The Gut Brain

Connection
Diet, the Microbiome and Brain Function
Zenovia Ursuliak MD, PhD, FRCPC
 What is the microbiome?
 “The characteristic microbial community occupying a
reasonably well defined habitat which has distinct physico-
chemical properties…encompasses their theatre of
activities” – M. N. Burge, 1988 “Fungi in Biological Control
Systems”
 “biome” – community
 “ome” – genome
 National Institutes of Health (NIH) established the Human
Microbiome Project in 2008 to characterize microbial
communities found at multiple human body sites and to look
for correlations between changes in the microbiome and
human health. http://hmpdacc.org
 Objectives
 To know how gut microbiota may participate in
bidirectional communication within the gut brain axis.

 To recognize factors that impact our gut microbiome.

 To become familiar with the pre-clinical data that
suggests diet and the gut microbiome may play a role
in mental illness.
“Let food be thy medicine and medicine be
thy food.”
- Hippocrates
 Let’s meet our 100 trillion friends
 10x the number of human cells
 150X the number of human genes
 1000 species, 7000 strains
 Most bacteria live in our large intestine
 Bacteria communicate with and affect our brain via:
 Products secreted into the bloodstream (5-HT, LPS, B12)
 Trigger cytokine production by gut mucosal immune cells
 Stimulating the vagal nerve through chemical
messengers

Evrensel et al. The gut-brain axis:The missing link in depression. Clin. Psychopharm. Neuro. 2015; 13(3) 239-244
 Roles of the Microbiome
 Bacteria ferment polysaccharides in the proximal colon
(no digestive enzymes there) into short chain fatty
acids – supply 10% of our energy needs1
 Control the population of pathogenic bacteria2
 Main source of vitamin K, and to a lesser extent the
vitamin B complex2
 Help regulate our immune response2
 Regulate tryptophan metabolism- impacts 5-HT and
kynurenine levels – brain effects3

1) Maukonen and Saarela. Proceedings of the Nutrition Society (2015), 74, 23–36;
2) Ghaisis et al. Pharmacology and Therapeutics (2015), doi: 10.1016/j.pharmthera.2015.11.012;
3) Anglin et al. Can J Psychiatry 2015;60(10):460–463
 Roles of the Microbiome
 Produce neurotransmitters:
 Lactobacillus and Bifidobacterium GABA
 E. coli, Bacillus and Saccharomyces norepinephrine
 Candida, Streptococcus, Escherichia and Enterococcus
serotonin
 Bacillus and Serratia dopamine

 Affect brain development and plasticity by secreting

BDNF, synaptophysin, post-synaptic density-95
 Roles of the Microbiome
 Mice born in germ free conditions (no gut microbes) have1:
 Altered expression and turnover of neurotransmitters in
the enteric and central nervous system (5-HT and BDNF
in the hippocampus). Introducing bacteria into these
animals can reverse these abnormalities
 Increased stress response and ACTH/cortisol levels.
Bacterial colonization of the gut can reverse these
abnormalities but only in very young mice.

This suggests there is a critical period during which

the plasticity of neural regulation is sensitive to input
from microbiota

1) Carabotti et al. Ann Gastroenterol 2015; 28 (2): 203-209

 Impacts on the microbiome
 Mode of delivery: immediately after birth: Vaginal births
– Lactobacillus (resemble mothers’ vagina), C-sections
– Staphylococcus (resemble mother’s skin)1

 Early nutrition and mode of delivery: Sampled babies

stool at 4 months: Formula fed – increased Clostridium
difficile, C-section – decreased diversity2

 Early life stress (maternal separation) in rhesus

monkeys and rats affects composition of gut
microbiota3

1) Dominguez-Bello et al. PNAS. 2010. 107(26): 11971–11975; 2)Azad MB et al. CMAJ, March 19,
2013, 185(5): 385-394;3) O’Mahony et al. Neuroscience. 2015 Oct 1. pii: S0306-4522(15)00895-
7doi: 10.1016/j.neuroscience.2015.09.068.
 Diet impacts the microbiome
 African children (vegetable fiber) – Actinobacteria, Prevotella
and Bacteroidetes , European children (starch and protein) –
Firmicutes and Proteobacteria1. Prevotella produce high
levels of SCFAs and have a protective role against gut
inflammation.
 Adding plant fiber can shift microbiota but shift depends on
initial microbiota composition2
 Enhanced gut microbial diversity was correlated with
increased exercise and dietary protein intake in athletes
compared with size, age and gender-matched nonathletic
control groups. Athletes also exhibited lower inflammatory
markers and improved metabolic markers3.
1) De Filippo C et al. Proc Natl Acad Sci U S A. 2010;107:14691-14696;
2) Flint, H. J. et al. Nat. Rev. Gastroenterol. Hepatol. 9, 577–589 (2012);
3) Clarke SF, et al. Gut. 2014;64:1913-1920.
Chemicals impact the microbiome
 Pharmaceuticals:
 Antibiotics can have lasting impacts in decreasing diversity1
 Bifidobacteria and Bacteroides are esp. sensitive
 Olanzapine: 21 days decreases proteobacteria and
actinobacteria and increases firmicutes2

 Pesticides:
 Glyphosate (Round up – applied to wheat) known to affect
microbiome of poultry, cattle and swine: increases pathogenic
bacteria, Salmonella and Clostridium and decreases beneficial
bacteria, Lactobaccilus, Bifidobacterium and Enterococcus3
 Can glyphosate affect the human microbiome?

1) Jernberg C. et al. ISME J. 2007;1:56-66; 2) Davey KJ et al. Psychopharm. (Berl) 2012;221:155-169;

3) Samsel and Seneff. Interdiscip Toxicol. 2013 Dec;6(4):159-84
 What mental illnesses could the
gut microbiome affect?
 Preclinical and initial clinical research supports the role
of the microbiome in the following illnesses:
 Autism
 Depression
 Schizophrenia
 Anxiety
 ADHD
 Dementia’s (AD, PD)
 Autism
 C-sections, hospitalization, early infections, and
associated antibiotic exposure are risk factors for ASD1
 These alter the developing gut microbiome2
 NIH study 2012 (n=121) – 85% of children with autism
have constipation, 92% report GI distress.3

 autism onset sometimes follows prolonged antibiotic

usage (which, for some antibiotics, is known to result in
the emergence of Clostridium strains)4

1) Niehus R, Lord C. J Dev Behav Pediatr 2006; 27: S120-7; 2) Clemente JC et al. Cell 2012; 148: 1258-70;
3) Gorrindo, P. et al.. Autism Research, 2012, Vol.5(2), 101-108;
4) Finegold, S. M. et al. Anaerobe 18, 260–262 (2012).
 Autism
 Molecular-based analysis of stool samples found
Desulfovibrio and Clostridial spp. more frequently in
children with regressive autism than in healthy
controls.1
 Note that anxiety and peculiar dietary habits associated
with autism may account for microbiome shifts

 Oral vancomycin (antibiotic not absorbed from the GI

tract) administration in children with regressive autism
showed short-term benefit in 8/10 with blinded raters2

1) Finegold, S. M. et al. Anaerobe 18, 260–262 (2012);

2) Sandler, R. H. et al. J. Child Neurol. 15, 429–435 (2000).
 Autism- GI permeability
 Study of 90 children with autism, 146 first degree relatives
and 64 control children, 146 control adults1:
 Intestinal permeability (measured by urinary excretion of
metabolically inert sugars after oral dosing) was abnormal
in autistic children (36.7%) and 1st degree relatives
(21.2%) vs. controls (4.8%)
 Fecal calprotectin (measure of GI inflammation) was
elevated in children with autism (24.4%), relatives
(11.6%) and controls (o%) (only those with abnormal IPT
were tested– budgetary issue)

1) De Magistris, L. et al. Journal of Pediatric Gastroenterology and Nutrition, 2010, Vol.51(4), p.418-424
 Autism
 Short chain fatty acids are metabolic products of gut bacteria when
they ferment dietary fiber.
 3 types made: acetic, propionic and butyric.
 can be excreted or absorbed to use as a source of energy. Butyric
acid is an important source of energy for cells lining the colon.
Different bacteria produce different SCFA.
 Propionic acid is a key fermentation product of Clostridia,
Bacteroides, Desulfovibrio – bacterial species associated with
autism1
 intracerebroventricular administration of propionic acid, a microbial
metabolite, produces autism-like behaviour in rats (repetitive
behavours, hyperactivity, anxiety and decreased socialization)2

1) Finegold SM.. Anaerobe 2011; 17: 367-8; 2)MacFabe, D. F. et al. Behav. Brain. Res. 176, 149–169 (2007).
 Autism
 Increased mean levels of propionic acid in stool of ASD
children have been shown1.

 Lipopolysaccharide (outer cell wall component of gram

negative bacteria) significantly higher in blood of adults
patients with severe autism compared to healthy
controls and inversely predicted scores on socialization
domains2

1) Wang L. et al. Dig Dis Sci 2012; 57: 2096-102;

2) Emanuele, E.; et al. Neuroscience Letters, 2010, Vol.471(3), pp.162-165;
 Autism
 Mouse model of autism – Maternal Immune Activation
(inject pregnant mothers with the viral mimic poly (I:C))
yields offspring with the core communicative, social, and
stereotyped impairments similar to autism, as well as a
common autism neuropathology—a localized deficiency in
cerebellar Purkinje cells
 2013 study1 showed increased intestinal permeability and
cytokine levels and microbiome shifts similar to humans with
autism in the adult MIA offspring vs. controls
 Oral treatment of MIA offspring with Bacteroides fragilis
corrected gut permeability, restored microbial composition, and
improved communicative, stereotypic, anxiety-like and
sensorimotor behaviors but retained deficits in sociability.

Hsiao et al. Cell 155, 1451–1463, December 19, 2013

 Autism: Clinical studies
 12 week double-blind, placebo-controlled study
Lactobacillus plantarum given to children with autism
resulted in1:
 significant increases in beneficial bacteria lactobacilli and
enterococci
 significant reduction in Clostridium
 reduced GI problems
 improved behaviour scores
 Gluten-free/casein-free diet (GF/CF) studies yield
mixed results although some that have shown benefit
have also have shown reduced GI permeability2
1) Parracho HMRT et al. (2010). Int J Probiot Prebiot 5, 69 – 74;
2) Mayer at al. Bioessays 36: 933–939,
 Autism: Summary of the research
 Risk factors for autism also known to impact the gut
microbiome

 People with autism have altered microbiome (Clostridium,

Desulfovibrio), high rates of GI complaints and increased GI
permeabilty

 Administration of propionic acid (metabolite of Clostridium)

into brains of mice induces autistic behaviour and high levels
of propionic acid found in stool of people with autism

 Probiotics can improve autism behaviours in a mouse model

and small human RCT
 Depression
 In double-blind, randomized crossover trials administration
of LPS to healthy humans:
 transiently induced increased cytokine and cortisol secretion,
anxiety and depressed mood and decreased verbal and
nonverbal memory. Levels of cytokines positively correlated
with impacts on anxiety, mood and memory1
 Similar results and increased visceral pain sensitivity2
 Increased activation of the right inferior orbital frontal cortex in
response to emotional visual stimuli3
 pretreatment with citalopram but not bupropion, decreased
LPS-induced anhedonia and fatigue but cytokine levels were
unchanged4.
1) Reichenberg et al. Arch Gen Psychiatry 2001, 58:445–452;
2) Grigoleit et al. PLoS One 2011, 6:e28330;
3) Kullmann et al. Hum Brain Mapp 2012. doi:10.1002/ hbm.22063;
4) Dellagioia et al. Brain Behav Immun 2013;31: 197-204
 Depression
 Mice: previous experience with physical and/or
psychological stress (e.g. tail shock, social defeat)
leads to an even more pronounced inflammatory
cytokine release subsequent to LPS administration1

 Rats: non-desirable gut microbes (e.g. Campylobacter

jejuni), at quantities too low to produce a detectable
immune response, influenced animal behavior
indicative of human anxiety2

1) Audet et al. Brain Behav Immun 2011, 25:1197–120;

2) Lyte et al. Physiol Behav 1998, 65:63–68;
 Depression
 In 1641 people referred to an internal medicine clinic
for GI complaints, 84.1% state anxiety, 67% trait
anxiety and 27% depressive symptoms1

 DNA sequencing of fecal samples from 55 depressed

and non-depressed people showed that Bacteroidales
was overrepresented (p = 0.05), Lachnospiraceae was
underrepresented (p = 0.02) in group with depression2

1) Addolorato et al. Int J Clin Pract 2008, 62:1063–1069;

2) Naseribafrouei et al. Neurogastroenterol Motil (2014) 26, 1155–1162;
 Depression
 Bifidobacterium can
 inhibit LPS-induced inflammation, by blocking NF-kB
activation in intestinal epithelial cell lines1
 Reverse elevated HPA axis response, cytokines and
depressive symptoms in rats separated from mothers at
birth2

 In a RCT of healthy women, the group that consumed a

fermented milk product for 4 weeks showed reduced
reactivity to a task probing attention to negative context
on fMRI
 Changes seen in brain regions that control central
processing of emotion and sensation3
1) Riedel et al. World Journal of Gastroenterology 2006;12(3):3729–55;
2) Desbonnet et al. Neuroscience 170 (2010) 1179–1188;
3) Tillisch et al. Gastroenterology 2013;144:1394–1401;
 Depression
 In double-blind RCTs with healthy people:
 People who were given a mixture of probiotics, containing L.
helveticus and B. longum, for 30 days demonstrated
significantly less psychological distress than their matched
placebo counterparts1
 healthy subjects who scored in the lower third for depressed
mood showed significant improvement, after being fed a
probiotic-containing milk drink for 3 weeks, as compared to their
placebo counterparts2

 The Mediterranean diet (increased fruits, vegetables and

fish, decreased meat and sugars/starch) has shown benefit
in reducing risk of depression3
 Could some of the impact be on dietary influence on the
microbiome?

1) Messaoudi et al. Br J Nutr. 2011;105(5):755–764;

2) Barrett et al. J Appl Microbiol. 2012;113(2):411–417.
3) Sánchez-Villegas et al. BMC Medicine 2013, 11:208
 Depression – Summary of the
research
 People with depression have altered microbiomes and
increased rates of GI complaints
 Bacterial products (LPS) can induce depressive
symptoms in humans that can be prevented by
pretreatment with antidepressants and probiotics
 Probiotics in rats can reverse the HPA and cytokine
abnormalities and depressive symptoms induced by
maternal separation
 Probiotics in human show benefit in mood, distress and
reactivity to negative stimuli
 Where to go from here?
 Anecdotal reports of recoveries from autism with the
GAPS diet and fecal microbial transplants and
recoveries from depression with dietary changes plus
probiotics but clinical trials are needed to address
these questions:
 Can fecal microbial transplants and dietary and probiotic
interventions be useful in clinical populations?
 Can biomarkers of bacterial metabolites, inflammation
and food antigens be used to select individuals that will
benefit from dietary and probiotic interventions?
 Summary
 The microbiome plays a role in the bidirectional gut-
brain axis through bacterial metabolites, immune cells,
the vagus nerve and the HPA axis.

 Preclinical data suggests that the gut microbiome and

diet may play a role in the pathogenesis of several
psychiatric illnesses including autism, depression and
schizophrenia.

 Clinical trials are needed to evaluate whether fecal

microbial transplant, dietary inventions and probiotics
are viable therapeutic options.