Quadruple colocalization of calretinin,calcitonin gene-related peptide,vasoactive intestinal peptide,and substance P in fibers within the villi of the rat intestine

Double-labeling immunofluoresenct histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin-containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine. The coexistence of calretinin in calcitonin-gene-related-peptide-containing cells that also contained substance P and vasoactive intestinal polypeptide in the dorsal root ganglia suggest that these ganglia are the source of the quadruple colocalization within the sensory fibers of the villi. Although the function of calretinin in these nerves is unknown, it is hypothesized that the coexistence of three potent vasodilatory peptides influences the uptake of metabolized food products within the vasculature of the villi.     

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

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

Proteomic analysis of colonic mucosa in a rat model of irritable bowel syndrome

Irritable bowel syndrome (IBS) is one of the most common functional disorders of the gastrointestinal tract. It is characterized by abdominal pain and changes in bowel habits. Various studies have investigated the pathophysiologic processes underlying IBS, but the mechanism remains poorly understood. In the present study, we established an IBS model and identified differentially expressed proteins in colon tissue of IBS rats compared with healthy controls by 2‐D gel electrophoresis, MALDI‐TOF‐MS, and Western blot analysis. Our results showed that 13 of the 1396 protein spots on 2‐D gel were differently expressed between the IBS and control groups. Ontological analysis of these proteins revealed primary roles in catalytic activity (protein disulfide‐isomerase A3, glyoxalase I, cathepsin S, α‐enolase), structural support (cytokeratin 8), antioxidant activity (peroxiredoxin‐6), protein binding (transgelin, serpin peptidase inhibitor B5), and signal transduction (40S ribosomal protein SA). Protein disulfide‐isomerase A3 and cytokeratin 8 overexpression in IBS were confirmed by Western blot. The findings indicate that multiple proteins are involved in IBS processes that influence intestinal tract immunity, inflammation, and nerve regulation. Our study provides useful candidate genes and proteins for further investigation.     

Localization and function of adenosine receptor subtypes at the longitudinal muscle--myenteric plexus of the rat ileum

Adenosine plays a dual role on acetylcholine (ACh) release from myenteric motoneurons via the activation of high-affinity inhibitory A₁ and facilitatory A_2A receptors. The therapeutic potential of adenosine-related compounds for controlling intestinal motility and inflammation, prompted us to investigate further the role of low-affinity adenosine receptors, A_2B and A₃, on electrically-evoked (5 Hz, 200 pulses) [³H]ACh release from myenteric neurons. Immunolocalization studies showed that A_2B receptors exhibit a pattern of distribution similar to the glial cell marker, GFAP. Regarding A₁ and A₃ receptors, they are mainly distributed to cell bodies of ganglionic myenteric neurons, whereas A_2A receptors are localized predominantly on cholinergic nerve terminals. Using selective antagonists (DPCPX, ZM241385 and MRS1191), data indicate that modulation of evoked [³H]ACh release is balanced through tonic activation of inhibitory (A₁) and facilitatory (A_2A and A₃) receptors by endogenous adenosine. The selective A_2B receptor antagonist, PSB603, alone was devoid of effect and failed to modify the inhibitory effect of NECA. The A₃ receptor agonist, 2-Cl-IB MECA (1–10 nM), concentration-dependently increased the release of [³H]ACh. The effect of 2-Cl-IB MECA was attenuated by MRS1191 and by ZM241385, which selectively block respectively A₃ and A_2A receptors. In contrast to 2-Cl-IB MECA, activation of A_2A receptors with CGS21680C attenuated nicotinic facilitation of ACh release induced by focal depolarization of myenteric nerve terminals in the presence of tetrodotoxin. Tandem localization of excitatory A₃ and A_2A receptors along myenteric neurons explains why stimulation of A₃ receptors (with 2-Cl-IB MECA) on nerve cell bodies acts cooperatively with prejunctional facilitatory A_2A receptors to up-regulate acetylcholine release. The results presented herein consolidate and expand the current understanding of adenosine receptor distribution and function in the myenteric plexus of the rat ileum, and should be taken into consideration for data interpretation regarding the pathophysiological implications of adenosine on intestinal motility disorders.     

Structural organization and neuropeptide distributions in the equine enteric nervous system: an immunohistochemical study using whole-mount preparations from the small intestine

The architecture and neurochemistry of the enteric nervous system was studied by use of whole-mount preparations obtained by microdissection of the horse jejunum. A myenteric plexus and two plexuses within the submucosa were identified. The external submucosal plexus lying in the outermost region of the submucosa had both neural and vascular connections with the inner submucosal plexus situated closer to the mucosa. Counts of neurones stained for NADH-diaphorase demonstrated the wide variation in size, shape and neurone content of individual ganglia in both the external and internal submucosal plexuses. The average number of cells/ganglion was similar in each plexus (about 25 cells). Immunoreactivities for galanin, vasoactive intestinal peptide and neuropeptide Y were observed in nerve cell bodies and fibres of each of the plexuses. Immunoreactivity for substance P was extensive and strong in nerve fibres of all plexuses but was weaker in cell bodies of the submucosal neurones and absent in the cell bodies of the myenteric plexus. Comparative quantitative analysis of immunoreactive cell populations with total cell numbers (enzyme staining) was indicative of neuropeptide colocalization in the external submucosal plexus.     

Structure of the tertiary component of the myenteric plexus in the guinea-pig small intestine

The tertiary component of the myenteric plexus consists of interlacing fine nerve fibre bundles that run between its principal ganglia and connecting nerve strands. It was revealed by zinc iodide-osmium impregnation and substance P immunohistochemistry at the light-microscope level. The plexus was situated against the inner face of the longitudinal muscle and was present along the length of the small intestine at a density that did not vary markedly from proximal to distal. Nerve bundles did not appear to be present in the longitudinal muscle as judged by light microscopy, although numberous fibre bundles were encountered within the circular muscle layer. At the ultrastructural level, nerve fibre bundles of the tertiary plexus were found in grooves formed by the innermost layer of longitudinal smooth muscle cells. In the distal parts of the small intestine, some of these nerve fibre bundles occasionally penetrated the longitudinal muscle coat. Vesiculated profiles in nerve fibre bundles of the tertiary plexus contained variable proportions of small clear and large granular vesicles; they often approached to within 50–200 nm of the longitudinal smooth muscle cells. Fibroblast-like cells lay between strands of the tertiary plexus and the circular muscle but were never intercalated between nerve fibre varicosities and the longitudinal muscle. These anatomical relationships are consistent with the tertiary plexus being the major site of neurotransmission to the longitudinal muscle of the guinea-pig small intestine.     

Shapes of nerve cells in the myenteric plexus of the guinea-pig small intestine revealed by the intracellular injection of dye

Summary The shapes of myenteric neurons in the guineapig small intestine were determined after injecting living neurons with the dye Lucifer yellow via a microelectrode. The cells were fixed and the distribution of Lucifer yellow rendered permanent by an immunohistochemical method. Each of 204 nerve cells was examined in whole-mount preparations of the myenteric plexus and drawn using a camera lucida at 1250 x magnification. Four cell shapes were distinguished: (1) neurons with several long processes corresponding to type II of Dogiel; (2) neurons with a single long process and lamellar dendrites corresponding to type I of Dogiel; (3) neurons with numerous filamentous dendrites; and (4) small neurons with few processes. About 15% of the neurons could not be placed into these classes or into any single class. The type II neurons (39% of the sample) had generally smooth somata and up to 7 (average 3.3) long processes, most of which ran circumferentially. Dogiel type I neurons (34% of sampled neurons) had characteristic lamellar dendrites, i.e., broad dendrites that were flattened in the plane of the plexus. The filamentous neurons (7% of the sample), had, on average, 14 fine processes up to about 50 m in length. Small neurons with smooth outlines and a few fine processes made up 5% of the neurons encountered. We conclude that myenteric neurons that have been injected with dye can be separated into morphologically distinct classes and that the different morphological classes probably correspond to different functional groupings of neurons.     

Basic fibroblast growth factor, neurofilament, and glial fibrillary acidic protein immunoreactivities in the myenteric plexus of the rat esophagus and colon

The enteric nervous system consists of a number of interconnected networks of neuronal cell bodies and fibers as well as satellite cells, the enteric glia. Basic fibroblast growth factor (bFGF) is a mitogen for a variety of mesodermal and neuroectodermal-derived cells and its presence has been described in many tissues. The present work employs immunohistochemistry to analyze neurons and glial cells in the esophageal and colic enteric plexus of the Wistar rat for neurofilament (NF) and glial fibrillary acidic proteins (GFAP) immunoreactivity as well as bFGF immunoreactivity in these cells. Rats were processed for immunohistochemistry; the distal esophagus and colon were opened and their myenteric plexuses were processed as whole-mount preparations. The membranes were immunostained for visualization of NF, GFAP, and bFGF. NF immunoreactivity was seen in neuronal cell bodies of esophageal and colic enteric ganglia. GFAP-immunoreactive enteric glial cells and processes were present in the esophageal and colic enteric plexuses surrounding neuronal cell bodies and axons. A dense net of GFAP-immunoreactive processes was seen in the ganglia and connecting strands of the myenteric plexus. bFGF immunoreactivity was observed in the cytoplasm of the majority of the neurons in the enteric ganglia of esophagus and colon. The two-color immunoperoxidase and immunofluorescence methods revealed bFGF immunoreactivity also in the nucleus of GFAP-positive enteric glial cells. The results suggest that immunohistochemical localization of NF and GFAP may be an important tool in the study of the plasticity in the enteric nervous system. The presence of bFGF in neurons and glia of the myenteric plexus of the esophagus and the colon indicates that this neurotrophic factor may exert autocrine and paracrine actions in the enteric nervous system.     

Characterisation of calcitonin gene-related peptide-immunoreactive neurons in the myenteric plexus of rat colon

A mechanical or chemical stimulus applied to the intestinal mucosa induces motility reflexes in the rat colon. Enteric neurons containing calcitonin gene-related peptide (CGRP) have been suggested as intrinsic primary afferent neurons responsible for mediating such reflexes. In the present study, immunohistochemistry was performed on whole-mount stretch preparations to investigate chemical profiles, morphological characteristics and projections of CGRP-containing neurons in the myenteric plexus of the rat colon. CGRP-positive neuronal cell bodies were detected in preparations incubated with colchicine-containing medium, whereas CGRP-positive nerve fibres were found in colchicine-untreated preparations. These neurons had large oval or round cell bodies that were also immunoreactive for the calcium-binding protein calretinin and neurofilament 200. Myenteric neurons positive for both calretinin and neurofilament 200 had several long processes that emerged from the cell body, consistent with Dogiel type II morphology. Application of the neural tracer DiI to the intestinal mucosa revealed that DiI-labelled myenteric neurons each had an oval or round cell body immunoreactive for calretinin. Thus, CGRP-containing myenteric neurons are Dogiel type II neurons and are immunoreactive for calretinin and neurofilament 200 in the rat colon. These neurons probably project to the intestinal mucosa. This study was supported by a Waseda University Grant for Special Research Projects (2008A-889).     

The ultrastructure and distribution of 5-HT-containing neurons in the intestine of a teleost fish,Aldrichetta forsteri

Summary The intestine of Aldrichetta forsteri was examined ultrastructurally for evidence of 5-HT-containing intrinsic neurons. A population of nerve cell bodies and processes characterized by the presence of amine-containing vesicles was found after the destruction of adrenergic processes with 6-hydroxydopamine. The distribution of the neurons matched that of the 5-HT-containing neurons previously seen with formaldehyde-induced fluorescence and 5-HT immunohistochemistry in A. forsteri and other teleosts. The axons made close contacts with the circular smooth muscle layer and the muscularis mucosae. The axons also lay exposed to the epithelial cells of the mucosa but did not make contact with neurons or with the longitudinal smooth muscle layer.