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The gastrointestinal tract operates in a highly dynamic environment. The gut is typically exposed to continually changing and highly convoluted luminal compositions comprising not only ingested content but also a multitude of resident microbes and microbial factors. It is therefore critical that the gut is capable of distinguishing between nutritious components from noxious substances. This is facilitated by specialized cellular sensory machinery that are in place in the intestinal epithelium and the ENS. However, the specific chemosensory processes and enteric neuronal pathways that enable the gut to discern and respond appropriately to different chemicals remain unclear. A major hurdle in studying the neural processing of luminal information has been the complex spatial organization of the mucosal structures and their innervation along the radial axis. Much of our current knowledge of enteric neuronal responses to luminal stimuli stems from studies that used semi-dissected guinea pig small intestine preparations with the mucosa and submucosa removed in one-half in order to record electrical activity from exposed myenteric neurons or in the circular muscle. Building on this, we ultimately strive to work towards integrated systems with all the gut layers intact. With advanced microscopy techniques including multiphoton intravital imaging, together with transgenic technologies utilizing cell-type specific activity-dependent reporters, we stand in good stead for studying the ENS in more intact preparations and even in live animals. In this chapter, we highlight recent contributions to the knowledge of sensory gut innervation by the developing and mature ENS. We also revisit established work examining the functional connectivity between the myenteric and submucosal plexus, and discuss the methodologies that can help advance our understanding of the enteric circuitry and signaling along the mucosa-serosa axis.

Background Gastrointestinal (GI) function is critically dependent on the control of the enteric nervous system (ENS), which is situated within the gut wall and organized into two ganglionated nerve plexuses: the submucosal and myenteric plexus. The ENS is optimally positioned and together with the intestinal epithelium, is well-equipped to monitor the luminal contents such as microbial metabolites and to coordinate appropriate responses accordingly. Despite the heightened interest in the gut microbiota and its influence on intestinal physiology and pathophysiology, how they interact with the host ENS remains unclear. Methods Using full-thickness proximal colon preparations from transgenic Villin-CreERT2;R26R-GCaMP3 and Wnt1-Cre;R26R-GCaMP3 mice, which express a fluorescent Ca2+ indicator in their intestinal epithelium or in their ENS, respectively, we examined the effects of key luminal microbial metabolites (SCFAs and 5-HT) on the mucosa and underlying enteric neurons. Key results We show that the SCFAs acetate, propionate, and butyrate, as well as 5-HT can, to varying extents, acutely elicit epithelial and neuronal Ca2+ responses. Furthermore, SCFAs exert differential effects on submucosal and myenteric neurons. Additionally, we found that submucosal ganglia are predominantly aligned along the striations of the transverse mucosal folds in the proximal colon. Conclusions & inferences Taken together, our study demonstrates that different microbial metabolites, including SCFAs and 5-HT, can acutely stimulate Ca2+ signaling in the mucosal epithelium and in enteric neurons.

During embryonic development, the gut is innervated by intrinsic (enteric) and extrinsic nerves. Focusing on mammalian ENS development, in this Review we highlight how important the different compartments of this innervation are to assure proper gut function. We specifically address the three-dimensional architecture of the innervation, paying special attention to the differences in development along the longitudinal and circumferential axes of the gut. We review recent information about the formation of both intrinsic innervation, which is fairly well-known, as well as the establishment of the extrinsic innervation, which, despite its importance in gut-brain signaling, has received much less attention. We further discuss how external microbial and nutritional cues or neuroimmune interactions may influence development of gut innervation. Finally, we provide summary tables, describing the location and function of several well-known molecules, along with some newer factors that have more recently been implicated in the development of gut innervation.

Nutrient signals sensed by enteroendocrine cells are conveyed to the enteric nervous system (ENS) to initiate intestinal reflexes. We addressed whether there are specific enteric pathways dedicated to detecting different luminal nutrients. Calcium imaging was performed on intact jejunal preparations from Wnt1-cre;R26R-GCaMP3 and Villin-cre;R26R-GCaMP3 mice which express a fluorescent calcium indicator in their ENS or intestinal epithelium, respectively. Glucose, acetate, and L-phenylalanine were perfused onto the mucosa whilst imaging underlying enteric neurons. Nutrient transport or diffusion across the mucosa was mimicked by applying nutrients onto sensory nerve endings in a villus, or onto myenteric ganglia. The nutrients perfused onto the mucosa each elicited Ca2+transients in submucosal neurons and in distinct patterns of myenteric neurons. Notably, the neurochemical subtypes of myenteric neurons that responded differed between the nutrients, while submucosal responders were predominantly cholinergic. Nutrients applied into villi or onto ganglia did not elicit specific neuronal responses but did stimulate Ca2+signaling in the mucosal epithelium. These data suggest that nutrients are first detected at the level of the epithelium and that the ENS is capable of discriminating between different compositions of luminal content. Furthermore, our data show that responses to mucosal stimulation are primarily in the myenteric plexus and submucosal neurons respond secondarily.

The enteric nervous system (ENS) is an extensive network comprising millions of neurons and glial cells contained within the wall of the gastrointestinal tract. The major functions of the ENS that have been most studied include the regulation of local gut motility, secretion, and blood flow. Other areas that have been gaining increased attention include its interaction with the immune system, with the gut microbiota and its involvement in the gut-brain axis, and neuro-epithelial interactions. Thus, the enteric circuitry plays a central role in intestinal homeostasis, and this becomes particularly evident when there are faults in its wiring such as in neurodevelopmental or neurodegenerative disorders. In this review, we first focus on the current knowledge on the cellular composition of enteric circuits. We then further discuss how enteric circuits detect and process external information, how these signals may be modulated by physiological and pathophysiological factors, and finally, how outputs are generated for integrated gut function. ispartof: CELLULAR AND MOLECULAR LIFE SCIENCES vol:77 issue:22 pages:4505-4522 ispartof: location:Switzerland status: published

Prior studies suggest that β-nicotinamide adenine dinucleotide (β-NAD) is an important inhibitory motor neurotransmitter in the enteric nervous system. Metabolism of β-NAD at the neuroeffector junction (NEJ) is likely to be necessary for terminating inhibitory neurotransmission and may also produce bioactive metabolites. The enteric NEJ consists of enteric neurons and postjunctional cells of the SIP syncytium, smooth muscle cells (SMCs), interstitial cells of Cajal (ICC), and cells expressing platelet-derived growth factor receptor α (PDGFRα(+) cells). We examined possible specialized functions of the NEJ in β-NAD metabolism by determining the degradation of 1,N(6)-etheno-NAD (eNAD) in colonic tunica muscularis of wild-type, Cd38(−/−), Nt5e(−/−), Enpp1(−/−), and Cd38(−/−)/Nt5e(−/−) mice and in SIP cells from mice expressing cell-specific fluorescent reporters purified by FACS. We measured eNAD and its metabolites eADP-ribose, eAMP and e-adenosine (eADO) from tissues and sorted SIP cells using liquid chromatography. eNAD exposed to colonic muscularis of wild-type mice produced eADPR, eAMP, and eADO. CD38 mediated the conversion of eNAD to eADPR, whereas ENPP1 mediated degradation of eNAD and eADPR to eAMP. NT5E (aka CD73) was the primary enzyme forming eADO from eAMP. PDGFRα(+) cells and SMCs were involved in production of eADO from eNAD, and ICC were not involved in extracellular metabolism of eNAD. CD38 mediated the eNAD metabolism in whole tissues, but CD38 did not appear to be functionally expressed by SMCs or ICC. NT5E was expressed in SMCs > PDGFRα(+) cells. Our data show that extracellular metabolism of β-NAD in the colon is mediated by multiple enzymes with cell-specific expression.

Detection of nutritional and noxious food components in the gut is a crucial component of gastrointestinal function. Contents in the gut lumen interact with enteroendocrine cells dispersed throughout the gut epithelium. Enteroendocrine cells release many different hormones, neuropeptides, and neurotransmitters that communicate either directly or indirectly with the central nervous system and the enteric nervous system, a network of neurons and glia located within the gut wall. Several populations of enteric neurons extend processes that innervate the gastrointestinal lamina propria; however, how these processes develop and begin to transmit information from the mucosa is not fully understood. In this study, we found that Tuj1-immunoreactive neurites begin to project out of the myenteric plexus at embryonic day (E)13.5 in the mouse small intestine, even before the formation of villi. Using live calcium imaging, we discovered that neurites were capable of transmitting electrical information from stimulated villi to the plexus by E15.5. In unpeeled gut preparations where all layers were left intact, we also mimicked the basolateral release of 5-HT from enteroendocrine cells, which triggered responses in myenteric cell bodies at postnatal day (P)0. Altogether, our results show that enteric neurons extend neurites out of the myenteric plexus early during mouse enteric nervous system development, innervating the gastrointestinal mucosa, even before villus formation in mice of either sex. Neurites are already able to conduct electrical information at E15.5, and responses to 5-HT develop postnatally.NEW & NOTEWORTHY How enteric neurons project into the gut mucosa and begin to communicate with the epithelium during development is not known. Our study shows that enteric neurites project into the lamina propria as early as E13.5 in the mouse, before development of the submucous plexus and before formation of intestinal villi. These neurites are capable of transmitting electrical signals back to their cell bodies by E15.5 and respond to serotonin applied to neurite terminals by birth. ispartof: AMERICAN JOURNAL OF PHYSIOLOGY-GASTROINTESTINAL AND LIVER PHYSIOLOGY vol:318 issue:1 pages:G53-G65 ispartof: location:United States status: published

Alpha-Synuclein (α-Syn) is expressed in the central nervous system and the nervous system of the gut (enteric nervous system, ENS), and is well known to be the major constituent of Lewy bodies which are the hallmark of Parkinson's disease. Gastrointestinal disorders frequently manifest several years before motor deficits develop in Parkinson's patients. Despite extensive research on pathological rodent models, the physiological role of α-Syn in the normal ENS is unclear hampering analysis of its neuropathology. We compared the ENS in colons of α-Syn-knockout (α-Syn KO) and wild-type mice using immunohistochemistry and calcium-imaging of responses to synaptic input. We found that α-Syn is predominantly expressed in cholinergic varicosities, which contain vesicular acetylcholine transporter. α-Syn KO mice had higher enteric neuron density and a larger proportion of cholinergic neurons, notably those containing calretinin, demonstrating a role for α-Syn in regulating development of these neurons. Moreover, α-Syn deletion enhanced the amplitude of synaptically activated [Ca2+]i transients that are primarily mediated by acetylcholine activating nicotinic receptors suggesting that α-Syn modulates the availability of acetylcholine in enteric nerve terminals.

Neural control of the function of visceral organs is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence, and is often dysregulated in gastrointestinal disorders

Coordination of gastrointestinal function relies on joint efforts of enteric neurons and glia, whose crosstalk is vital for the integration of their activity. To investigate the signaling mechanisms and to delineate the spatial aspects of enteric neuron-to-glia communication within enteric ganglia we developed a method to stimulate single enteric neurons while monitoring the activity of neighboring enteric glial cells. We combined cytosolic calcium uncaging of individual enteric neurons with calcium imaging of enteric glial cells expressing a genetically encoded calcium indicator and demonstrate that enteric neurons signal to enteric glial cells through pannexins using paracrine purinergic pathways. Sparse labeling of enteric neurons and high-resolution analysis of the structural relation between neuronal cell bodies, varicose release sites and enteric glia uncovered that this form of neuron-to-glia communication is contained between the cell body of an enteric neuron and its surrounding enteric glial cells. Our results reveal the spatial and functional foundation of neuro-glia units as an operational cellular assembly in the enteric nervous system. ispartof: GLIA vol:67 issue:6 pages:1167-1178 ispartof: location:United States status: Published online

γ-Aminobutyric Acid (GABA) and its receptors, GABAA,B,C, are expressed in several locations along the gastrointestinal tract. Nevertheless, a role for GABA in enteric synaptic transmission remains elusive. In this study, we characterized the expression and function of GABA in the myenteric plexus of the mouse ileum. About 8% of all myenteric neurons were found to be GABA-immunoreactive (GABA+) including some Calretinin+ and some neuronal nitric oxide synthase (nNOS+) neurons. We used Wnt1-Cre;R26R-GCaMP3 mice, which express a genetically encoded fluorescent calcium indicator in all enteric neurons and glia. Exogenous GABA increased the intracellular calcium concentration, [Ca2+]i of some myenteric neurons including many that did not express GABA or nNOS (the majority), some GABA+, Calretinin+ or Neurofilament-M (NFM)+ but rarely nNOS+ neurons. GABA+ terminals contacted a significantly larger proportion of the cell body surface area of Calretinin+ neurons than of nNOS+ neurons. Numbers of neurons with GABA-induced [Ca2+]i transients were reduced by GABAA,B,C and nicotinic receptor blockade. Electrical stimulation of interganglionic fiber tracts was used to examine possible effects of endogenous GABA release. [Ca2+]i transients evoked by single pulses were unaffected by specific antagonists for each of the 3 GABA receptor subtypes. [Ca2+]i transients evoked by 20 pulse trains were significantly amplified by GABAC receptor blockade. These data suggest that GABAA and GABAB receptors are not involved in synaptic transmission, but suggest a novel role for GABAC receptors in modulating slow synaptic transmission, as indicated by changes in [Ca2+]i transients, within the ENS. ispartof: FRONTIERS IN PHYSIOLOGY vol:9 issue:FEB ispartof: location:Switzerland status: published

The enteric nervous system (ENS) situated within the gastrointestinal tract comprises an intricate network of neurons and glia which together regulate intestinal function. The exact neuro-glial circuitry and the signaling molecules involved are yet to be fully elucidated. Vasoactive intestinal peptide (VIP) is one of the main neurotransmitters in the gut, and is important for regulating intestinal secretion and motility. However, the role of VIP and its VPAC receptors within the enteric circuitry is not well understood. We investigated this in the submucosal plexus of mouse jejunum using calcium (Ca2+)-imaging. Local VIP application induced Ca2+-transients primarily in neurons and these were inhibited by VPAC1- and VPAC2-antagonists (PG 99-269 and PG 99-465 respectively). These VIP-evoked neural Ca2+-transients were also inhibited by tetrodotoxin (TTX), indicating that they were secondary to action potential generation. Surprisingly, VIP induced Ca2+-transients in glia in the presence of the VPAC2 antagonist. Further, selective VPAC1 receptor activation with the agonist ([K15, R16, L27]VIP(1-7)/GRF(8-27)) predominantly evoked glial responses. However, VPAC1-immunoreactivity did not colocalize with the glial marker glial fibrillary acidic protein (GFAP). Rather, VPAC1 expression was found on cholinergic submucosal neurons and nerve fibers. This suggests that glial responses observed were secondary to neuronal activation. Trains of electrical stimuli were applied to fiber tracts to induce endogenous VIP release. Delayed glial responses were evoked when the VPAC2 antagonist was present. These findings support the presence of an intrinsic VIP/VPAC-initiated neuron-to-glia signaling pathway. VPAC1 agonist-evoked glial responses were inhibited by purinergic antagonists (PPADS and MRS2179), thus demonstrating the involvement of P2Y1 receptors. Collectively, we showed that neurally-released VIP can activate neurons expressing VPAC1 and/or VPAC2 receptors to modulate purine-release onto glia. Selective VPAC1 activation evokes a glial response, whereas VPAC2 receptors may act to inhibit this response. Thus, we identified a component of an enteric neuron-glia circuit that is fine-tuned by endogenous VIP acting through VPAC1- and VPAC2-mediated pathways.

In the gastrointestinal tract, vasoactive intestinal peptide (VIP) is found exclusively within neurons. VIP regulates intestinal motility via neurally mediated and direct actions on smooth muscle and secretion by a direct mucosal action, and via actions on submucosal neurons. VIP acts via VPAC1 and VPAC2 receptors; however, the subtype involved in its neural actions is unclear. The neural roles of VIP and VPAC1 receptors (VPAC1R) were investigated in intestinal motility and secretion in guinea pig jejunum. Expression of VIP receptors across the jejunal layers was examined using RT-PCR. Submucosal and myenteric neurons expressing VIP receptor subtype VPAC1 and/or various neurochemical markers were identified immunohistochemically. Isotonic muscle contraction was measured in longitudinal muscle-myenteric plexus preparations. Electrogenic secretion across mucosa-submucosa preparations was measured in Ussing chambers by monitoring short-circuit current. Calretinin+ excitatory longitudinal muscle motor neurons expressed VPAC1R. Most cholinergic submucosal neurons, notably NPY+ secretomotor neurons, expressed VPAC1R. VIP (100 nM) induced longitudinal muscle contraction that was inhibited by TTX (1 μM), PG97–269 (VPAC1 antagonist; 1 μM), and hyoscine (10 μM), but not by hexamethonium (200 μM). VIP (50 nM)-evoked secretion was depressed by hyoscine or PG97–269 and involved a small TTX-sensitive component. PG97–269 and TTX combined did not further depress the VIP response observed in the presence of PG97–269 alone. We conclude that VIP stimulates ACh-mediated longitudinal muscle contraction via VPAC1R on cholinergic motor neurons. VIP induces Cl− secretion directly via epithelial VPAC1R and indirectly via VPAC1R on cholinergic secretomotor neurons. No evidence was obtained for involvement of other neural VIP receptors.

Cholera toxin (CT) is well established to produce diarrhea by producing hyperactivity of the enteric neural circuits that regulate water and electrolyte secretion. Its effects on intestinal motor patterns are less well understood. We examined the effects of luminal CT on motor activity of guinea-pig jejunum in vitro. Segments of jejunum were cannulated at either end and mounted horizontally. Their contractile activity was video-imaged and the recordings were used to construct spatiotemporal maps of contractile activity with CT (1.25 or 12.5 μg/ml) in the lumen. Both concentrations of CT induced propulsive motor activity in jejunal segments. The effect of 1.25 μg/ml CT was markedly enhanced by co-incubation with granisetron (5-HT(3) antagonist, 1 μM), which prevents the hypersecretion induced by CT. The increased propulsive activity was not accompanied by increased segmentation and occurred very early after exposure to CT in the presence of granisetron. Luminal CT also reduced the pressure threshold for saline distension evoked propulsive reflexes, an effect resistant to granisetron. In contrast, CT prevented the induction of segmenting contractions by luminal decanoic acid, so its effects on propulsive and segmenting contractile activity are distinctly different. Thus, in addition to producing hypersecretion, CT excites propulsive motor activity with an entirely different time course and pharmacology, but inhibits nutrient-induced segmentation. This suggests that CT excites more than one enteric neural circuit and that propulsive and segmenting motor patterns are differentially regulated. ispartof: FRONTIERS IN NEUROSCIENCE vol:4 issue:SEP ispartof: location:Switzerland status: published