Morphological, molecular, and functional characterization of mouse glutamatergic myenteric neurons.

The enteric nervous system (ENS) functions largely independently of the central nervous system (CNS). Glutamate, the dominant neurotransmitter in the CNS and sensory afferents, is not a primary neurotransmitter in the ENS. Only a fraction (∼2%) of myenteric neurons in the mouse distal colon and rectum (colorectum) are positive for vesicular glutamate transporter type 2 (VGLUT2), the structure and function of which remain undetermined. Here, we systematically characterized VGLUT2-positive enteric neurons (VGLUT2-ENs) through sparse labeling with adeno-associated virus, single-cell mRNA sequencing (scRNA-seq), and GCaMP6f calcium imaging. Our results reveal that the majority of VGLUT2-ENs (29 of 31, 93.5%) exhibited Dogiel type I morphology with a single aborally projecting axon; most axons (26 of 29, 89.7%) are between 4 and 10 mm long, each traversing 19 to 34 myenteric ganglia. These anatomical features exclude the VGLUT2-ENs from being intrinsic primary afferent or motor neurons. The scRNA-seq conducted on 52 VGLUT2-ENs suggests different expression profiles from conventional descending interneurons. Ex vivo GCaMP6f recordings from flattened colorectum indicate that almost all VGLUT2-EN (181 of 215, 84.2%) are indirectly activated by colorectal stretch via nicotinic cholinergic neural transmission. In conclusion, VGLUT2-ENs are a functionally unique group of enteric neurons with single aborally projecting long axons that traverse multiple myenteric ganglia and are activated indirectly by colorectal mechanical stretch. This knowledge will provide a solid foundation for subsequent studies on the potential interactions of VGLUT2-EN with extrinsic colorectal afferents via glutamatergic neurotransmission.NEW & NOTEWORTHY We reveal that VGLUT2-positive enteric neurons (EN), although constituting a small fraction of total EN, are homogeneously expressed in the myenteric ganglia, with a slight concentration at the intermediate region between the colon and rectum. Through anatomic, molecular, and functional analyses, we demonstrated that VGLUT2-ENs are activated indirectly by noxious circumferential colorectal stretch via nicotinic cholinergic transmission, suggesting their participation in mechanical visceral nociception.

Lonicerin alleviates intestinal myenteric neuron injury induced by hypoxia/reoxygenation treated macrophages by downregulating EZH2.

Intestinal ischemia-reperfusion (IR) injury is a common gastrointestinal disease that induces severe intestinal dysfunction. Intestinal myenteric neurons participate in maintaining the intestinal function, which will be severely injured by IR. Macrophages are widely reported to be involved in the pathogenesis of organ IR injury, including intestine, which is activated by NLRP3 signaling. Lonicerin (LCR) is a natural extracted monomer with inhibitory efficacy against the NLRP3 pathway in macrophages. The present study aims to explore the potential protective function of LCR in intestinal IR injury. Myenteric neurons were extracted from mice. RAW 264.7 cells were stimulated by H/R with or without 10 µM and 30 µM LCR. Remarkable increased release of IL-6, MCP-1, and TNF-α were observed in H/R treated RAW 264.7 cells, along with an upregulation of NLRP3, cleaved-caspase-1, IL-1ß, and EZH2, which were sharply repressed by LCR. Myenteric neurons were cultured with the supernatant collected from each group. Markedly decreased neuron number and shortened length of neuron axon were observed in the H/R group, which were signally reversed by LCR. RAW 264.7 cells were stimulated by H/R, followed by incubated with 30 µM LCR with or without pcDNA3.1-EZH2. The inhibition of LCR on NLRP3 signaling in H/R treated RAW 264.7 cells was abolished by EZH2 overexpression. Furthermore, the impact of LCR on neuron number and neuron axon length in myenteric neurons in the H/R group was abated by EZH2 overexpression. Collectively, LCR alleviated intestinal myenteric neuron injury induced by H/R treated macrophages via downregulating EZH2.

Biphenotypic Cells and α-Synuclein Accumulation in Enteric Neurons of Leucine-Rich Repeat Kinase 2 Knockout Mice.

BACKGROUND: Leucine-rich repeat kinase 2 is a molecule that is responsible for familial Parkinson's disease. Our previous findings revealed that leucine-rich repeat kinase 2 is expressed in the enteric nervous system. However, which cells in the enteric nervous system express leucine-rich repeat kinase 2 and whether leucine-rich repeat kinase 2 is associated with the structure of the enteric nervous system remain unclear. The enteric nervous system is remarkable because some patients with Parkinson's disease experience gastrointestinal symptoms before developing motor symptoms. AIMS: We established a leucine-rich repeat kinase 2 reporter mouse model and performed immunostaining in leucine-rich repeat kinase 2 knockout mice. METHODS: Longitudinal muscle containing the myenteric plexus prepared from leucine-rich repeat kinase 2 reporter mice was analyzed by immunostaining using anti-green fluorescent protein (GFP) antibody. Immunostaining using several combinations of antibodies characterizing enteric neurons and glial cells was performed on intestinal preparations from leucine-rich repeat kinase 2 knockout mice. RESULTS: GFP expression in the reporter mice was predominantly in enteric glial cells rather than in enteric neurons. Immunostaining revealed that differences in the structure and proportion of major immunophenotypic cells were not apparent in the knockout mice. Interestingly, the number of biphenotypic cells expressing the neuronal and glial cell markers increased in the leucine-rich repeat kinase 2 knockout mice. Moreover, there was accumulation of α-synuclein in the knockout mice. CONCLUSIONS: Our present findings suggest that leucine-rich repeat kinase 2 is a newly recognized molecule that potentially regulates the integrity of enteric nervous system and enteric α-synuclein accumulation.

Accelerated Electron Ionization-Induced Changes in the Myenteric Plexus of the Rat Stomach.

The influence of accelerated electrons on neuronal structures is scarcely explored compared to gamma and X-rays. This study aims to investigate the effects of accelerated electron radiation on some pivotal neurotransmitter circuits (cholinergic and serotonergic) of rats' myenteric plexus. Male Wistar rats were irradiated with an electron beam (9 MeV, 5 Gy) generated by a multimodality linear accelerator. The contractile activity of isolated smooth muscle samples from the gastric corpus was measured. Furthermore, an electrical stimulation (200 µs, 20 Hz, 50 s, 60 V) was performed on the samples and an assessment of the cholinergic and serotonergic circuits was made. Five days after irradiation, the recorded mechanical responses were biphasic-contraction/relaxation in controls and contraction/contraction in irradiated samples. The nature of the contractile phase of control samples was cholinergic with serotonin involvement. The relaxation phase involved ACh-induced nitric oxide release from gastric neurons. There was a significant increase in serotonergic involvement during the first and second contractile phases of the irradiated samples, along with a diminished role of acetylcholine in the first phase. This study demonstrates an increased involvement of serotonergic neurotransmitter circuits in the gastric myenteric plexus caused by radiation with accelerated electrons.

Functional Intraregional and Interregional Heterogeneity between Myenteric Glial Cells of the Colon and Duodenum in Mice.

Enteric glia are a unique population of peripheral neuroglia that regulate homeostasis in the enteric nervous system (ENS) and intestinal functions. Despite existing in functionally diverse regions of the gastrointestinal tract, enteric glia have been approached scientifically as a homogeneous group of cells. This assumption is at odds with the functional specializations of gastrointestinal organs and recent data suggesting glial heterogeneity in the brain and ENS. Here, we used calcium imaging in transgenic mice of both sexes expressing genetically encoded calcium sensors in enteric glia and conducted contractility studies to investigate functional diversity among myenteric glia in two functionally distinct intestinal organs: the duodenum and the colon. Our data show that myenteric glia exhibit regionally distinct responses to neuromodulators that require intercellular communication with neurons to differing extents in the duodenum and colon. Glia regulate intestinal contractility in a region-specific and pathway-specific manner, which suggests regionally diverse engagement of enteric glia in local motor patterns through discrete signaling pathways. Further, functional response profiles delineate four unique subpopulations among myenteric glia that are differentially distributed between the colon and duodenum. Our findings support the conclusion that myenteric glia exhibit both intraregional and interregional heterogeneity that contributes to region-specific mechanisms that regulate digestive functions. Glial heterogeneity adds an unexpected layer of complexity in peripheral neurocircuits, and understanding the specific functions of specialized glial subtypes will provide new insight into ENS physiology and pathophysiology.SIGNIFICANCE STATEMENT Enteric glia modulate gastrointestinal functions through intercellular communication with enteric neurons. Whether heterogeneity exists among neuron-glia interactions in the digestive tract is not understood. Here, we show that myenteric glia display regional heterogeneity in their responses to neuromodulators in the duodenum and the colon, which are functionally distinct organs. Glial-mediated control of intestinal motility is region and pathway specific. Four myenteric glial subtypes are present within a given gut region that are differently distributed between gut regions. These data provide functional and regional insights into enteric circuit specificity in the adult enteric nervous system.

Effects of experimental ulcerative colitis on myenteric neurons in P2X7-knockout mice.

This study aimed to investigate the distal colon myenteric plexus and enteric glial cells (EGCs) in P2X7 receptor-deficient (P2X7-/-) animals after the induction of experimental ulcerative colitis. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) was injected into the distal colon of C57BL/6 (WT) and P2X7 receptor gene-deficient (P2X7-/-, KO) animals. Distal colon tissues in the WT and KO groups were analyzed 24 h and 4 days after administration. The tissues were analyzed by double immunofluorescence of the P2X7 receptor with neuronal nitric oxide synthase (nNOS)-immunoreactive (ir), choline acetyltransferase (ChAT)-ir, and PGP9.5 (pan neuronal)-ir, and their morphology was assessed by histology. The quantitative analysis revealed 13.9% and 7.1% decreases in the number of P2X7 receptor-immunoreactive (ir) per ganglion in the 24 h-WT/colitis and 4 day-WT/colitis groups, respectively. No reduction in the number of nNOS-ir, choline ChAT-ir, and PGP9.5-ir neurons per ganglion was observed in the 4 day-KO/colitis group. In addition, a reduction of 19.3% in the number of GFAP (glial fibrillary acidic protein)-expressing cells per ganglion was found in the 24 h-WT/colitis group, and a 19% increase in the number of these cells was detected in the 4 day-WT/colitis group. No profile area changes in neurons were observed in the 24 h-WT and 24 h-KO groups. The 4 day-WT/colitis and 4 day-KO/colitis groups showed increases in the profile neuronal areas of nNOS, ChAT, and PGP9.5. The histological analysis showed hyperemia, edema, or cellular infiltration in the 24 h-WT/colitis and 4 day-WT/colitis groups. Edema was observed in the 4 day-KO/colitis group, which showed no histological changes compared with the 24 h-KO/colitis group. We concluded that ulcerative colitis differentially affected the neuronal classes in the WT and KO animals, demonstrating the potential participation and neuroprotective effect of the P2X7 receptor in enteric neurons in inflammatory bowel disease.

Effect of Calcium-Sensitive Receptor Agonist R568 on Gastric Motility and the Underlying Mechanism.

INTRODUCTION: Calcium-sensitive receptor (CaSR) is expressed in the enteric nervous system of gastrointestinal tract. However, its role in the regulation of gastrointestinal motility has not yet been fully elucidated. We aimed to investigate the effect of the CaSR agonist - R568 on gastric motility and its potential mechanism. METHODS: In vivo, R568 was given by gavage to explore gastric emptying with or without capsaicin which specifically blocks the function of vagal afferents; neurotransmitters synthetized in the myenteric plexus of the gastric corpus and antrum were analysed by ELISA and immunofluorescence staining; gastric muscle strips contraction recording and intracellular single unit firing recording were used to study the effect of R568 on muscle strips and myenteric interstitial cells of Cajal (ICCs) ex vitro. RESULTS: Gastric emptying was inhibited by R568 in Kunming male mice, and capsaicin weakened this effect. The expression of c-fos-positive neurons increased in the nucleus tractus solitarius when R568 was treated. R568 decreased the expression of cholinergic neurons and reduced the synthesis of acetylcholine. Conversely, R568 increased the expression of nitrogenic neurons and enhanced the synthesis of nitric oxide in the myenteric plexus. Ex vitro results showed that R568 inhibited the contraction of the gastric antral muscle strip and suppressed the spontaneous firing activity of pacemaker ICCs. CONCLUSION: Activation of the gastrointestinal CaSR inhibited gastric motility in vivo and ex vitro. Transmitting nutrient signals to the brain through the vagal afferent nerve, modulating the cholinergic and nitrergic system in the enteric nervous system, and inhibiting activity of pacemaker ICCs in the myenteric plexus are involved in the mechanism of CaSR in gastric motility suppression.

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.

Enteric glia: extent, cohesion, axonal contacts, membrane separations and mitochondria in Auerbach's ganglia of guinea pigs.

Studied by electron microscopy and morphometry, Auerbach's ganglia comprise nerve cell bodies that occupy ~ 40% of volume; of the neuropil, little over 30% is neural processes (axons, dendrites) and little less than 30% is glia (cell bodies, processes). The amount of surface membrane of neural elements only marginally exceeds that of glia. Glial cells extend laminar processes radially between axons, reaching the ganglion's surface with specialized membrane domains. Nerve cells and glia are tightly associated, eliminating any free space in ganglia. Glia expands maximally its cell membrane with a minimum of cytoplasm, contacting a maximal number of axons, which, with their near-circular profile, have minimal surface for a given volume. Shape of glia is moulded by the neural elements (predominantly concave the first, predominantly convex the second); the glia extends its processes to maximize contact with neural elements. Yet, a majority of axons is not reached by glia and only few are wrapped by it. Despite the large number of cells, the glia is not sufficiently developed to wrap around or just contact many of the neural elements. Mitochondria are markedly fewer in glia than in neurons, indicating a lower metabolic rate. Compactness of ganglia, their near-circular profile, absence of spaces between elements and ability to withstand extensive deformation suggest strong adhesion between the cellular elements, holding them together and keeping them at a fixed distance. Many axonal varicosities, with vesicles and membrane densities, abut on non-specialized areas of glia, suggesting the possibility of neurotransmitters being released outside synaptic sites.

Nitrergic and Purinergic Nerves in the Small Intestinal Myenteric Plexus and Circular Muscle of Mice and Guinea Pigs.

ATP is an excitatory and inhibitory neurotransmitter, while nitric oxide (NO) is an inhibitory neurotransmitter in the enteric nervous system (ENS). We used a vesicular nucleotide transporter (SLC17A9, VNUT) antibody and a nitric oxide synthase (NOS) antibody to identify purinergic and nitrergic nerves in mouse and guinea ileum. Mouse: VNUT-immunoreactivity (ir) was detected in nerve fibers in myenteric ganglia and circular muscle. VNUT-ir fibers surrounded choline acetyltransferase (ChAT), nitric oxide synthase (nNOS), and calretinin-ir neurons. VNUT-ir nerve cell bodies were not detected. Tyrosine hydroxylase (TH)-ir nerves were detected in myenteric ganglia and the tertiary plexus. Guinea pig: VNUT-ir was detected in neurons and nerves fibers and did not overlap with NOS-ir nerve fibers. VNUT-ir was detected in nerve fibers in ganglia but not nerve cell bodies. VNUT-ir nerve fibers surrounded NOS-ir and NOS- neurons. NOS-ir and VNUT-ir nerve fibers did not overlap in myenteric ganglia or circular muscle. VNUT-ir nerves surrounded some ChAT-ir neurons. VNUT-ir and ChAT-ir were detected in separate nerves in the CM. VNUT-ir nerve fibers surrounded calretinin-ir neurons.Conclusions: VNUT-ir neurons likely mediate purinergic signaling in small intestinal myenteric ganglia and circular muscle. ATP and NO are likely released from different inhibitory motorneurons. VNUT-ir and ChAT-ir interneurons mediate cholinergic and purinergic synaptic transmission in the myenteric plexus.

Identifying Types of Neurons in the Human Colonic Enteric Nervous System.

Distinguishing and characterising the different classes of neurons that make up a neural circuit has been a long-term goal for many neuroscientists. The enteric nervous system is a large but moderately simple part of the nervous system. Enteric neurons in laboratory animals have been extensively characterised morphologically, electrophysiologically, by projections and immunohistochemically. However, studies of human enteric nervous system are less advanced despite the potential availability of tissue from elective surgery (with appropriate ethics permits). Recent studies using single cell sequencing have confirmed and extended the classification of enteric neurons in mice and human, but it is not clear whether an encompassing classification has been achieved. We present preliminary data on a means to distinguish classes of myenteric neurons in specimens of human colon combining immunohistochemical, morphological, projection and size data on single cells. A method to apply multiple layers of antisera to specimens was developed, allowing up to 12 markers to be characterised in individual neurons. Applied to multi-axonal Dogiel type II neurons, this approach demonstrated that they constitute fewer than 5% of myenteric neurons, are nearly all immunoreactive for choline acetyltransferase and tachykinins. Many express the calcium-binding proteins calbindin and calretinin and they are larger than average myenteric cells. This methodology provides a complementary approach to single-cell mRNA profiling to provide a comprehensive account of the types of myenteric neurons in the human colon.

Evaluation of myenteric neurons in the colon of rats exposed to 2,4 dichlorophenoxyacetic acid herbicide.

The assessment of the enteric nervous system provides a better understanding of the effects that contaminants can have on the health and well-being of organisms. It has been reported that 2,4-dichlorophenoxyacetic acid (2,4-D) is a highly persistent herbicide in the environment that is responsible for neurotoxic changes in different myenteric neuronal subpopulations. The current study aimed to evaluate the effects of 2,4-D on myenteric neurons in the colon of Rattus norvegicus for the first time. A dose of 2,4-D (5 mg/kg/day) was administered to the experimental group (2,4-D) for 15 days. Then, the proximal colon was collected and submitted to Giemsa and NADPH-d histochemical techniques for the disclosure of total and nitrergic neurons. The 2,4-D group presented a higher density of total neurons (p = 0.05, t-test), which together with the maintenance of nitrergic neuronal density, may be related to the increase in the expression of the neurotransmitter acetylcholine by colocalization, responsible for stimulating the intestinal smooth muscle and increasing the chances of the expulsion of the harmful content present in the lumen. Over 15 days, the neurotoxic effects of 2,4-D in the myenteric plexus influenced an increase in the general population of myenteric neurons in the colon.

Optogenetic activation of the distal colon epithelium engages enteric nervous system circuits to initiate motility patterns.

Digestive functions of the colon depend on sensory-motor reflexes in the enteric nervous system (ENS), initiated by intrinsic primary afferent neurons (IPANs). IPAN terminals project to the mucosal layer of the colon, allowing communication with epithelial cells comprising the colon lining. The chemical nature and functional significance of this epithelial-neural communication in regard to secretion and colon motility are of high interest. Colon epithelial cells can produce and release neuroactive substances such as ATP and 5-hydroxytryptamine (5-HT), which can activate receptors on adjacent nerve fibers, including IPAN subtypes. In this study, we examined if stimulation of epithelial cells alone is sufficient to activate neural circuits that control colon motility. Optogenetics and calcium imaging were used in ex vivo preparations of the mouse colon to selectively stimulate the colon epithelium, measure changes in motility, and record activity of neurons within the myenteric plexus. Light-mediated activation of epithelial cells lining the distal, but not proximal, colon caused local contractions and increased the rate of colonic migrating motor complexes. Epithelial-evoked local contractions in the distal colon were reduced by both ATP and 5-HT receptor antagonists. Our findings indicate that colon epithelial cells likely use purinergic and serotonergic signaling to initiate activity in myenteric neurons, produce local contractions, and facilitate large-scale coordination of ENS activity responsible for whole colon motility patterns.NEW & NOTEWORTHY Using an all-optical approach to measure real-time cell-to-cell communication responsible for colon functions, we show that selective optogenetic stimulation of distal colon epithelium produced activity in myenteric neurons, as measured with red genetically encoded calcium indicators. The epithelial-induced neural response led to local contractions, mediated by both purinergic and serotonergic signaling, and facilitated colonic motor complexes that propagate from proximal to distal colon.

High-fat diet impairs duodenal barrier function and elicits glia-dependent changes along the gut-brain axis that are required for anxiogenic and depressive-like behaviors.

BACKGROUND: Mood and metabolic disorders are interrelated and may share common pathological processes. Autonomic neurons link the brain with the gastrointestinal tract and constitute a likely pathway for peripheral metabolic challenges to affect behaviors controlled by the brain. The activities of neurons along these pathways are regulated by glia, which exhibit phenotypic shifts in response to changes in their microenvironment. How glial changes might contribute to the behavioral effects of consuming a high-fat diet (HFD) is uncertain. Here, we tested the hypothesis that anxiogenic and depressive-like behaviors driven by consuming a HFD involve compromised duodenal barrier integrity and subsequent phenotypic changes to glia and neurons along the gut-brain axis. METHODS: C57Bl/6 male mice were exposed to a standard diet or HFD for 20 weeks. Bodyweight was monitored weekly and correlated with mucosa histological damage and duodenal expression of tight junction proteins ZO-1 and occludin at 0, 6, and 20 weeks. The expression of GFAP, TLR-4, BDNF, and DCX were investigated in duodenal myenteric plexus, nodose ganglia, and dentate gyrus of the hippocampus at the same time points. Dendritic spine number was measured in cultured neurons isolated from duodenal myenteric plexuses and hippocampi at weeks 0, 6, and 20. Depressive and anxiety behaviors were also assessed by tail suspension, forced swimming, and open field tests. RESULTS: HFD mice exhibited duodenal mucosa damage with marked infiltration of immune cells and decreased expression of ZO-1 and occludin that coincided with increasing body weight. Glial expression of GFAP and TLR4 increased in parallel in the duodenal myenteric plexuses, nodose ganglia, and hippocampus in a time-dependent manner. Glial changes were associated with a progressive decrease in BDNF, and DCX expression, fewer neuronal dendritic spines, and anxiogenic/depressive symptoms in HFD-treated mice. Fluorocitrate (FC), a glial metabolic poison, abolished these effects both in the enteric and central nervous systems and prevented behavioral alterations at week 20. CONCLUSIONS: HFD impairs duodenal barrier integrity and produces behavioral changes consistent with depressive and anxiety phenotypes. HFD-driven changes in both peripheral and central nervous systems are glial-dependent, suggesting a potential glial role in the alteration of the gut-brain signaling that occurs during metabolic disorders and psychiatric co-morbidity.

Immunohistochemical expression and neurochemical phenotypes of huntingtin-associated protein 1 in the myenteric plexus of mouse gastrointestinal tract.

Huntingtin-associated protein 1 (HAP1) is a neural huntingtin interactor and being considered as a core molecule of stigmoid body (STB). Brain/spinal cord regions with abundant STB/HAP1 expression are usually spared from neurodegeneration in stress/disease conditions, whereas the regions with little STB/HAP1 expression are always neurodegenerative targets. The enteric nervous system (ENS) can act as a potential portal for pathogenesis of neurodegenerative disorders. However, ENS is also a neurodegenerative target in these disorders. To date, the expression of HAP1 and its neurochemical characterization have never been examined there. In the current study, we determined the expression of HAP1 in the ENS of adult mice and characterized the morphological relationships of HAP1-immunoreactive (ir) cells with the markers of motor neurons, sensory neurons, and interneurons in the myenteric plexus using Western blotting and light/fluorescence microscopy. HAP1-immunoreaction was present in both myenteric and submucosal plexuses of ENS. Most of the HAP1-ir neurons exhibited STB in their cytoplasm. In myenteric plexus, a large number of calretinin, calbindin, NOS, VIP, ChAT, SP, somatostatin, and TH-ir neurons showed HAP1-immunoreactivity. In contrast, most of the CGRP-ir neurons were devoid of HAP1-immunoreactivity. Our current study is the first to clarify that HAP1 is highly expressed in excitatory motor neurons, inhibitory motor neurons, and interneurons but almost absent in sensory neurons in myenteric plexus. These suggest that STB/HAP1-ir neurons are mostly Dogiel type I neurons. Due to lack of putative STB/HAP1 protectivity, the sensory neurons (Dogiel type II) might be more vulnerable to neurodegeneration than STB/HAP1-expressing motoneurons/interneurons (Dogiel type I) in myenteric plexus.

Phosphorylated Tau protein in the myenteric plexus of the ileum and colon of normothermic rats and during synthetic torpor.

Tau protein is of primary importance for neuronal homeostasis and when hyperphosphorylated (PP-Tau), it tends to aggregate in neurofibrillary tangles, as is the case with tauopathies, a class of neurodegenerative disorders. Reversible PP-Tau accumulation occurs in the brain of hibernating rodents and it was recently observed in rats (a non-hibernator) during synthetic torpor (ST), a pharmacological-induced torpor-like condition. To date, the expression of PP-Tau in the rat enteric nervous system (ENS) is still unknown. The present study immunohistochemically investigates the PP-Tau expression in the myenteric plexus of the ileum and colon of normothermic rats (CTRL) and during ST, focusing on the two major subclasses of enteric neurons, i.e., cholinergic and nitrergic.Results showed that both groups of rats expressed PP-Tau, with a significantly increased percentage of PP-Tau immunoreactive (IR) neurons in ST vs. CTRL. In all rats, the majority of PP-Tau-IR neurons were cholinergic. In ST rats, the percentage of PP-Tau-IR neurons expressing a nitrergic phenotype increased, although with no significant differences between groups. In addition, the ileum of ST rats showed a significant decrease in the percentage of nitrergic neurons. In conclusion, our findings suggest an adaptive response of ENS to very low core body temperatures, with changes involving PP-tau expression in enteric neurons, especially the ileal nitrergic subpopulation. In addition, the high presence of PP-Tau in cholinergic neurons, specifically, is very interesting and deserves further investigation. Altogether, these data strengthen the hypothesis of a common cellular mechanism triggered by ST, natural hibernation and tauopathies occurring in ENS neurons.

COLQ and ARHGAP15 are Associated with Diverticular Disease and are Expressed in the Colon.

BACKGROUND: Diverticular disease is a common but poorly understood disease of the gastrointestinal tract. Recent studies have identified several single nucleotide polymorphisms (SNPs) that are associated with diverticular disease. MATERIALS AND METHODS: The genotypes of three SNPs (rs4662344 in ARHGAP15, rs7609897 in COLQ, and rs67153654 in FAM155A) were identified by Taqman assay in 204 patients with diverticular disease. Clinical characteristics were obtained from the medical record to study association with genotype. To evaluate gene expression in colon tissue, qPCR was performed on 24 patients with diverticulitis, and COLQ was localized using immunohistochemistry. RESULTS: The ARHGAP15 and COLQ SNPs were significantly associated with both diverticular disease and specifically diverticulitis, while the FAM155A was not associated with either. No association was found with clinical disease characteristics. Heterozygous genotypes at the ARHGAP15 SNP was associated with lower ARHGAP15 expression in colon tissues. COLQ protein localized to the myenteric plexus in the colon. CONCLUSIONS: This study confirmed association of the ARHGAP15 and COLQ SNPs with diverticular disease in our patients but could not confirm FAM155A SNP association. Neither of these SNPs appeared to associate with more severe disease, but genotype at the ARHGAP15 SNP did impact expression of ARHGAP15 in the colon. Additionally, this study is the first to localize COLQ in the colon. Its presence in the myenteric nervous system suggests COLQ SNP variants may contribute to diverticular disease by altering motility.

Cocaine and Amphetamine Regulated Transcript (CART) Expression Changes in the Stomach Wall Affected by Experimentally Induced Gastric Ulcerations.

Cocaine- and amphetamine-regulated transcript (CART) is a peptide suggested to play a role in gastrointestinal tract tissue reaction to pathology. Gastric ulceration is a common disorder affecting huge number of people, and additionally, it contributes to the loss of pig livestock production. Importantly, ulceration as a focal disruption affecting deeper layers of the stomach wall differs from other gastrointestinal pathologies and should be studied individually. The pig's gastrointestinal tract, due to its many similarities to the human counterpart, provides a valuable experimental model for studying digestive system pathologies. To date, the role of CART in gastric ulceration and the expression of the gene encoding CART in porcine gastrointestinal tube are completely unknown. Therefore, we aimed to verify the changes in the CART expression by Q-PCR (gene encoding CART in the tissue) and double immunofluorescence staining combined with confocal microscopy (CART immunofluorescence in enteric nervous system) in the porcine stomach tissues adjacent to gastric ulcerations. Surprisingly, we found that gastric ulcer caused a significant decrease in the expression of CART-encoding gene and huge reduction in the percentage of CART-immunofluorescent myenteric perikarya and neuronal fibers located within the circular muscle layer. Our results indicate a unique CART-dependent gastric response to ulcer disease.

Histamine-dependent interactions between mast cells, glia, and neurons are altered following early-life adversity in mice and humans.

Early-life adversity contributes to the development of functional bowel disorders later in life through unresolved mechanisms. Here, we tested the hypothesis that early-life adversity alters anatomical and functional interactions between mast cells and enteric glia. The effects of early-life stress were studied using the neonatal maternal separation (NMS) stress mouse model. Anatomical relationships between mast cells and enteric glia were assessed using immunohistochemistry and mast cell reporter mice (Mcpt5Cre;GCaMP5g-tdT). Immunohistochemistry was used to assess the expression of histamine, histamine 1 (H1) receptors, and glial fibrillary acidic protein. Functional responses of glia to mast cell mediators were assessed in calcium imaging experiments using Sox10CreERT2;GCaMP5g-tdT mice and cultured human enteric glial cells. NMS increases mast cell numbers at the level of the myenteric plexus and their proximity to myenteric ganglia. Myenteric glia respond to mediators released by activated mast cells that are blocked by H1 receptor antagonists in mice and humans and by blocking neuronal activity with tetrodotoxin in mouse tissue. Histamine replicates the effects of mast cell supernatants on enteric glia, and NMS increases histamine production by mast cells. NMS reduces glial responses to mast cell mediators in mouse tissue, while potentiating responses in cultured human enteric glia. NMS increases myenteric glial fibrillary acidic protein expression and reduces glial process length but does not cause neurodegeneration. Histamine receptor expression is not altered by NMS and is localized to neurons in mice, but glia in humans. Early-life stress increases the potential for interactions between enteric glia and mast cells, and histamine is a potential mediator of mast cell-glial interactions through H1 receptors. We propose that glial-mast cell signaling is a mechanism that contributes to enteric neuroplasticity driven by early-life adversity.NEW & NOTEWORTHY Early-life adversity places an individual at risk for developing functional gastrointestinal disorders later in life through unknown mechanisms. Here, we show that interactions between mast cells and glia are disrupted by early-life stress in mice and that histamine is a potential mediator of mast cell-glial interactions.

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.