Gut instincts: Multi-level control and neural pathways involved in gastrointestinal interoception.

The ENS serves as the gut’s intrinsic and local control system, responsible for executing core GI functions, e.g., motility and secretions, that facilitate digestion ( Figure 1 ). Known as the ‘gut’s brain,’ the ENS contains sensory, motor, and interneurons locally organized into reflex circuits capable of detecting and responding to luminal contents or other changes in the local environment independent of the CNS ( 17 ). Motility and secretory reflexes are initiated by a diverse set of gut-intrinsic sensory cells, including intrinsic primary afferent neurons (IPANs) ( 18 – 21 ) in the myenteric and submucosal plexuses ( 22 – 25 ), as well as enteroendocrine cells (EECs) that line the epithelium ( 26 – 31 ) . Together, these cells detect a wide range of mechanical and chemical sensory stimuli and transduce diverse sensory inputs into neural signals that activate enteric motor networks to engage a proper response. Mechanical stimuli encountered and detected in the gut include mucosal displacement, shear forces, tension, longitudinal stretch, and distension that may result from a bolus or osmotic-induced shift of fluid into the lumen ( 17 , 23 , 32 , 33 ). These mechanical forces are detected by mechanically-gated ion channels, Piezo1 ( 22 , 25 ) and Piezo2 ( 22 , 30 , 34 – 36 ), as well as other mechanosensitive receptors like TRPA1 ( 28 ) expressed on IPANs and/or EECs. Thus, mechanotransduction in the ENS may be directly mediated through IPANs or indirectly through EEC-to-IPAN communication to facilitate downstream responses. In vivo and ex vivo optogenetic manipulation of Piezo1-expressing enteric neurons significantly altered colon motility ( 22 ). Similarly, it was shown that Piezo2-expressing EECs detect mechanical forces and release neurotransmitters such as serotonin (5-HT) ( 30 ) to stimulate downstream ENS motor activity ( 27 , 37 ). Chemical cues including nutrients, bile acids, pH changes, microbial metabolites, and inflammatory mediators are detected by numerous types of chemoreceptors (both GPCRs and ligand-gated ion channels) expressed on sensory cells in the gut ( 38 ). EECs in the small intestine, for example, can detect nutrients from the lumen through glucose transporters (SGLT1) or short-chain fatty acid receptors (FFAR2) and release neurotransmitters 5-HT and ATP to activate enteric neurons and initiate secretomotor processes ( 39 ). Intrinsic ENS pathways are equipped with the molecular machinery and connectivity to detect and respond to diverse luminal cues and challenges that vary significantly along the proximal-distal axis of the GI tract. The expression of receptors on subclasses of enteric neurons and EECs exhibits region-specificity, reflecting the types of stimuli encountered in a particular region, enabling optimal detection and monitoring of the local environment ( 34 – 36 , 40 ). EECs exhibit strong regional specificity ( 35 , 41 ), where proximal intestinal EECs are tuned for chemosensory detection of nutrients via diverse GPCRs (e.g. taste receptors – T1R2 and T1R3) ( 39 ), and colorectal EECs are enriched for mechanosensitive Piezo2 channels that enable detection of luminal distension and initiation of defecatory reflexes ( 32 ). Regional differences in the ENS circuits also prime certain areas of the GI tract to handle luminal contents with specific properties and initiate distinct efferent responses that control gut function ( 18 , 42 – 45 ). Enteric neuron subtypes and ENS circuits have been well-defined in the small intestine and distal colon, focusing primarily on their role in motility and fluid secretion ( 9 , 46 ). In contrast to the intestine, where reflex control is largely mediated by intrinsic neural circuits within the organ, gastric functions including the breakdown and processing of ingested food ( 47 ), are predominantly controlled through vagal input. For example, the stomach has specialized enteric neurons that promote the secretion of acid, pepsinogen, and gastrin from various types of cells lining the mucosa, but these secretomotor neurons are activated by vagal nerve activity rather than local gastric reflexes initiated by IPANs, which are rare in the stomach ( 46 , 47 ). In the distal colon, local self-sustained peristaltic reflexes generate propulsive contractions that allow for the forward movement of fecal content. Peristalsis is initiated by distension-sensitive IPANs that engage ascending excitatory and descending inhibitory enteric neural networks to induce oral contraction and aboral relaxation of smooth muscle, respectively ( 17 – 20 ). Unlike the distal colon, colon motor complexes (CMCs) in the proximal colon appear to be involved in the formation of fecal pellets ( 45 , 48 ), are spontaneously and rhythmically generated, and do not rely on IPAN activation via distension. The mechanisms underlying this motor pattern are incompletely understood, however recent evidence suggests they may be the result of self-regenerating interactions between the ENS and interstitial cells of Cajal that are mediated, in part, by a unique subclass of IPANs specific to the proximal colon ( 42 , 49 ). Thus, the heterogeneity of the ENS and connectivity to other intrinsic cells along the GI tract reflects region-specific functions. In contrast to the locally acting ENS, the PVG (including the celiac, inferior mesenteric and pelvic ganglia) function as long-distance coordinators that integrate sensory information from multiple gut regions, regulating activity across different segments along the GI tract ( 50 – 58 ). Postganglionic neurons in PVG receive peripheral input from intestinofugal afferent neurons (IFANs) that are activated by distension and other mechanical cues originating from the gut wall and relay signals via cholinergic transmission ( 56 , 59 – 70 ). Recent single-cell sequencing of the celiac ganglion has revealed that PVG neurons are organized into transcriptionally distinct subclasses that likely segregate according to the specific organs they innervate ( 12 ). These molecularly defined groups align with known organ-to-organ reflex pathways, suggesting that target-specific output is hard-wired at the level of the PVG and may explain how individual reflex circuits maintain specificity despite converging sensory inputs. Within this circuitry, distension detected in distal gut regions activates IFAN to PVG pathways, which in turn send inhibitory feedback to the ENS to slow motility in more proximal segments ( Figure 2 ). Specific organ-to-organ reflexes include the entero-enteric where stimulation of small intestinal IFANs by distension or acid results in the PVG-mediated inhibition of gastric motility ( 61 , 71 , 72 ). Certain neuropeptides and transmitters released from EECs can also activate IFANs to engage PVG circuits for organ-to-organ coordination. For example, the ileal brake is a reflex feedback mechanism by which nutrients in the ileum trigger the slowing of the proximal gut to allow additional time for their complete digestion and absorption ( 52 ). Recent work incorporating opto- and chemogenetics identified afferent signaling pathways involved in this reflex that was determined to rely on the distension-induced release of glucagon-like peptide 1 (GLP-1) from ileal L cells that stimulates local IFANs and decreased gastric emptying and feeding behaviors via PVG activation ( 14 ). Another reflex that has been described in both humans ( 73 , 74 ) and pre-clinical models ( 75 – 77 ) is the colo-gastric reflex where distension of the colorectum slows down gastric emptying and decreases myoelectric activity of the stomach. The precise neural circuits involved in this pathway are not well established, but existing studies that incorporate chemical and surgical nerve ablation suggest either vagal or spinal-splanchnic-PVG efferent pathways as playing a role in this reflex ( 78 – 81 ). Beyond their role in modulating gastrointestinal reflexes, the PVG contains postganglionic neurons that innervate non-GI organs like the liver, spleen and mesenteric lymph nodes ( 82 , 83 ), suggesting the possibility that PVG-mediated pathways participate in gut-to-immune reflexes which remains an intriguing open question with physiological and clinical implications.

Interoceptive functions in the GI tract are executed by multiple levels of neural control. Locally, the ENS engages intrinsic reflexes for secretion and motility. Intestinofugal afferent input recruits motor neurons in the PVG to coordinate activity across distant regions of the GI tract. Additional recruitment of lumbosacral spinal and vagal reflexes regularly occurs for other normal, interoceptive functions that are still below the level of conscious awareness. Conscious interoceptive functions in the gut involve (1) vagal pathways to the NTS and hypothalamus that mediate feeding behavior, as well as (2) the lumbosacral spinal pathways involved in defecation and voluntary elimination of waste. The thoracolumbar spinal innervation of the GI tract, by contrast, seems to only be engaged during times of ‘crisis’ or allostatic load (e.g., inflammation, infection, dysbiosis, chronic stress, etc.) and may contribute to pathological interoceptive awareness, visceral hypersensitivity, and other negative GI symptoms. This is also the pathway that has been least characterized anatomically, molecularly, and functionally, thus presenting an opportunity for innovation and research development. By mapping the unique receptors, central targets and functions of each pathway, we can begin to identify pathway-specific biomarkers or develop neuromodulatory strategies that can selectively manipulate activity in each pathway to control symptoms and GI function.

Most GI functions occur subconsciously without our awareness, with the exceptions of vagal-mediated signals related to food intake and LS spinal-mediated signals related to defecation. But dysfunction in the interoceptive pathways described above can become pathological, presenting as either decreased or increased awareness that is indicative of hypo- or hyper-sensitivity, respectively. However, we currently do not have a clear grasp on what is normal versus abnormal to feel in our gut, or the mechanisms driving abnormal gut feelings. Numerous conditions are linked to GI-related interoceptive dysfunction, most notably the disorders of gut-brain interaction, but even non-GI diseases have been associated with increased interoceptive awareness and autonomic dysregulation specific to the GI tract. Disorders of Gut Brain Interaction (DGBIs) are highly prevalent conditions with up to 40% of the population experiencing symptoms consistent with at least one DGBI ( 210 ). Abdominal pain is a common symptom in DGBIs throughout the luminal GI tract, presenting as (a) esophageal hypersensitivity and functional heartburn in the esophagus or (b) functional dyspepsia when related to food intake/epigastric discomfort, (c) irritable bowel syndrome when accompanied by stool changes, and (d) anorectal symptoms in cases of levator ani syndrome (Rome IV Criteria, 2016). Abdominal pain is thought to be generated by hypersensitivity (either allodynia or hyperalgesia) of the viscera and associated with interoceptive dysfunction. Our understanding of the mechanisms underlying these changes in DGBIs remains limited because, as discussed in this review, most of what is known about visceral hypersensitivity comes from studies that use colorectal distension even though hypersensitivity at all levels of the GI tract have been implicated as suspected sources of pain generation in DGBIs ( 209 ). Current clinical assessment of visceral hypersensitivity is possible at the level of the esophagus and the rectum, but not widely feasible at levels of bowel in between. At the level of the esophagus, pH and impedance testing captures esophageal symptoms and correlate them to the presence of acid or nonacidic reflux ( 211 ), differentiating reflux from functional heartburn or esophageal hypersensitivity. The rectal barostat testing utilizes an anally inserted balloon with distension to set threshold levels to identify hyper or hyposensitivity to pain. This has been traditionally used to assess pain in IBS, but there is poor correlation between the visceral pain thresholds utilized and abdominal pain, with some studies showing only 50% of patients with IBS show hypersensitivity by rectal measurements ( 212 ). Conversely, medications that improve rectal sensitivity do not reliably reduce IBS pain symptoms ( 213 ). Mechanistically, these clinical findings indicate that the LS spinal pathway is not the major driver of IBS symptoms, and this is further supported by results from preclinical studies. Efforts are ongoing to develop tests for assessing pain in parts of the GI tract that are less accessible. Gastric hypersensitivity has been approximated utilizing nutrient drink tests, whereby liquids are ingested at a set pace to assess hypersensitivity in functional dyspepsia. Although the assay is used in multiple pharmacological studies, protocols are disparate across institutions. Further validation studies are needed to standardize a protocol to serve as a biomarker for gastric hypersensitivity in functional dyspepsia ( 214 ). Small bowel hypersensitivity is a suspected source of pain generation in both gastroduodenal and colonic DGBIs with ex-vivo studies showing intestinal inflammation driven by mast cell activation and barrier dysfunction associated with hypersensitivity ( 215 ). These alterations in small bowel or colonic sensation have also been shown to accompany motility changes contributing to IBS symptomatology ( 216 ). Small bowel sensation has been shown clinically to be distinct from colon sensation with trials of small bowel gaseous distension generating more pain than colonic gaseous distension in patients with IBS ( 217 ). Currently, however, a clinical test does not exist to study small bowel hypersensitivity. While we have made significant progress in clinical testing of visceral hypersensitivity in specific portions of the luminal GI tract, research efforts need to be expanded to include the less accessible regions (that make up 90% of the GI tract) and begin to think about non-luminal sources of GI pain. Based on the large repertoire of response properties observed in GI sensory afferents, one would predict that not all GI-related pain will be evoked by luminal distension. In general, pain has been associated with tension, stiffness and inflammation (e.g., muscle spasms, arthritis, fibromyalgia), and it is likely these pain qualities are conserved across somatic and visceral sensory systems. Fibrosis, serosal adhesions, and muscle stiffening occurs in the GI tract in conditions like IBD and bowel endometriosis that are associated with intense abdominal and pelvic pain and a host of other autonomic issues, but nothing is known about how physical changes in the tissue impact sensory processing or any long-term changes in sensitivity. Interestingly, patients with endometriosis experience pain, constipation, and GI neuromuscular dysfunction regardless of the presence of lesions on the bowel ( 218 – 220 ), highlighting the interconnectedness of systems involved in visceral interoception. To address these clinical needs, we must have complete understanding of the sensory pathways innervating the entire GI tract, what they detect, how nociceptive signals are transmitted to the brain, and their role in driving visceral hypersensitivity. This will first require preclinical studies, but defining the properties and pain behaviors associated with sensory afferents in animal models will then inform how to best assess, and eventually treat, the different forms of hypersensitivity and chronic abdominal pain observed in the clinic. Having a full battery of tests to identify the source of hypersensitivity will present additional opportunities to develop new treatments for GI pain with increased specificity and efficacy.

Communication between vagal afferents of the GI tract and the CNS plays a crucial role in regulating hunger and satiety ( 84 – 88 ), with individual vagal afferents discriminating diverse sensory features that become integrated for a comprehensive assessment on the organism’s metabolic state ( 89 – 92 ) ( Figure 3 ). Vagal afferents were initially characterized by their response properties to mechanical and chemical stimuli within the GI tract, including distension, tension, stretch and mucosal deformation ( 89 , 93 – 96 ), as well as nutrients, microbial metabolites, and other chemical agents. Additional classification based on peripheral terminal morphology and/or expression of mechano- and/or chemo-receptors began to reveal the complexities and vast heterogeneity of the vagal afferent system, even in sensory transduction mechanisms. Some afferents express chemoreceptors that directly sense specific nutrients, such as, glucose ( 97 , 98 ) and fatty acids ( 99 ) as well as bacterial metabolites ( 100 ). Other nutrients are indirectly sensed by vagal afferents through communication with EECs, which detect and transmit signals via gut hormones (e.g., CCK ( 101 ), PYY ( 102 ), GLP-1 ( 103 – 105 ), ghrelin ( 104 , 106 ) or neurotransmitters (5-HT ( 107 )). Recent single cell RNA sequencing studies performed on back-labeled or barcoded nodose ganglion neurons have revealed novel insights into how the vagal afferent system decodes and interprets complex interoceptive signals from all over the body. Multiple dimensions of sensory input – innervated-organ, tissue-layer, and stimulus modality – are encoded by the molecular identity of vagal afferents ( 108 ), defining their individual properties while ensuring the collective ability to detect the complete repertoire of sensory information along the GI tract. Stomach-innervating vagal afferents with endings that form intramuscular arrays (IMAs) are enriched for Calca ( 108 , 109 ), respond to stretch with a transient response pattern ( 108 ), and make extensive connections with interstitial cells of Cajal (ICC) ( 110 ). These IMA-ICC complexes are speculated to function analogously to muscle spindle organs, serving as sites of sensorimotor integration to monitor and control the degree of stomach filling ( 111 ). Vagal afferents with mucosal endings in the stomach express Calca, Sst, and Gpr65, detecting the chemical properties of ingested food, whereas mucosal endings in the small intestine express Gpr65, Vip, Uts2b and Glp1r and exhibit sensitivity to both chemical and mechanical properties. Vagal afferents with intraganglionic laminar endings (IGLEs) in the myenteric plexus were originally classified as tension receptors ( 112 ) but appear to make up several subpopulations that use different signaling mechanisms to regulate feeding behavior. For example, IGLEs in the stomach are tuned to detect mechanical forces and are enriched for Glp1r , whereas IGLEs in the small intestine are tuned to detect chemical signals and enriched for Oxtr, but both populations play prominent roles in driving feeding responses ( 109 ). Unlike the transient responses to stretch given by IMAs, Piezo2-expressing IGLEs in the stomach respond to stretch with a sustained response pattern ( 108 ). IGLEs that lack Piezo2, but respond to mechanical stimulation, express receptors for enteroendocrine hormones, e.g., CCK and 5-HT, ( 108 ), suggesting that some mechanical signals are relayed through epithelial-neuron communication. Thus, the afferent arm of the vagal pathway utilizes diverse mechanisms to deliver complex sensory signals the CNS, where multiple coding dimensions about stomach fullness, the physical and chemical properties of ingested food, and the current metabolic demands of the organism must be integrated for a complete assessment of an organism’s fed versus fasted state. Gut sensory information, both mechanical and chemical, reaches the brain through vagal afferents and is processed along a multi-synaptic pathway centered on the nucleus of solitary tract (NTS). The NTS acts as a major convergence hub for inputs carrying visceral sensory information, especially those related to food intake, with considerable overlap of incoming signals from different organs and sensory systems ( 113 ). For example, the rostral NTS receives direct input from taste buds ( 114 ), as well as indirect input from the olfactory bulb ( 115 , 116 ) , demonstrating the NTS as a key site for coordinated processing of sensory cues related to the ingestion of food e.g., taste, smell, and hunger. From the NTS, sensory information is relayed to the parabrachial nucleus (PBN), which integrates gustatory, olfactory and GI signals ( 117 , 118 ). The PBN then projects to various higher-order brain regions for further processing, including the insular cortex, the arcuate nucleus (ARC) of the hypothalamus and central nucleus of the amygdala (CeA) ( 117 , 119 , 120 ). These higher brain structures also send reciprocal connections back to the PBN ( 121 ), which in turn projects back to the NTS ( 120 ). Through the NTS, this information engages efferent pathways such as those arising from the dorsal motor nucleus of the vagus (DMV), allowing the brain to regulate visceral organ function ( 122 ). Vagal efferent neurons arising from the DMV provide parasympathetic motor and secretory control to the esophagus, stomach, small intestine, and colon, following a clear proximal-to-distal innervation gradient ( 123 , 124 ). DMV motor neurons modulate gastrointestinal motility through the actions of two different pathways. More localized to the medial and rostral DMV, excitatory cholinergic pathways mediate smooth muscle contraction through muscarinic receptors, whereas inhibitory pathways promote smooth muscle relaxation through nitric oxide (NO) or vasoactive intestinal peptide (VIP) and tend to arise from the caudal DMV ( 123 ). In the stomach, regional differences in vagal efferent pathways allows for fine-precision control of gastric motility. Excitatory vagal pathways in the antrum enhance contractions for mixing, while inhibitory pathways engage nitrergic neurons in the fundus for muscle relaxation to accommodate ingested food ( 123 ). In preclinical models, vagal nerve stimulation (VNS) accelerates gastric emptying ( 125 , 126 ) and enhances migrating motor complexes in the small intestine ( 127 ). Vagal efferent neurons likely modulate these motor behaviors via interactions with enteric neurons ( 125 ) and ICC ( 128 ), key regulators of gut motility. In vivo imaging studies show that VNS elicits synchronized Ca 2+ responses in stomach myenteric neurons that correlate with tissue displacement ( 125 ). Interestingly, Ca 2+ activity in colonic myenteric neurons was also evoked by vagal stimulation but without producing measurable tissue movement, highlighting the possibility that vagal-mediated activation of the ENS in distal GI regions may contribute to non-motility related functions ( 125 ). Beyond their motor effects, vagal efferents also regulate secretory activity by modulating gastric acid secretion, mucosal blood flow, and intestinal fluid secretion ( 129 ). Through these spatially organized, functionally distinct efferent pathways, the DMV exerts fine-tuned control over gastric tone, contractility, emptying, intestinal transit, and absorptive function that collectively influence nutrient handling, feeding behavior and metabolism. Unlike the well-defined role of the vagus nerve in nutrient homeostasis and metabolism, its contributions to visceral pain are less clear. Spinal afferents have generally been assumed to solely transmit nociceptive signals perceived as ‘pain’ in the brain, whereas vagal afferents were linked to the emotional components of visceral pain, such as fear, nausea, anxiety, and depression ( 130 – 132 ). Recent studies, however, indicate that vagal afferents may plan a significant role in nociception and pain modulation. Vagal afferents in the stomach ( 133 – 135 ) and esophagus ( 136 – 139 ) are capable of detecting noxious agents, and some undergo sensitization when exposed to inflammatory mediators ( 133 , 135 , 136 , 139 ), indicating potential contributions to the development of visceral hypersensitivity. Further evidence comes from clinical observations in patients with complete cervical spinal cord injuries, who often report poorly localized, cramping abdominal pain ( 140 – 143 ), sensations traditionally associated with pain mediated by spinal afferents ( 144 ). However, since the signals from vagal afferents are the only intact signals to reach the brain in these patients, it suggests that vagal afferents can contribute to pain perception in certain conditions. The precise extent and circumstances under which vagal afferents transmit painful sensations in healthy individuals, or how their function might be affected by more common issues like inflammation, remains unknown. Another major interoceptive function in the GI tract that involves conscious awareness is the elimination of digestive waste, the processes for which are initiated and executed by the lumbosacral (LS) spinal pathway via the pelvic nerves ( Figure 4 ). Innervation of the GI tract by afferents in LS dorsal root ganglia via pelvic nerves is restricted to the distal colon and rectum ( 15 , 145 ), likely occupying areas that receive minimal or no vagal afferent innervation. Teased fiber recordings in ex vivo colon preparations have shown that pelvic nerve or LS afferents respond to a diversity of mechanical stimuli ranging from low to high intensity, including circumferential stretch, probe and mucosal brush ( 146 ). LS afferents also exhibit chemosensitivity to noxious chemical agents and inflammatory mediators, e.g., bradykinin, 5-HT, purines, and some previously “silent” afferents develop sensitivity to mechanical stimuli after brief exposure ( 147 ), suggesting their involvement in acute visceral hypersensitivity. In these foundational studies (reviewed in ( 148 )), colorectal afferents were classified using an anatomical-based naming scheme (e.g., “mucosal” or “serosal”) that depended on their response profiles to a designated set of mechanical stimuli. For example, afferents that responded to mucosal probe were called “mucosal” afferents; “muscular” afferents displayed sensitivity to circumferential stretch; whereas “serosal” afferents exhibited little mucosal- or stretch-sensitivity but responded to poking the serosal surface of the tissue, likely activating axons rather than receptive fields. Subsequent neural tracing ( 149 ) and/or genetic studies ( 150 ) have confirmed the presence of LS afferent endings in the myenteric plexus, circular muscle, submucosa, and mucosa, with afferents innervating one or multiple layers, but endings have not been observed in the serosa using these approaches. Like vagal afferents, the location of endings within the tissue does not well-predict functional responses; rather, the spinal origin and (to a lesser degree) the molecular profile are strong indicators of response properties. In the last decade, several key studies have revealed insights into distinct subtypes of LS afferent neurons, their molecular and functional properties, and how they are unique from afferents originating from thoracolumbar (TL) spinal levels. In vivo studies confirm that sensory neurons in L6-S1 DRG respond to colorectal distension (CRD) across a wide range of pressures ( 147 , 150 – 152 ) and contribute to the physiological responses evoked by noxious levels of distension ( 153 ). Distension responses appear to be partially mediated by Piezo2 ( 150 , 154 ), which is more enriched in colon-innervating LS afferents compared to TL and nodose afferents innervating the same colon region ( 15 ); however, although the responses to distension were lower, they were not abolished in Piezo2 knockout mouse models ( 150 , 154 ), suggesting additional channels and receptors contribute to mechanosensitivity in the distal colon. Studies have begun to correlate molecular profile, response properties and anatomical location of peripheral and central terminals, but there are discrepancies likely due to differences in approach and experimental design ( 15 , 150 , 155 ). Two independent studies that compared the transcriptional profiles of back-labeled colon afferents from different spinal pathways ( 15 , 155 ) have reached similar conclusions regarding molecular subpopulations of LS and TL afferents. In contrast, the markers for distal colon afferents used in Wolfson et al (2023) were originally identified and validated using all neurons within the DRG ( 150 ), where somatic-tissue innervating neurons far outnumber those innervating visceral organs. The molecular make-up of somatic versus visceral sensory neurons is known to be distinct; in the skin, Th is mostly restricted to C-LTMRs with virtually no overlap with Calca , whereas nearly all colonic afferents express Calca and a large proportion co-express Th . Indeed, none of the five markers identified in this study ( Adra2a, Th, Bmpr1b, Sstr2, TrkB ) appear to be uniquely expressed when comparing to datasets restricted to colon afferents, even when limiting the search to LS DRG neurons, which were the focus of the calcium imaging experiments ( 150 , 155 ). For example, the pelvic peptidergic group ‘pPEP’ from Hockley et al. is enriched for Adra2a, Th, Bmpr1b, and Sstr2 , (i.e., 4 out of 5 markers), and the other pelvic group (pNF) is enriched for Th , Bmpr1b , and TrkB . The same holds true for the reverse, where each of the five markers is highly expressed in at least 2 molecularly-distinct clusters of colon-innervating sensory neurons ( 155 ), highlighting the value of incorporating back-labeling approaches to narrow in on the target population for molecular profiling. Regardless of their molecular identity, these wide dynamic range LS mechanoreceptors proportionally activate second-order projection neurons in the dorsal horn of the LS spinal cord in response to innocuous and noxious distension ( 156 ). In contrast to the very acute effect of inflammatory mediators on LS afferent sensitivity in ex vivo experiments ( 147 ), in vivo models of inflammation indicate that the sensitivity of DRG and dorsal horn neurons at LS spinal levels remains unchanged ( 157 ), further supporting the LS afferent pathway’s primary role of reporting the degree of colorectal distension or rectal filling to the CNS, regardless of inflammatory state. Signals from the LS spinal cord reach the brain via ascending pathways of the spinothalamic tract, where the urge to defecate is perceived and descending motor pathways under voluntary control become engaged ( 158 – 160 ). Along with the expected areas of activation in the insular, somatosensory, and anterior cingulate cortices that have been associated with visceral sensation and pain, regions of the brainstem are uniquely activated by the LS pathway. Wang et al. ( 158 ) compared neuronal activation in response to noxious CRD in mice with 1) all sensory pathways intact, 2) TL DRGs removed, or 3) LS DRGs removed. Results showed that the LS pathway specifically engages brainstem regions associated with defecation ( 161 – 163 ), but male and female mice exhibited differences that may have important clinical relevance. Compared to pre-CRD, there was a significant increase in activated pERK+ neurons in Barrington’s nucleus in response to CRD, but only in male mice ( 158 ). However, rather than being due to hypo-excitability, the lack of response in female mice appears to be caused by increased basal activity. In other words, the defecatory circuits normally engaged by CRD in males may already be maximally activated in females with no room for further increases, but this will require additional studies to determine underlying mechanisms involved. The motor efferent outflow from the LS spinal pathway is primarily involved in initiating propulsive motility and executing defecatory reflexes for the elimination of waste. Descending motor pathways synapse onto preganglionic neurons in the ventral horn that send signals to postganglionic neurons via the pelvic nerve, initiating a multi-synaptic relay from the CNS to the ENS circuits responsible for producing propulsive colon motor complexes ( 159 , 160 , 164 ). LS afferents also mediate spinal reflexes that do not reach the brain to facilitate the aboral movement of fecal contents ( 165 , 166 ). However, the act of defecation requires the engagement of additional voluntary motor pathways to the pelvic floor and abdominal muscles to generate anorectal pressure gradients necessary for a bowel movement. For a more in-depth review of the LS spinal circuits involved in colorectal motility and defecation, please see ( 159 ). The TL spinal (splanchnic) pathway is a key component of the sympathetic nervous system (SNS), redirecting energy and resources to areas of the body that needs it most. Under normal homeostatic conditions, this pathway remains largely inactive, as most of the sensory-motor functions associated with sympathetic outflow can be carried out by intestinofugal-PVG circuits. However, the TL pathway becomes responsive under certain conditions, typically associated with inflammation, injury or stress, and therefore, acts as the body’s “alert system” ( 167 , 168 ) ( Figure 5 ). Of the three sensory pathways to the gut, the least is known about afferents originating from TL DRGs, and although this pathway innervates every GI organ, most functional studies have characterized properties of TL afferents in the distal colon. While the limitations of focusing on a specific GI region are obvious, by directly comparing pelvic colonic afferents from LS DRGs to splanchnic colonic afferents from TL DRGs, studies have revealed important insights into the distinct functions of these two pathways. TL/splanchnic colon afferents are mechanosensitive but less likely to respond at a lower stimulus intensities, stretch and probing compared to pelvic afferents under normal conditions ( 146 ). TL splanchnic afferents exhibit chemosensitivity to several mediators, including bradykinin, capsaicin and ATP, supporting their involvement in post-inflammatory hypersensitivity ( 169 , 170 ). A study by Hughes et al. ( 171 ) showed that splanchnic afferents from TL spinal levels had sustained increases in responses to mechanical stimulation during and following inflammation, while pelvic afferents displayed increased sensitivity after recovery from inflammation. These inflammatory mediators not only sensitize peripheral endings but also amplify nociceptive transmission within the spinal dorsal horn, increasing neuron activity ( 172 ). As a result, a pathway that is largely subdued during homeostasis becomes the principal conduit for and dominant driver of visceral pain and hypersensitivity. Importantly, such inflammation-induced sensitization may also serve a broader adaptive purpose, potentially engaging autonomic motor circuits of the CNS that are necessary to orchestrate systemic immune responses required for protection, healing and recovery. The CNS pathways and brain regions responsible for transmitting and processing pain signals that originate from visceral and somatic tissues have similarities and differences that influence perceptual qualities ( 173 ). Pain signals coming into the spinal cord from the GI tract reach the brain via the spinothalamic tract, also designated for carrying nociceptive and thermal input from somatic tissues. Imaging studies indicate that although visceral and somatic pain are processed in the same brain regions, visceral pain is more strongly associated with activation of emotional, affective areas with less activation of areas involved in sensory discrimination, contributing to its poor localization ( 173 , 174 ). Patients with various causes of visceral hypersensitivity have described their abdominal pain with a range of descriptors, such as burning, stabbing, pressure, bloating, cramping, and the quality may depend on origin, cause, duration, or other factors. But unlike feeding behavior and defecation, the physiological or adaptive purpose of experiencing abdominal pain remains unknown. The pain is likely meant to incite actions in the organism that facilitate protection of gut tissue from further harm or damage while it heals. In mice with colitis, the caudal ventrolateral medulla (cVLM) appears to be involved in visceral hypersensitivity ( 175 ). The cVLM is responsible for integrating visceral sensory information to coordinate autonomic outflow from the CNS, and interestingly, the predominant sensory input from the colon to the cVLM arises from the TL but not LS spinal pathway ( 158 ). Based on the collective evidence, activation of the TL pathway seems to alert the brain that there is a problem that requires the attention, resources, and demands of the entire body. The autonomic motor outflow from the TL spinal cord has been traditionally associated with the SNS, described as promoting physiological processes and mobilizing energy resources necessary to invoke a “fight” or “flight” response to threats or danger In other organ systems this involves coordinating local and systemic responses to redirect focus to an area in that requires attention, and the same holds true for the gut. For example, sympathetic vasoconstriction works in tandem with sensory neurons involved in vasodilation to control blood flow for thermoregulation and as part of the exercise pressor reflex, ensuring blood flow is redirected to the organs and tissues in need. Similarly, stressors in the GI tract prompt the SNS, likely by “waking up” TL afferents that engage TL autonomic motor outflow, to redirect resources, energy, and attention to an area in need. The SNS has the remarkable ability to have simultaneous actions on multiple cell types in different regions of the body. Locally, these effects may appear unrelated or even opposing of each other, but in reality, are working together to achieve a common goal. The sympathetic control over colon motility, for example, exhibits regional differences ( 51 ) – when activated, sympathetic neurons inhibit ENS activity in distal regions to suppress the movement of fecal contents, while also promoting myogenic contractions in proximal regions to facilitate further water reabsorption. Similarly in the stomach, activation of the SNS inhibits gastric motility and simultaneously stimulates contraction of the pyloric sphincter, thereby restricting the movement of contents between regions ( 47 , 176 , 177 ). To accomplish this kind of dynamic regulation, the SNS has a wide repertoire of neurotransmitters and receptors that act on numerous cell types (e.g., endothelial cells, immune cells, smooth muscle, etc.) and can have different downstream effects depending on the physiological context, especially in circumstances requiring neuroimmune regulation, as discussed further below.

The gut microbiota communicates bidirectionally with the host neuro-immune system through its metabolites. These metabolites influence immune cell function, regulate inflammatory responses and impact the activity of the enteric and extrinsic neurons. In turn, the host’s nervous and immune systems shape the composition and function of the microbiota, creating a dynamic feedback loop that is critical for maintaining gut homeostasis. Disruption of this communication, whether through microbial imbalance or impaired host signaling, can contribute to GI disorders, visceral hypersensitivity, altered feeding behavior, and exacerbate stress responses. Growing evidence suggests that gut microbiota can regulate feeding behavior of the host. In a recent study, it was found that bacterial flagellin in mouse colon activates Toll-like receptor 5 (TLR5) on peptide YY (PYY)-expressing colonic neuropod cells. This activation triggers PYY release onto NPY2R-expressing vagal nodose neurons, thereby regulating feeding ( 203 ). This is supported by a previous study where TLR5-deficient mice exhibited features of metabolic syndrome and changes in the gut microbiota composition ( 204 ). Furthermore, germ free mice colonized with fecal microbiota from obese co-twin exhibited significantly greater increase in body mass and adiposity than those colonized with microbiota from the lean co-twin ( 205 ). Notably, cohousing obese- and lean-colonized mice prevented the obese cage mates from gaining body mass and adiposity ( 205 ). Therefore, there is a link between gut microbiota and host feeding behavior and metabolic health. The gut microbiota also influences the stress response through the HPA axis. In response to restraint stress, germ free mice had elevated plasma adrenocorticotropic (ACTH) and corticosterone levels compared to specific pathogen free mice ( 206 ). This stress response was reversed by reconstitution with Bifidobacterium infantis in germ free mice ( 206 ). It was also recently uncovered that stress and circadian systems are interconnected, where gut microbiota is a regulator of HPA-axis rhythmicity. Depletion of the microbiota alters the brain’s transcriptome and metabolome in stress-responding pathways in the hippocampus and amygdala during the day ( 207 ). The group also reported that microbially depleted mice displayed impaired hypothalamic blood-brain barrier and an altered behavioral response to stress, reflected in the reduced social interaction time ( 207 ). Dysbiosis of the gut microbiota has been shown to increase sensitivity of colonic distension and induce hyperexcitability in DRG neurons ( 208 ). Interestingly, DRG neurons from naïve mice exhibit similar hyperexcitability when exposed to serum from vancomycin-treated mice, suggesting that microbial dysbiosis changes circulating mediators that influence nociception ( 208 ). In addition, germ-free mice colonized with intestinal aspirates from GI patients with abdominal pain as their predominant symptom had higher responses to distension, indicating visceral hypersensitivity ( 209 ). Thoracic DRG neurons exposed to intestinal aspirates from the pain group showed increased neuronal activation and triggered higher calcium responses ( 209 ). Together these findings suggest that gut microbiota may regulate pain signaling through the activation of DRG neurons.

There is a bidirectional communication between neural pathways and the immune system to maintain gut homeostasis, and each pathway of the gut-brain axis has its own signaling mechanisms to mediate immune function ( 178 – 180 ). Notably, because of the breadth of studies, this review does not cover all aspects of neuroimmune regulation, but we highlight below some of the key roles of different neural control pathways in modulating immune responses. Starting at the level of the ENS, enteric neurons can interact with immune cells through the release of cytokines and neuropeptides. A group has demonstrated that muscularis macrophages (MMs) and enteric neurons interact with each other to regulate intestinal peristalsis, where MMs produce bone morphogenetic protein 2 (BMP2) to support enteric neurons, while neurons maintain MM homeostasis by producing macrophage specific growth factor, colony stimulating factor 1 (CSF1) ( 181 ). Enteric neurons also release IL-18 cytokine which signals to goblet cells to protect the epithelial barrier against pathogens, and deficiency in enteric-derived IL-18 results in microbial dysbiosis ( 182 , 183 ). Moreover, enteric neurons have an important role in regulating the activity of innate lymphoid cells (ILCs) in the gut. VIP expressing enteric neurons have been shown to mediate type 3 ILC function both at homeostasis and during host defense ( 184 , 185 ). Modulation of this pathway maintains intestinal barrier function and decreases susceptibility to inflammation-induced gut injury. Sensory neurons locally communicate with and modulate the function of the immune system, through the release of neuropeptides from peripheral terminals, such as calcitonin gene-related peptide (CGRP) and Substance P (SP). CGRP promotes mucus production through goblet cells via the CGRP-Ramp1 pathway, protecting intestinal barrier function ( 186 ). Ablation of nociceptors increases susceptibility to DSS-induced colitis in mice, and activating nociceptors through CGRP administration enhances mucus production and protects against colitis in mice ( 186 ). A previous study also highlighted the protective role of CGRP, demonstrating that CGRP-deficient mice are more susceptible to colitis compared to wildtype mice ( 187 ), whereas SP promotes the secretion of pro-inflammatory cytokines by immune cells ( 188 , 189 ). Some studies indicate a detrimental role of SP in DSS- and TNBS-induced colitis, where increased SP release may drive intestinal inflammation ( 190 , 191 ). However, there are studies showing the protective role of SP in maintaining gut microbiota homeostasis and preserving epithelial barrier function ( 192 , 193 ). Therefore, additional studies are needed to investigate the dynamic relationships between sensory neuropeptides, like CGRP and SP, and immune activation. Parasympathetic immune regulation occurs through vagal efferent pathways. Activation of the cholinergic anti-inflammatory pathway by stimulation of the efferent vagal nerve attenuated systemic inflammation in response to endotoxin shock ( 194 , 195 ). VNS may also recruit additional cholinergic activity by engaging the ENS, and together, these circuits regulate gut immune responses appear to target α7 nicotinic acetylcholine receptors (α7nAChR) on muscularis macrophages (MMs) to alter levels of phagocytotic activity ( 196 , 197 ). It is important to note, as highlighted in another review ( 198 ), tight regulation of VNS is vital as uncontrolled stimulation could promote sustained excessive inflammatory responses and potentially give rise to autoimmune disorders. Depending on the context, sympathetic pathways can exert both pro- and anti-inflammatory actions in the GI tract with the ultimate goal of healing, recovery, and return to homeostasis. In general, the SNS responds acutely in a proinflammatory manner, potentially acting as a beacon honing on the area in need, whereas chronically, the SNS has anti-inflammatory actions meant to protect the GI tract from potential harm or damage. For example, adrenergic signaling in muscularis macrophages (MMs) protects against infection-induced enteric neuronal loss and dysmotility, through an arginase 1-polyamine axis ( 199 ); ablation of β2 adrenergic receptors in MMs exacerbated neuronal loss following enteric infection, whereas sympathetic activation mediates enteric neuron protection during infection ( 199 ). Complementing these findings, sympathetic denervation led to decreased norepinephrine concentrations in intestinal tissue and the development of colitis in mice ( 200 ). Further highlighting its protective role, sympathetic nerves regulate intestinal epithelial proliferation and improve mucosal healing through α 2a adrenergic receptor signaling, where the treatment of α 2 agonist increased stem cell and proliferation markers ( 201 , 202 ). However, there are still numerous open-ended questions regarding the role of the SNS in driving long-lasting, pathophysiological changes that lead to GI disease and disorders of gut-brain interaction. Clinical evidence strongly supports that chronic stress and high levels of sympathetic tone have negative impacts on health and may even be a causative agent for disease, but preclinical studies will be required to define the mechanisms at play.

Interoception refers to the process by which the nervous system senses, interprets, and responds to changes in the body’s internal state, thereby maintaining homeostasis and regulating behavior. Much of the interoceptive processing occurs subconsciously through sensory-autonomic neural reflexes and humoral (endocrine) signaling that regulate essential functions, but internal signals may also result in conscious perceptions that invoke subsequent responses and/or behavioral adaptations ( 1 – 3 ) The awareness of certain interoceptive signals from the gut is a normal part of gastrointestinal physiology, typically involved in functions that require voluntary actions. However, dysfunction can occur in interoceptive processes, leading to pathological interoceptive awareness that manifests as hypo- or hypersensitivity, and is prevalent across a diverse range of gastrointestinal diseases and disorders of gut-brain interaction, including irritable bowel syndrome, panic disorder, anorexia nervosa, and autism ( 4 – 7 ). Despite great advances in research on interoception, mechanisms of visceral sensation, and the gut-brain axis, there are significant gaps in our understanding of the neural circuits and mechanisms involved in normal and abnormal interoception in the gut. Homeostatic functions in the gastrointestinal (GI) tract are carried out by multiple levels of sensory-motor neural control, mostly below the level of consciousness, but higher-level circuits may be “called upon” to engage in specific responses that are needed at that time. Accordingly, the GI tract is home to a large and diverse set of sensory cells that, collectively, detect and respond to a wide range of stimuli encountered in the gut ( 8 ). Many of these things can be handled by local control systems, such as the enteric nervous system (ENS; the gut’s brain) capable of initiating and sustaining activity required for peristalsis ( 9 ). However, other stressors in the gut require responses that depend on well-timed coordination with distant GI regions, and thus the ENS recruits the next level of command – the prevertebral ganglia (PVG), where postganglionic motor neurons innervating abdominal and pelvic organs are bundled together and integrate sensory signals to coordinate motility, secretions, and other functions ( 10 – 14 ). Higher control involves engagement of the central nervous system (CNS) via vagal and spinal pathways, which appear to oversee distinct sensory-motor functions ( 15 ). Thus, in addition to hierarchical control, incoming input to the CNS exhibits functional segregation among pathways allowing for parallel processing. This circuit architecture is particularly prominent in sensory systems that require simultaneous integration of multiple, complex features, e.g., the visual system ( 16 ). Ascending signals that reach cortical brain regions form sensory perceptions involved in conscious interoceptive processing ( 3 ). For the gut, conscious interoception pertains to (1) feeding behavior mediated by vagal pathways to the nucleus of solitary tract (NTS) and hypothalamus, as well as (2) the lumbosacral (LS) spinal pathways involved in defecation and voluntary elimination of waste. The thoracolumbar (TL) spinal pathways, by contrast, seem to become engaged during times of ‘crisis’ or allostatic load (e.g., inflammation, infection, dysbiosis, chronic stress, etc.) and may contribute to pathological interoceptive awareness, visceral hypersensitivity, and other negative GI symptoms observed in disorder of gut-brain interaction. Distinct neural pathways of the gut-brain axis appear to have unique sensory and motor roles in maintaining homeostasis, regulating gut functions under normal conditions and alerting to damage, infection or inflammation. With the development of new approaches, increasing evidence supports that individual pathways express distinct types of receptors, respond to different sensory modalities, have different CNS targets, and lead to distinct motor output commands and behavioral responses. The aim of this review is to dissect the distinct neural pathways of the gut-brain axis, their specific physiological roles, and how their dysfunction relates to visceral hypersensitivity, pathological interoceptive awareness, and other GI-related symptoms.