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Gut Emotions - Mechanisms of Action of

Probiotics as Novel Therapeutic Targets for
Depression and Anxiety Disorders
ARTICLE in CNS & NEUROLOGICAL DISORDERS - DRUG TARGETS (FORMERLY CURRENT DRUG TARGETS - CNS &
NEUROLOGICAL DISORDERS) NOVEMBER 2014
Impact Factor: 2.7 DOI: 10.2174/1871527313666141130205242 Source: PubMed
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1770
CNS & Neurological Disorders - Drug Targets, 2014, 13, 1770-1786
Gut Emotions - Mechanisms of Action of Probiotics as Novel Therapeutic

Targets for Depression and Anxiety Disorders
Anastasiya Slyepchenko1,, Andre F. Carvalho2,, Danielle S. Cha1, Siegfried Kasper3 and
Roger S. McIntyre*,1,4
1
Mood Disorders Psychopharmacology Unit, University of Toronto, Toronto, ON, Canada
Translational Psychiatry Research Group, Faculty of Medicine, Federal University of Ceara, Fortaleza, CE, Brazil
Department of Psychiatry and Psychotherapy, Medical University of Vienna, Whringer Grtel 18-20, Vienna, Austria
Departments of Psychiatry and Pharmacology, University of Toronto, Toronto, ON, Canada

Abstract: A priority clinical and research agenda in mood and anxiety disorders is to identify determinants that influence
illness trajectory and outcome. Over the past decade, studies have demonstrated a bidirectional relationship between the
gut microbiome and brain function (i.e., the microbiota-gut-brain axis). Probiotic treatments and developmental analysis
of the microbiome may provide potential treatments and preventative measures for depressive and anxiety disorders. This
systematic literature review aims to identify original studies linking the gut microbiota to major depressive disorder and
anxiety disorders. Furthermore, this review searched for original reports focusing on possible therapeutic and preventative
effects of probiotics for these debilitating conditions. Accumulating data indicate that the gut microbiota communicates
with the CNS through neural, endocrine and immune pathways. Studies in germ-free animals indicate that the microbiota
is involved in the regulation of the stress response (e.g., hypothalamic-pituitary-adrenal axis) and in CNS development at
critical stages. Probiotics attenuate anxiety and depressive-like behaviors in experimental animal models. Notwithstanding
some inconsistencies and methodological limitations across trials, clinical studies suggest that probiotics may mitigate
anxiety symptoms. However, future studies should investigate the anxiolytic and antidepressant effects of probiotics in
more phenotypically homogeneous populations. In conclusion, the emerging concept of a gut microbiota-brain axis
suggests that the modulation of the gut microbiota may provide a novel therapeutic target for the treatment and/or
prevention of mood and anxiety disorders.
Keywords: Anxiety, depression, gut microbiota, probiotics, review, treatment.

1. INTRODUCTION
During the past decade, studies evaluating the
physiological role of gut commensal microbiota
demonstrated their involvement in vital processes, including
but not limited to the development of the immune system
[1], nutrient processing and mucosal barrier fortification [2].
The gut is estimated to contain approximately 1-2 kilograms
of bacteria, comprised of approximately 15,000 - 36,000
species and 1,800 genera [3]. Bacteroides, Ruminococcus,
Faecalbibacterium, Clostridium and Eubacterium genera
compose the majority of the human gut microbiome. The
microbiome has been reported to be involved in critical
digestive and immune processes (e.g., nutrient extraction,
immune function and enteropathogen protection) [4].
Development of the human microbiome begins after birth, at
which point the digestive tract is sterile, with colonisation
commencing within hours of the infants life [5]. The
microbiome remains relatively stable after the age of three,
*Address correspondence to this author at the Mood Disorders
Psychopharmacology Unit, University Health Network, 399 Bathurst Street,
Toronto, Ontario, M5T 2S8, Canada; Tel: +1 416 603 5279;
Fax: +1 416 603 5368; E-mail: Roger.McIntyre@uhn.ca
These authors contributed equally to the present work.
1996-3181/14
$58.00+.00
although a number of factors influence its composition

throughout an individuals lifespan, including the
environment, diet, drug exposure and genetic factors.
Notwithstanding the similarities in genetic profile for the
combination of microbiota present in humans, significant
inter-individual variability exists in the composition of the
microbiome. The foregoing observations provide the basis
for projects, such as the Human Microbiome Project, which
aim to explore and characterize the microbiome [4].
Animal models evaluating the relationship between host
organisms and their microbiomes suggest that a symbiotic
reciprocity exists. A prominent example of this is a study of
germ-free mice colonised with the microbiota of obese mice
during adulthood. Germ-free mice colonized with microbiota
of obese mice gained fat more quickly than did mice
colonised with the microbiome of a lean mouse [6],
illustrating the significant impact a hosts microbiome can
have on host metabolism. Disturbed microbiomes have been
observed for a wide range of disorders, including irritable
bowel syndrome, obesity and Crohns disease [5].
The bidirectional relationship between the gut and the
brain has been referred to as the gut-brain axis, which is
mediated through the autonomic nervous and enteric nervous
systems (ENS), respectively [7]. Furthermore, monoaminergic pathways, the sympatho-adrenal axis and the hypothala 2014 Bentham Science Publishers
Probiotics, Anxiety and Depression
mic-pituitary-adrenal (HPA) axis mediate the cross-talk

between the gut and the brain [8]. The outputs to the gut
from the brain are generated by the amygdala and
hypothalamus, which in turn receive signals from a cortical
network which includes the medial prefrontal cortex and the
anterior cingulate cortex [9, 10]. This network is responsible
for the integration and transmission of information about
homeostasis in the gastrointestinal tract, visceral pain and
food intake [8]. The ENS is comprised of 200-600 million
neurons, as a mechanism of communicating homeostatic
needs to the brain, including mechanical distension and
nutrient needs [11]. Primary afferent neurons, enteroendocrine cells and immune cells comprise the mechanisms
through which information is encoded in the gut [12]. The
gut is innervated by vagal afferents, many of which
terminate near enteroendocrine cells and respond to
neuropeptides; these nerve terminals discharge in response to
mechanical and chemical events in the lumen, causing satiety
or hunger [13]. The gut is also innervated by spinal afferents,
which provide information that is necessary to carry out
peristalsis and other spinal reflexes [8].
Intrinsic primary afferents are the principal communication
pathway within the gut. In turn, the brains modulation of gut
physiology is extensive, with a number of pathways, for
example nociceptive pathways, pathways which are responsible
CNS & Neurological Disorders - Drug Targets, 2014, Vol. 13, No. 10
1771
for homeostasis, and food consumption [12]. Enteroendocrine

cells are the first to integrate information from various stimuli
coming from mechanical distension of the gut and chemical
alterations, providing important regulatory output to digestive
ENS and CNS processes [14]. Immune-related signalling has an
important balancing function of being responsive to foreign,
pathogenic organisms, yet being tolerant to commensal bacteria
[15]. Certain vagal afferents are endowed with receptors which
allow them to respond to products which come from mast cells
and macrophages, leading to activation of the immune response
[7]. These include histamine, serotonin and corticotropin
releasing hormone (CRH) as well as different cytokine subtypes
[8]. Furthermore, an increased production of inflammatory
cytokines (e.g., interleukin-6 [IL-6], tumour necrosis factor-
[TNF-] and interferon- [IFN-]) results in the activation of
indoleamine-2,3-deoxygenase, while glucocorticoids induce
tryptophan-2,3-dioxygenase activation [16]. Both tryptophan2,3-dioxygenase and indoleamine-2,3-deoxygenase degrade
tryptophan, which is the precursor of serotonin (5-HT), limiting
5-HT synthesis. In addition a number of tryptophan catabolites,
such as kynurenine and quinolinic acid may have
depressogenic, anxiogenic and neurotoxic effects [16]. See Fig.
(1) for a schematic depiction of physiological processes related
to the gut-brain axis, which require the gut microbiota.
Fig. (1). A schematic representation of the effects of chronic stress, anxiety and depression on the gut-brain axis. The reciprocal
communication depends on a physiological gut microbiota. Mammals with a disrupted microbiome have a hypersecretion of corticotrophinreleasing hormone (CRH) and an overall activation of the hypothalamic-pituitary-adrenal (HPA) axis. Furthermore, an increase in vagal
drive which activates the immune system and alters the gastrointestinal barrier occurs. An increase in inflammatory cytokines leads to an
activation of the kynurenin pathway and to an abnormal monoaminergic neurotransmission. A desensibilization of central nervous system
(CNS) cortisol receptors also leads to impaired norepinephrine (NE) and serotonin (5-HT) transmission. An elevation in CRH alters colonic
motility. Abnormalities in the integrity of the gut microbiota may contribute to the pathophysiology of depression and anxiety and initiate a
pathological cross-talk between the central nervous system and the gastrointestinal tract.
1772
In recent years, accumulating evidence indicates that the

gut microbiome may play a role in the pathophysiology of
mood and anxiety disorders [17-19]. The overarching aims
of this literature review are two-fold: (i) to summarize
evidence linking the gut microbiota to the pathophysiology
of mood and anxiety disorders and (ii) to synthesize
emerging data on probiotics as novel therapeutic targets for
mood and anxiety disorders. Finally, the present paper will
discuss limitations of current evidence and propose
directions for further research.
2. METHODS
2.1. Identification of Papers
This systematic review searched the Medline/PubMed,
Scopus and PsycINFO databases, including citations through
April 1st, 2014. The search strategy used involved using
keywords from two categories in various combinations, the
first of these being one of the following: microbiota,
probiotic, probiotics, and the second characterizing behavior
or one of three neurotransmitters related to mood and anxiety
disorders: mood, depression, anxiety, behavior, cognition,
brain-derived neurotrophic factor (BDNF), gammaaminobutyric acid (GABA) and 5-HT. A hand-search of the
reference lists of included articles augmented the search
strategy.
2.2. Selection Criteria
Included articles were original reports on the role of the
gut microbiota and probiotics on the pathophysiology and/or
treatment of mood and anxiety disorders. Overall
methodological quality was considered for inclusion. Two
authors had evaluated all articles (AS and AFC). A total of
34 studies of the 728 unique references screened were
ultimately selected for the purpose of this literature review.
Studies were selected if they involved the administration of
probiotics to human or animal subjects and were related to
depression, anxiety or stress-related behavior; studies
involving a vagotomy combined with probiotic
administration were also included. Several studies involved
the administration of prebiotics, antimicrobials or bacterial
infection in order to examine stress, anxiety and/or
depression. Six of these studies involved human participants,
and 30 were performed on rats or mice.
Slyepchenko et al.
(GF) rodents are raised in an environment such that any

microbiota is absent, while gnotobiotic rodents refer to
animals whose complete microbial composition is known,
i.e. either GF or GF colonised with a known microorganism
[29]. Finally, several of the studies used SPF rodent fecal
matter to colonise GF rodent digestive tracts [21, 23, 25].
3.2. Differences in GF Versus SPF Rodents
Five studies compared germ free rodents to specific
pathogen free rodents on behavioral dimensions, as well as
HPA axis response, mRNA differences, and several other
measures [20-23, 25]. (See Supplementary Material for
behavioral testing methodology).
3.2.1. Behavior and HPA Axis Response
Between the studies, GF mice showed a lower-anxiety
phenotype, as displayed by performance on behavioral tests,
however the results of these tests themselves were not
consistent, other than a consensus of lower-anxiety on the
elevated plus maze by mice [23, 24]. For the light-dark box
preference test, two studies found anxiety phenotypes to be
decreased in GF mice [21, 23], while another found no
difference in anxiety-like behavior for this test [22, 23]. For
performance on the open field test, one study found GF mice
to display more exploratory behavior [23], while another did
not observe differences between GF and SPF mice in this
paradigm [24]. A difference was also found for GF mice in
terms of memory, with GF mice performing poorly on both
the T-maze and novel object test [22]. Evidence of increased
serum [22] and plasma corticosterone [21, 24, 26] for GF
mice was also confirmed by four studies, indicating an
alteration in the manner in which the HPA axis functions in
these animals. Further support for an over-activation of the
HPA axis function is the observation of higher plasma
adrenocorticotropic hormone in GF mice [26].
3.2.2. Neural Correlates
3.1. Comparative Studies of Microbiota Effects on

Murine Behavior and Stress-Associated Phenotype
Brain-derived neurotrophic factor levels were shown to

be lower for male GF rats in the hippocampus across all four
of these studies. BDNF levels were shown to be low in the
male mouse cortex [26] and specifically in the amygdala and
cingulate cortex [23]. Male GF mice had lower BDNF
mRNA in the hippocampus [26]. As well, Neufeld et al. [24]
found higher expression of the NR2B, but not the NR1 or
NR2A, N-methyl-D-aspartate (NMDA) receptor mRNA in
the central region of the amygdala for female mice, while
Sudo et al. [26] found lower NR1 and NR2A mRNA levels
in the cortex, and lower hippocampal NR2A mRNA
expression in male mice.
Of the selected studies, six examined the correlations

between microbiota composition and behavioral phenotypes
(Table 1) [20-26]. A further two examined the correlations of
mice raised in stressful conditions, i.e. either being exposed
to stress in their early lives or a stress-sensitive strain [27,
28]. Rodents used in these studies generally had one of 4
subtypes of altered microbiota. The first, specific pathogen
free (SPF) rodents are raised in conditions such that a normal
microbiota composition is observed, aside from any
pathogenic
organisms.
Germ-free
Monoamine turnover was faster in GF mice [23], and a

higher metabolism of tryptophan, combined with higher
serotonin levels was observed in another study [21].
Meanwhile, Neufeld et al. [24] found 5HT1A receptor
mRNA expression to be lowered in the dentate gyrus but not
the cornu ammonis 1 (CA1) region of the hippocampus.
Heijtz et al. [23] found D1 dopamine receptor mRNA to be
at higher levels in GF mice hippocampi; and at lower levels
in their striata and nuclei accumbens. In a study involving
stress-sensitive rats, slower turnover of dopamine was
3. RESULTS
Table 1.
1773
Comparative studies of microbiota effects on the development of murine phenotypes.
Reference
Strain
Bercik et al.,
2011a [20]
BALB/
c, NIH
Clarke et al.,
2013 [21]
SwissWebster
Sex
M, F
Diaz Heijitz,
et al., 2011
[23]
NMRI
Gareau et al.,
2011 [22]
SwissWebster,
C57BL/6
Neufeld et al., Swiss2011 [24]

Webster
Nishino et al.,
BALB/c
2013 [25]
Sudo et al.,
2004 [26]
Bendtsen,
et al., 2012
[27]
CrumeyrolleArias et al.,
2014 [28]
BALB/c
BALB/c
F344
Microorganisms
Model
SPF, GF
Vagotomy,
chemical
sympathectomy,
antimicrobials
GF, CC,
colonised GF
Test
Phenotype
Anxiety
NIH microbiota colonisation of GF mice had similar effects as

antimicrobial on SPF other than amygdala BDNF reduction;
BALB/c microbiota colonisation of GF intestine produces
anxious behavior, independent of vagus nerve and sympathetic
connections.
L/DB
GF have lower anxiety-like behavior in L/DB, lower levels of

TNF-, higher post-stress plasma corticosterone, lower
kyurenine:tryptophan ratio than CC; M GF mice had lower
BDNF mRNA, higher 5HT, 5-HIAA in HC, higher tryptophan
availability; colonisation GF normalized L/DB test performance,
tryptophan metabolism and availability.
GF colonisation
with SPF
bacteria
Anxiety,
motor
activity
GF mice show more exploratory behavior in OFT, EPM; more

anxiety-like behavior in EPM; GF colonised with SPF in
childhood initially behave like GF then like SPF mice; no
OFT, L/DB, difference in adult colonisation condition; GF have faster NE, DA,
EPM
5-HT turnover in St; GF have lowered expression of NGFI-A
mRNA in FC, S, HC, A; GF mice have lower BDNF mRNA in
HC, A, cingulate cortex; D1 mRNA higher in GF hippocampus
and lower in St, NAcc; increased synaptophysin, PSD-95.
GF, SPF; L.
Citerobacter
rhamnosus
rodentium
R0011, L.
infection, WAS
helveticus R0052
Anxiety,
memory
GF mice have normal anxiety levels but have worse memory

Novel object
than SPF mice with or without stress; low BDNF in CA1, lower
task, L/DB,
c-Fos positive HC cells after WAS; higher baseline serum
T-maze
corticosterone levels for GF mice.
Anxiety
EPM, OFT
GF mice have lower anxiety-like behavior in EPM; no

differences in OFT; GF have higher NR2B mRNA expression
in CeA, higher BDNF mRNA in DG, decreased 5HT1A mRNA
in DG; GF mice have higher plasma corticosterone.
OFT, MB
GF exposed to external environment had decreased anxiety

(MB); colonisation decreased locomotor activity (OFT) and
anxiety (MB); B. coccoides lowered anxiety, no change in OFT;
B. infantis changed locomotor behavior, no effect on MB; ExGF mice had higher 5-HIAA/5-HT turnover rate in St and
lowered 5-HIAA levels in brainstem; no tryptophan differences;
EX-GF mice have lower DA in PFC, brainstem and lower
DOPAC in St; lower HVA in cortex; higher turnover of
DOPAC/DA in brainstem, mPFC, HVA/DA in brainstem, St,
mPFC; MHPG levels higher in St, brainstem for EX-GF, while
MHPG levels higher in S and brainstem, MHPG/NE turnover
increased in brainstem, St, HC.
Stress
Heightened HPA response for GF mice: higher plasma ACTH.

corticosterone; GF mice have lower BDNF in cortex, HC, lower
NR-1 and NR-2a mRNA levels in cortex, lower NR-2a mRNA
in HC, normal NT-3, NGF; gnotobiotic mice had normalized
HPA response. Increased IL-6 plasma levels, c-Fos mRNA level
in PVN; increased plasma corticosterone; pretreatment with IL6 antibody failed to affect c-Fos, corticosterone response; SPF
flora colonisation partly attenuated HPA response of GF mice.
Anxiety
depression
Microbiota
composition
, TST,
Triple test
(EPM,
L/DB, OFT)
Difference in Odoribacter, Alistipes, Coriobacteriaceae

composition for grid floor mice; increased depression-like
behavior for grid floor mice, time spent in triple test sections
correlated to levels of IL-1, IFN-, microbiota composition
(Firmicutes); no cytokine level differences; higher glycated
hemoglobin, low blood glucose.
OFT, social
interaction
GF rats had less social interactions, increased anxiety-like

behavior, triple amount of serum corticosterone as stress
response, high CRF mRNA in hypothalamus, low
glucocorticoid receptor mRNA in HC (heightened HPA
response), low DA turnover in FC, S, HC, but no differences in
NE; no sensorimotor dysfunction found.
SPF, GF
SPF, GF
Observation
SPF, GF, GF
reconstituted
with SPF or
gnotobiotic with
B. infantis
Anxiety
Anxiety,
GF colonisation
novel
environment
with CC
stress
Contaminationfree
GF, colonised
environment,
GF, gnotobiotic
colonisation
with B. infantis,
with SPF
Blautia coccoides
bacteria,
gnotobiotic with
probiotics
Analyzed
Behavior
WT
GF, SPF
Acute restraint
stress
Grid floor
(stress)
F344 stress
sensitive rats
Anxiety
Anxiety
Abbreviations: GF - Germ-Free; SPF - Specific Pathogen Free; BDNF - Brain Derived Neurotrophic Factor; L/DB - Light/Dark Box Preference Test; CC - Conventionally Colonized;
TNF- - Tumour Necrosis Factor , 5-HT - 5 Hydroxytryptamine (Serotonin); 5-HIAA- 5-Hydroxyindoleacetic Acid; HC - Hippocampus; OFT - Open Field Test; EPM - Elevated Plus
Maze; NE - Norepinephrine; DA - Dopamine; NGFI-A - Nerve Growth Factor-Inducible Protein A; FC - Frontal Cortex; S - Striatum; A - Amygdala; D1 - Dopamine Receptor D1; NAcc Nucleus Accumbens; PSD-95 - Postsynaptic Density Protein 95; WAS - Water Avoidance Stress; MB - Marble Burying Task; OFT - Open Field Test; DOPAC - Dihydroxyphenylacetic
Acid; HVA - Homovanillic Acid; mPFC - Medial Prefrontal Cortex; MHPG- 3-Methoxy-4-Hydroxyphenylglycol; NR-1 - N-Methyl D-Aspartate Receptor Subtype 1; NR-2a -- N-Methyl
D-Aspartate Receptor Subtype 2a; NT-3 - Neurotrophin 3; NGF - Nerve Growth Factor; HPA Axis - Hypothalamic-Pituitary-Adrenal Axis; ACTH - Adrenocorticotropic Hormone; IL-6 Interleukin-6; PVN - Paraventricular Nucleus of the Hypothalamus; IL-1 - Interleukin-1; IFN- - Interferon-; CRF - Corticotropin Releasing Factor.
1774
Slyepchenko et al.
observed in the frontal cortices of GF rats [28]. This suggests

that microbiota composition does in fact affect turnover of
monoamines, which play a vital role in the regulation of
affective and stress-related behaviors and phenotypes.
interaction with the way in which the mice were raised. A

curious finding is that a high level of glycated hemoglobin
was observed in the stress-condition raised mice, however
the blood glucose was observed to be low [27].
Heijtz et al. [23] found lower expression of nerve growth

factor (NGFI-A) mRNA in frontal cortices, striata,
hippocampi and amygdali of GF rats, as well as increased
synaptophysin and PSD-95 (postsynaptic density protein95). C-Fos protein expression was shown to be lower in the
hippocampi [22] of GF mice, as opposed to SPF mice.
A study involving a stress- sensitive strain of rats showed

GF rats to have several dysfunctions in stress-related
behaviors. The lack of a microbiome increased anxiety-like
behavior and tripled the levels of plasma corticosterone,
while elevating CRH mRNA expression in the
hypothalamus, indicating a sharp HPA axis response to
stress. Accordingly, a decrease in glucocorticoid receptor
expression in the hippocampus was also observed. In these
animals, a higher dopamine turnover was observed for
certain cortical areas as well [28].
3.3. Colonisation Effects

Four of these studies looked at the effects of colonising
GF mice with SPF or conventionally colonised microbiota,
or microbiota from different strains of mice [20, 21, 23, 26].
Colonisation of GF mice with the conventionally colonised
microbiota led to a normalization of performance on an
anxiety task (light/dark box), as well as the altered
tryptophan metabolism observed in GF animals [21]. In
another study, colonisation of BALB/c GF mice with NIH
microbiota led to an anxiolytic behavior, with an
accompanying hippocampal BDNF increase, while
colonising NIH GF mice with BALB/c microbiota led to
anxiety-like behavior [20]. In another experiment, GF mice
colonised with SPF organisms in their young age behaved
initially like GF mice, then like SPF mice, with no detectable
difference found between the mice colonised in adulthood
and GF mice [23]. Another experiment observed SPF
microbiota colonisation of GF mice to partly attenuate the
heightened HPA response [26].
3.4. Gnotobiotic mice with Bifidobacterium infantis or
Blautia coccoides
Germ-free mice reconstituted with B. infantis had a
normalized stress response, as opposed to GF mice, as well
as an increase in plasma levels of IL-6 and corticosterone,
and an increased c-Fos mRNA expression in the
paraventricular nucleus. When the mice had been
administered a IL-6 antibody prior to probiotic treatment, the
corticosterone and c-Fos response was not affected [26].
This indicates that the corticosterone and c-Fos response
(thus activation of the examined brain areas) were not
mediated by the IL-6 increase, and rather other mechanisms
may account for the behavioral responses of this probiotic
[26].
Another study reconstituted GF mice with either B.
infantis or Blautia coccoides. The former had an effect of
normalizing locomotor behavior, but not on marble burying
task, while the latter had the opposite effect [25]. This
suggests varying behavioral effects of probiotics.
3.4.1. Stress-Raised/Stress-Sensitive Rodents
Mice which were raised in stressful conditions (grid
floor) showed an altered composition of microbiota,
particularly in Odoribacter, Alistipes and Coriobacteriaceae,
as well as increased depression-like behavior [27].
Microbiota composition (Firmicutes) and pro-inflammatory
cytokine levels (IL-1 and IFN-) related to performance in
the anxiety tests; however, this did not have a significant
3.5. Effects of Antimicrobials, Ambient Bacteria and

Bacterial Infection on Murine Phenotypes
Six studies analyzed these effects on murine phenotypes
[20, 22, 30-33] (Table 2).
A study which disturbed microbiota composition of SPF
mice through antimicrobials observed anxiolytic behavior
expressed through light/dark box preference test, and step
down test performance. These reversible behavioral
alterations were accompanied by an increase of BDNF
mRNA expression in the hippocampus and a decrease
thereof in the amygdala. The second portion of this
experiment colonised GF mice with NIH microbiota, which
displayed similar phenotypes to the antimicrobial disturbing
of SPF mice, aside from BDNF mRNA reduction.
Antimicrobial administration to GF mice had no effect on
phenotype, giving further support to microbiota as the major
mediating factor of these effects. Meanwhile, when
colonisation of GF mice was performed with BALB/c
microbiota, anxious behavioral phenotypes were observed.
This study did not observe vagal, sympathetic or cytokine
mediation of the effects of antimicrobials. Cytokine
differences were associated with anxiety-related test
behaviors (IL-1, IFN-) [20].
Three studies explored the impact of bacterial infection
on phenotype [22, 30, 31] (Table 2). Two of these performed
the behavior analysis within 8 hours of the infection, prior to
the inflammatory reaction and the presence of sickness
behavior, indicating that behavior effects are not a direct
consequence of inflammatory stress [30, 31]. Both studies
reported an increase in the anxiety-like behavior, through the
open field [31] and holeboard tests [30]. C-Fos levels were
altered in both studies, with an increase of c-Fos mRNA
expression in vagal sensory neurons [31] and expression
thereof in the bed nucleus of the stria terminalis
corresponding to anxiety-like behavior for infected mice
[30]. On the other hand, c-Fos mRNA levels were predictive
of anxiety-like behavior in the central amygdala and
basolateral amygdala for non-infected mice. High c-Fos
levels were also observed in the paraventricular nucleus,
medial prefrontal cortex, the basolateral amygdala and bed
nucleus of the stria terminalis for a combination of infection
and anxiety induction, although these did not seem to predict
behavioral alterations [30]. These studies used different
infectious bacteria (Citerobacterum rodentium and
Campylobacter jejuni).
Table 2.
1775
Studies investigating bacterial infection, antimicrobials and administration of probiotics on murine phenotypes.
References
Strain
Sex
Microorganisms
Model
Analyzed
Behavior
Test
Phenotype
Novel
object
task,
L/DB,
T-maze
No behavior difference from infection observed, but

after exposure to WAS memory dysfunction occurs;
memory dysfunction prevented by probiotics; change in
microbiota (increase in Firmicutes, Enterobacteriaceaae,
Eubacteria retale) induced by infection; all ameliorated
by probiotics, other than Bacteroides levels; BDNF in
HC reduced, partly reversed by probiotic treatment.
Hole
Board
Anxiety-like behavior increased from infection before

sickness behavior; c-Fos levels in BST predict anxietylike behavior in infected mice; c-Fos in CeA and BLA
predicted anxiety for non-infected mice; c-Fos in PVN,
BLA, BST, mPFC respond to infection and anxiety
induction, however do not correlate with behavior.
Bacterial Infection
Gareau et al., 2011

[22]
SwissWebster,
C57BL/6
GF, SPF; L.
Citerobacter
rhamnosus R0011,
rodentium
L. helveticus
infection, WAS
R0052
Anxiety
Memory
Goehler et al., 2013

[30]
CF-1
WT
Campylobacter
jejuni infection
Anxiety
Lyte et al., 2006

[31]
CF-1
WT
Citrobacter
rodentium
Anxiety
Mycobacterium
vaccae
Ambient
bacteria
exposure 1 x (2
+12) across 7
weeks
Learning
Anxiety
Increased anxiety-like behavior prior to sickness

behavior; no change for IFN-, TNF- and IL-12 levels;
increased c-Fos expression in vagal sensory neurons.
Ambient Bacteria Administration
Matthews and Jenks,

2013 [32]
BALB/c
SPF
HebbWilliams
complex
maze
Reduced anxiety-like behaviors, improved maze

performance in Hebb-Williams complex maze.
L/DB,
SDT
Antimicrobials changed microbiome composition,

increased exploratory behavior in L/DB and SDT in
SPF mice; BDNF in HC increased, decreased BDNF in
A; no cytokine changes observed; effects of
antimicrobials in SPF were reversible; no effect of
antimicrobials for GF mice; NIH microbiota
colonisation of GF mice had similar effects as
antimicrobial on SPF other than amygdala BDNF
reduction; BALB/c microbiota colonisation of GF
intestine produces anxious behavior, regardless of vagal
or sympathetic drive.
Antimicrobial Administration
Bercik et al., 2011a

[20]
BALB/c,
NIH
SPF, GF
Vagotomy,
chemical
sympathectomy,
antimicrobials
Anxiety
Hole
Board
Anxiety-like behavior increased from infection before

sickness behavior; c-Fos levels in BST predict anxietylike behavior in infected mice, CeA and BLA for noninfected mice; PVN, BLA, BST, mPFC respond to
infection and anxiety induction, however do not
correlate with behavior.
Anxiety
OFT,
Hole
board
Increased anxiety-like behavior prior to sickness

behavior; no change for IFN-, TFN- and IL-12 levels;
increased c-Fos expression in vagal sensory neurons.
BDNF and NR1 mRNA expression increased in HC of

FOS-eating animals; NR1, NR2a, increased in GOSeating animals; low BDNF mRNA in CA3, high HC Dserine, high plasma peptide YY in GOS-treated
animals, plasma from GOS-treated animals increased
BDNF release from SH-SY5Y cells.
Goehler et al., 2013

[30]
CF-1
WT
Campylobacter
jejuni infection
(prior to
sickness
behavior)
Lyte et al., 2006 [31]
CF-1
WT
Citrobacter
rodentium
WT
Fructooligosaccharides
(FOS) and
galactooligosaccharides
(GOS) for 5
weeks
Anxiety
Probiotic Administration
Savignac et al., 2013

[33]
SpragueDawley
Abbreviations: GF -Germ Free; SPF - Specific Pathogen Free; WAS - Water Avoidance Stress; L/DB - Light/Dark Box Preference Test; BDNF - Brain Derived Neurotrophic
Factor; HC - Hippocampus; WT - Wild Type; BST - Basal Striatum; CeA - Central Amygdala; BLA - Basolateral Amygdala; PVN - Paraventricular Nucleus; mPFC - Medial
Prefrontal Cortex; IFN- - Interferon-; TNF- - Tumour Necrosis Factor-; IL-12 - Interleukin-12; SDT- Passive Avoidance Step-Down Test; A - Amygdala; NR1 - N-Methyl-DAspartate Receptor Subtype 1; NR2a - N-Methyl-D-Aspartate Receptor Subtype 2a; GOS - Galacto-Oligosaccharides; FOS - Fructo-Oligosaccharides; CA3 - Cornu Ammonis Area
3 of the Hippocampus; HC - Hippocampus.
Ten and thirty days after infection with Citerobacter

rodentium, another group of mice had normal anxiety
behaviors. However when these mice had been exposed to
acute stress (through a water avoidance stress method), a

memory impairing effect was verified as analyzed by the
novel object recognition task. Furthermore, reduced levels of
1776
BDNF mRNA expression in their hippocampi paralleled

these cognitive abnormalities. A change in microbiota
composition was also demonstrated, with an increase in the
levels of Firmicutes, Enterobacteriaceae, as well as
Eubacteria retale, and a decrease in the levels of Bacteroides
[22].
Ambient bacteria are mircroorganisms which reside in
the environment and are non-pathogenic. A study used a
bacterium found in temperate environments which is not
capable of colonisation of a digestive tract (Mycobacterium
vaccae). Administration of these bacteria led to a reduction
of anxiety-like behaviors and an improved performance in
the Hebb-Williams complex maze. The authors propose an
effect of antigens on the immune system, such that serotonin
pathways are altered by ingestion of these bacteria, leading
to the improved anxiety phenotype, and improved
performance in a land maze [32].
Slyepchenko et al.
decreased in a stress response paradigm after 17 days of L.

rhamnosus R0011 treatment [22] and 4 weeks of L.
rhamnosus JB-1 treatment [36]. No effect was observed for
any of corticotropin releasing factor or vasopressin levels,
after 2 weeks of B. infantis 35624 treatment [35].
Brain-derived neurotrophic factor mRNA levels that had
been elevated from an acute stress had been attenuated in the
CA1 region of the hippocampus, as well as the c-Fos levels
in this region through treatment by L. rhamnosus R0011and
L. helveticus R0052 [22]. Treatment with probiotics
improved IFN- levels in both healthy and infected mice for
one of the aforementioned studies [22]. As well, another
study observed a decrease in IFN-, IL-6, IL-10 and TNF-
as a result of probiotic treatment of healthy mice [35].
Alterations were observed for serotonin turnover, as well as
a decrease in a dopamine metabolite in the amygdaloid
cortex as a result of B. infantis 35624 treatment [35]. An
alteration was observed for the expression of GABA receptor
mRNA in several brain areas related to emotion and memory
regulation [36].
Another study administered fructooligosaccharides

(FOS) and galactooligosaccharides (GOS) to rats for the
span of five weeks. Lactobacilli and Bifidobacteria consume
these soluble substances, leading to an abundance of their
presence in the gastrointestinal tract. The administration of
FOS produced an increase in the mRNA expression of
hippocampal BDNF and NMDA NR1 receptors.
Administration of GOS resulted in lowered BDNF
expression in the CA4 hippocampal subregion, as well as
higher levels of hippocampal D-serine, along with a higher
amount of BDNF released from GOS-treated SH-SY5Y
cells. Plasma peptide YY was also elevated as a result of
GOS administration [33].
Anxiolytic effects and mRNA expression alterations as a

result of 28-day long L. rhamnosus JB-1 administration to
healthy mice were not observed in vagotomised mice [36].
Moreover, in an ex-vivo cannulation of the jejunum in Swiss
Webster rats, increased vagal afferent firing frequency was
observed upon administration of L. rhamnosus JB-1, but not
L. salivarus. This difference was observed in both single and
multiple units, with vagotomy eliminating the firing
increase, and thus abolishing the effects of the probiotic
administration [37].
3.6. Effects of Probiotic Administration on Murine

Phenotypes
3.7. Administering Probiotics

Gastrointestinal Inflammation
Of the studies examined, 18 studies that administered

probiotics to rodents were identified; measuring behavioral
phenotypes and/or related CNS correlates (Table 3). Some of
these studies used probiotics as a means to alleviate the
effects of pathology-related behavioral phenotypes, while
others simply examined the effect of probiotics on relieving
short-term stress exposure effects, or their effects in healthy
rodents.
The majority of research examining probiotics is

associated with evaluating their role in gastrointestinal
dysfunction. Several studies have been performed on the
effects of probiotics on models of colitis in terms of CNS
functions, finding B. longum to attenuate anxiety-like
behavior which results from chronic colitis model [38, 39].
The effects of this probiotic were not present, aside from
lower enteric neuron excitability, when a severing of the
vagus nerve was performed prior to probiotic administration.
BDNF levels in neuroblastoma cells were not shown to be
affected by probiotic administration [39]. In an acute model
of chronic colitis, administration of B. infantis or L. reuteri
decreased TNF and MCP-1 levels. IL-6 levels were
decreased by L. reuteri in all mice, while B. infantis only
decreased IL-6 in vagotomised animals. When an in vitro
test was performed, B. infantis was reported to be more
efficacious in lowering cytokine secretion, and was found to
have better performance after a vagotomy. In a chronic
model, B. infantis reduced TNF-, IL-6 and IFN- in
vagotomised mice [40].
3.6.1. Effects of Probiotics on Healthy Rodents

Five studies examined the effects of probiotics on
behavioral and behavior-related phenotypes in rodents.
Administration of L. helveticus R0052 and B. longum R0175
for 2 weeks led rodents to express less anxiety-like behavior
in response to a defensive conditioned burying test [34]. No
effect on alleviating depression-like behavior was observed
for the forced swim test, after administering B. infantis
35624 for a 2 week span [35]. However, after a 4 week
period of L. rhamnosus JB-1 administration, mice had less
anxiety-like behavior in the elevated plus maze and less
depression-like behavior in the forced swim test [36]. A
preventative effect on memory decline from probiotic
administration had also been observed, as well as attenuated
epithelial cell hyperplasia and microbiota alteration resultant
from infection [22]. Corticosterone levels were shown to be
to
Rodents
with
3.8. Administering Probiotics to Rodents in Models of

Depression and Depression-Comorbid Disorders
The maternal separation model of depression involves
exposing rodents to early life stress. Two studies looked at
Table 3.
1777
Studies administering probiotics to alter murine behaviors and phenotypes.
References
Strain
Sex
Probiotic
(Treatment
Duration)
Model
Analyzed
Behavior
Test/Parameter
Phenotype from
Probiotic
None
Plasma, tissue
analysis, PCR
focusing on HPA
plasticity, in vivo
intestinal
permeability,
protein extraction,
western blot
Probiotic lessens increments in c-Fos

mRNA in PVN, AN, CA3, but not in the
lateral parabrachial nucleus, LC, NTS or
DG; increases c-Fos in WAS when
contrasted to controles; increased stressreduced BDNF, whereas decreased
Gap43, Gfap, Vim, Syt4, Snap25, Reln,
Sema; restored occludin and JAM-A
reduced by WAS in gut permeability.
Social interaction
test, FST, SDT
Modulates social interaction time,

improves swimming time and escape time,
attenuates performance in step-down test;
attenuates intestinal barrier permeability
defects; decrease plasma IL-1 (not
affected by AMI).
Anxiety
SDT
Normalizing effect of probiotic on SDT

results as altered by induced chronic
colitis; probiotic given to mice with
vagotomies performed before treatment
had no effect except lower enteric neuron
excitability; no effect on BDNF.
Anxiety
L/DB, SDT, in situ

hybridization,
staining + tissue
assessment of
colon, plasma
analysis
B. longum attenuated anxiety-like

behavior resultant from infection, CA1
BDNF levels but not cytokine or
kynurenine; vagus nerve played no effect;
L. rhamnosus had no effect.
Social interaction
test, FST, SDT
Performance improved on elevated plus

maze and forced swim test, lower
corticosterone levels in response to stress;
up-regulation of GABA2 mRNa in DG,
GABAB1b mRNA in cingulate, prelimbic
cortices; down-regulation of GABA2
mRNA in cingulate, prelimbic, infralimbic
cortices, CeA, BLA; down-regulation of
GABAB1b mRNA in BLA, CeA, LC,
DG, CA1, CA3; effects not present in
vagotomised mice.
FST, plasma
corticosterone,
flow cytometry,
HPLC, RT-PCR
No difference for FST; IFN-, IL-6, and

TNF- decrease, IL-10 decrease (unusual
for antidepressants); increase in plasma
tryptophan, kyurenic acid; low 5-HIAA in
FC; low DOPAC in amygdaloid cortex;
lower MAO activity; no effect on CRH,
plasma corticosterone, AVP.
L. helveticus
R0052
AitBeignaoui et
al., 2014 [43]
CS7BI6
B. longum
R0175
WAS (stress)
L. salivarius
(control)
(2 weeks)
ArseneaultBreard et al.,
2012 [45]
L. helveticus
R0052
SpragueDawley
Not
informed
B. longum
R0175
Post-AMI
depression
Depression
Social
interaction
(7 + 10 days)
Bercik et al.,
2011b [39]
AKR
B. longum
NCC3001
(2 weeks)
L. rhamnosus
NCC4007
Bercik et al.,
2010 [38]
BALB/c, AKR
SPF
B. longum
NCC3001
Chronic
colitis,
vagotomy
Trichiris muris
infection,
vagotomy
(10 days)
L. rhamnosus
Bravo et al.,
2011 [36]
BALB/c
JB-1
(28 days)
Desbonnet et
al., 2008 [35]
Desbonnet et
al., 2010 [41]
SpragueDawley
SpragueDawley
B. infantis
35624 (14 days)
B. infantis
35634 (45 days)
L. rhamnosus
R0011
Gareau et al.,
2007 [42]
SpragueDawley
Not
informed
L. helveticus
R0052
(16 days)
Healthy
animals,
vagotomy
Healthy
animals
Maternal
separation
Maternal
separation,
WAS
Anxiety
Stress
Depression
Depression
MS-induced FST alteration either

FST, corticosterone eliminated or mediated (immobility, swim
time respectively); NA but not 5-HT
levels, whole blood
Depression
decreases in brainstem was attenuated by
culture, flow
probiotic; elevated IL-6 levels mitigated
cytometry, HPLC,
by probiotic; CRH levels were not
RT-PCR
attenuated by probiotic.
Stress
Novel object test,

T-maze, L/DB,
corticosterone,
histology,
immunohistochemi
stry, qPCR
L. rhamnosus strain restored depleted

quantities of Lactobacillus organisms in
MS pups; restore ion transport,
macromolecular permeability; restored
normal levels of adherence/penetration of
commensal organisms; restore normal
corticosterone levels; persistent effects
until rats are older.
1778
Slyepchenko et al.
(Table 3) contd..
References
Gareau et al.,
2011 [22]
Strain
SpragueDawley
Sex
Probiotic
(Treatment
Duration)
GF, SPF; L.
rhamnosus
R0011, L.
helveticus
R0052
Model
Citerobacter
rodentium
infection,
WAS
Analyzed
Behavior
Test/Parameter
Phenotype from
Probiotic
Anxiety,
Memory
Novel object test,

T-maze, L/DB,
corticosterone,
histology,
immunohistochemi
stry, qPCR
Attenuated corticosterone release, colonic

epithelial cell hyperplasia, microbiota
alteration; IFN- levels improved in both
control and infected mice; attenuated
BDNF, c-Fos expression in CA1 as
induced by WAS; preventative effect on
memory decline.
Depression
FST, social
interaction test,
SDT, biochemical
analysis, rat
cytokine/chemokin
e assay
Improved impaired social interaction, FST

test results with and without high-PUFA
diet; lessened attempts to succeed SDT
test with and without diet change; prevent
apoptosis (caspase-3 activity) in DG and
MA, but not CA1, CA3, LA; no additive
effects observed.
(17 days)
L. helveticus
R0052
Gilbert et al.,
2013 [46]
SpragueDawley
B. longum
R0175
high n-3 PUFA
diet
Post-AMI
depression
(2 weeks)
Kantak et al.,
2014 [48]
Luo et al.,
2014 [49]
BALB/cJ
L. rhamnosus
GG
(2, 4 weeks)
SpragueDawley (SPF)
L. helveticus
NS8
(2 weeks)
Obsessivecompulsive
disorder model
(RU24969)
Hyperammone
mia-induced
neuroinflamma
tion (hepatic
encepalopathy)
OFT, social
experience, MB,
ultrasonic
Anxiety
vocalization, interLocomotion male aggression
Social
Experience
Anxiety
Memory
Decreased locomotor impairments for

both pre-treatment durations (OFT);
decrease in MB; no aggression
differences; comparable to fluoxetine
treatment.
EPM, MWM
Improved EPM and MWM performances;

no effect on hyperammmonemia-induced
iNOS changes; ameliorated elevated
PGE2 levels in cerebellum, HC; reduced
elevated IL-6 levels in cerebellum, HC,
PFC; reduced raised levels of 5-HT in HC
and cerebellum, but not 5-HIAA;
increased lowered kynurenine:tryptophan
ratio, decreased heightened kynurenic
acid:kynurenine ratio.
OFT
No overall effect on OFT; no effect on

corticosterone; B. infantis 35624
diminished pain reaction for
normosensitive and hypersensitive rats.
Conditioned
defensive burying
test
Lower scores in probiotic-treated rats for

anxiety and stress; similar effects
observed with diazepam treatment.
Barnes maze
Attenuated Western diet-induced anxiety

and memory dysfunction in WT mice;
decreased anxiety-like behavior in mice
fed with standard diet; IL-10 deficient
mice not affected compared to WT
animals other than an increase IL-
expression; no differences observed for
IFN, IL-2, IL-4, IL-5, IL-10, IL-12,
TNF.
OFT
No overall effect on OFT; no effect on

corticosterone; B. infantis 35624
diminished pain reaction for
normosensitive and hypersensitive rats.
Normalized HPA response. increased IL-6

plasma levels, c-Fos mRNA level in PVN;
increased plasma corticosterone;
pretreatment with IL-6 antibody failed to
affect c-Fos, corticosterone response.
L. salivarius
UCC118
McKeman et
al., 2010 [50]
SpragueDawley
Wistar-Kyoto
Not
informed
B. infantis
35624
B. breve
UCC2003
Visceral
normosensitive
and visceral
hypersensitive
Visceral
Pain
Anxiety
( 2 weeks)
L. helveticus
R0052
Messaoudi et
al., 2011 [34]
Wistar
B. longum
R0175
WT
Anxiety
Stress
(2 weeks)
Ohland et al.,
2013 [47]
IL-10
Not
deficient, WT
informed
129/SvEv. SPF
Perez-Burgos
et al, 2013
Swiss Webster
[37]
L. helveticus
R0052
(3 weeks)
L. rhamnosus
JB-1
M
L salivarus
(control)
Standard
versus
Western-type
diet
Visceral
normosensitive
and visceral
hypersensitive
Anxiety
Memory
Visceral
Pain
Anxiety
(single)
Sudo et al.,
2004 [26]
BALB/c
B. infantis
(single)
GF, Acute
restraint stress
1779
(Table 3) contd..
References
Strain
Sex
CB17/IcrHanHsd
SCID,
Van der Kleij
Not
et al., 2008 CB6F1/OlaHsd
informed
[40]
(BALB/c
background),
BALB/c
Probiotic
(Treatment
Duration)
L. reuteri,
B. infantis (9
days, 2 weeks,
10 weeks)
Model
Vagotomy,
acute and
chronic colitis
Analyzed
Behavior
Test/Parameter
Phenotype from
Probiotic
Acute: B. infantis and L. reuteri decrease
TNF. MCP-1 level in all mice; L. reuteri
decreases IL-6 levels in all mice; B.
infantis decreases IL-6 in vagotomised
animals; in vitro B. infantis more effective
at decreasing cytokine secretion;
Chronic: no cytokine effects except
reduction of secretion in mice with
vagotomies for TNF, IL-6, IFN-.
Abbreviations: WAS - Water Avoidance Stress; PCR - Polymerase Chain Reaction; HPA - Hypothalamic Pituitary Adrenal Axis; PVN - Paraventricular Nucleus; AN- Amygdaloid
Nucleus; CA3 - Cornu Ammonis Area 3 of the Hippocampus; LC - Locus Coerulus; NTS - Nucleus Tractus Solitarus; DG - Dentate Gyrus; BDNF - Brain Derived Neurotrophic
Factor; Gap43 - Growth Associated Protein 43; Gfap - Glial Fibrillary Acidic Protein; Vim - Vimentin; Syt4 - Synaptotagmin-4; Snap25 - Synaptosomal-Associated Protein 25; Reln
- Reelin; Sema - Semaphorin; JAM-A- Junctional Adhesion Molecule A; AMI - Acute Myocardial Infarction; FST - Forced Swim Test; SDT - Passive Avoidance Step Down Test;
CA1 - Cornu Ammonis Area 1 of the Hippocampus; GABA2 - Gamma Aminobutyric Acid Receptor Subunit 2; CeA - Central Amygdala; BLA - Basolateral Amygdala;
GABAB1b - Gamma Aminobutyric Acid Receptor Subunit B1b; HPLC - High Performance Liquid Chromatography; RT-PCR- Real-Time Polymerase Chain Reaction; IFN- Interferon-; IL-6 - Interleukin-6; TNF- - Tumor Necrosis Factor ; IL-10 - Interleukin - 10; 5-HIAA -5-Hydroxyindoleacetic Acid; FC - Frontal Cortex; DOPAC -3,4Dihydroxyphenylacetic Acid; MAO - Monoamine Oxidase; CRF- Corticotropin Releasing Factor; AVP - Arginine Vasopressin; MS - Maternal Separation; NA - Noradrenaline;
qPCR - Quantitative Real-Time Polymerase Chain Reaction; PUFA - Polyunsaturated Fatty Acids; MA - Medial Amygdala; LA - Lateral Amygdala; OFT - open field test; MB marble burying task; EPM - elevated plus maze; MWM - Morris Water Maze; iNOS - inducible nitric oxide synthase; PGE2 - prostaglandin E2; PFC -Prefrontal Cortex; 5-HT - 5Hydroxytryptamine (Serotonin); IL- - Interleukin-; IL-2 - Interleukin-2; IL-4 - Interleukin-4; IL-5 - Interleukin-5; MCP-1 - Monocyte Chemoattractant Protein-1.
the effects of probiotic treatment on behavioral responses to

maternal separation [41, 42]. The rats performance on the
FST was normalized by treatment with B. infantis, also
restoring noradrenaline levels in the brainstem, and lowering
levels of IL-6. However, CRH levels remained elevated,
while 5-HT levels in the brainstem remained decreased [41].
Furthermore, treatment with L. rhamnosus in another study
restored Lactobacillus levels in the gut of animals, as well as
several molecular mechanisms that were dysfunctional in the
gut, including adherence and penetration of bacteria in the
gut. Corticosterone levels were reduced to normal, with
effects lasting into the adulthood of rats [42]. Mice under a
chronic stress model (water avoidance stress) were observed
to benefit from probiotic effects. The administered probiotic
allowed for a decrease in elevated c-Fos levels in several
areas of the brain. Moreover, BDNF levels that had been
reduced following the stress-inducing paradigm were
restored. The probiotic also affected the expression of
several tight junction proteins, among others, which had
been low as a result of the stress paradigm [43].
Depressive symptoms and syndromes are frequently
reported following myocardial infarction [44, 45]. Two
studies analyzed the effects of administering probiotics to
post-myocardial infarction rodents. One of these studies
displayed an attenuation of previously decreased social
interaction time, and attenuating the rats anxiety-like
behavior, as well as accompanying permeability issues in the
gut. In this particular study, plasma IL- was also reduced,
although it was not affected by myocardial infarction [42].
Another study attempted to treat post-myocardial depression
with probiotics and/or a high n-3 polyunsaturated fatty acid
diet. The probiotics acted to improve anxiety-like behavior
and depression-like behavior. Furthemore, caspase-3 activity
characteristic of post-myocardial states was prevented in the
dentate gyrus and medial amygdala, demonstrating inhibition
of apoptosis in these areas. No additive effects of the diet
and probiotics were observed [46].
A study examined the effects of standard and Western
diet, with L. helveticus administration on anxiety and
memory performance in SPF and IL-10 deficient mice. In
Western diet-induced anxiety and memory impairment,

probiotic treatment was able to attenuate these behavioral
abnormalities, and decrease anxiety-like behavior with mice
with a normal diet. However, in IL-10 deficient mice, the
probiotics affected nothing but the expression of IL- with
an increasing effect [47].
Anxiety is present in a myriad of psychiatric syndromes
(for example, obsessive compulsive disorder). A recent study
observed the effects of probiotic on an OCD mouse model,
demonstrating that probiotics decrease locomotor impairments in the open field test, as well as alterations in marble
burying behavior [48]. Anxiety and memory deficits that are
part of hyper-ammonemia-induced neuroinflammation
model of hepatic encephalopathy were also attenuated by
probiotics, as well as some associated serotonin pathway
alterations [49].
Pain reaction has also been attenuated for visceral
normosensitive and hypersensitive rats by probiotic use,
however no alteration was found for the open field test [50].
3.9. Clinical Studies
To date, only a few studies have been performed on
human participants. However, probiotic administration has
been associated with positive psychological behavior
modifications in individuals not affected by mental disorders
(Table 4). Unfortunately, these studies have not been
consistent in their methodology, with varying durations of
probiotic administration and varying strains of bacteria being
administered to participants. The majority of these studies
were double-blind randomized control trials, though small
sample size in some should be taken into consideration.
Studies have been performed on healthy subjects as well as
subjects undergoing stress, as well as in subjects with
chronic fatigue syndrome.
Recently, the probiotic Clostridium butyricum was
administered in a RCT to patients awaiting surgery for
laryngeal cancer [51]. After two weeks of treatment,
participants of the probiotic group displayed a significant
1780
Table 4.
Slyepchenko et al.
Clinical trials administering probiotics to alter behavior in humans.
References
Benton et al.,
2007 [53]
Diop et al.
2007 [54]
Rao et al.,
2009 [52]
Messaoudi et
al., 2011 [34]
Probiotics
Inclusion Criteria
Sample Size
(Gender
Distribution)
Design
(Duration)
Outcomes
Results
Lactobacillus casei
Shirota
Healthy volunteers
recruited from general
population
N=132 (not
available)
RCT (20 days)

double-blind
Profile of mood
states (POMS);
memory/verbal
fluency test
No overall difference in
POMS scores; memory worse
in the probiotic group at
endpoint.
A 62-item stress
questionnaire
Inconsistent overall beneficial

effects of the probiotic on
stress.
L. acidophilus
R0052
Bifidobacteria
longum R0175
Volunteers with at
least two symptoms of N=75 (21 M and 54
stress; not taking
F)
psychotropic drugs
RCT (3 weeks)
double blind
L. casei Shirota
Patients meeting
criteria for chronic
fatigue syndrome; no
mental disorders
except anxiety and
depression
RCT (8weeks)
double blind
L. helveticus
B. longum
Healthy volunteers; A
score 12 in both
dimensions of the
hospital anxiety and
depression scale
(HADS); not taking
psychotropic in the
month prior to the
study
N=39 (not
available)
Beck depression
inventory
Beck anxiety
inventory
Hopkins symptom
checklist-90 (HSCL90)
HADS
N=55 (14 M and 41

F)
RCT (30 days)

double-blind
Perceived stress
scale
Coping checklist
Tillisch et
al., 2013
[55]
Yang et al.,
2014 [51]
Clostridium
butyricum
Healthy female
volunteers
No significant GI
complaints; no
mental disorder; not
taking
Patients scheduled to
partial laryngectomy
due to laryngeal
cancer
Overall improvement in the

HSCL-90 and HADS-anxiety
scores in the probiotic group;
no significant differences in
cortisol levels.
Urinary free cortisol
RCT (4 weeks)
B. animalis
LActis,
Streptococcus
thermophiles, L.
bulgaricus,
Lactococcus
lactis Lactis
Improved anxiety scores in

the probiotic group at
endpoint.
N=36
N=20 (10 M and

10 F)
Control group
1: no
intervention
3T functional
resonance magnetic
imaging
Control group
2: nonfermented
milk product
Emotional attention
task
RCT (2 weeks)
double blind
CRH plasma levels

Hamilton anxiety
rating scale
Women from probiotic

group had decreased taskrelated activity in network
involving affective,
interoceptive and
somatosensory regions,
including insular cortex and
amygdala. At resting state,
participants from the
probiotic group had changes
in midbrain connectivity.
Significant reduction in
anxiety scores in cancer
patients taking the probiotic.
Reduction in pre-surgery
CRH plasma levels to
control levels in cancer
patients in the probiotic
group.
Abbreviations: RCT- Randomized Controlled Trial; POMS - Profile Of Mood States; HSCL-90 - Hopkins Symptom Checklist-90; HADS - Hospital Anxiety and Depression Scale;
GI - Gastrointestinal; CRH - Corticotropic Releasing Hormone.
decrease in Hamilton Anxiety Scale scores, as well as a

decrease in serum CRH, as induced by pre-surgical stress.
Another RCT involving patients with chronic fatigue
syndrome demonstrated a decrease in anxiety scores, but not
in depression scores after 2 months of treatment with L.
casei Shirota [52]. The fecal concentration of Lactobacillus
and Bifidobacteria are lower in chronic fatigue syndrome,
but these concentrations had been increased in those patients
randomized to the probiotic group [52]. A study investigated
the effects of a 20-day treatment with L. casei Shirota on
mood symptoms and memory performance [53]; treatment
with the probiotic improved mood scores for individuals
reporting low baseline depressed/elated dimension scores;
however, overall memory seemed to be impaired by the
probiotic.
On the other hand, Messaoudi et al. showed 30-day-long

administration of L. helveticus and B. longum to display
positive effects on performances on the Hopkins Symptom
Checklist, on the anxiety subscale of the Hospital Anxiety
and Depression Scale, the coping checklist and cortisol
levels. However, no effect was seen for the perceived stress
scale [34]. Meanwhile, a study performed by Diop et al.
administered a probiotic mixture of L. acidophilus and B.
longum to participants undergoing stress, showing certain
physiological but not psychological stress symptoms to be
alleviated by probiotic treatment [54].
Another investigation explored the effects of
administering four bacterial strains (B. animalis Lactis,
Streptococcus thermophiles, L. bulgaricus, Lactococcus
lactis Lactis) to female participants for a span of four weeks
on emotional attention tasks in a functional magnetic

resonance imaging study. Lower activity in a network which
involves affective, interoceptive and somatosensory regions
often dysregulated in individuals with anxiety dysfunctions
was observed in this RCT (Table 4) [55].
4. DISCUSSION
Stressful environmental conditions in childhood have
been linked to major depressive disorder (MDD) and anxiety
disorders, particularly when combined with genetic
assessments [56]. Chronic hyperactivation of the stress
response has also been linked to MDD [57, 58], indicating
early environmental stress to be an important factor to
consider while discussing the relationship of gut microbiota
and mood and anxiety disorders.
Gut microbiota composition was impacted by stressful
environmental conditions during upbringing of mice [27],
while the possession of a microbiome seems to have
mediated the effects of stress on a stress-sensitive strain [28].
A lack of gut microbiota results in an overactive HPA axis
[21, 22, 24, 26, 38], and alterations in behavioral
phenotypes. Most studies found that rodents lacking the gut
microbiome have a phenotype of reduced anxiety [21, 2325]. However, two studies did not replicate these findings in
the light-dark box preference test [22, 23]. Some of the
discrepancies observed between the results of the tests,
particularly the open field test [23, 24] can be accounted for
by sex differences between the groups of subjects used by
the researchers. The sex differences were not present for the
EPM results [23, 24], suggesting these sex differences on the
effects of the gut microbiome may be task specific.
Assessments of the mice on the light-dark box preference
test also varied across studies [21-23]. The overactivity of
the HPA axis is indicative of increased stress responsivity,
thus the increase in activity of the HPA axis in mice lacking
microbiota and the change in microbiota composition
following early life stress indicates that the relationship of
the gut and the brain are developmentally intertwined.
A decrease in BDNF expression was found for various
cortical areas related to emotion and memory pathways for
male mice with an absent gut microbiota [21-23, 26] with a
potential sex difference for female mice, as BDNF levels
were either unchanged [21] or elevated [24]. BDNF is
important for neurodevelopment, particularly for synapse
formation and axon guidance [59]. Low serum BDNF are
observed in patients with MDD [60], and prevention of
BDNF expression in the hippocampus has been shown to
induce depressive-like behaviors accompanied with a
reduction in neurogenesis [61]. Other neuroplasticity related
changes were observed in mice, with a difference observed
in NMDA receptor expression alteration observed for both
sexes [24, 26], and alteration in the levels of key proteins
related to synapse function (i.e., synaptophysin and PSD-95)
[23]. Overall, this evidence indicates that neuroplasticity
may in part be dependent on the presence of a physiological
gut microbiota. Microbiome presence does seem to have an
effect on monoamine turnover [23, 28] as well, suggesting
the possibility of multiple mechanisms by which behavior
might be altered as a result of microbiome composition.
Therefore, alterations in BDNF, NMDA receptor, and
monoaminergic functions may play a role in the regulatory
1781
effects exerted by the gut microbiota in stress-related

behaviors.
Mice which had been colonised with SPF microbiota had
displayed a normal phenotype, if the colonisation was
performed at a young age [23, 26], or to the parents of the
mice used [25]. Adult colonisation had no effect on animal
behavioral phenotypes [23], suggesting a critical period for
the effects of gut microbiota on brain development. These
results indicate that a gut microbiota is required for brain
maturation and the development of a physiological response
to stress. Thus, a disruption of the gut microbiota
composition at critical developmental stages may lead to a
higher propensity for disorders related to a dysfunctional
response to stress (e.g., MDD and anxiety disorders).
Antimicrobial administration studies offers further
evidence of BDNF levels being affected by disturbing
microbiota, however these effects seemed to not be mediated
by vagal, sympathetic or cytokine pathways [20]. When the
gut microbiome was altered in another way, through
bacterial infection, anxiety-like behavior and activation in
vagal sensory neurons and the BST were observed prior to
sickness behavior [30, 31]. This suggests the presence of a
rapid neural response to induced alterations in gut
microbiome composition. Furthermore, bacterial infection
may have long-lasting detrimental effects on memory
formation in animals exposed to stress [22].
When
ambient,
non-colonising
bacteria
were
administered to mice, an anxiolytic effect was observed,
combined with improvements in spatial learning [32]. This
may indicate that ubiquitous ambient bacteria act to
modulate our behavior as well. The authors suggested that an
immune-mediated effect, resulting in a change in serotonin
pathways [32]. An increase in BDNF release was one of the
results of probiotic administration, as well as its expression
elevated in sub-regions of the hippocampus. The NDMA
NR1 subunit receptor expression in the hippocampus was
also increased by one of the probiotics [33].
Consistent with the aforementioned neurobiological
findings, probiotic administration seems to alleviate some
anxiety-like behaviors in healthy mice [34, 36], and
depressive-like behavior in healthy mice is either lowered
[36] or unaffected [35]. Accompanying behavioral effects,
stress-related activation of the hippocampus seems to be
reduced by probiotic pre-treatment, along with an increment
in BDNF mRNA in this area [22]. HPA axis activity also
seems to be reduced by probiotic treatment [22, 36],
although these effects are only present for corticosterone, but
not CRH or vasopressin levels [35]. Turnover of some
monoamines seems to be affected by probiotic treatment as
well [35], with alterations in GABA receptor expression
observed in memory and emotion areas [36]. Anxiolytic
effects of L. rhamnosus JB-1 seem to be dependent on vagal
discharge [37]. Probiotic treatment also reduced levels of
several cytokines [22, 36], promoting an overall antiinflammatory effect. A decrease in a cytokines observed to
be elevated in MDD (IL-6 and TNF-) [62] was seen after
the administration of probiotics.
Probiotics attenuate depressive-like behaviors in rodents
[41]; several other correlates observed the model, such as gut
dysfunctions and microbiota imbalance [42], along with a
1782
restoration of brainstem noradrenaline balance and a

reduction in IL-6 levels in the maternal separation depression
model [41]. The effects on the HPA axis were of nonuniform nature, with corticosterone levels being attenuated
in one study through adulthood [42], but the CRH levels
continuing to be elevated in another [41]. A chronic stress
mouse model of depression also benefitted from probiotic
effects, decreasing hyperactivation in some brain areas, and
restoring BDNF levels that were decreased by the stress [43].
4.1. Clinical Effects of Probiotics
Clinical studies of the effect of probiotics on anxiety and
depression have been limited, varying effect on behavior and
in length, ranging from 2 weeks to 2 months. In two of the
studies that have involved anxiety or stress as a comorbidity,
the administration of probiotics has been effective in
alleviating symptoms of anxiety [51, 52]. No studies have, as
of yet, been performed on patients with MDD or anxiety
disorders. Overall, results so far are mixed and there were
significant variations in protocolos. Furthermore, no studies
have examined the behavioral effects of the long-term
administration of probiotics in humans. As well,
investigation of anxiolytic and anti-depressant effects of
probiotics in younger populations is warranted, considering
that a critical period does exist for the microbiomes effects
on CNS development. A recent study examined the
relationship between microbiome composition and
depression, reporting high levels of Bacteroidales, and low
levels of Lachnospiraceae in participants with MDD [63].
Further, large-scale studies on microbiome composition in
patients with mood and anxiety disorders could yield
additional information on the brain-gut axis effects, as
probiotics involve the admission of a single strain into a vast
and diverse microbiome. Examination of specific
manifestations of neuropsychiatric disorders (e.g. cognitive,
mood, neurotransmitter expression) as correlated to
microbiome composition may provide insight to the
pathophysiological development of these disorders.
Considering the literature which has identified effects of
the gut microbiota on CNS development, it is important to
investigate the specific mechanisms by which the
microbiome may affect behavior. Behavioral effects are
evidently modulated by a physiological gut microbiota, and
with increasing exposure to Western-type diets it is possible
to speculate that our microbiome is changing for the worse,
which may contribute to the pathophysiology of mood and
anxiety disorders [47]. Diet thus may confound some of the
results discussed above, as it influences composition of the
gut microbiota, potentially leading to altered production of
immunomodulatory substances [64] in addition to
influencing neurogenesis and the stress response [65].
CONCLUSION
Taken together, the presence of a microbiome may
modulate the development of anxiety and depressive-like
behaviors through multiple mechanisms, including but not
limited to effects on the HPA axis and neuroplasticity-related
mechanisms in the brain, as well as possible monoamine
turnover alterations. This modulation seems to have a critical
developmental period. Potential beneficial effects of probiotics
Slyepchenko et al.
on depressive and anxiety-like behaviors may be strainspecific, and in certain circumstances seem to be modulated
by the vagus nerve. Clinical studies of probiotic administration
thus far provide inconsistent findings. Future clinical trials
should determine which subpopulations could potentially
respond better to probiotic treatment of depression and
anxiety. In this regard, potential therapeutic effects of different
probiotics may be investigated in mental disorders associated
with a deregulation of the stress system regardless of classical
diagnostic boundaries (e.g., anxiety, MDD, and posttraumatic
stress disorders) [66]. Given the fact that the intestinal
microbiota play a role in neurodevelopment of brain networks
related to emotion regulation, the microbiota may be a
potential target for the prevention of anxiety and depressive
disorders in at-risk populations.
LIST OF ABBREVIATIONS
5-HT
= 5-Hydroxytryptamine
AMI
= Acute Myocardial Infarction
BDNF
= Brain-Derived Neurotrophic Factor
CA1, CA3, CA4 = Cornu Ammonis Area 1, 3, 4

CNS
= Central Nervous System
CRH
= Corticotropin Releasing Hormone
DA
= Dopamine
ENS
= Enteric Nervous System
EPM
= Elevated Plus Maze
FC
= Frontal Cortex
FOS
= Fructo-Oligosaccharides
FST
= Forced Swimming Test
GABA
= Gamma-Amino Butyric Acid
GF
= Germ-Free
GOS
= Galacto-Oligosaccharides
HC
= Hippocampus
HPA
= Hypothalamic-Pituitary-Adrenal Axis
L/DB
= Light-Dark Preference Test
MB
= Marble Burying
MWM
= Morris Water Maze
NA
= Noradrenaline
NAcc
= Nucleus Accumbens
NE
= Norepinephrine
NMDA
= N-Methyl-D-Aspartate
OFT
= Open-Field Test
RCT
= Randomized Controlled Trial
SDT
= Passive Avoidance Step-Down Test
SIH
= Stress-Induced Hyperthermia
SPF
= Specific Pathogen-Free
TNF-
= Tumour Necrosis Factor
WAS
= Water Avoidance Test
CONFLICT OF INTEREST
Roger S. McIntyre has received research grants from
Stanley Medical Research Institute, National Alliance for
Research on Schizophrenia and Depression(NARSAD),
National Institutes of MentalHealth, Eli Lilly, Janssen-Ortho,
Shire, Astra-Zeneca, Pfizer, Lundbeck, Forest, Sepracor, and
BMS; has served on advisory boards for Astra-Zeneca,
Bristol-Myers Squibb, Janssen-Ortho, Eli Lilly, Lundbeck,
Pfizer, Shire, Merck, Sepracorand Otsuka; has served on
speakers bureaus for Janssen-Ortho, Astra Zeneca, Eli Lilly,
Lundbeck, Merck, Pfizer, and Otsuka; and CME activities
for Astra Zeneca, Bristol-Myers Squibb, Physicians
Postgraduate Press, CME Outfitters, Merck, Eli Lilly, Pfizer,
Lundbeck and Otsuka.
Siegfried Kasper received grants/research support,
consulting fees and honoraria within the last three years from
Alkermes, Angelini, AOP-Pharma, AstraZeneca, BristolMyers Squibb, Eli Lilly, Janssen, Lundbeck, Merck Sharp
and Dome (MSD), Neuraxpharm, Pfizer, Pierre Fabre,
Schwabe, Servier
ACKNOWLEDGEMENTS
AFC is the recipient of a research fellowship from
Conselho Nacional de Desenvolvimento Cientifico e
Tecnologico (CNPq; level II; Brazil). Authors AS and AFC
equally contributed as first authors to the present paper.
APPENDIX
Behavior Testing Methodology of Included Animal
Studies
Elevated Plus Maze (Fig. A1a)
As a long-established measure for anxiety-like behavior
in mice and rats, the elevated plus model has been in use for
nearly 30 years. The maze is composed of four arms, two of
which are open, and two are closed. Time spent in the open
arms and number of entries into the open arms are the
measures used in quantifying the behavior, with more time
spent in closed arms, and less entries into open arms
signifying more anxious behavior [67]. Modern studies are
able to use video camera recordings and later scoring these
behaviors [23].
Open Field Test (Fig. A1b)
The manner an animal behaves in a novel environment is
reflective of its emotional state. The open field takes
advantage of this fact, and exposes rats to an environment of
the acute, mild stress of light and noise [68], normally in a
box with a detection system in form of a camera or motion
sensor. Spontaneous motor activity is quantified, analyzing
the amount of time the mouse spends in the centre of the
box, the periphery and overall; as well as slow or fast
movement of the mouse [23]. This test is used to measure
locomotor and anxiety-like behavior.
Hole Board Test (Fig. A1c)
An automatic hole board apparatus can be used to
analyze exploratory behaviors in a manner such that they are
1783
able to indicate anxiety-like behavior. In this test, a mouse is

placed in a box with a number of equidistant holes in the
floor, with a sensor which detects rearing, head-dipping, and
head-dipping latency. Increased number of head-dips and
lengths of these are typical of non-anxiety-like behavior,
while anxiety-like behavior is described by a lower number
of head-dips, shorter spans of these and head-dipping latency
[69].
Forced Swim Test (Fig. A1d)
The forced swim test is used to test for depression-like
behavior in rodents. In this test, the animal is placed into a
cylinder partially filled with water, so that it is required to
swim to stay above the water and not drown. This is
recorded by a video camera for further analysis. Climbing,
swimming and immobility behaviors are scored, with
immobility indicating the experimental animal to have
developed learned helplessness, and thus indicating a
depression-like phenotype [70].
Barnes Maze (Fig. A1e)
The Barnes maze consists of a circular platform with
holes around the circumference of the platform, one of which
has an escape route for the animal. The animal is placed onto
this platform, with a camera monitoring its movements for
later motion-detecting software analysis, and attempts to find
the escape route. Normally, the distance travelled,
speed/velocity, amount of time spent in the appropriate areas
of the maze, amount of time spent mobile and latency are
measured, with improvement on the test being reflective of
spatial memory and learning [71].
Light/Dark Box Preference Test (Fig. A1f)
This is another test which exposes rodents to a novel
environment, giving the animal the option of exploring a
bright, open compartment, or staying in the safe, dark
compartment of the apparatus. Time spent in each
compartment quantifies the anxiety-like behavior displayed
by the rodent [72].
Tail Suspension Test (Fig. A1g)
The tail suspension test consists of suspending a rodent
by its tail, thus inducing short-term stress and eventually,
immobility in the animal (behavioral despair). The span of
immobility, as well as the number of immobile instances, as
recorded by scorers from a videotape, indicate the degree of
depression-like behavior [73].
Passive Avoidance Step-Down Test (Fig. A1h)
In this test, a rodent is placed on a raised platform located
in a box lined, with the latency to step down measured by the
presence of all paws off the platform. This is used to assess
anxiety-like behavior, as being on a raised platform is
generally dangerous for an animal in natural habitat [39].
Conditioned Defensive Burying Test (Not Pictured)
In the conditioned defensive burying test, a rodent is
exposed to an electric shock, resulting in the mouses natural
defensive behavior of piling material atop the aversive
stimulus. Anxiety-like behavior is measured through the
1784
Slyepchenko et al.
Fig. (A1). Behavioral testing apparatus of included pre-clinical investigations.
percentage of approaches and escapes, temporal length of

burying behavior, amount of head stretchings in the direction
of the shock [34].
Stress-Induced Hyperthermia (Not Pictured)
When exposed to stress, a part of the autonomic response
acts to increase core body temperature. The protocol is used to
evaluate anxiety-like behavior, and consists of first administering the drug, letting the animal calm down in a less stressful
environment for some time. The body temperature of the animal
is then measured twice, with a 10-15 minute difference between
the measurements. The elevation in temperature between these
two measurements is the stress induced hyperthermia, and this
is reduced by anxiolytic drugs [74].
Morris Water Maze (Not Pictured)
The Morris water maze is used to evaluate memory and
spatial learning in rodents. The test consists of a circular
pool with an invisible platform located somewhere in the
pool. The rodent is placed in the water and tries to find the
platform that helps it escape from the water. Performance on
this task is evaluated by measuring escape latency as a
measure of memory. Spatial memory is evaluated by amount
of time spent swimming in the targeted area of the pool.
Swimming ability and motivation may also be tested [75].
T-Maze (Not Pictured)

The T-maze uses the exploratory tendencies of rodents in
new environments. This test is used as a measure of
working/spatial memory, as the rodent is placed into one arm
of the T-shaped maze, and then chooses whether to head into
the left-side or right-side arm of the maze. Working memory
is evaluated on the equality of turns made by the rodent into
both arms, with an unbalanced ratio indicating dysfunctional
working memory [21].
Novel Object Test (Not Pictured)
The novel object test is used to evaluate spatial working
memory. Rodents naturally are more likely to inspect a novel
object, rather than one they have seen previously. Mice are
exposed to two objects, one of which is a novel object,
assessing the mouses tendency to explore the novel object.
A lack of discrimination between the two indicates a lack of
memory for the object [21].
Marble Burying (Not Pictured)
As a model of anxiety and obsessive compulsive
disorder, this test is used to evaluate anxiety-like behavior in
rodents. In this test, a number of marbles is placed in a box,
the bottom of which is covered by sawdust. The increasing
number of marbles buried by the mice is indicative of
anxiety- like behavior [20].
Hebb-Williams Maze (Not Pictured)

This maze is used to evaluate spatial learning and
working memory performance in rodents. The maze has
several difficulty levels, with mice trying to attain a food
reward at the end of the maze. Anxiety-like behaviors are
assessed within the maze, with the following being
indicators of anxiety: defecation, elongation, grooming,
immobilization, latency to start, return to start, wall climbing
[31].
[19]
[20]
[21]
[22]
Fear Conditioning (Not Pictured)

This paradigm is used to test memory through the
diminishing of the fear response induced by conditioning
[35].
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Revised: August 14, 2014
PMID: 25470391
Accepted: August 14, 2014

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Gut Emotions - Mechanisms of Action of

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Available from: Andre Carvalho

Send Orders for Reprints to reprints@benthamscience.ae

CNS & Neurological Disorders - Drug Targets, 2014, 13, 1770-1786

Gut Emotions - Mechanisms of Action of Probiotics as Novel Therapeutic

Mood Disorders Psychopharmacology Unit, University of Toronto, Toronto, ON, Canada

Departments of Psychiatry and Pharmacology, University of Toronto, Toronto, ON, Canada

Keywords: Anxiety, depression, gut microbiota, probiotics, review, treatment.

These authors contributed equally to the present work.

although a number of factors influence its composition

Probiotics, Anxiety and Depression

mic-pituitary-adrenal (HPA) axis mediate the cross-talk

for homeostasis, and food consumption [12]. Enteroendocrine

In recent years, accumulating evidence indicates that the

(GF) rodents are raised in an environment such that any

3.1. Comparative Studies of Microbiota Effects on

Brain-derived neurotrophic factor levels were shown to

Of the selected studies, six examined the correlations

Monoamine turnover was faster in GF mice [23], and a

Probiotics, Anxiety and Depression

Comparative studies of microbiota effects on the development of murine phenotypes.

Neufeld et al., Swiss2011 [24]

NIH microbiota colonisation of GF mice had similar effects as

GF have lower anxiety-like behavior in L/DB, lower levels of

GF mice show more exploratory behavior in OFT, EPM; more

GF mice have normal anxiety levels but have worse memory

GF mice have lower anxiety-like behavior in EPM; no

GF exposed to external environment had decreased anxiety

Heightened HPA response for GF mice: higher plasma ACTH.

Difference in Odoribacter, Alistipes, Coriobacteriaceae

GF rats had less social interactions, increased anxiety-like

observed in the frontal cortices of GF rats [28]. This suggests

interaction with the way in which the mice were raised. A

Heijtz et al. [23] found lower expression of nerve growth

A study involving a stress- sensitive strain of rats showed

3.3. Colonisation Effects

3.5. Effects of Antimicrobials, Ambient Bacteria and

Probiotics, Anxiety and Depression

No behavior difference from infection observed, but

Anxiety-like behavior increased from infection before

Gareau et al., 2011

Goehler et al., 2013

Lyte et al., 2006

Increased anxiety-like behavior prior to sickness

Ambient Bacteria Administration

Matthews and Jenks,

Reduced anxiety-like behaviors, improved maze

Antimicrobials changed microbiome composition,

Bercik et al., 2011a

Anxiety-like behavior increased from infection before

Increased anxiety-like behavior prior to sickness

BDNF and NR1 mRNA expression increased in HC of

Goehler et al., 2013

Lyte et al., 2006 [31]

Savignac et al., 2013

Ten and thirty days after infection with Citerobacter

acute stress (through a water avoidance stress method), a

BDNF mRNA expression in their hippocampi paralleled

decreased in a stress response paradigm after 17 days of L.