VIP is involved in peripheral CRF-induced stimulation of propulsive colonic motor function and diarrhea in male rats.

We investigated whether vasoactive intestinal peptide (VIP) and/or prostaglandins contribute to peripheral corticotropin-releasing factor (CRF)-induced CRF1 receptor-mediated stimulation of colonic motor function and diarrhea in rats. The VIP antagonist, [4Cl-D-Phe6, Leu17]VIP injected intraperitoneally completely prevented CRF (10 µg/kg ip)-induced fecal output and diarrhea occurring within the first hour after injection, whereas pretreatment with the prostaglandins synthesis inhibitor, indomethacin, had no effect. In submucosal plexus neurons, CRF induced significant c-Fos expression most prominently in the terminal ileum compared with duodenum and jejunum, whereas no c-Fos was observed in the proximal colon. c-Fos expression in ileal submucosa was colocalized in 93.4% of VIP-positive neurons and 31.1% of non-VIP-labeled neurons. CRF1 receptor immunoreactivity was found on the VIP neurons. In myenteric neurons, CRF induced only a few c-Fos-positive neurons in the ileum and a robust expression in the proximal colon (17.5 ± 2.4 vs. 0.4 ± 0.3 cells/ganglion in vehicle). The VIP antagonist prevented intraperitoneal CRF-induced c-Fos induction in the ileal submucosal plexus and proximal colon myenteric plexus. At 60 min after injection, CRF decreased VIP levels in the terminal ileum compared with saline (0.8 ± 0.3 vs. 2.5 ± 0.7 ng/g), whereas VIP mRNA level detected by qPCR was not changed. These data indicate that intraperitoneal CRF activates intestinal submucosal VIP neurons most prominently in the ileum and myenteric neurons in the colon. It also implicates VIP signaling as part of underlying mechanisms driving the acute colonic secretomotor response to a peripheral injection of CRF, whereas prostaglandins do not play a role. NEW & NOTEWORTHY Corticotropin-releasing factor (CRF) in the gut plays a physiological role in the stimulation of lower gut secretomotor function induced by stress. We showed that vasoactive intestinal peptide (VIP)-immunoreactive neurons in the ileal submucosal plexus expressed CRF1 receptor and were prominently activated by CRF, unlike colonic submucosal neurons. VIP antagonist abrogated CRF-induced ileal submucosal and colonic myenteric activation along with functional responses (defecation and diarrhea). These data point to VIP signaling in ileum and colon as downstream effectors of CRF.

Abdominal surgery induced gastric ileus and activation of M1-like macrophages in the gastric myenteric plexus: prevention by central vagal activation in rats.

Inflammation plays a role in abdominal surgery (AS)-induced intestinal ileus that is alleviated by electrical vagal stimulation. Intracisternal injection of RX-77368, the stable thyrotropin-releasing hormone agonist, activates dorsal motor nucleus neurons and gastric vagal efferent discharges. We investigated the gastric inflammation induced by AS and the modulation by intracisternal RX-77368 in rats. RX-77368 (50 ng/rat) or saline was injected followed, 1 h later, by laparotomy and small intestinal/cecal manipulation. The sham group had anesthesia alone. After 6 h, gastric emptying (GE) and the inflammation in gastric corpus were determined. AS inhibited GE by 72% vs. control and doubled the number of M1-like macrophage immunoreactive for major histocompatibility complex class II (MHCII; M1 marker) but not for cluster of differentiation 206 (CD206; M2 marker) (MHCII+/CD206-) while there was no change in M2-like macrophages (MHCII-/CD206+). AS increased mRNA levels of interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) by 1.7- and 1.5-fold, respectively, in the gastric submucosa plus muscle layers and the infiltration of neutrophils labeled by myeloperoxidase by 9.5-fold in the muscularis externa. RX-77368 inhibited AS-related gastric changes while not altering these parameters in the sham group. There was a significant negative correlation between GE and IL-1ß (r = -0.46), TNF-α (r = -0.44), M1 macrophage (r = -0.82), and neutrophils (r = -0.91). The M2-like macrophages and IL-10 expression were unchanged by AS with intracisternal saline or RX-77368. These data indicate that AS activates gastric M1 macrophages and increases proinflammatory cytokines expression, which are prevented by central vagal activation and may contribute to the correlated dampening of postoperative gastric ileus.NEW & NOTEWORTHY MHCII+/CD206- (M1) and MHCII-/CD206+ (M2) constitute two distinct populations of macrophages that are in close apposition to the cholinergic neurons in the rat gastric myenteric plexus (MP). Abdominal surgery (6 h) activates M1 macrophage leading to inflammation in the gastric MP correlated with the delayed gastric emptying, which was abolished by central vagal stimulation via intracisternal injection of RX-77368. Vagal stimulation linked with the cephalic phase may have potential beneficial effects to curtail postoperative gastric ileus.

High-protein diet improves sensitivity to cholecystokinin and shifts the cecal microbiome without altering brain inflammation in diet-induced obesity in rats.

High-protein diet (HPD) curtails obesity and/or fat mass, but it is unknown whether it reverses neuroinflammation or alters glucose levels, CCK sensitivity, and gut microbiome in rats fed a Western diet (WD)-induced obesity (DIO). Male rats fed a WD (high fat and sugar) for 12 wk were switched to a HPD for 6 wk. Body composition, food intake, meal pattern, sensitivity to intraperitoneal CCK-8S, blood glucose, brain signaling, and cecal microbiota were assessed. When compared with a normal diet, WD increased body weight (9.3%) and fat mass (73.4%). CCK-8S (1.8 or 5.2 nmol/kg) did not alter food intake and meal pattern in DIO rats. Switching to a HPD for 6 wk reduced fat mass (15.7%) with a nonsignificantly reduced body weight gain, normalized blood glucose, and decreased feeding after CCK-8S. DIO rats on the WD or switched to a HPD showed comparable microbial diversity. However, in HPD versus WD rats, there was enrichment of 114 operational taxonomic units (OTUs) and depletion of 188 OTUs. Of those, Akkermansia muciniphila (enriched on a HPD), an unclassified Clostridiales, a member of the RF39 order, and a Phascolarctobacterium were significantly associated with fat mass. The WD increased cytokine expression in the hypothalamus and dorsal medulla that was unchanged by switching to HPD. These data indicate that HPD reduces body fat and restores glucose homeostasis and CCK sensitivity, while not modifying brain inflammation. In addition, expansion of cecal Akkermansia muciniphila correlated to fat mass loss may represent a potential peripheral mechanism of HPD beneficial effects.

Urocortins and CRF receptor type 2 variants in the male rat colon: gene expression and regulation by endotoxin and anti-inflammatory effect.

Urocortins (Ucns) 1, 2, and 3 and corticotropin-releasing factor receptor 2 (CRF2) mRNA are prominently expressed in various layers of the upper gut. We tested whether Ucns and CRF2 variants are also expressed in the different layers of the rat colon, regulated by LPS (100 µg/kg ip) and play a modulatory role in the colonic immune response to LPS. Transcripts of Ucns and CRF2b, the most common isoform in the periphery, were detected in all laser microdissected layers, including myenteric neurons. LPS increased the mRNA level of Ucn 1, Ucn 2, and Ucn 3 and decreased that of CRF2b in both the colonic mucosa and submucosa + muscle (S+M) layers at 2, 6, and 9 h after injection with a return to basal at 24 h. In addition, CRF2a, another variant more prominent in the brain, and a novel truncated splice variant CRF2a-3 mRNA were detected in all segments of the large intestine. LPS reciprocally regulated the colonic expression of these CRF2 variants by decreasing both CRF2a and CRF2b, while increasing CRF2a-3 in the mucosa and S+M. The CRF2 antagonist astressin2-B further enhanced LPS-induced increase of mRNA level of interleukin (IL)-1ß, TNF-α, and inducible nitric oxide synthase in S+M layers and IL-1ß in the mucosa and evoked TNF-α expression in the mucosa. These data indicate that Ucns/CRF2 variants are widely expressed in all colonic layers and reciprocally regulated by LPS. CRF2 signaling dampens the CD14/TLR4-mediated acute inflammatory response to Gram-negative bacteria in the colon.

Vasculature in the mouse colon and spatial relationships with the enteric nervous system, glia, and immune cells.

The distribution, morphology, and innervation of vasculature in different mouse colonic segments and layers, as well as spatial relationships of the vasculature with the enteric plexuses, glia, and macrophages are far from being complete. The vessels in the adult mouse colon were stained by the cardiovascular perfusion of wheat germ agglutinin (WGA)-Alexa Fluor 448 and by CD31 immunoreactivity. Nerve fibers, enteric glia, and macrophages were immunostained in the WGA-perfused colon. The blood vessels entered from the mesentery to the submucosa and branched into the capillary networks in the mucosa and muscularis externa. The capillary net formed anastomosed rings at the orifices of mucosa crypts, and the capillary rings surrounded the crypts individually in the proximal colon and more than two crypts in the distal colon. Microvessels in the muscularis externa with myenteric plexus were less dense than in the mucosa and formed loops. In the circular smooth muscle layer, microvessels were distributed in the proximal, but not the distal colon. Capillaries did not enter the enteric ganglia. There were no significant differences in microvascular volume per tissue volume between the proximal and distal colon either in the mucosa or muscularis externa containing the myenteric plexus. PGP9.5-, tyrosine hydroxylase-, and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers were distributed along the vessels in the submucosa. In the mucosa, PGP9.5-, CGRP-, and vasoactive intestinal peptide (VIP)-immunoreactive nerves terminated close to the capillary rings, while cells and processes labeled by S100B and glial fibrillary acidic protein were distributed mainly in the lamina propria and lower portion of the mucosa. Dense Iba1 immunoreactive macrophages were closely adjacent to the mucosal capillary rings. There were a few macrophages, but no glia in apposition to microvessels in the submucosa and muscularis externa. In conclusion, in the mouse colon, (1) the differences in vasculature between the proximal and distal colon were associated with the morphology, but not the microvascular amount per tissue volume in the mucosa and muscle layers; (2) the colonic mucosa contained significantly more microvessels than the muscularis externa; and (3) there were more CGRP and VIP nerve fibers found close to microvessels in the mucosa and submucosa than in the muscle layers.

The ghrelin agonist, HM01 activates central vagal and enteric cholinergic neurons and reverses gastric inflammatory and ileus responses in rats.

BACKGROUND: Electrical vagal stimulation alleviates abdominal surgery (AS)-induced intestinal inflammation. Ghrelin receptors (GHS-Rs) are expressed in the brain and peripheral tissues. We investigated the influence of HM01, an orally active ghrelin agonist crossing the blood-brain barrier, on AS-induced gastric inflammation and emptying (GE) in rats. METHODS: HM01 (6 mg/kg) or saline pretreatment was administered per orally (po) or intraperitoneally (ip). We assessed GE, gastric cytokine mRNA, and Fos positive cells in the dorsal motor nucleus of the vagus (DMN) and gastric corpus myenteric plexus (MP) in sham (anesthesia alone) and AS groups. The transcripts of GHS-R1 variants were determined in the medulla oblongata and gastric corpus of naïve rats. KEY RESULTS: In vehicle pretreated rats, HM01 (ip) significantly increased the number of Fos immunoreactive cells in the MP and DMN in 55% and 52% of cholinergic neurons respectively. Hexamethonium did not modify HM01-induced Fos expression in the DMN while reducing it in the MP by 2-fold with values still significantly higher than that in control groups. AS upregulated gastric IL-1ß and TNFα expression and inhibited GE by 66.6%. HM01 (po) abolished AS-induced gastric ileus and increased cytokine expression and elevated IL-10 by 4.0-fold versus vehicle/sham. GHS-R1a mRNA level was 5.4-fold higher than the truncated GHS-R1b isoform in the brain medulla and 40-fold higher in the gastric submucosa/muscle layers than in the mucosa. CONCLUSIONS AND INFERENCE: Peripheral HM0 activates central vagal and myenteric cholinergic pathways that may influence both central and peripheral targets to prevent AS-induced gastric inflammatory and ileus.

New insight on the enteric cholinergic innervation of the pig colon by central and peripheral nervous systems: reduction by repeated loperamide administration.

Introduction: The central and peripheral nervous systems provide cholinergic innervation in the colon. The ability to assess their neuroanatomical distinctions is still a challenge. The pig is regarded as a relevant translational model due to the close similarity of its enteric nervous system (ENS) with that of human. Opioid-induced constipation is one of the most common side effects of opioid therapy. Methods: We developed an approach to differentiate the central and peripheral cholinergic innervation of the pig colon using double immunolabeling with a novel mouse anti-human peripheral type of choline acetyltransferase (hpChAT) antibody combined with a rabbit anti-common type of ChAT (cChAT) antibody, a reliable marker of cholinergic neurons in the central nervous system. We examined their spatial configurations in 3D images of the ENS generated from CLARITY-cleared colonic segments. The density was quantitated computationally using Imaris 9.7. We assessed changes in the distal colon induced by daily oral treatment for 4 weeks with the µ opioid receptor agonist, loperamide (0.4 or 3 mg/kg). Results: The double labeling showed strong cChAT immunoreactive (ir) fibers in the cervical vagus nerve and neuronal somata and fibers in the ventral horn of the sacral (S2) cord while hpChAT immunoreactivity was visualized only in the ENS but not in the vagus or sacral neural structures indicating the selectivity of these two antibodies. In the colonic myenteric plexus, dense hpChAT-ir neurons and fibers and varicose cChAT-ir fibers surrounding hpChAT-ir neurons were simultaneously visualized in 3D. The density of cChAT-ir varicose fibers in the outer submucosal plexus of both males and females were higher in the transverse and distal colon than in the proximal colon and in the myenteric plexus compared to the outer submucosal plexus and there was no cChAT innervation in the inner submucosal plexus. The density of hpChAT in the ENS showed no segmental or plexus differences in both sexes. Loperamide at the highest dose significantly decreased the density hpChAT-ir fibers + somata in the myenteric plexus of the distal colon. Discussion: These data showed the distinct density of central cholinergic innervation between myenteric and submucosal plexuses among colonic segments and the localization of cChAT-ir fibers around peripheral hpChAT neurons in 3D. The reduction of cholinergic myenteric innervation by chronic opiate treatment points to target altered prokinetic cholinergic pathway to counteract opiate constipation.

Comparative transcriptomics reveals highly conserved regional programs between porcine and human colonic enteric nervous system.

The porcine gut is increasingly regarded as a useful translational model. The enteric nervous system in the colon coordinates diverse functions. However, knowledge of the molecular profiling of porcine enteric nerve system and its similarity to that of human is still lacking. We identified the distinct transcriptional programs associated with functional characteristics between inner submucosal and myenteric ganglia in porcine proximal and distal colon using bulk RNA and single-cell RNA sequencing. Comparative transcriptomics of myenteric ganglia in corresponding colonic regions of pig and human revealed highly conserved programs in porcine proximal and distal colon, which explained >96% of their transcriptomic responses to vagal nerve stimulation, suggesting that porcine proximal and distal colon could serve as predictors in translational studies. The conserved programs specific for inflammatory modulation were displayed in pigs with vagal nerve stimulation. This study provides a valuable transcriptomic resource for understanding of human colonic functions and neuromodulation using porcine model.

Urocortins and CRF type 2 receptor isoforms expression in the rat stomach are regulated by endotoxin: role in the modulation of delayed gastric emptying.

Peripheral activation of corticotropin-releasing factor receptor type 2 (CRF(2)) by urocortin 1, 2, or 3 (Ucns) exerts powerful effects on gastric function; however, little is known about their expression and regulation in the stomach. We investigated the expression of Ucns and CRF(2) isoforms by RT-PCR in the gastric corpus (GC) mucosa and submucosa plus muscle (S+M) or laser captured layers in naive rats, their regulations by lipopolysaccharide (LPS, 100 µg/kg ip) over 24 h, and the effect of the CRF(2) antagonist astresssin(2)-B (100 µg/kg sc) on LPS-induced delayed gastric emptying (GE) 2-h postinjection. Transcripts of Ucns and CRF(2b,) the most common wild-type CRF(2) isoform in the periphery, were expressed in all layers, including myenteric neurons. LPS increased Ucn mRNA levels significantly in both mucosa and S+M, reaching a maximal response at 6 h postinjection and returning to basal levels at 24 h except for Ucn 1 in S+M. By contrast, CRF(2b) mRNA level was significantly decreased in the mucosa and M+S with a nadir at 6 h. In addition, CRF(2a), reportedly only found in the brain, and the novel splice variant CRF(2a-3) were also detected in the GC, antrum, and pylorus. LPS reciprocally regulated these variants with a decrease of CRF(2a) and an increase of CRF(2a-3) in the GC 6 h postinjection. Astressin(2)-B exacerbated LPS-delayed GE (42-73%, P < 0.001). These data indicate that Ucn and CRF(2) isoforms are widely distributed throughout the rat stomach and inversely regulated by immune stress. The CRF(2) signaling system may act to counteract the early gastric motor alterations to endotoxemia.

Activation of corticotropin-releasing factor receptor 2 mediates the colonic motor coping response to acute stress in rodents.

BACKGROUND & AIMS: Corticotropin-releasing factor receptor-1 (CRF(1)) mediates the stress-induced colonic motor activity. Less is known about the role of CRF(2) in the colonic response to stress. METHODS: We studied colonic contractile activity in rats and CRF(2)-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute partial-restraint stress (PRS), and/or intraperitoneal injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF(1) and CRF(2) using immunohistochemical and immunoblot analyses. We measured phosphorylation of extracellular signal-regulated kinase 1/2 by CRF ligands in primary cultures of LMMP neurons (PC-LMMPn) and cyclic adenosine monophosphate (cAMP) production in human embryonic kidney-293 cells transfected with CRF(1) and/or CRF(2). RESULTS: In rats, a selective agonist of CRF(2) (urocortin 2) reduced CRF-induced defecation (>50%), colonic contractile activity, and Fos expression in the colonic LMMP. A selective antagonist of CRF(2) (astressin(2)-B) increased these responses. Urocortin 2 reduced PRS-induced colonic contractile activity in wild-type and CRF-overexpressing mice, whereas disruption of CRF(2) increased PRS-induced colonic contractile activity and CRF-induced defecation. CRF(2) colocalized with CRF(1) and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of extracellular signal-regulated kinase in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF(1) (NBI35965) or astressin(2)-B, respectively. The half maximal effective concentration, EC(50), for the CRF-induced cAMP response was 8.6 nmol/L in human embryonic kidney-293 cells that express only CRF(1); this response was suppressed 10-fold in cells that express CRF(1) and CRF(2). CONCLUSIONS: In colon tissues of rodents, CRF(2) activation inhibits CRF(1) signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF(2) function impairs colonic coping responses to stress.

Serum and colonic mucosal immune markers in irritable bowel syndrome.

OBJECTIVES: Low-grade colonic mucosal inflammation has been postulated to have an important role in the pathophysiology of irritable bowel syndrome (IBS). The objectives of this study were (i) to identify serum and tissue-based immunological and neuroendocrine markers associated with mucosal inflammation in male (M) and female (F) patients with non-post-infectious IBS (non-PI-IBS) compared with healthy controls and (ii) to assess possible correlations of such markers with IBS symptoms. METHODS: Sigmoid mucosal biopsies were obtained from 45 Rome II positive IBS patients without a history of PI-IBS (26 F, 35.5% IBS-C, 33.3% IBS-D, 31.1% IBS-A/M) and 41 healthy controls (22 F) in order to measure immunological markers (serum cytokine levels, colonic mucosal mRNA levels of cytokines, mucosal immune cell counts) and neuroendocrine markers associated with mucosal inflammation (corticotropin releasing factor- and neurokinin (NK)-related ligands and receptors, enterochromaffin cells). Symptoms were measured using validated questionnaires. RESULTS: Of all the serum and mucosal cytokines measured, only interleukin-10 (IL-10) mRNA expression showed a group difference, with female, but not male, patients showing lower levels compared with female controls (18.0±2.9 vs. 29.5±4.0, P=0.006). Mucosal mRNA expression of NK-1 receptor was significantly lower (1.15±0.19 vs. 2.66±0.56, P=0.008) in female, but not male, patients compared with healthy controls. No other significant differences were observed. CONCLUSIONS: Immune cell counts and levels of cytokines and neuropeptides that are associated with inflammation were not significantly elevated in the colonic mucosa of non-PI-IBS patients, and did not correlate with symptoms. Thus, these findings do not support that colonic mucosal inflammation consistently has a primary role in these patients. However, the finding of decreased IL-10 mRNA expression may be a possible biomarker of IBS and warrants further investigation.

Transduction of Systemically Administered Adeno-Associated Virus in the Colonic Enteric Nervous System and c-Kit Cells of Adult Mice.

Systemic delivery of adeno-associated virus (AAV) vectors transduces the enteric nervous system. However, less is known on the mapping and morphological and neurochemical characterization in the adult mouse colon. We used AAV9-CAG-GFP (AAV9) and AAV-PHP.S-hSyn1-tdTomato farnesylated (PHP.S-tdTf) to investigate the segmental distribution, morphologies and neurochemical coding of the transduction. The vectors were retro-orbitally injected in male and female adult mice, and 3 weeks later, the colon was prepared for microcopy with or without immunohistochemistry for neuronal and non-neuronal markers. In contrast to the distributions in neonatal and juvenile rodents, the AAV transduction in neurons and/or nerve fibers was the highest in the proximal colon, decreased gradually in the transverse, and was sparse in the distal colon without difference between sexes. In the proximal colon, the AAV9-transduced myenteric neurons were unevenly distributed. The majority of enteric neurons did not have AAV9 expression in their processes, except those with big soma with or without variously shaped dendrites, and a long axon. Immunolabeling demonstrated that about 31% neurons were transduced by AAV9, and the transduction was in 50, 28, and 31% of cholinergic, nitrergic, and calbindin-positive myenteric neurons, respectively. The nerve fiber markers, calcitonin gene-related peptide alpha, tyrosine hydroxylase or vasoactive intestinal polypeptide co-localized with AAV9 or PHP.S-tdTf in the mucosa, and rarely in the myenteric plexus. Unexpectedly, AAV9 expression appeared also in a few c-Kit immunoreactive cells among the heavily populated interstitial cells of Cajal (ICC). In the distal colon, the AAV transduction appeared in a few nerve fibers mostly the interganglionic strands. Other types of AAV9 and AAV-PHP vectors induced a similar colonic segmental difference which is not colon specific since neurons were transduced in the small intestine and gastric antrum, while little in the gastric corpus and none in the lower esophagus. Conclusion: These findings demonstrate that in adult mice colon that there is a rostro-caudal decrease in the transduction of systemic delivery of AAV9 and its variants independent of sex. The characterization of AAV transduction in the proximal colon in cholinergic and nitrergic myenteric neurons along with a few ICC suggests implications in circuitries regulating motility.

Activation of CRF1 receptors expressed in brainstem autonomic nuclei stimulates colonic enteric neurons and secreto-motor function in male rats.

BACKGROUND: Hypothalamic corticotropin-releasing factor (CRF) receptor 1 (CRF1 ) plays a role in acute stress-related stimulation of colonic motor function. Less is known on CRF1 signaling in the brainstem. METHODS: We investigate CRF1 expression in the brainstem and the colonic response to 4th ventricle (4V) injection of CRF and urocortin (Ucn) 2 (3 µg/rat) in chronically cannulated male rats. KEY RESULTS: Transcripts of CRF1 wild-type 1a and splice variants 1c, 1e, 1f, 1o along with three novel variants 1a-2 (desK-110 in exon 5), 1p (-exon 7), and 1q (exon 5 extension) were identified in the pons and medulla. The area postrema, nucleus tractus solitarius, dorsal motor nucleus of the vagus, locus coeruleus, and Barrington's nucleus isolated by laser capture microdissection expressed 1a, 1a-2, and 1p but not 1q. Compared to 4V vehicle, 4V CRF induced fecal pellet output (FPO) and diarrhea that were blocked by the CRF antagonist, astressin-B. CRF2 agonist, Ucn2 had no effect on basal or CRF-induced FPO. CRF actions were correlated with the induction of c-Fos immunoreactivity in myenteric neurons of the proximal and distal colon (pC, dC) and submucosal neurons of dC. c-Fos immunoreactivity occurred in 39% and 37% of myenteric cholinergic and 7% and 58% of nitrergic neurons in the pC and dC, respectively. CONCLUSIONS & INFERENCES: CRF1a and its splice variants are expressed in brainstem nuclei, and activation of CRF1 signaling at the level of the brainstem stimulates colonic secretory-motor function through activation of colonic enteric neurons.

Intrinsic cholinergic innervation in the human sigmoid colon revealed using CLARITY, three-dimensional (3D) imaging, and a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum.

BACKGROUND: We previously reported the specificity of a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum for immunostaining of cholinergic neuronal cell bodies and fibers in the human colon. In this study, we investigate 3D architecture of intrinsic cholinergic innervation in the human sigmoid colon and the relationship with nitrergic neurons in the enteric plexus. METHODS: We developed a modified CLARITY tissue technique applicable for clearing human sigmoid colon specimens and immunostaining with hpChAT antiserum and co-labeling with neuronal nitric oxide synthase (nNOS) antibody. The Z-stack confocal images were processed for 3D reconstruction/segmentation/digital tracing and computational quantitation by Imaris 9.2 and 9.5. KEY RESULTS: In the mucosa, a local micro-neuronal network formed of hpChAT-ir fibers and a few neuronal cell bodies were digitally assembled. Three layers of submucosal plexuses were displayed in 3D structure that were interconnected by hpChAT-ir fiber bundles and hpChAT-ir neurons were rarely co-labeled by nNOS. In the myenteric plexus, 30.1% of hpChAT-ir somas including Dogiel type I and II were co-labeled by nNOS and 3 classes of hpChAT-ir nerve fiber strands were visualized in 3D images and videos. The density and intensity values of hpChAT-ir fibers in 3D structure were significantly higher in the circular than in the longitudinal layer. CONCLUSIONS AND INFERENCES: The intrinsic cholinergic innervation in the human sigmoid colon was demonstrated layer by layer for the first time in 3D microstructures. This may open a new venue to assess the structure-function relationships and pathological alterations in colonic diseases.

Multicolor sparse viral labeling and 3D digital tracing of enteric plexus in mouse proximal colon using a novel adeno-associated virus capsid.

BACKGROUND: Intravenous administration of adeno-associated virus (AAV) can be used as a noninvasive approach to trace neuronal morphology and links. AAV-PHP.S is a variant of AAV9 that effectively transduces the peripheral nervous system. The objective was to label randomly and sparsely enteric plexus in the mouse colon using AAV-PHP.S with a tunable two-component multicolor vector system and digitally trace individual neurons and nerve fibers within microcircuits in three dimensions (3D). METHODS: A vector system including a tetracycline inducer with a tet-responsive element driving three separate fluorophores was packaged in the AAV-PHP.S capsid. The vectors were injected retro-orbitally in mice, and the colon was harvested 3 weeks after. Confocal microscopic images of enteric plexus were digitally segmented and traced in 3D using Neurolucida 360, neuTube, or Imaris software. KEY RESULTS: The transduction of multicolor AAV vectors induced random sparse spectral labeling of soma and neurites primarily in the myenteric plexus of the proximal colon, while neurons in the submucosal plexus were occasionally transduced. Digital tracing in 3D showed various types of wiring, including multiple conjunctions of one neuron with other neurons, neurites en route, and endings; clusters of neurons in close apposition between each other; axon-axon parallel conjunctions; and intraganglionic nerve endings consisting of multiple nerve endings and passing fibers. Most of digitally traced neuronal somas were of small or medium in size. CONCLUSIONS & INFERENCES: The multicolor AAV-PHP.S-packaged vectors enabled random sparse spectral labeling and revealed complexities of enteric microcircuit in the mouse proximal colon. The techniques can facilitate digital modeling of enteric micro-circuitry.

Peripheral peptide YY inhibits propulsive colonic motor function through Y2 receptor in conscious mice.

Peptide YY (PYY) antisecretory effect on intestinal epithelia is well established, whereas less is known about its actions to influence colonic motility in conscious animals. We characterized changes in basal function and stimulated colonic motor function induced by PYY-related peptides in conscious mice. PYY(3-36), PYY, and neuropeptide Y (NPY) (8 nmol/kg) injected intraperitoneally inhibited fecal pellet output (FPO) per hour during novel environment stress by 90%, 63%, and 57%, respectively, whereas the Y(1)-preferring agonists, [Pro(34)]PYY and [Leu(31),Pro(34)]NPY, had no effect. Corticotrophin-releasing factor 2 receptor antagonist did not alter PYY(3-36) inhibitory action. PYY and PYY(3-36) significantly reduced restraint-stimulated defecation, and PYY(3-36) inhibited high-amplitude distal colonic contractions in restrained conscious mice for 1 h, by intraluminal pressure with the use of a microtransducer. PYY suppression of intraperitoneal 5-hydroxytryptophan induced FPO and diarrhea was blocked by the Y(2) antagonist, BIIE0246, injected intraperitoneally and mimicked by PYY(3-36), but not [Leu(31),Pro(34)]NPY. PYY(3-36) also inhibited bethanechol-stimulated FPO and diarrhea. PYY(3-36) inhibited basal FPO during nocturnal feeding period and light phase in fasted/refed mice for 2-3 h, whereas the reduction of food intake lasted for only 1 h. PYY(3-36) delayed gastric emptying after fasting-refeeding by 48% and distal colonic transit time by 104%, whereas [Leu(31),Pro(34)]NPY had no effect. In the proximal and distal colon, higher Y(2) mRNA expression was detected in the mucosa than in muscle layers, and Y(2) immunoreactivity was located in nerve terminals around myenteric neurons. These data established that PYY/PYY(3-36) potently inhibits basal and stress/serotonin/cholinergic-stimulated propulsive colonic motor function in conscious mice, likely via Y(2) receptors.

A Novel Antiserum Against a Predicted Human Peripheral Choline Acetyltransferase (hpChAT) for Labeling Neuronal Structures in Human Colon.

Choline acetyltransferase (ChAT), the enzyme synthesizing acetylcholine (ACh), has an exon-skipping splice variant which is expressed preferentially in the peripheral nervous system (PNS) and thus termed peripheral ChAT (pChAT). A rabbit antiserum previously produced against rat pChAT (rpChAT) has been used for immunohistochemistry (IHC) to study peripheral cholinergic structures in various animals. The present study was undertaken to develop a specific antiserum against a predicted human pChAT (hpChAT) protein. A novel mouse antiserum has been successfully raised against a unique 14-amino acid sequence of hpChAT protein. Our Western blot using this antiserum (termed here anti-hpChAT serum) on human colon extracts revealed only a single band of 47 kDa, matching the deduced size of hpChAT protein. By IHC, the antiserum gave intense staining in many neuronal cells and fibers of human colon but not brain, and such a pattern of staining seemed identical with that reported in colon of various animals using anti-rpChAT serum. In the antibody-absorption test, hpChAT-immunoreactive staining in human colon was completely blocked by using the antiserum pre-absorbed with the antigen peptide. Double immunofluorescence in human colon moreover indicated that structures stained with anti-hpChAT were also stained with anti-rpChAT, and vice versa. hpChAT antiserum allowed the identification of cell types, as Dogiel type cells in intramural plexuses, and fiber innervation of colon muscles and mucosae. The present results demonstrate the specificity and reliability of the hpChAT antiserum as a novel tool for immunohistochemical studies in human colon, opening venues to map cholinergic innervation in other human PNS tissues.

Brain and Gut CRF Signaling: Biological Actions and Role in the Gastrointestinal Tract.

BACKGROUND: Corticotropin-releasing factor (CRF) pathways coordinate behavioral, endocrine, autonomic and visceral responses to stress. Convergent anatomical, molecular, pharmacological and functional experimental evidence supports a key role of brain CRF receptor (CRF-R) signaling in stress-related alterations of gastrointestinal functions. These include the inhibition of gastric acid secretion and gastric-small intestinal transit, stimulation of colonic enteric nervous system and secretorymotor function, increase intestinal permeability, and visceral hypersensitivity. Brain sites of CRF actions to alter gut motility encompass the paraventricular nucleus of the hypothalamus, locus coeruleus complex and the dorsal motor nucleus while those modulating visceral pain are localized in the hippocampus and central amygdala. Brain CRF actions are mediated through the autonomic nervous system (decreased gastric vagal and increased sacral parasympathetic and sympathetic activities). The activation of brain CRF-R2 subtype inhibits gastric motor function while CRF-R1 stimulates colonic secretomotor function and induces visceral hypersensitivity. CRF signaling is also located within the gut where CRF-R1 activates colonic myenteric neurons, mucosal cells secreting serotonin, mucus, prostaglandin E2, induces mast cell degranulation, enhances mucosal permeability and propulsive motor functions and induces visceral hyperalgesia in animals and humans. CRF-R1 antagonists prevent CRF- and stressrelated gut alterations in rodents while not influencing basal state. DISCUSSION: These preclinical studies contrast with the limited clinical positive outcome of CRF-R1 antagonists to alleviate stress-sensitive functional bowel diseases such as irritable bowel syndrome. CONCLUSION: The translational potential of CRF-R1 antagonists in gut diseases will require additional studies directed to novel anti-CRF therapies and the neurobiology of brain-gut interactions under chronic stress.

Identification and characterization of multiple corticotropin-releasing factor type 2 receptor isoforms in the rat esophagus.

The rat esophagus shares some cellular features with skin squamous epithelium and striated muscle that express high levels of corticotropin-releasing factor type 2 (CRF2) receptors or their cognate ligand urocortin (Ucn) 1, 2, and 3. We investigated the expression and cell signaling of CRF2 receptors and ligands in the rat esophagus and lower esophageal sphincter (LES) by RT-PCR and quantitative PCR in normal and corticosterone-treated whole esophageal tissue, laser capture microdissected layers, and isolated esophageal cells. The expression of CRF2 receptor protein and intracellular cAMP and ERK1/2 responses to CRF agonists and CRF2 antagonist were determined in cultured esophageal cells and HEK-293 cells transfected with CRF2b receptors. CRF2 was abundantly expressed in the mucosa and longitudinal muscle layers of the esophagus and LES, whereas CRF1 expression was scarce. CRF2b wild-type transcript was predominantly expressed in the esophagus, and in addition, several new CRF2 splice variants including six CRF2a isoforms were identified. Expression of Ucn 1, Ucn 2, and to a smaller extent Ucn 3, but not CRF mRNA, was detected in the esophagus and LES. Ucn 1 and Ucn 2 stimulated dose-dependent cAMP production and ERK1/2 phosphorylation in the esophageal cells, whereas CRF and CRF1 agonist, cortagine, had less potent effects. In addition, Ucn 2-stimulated cAMP and ERK responses were blocked by the CRF2 antagonist, astressin2-B. These data established the presence of a prominent CRF2 signaling system in the esophagus and LES-encompassing multiple CRF2 receptor variants and Ucn, suggesting a functional role in secretomotor activity and epithelial and muscle cell proliferation.

Water avoidance stress activates colonic myenteric neurons in female rats.

Stress stimulates colonic motor function and plays a role in functional bowel disorders, prevalently in women. We examined, in conscious female rats, the influence of water avoidance stress for 60 min on colonic myenteric neuron activity using immunohistochemical detection of Fos as a marker of neuronal activity. In control rats, Fos immunoreactive nuclei were rare in proximal and distal colon and no defecation was observed. Water avoidance stimulated fecal pellet output, which was associated with Fos expression in myenteric ganglia of proximal and distal colon including in a population of peripheral choline acetyltransferase-immunoreactive neurons. Atropine blocked fecal pellet output but not Fos expression in myenteric ganglia. These results indicate that psychological stress stimulates the activity of colonic cholinergic myenteric neurons.