EXPOSURE
Because 80-90% of food energy is used to maintain thermal homeostasis
(Bartholomew, 1977), food intake is frequently increased by cold-exposed
small mammals including laboratory rats (Brobeck, 1948; Johnson
and Cabanac, 1982), Syrian hamsters (Bartness, Ruby, and Wade, 1984),
Food Hoarding 73
Siberian hamsters (Masuda and Oishi, 1988) and house mice (Mus musculus
(Perrigo and Bronson, 1985)) to name a few species. Cold exposure also
increases food hoarding by most of these species. For example, there is an
inverse relation between decreasing ambient temperature and food hoard
size by laboratory rats (McCleary and Morgan, 1946; Fantino and Cabanac,
1984). The percentage of animals hoarding food (hoarding itself was not
quantified) increases in cold-exposed Siberian hamsters, an effect exagger-
ated when combined with short days (Masuda and Oishi, 1988). Similarly,
when deer mice (Peromyscus maniculatus bairdii) are exposed to short days
and cold ambient temperatures food hoard size also is increased, but the
short photoperiod, not the cold, is the primary controller of this response,
despite the origin of these wild-trapped animals from central Michigan
(Barry, 1976). In contrast to deer mice, food hoarding of white-footed mice
(Peromyscus leucopus noveboracensis) is driven more by the cold than the
photoperiod (Barry, 1976). Oddly, cold exposure inhibits food hoarding by
laboratory mice at temperatures as mild as 59F (Ross and Smith, 1953),
whereas cold-exposed pouched mice (Saccostomus camestris) do not alter
their hoarding (Ellison, 1996). Thus, across a wide-range of mouse species,
cold-induced food hoarding is not as robust as in hamster or rat species.
C. FOOD SHORTAGE (FASTING/FOOD RESTRICTION)

Food availability in nature is often less than optimal, unlike the utopian
conditions of the laboratory, and therefore requires significant allocations
of time and effort to find and capture food energy. The response to
food shortages in the wild is emulated in the laboratory by withdrawing
food completely (fasting) or by decreasing the food allotment (food
restriction). The first laboratory studies of food hoarding were done using
rats (see above) and these nonnatural hoarders are somewhat reluctant to
exhibit hoarding unless food deprived (Morgan, Stellar, and Johnson, 1943;
Stellar and Morgan, 1943; Porter, Webster, and Licklider, 1951; Herberg
and Blundell, 1967; Blundell and Herberg, 1973; Fantino and Cabanac,
1980). Surprisingly, food hoarding in rats occurs relatively independently of
the absolute level of body fat as evidenced by approximately equivalent
fasting-induced increases in food hoarding by Zucker obese and lean rats
(Herberg and Winn, 1982). Similarly, dietary obese rats hoard about as
much as their chow-fed counterparts, despite being heavier (fatter) at the
time of food restriction (Winn and Herberg, 1985). Although the above data
suggest that fasting generates an energy deficit, or at least moves
animals toward a negative energy balance, the nature of the deficit has
been difficult to pin down; moreover, the behavior of the postfast animal is
not always congruent with this view. For example, fasted rats prefer to
hoard inedible objects to food pellets (Wallace, 1979)! The deficit hypothesis
(Morgan, Stellar, and Johnson, 1943) and its implications for the control of
food hoarding based on changes in metabolic fuel utilization will be
discussed below.
In addition to the postfast increases in hoarding by laboratory rats,
they also overeat (Baker, 1955), as do most animals across many taxa
after food deprivation. In rats, there is a behavioral competition between
feeding and hoarding postfast such that hoarding is more reliably shown
if the animals are first allowed to eat and then given a hoarding test
(Stellar, 1947). By contrast to laboratory rats, an energetic regulatory puzzle
exists for all hamster species tested because they fail to overeat after a fast
(Syrian [Mesocricetus auratus], (Silverman and Zucker, 1976); Turkish
[Mesocricetus brandtl], (Rowland, 1982); Siberian [Phodopus sungorus],
(Bartness and Clein, 1994)). Although they do not exhibit a postfast
hyperphagia, this is not because they physically cannot overeat since they do
so under some conditions (e.g., calorically diluted diets (Rowland, 1982);
lesions of the paraventricular or ventromedial VMH nuclei of the hypothal-
amus (PVN and VMH, respectively; (Bartness, Bittman, and Wade, 1985;
Rowland et al., 1986; Bittman et aI., 1991)). Silverman and Zucker (1976)
speculated that the lack of a postfast increase in food intake by hamsters
might be because selection pressures for building food caches to counteract
shortfalls in foragable food. We repeatedly tested this notion that hamsters
hoard food rather than overeat in response to a fast using Siberian hamsters
and found that food hoarding, but not food intake, is markedly increased
postfast (Bartness and Clein, 1994; Wood and Bartness, 1996a, b; Bartness,
1997; Day and Bartness, 2003; Day, Mintz, and Bartness, 1999;). The same
postfast increase in food hoarding, but not food intake, occurs in Syrian
hamsters, albeit at somewhat lower levels ((Lea and Tarpy, 1986);
cf. (Wong and Jones, 1985)). Thus, it appears that the control of
food intake and food hoarding are separable in hamsters, making
it possible, in principle, to determine the underlying mechanisms for
each response. We will return to this idea later in this review.
Finally, by contrast to laboratory rats and hamsters that increase
food hoarding postfast (see above), a few species do not, but instead only
overeat such as Shaw's jird (Meriones shawi; (Demas and Bartness, 1999)),
Mongolian gerbils (also a jird not a gerbil - Meriones unguiculatus (Nyby
and Thiessen, 1980; Wong and Jones, 1985)) and laboratory mice (Ross and
Smith, 1953). For the latter, food hoarding is suppressed after fasting (Ross
and Smith, 1953).
D. FOOD TYPE
It would seem adaptive for animals to seek, store, and eat foods that
maximize their foraging effort; that is calorically dense foods. These typically
Food Hoarding 75
are high in fat content and in nature typically seeds (e.g. sunflower seeds
"~50% calories are lipid by weight). Foods are chosen for other reasons too
such as appetites for macronutrients (e.g., protein for growth; (Richter and
Barelare, 1938)) or for micronutrients (e.g., vitamin B (Richter, Holt, and
Barelare, 1937)). The notion that animals will seek and consume diets that
will restore nutritional or metabolic imbalances was championed by Curt
Richter (1922) - so-called 'dietary wisdom'. Although not studied extensively
in the context of food hoarding, some data exist. In separate studies, we tested
for changes in food intake and food hoarding preferences in fasted (Day,
Mintz, and Bartness, 1999), or in pregnant or lactating Siberian hamsters
(Day, Mintz, and Bartness, 2002). Briefly, hamsters were given a choice of
composite diets varying in caloric density as well as in macronutrient
composition (i.e., sunflower seeds [SS], pellet chow [PC] and rabbit chow
[RC]). Following fasting, food intake (calories) was not increased, however,
food hoarding was increased especially on the first day of refeeding and was
primarily due to increases in SS hoarding. The order of food intake and
hoarding preferences was not changed after a fast (SS > PC > RC), but the
degree of food hoarding preference for SS was exaggerated (Day, Mintz, and
Bartness, 1999). These data suggest that with decreases in lipid stores
(internally stored energy), such as occurs with fasting (Day and Bartness,
2003), hoarding of lipid-rich foods (externally stored energy) increases. We
will return to this apparent inverse relation between internal (body fat) and
external (food hoard) energy stores below. Finally, pregnant self-selecting
Siberian hamsters ate relatively more carbohydrate and less fat, and hoarded
less carbohydrate and more fat than their virgin counterparts (protein not
affected). In contrast, lactating and virgin self-selecting hamsters both ate and
hoarded relatively more carbohydrate than protein or fat compared with PC-
fed hamsters, but were not different from each other. The pregnancy-induced
increased eating and hoarding of carbohydrate may have helped meet
immediate energy needs sparing dwindling lipid reserves because Siberian
hamsters lose ~50% of their body fat during pregnancy. Interestingly, the
opportunity to self-select their diet eliminated pup cannibalism, a behavior
that was rampant by the PC-fed lactating (~60% eaten, 8/10litters; (Day,
Mintz, and Bartness, 2002)). As with self-selection food intake studies, the
interpretation of food choice data for hoard composition is fraught with
problems associated with the diets themselves as well as with other problems
(for review see Friedman, 2000).
E. FOOD AVAILABILITY(FORAGING EFFORT)

The relation between foraging effort and food hoarding has been
infrequently studied in the laboratory. From an energetic standpoint, one
might expect an inverted 'U' or 'V' function relating foraging effort to food
hoard size. That is, food hoarding should increase at low foraging efforts,
stimulated by the deviation in food availability from the utopian condition
of free-food to that requiring some effort to obtain food, but decline when
the foraging effort is so great so as to impose an exercise-induced energy
deficit where food would be eaten rather than stored for latter consumption.
In laboratory rats, food is taken equally from food bins located at varying
lengths from the home cage suggesting that food energy is not optimally
acquired because it should have been taken solely from the closest bin until
that supply was exhausted (Miller and Viek, 1950). Rats sometimes do
behave in a more energy efficient manner relative to foraging effort,
for example by decreasing food hoard size as the tube leading from
their home cage to the food source is lengthened (Cabanac and Swiergiel,
1989). Analogous findings occur when Syrian hamsters work for food by
bar pressing where food hoarding decreases as the bar press response
requirement for pellet delivery increases (Lea and Tarpy, 1986). In both of
these examples, however, the effort required to obtain the food does not
seem very demanding. Therefore, we aimed to study the relation between
foraging effort and food hoard size by requiring more substantial energy
expenditure to obtain food - perhaps more closely mimicking foraging
in nature.
We adapted the foraging effort paradigm of Perrigo and Bronson (1985)
to our food hoarding paradigm (Bartness and Clein, 1994) so as to
incorporate two characteristics of foraging and hoarding in the wild - effort
and distance. We tested the effects of increased foraging effort on foraging
(pellets earned), food intake (pellets eaten) and hoarding (pellets hoarded)
by Siberian hamsters housed in this foraging/hoarding system. Because we
will continue to focus on the effects of various manipulations on foraging,
food hoarding, and intake from Siberian hamsters housed in this foraging/
hoarding system throughout this review, we will briefly describe it. Two
cages are positioned one above the other and are connected by ~1.52m
tubing that has corners and straightways for both horizontal and vertical
climbs. The bottom cage represents an underground burrow, is dark, and
contains bedding and nesting material. The top cage represents the foraging
area located above ground in the wild, is lit, contains a running wheel and
a water bottle. Food pellets (75mg) are presented contingent upon the
completion of a programmed number of wheel revolutions in the upper
cage. In our first experiments, we had foraging efforts ranging from
10 revolutions/pellet (5.24m/pellet) to 200 revolutions/pellet (104.8m/
pellet). We also included a running wheel with noncontingently available
food (Free Wheel/Free Food; a control for exercise and foraging
opportunity) as well as a sedentary control group where food also was
available noncontingently, but the wheel was blocked from turning
(Immobilized Wheel/Free Food). Food intake remained mostly constant
Food Hoarding 77
with the exception of a slight increase at 10 revolutions/pellet (Figure 1).

Food hoard size exhibited the inverse 'U' or 'V' function with increases
in foraging effort. That is, food hoard size increased four-fold with non-
contingent wheel running (Free Wheel/Free Food) compared with sedentary
conditions (Immobilized Wheel/Freed Food group), remained increased
but to a lesser degree (three-fold) at the lower foraging efforts (10 and 50
revolutions), but decreased to or below sedentary control at the highest
efforts (100 and 200 revolutions, respectively; Figure 1). Despite the wheel
running requirement for most groups, total carcass lipid only decreased
Foraging Food Intake

(Pellets Earned) (Pellets Eaten)
500 ' 60
450 '
50,
400 '
350 ' 40
.~
=
Q.
300 '
250 ,
200 '
_-,'3o
20
150 ,
100 '
10
50'
0 0
,, ~ ~ Immobilized Wheel
, ~1 Free Wheel
m 10 Revolutions/Pellet
..... 50 Revolutions/Pellet
........ 100 Revolutions/Pellet
.... 200 Revolutions/Pellet
Hoarding
(Pellets Hoarded) Gonadal WAT Mass
~t
20 ~'~ ,~8 0.16 :~ ~8

0.14
lli i
~ = 010
0 L [~ 0.02
. - - 0.00 . . . . . .
Time 0
FIGURE 1 M e a n S E M daily pellets (g) earned, eaten and hoarded, as well as gonadal
white adipose tissue (WAT) mass (g) of Siberian hamsters in an experiment where foraging
effort was varied. * p < 0.05 vs. all groups; ** p < 0.05 vs. Immobilized Wheel, 100, 200 revs/
pellet groups; t P < 0.05 vs. 200 revs/pellet group; ~ p < 0.05 vs. 50, 100, a n d 200 revs/pellet
groups. (Adapted from Day and Bartness, 2001).
at the highest foraging effort (200 revolutions) along with the masses of all
individual white adipose tissues (WAT; parametrial [gonadal], inguinal, and
retroperitoneal WAT), data not consistent with the proposed inverse
relation between food hoard size and body fat levels (Herberg and Blundell,
1970; Fantino and Cabanac, 1980; Bartness and Clein, 1994) discussed
below. A fat pad-specific decrease in mass was seen across the foraging
efforts for the gonadal WAT, but not for the other fat pads, with decreases
in mass beginning with hamsters given access to a running wheel (Free
Wheel/Free Food) and continuing as the foraging effort reached 100
revolutions (Figure 1). Interestingly, these decreases in gonadal WAT
mass were associated with increases in food hoarding above sedentary
control levels until hoard size returned to sedentary control levels at the
higher foraging efforts (Figure 1). These and other data (epididymal WAT
[EWAT] removal decreases spermatogenesis in rat testes (Srinivasan et al.,
1986)) suggests that gonadal fat is vital for reproduction and hence
monitored closely with appropriate energy saving/acquiring responses
occurring when stored lipid in these pads decrease. Therefore, despite the
apparent dissociation between total body fat decreases with food hoarding
increases, the importance of changes in the lipid stores of specific fat pads
may represent a fundamentally new way to view changes in body fat and
their consequent changes in behavior/physiology, not just for foraging/food
hoarding, but in the field of energy regulation in general.
IV. Some Peripheral Physiological Controls of Food Hoarding

We have made a somewhat arbitrary dichotomy of the potential
mechanisms underlying foraging, food hoarding, and intake - peripheral
physiological factors versus central factors. Obviously peripheral factors,
such as gonadal steroids or leptin, have central effects and central factors
like neuropeptide Y or the melanocortins have peripheral effects, but right
or wrong, they typically are studied separately. For convenience only here,
we present the following section on peripheral factors, followed by the
related issue of body fat levels and then a section on central factors. When
possible and enlightening, however, we will discuss their interrelations.
A. METABOLIC FUEL CHALLENGES/CHANGESIN METABOLIC RATE

The first attempts at testing the 'deficit hypothesis' of Morgan and Stellar
(1943), was by Elliott Stellar where he sought to alter the metabolism of
laboratory rats and assess the consequent changes in food hoarding in his
dissertation studies (Stellar, 1947). In an attempt to inhibit hoarding by
increasing circulating metabolic fuels, epinephrine was injected peripherally
Food Hoarding 79
to increase mobilization of lipid fuels via lipolysis and to increase

mobilization of carbohydrate fuels via glycogenolysis. Glucose was injected
to increase circulating carbohydrate fuels more directly. These manipula-
tions largely did nothing to food hoarding except for a small decrease in
hoarding with the highest dose of epinephrine and this likely was due to
nonspecific debilitating effects (Morgan, Stellar, and Johnson, 1943).
Furthermore, injections of insulin given to clear the circulation of all
metabolic fuels, were without effect on hoarding (Stellar, 1943). We also
were interested in testing the effects of metabolic fuel utilization on food
hoarding. Therefore, we injected Siberian hamsters with long-lasting insulin
and, in separate tests, we also injected the nonmetabolizable glucose analog,
2-deoxy-D-glucose that occupies the enzymes of glycolysis to induce a
temporary decrease in glucose utilization ('glucoprivation'). Both energetic
crises stimulate food intake in laboratory rats (McClure, 1987) and, but to a
much lesser degree, in Siberian hamsters (Bartness, Morley, and Levine,
1995), but not Syrian hamsters (Ritter and Balch, 1978; Rowland, 1978).
Neither treatment, however, affected food hoarding by Siberian hamsters,
nor did the fatty acid utilization blocker methyl palmoxirate that engenders
lipoprivation and can increase food intake in laboratory rats (Friedman and
Tordoff, 1986). Because these treatments were given just before the dark
period and food hoarding was measured at the beginning of the light period,
we may have missed an early effect of these substances on hoarding
(Bartness, Morley, and Levine, 1995). A similar lack of effect of insulin and
2-deoxy-u-glucose on hoarding was seen in Shaw's jird (Demas and
Bartness, 1999). As an alternative to the view that food hoarding may be
triggered by short-term metabolic challenges, it may be that food hoarding
is a long-term energy strategy that is not sensitive to short duration
alterations in metabolic fuel utilization. Therefore, it may be that if a long-
lasting, chronic state of glucoprivation or lipoprivation was induced, then
increases in food hoarding might be triggered. This remains to be tested.
Besides the tests of insulin and epinephrine on food hoarding, relatively
little is known about other hormones that affect energy utilization or
metabolism. Increases in metabolic rate generated through thyroxine
injections, and decreases in metabolic rate generated through thyroidectomy
or injections of thiouracil do not affect food hoarding (Stellar, 1951).
B. GONADAL STEROIDS
Gonadal steroids have received the most study of the hormones tested for
effects on food hoarding, likely because female laboratory rats hoard more
than males, even when non-food deprived (e.g., ((Herberg, Pye, and
Blundell, 1972; Coling and Herberg, 1982); cf. (Kalsbeek et al., 1988))), as
do female Mongolian gerbils (Nyby et al., 1973). The effects of changes in
ovarian steroids across the estrous cycle on food hoarding have been studied
in several species. Female Syrian hamsters decrease food hoarding on the
day of behavioral estrus and this decrease is correlated with high
concentrations of circulating estradiol and progesterone with no difference
in hoarding among the other stages of the estrous cycle (Estep, Lanier, and
Dewsbury, 1978). A similar finding occurs in female rats where food
hoarding is greatest during diestrus and decreases during proestrus and
estrus (Herberg, Pye, and Blundell, 1972; Fantino and Brinnel, 1986). This
contrasts with the results of a test of the 'drive' to hoard food in female rats.
There, rats had to cross an electrified grid to get to the food source during
hoarding tests and a higher drive to hoard food, as indicated by the higher
current of electric shock tolerated, occurred at proestrus and a lower drive
during diestrus (Borker, Dhume, and Gogate, 1985). When female rats are
ovariectomized to eliminate the primary source of circulating estrogen and
progesterone, body mass increases (Bogart, Lasley, and Mayer, 1944; Wade
and Zucker, 1970), reflected as increases in body fat (Leshner and Collier,
1973). Surprisingly, in spite of the apparent inverse relation between body
fat levels and food hoarding (vide infra), ovariectomized rats increase food
hoarding in parallel with the increase in body mass/fat (Coling and Herberg,
1982). These effects are reversed by peripheral injections of estradiol
benzoate, but not progesterone or testosterone (Coling and Herberg, 1982),
although 17-alpha or 17-beta estradiol did not affect food hoarding by
ovariectomized rats in another study (Donohoe et al., 1984). Finally, little is
known about the role of androgens in food hoarding. Castration of
Mongolian gerbils increases food hoarding, as did ovariectomy of rats
above, whereas castration plus testosterone propionate injections reverses
this effect in gerbils (Nyby et al., 1973).
Collectively, food hoarding generally is inhibited when females of various
species would be involved in seeking a mate or in mating itself, which makes
sense from an adaptive significance standpoint. The underlying role of
gonadal steroids and other factors such as prolactin during pregnancy and
lactation are discussed in light of the body fat changes seen across these
reproductive conditions below.
V. Inverse Relation between Adiposity and Food Hoarding

Some of the data presented above, especially those concerned with fasting
and food restriction, suggest that body fat levels are inversely related to food
hoarding levels. That is, when internally stored energy in the form of body
fat decreases, externally stored energy in the form of a food hoard increases
(Herberg and Blundell, 1970; Fantino and Cabanac, 1980; Bartness and
Clein, 1994). This hypothesis not only is supported by the deprivation/food
restriction studies of normal laboratory rats discussed above, but also holds
Food Hoarding 81
for dietary obese ('supermarket diet'-fed; (Winn and Herberg, 1985)) and for
genetic obese (Zucker fa/fa) rats that are body mass reduced via food
restriction (Herberg and Winn, 1982). In these obese animals, food hoarding
is triggered by the same percent loss of body mass (fat) as for the chow-fed
controls (Winn and Herberg, 1985) and lean (Fa/Fa) controls (Herberg and
Winn, 1982).
In addition to this inverse relation between food hoarding and body fat
in laboratory rats, similar findings and support for this notion comes
from other species. For example, Mongolian gerbils fed for only 1.5h
('schedule-fed') for several weeks have reduced lumbar fat (inguinal WAT;
(Nyby and Thiessen, 1980)) and associated increases in food hoarding.
Unlike some species of rat that fatten during pregnancy (e.g., eastern wood
rats [Nemotoma floridana];(McClure, 1987)) and human females, Syrian
(Wade, Jennings and Trayhurn, 1986) and Siberian (Schneider and Wade,
1987) hamsters lose 40-50% of their body fat during pregnancy and both
Syrian (Miceli and Malsbury, 1982) and Siberian (Bartness, 1997; Day,
Mintz, and Bartness, 2002) hamsters increase food hoarding during
pregnancy. Both species lose additional body fat during lactation and
show associated increases in food hoarding (Bartness, 1997; Miceli and
Malsbury, 1982; Day, Mintz, and Bartness, 2002) compared with their
nulliparous counterparts.
From the converse side of this putative inverse relation, increases in
internal energy stores (body fat levels) should cause decreases in food
hoard size (external energy stores). There is some supporting evidence for
this hypothesis in that mice fed lipid-rich supplements are heavier (fatter)
than non-supplemented mice and hoard less food (Ross and Smith, 1953). In
addition, Siberian hamsters fed a high fat diet that increases their body mass
(Wood and Bartness, 1996a) and body fat (Wood, A. D. and Bartness, T. J.,
unpublished observations) decrease their food hoarding compared with
thinner, chow-fed controls (Wood and Bartness, 1996a).
The most direct evidence for the putative relation between body fat levels
and food hoarding is seen after surgical removal of body fat (partial
lipectomy termed 'lipectomy' here). Removal of the epididymal WAT pads
in male Siberian hamsters stimulates food hoarding (Wood and Bartness,
1997) supporting the inverse relation between body fat levels and food
hoarding. This increase, however, is not permanent because many animals,
including Siberian hamsters, show compensatory increases in the remaining
nonexcised WAT pads after a lipectomy-induced lipid deficit (for review see
Mauer, Haeris, and Bartness, 2001). Thus, across time, as the reparation of
the lipid deficit occurs, food hoarding returns to prelipectomy levels (Wood
and Bartness, 1997). These results contrast with a lack of increased food
hoarding lipectomized rats (Michel and Cabanac, 1999). In terms of the
latter, several fat pads were removed including the gonadal pad (epididymal
82 Timothy J. Bartnessand Diane E. Day
WAT), perirenal, abdominal WAT (unclear as to what fat depot the latter is
as rats do not possess abdominal fat per se), and ventral subcutaneous fat
(most likely inguinal WAT). Removal of this much fat from an animal is
traumatic, as acknowledged by the authors (Michel and Cabanac, 1999),
therefore making it difficult to interpret these findings. Moreover, the
method used to test food hoarding by these severely lipectomized rats also
requires sequential food restriction to produce a within-animal determina-
tion of body mass versus food hoarding (Michel and Cabanac, 1999). Thus,
the rats bearing this large lipid deficit are then further stressed by restricted
feeding making it even more difficult to interpret their findings. Siberian
hamsters, made obese due to PVN lesions, and that are subsequently
lipectomized do not increase food hoarding (Wood and Bartness, 1997),
however, as would be predicted by this inverse relation. This may be because
a sufficiently large lipid deficit was not created in these fat animals with only
the removal of the epididymal WAT pads. This finding is reminiscent of the
failure of food deprivation to increase food hoarding in rats made obese by
VMH lesions unless they are first body mass (fat) reduced to their prelesion
levels and then fasted (Herberg and Blundell, 1970). Collectively, consider-
able support exists for the posited inverse relation between internal energy
stores (fat) and external energy stores (food hoard). These supportive data
beg the question as to how the lipid deficits are sensed by the brain to
ultimately trigger increases in food hoarding.
One means by which the brain could be informed of body fat levels, or
some corollary of this factor, is by leptin, a primarily but not exclusively,
adipose-derived factor (for review see Harris, 2000). That is, one notorious
presumed role of leptin is to inform the brain of body fat levels (for review
see Kaiyala, Woods, and Schwartz, 1995) because of the positive correlation
between the degree of adiposity and circulating concentrations of leptin
(e.g., (Klein et al., 1996)) and the ability of leptin treatment to decrease
body fat (e.g., (Halaas et al., 1995; Pelleymounter et al., 1995)). Because of
the inverse relation between body fat levels and food hoarding (Fantino
and Cabanac, 1980; Phillips, Robinson, and Davey, 1989; Bartness, 1997;
Wood and Bartness, 1997), we (Day, D.E. and Bartness, T. J., unpublished
observations) and others (Schneider and Buckley, 2001) reasoned that the
decrease in body fat associated with fasting should cause a decrease in leptin
gene expression in WAT and consequently a decrease in circulating leptin
concentrations (Maffei et al., 1995; Trayhurn et al., 1995). These reduced
concentrations of circulating leptin would, in turn, signal the brain that lipid
energy stores were reduced and therefore trigger the postfast-induced
increase in food hoarding. If circulating leptin concentrations are artificially
elevated by exogenous administration, however, then the brain should
receive a signal indicating normal or elevated body fat levels when they
actually are being depleted by the fast.
Food Hoarding 83
In our experiment designed to test this prediction, Siberian hamsters were

housed in the foraging/hoarding system and, after adaptation and stable
food hoarding and intake, had minipumps implanted subcutaneously for
constant delivery of leptin (1.3 ggAtl/hr). Leptin administration during this
nonfasting condition slowly decreased food hoarding (although there also
was a slight decrease in hoarding by vehicle-treated hamsters, but not as
marked; Figure 2, top). This leptin-induced decrease in food hoarding
suggests that the brain was receiving a signal indicating a surfeit of lipid
stores that triggered a decrease in food hoarding. Food intake was not
affected by leptin (Figure 2). Following a 32h fast, food intake was not
affected by leptin or vehicle treatment hamsters as expected (Figure 2), but
food hoarding increased postfast despite the leptin infusion. This last result
may have occurred for a number of reasons including an insufficient dose of
the hormone or because of the route of administration. In terms of the
latter, peripheral bolus injections of leptin are more effective in decreasing
food intake in laboratory rats than peripheral continuous infusions (e.g.,
(Harris et al., 2001)). Support for a role of leptin in controlling food
hoarding and therefore indirect support for the role of body fat in
controlling this appetitive ingestive behavior, however, is stronger for Syrian
hamsters given bolus leptin injections during fasting. (Schneider et al.,
1998). Specifically, fasting-induced infertility in Syrian hamsters is blocked
by giving leptin injections during the fast (Schneider et al., 1998) and
consequently the same protocol was tried in an attempt to block fasting-
induced increases in food hoarding (Schneider and Buckley, 2001).
Peripheral leptin injections (5mg/kg) given every 12h during a 48h fast
blocked the postfast-induced increase in food hoarding. These data
especially, as well as our preliminary data to a lesser extent, suggest a role
of leptin in food hoarding. Our failure to block postfast-induced increases
in hoarding by leptin administration may be due to the route of leptin
administration we used (constant rather than bolus) and/or that we gave
leptin prefast, during the fast and postfast, whereas in the Syrian hamster
study leptin only was given during the fast (Schneider and Buckley, 2001).
Alternatively, there may be bona fide species differences in the response to
leptin. Note, however, that fasted fa/fa obese rats that do not synthesize
leptin, increase food hoarding after a fast, suggesting that fasting-induced
decreases in leptin are not necessary to stimulate postfast food hoarding
(Herberg and Winn, 1982). Thus, the role of leptin in food hoarding is not a
simple one and may vary across species.
Finally, there is another mechanism by which changes in body fat could
be conveyed to the brain. WAT has sensory innervation, initially suggested
by the discovery of substance P in WAT (Fredholm, 1985), a neurotrans-
mitter typically associated with sensory innervation. Later studies demon-
strated sensory innervation of WAT neuroanatomically using retrograde
Hoard Size
7,0. --e-- Vehicle
6.5. --o-- Leptin
6.0-
5.5-
5.0-
A 4.5-
4.0-
21 3.5-
O 3.0-
"r
2.5-
1.5-
1.0-
0.5-
0.0 . . . . . . . . . . . . . . . . . . . . . . , , , ,
10EBB887B685B48382BIB -11-10-9 -8 -7 -6 -,5 -4 -3 -2 -1 S 1 2 3
Days
Food Intake
7.0
Vehicle
6.5
--c-- Leptin
6.0
5.5
5.0
A 4.5
O}
v 4.0
3,5
3.0
m 2.5
2.0
1.5
1.0
0.5
0.0 . . . . . . . . . . . . . . . . . . . . . . . . . .
lOE96887B6B,SB4535251B -11-10.-9 -8 -7 -6 -5 .-4 -3 -2 -1 S 1 2 3
Days
FIGURE2 Mean (4- SEM) g of hoarded food (Hoard Size, top) and g of eatern food (Food
Intake, bottom) by Siberian given s.c. infusions of leptin (1.3 ~tg/gl/hr, open circles). No pumps
were in place for either group during baseline (Day 10B-Day 1B). At that time, pumps were
implanted and remained in place throughout the rest of the experiment. At Day S, a 32h fast
was started for all hamsters, followed by days (1 to 3) of postfast measures (Day, D.E.,
Rooks, C. and Bartness, T.J., unpublished observations).
tract tracers injected into W A T and labeling bipolar sensory neurons in the
dorsal root ganglia (Fishman and Dark, 1987). Although we do not know
what is being sensed by these nerves, there is evidence that the sensory
innervation of W A T m a y interact with the sympathetic innervation of W A T
(Youngstrom and Bartness, 1995) to regulate the level of the sympathetic
Food Hoarding 85
drive (Luthman et al., 1989; Ralevic, Karaon, and Burnstock, 1995) and
thus modify lipid mobilization and fat pad size (for review see Bartness and
Bamshad, 1998).
There are conflicting data with this proposed inverse relation between
body mass/fat and food hoarding, however. For example, from the
decreasing side of body mass/fat continuum, cold exposed laboratory rats
immediately increase food hoarding before their body mass (fat) decreases
(Fantino and Cabanac, 1984). Similarly, Siberian hamsters increase their
food hoarding the first day they are fed a calorically-diluted diet before their
body mass (fat) decreases (Wood and Bartness, 1996a). When Siberian
hamsters are required to forage for their food by running a prescribed
number of wheel revolutions (see above and Day and Bartness, 2001), food
hoarding increases at low levels of foraging effort without a change in body
mass or total carcass lipid. In addition, central administration of certain
neuropeptides can affect food hoarding (see below) and some of these cause
increases in food hoarding without concomitant decreases in body mass
(fat). For example, intracerebroventricularly (icv) administered corticotro-
pin-releasing hormone (CRH) in laboratory rats (Cabanac and Richard,
1995), or either icv neuropeptide Y (NPY) or agouti-related protein (AgRP)
in Siberian hamsters, increase food hoarding with no change in body mass
(fat; (Day and Bartness, 2002)). Similarly, electrical stimulation of sites in
the lateral hypothalamus (LH) that trigger food intake, but not other sites,
trigger food hoarding when rats are given the opportunity to hoard without
affecting body mass/fat (Herberg and Blundell, 1967).
On the increasing side of the body mass/fat continuum, there also are data
that conflict with this simple inverse relation between internal energy stores
(fat) and external energy stores (hoard size). Thus, the body mass and fat
increases resulting from lesions of the PVN or of the VMH are associated
with normal levels of food hoarding rather than decreases in Siberian
hamsters (Wood and Bartness, 1997) and laboratory rats (Herberg and
Blundell, 1970), respectively. Zucker obese fats (fa/fa) that are ~1.5 times
the weight of their lean counterparts (Fa/Fa), an increase primarily due to
enhanced body fat, hoard the same amount of food despite the differences in
internal energy stores rather than decreasing food hoard size (Gosselin and
Cabanac, 1996). In addition, high fat diet-fed Siberian hamsters have
increases in body mass and a associated decrease in food hoarding (Wood
and Bartness, 1996a) as mentioned above, but the decrease occurs on the
first day of high fat diet feeding before their body mass changes (Wood and
Bartness, 1996a). Therefore, increases or decreases in food hoarding are not
always associated with opposite changes in body mass/fat and vice versa.
One possible resolution to some of the above mentioned discrepancies
with the hypothesized inverse relation between food hoarding and body
mass/fat is that the measures of body mass especially, but also of body fat
(whole animal carcass composition) are not sensitive enough to detect shifts
in lipid mobilization or accumulation. Therefore, an alternative view, and
one borrowing liberally from the metabolic control of food intake
(Friedman, 1991; Friedman and Rawson, 1994) and of fertility (Wade and
Schneider, 1992; Wade, Schneider, and Li, 1996), may be that 'energy
balance' or 'energy flux' (i.e., the net storage/utilization of internal energy
stores) regulates food hoard size. Thus, with a negative energy balance or
increases in energy flux away from storage (i.e., mobilization of fuel), food
hoarding increases. This view supports most of the existing data on food
hoarding in this species. Not only does this view help explain the increases in
food hoarding associated with: (1) the negative energy balances of
pregnancy and lactation (Bartness, 1997), (2) food restriction-induced
decreases in body mass/fat (Phillips, Robinson, and Davis, 1989; Mauer and
Bartness, 1994; Wood and Bartness, 1996b; Day, Mintz, and Bartness, 1999)
and (3) surgically induced lipid deficits (Wood and Bartness, 1997), but it
also helps explain the increases in food hoarding that are not associated with
reductions in total body as seen with small increases in foraging effort
discussed above (Day and Bartness, 2001). Therefore, it may be that there
are common underlying processes for the metabolic control of food
hoarding that are shared with those controlling food intake (Friedman
and Stricker, 1976) and fertility (Wade and Schneider, 1992) as we have
postulated previously (Day and Bartness, 2001).
VI. Central Control of Food Hoarding

The neural substrates underlying the appetitive ingestive behaviors of
foraging and food hoarding are virtually unknown. Of course, the neural
substrates underlying the consummatory ingestive behavior of feeding
abound, even in Siberian hamsters (Bittman et al., 1991; Boss-Williams
and Bartness, 1996; Mauer and Bartness, 1997; Purvis and Duncan, 1997;
Wood and Bartness, 1997; Mercer et al., 2000a). In this review, we have
made a somewhat arbitrary dichotomy of the mechanisms underlying these
behaviors peripheral factors (largely discussed above) and central factors.
We recognize the important interactions between the brain and periphery,
but feel that separate approaches may yield a common system. Indeed, this
was our successful approach in determining the mechanisms underlying the
short photoperiod (melatonin)-induced decrease in body fat of Siberian
hamsters. There we identified important brain areas and peripheral targets
and then literally found a connection between them (Song and Bartness,
2001). At this point, our lack of knowledge of central sites/neurotransmit-
ters involved in foraging/food hoarding, as well as the role of key peripheral
organs such as body fat, makes this approach impossible. Thus, at some
Food Hoarding 87
point, a merging of the known central and peripheral modulators or

foraging/food hoarding must occur, but for simplicity of discussion, we now
consider some possible central mechanisms underlying these appetitive
ingestive behaviors.
Woods et al. (1998) have proposed that some of the neuropeptides shown
to stimulate food intake in home-cage feeding tests of laboratory rats and
mice may instead bring animals into contact with food (foraging or the
appetitive phase of the ingestive sequence described above (Craig, 1918)).
That is, neuropeptides might trigger food-seeking behavior and, if the food
is readily present requiring little or no effort, as in home-cage testing, then
the consummatory phase would proceed automatically (Woods et al., 1998).
Indeed, this seems to be the case for icv injected NPY in rats being fed via an
intraoral cannula requiring no appetitive response (Seeley, Payne, and
Woods, 1995). Specifically for this ingestive behavior model, laboratory rats
passively get food infused into their mouth and swallow except when full
(e.g., (Grill and Norgren, 1978; Grill, Berridge, and Ganster, 1984)), at
which time the food dribbles out of the mouth (an all too familiar sight by
human parents when trying to feed their full babies). Fasted rats, or rats
injected with insulin to engender 'hunger,' consume more food this way than
do ad libitum-fed or vehicle injected rats (Flynn and Grill, 1983; Seeley,
Payne, and Woods, 1995). In contrast to the voracious feeding by rats
receiving icv NPY in conventional home cage tests (Clark et al., 1984;
Levine and Morley, 1984; Stanley and Leibowitz, 1984), these passively fed
rats do not increase their food intake when given icv NPY (Seeley et al.,
1995). If these same animals have to move to drink a calorically laden liquid
(sucrose solution), however, then icy NPY increases caloric intake (Seeley,
Payne, and Woods, 1995). Thus, it appears that NPY does not stimulate
food consumption per se in the absence of appetitive behavior (foraging and
acquiring food). These findings, and those of others suggesting NPY-
induces foraging-like behavior in rats (Harland, Bennet, and Gardiner,
1988; VanNess, DeMaria, and Overton, 1999), led us to hypothesize that
relatively separate neural systems regulate foraging and food hoarding (i.e.,
the appetitive responses) and food intake (consummatory response). This
almost has to be true at some level because the behaviors are different.
Furthermore, we believe that some of the neuropeptides thought to be
regulators of food intake per se, such as NPY above, also, or instead, may
be regulators of appetitive responses driving animals toward food.
Conversely, some of the neuropeptide inhibitors of food intake (e.g.,
(oe-melanocyte-stimulating hormone [(a-MSH; e.g., (Kim et al., 2000)],
urocortin; (Spina et al., 1996; Smagin et al., 1998)) may also, or instead,
inhibit foraging/food hoarding. Finally, we would be neurochemically and
neuroanatomically naive to approach this problem as 'one peptide - one
behavior' and/or 'one brain structure - one behavior.' That said, we chose to
test potential neuropeptide candidates one at a time, and initially to use the
icv route of administration because we were uncertain where precisely to
inject these substances.
Therefore, we recently began a series of preliminary studies testing
peptides that would be likely candidates to control foraging/food hoarding,
some of the preliminary results of which, are shown and/or discussed here.
Because we are using the foraging/hoarding apparatus discussed above (Day
and Bartness, 2001), and because food intake and food hoarding by Siberian
hamsters are largely independent of one another (i.e., increases in one
behavior usually are not accompanied by increases in the other behavior;
e.g., (Bartness and Clein, 1994; Bartness, 1997; Wood and Bartness, 1997;
Day, Mintz, and Bartness, 2002)), we may be in a unique position to
determine if there are neuropeptide controllers of these appetitive ingestive
behaviors. We began these experiments by testing neuropeptides that
conformed to two criteria. For the first, we sought candidate peptides whose
gene expression and/or content increased with fasting, a condition
promoting marked increases in food hoarding, but not food intake in this
species as discussed above (Bartness and Clein, 1994; Wood and Bartness,
1996b; Bartness, 1997; Day, Mintz, and Bartness, 1999). Secondly, because
it is easier to interpret increases rather than decreases in behavior, we chose
peptides that increase food intake when given exogenously to Siberian
hamsters or other small rodents. Below we discuss the results of three such
preliminary tests as examples of the potential to determine the central
control of food hoarding.
Fasting engenders a host of central and peripheral changes in hamster
physiology and includes alterations in neuropeptides involved with energy
balance. Specifically, fasting induces increases in NPY and agouti-related
protein (AgRP) mRNA levels in the arcuate nucleus of Siberian hamsters,
with no changes in orexin, and decreases in proopiomelanocortin gene
expression (Reddy et al., 1999; Mercer et al., 2000a) - responses consistent
with similar studies of laboratory rats or mice (i.e., (Sanacora et al., 1990;
Schwartz et al., 1993; Ebihara et al., 1999)). Although changes in gene
expression do not necessarily reflect changes in neuropeptide release in the
terminal fields, they often are consistent with such changes (e.g., NPY
release in the PVN with fasting; (Sahu, Kalra, and Kalra, 1988; Beck et al.,
1990; Jain et al., 1998)). Finally, icv- or PVN-injected NPY causes
impressive increases in food intake (Levine and Morley, 1984; Stanley and
Leibowitz, 1984) in laboratory rats (Clark et al., 1984; Levine and Morley,
1984; Stanley and Leibowitz, 1984) and other species (Larsen et al., 1999;
Morris and Crews, 1990) including Siberian hamsters (Boss-Williams and
Bartness, 1996). As mentioned above, NPY injected into the PVN of rats
also stimulates behaviors suggestive of foraging (Harland et al., 1988;
VanNess et al., 1999).
Food Hoarding 89
NPY administered into the third ventricle markedly stimulates food

intake in Siberian hamsters in standard home cage tests (Boss-Williams and
Bartness, 1996). Using the foraging/hoarding apparatus, the results of
preliminary studies also show that foraging is increased by NPY (Day
and Bartness, 2002). In addition, NPY increased food intake substantially
(200-300%), but these increases were not as impressive as the increases in
food hoarding (300 1100% increases; (Day and Bartness, 2002)). Moreover,
the NPY-induced increases in food intake waned as foraging effort
increased, but food hoarding either remained high or decreased less than
did food intake (Day and Bartness, 2002). This contrasts to what happens
with vehicle injection or in nonoperated hamsters in our recent study (Day
and Bartness, 2001) where food hoarding decreases as foraging effort
increases (animals essentially eat all the food they earn). This makes the
greater NPY-induced increases in food hoarding compared with food intake
even more impressive in these hamsters. The NPY receptor subtype
underlying these increases in food hoarding intake and foraging remains
to be determined.
In a second preliminary study, we tested the effects of AgRP on foraging,
food hoarding and intake in Siberian hamsters (Day and Bartness, 2002).
AgRP is part of the melanocortin (MC) system acting as an endogenous
MC receptor antagonist (Ollmann et al., 1997) and is almost exclusively
co-synthesized by NPY neurons in laboratory rats (Hahn et al., 1998) and
Siberian hamsters (Hahn et al., 1998; Mercer et al., 2000a). The agonist for
the MC receptor subtypes MC-3 and MC-4 is (o~-MSH, a byproduct of
cleavage of a large precursor peptide, proopiomelanocortin (Chronwall,
1985) and an inhibitor of food intake in rats (Poggioli, Vergoni,
and Bertolini, 1986). AgRP gene expression increases with fasting in the
arcuate nucleus of rats (Hahn et al., 1998; Mizuno et al., 1999) and of
Siberian hamsters (Mercer et al., 2000b). In addition, icv, PVN or
dorsomedial hypothalamic nucleus injections of AgRP alone in rats
profoundly stimulate food intake and does so for several days (Hagan
et al., 2000; Kim et al., 2000; Wirth and Giraudo, 2000, 2001). Therefore,
we injected AgRP into the third ventricle of Siberian hamsters acclimated
to our foraging/hoarding apparatuses at the start of the dark period.
AgRP significantly increased food hoarding and foraging, but had little
or no effect on food intake. Thus, based on these preliminary data, both
NPY nor AgRP stimulated foraging for food, and both were potent
stimulators of food hoarding. In addition to stimulators of foraging,
there may also be inhibitors and their waxing and waning help to trigger
or suppress foraging, respectively. As suggested above, some of these
may belong to the ever-growing list of food intake inhibitors classified
as such based on their ability to decrease food intake in home cage testing
conditions.
Finally, we began a series of studies examining the role of opiates

on foraging, food hoarding and intake. A role for opiates in the central
control of food intake has been established for some time and for many
species under a variety of conditions (for review see Levine et al., 1985),
including some experiments where food hoarding was examined (Kavaliers
and Hirst, 1985, 1986). Deer mice (Perornyscus maniculatus) selectively
increase food hoarding when mu opiate receptor subtype agonists, such
as morphine, are given whereas stimulation of kappa opiate receptor subtype
agonists, such as U-50,488H, selectively stimulate food intake (Kavaliers and
Hirst, 1985, 1986). Giving a mixed mu and kappa receptor subtype agonist
(ketocyclazocine) or combinations o f m u and kappa agonists (morphine and
U-50,488H) stimulate both behaviors (Kavaliers and Hirst, 1985, 1986).
Therefore, we gave Siberian hamsters acclimated to our foraging/hoarding
apparatus, morphine (mu receptor subtype agonist), U-50,488H (kappa
receptor subtype agonist) or the broad-spectrum, long-lasting opiate
receptor blocker, naltrexone. Unlike the deer mice above, at the 10
revolution/pellet foraging effort, food intake was not affected by the
kappa receptor subtype agonist U-50,488 at any time point, whereas food
intake was stimulated by morphine, but only during the first hour (Figure 3).
Morphine significantly stimulated food hoarding across all time points,
however (Figure 3), whereas naltrexone did not significantly affect food
intake or hoarding, but tended to inhibit both behaviors at all measurement
intervals (Figure 3). With increases in foraging effort (50 revolutions/pellet),
neither of the receptor agonists, or the receptor antagonist affected
food intake or hoarding (data not shown). Therefore, the same selectivity
for the opiate receptor subtype stimulation of food intake (kappa) and food
hoarding (mu) by deer mice (Kavaliers and Hirst, 1985, 1986) is not seen in
Siberian hamsters. Indeed, the only selectivity seen by Siberian hamsters was
an increase in food hoarding, albeit a small one, compared with the huge
increases seen by NPY and AgRP (see above). There are many possible
reasons for the differences between the deer mice and Siberian hamsters
experiments including species, the kappa agonist tested, and the testing
apparatus among them.
It should be clear that the study of the central controllers of the appetitive
phase of ingestive behaviors - foraging and food hoarding - are in their
infancy, especially when compared to what is known about the central
controllers of the consummatory phase of ingestive behaviors. We feel that
as we deepen our understanding of the central controllers of appetitive
ingestive behaviors, however, we also might add to our understanding of
consummatory ingestive behaviors. That is, perhaps some of the long list
of food intake inhibitors might be parceled out between those that inhibit
foraging, and thus ultimately inhibit food intake, and those that only inhibit
food intake but not foraging.
Food Hoarding 91
Percent of Pellets Earned

MORPHINE % Eaten
% Hoarded
lo0
80 80
60 60
E .

~2 N 20
o
0 - 1 Hr 1 - 2 Hr 2 - 4 Hr 4 - 24 Hr Total 0 - 1 Hr 1 -' 2 Hr 2 " 4 Hr 4-'24 Hr Total
Hours Post Injection Hours Post Injection

NAL TREXONE % Eaten
% Hoarded
100. m Naltrexone lOO
Saline
f- 80- i NaFatatone
60- 60
e
~/. 40- ~. ~
20-
0
0~1 Hr 1 " 2 H r 2 " 4 H r 4 - 2 4
Hours Post Injection

Hr Total
U50488
o f
0-'1 Hr 1 -'2Hr 2 - ' 4 H r 4 - ' 2 4 H r Total
Hours Post Injection
% Hoarded % Eaten
100 100
lU 60 60
N 2
t -u-- 1
0 - 1 Hr 1 - 2 H r 2 - 4 H r 4 - 2 4 H r Total
N 2o
o . . . . .
0 - 1 Hr 1 - 2 H r 2 - 4 H r 4 - 2 4 H r Total
Hours Post Injection Hours Post Injection
FIGURE 3 Mean (+ SEM) percentages of pellets earned that were hoarded (left side) and
eaten (right side) by Siberian hamsters foraging for their food (I0 revolutions/pellet) after s.c.
injections of morphine (10mg/kg), naltrexone (10mg/kg) and U50488 (lmg/kg). Data are
plotted in bins between 0-1, 1-2, 2-4, 2-24 h after injection and total across the 24 h period.
*=p < 0.05 vs. saline.
VII. Summary and Concluding Remarks

W e h a v e reviewed m u c h o f the l i t e r a t u r e o n f o o d h o a r d i n g in S i b e r i a n a n d
S y r i a n h a m s t e r s as well as l a b o r a t o r y rats, mice, a n d s o m e o t h e r small r o d e n t
species in a n a t t e m p t to synthesize w h a t is k n o w n a b o u t the e n v i r o n m e n t a l
a n d p h y s i o l o g i c a l c o n t r o l l e r s o f a p p e t i t i v e ingestive b e h a v i o r s such as f o o d
hoarding and, to a lesser extent, foraging. We made a somewhat arbitrary

distinction between central and peripheral factors describing the roles of
many factors in each category, but we again emphasize that this does not
imply a true distinction between these factors; rather, it was one of
convenience of description. The apparent inverse relation between body fat
levels and food hoarding (Herberg and Blundell, 1970; Fantino and Cabanac,
1980; Bartness and Clein, 1994) appears to be just that-apparent, because of
the many exceptions to this rule. One possible way to unify the discordant
data that seems to contradict this relation is to view the events that trigger
foraging/food hoarding as changes in energy flux or balance rather than
measurable changes in body fat p e r se. Clearly, more intensive investigations
using metabolic blockers are needed to propel this notion further such as has
been done for the study of the metabolic controls of food intake (Friedman,
1995; Scheurink and Nolan, 1996) and of reproduction (for review see Wade,
Schneider, and Li, 1996; Schneider and Wade, 1999).
We also hypothesized that some of the bewilderment regarding the large
number of peptides that appear to inhibit food intake compared with the
relatively small number that stimulate it, may be due to the role of some of
the inhibitors in suppressing foraging. It makes sense from an adaptive
significance view that although foraging for food is important, it also
is energetically costly and often a dangerous journey due to predation. Thus,
it would make sense to have several mechanisms to suppress the urge to
forage for food at the slightest sensation of hunger in inclement weather, or
during times when predation would be likely. Therefore, because of the
sequential order of searching for food and then ingesting it, if foraging
is inhibited, food intake will consequently be inhibited. We presented
preliminary data supporting the role of NPY and AgRP in primarily
stimulating food hoarding rather than food intake in our paradigm where
animals have to forage for their food and have the opportunity to hoard it.
As we noted and emphasize again here, it would be naive to expect that
there are peptides that only control foraging, food hoarding or food intake
and that do so only in one brain area.
Collectively, we hope that this review will stimulate thinking and more
emphasis on the control of each phase of the ingestive behavior sequence -
appetitive and consummatory phases, resulting in new models and
experiments to yield a broader understanding of the search for, storage
of, and consumption of food.
Acknowledgment
This work was supported in part by the National Science Foundation IBN 9876495 to TJB.
The authors thank Drs. Jill Schneider, Stephan Woods, Allen Levine, Randy Seeley, Eric Corp
and George Wade for their helpful discussions of food hoarding and of neuropeptides.
Food Hoarding 93
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The Functional Organization of the Peripheral

Gustatory System: LessonsFrom Behavior
Alan C. Spector
Department of Psychology and Centerfor Smell and Taste
University of Florida, Gainesville, Florida 32611
I. Introduction
The substances an animal consumes can be lethal either immediately or
after a prolonged period of intermittent ingestion. At the same time, what
an animal fails to eat or drink can also lead to its demise. Inappropriate
ingestion need not impact upon survival directly, but can affect an animal's
ability to function optimally in its environment. It remains uncontested that
throughout the animal kingdom, what and how much an animal ingests
is determined in large part by the chemical properties of the substrate
interacting with preingestive receptor systems. 1 It is therefore likely that
chemosensory systems are complicit in many of the abnormalities associated
with eating and drinking.
The chemosensory features of food and fluid are complex, and in
vertebrates engage three cephalic sensory modalities including the olfactory,
trigeminal, and gustatory systems. The perceptual integration of the
sensations arising from input channeled through these different chemosen-
sory systems is what is referred to asflavor. From an analytical perspective,
the term taste is reserved for chemicals that are relatively nonvolatile and are
in a physical state in which they can contact receptor elements on the
membranes of taste receptor cells in the oropharyngeal cavity resulting in
cellular stimulation and ultimately leading to alterations in behavior 2 or
physiology.
1For vertebrates, the use of the term preingestive signifies the stimulation of preesophageal
chemoreceptors. Given that once foods and fluids are swallowed, they come into contact with a
variety of different forms of chemoreceptors strategically placed at key locations in different
body compartments, the preingestive distinction is not trivial.
2perception is an inferred process and can never be measured directly. I therefore use the term
behavior to focus on what is actually measured. T h a t said, I take liberties later in the chapter
with m y use of the term perception.
101
Copyright2003, ElsevierInc.
0363-0951/03 $35.00
102 Alan C. Spector
In vertebrates, taste receptor cells are grouped in structures called

taste buds. Taste buds are distributed in distinct fields in the oral cavity
of many mammals including rats and humans (Bradley, 1971). Each
taste bud field is innervated by a specific branch arising from one of three
cranial nerves. What is the functional significance of this anatomical
arrangement? Arguably, it is possible that each nerve is more or less specia-
lized for a particular function. Assuming that this may be true, the next
question is what might those functions be? My goal in this chapter is to
review the evidence, some of which was collected over the last decade in my
laboratory, supporting the hypothesis that the gustatory nerves are
functionally specialized in the rat, which is the most widely used animal
model in taste research. The primary findings upon which this hypothesis
is based were generated in behavioral experiments designed to assess the loss
and return of function following the selective transection and regeneration
of gustatory nerves. Such experiments help reveal what portions of the
system are necessary and/or sufficient for function to be maintained.
II. Anatomy, Electrophysiology, and Molecular Biology of the

Peripheral Gustatory System
A. ANATOMY
Before exploring the behavioral ramifications of manipulations of the
peripheral gustatory system, it would be useful to discuss the "hardware."
Each taste bud contains about 50 epithelial cells, some of which serve as
chemoreceptors. These cells are joined by tight junctions and contain
microvilli that protrude into an opening in the cornified epithelial cell layers
called the taste pore. Taste stimuli gain access to the taste bud through its
pore. The receptor sites for the initial interaction between the stimulus
compound and the taste receptor cell are generally ion channels or seven-
transmembrane G-protein coupled receptors usually located in the apical
membrane (see Herness and Gilbertson, 1999; Gilbertson, Damak, and
Margolskee, 2000). 3
In the rat, there are approximately 1400 taste buds distributed in several
regions or fields in the oral epithelium (Miller, 1977). The anterior two
thirds of the tongue contains approximately 13% of the total with a dense
distribution on the dorsal and ventral tip. These taste buds are housed in
nipple-like protrusions called fungiform papillae. Each fungiform papilla in
the rat contains a single taste bud that is innervated by the chorda tympani
sit is possible that some receptor sites are located in the basolateral membrane as well. For
compounds to gain access to these sites they would have to penetrate the tight junctions
(Herness and Gilbertson, 1999).
The Functional Organization of the Peripheral Gustatory System 103
(CT) branch of the 7th cranial nerve. The taste buds of the palate,
representing about 16% of the total, are found in three regions. The most
anterior location is the incisive papilla surrounding the opening of the
nasoincisor ducts which connect the oral cavity with the vomeronasal organ.
Moving caudally, there is a strip of taste buds at the border between the
hard and soft palates referred to as the "geschmacksstreifen" which
translates into "taste stripe" (Miller, 1977). The remaining palatal taste
buds are found in the soft palate, which is referred to as the posterior
palatine field. The palatal taste buds are innervated by the greater superficial
petrosal (GSP) branch of the facial nerve (7th cranial nerve). The taste
buds in the posterior tongue are found primarily in the single circumvallate
papilla along the midline of the dorsal surface of the tongue and in the
foliate papillae found on both sides. These papillae are trench-like with
taste buds lining the walls. In the rat, there are about 4-5 foliate trenches
per side (sometimes more) but there is interanimal variation in the number.
These posterior tongue taste buds represent approximately 56% of the total
and are innervated by the lingual-tonsillar branch of the glossopharyngeal
(GL) nerve (gth cranial nerve). The taste buds in the most anterior trench
of the foliate are also partially supplied by the CT (Whiteside, 1927). About
10% of the total buds are found in the nasopharynx and the laryngeal
epithelium including the epiglottis. These are innervated by the superior
laryngeal (SLN) branch of the vagus nerve (10th cranial nerve). Based
on their anatomical position and response properties these laryngeal
taste receptors are thought to be involved in the protection of the airways
(see Travers and Nicklas, 1990). The remaining ~5% of the taste buds are
scattered in the buccal wall and sublingual organ. The exact numbers and
proportions of taste buds noted above vary across subjects and studies, but
the organization represented in Figure 1 is reasonably faithful (see Bradley,
1971; Miller, 1977; Travers and Nicklas, 1990).
The axons of the CT and GSP have their soma settled in the geniculate
ganglion and terminate in the lateral portion of the rostrocentral (RC)
subdivison of the most rostral pole of the nucleus of the solitary tract (NST).
The axons of the GL, the soma of which reside in the petrosal ganglion,
also terminate in the RC of the NST but more caudally. Finally, the axons of
the SLN, which have their soma located in the nodose ganglion, terminate
even more caudally in the NST. Although there appears to be some degree
of orotopic organization of these terminal projection zones there is also
extensive overlap (Hamilton and Norgren, 1984; Norgren, 1995).
B. ELECTROPHYSIOLOGY
All of the gustatory nerves in the rat apparently respond to representative
stimuli from all of the human prototypical psychophysical classes
104 Alan C. Spector
TB% ORAL RECEPTOR FIELD NERVE
OR >
-16% PALATE
CHORDATYMPANI(VII}
-13% ANTERIOR TONGUE
I GLOSSOPHARYNGEAL(IX)~=
-56% [ POSTERIOR TONGUE
-10% LARYNGEAL EPITHELIUM SUPERIORLARYNGEAL (X)~=
FIGURE 1 The basic anatomical organization of the peripheral gustatory system in the rat,
The approximate percentage of the total taste buds found in each oral receptor field is noted.
These percentages are based on summaries provided by Miller (1977) and Travers and Nicklas
(1990). The exact number of taste buds in each field varies across animals and studies and thus
the proportions listed are rough estimates. The percentage for the laryngeal epithelium includes
some taste buds found in the nasopharynx. The remaining 5% of taste buds are scattered on the
buccal wall and in the sublingual organ. The gustatory nerves project to the rostral nucleus of
the solitary tract in a roughly orotopic pattern but with considerable overlap.
(i.e., "sweet," "salty," "sour," and "bitter"), but the nerves differ markedly
with respect to their relative sensitivity to such taste compounds. The CT
responds best to salts and acids (Pfaffmann, 1955; Ogawa, Sato and
Yamashita, 1968; Boudreau et al., 1983, 1985; Frank, Contreras, and
Hettinger, 1983). The GSP responds best to sugars and moderately to salts
(Nejad, 1986). The G L responds best to alkaloids, such as quinine, and other
compounds described as bitter by humans, but also shows appreciable
responsiveness to acids, salts, and sugars (Boudreau et al., 1987; Frank, 1991;
Dahl, Erickson, and Simon, 1997). The response properties of the S L N in the
rat have not been extensively studied (see, Andrew, 1956; Shingai, 1980). In
the hamster, however, this nerve responds well to water, HC1, and extremely
hypertonic NaC1 consistent with its postulated role of airway protection
(Dickman and Smith, 1988; Smith and Hanamori, 1991).
Single fiber examinations of the rat CT and G L have revealed physio-
logically defined classes of taste afferents that differ with respect to the
selectivity of their tuning. The CT contains a population of fibers, referred to
by Frank, Contreras, and Hettinger (1983) as N-fibers, that are narrowly
tuned to respond to sodium (and lithium) salts. Another group of CT fibers is
referred to as H-fibers; these fibers respond best to acids and are broadly
tuned in that they also respond to both sodium and nonsodium salts and
to quinine (Frank, Contreras, and Hettinger, 1983). Others have found
similar classes of units recording in the geniculate ganglion of the CT

(Boudreau et al., 1983, 1985; Lundy and Contreras, 1999; see also, Ninomiya
and Funakoshi, 1988; Contreras and Lundy, 2000). The CT responds only
modestly, at best, to sucrose and accordingly there appears to be few single-
fibers that respond to this sugar (Frank, Contreras, and Hettinger, 1983).
On the basis of neurophysiological response profiles, Frank (1991) has
identified three major classes of taste fibers in the GL: A-fibers, S-fibers,
and Q-fibers. The A-fibers are broadly tuned responding to acids and salts.
The S-fibers are narrowly tuned responding to sugars and some synthetic
"sweeteners." The Q-fibers are also narrowly tuned and respond best to
quinine and other compounds described as "bitter" by humans (also see
Dahl, Erickson, and Simon, 1997). Single units in the rat petrosal ganglion
(ganglion of the GL) are also very sensitive to alkaloids (Boudreau et al.,
1997). A comprehensive single-fiber analysis of taste responses in the GSP
and SLN in the rat has yet to be published.
C. BIOPHYSICSAND MOLECULARBIOLOGY
The presence or absence of ion channels or protein receptor sites on the
surface of a taste receptor cell determines the potential for that cell to signal
the presence or absence of a stimulus. Accordingly, it is reasonable to ask
whether there is a pattern to the anatomical distribution of particular types
of protein receptors or ion channels across fields of taste buds. Without
going into too much detail about the mechanics of various transduction
pathways, in the rat, there is some evidence of anatomical segregation of
certain receptive elements, but there is also evidence for an overlapping
distribution of others.
Epithelial sodium channels (ENaCs) are found in a variety of sodium
absorbing tissues including taste receptor cells (e.g. Heck, Mierson, and
DeSimone, 1984; Simon et al., 1993; Garty and Palmer, 1997; Lin et al.,
1999; Lindemann, Gilbertson and Kinnamon, 1999). Based on the effects of
the ENaC blocker amiloride, ENaCs appear to be involved in a large part of
sodium taste transduction and are thought to be responsible for the narrow
tuning characteristics of the sodium-responsive N-fibers of the CT; the
sodium responses of H-fibers are unaffected by amiloride treatment
(Ninomiya and Funakoshi, 1988; Hettinger and Frank, 1990; Lundy and
Contreras, 1999). Although ENaCs have been immunohistochemically
identified to exist in all of the taste bud fields on the tongue, those found in
the posterior tongue taste buds are apparently not functional in the rat (Li,
Blackshaw, and Snyder, 1994; Lindemann et al., 1998; Lindemann,
Gilbertson, and Kinnamon, 1999; Lin et al., 1999). That is, lingual amilo-
ride treatment does not alter the responsiveness of the GL or its associated
taste receptor cells to NaC1 applied to the posterior tongue (Formaker and
106 Alan C. Spector
Hill, 1991; Doolin and Gilbertson, 1996; Gilbertson and Zhang, 1998;
Kitada, Mitoh, and Hill, 1998). This shows that simply the mere presence of
mRNA or protein representing a given channel or receptor does not
necessarily imply functionality (see Doolin and Gilbertson, 1996;
Lindemann et al., 1999). Amiloride has also been shown to affect sodium
signal tranduction in palatal taste receptor cells (Doolin and Gilbertson,
1996; Gilbertson and Zhang, 1998; Sollars and Hill, 1998). Thus, the ENaC
channel that participates in normal sodium taste transduction appears to be
limited to the taste bud fields of the 7th cranial nerve, at least in the rat. 4
Another important transduction element is the G-protein subunit
ot-gustducin expressed somewhat selectively in taste receptor cells. 5 It is
thought to be part of the transduction pathway for both "bitter-tasting"
and some "sweet-tasting" compounds (McLaughlin, McKinnon, and
Margolskee, 1992; Wong, Gannon, and Margolskee, 1996; Wong et al.,
1996). It is predominantly expressed in the taste buds of the posterior tongue
and palate, but can also be found in taste receptor cells of the anterior
tongue (Boughter et al., 1997).
Recently, a family of ~30 different genes were identified in rodents
and humans encoding for seven-transmembrane G-protein coupled recep-
tors that bind with ligands that are bitter tasting to humans and avoided
by animals (Adler et al., 2000; Chandrashekar et al., 2000; Matsunami,
Montmayeur, and Buck, 2000). Receptors belonging to this family are
referred to as T2Rs. These evidently serve as taste receptors and each is
thought to bind rather selectively with a specific ligand. The mRNAs of
T2Rs are coexpressed in taste receptor ceils such that a given cell can have
many subtypes of receptors from the T2R family. This has been interpreted
as allowing the gustatory system to respond to a diverse set of potentially
harmful compounds but with limited discriminability (Chandrashekar et al.,
2000; see Caicedo and Roper, 2001; Spector and Kopka, 2002 for alternative
views). Interestingly, all cells that express a T2R also express ot-gustducin
(Adler et al., 2000). The converse, however, is not true. It has been reported
that mRNA for T2Rs are found in about 15% of the taste receptor cells
in the taste buds of the circumvallate papillae, foliate papillae, epiglottis,
and geschmacksstreifen, and are much less frequently seen in the fungiform
taste buds of the anterior tongue (the posterior palatine field and NID
were not examined). These findings suggest that the CT should be less
responsive to T2R ligands than the other taste nerves. As noted above, it is
fair enough to say that the GL is more responsive to quinine and other
4It should be noted that aldosteronetreatment or sodium deprivationcan induce amiloride-

sensitiveNa+ currentsin somecircumvallatetaste receptor cells (Linet al., 1999).
5ee-Gustducinhas also been foundin somecellsof the gastrointestinaltract (Hofer, 1996).
bitter-tasting ligands compared with the CT, but as will be shown below,
exactly how this translates into behavior is not as straightforward as
might be expected.
A second family of genes encoding for candidate G-protein coupled taste
receptors binding with sugars, synthetic sweeteners, and amino acids has
also recently been discovered (Hoon et al., 1999; Bachmanov et al., 2001;
Kitagawa et al., 2001; Max et al., 2001; Montmayeur et aI., 2001; Nelson
et al., 2001). The receptors from this family are referred to as TIRs. So far,
three receptors have been identified in this family (T1R1, T1R2, and T1R3)
in mouse, rat, and human tissue. These receptors are thought to form
heterodimers with each other that determine their ligand binding chara-
cteristics. It is interesting to note that T1Rs are not coexpressed with T2Rs
in taste receptor cells despite the fact that some taste receptors display
changes in K+-current and intracellular Ca 2+ levels in response to both
sucrose and quinine (Gilbertson et al., 2001; Caicedo, Kim, and Roper,
2002). As a group, the T1Rs are apparently more uniformly expressed in the
rodent oral cavity compared with the T2Rs, although individual stibtypes of
the former family are differentially distributed. For example, T1R1 is
uncommon in the taste buds of the circumvallate papillae (innervated by the
GL), whereas T1R2 is very rare in the fungiform papillae (innervated by the
CT) (Hoon et al., 1999; Nelson et al., 2001). In a heterologous expression
system the T1R2+T1 R3 heterodimer binds with a variety of "sweet" tasting
compounds including sucrose, fructose, saccharin, dulcin, acesulfame-K,
and some D-amino acids. The T1RI+T1R3 heterodimer binds with many
L-amino acids and activation of this receptor complex is enhanced when
these stimuli are presented in the presence of the purine nucleotide
inosine monophosphate (Nelson et al., 2001, 2002; Li et al., 2002).
Another candidate taste receptor that has been identified is a splice
variant of the mGluR4 metabotropic glutamate receptor subtype found on
the postsynaptic membranes of some neurons in the brain (Chaudhari,
Landin, and Roper, 2000). The taste-mGluR4 receptor binds with
glutamate, the stimulus that is thought to be the prototypical representative
of the somewhat controversial "fifth" taste quality called umami. Although
its expression pattern in the oral cavity has yet to be comprehensively
described, the taste-mGluR4 receptor has been found in rat foliate and
circumvallate taste buds. The relative contributions of T 1R 1+T 1R3 and the
mGluR4 receptors to taste responses associated with glutamate remains
to be resolved.
The discovery of these taste receptor and G-protein genes has been truly
remarkable and offer very promising experimental opportunities to
manipulate the peripheral gustatory system. However, one must exercise
some restraint in interpreting these expression patterns especially with
regard to functionality. The presence of mRNA for a given taste receptor
108 Alan C. Spector
in a cell does not necessarily mean that the receptor is functionally expressed
(Lindemann, Gilbertson, and Kinnamon, 1999; cf. Caicedo and Roper,
2001; Caicedo, Kim, and Roper, 2002; Gilbertson et al., 2001; Spector and
Kopka, 2002).
III. Tongue Maps and Their Meaning

The apparent relative differences in nerve responsiveness to various classes of
taste compounds (e.g., salts, sugars, alkaloids, acids) coupled with the
differential distribution of certain functional fiber types and expression
patterns of taste receptors leads to the question of whether there are regional
differences in taste sensitivity in the oral cavity as assessed psychophysically.
In fact, many textbooks present human tongue maps claiming that the
posterior tongue is most sensitive to bitter, the tip of the tongue is most
sensitive to sweet and salty, and the side of the tongue is most sensitive to
sour. Although this notion has been propagated for many years, it is not
entirely consistent with the psychophysical research that has been conducted
(Collings, 1974; Miller and Bartoshuk, 1991; Smith and Margolis, 1999).
First, these tongue maps ignore the presence of the palatal receptor field.
Second, if anything, in humans the threshold concentration for quinine is
lower on the front of the tongue than the back of the tongue and lowest on the
palate (Collings, 1974). Third, although it is true that the growth of sensation
magnitude for bitter tasting compounds increases more steeply as concen-
tration is raised when these stimuli are applied to the back of the tongue
(circumvallate and foliate papillae) as opposed to the front of the tongue,
this is true for other compounds as well such as NaC1 (Collings, 1974).
The electrophysiological findings in the rat lend some support to the
notion that there could be regional differences in psychophysical sensitivity
to certain classes of taste compounds. However, extrapolating this to
humans is a conceptually risky venture. Indeed, there are differences in
the electrophysiological response profiles of the gustatory nerves across
species of rodents. For example, although the rat CT responds to sucrose,
it does so only poorly, whereas this nerve in the hamster responds
exceptionally well to the disaccharide (Pfaffmann, 1955; Hagstrom and
Pfaffmann, 1959; Frank, Contreras, and Hettinger, 1983; Frank, Bieber,
and Smith, 1988). The CT response to NaC1 in the rat and the C57BL/6J
mouse strain is significantly attenuated by lingual treatment with the
ENaC blocker amiloride, whereas the NaC1 response of the CT in the
DBA/2, 129/J, and BALB mouse strains is unaffected by the drug
(Gannon and Contreras, 1995; Ninomiya et al., 1996).
Perhaps the most important criticism about the notion of tongue maps is
that they are based on an oversimplified one-dimensional view of taste
function. As is discussed in the next section, taste signals contribute to a
variety of functions including stimulus identification, taste-elicited oral

motor reflexes, promotion or discouragement of ingestion (i.e., affect), and
physiological/secretory responses. I do not mean to suggest that regional
differences in sensitivity to certain taste compounds do not exist. Rather,
these differences likely vary across mammalian species and pertain to a
rather constricted view of taste function. As I hope to demonstrate in the
following paragraphs, the data that has accumulated regarding the
functional consequences of selective nerve transection paint a more
complex, yet intriguing, picture of the relative contributions of the various
taste bud fields to taste-related behavior.
IV. D o m a i n s of Taste Function

Taste function can be categorized into at least three general domains (see
Pfaffmann, Norgren and Grill, 1977; Pfaffmann, Frank, and Norgren, 1979;
Norgren, 1985; Scott and Mark, 1986). First, the sensory-discriminative
domain relates to neural processes that lead to the identification of the
stimulus with regard to both quality (metathetic, see Stevens and Galanter,
1957) and intensity (prothetic, see Stevens and Galanter, 1957). Second, the
affective domain relates to neural processes that promote or discourage
ingestion of the taste stimulus. Approach and avoidance behavior as well as
taste-elicited oral motor and somatic reflexes fall into this category as well.
The hedonic characteristics of a taste stimulus are the manifestation of such
neural processes. Third, the physiological domain relates to neural processes
subserving cephalic phase physiological reflexes such as salivation (for more
discussion on these domains, see Spector, 2000).
A. SENSORY-DISCRIMINATIVEDOMAIN
The sensory-discriminative domain is sometimes confused with, or at least
not distinguished from, the affective domain in the literature. Humans and
animals can discriminate among taste stimuli regardless of their hedonic
characteristics. There are many taste compounds that rats can discriminate
but are equally preferred or avoided. Accordingly, the equal preference or
aversion (as assessed by any procedure) between two or more taste
compounds does not necessarily imply that the stimuli are indiscriminable.
Likewise, failure to observe a preference or aversion to a taste solution
relative to water (the solvent) does not necessarily mean that the compound
is undetectable. For example, C57BL/6J mice are indifferent to even high
concentrations of sucrose-octacaetate (SOA) as assessed by two-bottle
intake tests, but these mice can, nonetheless, acquire a conditioned taste
aversion to SOA indicating that they can detect the stimulus at least at high
concentrations (Harder et al., 1984).
110 Alan C. Spector
Methodologically speaking, function in the sensory-discriminative domain

can be dissociated from the affective/hedonic domain by employing a variety
of operant and classical conditioning procedures to measure both detection
thresholds and quality discriminations in intact rats (e.g., Carr, 1952; Koh
and Teitelbaum, 1961; Morrison and Norrison, 1966; Morrison, 1969, 1974;
Slotnick, 1982; Brosvic and Hoey, 1990; Spector, Schwartz, and Grill, 1990;
Spector and Grill, 1992; Spector, Guagliardo, and St. John, 1996a; St. John
et al., 1997; St. John and Spector, 1998). These procedures have the
advantage of not relying on the hedonic properties of the taste solution to
motivate the behavior. In other words, taste serves as a signal for other
reinforcing or punishing events (e.g., food, water, shock). In addition, in
many of these paradigms, taste samples consist of small volumes and
immediate responses are measured, thus increasing the confidence that the
behavior is guided by oral sensory cues.
B. AFFECTIVEDOMAIN
Other procedures are designed to assess the affective component of taste
stimulation. The most common of these is a simple intake test in which the
animal is presented with a taste solution for a period of time and the amount
ingested is recorded. The longer an intake test is, however, the more likely
stimulation of postingestive receptor systems can influence the results. Some
investigators have applied procedures designed to minimize the effects of
such postingestive factors on the assessment of the motivational properties
of taste solutions. One such procedure, referred to as the brief-access
taste test, involves the presentation of very small samples of taste stimuli for
very brief duration trials and the animal's unconditioned licking responses
are measured (e.g., Young and Trafton, 1964; Davis, 1973; Smith, Davis,
and O'Keefe, 1992; Glendinning, Gresack, and Spector, 2002). Several
concentrations can be presented in a random order and a concentration-
response function can be derived. In cases where the taste compounds are
naturally aversive, water-deprivation can be used to motivate stimulus
sampling and provide a high baseline of licking from which concentration-
dependent decreases can be quantified. In the case that a given manipulation
alters the concentration-licking functions, the basis of the variance may
be attributable to either the strength of the signal emanating from the
periphery or in the way that central "reward" circuits process the signal.
Experimentally distinguishing between these two possibilities, which are
not necessarily mutually exclusive, is difficult but not impossible. The
dissociation could be examined by the comparison of behavioral profiles
emerging from tasks in which a relevant taste compound serves as a signal
(such as those mentioned above), irrespective of its hedonic properties, with
profiles associated with tasks designed to assess affective responses. With
The FunctionalOrganizationof the Peripheral GustatorySystem lll
regard to the latter, it is important to consider that affective responses to

taste fall into two subclasses: appetitive and consummatory responses
(Craig, 1918). Appetitive responses refer to the behavior that serves to bring
the animal to the goal object (i.e., food and fluid). This would include
operant tasks in which the animal must perform a particular response to
obtain a taste reinforcer. 6 Consummatory responses refer to reflex-like
behavior that is elicited by stimulus contact with sensory receptors (Grill
and Berridge, 1985; Grill et al., 1987). A relatively pure way to measure
consummatory responses is by delivering a taste stimulus, under complete
experimenter control, through an intraorally implanted cannula (e.g., Grill
and Norgren, 1978a; Grill et al., 1987; Spector, Breslin, and Grill, 1988;
Berridge, 1996). These two subclasses of affective responsiveness can
apparently be dissociated neurally (e.g., Grill and Norgren, 1978b, c; Flynn
and Grill, 1988; Parker, 1995; Berridge, 1996). Intake tests and the brief-
access taste test possess both appetitive and consummatory components (see
Hulse, 1967).
C. PHYSIOLOGICALDOMAIN
The physiological domain refers to physiological responses elicited by taste
stimuli. The most notable of these is salivation, but there is evidence
for others as well (e.g., Pavlov, 1902; Nicolaidis, 1969; Louis-Sylvestre,
1976; Powley, 1977; Berthoud et al., 1980; Berthoud, and Jeanrenaud, 1982;
Bradley, 1991; Teff, Mattes, ar:d Engelman, 1991; Mattes, 1997; Teff, 1999;
Mattes, 2001a, b). For example, certain sugars and synthetic "sweeteners"
can lead to preabsorptive increases in serum insulin concentration
(Berthoud et al., 1980; Berthoud and Jeanrenaud, 1982; Grill, Berridge,
and Ganster, 1984). There is a delayed increase in blood triglyceride levels
after restricted oral stimulation with fat (Mattes, 1996, 1997, 2001a, b).
These taste-stimulated physiological responses display some impressive
chemospecificity. This domain of taste function has not been comprehen-
sively explored, but its existence leads to the question of what types of
manipulations in the peripheral gustatory system would affect it.
V. Functional Consequences of Gustatory Nerve Transection

The theoretical framework depicted in the prior section defines the general
functions, the neural basis of which, we are seeking to understand. This said,
there are several approaches that can be adopted to link neural processes
with function (see Spector, 2000). One strategy to discern the organization
6The use of taste compounds as reinforcers should not be confused with their use as
discriminative signals in operant tasks.
112 Alan C. Spector
of a neural system is to selectively remove its components and assess the

consequences. In the peripheral gustatory system, this can be done in a
variety of ways. For example, ENaCs, found in taste receptor cells, can be
blocked to determine what contribution signals arising from these channels
play in taste function (see Schiffman, Lockhead and Maes, 1983; Bernstein
and Hennessy, 1987; McCutcheon, 1991; McCutcheon, 1992; Tennissen,
1992; Smith and Ossebaard, 1995; Ossebaard and Smith, 1996; Spector,
Guagliardo, and St. John, 1996a; Ossebaard, Polet and Smith, 1997;
Roitman and Bernstein, 1999; Brot, Watson and Bernstein, 2000; Geran and
Spector, 2000a, b). Likewise, different agonists and antagonists for receptors
can be used as has been done to study the role of mGluR4 taste receptors in
taste-related behavior (Chaudhari et al., 1996; Stapleton, Roper and Delay,
1999; Stapleton et al., 2002). Also, gene deletion and rescue experiments
can be used to selectively examine the contribution of particular elements
of the transduction cascade in taste perception (Wong, Ruiz-Avila and
Margolskee, 1999; Nelson et al., 2001; Ruiz-Avila et al., 2001).
The approach that my laboratory and others have adopted is to examine
the relative contribution of whole nerves innervating the oral cavity by
measuring taste-related behavior before and after selective neurotomy.
These nerve transection experiments test the hypothesis that there is
some degree of anatomical segregation of function that emerges early in
the gustatory system. This is not a new idea and it has been previously
proposed in various incarnations in the literature (Atema, 1971; Nowlis,
1977; Finger and Morita, 1985; Frank, 1991, Finger, 1997b; St. John and
Spector, 1998). The challenge has been to discern what the organizational
principles are. The author hopes to provide some insight into what that
organization might be in the rat, but before this case is presented, it is
important to review some caveats.
A. GENERAL CAVEATS: NECESSARY VERSUS SUFFICIENT

AND FUNCTIONAL SPECIFICITY
When a component of a neural system is removed and a behavior is
measurably affected, then the necessity of the missing part is demonstrated
(Spector, 2000). The interpretive strength of such a conclusion depends
on how selective the removal was, what is known about the properties of
the removed component, and what behavior was measured. If a nerve is
transected and an animal drinks less taste solution from a bottle, one
can confidently attribute the result to the specific loss of the nerve because
the transection was presumably selective. It is important, however, to
consider that the altered behavior may not stem from the loss of taste
input provided by the nerve. For example, the gustatory nerves are mixed in
that they not only contain afferent fibers that respond to taste compounds,
but also contain some afferent fibers that respond to temperature and
mechanical stimulation. Moreover, these nerves also contain parasympa-
thetic preganglionic efferent fibers destined for vascular and glandular
structures (e.g., Ogawa, Sato, and Yamashita, 1968; Hellekant, 1971;
Hellekant and Kasahara, 1973; Young and Van Lennep, 1978; Gurkan and
Bradley, 1987; Bradley, 1991; Matsuo et al., 1995). Notably, the glosso-
pharyngeal nerve provides the innervation of the von Ebner's glands, the
secretions of which are emptied into the troughs of the circumvallate
and foliate papillae. The chorda tympani nerve supplies some of the
parasympathetic innervation of the sublingual and submaxillary salivary
glands. Thus, if a behavior is altered after transection of a gustatory nerve,
the possibility that the outcome is based on the loss of nontaste fiber types
must be considered. It also must be recognized that the result pertains
specifically to the behavior measured. The generalizability of the outcome to
other types of behavior or even the same type of behavior measured in a
slightly different fashion needs to be verified.
Sufficiency of a component in a neural system is a much more difficult
condition to demonstrate because there is generally a limit to the specificity of
the test that can be applied. For example, if a taste nerve is transected and
there is no measurable change in some taste-related behavior, then obviously
the removed component is unnecessary. It remains unclear, however, how
much of the remaining system is truly sufficient. Perhaps only one of the
remaining nerves is sufficient and the others are irrelevant. Perhaps none of
the gustatory nerves are necessary nor sufficient, but the behavior is under the
influence of nontaste cues.
Many of these issues can be addressed empirically. Others can be
dismissed or at least weakened by the profile of outcomes generated in an
experiment. In some experiments, however, they remain caveats that place
limitations on the interpretation of results from nerve transection studies.
B. EARLY HISTORY AND INTAKE TESTS
Early notions of the functional role of gustatory nerves in taste-guided

behavior were based almost exclusively on findings from intake tests
conducted with nerve-transected rats. Typically, in these tests, two bottles,
one containing a taste solution (usually sodium chloride, quinine, or sucrose)
and the other containing water, were presented to an animal for 2z1~48 h and
the relative intake was measured. Different concentrations of a taste
compound were tested and a preference/aversion function was derived.
The results of these studies were surprising to many researchers because
of the relative lack of effect gustatory nerve transection had on taste
preference and aversion. For example, bilateral transection of the CT, a
nerve which innervates ~ 1 3 % of the total oral taste buds located in the
114 Alan C. Spector
anterior tongue and is exceptionally responsive to NaC1, had very little

effect on NaC1 preference (Richter, 1939; Akaike, Hiji, and Yamada, 1965;
Pfaffmann, 1952; Vance, 1967; Grill, Schwartz, and Travers, 1992b).
Bilateral transection of the GL, a nerve which innervates ~60% of the total
oral taste buds located in the posterior tongue and is exceptionally respon-
sive to quinine, had no effect on quinine aversion (Akaike, Hiji, and Yamada,
1965; Kawamura, Okamoto, and Funakoshi, 1968; Grill, Schwartz, and
Travers, 1992). The quinine aversion function was, however, substantially
shifted to the right after complete gustatory dennervation of the tongue
(combined transection of the CT and GL), but despite the removal of ~70%
of the receptor population, the rats were still responsive to quinine, albeit at
significantly higher concentrations (Pfaffmann, 1952; Vance, 1967). Other
studies in a variety of rodents have collectively produced mixed results that
seem to be based on the strain or species used, the nerves transected, and the
taste compound tested (Kawamura, Okamoto, and Funakoshi, 1968;
Jacquin, 1983; Sollars, Sollars, and Bernstein, 1991; Grill and Schwartz,
1992; Grill, Schwartz, and Travers, 1992; Tonosaki and Uebayashi, 1993;
Barry, Larson, and Frank, 1996; Chappell, St. John, and Spector, 1998). In
all of these studies, the GSP and the SLN were left intact and thus it was
generally assumed that the remaining function was attributable to palatal
and laryngeal taste receptors (~30% of the total).
It has been over 60 years since the first experiment of this kind was
reported by Curt Richter and it now appears that, for a variety of reasons,
the profile of results produced by these preference studies with nerve-
transected rats are not as paradoxical as it may have initially appeared.
First, although the long-term preference test is a useful first approximation
of an animal's taste responsiveness, it is not necessarily a rigorous
examination of gustatory function. Certain compounds such as NaC1 and
sucrose can stimulate postingestive receptor systems especially at the high
concentrations. At low concentrations, an intact animal may not display a
preference or aversion for the test compound relative to water even though
the taste quality of the stimulus is discernable. This could obscure significant
effects that nerve transection has on taste recognition or detection in the low
concentration range; a range that is probably the most ecologically relevant
to the animal. Second, to the extent that preference/aversion behavior is
guided by taste, postingestive factors notwithstanding, it is the hedonic
properties of the stimulus that drive the behavior. Thus, nerve transection
could potentially lead to significant changes in sensory/discriminative
features of a taste stimulus without affecting its affective potency. Finally,
there does appear to be some convergence of input from various receptor
fields in the oral cavity (Ogawa and Kaisaku, 1982; Travers, Pfaffmann, and
Norgren, 1986; Sweazey and Smith, 1987; Ogawa and Nomura, 1988;
Travers and Norgren, 1995; Grabauskas and Bradley, 1996; Halsell and
Travers, 1997). This might explain why, say, transection of the CT or G L

alone does not affect quinine aversion in a two-bottle intake test, but
combined transection of these nerves has a significant effect.
The findings from two-bottle preference tests conducted with nerve-
transected rats inspired additional behavioral work more directly aimed at
specific aspects of taste function. When researchers started employing
more psychophysically rigorous tasks to assess the taste capacities of animals
after selective nerve transection, unequivocal impairments were discovered.
C. EFFECTS OF CHORDA TYMPANI NERVE TRANSECTION

ON TASTE-RELATED BEHAVIOR
Signal Detection
The most notable effect of CT transection on taste-guided behavior is that it

interferes with salt taste. The degree of impairment caused by transection of
the nerve depends heavily on the task used to assess function, as is clear
from the two-bottle test findings already discussed. Spector et al. (1990) used
a shock avoidance paradigm to train thirsty rats to suppress licking when a
taste solution was presented to avoid a mild but annoying foot-shock. When
water was presented, rats were permitted to maintain licking and if they
suppressed responding, they were punished with a time-out, further delaying
the opportunity to rehydrate. In this experiment, sucrose and NaC1 trials at
various concentrations were interspersed in a quasi-random fashion
throughout the session and psychometric functions were derived over
days. Transection of the CT raised NaC1 thresholds between 1 and 2 orders
of magnitude, but had marginal, if any, effects on sucrose threshold
(Figure 2). This finding was replicated with other operant conditioning
procedures (Slotnick, Sheelar, and Rentmeister-Bryant, 1991; K o p k a and
Spector, 2001). Using a similar procedure, St. John and Spector (1996)
found that quinine detection thresholds were unchanged after CT
transection. This result had to be qualified by the fact that the sham-
operated rats significantly decreased their quinine thresholds postsurgically,
but the fact remains that the CT-transected rats were just as sensitive after
surgery as they were before. Geran, Guagliardo, and Spector (1999) found
that KC1 thresholds were shifted by about 0.6 log10 units after CT
transection demonstrating that the neurotomy can affect sensitivity to
nonsodium salts as well. So with respect to perithreshold sensitivity, CT
transection has no remarkable effects on the detection of sucrose and
quinine, but significantly impairs sensitivity to NaC1 and KC1. It is impor-
tant to stress that while transection of the CT compromises the rat's taste
detection of some salts at low concentrations, these animals can still detect
higher concentrations of these stimuli. The higher the salt concentration,
116 Alan C. Spector
NaCI
P~mBefore-1 ve Before-2
am Before-2 ve After CTX
3.0J
~ 2.si
bl
2.0
n,
Z
1.5
t-
O 1.0
o, 0.5
z
.~ 0.0
o
+ -7o -1
-1.5
1A 2* SA ~, lo 2o 3o 4o ~o
EXPI[RIMrNT 1 EXPERIMENT2
SUCROSE
[~Before-1 ve Before-2
m"Before-2 vs After CTX
~ 2.5
W 3"01
2.o
1.5
1.0
o. 0.s
z
0.0
~ 0.5
~ - 1 0
-1.5
1A 2 , SA 1D 2D 5D ,D
EXPERIMENT1 EXPERIMENT2
FIGURE 2 Change in the log10 concentration of the detection threshold for NaC1 and
sucrose as a function of bilateral chorda tympani transection (CTX) assessed by a conditioned
shock avoidance procedure. Bars going up represent a decrease in sensitivity (increase in
threshold) and bars going down represent an increase in sensitivity. Hatched bars represent
the difference across two threshold determinations made before surgery. Solid bars represent
the difference between the second presurgical threshold determination and that conducted
after CTX. NaC1 and sucrose thresholds were measured concurrently in the same animals. Error
bars on subjects represent the asymptotic standard error of the locus of rise parameter (which
served as the definition of threshold) from curve fits. Note that CTX caused unequivocal
increases in threshold for all animals. In contrast, with the exception of 1 rat, CTX had
marginal effects on sucrose sensitivity. Reprinted with permission from Spector, Schwartz, and
Grill (1990).
the more likely that trigeminal fibers will be stimulated, so it is somewhat

unclear how much of the remaining stimulus detectability is gustatory in
origin (Wang, Erickson, and Simon, 1993; Pittman and Contreras, 1998).
Unconditioned Licking Responses
The assessment of taste intensity in the suprathreshold range in nerve-

transected rats has relied primarily on the brief-access taste test in which rats
are presented with short periods (on the order of several seconds) of access to
taste stimuli and unconditioned licking responses are measured (e.g., Young
and Trafton, 1964; Davis, 1973; Krimm et al., 1987; Smith, Davis, and
O'Keefe, 1992; Breslin, Spector, and Grill, 1993; Spector, Grill, and Norgren,
1993; O'Keefe, Schumm, and Smith, Davis, and O'Keefe, 1994; St. John,
Garcea, and Spector, 1994; Markison, St. John, and Spector, 1995; Spector,
1995; Spector, Redman, and Garcea, 1996b). Because the responses are
driven by the hedonic characteristics of the taste solutions, it is impossible to
dissociate neurotomy-induced effects that are based on alterations in
perceived suprathreshold intensity from those that are based on alterations
in affective processing. This is of no consequence interpretively, however,
because as it turns out, CT transection causes remarkably little change in
responsiveness to a variety of taste compounds including NaC1, sucrose,
maltose, and quinine hydrochloride (Yamamoto and Asai, 1986; Spector,
Travers, and Norgren, 1993; Cauthon, Garcea, and Spector, 1994; Spector,
Redman, and Garcea, 1996b; St. John, Garcea, and Spector, 1994) (Figures 3
and 4). Of course, the findings with sucrose, maltose, and quinine should not
be surprising given the results on threshold testing discussed above, but the
lack of effect on NaC1 licking requires some explanation. In brief-access tests
with NaC1, animals must be tested in a water-deprived state otherwise they
do not sample the stimuli. When testing sucrose, no deprivation state is
necessary because of the unconditioned reinforcing properties of the
compound. 7 Under water-deprivation conditions, animals lick at near
maximal rates when water and low to midrange NaC1 concentrations are
presented. Rats do not start to suppress their licking until the concentration
reaches hypertonic values. Thus even if a manipulation made hypotonic
concentrations of NaC1 tasteless to a rat, the brief-access test would not
be able to indicate the altered sensitivity because of ceiling effects.
7It is interesting to note that in the Spector et al. (1996b) study a certain proportion of rats did
not initiate a sufficient n u m b e r o f sucrose trials in a brief-access test before surgery under non-
deprivation conditions to be included in the later phases of the experiment. Apparently the
reinforcing efficacy of the various sucrose solutions was not sufficient to support sampling
behavior in these rats. It would be worthwhile to examine whether this phenotype correlates
with other taste-related affective responses.
118
i"
Ul
2O
2O
" 15
55
5O
40 oy
~o~
CON
~
......~
~.
Alan C. Spector
CTX GL.X ~-~rX+GI~
25
0
-5 I J J , .... ,, .-,,, . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . .
GSPX+CTX GSPX+C'rX+GLX 10 100 1000

,50
"-'o
30
20
-5
10
i . . '~
100 1000 10 100 1000 t0
tf I
t00 1000
SUCROSE CONCENTRATION(~II)
FIGURE 3 The effect of various gustatory nerve transections on unconditioned licking to

sucrose in brief-access taste tests in nondeprived rats. The responses to water were subtracted
out. Each stimulus trial was 10 s and stimulus concentrations were randomized within blocks.
Closed circles: before surgery; open circles: after surgery. CON: sham-operated control; CTX:
bilateral chorda tympani nerve transection; DSAL: extirpation of the sublingual and
submaxillary salivary glands; GLX: bilateral glossopharyngeal nerve transection; GSPX:
bilateral greater superficial petrosal nerve transection. Reprinted with permission from Spector,
Redman, and Garcea (1996). Copyright 1996 by the American Psychological Association.
Nevertheless, it is still noteworthy that the rat's unconditioned responsive-

ness to hypertonic NaC1 concentrations is in general unaffected by CT
transection indicating that this nerve is unnecessary for competence in this
test to be maintained (Yamamoto and Asai, 1986; Cauthon et al., 1994).
Oral Motor Taste Reactivity

Taste-elicited oral motor ingestive responses to certain intraorally infused
taste compounds decrease in frequency after CT transection. The number of
ingestive responses (e.g., tongue protrusions, lateral tongue protrusions,
mouth movements) elicited by brief infusions of NaC1, quinine, and MgC12
(a "bitter" tasting salt to humans) at suprathreshold concentrations is
reduced by CT transection (Grill, Schwartz, and Travers, 1992). In contrast,
the frequency of occurrence of aversive responses to these compounds
"(
CONTROL DESAUVATED CTx
1.2 BEFOREI
_o
1.0
0.8
< 0.6
3:
0.4
~< 0.2
0.0 .... ~ ,,-..1 ..... ~ ....
0.01 0.1 1 0,01 0,1 I 0.01 0.1 I

CONCENTRATION (mM)
GLx GSPx+CTx GLx+CTx

1.2
0
1.0
"'~"
0.8
I-
< 0.6
0.4
0.2
0 . 0 .... ~ , ,-,7 , ,,,,-1 . . . . . . . ,-n ,,,7 . ,..~ .......... ~ , ,,,7 , - 7 ..,
0.01 0.1 1 0.01 0.1 1 0,01 0.1 1

CONCENTRATION ( m M )
FIGURE 4 The effect of various gustatory nerve transections on unconditioned licking to

quinine hydrochloride in brief-access taste tests in water-deprived rats. Licks to quinine were
divided by licks to water. Closed circles: before surgery; open circles: after surgery. CONTROL:
sham-operated control; CTx: bilateral chorda tympani nerve transection; DESALIVATED:
extirpation of the sublingual and submaxillary salivary glands; GLx: bilateral glossopharyngeal
nerve transection; GSPx: bilateral greater superficial petrosal nerve transection. Reprinted with
permission from St. John, Garcea, and Spector (1994). Copyright 1994 by the American
Psychological Association.
(e.g., gapes, chin rubs) is relatively unaffected by the transection (Travers,

Grill, and Norgren, 1987; Grill, Schwartz, and Travers, 1992). The
effectiveness of sucrose, which is a very potent stimulus for the elicitation
of ingestive taste reactivity responses, is unaltered by CT transection
(Travers et al., 1987; Grill, Schwartz, and Travers, 1992).
Stimulus Discrimination
In addition to raising the detection thresholds for NaC1 and KC1, CT
transection also impairs discrimination between the salts. Spector and Grill
(1992) reported that CT transection severely disrupted NaC1 versus KC1
discrimination performance on a conditioned shock avoidance task. This
finding was subsequently replicated using similar and different operant
120 Alan C. Speetor
CON GLX DSAL CTX GLX+CTX Ti'H TRIPLE

SURGICAL GROUP
POSTSURGICAL
-- CHANCE PERFORMANCE
FIGURE 5 Overall performance on a quinine hydrochloride versus KC1 taste discrimination

task collapsed across concentration and taste stimulus before (solid bar) and after (hatched bar)
surgery. The quinine hydrochloride concentrations were 0.1, 0.3, and 1.0 mM; the KC1 conce-
ntrations were 0.1, 0.3, and 1.0 M. Chance performance was 50%. *signifies statistically
significant decrease in performance compared with before surgery; # signifies the lack of a
statistically significant difference from chance performance (p > .05). CON: sham-operated
control; CTX: bilateral chorda tympani nerve transection; DSAL: extirpation of the sublingual
and submaxillary salivary glands; GLX: bilateral glossopharyngeal nerve transection; TRIPLE:
bilateral transection of the greater superficial petrosal, chorda tympani, and glossopharyngeal
nerves; 7TH: bilateral transection of the greater superficial petrosal and chorda tympani branches
of the seventh cranial nerve. Reprinted with permission from St. John and Spector (1998).
Copyright 1998 by the Society for Neuroscience.
conditioning procedures (St. John, Markison, and Spector, 1995; Kopka,

Geran, and Spector, 2000b). St. John and Spector (1998) found that CT
transection also caused significant drops in performance on a quinine versus
KC1 taste discrimination task (Figure 5). Although a neurotomy-induced
decrease in sensitivity to these stimuli likely contributes to some loss of
performance, discriminability of stimulus concentrations above the detec-
tion threshold for CT-transected rats is also impaired. The impairment of
taste discriminability caused by CT transection displays some degree of
chemospecificity as such rats show no impairments in maltose versus sucrose
or quinine versus sucrose discriminations in a conditioned shock avoidance
task (Spector and Gill, 1992; Spector et al., 1997). As a brief methodological
digression, it is worth emphasizing that in all of these discrimination
experiments concentration was varied in an attempt to render intensity an

irrelevant cue. This procedural feature helps minimize the potential for
the animal to discriminate solely on the basis of a strong versus weak taste
irrespective of quality (see Spector, 2003).
Sodium Appetite
Another example of the decreased discriminability of sodium relative to
nonsodium salts is provided by the compromised expression of sodium
appetite in rats with CT transection. When acutely depleted of sodium by
adrenalectomy or natriuretic treatment (e.g., furosemide injection), or
placed on a sodium-deficient diet, intact rats will increase their ingestion of
sodium salts, even at concentrations not normally preferred (e.g., Richter,
1936; Wolf, 1969; Denton, 1982; Epstein, 1984; Fregly and Rowland, 1985;
Schulkin, 1991). The potentiated responsiveness is relatively specific for salts
containing the sodium cation regardless of the anion (Nachman, 1962;
Handal, 1965; Wolf, 1969; Geran and Spector, 2001). This apparently innate
phenomenon offers an opportunity to assess whether the natural ability of
the rat to recognize Na + is altered by manipulations of the gustatory system
(Wolf, 1969). Transection of the CT decreases the depletion-induced intake
of NaC1 in both normal and sham-drinking tests, in which the ingested
solution drains out of a chronically implanted gastric cannula to minimize
postingestive stimulation (Sollars and Bernstein, 1992; Frankmann, Sollars,
and Bernstein, 1996). O'Keefe, Schumm, and Smith (1994) reported that
after CT transection, rats maintained on a sodium-deficient diet decreased
their licking to low concentrations of NaC1 relative to that seen in presur-
gical testing. Breslin, Spector, and Grill (1993, 1995) and others (Markison,
St. John, and Spector, 1995) found that sodium-depleted rats with CT
transections decreased their licking response to NaC1 in brief-access tests but
increased licking to low concentrations of KC1 relative to intact controls.
Interestingly, although CT transection severely compromises the cation
specificity and potentiated intake associated with depletion-induced sodium
appetite and impairs the ability to discriminate NaC1 from KC1, it does not,
in general, entirely eliminate these functional capacities in rats. In contrast,
treatment with the ENaC blocker amiloride completely abolishes the ability
of the rat to discriminate NaC1 from KC1 (Spector, Guagliardo, and St.
John, 1996a) (Figure 6) and eliminates the cation specificity of sodium
appetite at least at low to midrange concentrations (Geran and Spector,
2001). This latter finding suggests that the partial effects of CT transection
on sodium sensibility are due to a decrease in the total number of ENaC-
possessing taste receptor cells. Because functional ENaCs do not appear to
exist in the posterior tongue taste buds (Formaker and Hill, 1991;
Gilbertson and Zhang, 1998; Kitada, Miotoh, and Hill, 1998; Lindemann,
122 Alan C. Spector
NaCI vs. KCI DISCRIMINATION

OVERALL PERFORMANCE]
90 O---(~3----<)---0----
nl
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nl
}- 60
9
uJ
r~ 50 ........................................................................
rY AMILORIDESESSIONS
O 40
O CONTROLSESSIONS
30
.J b = 0.98
<
20 C = 4.57 ~tM
O d = 49.84
10 r2 = 0.99
0 '"1 ' ' ' '''"1 ' ' ' '''"1
1 10 100
[AMILORIDE (#M)]
FIGURE 6 Overall performance on a NaCI versus KC1 taste discrimination task collapsed
across concentration and taste stimulus as a function of the concentration of amiloride
hydrochloride used as the solvent. Amiloride, which is apparently tasteless to rats, is an
epithelial sodium channel blocker and selectively interferes with sodium taste transduction.
The concentrations of NaC1 and KCI were 0.05, 0.1, and 0.2 M. Chance performance was
50%. A 3 parameter logistic function If(x)= ((a-d)/(l+(x/c)b)))-d] was fit to the data, where:
a = constant determined by control session performance, b = slope parameter, c = parameter
representing the midpoint concentration between the constant (a) and the minimum asymptote
parameter (d), r== proportion of variance accounted for by the fit. Reprinted with permission
from Spector, Guagliardo, and St. John (1996a). Copyright 1996 by the Society of
Neuroscience.
G i l b e r t s o n , a n d K i n n a m o n , 1999), the taste r e c e p t o r s o f the p a l a t e are

i m p l i c a t e d as c o n t r i b u t i n g to salt d i s c r i m i n a t i o n function, a c o n c l u s i o n
s u p p o r t e d b y the o b s e r v a t i o n t h a t a m i l o r i d e t r e a t m e n t reduces the
e l e c t r o p h y s i o l o g i c a l l y m e a s u r e d r e s p o n s e o f the G S P to NaC1 ( H a r a d a
et al., 1997; see also G i l b e r t s o n a n d Z h a n g , 1998). I n d e e d , the a t t e n u a t i n g
effect o f C T t r a n s e c t i o n o n s o d i u m a p p e t i t e is further e n h a n c e d b y a m i l o r i d e
t r e a t m e n t ( R o i t m a n a n d Bernstein, 1999). T h e fact t h a t the c h o r d a t y m p a n i
fibers (or s o m a ) t h a t are n a r r o w l y t u n e d to r e s p o n d to N a + ( a n d Li +) salts
have their s o d i u m responses selectively s u p p r e s s e d b y a m i l o r i d e relative to
o t h e r b r o a d l y t u n e d , salt-responsive fibers is c o n c e p t u a l l y c o n s i s t e n t
w i t h the findings discussed a b o v e ( N i n o m i y a a n d F u r a k o s h i , 1988;
H e t t i n g e r a n d F r a n k , 1990; L u n d y a n d C o n t r e r a s , 1999).
Conditioned Taste Aversion
One way to assess whether the perceptual characteristics of taste stimuli

change as a function of an experimental manipulation is through the use of
conditioned taste aversion generalization tests (e.g., Nachman, 1963; Tapper
and Halpern, 1968; Smith, Travers, and VanBuskirk, 1979; Nowlis, Frank,
and Pfaffmann, 1980; Spector and Grill, 1988). In these paradigms,
ingestion of a taste solution during a circumscribed time period (e.g.,
15min) is immediately followed by or concurrently occurs with adminis-
tration of an agent that causes visceral malaise (the emetic LiC1 is commonly
used). On subsequent occasions, animals will decrease their consumption of
the conditioned taste stimulus. In addition, conditioned stimuli that
previously elicited ingestive profiles of oral motor taste reactivity (e.g.,
tongue protrusions to sucrose) will then trigger aversive responses (e.g.,
gapes) (see Grill and Norgren, 1978a; Spector, Breslin, and Grill, 1988). One
can assess the degree of perceptual similarity between the conditioned taste
stimulus and other compounds by including an array of test stimuli in the
procedure. Stimulus generalization, however, can occur in both the intensive
and qualitative dimensions, so it is important that the concentrations be
carefully considered in the experimental design (see Spector and Grill, 1988;
Spector, 2003).
The conditioned taste aversion paradigm is a bit unique with regard to the
functional framework detailed earlier in the chapter. It taps into sensory/
discriminative processes because a response is conditioned to a specific
taste stimulus with a particular qualitative and intensive signature.
It also, however, engages affective/hedonic processes because the motiva-
tional properties of the stimulus are actually changed through conditioning.
In fact, it apparently taps into the physiological reflex domain as well in
that sugars that are reportedly effective preabsorptive secretagogues
of insulin lose their potency after conditioning (Berridge, Grill, and
Norgren, 1981).
The effect of CT transection on the acquisition of conditioned taste
aversions is somewhat equivocal across studies. Yamamoto et al. (1994)
reported that transection or anesthesization of the CT substantially
impaired the acquisition of a conditioned NaC1 aversion. In contrast,
St. John, Markison, and Spector (1997b) found only a modest effect of the
neurotomy on the expression of a NaC1 aversion as assessed in both an
intake and brief-access test. The transection of the CT caused much more
substantial interference in the acquisition of a conditioned taste aversion to
0.1 M KC1, likely because this concentration was weak based on the effect
cutting this nerve has on KC1 detection thresholds (cf., Geran, Guagliardo,
and Spector, 1999). An aversion to KC1 was nonetheless significantly
conditioned in these rats after two conditioning trials. Somewhat more
124 Alan C. Spector
surprising, the NaC1 aversion in the CT-transected rats did not generalize to
KC1. Apparently the remaining peripheral signal is sufficient to allow the rat
to differentially respond to these two salts in this experimental paradigm.
Given that amiloride treatment during conditioning has been shown to lead
to substantial generalization of a NaC1 aversion to nonsodium salts in
subsequent testing (Hill, Formaker, and White, 1990), it seems likely that
the ENaC-possessing taste receptor cells remaining in the palate are
responsible for the maintenance of near normal salt aversion generalization
profiles in CT-transected rats.
Sollars, Tracy, and Bernstein (1996) conditioned an aversion to 0.15M
NaC1 and then transected the CT in F344 and Wistar rats. Subsequent
postsurgical testing in 30 min single-bottle drinking tests revealed that the
conditioned rats with CT transections retained the aversion, but did show a
steeper generalization decrement at concentrations lower than the condi-
tioned stimulus (i.e., < 0.15 M). The authors concluded that the perceptual
characteristics of the stimulus were not sufficiently altered by the neurotomy
to impair its recognition on the postsurgical test. Once again, it would
appear that there was enough of a peripheral signal remaining after CT
transection to maintain performance in this particular task. In passing, it
should be noted that a presurgically conditioned aversion to 0.1 M NaC1 is
abolished by CT nerve crush in hamsters (Barry, Larson, and Frank, 1993).
In addition, CT transection in hamsters attenuates the expression of a
presurgically conditioned aversion to sucrose and monosodium glutamate
(Yamamoto et al., 1988; Harada, 1992).
The Role of Chorda Tympani Efferents Innervating Salivary Glands

As noted above, the CT contains preganglionic parasympathetic efferent
fibers destined for glandular and vascular structures associated with the
oral cavity. The CT supplies a large portion of the innervation of the
sublingual and submaxillary salivary glands. The presence of these efferent
fibers raises the question whether the effects of CT transection are due to
the removal of taste input from the anterior tongue or are attributable to
neurotomy-induced alterations in salivary function affecting the remaining
taste receptors. Indeed, CT transection causes decreases in feeding
efficiency in rats, a characteristic of desalivated animals (Smith et al.,
1988). Nevertheless, the relative specificity of the taste-related behavioral
consequences of CT transection weakens the hypothesis that the effects
were due to a general interference of oral taste receptor function
throughout the oral cavity caused by an interruption of the efferent
fibers. It is true that in some cases, extirpation of the sublingual and
submaxillary glands (a manipulation much more extreme than partial
denervation) has effects on taste-guided responses (Catalanotto and
Sweeney, 1973; Thrasher and Fregly, 1988; Brosvic and Hoey, 1990;
Markison et al., 1995; Spector, Redman, and Garcea, 1996b; St. John
et al., 1997; Kopka, Garcea, and Spector, 2000a), but in other cases such
a manipulation is without consequence (Brosvic and Hoey, 1990; St. John,
Garcea, and Spector, 1994; St. John et al., 1998). Depending on the
measure, some of the observed "taste" effects may actually have had a
motor origin in that licking behavior was generally disrupted (Markison
et al., 1995; Spector, Redman, and Garcea, 1996b). Moreover, removal of
the sublingual and submaxillary salivary glands could lead to functional
impairments specifically in anterior tongue taste buds (Cano and
Rodriguez-Echandia, 1980; Morris-Wiman et al., 2000). In sum, the
degree to which the taste-related behavioral effects of CT transection are
attributable to the loss of CT efferents remains unclear, but based on the
bulk of evidence any impairments in salivary gland function appear to be
superfluous to the primary deafferentation-induced loss of sensory input
from anterior tongue taste buds.
The consequences of CT transection on taste-related behavior hardly
paint a simple picture of function. As the paragraphs below will explain,
however, when the behavioral effects of the transections of other gustatory
nerves are considered, interesting functional principles begin to emerge.
Nevertheless, the complexity of effects generated by a single nerve
transection highlights the importance of applying a variety of taste-related
tasks to provide a more comprehensive view of taste function following
manipulations of the gustatory system.
D. EFFECTS OF GLOSSOPHARYNGEALNERVE TRANSECTION

ON TASTE-RELATED BEHAVIOR
Although electrophysiological measures indicate that the GL is very
responsive to orally applied quinine relative to the other gustatory nerves
in rats, its transection causes no change in quinine detection thresholds,
notwithstanding the caveat that the control group in this study decreased
its threshold after sham surgery (St. John and Spector, 1996). Likewise,
concentration-response functions (measured in within-subjects designs)
produced by water-deprived rats licking quinine during brief-access tests are
unaffected by GL transection (St. John, Garcea, and Spector, 1994). It
should be noted, however, that GL-transected rats without presurgical
testing experience (i.e., in between-subjects designs) do show some modest
decrement in unconditioned responsiveness to midrange-high concentra-
tions of quinine (Spector and St. John, 1998; Markison, St. John, and
Spector, 1999). Transection of the GL produces no remarkable effects on
126 Alan C. Spector
unconditioned responsiveness to sucrose, maltose, or NaC1 in brief-access

tests (Cauthon et al., 1994; Spector, Redman, and Garcea, 1996b).
Combined transection of the CT and GL does, however, result in signifi-
cant effects on some taste-related behavior. Although transection of either
nerve alone has little effect on quinine sensitivity, combined transection of
the CT and GL has a synergistic effect, raising the detection threshold by
over an order of magnitude (St. John and Spector, 1996). Likewise, in brief-
access taste tests, combined transection of the CT and the GL results in
substantial rightward shifts in the quinine licking avoidance curve and a
modest suppression of unconditioned licking for high concentrations of
sucrose and maltose (Yamamoto and Asai, 1986; St John, Garcia, and
Spector, 1994; Spector, Redman, and Garcea, 1996b). This suggests that
there is central convergence between input of the CT and the GL with
respect to processes governing stimulus acceptability and simple chemical
detection. Alternatively, perhaps changes in these particular taste-related
behaviors are not observed until the input from a certain criterion number
of taste receptor cells is removed from the peripheral signal. These two
hypotheses are not mutually exclusive.
Performance in a variety of operantly conditioned taste discriminations
including maltose versus sucrose, NaC1 versus KC1, NaC1 versus NHaC1,
NH4C1 versus KC1, and quinine versus KC1 remains relatively unaltered
after GL transection (St. John and Spector, 1998; Spector and Gill, 1992;
Spector et al., 1997; Geran, Garcea, and Spector, 2002a). It was the latter
experiment, conducted by my former student Steven St. John for his
dissertation, that forced the reevaluation of the potential role of the GL in
taste function (St. John and Spector, 1998). In this study, rats were trained
to discriminate quinine from KC1, and as is typical in our discrimination
procedures, concentration was varied to render intensity cues irrelevant.
Quinine and KC1 were chosen because the GL appears to contain afferent
fibers that respond differentially to these stimuli (Frank, 1991). The Q-fibers
respond rather selectively to quinine (and some other "bitter-tasting"
compounds) and respond poorly to KC1. In contrast, KC1 stimulates
activity in A-fibers, which respond very well to acids and salts but do not
respond at all to quinine. Although the CT responds to both stimuli, this
activity appears to be attributable to broadly tuned fiber types that are
stimulated by both compounds (Frank, Contreras, and Hettinger, 1983;
Lundy and Contreras, 1999). Thus, the GL has units that differentially
respond to the two taste compounds in question and the CT does not.
Accordingly, we reasoned that GL transection should disrupt the
discrimination, whereas CT transection should have no effect. To our
The Functional Organization of the Peripheral Gustatory. System 127
surprise, the opposite occurred (Figure 5). Transection of the GL was

unequivocally without effect, whereas CT transection significantly disrupted
discrimination performance. Animals that had both the CT and the GL
transected performed equally to those that had the CT transected alone,
further suggesting that the input of the GL was unnecessary for competence
in this discrimination. The disparity between the behavioral outcomes and
those predicted from the electrophysiological findings remains to be
resolved, but quinine and KC1 stimulation must lead to some differential
signal, either spatially or temporally based, in the CT (and GSP, see below).
One potential example of a temporally based explanation would be
differences in the rise time of the respective taste sensations relative to
somatosensory stimulation by the fluid. Differences in rise time could derive
from differences in the respective transduction processes engaged by the two
stimuli in taste receptor cells (e.g., second messenger versus ion channels).
To date, performance on any task that requires taste recognition or
qualitative taste discrimination is unaffected by GL transection in rats.
Transection of the GL has no effect on the acquisition of conditioned taste
aversions to NaC1 or sucrose and does not compromise the robustness or
cation specificity of a depletion-induced sodium appetite (Yamamoto et aI.,
1994; Markison et al., 1995; Eylam, Garcea, and Spector, 2000). Of course,
only a relatively limited number of taste compounds have been tested, but
given that the GL innervates ~60% of the total taste buds in the oral cavity
of the rat, the relative lack of consequences on taste-guided licking behavior
when this nerve is individually severed is remarkable.
Oral Motor Taste Reactivity

The findings just reviewed lead one to question the primary role of the GL in
taste function. Some insight into GL function has been achieved through the
assessment of unconditioned protective oral motor rejection reflexes, espe-
cially the gape. Transection of the GL markedly decreases the number of
gapes elicited by intraorally infused quinine, whereas transection of the CT
has relatively marginal effects (Travers, Grill, and Norgren, 1987; Grill,
Schwartz, and Travers, 1992; King, Garcea, and Spector, 2000). The
frequency of occurrence of aversive oral motor responses to quinine,
MgC12, and NaC1 has also been reported to decrease after GL transection.
It is somewhat counterintuitive that transection of the GL markedly
compromises quinine-induced gaping, but has no effect on quinine detection
threshold, and modest, if any, effects on unconditioned avoidance responses
to quinine, depending on how it is measured. It is precisely these types of
findings that point to differences in the functional roles of the gustatory
nerves. Keep in mind that spout licking in brief-access taste tests involves
both appetitive and consummatory components, but taste reactivity to intra-
128 Alan C. Spector
orally infused stimuli is purely consummatory. The failure of the decerebrate

rat preparation to spontaneously eat or drink but nonetheless to display
the full range of concentration-dependent oromotor responses to taste stimuli
clearly demonstrates that appetitive taste responses can be neurally
dissociated from consummatory behavior (Grill and Norgren, 1978b, c).
Although transection of the GL substantially decreases the frequency
of occurrence of gapes elicited by quinine, it does not eliminate them. If the
CT is transected in addition to the GL, however, gaping to quinine, even
at a concentration of 3.0 mM (which is high), is virtually eliminated (Grill,
Schwartz, and Travers, 1992). Thus, it appears that while the GL is
necessary for normal quinine-elicited gaping to occur and the CT is not, the
CT, nonetheless, makes a contribution to this behavioral response that is
only revealed in the absence of GL input (i.e., CT transection -I- GL
transection eliminates gapes). This also suggests that the input of the GSP
(and SLN) is not sufficient to support quinine-elicited gaping in the rat. It is
also worth noting that although neither GL nor CT transection alters
ingestive oral motor responses to sucrose, combined transection of the
nerves substantially suppresses this behavior and flattens the sucrose
concentration-response function. Once again, this suggests that ingestive
oral motor taste reactivity to sucrose is supported by converging inputs of
the CT and GL or that a threshold level of receptor loss must be reached
before deficits are expressed.
In addition to markedly reducing the occurrence of gapes elicited in
response to quinine, GL transection also changes the topographic pattern of
quinine-induced c-Fos-like immunoreactive (FLI) neurons in the rostral NST
of the rat (King et al., 1999; King, Garcea, and Spector, 2000). The
expression of the immediate early gene product c-Fos is thought to represent
neurally excited cells, at least in a subpopulation of neurons, and thus is
useful for constructing spatial maps of neuronal activity in the brain under
various stimulus conditions (see Morgan and Curran, 1989; Sheng and
Greenberg, 1990; Morgan and Curran, 1991; Sagar, Sharp, and Curran,
1991; Sharp, Sagar, and Swanson, 1993). The distribution and number of
FLI neurons under conditions of quinine stimulation in GL-transected rats is
indistinguishable from that observed in intact rats stimulated with water
(Figures 7 and 8). Although CT transection reduces the number of FLI
neurons stimulated by quinine, the topographic pattern within the rostral
NST is unaffected by the neurotomy. These findings suggest that the rostral
NST neurons expressing c-Fos in response to orally infused quinine are
related in some way to circuits involved in the production of unconditioned
oral motor rejection responses to taste stimuli.
The fact that GL transection reduces oral motor rejection responses
unconditionally elicited by quinine and certain other aversive stimuli, raises
the question about whether an intact GL is required for gaping under any
FIGURE 7 Representative photomicrographs of cross-sections taken through the gustatory

zone of the nucleus of the solitary tract (NST) depicting Fos-like immunoreactivity (FLI). These
sections were taken at a level about 150300/am rostral to where the NST first abuts the 4th
ventricle. The sections were divided into six portions referred to as subfields for quantification.
A. FLI produced by intraoral quinine (3.0 raM) stimulation in sham-operated rats. B. FLI
produced by intraoral quinine (3.0 mM) stimulation in rats with regenerated glossopharyngeal
nerves. C. FLI produced by water stimulation in sham-operated rats. D. FL1 produced by
intraoral quinine (3.0 mM) stimulation in rats in which regeneration was prevented following
bilateral glossopharyngeal nerve transections. Compare with Figure 8. Scale bar: 100 gm.
Reprinted with permission from King, Garcea, and Spector (2000). Copyright 2000 by the
Society for Neuroscience.
circumstance. T o address this, E y l a m et al. (2000) transected the G L a n d

then c o n d i t i o n e d a taste aversion to 0.3 M sucrose. The G L - t r a n s e c t e d
rats acquired the aversion j u s t as well as s h a m - o p e r a t e d controls as assessed
by a 15-rain single-bottle intake test. These a n i m a l s were then tested for
their oral m o t o r responses to a 1-min i n t r a o r a l i n f u s i o n of 0.3 M sucrose.
There was absolutely n o difference between the two groups in terms of
gaping or a n y other oral m o t o r response. This finding d e m o n s t r a t e s that
rats with G L t r a n s e c t i o n are capable o f g a p i n g a n d that i n p u t from the
r e m a i n i n g g u s t a t o r y nerves can sufficiently serve for the c o n d i t i o n i n g of
rejection responses to a n otherwise p a l a t a b l e taste stimulus. 8
8The possibility that retronasal olfaction or trigeminal stimulation contributed to the

maintenance of the function in the absence of the GL cannot be entirely dismissed.
130 Alan C. Speetor
n0,35 ~ A Q=siimutatsd C W-stimulated

SHAMsubjects SHAMsubjects
o.3o 1
o.2st
~o.2o t
~.,o]
0,3,
~ 0.30 'i
e_ 0,25
.]
I
REGsubjects
O Q*stimutated
GLX& PRE subje~
0,0 t
1 2 3 4 5 6 1 2 3 4 5 6
Subfield Subfield
FIGURE 8 Pattern of Fos-like immunoreactivity (FLI) in the six cross-sectional subfields

averaged across four levels spanning the rostral nucleus of the solitary tract (the gustatory
zone). Each cross-section was divided into six portions referred to as subfields for quantification
as shown in Figure 7. The vertical axes represent the proportion of the total FLI cells in each
subfield. This measure allows the pattern of the response to be revealed independent of the total
number of FLI cells expressed. A. pattern of FLI produced by intraoral quinine (3.0 raM)
stimulation in sham-operated rats. B. pattern of FLI produced by intraoral quinine (3.0 raM)
stimulation in rats with regenerated glossopharyngeal nerves. C. pattern of FLI produced by
water stimulation in sham-operated rats. D. pattern of FLI produced by intraoral quinine (3.0
mM) stimulation in rats in which regeneration was prevented following bilateral glossophar-
yngeal nerve transections. Subfield 1: dorsal-lateral portion, Subfield 2: dorsal-central portion,
Subfield 3: ventral-lateral portion, Subfield 4: ventral-central portion, Subfield 5, dorsal-medial
portion, Subfield 6, ventral-medial portion. Reprinted with permission from King, Garcea, and
Spector (2000). Copyright 2000 by the Society for Neuroscience.
E. EFFECTS OF COMBINED TRANSECTION OF THE C H O R D A TYMPANI

AND G R E A T E R SUPERFICIAL PETROSAL NERVES

Together, the CT and the GSP comprise the collective gustatory input
provided by the 7th cranial nerve. Thus, it is informative to compare the
relative consequences of 7th versus 9th nerve transection on taste-guided
behavior, assuming that the SLN plays a minor, if any, role. Combined
transection of the CT and GSP significantly depresses unconditioned
responsiveness to sucrose and maltose, but does not eliminate it as assessed
in brief-access tests (Krimm et al., 1987; Spector, Redman, and Garcea,
1996b). The additional removal of the GL essentially flattens the
concentration-response function, suggesting that the SLN does not make
a significant contribution to this particular behavior.
These results raise the question of what the effects of GSP transection
alone would be. In our work, we have not yet included this manipulation
because we are not confident that the GSP can be transected without
potentially damaging the CT, at least using our current surgical approach. 9
Krimm et al. (1987), reported that GSP transection above was just as
effective as when it was combined with CT transection in reducing sucrose
responsiveness measured in brief-access tests in rats. Histological examina-
tion of the anterior tongue, however, was only qualitatively evaluated,
leaving open the possibility that the CT was inadvertently damaged at least
in some of the rats that received GSP transection alone. Spector et al. (1993)
found that cautery of the nasoincisor ducts (NID; supplied by the GSP)
alone did not attenuate unconditioned sucrose responsiveness in rats unless
it was combined with bilateral CT transection, which by itself produced
a marginal decrease, supporting the existence of centrally converging inputs
from the two nerve branches.
Combined transection of the CT and GSP also significantly attenuates
the unconditioned lick avoidance of NaC1 and quinine in brief-access tests,
but does not eliminate it (Spector et al., 1994; Spector, 1996). The G L is
apparently capable of maintaining some degree of lick avoidance with respect
to high concentrations of NaC1 and quinine. St. John et al. (1994) reported
results from a single rat that had the G L transected in addition to transection
of the CT and GSP; this animal had a flat quinine concentration-response
curve in the brief-access test and responded to concentrations as high as
3.0 m M as if they were water. Although only a sample size of one, this result
once again suggests that the input of the SLN is not sufficient to maintain
any unconditioned responsiveness to these taste compounds.
One of the most interesting findings is that regardless of the compound
tested, performance in sensory-discriminative tasks in which the taste
stimulus serves as a signal is severely impaired by combined transection of
the CT and the GSP, but is absolutely unaffected by transection of the GL.
This includes a sucrose versus maltose discrimination and a quinine versus
KC1 discrimination (Spector et al., 1997b; Spector and St. John, 1998). In
both of these examples, discrimination performance was substantially
impaired by the removal of the 7th nerve taste input, but some degree of
discriminability did remain, albeit extremely blunted. In other discrimina-
tion tests, including NaC1 versus NH4C1 and KC1 versus NH4C1,
9David Hill has communicatedthat he uses a ventral approach through the bulla to exposethe
GSP with minimal risk to the integrity of the CT. This also might be more favorable for GSP
regeneration.
132 Alan C. Spector
performance dropped to chance levels after combined transection of the

gustatory branches of the 7th nerve, but was relatively unaffected by GL
transection (Geran, Garcea, and Spector, 2002b). It is true that only a
limited number of tasks and a small number of taste stimuli have been tested
to date, but the data available consistently support the hypothesis that
sensory/discriminative taste function relies heavily on the collective
gustatory input of the 7th nerve, but not on that of the 9th nerve (see
St. John and Spector, 1998).
F. SUMMARYAND FUNCTIONAL IMPLICATIONS
One lesson from the tapestry of findings generated across taste-related

behavioral experiments with nerve-transected rats is that a single testing
method is insufficient to capture all of the functional consequences of the
neurotomy on gustatory function. In some cases, a given nerve transection
can lead to unequivocal deficits in responsiveness to a given taste stimulus
as measured by some tasks and then have absolutely no effect on
responsiveness as measured by other tasks. For example, CT transection
causes substantial deficits in signal detection of low concentrations of
NaCI, disrupts the cation specificity and robustness of sodium appetite, alters
salt discriminability, yet has no effect on NaC1 preference in two-bottle
intake tests or on NaC1 lick avoidance in brief-access taste tests. Of course, it
is these kinds of dissociations of effects that buttress the functional
framework set forth earlier in this chapter.
Within the context of that functional framework, there are several
implications emerging from the collective experimental results discussed
above.
The gustatory branches of the 7th cranial nerve (i.e., CT and GSP)
provide critical input that is channeled into neural circuits subserving
taste signal discrimination (i.e., input that allows the animal to
discriminate one taste from another). The input of the 9th nerve is
unnecessary with regard to this function.
The 9th cranial nerve appears to be critical for normal unconditioned
oral motor rejection reflexes such as gaping. The input of the CT
(and perhaps the GSP) is unnecessary with regard to this function.
Detection of the presence (or absence) of a taste stimulus regardless of its
qualitative features and unconditioned approach-avoidance behavior
appear to be supported by inputs from at least two of the three major
gustatory nerve branches (CT, GL, and GSP) depending on the stimulus.
It is noteworthy that there is electrophysiological evidence for a central
convergence of signals from segregated oral taste receptor fields (Ogawa
and Kaisaku, 1982; Ogawa and Nomura, 1988; Travers, Pfaffman, and
Norgen, 1986; Sweazey and Smith, 1987; Travers and Norgen, 1995;
Grabauskas and Bradley, 1996; Halsell and Travers, 1997).
The above implications require some qualification. First, more taste
compounds must be tested with a wider variety of tasks that are designed to
assess behavior with regard to the postulated functional domains. Even
within a task there are a number of procedural parameters (e.g., number of
sample licks, timing of trial components, reinforcement size, deprivation
state, number of conditioning trials, presurgical experience) that if varied
might reveal functional competence or deficits that were previously
obscured.
Second, the distinction between which nerves are necessary and which
nerves are sufficient in the maintenance of function deserves some
consideration. It is clear that in every case tested, to my knowledge,
the 7th nerve is necessary for normal taste discrimination or recognition to
be behaviorally expressed, whereas the 9th nerve is not. Likewise, the 9th
nerve is necessary for normal unconditioned gaping to occur in response to a
limited number of tested compounds that are normally avoided by rats,
whereas the CT, at least, is not. The degree to which these respective nerves
are sufficient for the proposed functions is not absolute. Thus, in the absence
of a nerve necessary for normal function to be expressed, slight partial
competence sometimes remains with respect to the disrupted behavior,
depending on the taste compounds and the task. Some of the remaining
function may be due to the contribution of the SLN which is left intact, or to
the involvement of trigeminal or olfactory input. This possibility, however,
is weakened in cases where transection of the CT, GL, and GSP completely
eliminates behavioral competence (e.g., St. John and Spector, 1998).
Third, there is not such a clear-cut distinction between the contribution
of 7th and 9th cranial nerves with respect to taste detection and approach-
avoidance behavior that universally applies to all taste stimuli.
A prototypical example of this point is the fact that transection of the CT
or GL alone does not cause substantial impairments in taste sensitivity or
avoidance behavior with respect to quinine, but their combined transection
does. It is important to note, however, that detection thresholds and
approach-avoidance behavior do not necessarily reveal anything about a
chemical's discriminative qualitative features which appear to depend on the
signals carried in the branches of the seventh cranial nerve, at least based on
the experiments conducted to date.
Fourth, none of the experiments discussed above measured function in
the physiological domain and it is tempting to speculate that the 9th nerve
might play a role in governing such reflexes (e.g., taste-elicited salivation) as
has been proposed by others (Frank, 1991).
134 Alan C. Spector
Fifth, in some cases there is a satisfying correspondence between the

known electrophysiological properties of the transected nerves and the
behavioral consequences of the neurotomy, but other times the link is more
elusive. A greater effort to characterize the response properties of the taste
fibers comprising these nerves within the context of the behavioral results
discussed above might help to resolve some of these apparent paradoxes
while at the same time provide insight into the principles of neural coding at
this primary stage of gustatory processing. More to the point, the behavioral
consequences of gustatory nerve transection do not reveal the details of
the neural mechanisms subserving the effects. For example, transection
of a nerve removes input that can potentially be excitatory or inhibitory
on second order neurons. This can obviously alter the pattern of down-
stream activity in the local and ascending connections of the gustatory
system ultimately leading to the functional consequences observed.
However, the organizational principles underlying processing in the central
gustatory system can be revealed through efforts to link the behavioral
outcomes of nerve transection with changes in patterns of taste-stimulated
activity at the cellular and intercellular level (cf., King et al., 1999; King,
Garcea, and Spector, 2000).
VI. Functional Consequences of Nerve Regeneration

After nerve crush or nerve transection, the taste buds of the denervated
receptor field degenerate (e.g., Vintschgau and Honingschmid, 1876;
Whiteside, 1927; Guth, 1957; Fujimoto and Murray, 1970; Cleaton-Jones,
1971; el Eishi and State, 1974; Cheal and Oakley, 1977b; Miller and Spangler,
1982; Hosley, Hughes, and Oakley, 1987; Ganchrow and Ganchrow, 1989;
Hard af Segerstad, Hellekant, and Farbman, 1989; Barry and Frank, 1992;
Oakley et al., 1993; St. John, Markison, and Spector, 1995; Ninomiya, 1998).
Following bilateral GL transection, the taste buds in the circumvallate
papilla and in most of the foliate trenches entirely disappear (some taste
buds of the anterior trenches of the foliate are innervated by the CT). After
CT transection some taste buds remain, depending on the species, but many
of these are "abnormal" morphologically (Cheal and Oakley, 1977;
Whitehead et al., 1987; Hard af Segerstad, Hellekant, and Farbman, 1989;
Barry and Frank, 1992; Parks and Whitehead, 1998). After GSP transection
in rats the majority of taste buds in the nasoincisor ducts and geschmacks-
streifen disappear, but in some cases, a few taste buds in the posterior
palatine field survive transection (Cleaton-Jones, 1971; Miller, 1977; Spector,
Redman, and Garcea, 1996b; Spector et al., 1997; St. John and Spector, 1998;
St. John, Garcea, and Spector, 2002).
It is well documented that taste buds reappear following regeneration of
the crushed or transected nerve (Oakley, 1967a, b; Oakley and Cheal, 1975;
Cheal et al., 1977; Barry and Frank, 1992; St. John et al., 1995; Cheal and
Oakley, 1997). These results, coupled with the degeneration seen after nerve
damage, demonstrate that the presence of morphologically intact taste buds
is trophically dependent on gustatory innervation. Regenerating taste fibers
find their way to their appropriate receptor fields. That is, regenerating GL
fibers innervate the posterior tongue and regenerating CT fibers innervate
the anterior tongue. Because the peripheral processes of the remaining intact
taste axons apparently do not sprout, the reappearance of morphologically
intact taste buds is attributable to epithelial reinnervation by the transected
nerve (Kinnman and Adlskogius, 1988; Barry and Frank, 1992). A finding
buttressed by the observation that when the nerve is severed in a manner
that prevents regeneration (e.g., removal of a long segment), taste buds do
not reappear, at least within the time frame of the experiments that have
examined this (cf., King, Garcea, and Spector, 2000; Kopka et al., 2000b,
2001). Although the CT and GL appear to regenerate with ease, the GSP
does not do so as readily. St. John et al. (2002) found that at 112 days after
bilateral GSP transection less than 25% of the palatal taste buds reappeared
and this number did not increase appreciably by 224 days. It remains
unclear why this is so, but it could be related to the surgical approach. 9 It is
noteworthy that in gerbils and cats, CT nerve crush promotes faster and
more effective reinnervation of the lingual epithelium compared with CT
transection, and perhaps a similar situation is true for the rat GSP (Cheal
and Oakley, 1977; Robinson, 1989; Robinson and Winkles, 1991). When CT
regeneration is allowed following transection in the middle ear in rats, taste
buds return starting between 14 and 28 days after surgery and reach
asymptote at about 42 days. The number of morphologically intact taste
buds at asymptote is 75% the normal complement (St. John et al., 1995) and
appears to be related to a concomitant degeneration of fungiform papillae
which, when denervated, develop ectopic filliform spines (Figure 9) (cf.,
Oakley et aI., 1993). There is evidence that the return of circumvallate taste
buds after GL transection reaches a maximum that is also ~25% less than
the number in intact rats ((King, Garcea, and Spector, 2000; St. John, Garcea,
and Spector, 2003), Figure 10). In humans, CT regeneration following nerve
transection without repair appears to be relatively uncommon, but can be
promoted by coaptation of the proximal and distal stumps upon which some
reinnervation of the affected receptor field occurs (Chilla, Nicklatsch, and
Arglebe, 1982; Grant et al., 1989; Zuniga, Chen, and Miller, 1994; Zuniga,
Chen, and Phillips, 1997; Saito et al., 2001).
After CT nerve crush and regeneration in hamsters, only 33% of the
myelinated fibers remain, even at 16 weeks, and the area and density of
axonally transported label in the terminal projection field for this nerve
in the NST is smaller by 23 and 37%, respectively (Cain, Frank, and Barry,
1996; Barry, 1999). Interestingly, transection of the CT in the hamster
136 Alan C. Spector
A C
180
[
180
12o
so
9o,"]2i E:"~~
~?:-?E~E~?ZE~;5~E~
1 . 0 - ~ . ~ , . ~ . . . . . . . . . . . . . . . . . . . . . . . . . ~ ......
/
0.8-
120
0.6-
90
o 0.4-
60
60 -] ---COMBINED CON
..........STANDARD ERROR
t
0.2-
~J 50 50 ~. CONTROLGROUPS
< 0 CTX GROUPS
0 i l L i i 0.0 ~ , ~ t , 0 L I I t
14 28 42 56 70 14 28 42 56 70 14 28 42 56 70
DAYS S I N C E S U R G E R Y DAYS S I N C E S U R G E R Y D A Y S S I N C E SURGERY
FIGURE 9 A. Regeneration of anterior tongue taste buds in various groups of rats as a

function o f days since bilateral chorda tympani nerve transection in the middle ear. B. Ratio of
taste pores per fungiform papillae in the same animals. C. Number of fungiform papillae in the
same animals. Closed circles: means (-4- se) for nerve-transected groups; Open triangles: means
(4- se) for sham-operated control groups. Dashed and dotted lines represent the mean and se
respectively of the two control groups combined. Reprinted with permission from St. John,
Markison, and Spector (1995).
causes minimal loss of geniculate ganglion cells, but does result in persistent
evidence of NST degeneration even at 161 days postsurgery when the nerve
has regenerated (Whitehead, McGlathery, and Manion, 1995). Thus,
although the CT regenerates in hamsters, the associated peripheral and
central anatomy apparently do not return completely to normal and this
could likely be true for the rat as well.
Given that the CT and GL have such a great proclivity to regenerate in
rodents leads one to question to what extent function returns upon
regeneration of a transected gustatory nerve. In hamsters, a presurgically
conditioned NaC1 aversion abolished by CT nerve transection was
reacquired upon CT regeneration (Barry, Larson, and Frank, 1993). In
fact, in most instances to date in which transection of a taste nerve impaired
performance on a taste-related task, once the nerve regenerated, function
returned to normal, despite that only about 3/4ths of the normal number
of taste buds reappeared (St. John, Markison, and Spector, 1995; King,
Garcea, and Spector, 2000; Kopka et al., 2000a, b; Kopka and Spector,
2001). For example, bilateral transection of the CT increases NaC1 threshold
by over an order of magnitude and significantly impairs a NaC1 versus KC1
discrimination in rats (Spector, Schwartz, and Grill, 1990; Spector and Grill,
1992; Slotnick et al., 1991; St. John et al., 1995, 1997a). As illustrated in
Figures 11 and 12, in both of these tasks function returns to normal once
the nerve regenerates (Kopka et al., 2000a, b; Kopka and Spector, 2001).
A 500 94
400.
m
o 300
O3
AH
0~
F- 200
AN
> AN
0100
B
5O
40
~9
20
10
C
~50
E
"~5 40
o
30
03
g20
. p < .002
tI
10
|
I
--7/// . . . . , ~
380 400 420 440 460 480 500 520
C V Taste Buds
FIGURE 10 The mean (+ se) number of taste buds in the circumvaltate papilla (panel A)
and frequency of occurrence of gapes during the first min of a 30-min intraoral infusion
(panel B) in different groups of rats at 17, 52, and 94 days (numbers at top of each panel) after
surgery. Shaded hatched bars: groups that were infused with 3.0 mM quinine hydrochloride;
unshaded hatched bars: groups that were infused with water. SHAM: sham-operated control;
GLX: short survival time, bilateral glossopharyngeal nerve transection; REG: longer survival
time, bilateral glossopharyngeal nerve transection with regeneration promoted; PRE: longer
survival time, bilateral glossopharyngeal nerve transection with regeneration prevented. Panel C:
Correlation between gapes to intraoral infusions of 3.0 mM quinine hydrochloride and
circumvallate taste bud number in sham-operated rats. See King et al. (2000) for further
explanation of symbols related to statistical analysis. Reprinted with permission from King,
Garcea, and Spector (2000). Copyright 2000 by the Society for Neuroscience.
138 Alan C. Specter
Z,'!'
100
LU SHAM-7 SHAM-62 CTX-62R

~ 80.
F-
-.~ 60
~ 40
20
~ o
0.001 0.01 0.1 0.001 0.01 0.1 0.001 0.01 0.1 1

100
/l,ii
i PRESURG]CAL
W POSTSURGICAL
AMILORIDE
I.-
--
-r 60
(:3
~_ 40
Ul
rY 20
n~
0
~ 0
0.001 0.01 0.1 0.001 0.01 0.1
NaCI CONCENTRATION (M)
FIGURE 11 Mean (-4- se) corrected hit rate in a taste signal detection task as a function
of NaC1 concentration in various groups of rats. The corrected hit rate was based on
the actual hit rate (correct NaC1 detections) adjusted by the false alarm rate (reporting the
presence of NaC1 during water presentations). Threshold was arbitrarily defined as the
concentration at one-half the asymptotic corrected hit rate. Closed circles: presurgical
threshold determination; open triangles: postsurgical threshold determination; closed squares:
postsurgical threshold determination with 100 gM amiloride hydrochloride adulterating the
solutions. SHAM-7: sham surgery with postsurgical testing starting at seven days; SHAM-
62: sham surgery with postsurgical testing starting at 62 days; CTX-62R: bilateral chorda
tympani nerve transection with regeneration promoted and postsurgical testing starting at
62 days; CTX-7P: bilateral chorda tympani nerve transection with regeneration prevented
and postsurgical testing starting at seven days; CTX-62P: bilateral chorda tympani nerve
transection with regeneration prevented and postsurgical testing starting at 62 days. Notice
that threshold sensitivity to NaC1 returns to normal following chorda tympani nerve
regeneration. The effectiveness of amiloride to raise threshold is eliminated by chorda
tympani nerve transection provided the nerve does not regenerate. If the nerve does
regenerate then the effectiveness of amiloride to compromise NaC1 sensitivity returns to
normal suggesting that functional ENaCs are present in regenerated anterior tongue taste
buds. Reprinted with permission from Kopka and Spector (2001). Copyright 2000 by the
American Psychological Association.
I m p o r t a n t l y , in a n i m a l s in w h i c h t h e C T w a s p r e v e n t e d f r o m r e g e n e r a t i n g
(by c a u t e r y - i n d u c e d c e r u m i n p r o d u c t i o n filling t h e m i d d l e ear), f u n c t i o n
remained impaired even though the rats were tested after the same
p o s t s u r g i c a l t i m e p e r i o d as t h e a n i m a l s w i t h r e g e n e r a t e d nerves. T h i s
r e s u l t d e m o n s t r a t e s t h a t t h e r e c o v e r y o f f u n c t i o n is d u e t o r e g e n e r a t i o n o f
SHAM-7 (n = 2) SHAM-62 (n = 5) CTX-62R (n = 5)

1.0
__'%
t"- 0.9
o
UJ
n," 0.8
n-
O
tO 0.7
Z
O 0,6
I--
O O.5
C1. Means ef ~/I- Means of Means of
o Mean Curve Individual Curves. Mean Curve Individual Curves Mean Curve Individual Curves
n" 0.4 b = 0.93 b = 1.018 (0.082) b = 0.96 b = 0.998 (0.105) - b = 1.48 b = 1.686 (0.470)
13... C = 6.61taM c = 6.68gM (1.66) c = 4.71gM C = 4.65taM (1.29) C = 5.97taM c = 6.05pM (1.12)
,._1
-- 0.3 d = 0.46 d = 0.461 (0.029) d = 0.48 d = 0.478 (0.015) - d = 0.49 d = 0.480 (0.024)
< r 2 = 0.99 r2 = 0.975 (0,005) r2 = 0.99 r2 = 0.948 (0.021) r2 = 0.99 r2 = 0.980 (0,012)
n*
LLI 0,2
>
0
0.1 I 0 em]lor[de ]
control (no amiloride)
0.0
1 10 100 1 10 100 1 10 100
AMILORIDE CONCENTRATION (uM)
FIGURE 12 Overallperformance on a NaC1 versus KC1 taste discrimination task collapsed

across concentration and taste stimulus as a function of the concentration of amiloride
hydrochloride used as the solvent in three groups of rats. The concentrations of NaC1 and KC1
were 0.05, 0.1, and 0.2 M. Chance performance was 50%. See Figure 6 for the definition of the
parameters from the logistic curve fit. SHAM-7: sham surgery with postsurgical testing starting
at seven days; SHAM-62: sham surgery with postsurgical testing starting at 62 days; CTX-62R:
bilateral chorda tympani nerve transection with regeneration promoted and postsurgical testing
starting at 62 days. Note that the effectiveness of amiloride to disrupt a NaC1 versus KC1 taste
discrimination is normal in rats with regenerated chorda tympani nerves. Reprinted with
permission from Kopka, Geran, and Spector (2000b).
the nerve and not solely to some other c o m p e n s a t o r y process that occurs
over time such as some type o f reorganization o f central circuits. Also,
animals with regenerated CTs are just as sensitive to the p e r f o r m a n c e
disrupting effects o f amiloride (Figure 12). Remember, amiloride is an
E N a C blocker, virtually tasteless to rats, that suppresses (but does not
eliminate) C T (and GSP) responses to NaC1 applied to the anterior tongue
(or palate) (e.g., Hock, Mierson, and DeSimone, 1984; Simon et al.,
1993; M a r k i s o n and Spector, 1995; G a r r y and Palmer, 1997; Sollars and
Hill, 1998; L i n e t al., 1999; Lindemann, Gilbertson and K i n n a m o n , 1999).
The partial inhibition is t h o u g h t to be due to the fact that it blocks the
relatively cation-selective apically positioned transcellular sodium transduc-
tion p a t h w a y , while not affecting less cation-selective transduction p a t h w a y s
(e.g., Heck, Mierson, and DeSimone, 1984; Ye, Heck, and DeSimone, 1993;
140 Alan C. Spector
Stewart, DeSimone, and Hill, 1997; Lindemann, Gilbertson, and

Kinnamon, 1999). Behaviorally, in intact rats, stimulus adulteration raises
the NaC1 detection threshold by close to an order of magnitude and severely
impairs NaC1 versus KC1 discrimination performance (Geran and
Spector, 2000a, b; Kopka et al., 2000b; Kopka and Spector, 2001). As is
shown in Figures 11 and 12, amiloride adulteration of the stimuli disrupted
NaC1 sensitivity and salt discrimination performance in rats with
regenerated CTs just as much as it did in intact rats. Thus, it would
appear that the functional status of the amiloride-sensitive transduction
pathway of the anterior tongue taste buds innervated by the regenerated CT
is normal as assessed psychophysically.
The psychophysical recovery of taste function following regeneration of
gustatory nerves is consistent with whole-nerve and single-fiber electro-
physiology studies in rodents. Regenerated nerves appear to have the
same response profiles as intact nerves and regenerated anterior tongue taste
buds are sensitive to the NaC1 response-suppressing effects of amiloride
(Oakley, 1967a, b; Oakley and Cheal, 1975; Cheal et al., 1977; Hill and
Phillips, 1994; Cain, Frank, and Barry, 1996; Ninomiya, 1998). In the
central nervous system, the pattern of responsiveness of NST neurons to an
array of taste stimuli placed on the anterior tongue in hamsters with
regenerated CTs is also relatively normal with some interesting exceptions,
but in some cases the response rates are lower (Barry, 1999). Congruent with
the theme of this chapter, it is important to show that such neurophysio-
logical effects translate into behavioral ones.
If the GL is allowed to regenerate, the pattern of quinine-induced neural
activity in the NST returns to normal, as does the occurrence of gapes
(Figures 7, 8, and 10). This is true despite the fact that only ~75% of the
normal complement of circumvallate taste buds return (King, Garcea, and
Spector, 2000). If both the GL and the CT are transected, which leads to
severely blunted unconditioned responsiveness to quinine in brief-access
taste tests, the concentration-response function returns to normal upon
regeneration of both nerves (Geran, Garcea, and Spector, 2001).
Clearly, in the face of sometimes massive loss of gustatory input that
leads to unequivocal behavioral deficits in taste-related tasks and some
possibly permanent anatomical changes, the gustatory system in rats main-
tains the capacity to restore function provided the nerve regenerates. The
fact that control groups in which regeneration of the nerve(s) is prevented
do not display any recovery of function suggests that the brain cannot
sufficiently compensate for the loss. In addition to the return of behavioral
competence upon nerve regeneration, the scant evidence that exists does
suggest that patterns of neural activity at least in the NST also return to
normal.
The FunctionalOrganizationof the Peripheral Gustatory System 141
VII. Cross-Regeneration of Gustatory Nerves

In a set of fascinating and innovative experiments, Oakley (1967a, b)
demonstrated that taste nerves can be cross-regenerated. In those
experiments, the peripheral stump of the CT (CTp) was sutured to the
central stump of the GL (GLc). Consequently, after regeneration, input
from the anterior tongue taste receptors was routed through the GL to the
brain. The converse experiment was also conducted; the peripheral GL
(GLp) was sutured to the central CT (CTo). The resultant response
characteristics of these nerves were consistent with the receptor field that
they innervated. Thus, the cross-innervated GLp-CT responded similar to
an intact GL with respect to taste solutions applied to the posterior tongue.
The cross-innervated CTp-GLc responded similar to an intact CT.
Moreover, it has been shown that the molecular characteristics of the
cross-regenerated anterior tongue taste buds (innervated by the GL), as
assessed by the expression of a-gustducin and A-blood group antigen, are
similar to the normal anterior tongue taste buds (Boughter et al., 1997).
Nejad and Beidler (1994) performed a similar experiment except that they
formed a cross-anastomoses between the CT and the GSP. The rat GSP
displays superior responsiveness to sucrose relative to the CT. In agreement
with Oakley's findings, when the central stump of the GSP regenerated into
the anterior tongue (as opposed to the palate), the GSP fibers lost their
exceptional responsiveness to sucrose, responding in a much reduced
fashion as the normal CT does. When the central stump of the CT
regenerated into the palate, the CT became just as responsive to sucrose as
the normal GSP. Collectively, these findings were interpreted as indicating
that the regenerating nerve fibers do not dictate the chemical sensitivity of
the receptor field they are innervating. It would be instructive to conduct
a single fiber examination of cross-regenerated nerves in the rat, especially
given that the response profiles of the GL taste fibers differ substantially
from those in the CT as noted above.
Although a single-fiber analysis of cross-regenerated nerves has yet to be
done in the rat, it has been performed in the C57BL/KsJ mouse (Ninomiya,
1998). Ninomiya's single-fiber study was remarkable not only for its
conceptual value but for the technical virtuosity displayed in the extremely
difficult surgical manipulation performed in the mouse. This study took
advantage of the fact that the GL in this strain has very few amiloride-
sensitive sodium-responsive taste fibers, whereas the CT has many. In the
GLp-CTc mice, the CTc had a similar number of amiloride-sensitive
sodium-responsive taste fibers as compared with the intact CT despite that it
innervated the posterior tongue taste receptor field. Similarly, in the mice
that had the central portion of the GL cross-reinnervating the anterior
tongue (CTp-GLc), there were very few sodium-responsive taste fibers that
142 Alan C. Spector
were amiloride sensitive. Thus, in contrast to what was concluded based on

the early whole-nerve studies with rats, the central portion of the cross-
regenerated nerve, not the receptor field it was innervating, dictated the
composition of taste-responsive fiber types it possessed. Nevertheless, there
are some important caveats to consider. First, the possibility of species
differences cannot be completely dismissed, although admittedly it would
be strange if such a fundamental process as synaptic selectivity during
reinnervation was different across rodent species. Second, the Ninomiya
(1998) study focused primarily on salt responsiveness and employed a
limited taste stimulus array that included mostly salts. This is not a criticism,
as the choice of stimuli was thoughtful and optimized the interpretive power
of the experimental design. It is unclear, however, what would happen if
other physiologically defined fiber types were examined (e.g., Q-fibers, S-
fibers). Third, it remains to be determined whether the central portion of the
taste nerve is inducing the chemical sensitivity of taste bud progenitor cells
when the regenerating axons reach the lingual epithelium or whether the
fibers are simply attracted to the matching receptor type that already exists
in the receptor field once the taste buds have regenerated. For the latter to
be true, there would have to be a minimum number of all relevant receptor
cell types in the respective receptor fields. Finally, it remains to be seen
whether animals with cross-regenerated nerves would display many of the
normal taste-related behaviors discussed in the previous paragraphs.
With regard to the behavioral consequences of taste nerve cross-
regeneration, Oakley demonstrated that preference-aversion functions as
measured by 24-h two-bottle intake tests (taste stimulus versus water) were
relatively normal in animals that had cross-reinnervated taste buds (Oakley,
1969). In that study, the CT and GL were transected unilaterally and the
peripheral portion of the CT was sutured to the central portion of the GL
such that the GL fibers regenerated to reinnervate the taste buds of the
anterior tongue; the remaining central portion of the CT was avulsed and
the contralateral CT and GL were transected shortly before testing. Despite
the massive loss of taste input from the tongue and that signals from half of
the anterior tongue were being routed through circuits normally associated
with the GL (posterior tongue), animals displayed relatively normal
ingestive behavior to sucrose, NaC1, quinine, and saccharin. This finding
must be qualified, however, by the fact that the long-term two-bottle intake
test provides a rather limited assessment of gustatory function (Grill et al.,
1987; Spector, 2000, 2003). In fact, in many cases nerve transection itself
does not alter such behavior (Richter, 1939; Pfaffmann, 1952; Akaike, Hiji,
and Yamada, 1965; Vance, 1967; Grill and Schwartz, 1992; Grill, Schwartz,
and Travers, 1992), so it was probably not the optimal assay to use to
assess the functional consequences of cross-regeneration of taste nerves.
Nevertheless, these results are provocative and suggest that the central
The FunctionalOrganizationof the Peripheral Gustatory System 143
gustatory system may have a tremendous capacity to adapt to altered

peripheral inputs. Alternatively, if cross-regenerated taste fibers find their
matching receptor cell type as suggested by Ninomiya's findings (Ninomiya,
1998), it could allow for the pattern of the peripheral signal to be relatively
unchanged and permit many taste-related behaviors to be normally
maintained.
VIII. Functional Organization of the Human

Peripheral Gustatory System
The behavioral work assessing the effects of gustatory nerve transection in
rodents raises the question of whether a similar profile of consequences is
produced in humans sustaining injury to nerves innervating the oral cavity.
The CT is the most commonly damaged of the gustatory nerves because
of its precarious route from the tongue to the brain. Passing near the
mandibular third molars in association with the lingual nerve and then
crossing alone through the middle ear, the CT is vulnerable to injury in third
molar extraction procedures and otological surgery. The consensus in the
literature is that humans with CT damage do not complain of overall
alterations in taste perception even though they have complete localized
loss of taste sensation in the denervated area. However, the extent of
altered taste function in such patients remains to be comprehensively
assessed. Moreover, reports of humans presenting with complaints of taste
dysfunction after CT damage do exist. It appears that the most common
symptom of unilateral or bilateral CT injury is the reported presence of
a persistent "metallic" taste, sometimes also described as "salty" or "bitter"
(e.g. Rice, 1963; Bull, 1965). In addition, due to the presence of the
parasympathetic secretomotor fibers in the CT, damage to this nerve,
especially when bilateral, results in dryness of the mouth. Interestingly, Bull
(Bull, 1965) noted that some patients with unilateral CT damage reported
that only certain foods tasted different. Apparently, many subjects reported
having difficulty discriminating tea from coffee. In addition to the
symptoms listed above, patients with bilateral CT damage reported
having poorer taste in general.
In the laboratory, the CT can be anesthetized either by injection into the
tissue of the posterior ear canal through which the anesthetic penetrates to
reach the nerve in the middle ear or by injection within the pterygomandibular
space after its junction with the lingual nerve. In the latter case the ipsilateral
anterior tongue is completely numb of sensation because the lingual nerve
is affected. Although nerve block with anesthesia is not the same as nerve
transection, it does provide an opportunity to assess the consequences
of removal of specific nerve input on taste perception. Bilateral CT or
CT/lingual anesthesia decreased magnitude estimates of the intensity of
144 Alan C. Spector
suprathreshold concentrations of NaC1 applied to the whole mouth

(Catalanotto et al., 1993; Yanagisawa et al., 1998). After bilateral CT/lingual
anesthetic nerve block, the supratheshold intensity of sucrose and citric
acid were much less affected and quinine was not affected at all (Catalanotto
et at., 1993). In the case of middle ear CT anesthesia, the perceived intensity
of sucrose, citric acid, and quinine all significantly decreased under whole
mouth stimulation CYanagisawa et al., 1998).
There are two very peculiar findings arising from the anesthetic nerve
block experiments involving the CT. First, in one study about half the
subjects reported taste phantoms (Yanagisawa et al., 1998). This, of course,
resembles the complaints of some patients with CT damage. An explicit
sham treatment group was not included in this study, but presumably the
incidence of reports of phantoms would be low in such subjects.
Nevertheless, these phantoms were described as being rather intense and
often occurring on the posterior tongue near the foliate papillae suggesting
that these sensations were caused by CT nerve block and were not an
artifact of the test context or injection procedure.
The second finding of interest is based on the psychophysical results
of localized stimulus application after anesthetic CT block in humans
(Lehman et al., 1995; Yanagisawa et aI., 1998). When the CT was
unilaterally anesthetized (middle ear), there was a significant increase in
the perceived intensity of quinine on the contralateral field of the posterior
tongue (innervated by the GL). Bilateral CT block increased the perceived
intensity of quinine on both sides of the posterior tongue (Yanagisawa et al.,
1998). Based on these findings, it has been suggested that the removal of
the input from one taste nerve may release the input of other taste nerves
from central inhibition (i.e., disinhibition) (Lehman et al., 1995; Yanagisawa
et al., 1998). These results have also been marshaled to explain the relative
"constancy" of taste perception following CT transection. In other words,
although there is a loss of excitatory input after CT injury there could be a
compensatory loss of inhibitory input as well. Although there are some
electrophysiological data that are consistent with this view (Halpern and
Nelson, 1965), a recent comprehensive examination of the effects of CT
anesthetic block on the taste responsiveness of neurons in the rostral NST of
the rat concluded that although cases of disinhibition were found, they were
uncommon and small in magnitude (Dinkins and Travers, 1998). It is also
important to note that increases in the perceived intensity of NaC1, citric
acid, or sucrose in any oral taste bud field following unilateral or bilateral
CT anesthetic nerve block have not been consistently observed across
studies and have been small when they occur; in fact, some decreases have
been observed (Lehman et al., 1995; Yanagisawa et al., 1998). Given these
considerations, the generality of the hypothesis that disinhibition is the basis
of the maintenance of function after specific gustatory nerve injury remains
tentative. Nevertheless, the possibility that dishinhibition occurs emphasizes

the point that the neural consequences of selective loss of gustatory nerve
input can potentially be quite complex.
In an early study, sucrose and NaC1 detection and recognition thresholds
were shown to be significantly increased by bilateral anesthesia of the tongue
if subjects were prevented from swallowing, but were relatively unaffected
by palatal anesthesia (Henkin and Christiansen, 1967). In contrast, HC1
detection thresholds and recognition thresholds for urea and HCL were
significantly increased by palatal anesthesia but only marginally affected, if
at all, by anesthesia of the tongue. What is unusual, is that when the subjects
that had both the palate and tongue anesthetized were allowed to swallow,
thus stimulating laryngeal taste receptors, detection, and recognition
thresholds to all four tastants were relatively normal. In light of all of the
accumulated data regarding nerve transection in animals and nerve
anesthesia in humans, the results from this experiment are difficult to
reconcile.
In general, researchers seem surprised by the relative lack of effect that
CT nerve injury or anesthesia has on the perceived intensity of standard
taste compounds delivered to the whole mouth. This is very reminiscent of
the early animal work in which researchers were struck by the finding that
taste preferences in two-bottle intake tests were left relatively undisturbed by
certain gustatory nerve transections. It is important to note, however, that
removal of CT input either by anesthesia or injury is not entirely without
effect in humans as documented above. In fact, the data collected with
human subjects is not grossly inconsistent with the findings from the rat
model, notwithstanding the noted exception. As discussed, CT transection is
frequently without effect in certain taste tasks with rats (e.g., the brief-access
taste test). Yet, on other taste-related tasks involving the same compounds
there are notable effects of the neurotomy.
On the surface, it might appear that the implications presented above
regarding the respective roles of the 7th and 9th cranial nerves in taste
function based on findings with the rat model might not pertain to humans.
After all, humans appear to be able to identify taste compounds when
placed in various locations of the oral cavity, even those innervated by the
glossopharyngeal nerve (Collings, 1974). Indeed, it is possible that the
gustatory nerves are less functionally segregated in humans. However, such
a conclusion would be premature based on the limited nature of the data
available. There are significant methodological differences between the very
few experiments looking at regional taste recognition in humans compared
with the discrimination experiments conducted in rats. Perhaps the
most important of these is that the taste discrimination tests conducted
with rats often involve more closely related compounds (e.g., sucrose versus
maltose, NaC1 versus KC1) and thus may represent more challenging signal
146 Alan C. Spector
discrimination tasks. Moreover, the nerve anesthesia experiments in

humans, have generally not focused on taste quality discrimination but
rather suprathreshold intensity as assessed by magnitude estimation or
related scaling procedures (Catalanotto et al., 1993; Lehman et al., 1995;
Yanagisawa et al., 1998). Although it is impossible to conduct magnitude
estimation experiments in nonverbal animal subjects, it would be instructive
to design experiments in humans that emulate the psychophysical
procedures that have been conducted in animal subjects. Such experiments
would provide an analytical bridge between gustatory neurobiology based
on animal models and human sensory report (see Spector, 2000).
IX. Final Remarks

The idea that neural input from the various gustatory nerves subserves
different taste functions is not new. In teleost fish, the facial lobe, which
is the homologue of the rostral NST, receives gustatory input from the 7th
nerve, which in turn innervates extraoral taste buds. The vagal lobe, which is
a homologue of the caudal NST, receives gustatory input from intraorally
distributed taste buds (Herrick, 1903, 1905; Bardach and Atema, 1971;
Goehler and Finger, 1992; Finger, 1997a). When the facial lobe is ablated,
fish have difficulty searching for food (e.g., approach behavior), but will
initiate consummatory responses if the substrate is placed in the oral cavity.
In contrast, when the vagal system is disrupted, food search is unaffected
but the fish display impaired consummatory behavior (Atema, 1971).
Although it is true that the functional organization of the gustatory nerves
in the rat does not exactly emulate that described for the fish, there is no
strong reason that it should, given that the selective pressures for aquatic
species certainly differ from those for terrestial animals. The point is that
there is a phylogenetic precedent supporting some degree of functional
specialization among the gustatory nerves (see Atema, 1971; Finger and
Morita, 1985; Finger, 1997b).
In the mammalian model, Nowlis first postulated that the gustatory input
of the 9th cranial nerve is critical in the generation of reflex rejection
behavior and that the input of the 7th nerve contributes to the generation
of reflex ingestive behavior (Nowlis, 1977). The review of findings across all
of the experiments to date assessing the effects of selective neurotomy on
unconditioned taste-guided licking and on taste-elicited oromotor respon-
ses challenge this somewhat oversimplified view (see Grill et al., 1992).
Nevertheless, it was a sensible hypothesis, based on what has been shown in
fish, and was not entirely without merit given that the input of the GL does
appear to be necessary and perhaps sufficient for the generation of normal
taste-elicited oromotor rejection responses in the rat.
The suggestion that the gustatory nerves are relatively specialized should
not be conceptually unpalatable. Indeed, the vertebrate nervous system
seems to be organized on the principle of functional specialization of nerves,
as any neuroanatomy student who must memorize the cranial nerves will
tell you. There are arguably ample behavioral data to demonstrate that the
gustatory nerves are not functionally equivalent. In many ways, the
suggestion that the gustatory nerves are relatively specialized can be
viewed as an intrarnodal application of the doctrine of specific nerve
energies. Perhaps, this could be extended to fiber types within nerves as well,
and may be at the root of the potential differences between the organization
of the peripheral gustatory system in rodents and humans. For example, the
primate visual system has two classes of retinal ganglion cells that contribute
to two functionally distinguishable systems (the M and P system), but these
are both found in a single nerve. If the input from gustatory afferent fibers
contributes differentially to various taste functions as suggested, then it
becomes a question of what those functions are and how those afferent
fibers are distributed among the various taste nerves. The pattern of that
distribution might vary across species. Whether all or some of the features
of the functional organization of the peripheral gustatory system proposed
in the above paragraphs survive more detailed experimental scrutiny, only
time will tell.
Acknowledgments
I thank Shachar Eylam, Laura C. Geran, Connie Colbert, and Cedrick D. Dotson for
commenting on an earlier draft. I extend my sincere gratitude to Harvey Grill not only for his
excellent editorial feedback on this chapter, but also for his support and tutelage during the
beginning stages of my career and beyond. A large part of the work presented in this chapter
was funded in part by grants from the National Institute on Deafness and Other
Communication Disorders (KO4-DC00104, R01-DC01628 and R01-DC04574).
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Author Index
A Atema, J., 145, 147

Auger, A. P., 46, 48, 51, 59
Abdelgadir, S. E., 48, 66 Auvinen, S., 20, 33
Abe, H., 12, 15, 23, 24, 25, 35
B
Abe, M., 3, 9, 23, 37
Abusnana, S., 87, 89, 96 Bachmanov, A. A., 107, 147
Adler, E., 106, 107, 146, 148, 152, 153 Bae, K., 22, 25
Adler, N. T., 34 Baile, C. A., 74, 98
Adlskogius, H., 134, 152 Baker, M. B., 82, 98
Airaksinen, M. S., 20, 33 Baker, R. A., 74, 93
Aizawa-Abe, M., 88, 94 Balch, O. K., 79, 98
Ajpru, S., 12, 30 Baldino, F., Jr., 41, 63
Akaike, N., 47, 63, 114, 141, 146 Bale, T. L., 51, 59
Akaishi, T., 43, 49, 51, 59, 66, 68 Ball, G. F., 44, 63
Akesson, T. R., 42, 43, 59 Bamshad, M., 85, 93
Akiyama, M., 13, 17, 19, 29, 32 Bandler, R., 42, 59
Albers, H. E., 12, 16, 17, 18, 25, 28, Barbacka-Surowiak, G., 7, 25
29, 31 Barbry, P., 105, 153
Albrecht, U., 22, 25, 26, 35, 37 Bardach, J. E., 145, 147
Amir, S., 15, 27 Barelare, B., Jr., 75, 98
Anderson, P., 55, 64 Barfield, R. J., 40, 50, 61, 65
Anderson, S. T., 55, 59 Barker, S., 80, 94
Andrew, B. L., 104, 146 Barrera, J., 18, 34
Antle, M. C., 5, 7, 12, 18, 19, 25, 28, Barry, M. A., 114, 124, 133, 134, 135, 139,
31, 33 147, 148
Aoki, A., 48, 49, 60, 66 Barry, W. J., 72, 73, 93
Aou, S., 40, 57, 59 Barsh, G. S., 89, 98
Arai, Y., 48, 49, 64, 68 Bartholomew, G. A., 72, 93
Argamaso, S., 12, 27 Bartness, T. J, 71, 72, 74, 75, 76, 77, 78, 79,
Arglebe, C., 135, 148 80, 81, 82, 84, 85, 86, 88, 89, 90, 92, 93,
Asaba, K., 43, 64 94, 96, 97, 99, 100
Asai, K., 117, 118, 125, 159 Bartoshuk, L. M., 108, 143, 145, 148, 152,
Asai, M., 3, 9, 25 153, 159
Asarian, L., 56, 62 Baskin, D. G., 88, 89, 95, 99
Aschoff, J., 2, 3, 4, 5, 25 Basso, A. M., 87, 99
Ashcroft, F., 55, 65 Battey, J. F., 107, 152
Askew, G. R., 50, 67 Bauman, L., 4, 36
164 Author Index
Bean, B., 83, 99 Borg, M. A., 55, 59

Beato, M., 50, 59 Borg, W. P., 55, 59
Beauchamp, G. K., 107, 108, 147, 154 Borker, A. S., 80, 93
Beck, B, 88, 93 Bos, N. P. A., 8, 31
Beidler, L. M., 117, 124, 130, 140, 152, Boschma, Y., 43, 61
154, 157 Boss-Williams, K. A., 86, 88, 89, 94
Beikirch, R. J., 56, 60 Bossert, J. M., 19, 31
Beinfeld, M. C., 41, 68 Boudreau, J. C., 104, 105, 147
Beitz, A. J., 42, 59 Boughter, J. D., Jr., 106, 107, 108, 140,
Belcourt, J., 7, 31 147, 150
Belenky, M., 18, 33 Boulos, Z., 4, 7, 26
Bennett, T., 87, 88, 95 Brabet, P., 15, 28
Benoit, R., 56, 65 Bradbury, M. J., 18, 26
Benos, D. J., 105, 138, 156 Bradley, A., 22, 37
Bereiter, D. A., 111, 147 Bradley, R. M., 102, 103, 111, 113, 114, 132,
Bernstein, I. L., 112, 114, 121, 123, 147, 148, 147, 150, 151
149, 156, 157 Brawer, J. R., 43, 44, 68
Berridge, K., 123, 147 Breininger, J. F., 89, 95
Berridge, K. C., 87, 95, 111, 147, 150 Brennan, C., 48, 67
Berson, D. M., 13, 25, 29 Breslin, P., 111, 123, 157
Berthoud, H. R., 111, 147 Breslin, P. A. S., 117, 121, 147
Bertolini, A., 89, 98 Brewer, J. M., 17, 27
Bethea, C. L., 47, 65 Brink, E. E., 39, 59
Bhatti, J. R., 87, 89, 96 Brinkley, L., 124, 154
Bieber, S. L., 108, 149 Brinnel, H., 80, 94
Biello, S., 15, 29 Brobeck, J. R., 55, 60, 72, 94
Biello, S. M., 5, 12, 16, 17, 18, 21, 25, 28, Brodin, E., 84, 96
30, 35 Bronson, F. H., 73, 76, 98
Billington, C. J., 90, 96 Brosvic, G. M., 110, 124, 148
Bina, K. G., 19, 20, 26 Brot, M. D., 112, 148
Bindra, D., 71, 93 Brown, H. E., 52, 66
Bittman, E. L., 17, 27, 74, 86, 93 Brown, T. J., 40, 44, 48, 50, 60, 65
Blache, D., 40, 59 Buchanan, G. F., 15, 26
Blackshaw, S., 105, 152 Buck, L. B., 106, 107, 153, 154
Blanchard, J., 18, 32 Buckley, C. A., 82, 83, 99
Blaustein, J., 43, 59 Bueno, J., 46, 51, 60
Blaustein, J. D., 39, 44, 46, 51, 57, 59, 61, Bull, T. R., 142, 148
67, 68 Burlet, A., 88, 93
Blessing, W. W., 41, 68 Burlet, C., 88, 93
Block, G., 3, 9, 37 Burley, S. K., 82, 95
Block, G. D., 8, 23, 24, 29, 37 Burnstock, G., 84, 98
Bloom, S. R., 87, 89, 96 Butera, P. C., 56, 60
Blundell, J. E., 73, 78, 79, 80, 82, 85, 92, 93,
C
95, 96
Bobrzynska, K. J., 19, 26 Cabanac, M., 72, 73, 76, 78, 80, 82, 85, 92,
Bock, R., 105, 153 94, 95, 96, 97
Bogart, R., 80, 93 Caicedo, A., 106, 108, 148
Bogic, L., 48, 59 Cain, P., 135, 139, 148
Bone, I., 135, 150 Cajal, S., 43, 60
Bonsall, R. W., 57, 60 Calhoun, J. B., 70, 94
Boone, T., 82, 98 Calizo, L. H., 42, 44, 48, 53, 54, 60, 61
Author Index 165
Cambras, T., 7, 26 Clark, A. S., 48, 52, 66, 67

Cameron, J. L., 88, 96 Clark, J. T., 87, 88, 94
Campagne, F., 107, 153 Clarke, I. J., 48, 55, 59, 66
Campuzano, A., 7, 26 Cleaton-Jones, P., 133, 134, 148
Cano, J., 124, 148 Clein, M. R., 74, 76, 78, 80, 88, 92, 93
Canteras, N. S., 42, 43, 44, 56, 60 Cohen, S. L., 82, 95
Cao, V. H., 21, 33 Coirini, H., 44, 66
Caputo, F. A., 74, 98 Coirini, J., 44, 63
Caraty, A., 55, 62 Coleman, S., 12, 27
Card, J. P., 12, 15, 32 Coling, J. G., 80, 94
Carr, W. J., 110, 148 Collier, G., 80, 96
Carrer, H. F., 48, 60 Collings, V. B., 108, 144, 148
Carson-Jurica, M. A., 50, 60 Collins, F., 82, 98
Cartford, C., 112, 148 Colwell, C. S., 8, 9, 12, 18, 21, 26, 27, 31, 34
Catalanotto, F. A., 124, 143, 145, Commons, K. G., 41, 47, 60
148, 159 Cone, R. D., 55, 68
Catalanotto, F. C., 143, 145, 152 Corm, P. M., 41, 63
Cauthon, R., 117, 118, 125, 148 Contreras, R. J., 104, 105, 108, 117, 122,
Celentano, D. C., 46, 67 126, 148, 149, 153, 155
Cepoi, D., 55, 68 Coogan, A. N., 12, 30
Chait, B. T., 82, 95 Coolen, L. M., 46, 60
Chakrabarty, A. S., 99 Coppack, S. W., 82, 96
Challet, E., 15, 18, 26 Cote, N. K., 21, 22, 26, 27
Chan, J., 51, 65 Cox, V. C., 56, 63
Chan, R. K., 87, 99 Craig, W., 69, 87, 94, 111, 148
Chandrashekar, J., 106, 107, 112, 146, Crews, D., 40, 63, 66, 88, 97
148, 154 Cronin, A. S., 88, 98
Chang, A-M., 9, 36 Crowley, W. R., 41, 60, 87, 88, 94
Chang, C., 40, 43, 50, 67 Cuello, A. C., 56, 64
Chapleur-Chateau, M., 88, 93 Cullen, M. J., 82, 98
Chappell, J. P., 114, 148 Curlewis, J. D., 55, 59
Chase, P. A., 12, 26 Curran, G. H., 47, 64
Chatterjee, A., 107, 147 Curran, T., 128, 154, 156
Chaudhari, N., 107, 112, 148 Cutrera, R. A., 16, 18, 19, 26
Chaves, I., 9, 34
Cheal, M., 133, 134, 139, 148, 155 D
Chehab, F., 55, 68 Daan, S., 4, 5, 22, 26, 27, 33
Chen, D., 15, 26, 27, 28 Dagnault, A., 56, 60
Chen, N., 135, 160 Dahl, M., 104, 148
Chen, Y., 89, 98 Damak, S., 102, 107, 112, 150, 153, 156
Chen, Z., 107, 147 Daniels, D., 42, 44, 45, 46, 54, 60, 61
Chester, A. E., 51, 65 Dark, J., 84, 94
Chilla, R., 135, 148 Davey, G. C., 82, 86, 98
Chorsky, R. L., 57, 61 David-Gray, Z., 13, 27
Chowen, J. A., 52, 62 Davis, A. M., 47, 51, 59, 61
Christiansen, R. L., 144, 151 Davis, J. D., 110, 117, 148, 156
Christy, R. C., 106, 140, 147 Davis, P. G., 40, 50, 61, 66
Chronwall, B. M., 89, 94 Dawut, L., 9, 36
Chu, S., 48, 66 Day, D. E., 71, 74, 75, 77, 81, 85, 86, 88,
Cirrito, J., 55, 66 89, 94
Clancy, A. N., 57, 60, 65 De Bruin, J. P., 80, 96
166 Author Index
de Jong, P. J., 107, 147 Dunlap, J. C., 9, 13, 27, 35

De Kloet, E. R., 43, 61 Dunn, F. A., 13, 25
de la Iglesia, H. O., 22, 29 Durick, K., 107, 153
De Souza, E. B., 87, 99 During, M. P., 55, 59
de Wit, J., 9, 36 Dwyer, S. M., 6, 7, 8, 14, 21, 27, 34
Dean, R. R., 18, 27
DeCoursey, P. J., 4, 36 E
Delay, E., 112, 148 Earnest, D. J., 20, 27, 29
Delay, E. R., 112, 158 Eaton, S. J., 22, 27
Della-Fera, M. A., 74, 98 Ebihara, K., 88, 94
Dellovade, T. L., 41, 47, 50, 61, 67 Ebihara, S., 23, 25
DeMaria, J. E., 87, 88, 99 Ebling, F. J., 88, 98
Demas, G. E., 74, 79, 94 Ebling, F. J. P., 6, 30
Dement, W. C., 6, 18, 19, 26, 27 Edelstein, K., 15, 27
Denk, W., 53, 68 Edgar, D. M., 6, 18, 19, 21, 26, 27, 33
Dernbach, H., 22, 35 Edwards, C. M., 87, 89, 96
DeSimone, J. A., 105, 138, 139, 151, Edwards, D. A., 40, 50, 64
158, 160 Ehlen, J. C., 18, 19, 27, 28
DeVries, G. J., 74, 86, 93 Eichele, G., 22, 25, 37
Dewsbury, D. A., 80, 94 Eichler, V. B., 8, 32
Dhume, R. A., 80, 93 Eker, A. P. M., 9, 36
Dickey, W. P., 134, 139, 148 el Eishi, H. I., 133, 149
Dickman, J. D., 104, 149 Elands, J., 43, 61
Dieckmann, G., 57, 61 Ellis, G. B., 7, 27
Diekman, M. A., 40, 67 Ellison, G. T. H., 73, 94
Dietz, M., 20, 30 Eng, V., 50, 65
Diez-Noguera, A., 7, 26 Engelman, K., 111, 158
DiNardo, L. A., 18, 28 Eoh, S., 22, 27
Ding, J. M., 15, 21, 26, 27, 28, 30 Epstein, A. N., 121, 149
Dinkins, M. E., 143, 149 Erickson, R. P., 104, 117, 148, 159
Dirden, J. C., 17, 27 Erskine, M. S., 40, 44, 46, 66
Do, L. T., 104, 105, 147 Estep, D. Q., 80, 94
Dobbins, R. L., 55, 64 Etgen, A. M., 40, 41, 50, 52, 61, 65, 68
Dolce, C., 124, 154 Etoh, T., 3, 20, 27, 32
Don Carlos, L. L., 40, 43, 50, 61 Everitt, B. J., 41, 62
Donahue, J. E., 57, 61 Eyigor, O., 41, 63
Donohoe, T. P., 80, 94 Eylam, S., 126, 128, 149
Doolin, R. E., 105, 106, 149
Dornan, W. A., 42, 61 F
Dorner, G., 48, 61 Fabre-Nys, C. J., 40, 59
Dorsa, D. M., 49, 51, 59, 67 Fahrbach, S. E., 40, 43, 61
Dove, W. F., 9, 36 Fahrenkrug, J., 12, 13, 15, 28, 33
Dreifuss, J. J., 57, 64 Faiman, L. E., 21, 27, 30
Dube, M. G., 88, 89, 94, 96 Fantino, M., 73, 78, 80, 82, 85, 92, 94
Dubois-Dauphin, 57, 64 Farbman, S., 133, 134, 151
Ducan, J. S., 99 Fast, K., 143, 145, 148
Dudek0 F. E., 18, 33, 35 Feder, H. H., 41, 47, 64, 68
Dudley, C. A., 41, 61 Feenstra, M. G., 80, 96
Duenas, M., 52, 62 Fei, H., 82, 97
Dugovic, C., 26 Feng, L., 106, 107, 148, 154
Duncan, M. J., 86, 98 Ferguson, C., 47, 61
Author Index 167
Fernandez-Guasti, A., 57, 61 Ganchrow, D., 133, 149

Ferreira-Silva, L., 43, 44, 48, 53, 64 Ganchrow, J. R., 133, 149
Field, M. D., 17, 30 Gannon, K. S., 106, 108, 149, 159
Figlewicz, D. P, 87, 100 Gannon, R. L., 18, 27, 34, 36
Finger, T., 112, 145, 149 Ganster, D. J., 87, 95, 111, 150
Finger, T. E., 105, 138, 145, 149, 150, 153 Gantz, I., 89, 98
Finkelstein, J. A., 88, 98 Garcea, M., 117, 118, 119, 120, 121, 124,
Fishman, R. B., 84, 94 125, 126, 127, 128, 129, 130, 131, 133,
Flanagan, L. M., 41, 44, 46, 61 134, 135, 136, 138, 139, 148, 149, 150,
Flanagan-Cato, L. M., 42, 44, 45, 46, 48, 53, 152, 157, 158
54, 60, 61 Garcia-Segura, L. M., 52, 62
Flannelly, D. J., 70, 96 Gardiner, S. M., 87, 88, 95
Fleischmann, A., 52, 61 Garty, H., 105, 138, 149
Fleming, A., 72, 95 Gaylinn, B. D., 21, 34
Flerko, B., 41, 44, 64 Geary, N., 56, 62
Fliers, E., 56, 61 Gent, J. F., 143, 145, 148
Flynn, F. W., 87, 95, 111, 141, 149, 151 Georg, B., 13, 28
Fodor, M., 57, 61 Gerall, A. A., 39, 65
Ford, H., 88, 98 Geran, L. C., 112, 115, 119, 121, 123,
Formaker, B. K., 105, 121, 123, 149, 151 126, 131, 134, 135, 138, 139, 149,
Foster, R., 13, 27 150, 152
Foster, R. G., 12, 27 Gerlach, J. L., 48, 59
Frank, M., 109, 155 Getzinger, M. J., 44, 68
Frank, M. E., 104, 105, 108, 112, 114, 122, Geusz, M. E., 8, 29
124, 126, 133, 134, 135, 139, 147, 148, Ghatei, M. A., 87, 89, 96
149, 151, 155, 159 Gibbs, R. B., 40, 44, 46, 65
Frankfurt, M., 44, 48, 53, 62, 64, 66 Gilbertson, T. A., 102, 105, 106,
Frankmann, S. P., 149 107, 108, 121, 138, 139, 149, 150,
Fredholm, B. B., 83, 95 151, 153
Freedman, M. S., 13, 27 Gillespie, C. F., 16, 17, 18, 28, 29, 31
Fregly, M. J., 121, 124, 149, 159 Gillette, M. U., 3, 13, 15, 21, 27, 28, 30
Friedman, J. M., 82, 95, 97 Gingerich, R. L., 74, 98
Friedman, M. I., 75, 79, 83, 86, 92, 95, 99 Ginty, D. D., 13, 30
Frye, P., 109, 151 Giraudo, S. Q., 89, 100
Fuji, K., 17, 19, 29 Glass, J. D., 18, 19, 27, 28, 31, 34
Fujimoto, S., 133, 149 Glendinning, J. I., 110, 117, 150
Fukada, Y., 9, 33 Godfrey, M. H., 19, 26
Fukami, Y., 108, 154 Goehler, L. E., 145, 150
Fukuda, A., 51, 65 Gogate, M. G., 80, 93
Fukuhara, C., 16, 17, 27, 35 Gold, P. W., 43, 64
Fukui, H.0 23, 34 Gold, R. M., 55, 62
Fukuoka, Y., 135, 156 Goldman, B. D., 72, 74, 86, 93
Fukushima, M., 16, 30 Goldman, M. D., 83, 99
Fuller, P. J., 48, 66 Golombek, D., 15, 29
Fulwiler, C. E., 41, 62 Golombek, D. A., 16, 17, 25, 28
Funakoshi, M., 105, 114, 122, 152, 154 Gomez-Sanchez, E., 55, 64
Furukawa, T., 20, 27 Gores, T., 41, 44, 64
Gosnell, B. A., 90, 96
G
Gosselin, C., 85, 95
Gajiwala, K. S., 82, 95 Goto, K., 89, 100
Galanter, E. H., 109, 158 Goubillon, M.-L., 55, 62
168 Author Index
Gould, E., 48, 53, 62 Harlan, R. E., 42, 43, 51, 62

Goulding, E., 55, 68 Harland, D., 87, 88, 95
Goy, R. W., 39, 40, 41, 62, 65, 68 Harrington, M. E., 3, 15, 16, 17, 21, 22,
Grabauskas, G., 114, 132, 150 25, 26, 27, 28, 29, 30, 31, 34, 37
Grant, R., 135, 150 Harris, K. M., 53, 62
Gratten, D. R., 46, 49, 62 Harris, R. B., 83, 87, 95, 99
Greenberg, M. E., 13, 30, 128, 156 Harris, R. B. S., 81, 82, 95, 97
Greene, G., 51, 59 Hart, B. L., 40, 63
Gresack, J., 110, 117, 150 Hartman, B. K., 67
Gribkoff, V. K., 17, 28 Hashimoto, K., 43, 64
Grill, H. J., 87, 95, 109, 110, 111, 114, 115, Hashimura, E., 4I, 62
116, 117, 118, 119, 120, 121, 122, 123, Hastings, M. H., 5, 9, 16, 17, 30, 34
126, 127, 130, 137, 141, 145, 147, 149, Hastings, M. H. V., 6, 30
150, 151, 155, 157, 158, 159 Hastings, M. S., 22, 25
Gross, R. A., 9, 34 Hattar, S., 13, 29
Grossman, G. H., 18, 19, 27, 28 Hayase, M., 88, 94
Grubin, C. E., 88, 99 Heath, M. M., 87, 89, 96
Guagliardo, N. A., 110, 112, 115, 121, 122, Hecht, R., 82, 98
123, 124, 137, 150, 157, 158 Heck, G. I., 105, 138, 139, 151
Guo, W., 106, 148 Heck, G. L., I39, 160
Gurkan, S., 113, 151 Heimer, L., 41, 68
Gustafsson, J. A., 51, 65 Hellekant, G., 113, 133, 134, 15i
Guth, L., 133, 151 Heller, H. C., 18, 21, 33, 34, 36
Guyenet, P. G., 21, 34, 36 Henderson, L. P., 48, 67
Henderson, R. G., 47, 68
H Henkin, R. I., 144, 151
Hackenberg, T. D., 110, 123, 124, 137, 158 Hennessy, C. J., 112, 147
Hagan, M. M., 89, 95 Herberg, L. J., 73, 78, 79, 80, 81, 82, 83, 85,
Hagstrom, E. C., 108, 151 92, 93, 94, 95, 96, 100
Hahn, T. M., 89, 95 Herbert, J., 6, 30
Hajszan, T., 56, 62 Herbison, A. E., 55, 62
Halaas, J. L., 82, 95, 97 Herkenham, M. A., 20, 37
Hall, A., 15, 29 Herness, M. S., 102, 151
Hall, A. C., 22, 30 Herrick, J. C., 145, 151
Hall, S. J., 5, 16, 30 Herzog, E. D., 3, 8, 9, 24, 29, 37
Halpern, B. P., 122, 143, 151, 158 Hettinger, T. P., 104, 105, 108, 122, 126,
Halpern, M., 39, 66 134, 149, 151, 159
Halsell, C. B., 114, 132, 151 Hexter, D. P., 48, 59
Hamada, T., 3, 11, 15, 18, 28, 35, 36 Hida, A., 23, 37
Hamaoka, T., 41, 62 Hiji, Y., 114, 141, 146
Hamilton, J. M., 72, 93 Hill, D. L., 105, 106, 121, 123, 139, 149, 151,
Hamilton, R. B., 103, 151 152, 157, 158
Han, H. C., 15, 29 Hinch, D., 6, 36
Hanamori, T., 104, 156 Hindersson, P., 13, 28
Handal, P. J., 121, 151 Hino, A., 107, 152
Hannibal, J., 12, 13, 15, 26, 28, 33 Hirst, M., 90, 96
Hansen, S., 41, 55, 62 Hoang, N. K., 104, 105, 147
Harada, S., 124, 151 Hochberg, R. B., 44, 48, 60
Hard af Segerstad, C., 133, 134, 151 Hoeijmakers, J. H. J., 9, 36
Harder, D. B., 109, 151 Hoey, N. E., 110, 124, 148
Harlan, R., 47, 63 Hofer, D., 152
Author Index 169
Hoffman, R. A., 72, 96 J

Hoggard, N., 86, 88, 89, 97 Jacobmeier, B., 22, 35
Holland, V. F., 105, 138, 156 Jacquin, M. F., 114, 152
Holmes, M. M., 7, 31 Jagota, A., 22, 29
Holt, L. E., 75, 98 Jain, M. R., 88, 96
Homanics, G. E., 47, 61 Jamen, F., 15, 28
Homes, M. M., 19, 31 Jang, I.-S., 47, 63
Hong, S. K., 15, 29 Janik, D., 16, 20, 25, 29
Honingschmid, J., 133, 159 Janovick, J. A., 41, 63
Honma, K.-I., 8, 9, 11 12, 12, 15, 22, 23, 25, Jasnow, A. M., 16, 17, 31
32, 35 Jeanrenaud, B., 111, 147
Honma, S., 8, 9, 11 12, 12, 15, 22, 23, 25, Jenab, S., 52, 66
32, 35 Jennes, L , 41, 63
Honmura, A., 49, 68 Jennings, G., 81, 100
Honrado, G. I., 5, 32 Jhanwar-Uniyal, M., 88, 93
Hoon, M. A., 106, 107, 112, 146, 148, Ji, H., 83, 99
152, 154 Jin, X., 22, 25
Hopf, A., 56, 64 Johnson, A. E., 44, 63
Hoque, S., 15, 22, 27, 29 Johnson, K. G., 72, 96
Horikawa, K., 13, 17, 19, 23, 29, 32, 34 Johnson, N. J., 80, 94
Horiuchi, M., 55, 65 Johnson, O., 71, 73, 74, 78, 79, 97
Horton, L. E., 48, 66 Johnson, R. F., 12, 16, 29
Hosley, M. A., 133, 152 Jones, C. H., 74, 100
Hou, L.-T., 134, 159 Jones, K. J., 50, 63
Houpt, T. A., 5-6, 31 Jones, K. R., 55, 68
Howell, L. A., 87, 99 Jones, L. B., 134, 139, 148
Hrabovszky, E., 56, 62 Jonouchi, M., 3, 9, 25
Hrupka, B. J., 56, 62 Jonsson, G., 84, 96
Hsu, D. S., 9, 36 Journot, L , 15, 28
Huang, L. Q., 107, 153 Joy, J. E., 17, 29
Hughes, S. E., 133, 152
Huhman, K. L., 16, 17, 18, 28, 29, 31 K
Hulse, S. H., 111, 152 Kaasik, K., 22, 37
Humby, T., 6, 30 Kaisaku, J., 114, 132, 155
Hunt, J. Mc. V., 71, 96 Kaiyala, K., 82, 96
Kako, K., 23, 34
I Kallo, I., 56, 62
Igawa, H., 135, 156 Kaha, P. S., 87, 88, 89, 94, 96, 98
Ikeda, M., 23, 25 Kalra, S. P., 87, 88, 89, 94, 96, 98
Illnerova, H., 11, 22, 26, 36 Kalsbeek, A., 16, 19, 26, 80, 96
Inagaki, S., 41, 42, 43, 62, 68 Kang, H.-C., 15, 29
Ing, N. H., 48, 66 Kanno, S-I., 9, 36
Ingrain, S. M., 41, 63 Kaplan, J. M., 111, 141, 151
Inoue, T., 113, 153 Karoon, P., 84, 98
Inouye, S., 17, 35 Karrer, T. A., 143, 145, 159
Inouye, S. I., 36 Kasahara, Y., 113, 151
Inouye, S.-IT., 11, 16, 29, 35 Kashima, Y., 55, 65
Isejima, H., 3, 9, 25 Katayama, T., 16, 30
Ishida, N., 23, 34 Kater, S. B., 53, 62
Ishnnina, T. A., 57, 63 Katsumoto, T., 89, 100
Itowi, N., 21, 22, 29 Katsuno, Y., 8, 12, 35
170 Author Index
Katsuura, G., 88, 94 Korach, K. S., 50, 51, 56, 62, 65

Kaufman, C. M., 21, 26 Kornhauser, J. M., 9, 13, 30, 36
Kavaliers, M., 90, 96 Koster-Van Hoffen, G. C., 8, 31
Kawamura, Y., 114, 152 Kotz, C. M., 89, 96
Kazantsev, A., 9, 36 Kow, L.-M., 39, 40, 41, 47, 51, 60, 62, 63, 64
Keay, K. A., 42, 59 Krause, R. G., 41, 63
Keefe, D. L., 20, 29 Krebs, J. R., 69, 99
Keefer, D. A., 40, 67 Kretz, O., 105, 153
Keiner, M., 40, 43, 50, 65 Krey, L., 41, 61
Kelton, M., 49, 65 Krieger, M. S., 40, 50, 61
Kendrick, A. M., 40, 63 Krimm, R., 157
Kennedy, G. C., 71, 96 Krimm, R. F., 117, 124, 130, 152, 157
Kern, P. A., 82, 97 Kruijver, F. P. M., 57, 61
Kerns, J. A., 89, 98 Kruk, M. R., 55, 63
Kershaw, M., 88, 98 Kruse, F., 22, 35
Khalsa, S. B. S., 8, 29 Krust, A., 44, 66
Kilduff, T. S., 21, 33 Kume, K., 9, 34
Kim, D. Y., 15, 29 Kuroda, H., 3, 16, 30, 32
Kim, M. S., 87, 89, 96 Kusakabe, Y., 107, 152
Kim, S., 82, 97 Kveton, J. F., 143, 145, 152, 159
Kim, Y. I., 15, 29
King, C. T., 127, 128, 129, 133, 134, 136, L
139, 152 Lakhdar-Ghazal, N., 16, 19, 34
King, D. P., 9, 36 Lakshmanan, S., 99
King, J., 56, 63 Lallone, R. L., 82, 95
King, J. C., 43, 56, 57, 59, 61, 67 Lamey, P. J., 135, 150
Kinnamon, S. C., 105, 106, 108, 121, 138, Lamp, C., 112, 148
139, 153 Landin, A. M., 107, 148
Kinnman, E., 134, 152 Landt, M., 82, 96
Kirby, J. D., 20, 37 Lane, M. V., 50, 67
Kita, H., 40, 63 Lanier, D. L., 80, 94
Kitada, Y., 105, 121, 152 Larkin, D., 22, 25
Kitagawa, M., 107, 152 Larriva-Sahd, J., 48, 49, 63
Kitamura, R., 124, 159 Larsen, P. J., 15, 28, 88, 96
Kito, S., 42, 43, 68 Larson, D. C., 114, 124, 135, 147
Kiyomitsu, Y., 124, 159 Lasley, J. F., 80, 93
Klein, D. C., 8, 30 Latlone, R., 82, 97
Klein, S., 82, 96 Lauber, A. H., 48, 63
Kleopoulos, S. P., 40, 44, 46, 65 Lavery, R. J., 7, 34
Klosterman, S. A., 48, 66 Lawton, A., 133, 134, 155
Knudsen, S. M., 13, 15, 28, 33 Lax, P., 7, 30
Kobayashi, K., 9, 36 Lea, S. E. G., 74, 76, 96
Kobayashi, M., 3, 9, 25 Lederman, A., 12, 27
Koh, E. T., 56, 67 Lee, A., 21, 34, 36
Koh, S. D., 110, 152 Lee, C. C., 9, 22, 25, 34, 37
Kolakowski, L. F., Jr., 9, 34 Lee, G. H., 82, 97
Komisaruk, B. R., 41, 60 Lee, K. J., 15, 29
Kondo, Y., 51, 67 Leedy, M. G., 40, 63
Koob, G. F., 87, 99 Lehman, C. D., 143, 145, 152
Kopka, S. L., 106, 108, 115, 119, 124, 134, Lehman, M. N., 51, 59
135, 137, 138, 139, 152, 157 Lei, H., 43, 47, 67
Author Index 171
Leibowitz, S. F., 87, 88, 93, 99 Lundy, R. F., Jr., 105, 122, 126, 153
Lenn, N. J., 12, 32 Luthman, J. L., 84, 96
Leranth, C., 44, 47, 63 Lyman, C. P., 72, 96
LeSauter, J., 11, 28 Lynch, K. R., 21, 34, 36
Leshner, A. I., 80, 96 Lynn, D. M., 52, 66
Levine, A. S., 79, 87, 88, 90, 93, 96
Levine, J. A., 89, 96 M
Levine, J. D., 12, 30 MacIntyre, D. E., 15, 18, 26
Lewis, C., 48, 64 MacLuskey, N. J., 44, 48, 60
Lewis, M. E., 41, 63 MacLusky, N. J., 40, 50, 65
Li, H-Y., 86, 92, 100 Madden, L., 87, 100
Li, Q., 22, 37 Madiera, M. D., 43, 44, 48, 53, 64
Li, S., 107, 147 Madrid, J. A., 7, 30
Li, X., 107, 147 Madsen, K., 88, 96
Li, X. D., 107, 153 Maes, F. W., 112, 156
Li, X.-J., 105, 152 Maffei, M., 82, 95, 97
Liao, H. W., 13, 29 Mai, J. K., 56, 64
Liberles, S. D., 107, 154 Majdic, G., 55, 64
Licklider, J. C. R., 73, 98 Makimura, H., 89, 97
Lin, W. H., 105, 138, 153 Makino, S., 43, 64
Lindemann, B., 105, 106, 108, 121, 138, Malik, K. F., 47, 64
139, 153 Malsbury, C. W., 40, 42, 61, 64, 74, 81,
Lindemeier, J., 107, 152 97, 98
Liou, S. Y., 12, 25 Manabe, Y., 135, 156
Liposits, Z., 56, 62 Manion, B. G., 135, 159
Lisciotto, C. A., 43, 64 Manogue, K., 51, 64
Lisk, R. D., 40, 67 Marchant, E. G., 6, 16, 19, 25, 30, 31
Liss, B., 55, 65 Margolis, F. L., 108, 156
Liu, C., 21, 30 Margolskee, R. F., 102, 106, 107, 112, 150,
Liu, Z., 107, 153 153, 156, 159
Lockhead, E., 112, 156 Mark, G. P., 109, 156
Logothetis, D. E., 8, 36 Markison, S., 110, 117, 119, 120, 121, 123,
Long, C. N. H., 55, 60 124, 125, 126, 131, 133, 134, 135, 137,
Lonstein, J. S., 42, 44, 64 138, 153, 157, 158
Lopingco, T., 89, 97 Martin, C. E., 6, 27
Lore, R. K., 70, 96, 99 Marvel, C. L., 16, 17, 18, 28, 29, 31
Loros, J. L., 13, 35 Mason, R., 16, 25
Losee-Olsen, S., 4, 5, 36 Masubuchi, S., 23, 25
Louis-Sylvestre, J., 53, 111 Masuda, A., 72, 73, 97
Loup, F., 57, 64 Masuda, Y., 113, 153
Low-Zeddies, S. S., 8, 9, 30 Masuzaki, H., 88, 94
Lowery, P. L., 9, 36 Mathews, D., 40, 50, 64
Lowlicht, R. A., 143, 145, 152 Matsumoto, A., 48, 49, 64
Lu, W., 22, 37 Matsunami, H., 106, 107, 153, 154
Lubahn, D. B., 50, 65 Matsuo, R., 113, 124, 153, 159
Lucas, R. J., 13, 27 Matsuoka, N., 88, 94
Luellig, M., 112, 158 Matsuzaki, T., 42, 43, 68
Luine, V. N., 49, 65 Mattes, R. D., 111, 153, 158
Luiten, P. G. M., 40, 43, 64, 67 Matthijssen, M. A., 80, 96
Lumia, A. R., 55, 64 Mauer, M. M., 81, 86, 97
Lundy, R. F., 105, 148 Max, M., 107, 153
172 Author Index
Mayer, D. T., 80, 93 Miller, S., 135, 150

Mayo, K. E., 13, 30 Millette, M. U., 15, 26
Maywood, E. S., 5, 6, 9, 16, 17, 22, 25, Millhouse, O. E., 43, 44, 65
30, 34 Milner, T. A., 41, 47, 60
McArthur, A. J., 12, 30 Minami, K., 55, 65
McCarthy, M. M., 46, 47, 48, 51, 59, 64 Minami, T., 51, 65
McCleary, R. A., 73, 97 Ming, D., 106, 159
McClure, P. A., 79, 81, 97 Minokoshi, Y., 55, 65
McCutcheon, N. B., 112, 153 Mintz, E. M., 16, 17, 18, 28, 31, 71, 74, 75,
McDonald, J. D., 9, 36 81, 86, 88, 94
McDonald, T. J., 57, 68 Mirkes, S. J., 47, 65
McEwen, B. S., 40, 41, 44, 46, 48, 49, 50, 53, Mirmiran, M., 8, 31
59, 61, 62, 63, 64, 65, 66, 68 Miselis, R. R., 12, 30, 42, 44, 45, 46, 60, 61
McGarry, J. D., 55, 64 Mistlberger, R. E., 5, 5-6, 6, 7, 16, 18, 19,
McGinnis, M. Y., 50, 55, 64, 66 20, 25, 28, 30, 31
McGlathery, S. T., 135, 159 Mistretta, C. M., 114, 159
McKinnon, P. J., 106, 153 Mitchell, T. D., 83, 95
McKlveen, R. E., 7, 27 Mitoh, Y., 105, 121, 152
McLaughlin, S. K., 106, 153 Miura, H., 107, 152
Mead, S. M., 6, 30 Miyake, Y., 23, 34
Meelis, W., 55, 63 Miyamoto, Y., 9, 31, 36
Meijer, J. H., 17, 20, 30, 34 Miznno, T. M., 89, 97
Meisel, R. L., 40, 67 Moar, K. M., 86, 88, 89, 97
Meister, M., 8, 36 Mobbs, C. V., 40, 44, 46, 65, 89, 97
Menaker, M., 3, 4, 5, 9, 15, 21, 23, 24, 26, Mochizuki, T., 22, 29
32, 33, 34, 36, 37 Moffatt, C. A., 46, 59
Mercer, J. G., 86, 88, 89, 97 Moga, M. M., 17, 20, 31
Merchenthaler, I., 41, 44, 50, 56, 62, 64, 67 Mohamed-Ali, V., 82, 96
Meredith, J. M., 51, 59 Monroy, E., 40, 43, 50, 61
Merlo-Pich, E., 87, 99 Montmayeur, J. P., 106, 107, 153, 154
Merryday, D., 157 Moore, R. Y., 8, 9, 10, 11, 12, 15, 16, 17, 18,
Mesulam, M. M., 56, 65 20, 26, 29, 30, 31, 32, 35
Metzger, J. M., 15, 18, 26 Moore-Ede, M. C., 5 4 , 31
Meyer, E. L., 16, 31 Moreines, J., 49, 65
Meyer, J. L., 22, 27, 30 Morgan, C. T., 71, 73, 74, 78, 79, 97, 99
Meyer-Bernstein, E. L., 17, 18, 19, 31 Morgan, D. G., 87, 89, 96
Miceli, M. 0., 74, 81, 97, 98 Morgan, J. I., 128, 154
Micevych, P. E., 42, 43, 59 Morgan, P. J., 86, 88, 89, 97
Michael, R. P., 57, 60, 65 Morimoto, T., 113, 153
Miche, S., 9, 31 Morin, L. P., 6, 12, 15, 16, 17, 18, 19, 29, 30,
Michel, C., 82, 97 31, 32, 35
Michels, K. M., 18, 31, 35 Morita, Y., 112, 145, 149
Mierson, S., 105, 138, 139, 151 Moriya, T., 3, 9, 13, 17, 19, 25, 29, 32, 35
Miki, T., 55, 65 Morley, J. E., 79, 87, 88, 90, 93, 96
Mikkelsen, J. D., 15, 16, 20, 28, 29 Morohashi, K.-I, Li, E., 43, 47, 67
Mikkelson, J. D., 11, 36 Morrell, J. I., 39, 40, 43, 50, 59, 61, 64
Miller, G. A., 71, 76, 97 Morris, Y. A., 88, 97
Miller, I. J., 108, 133, 153 Morris-Wiman, J., 124, 154
Miller, I. J., Jr., 102, 103, 104, 117, 124, 130, Morrison, G. R., 110, 154
134, 135, 152, 154, 157, 160 Morton, M. T., 9, 34
Miller, J. D., 18, 19, 21, 27, 31, 33, 34, 36 Mos, J., 55, 63
Author Index 173
Moss, R. L., 41, 61 Noppen, N. W. A. M., 56, 61

Mrosovsky, N., 2, 4, 5, 6, 7, 16, 17, 19, 20, Norgren, R., 87, 95, 103, 109, 111, 114, 117,
25, 26, 29, 30, 32, 34 118, 119, 123, 127, 130, 132, 147, 150,
Mueller, K. L., 106, 146, 148 151, 154, 155, 157, 158, 159
Mufson, E. J., 56, 65 Norrison, W., 110, 154
Muijtjens, M., 9, 36 Nowlis, G. H., 122, 145, 154, 155
Muller, D., 55, 57, 65 Numan, M., 55, 66
Miiller, J., 146, 154 Numan, M. J., 55, 66
Munoz, M., 13, 27 Numano, R., 23, 37
Murakami, N., 3, 16, 20, 27, 30, 32 Numata, Y., 88, 94
Muramatsu, M., 40, 43, 50, 67 Nyby, J., 74, 80, 81, 97
Murphy, M. R., 71, 97
Murray, R. G., 133, 149 0
Oakley, B., 133, 134, 139, 140, 141, 148,
N
152, 155
Na, H, S., 15, 29 Ogawa, H., 113, 114, 132, 155
Nabekura, J., 51, 65 Ogawa, S., 50, 51, 56, 62, 65
Nachman, M., 121, 122, 154 Ogawa, Y., 88, 94
Naftolin, F., 44, 47, 52, 62, 63 Oguchi, H., 8, 35
Nagano, M., 43, 47, 67 O'Hara, B. F., 21, 33
Nagase, T., 23, 34 Ohara, O., 23, 34
Nagia, K., 21, 29 Ohi, K., 6, 37
Nah, H.-D., 134, 159 Ohi, K. X., 38
Nakagawa, H., 21, 29 Ohmen, J. D., 107, 147
Nakai, M., 16, 30 Ohtsubo, T., 135, 156
Nakamura, O., 113, 153 Oishi, T., 72, 73, 97
Nakamura, W., 8, 11-12, 22, 32 Okada, T., 23, 34
Nakao, K., 88, 94 Okamoto, J., 114, 152
Namihira, M., 23, 25 Okamura, H., 3, 9, 13, 17, 19, 25, 29,
Nathanielsz, P. W., 57, 68 35, 36
Naylor, E., 15, 18, 26 Okano, T., 9, 33
Nejad, M. S., 104, 117, 124, 130, 140, 152, O'Keefe, G. B., 110, 117, 121, 155, 156
154, 157 Okumura, H., I1, 37
Nelson, D., 20, 29 Olivier, B., 55, 63
Nelson, G., 107, 112, 154 Ollmann, M. M., 89, 98
Nelson, L. M., 143, 151 O'Malley, B. W., 50, 60
Nicklas, K., 103, 104, 159 Oomura, Y., 40, 51, 55, 57, 59, 63, 65
Nicklatsch, J., 135, 148 Oravec, J., 104, 105, 147
Nicolaidis, S., 111, 154 Orthner, H., 55, 57, 65
Niel, L., 19, 25 Osawa, Y., 43, 47, 67
Nielsen, H. S., 15, 28, 33 Ossebaard, C. A., 112, 155, 156
Nielson, H. S., 12, 33 Ostrum, K. M., 143, 145, 148
Ninomiya; Y., 43, 47, 67, 105, 107, 108, 122, Otori, Y., 16, 35
133, 139, 140, 142, 152, 154 Overton, J. M., 87, 88, 99
Nishiyama, M., 43, 64 Owen, K., 80, 97
Nishizuka, M., 46, 53, 65
Niwa, O., 43, 47, 67 P
Noda, I., 135, 156
Palkovits, M., 41, 68
Nolan, L. J., 92, 98
Palmer, L. G., 105, 138, 149
Nomura, M., 43, 47, 67
Panula, P., 20, 21, 28, 33
Nomura, T., 114, 132, 155
Parker, K. L., 55, 64
174 Author Index
Parker, L. A., 111, 155 Purvis, C. C., 86, 98

Parks, J. D., 134, 155 Pye, J. G., 79, 80, 96
Parsons, B., 48, 66
Paula-Barbosa, M. M., 43, 44, 48, 53, 64 O
Pavlov, I. P., 111, 155 Qiu, J., 55, 68
Payne, C. J., 87, 99 Quinones-Jenab, V., 52, 66
Pelleymounter, M. A., 82, 98
Pellowski, M. W., 21, 34 R
Penev, P. D., 19, 33 Rabinowitz, D., 82, 95
Penney, R. B., 42, 61 Radcliffe, L. A., 3, 9, 37
Perrigo, G., 73, 76, 98 Rahmani, T., 16, 31
Peters, H. J. P. W., 46, 60 Rainbow, T. C., 48, 50, 66
Petit, C., 9, 36 Rajendren, G., 41, 61
Petrusz, P., 41, 44, 64 Ralevic, V., 84, 98
P6vet, P., 16, 18, 19, 26, 34 Ralph, M., 4, 5, 32
Pfaff, D. W., 39, 40, 41, 42, 43, 44, 46, 47, Ralph, M. R., 9, 15, 33, 34
48, 49, 50, 51, 52, 53, 56, 59, 60, 61, 62, Ramirez-Degollado, M., 48, 49, 63
63, 64, 65, 66 Ramon, Y., 43, 60
Pfaffmann, C., 108, 109, 114, 122, 132, 141, Rance, N. E., 56, 67
151, 155, 159 Rand, M. S., 40, 63, 66
Pfaus, J. G., 40, 41, 42, 44, 46, 61, 65 Ranganathan, S., 82, 97
Phillips, C. L., 135, 160 Rashotte, M. E., 109, 151
Phillips, J. H., 82, 86, 98 Ravussin, E., 82, 97
Phillips, L. M., 139, 151 Rawson, J. A., 48, 66
Phoenix, C. H., 39, 40, 62, 65 Rawson, N. E., 86, 95
Pickard, G. E., 15, 18, 33, 34, 35 Rayner, D. V., 99
Pieschi, R. L., 17, 28 Rea, M. A., 16, 18, 27, 28, 33, 34, 36
Piggins, H. D., 12, 15, 30, 33 Reddy, A. B., 88, 98
Pinto, L. H., 9, 36 Redman, R., 117, 118,124, 125, 130, 134, 157
Pirvola, U., 20, 33 Redmann, S. M., Jr., 83, 95
Pisano, R. G., 70, 98 Reebs, S. G., 5, 7, 32, 34
Pitrosky, B., 16, 19, 34 Reed, D. R., 107, 147
Pittendrigh, C. S., 2, 3, 4, 5, 27, 33 Reeves, A. G., 55, 57, 66
Pittman, D. W., 117, 155 Reichardt, L. F., 55, 68
Pleim, E. T., 40, 50, 65 Reichlin, S., 57, 61
Plum, F., 55, 57, 66 Reid, M. S., 19, 27
Poggioli, R., 89, 98 Reiter, R. J., 72, 96
Polet, I. A., 112, 155 Rendon, R. A., 17, 28
Polston, E. K., 40, 44, 46, 66 Rentmeister-Bryant, H., 115, 137, 156
Pool, C. W., 57, 63 Reppert, S. M., 8, 9, 22, 23, 25, 30, 34,
Porte, D., Jr., 87, 100 36, 37
Porter, J. H., 73, 98 Resko, J. A., 48, 66
Powley, T. L., 55, 66, 111, 155 Rhee, J. S., 47, 63
Pozzo Miller, L. D., 48, 49, 66 Riberdy, A. F., 18, 33
Pratley, R. E., 82, 97 Rice, J. C., 142, 155
Price, D. L., 56, 67 Richard, D., 56, 60, 85, 94
Price, K. M., 31 Richter, C. P., 75, 98, 114, 121, 141, 156
Pritchard, L. M., 89, 95 Riddle, D. R., 133, 134, 155
Prosser, R. A., 3, 6, 18, 19, 27, 33, 34 Ritter, R. C., 79, 98
Provencio, I., 12, 27 Rivier, J., 87, 99
Pumplin, D. W., 106, 140, 147 Roberts, J. L., 89, 97
Author Index 175
Robinson, A., 82, 86, 98 Sakurai, T., 89, 100

Robinson, P. P., 134, 156 Salmon, P. A., 4, 5, 32
Rodriguez, C. A., 104, 105, 147 Sanacora, G., 88, 98
Rodriguez-Echandia, E. L., 124, 148 Sanear, A., 9, 31
Rodriquez-Sierra, J. F., 41, 60 Sancar, S., 9, 36
Roeder, F., 55, 57, 65 Saper, C. B., 41, 62
Roenneberg, T., 22, 26 Sat, M., 40, 67
Roeper, J., 55, 65 Saraya, A., 55, 65
Rogers, W. T., 56, 67 Sasaki, J., 43, 47, 67
Roitman, M. F., 112, 121, 156 Sasaki, M., 9, 33
Romano, G. J., 44, 48, 63, 66 Sato, K., 23, 34
Rondan-Zarate, A., 48, 49, 63 Sato, M., 113, 155
Rong, M., 107, 153 Satoh, N., 88, 94
Ronnekleiv, O. K., 48, 66 Scardicchio, D. S, 72, 95
Room, P., 40, 64 Scardicchio, L. G., 72, 95
Roper, S., 112, 148 Schak, K. M., 3, 17, 28
Roper, S. D., 106, 107, 108, 112, 148, 158 Scherbarth, F , 22, 35
Roselli, C. E., 48, 66 Scheurink, A. J. W , 92, 98
Rosenwasser, A. M., 6, 7, 8, 12, 14, 21, 27, Schiffman, S. S., 112, 156
30, 34 Schipper, H. M., 57, 61
Rosin, D. L., 21, 34, 36 Schneider, H., 57, 61
Ross, A. W., 86, 88, 89, 97 Schneider, J. E, 81, 82, 83, 86, 92, 99, 100
Ross, D. A., 107, 147 Schneider-Jonietz, B., 57, 61
Ross, I., 73, 74, 81, 98 Schrader, W. T., 50, 60
Rossi, M., 87, 89, 96 Schuhler, S., 16, 19, 34
Rossier, B. C., 105, 138, 153 Schulkin, J., 121, 156
Rowland, N. E., 74, 79, 98, 121, 127, 133, Schumacher, M., 44, 66
149, 152 Schumm, J., 117, 121, 155
Roy, E. J., 52, 66 Schwaber, J. S, 56, 67
Ruby, N. F., 72, 93 Schwartz, G. J., 110, 111,114, 115, 116, 118,
Rniz-Avila, L., 106, 112, 156, 159 119, 122, 127, 137, 141, 145, 150, 151, 158
Rusak, B., 4, 7, 12, 15, 17, 19, 20, 26, 28, 31, Schwartz, M. W., 82, 88, 89, 95, 96, 99
33, 34 Schwartz, W. J., 9, 22, 26, 29, 34
Rushing, P. A., 89, 95 Schwartz-Giblin, S., 39, 66
Ryan, D. H., 87, 99 Scott, C. J., 48, 66
Ryba, N. J., 106, 107, 146, 148, 152, 154 Scott, P. A., 18, 33
Ryba, N. J. P., 107, 112, 154 Scott, T. R , 109, 156
Seal, L. J., 87, 89, 96
S Seeley, R., 87, 99
Saboureau, M., 16, 18, 19, 26, 34 Seeley, R. J., 87, 89, 95, 100
Sagar, S. M., 128, 156 Segal, M., 53, 66
Sage, M., 22, 37 Sego, R., 124, 154
Sahu, A., 88, 98 Seiden, L. A., 12, 26
Saito, H., 135, 156 Seino, S, 55, 65
Saito, T., 135, 156 Selby, C. P., 9, 36
Sakai, N., 123, 126, 159 Selim, M., 18, 28
Sakaki, Y., 23, 24, 36, 37 Selmanoff, M., 46, 49, 62
Sakamoto, K., 23, 34 Semba, K , 19, 26
Sako, N., 123, 126, 159 Setalo, G., 41, 44, 64
Sakuma, Y., 40, 43, 49, 50, 51, 59, 65, 66, Shanabrough, M., 44, 47, 63
67, 68 Shanker, Y. G., 107, 153
176 Author Index
Sharp, F. R., 128, 156 Smith, G. P., 56, 62

Shearman, L. P., 9, 23, 34, 37 Smith, J. C., 109, 110, 117, 121, 124, 130,
Sheehan, T. P., 55, 66 151, 152, 155, 156, 157
Sheelar, S., 115, 137, 156 Smith, M. S., 88, 96
Sheng, M., 128, 156 Smith, R. D., 6, 17, 35
Sherwin, R. S., 55, 59 Smith, S. T., 48, 67
Shiba, H., 43, 47, 67 Smith, W. I., 73, 74, 81, 98
Shibamori, Y., 135, 156 Smithies, 0., 9, 36
Shibata, S., 3, 9, 13, 15, 17, 18, 19, 25, 28, Snyder, G. L., 51, 59
29, 32, 35, 36 Snyder, S. H., 105, 152
Shigeyoshi, Y., 11, 13, 35, 37 Sollars, P. J., 18, 33, 35, 114, 157
Shimoda, K., 6, 37, 38 Sollars, S., 106, 121, 157
Shimura, T., 123, 126, 159 Sollars, S. I., 114, 121, 123, 149, 157
Shin, H. C., 15, 29 Song, C. K., 86, 99
Shingai, T., 104, 156 Song, E. J., 3, 9, 37
Shinoda, K., 43, 47, 67 Soni, B., 13, 27
Shinohara, K., 12, 15, 16, 25, 35 Spangler, K. M., 133, 153
Shinohara, Y., 42, 43, 68 Speetor, A. C., 106, 108, 109, 110, 111, 112,
Shioiri, T., 19, 35 114, 115, 116, 117, 118, 119, 120, 121,
Shiosaka, S., 41, 62 122, 123, 124, 125, 126, 127, 128, 129,
Shiotani, Y., 41, 62 130, 131, 132, 133, 134, 135, 136, 137,
Shirakawa, T., 8, 9, 11-12, 22, 32, 35 138, 139, 141, 145, 147, 148, 149, 150,
Shiuchi, T., 55, 65 151, 152, 153, 157, 158
Shivers, B. D., 42, 43, 47, 51, 62, 63 Speh, J. C., 12, 32
Shughrue, P. J., 49, 50, 56, 62, 67 Spina, M., 87, 99
Shulman, G. I., 55, 59 Spitznagel, E., 3, 9, 37
Siegel, E. G., 111, 147 Srinivasan, V., 99
Sieghart, W., 47, 61, 68 Sriram, S., 9, 34
Silver, R., 10, 11, 28, 32 St. John, S. J., 110, 112, 114, 115, 117, 119,
Silverman, H. J., 74, 99 120, 121, 122, 123, 124, 125, 126, 130,
Silverstein, J., 89, 97 131, 132, 133, 134, 135, 137, 148, 153,
Simerly, R. B., 40, 42, 43, 44, 50, 56, 60, 67 157, 158
Simmons, D. M., 48, 66 Stanfield, E. J., 41, 62
Simon, S. A., 104, 105, 117, 138, 148, Stanley, B. G., 87, 88, 99
156, 159 Stanley, S. A., 87, 89, 96
Simpson, D., 135, 150 Stapleton, J. R., 112, 158
Simpson, J. S., 83, 95 Staszewski, L., 107, 153
Sipols, A. J., 87, 88, 99, 100 State, F. G., 133, 149
Sivakumar, L., 104, 105, 147 Staudt, J., 48, 61
Skeen, L. C., 41, 63 Steinlechner, S., 22, 35
Slater, N. T., 35 Stellar, E., 71, 73, 74, 78, 79, 97, 99
Slotnick, B. M., 110, 115, 137, 156 Stephan, F. K., 12, 35
Smagin, G. N., 87, 99 Stephens, D. W., 69, 99
Smale, L., 16, 18, 29, 35 Stephens, P. H., 56, 64
Small, C. J., 87, 89, 96 Stern, J. M., 42, 44, 64
Smart, C. M., 18, 35 Sterner, M. R., 40, 67
Smith, B. N., 18, 33, 35 Stevens, R., 80, 94
Smith, D. M., 87, 89, 96 Stevens, S. S., 109, 158
Smith, D. V., 104, 106, 107, 108, 112, 114, Stewart, R. E., 139, 158
122, 132, 140, 147, 149, 150, 155, 156, 158 Stidsen, C. E., 88, 96
Smith, E., 5, 16, 30 Stokkan, K. A., 23, 36
Author Index 177
Stopa, E. G., 12, 25, 56, 57, 61, 67 Teff, K. L., 111, 158
Storer, T. I., 70, 98 Tei, H., 13, 23, 24, 35, 36, 37
Stornetta, R. L., 21, 34 Teitelbaum, P., 110, 152
Strack, A. M., 89, 95 Tennissen, A. M., 112, 158
Straume, M., 8, 24, 29 Ter Horst, G. J., 43, 67
Stricker, E. M., 86, 95 Teske, J. A., 89, 96
Stromberg, E., 84, 96 Tetel, M. J., 44, 46, 67, 68
Stumpf, W. E., 40, 67 Than, T., 112, 148
Sugden, D., i2, 30 Thiessen, D. D., 74, 80, 81, 97
Sukhov, R. R., 56, 67 Thomas, M. E. A., 99
Sumova, A., 11, 36 Thombre, D. P., 99
Sun, Z. S., 22, 25, 37 Thornton, J. E., 41, 68
Sunter, D., 87, 89, 96 Thrasher, T. N., 124, 159
Svendsen, C. N., 56, 67 Thresher, R. J., 9, 36
Swaab, D. F., 56, 57, 61, 63, 67 Tilbrook, A. J., 48, 66
Swanson, L. W., 40, 42, 43, 44, 50, 56, Tillet, Y., 55, 59
60, 67 Tobet, S. A., 47, 57, 61, 68
Swanson, R. A., 128, 156 Toft, D. O., 43, 59
Sweazey, R. D., 114, 132, 158 Tohyama, M., 41, 42, 43, 62, 68
Sweeney, E. A., 124, 148 Tokuriki, M., 135, 156
Swiergiel, A. H., 76, 94 Tominaga, K., 3, 16, 18, 35, 36
Szczepaniak, L. S., 55, 64 Tonosaki, K., 114, 159
Toran-Allerand, C. D., 44, 60
T Tordoff, M. G., 79, 95, 107, 147
Taguchi, K., 13, 35 Torres-Aleman, I., 52, 62
Takagi, H., 41, 62 Tosini, G., 17, 23, 27, 36
Takahashi, J. S., 3, 4, 6, 8, 9, 13, 16, 20, 26, Trachsel, L., 21, 36
29, 30, 35, 36, 37 Tracy, C. J., 123, 157
Takahashi, K., 3, 6, 19, 20, 27, 32, 35, 37, 38 Trafton, C. L., 110, 117, 160
Takahashi, L. K., 40, 67, 70, 99 Travers, J. B., 114, 118, 119, 122, 127, 141,
Takahashi, R., 23, 37 145, 150, 156, 159
Takahashi, S., 6, 19, 35, 37, 38 Travers, S. P., 103, 104, 114, 117, 127, 132,
Takamure, M., 3, 32 133, 143, 149, 151, 152, 158, 159
Takano, R., 9, 36 Travnickova, Z., 11, 36
Takao, M., 9, 36 Trayhurn, P., 81, 99, 100
Takekida, S., 11, 13, 35, 37 Tribollet, E., 57, 64
Takhashi, K., 6, 37 Trimble, E. R., 111, 147
Takoa, M., 13, 25 Tsuneyoshi, A., 18, 35
Talley, E. M., 21, 34, 36 Turcotte, J., 43, 59
Tamaki, M., 88, 94 Turcotte, J. C., 51, 59
Tamborlane, W. V., 55, 59 Turek, F. W., 4, 5, 6, 7, 9, 15, 16, 17, 18, 19,
Tanaka, H., 23, 34 20, 26, 27, 29, 33, 35, 36, 37
Tang, S., 83, 99
Tang-Christensen, M., 88, 96 U
Tapper, D. N., 122, 158 Uebayashi, H., 114, 159
Tarpy, R. M., 74, 76, 96 Ueda, M., 23, 37
Tashiro, S., 51, 67 Ueki, S., 18, 36
Tateishi, K., 41, 62 Ulibarri, C., 50, 68
Taylor, J., 50, 65 Unmehopa, U. A., 57, 63
Tecco, J. M., 6, 36 Utunomiya, K., 3, 32
Tecott, L. H., 55, 68 Uylings, H. B., 80, 96
178 Author Index
V Weaver, D. P., 23, 37

Vaishnav, S., 22, 37 Weaver, D. R., 9, 22, 25, 34
Vale, W., 87, 99 Weber, E. T., 16, 18, 27, 33, 36
van den Pol, A. N., 17, 28 Webster, F. A., 73, 98
van der Horst, G. T. J., 9, 22, 26, 34, 36 Wee, B. E., 20, 36
Van Der Laan, C. E., 55, 63 Wehr, T. A., 22, 26
van der Plas, J., 41, 68 Weinstein, H., 107, 153
Van Der Ploeg, L. H., 89, 95 Weiss, M. L., 12, 30
Van Der Poel, A. M., 55, 63 Welsh, D. K., 8, 36
van der Zee, E., 20, 30 West, D. B., 107, 147
van Heerikhuize, J. J., 57, 63 Whishaw, G. E., 70, 100
Van Houten, M., 43, 44, 68 Whishaw, I. Q., 70, 100
Van Lennep, E. W., 113, 160 White, J. D., 88, 98
van Reeth, 0., 5, 6, 7, 16, 20, 26, 36, 37 White, K. S., 123, 151
VanBuskirk, R. L., 122, 156 White, T. D., 104, 105, 147
Vance, W. B., 114, 141, 159 Whitehead, M. C., 134, 135, 155, 159
Vander Wall, S. B., 70, 99 Whiteside, B., 103, 133, 159
VanNess, J. M., 87, 88, 99 Whiting, P. J., 47, 68
Vathy, I., 41, 68 Whitney, G., 109, 151
Veening, J. G., 46, 60 Whorl, R. C., 47, 68
Venier, G., 40, 59 Wickland, C. R., 5, 16, 37
Venuti, J. M., 11, 28 Wiersinga, W. M., 56, 61
Vergoni, A. V., 89, 98 Wiler, S. W., 21, 33
Verkerk, A., 9, 36 Williams, G. W., 55, 64
Viek, P., 76, 97 Willoughby, R. R., 71, 96
Vilaplana, J., 7, 26 Willoughby, W. O., 41, 68
Vincent, P. A., 41, 52, 61, 68 Wilsbacher, L. D., 3, 9, 37
Vintschgau, M. V., 133, 159 Wilson, B. D., 89, 98
Visser, T. J., 56, 61 Wilson, L., 157
Vitaterna, M. H., 9, 36 Winkles, P. A., 134, 156
Vogt, L. J., 21, 34 Winn, P., 73, 81, 83, 95, 100
Vogt, M. B., 114, 159 Winters, D., 82, 98
yon Schantz, M., 13, 27 Wirth, M. M., 89, 100
Voorhuis, D. A. M., 43, 61 Wisialowski, T. A., 17, 28
Vrang, N., 19, 26 Wolf, G., 121, 159
Wolfe, J. B., 70, 71, 100
W Wong, G. T., 106, 112, 156, 159
Wong, R., 74, 100
Wada, H., 21, 22, 29
Wood, A. D., 72, 74, 81, 82, 85, 86, 88, 100
Wade, G. N., 39, 56, 59, 68, 72, 74, 80, 81,
Woods, S. C., 82, 87, 89, 95, 96, 99, 100
86, 92, 93, 99, 100
Woolley, C. S., 48, 53, 62
Walker, L. C., 56, 67
Wu, C., 107, 147
Wallace, P., 80, 97
Wu, L.-H., 133, 134, 155
Wallace, R. J., 73, 100
Wu, S. S., 57, 68
Walsh, J. P., 55, 59
Wang, C., 89, 96 X
Wang, Y., 117, 159 Xu, B., 55, 68
Watanabe, A., 15, 28 Xu, H., 107, 153
Watanabe, S., 3, 15, 18, 28, 35, 36
Watanabe, T., 47, 63 Y
Watson, C. H., 112, 148 Yahr, P., 50, 68
Watson, N. V., 16, 19, 30 Yamada, K., 114, 141, 146
Author Index 179
Yamada, N., 6, 19, 35, 37, 38 Young, M., 55, 64

Yamagishi, T., 135, 156 Young, M. W., 9, 37
Yamaguchi, S., 3, 9, 25 Young, P. T., 110, 117, 160
Yamamoto, S., 13, 35 Young, W. C., 39, 65
Yamamoto, T., 117, 118, 123, 124, 125, Young, W. S., III., 56, 67
126, 159 Youngstrom, T. G., 84, 100
Yamanaka, A., 89, 100 YU, C. S., 106, 140, 147
Yamano, M., 41, 42, 43, 62, 68 Yuste, R., 53, 68
Yamanouchi, K., 49, 68
Yamanouchi, S., 15, 28 Z
Yamashita, S., 113, 155
Yamatodani, A., 21, 22, 29 Zaborszky, L., 41, 68
Yamauchi, Y., 113, 153 Zamora, S., 7, 30
Yamazaki, K., 108, 154 Zampighi, G. A., 105, 138, 156
Yamazaki, S., 3, 9, 23, 24, 36, 37 Zang, K., 55, 68
Yan, L., 11, 13, 35, 37 Zatz, M., 20, 37
Yan, X., 83, 95 Zee, P. C., 16, 19, 20, 33, 37
Yanagisawa, K., 143, 145, 159 Zelman, F. P., 39, 63
Yanagisawa, M., 89, 100 Zhang, C., 52, 66
Yanase, M., 49, 68 Zhang, H., 105, 106, 107, 108, 121, 150
Yang, H., 112, 148 Zhang, Y., 16, 20, 37, 82, 97
Yang, Y. K., 89, 98 Zhang, Y. F., 107, 112, 154
Yannielli, P. C., 16, 37 Zhao, G., 107, 154
Yasoshima, Y., 123, 126, 159 Zheng, B., 9, 22, 25, 34, 37
Yasui, A., 9, 36 Zhu, Y.-S., 41, 61
Yau, K. W., 13, 29 Zielinski, J. E., 44, 48, 60
Ye, Q., 139, 160 Zoeller, R. T., 12, 25
Yocca, F. D., 17, 28 Zoller, M., 107, 153
Yokota, S., 17, 19, 29 Zucker, I., 12, 35, 56, 68, 74, 80, 99, 100
Yoon, Y. W., I5, 29 Zuker, C. S., 106, 107, 112, 146, 148,
Yoshii, H., 43, 47, 67 152, 154
Yoshimasa, Y., 88, 94 Zumpe, D., 57, 65
Yoshimatsu, H., 40, 57, 59 Zuniga, J. R., 135, 160
Yoshioka, T., 88, 94 Zylka, M., 23, 37
Young, J. A., 113, 160 Zylka, M. J., 9, 34
Subject Index
5HT receptors, 18 19 Chorda tympani (CT) nerve, 103

8-OH-DPAT, 18-19 effects of combined CT and GSP
transection, 130-131
A
stimulus discrimination, 131
A-fibers, 105, 126 unconditioned licking responses,
Acetylcholine, 20 130-131
Agouti-related protein (AgRP), 85, effects of CT transection, 115 125,
88, 89 133-135, 137-138
Alpha-2 adrenergic receptors, 21 conditioned taste aversion, 122-124
c~-gustducin, 106, 140 oral motor taste reactivity, 118-119
Amiloride, 105 106, 121, 122, 123 signal detection, 115-117, 137
effectiveness of, 137, 138, 139 sodium appetite, 121-122
Androgen receptors, 43, 48, 55, 57 stimulus discrimination, 119-120, 126,
Androgens, role in food hoarding, 80 137, 138
Anesthesia, 142-144, 145 on taste buds, 133-135
palatal, 144 unconditioned licking responses,
of tongue, 144 117-118
Appetitive behaviors, 69, see also innervation of salivary glands by, 113, 124
Ingestive behavior, appetitive regeneration of, 134-139
phase CT and GL cross-regeneration,
Aromatase activity, 48 140-142
B CT and GSP cross-regeneration, 140
Behavior, definition of, 101 in humans, 134-135
Benzodiazepines, 5-6, 16 Circadian oscillators, 22-23
Bmal gene, 9
Circadian pacemaker
Body fat levels activity-dependent feedback effects, 6-7
changes in pregnancy, 81 dark pulse effects, 7-8
inverse relation to food hoarding, neurobiology of, 8-12
80 86 nonphotic effects, 5-7, 13, 14
Brief-access taste tests, 110, 111, 117, 118, novelty-induced effects, 5, 16, 17, 19-20
119, 130 phase shifting, 14
after GL and CT transection, 139 carbachol-induced, 2~21
clonidine-induced, 21
C histamine-induced, 21-22
C-fos gene, 13 serotonergic, 18
Carbachol, 15-16, 20 triazolam-induced, 19
182 Subject Index
photic effects, 3-5, 13, 14 receptors

photic entrainment, 12-13, 15, 16 expressed by VMH afferents, 52
sleep-deprivation effects, 5 expressed by VMH neurons, 43, 44,
Circadian system, see also Circadian 45M6, 52
oscillators; Circadian pacemaker spine density regulator, 48
free-running, 2, 15 Estrus cycle, effects on food hoarding, 80
behavioral studies of, 3
period affected by continuous F
illumination, 4-5
Circumvallate papillae, 103, 106, 107, Fasting, 73-74, 88
133, 136 in photoperiod, 72
C K I e gene, 9, 10 Fish, 145
C l o c k genes, 9 Flank stimulation, 46
Clock-controlled genes (CCGs), 9 Flavor, 101
Clonidine, 21 FLI, see Fos-like immunoreactivity
Cold exposure, 72-73, 85 Foliate papillae, 103, 106
Consummatory behaviors, 69, see also Food-carrying behavior, 70-71
Ingestive behavior, consummatory Food hoarding, 69-92
phase central control of, 86-91
Corticotropin-releasing hormone deficit hypothesis, 71, 74
(CRH), 85 early laboratory studies, 70-71
Cry genes, 9, 10 environmental influences, 71-78
cold exposure, 72-73, 85
food shortage, 73 74
D
food type, 74-75
DARPP-32, 51 foraging effort, 75-78
Dendrites, 54 photoperiod, 72
Dietary wisdom, 75 inverse relation to adiposity, 80-86
Disinhibition, 143-144 lipostatic theory, 71
Dopamine receptors, 51 in nature, 70-71
Drosophila clock genes, 9 peripheral physiological controls of,
78 80
gonadal steroids, 79-80
E
metabolic fuel challenges, 78-79
ENaCs, see Epithelial sodium channels Food intake, role of VMH in, 56
Energy acquisition, 70 Food restriction, 73
Energy balance, 86, 92 Foraging, 69, 87 90
Energy flux, 86, 92 effort, 75 78
Energy stores, 75, 81, 85 Fos induction, 46
Enkephalin, 41, 42, 43 Fos-like immunoreactivity (FLI),
Epiglottis, 106 122129
Epinephrine, 78, 79 Frustration, 71
Epithelial sodium channels (ENaCs), Fungiform papillae, 102, 107, 134
105-106, 112, 121, 123, 138
ERa, 48, 5~51, 56 G
ERr, 48, 50-51, 56 G-protein genes, 107
Estradiol, 53, 80 GABA, 10, 11, 15, 16, 17
Estrogen in VMH, 46, 47, 48, 49, 52
cellular effects of, 50-51 GAD, 47
neonatal exposure to, 49 GAP-43, 49
neuronal activity effects of, 51-52 Gaping, 127-129, 132, 136
Subject Index 183
Geniculohypothalamic tract (GHT), 15-17 early history, 113 115

Geschmacksstreifen, 103, 106, 134 general caveats, 112-113
GHT, s e e Geniculohypothalamic tract GL transection, s e e
Glossopharyngeal (GL) nerve, 103 Glossopharyngeal
effects of GL transection, 125 129, 131 (GL) nerve, effects of GL
oral motor taste reactivity, transection
127-129 summary and functional
sodium appetite, 126 implications, 131 133
stimulus discrimination, 120, H
125-126, 131
on taste buds, 133, 134 H-fibers, 104, 105
unconditioned licking responses, 125 Hibernation, 72
Histamine, 21-22
human, 144
innervation of von Ebner's glands Hunger, 71
Hyperphagia, 74
by, 113
regeneration of, 134-136, 139 I
GL and CT cross-regeneration, IGL, s e e Intergeniculate leaflet
140-142 Immediate early-response genes
Glucoprivation, 79 (IEGs), 13, 16
Glutamate, 13, 16, 17, 21, 22 Infertility, fasting-induced, 83
in VMH, 46, 47, 52 Ingestive behavior
Goal-lessness, 71 appetitive phase of, 69, 87, 88, 90, 111,
Golgi impregnation, 53 127, s e e a l s o Food hoarding;
Gonadal fat, 78 Foraging
Gonadal steroids, 79-80 consummatory phase of, 69, 87, 90-91,
Greater superficial petrosal (GSP) 111, 127
nerve, 103, 114 Inosine monophosphate, 107
effects of combined CT and GSP Instinct, 71
transection, 130-131 Insulin, 79, 111, 123
stimulus discrimination, 131 Intake tests, 110, 111, 113 115, 141
unconditioned licking responses, Intergeniculate leaflet (IGL), 11,
130-131 15-17, 18, 19
regeneration of, 134
GSP and CT cross-regeneration, 140 K
Gustatory system, peripheral, 101-146 Kinase C, 51
anatomy, 102-103, 104
biophysics and molecular biology, 105 L
electrophysiology, 103-105 Lateral hypothalamus (LH), 85
human, 142-145 Leptin, 82-83
compared with rodents, 146 Licking responses, 110, 117-118, 119, 125,
nerve cross-regeneration, 140-142 130-131, 145
nerve regeneration consequences, Lipectomy, 81
133-139 Lipid deficits, 81 82, 86
uerve transection consequences, sensed by brain, 82-83
111-133 Lipoprivation, 79
CT and GSP transection, s e e Chorda Lordosis response, 39~40
tympani (CT) nerve, effects of estrogen mediated, 50-51
combined CT and GSP in male rats, 49
transection role of GABA in, 46, 47
CT transection, s e e Chorda tympani (CT) role of glutamate in, 47
nerve, effects of CT transection role of VMH in, 44, 47, 58
184 Subject Index
M Periaqueductal gray (PAG), 40, 41,

Macronutrients, 75 42, 54
Masking, 2 role in lordosis reflex, 44
Maternal aggression, 55 subdivisions of, 42
Maternal behavior role, 55 Peripheral gustatory system, s e e Gustatory
Maternal nurturing, 55 system, peripheral
Mecamylamine, 20 Phase-response curves (PRCs), 2
Melanocortin (MC), 89 for carbachol-induced phase-
Melanopsin, 13 shifting, 20
Melatonin, 23, 72, 86 dark pulse, 7-8
Metabolic fuel utilization, 79 nonphotic, 6, 14, 20
mGluR4 receptors, 107, 112 for novelty-induced activity, 4, 5, 7
Micronutrients, 75 photic, 3~4, 6, 14, 20
MK-801, 21 for serotonergic stimulation, 18
Morphine, 90, 91 triazolam effects, 4, 5, 7
Photoperiod, 72, 86
N Pineal gland, 72
Pituitary adenyl cyclase activating peptide
N-fibers, 104, 105 (PACAP), 11, 15
Naltrexone, 90, 91 Posterior palatine field, 103, 134
Nasoincisor ducts (NID), 130, 134 PRCs, s e e Phase-response curves
Neuropeptide Y (NPY), 11, 15, 16, 17, 88 Predation, 92
injected, 85, 87, 88, 89 Progesterone, 80
Neuropeptides Progestin receptors, 44, 46, 48
as controllers of appetitive ingestive Prolactin, 42, 43, 80
behaviors, 88-90 PRV detected in, 45
as controllers of food intake, 87 Proopiomelanocortin gene, 88
Neuropil, in VMH, 48 Pseudorabies virus (PRV), 44-46
N P Y , s e e Neuropeptide Y Pup cannibalism, 75
Nucleus of the solitary tract (NST), 103, P V N , s e e Paraventricular nucleus
127-128, 135, 139, 143, 145
Q
O
Q-fibers, 105, 126
Opiates, 90 Quinine aversion, 114, 115
Oral motor taste reactivity, 118 119,
127-129 R
Orexin, 88, 89
Oxytocin receptors, 43, 44, 51 Raphe nuclei, mesencephalic, 11, 17-20
Reproduction, and food hoarding, 80,
P 86, 92
Retinohypothalamie tract (RHT),
PAG, s e e Periaqueductal gray
1~15, 17
Paraventricular nucleus (PVN), 40-41
P e r genes, 9, 10, 14, 17
S
expression effects
of 8-OH-DPAT, 19 S-fibers, 105
of glutamate, 13 Salivary glands, innervation of, 113,
of light exposure, 17 124-125
of PACAP, 15 Salivation, 111, 133
expression in peripheral tissues, 23 SCN, s e e Suprachiasmatic nucleus
functional specialization, 22 Security, 71
Perception, 101 Serotonin, 17-20
Subject Index 185
Sexual behavior Temperature rhythm, daily, 2

human, 57 Testosterone, 80
importance of VMH for, 40, 55 neonatal exposure to, 49
rodent, 39 Thermal homeostasis, 72
SF-1 gone, 55 Thiouracil, 79
SLN, see Superior laryngeal (SLN) nerve Thyroxine, 79
Sodium appetite, 121 122, 126 Tim gone, 9
Spines, dendritic, 53, 54, 58 Tongue maps, 108-109
Splitting, 22 Triazolam, 5 6, 16, 17, 19-20
Substance P (SP), 11, 15, 42, 43, 46, 56 Triglyceride, 111
in WAT, 83
Superior laryngeal (SLN) nerve, 103, U
114, 132 U-50,488H, 90, 91
minor role in taste-guided behavior, 130 Umami, 107
Suprachiasmatic nucleus (SCN)
circadian oscillation in, 8 4 V
core, 10-12 Vaginal cervix stimulation (VCS), 39, 46
GRP-positive neurons in, 10 Ventromedial nucleus of the hypothalamus
VIP-positive neurons in, 10
(VMH), 39-58
SCN afferent systems, 11, 12-22 afferents, 4(~41, 49, 52
IGL, 11, 15 17 calorie regulation role, 55-56
raphe, 11, 17-20 cellular effects of estrogen in, 50-51
retinal, 11, 1~15
central zone, 43
shell afferents, 2(~22 dorsomedial zone, 43
shell, 10-12
spine density in, 53, 54
AVP-positive neurons in, 10
efferents, 42
T human, 56 57
T1Rs, 107 intrinsic circuitry, 43~47
T2Rs, 106, 107 male sexual behavior role, 55
Taste, 101 maternal behavior role, 55
Taste aversion, conditioned, 12~124 neuronal activity effects of estrogen in,
Taste buds, 102-103, 104 51-52
after nerve transaction, 133-134 rostral zone, 43, 45
regeneration of, 134, 135, 136, 139 sexual dimorphisms of, 47-50
Taste discrimination tasks, 11%120, synaptic organization, 44, 48
122, 125-126, 131, 137-138, estrogen-induced changes, 52-55
144-145 ventrolateral zone, 43, 44
Taste dysfunction, human, 142-145 neurons in, 45M6, 54
Taste function, t08-109 role in lordosis response, 44, 47, 58
domains of, 109-111 spine density in, 53 54
affective, 109, 111~111 Visual system, primate, 146
physiological domain, 111 VMH, see Ventromedial nucleus of the
sensory-discriminative, 109 110 hypothalamus
Taste phantoms, 143
Taste pore, 102 W
Taste receptor cells, 101, 102 White adipose tissues (WAT), 78, 81 82,
ion channels on, 105 83-85
laryngeal, 102, 114 epididymal (EWAT), 78, 81
palatal, 106, 114 gonadal, 77, 78
protein receptor sites on, 105 sensory innervation, 83-85

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Progress in

Numbersin parenthesesindicatethe pageson whichthe authors' contributionsbegin.

Alan M. Rosenwasser, Department of Psychology, University of Maine,

In Volume 18 of Progress in Psychobiology and Physiological Psychology,

Volume 18 consists of four original chapters covering a broad range of

is contributed by Alan Rosenwasser who revisits a topic he wrote about in

neurochemical systems in brain that are regulated by leptin. Almost all of

Volume 7 Behavioral Modulation of Visual Responses in

David A. McCormick, David G. Lavond, Mechanisms of Brain-Stimulation Reward

Fear and Its Neuroendocrine Basis Rita J. Valentino, Andre L. Curtis,

Neurobiology of the Mammalian Circadian System:

II. Conceptual and Methodological Foundations

Copyright 2003, Elsevier Inc.

In principle, stimuli that alter the overt expression of a given circadian

In either case, these experimental methods usually involve relatively long

Ill. Photic Effects on the Mammalian Circadian Pacemaker

the relatively precise timing of free-running activity rhythms in this species.

FIGURE 1 Selected phase-response curves (PRCs) obtained from free-running Syrian

average per-cycle delay or an increase in the average per-cycle advance).

IV. Nonphotic Effects on the M a m m a l i a n Circadian P a c e m a k e r

daytime locomotor activity can modulate the steady-state phase of mice

V. The Strange Case of Dark Pulses

VI. Neurobiology of the Circadian Pacemaker

apparently remain synchronized even in the absence of sodium-dependent

this anatomical scheme has been reconceptualized as comprising distinct

Hypothalarnus, Thalamus, Basal Forebrain

Post Hyp (HA)

IGL (NPY, GABA)

Retina (GLU, SP, PACAP)

A l t h o u g h the specific f u n c t i o n s o f these chemically-defined S C N cell

VII. Pacemaker Inputs: The Retinohypothalamic Tract

Mediation o -photic input

noncone photoreceptor (Freedman et al., 1999). Indeed, it now appears that

Subjective night Subjective day Subjective night Subjective day

In addition to glutamate, RHT terminals also release two identified

VIII. Pacemaker Inputs: The Geniculohypothalamic Tract

Pickard, Ralph, and Menaker, 1987). The complexity of the reported

IX. Pacemaker Inputs: Role of the Mesencephalic Raphe Nuclei

and (2) mediation of nonphotic, behavioral state-related effects on the

destruction of serotonin terminals within the SCN via local application of

expression is stimulated in IGL neurons by novelty-induced but not by

X. Other Afferent Systems: Acetylcholine,

muscarinic but not nicotinic antagonists blocks carbachol-induced phase

pharmacological inhibition of histamine synthesis reduces the phase shifting

XI. Multiple Oscillators

In addition, it is not known if hypothesized intra-SCN morning and evening

XII. S u m m a r y and Conclusions

intra-SCN oscillators, which may correspond with anatomically recognized

Daan, S., & Pittendrigh, C. S. (1976). A functional analysis of circadian pacemakers

Pittendrigh, C. S., & Daan, S. (1976). A functional analysis of circadian pacemakers in

Hypothalamic Neural Circuitry: ATarget

II. Neural Connectivity of the V M H

nucleus (PVN), ventral premamillary nuclei, and the periaqueductal gray.

C. INTRINSIC CIRCUI:FRY OF VMH

Another special feature of neurons within the ventrolateral subdivision is

transported retrogradely and can be detected immunohistochemically. A

neurons from the estrogen receptor-containing neurons suggests that

I l l . Sexual Dimorphisms of the V M H

twice the number of cells containing phosphorylated CREB compared with

There also are sex differences in neurotransmission in the VMH. For

Parameters that are greater in males

IV. The Effect of Estrous Cycle on V M H Activity

B. EFFECTS ON NEURONAL ACTIVITY