Morphologies, dimensions and targets of gastric nitric oxide synthase neurons.

We investigated the distributions and targets of nitrergic neurons in the rat stomach, using neuronal nitric oxide synthase (NOS) immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Nitrergic neurons comprised similar proportions of myenteric neurons, about 30%, in all gastric regions. Small numbers of nitrergic neurons occurred in submucosal ganglia. In total, there were ~ 125,000 neuronal nitric oxide synthase (nNOS) neurons in the stomach. The myenteric cell bodies had single axons, type I morphology and a wide range of sizes. Five targets were identified, the longitudinal, circular and oblique layers of the external muscle, the muscularis mucosae and arteries within the gastric wall. The circular and oblique muscle layers had nitrergic fibres throughout their thickness, while the longitudinal muscle was innervated at its inner surface by fibres of the tertiary plexus, a component of the myenteric plexus. There was a very dense innervation of the pyloric sphincter, adjacent to the duodenum. The muscle strands that run between mucosal glands rarely had closely associated nNOS nerve fibres. Both nNOS immunohistochemistry and NADPH histochemistry showed that nitrergic terminals did not provide baskets of terminals around myenteric neurons. Thus, the nitrergic neuron populations in the stomach supply the muscle layers and intramural arteries, but, unlike in the intestine, gastric interneurons do not express nNOS. The large numbers of nNOS neurons and the density of innervation of the circular muscle and pyloric sphincter suggest that there is a finely graded control of motor function in the stomach by the recruitment of different numbers of inhibitory motor neurons.

5-HT containing enteroendocrine cells characterised by morphologies, patterns of hormone co-expression, and relationships with nerve fibres in the mouse gastrointestinal tract.

5-HT containing enteroendocrine cells (EEC), the most abundant type of EEC in the gut, regulate many functions including motility, secretion and inflammatory responses. We examined the morphologies of 5-HT cells from stomach to rectum, patterns of hormone co-expression in the stomach and colon, and the relationship of 5-HT cells with nerve fibres. We also reviewed some of the relevant literature. The morphologies of 5-HT cells were distinct, depending on their location in the gut. A noticeable feature of some 5-HT cells in the antrum and colon was their long basal processes, which resembled processes of neurons, whereas 5-HT cells in the small intestinal mucosa lacked basal processes. In the stomach, numerous 5-HT cells, including cells with basal processes, were identified as enterochromaffin-like cells by their expression of histidine decarboxylase. In the colon, we observed a small number of 5-HT cells that were in close contact with, but distinct from, oxyntomodulin (OXM) and PYY immunoreactive EEC. We did not find specific relationships between nerve fibres and the processes of colonic 5-HT cells. We conclude that five major features, i.e., gut region, morphology, hormone content, receptor repertoire and cell lineage, can be used to define 5-HT cells.

The distribution and chemical coding of enteroendocrine cells in Trypanosoma cruzi-infected individuals with chagasic megacolon.

Chagas disease is caused by the parasite, Trypanosoma cruzi that causes chronic cardiac and digestive dysfunction. Megacolon, an irreversible dilation of the left colon, is the main feature of the gastrointestinal form of Chagas disease. Patients have severe constipation, a consequence of enteric neuron degeneration associated with chronic inflammation. Dysmotility, infection, neuronal loss and a chronic exacerbated inflammation, all observed in Chagas disease, can affect enteroendocrine cells (EEC) expression, which in turn, could influence the inflammatory process. In this study, we investigated the distribution and chemical coding of EEC in the dilated and non-dilated portion of T. cruzi-induced megacolon and in non-infected individuals (control colon). Using immunohistochemistry, EECs were identified by applying antibodies to chromogranin A (CgA), glucagon-like peptide 1 (GLP-1), 5-hydroxytryptamine (5-HT), peptide YY (PYY) and somatostatin (SST). Greater numbers of EEC expressing GLP-1 and SST occurred in the dilated portion compared to the non-dilated portion of the same patients with Chagas disease and in control colon, but numbers of 5-HT and PYY EEC were not significantly different. However, it was noticeable that EEC in which 5-HT and PYY were co-expressed were common in control colon, but were rare in the non-dilated and absent in the dilated portion of chagasic megacolon. An increase in the number of CgA immunoreactive EEC in chagasic patients reflected the increases in EEC numbers summarised above. Our data suggests that the denervation and associated chronic inflammation are accompanied by changes in the number and coding of EEC that could contribute to disorders of motility and defence in the chagasic megacolon.

Morphologies and distributions of 5-HT containing enteroendocrine cells in the mouse large intestine.

Serotonin (5-HT)-containing gastrointestinal endocrine cells contribute to regulation of numerous bodily functions, but whether these functions are related to differences in cell shape is not known. The current study identified morphologies and localization of subtypes of 5-HT-containing enteroendocrine cells in the mouse large intestine. 5-HT cells were most frequent in the proximal colon compared with cecum and distal colon. The large intestine harbored both open (O) cells, with apical processes that reached the lumen, and closed (C) cells, not contacting the lumen, classified into O1, O2, and O3 and C1, C2, and C3 cells, by the lengths of their basal processes. O1 and C1 cells, with basal processes sometimes longer that 100 µm, were most common in the distal colon. Their long basal processes ran against the inner surfaces of the mucosal epithelial cells and were strongly immunoreactive for 5-HT; these processes are ideally placed to communicate with the epithelium and to react to mechanical forces. O2 and C2 cells that had similar but shorter basal processes were also most common in the distal colon. O3 and C3 cells had no or very short basal processes. The O3 open type 5-HT cells were abundant in the proximal colon, particularly at the luminal surface, where they could release 5-HT into the lumen to act on luminal 5-HT receptors. Numerous O3 type 5-HT cells occurred in the lower (submucosal) region of the crypts in all segments and might release 5-HT to influence cell renewal in the crypt proliferative zones.

Design, synthesis and characterization of a fluorescently labeled functional analog of full-length human ghrelin.

Human ghrelin receptor (GHSR) is a recognized prospective target in the diagnosis and therapy of multiple cancer types. To gain a better understanding of this receptor signaling system, we have synthesized a novel full-length ghrelin analog that is fluorescently labeled at the side-chain of a C-terminal cysteine extension. This analog exhibited nanomolar affinity and potency for the ghrelin receptor. It shows comparable efficacy with that of endogenous ghrelin. The fluorescently-labeled ghrelin analog is a valuable tool for in vitro imaging of cell lines that express ghrelin receptor.

Quantitation and chemical coding of enteroendocrine cell populations in the human jejunum.

Recent studies reveal substantial species and regional differences in enteroendocrine cell (EEC) populations, including differences in patterns of hormone coexpression, which limit extrapolation between animal models and human. In this study, jejunal samples, with no histologically identifiable pathology, from patients undergoing Whipple's procedure were investigated for the presence of gastrointestinal hormones using double- and triple-labelling immunohistochemistry and high-resolution confocal microscopy. Ten hormones (5-HT, CCK, secretin, proglucagon-derived peptides, PYY, GIP, somatostatin, neurotensin, ghrelin and motilin) were localised in EEC of the human jejunum. If only single staining is considered, the most numerous EEC were those containing 5-HT, CCK, ghrelin, GIP, motilin, secretin and proglucagon-derived peptides. All hormones had some degree of colocalisation with other hormones. This included a population of EEC in which GIP, CCK and proglucagon-derived peptides are costored, and four 5-HT cell populations, 5-HT/GIP, 5-HT/ghrelin, 5-HT/PYY, and 5-HT/secretin cell groups, and a high degree of overlap between motilin and ghrelin. The presence of 5-HT in many secretin cells is consistent across species, whereas lack of 5-HT and CCK colocalisation distinguishes human from mouse. It seems likely that the different subclasses of 5-HT cells subserve different roles. At a subcellular level, we examined the vesicular localisation of secretin and 5-HT, and found these to be separately stored. We conclude that hormone-containing cells in the human jejunum do not comply with a one-cell, one-hormone classification and that colocalisations of hormones are likely to define subtypes of EEC that have different roles.

The identification of neuronal control pathways supplying effector tissues in the stomach.

The stomach acts as a buffer between the ingestion of food and its processing in the small intestine. It signals to the brain to modulate food intake and it in turn regulates the passage of a nutrient-rich fluid, containing partly digested food, into the duodenum. These processes need to be finely controlled, for example to restrict reflux into the esophagus and to transfer digesta to the duodenum at an appropriate rate. Thus, the efferent pathways that control gastric volume, gastric peristalsis and digestive juice production are critically important. We review these pathways with an emphasis on the identities of the final motor neurons and comparisons between species. The major types of motor neurons arising from gastric enteric ganglia are as follows: immunohistochemically distinguishable excitatory and inhibitory muscle motor neurons; four neuron types innervating mucosal effectors (parietal cells, chief cells, gastrin cells and somatostatin cells); and vasodilator neurons. Sympathetic efferent neurons innervate intramural arteries, myenteric ganglia and gastric muscle. Vagal efferent neurons with cell bodies in the brain stem do not directly innervate gastric effector tissues; they are pre-enteric neurons that innervate each type of gastric enteric motor neuron. The principal transmitters and co-transmitters of gastric motor neurons, as well as key immunohistochemical markers, are the same in rat, pig, human and other species.

Distribution and co-expression patterns of specific cell markers of enteroendocrine cells in pig gastric epithelium.

Although the pig is an accepted model species for human digestive physiology, no previous study of the pig gastric mucosa and gastric enteroendocrine cells has investigated the parallels between pig and human. In this study, we have investigated markers for each of the classes of gastric endocrine cells, gastrin, ghrelin, somatostatin, 5-hydroxytryptamine, histidine decarboxylase, and PYY cells in pig stomach. The lining of the proximal stomach consisted of a collar of stratified squamous epithelium surrounded by gastric cardiac glands in the fundus. This differs considerably from human that has only a narrow band of cardiac glands at its entrance, surrounded by a fundic mucosa consisting of oxyntic glands. However, the linings of the corpus and antrum are similar in pig and human. Likewise, the endocrine cell types are similar and similarly distributed in the two species. As in human, gastrin cells were almost exclusively in the antrum, ghrelin cells were most abundant in the oxyntic mucosa and PYY cells were rare. In the pig, 70% of enterochromaffin-like (ECL) cells in the antrum and 95% in the fundus contained 5-hydroxytryptamine (5-HT), higher proportions than in human. Unlike the enteroendocrine of the small intestine, most gastric enteroendocrine cells (EEC) did not contain colocalised hormones. This is similar to human and other species. We conclude that the pig stomach has substantial similarity to human, except that the pig has a protective lining at its entrance that may reflect the difference between a pig diet with hard abrasive components and the soft foods consumed by humans.

Cellular and sub-cellular localisation of oxyntomodulin-like immunoreactivity in enteroendocrine cells of human, mouse, pig and rat.

We use a monoclonal antibody against the C-terminal of oxyntomodulin (OXM) to investigate enteroendocrine cells (EEC) in mouse, rat, human and pig. This antibody has cross-reactivity with the OXM precursor, glicentin (Gli) but does not recognise glucagon. The antibody stained EEC in the jejunum and colon of each species. We compared OXM/Gli immunoreactivity with that revealed by antibodies against structurally related peptides, GLP-1 and glucagon and against GIP and PYY that are predicted to be in some EEC that express OXM/Gli. We used super-resolution to locate immunoreactive vesicles. In the pancreas, OXM/Gli was in glucagon cells but was located in separate storage vesicles to glucagon. In jejunal EEC, OXM/Gli and GIP were in many of the same cells but often in separate vesicles, whereas PYY and OXM/Gli were commonly colocalised in the same storage vesicles of colonic EEC. When binding of anti-GLP-1 to the structurally related GIP was removed by absorption with GIP peptide, GLP-1 and OXM/Gli immunoreactivities were contained in the same population of EEC in the intestine. We conclude that anti-OXM/Gli is a more reliable marker than anti-GLP-1 for EEC expressing preproglucagon products. Storage vesicles that were immunoreactive for OXM/Gli were almost always immunoreactive for GLP-1. OXM concentrations, measured by ELISA, were highest in the distal ileum and colon. Lesser concentrations were found in more proximal parts of small intestine and pancreas. Very little was in the stomach. In EEC containing GIP and OXM/Gli, these hormones are packaged in different secretory vesicles. Separate packaging also occurred for OXM and glucagon, whereas OXM/Gli and PYY and OXM/Gli and GLP-1 were commonly contained together in secretory vesicles.

Diversity of enteroendocrine cells investigated at cellular and subcellular levels: the need for a new classification scheme.

Enteroendocrine cells were historically classified by a letter code, each linked to a single hormone, deduced to be the only hormone produced by the cell. One type, the L cell, was recognised to store and secrete two products, peptide YY (PYY) and glucagon-related peptides. Many other exceptions to the one-cell one-hormone classifications have been reported over the last 40 years or so, and yet the one-hormone dogma has persisted. In the last 6 years, a plethora of data has appeared that makes the concept unviable. Here, we describe the evidence that multiple hormone transcripts and their products reside in single cells and evidence that the hormones are often, but not always, processed into separate storage vesicles. It has become clear that most enteroendocrine cells contain multiple hormones. For example, most secretin cells contain 5-hydroxytryptamine (5-HT), and in mouse many of these also contain cholecystokinin (CCK). Furthermore, CCK cells also commonly store ghrelin, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1), neurotensin, and PYY. Several hormones, for example, secretin and 5-HT, are in separate storage vesicles at a subcellular level. Hormone patterns can differ considerably between species. Another complication is that relative levels of expression vary substantially. This means that data are significantly influenced by the sensitivities of detection techniques. For example, a hormone that can be detected in storage vesicles by super-resolution microscopy may not be above threshold for detection by conventional fluorescence microscopy. New nomenclature for cell clusters with common attributes will need to be devised and old classifications abandoned.

Distribution and characterisation of CCK containing enteroendocrine cells of the mouse small and large intestine.

There is general consensus that enteroendocrine cells, EEC, containing the enteric hormone cholecystokinin (CCK) are confined to the small intestine and predominate in the duodenum and jejunum. Contrary to this, EEC that express the gene for CCK have been isolated from the large intestine of the mouse and there is evidence for EEC that contain CCK-like immunoreactivity in the mouse colon. However, the human and rat colons do not contain CCK cells. In the current study, we use immunohistochemistry to investigate CCK peptide presence in endocrine cells, PCR to identify cck transcripts and chromatography to identify CCK peptide forms in the mouse small and large intestine. The colocalisation of CCK and 5-HT, hormones that have been hypothesised to derive from cells of different lineages, was also investigated. CCK immunoreactivity was found in EEC throughout the mouse small and large intestine but positive cells were rare in the rectum. Immunoreactive EEC were as common in the caecum and proximal colon as they were in the duodenum and jejunum. CCK gene transcripts were found in the mucosa throughout the intestine but mRNA for gastrin, a hormone that can bind some anti-CCK antibodies, was only found in the stomach and duodenum. Characterisation of CCK peptides of the colon by extraction, chromatographic separation and radioimmunoassay revealed bioactive amidated and sulphated forms, including CCK-8 and CCK-33. Moreover, CCK-containing EEC in the large intestine bound antibodies that target the biologically active sulfated form. Colocalisation of CCK and 5-HT occurred in a proportion of EEC throughout the small intestine and in the caecum but these hormones were not colocalised in the colon, where there was CCK and PYY colocalisation. It is concluded that authentic, biologically active, CCK occurs in EEC of the mouse large intestine.

Analysis of enteroendocrine cell populations in the human colon.

Recent studies have shown that patterns of colocalisation of hormones in enteroendocrine cells are more complex than previously appreciated and that the patterns differ substantially between species. In this study, the human sigmoid colon is investigated by immunohistochemistry for the presence of gastrointestinal hormones and their colocalisation. The segments of colon were distant from the pathology that led to colectomy and appeared structurally normal. Only four hormones, 5-hydroxytryptamine (5-HT), glucagon-like peptide 1 (GLP-1), peptide YY (PYY) and somatostatin, were common in enteroendocrine cells of the human colon. Cholecystokinin, present in the colon of some species, was absent, as were glucose-dependent insulinotropic peptide, ghrelin and motilin. Neurotensin cells were extremely rare. The most numerous cells were 5-HT cells, some of which also contained PYY or somatostatin and very rarely GLP-1. Almost all GLP-1 cells contained PYY. It is concluded that enteroendocrine cells of the human colon, like those of other regions and species, exhibit overlapping patterns of hormone colocalisation and that the hormones and their patterns of expression differ between human and other species.

The chemical coding of 5-hydroxytryptamine containing enteroendocrine cells in the mouse gastrointestinal tract.

The majority of 5-HT (serotonin) in the body is contained in enteroendocrine cells of the gastrointestinal mucosa. From the time of their discovery over 80 years ago, the 5-HT-containing cells have been regarded as a class of cell that is distinct from enteroendocrine cells that contain peptide hormones. However, recent studies have cast doubt on the concept of there being distinct classes of enteroendocrine cells, each containing a single hormone or occasionally more than one hormone. Instead, data are rapidly accumulating that there are complex patterns of colocalisation of hormones that identify multiple subclasses of enteroendocrine cells. In the present work, multiple labelling immunohistochemistry is used to investigate patterns of colocalisation of 5-HT with enteric peptide hormones. Over 95 % of 5-HT cells in the duodenum also contained cholecystokinin and about 40 % of them also contained secretin. In the jejunum, about 75 % of 5-HT cells contained cholecystokinin but not secretin and 25 % contained 5-HT plus both cholecystokinin and secretin. Small proportions of 5-HT cells contained gastrin or somatostatin in the stomach, PYY or GLP-1 in the small intestine and GLP-1 or somatostatin in the large intestine. Rare or very rare 5-HT cells contained ghrelin (stomach), neurotensin (small and large intestines), somatostatin (small intestine) and PYY (in the large intestine). It is concluded that 5-HT-containing enteroendocrine cells are heterogeneous in their chemical coding and by implication in their functions.

Selenium and vitamin E together improve intestinal epithelial barrier function and alleviate oxidative stress in heat-stressed pigs.

What is the central question of this study? Oxidative stress may play a role in compromising intestinal epithelial barrier integrity in pigs subjected to heat stress, but it is unknown whether an increase of dietary antioxidants (selenium and vitamin E) could alleviate gut leakiness in heat-stressed pigs. What is the main finding and its importance? Levels of dietary selenium (1.0 p.p.m.) and vitamin E (200 IU kg(-1) ) greater than those usually recommended for pigs reduced intestinal leakiness caused by heat stress. This finding suggests that oxidative stress plays a role in compromising intestinal epithelial barrier integrity in heat-stressed pigs and also provides a nutritional strategy for mitigating these effects. Heat stress compromises the intestinal epithelial barrier integrity of mammals through mechanisms that may include oxidative stress. Our objective was to test whether dietary supplementation with antioxidants, selenium (Se) and vitamin E (VE), protects intestinal epithelial barrier integrity in heat-stressed pigs. Female growing pigs (n = 48) were randomly assigned to four diets containing from 0.2 p.p.m. Se and 17 IU kg(-1) VE (control, National Research Council recommended) to 1.0 p.p.m. Se and 200 IU kg(-1) VE for 14 days. Six pigs from each dietary treatment were then exposed to either thermoneutral (20°C) or heat-stress conditions (35°C 09.00-17.00 h and 28°C overnight) for 2 days. Transepithelial electrical resistance and fluorescein isothiocyanate-dextran (4 kDa; FD4) permeability were measured in isolated jejunum and ileum using Ussing chambers. Rectal temperature, respiratory rate and intestinal HSP70 mRNA abundance increased (all P < 0.001), and respiratory alkalosis occurred, suggesting that pigs were heat stressed. Heat stress also increased FD4 permeability and decreased transepithelial electrical resistance (both P < 0.01). These changes were associated with changes indicative of oxidative stress, a decreased glutathione peroxidase (GPX) activity and an increased glutathione disulfide (GSSG)-to-glutathione (GSH) ratio (both P < 0.05). With increasing dosage of Se and VE, GPX-2 mRNA (P = 0.003) and GPX activity (P = 0.049) increased linearly, the GSSG:GSH ratio decreased linearly (P = 0.037), and the impacts of heat stress on intestinal barrier function were reduced (P < 0.05 for both transepithelial electrical resistance and FD4 permeability). In conclusion, in pigs an increase of dietary Se and VE mitigated the impacts of heat stress on intestinal barrier integrity, associated with a reduction in oxidative stress.

A comparative analysis of gastrointestinal tract barrier function and immune markers in gilt vs. sow progeny at birth and weaning.

Progeny born to primiparous sows (gilt progeny; GP) have lower birth, weaning and slaughter weights than sow progeny (SP). GP also have reduced gastrointestinal tract (GIT) development, as evidenced by lower organ weights. Therefore, the aim of this experiment was to quantify changes in GIT barrier function that occur in birth and weaning, representing two major challenges to the young piglet. The effects of parity (GP vs. SP) in GIT barrier integrity function were quantified at four timepoints: birth (~0 h), 24 h after birth (24 h), 1-d preweaning (PrW), and 1-d postweaning (PoW) in commercially reared piglets. Due to inherent differences between newborn and weanling pigs, the results were analyzed in two cohorts, birth (0 vs. 24 h, n = 31) and weaning (PrW vs. PoW, n = 40). Samples of the stomach, jejunum, ileum, and colon were excised after euthanasia and barrier integrity was quantified by measuring transepithelial resistance (TER), macromolecular permeability, the abundance of inflammatory proteins (IL-8, IL-1ß, and TNF-α) and tight junction proteins (claudin-2 and -3). Papp was characterized using a dual tracer approach comprising 4 KDa fluorescein isothiocyanate (FD4) and 150 kDa tetramethyl rhodamine isothiocyanate (T150)-labeled dextrans. Characteristic effects of the initiation of feeding and weaning were observed on the GIT with the initiation of feeding, such as increasing TER and reducing Papp at 24 h, consistent with mucosal growth (P = 0.058) This was accompanied by increased cytokine abundance as evidenced by elevations in TNF-α and IL-1ß. However, GP had increased IL-8 abundance (P = 0.011 and 0.063 for jejunum and ileum respectively) at birth than 24 h overall. In the weaning cohort, jejunal and ileal permeability to FD4 was higher in GP (P = 0.05 and 0.022, respectively) while only higher ileal T150 was observed in GP (P = 0.032). Ileal claudin-2 abundance tended to be higher in SP overall (P = 0.063), but GP ileal claudin-2 expression was upregulated weaning while no change was observed in SP (P = 0.043). Finally, other than a higher jejunal TNF-α abundance observed in SP (P = 0.016), no other effect of parity was observed on inflammatory markers in the weaning cohort. The results from this study indicate that the GIT of GP have poorer adaptation to early life events, with the response to weaning, being more challenging which is likely to contribute to poorer postweaning growth.

The progeny of primiparous sows (gilt progeny; GP) have poorer lifetime growth performance in comparison to progeny from multiparous sows (sow progeny; SP). Previous research suggests that there is an underlying biological basis for reduced growth performance which is attributed to differences in gastrointestinal tract (GIT) barrier development during early life. This study aimed to clarify the timeframes of when these differences are in effect by investigating GIT development during two major events of a piglet's life: birth and weaning. To do this, GIT tissue was collected from GP and SP at four time points; birth, 24 h after birth, 1-d preweaning, and 1-d postweaning and assessed for functional development. The main findings from this study indicate there are early signs of variation in GIT development within the first 24 h of life between GP and SP, and that these differences increase through the preweaning period, with GP entering weaning with poorer GIT development and function. Possible explanations for the reduced GI development may be reduced maternal nutrition during the suckling period.

Aging induces cell loss and a decline in phagosome processing in the mouse retinal pigment epithelium.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss and dysfunction in the retinal pigment epithelium (RPE) with age is known to contribute to disease development. The aim of this study was to investigate how the C57BL/6J mouse RPE changes with age. RPE structure was found to change with age and eccentricity, with cell size increasing, nuclei lost, and tight junctions altered in the peripheral retina. Phagocytosis of photoreceptor outer segments (POS) by the RPE was investigated using gene expression analysis and histology. RNA-Seq transcriptomic gene profiling of the RPE showed a downregulation of genes involved in phagosome processing and histological analysis showed a decline in phagosome-lysosome association in the aged tissue. In addition, failures in the autophagy pathway that modulates intracellular waste degradation were observed in the aged RPE tissue. These findings highlight that RPE cell loss and slowing of POS processing contribute to RPE dysfunction with age and may predispose the aging eye to AMD development.

Development of the aganglionic colon following surgical rescue in a cell therapy model of Hirschsprung disease in rat.

Patients with Hirschsprung disease lack enteric ganglia in the distal colon and propulsion of colorectal content is substantially impaired. Proposed stem cell therapies to replace neurons require surgical bypass of the aganglionic bowel during re-colonization, but there is inadequate knowledge of the consequences of bypass. We performed bypass surgery in Ednrb-/- Hirschsprung rat pups. Surgically rescued rats failed to thrive, an outcome reversed by supplying electrolyte- and glucose-enriched drinking water. Histologically, the bypassed colon had normal structure, but grew substantially less in diameter than the functional region proximal to the bypass. Extrinsic sympathetic and spinal afferent neurons projected to their normal targets, including arteries and the circular muscle, in aganglionic regions. However, although axons of intrinsic excitatory and inhibitory neurons grew into the aganglionic region, their normally dense innervation of circular muscle was not restored. Large nerve trunks that contained tyrosine hydroxylase (TH)-, calcitonin gene-related peptide (CGRP, encoded by Calca or Calcb)-, neuronal nitric oxide synthase (nNOS or NOS1)-, vasoactive intestinal peptide (VIP)- and tachykinin (encoded by Tac1)-immunoreactive axons occurred in the distal aganglionic region. We conclude that the rescued Ednrb-/- rat provides a good model for the development of cell therapies for the treatment of Hirschsprung disease.

Stratification of enterochromaffin cells by single-cell expression analysis.

Dynamic interactions between gut mucosal cells and the external environment are essential to maintain gut homeostasis. Enterochromaffin (EC) cells transduce both chemical and mechanical signals and produce 5-hydroxytryptamine (5-HT) to mediate disparate physiological responses. However, the molecular and cellular basis for functional diversity of ECs remains to be adequately defined. Here, we integrated single-cell transcriptomics with spatial image analysis to identify fourteen EC clusters that are topographically organized along the gut. Subtypes predicted to be sensitive to the chemical environment and mechanical forces were identified that express distinct transcription factors and hormones. A Piezo2+ population in the distal colon was endowed with a distinctive neuronal signature. Using a combination of genetic, chemogenetic and pharmacological approaches, we demonstrated Piezo2+ ECs are required for normal colon motility. Our study constructs a molecular map for ECs and offers a framework for deconvoluting EC cells with pleiotropic functions.

Dopamine and ghrelin receptor co-expression and interaction in the spinal defecation centers.

BACKGROUND: Dopamine receptor 2 (DRD2) and ghrelin receptor (GHSR1a) agonists both stimulate defecation by actions at the lumbosacral defecation center. Dopamine is in nerve terminals surrounding autonomic neurons of the defecation center, whereas ghrelin is not present in the spinal cord. Dopamine at D2 receptors generally inhibits neurons, but at the defecation center, its effect is excitatory. METHODS: In vivo recording of defecation and colorectal propulsion was used to investigate interaction between DRD2 and GHSR1a. Localization studies were used to determine sites of receptor expression in rat and human spinal cord. KEY RESULTS: Dopamine, and the DRD2 agonist, quinpirole, directly applied to the lumbosacral cord, caused defecation. The effect of intrathecal dopamine was inhibited by the GHSR1a antagonist, YIL781, given systemically, but YIL781 was not an antagonist at DRD2. The DRD2 agonist, pramipexole, administered systemically caused colorectal propulsion that was prevented when the pelvic nerves were cut. Drd2 and Ghsr were expressed together in autonomic preganglionic neurons at the level of the defecation centers in rat and human. Behaviorally induced defecation (caused by water avoidance stress) was reduced by the DRD2 antagonist, sulpiride. We had previously shown it is reduced by YIL781. CONCLUSIONS AND INFERENCES: Our observations imply that dopamine is a transmitter of the defecation pathways whose actions are exerted through interacting dopamine (D2) and ghrelin receptors on lumbosacral autonomic neurons that project to the colorectum. The results explain the excitation by dopamine agonists and the conservation of GHSR1a in the absence of ghrelin.