‘Neuropeptide tyrosine’ (NPY) is co-stored with noradrenaline in vascular but not in parenchymal sympathetic nerves of brown adipose tissue

By using immunohistochemistry it is shown that both the parenchymal and vascular sympathetic innervation in the interscapular depot of brown adipose tissue in the rat contain the catecholamine-synthesizing enzyme tyrosine-hydroxylase (TH). In contrast, 'neuropeptide tyrosine' (NPY) is selectively present in the vascular sympathetic nerves of the tissue--but not in nerves around brown fat cells. This is consistent with the presence of two populations of neurons (containing either TH alone or TH plus NPY) in the stellate ganglion, which is the probable origin of the sympathetic nerves in the interscapular brown adipose tissue. Furthermore, the perivascular NPY-positive nerves in the brown adipose tissue disappeared after 6-hydroxydopamine treatment, demonstrating their noradrenergic nature. Taken together, these findings suggest that sympathetic nerves to blood vessels and brown fat cells represent two separate subpopulations of autonomic neurons.     

The age specifics of the neurotransmitter composition of the stellate ganglion neurons

The stellate ganglion neurons' transmitter composition in the rat siblings of different age (newborn, 10-, 20-, 30-, 60-day old) revealed that the greatest amount of the neurons in all the rats contained thyrosinhydroxylase. Most cholinacetyltransferasepositive. In 30- and 60-day old rats, just a few cells contained both above enzymes. Since birth, the percentage of cells containing thyrosinhydroxylase and neuropeptide Y has been growing. Along with that, the share of somatostatin-positive neurons was decreasing. The percentage of the VIP-positive cells and the neurons containing cholinacetyltransferase was increasing until the 10th day of life and then dropped again. The somatostatin-positive neurons in all the rat siblings were represented by small cells, whereas the neurons containing cholinacetyltransferase were represented by large cells. The maturation of the neurotransmitter set in the rat stellate ganglion terminates by the end of the second month of life.     

Immunohistochemical characterisation of cholinergic neurons in the anterior pelvic ganglion of the male pig

This study investigated immunohistochemical properties of cholinergic neurons in the anterior pelvic ganglion (APG) of juvenile male pigs (n=7). Cholinergic neurons were identified using antibodies against choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT). Immunoblotting was applied to verify the specificity of ChAT-immunostaining. Western blotting performed on APG tissue homogenates detected single immunoreactive protein with a molecular weight matching that of ChAT (71.6 kDa). It was found that many APG neurons expressed immunoreactivity to ChAT or VAChT (40% and 39% of the neurons, respectively). The analysis of adjacent sections from the ganglion revealed complete colocalization of ChAT and VAChT in these nerve cells. Furthermore, virtually all the ChAT-positive neurons were tyrosine hydroxylase (TH)-negative (non-adrenergic) but many of them displayed immunoreactivity to nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or somatostatin (SOM). There were also single nerve cell bodies that stained for neither ChAT nor TH. The comparison of the adjacent sections revealed that NOS, VIP, NPY and SOM were simultaneously co-expressed in the majority of the cholinergic somata. ChAT- or VAChT-positive varicose nerve terminals supplied nearly all neuronal profiles within the ganglion often forming loose basket-like formations surrounding the particular nerve cell bodies. The present study for the first time has revealed that nearly all non-adrenergic neurons in the porcine APG are cholinergic in nature, i.e. express immunoreactivity for ChAT and VAChT. Considering a high coincidence between the chemical coding of non-adrenergic (cholinergic) nerve fibres supplying some porcine male reproductive organs described in earlier papers and that of cholinergic pelvic neurons found in this study it is further concluded that pelvic ganglia are probably the major source of cholinergic innervation for the porcine urogenital system.     

Intrinsic choroidal neurons in the chicken eye: chemical coding and synaptic input

Intrinsic choroidal neurons (ICNs) exist in some primates and bird species. They may act on both vascular and non-vascular smooth muscle cells, potentially influencing choroidal blood flow. Here, we report on the chemical coding of ICNs and eye-related cranial ganglia in the chicken, an important model in myopia research, and further to determine synaptic input onto ICN. Chicken choroid, ciliary, superior cervical, pterygopalatine, and trigeminal ganglia were prepared for double or triple immunohistochemistry of calcitonin gene-related peptide (CGRP), choline acetyltransferase (ChAT), dopamine-β-hydroxylase, galanin (GAL), neuronal nitric oxide synthase (nNOS), somatostatin (SOM), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP), vesicular monoamine-transporter 2 (VMAT2), and α-smooth muscle actin. For documentation, light, fluorescence, and confocal laser scanning microscopy were used. Chicken ICNs express nNOS/VIP/GAL and do not express ChAT and SOM. ICNs are approached by TH/VMAT2-, CGRP-, and ChAT-positive nerve fibers. About 50% of the pterygopalatine ganglion neurons and about 9% of the superior cervical ganglion neurons share the same chemical code as ICN. SOM-positive neurons in the ciliary ganglion are GAL/NOS negative. CGRP-positive neurons in the trigeminal ganglion lack GAL/SOM. The neurochemical phenotype and synaptic input of ICNs in chicken resemble that of other bird and primate species. Because ICNs lack cholinergic markers, they cannot be readily incorporated into current concepts of the autonomic nervous system. The data obtained provide the basis for the interpretation of future functional experiments to clarify the role of these cells in achieving ocular homeostasis.     

NPY-, galanin-, VIP/PHI-, CGRP- and substance P-immunoreactive neuronal subpopulations in cat autonomic and sensory ganglia and their projections

Summary The neuronal subpopulations in the cat stellate, lower lumbar and sacral sympathetic ganglia were studied with regard to the cellular distribution of immunoreactivity to tyrosine hydroxylase (TH), acetylcholinesterase (AChE) and various neuronal peptides. Coexistence of neuropeptide Y (NPY)- and galanin (GAL)-like immunoreactivity (LI) was found in a high proportion of the neuronal cell bodies; these cells also contained immunoreactivity to TH, confirming their presumably noradrenergic nature. Some TH- and GAL-immunoreactive principal ganglion cells lacked NPY-LI. Two populations (scattered and clustered) of vasoactive intestinal polypeptide (VIP)- and peptide histidine isoleucine (PHI)-positive cell bodies were found in the sympathetic ganglia studied. The scattered VIP/PHI neurons also contained AChE-LI, calcitonin gene-related peptide (CGRP)-and, following culture, substance P (SP)-LI. The clustered type only contained AChE-LI. In the submandibular and sphenopalatine ganglia, neurons were AChE- and VIP/ PHI-immunoreactive but lacked CGRP- and SP-LI. Many GAL- and occasional TH-positive neurons were found in these ganglia. In the spinal ganglia, single NPY-immunoreactive sensory neuronal cells were observed, in addition to CGRP- and SP-positive neurons. The present results show that there are at least two populations of sympathetic cholinergic neurons in the cat. Retrograde tracing experiments indicate that the scattered type of cholinergic neurons contains four vasodilator peptides (VIP, PHI, CGRP, SP) and provides an important input to sweat glands, whereas the clustered type (containing VIP and PHI) mainly innervates blood vessels in muscles.     

Age-Related Changes in Tyrosine Hydroxylase and Choline Acetyltransferase in Sympathetic Ganglia of a Rat Strain with Reduced Sympathetic Neuron Numbers

Abstract: In Wistar rats, a subpopulation of sympathetic ganglionic neurons dies during ageing, but in the GH strain, these same neurons die during the period of perinatal maturation. We have compared tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in superior cervical ganglia of GH and control rats at different ages. Ganglionic TH rose to near adult levels between postnatal weeks 1 and 2. No significant differences in TH values were seen between GH and control ganglia at any age, indicating that reduced neuron numbers are compensated for by increased cellular activity, Ganglionic ChAT rose initially in parallel with TH and then more slowly over postnatal weeks 3–4, reaching adult levels that were about 20° lower in GH than in normal ganglia. During ageing, TH remained constant but ChAT continued to rise slowly in GH ganglia, whereas ChAT in normal ganglia fell by about 10°. Both the strain difference in ChAT during development and the fall in ChAT during ageing in normal animals parallel the differences in ganglion cell numbers seen under these circumstances.     

Identification of neuron types in the submucosal ganglia of the mouse ileum

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found. This work was supported by a grant from the National Health and Medical Research Council of Australia (grant no. 400020) and an Australian Research Council international linkage grant (no. LZ0882269) for collaboration between the Melbourne and Bologna laboratories.     

Immunohistochemical analysis of intracardiac ganglia of the rat heart

The neurochemistry of intracardiac neurons in whole-mount preparations of the intrinsic ganglia was investigated. This technique allowed the study of the morphology of the ganglionated nerve plexus found within the atria as well as of individual neurons. Intracardiac ganglia formed a ring-like plexus around the entry of the pulmonary veins and were interconnected by a series of fine nerve fibres. All intracardiac neurons contained immunoreactivity to PGP-9.5, choline acetyl transferase (ChAT) and neuropeptide Y (NPY). Two smaller subpopulations were immunoreactive to calbindin or nitric oxide synthase. Furthermore, a subpopulation (approximately 6%) of PGP-9.5/ChAT/NPY-immunoreactive cells lacking both calbindin and nitric oxide synthase (NOS) was surrounded by pericellular baskets immunoreactive to ChAT and calbindin. Vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activated peptide (PACAP), substance P and tyrosine hydroxylase (TH) immunoreactivity was observed in nerve fibres within the ganglion, but never in neuronal somata. Furthermore, immunoreactivity for NPY was not observed in pericellular baskets surrounding intracardiac neurons, despite being present in all intrinsic neuronal cell bodies. Taken together, the results of this study indicate a moderate level of chemical diversity within the intracardiac neurons of the rat. Such chemical diversity may reflect functional specialisation of neurons in the intracardiac ganglia.This work was supported by a grant-in-aid (G00M0670) from the National Heart Foundation of Australia     

Developmental regulation of tyrosine hydroxylase expression in primary sensory neurons of the rat

The regulation of transmitter phenotype in primary sensory neurons remains poorly understood. However, recent studies of catecholaminergic (CA) sensory neurons suggest that expression of this particular phenotype may be related to innervation of specific peripheral tissues. In the glossopharyngeal petrosal ganglion (PG) of adult rats, for example, the vast majority of CA sensory neurons innervate a single target, the carotid body. The present study was undertaken, therefore, to begin investigating factors that underlie CA differentiation in sensory neurons, using the rat PG as a model system. Immunocytochemical, biochemical, and morphometric methods were used to investigate the normal time course of CA development in the PG in vivo, employing tyrosine hydroxylase (TH) as a phenotypic marker. These studies revealed two temporally distinct waves of TH expression during embryogenesis. TH immunoreactivity was initially detectable on Embryonic Day (E) 11.5; the number of stained cells increased markedly by E12.5 and then fell off sharply to near 0 by E15.5. Simultaneous immunostaining for TH and neurofilament proteins revealed a high proportion of double-labeled perikarya on E12.5, indicating that the transiently TH-positive cells are neurons. A second, sustained phase of TH expression began on E16.5, and by Postnatal Day 1 adult numbers of TH-containing ganglion cells were present. Western blot analysis demonstrated that TH levels per cell rose 3.5-fold in the perinatal period, indicating that maturation of this particular catecholaminergic trait in PG sensory neurons is highly regulated around birth. Morphometric techniques were used to define the relationship between neurons that transiently exhibit TH immunoreactivity early in gangliogenesis and those that maintain enzyme expression in the mature PG. These studies revealed separate and distinct growth curves for the early and late TH cells, respectively, demonstrating that the appearance, disappearance, and reappearance of immunoreactive cells reflects the differentiation of two separate populations of PG neurons. Moreover, these data indicate that TH expression in the population of CA cells that persists in the mature PG begins around E16.5. This is after peripheral target innervation has begun, raising the possibility that neuron-target interactions regulate biochemical differentiation of these CA sensory neurons.     

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes,neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.     

Intrinsic neurons in the rat ovary: an immunohistochemical study

Previous studies have shown the presence of neuronal perikarya in the primate ovary, but not in the ovary from Sprague-Dawley rats. We report here that while such intrinsic neurons are indeed absent in this strain of rats, they can be visualized in the ovary from Wistar rats. The neurons, identified by their morphology and by the expression of NeuN (a neuron-specific nuclear protein), were detected at all postnatal intervals examined, from 14 h after birth to 50 days of age. While they were present in the ovarian hilum and medulla at all ages studied, neurons first appeared in the ovarian cortex during the juvenile period (postnatal days 10-20). In all cases, the size of the neuronal soma increased significantly during prepubertal development, reaching maximal values before puberty. Some neurons were catecholaminergic, as indicated by their content of immunoreactive tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. Some showed neuropeptide Y (NPY) immunoreactivity. TH-positive neurons were seen either in isolation or clustered in ganglion-like structures in both the ovarian cortex and medulla. These results indicate that ovarian neurons are not present in all strains of rats, but when present, the chemical phenotype of some of them is of a sympathetic nature, similar to that described in primates.     

Biosynthesis, Development, and Regulation of Neuropeptide Y in Superior Cervical Ganglion Culture

The biosynthesis of neuropeptide Y (NPY) and norepinephrine (NE) has been examined in dissociated neuronal cultures from newborn rat superior cervical ganglion (SCG). NPY synthetic rate was measured by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after incubation in medium containing a labeled amino acid. The authenticity of the NPY was confirmed by reverse-phase HPLC analyses of tryptic peptides. The NPY synthetic rate in cultures grown in complete serum free medium increased 30-fold after plating, in parallel to catecholamine synthesis; both NPY and the catecholamines reached the rate for adult SCG neurons. This development in culture is seen without spinal cord input, target organs, or significant numbers of glial cells. NPY synthesis was maintained in the face of a major decrease in the rate of NE production after cholinergic induction.     

Neuropeptide Y and vasoactive intestinal peptide coexist in rat thyroid nerve fibers emanating from the thyroid ganglion

Neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) occur in nerve fibers around blood vessels and between follicles in the thyroid gland of the mouse and rat. VIP-immunoreactive fibers are numerous, while NPY-immunoreactive fibers are fewer. Most of the latter fibers contain noradrenaline (NA) as well as NPY, while a subpopulation was found to contain VIP instead of NA. We have determined the origins of rat thyroid nerve fibers containing NPY, VIP or NPY/VIP by investigating 3 conceivable sources, i.e. the superior cervical ganglion, the nodose ganglion and the thyroid ganglion. Chemical sympathectomy or removal of the superior cervical ganglion did not affect the frequency of VIP-immunoreactive fibers but eliminated most of the NPY-immunoreactive fibers as well as all NA-containing nerve fibers (recognized by antibodies to dopamine-beta-hydroxylase). The NPY-immunoreactive fibers that remained after sympathectomy occurred around blood vessels and between follicles and contained VIP. Cervical vagotomy (removal of the nodose ganglion including the adjacent vagus) did not overtly affect the frequency of NPY/VIP-, VIP-, or NPY/NA-containing fibers in the thyroid. In contrast, extirpation of the thyroid ganglion, which is situated immediately outside the thyroid capsule, greatly reduced the number of VIP- and NPY/VIP-containing fibers in the rat thyroid. On the whole, the results of radioimmunoassay of NPY and VIP agreed well with the immunocytochemical findings. High performance liquid chromatography confirmed the identity of NPY and VIP. The present findings suggest the existence in the rat thyroid of one NPY-containing nerve fiber population that harbours NA and emanates from the superior cervical ganglion; one NPY-containing fiber population that is non-adrenergic, harbours VIP and originates in the thyroid ganglion; and a second VIP-containing fiber population that is devoid of NPY and appears to derive from the thyroid ganglion.     

Leptin receptors,NPY, and tyrosine hydroxylase in autonomic neurons supplying fat depots in a pig

The goal of this study was to determine the immunohistochemical characteristics of peripheral adrenergic OBR-immunoreactive (OBR-IR) neurons innervating adipose tissue in a pig. The retrograde tracer, Fast Blue (FB), was injected into either the subcutaneous, perirenal, or mesentery fat tissue depots of three male and three female pigs each with approximately 50 kg body weight. Sections containing FB(+) neurons were stained for OBR, tyrosine hydroxylase (TH) or neuropeptide Y (NPY) using a double labeling immunofluorescence method. OBR, TH, and NPY immunoreactivities were present in the thoracic (T) and lumbar (L) ganglia of the sympathetic chain, as well as in the coeliac superior mesenteric ganglion (CSMG), inferior mesenteric ganglion (IMG), intermesenteric ganglia (adrenal-ADG, aorticorenal-ARG, and ovarian-OG or testicular-TG ganglion). These results indicate that, in addition to neuroendocrine functions, leptin may affect peripheral tissues by acting on receptors located in sympathetic ganglion neurons.     

Recombinant Cholinergic Differentiation Factor (Leukemia Inhibitory Factor) Regulates Sympathetic Neuron Phenotype by Alterations in the Size and Amounts of Neuropeptide mRNAs

The cholinergic differentiation factor (CDF) in heart cells is identical to leukemia inhibitory factor (LIF). Recombinant CDF/LIF was shown to alter dramatically neurotransmitter production as well as the levels of several neuropeptides in cultured rat sympathetic neurons. Here it is shown that these changes are likely to be caused by alterations in the mRNA for these proteins and peptides. Growth in 1 nM recombinant CDF/LIF induces mRNA for acetyl CoA: choline-O-acetyltransferase [EC 2.3.1.6; choline acetyltransferase (ChAT)], somatostatin (SOM), substance P, and vasoactive intestinal polypeptide while lowering mRNA levels of tyrosine hydroxylase (EC 1.14.16.2) and neuropeptide Y (NPY). In addition, the sizes of the mRNAs for ChAT, SOM, and NPY are larger after recombinant CDF/LIF treatment.     

Distribution of neuropeptide Y and vasoactive intestinal polypeptide immunoreactive nerves in normal and transplanted pancreatic tissue

Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactive nerves were demonstrated in 21-day-old embryonic pancreatic tissue fragments transplanted into the anterior eye chamber of rats for 22, 45 and 109 days and in 60-day-old normal adult pancreas using immunohistochemical technique. In normal adult tissue, NPY-positive neurons lie close to the basal and lateral walls of the acinar cells. NPY-containing nerve fiber plexuses were found around blood vessels. VIP-immunopositive nerves were also discernible in the outer parts of the islets of Langerhans and on pancreatic ducts. In the transplants, it is not only the neural elements that survived but also the pancreatic ducts and the endocrine cells. VIP- and NPY-positive neurons were found in the stroma of the surviving pancreatic tissue. The distribution of these neural elements is similar to that of normal tissue in the surviving pancreatic ducts but different with regards to the acinar tissue. This study confirms that intrinsic nerves can survive and synthesize polypeptides even after 109 days of transplantation into the anterior eye chamber.     

Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat

Summary The distribution and relative density of peptide-containing nerves was studied in the rat in order to assess the progression of neuronal changes during the postnatal development of the male genital system from the prepubertal age to adulthood. Testis, caput and cauda epididymis, ductus deferens, seminal vescicles, prostate and penis from 8-, 20-, 38-, and 70-day-old rats were sectioned and were immunostained with antisera to the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and to a general neuronal marker, protein gene product 9.5 (PGP 9.5). The testicular parenchyma and caput epididymis did not show any immunoreactivity. Very scattered CGRP-containing nerves were present in 8-day-old rats; numerous VIP-, CGRP-, and NPY-peptide-containing nerves were observed in the cauda epididymis, ductus deferens, accessory glands and penis of 20-day-old rats. The number of nerves increased in 35-day-old rats while no changes were observed in more adult rats. A parallel increase was seen for the immunostain for PGP 9.5. These data suggest that peptide-containing nerves appear in the genital system after birth and reach a full development before the completion of puberty. Peptide-containing nerves were visible first in the interstitial area and then spread in the muscular coat of the ducts, glands and of the blood vessels. While CGRP- and NPY-containing nerves were distributed in the vicinity of the muscle cells, VIP-containing nerves were also observed in the subepithelial regions, suggesting a possible role of this neuropeptide in the control of epithelial functions.     

Postnatal development and distribution of peptide-containing nerves in the genital system of the male rat. An immunohistochemical study.

The distribution and relative density of peptide-containing nerves was studied in the rat in order to assess the progression of neuronal changes during the postnatal development of the male genital system from the prepubertal age to adulthood. Testis, caput and cauda epididymis, ductus deferens, seminal vesicles, prostate and penis from 8-, 20-, 38-, and 70-day-old rats were sectioned and were immunostained with antisera to the neuropeptides calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY), and to a general neuronal marker, protein gene product 9.5 (PGP 9.5). The testicular parenchyma and caput epididymis did not show any immunoreactivity. Very scattered CGRP-containing nerves were present in 8-day-old rats; numerous VIP-, CGRP-, and NPY-peptide-containing nerves were observed in the cauda epididymis, ductus deferens, accessory glands and penis of 20-day-old rats. The number of nerves increased in 35-day-old rats while no changes were observed in more adult rats. A parallel increase was seen for the immunostain for PGP 9.5. These data suggest that peptide-containing nerves appear in the genital system after birth and reach a full development before the completion of puberty. Peptide-containing nerves were visible first in the interstitial area and then spread in the muscular coat of the ducts, glands and of the blood vessels. While CGRP- and NPY-containing nerves were distributed in the vicinity of the muscle cells, VIP-containing nerves were also observed in the subepithelial regions, suggesting a possible role of this neuropeptide in the control of epithelial functions.     

Adrenergic, nitrergic and peptidergic innervation of the urethral muscle in the boar

In this study, the innervation of the urethral muscle in adult male pigs was investigated using combined NADPH-diaphorase (NADPH-d) histochemistry and immunocytochemistry. Nerve fibres supplying the urethral muscle were found to show NADPH-d activity and they also expressed immunoreactivity to catecholamine synthesising enzymes including tyrosine hydoxylase (TH) and dopamine-beta-hydroxylase (DbetaH) as well as to: vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Different subpopulations of the nerve fibres (NADPH-d positive, TH-, DbetaH-, VIP- and NPY-immunoreactive (IR), but also NADPH-d/VIP- and NADPH-d/NPY-IR) were disclosed. These nerve fibres were observed not only to run among muscle fibres of the urethral muscle, but also within extrinsic nerve trunks. Moreover, in the organ studied, numerous ganglia were found. The intramural ganglia, composed of a few to 30 neurons were located in the proximal, middle and distal regions of the pelvic urethra. In the vicinity of the urethral muscle, there were mainly small ganglia containing two to several neurons, but also larger ganglia consisting of up to tens neurons were encountered in the connective tissue surrounding the pelvic urethra. In the ganglia observed in the neighbourhood of the urethral muscle, different subpopulations of nerve cells were found, namely: catecholaminergic, nitrergic, VIP-IR, NPY-IR and also NADPH-d/DbetaH-, NADPH-d/VIP- and NADPH-d/NPY-positive. Possible sources of the innervation for this muscle were also discussed.