Immunohistochemical features of substance P-immunoreactive chromaffin cells and nerve fibers in the rat adrenal gland

The distribution of substance P (SP) immunoreactivity and the colocalization of SP with other bioactive substances in chromaffin cells and nerve fibers were investigated in the rat adrenal gland at the light microscopic level. In the capsule and cortex, SP immunoreactivity was seen in some nerve fibers around blood vessels and in thick nerve bundles passing through the cortex directly into the medulla. In the medulla, the SP immunoreactivity was observed in a small number of chromaffin cells; these SP-immunoreactive chromaffin cells were either phenylethanolamine N-methyltransferase (PNMT) immunoreactive or immunonegative, indicating that they were either adrenaline cells or noradrenaline (NA) cells. SP-immunoreactive varicose nerve fibers were also found in the medulla and were in contact with a cluster of the NA cells showing catecholamine fluorescence, which suggests that SP from medullary nerve fibers may regulate the secretory activity of the NA cells. Because no SP-immunoreactive ganglion cell was present in the rat adrenal gland, the intra-adrenal nerve fibers were considered to be extrinsic in origin. The double-immunostaining method further revealed that the SP-immunoreactive chromaffin cells also exhibit immunoreactivities for calcitonin gene-related peptide (CGRP), and neuropeptide tyrosine (NPY), suggesting that these peptides can also be released from the chromaffin cells by certain stimuli. The intra-adrenal nerve fibers in the medulla were composed of SP-single immunoreactive, and SP/CGRP-, SP/choline acetyltransferase (ChAT)-, SP/nitric oxide synthase (NOS)-, SP/pituitary adenylate cyclase activating polypeptide (PACAP)-, ChAT/NOS-, and ChAT/PACAP-immunoreactive nerve fibers, which may affect the secretory activity of the NA cells. In the adrenal capsule, the nerve fibers were present around blood vessels and showed immunoreactivities for SP/ CGRP, SP/NPY, SP/NOS, and SP/vasoactive intestinal polypeptide, suggesting that the origin of nerve fibers in the capsule may differ from those in the medulla.     

The co-localization of neuropeptides in the submucosa of the small intestine of normal Wistar and non-diabetic BB rats.

Immunocytochemical double and triple staining techniques were employed on whole mounts of the submucosal plexus from normal Wistar and non-diabetic BB rat jejunum and ileum, to determine the patterns of co-localization of vasoactive intestinal polypeptide-, peptide histidine-isoleucine-, somatostatin-, neuropeptide Y-, calcitonin gene-related peptide-, substance P-, and galanin-immunoreactive nerves. Neuropeptide Y immunoreactivity was found in 38% of submucosal plexus neurons, within the same neuronal elements as vasoactive intestinal polypeptide immunoreactivity (39% of submucosal plexus neurons) and peptide histidine-isoleucine immunoreactivity. A small population (1% of submucosal plexus neurons) containing vasoactive intestinal polypeptide- and peptide histide isoleucine-like immunoreactivity without NPY-like immunoreactivity was also observed. A significant population of fibers containing vasoactive intestinal polypeptide and galanin immunoreactivity were observed in the mucosa and submucosa, although no cell bodies were detected which contained both neuropeptides. Galanin-like immunoreactivity was seen in a small (2% of submucosal plexus neurons) population, not co-localized with any of the other neuropeptides examined. All somatostatin-immunoreactive neuronal elements (18% of submucosal plexus neurons) contained calcitonin gene-related peptide immunoreactivity, just over half of which also contained substance P immunoreactivity. An additional 25% of submucosal plexus neurons contained calcitonin gene-related peptide- without somatostatin-like immunoreactivity and 28% of submucosal plexus neurons contained substance P without somatostatin-like immunoreactivity. Some degree of co-localization was seen between calcitonin gene-related peptide- and substance P-like immunoreactivity, however, this could not be directly quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuronal pathways to the rat thyroid revealed by retrograde tracing and immunocytochemistry

The distribution and origin of the nerve fibres innervating the rat thyroid were studied by immunocytochemistry, retrograde tracing and denervation experiments. Immunocytochemistry revealed nerve fibres containing noradrenaline, neuropeptide Y, vasoactive intestinal peptide, peptide histidine-isoleucine, galanin, substance P, neurokinin A and calcitonin gene-related peptide around blood vessels and follicles. Many of these transmitter candidates were found to co-exist with each other in different combinations in different subpopulations of neurons. Sympathectomy eliminated all noradrenaline- and noradrenaline/neuropeptide Y-containing fibres in the thyroid. Cervical vagotomy eliminated about 50% of the galanin-, substance P- and calcitonin gene-related peptide-containing fibres. Local denervation (removal of the thyroid ganglion and the thyroid nerve) eliminated all galanin- and substance P-immunoreactive fibres and the majority of noradrenaline-, noradrenaline/neuropeptide Y-, vasoactive intestinal peptide- and calcitonin gene-related peptide-containing fibres in the thyroid gland. Injection of True Blue into the thyroid gland labelled cell bodies in the thyroid ganglion, the laryngeal ganglion, the superior cervical ganglion, the jugular-nodose ganglionic complex, the dorsal root ganglia (C2-C5) and the trigeminal ganglion. Judging from the number of labelled nerve cell bodies, the superior cervical ganglion and the thyroid ganglion contribute most to the thyroid innervation, while the laryngeal ganglion and the trigeminal ganglion contribute least. The True Blue-labelled ganglia were examined for the presence of various populations of nerve cell bodies (only major populations are listed). The thyroid ganglion harboured neuropeptide Y, vasoactive intestinal peptide and galanin/vasoactive intestinal peptide cell bodies (in order of predominance); the laryngeal ganglion galanin/vasoactive intestinal peptide, vasoactive intestinal peptide and calcitonin gene-related peptide cell bodies; the superior cervical ganglion noradrenaline/neuropeptide Y and noradrenaline cell bodies; the jugular ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies; the nodose ganglion vasoactive intestinal peptide and vasoactive intestinal peptide/galanin cell bodies; the dorsal root ganglia (C2-C5) and the trigeminal ganglion calcitonin gene-related peptide, substance P/calcitonin gene-related peptide and galanin/substance P/calcitonin gene-related peptide cell bodies.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Ultrastructural localization of increased neuropeptide immunoreactivity in the axons and cells of the gracile nucleus following chronic constriction injury of the sciatic nerve.

Neuropeptide plasticity in the gracile nucleus is thought to play a role in the development of neuropathic pain following nerve injury. Two weeks after chronic constriction injury of adult rat sciatic nerve, galanin, neuropeptide Y and calcitonin gene-related peptide immunoreactivities were increased in fibers and cells in the gracile nucleus ipsilateral to injury. At the electron microscopic level, this increased neuropeptide immunoreactivity was localized in myelinated axons, boutons, dendrites, neurons and glial cells. Galanin-, neuropeptide Y- and calcitonin gene-related peptide-immunoreactive boutons were frequently presynaptic to dendrites of both immunoreactive and non-immunoreactive neurons. However, no neuropeptide Y, galanin and calcitonin gene-related peptide messenger RNA was detected in the injured side gracile nuclei by in situ hybridization. These results show that partial nerve injury to the sciatic nerve induces increases in the content of galanin, neuropeptide Y and calcitonin gene-related peptide immunoreactivities in synaptic terminals within the gracile nucleus, which suggests that there may be increased release of these neuropeptides following sensory or spontaneous stimulation of large-diameter primary afferents following partial nerve injury, perhaps one mechanism involved in neuropathic pain. We also show an apparent transfer of these neuropeptides to the cells of the gracile nucleus, both neurons and glial cells, an intriguing phenomenon of unknown functional significance.     

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.     

Motor response of the human isolated colon to capsaicin and its relationship to release of vasoactive intestinal polypeptide

The aim of this study was to obtain indirect evidence of the presence of capsaicin-sensitive afferents in the human colon by studying the motor response to capsaicin of longitudinal strips from the human isolated taenia coli in parallel to the ability of capsaicin or KCl to induce peptide release from the human superfused colon. Capsaicin (1 microM) evoked a relaxation of the taenia, approaching 60-80% of the response to isoprenaline. Tachykinins evoked contractions of the taenia, while calcitonin gene-related peptide induced a relaxation. Neither tachyphylaxis to calcitonin gene-related peptide nor preincubation with an anti-calcitonin gene-related peptide serum did block the response to capsaicin which was also unaffected by tetrodotoxin, apamin, naloxone or an anti-galanin serum. Vasoactive intestinal polypeptide produced a concentration-dependent tetrodotoxin-resistant relaxation which was shifted rightward in the presence of anti-vasoactive intestinal polypeptide serum. The anti-vasoactive intestinal polypeptide serum reduced the response to capsaicin and application of capsaicin prevented the ability of anti-vasoactive intestinal polypeptide serum to block exogenous vasoactive intestinal polypeptide. Capsaicin (1 microM) evoked a significant release of vasoactive intestinal polypeptide-like immunoreactivity from the superfused muscle but not mucosa of the human colon. A significant vasoactive intestinal polypeptide-like immunoreactivity release was also observed in response to KCl (80 mM). KCl but not capsaicin evoked a significant release of neurokinin A-like immunoreactivity from colonic muscle and mucosa. No significant release of either substance P-, neuropeptide Y-, galanin- or calcitonin gene-related peptide-like immunoreactivity was detected in response to capsaicin or KCl although detectable levels of each peptide were evident in tissue extracts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and origins of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibers in the pineal gland of gerbils

In the pineal gland of gerbils, substance P (SP)-, calcitonin gene-related peptide (CGRP)-, vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibers were demonstrated immunohistochemically. After intrapineal injection of biotin-wheat germ agglutinin, origins of fibers were examined by the combined technique of tracing method and immunohistochemistry. It was confirmed that SP- and CGRP-fibers originated from the trigeminal ganglion, VIP-fibers from the pterygopalatine ganglion and NPY-fibers from the superior cervical ganglion.     

The peptidergic innervation of human coronary and cerebral vessels

It is now well established that in addition to nerves containing classical transmitters, the mammalian vascular system is also supplied by nerve fibre subpopulations containing several vasoactive peptides. The precise function of these peptides (neuropeptide Y, calcitonin gene-related peptide, vasoactive intestinal polypeptide, somatostatin and the tachykinins) is still unknown, however, their widespread occurrence in perivascular nerves indicates that they are likely candidates for a role in the neurogenic regulation of the vascular system. It has been suggested that they may exert a direct vasomotor action via their own receptors and/or modulate the release and action of other vascular transmitters. Recently, several studies have focused on the supply of nerve fibres storing neuropeptides in the coronary and cerebral vasculature of laboratory animals, however, little is known on the distribution of these putative transmitters in human coronary and cerebral vessels. In this paper, the immunocytochemical evidence that several neuropeptides are localized in subpopulations of afferent and efferent nerve fibres supplying the human coronary and cerebral vasculature is focused.     

Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea-pig

Retrograde tracing of the fluorescent marker, True Blue, has been used together with immunohistochemistry employing antibodies to substance P, calcitonin gene-related peptide, somatostatin, vasoactive intestinal polypeptide and morphine-modulating peptide to study the afferent innervation of the stomach in rat, mouse and guinea-pig. Up to 85% of spinal afferents to the stomach in all three species contained immunoreactive calcitonin gene-related peptide, and up to 50% contained substance P. In all three species less than 10% of vagal afferents to the stomach reacted with antibodies to calcitonin gene-related peptide, or substance P. Cacitonin gene-related peptide-immunoreactive fibres were found in the myenteric plexus, circular muscle and around submucosal blood vessels in the stomach. In the rat, removal of the coeliac ganglion, splanchnic nerve section, or capsaicin treatment virtually abolished calcitonin gene-related peptide immunoreactivity in the stomach. Capsaicin and splanchnic section also abolished the staining of immunoreactive calcitonin gene-related peptide fibres in the coeliac ganglion. The same treatments abolished substance P staining of fibres around submucosal blood vessels, but in the myenteric plexus and circular smooth muscle there were still abundant immunoreactive fibres, presumably arising from intrinsic cell bodies. No somatostatin-containing visceral afferents could be found, although somatostatin was localized to cell bodies in rat dorsal root ganglia. Immunoreactive vasoactive intestinal polypeptide-containing dorsal root ganglia neurons were not found; although antibodies to morphine-modulatory peptide revealed immunoreactive nerve cell bodies, we were unable to exclude the possibility that this result is attributable to cross reactivity with calcitonin gene-related peptide. These results provide direct evidence that calcitonin gene-related peptide is a marker for a major subset of visceral primary afferent neurons and suggest that this population of spinal afferents makes a major contribution to the total gastric content of calcitonin gene-related peptide.     

Marked increases in calcitonin gene-related peptide-containing nerves in the developing rat following long-term sympathectomy with guanethidine

Changes in the innervation of the cardiovascular system, urinogenital tract and sympathetic and non-sympathetic ganglia have been examined following long-term sympathectomy. Patterns of innervation were investigated using histochemical and immunohistochemical techniques, while levels of noradrenaline and neuropeptides were measured by neurochemical assays. Large doses of guanethidine (50 mg/kg) were given daily for 3 weeks to 8-day-old rat pups, which were killed at 6 or 20 weeks of age. In both age groups noradrenergic nerves were severely depleted or absent, while in some regions dramatic increases of calcitonin gene-related peptide levels were demonstrated. This was revealed by an increase in the density of nerve fibres and in calcitonin gene-related peptide content (up to 18-fold), most notably in the right atrium and superior cervical ganglion. No changes in substance P- or vasoactive intestinal polypeptide-immunolabelled nerves were seen. Conversely, short-term sympathectomy by 6-hydroxy-dopamine treatment caused a depletion of noradrenaline which was not accompanied by an increase in the number or content of calcitonin gene-related peptide-immunolabelled nerves. The possibility that nerve growth factor is involved in the mechanism of hyperinnervation by calcitonin gene-related peptide-containing sensory nerves following long-term sympathectomy is discussed.     

Changes in nerves and neuropeptides in skin from 100 leprosy patients investigated by immunocytochemistry

The cutaneous innervation is now known to contain neuropeptides including substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory nerves, and vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY), principally in autonomic nerves. Skin biopsies from 100 leprosy patients and equivalent areas from 50 non-leprosy controls were fixed in p-benzoquinone solution for immunofluorescence staining and in Bouin's fluid for classification of leprosy type. Antisera to the neural markers, neurofilaments, and protein gene product 9.5 (PGP 9.5), and to neuropeptides were used. Cutaneous nerves and nerve endings immunoreactive for neuropeptides, neurofilaments, and PGP 9.5 were seen in all non-leprous control cases. In leprosy, PGP 9.5- and neurofilament-immunoreactive nerve fibres were seen in all 14 cases of the indeterminate (early) type and in the majority (33/43) of lepromatous cases, but in a smaller proportion (15/43) of tuberculoid cases. Neuropeptide immunoreactivity was seen in only 2/14 of the indeterminate leprosy specimens and was completely absent in other types. This early disappearance may be of diagnostic significance. Thus, cutaneous sensory and autonomic dysfunctions in leprosy are well reflected by changes in nerve fibres and neuropeptides.     

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.     

Uptake of 5-hydroxydopamine into non-sympathetic nerves of guinea-pig uterine artery in late pregnancy

Summary Perivascular nerve fibres of the uterine artery of virgin and late pregnant guinea-pigs were examined under the electron microscope following loading with 5-hydroxydopamine, a marker for catecholamine uptake, and immunohistochemistry for dopamine hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide. Varicosities, loaded with 5-hydroxydopamine labelled vesicles, and immunoreactive axons were counted in whole transverse sections of uterine arteries. Localization of the immunoreactivities in 5-hydroxydopamine-labelled vesicles was also studied. Colocalization of substance P and dopamine beta hydroxylase immunoreactivities was investigated at the light microscopic level. Both total and relative number of varicosities with 5-hydroxydopamine-labelled vesicles in a whole section of the artery increased in late pregnancy (61.2 ± 10.2 versus 24.5 ± 3.2 in virgin, representing 35% and 27% respectively, of all varicosities). Also the number of neuropeptide Y, vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons increased, but their relative proportion remained unchanged. In virgin guinea-pigs only calcitonin gene-related peptide and neuropeptide Y immunoreactivities were associated with varicosities loaded with small dense-cored vesicles, while in late pregnancy 5-hydroxydopamine-labelled vesicles were also seen in a number of vasoactive intestinal polypeptide, substance P and calcitonin gene-related peptide-immunoreactive axons. Double immunolabelling for dopamine hydroxylase and substance P immunoreactivity showed that substance P immunoreactivity was not present in dopamine hydroxylase-immunoreactive axons of the uterine artery, of neither virgin nor late pregnant guinea-pigs. It is concluded that vascular hypertrophy of the uterine artery in late pregnancy is associated with an increase in the number of perivascular nerve fibres, that involves many, if not all of the subpopulations of neurons supplying the uterine artery. Also 5-hydroxydopamine-labelled varicosities were increased, but the results of the present study indicate that some of the nerve fibres that are able to take up 5-hydroxydopamine in late pregnancy are not sympathetic (i.e are sensory and/or parasympathetic in origin). The relevance of these findings in pregnancy is discussed.     

Neuropeptides and neurotransmitter-synthesizing enzymes in intrinsic neurons of the human urinary bladder

Summary The expression of neuropeptides, and the enzymes nitric oxide synthase and tyrosine hydroxylase were examined in intramural ganglia of human urinary bladder using single label immunocytochemistry. Scattered ganglia composed of between 1–36 neurons (median 4) were observed in all layers of the lateral wall of the bladder. These contained immunoreactivity to vasoactive intestinal peptide, nitric oxide synthase, neuropeptide Y, and galanin. Neurons within the bladder were heterogeneous with regard to their content of these antigens, with the proportion of immunopositive cells ranging from 58–84%. Occasional neurons with immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, were also observed. No cell somata, however, were immunoreactive for enkephalin, substance P, calcitonin generelated peptide or somatostatin. Varicose terminals entering the ganglia were seen to form pericellular baskets surrounding some of the principal ganglion cells. The most prominent pericellular varicosities were those containing calcitonin gene-related peptide- or vasoactive intestinal peptide-immunoreactivity, followed by those with immunoreactivity for enkephalin, neuropeptide Y, or galanin. Less common were pericellular varicosities with substance P-immunoreactivity, which may represent collateral processes of unmyelinated primary sensory fibres, and presumptive noradrenergic processes containing tyrosine hydroxylase. Some calcitonin gene-related peptide-immunoreactive varicosities constituted a distinct type, terminating as large pericellular boutons 2–4 m in diameter. Fibres containing nitric oxide synthase- or somatostatin-immunoreactivity were not associated with the intramural neurons. The results demonstrate that intrinsic neurons within the human urinary bladder express a number of neuroactive chemicals, and could in principle form circuits with the potential to support integrative activity.     

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.     

Some nerve endings in the rat pelvic paracervical autonomic ganglia and varicosities in the uterus contain calcitonin gene-related peptide and originate from dorsal root ganglia

The pelvic paracervical autonomic ganglia of female rats were studied for a subpopulation of nerve endings that could be derived from sensory nerve fibers. Immunohistochemical staining using an antiserum against the synaptic-terminal protein synapsin I was used to identify terminal boutons, while an antiserum against the neuropeptide calcitonin gene-related peptide was used to reveal a subpopulation of sensory nerve fibers. The uterine cervix was also examined for the existence of calcitonin gene-related peptide and synapsin I immunoreactivity in nerve fiber varicosities. In addition, the location of nerve endings in the paracervical ganglion was compared to that in the superior cervical ganglion. Synapsin I immunoreactivity was present in the paracervical ganglion in abundant boutons around neuron somata and in the cervix in varicose nerve fibers of the myometrium, vasculature and epithelium. Double labeling immunocytochemistry revealed calcitonin gene-related peptide-like immunoreactivity in subpopulations of synapsin I-immunoreactive endings in ganglia and nerve varicosities in the cervix. Injection of a retrograde axonal tracer, fluorogold, into the paracervical ganglion produced labeled neurons in dorsal root ganglia and spinal cord; however, fluorogold-labeled neurons containing calcitonin gene-related peptide immunoreactivity were visualized only in dorsal root ganglia. Injections of fluorogold into the uterine cervix produced labeled neurons in the paracervical ganglion and dorsal root ganglia; however, only those in dorsal root ganglia contained immunoreactivity for calcitonin gene-related peptide. These results suggest that immunoreactivity for calcitonin gene-related peptide is present in a subpopulation of nerve endings in the paracervical ganglion and not merely in fibers of passage. The nerve endings in the ganglion and varicosities in the uterine cervix originate from sensory neurons in dorsal root ganglia. The arrangement of endings in the ganglia could play a role in sensory/autonomic interactions for modulation of visceral activity.     

Co-localization of calcitonin gene-related peptide- and substance P-like immunoreactivity in mucosal intra-epithelial nerves in the toad colon.

Mucosal intra-epithelial nerve terminals have been found in the colon of the toad, Bufo marinus. Co-localized in the fibres are calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and substance P (SP)-LI. The intraepithelial nerves are likely to be terminals of primary afferent sensory neurones. Fibres with vasoactive intestinal peptide (VIP)-LI or neuropeptide Y (NPY)-LI also supply the mucosa but do not penetrate the epithelium.     

Gamma‐aminobutyric acid B Receptor Immunoreactivity in the Mouse Adrenal Medulla

The present study examined gamma‐aminobutyric acid B (GABA_B) receptor, GABA, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) immunoreactivities in the mouse adrenal medulla. GABA_B receptor immunoreactivity was seen in numerous chromaffin cells and in a few ganglion cells of the adrenal medulla. By using a formaldehyde‐induced fluorescence (FIF) method, GABA_B receptor immunoreactivity was observed in numerous adrenaline (A) cells, but not in noradrenaline (NA) cells showing blue‐white fluorescence. This suggests that GABA_B receptors may be present in the A cells and be related to the secretory activity of A cells but not NA cells in the mouse adrenal medulla. GABA_B receptor immunoreactive ganglion cells were shown to be nNOS immunopositive by using a double immunostaining method. Weak GABA immunoreactivity was visible in some chromaffin cells and in the numerous nerve fibers of the medulla. By using the FIF method, weak GABA‐immunoreactive chromaffin cells were shown to be in the NA cells showing blue‐white fluorescence. GABA‐immunoreactive nerve fibers were in dense contact in A cells, but not NA cells. GABA‐immunoreactive nerve fibers closely contacted a few ganglion cells. Numerous GABA‐immunoreactive nerve fibers in the medulla showed ChAT immunoreactive. This result suggests that GABA and acetylcholine may be released from the same nerve fibers and may have a secretory effect on the A cells of the medulla. Anat Rec, 296:971–978, 2013. © 2013 Wiley Periodicals, Inc.