Mucosa of the guinea pig gastric corpus is innervated by myenteric neurones with specific neurochemical coding and projection preferences.

The present study identified and characterised myenteric neurones involved in the innervation of the gastric mucosa. We applied retrograde neuronal tracing methods by using the dye DiI (1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorat) in combination with the immunohistochemical demonstration of choline acetyltransferase (ChAT), enkephalin (ENK), neuropeptide Y (NPY), nitric oxide synthase (NOS), substance P (SP), and vasoactive intestinal peptide (VIP). This method showed distinct neurochemical coding of DiI-labelled neurones with projections to the mucosa (mucosa neurones): ChAT/- (indicating the presence of ChAT only, 32%), ChAT/NPY/ +/- VIP (22%), NOS/NPY/ +/- VIP (19%), ChAT/SP/ +/- ENK (12%), NOS/- (indicating the presence of NOS only, 8%), or ChAT/ENK (4.6%). DiI-labelled mucosa neurones did not contain calretinin, serotonin, or somatostatin. All ChAT population had primarily ascending projections, whereas the NOS populations had mainly descending projections. Both were further classified as longitudinally and circumferentially projecting neurones, the latter having projection preferences towards the lesser or greater curvature. All subpopulations exhibited projection preferences. Nitrergic projections primarily arose from cell bodies located at the lesser curvature. ChAT/- projections, which dominated the cholinergic pathway, mainly arose from cell bodies located at the greater curvature. The other major cholinergic pathway with the code ChAT/NPY/ +/- VIP consisted of neurones located mainly at the lesser curvature. The results suggest specific coding of gastric myenteric neurones with projections to the mucosa. Polarised projections consisted of ascending cholinergic and descending nitrergic neurones; the additional presence of NPY/VIP was a prominent feature in both pathways. Chemical coding, polarity, and projection preferences of enteric pathways to the gastric mucosa are remarkably different from those of other regions in the gut.     

Neurochemical coding of myenteric neurones in the human gastric fundus

The major functions of the stomach are under the control of the enteric nervous system (ENS), but the neuronal circuits involved in this control are largely unknown in humans. Enteric neurones can be characterized by their neuromediator or marker content, i.e. by neurochemical coding. The purpose of this study was to characterize the presence and co-localization of neurotransmitters in myenteric neurones of the human gastric fundus. Choline acetyltransferase (ChAT), neurone-specific enolase (NSE), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), substance P (SP) were detected by immunohistochemical methods in whole mounts of gastric fundus myenteric plexus (seven patients). Antibodies against ChAT and NOS labelled the majority of myenteric neurones identified by NSE (57.2 +/- 5.6% and 40.8 +/- 4.5%, respectively; mean +/- SD). The proportions of VIP- and SP-immunoreactive neurones were significantly smaller, constituting 19.6 +/- 6.9% and 16.0 +/- 3.7%, respectively. Co-localization studies revealed five major populations representing over 75% of the myenteric neurones: ChAT/-, 30.1 +/- 6.1%; NOS/-, 24.2 +/- 4.4%; ChAT/SP/-, 8.3 +/- 3.1%; NOS/VIP/-, 7.2 +/- 6.0%; ChAT/VIP/-, 4.9 +/- 2.6. Some similarities are apparent in the neurochemical coding of myenteric neurones in the stomach and intestine of humans, and between the stomach of humans and animals, but striking differences exist. The precise functional role of the neurochemically identified classes of neurones remains to be determined.     

Presence of putative neurotransmitters in the myenteric plexus of the gastrointestinal tract and in the musculature of the urinary bladder of the ferret

The innervation of the musculature in the ferret stomach, ileum, colon and urinary bladder was investigated using immunohistochemistry in noncolchicin-treated tissues. In the gastrointestinal tract two main subpopulations of myenteric neurones were found: cholinergic neurones expressing choline acetyltransferase (ChAT), which made up 68, 67 and 67% of the neurones in the stomach, ileum and colon, respectively, and nitrergic neurones containing nitric oxide synthase and NADPH-diaphorase (stomach: 23%, ileum: 21%, colon: 26%). In the stomach, cholinergic neurones expressed substance P (SP, 2% of all neurones), dopamine-β-hydroxylase (DBH, 19%) but not tyrosine hydroxylase (TH) or vasoactive intestinal polypeptide (VIP), while nitrergic neurones contained VIP and neuropeptide Y (NPY). TH- but not DBH-immunoreactivity was observed in 4% of gastric neurones. Intense immunoreactivity in the musculature suggests that part of ChAT/SP- and NOS/NPY/VIP-positive neurones function as motorneurones. In the ileum, a high number (32%) of DBH-positive neurones was demonstrated. About half of the SP-positive neurones in the ileum also contained calcitonin gene-related peptide (CGRP). In the urinary bladder, only few intramural ganglia were observed. The smooth muscle was densely innervated by ChAT, NPY and DBH immunoreactive fibres. The data showed that the innervation of the ferret viscera exhibited similarities but also differences as compared with other mammalian species. Some of the chemical coding of myenteric neurones is remarkably similar to that observed in other mammals.     

Characterization of circular muscle motor neurons of the duodenum and distal colon in the Australian brush-tailed possum.

The motor innervation of the duodenum and distal colon remains uncharacterized within the same species. Our aim was to compare the projections and neurochemical properties of duodenal and distal colon circular muscle motor neurons. Circular muscle motor neurons were retrogradely traced by using a neural tracer in vitro, processed for choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity and then visualized by using indirect immunofluorescence. A mean of 372 +/- 64 and 156 +/- 23 neurons (mean +/- SEM) were tracer-labeled within the duodenum and colon, respectively. The ChAT+/NOS- neurons comprised 57.6 +/- 6.6% and 39.6 +/- 4.4% of all labeled cells in the duodenum and colon, respectively, and projected mainly in the oral direction. Of all labeled cells, the ChAT-/NOS+ neurons comprised 8.5 +/- 2.3% in the duodenum and 46.6 +/- 5.0% in the distal colon and projected mainly in the anal direction. Of the remainder, 20.6 +/- 5.0% and 8.2 +/- 2.4% were ChAT+/NOS+ and 13.2 +/- 0.9% and 5.6 +/- 1.4% were ChAT-/NOS- in the duodenum and distal colon, respectively. Within both regions, the distribution of the ChAT+/NOS- and ChAT-/NOS+ neurons are consistent with the ascending excitatory and descending inhibitory reflexes. The proportion of ChAT-/NOS+ neurons is greater within the colon in comparison with the duodenum. A considerable proportion of duodenal motor neurons were ChAT+/NOS+ and ChAT-/NOS-. These two classes may underlie nonperistaltic motor patterns, which predominate within the duodenum. These findings demonstrate regional differences in the innervation of intestinal circular muscle.     

Quantification of subclasses of human colonic myenteric neurons by immunoreactivity to Hu, choline acetyltransferase and nitric oxide synthase

An accurate method to count human enteric neurons is essential to develop a comprehensive account of the classes of nerve cells responsible for gut function and dysfunction. The majority of cells in the enteric nervous system utilize acetyl choline, or nitric oxide, or a combination of these, as neurotransmitters. Antisera raised against the RNA-binding protein Hu, were used to identify nerve cell bodies in whole mounts of the myenteric plexus of human colon, and then were utilized to analyse cells immunoreactive for combinations of choline acetyltransferase and nitric oxide synthase. Antisera to Hu provided a reliable means to count apparently all enteric nerve cell bodies, revealing 10% more cell bodies than labelling with neuron specific enolase, and no labelling of glial cells as revealed by S100. ChAT+/NOS- neurons accounted for 48% (+/-3%) of myenteric neurons and ChAT-/NOS+ neurons accounted for 43% (+/-2.5%). ChAT+/NOS+ neurons comprised 4% (+/-0.5) of the total number of neurons, and a novel class of small ChAT-/NOS- neurons, making up 5% (+/-0.9%) of all cells, was described for the first time.     

Neurotransmitter coding of enteric neurones in the submucous plexus is changed in non-inflamed rectum of patients with Crohn's disease

Knowledge of the neurochemical coding of submucosal neurones in the human gut is important to assess neuronal changes under pathological conditions. We therefore investigated transmitter colocalization patterns in rectal submucosal neurones in normal tissue (n=11) and in noninflamed tissue of Crohn's disease (CD) patients (n=17). Neurone-specific enolase (NSE), choline acetyltransferase (ChAT), vasoactive intestinal polypeptide (VIP), substance P (SP), nitric oxide synthase (NOS) and calcitonin gene-related peptide (CGRP) were detected immunohistochemically in whole-mount preparations from rectal biopsies. The neuronal marker NSE revealed no differences in the number of cells per ganglion (controls 5.0; CD 5.1). Four cell populations with distinct neurochemical codes were identified. The sizes of the populations ChAT/VIP (58% vs. 55%), ChAT/SP (8% vs. 8%), and ChAT/- (22% vs. 22%) were similar in control and CD. The population VIP/- was significantly increased in CD (12% vs. 2% in controls). Unlike in controls, all NOS neurones colocalized ChAT in CD. Thickened CGRP-fibres occurred in CD. We identified neurochemically distinct populations in the human submucous plexus. The increase in the VIP/- population, extensive colocalization of ChAT and NOS and hypertrophied CGRP fibres indicated adaptive changes in the enteric nervous system in noninflamed rectum of CD patients.     

Intestinal dysmotility and enteric neurochemical changes in a Parkinson's disease rat model

Gastrointestinal disorders, constipation in particular, are the most common non-motor dysfunctions affecting Parkinson's disease (PD) patients. We have previously reported that rats bearing unilateral nigrostriatal lesion caused by 6-hydroxydopamine (6-OHDA) stereotaxic injection develop severe constipation together with a region-specific decrease of neuronal nitric oxide synthase (nNOS) in enteric neurons of the lower intestinal tract. Here, we extend these observations on other enteric neuronal subpopulations, investigating also the propulsive activity of isolated colonic specimens. Four weeks post 6-OHDA injection, lesioned rats showed a significant increase of vasoactive intestinal polypeptide (VIP) concomitant with the reduced expression of nNOS in the myenteric plexus of distal ileum and proximal colon; in particular VIP increased in a subpopulation of neurons actively expressing nNOS. On the other hand, choline acetyltransferase (ChAT) was not modified in any of the intestinal segments analyzed. Interestingly, we found a reduced expression of dopamine receptor type 2 (D2R) in proximal (-66.8%) and distal (-54.5%) colon, together with reduced peristalsis efficiency (decrease in intraluminal pressure and frequency of peristaltic events) in the 6-OHDA-lesioned rats. The selective depletion of dopaminergic nigrostriatal neurons is associated with changes in the expression of enteric inhibitory neurotransmitters, as well as of the D2R in intestinal specific regions. Moreover, 6-OHDA-lesioned rats demonstrated altered colon propulsive activity referable to the D2R decrease. Our findings unveil subtle mechanisms underlying the enteric neurochemical plasticity events evoked by disruption of the normal brain-gut cross-talk, giving a peculiar point of view on the pathophysiology of the severe constipation that frequently affects PD patients.     

Effects of the probiotic yeast Saccharomyces boulardii on the neurochemistry of myenteric neurones in pig jejunum

We studied the effects of food supplementation with Saccharomyces boulardii (S. boulardii; synonym S. cerevisiae HANSEN CBS 5926; 1 g per day for 9 days) on the presence and co-localization patterns of neuronal markers in myenteric neurones of the pig jejunum. The pan neuronal marker Hu revealed no change in the number of neuronal cell bodies per ganglion (37 +/- 7 in control vs 34 +/- 9 in the S. boulardii group). Ranked by size the following cell populations were identified: choline acetyltransferase (ChAT), calbindin-28k (CALB), substance P (SP), neurofilament 160 kD (NF-160), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP), calretinin (CALRET). We found a significant decrease in the number of CALB myenteric neurones in animals which received S. boulardii supplemented diet. None of the other neuronal markers revealed any difference between controls and S. boulardii treated animals. The study reports transmitter-localization patterns in the myenteric plexus of the pig jejunum and provides evidence that changes in the neurochemistry of enteric neurones occur with S. boulardii supplemented diet. Although only CALB expression was altered and the functional significance of this finding remains unknown, our study identified a possible new effector level of probiotics in the gut.     

Anatomical registration and three‐dimensional visualization of low and high‐resolution pan‐colonic manometry recordings

Background Colonic propagating sequences (PS) are important for the movement of colonic content and defecation, and aberrant PS patterning has been associated with slow transit constipation. However, because these motor patterns are typically recorded over long periods (24 h +), the visualization of PS spatiotemporal patterning is difficult. Here, we develop a novel method for displaying pan‐colonic motility patterns. Methods A 3D mesh representing the geometry of the human colon was created as follows: (i) Human colon images from the Visible Human Dataset were digitized to create a 3D data cloud, and (ii) A surface mesh was fitted to the cloud using a least‐squares minimization technique. Colonic manometry catheters were placed in the ascending colon of healthy controls and patients with slow transit constipation (STC), with the aid of a colonoscope. The colonic manometry data were interpolated and mapped to the model according to the following anatomical landmarks: cecum, hepatic flexure, splenic flexure, sigmoid‐descending junction, and anus. Key Results These 3D images clearly and intuitively communicate characteristics of normal and abnormal colonic motility. Specifically we have shown the reduced amplitude of the antegrade propagating pressure waves (PPW) throughout the colon and reduced frequency of PPWs at the mid‐colon in patients with STC. Conclusions and Inferences A novel method for the 3D visualization of PS is presented, providing an intuitive method for representing a large volume of physiological data. These techniques can be used to display frequency, amplitude or velocity data, and will help to convey regions of abnormally in patient populations.     

Enteric neuropathy evoked by repeated cisplatin in the rat

Background Acute administration of the antitumoral drug cisplatin can induce nausea/emesis and diarrhea. The long‐term effects of cisplatin on gastrointestinal motility, particularly after repeated administration, are not well known. Because cisplatin is highly neurotoxic, myenteric neurons can be affected. Our aim was to study the prolonged effects of repeated cisplatin administration in a rat model, focusing on gastrointestinal motor function and myenteric neurons. Methods Rats received saline or cisplatin (1 or 3 mg kg^?1, i.p.) once weekly for 5 weeks. One week after treatment, both upper gastrointestinal transit and colonic activity were evaluated, and tissue samples from ileum, colon and rectum were processed for histological analysis. Intestinal transit was measured invasively (charcoal method). Colonic activity was determined electromyographically. The gut wall structure was evaluated in sections using conventional histology and immunohistochemistry. Whole‐mount preparations from the distal colon were labeled for different markers, including nitric oxide synthase (NOS) and calcitonin‐gene related peptide (CGRP) to determine relative proportions of myenteric neurons vs the total neuronal population labeled with HuC/D. Key Results One week after repeated cisplatin exposure, the upper gastrointestinal transit rate and colonic activity were dose‐dependently reduced. The number of NSE‐ or HuC/D‐immunoreactive myenteric neurons per ganglion was decreased; the proportion of CGRP‐immunoreactive neurons was decreased, whereas that of NOS‐immunoreactive cells was increased. Conclusions & Inferences Chronic cisplatin may induce an enteric neuropathy characterized by changes in myenteric neurons associated with marked gastrointestinal motor dysfunction.     

Neuropeptides in idiopathic chronic constipation (slow transit constipation)

Tissue specimens from the large bowel of 18 patients with long-standing slow transit constipation were investigated to determine the distribution and density of several neuropeptides and amines in the enteric nerve system, and also of endocrine cells in comparison to normal individuals. CGRP (calcitonin gene-related peptide), galanin, glucagon, GRP (gastrin-releasing peptide), metenkephalin, motilin, neuropeptide Y (NPY), PACAP, peptide YY (PYY), serotonin, somatostatin, substance P and VIP were studied by immunohistochemistry. Tissue concentrations of VIP, substance P and galanin were also measured by radioimmunoassay. Significantly increased VIP, SP and galanin contents were found in specimens from the ascending colon. Levels of VIP and galanin were also increased in the transverse colon. Immunohistochemistry revealed only marginal changes with an increased density of PACAP nerve fibres in the smooth muscle and of VIP and PACAP nerves in the myenteric plexus of the transverse colon. In the descending colon substance P and NPY immunoreactivity were also increased in the myenteric plexus while the density of VIP nerve fibres was reduced in the mucosa/submucosa. The frequency of PYY-containing cells and the 5-HT-containing cells in the ascending colon was significantly increased in the constipated patients.     

Post-ischemic RSR13 amplifies the effect of dizocilpine on outcome from transient focal cerebral ischemia in the rat

Nitric oxide (NO) is a major non-adrenergic, non-cholinergic (NANC) inhibitory neurotransmitter in the gastrointestinal tract. NO released from the myenteric plexus enhances colonic transit and facilitates propulsion of the colonic contents by mediating descending relaxation. Although it has been suggested that colonic transit delays with aging, the mechanism of delayed colonic transit in aging remains unclear. We hypothesized that advanced age is associated with decreased expression of neuronal NO synthase (nNOS) and concomitant reduction in synthesis of NO in the rat colon. We studied nNOS mRNA expression, nNOS-immunohistochemistry, nNOS-immunoblotting and NOS catalytic activity in the mid-colon obtained from young (age 4–8 months) and aged (age 22–28 months) Fisher (F344×BN)F1 rats. Western blot analysis of PGP 9.5, a generic neuronal marker, of the colonic tissues were employed to study whether the total number of neurons of the myenteric plexus is reduced with aging. The number of nNOS-immunoreactive cells and nNOS synthesis in the colonic myenteric plexus were significantly reduced in aged rats. In contrast, expression of PGP 9.5 in colonic tissues was not affected in aged rats. Northern blot analysis demonstrated that the expression of neuronal nNOS mRNA was significantly reduced in the colonic tissues in aged rats. Basal and veratridine-induced release of -[³H]citrulline were significantly decreased in colonic tissues from aged rats, compared to young rats. It is suggested that advanced age is associated with diminished gene expression of nNOS, nNOS synthesis and catalytic activity of NOS. This may explain the mechanism of delayed colonic transit observed in advanced age.     

Regional differences in the sympathetic innervation of the guinea pig large intestine by neuropeptide Y- and tyrosine hydroxylase-immunoreactive nerves of divergent extrinsic origin.

Region-specific patterns of nerves with immunoreactivity to neuropeptide Y (NPY) have been described previously in the submucous plexus of guinea pig large intestine. Because these may have functional significance, the possibility of similar, characteristic variations of NPY-like immunoreactivity (NPY-ir) in the myenteric plexus was explored. Regional differences were found in the occurrence and pattern of distribution of NPY-ir in the myenteric plexus of the guinea pig large intestine. NPY-ir was present rarely within neuron somata in any region of the large intestine, and NPY-ir nerve fibers were present only within the distal large intestine, increasing progressively in density from the distal spiral to the rectum. Lesion of the colonic nerves, but not the hypogastric, intermesenteric, or lumbar splanchnic nerves, resulted in a loss of NPY-ir in the distal spiral and transverse colon but not in the descending colon or rectum. Ring myotomies in the descending colon resulted in a loss of NPY-ir proximal to the lesion. Dual-labeling immunohistochemical studies revealed that the NPY-ir nerve fibers rarely contained immunoreactivity for tyrosine hydroxylase (TH). Extrinsic nerve lesions resulted in an unequivocal reduction in NPY-ir in intraganglionic fibers of the submucosal plexuses of the transverse colon and a partial loss in the distal spiral and descending colon: the rectum was unaffected; in only a minority of guinea pigs, however, was any decrease in the NPY-ir innervation of submucosal blood vessels detected. The principal projections of NPY-ir nerves were from and through the inferior mesenteric ganglion; however, NPY-ir was not colocalized with TH-ir. It is proposed that nonnoradrenergic, NPY-containing neurons located in the inferior mesenteric ganglion project through the colonic nerves and that these proximally directed fibers innervate the transverse colon and the distal spiral. Nonnoradrenergic, NPY-ir neurons lying in the pelvic ganglia or sacral sympathetic chain may make an important contribution to the innervation of the myenteric plexus of the rectum and the descending colon.     

Characterization of the intrinsic and extrinsic innervation of the gall bladder epithelium in the Australian Brush-tailed possum (Trichosurus vulpecula)

Intrinsic neurones of the gall bladder modulate its function. Nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) are present in gall bladder neurones and nitric oxide and VIP modulate its epithelial functions. As an extensive extrinsic innervation of the gall bladder is also present, the source of the epithelial innervation is unclear. In this study the source of the gall bladder epithelial innervation is defined. Immunoreactivity for VIP, NOS, substance P (SP), calcitonin gene related peptide (CGRP) and tyrosine hydroxylase (TH) in organotypic cultured and freshly fixed gall bladder were compared. Retrograde tracing in vitro from the epithelium was used to identify putative intrinsic secretomotor neurones, which were then characterized by immunohistochemistry. Abundant spinal afferent and sympathetic innervation of the gall bladder epithelium was demonstrated by CGRP/SP and TH immunohistochemistry, respectively. The intrinsic secretomotor innervation of the epithelium is derived exclusively from neurones of the subepithelial plexus. A majority of these neurones were immunoreactive for NOS. Some of the NOS-immunoreactive neurones of the subepithelial plexus also contained VIP and/or SP. Gall bladder subepithelial plexus neurones, containing NOS and/or VIP/SP, innervate the epithelium, as do extrinsic neurones.     

Mucosal projections of enteric neurons in the porcine small intestine

In the present study, a combination of immunohistochemistry and retrograde 1,1;-didodecyl-3,3,3;,3;-tetramethylindocarbocyanine perchlorate (DiI) tracing was used to unravel the morphology, distribution, and neurochemical coding of submucous and myenteric neurons with axonal projections to the mucosa of the porcine small intestine. The majority of traced neurons was located in the inner submucous plexus (ISP; 78%), whereas the remaining part was distributed between the outer submucous plexus (OSP; 10%) and myenteric plexus (MP; 12%). Among these traced neurons, some distinct neuronal populations could be distinguished according to their morphologic and neurochemical properties. In the ISP, several types of traced neurons were detected: 1) morphologic type II neurons expressing choline acetyltransferase (ChAT) immunoreactivity, calcitonin gene-related peptide (CGRP) immunoreactivity, and substance P (SP) immunoreactivity; 2) ChAT/SP-immunoreactive (-IR) small neurons; 3) vasoactive intestinal polypeptide (VIP) -IR small neurons; and 4) multidendritic ChAT/somatostatin (SOM) -IR neurons. The traced neuronal populations of the OSP and MP were similar to each other. In both plexuses, the following DiI-labelled neurons were found: 1) ChAT/CGRP/(SP)-IR type II neurons; 2) multidendritic ChAT/SP-IR neurons; and 3) multidendritic ChAT/SOM-IR neurons. Comparison of the present findings with previously obtained data concerning the mucosal innervation pattern of the intestine of small mammals, revealed significant species differences with respect to the morphologic and neurochemical features of the involved enteric neuronal classes. Although not identical, a closer resemblance between pig and human enteric nervous system seems to be at hand, as far as the anatomic organization and the presence of neurochemically identified neuronal subtypes within the enteric nervous system are concerned.     

Morphometric evaluation of oesophageal wall in patients with nutcracker oesophagus and ineffective oesophageal motility

Abstract  The pathogenesis of nutcracker oesophagus (NE) and ineffective oesophageal motility (IEM) is unclear. Damage to the enteric nervous system or smooth muscle can cause oesophageal dysmotility. We tested the hypothesis that NE and IEM are associated with abnormal muscular or neural constituents of the oesophageal wall. Oesophageal manometry was performed in patients prior to total gastrectomy for gastric cancer. The oesophageal manometries were categorized as normal ( n  = 7), NE ( n  = 13), or IEM ( n  = 5). Histologic examination of oesophageal tissue obtained during surgery was performed after haematoxylin and eosin (H&E) and trichrome staining. Oesophageal innervation was examined after immunostaining for protein gene product-9.5 (PGP-9.5), choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS). There were no significant differences in inner circular smooth muscle thickness or degree of fibrosis among the three groups. Severe muscle fibre loss was found in four of five patients with IEM. The density of PGP-9.5-reactive neural structures was not different among the three groups. The density of ChAT immunostaining in the myenteric plexus (MP) was significantly greater in patients with NE ( P  < 0.05) and the density of nNOS immunostaining in the circular muscle (CM) was significantly greater in IEM patients ( P  < 0.05). The ChAT/nNOS ratio in both MP and CM was significantly greater in NE patients. NE may result from an imbalance between the excitatory and inhibitory innervation of the oesophagus, because more than normal numbers of ChAT-positive myenteric neurones are seen in NE. Myopathy and/or increased number of nNOS neurones may contribute to the hypocontractile motor activity of IEM.     

Arginosuccinate synthetase, arginosuccinate lyase and NOS in canine gastrointestinal tract: immunocytochemical studies

Nitric oxide synthase (NOS) requires the substrate L ‐arginine for NO production to support multiple gastrointestinal functions. We asked, ‘Where do enzymes to regenerate L ‐arginine from L ‐citrulline exist?’. We examined loci of immunoreactivities in the canine gastrointestinal tract for arginosuccinate synthetase and arginosuccinate lyase, enzymes that resynthesize L ‐arginine from L ‐citrulline, in relation to the distribution of nNOS immunoreactivity or NADPH‐diaphorase histochemistry. Arginosuccinate synthetase and lyase were present in many neurones and nerve fibres in the myenteric plexus of the lower oesophageal sphincter (LOS), antrum, pylorus, ileum and colon; in the submucosal plexus of ileum and colon; in longitudinal muscle of ileum and colon; and in nerve bundles in circular muscle everywhere. LOS muscle was also immunoreactive for both enzymes. Circular and longitudinal muscle cells of the ileum and colon and cells resembling interstitial cells of Cajal in the deep muscular plexus of the ileum and the submuscular plexus of the colon also appeared immunoreactive. In neurones, arginosuccinate synthetase and nNOS were usually co‐localized. NADPH diaphorase activity was present in LOS and likely in pylorus, but not in muscularis externa of ileum or colon. We conclude that resynthesis of L ‐arginine probably occurs in enteric nerves, interstitial cells of Cajal (ICC) and LOS muscle; also apparently in some cells without NOS to utilize it.     

Effect of age on the enteric nervous system of the human colon

Abstract  The effect of age on the anatomy and function of the human colon is incompletely understood. The prevalence of disorders in adults such as constipation increase with age but it is unclear if this is due to confounding factors or age-related structural defects. The aim of this study was to determine number and subtypes of enteric neurons and neuronal volumes in the human colon of different ages. Normal colon (descending and sigmoid) from 16 patients (nine male) was studied; ages 33–99. Antibodies to HuC/D, choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and protein gene product 9.5 were used. Effect of age was determined by testing for linear trends using regression analysis. In the myenteric plexus, number of Hu-positive neurons declined with age (slope = −1.3 neurons/mm/10 years, P  = 0.03). The number of ChAT-positive neurons also declined with age (slope = −1.1 neurons/mm/10 years of age, P  = 0.02). The number of nNOS-positive neurons did not decline with age. As a result, the ratio of nNOS to Hu increased (slope = 0.03 per 10 years of age, P  = 0.01). In the submucosal plexus, the number of neurons did not decline with age (slope = −0.3 neurons/mm/10 years, P  = 0.09). Volume of nerve fibres in the circular muscle and volume of neuronal structures in the myenteric plexus did not change with age. In conclusion, the number of neurons in the human colon declines with age with sparing of nNOS-positive neurons. This change was not accompanied by changes in total volume of neuronal structures suggesting compensatory changes in the remaining neurons.     

Substance P and vasoactive intestinal peptide are reduced in right transverse colon in pediatric slow‐transit constipation

Background Slow‐transit constipation (STC) is recognized in children but the etiology is unknown. Abnormalities in substance P (SP), vasoactive intestinal peptide (VIP) and nitric oxide (NO) have been implicated. The density of nerve fibers in circular muscle containing these transmitters was examined in colon from children with STC and compared to other pediatric and adult samples. Methods Fluorescence immunohistochemistry using antibodies to NO synthase (NOS), VIP and SP was performed on colonic biopsies (transverse and sigmoid colon) from 33 adults with colorectal cancer, 11 children with normal colonic transit and anorectal retention (NAR) and 51 with chronic constipation and slow motility in the proximal colon (STC). The percentage area of nerve fibers in circular muscle containing each transmitter was quantified in confocal images. Key Results In colon circular muscle, the percentage area of nerve fibers containing NOS > VIP > SP (6 : 2 : 1). Pediatric groups had a higher density of nerve fibers than adults. In pediatric samples, there were no regional differences in NOS and VIP, while SP nerve fiber density was higher in sigmoid than proximal colon. STC children had lower SP and VIP nerve fiber density in the proximal colon than NAR children. Twenty‐three percent of STC children had low SP nerve fiber density. Conclusions & Inferences There are age‐related reductions in nerve fiber density in human colon circular muscle. NOS and VIP do not show regional variations, while SP nerve fiber density is higher in distal colon. 1/3 of pediatric STC patients have low SP or VIP nerve fiber density in proximal colon.     

Variability in the occurrence of nitric oxide synthase immunoreactivity in different populations of rat sympathetic preganglionic neurons

The proportion of sympathetic preganglionic neurons (SPN) showing nitric oxide synthase (NOS) immunoreactivity appears to vary with innervation target and blood pressure level. For normotensive Sprague‐Dawley rats (SD), we evaluated peroxidase immunolabelling for choline acetyltransferase (ChAT) plus NOS in spinal cord segments T1–L2 and assessed NOS immunofluorescence in SPN retrogradely labelled with cholera toxin B subunit from the adrenal medulla (AM) or superior cervical (SCG), coeliac (CG), or major pelvic (MPG) ganglia. We also compared the distributions and numbers of NOS‐positive and NOS‐negative/ChAT‐positive lateral horn neurons in SD with those in normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR). In SD, WKY, and SHR, rostrocaudal, dorsoventral, and mediolateral differences occurred in the distributions of NOS‐positive and NOS‐negative/ChAT‐positive neurons in the intermediolateral cell column (IML), whereas the two groups were similarly distributed throughout the central autonomic area (CAA). Among the four retrogradely labelled populations of SPN, the percentages showing NOS immunoreactivity differed (CG‐projecting, 54.8% ± 0.7%; SCG‐projecting, 75.3% ± 1.2%; MPG‐projecting, 89% ± 1.1% and AM‐projecting, 98.6% ± 0.2%). Within each retrogradely labelled group of SPN, the NOS‐positive proportion also varied with subnuclear location (e.g., 25.5% ± 4.0% of CG‐projecting SPN in the CAA vs. 82.7% ± 7.6% of CG‐projecting SPN in the dorsolateral funiculus). The numbers of NOS‐positive and NOS‐negative/ChAT‐positive neurons in T9–T11 were the same in SD and SHR but differed in WKY. Our results show that the expression of NOS within SPN varies depending on the target that they innervate and also on their subnuclear location. Our data indicate that there are no anatomical differences between nitric oxide‐synthesizing SPN in normotensive SD and hypertensive SHR. J. Comp. Neurol. 514:492–506, 2009. © 2009 Wiley‐Liss, Inc.