Distribution and origin of secretoneurin-immunoreactive nerves in the female rat uterus

Secretoneurin is a 33-amino acid peptide derived from secretogranin II. Secretoneurin immunoreactivity has been localized in the peripheral nervous system where it exerts potent chemotactic activity for monocytes and may play a role in inflammation. Secretoneurin could play a role in this process, although the presence and distribution of secretoneurin-immunoreactive neurons in the female reproductive system has not been documented. Thus, this study was undertaken to examine secretoneurin immunoreactivity in nerves of the rat uterus and uterine cervix. A moderate plexus of secretoneurin-immunoreactive nerve fibers was present in the myometrium and endometrium of the uterus as well as in the smooth muscle and endocervix of the cervix. Many of these fibers were associated with the vasculature as well as the myometrium. Secretoneurin immunoreactivity was present in small- to medium-sized neurons of dorsal root and nodose ganglia. Retrograde tracing with FluoroGold indicated that some of these sensory neurons project axons to the cervix and uterine horns. Secretoneurin-immunoreactive terminal-like structures were associated with neurons in the sacral parasympathetic nucleus of the lumbosacral spinal cord. In addition, some secretoneurin terminals were apposed to pelvic parasympathetic neurons in the paracervical ganglia that projected axons to the uterus and cervix. Double-immunostaining indicated co-existence of calcitonin gene-related peptide or substance P with secretoneurin in some sensory neurons, in some terminals of the pelvic ganglia, as well as nerve fibers in the uterine horn and cervix. Finally, fibers in the uterus and cervix were depleted of secretoneurin by capsaicin treatment. This study indicates that secretoneurin is present in the uterus in C-afferent nerve fibers whose cell bodies are located in sensory ganglia. Some of these fibers contain both secretoneurin and calcitonin gene-related peptide or substance P. These substances have functions in inflammatory reactions. Further, secretoneurin could influence postganglionic parasympathetic "uterine-related" neurons in the pelvic ganglia and preganglionic parasympathetic neurons in the lumbosacral spinal cord.     

Distribution of NADPH-diaphorase-positive nerves in the uterine cervix and neurons in dorsal root and paracervical ganglia of the female rat.

Nitric oxide synthase (NOS) was selectively stained in nerve fibers of the uterine cervix and neurons of the paracervical (PG) and dorsal root ganglia (DRG) by NADPH diaphorase histochemistry. In the cervix, numerous NADPH-diaphorase-positive nerve fibers were observed in the myometrium, endometrium and around arteries. In addition, a subpopulation of neurons within ganglia that innervate the cervix, i.e., the PG and DRG, were NADPH-diaphorase positive; thus the fibers in the cervix could be sensory and/or autonomic. NADPH-diaphorase/NOS localization identifies sites where nitric oxide (NO) can be synthesized. Since NO relaxes vascular and nonvascular smooth muscle, the prevalence and anatomical localization of NADPH-diaphorase-positive fibers suggest that they could influence functions of the uterine cervix.     

Distribution of five peptides,three general neuroendocrine markers,and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: An immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I–II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I–III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I–II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

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.     

Calcitonin gene-related peptide-positive neurons and fibers in the cat dorsal column nuclei

An antiserum raised against the C-terminal region of rat alpha-calcitonin gene-related peptide has been used to investigate the morphology and topographical distribution of neurons and terminals containing calcitonin gene-related peptide in the cat dorsal column nuclear complex. Calcitonin gene-related peptide-positive fibers and axon terminals were denser in the cuneate nucleus than in the other dorsal column nuclei subdivisions and were observed throughout all rostrocaudal levels. They were densest in the dorsal and ventrolateral portions of the middle cuneate. Immunoreactive neurons were observed only in animals pre-treated with colchicine. In these cases, some calcitonin gene-related peptide-positive neurons were present in the cuneate and in the external cuneate. In double-labeling experiments, visualization of calcitonin gene-related peptide immunoreactivity in dorsal root ganglia neurons was combined with the retrograde transport of colloidal gold-labeled wheat germ agglutinin conjugated to inactive horseradish peroxidase injected in the cuneate nucleus. These experiments show that calcitonin gene-related peptide-positive fibers in the cuneate nucleus originate mostly from C3-C6 medium sized dorsal root ganglia neurons but also from some small and large neurons. These results suggest that calcitonin gene-related peptide-positive fibers may convey sensory information from a wide range of peripheral receptors.     

Distribution of TRPV1- and TRPV2-immunoreactive afferent nerve endings in rat trachea

Nociception in the trachea is important for respiratory modulation. We investigated the distribution, neurochemical characteristics, and origin of nerve endings with immunoreactivity for candidate sensor channels, TRPV1 and TRPV2, in rat trachea. In the epithelial layer, the intraepithelial nerve endings and dense subepithelial network of nerve fibers were immunoreactive for TRPV1. In contrast, TRPV2 immunoreactivity was observed mainly in nerve fibers of the tracheal submucosal layer and in several intrinsic ganglion cells in the peritracheal plexus. Double immunostaining revealed that some TRPV1-immunoreactive nerve fibers were also immunoreactive for substance P or calcitonin gene-related peptide, but neither neuropeptide colocalized with TRPV2. Injection of the retrograde tracer, fast blue, into the tracheal wall near the thoracic inlet demonstrated labeled neurons in the jugular, nodose, and dorsal root ganglia at segmental levels of C2-C8. In the jugular and nodose ganglia, 59.3% (70/118) and 10.7% (17/159), respectively, of fast blue-labeled neurons were immunoreactive for TRPV1, compared to 8.8% (8/91) and 2.6% (5/191) for TRPV2-immunoreactive. Our results indicate that TRPV1-immunoreactive nerve endings are important for tracheal nociception, and the different expression patterns of TRPV1 and TRPV2 with neuropeptides may reflect different subpopulations of sensory neurons.     

Differential expression of synaptoporin and synaptophysin in primary sensory neurons and up-regulation of synaptoporin after peripheral nerve injury

Synaptoporin and synaptophysin are integral membrane components of synaptic vesicles. The distribution of synaptoporin and its relationship with synaptophysin in sensory afferent fibers remain unclear. In the present study, we showed that in the rat dorsal root ganglia synaptoporin was expressed in subsets of small neurons that contain either calcitonin gene-related peptide or isolectin B4, and was distributed in their afferent terminals in laminae I-II of the spinal cord. Synaptophysin was expressed in 57% of synaptoporin-containing small dorsal root ganglion neurons and in large dorsal root ganglion neurons. In the spinal dorsal horn, synaptophysin-immunolabeling was weak in the afferent fibers in lamina I, outer lamina II and the dorsal part of inner lamina II, but strong in the afferent fibers in laminae III-IV. However, a subpopulation of isolectin B4-positive small dorsal root ganglion neurons expressed both synaptoporin and synaptophysin, and their afferent fibers were mainly distributed in the ventral part of inner lamina II. After peripheral nerve injury, synaptoporin expression was up-regulated in small dorsal root ganglion neurons, and synaptoporin level was increased in their afferent terminals. Thus, synaptoporin and synaptophysin have topographically distinct distributions in afferent fibers. Synaptoporin is a major synaptic vesicle protein in Adelta- and C-fibers in both physiological and neuropathic pain states.     

Long-term changes in the distribution of galanin in dorsal root ganglia after sciatic or spinal nerve transection in rats

The neuropeptide galanin is upregulated in primary afferent and sympathetic neurones and might be involved in the development of sympathetic perineuronal baskets ("rings") following nerve injury. Galanin, calcitonin gene-related peptide and tyrosine hydroxylase have been examined immunohistochemically in dorsal root ganglia and associated roots at times up to one year after transection of either sciatic or L5 spinal nerves in adult rats. Small diameter somata containing calcitonin gene-related peptide (with or without galanin) were reduced in number, whereas galanin (and, at later times, calcitonin gene-related peptide) appeared in medium to large diameter cells after both types of lesion. Galanin also appeared in axons in grey rami and somata in lumbar paravertebral ganglia. Within dorsal root ganglia, galanin-positive axons formed perineuronal rings of two types: (i) smooth coiled axons surrounded small (< 30 microm diameter) somata from which they probably arose; these were rare after 12 weeks, particularly after a spinal nerve lesion; and (ii) varicose terminals encircled medium to large galanin-positive somata; some arose from brightly immunofluorescent somata nearby and took nearly a year to disappear. About 30% of varicose galanin-positive rings had associated calcitonin gene-related peptide-positive terminals (partly colocalized) whereas nearly 45% had associated tyrosine hydroxylase-positive terminals (partly colocalized). Synaptophysin was present in swollen axons and in some varicosities of all types.We conclude that, after peripheral nerve lesions, varicose perineuronal rings around large diameter dorsal root ganglion cells may be formed by axotomized primary afferent neurones (some containing calcitonin gene-related peptide) and sympathetic neurones, both of which contain upregulated galanin. Exocytosis from the varicosities may modify the excitability of mechanosensitive somata. Small galanin-positive somata disappear over several months after both lesions as calcitonin gene-related peptide reappears in medium to large neurones.     

Ontogeny of acetylcholinesterase, substance P and calcitonin gene-related peptide-like immunoreactivity in chick dorsal root ganglia.

The distribution of acetylcholinesterase and of two neuropeptide (substance P and calcitonin gene-related peptide) immunoreactivities has been investigated in sensory neurons of lumbosacral dorsal root ganglia during chick embryo development, combining immunolocalization of neuropeptides with simultaneous histochemical detection of acetylcholinesterase, in order to study co-localization of the two peptides and their relations with acetylcholinesterase. Acetylcholinesterase at E7 of development appears in only a few neurons, usually the larger ones located in the lateroventral region of the ganglia. As development proceeds the number of neurons and intensity of staining increase. Until E12-13 acetylcholinesterase positivity is limited to the region of the ganglion containing larger neurons. At later stages (E20) it spreads progressively, leading to staining of cells over the whole ganglion. Substance P-like immunoreactivity appears at E6 and for calcitonin gene-related peptide at E7. These immunoreactivities progressively increase with development, remaining limited to the small neuron compartment of the dorsomedial region of the ganglion. Immunoreactivity for both neuropeptides reaches a maximum around E10-13 and then declines. Using simultaneous double immunostaining, calcitonin gene-related peptide and substance P-like immunoreactivities are largely co-localized, although their distribution is not completely coincident. Neuropeptide-positive cells are usually devoid of any acetylcholinesterase activity until E15. They become positive for the enzyme at later stages. The significance of acetylcholinesterase expression in sensory neurons and the possible relation of its appearance and neuron size is discussed.     

Calcitonin gene-related peptide in primary afferent neurons of rat: co-existence with fluoride-resistant acid phosphatase and depletion by neonatal capsaicin

Immunohistochemical and histochemical techniques were used to re-examine the extent to which neonatal capsaicin treatment depletes calcitonin gene-related peptide in the dorsal horn of the spinal cord, to determine the localization of calcitonin gene-related peptide in relation to that of fluoride-resistant acid phosphatase in lumbar dorsal root ganglia, and to compare the distribution of these primary afferent markers in the dorsal horn. A substantial depletion of calcitonin gene-related peptide was observed in the dorsal horn of adult rats treated neonatally with capsaicin suggesting that a large proportion of this peptide in the dorsal horn is contained within capsaicin-sensitive primary afferent fibers. In dorsal root ganglia 30% of all or 44% of small- and medium-sized calcitonin gene-related peptide-immunoreactive cells were positive for fluoride-resistant acid phosphatase. Conversely, 50% of cells positive for the phosphatase enzyme also displayed immunoreactivity for the peptide. In lamina II of the dorsal horn calcitonin gene-related peptide and fluoride-resistant acid phosphatase were found to have an overlapping distribution. The presence of fluoride-resistant acid phosphatase in a substantial proportion of neuropeptide-containing primary sensory neurons suggests a lack of segregation of sensory neuronal populations into peptide- and non-peptide-containing subgroups at least on the basis of non-peptide neurons defined as those containing fluoride-resistant acid phosphatase.     

Distribution of neurons expressing calcitonin gene-related peptide mRNAs in the brain stem, spinal cord and dorsal root ganglia of rat and guinea-pig

In situ hybridization histochemistry was used to localize calcitonin gene-related peptide mRNAs in spinal cord, brain stem and dorsal root ganglion neurons of the rat and guinea-pig. A 32P-labeled 23-base-long (23mer) oligodeoxyribonucleotide (oligomer) complementary to calcitonin gene-related peptide mRNA sequences encoding residues 23-30 of calcitonin gene-related peptide was used primarily as a probe (CGRP I probe). A 32mer complementary to mRNA sequences for residues 10-20 of calcitonin gene-related peptide (CGRP II probe) was also used as a positive control for specificity of the 23mer for calcitonin gene-related peptide mRNA. In both the guinea-pig and rat calcitonin gene-related peptide mRNA was localized specifically to neurons of the dorsal root ganglion, to spinal motoneurons and to motoneurons of the hypoglossal, facial and accessory facial motor nuclei. Differences in the distribution of calcitonin gene-related peptide mRNA between the rat and guinea-pig included a higher proportion of rat dorsal root ganglion neurons containing calcitonin gene-related peptide mRNA and the localization of calcitonin gene-related peptide mRNA to motoneurons of the ambiguus motor nucleus, parabrachial and peripeduncular nucleus of the rat but not the guinea-pig. In the guinea-pig, in contrast, calcitonin gene-related peptide mRNA was localized also to motoneurons of the abducens, trigeminal, trochlear and oculomotor nerves. The neuronal groups in the intact rat found here to contain calcitonin gene-related mRNA have also been shown previously to contain calcitonin gene-related peptide immunoreactivity in colchicine-treated rats. Colchicine-treated rats, however, have been found to contain additional groups of calcitonin gene-related peptide immunoreactive neurons which, in the intact rats used in the present study, showed no detectable hybridization with the calcitonin gene-related peptide probe.     

The coexistence of neuropeptides in feline sensory neurons

The coexistence of immunoreactivity to the peptides substance P, bombesin, calcitonin gene-related peptide and somatostatin has been determined in single, lumbar and sacral dorsal root ganglion cells in the cat. Colchicine pretreated L7 and S1 dorsal root ganglia were embedded in wax and cut into 5 microns sections. Groups of four, serially adjacent sections were reacted with antisera to one of four peptides using avidin-biotin immunocytochemistry. It was thus possible to determine the coincidence of the four peptides in single cell bodies by examining the immunoreactivity in a ganglion cell in one section and then locating the same cell in three adjacent sections. As a comparison, this procedure was repeated on a different population of ganglion cells using antiserum to substance P, bombesin and calcitonin gene-related peptide only. The results indicate that different combinations of three or four peptides may occur in single, small diameter sensory neurons in the cat. It would appear that immunoreactivity to bombesin and/or calcitonin gene-related peptide coexists with immunoreactivity to substance P in some dorsal root ganglion cells. However, immunoreactivity to each of these peptides was also found to occur alone in single cells. Immunoreactivity to calcitonin gene-related peptide but not to the other three peptides was found to occur in some medium-sized cell bodies (up to 70 microns). Somatostatin-like immunoreactivity was found to have a high level of coexistence with substance P-like immunoreactivity in cells which contained immunoreactivity to these two peptides only. Immunoreactivity to all the four peptides tested was found to occur in 18-26% of ganglion cells which contained at least one peptide.     

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.     

Histochemical characterization of sensory neurons with high-affinity receptors for nerve growth factor

Summary Approximately one half of the neurons in the lumbar dorsal root ganglion of adult rats display high-affinity receptors for nerve growth factor (NGF). To ascertain which types of sensory neurons are potentially responsive to NGF, adjacent cryostat sections of rat dorsal root ganglia were processed either for NGF-receptor using radioautography or by one of four histochemical procedures. Histograms of the densities of neuronal labelling by radioiodinated NGF were examined for subpopulations of lumbar sensory neurons with thiamine monophosphatase enzyme activity or with immunoreactivity for calcitonin gene-related peptide (CGRP), substance P, or somatostatin. Virtually all neurons with strong CGRP immunoreactivity had high-affinity NGF binding sites, although some neurons with faintly positive CGRP immunoreactivity lacked such NGF binding. A subpopulation of large neurons, approximately 5% of the total, had dense labelling by¹²⁵I-NGF but were not stained by this immunohistochemical technique for CGRP. Of the three major populations of small neurons those with substance P immunoreactivity were consistently and heavily labelled by radioiodinated NGF whereas those with somatostatin immunoreactivity or thiamine monophosphatase activity were not specifically labelled by radioautography. For these primary sensory neurons in mature rats the genes for substance P and CGRP seem to be strongly expressed only in neurons capable of responding to NGF. On the other hand, neurons containing somatostatin and thiamine monophosphatase invariably lack high-affinity NGF receptors.     

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.     

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)     

Comparative study on the innervation of nerves with calcitonin gene-related peptide, substance P and neurokinin A immunoreactivity in the walls of the cerebral arteries of small bats (Mammalia: Microchiroptera).

The distribution and origin of nerves with calcitonin gene-related peptide, substance P or neurokinin A immunoreactivity in the walls of the cerebral arteries were investigated in three microchiropteran species. The supply of nerves immunoreactive for substance P and neurokinin A to the bat cerebral arteries is confined mostly to the vertebral and basilar arteries. The density of innervation of calcitonin gene-related peptide-immunoreactive nerves and that of nerves with substance P or neurokinin A immunoreactivity in the vertebrobasilar system differ among species: the Japanese large footed bat is innervated with nerves with calcitonin gene-related peptide, substance P and neurokinin A immunoreactivity with about the same density, whereas in the greater horseshoe bat, there are many substance P-immunoreactive nerves with very weak or no calcitonin gene-related peptide immunoreactivity, and in the bent-winged bat, calcitonin gene-related peptide immunoreactivity is not found in all nerves with substance P immunoreactivity in the pial arteries of all parts of the brain. Nearly all cells immunoreactive for substance P, calcitonin gene-related peptide or both in the trigeminal and cervical dorsal root ganglia were small. In the greater horseshoe bat and the bent-winged bat, there is a correlation between the level of expression of substance P and calcitonin gene-related peptide immunoreactivity in the cervical dorsal root and trigeminal ganglia and the cerebral perivascular nerves supplying the vertebrobasilar system. The cerebrovascular innervation of nerves with substance P immunoreactivity in small bats is similar to that of cats and guinea-pigs in which the trigeminal ganglia have been destroyed. These observations, in addition to absence of nerve cells showing substance P or calcitonin gene-related peptide immunoreactivity in the pial artery and in the nerve bundles accompanying the extracranial internal carotid and vertebral arteries, suggest that substance P-immunoreactive nerves with or without immunoreactivity to calcitonin gene-related peptide in the vertebrobasilar system of small bats originate exclusively from the cervical dorsal root ganglia. The remaining fibres probably originate in the trigeminal ganglia.     

Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin

The temporal course of changes in peptide expression in the dorsal root ganglia L4 and L5 and in the dorsal horn of the spinal cord has been studied in rats subjected to a sciatic nerve transection at a mid-thigh level following different survival times. Galanin-, substance P-, vasoactive intestinal polypeptide-, peptide histidine-isoleucine- and calcitonin gene-related peptide-like immunoreactivities have been studied both by immunohistochemistry and radioimmunoassay. Galanin messenger ribonucleic acid has also been studied by in situ hybridization in the dorsal root ganglia of normal and lesioned animals. In addition, a group of animals with a sciatic nerve crush was studied to compare possible differences in peptide expression after both types of lesions. The results show that the transection induces an increase in the number of cell bodies expressing galanin-like immunoreactivity in the ganglia, and that the galanin levels rise about 120-fold after three and 14 days of survival. This increase reflected increased synthesis of the peptide, since there was a rise in the galanin messenger ribonucleic acid already at 24 h post-lesion, which was maintained for at least 60 days. In the spinal cord there was an increase of staining in the midportion of the outer layers of the dorsal horn that corresponded to fibers thought to arise from cells of the dorsal root ganglia affected by the transection. Also a depletion of substance P-like and an increase in vasoactive intestinal polypeptide- and peptide histidine-isoleucine-like immunoreactivities in the dorsal root ganglia were confirmed. These changes were shown to be rapidly detectable and were paralleled by similar changes in the dorsal horn of the spinal cord. For calcitonin gene-related peptide the immunohistochemistry was inconclusive, and the radioimmunoassay showed no detectable changes. After nerve crush a transient increase in the number of galanin immunoreactive neurons was observed, as well as a decrease in the number of neurons showing substance P-like immunoreactivity. These changes were most noticeable between six and 14 days of survival. After this, peptide expression seemed to return slowly to normal, that is by day 45 post-crush only a few cells showed galanin-like, and many sensory neurons expressed substance P-like immunoreactivity. The results demonstrate that when primary sensory neurons are peripherally lesioned they respond in a complex manner, altering their normal production of peptides by increasing or decreasing their synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neuropeptide expression in cultures of adult sensory neurons: modulation of substance P and calcitonin gene-related peptide levels by nerve growth factor

In contrast to developing sensory neurons, the survival of adult rat dorsal root ganglion neurons in pure neuronal culture is not dependent on specific neurotrophic factors such as nerve growth factor or brain-derived neurotrophic factor [Lindsay R. M. (1988) J. Neurosci. 8, 2394-2405]. In the present study we have examined possible modulatory effects of nerve growth factor on the neuropeptide content of sub-populations of adult rat dorsal root ganglion neurons in vitro. During the first 1-2 days in culture the neuropeptides substance P and calcitonin gene-related peptide could be detected by immunofluorescence staining in cultures grown in the presence or absence of nerve growth factor, but at longer times in nerve growth factor-deprived cultures there was loss of immunoreactive staining for both peptides. In the presence of nerve growth factor, however, the percentage of substance P- and calcitonin gene-related peptide-immunoreactive neurons remained relatively constant, for at least 14 days, at levels that were similar to the percentage of such peptide-containing neurons found in sections of adult rat dorsal root ganglia. Quantitation by radioimmunoassay of the levels of substance P and calcitonin gene-related peptide in cultures grown in the presence or absence of nerve growth factor agreed with the qualitative observations obtained by immunofluorescence: 10-15-fold higher levels of substance P and calcitonin gene-related peptide were found in cultures grown with nerve growth factor for 18 days, as compared to nerve growth factor-deprived cultures. In nerve growth factor-treated cultures increased levels of substance P and calcitonin gene-related peptide were observed within 3-6 days in vitro, and further steady increases in the levels of both peptides were found up to 18 days. A low basal level of both peptides could always be detected, even in the presence of an excess of antibodies to nerve growth factor. Up-regulation of the synthesis of substance P and calcitonin gene-related peptide did not depend on nerve growth factor being present at the initiation of the cultures, as elevated levels of both peptides could be induced in cultures even after up to 10 days' prior deprivation of nerve growth factor. Removal of nerve growth factor from the cultures resulted in reduced levels of peptide within 3 days.(ABSTRACT TRUNCATED AT 400 WORDS)