The gut-brain axis: Interactions between enteric microbiota, central and enteric nervous systems

Abstract

The gut-brain axis (GBA) consists of bidirectional communication between the central and the enteric nervous system, linking emotional and cognitive centers of the brain with peripheral intestinal functions. Recent advances in research have described the importance of gut microbiota in influencing these interactions. This interaction between microbiota and GBA appears to be bidirectional, namely through signaling from gut-microbiota to brain and from brain to gut-microbiota by means of neural, endocrine, immune, and humoral links. In this review we summarize the available evidence supporting the existence of these interactions, as well as the possible pathophysiological mechanisms involved. Most of the data have been acquired using technical strategies consisting in germ-free animal models, probiotics, antibiotics, and infection studies. In clinical practice, evidence of microbiota-GBA interactions comes from the association of dysbiosis with central nervous disorders (i.e. autism, anxiety-depressive behaviors) and functional gastrointestinal disorders. In particular, irritable bowel syndrome can be considered an example of the disruption of these complex relationships, and a better understanding of these alterations might provide new targeted therapies.

Full text

© 2015 Hellenic Society of Gastroenterology www.annalsgastro.gr
Annals of Gastroenterology (2015) 28, 203-209
REVIEW
The gut-brain axis: interactions between enteric microbiota,
central and enteric nervous systems
Marilia Carabotti
a
, Annunziata Scirocco
a
, Maria Antonietta Maselli
b
, Carola Severi
a
University Sapienza, Rome; S. De Bellis, Castellana Grotte, Bari, Italy
Introduction
Insights into the gut-brain crosstalk have revealed a complex
communication system that not only ensures the proper
maintenance of gastrointestinal homeostasis, but is likely to
have multiple eects on aect, motivation, and higher cognitive
functions. e complexity of these interactions is enclosed in
the denomination of “gut-brain axis” (GBA) [1]. Its role is to
monitor and integrate gut functions as well as to link emotional
and cognitive centers of the brain with peripheral intestinal
functions and mechanisms such as immune activation,
intestinal permeability, enteric reex, and entero-endocrine
signaling. e mechanisms underlying GBA communications
involve neuro-immuno-endocrine mediators.
is bidirectional communication network includes the
central nervous system (CNS), both brain and spinal cord,
the autonomic nervous system (ANS), the enteric nervous
system (ENS) and the hypothalamic pituitary adrenal (HPA)
axis (Fig.1). e autonomic system, with the sympathetic and
parasympathetic limbs, drives both aerent signals, arising from
the lumen and transmitted though enteric, spinal and vagal
pathways to CNS, and eerent signals from CNS to the intestinal
wall. e HPA axis is considered the core stress eerent axis that
coordinates the adaptive responses of the organism to stressors
of any kind [2]. It is a part of the limbic system, a crucial zone
of the brain predominantly involved in memory and emotional
responses. Environmental stress, as well as elevated systemic
pro-inammatory cytokines, activate this system that, through
secretion of the corticotropin-releasing factor (CRF) from
the hypothalamus, stimulates adrenocorticotropic hormone
(ACTH) secretion from pituitary gland that, in turn, leads
to cortisol release from the adrenal glands. Cortisol is a major
stress hormone that aects many human organs, including the
brain. us, both neural and hormonal lines of communication
combine to allow brain to inuence the activities of intestinal
functional eector cells, such as immune cells, epithelial cells,
enteric neurons, smooth muscle cells, interstitial cells of Cajal and
enterochroman cells. ese same cells, on the other hand, are
under the inuence of the gut microbiota [3] whose contributing
role in brain-gut reciprocal communications has recently been
assessed. e concept of a microbiome GBA is now emerging.
e enteric microbiota is distributed in the human
gastrointestinal tract and, although each person’s microbiota
prole is distinct, relative abundance and distribution along the
a
Department of Internal Medicine and Medical Specialties, University
Sapienza, Rome (Marilia Carabotti, Annunziata Scirocco, Carola
Severi);
b
Experimental Pharmacology Laboratory, Scientic Institute of
Gastroenterology S. de Bellis, Castellana Grotte, Bari (Maria Antonietta
Maselli), Italy
Conict of Interest: None
Correspondence to: Marilia Carabotti, Viale del Policlinico 155, 00161
Rome, Tel.: +390649 978376, Fax: +390644 63737,
e-mail: mcarabotti@yahoo.it
Received 5September 2014; accepted 7December 2014
e gut-brain axis (GBA) consists of bidirectional communication between the central and the
enteric nervous system, linking emotional and cognitive centers of the brain with peripheral intestinal
functions. Recent advances in research have described the importance of gut microbiota in inuencing
these interactions. is interaction between microbiota and GBA appears to be bidirectional, namely
through signaling from gut-microbiota to brain and from brain to gut-microbiota by means of
neural, endocrine, immune, and humoral links. In this review we summarize the available evidence
supporting the existence of these interactions, as well as the possible pathophysiological mechanisms
involved. Most of the data have been acquired using technical strategies consisting in germ-free animal
models, probiotics, antibiotics, and infection studies. In clinical practice, evidence of microbiota-
GBA interactions comes from the association of dysbiosis with central nervous disorders (i.e.autism,
anxiety-depressive behaviors) and functional gastrointestinal disorders. In particular, irritable bowel
syndrome can be considered an example of the disruption of these complex relationships, and a
better understanding of these alterations might provide new targeted therapies.
Keywords Gut-brain axis, enteric microbiota, central nervous system, enteric nervous system,
irritable bowel syndrome
Ann Gastroenterol 2015; 28 (2): 203-209
Abstract

204 M. Carabotti et al
Annals of Gastroenterology 28
intestine of these bacterial phylotypes is similar among healthy
individuals. e two more prominent phyla are Firmicutes and
Bacteroides accounting for at least ¾ of the microbiome[4].
is microbial community has important metabolic and
physiological functions for the host and contributes to its
homeostasis during life.
Role of microbiota in GBA
Both clinical and experimental evidence suggest that enteric
microbiota has an important impact on GBA, interacting not
only locally with intestinal cells and ENS, but also directly with
CNS through neuroendocrine and metabolic pathways.
In humans, the most compelling evidence of a
gastrointestinal microbe-brain interaction arose more than
20 years ago from the observation of the oen dramatic
improvement in patients with hepatic encephalopathy, aer the
administration of oral antibiotics [5]. In the meantime, emerging
data support the role of microbiota in inuencing anxiety and
depressive-like behaviors [6,7] and, more recently, of dysbiosis
in autism. In fact, autistic patients present specic microbiota
alterations according to the severity of the disease [8,9].
Dysbiosis occurs also in functional gastrointestinal disorders
(FGID) that are highly associated with mood disorders and
are linked to a disruption of GBA [10-12]. Data have been
provided that both brain-gut and gut-brain dysfunctions
occur, the former being dominant particularly in irritable
bowel syndrome (IBS) [13]. e disruption occurring in the
GBA determines changes in intestinal motility and secretion,
causes visceral hypersensitivity and leads to cellular alterations
of the entero-endocrine and immune system. Microbiota may
interplay with multiple of these dierent pathophysiological
IBS targets [14] and its role is supported by varying lines
of evidence: the presence in IBS patients of alterations in
microbiota composition with defects both in its stability and
diversity, the development of post-infectious IBS, the possible
coexistence with small intestinal bacterial overgrowth and the
ecacious treatment of certain probiotics and non-systemic
antibiotics [15-17]. Furthermore, the visceral hypersensitivity
phenotype, characteristic of IBS, can be transferred via the
microbiota of IBS patients to previously germ-free rats [18]. e
concomitant dysregulation of both GBA and gut microbiota in
the pathogenesis of IBS has lead to the proposal of considering
this FGID as a disorder of the microbioma-GBA [19].
From gut microbiota to brain
In the last years there has been a proliferation of
experimental works, conducted mainly on animals, aimed to
explore the contribution of the microbiota in modulating GBA.
Dierent technical strategies have been used, consisting in the
use of germ-free (GF) animals, probiotics, antibiotics and
infection studies [20].
Studies on GF animals have shown that bacterial
colonization of the gut is central to development and
maturation of both ENS and CNS [21,22]. e absence of
microbial colonization is associated to an altered expression
and turnover of neurotransmitters in both nervous
systems [21,23,24] and also to alterations of gut sensory-motor
functions, consisting in delayed gastric emptying and intestinal
transit [25,26] reduced migrating motor complex cyclic
recurrence and distal propagation [27,28] and enlarged cecal
size [29]. Neuromuscular abnormalities resulted associated
to a reduction in gene expression of enzymes involved in the
synthesis and transport of neurotransmitters, as well as in
that of muscular contractile proteins [30]. All these anomalies
are restored, aer animal colonization in a bacterial species-
specic manner.
(PRWLRQ
6WUHVV
$0* +,33
&)5
3,78,7$5<
$&7+
$GUHQDO
JODQGV
61$
DIIHUHQWV
61$
HIIHUHQWV
&257,62/
,17(67,1$/7$5*(76
027,/,7<
,0081,7<
3(50($%,/,7<
08&86
(16
086&/(
/$<(5
(3,7+(/,80
3$,102'8/$725(1'2*(12863$7+:$<6
(QWHULF
PLFWRELRWD
Figure1 Microbiome gut-brain axis structure
e central nervous system and in particular hypothalamic pituitary
adrenal (HPA) axis (in dashed line) can be activated in response to
environmental factors, such as emotion or stress. HPA is nalized to
cortisol release and is driven by a complex interaction between amygdala
(AMG), hippocampus (HIPP), and hypothalamus (HYP), constituting
the limbic system. HYP secretion of the corticotropin-releasing factor
(CRF) stimulates adrenocorticotropic hormone (ACTH) secretion from
pituitary gland that, in turn, leads to cortisol release from the adrenal
glands. In parallel, central nervous system communicate along both
aerent and eerent autonomic pathways (SNA) with dierent intestinal
targets such as enteric nervous system (ENS), muscle layers and gut
mucosa, modulating motility, immunity, permeability and secretion of
mucus. e enteric microbiota has a bidirectional communication with
these intestinal targets, modulating gastrointestinal functions and being
itself modulated by brain-gut interactions

Microbiota and gut-brain axis interactions 205
Annals of Gastroenterology 28
Studies conduced on GF animals have also demonstrated
that microbiota inuences stress reactivity and anxiety-like
behavior, and regulates the set point for HPA activity. ese
animals generally show a decreased anxiety [23,24,31-33] and
an increased stress response with augmented levels of ACTH
and cortisol [31,34]. Microbial colonization of the gut leads to
a normalization of the axis in an age-dependent manner, with
reversibility of the exaggerated stress response being observed
aer GF colonization only in very young mice, supporting the
existence of a critical period during which the plasticity of
neural regulation is sensitive to input from microbiota [34].
In parallel, in GF animals, also memory dysfunction
has been reported [35] probably to be ascribed to an altered
expression of brain-derived neurotrophic factor (BDNF),
one of the most important factors involved in memory. is
molecule is a neurotrophic factor, mainly located in the
hippocampus and cerebral cortex, which regulates dierent
aspects of brain activities and cognitive functions as well as
muscle repair, regeneration, and dierentiation [36]. Finally,
the presence of the microbiota results also to modulation of the
serotoninergic system, since an increase in serotonin turnover
and altered levels of related metabolites have been reported in
the limbic system of GF animals [24].
e impact of microbiota on GBA has been further
supported by studies nalized to the manipulation of gut
microbiota through the use of probiotics and/or antibiotics.
ese studies also conrm that microbiota aects anxiety
and HPA system by inuencing brain neurochemistry [37].
Chronic treatment with Lactobacillus rhamnosus JB-1 induced
region-dependent alterations in GABA mRNA in the brain.
In comparison to mice with controlled diet, GABA
B1b
increased in cortical cingulate and prelimbic regions while
concomitantly decreased in the hippocampus, amygdala, and
locus coeruleus. In turn GABA
Aα2
mRNA expression was
reduced in the prefrontal cortex and amygdala, but increased
in the hippocampus. e probiotics, in parallel, reduced stress-
induced release of cortisol, anxiety- and depression-related
behavior [38]. Similarly, transient alteration of microbiota
composition, obtained by administration of oral antimicrobials
(neomycin, bacitracin, and pimaricin) in specic-pathogen-
free mice, increased exploratory behavior and hippocampal
expression of BDNF [39]. Furthermore, change in microbiota
composition with the probiotics association VSL#3 leads to
an increase in BDNF expression, attenuation of age-related
alterations in the hippocampus [40], and reversion of neonatal
maternal separation-induced visceral hypersensitivity in a rat
model of IBS [41]. In this latter model of stress, a change in the
expression of subsets of genes involved in pain transmission
and inammation has also been described, that was reset by the
early life administration of probiotics.
Evidence indicates that microbiota communication with the
brain involves the vagus nerve, which transmits information
from the luminal environment to CNS. In fact, neurochemical
and behavioral eects were not present in vagotomized mice,
identifying the vagus as the major modulatory constitutive
communication pathway between microbiota and the
brain [38]. In a model of chronic colitis associated to anxiety-
like behavior, the anxiolytic eect obtained with a treatment
with Bidobacterium longum, was absent in mice that were
vagotomized before the induction of colitis [42].
Microbiota may interact with GBA through dierent
mechanisms (Table 1), the principal one likely being
modulation of the intestinal barrier, whose perturbation can
inuence all the underlying compartments. Probiotic species-
specic central eects are indeed associated with restoration
of tight-junction integrity and the protection of intestinal
barrier, as recently reported in an animal model of water
avoidance stress [43]. Pre-treatment of animals with probiotic
combined formulation of Lactobacillus helveticus R0052
and Bidobacterium longum R0175 restored tight junction
barrier integrity and attenuated HPA axis and autonomic
nervous system activities, assessed through plasma cortisol
and catecholamine measurements. Probiotics also prevented
changes in hippocampal neurogenesis and expression in
hypothalamic genes involved in synaptic plasticity.
Microbiota can interact with GBA also through the
modulation of aerent sensory nerves as reported for
Lactobacillus reuteri that, enhancing their excitability by
inhibiting calcium-dependent potassium channels opening,
modulates gut motility and pain perception [44]. Furthermore,
microbiota can inuence ENS activity by producing molecules
that can act as local neurotransmitters, such as GABA,
serotonin, melatonin, histamine and acetylcholine [45] and by
generating a biologically active form of catecholamines in the
lumen of the gut [46]. Lactobacilli also utilize nitrate and nitrite
to generate nitric oxide [47] and to produce hydrogen sulde
that modulates gut motility by interacting with the vanilloid
receptor on capsaicin-sensitive nerve bers [48].
e ENS represents also the target of bacterial metabolites.
One of the main product of bacterial metabolism are short-chain
fatty acid (SCFAs), such as butyric acid, propionic acid and acetic
acid, that are able to stimulate sympathetic nervous system [49],
mucosal serotonin release [50] and to inuence memory and
learning process [51,52]. In this context, it is interesting to report
that diet manipulation of microbiota may inuence behavior.
Mice fed with a diet containing 50% lean ground beef, have a
greater diversity of gut bacteria than those receiving standard
rodent chow, and presented an increase physical activity,
reference memory and less anxiety-like behavior [53].
Given the ability of gut microbiota to alter nutrient
availability and the close relationship between nutrient sensing
Table 1 Main principal mechanisms of the bidirectional brain-gut-
microbiota axis
From gut microbiota to brain:
Production, expression and turnover of neurotrasmitters
(i.e. serotonin, GABA) and neurotrophic factor (BDNF)
Protection of intestinal barrier and tight junction integrity
Modulation of enteric sensory aerents
Bacterial metabolites
Mucosal immune regulation
From brain to gut microbiota:
Alteration in mucus and biolm production
Alteration in motility
Alteration of intestinal permeability
Alteration in immune function

206 M. Carabotti et al
Annals of Gastroenterology 28
and peptide secretion by enteroendocrine cells, the interaction
of microbiota and GBA might also occur through the release
of biologically active peptides from enteroendocrine cells that
can aect the GBA [54]. For example, galanin stimulates the
activity of the central branch of the HPA axis (i.e.the release of
CRF and ACTH), thereby enhancing glucocorticoid secretion
from the adrenal cortex. Galanin also is able to stimulate
directly cortisol secretion from adrenocortical cells, and
norepinephrine release from adrenal medulla [55]. Ghrelin too
possesses a marked ACTH/cortisol-releasing eect in humans
and it is probably involved in the modulation of the HPA
response to stress and nutritional/metabolic variations [56].
Last but not least, microbiota aects mucosal immune
activation. e enhanced mucosal inammation induced
in mice aer treatment with oral antimicrobials, increases
substance P expression in ENS, an eect normalized by the
administration of Lactobacillus paracasei which also attenuates
antibiotic-induced visceral hypersensitivity [57]. e eects of
microbiota on immune activation might be in part mediated
by proteases. ese enzymes are upregulated in intestinal-
immune mediated disorders and become the end-stage
eectors of mucosal and enteric nervous damage [58-59].
Increased concentration of proteases have been detected in
fecal samples of IBS patients associated to specic intestinal
bacterial species [60,61]. e current working hypothesis in
IBS is that an abnormal microbiota activates mucosal innate
immune responses, which increase epithelial permeability,
activate nociceptive sensory pathways inducing visceral pain,
and dysregulates the enteric nervous system [62,63].
Similar mechanisms may be involved in the eects
induced by the gastric mucosa-colonizing microorganism,
Helicobacter pylori (H. pylori) on the GBA. e eects induced
by this microorganism may arise through both activation
of neurogenic inammatory processes and microelements
deciency secondary to functional and morphological
changes in the digestive tract [64]. Nevertheless, unequivocal
data concerning the direct and immediate eects of H.pylori
infection on the GBA are still lacking, and in clinical
practice the relationship between functional dyspepsia and
H. pylori infection is not well dened. In fact, the number
needed to treat to cure one case of dyspepsia is 14(95%CI
10-25 [65] suggesting a multifactorial etiology for the increase
in H.pylori-related upper FGID.
From brain to gut microbiota
Dierent types of psychological stressors modulate the
composition and total biomass of the enteric microbiota,
independently from duration. In fact, also the use of short
stressors impact the microbiota, being the exposure to
social stressor for only 2 h signicantly able to change the
community prole and to reduce the relative proportions of
the main microbiota phyla [66]. ese eects may be mediated,
through the parallel neuroendocrine output eerent systems
(i.e. autonomic nervous system and HPA), both directly via
host-enteric microbiota signaling and indirectly via changes in
the intestinal milieu (Table1). ese eerent neural pathways,
associated to the pain-modulator endogenous pathways,
constitute the so-called “emotional motor system” [1].
e direct inuence is mediated by the secretion, under
the regulation of brain, of signaling molecules by neurons,
immune cells and enterocroman cells, which might aect
microbiota. Communication between CNS eectors and
bacteria relies on the presence of neurotransmitter receptors
on bacteria. Several studies have reported that binding sites
for enteric neurotransmitters produced by the host are present
on bacteria and can inuence the function of components
of the microbiota, contributing to increase predisposition
to inammatory and infection stimuli [67]. High anity for
GABA system has been reported in Pseudomonas uorescens
with binding properties similar to those of a brain receptor [68].
Escherichia coli O157:H7 possesses a receptor for host-derived
epinephrine/norepinephrine that can be blocked specically
by adrenergic antagonists [69].
Besides, brain has a prominent role in the modulation of
gut functions, such as motility, secretion of acid, bicarbonates
and mucus, intestinal uid handling and mucosal immune
response, all important for the maintenance of the mucus
layer and biolm where individual groups of bacteria grow in
a multiplicity of dierent microhabitats and metabolic niches
associated with the mucosa [70]. Adysregulation of GBA can
then aect gut microbiota through the perturbation of the
normal mucosal habitat.
Stress induces variation in size and quality of mucus
secretion [71]. Acoustic stress aects gastric and intestinal
postprandial motility in dogs, delaying the recovery of the
migrating motor complex pattern and inducing a transient
slowing of gastric emptying [72]. Mental stress too increases
the frequency of cecocolonic spike-burst activity through
the central release of CRF [73]. Regional and global changes
in gastrointestinal transit can have profound eects on the
delivery of important nutrients, mainly prebiotics and dietary
bers, to the enteric microbiota.
Brain might also aect microbiota composition and
function by alteration of intestinal permeability, allowing
bacterial antigens to penetrate the epithelium and stimulate
an immune response in the mucosa. Acute stress increased
colonic paracellular permeability involving overproduction of
interferon-γ and decrease in mRNA expression of ZO-2 and
occluding [74]. Brain, through the ANS, may also modulate
immune function. e sympathetic branch modulates number,
degranulation and activity of mast cells with consequent
imbalance in tryptase and histamine release in stress-related
muscle dysfunction [75]. Other mast cell products, such as
CRF, in turn, can increase epithelial permeability to bacteria,
which facilitates their access to immune cells in the lamina
propria [1]. Also corticotropin releasing hormone receptors
are involved in colonic barrier dysfunction in response to mild
stress in neonatal maternal separation in adult rats that [76]
leads to depression and enhanced vulnerability to colitis [77].
Bilateral olfactory bulbectomy induced depression-like
behavior associated to elevated central CRF expression and
serotonin levels, associated to alterations in colonic motility
and intestinal microbial prole in mice [78]. Another possible
perturbation in the microbiota habitat induced by stress

Microbiota and gut-brain axis interactions 207
Annals of Gastroenterology 28
occurs through the enhancement in secretion of α-defensin,
an antimicrobial peptide, from Paneth cells [79].
Finally, it is important to remark that gut alterations
associated to stress facilitate the expression of virulent
bacteria. Norepinephrine released during surgery induces the
expression of Pseudomonas aeruginosa, which might result
in gut sepsis [80]. Besides, norepinephrine can also stimulate
proliferation of several strains of enteric pathogens and increase
the virulent properties of Campylobacter jejuni [81] and might
favor overgrowth of non-pathogenic isolates of Escherichia coli,
as well as of pathogenic Escherichia coli 0157:H7:3 [82,83].
Concluding remarks
Strong evidence suggests that gut microbiota has an important
role in bidirectional interactions between the gut and the nervous
system. It interacts with CNS by regulating brain chemistry and
inuencing neuro-endocrine systems associated with stress
response, anxiety and memory function. Many of these eects
appear to be strain-specic, suggesting a potential role of certain
probiotic strains as novel adjuvant strategy for neurologic
disorders. In addition, the eects of CNS on microbiota
composition are likely mediated by a perturbation of the normal
luminal/mucosal habitat that can also be restored by the use of
probiotics and possibly by diet. In clinical practice, an example
of this interaction is constituted by FGID, in particular IBS, now
considered a microbiome-GBA disorder.
Acknowledgment
e authors kindly thank Dr Laura Carabotti for the
artwork of the gures.
References
1. Rhee SH, Pothoulakis C, Mayer EA. Principles and clinical
implications of the brain-gut-enteric microbiota axis. Nat Rev
Gastroenterol Hepatol 2009;6:306-314.
2. Tsigos C, Chrousos GP. Hypothalamic-pituitary-adrenal axis,
neuroendocrine factors and stress. J Psychosom Res 2002;53:865-871.
3. Mayer EA, Savidge T, Shulman RJ. Brain-gut microbiome
interactions and functional bowel disorders. Gastroenterology
2014;146:1500-1512.
4. Eckburg PB, Bik EM, Bernstein CN, et al. Diversity of the human
intestinal microbial ora. Science 2005;308:1635-1638.
5. Morgan MY. e treatment of chronic hepatic encephalopathy.
Hepatogastroenterology 1991;38:377-387.
6. Foster JA, McVey Neufeld KA. Gut-brain axis: how the
microbiome inuences anxiety and depression. Trends Neurosci
2013;36:305-312.
7. Naseribafrouei A, Hestad K, Avershina E, et al. Correlation between
the human fecal microbiota and depression. Neurogastroenterol
Motil 2014;26:1155-1162.
8. Mayer EA, Padua D, Tillisch K. Altered brain-gut axis in autism:
comorbidity or causative mechanisms? Bioessays 2014;36:933-999.
9. Song Y, Liu C, Finegold SM. Real-time PCR quantitation of
clostridia in feces of autistic children. Appl Environ Microbiol
2004;70:6459-6465.
10. Simrén M, Barbara G, Flint HJ, et al. Rome Foundation Committee.
Intestinal microbiota in functional bowel disorders: a Rome
foundation report. Gut 2013;62:159-176.
11. Mayer EA, Tillisch K. e brain-gut axis in abdominal pain
syndromes. Annu Rev Med 2011;62:381-396.
12. Berrill JW, Gallacher J, Hood K, et al. An observational study
of cognitive function in patients with irritable bowel syndrome
and inammatory bowel disease. Neurogastroenterol Motil
2013;25:918-e704.
13. Koloski NA, Jones M, Kalantar J, Weltman M, Zaguirre J, TalleyNJ.
e brain-gut pathway in functional gastrointestinal disorders is
bidirectional: a 12-year prospective population-based study. Gut
2012;61:1284-1290.
14. Dupont HL. Review article: evidence for the role of gut microbiota
in irritable bowel syndrome and its potential inuence on
therapeutic targets. Aliment Pharmacol er 2014;39:1033-1042.
15. Spiller R, Lam C. An Update on Post-infectious Irritable Bowel
Syndrome: Role of Genetics, Immune Activation, Sero-tonin and
Altered Microbiome. JNeurogastroenterol Motil 2012;18:258-268.
16. Quigley EM. Small intestinal bacterial overgrowth: what it 77 is
and what it is not. Curr Opin Gastroenterol 2014;30:141-146.
17. Pimentel M, Lembo A, Chey WD, et al; TARGET Study Group.
Rifaximin therapy for patients with irritable bowel syndrome
without constipation. NEngl J Med 2011;364:22-32.
18. Crouzet L, Gaultier E, Del’Homme C, et al. e hypersensitivity to
colonic distension of IBS patients can be transferred to rats through
their fecal microbiota. Neurogastroenterol Motil 2013;25:e272-e282.
19. Kennedy PJ, Cryan JF, Dinan TG, Clarke G. Irritable bowel
syndrome: A microbiome-gut-brain axis disorder? World J
Gastroenterol 2014;20:14105-14125.
20. Bravo JA, Julio-Pieper M, Forsythe P, et al. Communication
between gastrointestinal bacteria and the nervous system. Curr
Opin Pharmacol 2012;12:667-672.
21. Barbara G, Stanghellini V, Brandi G, et al. Interactions between
commensal bacteria and gut sensorimotor function in health and
disease. Am J Gastroenterol 2005;100:2560-2568.
22. Stilling RM, Dinan TG, Cryan JF. Microbial genes, brain and
behaviour-epigenetic regulation of the gut-brain axis. Genes Brain
Behav 2014;13:69-86.
23. Clarke G, Grenham S, Scully P, et al. e microbiome-gut-brain axis
during early life regulates the hippocampal serotonergic system in a
sex-dependent manner. Mol Psychiatry 2013;18:666-673.
24. Diaz Heijtz R, Wang S, Anuar F, et al. Normal gut microbiota
modulates brain development and Behavior. Proc Natl Acad Sci
USA 2011;108:3047-3052.
25. Abrams GD, Bishop JE. Eect of the normal microbial ora on
gastrointestinal motility. Proc Soc Exp Biol Med 1967;126:301-304.
26. Iwai H, Ishihara Y, Yamanaka J, Ito T. Eects of bacterial ora on
cecal size and transit rate of intestinal contents in mice. Jpn J Exp
Med 1973;43:297-305.
27. Caenepeel P, Janssens J, Vantrappen G, Eyssen H, Coremans G.
Interdigestive myoelectric complex in germ-free rats. Dig Dis Sci
1989;34:1180-1184.
28. Husebye E, Hellström PM, Sundler F, Chen J, Midtvedt T. Inuence
of microbial species on small intestinal myoelectric activity and
transit in germ-free rats. Am J Physiol Gastrointest Liver Physiol
2001;280:G368-G380.
29. Wostmann E, Bruckner-Kardoss E. Development of cecal distention
in germ-free baby rats. Am J Physiol 1959;197:1345-1346.
30. Hooper LV, Wong MH, elin A, Hansson L, Falk PG, Gordon JI.
Molecular analysis of commensal host-microbial relationships in
the intestine. Science 2001;291:881-884.
31. Neufeld KM, Kang N, Bienenstock J, Foster JA. Reduced anxiety-

208 M. Carabotti et al
Annals of Gastroenterology 28
like behavior and central neurochemical change in germ-free mice.
Neurogastroenterol Motil 2011;23:255-264.
32. Neufeld KA, Kang N, Bienenstock J, Foster JA. Eects of intestinal
microbiota on anxiety-like behavior. Commun Integr Biol
2011;4:492-494.
33. Nishino, K. Mikami, H. Takahashi, et al. Commensal microbiota
modulate murine behaviors in a strictly contamination-
free environment conrmed by culture-based methods.
Neurogastroenterol Motil 2013;25:521-528.
34. Sudo N, Chida Y, Aiba Y, et al. Postnatal microbial colonization
programs the hypothalamic-pituitary-adrenal system for stress
response in mice. JPhysiol 2004;558:263-275.
35. Gareau MG, Wine E, Rodrigues DM, et al. Bacterial infection causes
stress-induced memory dysfunction in mice. Gut 2011;60:307-317.
36. Al-Qudah M, Anderson CD, Mahavadi S, et al. Brain-derived
neurotrophic factor enhances cholinergic contraction of longitudinal
muscle of rabbit intestine via activation of phospholipase C. Am J
Physiol Gastrointest Liver Physiol 2014;306:G328-G337.
37. Saulnier DM, Ringel Y, Heyman MB, et al. e intestinal
microbiome, probiotics and prebiotics in neurogastroenterology.
Gut Microbes 2013;4:17-27.
38. Bravo JA, Forsythe P, Chew MV, et al. Ingestion of Lactobacillus
strain regulates emotional behavior and central GABA receptor
expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA
2011;108:16050-16055.
39. Bercik P, Denou E, Collins J, et al. e intestinal microbiota aect
central levels of brain-derived neurotropic factor and behavior in
mice. Gastroenterology 2011;141:599-609.
40. Distrutti E, O’Reilly JA, McDonald C, et al. Modulation of
intestinal microbiota by the probiotic VSL#3 resets brain gene
expression and ameliorates the age-related decit in LTP. PLoS One
2014;9:e106503.
41. Distrutti E, Cipriani S, Mencarelli A, Renga B, Fiorucci S. Probiotics
VSL#3 protect against development of visceral pain in murine
model of irritable bowel syndrome. PLoS One 2013;8:e63893.
42. Bercik P, Park AJ, Sinclair D, et al. e anxiolytic eect of
Bidobacterium longum NCC3001 involves vagal pathways
for gut-brain Communication. Neurogastroenterol Motil
2011;23:1132-1139.
43. Ait-Belgnaoui A, Colom A, Braniste V, et al. Probiotic gut eect
prevents the chronic psychological stress-induced brain activity
abnormality in mice. Neurogastroenterol Motil 2014;26:510-520.
44. Kunze WA, Mao YK, Wang B, et al. Lactobacillus reuteri enhances
excitability of colonic AH neurons by inhibiting calcium-dependent
potassium channel opening. JCell Mol Med 2009;13:2261-2270.
45. Iyer LM, Aravind L, Coon SL, Klein DC, Koonin EV. Evolution of
cell-cell signaling in animals: did late horizontal gene transfer from
bacteria have a role? Trends Genet 2004;20:292-299.
46. Asano Y, Hiramoto T, Nishino R, et al. Critical role of gut microbiota
in the production of biologically active, free catecholamines in
the gut lumen of mice. Am J Physiol Gastrointest Liver Physiol
2012;303:G1288-G1295.
47. Sobko T, Huang L, Midtvedt T, et al. Generation of NO by
probiotic bacteria in the gastrointestinal Tract. Free Radic Biol Med
2006;41:985-991.
48. Schicho R, Krueger D, Zeller F, et al. Hydrogen sulde is a novel
prosecretory neuromodulator in the Guinea-pig and human colon.
Gastroenterology 2006;131:1542-1552.
49. Kimura I, Inoue D, Maeda T, et al. Short-chain fatty acids and
ketones directly regulate sympathetic nervous system via G
protein-coupled receptor 41 (GPR41). Proc Natl Acad Sci U S A
2011;108:8030-8035.
50. Grider JR, Piland BE. e peristaltic reex induced by short-chain
fatty acids is mediated by sequential release of 5-HT and neuronal
CGRP but not BDNF. Am J Physiol Gastrointest Liver Physiol
2007;292:G429-G437.
51. Vecsey CG, Hawk JD, Lattal KM, et al. Histone deacetylase
inhibitors enhance memory and synaptic plasticity via
CREB: CBP-dependent transcriptional activation. J Neurosci
2007;27:6128-6140.
52. Stefanko DP, Barrett RM, Ly AR, Reolon GK, Wood MA.
Modulation of long-term memory for object recognition via
HDAC inhibition. Proc Natl Acad Sci U S A 2009;106:9447-9452.
53. Li W, Dowd SE, Scurlock B, Acosta-Martinez V, Lyte M. Memory
and learning behavior in mice is temporally associated with diet-
induced alterations in gut bacteria. Physiol Behav 2009;96:557-567.
54. Uribe A, Alam M, Johansson O, Midtvedt T, eodorsson E.
Microora modulates endocrine cells in the gastrointestinal
mucosa of the rat. Gastroenterology 1994;107:1259-1269.
55. Tortorella C, Neri G, Nussdorfer GG. Galanin in the regulation of
the hypothalamic-pituitary-adrenal axis (Review). Int J Mol Med
2007;19:639-647.
56. Giordano R, Pellegrino M, Picu A, et al. Neuroregulation of the
hypothalamus-pituitary-adrenal (HPA) axis in humans: eects
of GABA-, mineralocorticoid-, and GH-Secretagogue-receptor
modulation. Sci World J 2006;17:1-11.
57. Verdú EF, Bercik P, Verma-Gandhu M, et al. Specic probiotic
therapy attenuates antibiotic induced visceral hypersensitivity in
mice. Gut 2006;55:182-190.
58. Saito T, Bunnett NW. Protease-activated receptors: regulation of
neuronal function. Neuromolecular Med 2005;7:79-99.
59. Biancheri P, Di Sabatino A, Corazza GR, MacDonald TT. Proteases
and the gut barrier. Cell Tissue Res 2013;351:269-280.
60. Gecse K, Róka R, Ferrier L, et al. Increased faecal serine protease
activity in diarrhoeic IBS patients: a colonic lumenal factor impairing
colonic permeability and sensitivity. Gut 2008;57:591-599.
61. Carroll IM, Ringel-Kulka T, Ferrier L, et al. Fecal protease activity
is associated with compositional alterations in the intestinal
microbiota. PLoS One 2013;8:e78017.
62. Collins SM, Bercik P. e relationship between intestinal microbiota
and the central nervous system in normal gastrointestinal function
and disease. Gastroenterology 2009;136:2003-2014.
63. eodorou V, Ait Belgnaoui A, Agostini S, Eutamene H. Eect
of commensals and probiotics on visceral sensitivity and pain in
irritable bowel syndrome. Gut Microbes 2014;5:430-436.
64. Budzyński J, Kłopocka M. Brain-gut axis in the pathogenesis
of Helicobacter pylori infection. World J Gastroenterol
2014;20:5212-5225.
65. Moayyedi P, Soo S, Deeks J, et al. Eradication of Helicobacter
pylori for non-ulcerdyspepsia. Cochrane Database Syst Rev
2006Apr19;(2):CD002096. Review. Update in: Cochrane Database
Syst Rev 2011;(2):CD002096.
66. Galley JD, Nelson MC, Yu Z, et al. Exposure to a social stressor
disrupts the community structure of the colonic mucosa-associated
microbiota. BMC Microbiol 2014;14:189.
67. Hughes DT, Sperandio V. Inter-kingdom signalling: communication
between bacteria and their hosts. Nat Rev Microbiol 2008;6:111-120.
68. Guthrie GD, Nicholson-Guthrie CS. Gamma-Aminobutyric
acid uptake by a bacterial system with neurotransmitter binding
characteristics. Proc Natl Acad Sci U S A 1989;86:7378-7381.
69. Clarke MB, Hughes DT, Zhu C, Boedeker EC, Sperandio V. e
QseC sensor kinase: a bacterial adrenergic receptor. Proc Natl Acad
Sci U S A 2006;103:10420-10425.
70. Macfarlane S, Dillon JF. Microbial biolms in the human
gastrointestinal Tract. J Appl Microbiol 2007;102:1187-1196.
71. Rubio CA, Huang CB. Quantication of the sulphomucin-
producing cell population of the colonic mucosa during protracted
stress in rats. In Vivo 1992;6:81-84.
72. Gué M, Peeters T, Depoortere I, Vantrappen G, Buéno L. Stress-
induced changes in gastric emptying, postprandial motility,
and plasma gut hormone levels in dogs. Gastroenterology
1989;97:1101-1107.

Microbiota and gut-brain axis interactions 209
Annals of Gastroenterology 28
73. Gué M, Junien JL, Bueno L. Conditioned emotional response
in rats enhances colonic motility through the central release of
corticotropin-releasing factor. Gastroenterology 1991;100:964-970.
74. Demaude J, Salvador-Cartier C, Fioramonti J, Ferrier L, BuenoL.
Phenotypic changes in colonocytes following acute stress or
activation of mast cells in mice: implications for delayed epithelial
barrier dysfunction. Gut 2006;55:655-661.
75. Santos J, Saperas E, Nogueiras C, et al. Release of mast cell mediators
into the jejunum by cold pain stress in humans. Gastroenterology
1998;114:640-648.
76. Söderholm JD, Yates DA, Gareau MG, Yang PC, MacQueen G,
Perdue MH. Neonatal maternal separation predisposes adult rats
to colonic barrier dysfunction in response to mild stress. Am J
Physiol Gastrointest Liver Physiol 2002;283:G1257-G1263.
77. Varghese AK, Verdú EF, Bercik P, et al. Antidepressants attenuate
increased susceptibility to colitis in a murine model of depression.
Gastroenterology 2006;130:1743-1753.
78. Park AJ, Collins J, Blennerhassett PA, et al. Altered colonic function
and microbiota prole in a mouse model of chronic depression.
Neurogastroenterol Motil 2013;25:733-e575.
79. Alonso C, Guilarte M, Vicario M, et al. Maladaptive intestinal
epithelial responses to life stress may predispose healthy women to
gut mucosal inammation. Gastroenterology 2008;135:163-172.e1.
80. Alverdy J, Holbrook C, Rocha F, et al. Gut-derived sepsis occurs
when the right pathogen with the right virulence genes meets
the right host: evidence for in vivo virulence expression in
Pseudomonas aeruginosa. Ann Surg 2000;232:480-489.
81. Cogan TA, omas AO, Rees LE, et al. Norepinephrine increases the
pathogenic potential of Campylobacter jejuni. Gut 2007;56:1060-
1065.
82. Freestone PP, Williams PH, Haigh RD, Maggs AF, Neal CP, LyteM.
Growth stimulation of intestinal commensal Escherichia coli by
catecholamines: a possible contributory factor in trauma-induced
sepsis. Shock 2002;18:465-470.
83. Freestone PP, Haigh RD, Williams PH, Lyte M. Involvement
of enterobactin in norepinephrine-mediated iron supply from
transferrin to enterohaemorrhagic Escherichia coli. FEMS
Microbiol Lett 2003;222:39-43.