Distribution and origin of peptide-containing nerve fibers in the celiac superior mesenteric ganglion of the guinea-pig

The origin of the peptidergic nerve fibers and terminals in the celiac superior mesenteric ganglion of the guinea-pig was studied. The distribution of immunoreactivity to enkephalin, substance P, calcitonin gene-related peptide, cholecystokinin, vasoactive intestinal polypeptide/peptide histidine isoleucine, bombesin and dynorphin was analysed in intact animals and in animals subjected to various denervation and ligation procedures. The present results show that each of the connected nerve trunks carries peptidergic pathways and contributes to the peptidergic networks in the celiac superior mesenteric ganglion. Thus, the thoracic splanchnic nerves contain enkephalin-, substance P- and calcitonin gene-related peptide-immunoreactivity of which substance P and calcitonin gene-related peptide coexist in the same nerve fibers. In addition, cholecystokinin-, vasoactive intestinal polypeptide/peptide histidine isoleucine- and dynorphin-immunoreactivity is present in some fibers. All of these immunoreactivities are present in sensory neurons except enkephalin which probably originates in the spinal cord. The mesenteric nerves carry enkephalin-, calcitonin gene-related peptide-, cholecystokinin-, vasoactive intestinal polypeptide/peptide histidine isoleucine-, bombesin- and dynorphin-immunoreactive fibers from the intestine and are the main source for cholecystokinin, vasoactive intestinal polypeptide/peptide histidine isoleucine, bombesin and dynorphin fibers. Double-staining experiments indicate that many of these peptides are synthesized in the same enteric neurons. Also the intermesenteric nerve contains peptide-immunoreactive fibers to the celiac superior mesenteric ganglion from different sources, probably including the distal colon as well as dorsal root ganglia and spinal cord at lower thoracic and lumbar levels. The results are discussed in relation to earlier morphological and physiological studies supporting the view of a role of the celiac superior mesenteric ganglion in local reflex mechanisms involved in regulation of gastrointestinal functions.     

The immunohistochemical localization of nine peptides in the sacral parasympathetic nucleus and the dorsal gray commissure in rat spinal cord

The sixth lumbar and first sacral spinal cord segments in the rat contain parasympathetic preganglionic neurons which innervate the pelvic viscera. There have been few studies, however, which have specifically considered the distribution of putative peptide neurotransmitters in these cord segments. The present paper describes and compares the immunohistochemical distribution of dynorphin (1-8)-, enkephalin-, somatostatin-, cholecystokinin octapeptide-, avian pancreatic polypeptide-, FMRF-NH2-, neurotensin-, and vasoactive intestinal polypeptide-like immunoreactivities in the dorsal gray commissure and sacral parasympathetic nucleus of the sixth lumbar and first sacral spinal cord segments in colchicine-treated rats. Antisera against all of the peptides, except avian pancreatic polypeptide, stained cells in the sacral parasympathetic nucleus. Dynorphin (1-8-), enkephalin-, and substance P-like immunoreactive cells were present in significantly greater numbers than somatostatin-, neurotensin-, cholecystokinin-, FMRF-NH2-, and vasoactive intestinal polypeptide-like immunoreactive cells. All of the antisera also stained fibers in the sacral parasympathetic nucleus in varying densities, and a fiber bundle which extended between the dorsal gray commissure and the sacral parasympathetic nucleus. Antisera against substance P and cholecystokinin stained a bundle of fibers that extended between the dorsal horn and the sacral parasympathetic nucleus. Antisera against somatostatin, cholecystokinin octapeptide, substance P and FMRF-NH2 stained an additional fiber bundle which extended between the lateral edge of the dorsal horn and the dorsal gray commissure. All the remaining antisera, except neurotensin, also stained fibers that extended between the sacral parasympathetic nucleus and the dorsal gray commissure, but in a sparser distribution. Immunoreactive cells were localized to the dorsal gray commissure in sections stained with each of the antisera. Dynorphin (1-8) and enkephalin antisera stained the greatest number of cells, followed by FMRF-NH2, neurotensin, somatostatin and avian pancreatic polypeptide. The smallest number of immunoreactive cells was present in substance P, cholecystokinin and vasoactive intestinal polypeptide immunostained sections. A significant difference was noted between the number of dynorphin, enkephalin, somatostatin, cholecystokinin, avian pancreatic polypeptide, FMRF-NH2, neurotensin and vasoactive intestinal polypeptide immunoreactive cells in the sacral parasympathetic nucleus and dorsal gray commissure.(ABSTRACT TRUNCATED AT 400 WORDS)     

Projections of peptide-containing neurons in rat colon

The distribution, origin and projections of nerve fibers containing vasoactive intestinal peptide, substance P, neuropeptide Y, galanin, gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin or enkephalin were studied in the midcolon of the rat by immunocytochemistry and immunochemistry. Most of these nerve fibers had an intramural origin as was established by extrinsic denervation (serving of mesenterial nerves). Extrinsic denervation eliminated neuropeptide Y-containing fibers of presumably sympathetic origin together with sensory nerve fibers containing both substance P and calcitonin gene-related peptide. Co-existence of two peptides in the same neuron was studied by double immunostaining. This revealed co-existence of neuropeptide Y and vasoactive intestinal peptide in one population of intramural neurons; an additional population of intramural neurons was found to contain vasoactive intestinal peptide but not neuropeptide Y. All somatostatin-containing neurons in the submucous ganglia were found to harbor calcitonin gene-related peptide. A much larger population of submucous neurons containing calcitonin gene-related but not somatostatin was also detected. Some perivascular calcitonin gene-related peptide-containing nerve fibers (of intrinsic origin) harbored vasoactive intestinal peptide while others (of extrinsic origin) harbored substance P. The polarities and projections of the various peptide-containing intramural neurons in the transverse colon were studied by analysing the loss of nerve fibers upon local disruption of enteric nervous pathways (myectomy or intestinal clamping). Myenteric neurons containing vasoactive intestinal peptide, galanin, gastrin-releasing peptide, calcitonin gene-related peptide, somatostatin or vasoactive intestinal peptide/neuropeptide Y gave off 5-10-mm-long descending projections while those containing substance P or enkephalin issued approx. 5-mm-long ascending projections. Submucous neurons containing calcitonin gene-related peptide, somatostatin/calcitonin gene-related peptide or gastrin-releasing peptide issued both ascending (2-6 mm) and descending (2-6 mm) projections, those containing vasoactive intestinal peptide issued ascending (approx. 2 mm) projections, while those containing galanin or vasoactive intestinal peptide/neuropeptide Y lacked demonstrable oro-anal projections. Enkephalin-containing fibers could not be detected in the mucosa and the mucosal substance P-containing nerve fibers were too few to enable us to delineate their projections.     

Receptor binding sites for cholecystokinin, galanin, somatostatin, substance P and vasoactive intestinal polypeptide in sympathetic ganglia.

Sympathetic ganglia are innervated by neuropeptide-containing fibers originating from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and in some cases, myenteric neurons. In the present report receptor autoradiography was used to determine whether sympathetic ganglia express receptor binding sites for several of these neuropeptides including bombesin, calcitonin gene-related peptide-alpha, cholecystokinin, galanin, neurokinin A, somatostatin, substance P, and vasoactive intestinal polypeptide. The sympathetic ganglia examined included the rat and rabbit superior cervical ganglia and the rabbit superior mesenteric ganglion. High levels of receptor binding sites for cholecystokinin, galanin, somatostatin, substance P, and vasoactive intestinal polypeptide were observed in all sympathetic ganglia examined, although only discrete neuronal populations within each ganglion appeared to express receptor binding sites for any particular neuropeptide. These data suggest that discrete populations of postganglionic sympathetic neurons may be regulated by neuropeptides released from pre- and postganglionic sympathetic neurons, dorsal root ganglion neurons, and myenteric neurons.     

Distribution of vasoactive intestinal polypeptide-, substance P-, enkephalin-and neurotensin-like immunoreactive nerves in the chicken gut during development

The ontogeny and distribution of nerve cell bodies and fibres which contain vasoactive intestinal polypeptide-, substance P-, enkephalin- and neurotensin-like immunoreactivity have been studied in the chicken gastrointestinal tract, using immunocytochemistry. All four peptides were found in nerve fibres, with characteristic distribution patterns, which, in the cases of vasoactive intestinal polypeptide, substance P and methionine enkephalin were similar to those described for the mammalian gut. In addition, many of these fibres were shown to arise from intrinsic neurons, since immunoreactive nerve cell bodies for each of the peptides studied were observed. Neurotensin-immunoreactive nerves were confined to the upper part of the tract and neurotensin immunoreactive cell bodies were only observed in embryonic and newly hatched chicken gut. All four peptides were first observed at 11 days of incubation, or Hamburger-Hamilton stage 37,²⁰ in the upper part of the tract, particularly in the gizzard. Substance P and methionine enkephalin were subsequently seen in more caudal regions, while vasoactive intestinal polypeptide developed from each end of the tract. Adult patterns of immunoreactivity in nerve fibres were achieved during the first week after hatching. A striking observation was that immunoreactive neuronal cell bodies were much more abundant in the gut of young chickens and chicken embryos than in that of adult birds.     

Immunohistochemical study of neuropeptides in vagal and glossopharyngeal afferent neurons in the rat

The presence and distribution of multiple neuropeptides in vagal and glossopharyngeal afferent ganglia of the rat were studied using immunohistochemistry. Substance P-, calcitonin-gene related peptide-, cholecystokinin-, neurokinin A-, vasoactive intestinal polypeptide-, and somatostatin-immunoreactive neurons were detected in each visceral afferent ganglion. Neurotensin-immunoreactive cells were not observed. In the nodose ganglion (inferior ganglion of the vagus nerve) occasional immunoreactive cells were scattered throughout the main (caudal) portion of the ganglion with small clusters of cells seen in the rostral portion. The pattern of distribution of the various peptides in the nodose ganglion was similar, with the exception of vasoactive intestinal polypeptide-immunoreactive neurons which exhibited a more caudal distribution. The relative numbers of immunoreactive cells varied, with the greatest numbers being immunoreactive for substance P or vasoactive intestinal polypeptide, and the lowest numbers being immunoreactive for neurokinin A and somatostatin. A build-up of immunoreactivity for each of the peptides, except somatostatin and neurotensin, was detected in vagal nerve fibers of colchicine-injected ganglia. Numerous peptide-immunoreactive cells were also found in the petrosal (inferior ganglion of the glossopharyngeal nerve) and jugular (superior ganglion of the vagus nerve) ganglia. No specific intraganglionic distribution was noted although the relative numbers of cells which were immunoreactive for the different peptides varied considerably. Substance P and calcitonin-gene related peptide were found in large numbers of cells, cholecystokinin was seen in moderate numbers of cells, and neurokinin A, vasoactive intestinal polypeptide and somatostatin were seen in fewer cells. These data provide evidence for the presence and non-uniform distribution of multiple peptide neurotransmitters in vagal and glossopharyngeal afferent neurons. In general, relatively greater numbers of immunoreactive cells were located in the rostral compared with caudal nodose ganglion, and in the petrosal and jugular ganglia compared with the nodose ganglion. Thus, multiple neuropeptides may be involved as afferent neurotransmitters in the reflexes mediated by vagal and glossopharyngeal sensory nerves.     

Projections of peptide-containing neurons in rat small intestine

The distribution, origin and projections of nerve fibers containing vasoactive intestinal peptide, neuropeptide Y, somatostatin, substance P, enkephalin and calcitonin gene-related peptide were studied in the rat jejunum by immunocytochemistry and immunochemistry. Their origin was determined by the use of various procedures for extrinsic denervation (chemical sympathectomy, bilateral vagotomy or clamping of mesenterial nerves). The terminations of the different types of intramural nerve fibers were identified by examination of the loss of nerve fibers that followed local disruption of enteric nervous pathways (intestinal myectomy, transection or clamping). The majority of the peptide-containing nerve fibers in the gut wall were intramural in origin, each nerve fiber population having its own characteristic distribution and projection pattern. Nerve fibers emanating from the myenteric ganglia terminated within the myenteric ganglia and in the smooth muscle layers: those storing vasoactive intestinal peptide/neuropeptide Y, somatostatin and substance P were descending, those storing enkephalin were ascending and those containing calcitonin gene-related peptide projected in both directions. Nerve fibers emanating from the submucous ganglia terminated mainly within the submucous ganglia and in the mucosa: those storing calcitonin gene-related peptide or vasoactive intestinal peptide/neuropeptide Y were ascending and those storing substance P or somatostatin were both ascending and descending. Enkephalin nerve fibers could not be detected in the mucosa.     

Lamina X of primate spinal cord: distribution of five neuropeptides and serotonin

The distribution of substance P, somatostatin, cholecystokinin, vasoactive intestinal polypeptide, enkephalin and serotonin in axons, terminals and neurons was compared in the area surrounding the central canal (lamina X) at five representative levels of the monkey spinal cord, using peroxidase-antiperoxidase immunocytochemistry. Immunoreactive neurons containing each of the neurochemicals were identified. At the cervical, thoracic and lumbar levels the area lateral to the canal had dense terminal fields immunoreactive for each neurochemical. The dorsal commissural region, the pericanal area, and the ventral commissural area were supplied by some but not all of the substances. In the lower thoracic cord innervation extended into the dorsal midline area and into the ventromedial commissural region. In contrast, in the sacral cord, the dorsal commissural region could be subdivided on the basis of innervation, and the lateral region was densely supplied by only cholecystokinin and serotonin, while the sacral ventral commissure and the pericanal area were supplied by all six neurochemicals. The immunocytochemical mappings were compared with published maps of functional classes of neurons and with the distribution of primary afferents and descending fibers in lamina X. The dense peptidergic and serotonergic innervation in the lateral area and the dorsal commissural area corresponded particularly with the location of projection neurons and primary afferents described in other studies.     

Distribution of enteric neurons showing immunoreactivity for substance P in the guinea-pig ileum

Substance P-like immunoreactivity has been localized in whole mount preparations of the isolated layers of the guinea-pig ileum. Axons containing substance P formed dense networks around the nerve cells and ran in the primary, secondary and tertiary nerve bundles of the myenteric plexus. 3.6% of the nerve cell bodies of the myenteric plexus and 11.3% of the cell bodies in the submucous plexus showed immunoreactivity for substance P. Axons ran in fine nerve bundles parallel to the longitudinal muscle, between this muscle and the myenteric plexus. Axons containing substance P also ran in small nerve trunks parallel to the circular muscle throughout its thickness and in the deep muscular plexus at the base of this muscle coat. In the submucosa, these axons ramified amongst ganglion cells of the plexus and ran in the internodal strands. In addition they formed a perivascular network around submucous arteries and contributed to the paravascular nerves following these arteries. Axons containing substance P formed a delicate plexus in the mucosa. After extrinsic denervation the nerves containing substance P that were associated with submucous arteries, and some in the submucous plexus, disappeared. The nerves in the other areas were not detectably different from normal.Comparison with the distribution of somatostatin, enkephalin and vasoactive intestinal polypeptide indicated the neurons containing substance P constitute a separate population within the enteric nervous system.     

Distribution of neuropeptides in the limbic system of the rat: The amygdaloid complex

The distribution of six neuropeptides (vasoactive intestinal polypeptide, cholecystokinin octapeptide, substance P, neurotensin, methionine-enkephalin and somatostatin) has been mapped in the amygdala using immunocytochemical methods. Cell bodies containing each peptide showed a differential distribution throughout the various subnuclei. Large numbers of vasoactive intestinal polypeptide and cholecystokinin-octapeptide-containing cell bodies were located in the lateral and cortical nuclei respectively, neurotensin- and methionine enkephalin-containing cell bodies in the central nucleus, and substance P-containing cell bodies primarily in the medial nucleus. Somatostatin-containing cell bodies were found in all nuclei. Neuropeptide-containing fibres were also differentially distributed. Substance P and cholecystokinin fibres formed dense plexuses in the medial nucleus whilst the greatest concentration of vasoactive intestinal polypeptide, neurotensin and methionine enkephalin fibres were seen in the central nucleus. Close observation of serial sections showed that all the neuropeptides studied had extensive intra-amygdaloid pathways and connections with other brain areas.The central nucleus and stria terminalis have particular importance in the organisation of peptides within the amygdala. The central nucleus acts as a focus for a number of converging/diverging peptide pathways and incoming catecholaminergic afferents. The stria terminalis contains all six peptides and represents the major efferent peptidergic system. The amygdala is thought to control a number of endocrine reponses and to regulate complex behavioural functions. The abundance of neuropeptides within the amygdala and their complex pattern of pathways imply that they may act to regulate endocrine responses to external events (e.g. stress) or alter emotional tone, functions thought to be controlled by the amygdala.     

Immunocytochemical identification of long ascending peptidergic neurons contributing to the spinoreticular tract in the rat

In the present study, we examined the peptidergic content of lumbar spinoreticular tract neurons in the colchicine-treated rat. This was accomplished by combining the retrograde transport of the fluorescent dye True Blue with the immunocytochemical labeling of neurons containing cholecystokinin-8, dynorphin A1-8, somatostatin, substance P or vasoactive intestinal polypeptide. After True Blue injections into the caudal bulbar reticular formation, separate populations of retrogradely labeled cells were identified as containing cholecystokinin-like, dynorphin-like, substance P-like or vasoactive intestinal polypeptide-like immunoreactivity. Retrogradely labeled somatostatin-like neurons were not identified in any of the animals examined. Each population of double-labeled cells showed a different distribution in the lumbar spinal cord. The highest yield of double-labeling occurred for cholecystokinin, with 16% of all intrinsic cholecystokinin-like neurons containing True Blue. These double labeled neurons were found predominantly at the border between lamina VII and the central canal region. About 11% of intrinsic vasoactive intestinal polypeptide-like neurons in the lumbar spinal cord were retrogradely labeled from the bulbar reticular formation. These neurons were found mostly in the lateral spinal nucleus, with only a few double-labeled cells located deep in the gray matter. Dynorphin-like double-labeled neurons were localized predominantly near the central canal; a smaller population was also seen in the lateral spinal nucleus. It was found that double-labeled dynorphin-like neurons made up 8% of all intrinsic dynorphin-like neurons. Retrogradely-labeled substance P-like neurons were rare; the few double-labeled neurons were found in the lateral spinal nucleus and lateral lamina V. These findings suggest a significant role for spinal cord peptides in long ascending systems beyond their involvement in local circuit physiology.     

The hypogastric nerve innervates a population of penile neurons in the pelvic plexus

Retrograde dye staining, enkephalin immunocytochemistry and nerve lesion paradigms were used to determine if penile neurons in the pelvic plexus are innervated by fibers in the hypogastric nerve. In the intact major pelvic ganglion of the rat, some 80% of penile neurons are enclosed by an enkephalin-positive fiber plexus. Following surgical interruption of the pelvic nerve, 20% of penile neurons were still surrounded by an enkephalin plexus. After interruption of the pelvic nerve and the hypogastric nerve, the enkephalin plexus in the ganglion was virtually absent, including the plexus around penile neurons. Therefore, possible intrinsic sources of the enkephalin fibers such as enkephalin-positive principal neurons and small intensely fluorescent cells, do not account for the delicate enkephalin fiber system in the pelvic ganglion. It is concluded that the pelvic nerve is the major source of preganglionic innervation to penile neurons in the major pelvic ganglion. However, it is significant that the hypogastric nerve is preganglionic to about 20% of penile neurons. The pathway through the hypogastric nerve may represent an alternate vasodilator system to penile erectile tissue.     

Distribution of [Met5]- and [Leu5]-enkephalin-, vasoactive intestinal polypeptide- and substance P-like immunoreactivities in human adrenal glands

There is recent evidence that the amine storing cells of mammalian adrenal medulla also contain bioactive peptides. In the present study we examined human adrenal glands with the immunoperoxidase-bridge method using specific antisera raised against [Met⁵]- and [Leu⁵]-enkephalin, vasoactive intestinal polypeptide hormone (VIP), and substance P. Approximately one-third of the cells in the adrenal medulla demonstrated enkephalin-like immunoreactivity. The intensity of the immunostain varied among individual cells but did not appear to correlate with amine content, as determined by the formaldehyde-induced fluorescence of catecholamines. An abundant network of varicose fibre-like structures and dots, representing preterminal and terminal nerves, demonstrated vasoactive intestinal polypeptide-like immunoreactivity and were found in close proximity to medullary gland cells. Substance P-like immunoreactivity was observed in a few fibres in the medulla and cortex. However, we could not detect cells containing vasoactive intestinal polypeptide- or substance P-like immunoreactivity in adrenal glands. p ]The present findings suggest that human adrenal medullary cells contain both [Met⁵]- and [Leu⁵]-enkephalin and are richly innervated by peptidergic nerves containing vasoactive intestinal polypeptide. These peptides may modulate the release and effects of catecholamines in the adrenal medulla. The nerves with substance P-like immunoreactivity may represent a separate peptidergic neuronal system.     

Neuropeptide Y co-exists with vasoactive intestinal polypeptide and acetylcholine in parasympathetic cerebrovascular nerves originating in the sphenopalatine, otic, and internal carotid ganglia of the rat

Neuropeptide Y co-exists with noradrenaline in the majority of the sympathetic nerves supplying cerebral blood vessels. However, after sympathectomy in the rat the number of cerebrovascular neuropeptide Y nerve fibers are only reduced in number despite a complete disappearance of the adrenergic markers. The origin of these non-sympathetic neuropeptide Y fibers was studied by nerve transections and retrograde axonal tracing utilizing True Blue. Three days after bilateral superior cervical sympathectomy, the number of neuropeptide Y-containing nerve fibers decreased to about 40% of that in non-treated animals. One week after True Blue application on the proximal portion of the middle cerebral artery, the tracer accumulated in neurons of the sphenopalatine, otic, and internal carotid ganglia. Of these cells 80%, 95% and 5%, respectively, were neuropeptide Y-positive. Some of the True Blue/neuropeptide Y-positive cells displayed immunoreactivity for vasoactive intestinal polypeptide and some were positive for choline acetyltransferase. Two weeks after bilateral removal of the sphenopalatine ganglion or transection of postganglionic fibers from the ganglion reaching the pial vessels through the ethmoidal foramen, together with subsequent sympathectomy, no neuropeptide Y-containing nerve fibers could be observed on the anterior cerebral and internal ethmoidal artery or the distal portion of the middle cerebral artery, whereas a few nerve fibers remained on the proximal portion of the middle cerebral artery, internal carotid artery, and the rostral portion of the basilar artery. In conclusion, neuropeptide Y in cerebrovascular nerves is co-stored not only with noradrenaline in sympathetic nerves from the superior cervical ganglion, but also with acetylcholine (reflected in the presence of choline acetyltransferase) and vasoactive intestinal polypeptide in parasympathetic nerves originating in the sphenopalatine, otic, and internal carotid ganglia.     

Target-related patterns of co-existence of neuropeptide Y, vasoactive intestinal peptide, enkephalin and substance P in cranial parasympathetic neurons innervating the facial skin and exocrine glands of guinea-pigs

The patterns of co-existence of neuropeptides in cranial autonomic neurons of guinea-pigs have been examined with quantitative double-labelling immunofluorescence and retrograde axonal tracing using Fast Blue. Within the sphenopalatine, otic, sublingual and submandibular ganglia, and a prominent intracranial ganglion associated with the glossopharyngeal nerve, most neurons contained immunoreactivity of vasoactive intestinal peptide, neuropeptide Y, enkephalin and substance P in combinations that were correlated with their projections. Hair follicles in the facial skin formed a major target of sphenopalatine ganglion cells. The combinations of peptides co-existing in these neurons depended upon the region of the skin where the follicles were located. The parotid gland was innervated by neurons with cell bodies in the otic ganglion or the intracranial ganglion. Most of these neurons contained immunoreactivity to all four peptides. The sublingual gland was innervated by local ganglion cells usually containing immunoreactivity to neuropeptide Y, vasoactive intestinal peptide and substance P. The submandibular gland was innervated by local ganglion cells containing enkephalin immunoreactivity and low levels of immunoreactivity to neuropeptide Y. Presumptive vasodilator neurons, containing immunoreactivity to vasoactive intestinal peptide but no other peptide examined here, comprised less than 10% of cranial autonomic ganglion cells. These results demonstrate that the patterns of co-existence of neuropeptides in cranial autonomic neurons show a high degree of target specificity. The discovery that hair follicles form a major parasympathetic target implies a broader range of actions of cranial autonomic neurons than has been suspected until now.     

Nitric oxide synthase, choline acetyltransferase, catecholamine enzymes and neuropeptides and their colocalization in the anterior pelvic ganglion, the inferior mesenteric ganglion and the hypogastric nerve of the male guinea pig

By the indirect immunofluorescence method, the distribution of nitric oxide synthase (NOS)-like immunoreactivity (LI) and its possible colocalization with neuropeptide immunoreactivities, with two enzymes for the catecholamine synthesis pathway, tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH), as well as the enzyme for the acetylcholine synthesis pathway, choline acetyltransferase (ChAT) were studied in the anterior pelvic ganglion (APG), the inferior mesenteric ganglion (IMG) and the hypogastric nerve in the male guinea pig. The analyses were performed on tissues from intact animals, as well as after compression/ligation or cut of the hypogastric nerve. In some cases the colonic nerves were also cut. Analysis of the APG showed two main neuronal cell populations, one group containing NOS localized in the caudal part of the APG and one TH-positive group lacking NOS in its cranial part. The majority of the NOS-positive neurons contained ChAT-LI. Some NOS-positive cells did not contain detectable ChAT, but all ChAT-positive cells contained NOS. NOS neurons often contained peptides, including vasoactive intestinal peptide (VIP), neuropeptide tyrosine (NPY), somatostatin (SOM) and/or calcitonin gene-related peptide (CGRP). Some NOS cells expressed DBH, but never TH. The second cell group, characterized by absence of NOS, contained TH, mostly DBH and NPY and occasionally SOM and CGRP. Some TH-positive neurons lacked DBH. In the IMG, the NOS-LI was principally in nerve fibers, which were of two types, one consisting of strongly immunoreactive, coarse, varicose fibers with a patchy distribution, the other one forming fine, varicose, weakly immunoreactive fibers with a more general distribution. In the coarse networks, NOS-LI coexisted with VIP- and DYN-LI and the fibers surrounded mainly the SOM-containing noradrenergic principal ganglion cells. A network of ChAT-positive, often NOS-containing nerve fibers, surrounded the principal neurons. Occasional neuronal cell bodies in the IMG contained both NOS- and ChAT-LI. Accumulation of NOS was observed, both caudal and cranial, to a crush of the hypogastric nerve. VIP accumulated mainly on the caudal side and often coexisted with NOS. NPY accumulated on both sides of the crush, but mainly on the cranial side, and ENK was exclusively on the cranial side. Neither peptide coexisted with NOS. Both substance P (SP) and CGRP showed the strongest accumulation on the cranial side, possibly partly colocalized with NOS. It is concluded that the APG in the male guinea-pig consists of two major complementary neuron populations, the cholinergic neurons always containing NOS and the noradrenergic neurons containing TH and DBH. Some NOS neurons lacked ChAT and could represent truly non-adrenergic, non-cholinergic neurons. In addition, there may be a small dopaminergic neuron population, that is containing TH but lacking DBH. The cholinergic NOS neurons contain varying combinations of peptides. The noradrenergic population often contained NPY and occasionally SOM and CGRP. It is suggested that NO may interact with a number of other messenger molecules to play a role both within the APG and IMG and also in the projection areas of the APG.     

Coexistence of peptide immunoreactivity in sensory neurons of the cat

The coexistence of the neuropeptides substance P, cholecystokinin, somatostatin and vasoactive intestinal polypeptide in cat sensory neurons has been examined using peroxidase-anti-peroxidase immunocytochemistry. Attempts were also made to locate cells containing bombesin, neurotensin, [Met]enkephalin and [Leu]enkephalin but no immunoreactivity was found when antisera to these peptides was used. Cells in the dorsal root ganglia were studied by cutting 5 microns serial wax sections or 15 microns cryostat sections. Coexistence was established by applying the antiserum to each peptide to serially adjacent 5 microns sections and establishing the presence of peptide-like immunoreactivity in each of 4 different sections through a single cell. Results showed that the distribution and combinations of coexistence of these neuropeptides in the cat is extremely complex; three and sometimes all four antisera showing immunoreactivity with a single cell. About 21% of all ganglion cells contained some immunoreactivity but there were certainly some small cells which did not contain any immunoreactivity. The coexistence of these peptides differed markedly from that previously reported in the rat suggesting that interspecific differences in the neuropeptide content of cells might be much greater than they are for classical neurotransmitters. The results are discussed in relation to the possible role of neuropeptides and the regulation of their production by sensory neurons.     

Parasympathetic innervation of cutaneous blood vessels by vasoactive intestinal polypeptide-immunoreactive and acetylcholinesterase-positive nerves: histochemical and experimental study on rat lower lip

The distribution and origin of perivascular acetylcholinesterase-active and vasoactive intestinal polypeptide-immunoreactive nerve fibers were studied in the rat lower lip by means of acetylcholinesterase histochemistry and vasoactive intestinal polypeptide immunohistochemistry. The perivascular nerve fibers stained intensely with both histochemical techniques and were widely distributed on small arteries and arterioles of the lower lip, especially in the transitional zone between the hairy skin and the mucous membrane. The distributions of the two types of fibers were very similar and most of them showed overlapping coloration, on consecutive staining for vasoactive intestinal polypeptide and acetylcholinesterase. Both acetylcholinesterase-positive and vasoactive intestinal polypeptide-immunoreactive fibers were completely lost on removal of the otic ganglion, while they were not affected by sympathetic ganglion removal or sensory nerve sectioning. In the otic ganglion, most cells exhibited acetylcholinesterase activity, and about 60% of the cells showed light to heavy vasoactive intestinal polypeptide immunoreactivity. These findings indicate that vessels in the rat lip are innervated by parasympathetic fibers originating from the otic ganglion and support the view that vasoactive intestinal polypeptide is present in cholinergic neurons. This may suggest the possible control by the parasympathetic nervous system of cutaneous blood vessels through vasoactive intestinal polypeptide-containing cholinergic neurons, in general or at least in the facial area.     

Target-specific projections of intrinsic ganglionic neurons with different chemical codes in the canine larynx.

The distribution and origin of peptide-containing intrinsic nerve fibers within the larynx were examined by immunohistochemistry and denervation experiments in the dog. In the normal larynx, a dense network of vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) fibers was seen around the acini of submucosal glands. VIP-, substance P (SP)-, or calcitonin gene-related peptide (CGRP)-IR fibers were seen in the walls of submucosal arteries, and VIP-, neuropeptide Y (NPY)-, or enkephalin (ENK)-IR fibers were seen around the arteries in the muscle tissue. Most of these peptide-IR fibers remained after bilateral denervation of the superior and inferior laryngeal nerves. Several small intrinsic ganglia were found along the peripheral branches of the laryngeal nerves. About 97% of the ganglionic neurons were VIP-IR; of these, 44% were immunoreactive to VIP alone, 22% to VIP and NPY, 13% to VIP and SP, 7% to VIP and ENK, and 14% to VIP, NPY and SP. These results reveal that the exocrine glands and blood vessels are innervated by the intrinsic ganglionic neurons and that subpopulations of ganglionic neurons with different chemical codes innervate specific target organs in the canine larynx.     

Neurotransmitters and neuropeptides within the mesencephalic trigeminal nucleus of the rat: an immunohistochemical analysis

In order to determine which neurotransmitters and neuropeptides are utilized by the neurons of the mesencephalic trigeminal nucleus and by the fibres making synaptic contact with these primary sensory cells, we have set up an immunohistochemical study using antibodies against 17 major neurotransmitters and neuropeptides in the rat. Apart from some intracellular immunostaining for glutamate, no immunoreactivity to any of the tested neurotransmitters and neuropeptides could be detected inside mesencephalic nucleus of the trigeminal nerve neurons. Our immunohistochemical observations indicate that mesencephalic nucleus of the trigeminal nerve neurons receive input from various nerve fibres that appear to utilize serotonin, GABA, dopamine, noradrenaline (and likely glutamate) as transmitters. The innervation appeared randomly distributed over all mesencephalic nucleus of the trigeminal nerve neurons. The presence of substance P, cholecystokinin, vasoactive intestinal polypeptide, bombesin/gastrin releasing peptide, [Leu]enkephalin and neuropeptide Y observed in some fibres that contact with mesencephalic nucleus of the trigeminal nerve neurons, presumably reflect the co-existence of these peptides with one of the neurotransmitters.