High- and medium-molecular-weight neurofilament proteins define specific neuron types in the guinea-pig enteric nervous system

Previous studies have demonstrated that neurofilament proteins are expressed by type II neurons in the enteric plexuses of a range of species from mouse to human. However, two previous studies have failed to reveal this association in the guinea-pig. Furthermore, immunohistochemistry for neurofilaments has revealed neurons with a single axon and spiny dendrites in human and pig but this morphology has not been described in the guinea-pig or other species. We have used antibodies against high- and medium-weight neurofilament proteins (NF-H and NF-M) to re-examine enteric neurons in the guinea-pig. NF-H immunoreactivity occurred in all type II neurons (identified by their IB4 binding) but these neurons were never NF-M-immunoreactive. On the other hand, 17% of myenteric neurons expressed NF-M. Many of these were uni-axonal neurons with spiny dendrites and nitric oxide synthase (NOS) immunoreactivity. NOS immunoreactivity occurred in surface expansions of the cytoplasm that did not contain neurofilament immunoreactivity. Thus, because of their NOS immunoreactivity, spiny neurons had the appearance of type I neurons. This indicates that the apparent morphologies and the morphological classifications of these neurons are dependent on the methods used to reveal them. We conclude that spiny type I NOS-immunoreactive neurons have similar morphologies in human and guinea-pig and that many of these are inhibitory motor neurons. Both type II and neuropeptide-Y-immunoreactive neurons in the submucosal ganglia exhibit NF-H immunoreactivity. NF-M has been observed in nerve fibres, but not in nerve cell bodies, in the submucosa. This work was supported by a grant from the National Health and Medical Council of Australia (grant number 400020).     

Distribution of pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity in neurons of the guinea-pig digestive tract and their projections in the ileum and colon

Pituitary adenylyl cyclase activating peptide (PACAP) is a novel hypothalamic peptide that is widely distributed in neurons, including those of the gastrointestinal tract. In this study, a polyclonal antiserum directed against PACAP-27 was used to investigate the localisation of PACAP throughout the gut and to determine the projections of PACAP-immunoreactive (IR) neurons in the guinea-pig small and large intestines. PACAP-IR fibres were seen in the myenteric and submucous plexuses, in the longitudinal and circular muscle layers and around blood vessels of the submucosa throughout the gut. In both the small and large intestine, PACAP-IR cell bodies, most with Dogiel type-I morphology, were seen in the myenteric ganglia following colchicine treatment. Lesion studies (myotomy and myectomy operations) revealed that PACAP-IR interneurons projected anally in the ileum and colon. Myectomy operations resulted in a loss of PACAP-IR fibres in the circular muscle under the operation, whereas PACAP-IR fibres remained in the submucosa and around blood vessels. Following extrinsic denervation of the ileum, the number of PACAP-IR fibres in the submucosal ganglia and around blood vessels decreased. This suggests that a portion of PACAP-IR fibres supplying the submucosal ganglia and blood vessels have an extrinsic source. To investigate this, immunohistochemical studies were performed on sympathetic and dorsal root ganglia. Numerous reactive cells were seen in the dorsal root ganglia, but none was seen in sympathetic pre- or paravertebral ganglia.     

Immunohistochemical localization of microtubule-associated proteins in the nervous system of the small intestine of guinea pig

Summary Layers containing Auerbach's and Meissner's plexuses were dissected from the small intestine of guinea pig and immunostained with affinity-purified antibodies against brain-specific microtubule-associated proteins (MAPs): MAP1, MAP2 and tau and a MAP with a molecular weight of 190000 dalton purified from bovine adrenal cortex (190-kDa MAP). MAP1 antibody stained the network of nerve fibers and the cell bodies of enteric neurons in both Auerbach's and Meissner's plexuses. Staining with anti-tau antibody gave the same results. Antibody against MAP2 stained neuronal cell bodies and short thin processes extending from them. Interganglionic strands composed mainly of long processes were unstained. Anti-190-kDa MAP antibody stained both the neuronal cell bodies and bundles of nerve fibers. However, the staining was less intense than that with anti-MAP1 and tau antibodies. Differentiation in the structure of the cytoskeleton probably exists in the neuronal processes of the enteric neurons as is shown in the dendrites and axons in some neurons of the central nervous system. Thus, enteric neurons possess axon-like processes containing MAP1, tau and probably lower amounts of 190-kDa MAP. Cell bodies and dendrite-like structures of these neurons contain MAP2 in addition to MAP1, tau and 190-kDa MAP.     

Calretinin immunoreactivity in cholinergic motor neurones,interneurones and vasomotor neurones in the guinea-pig small intestine

Summary Immunoreactivity for calretinin, a calcium-binding protein, was studied in neurones in the guinea-pig small intestine. 26±1% of myenteric neurones and 12±3% of submucous neurones were immunoreactive for calretinin. All calretinin-immunoreactive neurones were also immunoreactive for choline acetyltransferase and hence are likely to be cholinergic. In the myenteric plexus, two subtypes of Dogiel type-I calretinin-immunoreactive neurones could be distinguished from their projections and neurochemical coding. Some calretinin-immunoreactive myenteric neurones had short projections to the tertiary plexus, and hence are likely to be cholinergic motor neurones to the longitudinal muscle. Some of these cells were also immunoreactive for substance P. The remaining myenteric neurones, immunoreactive for calretinin, enkephalin, neurofilament protein triplet and substance P, are likely to be orad-projecting, cholinergic interneurones. Calretinin immunoreactivity was also found in cholinergic neurones in the submucosa, which project to the submucosal vasculature and mucosal glands, and which are likely to mediate vasodilation. Thus, calretinin immunoreactivity in the guinea-pig small intestine is confined to three functional classes of cholinergic neurones. It is possible, for the first time, to distinguish these classes of cells from other enteric neurones.     

Sources of inputs to longitudinal muscle motor neurons and ascending interneurons in the guinea-pig small intestine

Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guineapig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin-and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.     

Projections of 5-hydroxytryptamine-immunoreactive neurons in guinea-pig distal colon

The presence of 5-hydroxytryptamine in enteric neurons of the guinea-pig distal colon was demonstrated by immunohistochemistry and the projections of the neurons were determined. 5-Hydroxytryptamine-containing nerve cells were observed in the myenteric plexus but no reactive nerve cells were found in submucous ganglia. Varicose reactive nerve fibres were numerous in the ganglia of both the myenteric and submucous plexuses, but were infrequent in the longitudinal muscle, circular muscle, muscularis mucosae and mucosa. Reactivity also occurred in enterochromaffin cells. Lesion studies showed that the axons of myenteric neurons projected anally to provide innervation to the circular muscle and submucosa and to other more anally located myenteric ganglia. The results suggest that a major population of 5-hydroxytryptamine neurons in the colon is descending interneurons, most of which extend for 10 to 15 mm in the myenteric plexus and innervate both 5-hydroxytryptamine and non-5-hydroxytryptamine neurons.     

Immunohistochemical evidence for the presence of calcium-binding proteins in enteric neurons

Summary Immunoreactivity for vitamin D-dependent calcium-binding protein (CaBP) has been localized in nerve cell bodies and nerve fibres in the gastrointestinal tracts of guinea-pig, rat and man. CaBP immunoreactivity was found in a high proportion of nerve cell bodies of the myenteric plexus, particularly in the small intestine. It was also found in submucous neurons of the small and large intestines. Immunoreactive nerve fibres were numerous in the myenteric ganglia, and were also common in the submucous ganglia and in the intestinal mucosa. Immunoreactive fibres were rare in the circular and longitudinal muscle coats. In the myenteric ganglia of the guinea-pig small intestine the immunoreactivity is restricted to one class of nerve cell bodies, type-II neurons of Dogiel, which display calcium action potentials in their cell bodies. These neurons were also immunoreactive with antibodies to spot 35 protein, a calcium-binding protein from the cerebellum. From the distribution of their terminals and the electrophysiological properties of these neurons it is suggested they might be sensory neurons, or perhaps interneurons. The discovery of CaBP in restricted sub-groups of enteric neurons may provide an important key for the analysis of their functions.     

Galanin-immunoreactive neurons in the guinea-pig small intestine: their projections and relationships to other enteric neurons

Summary Galanin immunoreactivity was observed in nerve cell bodies and nerve fibres, but not in enteroendocrine cells, in the small intestine of the guinea-pig. Nerve terminals were found in the myenteric plexus, in the circular muscle, in submucous ganglia, around submucous arterioles, and in the mucosa. Lesion studies showed that all terminals were intrinsic to the intestine; those in myenteric ganglia arose from cell bodies in more orally placed ganglia. Myenteric nerve cells were also the source of terminals in the circular muscle. Galanin (GAL) was located in a population of submucous nerve cell bodies that also showed immunoreactivity for vasoactive intestinal peptide (VIP) and in a separate population that was immunoreactive for neuropeptide Y (NPY). Processes of the GAL/VIP neurons supplied submucous arterioles and the mucosal epithelium. Processes of GAL/NPY neurons ran to the mucosa. It is concluded that galanin immunoreactivity occurs in several functionally distinct classes of enteric neurons, amongst which are neurons controlling (i) motility, (ii) intestinal blood flow, and (iii) mucosal water and electrolyte transport.     

The distribution of P2X<Subscript>5</Subscript> purinergic receptors in the enteric nervous system of mouse

The distribution of the P2X₅ purinoceptor in the enteric nervous system of the mouse was studied by immunohistochemistry. P2X₅ receptor immunoreactivity was widely distributed in myenteric and submucosal plexuses throughout the gastrointestinal tract. In myenteric plexuses, immunoreactivity for the P2X₅ receptor was observed in nerve fibres that enveloped ganglion cell bodies, and possibly on glial cell processes. P2X₅ receptor immunoreactivity was colocalised with vasoactive intestinal peptide and surrounded ganglion cells that contained calretinin, calbindin or nitric oxide synthase. In the submucous plexus, P2X₅ receptor immunoreactivity occurred throughout the cytoplasm and on the surface membranes of the nerve cells. Double-labelling studies showed that 22%, 9%, 6% and 68% of P2X₅ receptor-immunoreactive neurones were also immunoreactive for calretinin, calbindin, nitric oxide synthase and vasoactive intestinal peptide, respectively. Thus, the P2X₅ receptor subunit is expressed in specific functional groups of neurones. P2X₂ and P2X₃ receptors were also present in the mouse enteric plexuses but no immunoreactivity for P2X₁, P2X₄ or P2X₆ receptors was found.     

Projections of neurons with neuromedin U-like immunoreactivity in the small intestine of the guinea-pig

Summary Neuromedin U immunoreactivity was located histochemically in the guinea-pig small intestine. Projections of immunoreactive neurons were determined by analysing patterns of degeneration following nerve lesions. The co-localization of neuromedin U immunoreactivity with immunoreactivity for substance P, neuropeptide Y, vasoactive intestinal peptide and calbindin was also investigated. Neuromedin U immunoreactivity was found in nerve cells in the myenteric and submucous plexuses and in nerve fibres in these ganglionated plexuses, around submucous arterioles and in the mucosa. Reactive fibres did not supply the muscle layers. Most reactive nerve cells in the myenteric ganglia had Dogiel type-II morphology and in many there was co-localization of calbindin, although some Dogiel type-II neuromedin U neurons were calbindin negative. Lesion studies suggest that these myenteric neurons project circumferentially to local myenteric ganglia. Projections from myenteric neurons also run anally in the myenteric plexus, while other projections extend to submucous ganglia, and still further projections run from the intestine to provide terminals in the coeliac ganglia. In the submucous ganglia neuromedin U was co-localized in three populations of nerve cells: (i) those with vasoactive intestinal peptide immunoreactivity, (ii) neurons containing neuropeptide Y, and (iii) neurons containing substance P. Each of these populations sends nerve fibres to the mucosa. Neuromedin U immunoreactivity is thus located in a variety of neurons serving different functions in the intestine and therefore probably does not have a single role in intestinal physiology.     

Distribution of enteric nerve cells that project to the coeliac ganglion of the guinea-pig

Summary The digestive tract of the guinea-pig, from the esophagus to the rectum, was examined in detail to determine the distribution and relative abundances of neurons in these organs that project to the coeliac ganglion and the routes by which their axons reach the ganglion. A retrogradely transported neuronal marker, Fast Blue, was injected into the coeliac ganglion. The esophagus, stomach, gallbladder, pancreas, duodenum, small intestine, caecum, proximal colon, distal colon and rectum were analysed for labelled neurons. Retrogradely labelled neurons were found only in the myenteric plexus of these organs, and in the pancreas. No labelled neurons were found in the gallbladder or the fundus of the stomach, or in the submucous plexus of any region. A small number of labelled neurons was found in the gastric antrum. An increasing density of labelled neurons was found along the duodenum. Similarly, an increasing density of labelled neurons was found from proximal to distal along the jejuno-ileum. However, the greates densities of labelled neurons were in the large intestine. many labelled neurons were found in the caecum, including a high density underneath its taeniae. An increasing density of labelled neurons was found along the length of the proximal colon, and labelled neurons were found in the distal colon and rectum. In total, more labelled cell bodies occurred in the large intestine than in the small intestine. The routes taken by the axons of viscerofugal neurons were ascertained by lesioning the nerve bundles which accompany vessels supplying regions of the digestive tract. Viscerofugal neurons of the caecum project to the coeliac ganglion via the ileocaeco-colic nerves; neurons in the proximal colon project to the ganglion via the right colic nerves, and neurons in the distal colon project to the ganglion via the mid colic and intermesenteric nerves. Neurons in the rectum project to the coeliac ganglion via the intermesenteric nerves. These nerves (except for the intermesenterics) all join nerve bundles from the small intestine that follow the superior mesenteric artery. All viscerofugal neurons of the caecum were calbindin-immunoreactive (calb-IR) and 94% were immunoreactive for vasoactive intestinal peptide (VIP-IR). In the proximal colon, 49% of labelled neurons were calb-IR and 85% were VIP-IR. In the distal colon, 80% of labelled neurons were calb-IR and 71% were VIP-IR.     

Muscarinic and Nicotinic Cholinergic Binding Sites in the Terminal Abdominal Ganglion of the Cricket (Acheta domesticus)

Cricket (Acheta domesticus) terminal abdominal ganglia (TG) contain high concentrations (approximately 2 pmol/mg protein) of muscarinic and nicotinic cholinergic binding sites, based on the capacity of TG to bind specifically the labelled ligands L-[3H]quinuclidinyl benzilate ([3H]QNB) and [125I]alpha-bungarotoxin ([125I]alpha-BGT) with high affinity. For both ligands, binding is saturable and reversible. Competitive displacement experiments indicate that the [3H]QNB and [125I]alpha-BGT binding sites probably represent pharmacologically distinct classes of putative TG acetylcholine receptors (AChRs). Results from physiological recording and autoradiographic localization experiments demonstrate that a portion of the putative nicotinic AChRs is localized in synaptic regions of the well-characterized cercal sensory-giant interneuron pathway in the TG, where they are likely to serve as functional synaptic AChRs. Unlike nicotinic ligands, muscarinic agents do not appear to be pharmacologically active in this pathway. Therefore, in the insect CNS, putative muscarinic and nicotinic AChRs coexist at high density, but can be pharmacologically distinguished from one another on the basis of criteria derived from both ligand binding and physiological methods.     

Topography of the enteric nervous system in Peyer's patches of the porcine small intestine

The mechanisms of intercommunication between the immune and nervous systems are not fully understood. In the case of the intestine, the enteric nervous system is involved in the regulation of immune responses. It was therefore decided to employ immunohistochemical techniques to investigate the structural organization of the enteric nervous system in Peyer's patches of the porcine small intestine. Using antibodies against various nervous system-specific markers (protein gene product 9.5, neuron-specific enolase, neurofilament 200, S-100 protein and the glial fibrillary acidic protein), an intimate and specific structural association could be demonstrated between enteric nerves and the compartments of Peyer's patches: follicles, interfollicular regions and domes. Peyer's patches have a close topographical relationship to the two submucosal plexuses. Enteric nerves are located around the follicle in the interfollicular area — the so-called traffic area-and in the dome area, which plays an important role in the uptake and presentation of antigens.     

Distribution of enteric nerve cells projecting to the superior and inferior mesenteric ganglia of the guinea-pig

Retrograde tracing, using Fast Blue dye, was employed to determine the distribution of enteric nerve cells that project to the superior mesenteric and inferior mesenteric ganglia of the guinea-pig. Retrogradely labelled neurons were found in the myenteric but not submucous ganglia. When the superior mesenteric ganglion was injected, labelled neurons were found in low frequencies (less than 5 nerve cell bodies/cm²) in the duodenum, jejunum, ileum, caecum and proximal colon. The distal colon was analysed in five segments of equal length (1–5; oral to anal). Segment 1 had about 4 labelled nerve cells/cm², whereas segments 2 to 5 displayed an average of about 25 nerve cells/cm². The rectum contained about 36 labelled neurons/cm². After injection of the inferior mesenteric ganglia with Fast Blue, no labelled neurons were found in the duodenum, jejunum, ileum or caecum. No labelled cells were observed in the gallbladder. A small number of labelled cells occurred in the proximal colon and in segment 1 of the distal colon. The frequency of labelled cells increased markedly in the more anal regions of the distal colon, and reached a peak in the rectum (138 cells/cm²). Both nerve lesions and immersion of the cut nerve in Fast Blue solution showed that the superior mesenteric nerve carries the axons of neurons located in the middle distal colon to the superior mesenteric ganglion. Almost half of the neurons in the rectum that project to the inferior mesenteric ganglia do so via the hypogastric nerves. Of neurons that projected to the inferior or superior mesenteric ganglia from the colon or rectum, similar proportions (about 75–80%) showed immunoreactivity for calbindin or VIP. For each of the prevertebral ganglia (coeliac, superior mesenteric and inferior mesenteric) the great majority of peripheral inputs arise from the large intestine.     

Neuron-specific enolase and S-100 protein immunohistochemistry for defining the structure and topographical relationship of the different enteric nerve plexuses in the small intestine of the pig

Summary The morphological and topographical features of the intramural enteric nervous system in the small intestine of the pig has been studied on whole mounts by means of neuron-specific enolase (NSE) and S-100 protein immu-nohistochemistry. A clear visualization of the myenteric plexus allows the recognition of its characteristic morphology, including the thin tertiary plexus coursing within the smooth muscle layers. In the tela submucosa two ganglionated plexuses, each with its own specific characteristics, can clearly be demonstrated: (1) the plexus submucosus externus (Schabadasch) located near the inner surface of the circular muscle layer at the abluminal side of the submucosal vascular arcades, and (2) the plexus submucosus internus (Meissner) close to the outer surface of the lamina muscularis mucosae at the luminal side of the submucosal vascular arcades. Due to the possibility to trace clearly the perivascular plexuses of these vascular arcades by use of immunohistochemical techniques with antibodies to NSE and S-100 protein, the two submucosal nerve plexuses can be demonstrated with exceptional clarity. This is the first report of an investigation of the intramural nerve plexuses of the small intestine of the pig using the NSE and S-100 immunostaining methods, which is sufficiently detailed to substantiate the characteristic topography and structure of the two submucosal plexuses and their relation to the smooth muscle layers and perivascular plexuses. The level of NSE immunoreactivity for enteric neurons displays great variation, a substantial proportion of the type-II neurons appearing strongly stained. Although little is known of the specific function of these enzymes, proposals are discussed.     

Dual embryonic origin of the mammalian enteric nervous system

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.     

The emergence of neural activity and its role in the development of the enteric nervous system

The enteric nervous system (ENS) is a vital part of the autonomic nervous system that regulates many gastrointestinal functions, including motility and secretion. All neurons and glia of the ENS arise from neural crest-derived cells that migrate into the gastrointestinal tract during embryonic development. It has been known for many years that a subpopulation of the enteric neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development. Recent studies have demonstrated that some enteric neurons exhibit electrical activity from as early as E11.5 in the mouse, with further maturation of activity during embryonic and postnatal development. This article discusses the maturation of electrophysiological and morphological properties of enteric neurons, the formation of synapses and synaptic activity, and the influence of neural activity on ENS development.     

Total numbers of neurons in myenteric ganglia of the guinea-pig small intestine

Two techniques that are thought to stain all of the neurons in the myenteric ganglia of the intestine are NADH diaphorase histochemistry and immunhistochemistry using a nerve cell body antiserum. However, this assumption has never been directly verified. In the present study myenteric ganglia of the guinea-pig ileum were prepared as whole-mounts and stained with either of these techniques. All nerve cells that could be identified in the whole-mounts were counted. The whole-mounts were then embedded flat in resin and serially sectioned at 1 m. Nerve cells were identified and counted from the serial sections, and the data compared to those obtained from the whole-mounts. NADH diaphorase histochemistry did not reveal all the neurons at incubation times that gave selective staining. In contrast, nerve cell body antiserum stained the entire neuronal population. To determine the total number of nerve cell bodies/ganglion and the proportion of nerve cell bodies with calbindin immunoreactivity, whole-mounts that had been processed for calbindin immunohistochemistry were serially sectioned and reconstructed. The total number of neurons per myenteric ganglion was 105±10 (SE). Calbindin-immunoreactive neurons comprised about 20% of the myenteric neurons, which is considerably less than previous estimates, because previously the total population has been underestimated. The spatial density of myenteric neurons in the undistended ileum of the guinea-pig is 17300 nerve cells/cm².     

The use of constitutive nuclear oncoproteins to count neurons in the enteric nervous system of the guinea pig

Immunohistochemical double labelling of the enteric nervous system of the guinea pig ileum was performed with a monoclonal antibody (anti-MYC 033) directed against a peptide sequence of the human c-Myc protein together with antibodies directed against either the neuron-specific antigens neuron-specific enolase or PGP 9.5 or the glia-specific marker S-100 to demonstrate that anti-MYC 033 labelled the nuclei of all enteric neurons but not glia. This strategy was also employed to demonstrate that another anti-c-Myc monoclonal anti-body, anti-MYC 070, labelled the nuclei of all neurons and glia, as well as perhaps all other cells in these preparations. A polyclonal antiserum raised against a peptide sequence of the human c-Fos protein (anti-FOS 4) was shown to label the identical nuclei as anti-MYC 033. The ganglionic density of nuclei labelled by anti-FOS 4 was found to be similar to previous measures of the ganglionic density of neurons. Double labelling with anti-MYC 033 and an antiserum directed against vasoactive intestinal polypeptide was performed to reexamine the ganglionic density of neurons that express this neuropeptide. Our results suggest that the ganglionic density of these neurons might be less than previously determined.     

The enteric nervous system of P2Y13 receptor null mice is resistant against high-fat-diet- and palmitic-acid-induced neuronal loss

Gastrointestinal symptoms have a major impact on the quality of life and are becoming more prevalent in the western population. The enteric nervous system (ENS) is pivotal in regulating gastrointestinal functions. Purinergic neurotransmission conveys a range of short and long-term cellular effects. This study investigated the role of the ADP-sensitive P2Y₁₃ receptor in lipid-induced enteric neuropathy. Littermate P2Y₁₃^+/+ and P2Y₁₃^−/− mice were fed with either a normal diet (ND) or high-fat diet (HFD) for 6 months. The intestines were analysed for morphological changes as well as neuronal numbers and relative numbers of vasoactive intestinal peptide (VIP)- and neuronal nitric oxide synthase (nNOS)-containing neurons. Primary cultures of myenteric neurons from the small intestine of P2Y₁₃^+/+ or P2Y₁₃^−/− mice were exposed to palmitic acid (PA), the P2Y₁₃ receptor agonist 2meSADP and the antagonist MRS2211. Neuronal survival and relative number of VIP-containing neurons were analysed. In P2Y₁₃^+/+, but not in P2Y₁₃^−/− mice, HFD caused a significant loss of myenteric neurons in both ileum and colon. In colon, the relative numbers of VIP-containing submucous neurons were significantly lower in the P2Y₁₃^−/− mice compared with P2Y₁₃^+/+ mice. The relative numbers of nNOS-containing submucous colonic neurons increased in P2Y₁₃^+/+ HFD mice. HFD also caused ileal mucosal thinning in P2Y₁₃^+/+ and P2Y₁₃^−/− mice, compared to ND fed mice. In vitro PA exposure caused loss of myenteric neurons from P2Y₁₃^+/+ mice while neurons from P2Y₁₃^−/− mice were unaffected. Presence of MRS2211 prevented PA-induced neuronal loss in cultures from P2Y₁₃^+/+ mice. 2meSADP caused no change in survival of cultured neurons. P2Y₁₃ receptor activation is of crucial importance in mediating the HFD- and PA-induced myenteric neuronal loss in mice. In addition, the results indicate a constitutive activation of enteric neuronal apoptosis by way of P2Y₁₃ receptor stimulation.