Role of microbiota in controlling hormone release associated with exercise-induced stress Elite athletes who train and compete for hours experience physical and emotional stress that causes shifts in physiological homeostasis stimulating the SAM and HPA axis [2] (Fig. 1). As reviewed by Ulrich-Lai et al [6], the SAM system, which is part of the sympathetic division of the autonomic nervous system, releases epinephrine from the medulla or center of the adrenal gland, which facilitates rapid mobilization of metabolic resources and regulation of the fight/flight response. It generally increases circulating levels of adrenaline (primarily from the adrenal medulla) and noradrenaline (primarily from sympathetic nerves), heart rate and force of contraction, peripheral vasoconstriction, and energy mobilization [6]. Parasympathetic tone can also be modulated during stress [6] (Fig. 1). On the other hand, stress stimuli activates the hypothalamic paraventricular nucleus (PVN) that interconnects with the bed nucleus of the stria terminalis (BNST) [6]. These neurons synthesize corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), which are released into the hypophysial portal circulation and transported to the anterior pituitary gland, where they stimulate the release of adrenocorticotropin (ACTH) into the systemic circulation [6]. ACTH interacts with receptors on the cortex of the adrenal gland to stimulate the production and release of glucocorticoids (GCs) into general circulation [6]. The effect of GCs depends upon the receptors to which they bind. There are two GC receptors: mineralocorticoid receptor (MR) and glucocorticoid receptor (GR). Outside the brain, GCs operate through GRs, whereas in the brain, GCs bind to both MR and GR (reviewed by Ulrich-Lai et al [6]). GRs mediate most of the stress effects of glucocorticoids (including metabolism and immunity). By binding to the GR, GCs inhibit the further release of CRH, thereby regulating both the basal HPA tone and the termination of the stress response [6]. MRs foster cellular activation (hippocampus) and mediate most of the basal effects, which include maintaining responsiveness of neurons to their neurotransmitters, maintaining the HPA circadian rhythm and maintaining blood pressure [29]. Acute physical exertion above 60% maximal oxygen uptake (V O2max ) is one of the physical stresses that stimulates the HPA axis and release of stress and catabolic hormones [30], whereas exercising below this intensity fails to cause such a spike in serum cortisol [31]. Exercising at 80% capacity has been shown to provoke a significant increase in ACTH levels pre- and post-exercise [31]. Furthermore, comprehensive studies in endurance athletes conducted by Lehmann [32, 33] during the past 20 years have shown that 60-80% of athletes in the early stage of chronic stress have higher pituitary CRH-stimulated ACTH response. Therefore, there is a clear connection between exercise-induced stress and increased stress hormone levels in athletes. Stress during exercise also activates the ANS [7], which increases the neuronal release of NE and other neurotransmitters in peripheral tissues such as the GI tract. Exercise and gut symptomatology have long been connected (reviewed by Cronin et al [34]). The bidirectional communication between the ANS and the ENS in the GI tract, the gut-brain axis, mainly occurs by way of the vagus nerve, which runs from the brainstem through the digestive tract (reviewed by Carabotti [35]). Beyond neuronal connection, other ways of communications between the gut-brain axes are via gut hormones [9] and gut microbiota molecules [10, 36]). There is increasing evidence that GI tract responds to stress by releasing hormones such as GABA, neuropeptide Y (NPY) and dopamine (reviewed by Holzer [37]). GABA, which is the body's dominant inhibitory neurotransmitter of the CNS, regulates blood pressure and heart rate and plays a major role in various gastrointestinal functions such as motility, gastric emptying and transient lower esophageal sphincter relaxation, as well as anxiety, depression, pain sensation and immune response [38]. Moderate exercise can increase GABA levels in the hypothalamus resulting in lower resting blood pressure, heart rate and sympathetic tone [39]. Under forced swimming in 25 °C water, de Groote and Linthorst [40] found that hippocampal GABA levels in rats decreased (70% of baseline). However, to discriminate between the psychological and physical aspects (i.e. the effects on body temperature) of forced swimming, another group of animals was forced to swim at 35 °C [40]. This later stressor, like novelty, caused an increase in hippocampal GABA (120% of baseline), suggesting a stimulatory effect of psychological stress [40]. NPY is also released in response to various stress stimuli, such as intense exercise, in the GI tract and plays a role in attenuates the HPA axis [41]. The NPY is a 36-amino acid peptide located throughout the gut-brain axis and is the most prevalent neuropeptide in the brain that plays a role in stress resilience, and inflammatory processes [42]. Rämson et al [41] studied NPY serum levels in 12 highly trained rowers and found that the post-exercise concentrations of NPY increased significantly. Though few studies have studied serum and hippocampal NPY levels in response to exercise, these results suggest it plays a role in reducing the stress response upon intense exercise [41]. Lastly, dopamine, the precursor to NE and epinephrine, can also be synthesized during stress in the GI tract. Dopamine production is dependent upon several factors: levels of its precursor tyrosine, enteric bacteria that directly produce dopamine, the type of stress experienced and sex [43]. There are several dopamine receptors throughout the intestines suggesting it plays a role in the gut-brain axis [43]. The GI tract, spleen and pancreas produce substantial amounts of dopamine [43]. The rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase, is found in human stomach epithelial cells showing its function exist beyond neurotransmission in the brain [43]. Habitual physical activity for about 1–2 hours a day has been shown to increase dopamine levels in the brain [44]. 45 21 46 47 47 1 48 48 8 49 50 8 Molecule Probiotic Strain, microbial metabolite Species Effects Tryptophan- precursor to 5-HT Bifidobacterium infantis Rats Aids in combating psychiatric disorders such as depression [84] Lactobacillus johnsonii In vitro model Reduces serum kynurenine concentrations and IDO activity in vitro in HT-29 colonic cells, which prevents the breakdown of tryptophan [138] Serotonin Lactococcus lactis subsp. Cremoris (MG 1363), Lactococcus lactis subsp. Lactis (IL 1403), Lactobacillus plantarum (FI8595), Streptococcus thermophilus (NCFB2392), Eschericchia coli K-12, Morganella morganii (NCIMB, 10466), Klebisella pneumoniae (NCIMB, 673), Hafnia alvei (NCIMB, 11999) In vitro model Produce serotonin [47, 139] Butyrate and acetate produced by bacteria Mice Induce serotonin synthesis in a dose-dependent manner by regulating the gene Tph1 that synthesizes serotonin [68] Dopamine Bacillus cereaus, Bacillus mycoides, Bacillus subtilis, Proteus vulgaris, Serratia marcescens, S. aureus, E.col, E.coli K-12, Morganella morganii (NCIMB, 10466), Klebisella pneumoniae (NCIMB, 673), Hafnia alvei (NCIMB, 11999) In vitro model Produce dopamine [140–142] GABA Lactobacillus rhamnosus (JB-1) Mice Regulates GABA receptor expression and reduced stress-induced corticosterone, anxiety and depression [143] Bifidobacterium dentium DPC6333, Bifidobacterium dentium NCFB2243, B. infantis UCC35624, Bifidobacterium adolescentis DPC6044, Lactobacillus brevis DPC6108, Bacillus mycoides, Bacillus subtiles, Proteus vulgaris, Lactobacillus rhamnosus YS9 Humans Lactobacillus brevis DPC6108 was the most effective at producing GABA [8]. Cortisol Lactobacillus helveticus R0052; Bacteroidetes longum R0175 Humans Reduce urinary free cortisol output [133, 144] Noradrenaline B. mycoides, B. subtilis, P. vulgaris, S. marescens, E. coli K-12 In vitro chromatography Regulates motility and secretions in the ENS. Elevated levels due to acute stress can cause the growth of pathogenic E. coli [140]. Recent studies and literature about gut-brain axes have focused on the role of microbiota and its molecules on controlling anxiety and depression (reviewed by Foster []). However, the role of microbiota in controlling exercise-induced stress adaptation remains unknown. The use of germ-free (GF) animals has provided one of the most significant insights into the role of the microbiota in regulating the development and function of the HPA axis in response to stress []. In GF mice, a mild restraint stress induced an exaggerated release of corticosterone and ACTH compared to the specific pathogen free (SPF) controls, thus elucidating a connection between the gut microbiota and HPA axis []. This aberrant stress response in GF mice was partially reversed by colonization with fecal matter from SPF animals and fully reversed by mono association of Bacillus infantis in a time dependent manner []. Thus, the gut microbial composition is critical to the development and function of an appropriate stress response and HPA axis []. In addition, there is increasing evidence shows that the commensal and resident community of gut microorganisms can regulate the HPA axis through the synthesis of hormones and neurotransmitters such as GABA, dopamine and serotonin (Table). Asano et al [] discovered that SPF mice had substantially higher levels of free, biologically active dopamine and NE in the gut lumen of the ileum and colon than GF mice. Moreover, GF mice treated with Clostridium species, fecal flora from the SPF mice or E. coli showed elevated levels of free catecholamines suggesting that the gut microbiota plays a role in their synthesis through dopamine regulation []. Moreover, other mouse studies suggest that the vagus nerve serves as some sort of 'hotline' by which gut microbes communicate directly with the CNS []. For instance, Bravo et al [] have found that Lactobacillus strain affects the CNS by regulating emotional behavior and central GABA receptor expression via the vagus nerve. Given the apparent link between early-life events and subsequent adult neurogenesis response to stress [], researchers need to understand whether the potential effects of disruptions to the microbiota in childhood might affect the neurobiology of stress and endocrine function of the microbiota. What is still missing is solid evidence that demonstrates gut microbiota causality in stress []. Of growing interest is how the gut microbiota interact directly with stress hormones in peripheral tissues such as the mucosal layer of the GI tract, which is called microbial endocrinology [10]. NE has shown to have a direct effect on gut Aeromonas hydrophila, Bordetella spp., Campylobacter jejuni, Helicobacter pylori, Listeria spp. and Salmonella enterica spp. among others. Some of the ways that NE can promote pathogenic bacterial growth is by facilitating E. coli to adhere to the intestinal wall by increasing the expression of its virulence factor K99 pilus adhesin as well as activating the expression of virulence-associated factors in Salmonella typhimurium, which then makes infection by these bacteria easier [10]. Additionally, NE has also been shown to increase levels of non-pathogenic E.coli and other gram-negative bacteria [51]. Currently, only one study has shown that exercise-induced stress directly modifies gut microbiota composition in non-GF or SPF animals. Allen et al [52] recently published the first study that increases the understanding of how the microbiome regulates the exercise-induced stress response, revealing unique microbiota-host interactions that are important for gastrointestinal and systemic health [52]. Voluntary wheel running for 6 weeks attenuated symptoms, whereas forced treadmill running exacerbates intestinal inflammation and clinical outcomes in a colitis mouse model [52]. Fecal and cecal levels of Turicibacter spp., which has been strongly associated with immune function and bowel disease, were significantly lower in voluntary runners compared to the 6 week forced treadmill running group. Additionally, Ruminococcus gnavus, which has well defined roles in intestinal mucus degradation, was increased in the forced group compared to sedentary group [52], together with Butyrivibrio spp., Oscillospira spp., and Coprococcus spp. This preliminary study in exercised and stressed-animals shows that physical activity can alter microbiota composition as well as metabolic function that could either positively or negatively affect performance.

Role of microbiota in controlling gastrointestinal symptoms associated with exercise-induced stress 53 54 54 54 2 55 56 56 57 2 57 58 59 Proper intestinal barrier function is crucial for maintaining immune and overall health []. There are more than 50 proteins that play an important role in regulating the tight junctions of the mucosal endothelial layer and thus intestinal permeability []. The tight junction complexes consist of 4 trans membrane proteins: occludin, claudins, junctional adhesion molecules and tricellulin that interact with the structural zonula occludens proteins (ZO1, ZO2 and ZO3) []. Under normal conditions, the tight junction complexes work to maintain the polarization of the intestinal barrier that controls the paracellular passage of only small molecules such as ions, water and leukocytes []. The intestinal barrier also serves as a doorway between the microorganisms and their byproducts, the enteric immune system response and the nutrient particles inside and outside the GI tract (Fig.) []. As detailed in a review based on acute effects of exercise on immune and inflammatory indices in untrained adults [], increased intestinal permeability, or “leaky gut” as it is commonly called, is a loosening of the tight junction protein structures. An excessive release of stress hormones induced by physical and psychological stress can cause lipopolysaccharides (LPS) translocation outside of the GI tract triggering immune and inflammatory responses often resulting in increased intestinal permeability []. The translocated LPS are detected by CD14 and toll-like receptor 4 (TLR4), which causes the release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), interferon alpha (IFNα), interferon-gamma (INFγ) and interleukins (IL1β or IL6), which can eventually result in endotoxemia [] (Fig.). These pro-inflammatory cytokines also increase the opening of the tight junctions through ZO1 and ZO2 pathways of tight junction protein complexes that can result in endotoxemia []. Additionally, the activation of the HPA axis may stimulate subepithelial mast cells to secrete immune mediators such as histamine, proteases and pro-inflammatory cytokines [], trigging intestinal permeability []. Depending on the type of exercise, intensity, age and other factors, between 20-50% of athletes suffer gastrointestinal symptoms, which have been shown to increase with exercise intensity [60]. In a study of 29 highly trained male triathletes, Jeukendrup et al [61] discovered that upon competition, 93% reported digestive disturbances and two participants had to abandon the race because of severe vomiting and diarrhea. According to expert review [53], hyperthermia, ischemia and hypoperfusion are other severe stimuli that can cause a loosening of the tight junctions during intense exercise. These are common occurrences among athletes as body temperature increases and blood pools away from the GI tract to periphery muscles and organs such as the heart and lungs during intense physical activity [62]. The redistribution of blood flow away from the intestines together with thermal damage to the intestinal mucosa can cause intestinal barrier disruption, followed by an inflammatory response [63]. In healthy young adult male cyclists who performed endurance sports activities during 4–10 hours per week, just one hour of physical activity at 70% maximum workload capacity produced splanchnic hypoperfusion, which can cause decreased GI circulation, increased intestinal permeability and damage to the small intestine [64]. Another study showed humans exercising at 70% V O2max presented a 60-70% reduction in splanchnic blood flow, and exercise-induced ischemia caused increased intestinal permeability when blood flow was reduced by 50% [65]. Ischemia also increases reactive oxygen species (ROS) production, which induces leaky gut as activated protein kinases phosphorylate tight junctions proteins resulting in hyperpermeability (Fig. 2) [57]. Hydrogen peroxide can also serve as a signaling molecule that activates transcription of several pro-inflammatory genes including nuclear factor kappa-light-chain-enhancer of activated B cells (NFKβ), TNFα, IL6, IFNγ, and IL1β [65] that can compromise barrier function. Therefore hypoperfusion and ischemia can result in increased intestinal permeability opening the door for LPS and enteric bacteria to circulate the bloodstream, possibly leading to endotoxemia. For instance, marathon, triathlete and ultra endurance athletes have been reported to have plasma LPS concentrations of 5 to 284 pg/mL along with up to 93% of athletes reporting digestive disturbances, which could be caused by the LPS-induced cytokine response [61]. Brock-Utne et al [66] discovered that 81% of randomly selected exhausted marathon runners showed endotoxemia (0,1 ng/mL), 2% presented lethal levels above 1 ng/mL and only 19% had normal levels. In addition, 58 out of the 72 runners who experienced high LPS levels also suffered from GI upset such as nausea, diarrhea and/or vomiting, whereas only 3 out of the 17 runners who had low plasma endotoxin concentrations reported such symptoms. The marathon participants who took more than 8 hours to complete the race suffered higher plasma endotoxin concentrations [66]. In a study in 18 triathletes who competed in an ultra triathlon that was 90 km long, their mean plasma LPS concentrations increased from 0.081 to 0.294 ng/mL, and their mean plasma anti-LPS immunoglobulin G concentrations decreased from 67.63 to 38.99 μg/mL. Starkie et al. [67] studied heat stress and immune response after intense cycling in seven healthy male athletes. Glucocorticoids released during intense exercise has also been shown to diminish the TLR expression, and therefore the capacity to produce anti-inflammatory cytokines and host antimicrobial defense [68]. All these studies show that intense exercise during prolonged periods not only can lead to increased intestinal permeability and thus increased plasma LPS levels, but it can also cause immunosuppression [69]. Given the gut microbiota’s diverse role in GI function, enteric immunity [70], endocrinology [11] as well as regulating oxidative stress [71–73] and hydration levels, it is not surprising that efforts to identify the mechanisms by which gut microbiota improves intestinal barrier function of elite athletes are increasing. In the colon and cecum, complex plant-derived polysaccharides are digested and subsequently fermented by gut microorganisms, such as Lactobacillus, Bifidobacterium, Clostridium, Bacteroides, into SCFAs and gases which are also used as carbon and energy sources by specialized bacteria such as reductive acetogens, sulfate-reducing bacteria and methanogens (reviewed by Marchesi et al [36] and by Flint et al [19]). Acetate, propionate, and N-butyrate are present at a molar ratio of approximately 60:20:20 in the colon and feces [74]. The composition of the gut microbiota, metabolic interactions between microbial species [52] and the amount and type of the main dietary macro- and micronutrients determine the types and amount of SCFAs produced by gut microorganisms [75, 76]. The more plant-derived polysaccharides, oligosaccharides, resistant starch and dietary fiber one eats, the more these bacteria can ferment these indigestible food sources into beneficial SCFA. The microbiota-produced SCFAs affect a range of host processes including control of colonic pH, with consequent effects on microbiota composition, intestinal motility, gut permeability and epithelial cell proliferation [77]. N-butyrate produced by gut bacteria regulates neutrophil function and migration, inhibits inflammatory cytokine-induced expression of vascular cell adhesion molecule-1, increases expression of tight junction proteins in colon epithelia and exhibits anti-inflammatory effects (reviewed by Nicholson [20]) . N-butyrate and propionate can increase transepithelial resistance which improves intestinal barrier function and decreases inflammation [78]. They also serve as a primary energy source, about 60-70%, for colonocytes [74], which prevents mucosal degradation [79] that can occur as a result of intense exercise due to hypoperfusion and ischemia for example. Matsumoto et al [80] performed a study in 14 male Wistar rats during five weeks. The control group was sedentary and the exercise group had access to an exercise wheel in their cage. They discovered through 16S rRNA gene sequencing that the rats that voluntarily exercised using the wheel presented higher cecum levels of SCFA than the sedentary control group. Levels of N-butyrate increased significantly between the exercise groups (8.14 ± 1.36 mmol/g of cecal contents) compared to the control group (4.87 ± 0.41 mmol/g of cecal contents) [80]. The cecum was approximately 1.5 times larger in the exercise group than in the control group, and the cecal tissue weights and contents were much greater in the exercise group than in the control group, indicating that a significant change had occurred in the cecal environment in response to voluntary wheel running [80]. Additionally, the cecal microbiota and SCFA profiles were much different between the exercise and control groups [80]. In general, exercise-induced stress can diminish intestinal barrier function and cause LPS translocation which results in GI upset, hydration imbalances, poor uptake of nutrients and electrolytes, as well as thermal damage of the intestinal mucosa, all of which negatively affect athletic performance [64]. Although there are few studies that show the effect exercise has on SCFA levels in the cecum and energy metabolism, those that do exist, illustrate how intense exercise affect SCFA production, which in turn affects the HPA axis, the GI health and may promote favorable athletic performance.

Role of microbiota in controlling the mood disturbances, fatigue, insomnia and depression associated with exercise-induced stress Many athletes who suffer from stress enter into a vicious cycle of over exerting themselves with strenuous training and competitions, which results in fatigue causing them to over train in order to overcome fatigue and decreased athletic performance [4]. Some scientists believe that evaluating the athlete's mood is the best way to tell if someone is suffering from stress as it is one of the most common symptoms [81]. To date, several biological mechanisms have been proposed to explain exercise induced mood disturbances, fatigue, insomnia and depression in athletes: (i) metabolic changes in muscle that ultimately lead to muscle exhaustion, and (ii) modifications in the CNS, which are termed central fatigue. 82 3 83 3 8 83 84 82 21 85 86 The central fatigue hypothesis states that increased of the neurotransmitter serotonin (5-hydroxytryptamine; 5-HTP) release is associated with sleep, drowsiness and central fatigue, which contribute to suboptimal physical performance (reviewed by Best et al [], Fig.). Furthermore, low serotonin in the brain also causes mood disorders and depression as well as changes in gut transit, blood pressure, cardiac function and platelet aggregation (reviewed by Evans et al [], Fig.). Approximately 95% of the body's serotonin is produced in the enterochromaffin cells (EC) of the intestines [], which plays a role in enteric motor and sensory functions such as visceral pain perception, further illustrating the gut-brain connection []. According to a review conducted by Best et al [], about 2% of ingested tryptophan is used for the synthesis of serotonin. However, during exercise, serotonin levels might also be increased through other known pathways: (i) kynurenine pathway [], and (ii) gut microbiota synthesis []. Once in the CNS, L-tryptophan is converted from tryptophan hydroxylase (TPH) into 5-HTP, the rate limiting step in brain serotonin synthesis [87]. The 5-HTP is then rapidly decarboxylated by the aromatic amino acid decarboxylase (AADC) to produce cytosolic serotonin [82]. For many years, a single gene encoding 5-TPH was believed to be responsible for serotonin biosynthesis in vertebrates. However, Walther and et al [88] have reported the existence of two distinct TPH genes in humans: TPH1 and TPH2. Of these enzymes, TPH1 is expressed in the periphery and in the pineal body, whereas TPH2 appears to be responsible for the synthesis of serotonin in the rest of the brain. Therefore, TPH could reflect an adaptation to different needs for regulation of serotonin production in the brain and peripheral organs [87]. Serotonin production may also occurs via the kynurenine pathway that is regulated by the tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO) [21]. IDO is stimulated by oxidative stress and pro-inflammatory cytokines such as IL6 and TFNα, which are released due to LPS-induced intestinal permeability experienced during intense exercise [89]. On the other hand, glucocorticoids can activate TDO [90, 91], and there is a growing body of evidence suggesting that a hyperactive HPA axis often co-occurs with depression due to increased levels of glucocorticoid hormones, systemic inflammation and increased production of pro-inflammatory cytokines [90] all of which are released due to exercise-induced stress and increased intestinal permeability. Consequently, glucocorticoids and pro-inflammatory cytokines induce TDO and IDO enzymes leading to less serotonin synthesis and possibly fatigue and depression that many athletes who suffer from stress. A very recent review on the microbiome [8] describes that microbiota also influence production of serotonin (Fig. 3). For instance, a study led by Yano et al. [92] has demonstrated in mice that indigenous spore-forming microbes directly stimulated intestinal serotonin synthesis and release. When focusing to the interaction between serotonin and exercise, running at low speed appears to increase cerebral serotonin levels and decrease depressive and anxious behavior, whereas high-speed running causes an increase in the gene expression of CRH [93]. Additionally, acute aerobic exercise has been shown to increase 5-HTP levels in the brain stem and hypothalamus in rats after swimming for 30 min/day for 6 days per week for 4 weeks [94]. The rise in brain tryptophan is claimed to result from exercise-induced elevations in serum of non-esterified fatty acid concentrations, which dissociate tryptophan from albumin in the blood and increase the serum free tryptophan (reviewed by Fernstrom and Fernstrom [95]). On the other hand, ample evidence shows that the serum free tryptophan does not dictate brain tryptophan uptake, nor do serum tyrosine levels and branched chain amino acids (BCAA) (i.e. leucine, isoleucine and valine), which is postulated to compete with tryptophan to cross the blood–brain barrier. However, Pechlivanis et al [96] reported different results when analyzing 22 serum metabolites of 14 young athletic men who responded to an intermittent sprint training program involving a very short recovery interval and another program with a longer recovery interval for eight weeks at 80% of V O2max . They discovered the leucine, valine and isoleucine decreased after pre-training exercise in both groups, suggesting that BCAA were probably taken up by the muscles during exercise possibly allowing more free tryptophan to cross the blood–brain barrier enabling serotonin synthesis. Of note is that increased lactate levels can also cause fatigue in athletes, not just serotonin synthesis caused by an influx of free tryptophan entering the brain. Nevertheless, Fernstrom et al [95] state that the central fatigue hypothesis is weak because there lacks evidence that shows what specifically causes an increase in brain tryptophan during exercise [95]. In regard to the central fatigue hypothesis, there is overwhelming evidence that the involvement of other molecules could contribute to central fatigue (reviewed by Foley and Fleshner [97] and by Foley [97]). It is suggested that altered dopaminergic pathways involving movement lead to fatigue (reviewed by Foley [97]). Fatigue can set in during exercise when dopamine levels start to drop while serotonin levels are still elevated [98]. The precise mechanisms for how a reduction in brain dopamine could impair exercise performance and influence central fatigue are yet not fully understood. Attempts have been made to prolong dopamine neurotransmission during exercise to fatigue. For example, manipulations of tyrosine and dihydroxyphenylalanine availability are just one instrument used to increase dopamine synthesis during exercise [97]. Further examples are depicted below (section “dietary recommendations to reduce exercise-induce stress behaviour”). Like 5-HTP, dopamine cannot easily cross the blood–brain barrier [97]; therefore, neurons must synthesize dopamine from its precursor tyrosine that’s ingested from the diet [97]. Tyrosine must compete with other amino acids for entry into the brain, including tryptophan and the BCAA, as they are mediated by the same carrier system (reviewed by Foley [97]). However, unlike tryptophan hydroxylase, the brain levels of tyrosine hydroxylase are saturated with substrate under normal conditions, and therefore, any attempt to increase the tyrosine concentrations cannot produce significant increases in dopamine [99]. Moreover, dopamine is an important neurotransmitter associated with motivation and reward [97]. Voluntary wheel running have been rewarding for rats in various experiments, but repeated exposure to natural rewards, like habitual exercise, can modify dopaminergic neuro circuitry negatively altering the motivation and reward centers in the brain associated with exercise resulting in fatigue [97]. On the other hand, moderate aerobic exercise has been shown to increase dopamine levels while reducing serotonin levels in the nigrostriatal tract [44], illustrating that exercise can greatly alter neurotransmitter metabolism. Other neuromodulators that may influence fatigue and mood during exercise include pro-inflammatory cytokines and ammonia. Increases in pro-inflammatory cytokines like IFNγ and IL6 have been associated with reduced exercise tolerance, acute viral or bacterial infection and increased tryptophan catabolism which could thus limit brain serotonin synthesis [100], leading to depressive behavior. Accumulation of ammonia in the blood and brain during exercise could also negatively affect the CNS function causing fatigue. Guezennec et al [101] investigated if exhaustive exercise increased ammonia detoxification in the brain mediated by glutamine synthesis which, subsequently would influence glutamate and GABA levels. They discovered that both trained and untrained rats that ran until exhausted presented an increase in serum ammonia, which can reduce brain energy by stimulating the Krebs cycle and glycolysis. The trained exercise group had levels of ammonia 50% higher than the untrained group and also presented lower levels of the excitatory neurotransmitter glutamate as well as a decrease in GABA in the striatum of the brain [101]. These findings show that exercise stimulates glutamine synthesis that’s used for ammonia detoxification resulting in decreased production of the excitatory neurotransmitter glutamate possibly causing fatigue in endurance athletes [101]. Glutamine, a nonessential amino acid that’s the most abundant in the human body [102], is crucial not only for the glutamate and GABA synthesis but also for optimal functioning of leukocytes such as lymphocytes and macrophages, T cell proliferation and function [104], intestinal enterocytes growth [102]. Therefore, prolonged intense exercise could negatively affect neurotransmitter homeostasis and immune response when glutamine levels are depleted [80, 81] in order to detoxify ammonia in the brain causing a more excitatory-glutamate driven response to exercise-induced stress and a decline in GABA-mediated inhibitory pathways. Additionally, depleted serum glutamine levels mean less uptake in the intestines leaving the enterocytes more susceptible to intestinal permeability [105]. The influence of gut microbiota on behavior is becoming increasingly evident [85]. As explained above, the gut microbiota serves as an endocrine organ in many ways facilitating the production and regulation of various neurotransmitters and hormones (Table 1), which can affect an athlete’s mood, motivation, and sensation of fatigue (Fig. 3). There is strong evidence that low levels or an absence of gut microbiota (i.e. GF animals) increase tryptophan and serotonin levels and modify central higher order behavior [106]. Desbonnet et al [107] administered the probiotic strain Bifidobacterium infantis during 14 days in naive rats who performed a forced swim test. Although the probiotic had no effect on swim performance, there was a significant reduction of IFNγ, TNFα and IL-6 in the probiotic-treated rats compared to controls, and there was a significant increase in plasma concentrations of tryptophan but also kynurenic acid in the bifidobacteria-supplemented rats. GF rats on the other hand had lower levels of tryptophan that increase after administering bifidobacteria species [107]. The authors concluded that this probiotic may have antidepressant effects and illustrates how gut bacteria can ultimately modulate serotonin levels [107]. Moreover, butyrate at the levels of 8 and 16 mM can indirectly affect serotonin synthesis in a dose-dependent manner by regulating the gene TPH1 in EC [108], which reinforce the role that bacteria has on behaviour regulation. More research is needed to show how certain bacteria strains can modulate neurotransmitters metabolism after strenuous activity in humans, while increasing the serum glutamine and the GABA production.