Self-Maintaining Gut Macrophages Are Essential for Intestinal Homeostasis.

Macrophages are highly heterogeneous tissue-resident immune cells that perform a variety of tissue-supportive functions. The current paradigm dictates that intestinal macrophages are continuously replaced by incoming monocytes that acquire a pro-inflammatory or tissue-protective signature. Here, we identify a self-maintaining population of macrophages that arise from both embryonic precursors and adult bone marrow-derived monocytes and persists throughout adulthood. Gene expression and imaging studies of self-maintaining macrophages revealed distinct transcriptional profiles that reflect their unique localization (i.e., closely positioned to blood vessels, submucosal and myenteric plexus, Paneth cells, and Peyer's patches). Depletion of self-maintaining macrophages resulted in morphological abnormalities in the submucosal vasculature and loss of enteric neurons, leading to vascular leakage, impaired secretion, and reduced intestinal motility. These results provide critical insights in intestinal macrophage heterogeneity and demonstrate the strategic role of self-maintaining macrophages in gut homeostasis and intestinal physiology.

Neuronal programming by microbiota regulates intestinal physiology.

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 disorders1. Luminal factors, such as diet and microbiota, regulate neurogenic programs of gut motility2-5, but the underlying molecular mechanisms remain unclear. Here we show that the transcription factor aryl hydrocarbon receptor (AHR) functions as a biosensor in intestinal neural circuits, linking their functional output to the microbial environment of the gut lumen. Using nuclear RNA sequencing of mouse enteric neurons that represent distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles that are controlled by the combined effects of host genetic programs and microbial colonization. Microbiota-induced expression of AHR in neurons of the distal gastrointestinal tract enables these neurons to respond to the luminal environment and to induce expression of neuron-specific effector mechanisms. Neuron-specific deletion of Ahr, or constitutive overexpression of its negative feedback regulator CYP1A1, results in reduced peristaltic activity of the colon, similar to that observed in microbiota-depleted mice. Finally, expression of Ahr in the enteric neurons of mice treated with antibiotics partially restores intestinal motility. Together, our experiments identify AHR signalling in enteric neurons as a regulatory node that integrates the luminal environment with the physiological output of intestinal neural circuits to maintain gut homeostasis and health.

Nano-positioning and tubulin conformation contribute to axonal transport regulation of mitochondria along microtubules.

Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules that fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin in neurons both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers like guanosine triphosphate (GTP)-tubulin. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin velocity and density on microtubules consisting of elongated dimers add to the increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin contributes to the regulation of mitochondrial motility and as such to the local distribution of mitochondria along axons.

Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2.

The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4-/- ) CRC models and an indirect co-culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4-/- ENS cell secretome, which is enriched for Nidogen-1 (Nid1) and Fibulin-2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS-derived Nidogen-1 and Fibulin-2 enhance colorectal carcinogenesis.

Simultaneous whole-cell patch-clamp and calcium imaging on myenteric neurons.

Live calcium imaging is often used as a proxy for electrophysiological measurements and has been a valuable tool that allows simultaneous analysis of neuronal activity in multiple cells at the population level. In the enteric nervous system, there are two main electrophysiological classes of neurons, after-hyperpolarizing (AH)- and synaptic (S)-neurons, which have been shown to have different calcium handling mechanisms. However, they are rarely considered separately in calcium imaging experiments. A handful of studies have shown that in guinea pig, a calcium transient will accompany a single action potential in AH-neurons, but multiple action potentials are required to generate a calcium transient in S-neurons. How this translates to different modes of cellular depolarization and whether this is consistent across species is unknown. In this study, we used simultaneous whole-cell patch-clamp electrophysiology together with calcium imaging to investigate how enteric neurons respond to different modes of depolarization. Using both traditional (4 Hz) and also high-speed (1,000 Hz) imaging techniques, we found that single action potentials elicit calcium transients in both AH-neurons and S-neurons. Subthreshold membrane depolarizations were also able to elicit calcium transients, although calcium responses were generally amplified if an action potential was present. Furthermore, we identified that responses to nicotinic acetylcholine receptor stimulation can be used to distinguish between AH- and S-neurons in calcium imaging.NEW & NOTEWORTHY Live calcium imaging is an important tool for investigating enteric nervous system (ENS) function. Previous studies have shown that multiple action potentials are needed to generate a calcium response in S-neurons, which has important implications for the interpretation of calcium imaging data. Here, we show that in mouse myenteric neurons, calcium transients are elicited by single action potentials in both AH- and S-neurons. In addition, nicotinic acetylcholine receptor stimulation can be used to distinguish between these two classes.

The enteric nervous system in gastrointestinal disease etiology.

A highly conserved but convoluted network of neurons and glial cells, the enteric nervous system (ENS), is positioned along the wall of the gut to coordinate digestive processes and gastrointestinal homeostasis. Because ENS components are in charge of the autonomous regulation of gut function, it is inevitable that their dysfunction is central to the pathophysiology and symptom generation of gastrointestinal disease. While for neurodevelopmental disorders such as Hirschsprung, ENS pathogenesis appears to be clear-cut, the role for impaired ENS activity in the etiology of other gastrointestinal disorders is less established and is often deemed secondary to other insults like intestinal inflammation. However, mounting experimental evidence in recent years indicates that gastrointestinal homeostasis hinges on multifaceted connections between the ENS, and other cellular networks such as the intestinal epithelium, the immune system, and the intestinal microbiome. Derangement of these interactions could underlie gastrointestinal disease onset and elicit variable degrees of abnormal gut function, pinpointing, perhaps unexpectedly, the ENS as a diligent participant in idiopathic but also in inflammatory and cancerous diseases of the gut. In this review, we discuss the latest evidence on the role of the ENS in the pathogenesis of enteric neuropathies, disorders of gut-brain interaction, inflammatory bowel diseases, and colorectal cancer.

Development of the intrinsic innervation of the small bowel mucosa and villi.

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.

Morphogenesis and motility of the Astyanax mexicanus gastrointestinal tract.

Through the course of evolution, the gastrointestinal (GI) tract has been modified to maximize nutrient absorption, forming specialized segments that are morphologically and functionally distinct. Here we show that the GI tract of the Mexican tetra, Astyanax mexicanus, has distinct regions, exhibiting differences in morphology, motility, and absorption. We found that A. mexicanus populations adapted for life in subterranean caves exhibit differences in the GI segments compared to those adapted to surface rivers. Cave-adapted fish exhibit bi-directional churning motility in the stomach region that is largely absent in river-adapted fish. We investigated how this motility pattern influences intestinal transit of powdered food and live prey. We found that powdered food is more readily emptied from the cavefish GI tract. In contrast, the transit of live rotifers from the stomach region to the midgut occurs more slowly in cavefish compared to surface fish, consistent with the presence of churning motility. Differences in intestinal motility and transit likely reflect adaptation to unique food sources available to post-larval A. mexicanus in the cave and river environments. We found that cavefish grow more quickly than surface fish when fed ad libitum, suggesting that altered GI function may aid in nutrient consumption or absorption. We did not observe differences in enteric neuron density or smooth muscle organization between cavefish and surface fish. Altered intestinal motility in cavefish could instead be due to changes in the activity or patterning of the enteric nervous system. Exploring this avenue will lead to a better understanding of how the GI tract evolves to maximize energy assimilation from novel food sources.

Structurally defined signaling in neuro-glia units in the enteric nervous system.

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.

A Novel Zebrafish ret Heterozygous Model of Hirschsprung Disease Identifies a Functional Role for mapk10 as a Modifier of Enteric Nervous System Phenotype Severity.

Hirschsprung disease (HSCR) is characterized by absence of enteric neurons from the distal colon and severe intestinal dysmotility. To understand the pathophysiology and genetics of HSCR we developed a unique zebrafish model that allows combined genetic, developmental and in vivo physiological studies. We show that ret mutant zebrafish exhibit cellular, physiological and genetic features of HSCR, including absence of intestinal neurons, reduced peristalsis, and varying phenotype expressivity in the heterozygous state. We perform live imaging experiments using a UAS-GAL4 binary genetic system to drive fluorescent protein expression in ENS progenitors. We demonstrate that ENS progenitors migrate at reduced speed in ret heterozygous embryos, without changes in proliferation or survival, establishing this as a principal pathogenic mechanism for distal aganglionosis. We show, using live imaging of actual intestinal movements, that intestinal motility is severely compromised in ret mutants, and partially impaired in ret heterozygous larvae, and establish a clear correlation between neuron position and organised intestinal motility. We exploited the partially penetrant ret heterozygous phenotype as a sensitised background to test the influence of a candidate modifier gene. We generated mapk10 loss-of-function mutants, which show reduced numbers of enteric neurons. Significantly, we show that introduction of mapk10 mutations into ret heterozygotes enhanced the ENS deficit, supporting MAPK10 as a HSCR susceptibility locus. Our studies demonstrate that ret heterozygous zebrafish is a sensitized model, with many significant advantages over existing murine models, to explore the pathophysiology and complex genetics of HSCR.

Spontaneous calcium waves in the developing enteric nervous system.

The enteric nervous system (ENS) is an extensive network of neurons in the gut wall that arises from neural crest-derived cells. Like other populations of neural crest cells, it is known that enteric neural crest-derived cells (ENCCs) influence the behaviour of each other and therefore must communicate. However, little is known about how ENCCs communicate with each other. In this study, we used Ca2+ imaging to examine communication between ENCCs in the embryonic gut, using mice where ENCCs express a genetically-encoded calcium indicator. Spontaneous propagating calcium waves were observed between neighbouring ENCCs, through both neuronal and non-neuronal ENCCs. Pharmacological experiments showed wave propagation was not mediated by gap junctions, but by purinergic signalling via P2 receptors. The expression of several P2X and P2Y receptors was confirmed using RT-PCR. Furthermore, inhibition of P2 receptors altered the morphology of the ENCC network, without affecting neuronal differentiation or ENCC proliferation. It is well established that purines participate in synaptic transmission in the mature ENS. Our results describe, for the first time, purinergic signalling between ENCCs during pre-natal development, which plays roles in the propagation of Ca2+ waves between ENCCs and in ENCC network formation. One previous study has shown that calcium signalling plays a role in sympathetic ganglia formation; our results suggest that calcium waves are likely to be important for enteric ganglia development.

Changes in Nicotinic Neurotransmission during Enteric Nervous System Development.

Acetylcholine-activating pentameric nicotinic receptors (nAChRs) are an essential mode of neurotransmission in the enteric nervous system (ENS). In this study, we examined the functional development of specific nAChR subtypes in myenteric neurons using Wnt1-Cre;R26R-GCaMP3 mice, where all enteric neurons and glia express the genetically encoded calcium indicator, GCaMP3. Transcripts encoding α3, α4, α7, ß2, and ß4 nAChR subunits were already expressed at low levels in the E11.5 gut and by E14.5 and, thereafter, α3 and ß4 transcripts were the most abundant. The effect of specific nAChR subtype antagonists on evoked calcium activity in enteric neurons was investigated at different ages. Blockade of the α3ß4 receptors reduced electrically and chemically evoked calcium responses at E12.5, E14.5, and P0. In addition to the α3ß4 antagonist, antagonists to α3ß2 and α4ß2 also significantly reduced responses by P10-11 and in adult preparations. Therefore, there is an increase in the diversity of functional nAChRs during postnatal development. However, an α7 nAChR antagonist had no effect at any age. Furthermore, at E12.5 we found evidence for unconventional receptors that were responsive to the nAChR agonists 1-dimethyl-4-phenylpiperazinium and nicotine, but were insensitive to the general nicotinic blocker, hexamethonium. Migration, differentiation, and neuritogenesis assays did not reveal a role for nAChRs in these processes during embryonic development. In conclusion, there are significant changes in the contribution of different nAChR subunits to synaptic transmission during ENS development, even after birth. This is the first study to investigate the development of cholinergic transmission in the ENS.

Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system.

Enteric glial cells are vital for the autonomic control of gastrointestinal homeostasis by the enteric nervous system. Several different functions have been assigned to enteric glial cells but whether these are performed by specialized subtypes with a distinctive phenotype and function remains elusive. We used Mosaic Analysis with Double Markers and inducible lineage tracing to characterize the morphology and dynamic molecular marker expression of enteric GLIA in the myenteric plexus. Functional analysis in individually identified enteric glia was performed by Ca(2+) imaging. Our experiments have identified four morphologically distinct subpopulations of enteric glia in the gastrointestinal tract of adult mice. Marker expression analysis showed that the majority of glia in the myenteric plexus co-express glial fibrillary acidic protein (GFAP), S100ß, and Sox10. However, a considerable fraction (up to 80%) of glia outside the myenteric ganglia, did not label for these markers. Lineage tracing experiments suggest that these alternative combinations of markers reflect dynamic gene regulation rather than lineage restrictions. At the functional level, the three myenteric glia subtypes can be distinguished by their differential response to adenosine triphosphate. Together, our studies reveal extensive heterogeneity and phenotypic plasticity of enteric glial cells and set a framework for further investigations aimed at deciphering their role in digestive function and disease.

A distinct vagal anti-inflammatory pathway modulates intestinal muscularis resident macrophages independent of the spleen.

The cholinergic anti-inflammatory pathway (CAIP) has been proposed as a key mechanism by which the brain, through the vagus nerve, modulates the immune system in the spleen. Vagus nerve stimulation (VNS) reduces intestinal inflammation and improves postoperative ileus. We investigated the neural pathway involved and the cells mediating the anti-inflammatory effect of VNS in the gut. The effect of VNS on intestinal inflammation and transit was investigated in wild-type, splenic denervated and Rag-1 knockout mice. To define the possible role of α7 nicotinic acetylcholine receptor (α7nAChR), we used knockout and bone marrow chimaera mice. Anterograde tracing of vagal efferents, cell sorting and Ca(2+) imaging were used to reveal the intestinal cells targeted by the vagus nerve. VNS attenuates surgery-induced intestinal inflammation and improves postoperative intestinal transit in wild-type, splenic denervated and T-cell-deficient mice. In contrast, VNS is ineffective in α7nAChR knockout mice and α7nAChR-deficient bone marrow chimaera mice. Anterograde labelling fails to detect vagal efferents contacting resident macrophages, but shows close contacts between cholinergic myenteric neurons and resident macrophages expressing α7nAChR. Finally, α7nAChR activation modulates ATP-induced Ca(2+) response in small intestine resident macrophages. We show that the anti-inflammatory effect of the VNS in the intestine is independent of the spleen and T cells. Instead, the vagus nerve interacts with cholinergic myenteric neurons in close contact with the muscularis macrophages. Our data suggest that intestinal muscularis resident macrophages expressing α7nAChR are most likely the ultimate target of the gastrointestinal CAIP.

Differences in enteric neuronal density in the NSE-Noggin mouse model across institutes.

The enteric nervous system (ENS) is a large and complex part of the peripheral nervous system, and it is vital for gut homeostasis. To study the ENS, different hyper- and hypo-innervated model systems have been developed. The NSE-Noggin mouse model was described as one of the few models with a higher enteric neuronal density in the colon. However, in our hands NSE-Noggin mice did not present with a hyperganglionic phenotype. NSE-Noggin mice were phenotyped based on fur appearance, genotyped and DNA sequenced to demonstrate transgene and intact NSE-Noggin-IRES-EGFP construct presence, and RNA expression of Noggin was shown to be upregulated. Positive EGFP staining in the plexus of NSE-Noggin mice also confirmed Noggin protein expression. Myenteric plexus preparations of the colon were examined to quantify both the overall density of enteric neurons and the proportions of enteric neurons expressing specific subtype markers. The total number of enteric neurons in the colonic myenteric plexus of transgenic mice did not differ significantly from wild types, nor did the proportion of calbindin, calretinin, or serotonin immunoreactive myenteric neurons. Possible reasons as to why the hyperinnervated phenotype could not be observed in contrast with original studies using this mouse model are discussed, including study design, influence of microbiota, and other environmental variables.

Mini-review: Enteric glial cell heterogeneity: Is it all about the niche?

Enteric glial cells represent the enteric population of peripheral glia. According to their 'glial' nature, their principal function is to support enteric neurons in both structural and functional ways. Mounting evidence however demonstrates that enteric glial cells crucially contribute to the majority of enteric nervous system functions, thus acting as pivotal players in the maintenance of gut homeostasis. Various types of enteric glia are present within the gut wall, creating an intricate interaction network with other gastrointestinal cell types. Their distribution throughout the different layers of the gut wall translates in characteristic phenotypes that are tailored to the local tissue requirements of the digestive tract. This heterogeneity is assumed to be mirrored by functional specialization, but the extensive plasticity and versatility of enteric glial cells complicates a one on one phenotype/function definition. Moreover, the relative contribution of niche-specific signals versus lineage determinants for driving enteric glial heterogeneity is still uncertain. In this review we focus on the current understanding of phenotypic and functional enteric glial cell heterogeneity, from a microenvironmental and developmental perspective.

An optimization and refinement of the whole-gut transit assay in mice.

BACKGROUND: Gastrointestinal motility measurements in mice are currently performed under suboptimal conditions, as these nocturnal animals are measured during light conditions. In addition, other stressors, like individual housing, placement in a new cage during observation, and lack of bedding and cage enrichment cause animal discomfort and might contribute to higher variability. Here we aimed to develop a refined method of the widely-used whole-gut transit assay. METHODS: Wildtype mice (N = 24) were subjected to the standard or refined whole-gut transit assay, either with or without a standardized slowing in gastrointestinal motility induced by loperamide. The standard assay consisted of a gavage with carmine red, observation during the light period and individual housing in a new cage without cage enrichment. For the refined whole-gut transit assay, mice were gavaged with UV-fluorescent DETEX®, observed during the dark period, while pairwise housed in their home cage with cage enrichment. Time until excretion of the first colored fecal pellet was assessed, and pellets were collected to assess number, weight, and water content. KEY RESULTS: The DETEX®-containing pellets were UV-detectable, allowing to measure the mice in their active period in the dark. The refined method caused less variation (20.8% and 16.0%) compared to the standard method (29.0% and 21.7%). Fecal pellet number, weight, and water content was significantly different between the standard and refined method. CONCLUSIONS & INFERENCES: This refined whole-gut transit assay provides a reliable approach to measure whole-gut transit time in mice in a more physiological context, with reduced variability compared to the standard method.

d-Serine agonism of GluN1-GluN3 NMDA receptors regulates the activity of enteric neurons and coordinates gut motility.

The enteric nervous system (ENS) is a complex network of diverse molecularly defined classes of neurons embedded in the gastrointestinal wall and responsible for controlling the major functions of the gut. As in the central nervous system, the vast array of ENS neurons is interconnected by chemical synapses. Despite several studies reporting the expression of ionotropic glutamate receptors in the ENS, their roles in the gut remain elusive. Here, by using an array of immunohistochemistry, molecular profiling and functional assays, we uncover a new role for d-serine (d-Ser) and non-conventional GluN1-GluN3 N-methyl d-aspartate receptors (NMDARs) in regulating ENS functions. We demonstrate that d-Ser is produced by serine racemase (SR) expressed in enteric neurons. By using both in situ patch clamp recording and calcium imaging, we show that d-Ser alone acts as an excitatory neurotransmitter in the ENS independently of the conventional GluN1-GluN2 NMDARs. Instead, d-Ser directly gates the non-conventional GluN1-GluN3 NMDARs in enteric neurons from both mouse and guinea-pig. Pharmacological inhibition or potentiation of GluN1-GluN3 NMDARs had opposite effects on mouse colonic motor activities, while genetically driven loss of SR impairs gut transit and fluid content of pellet output. Our results demonstrate the existence of native GluN1-GluN3 NMDARs in enteric neurons and open new perspectives on the exploration of excitatory d-Ser receptors in gut function and diseases.

Early emergence of neural activity in the developing mouse enteric nervous system.

Neurons of the enteric nervous system (ENS) arise from neural crest cells that migrate into and along the developing gastrointestinal tract. A subpopulation of these neural-crest derived cells express pan-neuronal markers early in development, shortly after they first enter the gut. However, it is unknown whether these early enteric "neurons" are electrically active. In this study we used live Ca(2+) imaging to examine the activity of enteric neurons from mice at embryonic day 11.5 (E11.5), E12.5, E15.5, and E18.5 that were dissociated and cultured overnight. PGP9.5-immunoreactive neurons from E11.5 gut cultures responded to electrical field stimulation with fast [Ca(2+)](i) transients that were sensitive to TTX and ω-conotoxin GVIA, suggesting roles for voltage-gated Na(+) channels and N-type voltage-gated Ca(2+) channels. E11.5 neurons were also responsive to the nicotinic cholinergic agonist, dimethylphenylpiperazinium, and to ATP. In addition, spontaneous [Ca(2+)](i) transients were present. Similar responses were observed in neurons from older embryonic gut. Whole-cell patch-clamp recordings performed on E12.5 enteric neurons after 2-10 h in culture revealed that these neurons fired both spontaneous and evoked action potentials. Together, our results show that enteric neurons exhibit mature forms of activity at early stages of ENS development. This is the first investigation to directly examine the presence of neural activity during enteric neuron development. Along with the spinal cord and hindbrain, the ENS appears to be one of the earliest parts of the nervous system to exhibit electrical activity.