We think of ourselves as autonomous individuals with a will of our own, but microbes in our gut turn out to have more say on how we feel and behave than we know.
Communication between the gut and the brain has long been acknowledged and has led to the gut being dubbed the ‘second brain’. This gut-brain communication was previously thought to be regulated by neural, endocrine and immunological signalling, but now research is focusing on how the gut microbiota impact such signalling in what is now being termed the microbiome-gut-brain axis (see below).
The role of the gut microbiota in gut-brain signalling is well evidenced by the simultaneous presence of mental health-related illnesses such as anxiety with gastrointestinal disorders such as irritable bowel syndrome (IBS) or inflammatory bowel disease (IBD) . Research dating back to the 1970s showed that stress alters the composition of the gut flora in adult mice . Since then, newborn mice suffering maternal separation-induced stress [3, 4] were found to have reduced level of Lactobacilli, which makes the animals more susceptible to infection. Further, hepatic encephalopathy (the occurrence of confusion, altered level of consciousness, and coma as a result of liver failure) is successfully treated with antibiotics or laxatives, thereby serving as a reminder that gut bacteria do send signals to the brain (albeit under pathological conditions in this case) . Indeed, the gut is thought to harbour the majority of the body’s microbes and recent work from the Human Microbiome Project reveals large variability in microbiota profiles between individuals, an average gut carrying around 1000 different species of microbes and more than 7000 strains.
How do gut microbes signal to the brain?
There are many ways in which the gut microbiota signal the brain (see ):
The composition of the micriobiota itself is one very dynamic factor that changes with diet, age, location, disease and so on. The microbiota composition determines competition for dietary ingredients as growth substrates, conversion of sugar into inhibitory fermentation products, production of growth substrates, release of bacteriocins (molecules toxic to other bacterial species), stimulation of the innate immune system, and competition against microbes colonizing the gut wall and gut-barrier function.
Another mechanism is through immune activation as the immune system signals bi-directionally with the brain. The circulation of pro- or anti-inflammatory cytokines is indirectly mediated by microbiotic influence on the innate immune system, which can then impact directly on brain function. The innate and adaptive immune system is also crucial for gut health in maintaining homeostasis of the intestinal host-microbial interface.
The vagus nerve or cranial nerve is the main nerve of the parasympathetic nervous system and mediates many organ functions including bronchial constriction, heart rate and gut mobility; transmitting information on the luminal environment such as hyperosmolarity, carbohydrate levels and presence of bacterial products. Vagotomy studies have linked certain gut-brain signalling to this nerve. Its activation also has anti-inflammatory effects.
Many microbes produce neurometabolites that are either neurotransmitters or modulators of neurotransmission including GABA (γ-aminobutyric acid) produced by Lactobacillus spp. and Bifidobacterium spp, noradrenaline produced by Escherichia spp, Bacillus spp and Saccharomyces spp, serotonin from Candida spp, Streptococcus spp, Escherichia spp and Enterococcus spp.], dopamine from Bacillus spp, and acetylcholine from Lactobacillus spp. These could act directly on nerve terminals in the gut or via ‘transducer’ cells such as enterochromaffin cells present throughout the intestinal tract and are accessible to microbes and in contact with afferent and efferent nerve terminals. Some of these cells may also signal and therefore modulate immune cell activity.
Microbial metabolites are produced by microbes when modulating the host metabolic reactions, including bile acid, short chain fatty acids and choline. Carbohydrates from dietary fibre are also broken down by microbes, resulting in the production of neuro-active chemicals such as n‑butyrate, acetate, hydrogen sulfide and propionate. Alterations or overproduction of certain metabolites are associated with brain disorders such as autism (see below).
Tryptophan metabolism is thought to be dysregulated in many digestive and brain disorders. Tryptophan is an essential amino acid that cannot be produced by the human body and must be provided by diet or gut bacteria. It is the precursor to many neuroactive agents including serotonin, which regulates gut motility and is also an important neurotransmitter that mediates mood and wellbeing. The kynurenine arm of tryptophan metabolism mediates 95 % of peripheral tryptophan levels. The kynurenine:tryptophan level is dysregulated in germ free animals as well as models of depression that are successfully treated with probiotics. Kynerenine metabolism can be induced by inflammatory mediators and stress hormones. A study has shown how probiotic bacteria can alter kynurenine concentrations.
Microbiome influence on behaviour
Many newer studies of the microbiome-gut-brain axis have looked at the regulation of the hypothalamic-pituitary-adrenal (HPA) that regulates the body’s reaction to stress. The HPA axis is a major part of the endocrine system that also regulates many other processes including digestion, immune response, mood and emotion, sexuality and energy storage & expenditure. Chronic over-activation of the HPA axis can have knock-on effects for learning and memory, anxiety and depression.
Early experiments comparing germ-free and germ-colonised mice showed that the activity of the HPA axis is exaggerated in germ-free mice, which is reversed following colonisation with the probiotic bacteria Bifidobacterium infantis ; and the earlier the colonisation, the fuller the reversal of effects.
However, the exact opposite observations have also been documented, with reduced anxiety being reported in germ-free mice [8, 9]. The reason for such discrepancies remains unclear, but may be due to the composition of the microbiota varying in the mice in different studies. At the behavioural level, studies suggest germ-free animals are bolder and show less anxiety on anxiety-tests such as the elevated plus maze or light-dark boxes, which test the animals’ aversion to open or light spaces . These changes were accompanied by altered NMDA and serotonin neurotransmitter receptor expression in the brain.
Studies have also been done by infecting animals with microbes and assessing physiology and behaviour. Infection with Trichuris muris, which is closely related to the human parasite Trichuris trichiura known as whipworm, leads to chronic inflammation . Infected mice showed increased anxiety-like behaviour, a rise in plasma kynurenine:tryptophan ratio and plasma levels of the pro-inflammatory cytokines tumour necrosis factor-α and interferon-γ. The mice also showed reduced expression of brain derived neuronal growth factor (BDNF) in the hippocampus, the area of the brain involved in learning and memory. Lactobacillus rhamnosus (JB-1) ingestion improved anxiety-like behaviour in mice, reduced the levels of the stress hormone corticosterone and also altered the expression of GABA neurotransmitter receptors in the brain, as consistent with findings in anxiety and depression-related disorders.
Anxiety levels can differ between strains of lab mice, a trait that has been exploited in microbiome studies. The Balb/c strain tends to be more timid and anxious compared with the NIH Swizz strain that is described as more gregarious. By generating germ-free animals of each strain and then infecting them with faecal content from colonised non-germ-free animals of the opposing strain, the behavioural phenotype was transferred between strains . This example of how behavioural traits can be transferred highlights the importance of the gut microbiota on behaviour and personality. Animal studies are being backed up by research on humans. A study published in 2013 showed that ingestion of a probiotic-containing fermented milk beverage by healthy women attenuated emotive stress-induced changes in brain activity and connectivity as assessed by functional magnetic resonance imaging (fMRI) . Another study of 66 healthy volunteers found that ingestion of probiotic combination for 30 days reduced psychological distress .
Learning and memory experiments have also shown how the gut microbiota can impact cognitive function. Germ-free mice showed stress-induced memory dysfunction  while diabetic mice with impairments in learning and memory were improved through probiotic supplementation . Whether these impairments in learning are a direct result of microbiome disturbance or an indirect result of stress or diabetes respectively remains unclear however.
Disruption of the microbiome and disease
With all this research revealing the importance of the gut microbiome on the gut-brain axis, it seems likely that many nervous system disorders such as pain, autism and multiple sclerosis may result from dysbiosis (microbial imbalance) in the gut.
Some of the most compelling evidence of the importance of the micriobiome-gut-brain axis comes from studies of the microbiota’s mediation of pain, particularly visceral pain. Probiotics were able to reduce pain symptoms associated with inflammatory bowel syndrome-induced pain as well as abdominal pain in animals [17, 18]. This has been associated with changes in the expression of opioid and cannabinoid receptors in the gut, which may be mediated by direct excitation of enteric neurones in the gut that control colonic motility.
With autism, observations of temporary improvements in symptoms following doses of antibiotics or dietary alterations have been noted since the 1990s. Autistic people show alterations in sulphur metabolism and urinary peptide profiles as well as increases in short chain fatty acids and ammonia in the gut. As production of short-chain fatty acids are by-products of anaerobic fermentation, it suggests an overgrowth of anaerobic bacteria such as Clostridia, Bacteriodetes, and Desulfovibrio. Clostridium has indeed been found in excess in the faeces of autistic children.
There is increasing evidence that exposure to Monsanto’s herbicide Roundup, may be an underlying cause of autism spectrum disorders (see ). Glyphosate, the active ingredient, acts through inhibition of the 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS synthase) enzyme in the shikimate pathway that catalyses the production of aromatic amino acids. This pathway does not exist in animals, but it does exist in bacteria, including those that live in the gut and are now known to be as much a part of our body as our own cells. A widely accepted dogma is that glyphosate is safe due to the lack of the EPSPS enzyme in our body. This however does not hold water now that the importance of our microbiota to our physiology is clear.
It has been found that autistic children in the United States are far more likely to be formula-fed, with some milk formulas containing soy and therefore it is most likely GM soy contaminated with glyphosate . Further, dysbiosis occurs in cattle and poultry exposed to glyphosate, with increases in pathogenic strains such as Salmonella and Clostridium and decreases in beneficial bacteria [21, 22]. Roundup has also been shown to be toxic to three beneficial bacterial starter cultures used for the dairy industry . Autistic patients show decreased tryptophan metabolism, while tryptophan depletion leads to exacerbation of autistic symptoms as well as feelings of reduced happiness and calmness. Increased ammonia in autistic patients may be explained by glyphosate’s ability to activate phenylalanine ammonia lyase (PAL), an enzyme in animals as well as gut bacteria, backing up the strong link between autism and hepatic encephalopathy that is also characterised by excessive ammonia. Glyphosate’s ability to impair liver function and therefore the clearance of xenobiotics from the body through inhibition of cytochrome P450 enzymes on top of impairment of serum sulphate transport could further exacerbate the problem. Glyphosate’s disruption of liver function, sulphate transport and the gut flora has also been suggested to contribute to many other diseases including Alzheimer’s, obesity, depression, cancer, infertility, diabetes and heart disease (see ).
Another disease of the nervous system that may be affected by the gut microbiota is multiple sclerosis (MS), a devastating autoimmune disease of the nervous system. Mouse autoimmune encephalomyelitis (EAE) models of MS, show reduced symptoms when they are kept germ-free, suggesting an underlying role of the gut microbiota . Further, mice susceptible to autoimmune encephalomyelitis (EAE) do not get the disease if they are kept in germ-free or specific pathogen-free conditions .
The gut may make up more than 98% of the genes in/on our body that are from the resident microbiota. The microbiota play a crucial role in our physiology. Disruption of their function and composition can have far reaching effects making it critical for us to understand exactly how environmental factors, whether it be antibiotic use, chemical exposure, diet and lifestyle are impacting this incredible symbiosis. The excessive use of antibiotics, particularly in agriculture, as well as harmful chemicals need to be reduced or even banned immediately if we are to curb the rise in chronic diseases in much of the world.