Vagal sensory neurons and gut-brain signaling

Abstract

Our understanding of the gut system has been revolutionized over the past decade, in particular regarding its role in immune control and psychological regulation. The vagus nerve is a critical link between gut and brain, transmitting diverse gut-derived signals, and has been implicated in many gastrointestinal, neurological, and immunological disorders. Using state-of-the-art technologies including single-cell genomic analysis, real-time neural activity recording, trans-synaptic tracing, and electron microscopy, novel physiological functions of vagal gut afferents have been uncovered and new gut-to-brain pathways haven been revealed. Here, we review the most recent findings on vagal sensory neurons and the gut-brain signaling, focusing on the anatomical basis and the underlying molecular and cellular mechanisms. Such new discoveries explain some of the old puzzling problems and also raise new questions in this exciting and rapidly growing field.

Introduction.

The gastrointestinal (GI) tract is home to ~500 million enteric neurons, ~70% of the body’s immune cells, and over 100 trillion microbes [1,2]. Numerous information needs to be monitored from the gut, including ingested food, energy homeostasis, inflammatory signals, and digestive progress, to ensure appropriate regulation of body physiological processes and behaviors to promote overall health. Food contents are constantly surveyed by specialized epithelial cells named enteroendocrine cells lining along the GI tract while mechanical changes associated with ingestion and digestion are detected by enteric neurons, enteroendocrine cells, and sensory nerve endings residing in GI walls. Absorbed nutrients, microbial metabolites, and secreted immune mediators like cytokines and chemokines are all actively monitored signals from the gut. Intriguingly, a clear association between many neurological disorders and digestive problems in human patients was noticed and extensively demonstrated in early studies [3], suggesting that the gut-brain axis is not only important for appetite control and intestinal immunity but also essential for brain cognitive functions.

The gut-to-brain axis contains two major pathways: afferents that originate from the nodose and dorsal root ganglia (DRG) transmit gut signals to the central nervous system (CNS) via the vagus and spinal sensory nerves respectively, while gut-derived hormones, neurotransmitters, inflammatory cues, and immune signals also enter the brain via circulation. As a major bidirectional connection between body and the brain, the vagus nerve has been a key focus in recent investigations of the gut-brain axis. The vagus nerve is the 10^th cranial nerve that supplies many GI organs including esophagus, stomach, small intestine, and colon, other essential organs in the digestive system such as liver, pancreas, and gallbladder, as well as cardiopulmonary organs like heart, lung, trachea, and aortic arch [4]. Sensory (afferent) and motor (efferent) fibers are intermingled in the vagus nerve, with their cell bodies reside in different locations: sensory neurons in the nodose/jugular ganglia adjacent to the jugular foramen and motor neurons in the dorsal motor nucleus of the vagus (DMV) and the nucleus ambiguus in the brainstem. Sensory neurons in the vagus nerve are genetically heterogeneous [5–7], equipped with a diversity of molecular machineries for detecting stretch, tension, and various chemical cues, or for communicating with other sensory cells such as enteroendocrine cells, neuroepithelia bodies, taste buds, and enteric neurons. Genetically distinct sensory neurons in the vagus nerve likely encode different body information [7]. However, the coding logic in vagal sensory ganglia has not been fully understood yet. What are the anatomical and molecular basis for sensing diverse gut inputs? How do distinct GI signals differentially regulate physiology, behavior, and brain functions? In this review, we will summarize recent progress on understanding the roles of vagal sensory neurons in mediating the gut-brain signaling and discuss potential future directions.

How do vagal sensory neurons receive diverse gut-brain signals?

Specialized sensory terminal endings evolve to better detect diversified sensory cues. Vagal sensory neurons form a diversity of mechano- and chemo-sensory terminals along the GI tract (Figure 1), including intraganglionic laminar endings (IGLEs) that communicate with clusters of myenteric neurons between longitudinal and circular muscle layers, intramuscular array endings (IMAs) which are branches of parallel telodendria running in parallel to smooth muscle fibers more strategically located near the sphincters, and mucosal endings arborizing within the mucosa layer with various morphologies [4,8–12]. In addition, vagal afferents also innervate intestinal glands, antral glands, and extreme posterior taste buds in the pharynx and upper esophagus [12,13]. Both IGLEs and IMAs are believed to detect mechanical changes [14–18], yet the underlying molecular mechanisms by which gut distension is sensed by these afferent endings are not well understood [19]. Mechanosensitive PIEZO channels mediate a number of body mechanosensory processes including pulmonary stretch, gentle touch, and blood pressure fluctuation [20–22]. It is unclear whether PIEZO channels also contribute to gut mechanosensation. Nevertheless, while PIEZO channels are Gd³⁺ sensitive [23], IGLEs’ mechano-sensitivity is Gd³⁺ insensitive [18], suggesting the existence of PIEZO independent mechanisms.

Figure 1. Diverse gut-brain connections via the sensory vagus nerve.

A, the gross anatomy of the sensory vagus nerve. Neurons with cell bodies within the nodose/jugular ganglia project to peripheral organs to monitor and send diverse body signals to a variety of brain regions including hypothalamus, subfornical organs, striatum, and hippocampus via the NTS to regulate food intake, fluid homeostasis, reward, memory, cognition, and other brain functions. B, vagal afferents and nutrient sensation in the gut. Anatomically, vagus afferents form IGLEs with enteric neurons, IMAs within both circular and longitudinal muscle layers, and free endings arborizing the villi. After being activated by specific nutrients, enteroendocrine cells release hormones to local or distal enteric neurons and vagal sensory endings. Some enteroendocrine cells named neuropods also form synapses onto vagal sensory terminals.

The gastrointestinal mucosal barrier, formed by a single layer of epithelial cells, prevents luminal entities from entering the underlying GI tissue. Enteroendocrine cells are the primary sensors for intestinal nutrients, and they function as an interface between luminal contents and afferent nerves [24]. A large number of GI neurohormones including cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and serotonin are released from enteroendocrine cells to regulate digestion, nutrient absorption, and food intake. Enteroendocrine cells were traditionally categorized alphabetically into 8 subpopulations based on the principal hormones they produce [24] and were recently shown to be more heterogeneous using single-cell transcriptomics [25,26], suggesting that the coding logic for intestinal nutrients in enteroendocrine cells is at best partially understood. Vagal afferents travel through the intestinal villus shaft, arborize along its apical pole, and form varicosities and swellings near the enteroendocrine cells [12]. It was believed, for a long time, that enteroendocrine cells lack physical contact with afferent nerves. Instead, they communicate in a paracrine fashion that released hormones will diffuse to signal adjacent afferent terminals. Receptors for many gut hormones, including CCKAR, GLP1R, NPY2R, and HTR3A are expressed in vagal sensory neurons [6,7,27,28]. Although the roles of some gut peptide/receptor pairs including GLP-1/GLP1R in this communication have been questioned [7,29], the importance of this paracrine connection in the gut-brain axis has been extensively described and well accepted. In addition to this classical model, recent studies using immunostaining of synaptic markers, monosynaptic rabies virus tracing, and electron microscopy have demonstrated that about two-thirds of enteroendocrine cells are able to make synapses with adjacent nerves [30,31] and a new connection mechanism between enteroendocrine cells and vagal afferents involving glutamatergic synapses was discovered [32] (Figure 1). Such synapses enable ultrafast millisecond transmission from enteroendocrine cells to vagal sensory neurons, which together with paracrine communication provide a complete time spectrum, from fast to slow, for gut-brain signaling. This breakthrough finding suggests that our understanding of the enteroendocrine-vagus interaction is far from complete and will open up new vistas in this important area.

How do vagal afferents communicate with the brain?

Vagal afferents primarily terminate in the nucleus of solitary tract (NTS) in the brainstem, where visceral information is further transmitted to higher brain regions such as the locus coeruleus (LC), the rostral ventrolateral medulla, the dorsal raphe nucleus, and the hypothalamus via multi-synaptic connections [4,33,34]. In fact, using large-scale neural mapping techniques, it has been shown that numerous brain regions are impacted by vagus nerve stimulation, and this connectivity has been termed the “vagus afferent network”. For example, the effects of vagus nerve stimulation on epilepsy and depression are thought to be at least in part through the NTS-LC pathway [33]. Recently, a novel gut-vagus-NTS-medial septum-hippocampus pathway has been characterized in detail, providing anatomical evidence for gut control of learning and memory [35].

Upon food consumption, vagal afferents from the gut send satiety signals to the hypothalamus via the NTS to regulate meal size in a negative-feedback manner [34,36,37]. In the hypothalamic arcuate nucleus, activation of AgRP neurons by food deprivation induces food craving, seeking, and consumption behaviors while POMC neurons are activated under fed condition and promote satiety [38]. During a meal, activity of AgRP neurons is dynamically regulated by multiple sensory inputs including visual and olfactory cues, nutrient detection in the mouth and upper GI tract, and homeostatic signals [39–41]. Simply presenting food to a hungry mouse rapidly and transiently inhibits AgRP neurons and activates POMC neurons even before food is consumed. Sustained inhibition of AgRP neurons requires not only food consumption but also calorie intake [42,43]. Consistently, AgRP neurons are inhibited in a fast and persistent manner by intragastric infusion of nutrients and intraperitoneal injection of gut-derived hormones (CCK, PYY, amylin, and serotonin) but not gastric distension. It is worth noting that AgRP neurons can distinguish calorie-free gel from caloric gel within 40 seconds after consumption, suggesting that a rapid gut-brain signaling pathway is involved. Considering the timescale of such acute responses, and the fact that both CCK and PYY receptors are expressed on vagal sensory neurons, the vagus nerve is likely responsible for transmitting this gut signal to AgRP neurons. Although it is commonly thought that calorie detection requires nutrient sensing, a recent report suggests that vagal interstinal IGLE mechanoreceptors but not mucosal endings are essential for inhibition of AgRP neurons and food intake [44].

Neurons in the subfornical organ (SFO) play an important role in fluid homeostasis and thirst satiation [45]. Like the rapid inhibition of AgRP neurons by food cues, ingestion of fluid and intragastric infusion of water rapidly inhibit Nos1⁺ excitatory neurons in the SFO in dehydrated animals [46,47]. Intragastric infusion of water also inhibits drinking behavior in dehydrated mice, suggesting gut signals are able to suppress thirst sensation. Both effects are greatly reduced in subdiaphragmatic vagotomized animals [47], indicating an essential role of this gut-vagus-SFO pathway in thirst control. Reduction of gut osmolarity may also activate SFO GLP1R neurons to inhibit water intake [48]. Vagal afferent responses to osmolarity challenges have been described in early studies [49] and are likely mediated by vagal GPR65 neurons, as these neurons exhibit afferent terminals arborizing the apical pore of intestinal villi and detect polymodal signals from the gut in vivo [7]. Direct neural projection from the NTS to the SFO has been reported earlier [50]; however, whether this connection is involved in the gut-to-SFO circuit still needs to be confirmed.

Increasing evidence has demonstrated the importance of the vagus nerve in neuropsychological functions. Vagal afferents are typically associated with suppression of food intake and negative valence but not the reward system. Using state-of-the-art viral tracing approaches, a novel gut-vagus-NTS-PBNdl (the dorsal lateral parabrachial nucleus)-SNc (the substantia nigra pars compacta) reward pathway was discovered [51]. Surprisingly, although both left and right vagus nerves carry satiety cues from the gut, this gut-SNc pathway is only mediated by the right vagus nerve. Optogenetic stimulation of this pathway induces a panel of reward behaviors, which is not mutually exclusive to satiation mediated by both vagus nerve branches. This striking finding not only provides evidence for a direct neural pathway that links the gut with the reward center in the brain, but also suggests functional asymmetry between left and right vagus nerves. Identifying the molecular signature of such neurons and the underlying anatomical and sensory basis would be informative to better understand this gut-brain reward pathway.

Vagal afferents, microbiota, and cognitive functions

Tens of trillions of microorganisms live in our gut, including over 1,000 known bacteria species. Together they play critical roles in development, gut physiology, immune functions, perception, cognition, memory, and other brain functions [2,52–54]. Numerous studies have suggested the existence of a microbiome-gut-brain axis (Figure 2). Gut microbiota are closely associated with many social behaviors and psychiatric disorders including anxiety, depression, Parkinson’s disease, Alzheimer’s disease, and autism spectrum disorder. In particular, germ-free animals receiving microbiota transplantation from animals or patients with such neurological diseases will develop similar symptoms [52–54]. Accumulating evidence is suggesting that the vagus nerve is one of the major routes in the microbiome-gut-brain axis. For example, the beneficial effects of chronic treatment with Lactobacillus rhamnosus, a lactic acid bacteria, on emotional behaviors are diminished in subdiaphragmatic vagotomized animals [55]. Similarly, Lactobacillus reuteri is able to rescue social deficits in mice with autism spectrum disorder by modulating synaptic plasticity in the ventral tegmental area (VTA) via oxytocin and the vagus nerve [56]. Microbes trigger secretion of gut-derived neurotransmitters, peptides and hormones as well as local immune mediators such as cytokines and chemokines. Metabolites produced by isolated gut microbes can signal enterochromaffin cells and regulate biosynthesis of serotonin [57], a crucial mediator for many physiological processes. In particular, serotonin can activate vagal afferents via serotonin receptor HTR3A [7], and therefore, may serve as a major signaling molecule between gut microbiota and the vagus nerve. Gut microbiota may also modulate brain functions via vagus nerve-independent pathways. For example, a recent study shows that gut bacteria that are significantly altered by ketogenic diet, such as Akkermansia and Parabacteroides, are essential for ketogenic diet mediated anti-seizure effect by regulating ketogenic gamma-glutamylated amino acids, critical fuels for glutamate and GABA biosynthesis [58].

Figure 2. The microbiome/immune-gut-brain pathway mediated by the vagus nerve.

Sensory neurons in the vagus nerve may communicate with microbiome and immune cells via multiple pathways. While some pathogenic microbes may activate vagal afferent terminals directly through a leaky intestinal barrier, most commensal microbiota interacts indirectly with the vagus nerve via enteroendocrine cells and/or enteric neurons, potentially through a serotonin dependent mechanism. Detection of inflammatory signals including TNF and IL-1 released from gut immune cells in the vagus nerve is likely mediated by a designated sensory neuron population that co-expresses multiple cytokine receptors, and cytokine information is specifically encoded with distinct neurograms. Such detection will trigger the ‘inflammatory reflex’ to regulate immune cell functions via the efferent arm of the vagus nerve.

Despite the consensus that the vagus nerve is a major component of the microbiota-gut-brain axis, it is less clear whether gut microbiota and/or microbial metabolites will activate vagal gut afferents directly. Sensory neurons innervating lung and skin are known to monitor and protect against bacterial/viral infection. Similar to this concept, an early study showed that Campylobacter jejuni, a diarrhea-causing pathogen, can induce neuronal activation in vagal ganglia and vagal central targets (NTS) [59]. On the other hand, a recent study using modern genetic tools clearly demonstrated that gut-projecting afferent neurons that directly respond to exposure of Salmonella enterica serovar Typhimurium (STm) and protect the host against this bacterial pathogen are nociceptors located in the dorsal root ganglia but not the vagus nerve [60]. As sensory fibers do not contact intestinal luminal contents under physiological conditions, direct activation of vagal afferents by gut microbes in vivo is likely due to altered intestinal permeability. In contrast, most commensal microbiota stays in the intestinal lumen and has to communicate with afferents in an indirect way. Gut microbiota produces numerous highly divergent metabolites and microbial products, including short-chain fatty acids (SCFAs), bile acids, lipopolysaccharides (LPS), and caseinolytic protease B (ClpB), many of which are potential neuronal modulators and are implicated in disease [61]. A recent chemical genetic screen systematically revealed numerous G-protein coupled receptors (GPCRs) as targets of human gut microbiota metabolites [62]. Gut microbiota can also modulate serotonin production in enterochromaffin cells [31,57], while 5-HT receptors are highly expressed in enteric neurons and gut vagal afferents [7,63]. Using electrophysiology, it was shown that Lactobacillus rhamnosus elicits mesenteric vagal afferent firing [64,65]. Nevertheless, whether this observation can be generalized to other psychoactive bacteria is not clear. Alternatively, commensal microbiota may influence the brain by modulating vagal neuron responses to other GI signals. In short, we still lack mechanistic understanding of the roles of vagal sensory neurons in the microbiota-gut-brain axis, calling for more cell-type specific ablation studies in the future.

Vagal afferents and inflammation

Inflammation is a tightly regulated protective response to invaders or injury. The intestinal immune system contains the majority of body’s immune cells and is constantly challenged by antigens from ingested food or gut microbiota. The vagus nerve plays an important role in neuronal regulation of intestine immunity. While the vagal efferent cholinergic anti-inflammatory pathway has been beautifully demonstrated through a series of seminal studies [66,67], how vagal afferents encode inflammatory cues and are involved in the ‘inflammatory reflex’ is less clear. Early work has suggested that vagal afferents may detect cytokines and other immune-derived signals via direct and indirect pathways [68]. Vagal sensory neurons express receptors for a number of inflammatory mediators, such as TNFR1 and IL1R, and can be activated by tumor necrosis factor (TNF) and interleukin 1 (IL-1) both in culture and in vivo [69–71]. Recent studies using computational approaches revealed that TNF and IL-1 elicit different firing patterns in vagal afferents in vivo, indicating immune signals are differentially encoded [70,71]. It is worth noting that TNFR1 and IL1R are largely expressed in the same vagal sensory neurons [70], suggesting that there is one designated neuron population for immune sensing in the vagus nerve that uses different cytokine receptors, potentially different intracellular signaling pathways, and distinct neurograms to encode cytokine-specific immune signals (Figure 2). It was hypothesized that vagal afferents within the intestinal villi could be primed for communicating with immune cells in the gut [68]. Alternatively, sensory endings targeting the portal vein or lymph nodes are also ideal candidates for monitoring immune signals. Nevertheless, the anatomical basis for immune sensing in the vagus nerve remains to be a mystery and needs to be addressed in the future.

Conclusion

New findings of the gut-brain axis are shaping the way we live and making important improvement for human health. The gut is not only considered as a system for digestion but a critical regulator for immune and cognitive functions as well. Vagal sensory neurons convey important gut signals to the brain, providing an excellent therapeutic target for treating digestive, immunological, and psychological diseases. However, our current understanding of this gut-brain pathway at the molecular and cellular level is far from complete. Many key issues including coding logic of gut signals in vagal sensory ganglia, the underlying sensory machineries and mechanisms, and function-specific neural circuits still need to be addressed. Applying state-of-the-art technologies such as single cell genomics, cell-type specific neural modulation, and real-time neural activity recordings in the future will greatly advance our understanding of this system and may provide essential information for improving existing vagus nerve stimulation based therapies and developing new therapeutic strategies.

Highlights.

The vagus nerve is a critical link between gut signals and the brain regulating various functions such as energy homeostasis, digestion, immune responses, reward, memory, and cognition.

Vagal afferents form different terminal endings in the gut, including direct synapses with some enteroendocrine cells named neuropods.

Vagal sensory neurons monitor ingested nutrients and water from the gut and provide fast regulation of food intake and fluid homeostasis.

Afferents in the left and the right vagus nerve exhibit asymmetric anatomical projections in the brain, e.g. a reward gut-to-brain circuit is specifically formed by right vagal afferents.