Immunohistochemical studies of the enteric nervous system in tissue culture and in situ: localization of vascoactive intestinal polypeptide (VIP), substance-P and enkephalin immunoreactive nerves in the guinea-pig gut

Immunofluorescence methods have been used to determine the detailed distribution of vasoactive intestinal polypeptide (VIP), substance-P and enkephalin nerve fibres in fixed cryostat sections from guinea-pig duodenum, jejunum, ileum, caecum at the site of the taenia coli, and proximal and distal colon. A novel method is used involving immunostaining of tissue culture preparations of both myenteric and submucous plexuses. These preparations allow each plexus to be studied in isolation from all axonal input for the first time, since they provide unequivocal extrinsic denervation together with severance of any intrinsic connections between the plexuses. In tissue sections the most prominent sites of VIP and substance-P immunoreactive fibres are the ganglia of the myenteric and submucous plexuses, the circular muscle layer and the longitudinal muscle of the taenia coli. In addition, VIP is prominent in the lamina propria of the submucosa except in the caecum. Enkephalin-immunopositive fibres are restricted to the ganglia of the myenteric plexus, the circular muscle layer and the longitudinal layer of the taenia coli. The culture preparations reveal that intrinsic ‘VIP neurons’ are common in the submucous plexus of the caecum and colon. They are also present, but in much lower numbers, in the myenteric plexus of the small intestine and colon but are not found in the myenteric plexus of the caecum. Intrinsic ‘substance-P neurons’ are present in the myenteric plexus from the small intestine, caecum and colon as well as in the submucous plexus of the colon; intrinsic ‘substance-P neurons’ are not found in the submucous plexus of the caecum. ‘Enkephalin neurons’ are numerous in the myenteric plexus of the small intestine, caecum and colon but are absent from the submucous plexus. Immunoreactivity is compared in the normal and denervated caecum by both the histochemical method and by radioimmunoassay of tissue extracts. In conjunction with the studies on tissue cultures, the results provide evidence for intrinsic reciprocal connections between the myenteric and submucous plexus of the caecum by neurons containing VIP and substance-P.An extensive comparison of these results with data from functional studies shows that the distribution of VIP, substance-P and enkephalin fibres in the gut is broadly in agreement with present knowledge of the action of these peptides on gut tissue, if it is assumed that they function as neurotransmitters or neuromodulators. In some instances, however, peptide-containing fibres and pathways are found which do not correlate with present knowledge obtained from functional studies. These observations provide new clues to the role of peptide neurons in gut function.     

On spirality in the intestinal wall

Methods of stripping did not demonstrate a significant spiral course of fibers within longitudinal muscle coats of the jejuno-ileum of the dog, cat, hog or man. Similar lack of spirality also characterized the large intestine of the dog. The presence of taeniae in the hog and man, and the extremely thin layer of muscle between them, permitted only the demonstration of directly longitudinal muscle in taeniae. Bands of muscle stripped from circular coats of the small intestine of the dog and hog came off overwhelmingly in complete rings. Stripping of the human intestine did not yield complete rings; breakage at anastomoses with the adjoining muscle sheet may indicate oblique bands that link up a fiber system, essentially circular, into a functionally spiral system. Bands stripped from circular coats of the large intestine of the dog came off overwhelmingly as circles. The taeniae of the hog and man prevent obtaining complete strips. Stripping of the submucosal stratum of the small intestine of the dog and man, and of the large intestine of the dog, revealed a latticed tube. Its fibers intersect in double spirals, coursing at angles of approximately 45° to the longitudinal axis of the intestine.     

Distribution of peripheral nerve terminals in the small and large intestine of congenital aganglionosis rats (Hirschsprung's disease rats)

The congenital aganglionosis rat is considered to be an animal model of Hirschsprung's disease. The mutants had a long constricted segment (from distal ileum to rectum) below the dilated distal ileum. In the dilated region, synaptophysin-immunoreactivity (IR) was almost preserved in all layers of the intestinal wall. In the constricted distal ileum and oral proximal colon, synaptophysin-IR was scarce in all layers, including the circular and longitudinal muscle layers. In the anal proximal and distal colon, synaptophysin-IR was almost scarce in the circular muscle layer (CML). An ultrastructural study confirmed that almost no terminals were found in the CML of any regions of constricted intestine. Therefore, the CML in any region of a constricted segment, is presumed to be poor innervation. However, a few synaptophysin-IR were found in the longitudinal muscle layer (LML) of an anal part of a constricted segment. An ultrastructural study also confirmed that some terminals were observed in the LML of this segment. The present study suggested that denervated CML is related to the production of constricted segment, irrespective of the presence or absence of terminals in the LML.     

Structure of the musculature of the chicken small intestine

Summary The small intestine of the chicken was studied by light and electron microscopy. The musculature, measuring about 180 m in thickness in the distended intestine, consists of four layers (outer longitudinal, outer circular, inner circular and inner longitudinal) which are directly apposed to one another. There is no layer of connective tissue equivalent to the submucosa of mammalian intestine, and the intestinal glands lie close to the inner longitudinal muscle. Mucosal folds are not formed during isotonic contraction of the intestine. The muscle cells of the chicken small intestine are characterized by large, numerous and sharply outlined dense bodies, by the presence of an extremely thin basal lamina, by prominent dense bands at the cell surface but relatively few intermediate junctions. There are many areas of direct apposition between cell membranes of adjacent cells and little collagen between the muscle cells. The four muscle layers have each distinctive structural features. Gap junctions between muscle cells occur only in the outer circular layer. The outer circular and outer longitudinal layers are closely apposed and numerous junctions of the adherens type link cells of the two layers. Intramuscular blood capillaries are rare and are found virtually only in the outer circular layer; their endothelial cells are joined by tight junctions. In the outer circular layer (but not in the other layers) there are two further cell types, fibroblasts and interstitial cells, which can be clearly distinguished from one another. The latter cells are intimately related to nerve bundles and are connected by gap junctions to some muscle cells.     

Electrophysiological study of the intestinal smooth muscle of the guinea-pig

The membrane properties of single cells of intestinal smooth muscle of duodenum, jejunum, ileum, caecum and rectum of guinea-pig have been studied.

1. The membrane potentials of longitudinal muscles of the duodenum, jejunum, ileum, caecum and rectum varied from 54 to 56 mV and those of circular muscles of jejunum, ileum, caecum and rectum varied from 57 to 60 mV. The ablated longitudinal muscle had a slightly lower value (50 mV) than the intact one.

2. The longitudinal muscle generated spontaneous discharges but these were rare in the circular muscles of the intestine except for the caecum. Overshoot potentials could be observed in all regions of the intestine. The maximum rate of rise of the spontaneously discharging longitudinal muscles varied from 11 to 18 V/sec.

3. Not all of the slow potential changes (but at least some) were generated by the nervous elements distributed between the muscle layers and in them.

4. The conduction velocities measured from the longitudinal muscles of jejunum and rectum in the presence of tetrodotoxin were 2·1 cm/sec and 4·0 cm/sec respectively.

5. Chronaxies of the longitudinal muscles of jejunum and rectum were 2-5 msec and 5-18 msec respectively.

6. Intracellular stimulation of the single cells of the duodenum and caecum could trigger a spike, similar to that observed in the taenia coli. The spikes were mostly graded ones; a spike of full size was rarely elicited. When the spikes were triggered, the after-hyperpolarization appeared consistently presumably caused by the increased potassium conductance.

7. The effective membrane resistance and the time constant were measured for the longitudinal muscles of the jejunum and rectum. When spikes were generated by intracellular stimulation the observed values were 40-50 MΩ and 3-5 msec in both tissues. These values were the same as those observed in the taenia coli.

8. When the time constant of the membrane was measured by the extracellular polarizing method, the longitudinal muscles of the jejunum especially and the rectum had smaller time constants than the taenia coli.

9. The differences of conduction velocity and chronaxie of the different regions of the intestine are discussed in relation to the cable properties of the tissues which are directly influenced by the morphological arrangements of the tissues.

     

Anatomy of the bilioduodenal junction of the opossum

Prevention of reflux is a major function of the terminal biliary duct system at its junction with the duodenum. We examined this area via scanning electron microscopy and light microscopy to explore anatomic features that might play such a role in the Virginia opossum, a species with a highly developed sphincter of Oddi (SO). The terminal apparatus, most of which consists of a dilated extramural ampulla, has a lumen with abundant folds. Mucus is produced by the lining epithelium and by a plethora of glands. Three muscle layers constitute the SO: an inner longitudinal, an outer circular, and a less consistent, outermost longitudinal. The terminal apparatus forms an acute angle and narrows as it enters the duodenum; at this point, the SO becomes continuous with the muscularis externa of the intestine. Four anatomical features with potential antireflux properties may be identified: mucus production, luminal folds, and the narrow opening and oblique course of the intramural duct.     

Anatomy of the bilioduodenal junction of the opossum.

Prevention of reflux is a major function of the terminal biliary duct system at its junction with the duodenum. We examined this area via scanning electron microscopy and light microscopy to explore anatomic features that might play such a role in the Virginia opossum, a species with a highly developed sphincter of Oddi (SO). The terminal apparatus, most of which consists of a dilated extramural ampulla, has a lumen with abundant folds. Mucus is produced by the lining epithelium and by a plethora of glands. Three muscle layers constitute the SO: an inner longitudinal, an outer circular, and a less consistent outermost longitudinal. The terminal apparatus forms an acute angle and narrows as it enters the duodenum; at this point, the SO becomes continuous with the muscularis externa of the intestine. Four anatomical features with potential antireflux properties may be identified: mucus production, luminal folds, and the narrow opening and oblique course of the intramural duct.     

Arrangement of the tunica muscularis of the ileum, cecum and colon at the transition between the small and the large intestines of swine.

The arrangement of the bundles of muscular fibers in the transition between the small and the large intestines was studied in 12 male adult crossbred swine by dissection, after immersion in 50% nitric acid solution. The connection between the small and the large intestines was formed by the association of the muscular tissue, the connective tissue and the fat tissue. The tunica muscularis of these organs was organized in thin thread-like bundles and wide ribbon-like bundles of variable width and thickness. The superficial bundles of the longitudinal stratum of the tunica muscularis of the ileum established the continuity with the large intestine; the deep bundles penetrated into the ileal papilla. The limit between the cecum and the ascending colon was externally marked by the sulcus cecocolicus dorsalis and ventralis. The connection between the cecum and the ascending colon was formed by bundles of muscular fibers coming from the ileum, and the taeniae ventralis, lateralis and medialis of the cecum. Some bundles of muscular fibers from the ascending colon and the cecum headed toward the sulcus cecocolicus. The median bundles of muscular fibers of the taenia ventralis of the cecum, near the termination of the ileum, were arranged to form a loop around the termination of the ileum, mixing with the musculature of the ascending colon at the level of this junction.     

Hexose transport across the apical and basolateral membrane of enterocytes from different regions of the chicken intestine

The properties of hexose transport across the apical and basolateral membranes of chicken enterocytes have been studied in the small and large intestine. Results show that (a) isolated epithelial cells from all segments except the coprodeum can accumulate 3-O-methylglucose (Glc3Me) against a concentration gradient, by a Na⁺-dependent and phloridzin-sensitive mechanism, (b) The cell cumulative capacity for Glc3Me (control/phloridzin-incubated cells) is lower in the small intestine than in the large intestine (rectum = proximal caecum = ileum > jejunum > duodenum). (c) Theophylline enhances the cell Glc3Me cumulative capacity 2.9-fold in the duodenum and 2.4-fold in the jejunum but has no effect in the other segments studied. (d) Analysis of sugar uptake indicates that net hexose influx rates decrease from proximal to distal regions: jejunum > duodenum > ileum = proximal caecum = rectum for the apical transport system (-methyl glucoside as substrate and phloridzin as inhibitor) and duodenum > jejunum > ileum = proximal caecum = rectum for the basolateral system (2-deoxyglucose; theophylline). (e) The duodenum and the jejunum show high apical and basolateral hexose transport rates, which confer a significant capacity for sugar absorption on the proximal intestine. More distal regions, including the ileum, the proximal caecum and the rectum, have transport systems analogous to those of the proximal intestine that keep a considerable potential capability to recover hexoses from the lumen.     

Acetylcholine and substance P responsiveness of intestinal smooth muscles in streptozotocin diabetic rats

We found previously that in in vitro tube form preparations of isolated intestine of streptozotocin (STZ) diabetic rats, frequency of spontaneous intraluminal pressure waves was significantly reduced in duodenum when compared with normal controls. In order to elucidate further the diabetic intestinal disorders, we examined the frequency and amplitude of spontaneous length changes and contractile responses to acetylcholine (ACh) and substance P (sP) in isolated intestinal segments of normal and experimental diabetic rats. In comparison with normal controls, we could confirm the significantly decreased frequency of spontaneous length changes in isolated longitudinal and circular muscle preparations of diabetic duodenum (1 month after STZ injection). Furthermore, amplitude of spontaneous length changes was significantly decreased in circular muscle preparations of duodenum, jejunum, and ileum but not in colon nor in longitudinal muscle preparations. Dose-response curves revealed that both ACh and sP responses were significantly decreased in longitudinal and circular muscle preparations of diabetic duodenum, jejunum, and ileum but not in colon. Mechanisms of reduced contractility of diabetic intestinal smooth muscle in response to ACh and sP were discussed.     

Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut.

The pattern of distribution and colocalization of nitric oxide-synthase (NOS) and NADPH-diaphorase in the myenteric plexus of whole-mount preparations of the antrum, duodenum, ileum, caecum, proximal colon and distal colon of the rat were investigated using immunohistochemical and histochemical staining techniques. Almost all the myenteric neurons that were NOS-positive in all regions of the gut examined were also stained for NADPH-diaphorase. However, in the stomach, duodenum and ileum, only a few of the NOS-positive nerve fibres in the tertiary and secondary plexuses and circular muscle layer were also stained for NADPH-diaphorase, whereas in the caecum and distal colon almost all the NOS-positive nerve fibres were also stained for NADPH-diaphorase. The results in the present study are consistent with the view that nitric oxide (NO) has a mediating role in gastrointestinal neurotransmission.     

An immunohistochemical study of the distribution of enteric GABA-containing neurons in the rat and guinea-pig intestine

gamma-Aminobutyric acid (GABA) antiserum was applied to sections of rat and guinea-pig intestine which were subsequently processed to reveal any immunoreactivity using either fluorescence or peroxidase techniques. Immunopositive fibres were demonstrated in stomach, duodenum, ileum and colon of rat and guinea-pig intestine. Myenteric ganglia and nerve bundles in the circular muscle contained immunopositive nerve fibres, while the longitudinal muscle, submucosa and mucosa were only rarely innervated. In favourable sections, immunopositive fibres could be seen running from the myenteric plexus into the circular muscle, thus suggesting that the GABA-immunopositive nerves in the circular muscle originate from neurons in the myenteric plexus. In both rat and guinea-pig, immunoreactive nerve cell bodies were most numerous in the myenteric plexus of the colon. In the rat, immunopositive fibres in the circular muscle were most abundant in the ileum, whereas in the guinea-pig it was the colon circular muscle that was most richly innervated. The results demonstrate that neurons which show GABA immunoreactivity are present along the length of the gastrointestinal tract. Their distribution in both myenteric ganglia and circular muscle is heterogeneous both within and between the two species studied. It is probable that this heterogeneity reflects the diversity and specificity of function of this class of enteric neurons.     

Structural Organization of Hepatic Portal Vein in Rat with Special Reference to Musculature,Intimal Folds,and Endothelial Cell Alignment

Structural organization of hepatic portal vein (HPV) was examined in adult rats by means of light and electron microscopy. Three characteristic features were found in the wall structure of rat HPV. (1) Tunica media consisted of two kinds of smooth muscle. The inner circular smooth muscle (CSM) was composed with one or two layer of smooth muscle cells, and was found in the entire length of the HPV and its tributaries. The outer longitudinal smooth muscle (LSM) was limited to a specific region of HPV; in particular it was well‐developed at distal half of HPV. CSM counteracts luminal hydrostatic pressure to prevent circumferential hyperextension of venous wall, whereas LSM is likely to counteract a tractive force produced by gravity and movement of small intestine. (2) Intima of HPV showed a unique feature, intimal folds, which protruded into the lumen and were aligned almost circumferentially. Intimal folds were found only at the same region where the LSM was well‐developed. Thus, LSM is presumably involved in the formation of intimal folds. (3) The endothelial cells between intimal folds were circumferentially aligned along the folds, although those in the other regions of HPV were arrayed along the longitudinal axis of HPV or the direction of blood flow as reported in other kinds of blood vessel. This finding implied that the circumferential blood flow locally occurs on the surface of intima between the intimal folds. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Structural and mechanical architecture of the intestinal villi and crypts in the rat intestine: integrative reevaluation from ultrastructural analysis

The ultrastructure of the rat intestinal interstitium was analyzed from the viewpoint of mechanical dynamics to stabilize the intestinal villi, crypts and mucosal folds. In the rat, the small intestine lacks circular folds, but the large intestine possesses spiral folds. The intestinal villi, the largest in the duodenum, decreased in size in the jejunum and ileum successively, and were absent in the large intestine. The intestinal interstitium consisted of lamina propria mucosae (LPM) and tela submucosa (TSM) separated by muscularis mucosae (MM), the LPM was subdivided into an upper part within the villi and a lower part among the crypts in the small intestine. The light microscopic density of interstitium in the intestinal wall was lowest in the upper LPM, moderately dense in the lower LPM and highest in the TSM, and that among the intestinal region was highest in the duodenum and decreased successively in the jejunum and ileum. In the large intestine, the TSM bulged to form spiral folds with very low density. The intestinal epithelium in the villi possessed wide intercellular spaces and that in the crypts had closed intercellular spaces. At electron microscopic level, the upper and lower LPM contained subepithelial supportive meshwork that consisted of collagen fibrils and myofibroblast processes. The lower LPM and TSM contained conspicuous bundles of collagen fibrils and, in addition, TSM contained minor populations of scattered collagen fibrils near the smooth muscle layer (SML). The diameter of collagen fibrils was the largest in the bundles of TSM, and decreased from the duodenum through the jejunum and ileum to the large intestine. On the basis of these observations, we hypothesize that the intestinal villi are mechanically stabilized by the balance between the expansive interstitial pressure and inward pull by the subepithelial supportive meshwork. This hypothesis explains the hitherto neglected fact that the intestinal epithelium possesses wide intercellular spaces only in the villi, and accounts for the counterforce against the perpendicular smooth muscle cells, which are supposed to contract the intestinal villi.     

Morphological changes of the myenteric plexus during early postnatal development of the rat.

The enteric nervous system needs to adapt itself constantly to the postnatal changes of the developing gut. The aim of this study was to examine the morphological changes between the distal and proximal segments of the gastrointestinal (GI) tract during the first two postnatal weeks. Myenteric plexus from the duodenum, proximal and distal colon of 1-, 7- and 14-day-old rat pups was dissected and examined under the scanning electron microscope. Wholemounts from the same regions and postnatal stages were stained with cuprolinic blue. Neuronal numbers per ganglionic area were counted and neuronal sizes were measured. Furthermore, segments of the above-mentioned areas were embedded in resin and semithin sections were cut. The thickness of the circular and longitudinal muscle layers was measured. The morphology of the myenteric plexus depends on localization as well as on the age of the animal. While in younger animals the myenteric plexus is usually densely packed, the network expands with increasing age. Similarly, the thickness of the circular and the longitudinal muscle layers increases. Nerve cell numbers per ganglionic area increase from duodenum to distal colon and decrease from the 1-day (P1) to the 14-day-old (P14) animal. The longest diameters and the area of the nerve cells decrease from duodenum to distal colon and increase with age of the animal. The intensity of the cuprolinic blue staining varies also according to age and segment of the gut. During the first two postnatal weeks the three-dimensional architecture of the myenteric plexus as well as the size and densities of the enteric neurons change according to the increasing gut length and the thickness of the muscle layer. The differences between duodenum and colon might reflect the different physiological properties of the proximal and distal gut as well as a varying grade of maturity, which is also supported by a variation in the cuprolinic blue staining intensity.     

Projections of substance P, vasoactive intestinal peptide and tyrosine hydroxylase immunoreactive nerve fibres in the canine intestine, with special reference to the innervation of the circular muscle.

Antisera raised against neuron specific enolase (NSE), substance P, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase (TH) were used to reveal nerve fibres in the wall of the canine small and large intestine. The circular muscle of the colon was innervated by nerve fibre bundles that ran parallel to the muscle throughout its thickness. A plexus of fibre bundles was found against the inner (submucosal) surface of the circular muscle. Fibres with substance P, VIP and TH immunoreactivity all contributed to this innervation. The circular muscle of the small intestine was distinctly separated into outer and inner layers by a dense plexus of nerve fibres, the deep muscular plexus. The outer and inner circular muscle were innervated by substance P, VIP and TH fibres. Extrinsic denervation through the severing of nerve fibres in the mesentery caused TH fibres in the intestine to degenerate, but had no detectable effect on the fibres with substance P or VIP immunoreactivity. Myectomy (the removal of the myenteric plexus from the full circumference of the intestine over a distance of 2-3 cm), performed 7-13 days before tissue was taken, resulted in an almost complete loss of substance P fibres from the circular muscle of the colon and the outer circular muscle of the small intestine. However, many fibres persisted in the deep muscular plexus of the small intestine, and most fibres remained in its inner circular muscle. The changes in distribution of VIP fibres were almost identical, except that a small proportion of reactive fibres remained in the circular muscle of the colon and the outer circular muscle of the small intestine. It is concluded that the circular muscle layers of the small intestine and colon have dual sources of intrinsic nerve supply: the myenteric ganglia supply fibres primarily to the outer part of the muscle and the submucous ganglia supply fibres to the inner muscle. The present study further demonstrated that VIP fibres ran anally in the myenteric plexus of both the small and large intestine, whereas substance P fibres ran orally in the large intestine and both orally and anally in the small intestine. The innervation of the muscularis mucosae and mucosa by substance P and VIP fibres was not affected by myectomy or extrinsic denervation, and these structures are therefore likely to be innervated by nerve cells in the submucous ganglia.     

Various structural and functional criteria of the organization of sphincters of hollow internal organs

Based on numerous data available in literature and authors' own findings, certain structural and functional criteria characterizing the organization of sphincters of the walls of hollow visceral organs were formulated. It is reasonable to attribute local thickening of the musculature circular layer (the main feature of a sphincter), changes in configuration of mucosal folds, cleft-like lumen of the organ in the zone of a sphincter, specificity of glandular and neurovascular mechanisms to the sphincter apparatus performing predominantly counterflux and coordinating functions.     

Morphological changes of the myenteric plexus during early postnatal development of the rat

The enteric nervous system needs to adapt itself constantly to the postnatal changes of the developing gut. The aim of this study was to examine the morphological changes between the distal and proximal segments of the gastrointestinal (GI) tract during the first two postnatal weeks. Myenteric plexus from the duodenum, proximal and distal colon of 1‐, 7‐ and 14‐day‐old rat pups was dissected and examined under the scanning electron microscope. Wholemounts from the same regions and postnatal stages were stained with cuprolinic blue. Neuronal numbers per ganglionic area were counted and neuronal sizes were measured. Furthermore, segments of the above‐mentioned areas were embedded in resin and semithin sections were cut. The thickness of the circular and longitudinal muscle layers was measured. The morphology of the myenteric plexus depends on localization as well as on the age of the animal. While in younger animals the myenteric plexus is usually densely packed, the network expands with increasing age. Similarly, the thickness of the circular and the longitudinal muscle layers increases. Nerve cell numbers per ganglionic area increase from duodenum to distal colon and decrease from the 1‐day (P1) to the 14‐day‐old (P14) animal. The longest diameters and the area of the nerve cells decrease from duodenum to distal colon and increase with age of the animal. The intensity of the cuprolinic blue staining varies also according to age and segment of the gut. During the first two postnatal weeks the three‐dimensional architecture of the myenteric plexus as well as the size and densities of the enteric neurons change according to the increasing gut length and the thickness of the muscle layer. The differences between duodenum and colon might reflect the different physiological properties of the proximal and distal gut as well as a varying grade of maturity, which is also supported by a variation in the cuprolinic blue staining intensity. Anat Rec 256:20–28, 1999. © 1999 Wiley‐Liss, Inc.     

Projections of chemically-specified neurons in the guinea-pig colon.

The arrangement of the enteric nerve plexuses in the colon of the guinea-pig and the distributions and projections of chemically specified neurons in this organ have been studied. Immunoreactivity for neuron specific enolase was used to examine the total population of neurons and individual subpopulations were studied using antibodies raised against calbindin, calcitonin gene-related peptide (CGRP), leu-enkephalin, gastrin releasing peptide (GRP), galanin, gamma aminobutyric acid, neurokinin A, neuropeptide Y (NPY), somatostatin, substance P, tyrosine hydroxylase and vasoactive intestinal peptide (VIP). Neuronal pathways within the colon were lesioned using myotomy and myectomy operations and extrinsic pathways running between the inferior mesenteric ganglia and the colon were also severed. Each of the antibodies revealed nerve cells and nerve fibres or only nerve fibres within the wall of the colon. VIP, galanin and GRP were in anally projecting pathways in the myenteric plexus, as they are in other species. In contrast, there are differences in the projection directions of enkephalin, substance P, NPY and somatostatin nerve fibres between regions and species. Surprisingly, somatostatin and NPY fibres have opposite projections in the small intestine and colon of the guinea-pig. The majority of nerve fibres that innervate the circular muscle, including fibres with immunoreactivity for VIP, enkephalin, substance P, NPY, galanin and GRP come from the myenteric ganglia. The mucosa is innervated by fibres from both the myenteric and submucous ganglia. The present results suggest that the guinea-pig distal colon is a suitable place in which to determine relations between structure, neurochemistry and functions of enteric neural circuits.     

Immunocytochemical analysis of potential neurotransmitters present in the myenteric plexus and muscular layers of the corpus of the guinea pig stomach

Recent electrophysiological studies of neurons of the myenteric plexus of the corpus of the guinea pig stomach have revealed that slow synaptic events are extremely rare. In contrast, they are commonly encountered in similar investigations of myenteric ganglia of the guinea pig small intestine. The current immunocytochemical analysis of the myenteric plexus and innervation of the muscularis externa of the corpus of the guinea pig stomach was undertaken in order to determine whether putative neurotransmitters capable of mediating slow synaptic events are present in gastric ganglia. A major difference between the small intestine and the stomach was found in the innervation of the musculature. Whereas the longitudinal muscle layer of the small intestine contains very few nerve fibers and is innervated mainly at its interface with the myenteric plexus, the longitudinal muscle of the corpus of the stomach contained as many varicose substance P (SP)-, vasocative intestinal polypeptide (VIP)-, and neuropeptide Y (NPY)-immunoreactive axons as the circular muscle layer. These putative neurotransmitters were also present in the ganglia of the myenteric plexus, where varicose SP-, VIP-, and NPY-immunoreactive fibers encircled nonimmunoreactive neurons. Varicose 5-hydroxytryptamine (5-HT)-immunoreactive terminal axons were essentially limited to the myenteric plexus and were found both in ganglia and in interganglionic connectives, where they were particularly numerous; 5-HT-immunoreactive neurons appeared to be more abundant in the stomach than in the small intestine. Tyrosine hydroxylase (TH)- and calcitonin-gene-related-peptide (CGRP)-immunoreactive axons were also more common in the myenteric plexus than in the musculature, but of these, only the TH-immunoreactive neurites tended, like those of the other putative transmitters, to encircle neurons in myenteric ganglia. Evidence was obtained that, as in the small intestine, at least some of the SP-, VIP-, NPY-, and 5-HT-immunoreactive fibers in the stomach are derived from intrinsic gastric myenteric neurons. In contrast, unlike the small intestine, gastric myenteric ganglia appeared to lack intrinsic CGRP-immunoreactive neurons; therefore, the CGRP-immunoreactive gastric axons are probably of extrinsic origin.(ABSTRACT TRUNCATED AT 400 WORDS)