Neurogastroenterology & Motility

Neurogastroenterol Motil (2014) 26, 510–520 doi: 10.1111/nmo.12295

Probiotic gut effect prevents the chronic psychological

stress-induced brain activity abnormality in mice

A. AIT-BELGNAOUI ,*,† A. COLOM ,* V. BRANISTE ,* L. RAMALHO ,* A. MARROT ,*,† C. CARTIER ,* E. HOUDEAU ,*

V. THEODOROU * & T. TOMPKINS †

*Neuro-Gastroenterologie et Nutrition team, TOXALIM, UMR 1331-INRA/INP/UPS, Toulouse, France

†Lallemand Health Solutions Inc., Montreal, QC, Canada

Key Messages

• This study provides the conclusive proof effect of probiotic formulation (Lactobacillus helveticus R0052 and
Bifidobacterium longum R0175) on chronic stress-induced brain activity dysregulation.
• This probiotic formulation treatment may contribute to its ability to ameliorate chronic stress-induced
abnormal brain plasticity, reduction in neurogenesis, and increase in stress hormone.
• These findings highlight the important role of bacteria in the bidirectional communication of the gut- brain
axis and suggest that probiotic treatment may prove to be a useful therapeutic alternative in stress-related
disorders such as anxiety and depression.

Abstract
Background A probiotic formulation (Lactobacillus
helveticus R0052 and Bifidobacterium longum R0175
combination, Probio’Stickâ) displays anxiolytic-like
activity and reduces apoptosis in the lymbic system in
animal models of depression. Based on the hypothesis
that modulation of gut microbiota by this probiotic
formulation has beneficial effects on brain activity in
stress conditions, we report a set of probiotic-evoked
physiological, cellular, and molecular events in the
brain of Probio’Stickâ pretreated mice submitted to
chronic psychological stress. Methods Water avoidance
stress (WAS) was applied or not (sham). Hypothalamic-
pituitary-adrenal (HPA) axis and autonomic nervous
system (ANS) responses to the chronic stress were
assessed through plasma corticosterone and catechol-
amine measurements. Specific markers for neuronal
activity, neurogenesis, and synaptic plasticity were
used to assess brain activity. In addition, gut perme-
ability and tight junction (TJ) proteins levels were also
determinated. Key Results We observed that a pretreat-
ment with the probiotic formulation attenuated HPA
axis and ANS activities in response to WAS, and
reduced cFos expression in different brain areas but
Lactobacillus salivarius (a negative control) treatment
was ineffective on these parameters. Moreover, probi-
otic pretreatment prevented the WAS-induced decrease
hippocampal neurogenesis and expression changes in
hypothalamic genes involved in synaptic plasticity.
These central effects were associated with restoration of
TJ barrier integrity in stressed mice. Conclusions &
Inferences These data suggest that chronic stress-
induced abnormal brain plasticity and reduction in
neurogenesis can be prevented by a pretreatment with
the Probio’Stickâ formulation, suggesting that probiot-
ics modulate neuroregulatory factors and various sig-
naling pathways in the central nervous system involved
in stress response.

Keywords brain-gut axis, chronic psychological

Address for Correspondence stress, intestinal barrier, probiotics.
Dr Afifa Ait-Belgnaoui, Neuro-Gastroenterology & Nutrition
Team, TOXALIM, UMR 1331-INRA/INP/UPS, 180 chemin de
Tournefeuille, BP 3, 31027 Toulouse Cedex 9, France.
Tel: 33 561 28 55 61; fax: 33 561 28 51 45; INTRODUCTION
e-mail: afifa@toulouse.inra.fr
Received: 30 July 2013 Chronic stress and acute traumatic experiences during
Accepted for publication: 29 November 2013 life are prevalent psychosocial factors worldwide,

510 © 2013 John Wiley & Sons Ltd

Volume 26, Number 4, April 2014
increasing susceptibility to functional gastrointestinal
(GI) disorders or exacerbating symptoms in inflamma-
tory bowel diseases. Patients with irritable bowel
syndrome (IBS) also show augmented sensitivity to
environmental stressors, often found comorbid with
psychiatric disorders including major depression and
anxiety. Of note, these patients displayed abnormal
activities of the hypothalamic-pituitary-adrenal (HPA)
axis and autonomic nervous system (ANS) associated
with exaggerated stress responses and cognitive disor-
ders.1–3 Recently, animal studies highlighted that
stress or chronic GI inflammation can alter the
composition of gut microbiota, which overall suggest
a link between change in enteric bacteria composition,
and stress-induced altered GI function4 or anxiety
behavior.5
Accumulating data indicate that enteric bacteria
communicate with the central nervous system (CNS).
It is proposed that the microbiota could be intimately
and constitutively involved in the modulation of both
CNS and peripheral nerve functions.6,7 For example,
the postnatal bacterial colonization of the gut programs
HPA axis function during development, and germ-free
mice display higher exploratory and lower anxiety-like
behavior compared to conventional animals.8,9 Diaz
Heijtz et al. extended the complex set of interactions
between intestinal microbiota and the CNS, in dem-
onstrating a modulatory role in central synaptogenesis
supporting normal cognitive function.10 Although the
causal link between CNS and microbiota remains
unknown, overall data support the notion that specific
changes in microbiota composition may help to ame-
liorate psychological behavior and quality of life in IBS
patients. According to the studies in laboratory rodents
underlying the benefit of probiotics on stress-evoked
GI disorders, it is now of importance to validate
specific probiotic formulations as therapeutic alterna-
tives in the management of anxiety and stress
responses linked to IBS symptoms. Indeed, some
probiotics have the potential to reduce mRNA expres-
sion of gamma-aminobutyric acid (GABA) receptors
associated with anxiety,11 or suppress mitogen stimu-
lation-induced increase in plasma tryptophan impli-
cated in depressive-like behaviors.12 Furthermore, the
modulation of intestinal microbiota by probiotics may
also prevent stress-associated gut leakiness, due to a
probiotic-mediated attenuation of HPA response in
rats exposed to acute psychological stress.13 Recently,
studies assessing the effect of a combination of
two probiotics (Bifidobacterium longum R0175 and
Lactobacillus helveticus R0052) not only high-
lighted their ability to reduce anxiety in rats, but
© 2013 John Wiley & Sons Ltd
Probiotic and Brain-gut axis
also in humans, where beneficial properties of the
probiotic formulation were correlated with decreased
levels of urinary free cortisol, suggesting attenuation of
HPA axis response to daily stressors.14 Reduction of
apoptosis in several brain areas or changes in memory
and anxiety-like behaviors were also observed in
animal models of depression after consumption of the
same probiotic strains,15 or diet supplemented by
L. helveticus R0052 only.16
Based on works to date, our aim was to investigate in
mice the central effect of the probiotic combina-
tion with B. longum R0175 and L. helveticus R0052
(Probio’Stickâ) on anxiety-like markers induced by
chronic psychological stress. The hypothesis was that
the probiotic treatment may target abnormal brain
activity, enhancement of HPA and ANS activities,
and intestinal barrier disruption in chronic stress
conditions.
MATERIALS & METHODS
Animal and bacteria preparation
Male 6–8 week old C57Bl6 mice (Janvier SA, Le Genest St Isle,
France) weighing 21–23 g were used. The animals were housed
individually under a constant temperature (21  1 °C) in a
pathogen-free animal facility, and maintained on a 12 h light/
dark cycle. Food (UAR pellets, Epinay, France) and water were
available ad libitum. All protocols were approved by the local
institutional animal (the Toulouse - Midi Pyr
en

ees Ethical
committee) care and use committee in compliance with the
European laws on the protection of animals (2010/63/EU). The
commercial probiotic given was a combination of freeze-dried
bacteria: L. helveticus R0052 and B. longum R0175 (Pro-
bio’Stickâ, provided by Lallemand Heath Solutions Inc., Blagnac,
France). L. salivarius HA113 (Lallemand Heath Solutions Inc.)
was used as control negative bacteria and grown at 37 °C in MRS
broth (VWR International, Fontenay-sous-Bois, France). After 17 h
of incubation, cultures were harvested by centrifugation at 4500 g
for 10 min. Bacterial solution was prepared every day in 0.9%
NaCl and administrated orally at a concentration of 109 CFU/day/
mice during 2 weeks.
Water avoidance stress
All stress sessions were performed at the same period of the day
(between 10:00 am and 12:00 pm) to minimize the influence of
circadian rhythms. The test apparatus consisted of a plexiglass
tank (42 cm length 9 42 cm width 9 19 cm height) with a block
(3 9 3 9 8 cm height) affixed in the center of the floor. The tank
was filled with fresh room temperature water (22 °C) to within
1 cm of the top of the block. The animals were placed on the
block for a period of 1 h daily for four consecutive days
corresponding to WAS. Sham consisted of placing the mice
similarly for 1 h daily for 4 days on the same platform in a
waterless container. This well characterized test represents a
potent psychological stressor with large elevations of ACTH and
corticosterone within 30 min.17
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A. Ait-Belgnaoui et al.
Experimental design
Three series of experiments were conducted to assess the effect of
probiotic formulation on WAS or sham. Eight groups of 8–10 male
mice received orally during 2 weeks either saline or probiotic
formulation (109 CFU/mouse/day). At the end of this period,
animals were submitted to WAS 1 h/day during four consecutive
days or sham. In the first series of experiments, four groups of
animals were used. After the chronic stress, blood was collected for
corticosterone, noradrenaline, and adrenaline plasmatic levels were
determined using Elisa kit. Mice were sacrificed and transcriptional
analyses of neuronal gene plasticity were investigated on the
hypothalamus. Colonic samples were also dissected for the
evaluation of the TJs protein expression levels. In the second series
of experiments, another four groups of animals were used and after
the chronic stress, the brain of animals was removed and the free
floating sections were immunostained for cFos and doublecortine
positive cells determination. And the in vivo intestinal perme-
ability was assessed in the last series of experiments.
The same experiments described below were conducted with
another probiotic L. salivarius HA113 to verify the probiotic
specificity on brain activity in response to chronic stress.
Tissue processing and staining
Mice were deeply anesthetized using a mixture of xylazine-
ketamine (10 mg/kg and 100 mg/kg i.p., respectively). Transcar-
dial perfusion was performed with 50 mL isotonic saline (0.9%
NaCl) followed by 60–100 mL of 4% paraformaldehyde (PAF)
fixation. Brain were dissected and removed, postfixed at 4 °C in
4% PAF in phosphate-buffered saline (PBS) for 4 h and cryo-
protected overnight in 30% sucrose at 4 °C. Frozen serial
sections (40 lm-thick) were collected in PBS and then rinsed
twice. For immunostainning, the brain slices were incubated at
room temperature in a blocking solution of 1% normal goat
serum in PBS with 0.25% Triton X-100 for 30 min and then
incubated overnight at 4 °C with rabbit polyclonal cFos anti-
body diluted in blocking solution (1 : 10 000; Ab-5, AbCys,
Paris, France) for 24 h at 4 °C. The incubated sections were
washed twice and incubated with biotinylated goat antirabbit
secondary antibody, diluted 1 : 1000 in blocking solution, and
then incubated with the avidin–biotin complex (Vectastain Elite
kit; Vector Laboratories, Paris, France). Peroxidase activity was
revealed using diaminobenzidine as chromogene (DAB substrate
kit; Vector Laboratories). The sections were then mounted on
gelatin-coated slides, dehydrated, and coverslipped with Prolong
Antifade kit (Life Technologies SAS, Saint Aubin, France). The
presence of cFos immunoreactivity was detected as a dark
brown reaction product in cell nuclei under a light microscope
(90i Nikon; Nikon France, Champigny-sur-Marne, France). For
immunofluorescence, sections were permeabilized in PBS-Tri-
ton X-100 and incubated at RT in a blocking solution of 3%
normal donkey serum in PBS with 0.3% Triton X-100 for
30 min. The following antibodies and final dilution were used.
Primary antibodies: goat polyclonal anti-a-Doublecortin, 1 : 250
(DCX C-18; Santa Cruz Laboratories, Santa Cruz, CA, USA)
antibodies; Fluorescent conjugated secondary antibodies (Alexa
488 and 594; Molecular Probes) were also used. Images were
acquired with a 90i Nikon microscope (Nikon France).
Cell counts and statistics
The anatomical localization of immuno-positive cells on the
planes of sections was standardized according to the Paxinos and
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Neurogastroenterology and Motility
Watson’s atlas.18 For quantitative assessment, the number of
immuno-positive cells was counted bilaterally in 8–10 sections of
selected nuclei using Lucia G4.8 software (Nikon France) in
different brain areas.
Plasma corticosterone, adrenaline, and
noradrenaline concentration
After chronic stress, blood was withdrawn from the facial vein in
heparinised tube, centrifuged, and plasma was recovered and
stored at 80 °C until assay. Corticosterone concentrations were
determined by a solid phase sandwich enzyme linked immuno-
sorbent assay (ELISA), specific for mice (Immunodiagnostic
system, Pouilly-en-Auxois, France). Noradrenaline and adrenaline
were assessed by 2-CAT Research ELISA kit (LDN, Nordhorm,
Germany) according to manufacturer’s instruction.
PCR array focused on HPA axis plasticity
Mice were decapitated and in aseptic conditions, brains were
removed from the skull and cooled on ice for 20 s followed by
coronal section. The hypothalamus was quickly dissected out
from a coronal midbrain section that included the anterior to
posterior hypothalamus, placed in sterile tubes, and frozen on
dry ice. Each sample was individually homogenized in a bead
beater for 2 min in a sterile microcentrifuge tube containing
1 mL QIAzol lysing reagent (Qiagen, GmbH, Hilden, Germany).
After the addition of 160 lL of chloroform, the samples were
vortexed for 15 s and left to stand for 2–3 min at room
temperature. Finally, the samples were centrifuged at 12 000 g
for 15 min at 4 °C and the supernatants collected. Total RNA
was reverse-transcribed with a high-capacity cDNA reverse
transcription kit (Applied Biosystems, Saint-Aubin, France).
Real time PCR was performed using the SYBR green IQ mix
(Bio-Rad, Hercules, CA, USA). Fluorescence was monitored and
analyzed in a CFX 96 detection system instrument (Thermal-
Cycler C1000; Bio-Rad). All data were normalized to b-actin for
each sample, all samples were run in duplicate and were
analyzed using LingRegPCR.19
In vivo intestinal permeability measurement
Evaluation of colonic paracellular permeability was performed
using 51Cr-EDTA (Perkin Elmer Life Sciences, Paris, France) as a
marker of paracellular permeation of apical TJ complexes. 51Cr-
EDTA (50 000 cpm/mice), diluted in 200 lL of saline was
administrated orally after WAS or sham. The animals were then
placed in metabolic cages, and feces and urine were collected
separately for 6 h. The radioactivity in the urine was measured on
a gamma counter (Cobra II, Packard Meriden, Connecticut, USA).
Permeability to 51Cr-EDTA was expressed as the percentage of the
total radioactivity administered.
Protein extraction and Western blot
Colons were homogenized in RIPA buffer (1% Igepal, 0.5%
deoxycholic acid, and 0.1% SDS in Tris buffered saline solution
19; pH 7.4) with protease inhibitors (Roche Diagnostics,
Meylan, France), and centrifuged at 10 000 g for 10 min
(4 °C). Protein concentrations were assessed using BC Assay
Uptima kit (Interchim, Montlucßon, France), and equal amounts
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Volume 26, Number 4, April 2014
of protein were separated by SDS/PAGE and transferred onto
nitrocellulose membranes. Membranes were blocked in Odyssey
blocker and then incubated with the polyclonal rabbit antijunc-
tion adhesion molecule (JAM-A; 1/500) or antioccludin (1/500;
Invitrogen, Vergy Pontoise, France) or 3-phosphate dehydroge-
nase (GAPDH 1/5000; Cell signaling Technology, Danvers, MA,
USA) overnight at 4 °C. After washing, the membranes were
incubated with fluorescent CF773 antirabbit 1 : 10 000 antibody
(Biotium, Hayward, CA, USA) with gentle rocking and protected
from light. Finally, they were scanned on infrared imaging
system Odyssey (Li-Cor, Lincoln, NE, USA). Band intensity was
analyzed by densitometry using the software ImajeJ (http://
rsbweb.nih.gov/ij). Occludin and JAMA expression was assessed
relative to GAPDH for each sample analyzed.
Statistical analysis
All data were expressed as means  SEM. Analyses were
performed by running Prism 4 software (GraphPad, San Diego,
CA, USA). Statistical comparisons between two groups were
performed using one-way ANOVA. Differences were considered
significant when post Student’s t-test (LSD) p < 0.05. Real
time RT-PCR values were analyzed by a two-sided Student’s t-test.
RESULTS
Effect of probiotic (Probio’Stickâ) treatment on
neuroendocrine and catecholamine response to
chronic stress
Compared to sham animals, corticosterone plasmatic
levels sharply increased in response to chronic stress
(184.5  20.2 vs 598.0  95.1 ng/mL in WAS mice,
p < 0.05; Fig. 1). A significant increase in plasma
Figure 1 Effect of oral pretreament by
Probio’Stickâ or vehicle (saline) on plasma
corticosterone, adrenaline, and
noradrenaline concentration in mice
submitted to chronic stress. (a and b)
p < 0.05, respectively, were significantly
different from sham + vehicle and WAS +
vehicle.
© 2013 John Wiley & Sons Ltd
Probiotic and Brain-gut axis
adrenaline and noradrenaline levels was also observed
in WAS mice (p < 0.05 vs sham; Fig. 1). There is no
effect of probiotic formulation on plasmatic levels of
corticosterone and catecholamines in basal conditions,
while the probiotic treatment prevented WAS-induced
increase in these plasma markers of chronic stress
(p < 0.05; Fig. 1).
Regarding L. salivarius pretreatment, we have
observed that this probiotic has no effect on
higher plasmatic levels of corticosterone, adrenaline,
and noradrenaline induced by WAS (Fig. 1). So, we
used this strain as a negative control in other
experiment.
Effect of probiotic (Probio’Stickâ) treatment on
chronic stress-induced neuronal activation in
brain
Compared to basal levels (sham), chronic stress (WAS)
increased cFos positive nuclei number in the following
brain areas: hypothalamic paraventricular nucleus
(PVN), amygdaloid nucleus (AN), lateral parabrachial
nucleus, locus coeruleus, nucleus tractus solitary, and
CA-1/3 of hippocampus (Table 1). In dentate gyrus
(DG) of the hippocampus, the number of cFos positive
nuclei was significantly lower than in sham groups
(Table 1). A two-week pretreatment with probiotic
significantly (p < 0.05) lessened the increase in cFos
positive neurons induced by WAS in the PVN, AN, and
CA-3 compared to sham. In contrast, the probiotic
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A. Ait-Belgnaoui et al. Neurogastroenterology and Motility

pretreatment significantly increased the number of Differential expression of genes involved in

cFos positive nuclei in WAS mice compared to vehicle synaptic plasticity in the hypothalamus after
groups (p < 0.05; Table 1). Probio’Stickâ treatment in stressed mice

Table 1 Number of Fos-immunoreactive nuclei per region in the brain In WAS conditions, genes involved in the neurotrophic
of mice submitted to WAS or sham after or not probiotic treatment signals displayed a lower expression level 40.7% for
BDNF (brain-derived neutrophic factor), a neutrophic
Sham WAS
factor, compared with control mice (sham). Similar
Vehicle Probiotic Vehicle Probiotic difference in gene expression was also observed for
serpin, Plau (urokinase-type plasminogen activator)
Forebrain
LPB 2 1 11 23  2 25  1 and Mmp9 (matrix metalloprotease; involved in tissue
LC 7 3 32 24  4 21  2 remodeling). The same gene expression profiling of the
NTS 5 4 34 83  11 62  15 cytoskeleton (Gap43: neuronal growth associated pro-
Hypothalamus
PVN 20 1 15  3 150  13 54  7* tein 43) and cell adhesion markers (tenascin-C, Reln:
Amygdala reelin and neural cell adhesion molecule) was observed
BLA 17 1 23  1 120  11 113  12 in stressed mice. Regarding the expression of genes
CeA 15 3 19  1 112  8 93  12
MeA 19 4 20  1 160  10 43  2* involved in microglia activation and synaptogenesis,
Hippocampus mRNA levels of Gfap (glial fibrillary acid protein), Vim
CA1 2 0 11 14  4 15  3 (vimentine), Syt4 (synaptotagmin), and Snap25 (synap-
CA3 1 0 31 20  4 7  2*
DG 5 3 41 23  0 46  2* tic-associated protein 25 kDa) were significantly
higher in WAS mice compared to sham (p < 0.05;
Values are given as the mean total number of Fos-IR nuclei per region Fig. 2).
of interest, and asterisk values are significant higher than correspond-
ing value in WAS treated with vehicle (one-way ANOVA, p < 0.05). When compared to WAS mice receiving the vehicle,
LBP, lateral parabrachial nucleus; LC, locus coeruleus; NTS, nucleus the probiotic treatment in stressed mice significantly
tractus solitary; PVN, paraventricular hypothalamic nucleus; BLA, baso- increased the expression of neurotrophic factor
lateral amygdaloid nucleus; CeA, central amygdaloid nucleus; MeA,
medial amygdaloid nucleus; CA1, hippocampal pyramidal cells, aera CA1; (BDNF) and decreased the expression of cytoskeleton
CA3, hippocampal pyramidal cells, aera CA3; DG, dentate gyrus. organization and microglial activation markers

Figure 2 Absolute percentage of expression of differences in each significant gene for the WAS (W) and WAS + probiotic (WP) compared with the
sham + vehicle. Values are means; n = 8 per group. *p < 0.05 and #p < 0.05 were considered significant from the Sham and WAS groups.

514 © 2013 John Wiley & Sons Ltd

Volume 26, Number 4, April 2014 Probiotic and Brain-gut axis

(Gap43, Gfap, Vim), synaptogenesis (Syt4, Snap25) and
cell adhesion markers (Reln, Sema [semaphorine];
Fig. 2).
Effect of probiotic (Probio’Stickâ) treatment on
neurogenesis in stressed mice
The impact of probiotic formulation on neurogenesis
was determined by quantifying in the DG of hippo-
campus the doublecortin (DCX)-positive cells, which is
one of earliest marker of neuronal density. In non-
treated animals, a significant reduction in DG neuro-
genesis occurred in response to WAS, with a 41.5%
decrease of DCX-positive cells in WAS-vehicle group
compared to sham (87.5  5.2 versus 51.2  3;
p < 0.05). This decrease appeared more pronounced in
the subgranular zone of the granular cell layer in the
DG (Fig. 3). Probiotic pretreatment before and during
WAS completely prevented the WAS-induced decrease
in neuronal growth (Fig. 3).
Probiotic (Probio’Stickâ) prevents gut barrier
impairment in stress mice
Probiotics treatment did not affect intestinal permeabil-
ity nor the expression of the TJ proteins occludin and
JAM-A in basal conditions (i.e., without chronic stress)—
two transmembrane proteins that seal intercellular

Figure 3 Quantification of neurogenesis by

detection of Doublecortin-expressing cells
after probiotic treatment (Probio’Stickâ) in
stressed mice. (A) Histogram representing
the number of DCX-expressing cells (SEM)
in the dentate gyrus of mice hippocampus
submitted to sham and WAS after vehicle or
probiotic treatment (Probio’Stickâ).
Statistical differences between WAS and
sham or WAS + probiotic are shown,
respectively, as ap < 0.05 and bp < 0.05. (B)
Confocal images of Doublecortin-expressing
in the dentate gyrus of sham and WAS
treated or not by probiotic (Probio’Stickâ).
White arrows point to labeled cells in the
inner granule cell layer in the dentate gyrus.

© 2013 John Wiley & Sons Ltd 515

A. Ait-Belgnaoui et al.
A esis induced by chronic stress. A transcriptional
B of brain regions involved in the HPA and ANS response
Figure 4 Effect of oral pretreatment by Probio’Stickâ of vehicle
(saline) on (A) hyperpermeability and (B) both colonic mucosal
occludin and JAM-A expression in mice submitted to chronic stress.
a,b
p < 0.05, respectively, were significantly different from sham +
vehicle and WAS + vehicle.
space at apical tight junctions.20 Water avoidance stress
procedure significantly (p < 0.05) increased intestinal
permeability to 51Cr-EDTA in comparison to non-
stressed mice (sham; Fig. 4), and this effect was abolished
after probiotic (Probio’Stickâ) pretreatment (Fig. 4A).
A reduction in the expression of occludin and JAM-A in
the colonic mucosa of WAS mice was also observed
(Fig. 4A). Probiotic (Probio’Stickâ) pretreatment signif-
icantly (p < 0.05) restored occludin and JAM-A expres-
sion to basal levels (i.e., sham+vehicule; Fig. 4B).
DISCUSSION
This study indicates that a 2-week treatment with the
probiotic formulation Probio’Stickâ (L. helveticus
R0052 and B. longum R0175) attenuated hypothal-
amo-pituitary-adrenal (HPA) axis and ANS response to
chronic stress as reflected by a decrease in plasmatic
levels of corticosterone, adrenaline, and noradrenaline
in stressed mice. Moreover, the probiotic prevented
changes in central neuronal activation and neurogen-
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Neurogastroenterology and Motility
analysis of the hypothalamus also suggested a benefi-
cial role of pretreatment with this probiotic in modu-
lating neuronal networks that coordinate synaptic
plasticity. We also showed that probiotic pretreatment
reduces the increase in intestinal permeability induced
by chronic stress, which is correlated by a prevention
of the stress-induced degradation in colonic mucosa of
TJ proteins.
It is well established that chronic stress exposure in
animals physically altered the structure and function
to stress.21 Chronic stress is often used to mimic
human depressive illness in animals and to elicit an
increase in noradrenaline in the hippocampus known
to be associated with deficits in learning and memory
tasks.22 Water avoidance stress is a well-described and
validated model for chronic psychological stress and
WAS animals displayed a more anxious and timid
behavior.23 In this study, the treatment of stressed
mice with the probiotic attenuated the HPA axis and
ANS responses to chronic stress, in diminishing cor-
ticosterone, noradrenaline and adrenaline circulating
levels. In contrast, L. salivarius treatment had no
effect on neuroendocrine response to chronic stress.
The lack of stress hormone response suggests that the
regulation of HPA and ANS response to chronic stress
is bacterial strain dependant. Thus, the normalization
of these stress hormones may be considered as an
important criterion in the assessment of antidepres-
sant activity of probiotics. In support of this, a recent
clinical study has shown that the same probiotic given
daily over 30 days significantly reduced psychological
distress in healthy volunteers, associated with a
decrease in urinary free cortisol levels.24 In rats,
Desbonnet et al., have shown a reduced noradrenaline
concentration in the hippocampus following chronic
administration of bifidobacteria treatment for
14 days.12 The administration of tricyclic antidepres-
sants to animals induced also a decrease in noradren-
aline levels in the hippocampus,25 suggesting that
bifidobacteria may affect the central neurochemical
under stress conditions by mechanisms comparable to
that observed in with chronic antidepressant drugs.
Germ-free mice exhibit a higher turnover rate of the
anxiety inducers, like noradrenaline, dopamine, and
serotonin in the striatum,10 which can influence the
development of behavior.9 It is probably that these
central neurochemical changes in the brain of stressed
mice were correlated with a marked neuroplasticity of
neurotransmitters in the PVN of the hypothalamus
that predicts enhanced HPA axis excitability in
response to chronic stress.26 Interestingly, recent
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Volume 26, Number 4, April 2014
studies have shown the ability of a probiotic treatment
to decrease hypothalamic corticotropin-releasing hor-
mone (CRF) expression in stressed rats13 or to modu-
late GABA neurotransmission implicated in anxiety
behavior in a cerebral area dependent manner.11 These
data suggest that the probiotics can affect neuronal
circuits implicated in the HPA axis response to stress
and we examined gene expression changes involved in
the neuronal plasticity in the hypothalamus. We report
herein the overall profiles of probiotic-triggered gene
expression with distinctive or common functions in
the regulation of neuronal networks. Transcriptional
analysis has shown that the neurotrophic factor BDNF
is upregulated after probiotic treatment in stressed
mice, a change could be link to the ability of
glucocorticoids to regulate the HPA axis. Indeed, it
has been shown that impairment in glucocorticoid
receptor function in the PVN led to upregulation of the
hypothalamic levels of BDNF, and disinhibition of the
HPA axis.27 Bercik et al. have demonstrated that a
transient perturbation of the gut microbiota increased
hippocampal BDNF and this change was correlated
with an altered exploratory behavior.28 Moreover, it is
interesting to note in this study that other genes
known to participate in neurotransmission and synap-
tic plasticity appeared also modulated in stressed mice
after probiotic treatment. For instance, the hypotha-
lamic expression of Syt4 and Snap25, a regulator of a
synaptic vesicule exocytosis in neurons, was decreased
after probiotic treatment in stressed mice. Previous
studies emphasized their expression in the brain
associated with associative and spatial memory29,30
and long-term potentiation,31 respectively. A decrease
in Snap25 and Syt4 knockout mouse are responsible for
presynaptic terminal damage in ischemic brain32, and
mice displays lower anxiety and less depression-like
behavior.29 On this basis, it is suggested in this study
that the probiotic treatment in stressed mice regulates
synaptic functions in the neuronal networks, hence
protects against stress-induced aberrant synaptic
changes in the brain. Another group of genes, such as
Mmp9, Gap43, Reln, Sema, Gfap and Vim highly
implicated in tissue remodeling, cytoskeleton organi-
zation, cell adhesion, and axonal elongation were also
modulated in stressed mice receiving probiotic treat-
ment. The regulation of stress activation and recovery
involves tight coordination between neuronal and glial
networks.33 In addition to their role of support and
nutrition of neurons, glial cells defend CNS from stress
insult through an up-regulation of the local inflamma-
tory processes. However, excessive or prolonged glial
activation enhances a chronic damage that eventually
propagates neuroinflammation suggesting the delicacy
© 2013 John Wiley & Sons Ltd
Probiotic and Brain-gut axis
of the balance between neuroprotection and neurotox-
icity.34 At a certain threshold of sensitivity, stress
exposure can evoke a neuroimmune response35 and
microglial remodeling36 able to modulate synaptic
function, impairing the normal establishment of neu-
rons connections. Our recent study reported the ability
of probiotic pretreatment to counteract the hypotha-
lamic neuroinflammation in stressed rats by a mech-
anism depending upon prevention of gut leakiness.13
Thus, one way hypothesized in the current study that
probiotic treatment may attenuate the neuronal and
glial disorganization-induced by chronic psychological
stress, hence normalizes neuronal networks.
Neuronal plasticity in the brain is a normal process
throughout life, both in the formation of new synaptic
connections, new neurons and glial cells. However,
stress can inhibit this plasticity and lead to cerebral
atrophy. In psychiatric disorders, such changes that
occurred in sensitive brain areas, which may play a role
in memory function and emotional states. In the
hippocampus, a remarkable capacity for structural
reorganization has been well described.37 Preexisting
neural circuit undergoes modifications in dendritic
complexity and synapse number, and entirely novel
neural connections are formed through a process of
neurogenesis. Stress exerts a significant brake in this
process as a potent negative regulator of neurogenesis.
Different chronic stress conditions suppress the rate of
adult DG proliferation in the hippocampus, and
decreases the size of newborn cell clusters.38 In the
hippocampus and the prefrontal cortex, chronic stress
can induce plasticity of stress circuitry through retrac-
tion of apical dendrites, reduction in spine density in
the CA-3 region of the hippocampus,39 suppression of
neurogenesis of DG granule neurons40 and a decrease in
glucocorticoid receptor expression.41 Chronic stress
also promotes changes in the PVN of the hypothalamus,
including increase in CRF, reduction in glucocorticoid
receptor42 and altered expression of numerous neuro-
transmitter receptor subunits.43,44 Finally, neurochem-
ical changes are seen in numerous stress regulatory
pathways which are projected in the PVN and increases
secretagogue synthesis in the PVN. These neurochem-
ical changes suggest that chronic stress enhances the
excitability of HPA to novel stress and decreases HPA
negative feedback at the level of the PVN and hippo-
campus. In this study, we found that chronic stress in
mice suppressed neurogenesis in the hippocampus
reflected by a decrease in doublecortin (DCX)-immu-
noreactive cells in the granule precursor cells of the DG,
and this number of DCX-positive cells was restored in
the probiotic-treated mice. These data suggest that
probiotic is able to enhance survival and differentiation
517
A. Ait-Belgnaoui et al. Neurogastroenterology and Motility

of cells into neurons. In the hippocampus, the same
probiotic in an animal model of depression reduces
apoptosis propensity through an Akt activity-depen-
dent mechanism.15 PI3K/Akt is a cell signaling path-
way that plays a key role in cellular homeostasis
through its role in regulation of apoptosis, cell growth,
and cell cycle.45 This signal transduction pathway
seems to be involved in the changes of synaptic
plasticity in depression states.46 This data support our
findings and suggests that the combination of two
probiotics promotes neurogenesis in the DG of the
hippocampus in stressed mice, by ameliorating the
functional recovery of such synaptic plasticity. We have
also shown that this probiotic treatment enhanced
neuronal activation changes in several areas in the
brain, and more particularly in the hippocampus which
can be correlated to axonal and dendrite neuronal
prolongment.47 More investigation was needed for
exploring the mechanistic relationship of this probiotic
formulation action toward cognitive improvement. An
assumed mechanism of action of probiotics on the
interaction between the brain and the gut could be
explained by the fact for example that B. longum is able
to modulate enteric neuron excitability48 involving the
vagal pathways in the gut-brain communication.49
While Perez-Burgos et al. have shown that other bacte-
ria such as L. salivarius did not alter firing rate of vagal
fibers.50 Consequently, these studies suggest firstly that
vagal afferent signaling pathways are required by
B. longum effect in the normalization of axiety-like
behavior. And secondly, these pathways are bacterial
strain specific in the brain-gut communication.
It is clear that probiotic can modulate various
aspects of microbiota-gut-brain axis.51 Interestingly,
both bacteria modulate enteric neuron excitability,
suggesting that enteric to vagus signaling is an impor-
tant means of communication along the microbiota-
gut-brain axis.49,52 An indirect role for microbiota in
stress response was recently demonstrated in an
animal model of stress-induced intestinal barrier
impairment. Indeed, a prevention of gut leakiness by
intestinal microbiota modulation with a probiotic
leads to attenuation of the HPA axis response to
stress.13 In the same way, we have shown that
probiotic formulation pretreatment reduces the
increase in intestinal paracellular permeability
induced by chronic stress correlated by the prevention
of the degradation in colonic mucosa expression of
tights junction’s proteins (occludin and JAMA). The
prevention of intestinal barrier integrity by this probi-
otic formulation has been also shown in model of
postmyocardial infarction depression. It has been
demonstrated that probiotic formulation (B. longum
and L. helveticus) prevents stress-induced memory
deficit in mice through a reduction in a cytokine-
dependant mechanism.53 Thus, targeting the microbi-
ota-gut-brain axis to modulate behavior by probiotic
seems to be relevant for the regulation of the stress
response.
In conclusion, our study shows that a 2-week
treatment by the probiotic formulation Probio’Stickâ
(B. longum and L. helveticus) attenuated HPA axis and
ANS activity in response to stress, and prevented the
stress-induced reduction in neurogenesis in the hippo-
campus. The attenuation of HPA axis by the probiotic
formulation protects neuronal plasticity at the hypo-
thalamic level, consequently maintains brain activity
against stress-mediated brain circuitry insult. We can
hypothesize that the probiotic promotion of neurogen-
esis in the hippocampus restored the HPA axis nega-
tive feedback.
FUNDING
No external funding was used for this study.
DISCLOSURE
None of the authors have conflicts of interest to declare.
AUTHOR CONTRIBUTION
AAB designed the research study, carried out the study, data
analysis and interpretation, and wrote the manuscript; VB
provided intellectual support to the study and contributed to
critical revised manuscript; AC, CC, and LR responsible for
material support and contribute to the data interpretation:
provide help in immunohistology and transcriptional analysis;
EH and VT provide critical revision of the manuscript; TT
obtained funding, contributed to data critical interpretation and
manuscript writing.

REFERENCES
1 Chang L, Sundaresh S, Elliott J, Anton
PA, Baldi P, Licudine A, Mayer M,
Vuong T et al. Dysregulation of the
hypothalamic-pituitary-adrenal (HPA)
axis in irritable bowel syndrome. Neu-
rogastroenterol Motil 2009; 21: 149–59.
2 Dinan TG, Quigley EM, Ahmed SM,
Scully P, O’Brien S, O’Mahony L,
O’Mahony S, Shanahan F et al. Hypo-
thalamic-pituitary-gut axis dysregula-
tion in irritable bowel syndrome:
plasma cytokines as a potential bio-
marker? Gastroenterology 2006; 130:
304–11.
3 Eriksson EM, Andren KI, Eriksson HT,
Kurlberg GK. Irritable bowel syn-

518 © 2013 John Wiley & Sons Ltd

Volume 26, Number 4, April 2014
drome subtypes differ in body aware-
ness, psychological symptoms and
biochemical stress markers. World J
Gastroenterol 2008; 14: 4889–96.
4 O’Mahony CM, Clarke G, Gibney S,
Dinan TG, Cryan JF. Strain
differences in the neurochemical
response to chronic restraint stress
in the rat: relevance to depression.
Pharmacol Biochem Behav 2011; 97:
690–9.
5 Bercik P, Verdu EF, Foster JA, Macri J,
Potter M, Huang X, Malinowski P,
Jackson W et al. Chronic gastrointes-
tinal inflammation induces anxiety-
like behavior and alters central
nervous system biochemistry in mice.
Gastroenterology 2010; 139: 2102–12,
e2101.
6 Collins SM, Bercik P. The relation-
ship between intestinal microbiota
and the central nervous system in
normal gastrointestinal function and
disease. Gastroenterology 2009; 136:
2003–14.
7 Grenham S, Clarke G, Cryan JF,
Dinan TG. Brain-gut-microbe com-
munication in health and disease.
Front Physiol 2011; 2: 94.
8 Sudo N, Chida Y, Aiba Y, Sonoda J,
Oyama N, Yu XN, Kubo C, Koga Y.
Postnatal microbial colonization pro-
grams the hypothalamic-pituitary-
adrenal system for stress response in
mice. J Physiol 2004; 558: 263–75.
9 Neufeld KM, Kang N, Bienenstock J,
Foster JA. Reduced anxiety-like
behavior and central neurochemical
change in germ-free mice. Neurogas-
troenterol Motil 2011; 23: 255–64,
e119.
10 Diaz Heijtz R, Wang S, Anuar F, Qian
Y, Bj€orkholm B, Samuelsson A, Hib-
berd ML, Forssberg H et al. Normal
gut microbiota modulates brain
development and behavior. Proc Natl
Acad Sci USA 2011; 108: 3047–52.
11 Bravo JA, Forsythe P, Chew MV,
Escaravage E, Savignac HM, Dinan
TG, Bienenstock J, Cryan JF. Inges-
tion of Lactobacillus strain regulates
emotional behavior and central
GABA receptor expression in a mouse
via the vagus nerve. Proc Natl Acad
Sci USA 2011; 108: 16050–5.
12 Desbonnet L, Garrett L, Clarke G,
Bienenstock J, Dinan TG. The probi-
otic Bifidobacteria infantis: an assess-
ment of potential antidepressant
properties in the rat. J Psychiatr Res
2008; 43: 164–74.
13 Ait-Belgnaoui A, Durand H, Cartier
C, Chaumaz G, Eutamene H, Ferrier
© 2013 John Wiley & Sons Ltd
L, Houdeau E, Fioramonti J et al.
Prevention of gut leakiness by a pro-
biotic treatment leads to attenuated
HPA response to an acute psycholog-
ical stress in rats. Psychoneuroendo-
crinology 2012; 37: 1885–95.
14 Messaoudi M, Lalonde R, Violle N,
Javelot H, Desor D, Nejdi A, Bisson
JF, Rougeot C et al. Assessment of
psychotropic-like properties of a pro-
biotic formulation (Lactobacillus
helveticus R0052 and Bifidobacteri-
um longum R0175) in rats and human
subjects. Br J Nutr 2011; 105: 755–64.
15 Girard SA, Bah TM, Kaloustian S,
Lada-Moldovan L, Rondeau I, Tomp-
kins TA, Godbout R, Rousseau G.
Lactobacillus helveticus and Bifido-
bacterium longum taken in combina-
tion reduce the apoptosis propensity
in the limbic system after myocardial
infarction in a rat model. Br J Nutr
2009; 102: 1420–5.
16 Ohland CL, Kish L, Bell H, Thiesen
A, Hotte N, Pankiv E, Madsen KL.
Effects of Lactobacillus helveticus on
murine behavior are dependent on
diet and genotype and correlate with
alterations in the gut microbiome.
Psychoneuroendocrinology 2013; 38:
1738–47.
17 Million M, Tache Y, Anton P.
Susceptibility of Lewis and Fischer
rats to stress-induced worsening of
TNB-colitis: protective role of brain
CRF. Am J Physiol 1999; 276: G1027–
36.
18 Paxinos G, Watson C. The Rat Brain
in Stereotaxic Coordinates, 2nd edn.
New York: Academic Press, 1986.
19 Ruijter JM, Ramakers C, Hoogaars
WM, Karlen Y, Bakker O, van den
Hoff MJ, Moorman AF. Amplification
efficiency: linking baseline and bias
in the analysis of quantitative PCR
data. Nucleic Acids Res 2009; 37: e45.
20 Braniste V, Leveque M, Buisson-Bre-
nac C, Bueno L, Fioramonti J, Hou-
deau E. Oestradiol decreases colonic
permeability through oestrogen
receptor beta-mediated up-regulation
of occludin and junctional adhesion
molecule-A in epithelial cells. J Phys-
iol 2009; 587: 3317–28.
21 Barha CK, Brummelte S, Lieblich SE,
Galea LA. Chronic restraint stress in
adolescence differentially influences
hypothalamic-pituitary-adrenal axis
function and adult hippocampal neu-
rogenesis in male and female rats.
Hippocampus 2011; 21: 1216–27.
22 Srikumar BN, Raju TR, Shankaran-
arayana Rao BS. The involvement of
519
Probiotic and Brain-gut axis
cholinergic and noradrenergic sys-
tems in behavioral recovery following
oxotremorine treatment to chroni-
cally stressed rats. Neuroscience
2006; 143: 679–88.
23 Bradesi S, Schwetz I, Ennes HS, Lamy
CM, Ohning G, Fanselow M, Potho-
ulakis C, McRoberts JA et al.
Repeated exposure to water avoid-
ance stress in rats: a new model for
sustained visceral hyperalgesia. Am J
Physiol Gastrointest Liver Physiol
2005; 289: G42–53.
24 Messaoudi M, Violle N, Bisson JF,
Desor D, Javelot H, Rougeot C. Ben-
eficial psychological effects of a pro-
biotic formulation (Lactobacillus
helveticus R0052 and Bifidobacteri-
um longum R0175) in healthy human
volunteers. Gut Microbes 2011; 2:
256–61.
25 Chung MY, Kim DG, Yoo KJ, Hong
SS. Regional differences in the levels
of biogenic amines and their metab-
olites in rat brain after tricyclic anti-
depressant treatments. Yonsei Med J
1993; 34: 266–77.
26 Flak JN, Ostrander MM, Tasker JG,
Herman JP. Chronic stress-induced
neurotransmitter plasticity in the
PVN. J Comp Neurol 2009; 517:
156–65.
27 Jeanneteau FD, Lambert WM, Ismaili
N, Bath KG, Lee FS, Garabedian MJ,
Chao MV. BDNF and glucocorticoids
regulate corticotrophin-releasing hor-
mone (CRH) homeostasis in the
hypothalamus. Proc Natl Acad Sci
USA 2012; 109: 1305–10.
28 Bercik P, Denou E, Collins J, Jackson
W, Lu J, Jury J, Deng Y, Blennerhassett
P et al. The intestinal microbiota
affect central levels of brain-derived
neurotropic factor and behavior in
mice. Gastroenterology 2011; 141:
599–609, 609 e591–593.
29 Ferguson GD, Herschman HR, Storm
DR. Reduced anxiety and depression-
like behavior in synaptotagmin IV
(-/-) mice. Neuropharmacology 2004;
47: 604–11.
30 Liu YF, Chen HI, Wu CL, Kuo YM,
Yu L, Huang AM, Wu FS, Chuang JI
et al. Differential effects of treadmill
running and wheel running on
spatial or aversive learning and
memory: roles of amygdalar brain-
derived neurotrophic factor and syn-
aptotagmin I. J Physiol 2009; 587:
3221–31.
31 Roberts LA, Morris BJ, O’Shaugh-
nessy CT. Involvement of two iso-
forms of SNAP-25 in the expression
A. Ait-Belgnaoui et al.
of long-term potentiation in the rat
hippocampus. NeuroReport 1998; 9:
33–6.
32 Ishimaru H, Casamenti F, Ueda K,
Maruyama Y, Pepeu G. Changes in
presynaptic proteins, SNAP-25 and
synaptophysin, in the hippocampal
CA1 area in ischemic gerbils. Brain
Res 2001; 903: 94–101.
33 Theodosis DT, Poulain DA, Oliet SH.
Activity-dependent structural and
functional plasticity of astrocyte-neu-
ron interactions. Physiol Rev 2008;
88: 983–1008.
34 Bailey SL, Carpentier PA, McMahon
EJ, Begolka WS, Miller SD. Innate and
adaptive immune responses of the
central nervous system. Crit Rev
Immunol 2006; 26: 149–88.
35 Hueston CM, Barnum CJ, Eberle JA,
Ferraioli FJ, Buck HM, Deak T.
Stress-dependent changes in neuroin-
flammatory markers observed after
common laboratory stressors are not
seen following acute social defeat of
the Sprague Dawley rat. Physiol
Behav 2011; 104: 187–98.
36 Tynan RJ, Beynon SB, Hinwood M,
Johnson SJ, Nilsson M, Woods JJ,
Walker FR. Chronic stress-induced
disruption of the astrocyte network
is driven by structural atrophy and
not loss of astrocytes. Acta Neuropa-
thol 2013; 126: 75–91.
37 Shors TJ, Foy MR, Levine S, Thomp-
son RF. Unpredictable and uncontrol-
lable stress impairs neuronal
plasticity in the rat hippocampus.
Brain Res Bull 1990; 24: 663–7.
38 Sapolsky RM. Is impaired neurogene-
sis relevant to the affective symptoms
of depression? Biol Psychiatry 2004;
56: 137–9.
39 Radley JJ, Rocher AB, Rodriguez A,
Ehlenberger DB, Dammann M, McE-
wen BS, Morrison JH, Wearne SL
et al. Repeated stress alters dendritic
spine morphology in the rat medial
prefrontal cortex. J Comp Neurol
2008; 507: 1141–50.
40 Gould E, McEwen BS, Tanapat P,
Galea LA, Fuchs E. Neurogenesis in
the dentate gyrus of the adult tree
shrew is regulated by psychosocial
stress and NMDA receptor activa-
tion. J Neurosci 1997; 17: 2492–8.
41 Magarinos AM, McEwen BS. Stress-
induced atrophy of apical dendrites of
hippocampal CA3c neurons: involve-
ment of glucocorticoid secretion and
excitatory amino acid receptors. Neu-
roscience 1995; 69: 89–98.
42 Makino S, Smith MA, Gold PW.
Increased expression of corticotro-
pin-releasing hormone and vasopres-
sin messenger ribonucleic acid
(mRNA) in the hypothalamic parav-
entricular nucleus during repeated
stress: association with reduction in
glucocorticoid receptor mRNA levels.
Endocrinology 1995; 136: 3299–
309.
43 Cullinan WE. GABA(A) receptor sub-
unit expression within hypophysio-
tropic CRH neurons: a dual
hybridization histochemical study. J
Comp Neurol 2000; 419: 344–51.
44 Ziegler DR, Cullinan WE, Herman JP.
Organization and regulation of parav-
entricular nucleus glutamate signal-
ing systems: N-methyl-D-aspartate
receptors. J Comp Neurol 2005; 484:
43–56.
45 Datta SR, Dudek H, Tao X, Masters S,
Fu H, Gotoh Y, Greenberg ME. Akt
phosphorylation of BAD couples sur-
vival signals to the cell-intrinsic
death machinery. Cell 1997; 91:
231–41.
46 Marsden WN. Synaptic plasticity in
depression: molecular, cellular and
functional correlates. Prog Neuropsy-
chopharmacol Biol Psychiatry 2013;
43: 168–84.
520
Neurogastroenterology and Motility
47 Chen S, Hillman DE. Transient c-fos
expression and dendritic spine plas-
ticity in hippocampal granule cells.
Brain Res 1992; 577: 169–74.
48 Khoshdel A, Verdu EF, Kunze W,
McLean P, Bergonzelli G, Huizinga
JD. Bifidobacterium longum
NCC3001 inhibits AH neuron excit-
ability. Neurogastroenterol Motil
2013; 25: e478–84.
49 Bercik P, Park AJ, Sinclair D, Khosh-
del A, Lu J, Huang X, Deng Y, Blen-
nerhassett PA et al. The anxiolytic
effect of Bifidobacterium longum
NCC3001 involves vagal pathways
for gut-brain communication. Neuro-
gastroenterol Motil 2011; 23: 1132–9.
50 Perez-Burgos A, Wang B, Mao YK,
Mistry B, McVeyNeufeld KA, Bie-
nenstock J, Kunze W. Psychoactive
bacteria Lactobacillus rhamnosus (JB-
1) elicits rapid frequency facilitation
in vagal afferents. Am J Physiol Gas-
trointest Liver Physiol 2013; 304:
G211–20.
51 Gareau MG, Sherman PM, Walker
WA. Probiotics and the gut microbi-
ota in intestinal health and disease.
Nat Rev Gastroenterol Hepatol 2010;
7: 503–14.
52 Kunze WA, Mao YK, Wang B, Huiz-
inga JD, Ma X, Forsythe P, Bienen-
stock J. Lactobacillus reuteri
enhances excitability of colonic AH
neurons by inhibiting calcium-depen-
dent potassium channel opening. J
Cell Mol Med 2009; 13: 2261–70.
53 Arseneault-Br
eard J, Rondeau I,
Gilbert K, Girard SA, Tompkins TA,
Godbout R, Rousseau G. Combina-
tion of Lactobacillus helveticus
R0052 and Bifidobacterium longum
R0175 reduces post-myocardial
infarction depression symptoms and
restores intestinal permeability in a
rat model. Br J Nutr 2012; 107:
1793–9.
© 2013 John Wiley & Sons Ltd