Gut Microbe to Brain Signaling: What Happens in Vagus…

The gut microbiota has emerged as a key player in health and disease. Here we discuss the vagus nerve, which connects the visceral organs and the brain, as an important communication pathway for the gut microbiota to influence brain and behavior. The gut microbiota has emerged as a key player in health and disease. Here we discuss the vagus nerve, which connects the visceral organs and the brain, as an important communication pathway for the gut microbiota to influence brain and behavior. Visceral organs and the central nervous system constantly communicate to provide a sense of the body’s physiological condition and to adapt accordingly to maintain homeostasis. One of the key players in this interoceptive feedback loop is the gut-brain axis, the complex bidirectional communication between the gastrointestinal (GI) tract and the central nervous system that involves endocrine, immune, and neural communication mechanisms, such as that through vagus nerve signaling. Although best studied in the context of food intake and satiety, it is clear that this axis can play a role across many aspects of physiology and behavior. More recently the microbiota (the trillions of microorganisms in and on our body) has been shown to be a key regulator of the gut-brain axis, and thus the microbiota-gut-brain axis has been proposed. However, the regulatory mechanisms occurring are only now being unravelled. In this NeuroView, we focus on one such pathway and describe how the gut microbiota may hijack vagus nerve signaling as a means to alter brain and behavior. The microbiota describes the consortium of different microbes, including bacteria, archaea, fungi, viruses, and parasites that live on our skin, in our lungs, and in the urogenital and GI tracts. Over the preceding decades, the microbiota has received increased attention for its involvement in various diseases ranging from obesity to cancer and has become one of the most reviewed topics in biomedical science. Within the different microbial communities, much attention has been drawn to the one residing in the GI tract, which is the largest and most diverse community of microorganisms. The initial microbiota is acquired at birth and develops alongside its host. Although rather easily perturbed in these early critical years, the microbiota acquires internal stability during the first years of development. This process is regulated by a combination of host genetics and environmental factors such as diet, stress, and exposure to other microbes or antibiotics. Hence, a well-functioning microbiota is highly adapted to the host and the host’s habitat and carries out metabolic and biochemical processes that are important for the host’s function. Although this description is broadly applicable to a stable human gut microbiota, the presence and abundance of bacterial species can vary significantly between individuals. This inter-individual difference is accounted for by the importance of the functions carried out by the microbiota, rather than the individual species, thus allowing unique microbiota composition without losing functionality. The adult microbiota is relatively stable but maintains a certain degree of flexibility to change in response to intrinsic and environmental factors (Figure 1A; Bastiaanssen et al., 2019Bastiaanssen T.F.S. Cowan C.S.M. Claesson M.J. Dinan T.G. Cryan J.F. Making sense of … the microbiome in psychiatry.Int. J. Neuropsychopharmacol. 2019; 22: 37-52Crossref PubMed Scopus (99) Google Scholar). It is important to stress that the gut microbiota is not uniform. Diversity and density of microorganisms increase along the GI tract according to nutritional, chemical, and immunological gradients (Figure 1C). Each individual species is therefore highly adapted to perform specific functions in defined environmental niches of the GI tract. As a whole, the gut microbiota produces an array of metabolites ranging from sugars and short chain fatty acids to neuroactive substances such as serotonin or γ-aminobutyric acid. These can serve as energy sources for the gut microbiota itself or the host, or can act on the host’s signaling pathways. Some of the most convincing evidence for the involvement of the microbiota in host physiology and psychology emerged from germ-free rodents, which have been deprived of any kind of microbe-host interactions from birth. The lack of microbiota in these animals resulted in a variety of altered physiological functions ranging from gut sensory-motor functions and gastric emptying to blood-brain barrier integrity and immune functions. Furthermore, germ-free rodents showed drastic alterations in brain physiology. Changes in hippocampal levels of the neurotransmitter serotonin, brain-derived neurotrophic factor (BDNF), and an altered level of neurogenesis, as well as structural changes to neurons in the amygdala, and variations in the level of myelination in the prefrontal cortex provide the physiological means to explain the differences in stress reactivity, anxiety-related, depressive-like, and social behaviors as well as cognition observed in germ-free animals (Figure 1B). While these studies indicated a relationship between the microbiota and brain and behavior, incontrovertible evidence for this link came from a study where anxiety phenotypes could be transmitted between strains of animals solely through the transplantation of the gut microbiota (Bercik et al., 2011Bercik P. Denou E. Collins J. Jackson W. Lu J. Jury J. Deng Y. Blennerhassett P. Macri J. McCoy K.D. et al.The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice.Gastroenterology. 2011; 141: 599-609, 609.e1–609.e3Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar). To date, a variety of preclinical and clinical studies have linked the gut microbiota to health and disease. Drastic changes in the gut microbiota have been observed in patients with autism spectrum disorder, schizophrenia, major depression, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. Interestingly, when transferring the gut microbiota of patients with these diseases to susceptible germ-free animals, many of the symptoms start to emerge which adds a causal element to the microbiota-gut-brain axis. Germ-free animals that had received the gut microbiota of patients with depression exhibited increased levels of anxiety-related and depressive-like behavior. Similarly, germ-free animals receiving the gut microbiota from patients with Parkinson’s disease or multiple sclerosis developed motor deficits accompanied by neuroinflammation and autoimmune encephalomyelitis, respectively. Strikingly, germ-free mice or antibiotic-treated transgenic mouse models of Alzheimer’s disease do not develop plaques. Furthermore, preclinical studies demonstrated beneficial effects of the supplementation of specific bacterial strains (probiotics) or so-called prebiotics, which serve as an energy source for beneficial bacteria, on anxiety-related, depressive-like as well as social behavior (Bastiaanssen et al., 2019Bastiaanssen T.F.S. Cowan C.S.M. Claesson M.J. Dinan T.G. Cryan J.F. Making sense of … the microbiome in psychiatry.Int. J. Neuropsychopharmacol. 2019; 22: 37-52Crossref PubMed Scopus (99) Google Scholar). Despite this large body of evidence from rodent and human studies supporting the influence of gut microbiota on physiology and psychology, we do not yet fully understand the mechanisms utilized by the gut microbiota to impact brain and behavior. What we know is that constant communication within the gut-brain axis regulates specific aspects of homeostasis, partly through signals coming from the gut microbiota. This exchange is bidirectional, thus, not only can the gut microbiota influence the host by changing aspects of homeostasis, but the opposite is also true. The gut-brain axis comprises different routes of communication including endocrine, immune, or neural mechanisms, through which the gut microbiota is capable of influencing the central nervous system. The gut microbiota can stimulate the release of gut peptides and hormones from enteroendocrine cells which are taken up by the bloodstream to affect centrally mediated events. Similarly, it induces cytokine and chemokine release that locally regulate bacterial concentrations, but these factors can also infiltrate the blood and lymphatic systems, ultimately allowing them to have direct or indirect central effects. Although the gut microbiota can communicate by endocrine and immune pathways, perhaps the fastest and most direct way for the microbiota to influence the brain is by hijacking vagus nerve signaling. The vagus nerve, which is the 10th cranial nerve, links the viscera with the brain. It is a paired nerve that consists of sensory (afferent) and motor (efferent) neurons. The vagus nerve tonically transmits information from the viscera to the brain and vice versa, and as part of the parasympathetic branch of the autonomic nervous system is involved in maintaining corporeal homeostasis. The vagus nerve has been extensively studied for its involvement in hunger, satiety, and stress response but also for its major role in the regulation of inflammation via neuronal motor efferents. Evidence for the involvement of gut vagal neurons in mental health and mood-related disorders surfaced as early as the beginning of the 20th century when gastrectomy (the partial or complete removal of the stomach) was applied to treat peptic ulcers. As a side effect, this resulted in the ablation of vagus nerve activity from the stomach downward, thereby preventing vagal communication between the lower GI tract and the brain. Patients that underwent this treatment demonstrated increased occurrences of psychiatric-related disorders. These findings are supported by similar animal studies that ablated gut-related vagal communication, resulting in changes in adult neurogenesis, stress reactivity, anxiety- and fear-related behavior, as well as cognition, which are indicative of brain changes observed in psychiatric disease. In contrast, vagus nerve stimulation, originally used for treating refractory epilepsy, resulted in mood improvement of patients and is now approved as a method to treat patients with refractory depression. Studies have shown that these vagal-mediated effects are also influenced by gut bacteria. Specific bacterial strains have been demonstrated to utilize vagus nerve signaling to communicate with the brain and to alter behavior. For example, administration of a subclinical dose of the diarrhea-causing pathogen Campylobacter jejuni resulted in increased levels of anxiety-related behavior and Fos immunoreactivity in cell bodies of vagal afferents as well as in the nucleus tractus solitarius (NTS), the main projection side of gut-related vagal afferents in the brain (Goehler et al., 2005Goehler L.E. Gaykema R.P. Opitz N. Reddaway R. Badr N. Lyte M. Activation in vagal afferents and central autonomic pathways: early responses to intestinal infection with Campylobacter jejuni.Brain Behav. Immun. 2005; 19: 334-344Crossref PubMed Scopus (276) Google Scholar). In addition, the beneficial effects of Lactobacillus rhamnosus JB1 (L. rhamnosus JB1) on anxiety-related and depressive-like behavior are blocked following vagotomy (Bravo et al., 2011Bravo J.A. Forsythe P. Chew M.V. Escaravage E. Savignac H.M. Dinan T.G. Bienenstock J. Cryan J.F. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve.Proc. Natl. Acad. Sci. USA. 2011; 108: 16050-16055Crossref PubMed Scopus (2203) Google Scholar), as are the cognitive and electrophysiological effects of a prebiotic which stimulates the production of beneficial bacteria (Vazquez et al., 2016Vazquez E. Barranco A. Ramirez M. Gruart A. Delgado-Garcia J.M. Jimenez M.L. Buck R. Rueda R. Dietary 2′-fucosyllactose enhances operant conditioning and long-term potentiation via gut-brain communication through the vagus nerve in rodents.PLoS ONE. 2016; 11: e0166070Crossref PubMed Scopus (44) Google Scholar). L. reuteri, which improves both social behavior in animal models of autism and wound healing, both via oxytocin signaling, has been shown to be dependent on vagus nerve regulated pathways (Sgritta et al., 2019Sgritta M. Dooling S.W. Buffington S.A. Momin E.N. Francis M.B. Britton R.A. Costa-Mattioli M. Mechanisms underlying microbial-mediated changes in social behavior in mouse models of autism spectrum disorder.Neuron. 2019; 101: 246-259.e6Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). To understand how specific bacteria within the gut microbiota utilize the vagus nerve to communicate with the brain, we need to understand where and how they can interact with the vagus nerve and when and how long they activate it to exert their effects on the brain. Finally, we need to be able to determine what circuits are altered in the brain. Within the small and large intestine, vagal afferents end in the muscle layer as well as in the mucosa. In the muscle layer, vagal afferents form intraganglionic laminar endings and intramuscular arrays, while some vagal endings synapse onto neurons from the enteric nervous system. The connection with the enteric nervous system, which governs the function of the GI tract, widens the scope of signals that can be transmitted by the vagus nerve. Different types of mucosal endings have been described in the small intestine: vagal villi and vagal crypt afferents. Vagal villi afferents, as the name suggests, terminate mostly in the apical part of the villus in close proximity to the epithelial layer. Vagal crypt afferent endings, on the other hand, encircle the luminal end of intestinal crypts. Although mucosal vagal afferents were thought to be free neuronal terminals, a recent study has demonstrated that at least a specific type of enteroendocrine cells are able to form glutamatergic synapses with what are likely to be vagal villus afferents in the small intestine (Figure 1C). Similar connections were also observed in the distal colon between enteroendocrine cells in the luminal part of the crypt and vagal afferents (Kaelberer et al., 2018Kaelberer M.M. Buchanan K.L. Klein M.E. Barth B.B. Montoya M.M. Shen X. Bohórquez D.V. A gut-brain neural circuit for nutrient sensory transduction.Science. 2018; 361: 361Crossref Scopus (358) Google Scholar). While muscular vagal afferents are implicated in detecting stretch and tension, mucosal vagal afferents are perfectly situated to detect chemicals absorbed across the epithelial layer or released by epithelial cells in response to luminal stimuli and to transmit this information within seconds. Electrophysiological experiments have demonstrated that vagal afferents respond to a variety of such stimuli including cytokines, nutrients, gut peptides, and hormones. Therefore, the gut microbiota can alter vagus nerve signaling by directly influencing the concentration or by inducing the release of either of these factors from enteroendocrine or gastrointestinal immune cells. Furthermore, some bacterial strains can produce neurotransmitters such as serotonin that, when absorbed, can act directly on vagus nerve endings or indirectly through activation of enteric neurons. Information from the vagus nerve is relayed in the brainstem, where gut vagal afferents mostly synapse onto neurons in the NTS. Gastrointestinal vagal afferents in the NTS are topographically organized, with vagal afferents from the stomach projecting to the medial and gelatinous nucleus and afferents from the intestines synapsing onto neurons in the medial and commissural nucleus. Although glutamate is the main neurotransmitter, evidence suggests that in addition to the topological organization, vagal afferents utilize different neurotransmitters depending on their origin. For example, the neuropeptide cocaine and amphetamine-regulated transcript, which is involved in satiety signaling, is mainly expressed in afferents from the proximal GI tract. Therefore, it is becoming clear that the location of the microbiota within the GI tract and the chemicals produced determine where and how within the NTS the information is relayed. When the equilibrium of the gut microbiota is altered with the supplementation of bacterial species, different incubation times seem to be needed to exert effects on brain and behavior. Whereas an acute, subclinical dose of pathogens can result in brain effects and altered behavior within hours, effects of probiotic strains (e.g., L. reuteri and L. rhamnosus) are usually described after weeks of chronic treatment. Nevertheless, electrophysiological studies have shown that direct infusion of L. rhamnosus into the small intestine increases the constitutive firing frequency of vagal afferents within minutes after luminal application (Perez-Burgos et al., 2013Perez-Burgos A. Wang B. Mao Y.K. Mistry B. McVey Neufeld K.A. Bienenstock J. Kunze W. Psychoactive bacteria Lactobacillus rhamnosus (JB-1) elicits rapid frequency facilitation in vagal afferents.Am. J. Physiol. Gastrointest. Liver Physiol. 2013; 304: G211-G220Crossref PubMed Scopus (160) Google Scholar). These observations raise the question of whether bacterial species affecting the vagus nerve induce neurobiological changes by simply activating the vagus nerve or by slowly altering the properties of vagus nerve signaling and related neuronal networks, or both. Changes to excitability, synapse remodeling, or alteration of the sensitivity for certain chemicals in response to microbiota-derived metabolites could therefore permanently affect vagus nerve signaling and in turn, result in changes to the vagal tone. Vagal tone describes the strength of vagal (parasympathetic) activation in the dynamic interaction between the parasympathetic and sympathetic branches of the autonomous nervous system. Cardiac vagal tone, as measured by heart rate variability, is often used to measure the strength of parasympathetic activation. When homeostasis is challenged in such instances as stress, changes in cardiac vagal tone can be observed. Interestingly, patients with Crohn’s disease or irritable bowel syndrome, two diseases with malfunctioning gut-brain communication and altered visceral interoception, also show alterations in cardiac vagal tone. Although it is yet to be determined whether changes in vagal tone in these instances are cause or consequence, these observations highlight the link between gut-related changes and the autonomic nervous system and suggest that the gut microbiota might be capable of influencing homeostasis by changing the properties of vagus nerve signaling and related neuronal networks (Bonaz et al., 2016Bonaz B. Sinniger V. Pellissier S. Vagal tone: effects on sensitivity, motility, and inflammation.Neurogastroenterol. Motil. 2016; 28: 455-462Crossref PubMed Scopus (66) Google Scholar). Understanding the neural circuitry underlying the effects of vagal stimulation on brain and behavior is a key goal for the field. Advances in optogenetic and chemogenetic tools are transforming how we delineate the relative contribution of these pathways to vagus-dependent behaviors. However, most of our knowledge to date has emerged from lesion and pharmacological studies coupled with sophisticated anatomical tracing techniques. From the NTS, information is relayed to various forebrain regions and other brainstem nuclei, most of which are also part of the central autonomic network. Projections from the NTS to the forebrain that also receive information from the GI tract have been identified by tracing studies. These include, among others, the parabrachial nucleus, locus coeruleus, hypothalamus, amygdala, bed nucleus of the stria terminalis, ventral tegmental area (VTA), and medial septum which provide the means to influence emotionality and motivation. In addition, multi-synaptic projections ascending from the NTS can transfer information from the vagus nerve to most likely any given structure in the brain. Studies identifying the specific neuronal networks involved in vagal gut-brain signaling and their effect on behavior have started to emerge. The paraventricular nucleus (PVN) of the hypothalamus is an important hub for relay signals emanating from the vagus nerve. Its projections to the pituitary and the VTA provide the means to directly influence the hypothalamic-pituitary-adrenal axis and the mesolimbic dopaminergic system, respectively allowing the vagus nerve to interact with stress response and reward circuitries. In addition, optogenetic tools have recently been used to identify the parabrachial nucleus (PBN) as a relay structure for vagus nerve signaling-dependent effects on reward and avoidance behavior. Gut vagal afferents transmit information from the NTS to the PBN and from there to the substantia nigra (SN) or the central nucleus of the amygdala (CeA). Activation of the PBN-SN pathway results in the induction of reward-related behavior whereas activation of the PBN-CeA pathway evokes avoidance behavior (Han et al., 2018Han W. Tellez L.A. Perkins M.H. Perez I.O. Qu T. Ferreira J. Ferreira T.L. Quinn D. Liu Z.W. Gao X.B. et al.A neural circuit for gut-induced reward.Cell. 2018; 175: 665-678.e23Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). In addition, a link between the NTS and the hippocampus via the medial septum has been demonstrated. This pathway provides the means by which information coming from the vagus nerve can impact hippocampal-dependent memory processes. Indeed, it was shown that lesioning of the vagus nerve or subdiaphragmatic deafferentation impaired episodic memory and altered BDNF concentrations in the hippocampus of rats (Suarez et al., 2018Suarez A.N. Hsu T.M. Liu C.M. Noble E.E. Cortella A.M. Nakamoto E.M. Hahn J.D. de Lartigue G. Kanoski S.E. Gut vagal sensory signaling regulates hippocampus function through multi-order pathways.Nat. Commun. 2018; 9: 2181Crossref PubMed Scopus (86) Google Scholar). Furthermore, vagus nerve stimulation is thought to at least in part impact depression by acting on noradrenergic neurons in the locus coeruleus (LC) which in turn can impact the serotonergic system in the dorsal raphe nucleus (DRN). The NTS-LC-DRN thereby provides another neuronal pathway that could be utilized by the gut microbiota to impact brain and behavior. Finally, integration of the NTS into the dorsal vagal complex, which is critical for interoceptive feedback, puts the NTS at a perfect relay hub for vagus nerve signaling to impact physiology and psychology. Proof that gut bacteria utilize these communication pathways has been uncovered in mouse models. Using optogenetic tools, it was demonstrated that L. reuteri utilizes the NTS-PVN-VTA pathway to alter social behavior in mouse models of autism spectrum disorder. By activating the vagus nerve, L. reuteri increased oxytocin levels in the PVN thereby rescuing social interaction-evoked long-term potentiation in dopaminergic neurons in the VTA, which in turn results in the normalization of social behavior (Sgritta et al., 2019Sgritta M. Dooling S.W. Buffington S.A. Momin E.N. Francis M.B. Britton R.A. Costa-Mattioli M. Mechanisms underlying microbial-mediated changes in social behavior in mouse models of autism spectrum disorder.Neuron. 2019; 101: 246-259.e6Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). In addition, direct projections from the NTS to the amygdala are likely to be involved in the effects of L. rhamnosus on anxiety-related behavior as administration decreased GABAAα2 receptor subunit expression in the amygdala (Bravo et al., 2011Bravo J.A. Forsythe P. Chew M.V. Escaravage E. Savignac H.M. Dinan T.G. Bienenstock J. Cryan J.F. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve.Proc. Natl. Acad. Sci. USA. 2011; 108: 16050-16055Crossref PubMed Scopus (2203) Google Scholar). Furthermore, the effects on BDNF levels in the hippocampus following the colonization of germ-free mice with gut bacteria (Bercik et al., 2011Bercik P. Denou E. Collins J. Jackson W. Lu J. Jury J. Deng Y. Blennerhassett P. Macri J. McCoy K.D. et al.The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice.Gastroenterology. 2011; 141: 599-609, 609.e1–609.e3Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar) might be mediated by the multi-synaptic pathway between the NTS and the hippocampus as described above. The same pathway could be responsible for changes in GABA receptor expression following L. rhamnosus administration (Bravo et al., 2011Bravo J.A. Forsythe P. Chew M.V. Escaravage E. Savignac H.M. Dinan T.G. Bienenstock J. Cryan J.F. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve.Proc. Natl. Acad. Sci. USA. 2011; 108: 16050-16055Crossref PubMed Scopus (2203) Google Scholar). However, many studies have also shown that the gut microbiota influences gene expression in the cortex but the exact neuronal pathways by which these changes are mediated have yet to be determined. Studies utilizing germ-free rodents or administration of specific bacterial strains have provided ample evidence for the role of the gut microbiota in physiology and psychology. Although most studies have focused on a specific behavioral readout when investigating the effects of single or multiple bacterial strains on brain and behavior, the effect of the gut microbiota is far more extensive when analyzed in the context of homeostasis. Thus, with the ability to impact the vagus nerve and vagus nerve-related functions, the gut microbiota offers the possibility to employ the manipulation of the gut microbiota as a non-invasive treatment option for various conditions.