Module 12

Lesson 1

by Dr. Irena O’Brien, PhD

GUT-BRAIN CONNECTION

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Welcome to this masterclass on the gut-brain connection where we look at how digestive health is intimately related to brain health, cognitive functioning and psychological wellbeing. Put simply, If you want to achieve optimal performance, you cannot ignore the health of your gut.

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“The manner in which the secretions of the alimentary canal and of certain other organs … are affected by strong emotions, is another excellent instance of the direct action of the sensorium on these organs, independently of the will or of any serviceable associated habit.”

The idea that gut health is related to brain health is not new. In the 19th century, Charles Darwin, in The Expression of the Emotions in Man and Animals (1872), wrote: “The manner in which the secretions of the alimentary canal and of certain other organs … are affected by strong emotions, is another excellent instance of the direct action of the sensorium on these organs, independently of the will or of any serviceable associated habit.” So Darwin recognized that emotions had a direct effect on the gut. Have you noticed that when you’re stressed or anxious, your digestion starts to act up?

Recently, interest in the relationship between gut health and brain health has become a hot topic and a growing body of research is showing that gut microbes play a significant role in the regulation of anxiety, mood, cognition, and even pain. And this communication between the gut and the brain is bidirectional, so that brain health also affects gut health. Scientists call this relationship the microbiota-gut-brain axis, or mgb.

Until very recently, much of the evidence for a microbiota-gut-brain axis had been anecdotal: psychiatrists had noted that stress-related psychiatric symptoms, such as anxiety, were often accompanied by gastrointestinal disorders, such as irritable bowel syndrome (IBS) and irritable bowel disease (IBD).

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“Overall, it is becoming increasingly apparent that behaviour, neuro-physiology and neuro-chemistry can be affected in many ways through modulation of the gut microbiota.”

In fact, a 2012 Nature Reviews Neuroscience article concluded that “Overall, it is becoming increasingly apparent that behaviour, neuro-physiology and neuro-chemistry can be affected in many ways through modulation of the gut microbiota.”

Cryan, J., & Dinan, T. (2012). Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nature Reviews Neuroscience, 13(10), 701–712. doi:10.1038/nrn3346

Photo by Dose Juice on Unsplash

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Let’s take a closer look at the gut and how it communicates with the brain. Here’s a slide from the Masterclass on Neuroanatomy where we looked at the enteric, or intestinal nervous system. The enteric nervous system (ENS) is one of the main divisions of the nervous system and consists of a system of neurons that governs the function of the gastrointestinal system. It is now usually referred to as separate from the autonomic nervous system since it has its own independent reflex activity. The ENS is also called the second brain. Both the central and enteric nervous systems are created from the same tissue during fetal development and both are connected via the vagus nerve, which extends from the brain to the abdomen.

The enteric nervous system consists of approximately 500 million neurons, which is one two-hundredth of the number of neurons in the brain, and 5 times as many as the one hundred million neurons in the spinal cord.

The enteric nervous system is embedded in the lining of the gastrointestinal system. The ENS is capable of autonomous functions such as the coordination of reflexes; although it receives considerable innervation from the autonomic nervous system, it can and does operate independently of the brain and the spinal cord. The enteric nervous system has been described as a "second brain" for several reasons. It can operate autonomously. It normally communicates with the central nervous system (CNS) and the brain through the parasympathetic (e.g., via the vagus nerve) and sympathetic (e.g., via the prevertebral ganglia) nervous systems, but also through our immune and endocrine systems.

The enteric nervous system includes efferent neurons (from the brain), afferent neurons (to the brain), and interneurons, all of which make the enteric nervous system capable of carrying reflexes and acting as an integrating centre without CNS input. The enteric nervous system also makes use of more than 30 neurotransmitters, most of which are identical to the ones found in the CNS, such as acetylcholine, dopamine, and serotonin. More than 90% of the body's serotonin lies in the gut, as well as about 50% of the body's dopamine.

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Image By OpenStax - https://cnx.org/contents/FPtK1zmh@8.25:fEI3C8Ot@10/Preface, CC BY 4.0, https://commons.wikimedia.org/w/index.php?curid=30147911

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The digestive system communicates with the CNS primarily through the vagus nerve, which runs from the brainstem to the gut. This communication is bidirectional, although the vast majority of the communication is afferent, meaning from the gut to the brain. The vagus nerve also relays essential stress-response signals. This bidirectional communication explains why CNS disorders are often accompanied by digestive problems. And abnormalities in the digestive system can directly shape both cognitive and emotional state.

The gut bacteria (or microbiota) influence the production of dopamine and epinephrine (or adrenaline). They also influence the production of serotonin, a neurotransmitter that plays a central part in gut motor function and digestion, as well as in various cognitive and mood disorders. Roughly 95% of the body’s serotonin is produced in the gut. The serotonin-producing cells in the gut can be viewed as the main hub for communication between the gut and brain, because they’re exposed to what we eat and to signals produced by gut microbes, and innervated by connections to the vagus nerve.

The brain can also affect the microbiota and potentially contribute to the onset of or exacerbate digestive disorders.

Eisenstein, M. (2016). Microbiome: Bacterial broadband. Nature, 533(7603), S104–S106. doi:10.1038/533s104a

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“…given the ability of the gut microbiota to influence serotonin and its precursor, tryptophan, regulate the stress response and modulate cognition and behaviour, the potential importance of the gut microbiota to psychiatry in general and to depression specifically is apparent.”

A 2015 article in Current Opinions in Psychiatry acknowledged that “…given the ability of the gut microbiota to influence serotonin and its precursor, tryptophan, regulate the stress response and modulate cognition and behaviour, the potential importance of the gut microbiota to psychiatry in general and to depression specifically is apparent.” This is interesting, and even essential, as it opens the door to an additional way of generating change, through optimizing gut health.

Many earlier studies on the gut-brain connection have used an animal model.

Dash, S., Clarke, G., Berk, M., & Jacka, F. (2015). The gut microbiome and diet in psychiatry: focus on depression. Current Opinion in Psychiatry, 28(1). doi:10.1097/YCO.0000000000000117

Photo by Noble Brahma on Unsplash

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Here’s one such study using mice. Now, Baltic mice are innately more anxious than Swiss mice. Swiss mice are more gregarious. In this study, the researchers put the mice in a lightbox and on a platform. Mice prefer dark spaces to light and because Baltic mice are more anxious than Swiss mice, you would expect them to leave the light box more quickly than the Swiss mice. And that is exactly what happened. The Baltic mice spent less time in the lightbox (this is the light bar) than the Swiss mice (this is the black bar), 60 seconds for the Baltic mice vs 250 seconds for the Swiss mice.

Mice also explore their surroundings. Because Baltic mice are more anxious than Swiss Mice, you would expect them to spend more time on the platform, reflecting their fear of stepping down to explore. And that’s exactly what they found. The Baltic mice spent more time on the platform than did the Swiss mice, 195 seconds for the Baltic mice to only 25 seconds for the Swiss mice.

Collins, S., Kassam, Z., & Bercik, P. (2013). The adoptive transfer of behavioral phenotype via the intestinal microbiota: experimental evidence and clinical implications. Current Opinion in Microbiology, 16(3), 240–245. doi:10.1016/j.mib.2013.06.004

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Then the researchers transferred gut bacteria between the Swiss mice and the Baltic mice.

In this slide, SPF refers to disease free mice and these are used as a control. In the left panel, germ free Swiss mice were given either Swiss gut bacteria or Baltic gut bacteria. When the Swiss GF mice were given Swiss gut bacteria, there was no difference in how long they stayed on the platform before they left to start exploring . However, when they were given Baltic gut bacteria, and remember that Baltic mice are naturally more anxious than Swiss mice, their behaviour changed. The Swiss mice became more fearful and stayed on the platform longer before they stepped down to start exploring.

In the panel on the right, when the Baltic germ free mice were given Baltic gut bacteria, there was no difference in how long they stayed on the platform before stepping down to explore . However, when they were given Swiss gut bacteria, and remember that Swiss mice are naturally less anxious than Baltic mice, they were faster to leave the platform .

The transfer of gut bacteria altered behaviour: Swiss mice become more fearful, similar to Baltic mice. And Baltic mice become less fearful, similar to Swiss mice.

This study shows how gut bacteria can affect behaviour. When the mice received new gut bacteria, their behaviour changed.

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Cortisol

Salivary cortisol did not differ significantly between groups atbaseline but was significantly lower following B-GOS com-pared with placebo (Fig. 1). This was shown by an ANOVAinteraction effect of pre- versus post-treatment, group(placebo, FOS or B-GOS) and sampling time point (inminutes post-waking) on salivary cortisol levels, followedup with separate group×time point ANOVAs for each dayof sampling (day 0: main effect of group F(2,41)=1.08, n.s.;day 21: main effect of group F(2,41)=4.20, p<0.05, followedup with Sidak-corrected contrasts: placebo vs. GOS, p=0.02,all others p>0.1). Main effects analyses of the day×group×time ANOVA confirmed that cortisol levels were increasedpost-waking 15, 30, 45 and 60 min after waking (significantmain effect of time F(2.41,98.63)=58.61, p<0.001, plannedfollow-up contrasts all significant at p<0.001). The maineffects of day of sampling and treatment group were notsignificant (p>0.1). Gender was not entered as a factor ofinterest due to insufficient power.

The lowered CAR in the B-GOS compared to the placebogroup on day 21 of supplement administration was also con-firmed when analysing area under the curve with respect toground (Fig. 2; day×group ANOVA on square-root-transformed salivary cortisol values: day×group interaction[F(2,41)=3.52, p=0.039], followed up with separate groupANOVAs for pre/day 0 [F(2,41)=1.24, n.s.] and post/day 21[F(2,41)=4.12, p=0.023, follow-up contrasts: placebo vs. B-GOS p=0.019, placebo vs. FOS p>0.1, FOS vs. B-GOSp>0.1]).

Emotional Test Battery

Attentional dot-probe task

There was a significant group×emotion×masking conditioninteraction in the visual dot-probe task (group×emotion×masking condition [F(2,41)=3.14, p=0.05]). As can be seenin Fig. 3, this effect was driven by decreased attentionalvigilance to negative versus positive information in theunmasked condition (Fig. 3b), with no significant main effects

or interactions in the masked condition (Fig. 3a; valence×group interaction in unmasked: F(2,41)=4.29, p=0.02;masked: F(2,41)=0.85, p>0.1). Follow-up analyses with sep-arate ANOVAs for prebiotic group compared with placebo inthe unmasked condition confirmed this effect as driven byincreased positive versus negative vigilance after B-GOScompared to placebo, while the FOS group did not performdifferently to placebo (B-GOS vs. placebo: valence×groupF(1,27)=6.94, p=0.014, FOS vs. placebo: valence×groupF(1,27)=3.20, n.s.).

FERT

There were no significant effects of prebiotic treatment onmeasures of accuracy (main effect of group: F(2,42)=1.71,n.s., emotion×group interaction F(7.83,164.52)=0.67, n.s.).Analysis of reaction time data revealed no significant interac-tion between group and emotion (main effect of group:F (2 ,42) = 0.53, n .s . ; emot ion × group interac t ionF(8.16,773.38)=1.10, n.s.).

Emotional categorisation, recall and recognition

Participants responded faster to positive (mean reactiontime=1031 ms, SD=228 ms) compared to negative(mean reaction time=1083 ms, SD=200 ms) self-referential personali ty words in the emotionalcategorisation task (valence×group ANOVA, main effectof valence: F(1,42)=12.82, p<0.01) and in the emotion-al word recognition task (mean reaction time to positivewords=1220.92, SD=263.03; mean reaction time tonegative words=1381.07, SD=348.13; main effect ofvalence: F(1,41)=23.34, p<0.001); however, there wasno significant main effect of prebiotic treatment group(F(2,42)=0.80, p>0.1) and the relative speeding forpositive words did not differ between groups (emo-tion×group F(2,42)=0.35, p>0.1). Positive words werealso remembered more often than negative words inboth the surprise recall task (mean accuracy, positivewords = 7.41 (SD = 2.45) , negat ive = 5.70 (2.29) ;F(1,41)=16.16, p<0.001) and in the recognition task

Fig. 1 Cortisol awakening response before and after administration of placebo, B-GOS or FOS. There were no differences in the salivary CAR pre-administration. Salivary cortisol awakening response was significantly lower after 3 weeks of B-GOS intake, but not FOS intake, compared with placebo

Psychopharmacology (2015) 232:1793–1801 1797

FOS: fructooligosaccharidesB-GOS: Bimuno®galactooligosaccharides

So what happens if we alter the gut bacteria of humans? Of course, in humans, we can’t introduce gut bacteria from anxious individuals into relaxed individuals. That would be unethical. But we can change gut bacteria by taking pre- or probiotics. In this study, the participants took one of two types of prebiotics, B-GOS or FOS, or a placebo daily for 3 weeks. Prebiotics are plant fibres that beneficially nourish the good bacteria in the gut. Probiotics introduce good bacteria into the gut whereas prebiotics act as a fertilizer for good bacteria that’s already there. Some prebiotic foods are green bananas, raw potato and legumes.

The chart here shows cortisol levels shortly after awakening. Cortisol levels at the beginning of the study, before any prebiotics were taken, are shown in black. Cortisol levels following 3 weeks of prebiotic use are shown in blue. We know that cortisol is a hormone released when we’re stressed or anxious.

For those who took the B-GOS form of prebiotics their cortisol levels had dropped but not so for the placebo or FOS group. Their cortisol levels remained unchanged. Just in case you’re wondering, a rich B-GOS source of prebiotics are legumes: lentils, chickpeas, green peas, lima beans, and kidney beans.

Schmidt, K., Cowen, P., Harmer, C., Tzortzis, G., Errington, S., & Burnet, P. (2014). Prebiotic intake reduces the waking cortisol response and alters emotional bias in healthy volunteers. Psychopharmacology, 232(10) under Creative Commons Attribution License

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(mean correct recognition, positive words=25.67 (SD=3.41), negative words=22.36 (SD=3.88); F(1,41)=53.54, p<0.001), but these effect did not differ betweengroups (p>0.1).

Self-report questionnaires

There were no significant effects of group on self-reportmeasures of state anxiety or perceived stress before or afterprebiotic/placebo administration (see Table 2). Furthermore,there were no group differences in the overall cognitive statusas assessed by digit span on the day of psychological testing.

Correlational analyses

To test the hypothesis that cortisol levels were associated withchanges in attentional dot-probe performance in the B-GOSgroup, we correlated difference scores of positive versus neg-ative reaction times in the unmasked condition with absolutecortisol levels upon waking on the day of testing and with thedifference in pre- versus post-prebiotics cortisol values (day0–day 21). There were no associations of cortisol with atten-tional performance (all p>0.1).

Discussion

The current study explored the neuroendocrine and affectiveeffects of two types of prebiotic supplements in healthy hu-man volunteers, using salivary CAR and a validated testbattery of emotional processing. Results revealed that B-GOS prebiotic intake was associated with decreased wakingsalivary cortisol reactivity and altered attentional bias com-pared to placebo. These results are consistent with previouslyfound anxiolytic-like effects of probiotics and reveal keydifferences between two different prebiotic supplements.

Our findings of lowered cortisol awakening reactivity inthe group receiving B-GOS prebiotics compared to the place-bo group indicate that prebiotic administration may modulateHPA activity in a similar fashion as the administration ofprobiotic strains directly seen in rodents (Sudo et al. 2004;Gareau et al. 2007) and humans (Messaoudi et al. 2011). Thecortisol awakening response is a reliable marker of HPA axisactivity which has been found to be increased by workstressors (Pruessner et al. 1997; Kunz-Ebrecht et al. 2004)and in individuals at high risk of depression (Mannie et al.2007). Insufficient or excessive cortisol reactivity may indi-cate dysfunctional HPA axis feedback mechanisms, whichmay provide useful targets for modulation by treatments incertain vulnerability or disease states (Pariante and Lightman2008; Dinan and Cryan 2012).

Participants receiving B-GOS supplements showed in-creased attentional vigilance to positive versus negative stim-uli on the dot-probe task. Our effects are similar to those seenfollowing administration of pharmacological agents such asthe selective serotonin reuptake inhibitor citalopram or thebenzodiazepine diazepam in healthy individuals (Browninget al. 2006; Murphy et al. 2009a, b). These effects have beeninterpreted as showing an early anxiolytic-like profile, wherethreatening stimuli are less likely to be attended to (Harmer2010). Interestingly, we found effects of the B-GOS prebioticadministration on altered attentional processing only in theunmasked condition (500 ms presentation) of the dot-probetask. Attentional vigilance to brief, masked presentations ofthreatening cues has primarily been interpreted as an

Fig. 2 Area under the curve (with respect to ground) of salivary cortisolawakening response pre- and post-prebiotic supplement/placebo intake.*p<0.05

Fig. 3 Vigilance reaction times in the attentional dot-probe task. aAttentional vigilance did not differ between groups during masked trialsof the attentional dot-probe task. b Participants showed decreasedattentional vigilance to negative versus positive words in the unmaskedcondition of the dot-probe task after B-GOS but not FOS intake comparedto placebo

1798 Psychopharmacology (2015) 232:1793–1801

(mean correct recognition, positive words=25.67 (SD=3.41), negative words=22.36 (SD=3.88); F(1,41)=53.54, p<0.001), but these effect did not differ betweengroups (p>0.1).

Self-report questionnaires

There were no significant effects of group on self-reportmeasures of state anxiety or perceived stress before or afterprebiotic/placebo administration (see Table 2). Furthermore,there were no group differences in the overall cognitive statusas assessed by digit span on the day of psychological testing.

Correlational analyses

To test the hypothesis that cortisol levels were associated withchanges in attentional dot-probe performance in the B-GOSgroup, we correlated difference scores of positive versus neg-ative reaction times in the unmasked condition with absolutecortisol levels upon waking on the day of testing and with thedifference in pre- versus post-prebiotics cortisol values (day0–day 21). There were no associations of cortisol with atten-tional performance (all p>0.1).

Discussion

The current study explored the neuroendocrine and affectiveeffects of two types of prebiotic supplements in healthy hu-man volunteers, using salivary CAR and a validated testbattery of emotional processing. Results revealed that B-GOS prebiotic intake was associated with decreased wakingsalivary cortisol reactivity and altered attentional bias com-pared to placebo. These results are consistent with previouslyfound anxiolytic-like effects of probiotics and reveal keydifferences between two different prebiotic supplements.

Our findings of lowered cortisol awakening reactivity inthe group receiving B-GOS prebiotics compared to the place-bo group indicate that prebiotic administration may modulateHPA activity in a similar fashion as the administration ofprobiotic strains directly seen in rodents (Sudo et al. 2004;Gareau et al. 2007) and humans (Messaoudi et al. 2011). Thecortisol awakening response is a reliable marker of HPA axisactivity which has been found to be increased by workstressors (Pruessner et al. 1997; Kunz-Ebrecht et al. 2004)and in individuals at high risk of depression (Mannie et al.2007). Insufficient or excessive cortisol reactivity may indi-cate dysfunctional HPA axis feedback mechanisms, whichmay provide useful targets for modulation by treatments incertain vulnerability or disease states (Pariante and Lightman2008; Dinan and Cryan 2012).

Participants receiving B-GOS supplements showed in-creased attentional vigilance to positive versus negative stim-uli on the dot-probe task. Our effects are similar to those seenfollowing administration of pharmacological agents such asthe selective serotonin reuptake inhibitor citalopram or thebenzodiazepine diazepam in healthy individuals (Browninget al. 2006; Murphy et al. 2009a, b). These effects have beeninterpreted as showing an early anxiolytic-like profile, wherethreatening stimuli are less likely to be attended to (Harmer2010). Interestingly, we found effects of the B-GOS prebioticadministration on altered attentional processing only in theunmasked condition (500 ms presentation) of the dot-probetask. Attentional vigilance to brief, masked presentations ofthreatening cues has primarily been interpreted as an

Fig. 2 Area under the curve (with respect to ground) of salivary cortisolawakening response pre- and post-prebiotic supplement/placebo intake.*p<0.05

Fig. 3 Vigilance reaction times in the attentional dot-probe task. aAttentional vigilance did not differ between groups during masked trialsof the attentional dot-probe task. b Participants showed decreasedattentional vigilance to negative versus positive words in the unmaskedcondition of the dot-probe task after B-GOS but not FOS intake comparedto placebo

1798 Psychopharmacology (2015) 232:1793–1801

© 2017 - 2020 The Neuroscience School

And then the researchers looked at behaviour. They asked the participants to classify emotional faces according to the emotion they exhibit. This is generally done by showing them pictures of individuals expressing basic emotions such as happiness, surprise, sadness, fear, anger, disgust, and asking them to press a key that identifies the emotion. The time it takes them to identify the emotion and press the key is measured. . The B-GOS group spent less time attending to negative words, shown in black, and more time attending to positive words, shown in blue, than the other two groups. Why this is important is that attention to negative words is a marker of anxiety and depression and the B-Gos group showed less attention to negative words.

The researchers noted that these effects here are similar to taking anti-depressants or anti-anxiety drugs.

So here you have a study showing that gut bacteria affect cortisol levels and anxiety.

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So what happens in the brain when the gut bacteria itself is altered?

In this study, the participants took probiotics for 4 weeks. After 4 weeks, their brains were scanned while they performed this emotional faces attention task. In this task, the participants see a face reflecting fear or anger. Here, for example, the emotion is fear. They then press a button to match the emotion they saw to one of the two faces below it. This task measures conscious and pre-conscious brain responses to emotional stimuli. It accesses subtle changes in emotional regulation.

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After 4 weeks when their brains were scanned, those who had taken the probiotics showed reduced activation to the emotional faces in the insula , the periaqueductal gray and somatosensory regions . The insula is involved in detecting the salience of incoming information and the PAG receives interoceptive, or internal, input and is involved in integrated brain responses to emotional stimuli.

And you can see in the bottom panel here that, compared to those who didn’t receive any probiotics (the NO IN group), or the controls who received an unfermented milk product, the group that received the probiotics - showed decreased activity across this network.

This study shows that probiotics alter brain activity: they can decrease the brain’s arousal to negative stimuli. And becoming less aroused by negative stimuli is a good thing.

Tillisch, K., Labus, J., Kilpatrick, L., Jiang, Z., Stains, J., Ebrat, B., … Mayer, E. (2013). Consumption of Fermented Milk Product With Probiotic Modulates Brain Activity. Gastroenterology, 144(7). doi:10.1053/j.gastro.2013.02.043

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Switching gut bacteria: reversed anxiety in micePre-biotics - lowered cortisol levels and reduced anxietyProbiotics - reduced brain activation to negative emotional stimuli

So far, we’ve seen evidence for the importance of gut microbiota to emotional processing. We saw that when gut bacteria were transferred between strains of mice, the gregarious mice became more anxious and the anxious mice became more gregarious. We’ve also seen that pre-biotics can lower cortisol levels and reduce anxiety. And finally, we’ve seen that probiotics reduced brain activation to negative emotional stimuli.

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