Bacteria–Enteric Nervous System Interactions

Gut bacteria regulate electrophysiological thresholds in enteric nervous system neurons. For example, myenteric neurons exposed to Bifidobacterium longum NCC3001-fermented substances showed reduced generation of action potentials when they were electrically stimulated. Similarly, colonic AH neurons (the chief sensory neurons in the colon) treated with Lactobacillus rhamnosus showed increased excitability, an effect that emerged from inhibition of calcium-controlled potassium gates. Other work showed that neurons from the dorsal root ganglion in the colon did not display hyperexcitability in response to noxious stimulation if they had been treated with Lactobacillus rhamnosus. Myenteric neurons are also in close proximity to the gut lumen, which would facilitate their contact with the microbiome. In germ-free mice, these neurons show lower levels of excitability compared to their normally-colonised counterparts. One study found evidence of intestinal neural abnormalities in the jejunum and ileum of germ-free mice in comparison to controls, with germ-free mice showing reduced nerve density, fewer nerves per ganglion, and a greater number of myenteric nitrergic neurons. Recent evidence also indicates that the microbiome affects ion transport controlled by cyclic adenosine monophosphate (cAMP).

Overall, these results provide striking evidence of direct, bacteria-induced modulation of the enteric nervous system. Moreover, the influence of the microbiome on the enteric nervous system extends beyond neurons, with recent findings demonstrating that gut bacteria also play a crucial role in the development and homeostasis of glial populations in the gut. Gut bacteria also produce a range of neurotransmitters through the metabolism of indigestible fibres. These include dopamine and noradrenalin by members of the Bacillus family, GABA by the Bifidobacteriafamily, serotonin by the Enterococcus and Streptococcus families, noradrenalin and serotonin by the Escherichia family, and GABA and acetylcholine by the Lactobacilli family. Though there is no direct evidence as of yet, it is likely that these neurotransmitters modulate synaptic activity in the proximal neurons of the enteric nervous system, and is an important avenue for future research.

Vagal Signalling

The vagus nerve plays an essential and wide-ranging role in coordinating parasympathetic activity, including regulation of heart rate and gut motility. It possesses an abundance of sensory fibres, and is able to convey rich information on organ function throughout the body to the brain. Vagal activity is sensitive to nutrition, exercise, and stress. Stimulating the vagus nerve exerts anti-inflammatory effects, and is used therapeutically for refractory depression, pain, and epilepsy. There is also evidence of both antidepressants and anxiolytics exerting vagal effects, suggesting that vagal modulation may be a common pathway for the effects of antidepressants, anxiolytics, and psychobiotics. Several animal studies have found that the vagus nerve mediates the relationship between psychobiotics and their psychophysiological effects, as severing the vagus nerve (vagotomy) abolishes responses to psychobiotic administration. However, one study has found that ingestion of antimicrobials increased intrinsic relative abundance of Lactobacilli in innately anxious male BALB/c mice, a change that was accompanied by increased exploratory behaviour and BDNF expression. Crucially, however, vagotomy did not eliminate these neural or behavioural benefits. Therefore, vagal signalling may be at most a partial mediator of bacterial effects.

Short-Chain Fatty Acids, Gut Hormones, and Bacteria-Derived Blood Metabolites

The human gut is incapable of digesting macronutrients such as plant polysaccharides. While these frequently appear in the diet, the human genome does not code the requisite enzymes for their digestion, which are supplied by the microbiome. The metabolisation of these fibres produces short-chain fatty acids (SCFAs), including acetate, butyrate, lactate, and propionate. SCFAs enter the circulatory system through the large intestine, where the greater proportion are directed into the liver and muscle. Although it is unclear to what extent the small fraction of SCFAs crossing into the central nervous system modulates neurotransmission, there is some evidence for their psychotropic properties at pharmacological concentrations. For instance, systemic sodium butyrate injections (200 mg/kg body weight) in rats produce antidepressant effects, and increase central serotonin neurotransmission and BDNF expression. Here, the action of butyrate as an epigenetic modifier is more likely compared to action as an agonist at a free fatty acid receptor (FFAR), given that there are few FFARs in the brain . However, it should be noted that the SCFAs display pleiotropy (independent effects produced by a single gene), and also stimulate the HPA axis or have direct effects on the mucosal immune system, which may indirectly affect central neurotransmission. A recent rodent investigation has also found that the SCFA acetate plays a causal role in obesity. Acetate generated by the gut bacteria in response to high-fat diets triggers parasympathetic activity and promotes increases in ghrelin, glucose-stimulated insulin, and further nutrition intake, creating a positive feedback loop that increases the likelihood of obesity.

SCFAs also influence secretion of satiety peptides, including cholecystokinin (CCK), peptide tyrosine tyrosine (PYY) and glucagon-like peptide-1 (GLP-1), from gut mucosal enteroendocrine cells which express FFARs. For instance, propionic acid mediates the release of GLP-1 and PYY through activation of FFAR2. Consistent with the concept that SCFAs are produced from the bacterial metabolism of dietary polysaccharides, prebiotic supplementation increases the production of intestinal SCFAs, which modulate enteroendocrine cells and their secretion of PYY and GLP-1. It is therefore reasonable that the satiety hormones may play a more significant role in the central effects of prebiotics compared to probiotics. Furthermore, circulating PYY and GLP-1 have brain-penetrant properties, and their administration to rodents have significant effects on neurotransmitters and behaviour.

The microbiome has also been shown to possess a substantial role in generating metabolites that enter circulation and exert a range of consequences outside the gut. A key study that compared germ-free mice to normally colonised mice found striking effects of the microbiome on the diversity and quantity of blood metabolites. For instance, germ-free mice had 40% greater plasma tryptophan concentrations than normal mice, but the normal mice had 2.8 times greater plasma serotonin levels than the germ-free mice. This suggests that gut bacteria crucially affect the metabolism of tryptophan into serotonin in Enterochromaffin cells (serotonin-secreting cells embedded in the luminal epithelium). Though the specific mechanism through which bacteria might control serotonin production in Enterochromaffin cells was unknown at that time, a recent study has attributed this role to indigenous spore-forming bacteria in the gut. There were similarly dramatic differences in other tryptophan metabolites, especially those containing indole, such as the antioxidant indole-3-propionic acid (IPA) and indoxyl sulphate, which were undetected in the germ-free mice and whose production was therefore interpreted as being fully mediated by gut bacteria. We speculate that these metabolites are sensitive to psychobiotic action. However, the relationships between the microbiome, bacteria-derived metabolites, and the central nervous system, as well as the role of psychobiotics in modulating this network, remain virtually unexplored.

Bacteria-Immune Interactions

A key function of the immune system is to detect and eliminate pathogens. Every microbe possesses a microbe-associated molecular pattern (MAMP, previously referred to as pathogen-associated molecular patterns). A range of microscopic elements may act as MAMPs, including microbial nucleic acids, molecular cell wall components (e.g., lipopolysaccharides), or bacterial flagella. Gut microbes can communicate with the enteric nervous system and the innate immune system via interactions between the MAMPs and pattern-recognition receptors embedded along the lumen. The family of pattern-recognition receptors includes Toll-like receptors (TLRs), C-type lectins, and inflammasomes. These receptors are able to detect the nature and potential effects of various microbes via the MAMPs and, at a broad level, transmit information about the microbial environment to the host, enabling specific immunological responses. The MAMPs of beneficial bacteria, by triggering pattern-recognition receptors, may precipitate secretion of anti-inflammatory cytokines such as interleukin-10. While rigorous mechanistic descriptions of the relationship between MAMPs, pattern-recognition receptors, and reductions in inflammation are lacking, one intriguing hypothesis is that beneficial bacteria might serve as physical barriers that block pathogenic MAMPs (e.g., lipopolysaccharides) from activating host pattern-recognition receptors such as TLR2 and TLR4 by binding to them instead, thereby preventing pro-inflammatory responses.

Prebiotics may act in a similar capacity, as there is evidence of direct interaction between oligosaccharides and the epithelium, independent of gut bacteria, with substantial reductions in pro-inflammatory cytokines. Prebiotics may prevent pathogenic MAMPs from accessing pattern-recognition receptors, either by acting as physical barriers to reduce the incidence of MAMP binding, or by directly binding to the receptor themselves. Thus, prebiotics need not exert all of their beneficial effects exclusively by growing commensal bacteria. One mechanism for psychobiotic effects is the mitigation of low-grade inflammation, typically observed as reductions in circulating pro-inflammatory cytokine concentrations. Pro-inflammatory cytokines are also capable of increasing the permeability of the blood–brain barrier, permitting access to potential pathogenic entities. Cytokines alter concentrations of several neurotransmitters that regulate communication in the brain, including serotonin, dopamine, and glutamate. Cytokines can also enter the brain through active uptake, stimulating secretion of pro-inflammatory substances such as prostaglandins , precipitating further inflammation. There is also emerging evidence of a lymphatic drainage system subserving the brain , which we speculate may allow cytokines to interact with neural tissue.

A parallel mechanism underlying psychobiotic-induced reductions in inflammation is the increase of anti-inflammatory cytokines such as interleukin-10. For example, in humans, Bifidobacterium infantis 35624 and Lactobacillus GG have been shown to enhance concentrations of interleukin-10. By reducing the total quantity of pro-inflammatory cytokines, either directly or by increasing anti-inflammatory cytokines, psychobiotics may be reducing the probability of cytokines gaining access to the central nervous system, and may also be restoring inflammation-induced permeability of the blood–brain barrier. Cytokine interactions at the blood–brain barrier are highly complex and more detailed discussions of those mechanisms are beyond the scope of this review. The reader is referred to existing research in this area.

A parasitic infection study yielded an important mechanistic insight regarding cytokine roles in microbiome–brain signalling. Healthy male AKR mice were infected with the Trichuris muris parasite, following which they were treated with Bifidobacterium longum NCC3001, Lactobacillus rhamnosusNCC4007, or vehicle. Infection increased anxious behaviour and reduced hippocampal BDNF mRNA levels. Bifidobacterium longum NCC3001 (but not Lactobacillus rhamnosus NCC4007) reduced anxious behaviour and normalised BDNF mRNA concentrations. However, these changes occurred in the absenceof prebiotic-induced reductions in any pro-inflammatory cytokines. This may be interpreted as evidence that psychobiotic effects also occur through mechanisms other than cytokine reduction. Pro-inflammatory cytokines are also known to compromise the integrity of the gut barrier . For example, Lactobacillus rhamnosus GG ameliorates gut barrier dysfunction by inhibiting the signalling potential of pro-inflammatory cytokines such as tumour necrosis factor-α.

The microbiome also plays a substantial role in broader immunological functions. For example, the presence of bacteria such as Bifidobacterium infantis35624 and Lactobacillus salivarius UCC118 in the gut has been shown to affect immunogenic responses toward pathogenic entities such as Salmonella typhimurium. The microbiome also contributes to the development of the immune system. For example, the expression of colonic effector pro-inflammatory genes that are sensitive to the action of interleukin-10 is bacteria-dependent. Exogenous bacteria can also trigger the development of immunogenic responses under certain conditions. For instance, the introduction of Helicobacter hepaticusin the presence of genetically-deficient interleukin-10 signalling systems led to an increase in pro-inflammatory marginal zone B cells (in the category of white blood cells, expressing antibodies) of the spleen. The microbiome also controls the development of appropriate immunosuppression in response to dietary antigens through the production of immunosuppressive regulatory T-cells. These cells prevent full immunogenic reactions to normal nutritional input, whereas germ-free mice do not possess this immunosuppressive activity and show exaggerated immune responses to dietary antigens. These bacteria–immune interactions illustrate boundary conditions for psychobiotics. For instance, certain genetic abnormalities that alter the immune system may result in unexpected psychobiotic effects.