Bacterial colonisation of the intestine has a major role in the post-natal development and maturation of the immune and endocrine systems. These processes are key factors underpinning central nervous system (CNS) signaling. Regulation of the microbiota-gut-brain axis is essential for maintaining homeostasis, including that of the CNS. Moreover, there is now expanding evidence for the view that commensal organisms within the gut play a role in early programming and later responsivity of the stress system. Research has focused on how the microbiota communicates with the central nervous system (CNS) and thereby influences brain function. The routes of this communication are not fully elucidated but include neural, humoral, immune and metabolic pathways. This view is underpinned by studies in germ-free animals and in animals exposed to pathogenic bacterial infections, probiotic agents or antibiotic agents which indicate a role for the gut microbiota in the regulation of mood, cognition, pain and obesity. Thus the concept of a microbiota-gut brain axis is emerging which suggests that modulation of the gut microflora may be a tractable strategy for developing novel therapeutics for complex stress-related CNS disorders where there is a huge unmet medical need.

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John F. Cryan Ph.D.
Professor & Chair
Department of Anatomy & Neuroscience
&
Principal Investigator,
Alimentary Pharmabiotic Centre
University College Cork
Ireland
Stress and the Brain-Gut-Microbiota Axis

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NeuroAids
A U.S. Army Educational Commission Poster
about Neurosypilis, 1918
Interfacing Microbiology and Neuroscience

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Brain & Body
Closely Interact
Time, Jan 2003
The Brain-Gut Axis

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Stressors

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Cryan et al., Trends in Pharmacol. Sci. 2002
The Rodent Psychiatrist: Behavioural Neuroscientist??
Gary Larson

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Human vs. Mouse Brain
Cryan and Holmes, Nature Rev Drug Discov. Sept 2005

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Altered Stress Axis

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Stress Alters Brain-Gut Axis

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Lupien et al., Nat Rev Neuroscience
Romanian Orphans

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“It’s not stress that kills us, it is our reaction to it”.
Hans Selye

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Irritable Bowel Syndrome (IBS) a disorder of
Brain-Gut Axis dysfunction
 Symptom complex including abdominal pain, altered bowel
function, bloating, mucosal inflammation, exaggerated stress
response, increases in plasma pro-inflammatory cytokines
 IBS affects approximately 10-20% of the general population
 It is the most common disease diagnosed by gastroenterologists
 Large co-morbidity with anxiety & depression
 Current therapies are frequently ineffective
 Visceral Pain is a key symptoms of IBS
 Stress (including early life stress) plays a major role in the onset
and exacerbation of symptoms in IBS
 Now viewed as a disorder of the Brain-Gut axis
Gastroenterologists: Gut disorder
Psychiatrist: Stress-related disorder
Neurobiologist-Neurologist: Chronic pain disorder

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Maternal Separation model of IBS
 Plotsky, Meaney and colleagues have long used
MS as an animal model of depression
 Neonatal psychosocial stress model where pups
are removed from the home cage for 3 hr/day
from pn day 2-12
Results in:
 Enhanced stress response (O’Mahony et al,
2006)
 Alterations in the serotonin system e.g.
alterations in pain modulating system, increase
in serotonin and its metabolite (O’Mahony et al.,
2008)
 Increased colonic transit, intestinal permeability
(Soderholm et al., 2002), mucosal
inflammation, altered microflora
O’Mahony et al. Psychopharmacology (2011)

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Open Field Endocrine
Group Mean Similarity
%
SEM
Non Separated 75.2 16.8
Maternally
Separated
59.9 * 21.0
Increased colonic transit, intestinal
permeability, mucosal inflammation,
colonic morphology
(O’Malley et al., 2010, Stress)
Diversity of Microbiota
Immune

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Maternal Separation results in visceral
hypersensitivity
O’Mahony et al., Biol. Psychiat 2008 In Press
O’Mahoney et al., 2009
Fasting CRD
T=0 T=24hr
0
80mmHg
8 mins

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Physiological Indicators of Chronic Social
Defeat and Overcrowding Stress
Chronic intermittent psychosocial stress (social defeat/overcrowding) for 19 days (Reber et al., 2006)
Tramullas et al., Stress 2012

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Repeated Social Interaction Stress
 Social stress can disrupt the brain-gut axis, inducing gut and psychiatric diseases;
 It is one of the first causes of stress in humans;
 Stronger immune reaction than other types of stress can be induced (Sheridan & al, 2000)
Chronic:6 days
++
Back alone home cage
2 Hours + 2 Hours
Killing
Chronic:6 days
++
Back alone home cage
2 Hours + 2 Hours
Killing
Male BALB/c
Home cage
Male C57BL/6
Agressive
intruder
Or acute
Sampling

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Repeated Social Interaction Stress
•Mice displayed 2 opposing
behavioural and physiological
subtypes after both stress
exposure, vs controls:
•Dominant/aggressive = lower
corticosterone and pro-
inflammatory cytokines levels
(IL-1β, IL-6 and mKC)
•Submissive/defensive = high
corticosterone and cytokine
levels
•Chronically stressed mice =
weight loss and colonic
microscopic damage increased
Corticosterone
0
50000
100000
150000
200000
*
Control Acute Dominant Submissive
Chronic
+
Concentrationpg/ml
Number of minor wounds a
potential confound
Savignac et al., Behav Brain Res 2010

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The Brain-Gut Axis
Spinal Cord
Gosselin et al. Trends Immunol,
under review

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Targeting Spinal Glial Glutamate Transport Reverses Early-
Life Stress-induced Visceral Hypersensitivity
Intrathecal
administration
of a glutamate
transport
blocker TBOA
produces
visceral
hypersensitivity
Glutamate uptake
activator riluzole
reduces maternal
separation-induced
visceral
hypersensitivity
Gosselin et al Gastroenterology June 2010
Maternal separation (MS) results in a selective reduction of the
expression of spinal glial glutamate transporter EAAT-1

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LPS-induced visceral hypersenitivity
O’Mahony and Cryan unpublished

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Toll-Like Receptors-
A role in stress regulation of brain-gut axis

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TLR’s and IBS
TLR4 (LPS Stimulation) TLR8 (ssRNA40 Stimulation)

26. apc.ucc.ieMcKernan et al., 2009 PLOS One
Increased TLR mRNA Expression in Distal Colon
of Two Models of Visceral Hypersensitivity
Maternally Separated Rat
Wistar Kyoto Rat

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TLR4 is essential for chronic psychosocial
stress-induced visceral hypersensitivity
Tramullas and Cryan unpublished
TLR4 antagonist
attenuates basal and
stress-induced
visceral hyperalgesia

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Gut Microbiome
• The human intestinal microbiota is composed of 1013
to 1014 microorganisms whose collective genome
("microbiome") contains at least 100 times as many
genes as our own genome
(Gill, S. R., et al(2006).Science 312, 1355-1359.)
An individuals flora generally remains constant,
however, the composition can fluctuate during
acute diarrhoeal illness, antibiotic treatments and
by diet changes.

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, ...
Malene et al., Early Human Development Volume 88, Supplement 1 2012 S41 - S49
Bacterial Colonization and Gut Development in Neonates

30. apc.ucc.ieCryan and Dinan, Nat Rev Neurosci Oct 2012
Signaling Along the Brain-Gut-Microbiota axis
Pain

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Cryan and Dinan, Nat Rev Neurosci Oct 2012
Strategies used to investigate the role of the
microbiota–gut–brain axis in health and disease.

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X
Germ Free Mice:
Understand impact of microbiota on brain function

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Germ Free Mice:
Understand impact of microbiota on brain function

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Decreased BDNF and Exaggerated Stress
Response
 BDNF is a neurotrophin
supporting neuronal
survival/growth
 Decreased BDNF in germ
free compared to control
animals
 Altered stress response
Corticosterone
0
50,000
100,000
150,000
200,000
250,000
Control
Germ Free
Baseline Stress
**
pg/ml
Clarke et al., Mol Psychiat 2012

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Microbiota controls visceral hypersensitivity
Tramullas et al., unpublished

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Does early-life antibiotic administration
affect behaviour in adulthood
O’Mahony et al., Submitted
Vancomycin 10-100 mg/kg
administered from postnatal
day 4 for 10 days in rats
No change in
• Cognition
• Anxiety
• Stress Response
• Immune Response

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Could potential probiotic administration in adulthood
affect stress-related anxiety and depression?
“PSYCHOBIOTICS”
Biol Psychiat 2013 In Press

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Probiotic Reduces Anxiety and Behavioural Despair
Probiotic Reduces
Stress-induced
Corticosterone Levels
Bravo et al., PNAS Sept 2011

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Lactobacillus Increases alter Cortical, Amygdalar
and Hippocampal GABAA Receptor Subunits

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Behavioural and Neurochemical effects of Probiotic are
abolished in Vagotomised animals
Bravo et al.,
PNAS Sept 2011

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Gut–Brain Axis:
What Happens In Vagus Affects Emotion.
Bravo et al. PNAS 2011;108:15661-15662
©2011 by National Academy of Sciences

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A distributed network of brain regions
showing decreases during an emotional
faces attention task
A resting state midbrain centered is negatively
correlated with midbrain activity after FMPP

53. apc.ucc.ieAndrea Levy, The Plain Dealer

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Bercik et al., Gastroenterology, 2011
Microbiota Transplantation Modifies Host Behaviour

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Autism spectrum disorders (ASD)
Neurodevelopmental disorder with high
heritability
1.1% general population
4:1 male/female ratio
3 symptom domains (DSM-IV)
Abnormal social interaction
Impaired communication
Repetitive behaviours
Other associated symptoms:
Cognitive impairment
Seizures
Anxiety
Hyperactivity
Sensory deficits
GI distress (<90% of patients)

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Can
modulation of
the gut
microbiota
affect
cognitive
performance in
humans?

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Cognitive Assessment
• Intra/Extradimensional Set
Shift (IED)
• Paired Associate Learning
(PAL)
• Spatial Working Memory
(SWM)
• Additional Tests:
• Classic Stroop

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Cognitive Tests
Intra/Extradimensional Set Shift (IED)
Executive Function: Reversal learning &
cognitive flexibility
Paired Associate Learning (PAL)
Visuspatial Episodic Memory
Spatial Working Memory (SWM)
Stroop
Attention/ Response Inhibition
1) 2)
3) 4)

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Am J Gastroenterol 2010; 105:1994–2002

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Visuospatial Memory
Deficit in IBS
Attentional Deficit in
Crohns Disease
Paul J Kennedy, Gerard Clarke, Ann O’Neill, John A Groeger, Eamonn MM
Quigley, Fergus Shanahan, John F Cryan, Timothy G Dinan Paper In Preparation

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Stress and the Microbiota-Gut–Brain Axis in
Health and Disease
Cryan and Dinan, Nat Rev Neurosci Oct 2012

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The Daily Mail, London Sept 2011

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