Selective association of nerve fibres immunoreactive for substance P or bombesin with putative cholinergic neurons of the male rat major pelvic ganglion

The male rat major pelvic ganglion contains both sympathetic and parasympathetic neurons that supply the lower urinary and digestive tracts, and the reproductive organs. The aim of this study was to describe the distribution and identify potential targets of sensory and intestinofugal axons in this ganglion. Two putative markers of these projections were chosen, substance P for primary sensory axons and bombesin for myenteric intestinofugal projections. Varicose substance P-immunoreactive axons were associated only with non-noradrenergic (putative cholinergic) somata, and most commonly with those that contained vasoactive intestinal peptide. Immunoreactivity for substance P was also present in a small group of non-noradrenergic somata, many of which were immunoreactive for enkephalins, neuropeptide Y or vasoactive intestinal peptide. Bombesin immunoreactivity was found only in preterminal and terminal (varicose) axons, the latter of which were exclusively associated with non-noradrenergic somata that contain neuropeptide Y-immunoreactivity. Some varicose axons containing either substance P-or bombesin-immunoreactivity were intermingled with clumps of small, intensely fluorescent cells. These studies indicate that substance P-and bombesin-immunoreactive axons are likely to connect with numerically small, but discrete, populations of pelvic neurons.     

Patterns of co-existence of peptides and differences of nerve fibre types associated with noradrenergic and non-noradrenergic (putative cholinergic) neurons in the major pelvic ganglion of the male rat

Summary The pelvic ganglia supply cholinergic and noradrenergic nerve pathways to many organs. Other possible transmitters are also present in these nerves, including peptides. Multiple labelling immunofluorescence techniques were used in this study of the male rat major pelvic ganglion (MPG) to examine: (1) the peptides present in noradrenergic (tyrosine hydroxylase (TH)-positive) and non-noradrenergic (putative cholinergic) neurons, and (2) the types of peptide-containing nerve fibres closely associated with these two groups of neurons. The distribution of the peptide galanin (GAL) within the MPG was also investigated. All of the TH-neurons contained neuropeptide Y (NPY), but none of the other tested peptides. However, many NPY neurons did not contain TH and may have been cholinergic. TH-negative neurons also displayed vasoactive intestinal peptide (VIP), enkephalin (ENK) or GAL. VIP and NPY formed the most common types of putative cholinergic pelvic neurons, but few cells contained both peptides. Many ENK neurons exhibited VIP, NPY or GAL. Varicose nerve terminals surrounding ganglion cells contained ENK, GAL, somatostatin (SOM) and cholecystokinin (CCK). These peptide-immunoreactive fibres were more often associated with the non-noradrenergic (putative cholinergic) than the noradrenergic neurons; two types (SOM and CCK) were preferentially associated with the non-noradrenergic NPY neurons. GAL was distributed throughout the MPG, in small neurons, scattered small, intensely fluorescent (SIF) cells, and both varicose and non-varicose nerve fibres. The nerve fibres were concentrated near the pelvic and penile nerves; most of the varicose fibres formed baskets surrounding individual GAL-negative somata.     

Distribution of neurons in the major pelvic ganglion of the rat which supply the bladder,colon or penis

Summary In male rats a large number of the postganglionic neurons which innervate the pelvic organs are located in the major pelvic ganglion. In the present study we have identified the location within this ganglion of neurons which project to either of three pelvic organs, the penis, colon or urinary bladder. Two fluorescent retrogradely-transported dyes, Fast Blue and Fluoro-Gold, were used. For most animals one dye was injected into the cavernous space of the penis, the wall of the distal colon or the wall of the urinary bladder. In a small number of animals two organs were injected, each with a different dye. One to six weeks after injection the major pelvic ganglia were fixed in buffered formaldehyde. The distribution of fluorescent dye-labelled cells was observed in whole mounts of complete ganglia and, in most cases, also in small accessory ganglia located between the ureter and the prostate. The studies showed a unique pattern of distribution for each organ-specific group of neurons. Most of the colon neurons are located in the major pelvic ganglion near the entrance of the pelvic nerve, whereas almost all of the penis neurons are near or within the penile nerve. Bladder neurons are relatively evenly distributed throughout the ganglion. These results demonstrate a distinct topographical organization of organ-specific neurons of the major pelvic ganglion of the male rat, a phenomenon which has also been observed in other peripheral ganglia.     

Synaptic organisation of the pelvic ganglion in the guinea-pig

Summary A semi-quantitative electron-microscopic study of neuronal cell bodies, nerve profiles and synapses in the anterior pelvic ganglia of the guinea-pig has been carried out following in vivo labelling of adrenergic nerve endings with 5-hydroxydopamine. Ganglion cells of three main types have been distinguished: 1) the majority (about 70%) not containing granular vesicles, probably cholinergic elements; 2) those containing large granular vesicles of uniform size (80–110 nm), with granules of medium density and prominent halos; and 3) those containing vesicles of variable size (60–150 nm), with very dense eccentrically placed granular cores. Some non-neuronal granule-containing cells were present, mainly near small blood vessels. Some 95% of the total axon profiles within the ganglia were cholinergic, the remaining 5% were adrenergic. However, 99% of synapses (i.e. axons within 50 nm of nerve cell membrane with pre-and post-synaptic specialisations) were cholinergic, and 1 % were adrenergic. Only three examples of nerve cell bodies exhibiting both cholinergic and adrenergic synapses were observed. Unlike the para-and prevertebral ganglia, the pelvic ganglia contained large numbers of axo-somatic synapses. As many as 20% of the nucleated neuronal cell profiles displayed two distinct nuclei.     

A dividing granule-containing cell in the pelvic ganglion of the guinea-pig

Summary A dividing granule-containing cell is described in the pelvic ganglion of the guinea-pig two days after pelvic nerve section. This appears to be the first report of a dividing granule-containing cell in adult tissue.     

Principal neurons projecting to the pineal gland in close association with small intensely fluorescent cells in the superior cervical ganglion of rats

Summary The localization in the superior cervical ganglia (SCG) of small, intensely fluorescent (SIF) cells and of principal nerve (PN) cells innervating the pineal gland was examined in adult male Sprague-Dawley rats. PN cells were demonstrated by means of the retrograde neuron-tracing method using the fluorescent tracer Fluoro-Gold (FG) injected into the pineal gland. SIF cells were visualized by the formaldehyde-induced fluorescence method. Twentynine percent of the FG-labeled PN cells were found closely associated with SIF cells. In the rostral half of the ganglion, 43% of the SIF cells were situated in juxtaposition to one or several labeled neurons. The possible influence of SIF cells on the regulation of pineal metabolism is discussed with respect to their role as both local endocrine cells and interneurons.     

Distribution of NADPH-diaphorase activity in rat paravertebral,prevertebral and pelvic sympathetic ganglia

Summary Paravertebral (superior cervical and stellate), prevertebral (coeliac-superior mesenteric, inferior mesenteric) and pelvic (hypogastric) sympathetic ganglia of the rat were investigated by enzyme histochemistry to ascertain the distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity. In the paravertebral ganglia the majority of the sympathetic neuronal perikarya contained lightly and homogeneously distributed formazan reaction product but there was a range of staining intensities amongst the neuron population. In contrast, in the prevertebral ganglia, intense NADPH-diaphorase staining was present in certain neurons. Firstly, a population of neurons of the coeliac-superior mesenteric ganglion complex were surrounded by densely NADPH-diaphorase-positive baskets of fibres and other stained fibres were seen in interstitial nerve bundles and in nerve trunks connected to the ganglion complex. Secondly, in both the inferior mesenteric ganglion and hypogastric ganglion there were many very intensely NADPH-diaphorase positive neurons. Stained dendritic and axonal processes emerged from these cell bodies. In both ganglia this population of neurons was smaller in size than the lightly stained ganglionic neurons and commonly had only one long (presumably axonal) process. The similarity of these highly NADPH-diaphorase-positive neurons with previously described postganglionic parasympathetic neurons in the hypogastric ganglion is discussed.     

Failure to demonstrate serotonergic neurons in the myenteric plexus of the rat

Summary Several recent studies suggested that serotonergic neuron-like elements are present in the guinea pig ileum. The present paper reports an extensive study of the digestive tract of the rat with the use of a histofluorescence technique. Administration of the serotonin precursor, tryptophan, associated with a monoamine oxidase inhibitor, did not allow histochemical demonstration of rapidly fading, yellow fluorescent, 6-hydroxydopamine-resistant neurons; conversely such neurons were readily detected in the brain. It is concluded that serotonergic neuron-like elements cannot be detected histochemically in the rat myenteric plexus area after chemical sympathectomy.     

The ultrastructure of paraganglia associated with the inferior mesenteric ganglia in the guinea-pig

Summary The paraganglia of the inferior mesenteric ganglia in the guinea-pig are composed of small chromaffin cells containing an abundance of granule-containing vesicles. The chromaffin cells are almost completely surrounded by satellite cells. In areas in which satellite cell processes do not intervene, the membranes of adjacent chromaffin cells are closely apposed and often form specialized attachment zones. The paraganglia contain a dense capillary network, the endothelial cells of which are often extremely attenuated and show areas of fenestration. The processes of chromaffin cells approach close to the capillary walls and are often bare of satellite cells covering on the side facing the capillary. Evidence has been obtained for the exocytotic release of the contents of chromaffin cell vesicles into pericapillary spaces. Synapses of cholinergic and noradrenergic axons are seen on the chromaffin cells. The cholinergic axons degenerate when the praganglia are decentralized, but the noradrenergic axons, which appear to arise from the local inferior mesenteric ganglia, remain intact. The results suggest that the paraganglia have an endocrine function.     

Electron-microscopic immunohistochemical study of the localization of immunoglobulin G in the choroid plexus of the rat

Summary The localization of autologous antiperoxidase immunoglobulin G (IgG) was studied in the choroid plexus of Lewis rats immunized against horseradish peroxidase (HRP). This experiment was performed to study the permeability of the choroid plexus to intravascular IgG. It was shown that autologous IgG was present in the extravascular spaces. The transendothelial transfer appeared to occur mainly via the fenestrations and some interendothelial junctions. No transfer of IgG at the level of epithelial cells toward the cerebrospinal fluid was demonstrated. Interstitial spaces in contact with the connective-tissue cells of the choroid stroma were strongly labeled. The significance of these spaces remains hypothetical and raises the question of the fate of IgG from the interstitial space.This work has been partly supported by Crédits Recherche Universitaire, Paris-val de Marne.     

Culture of major pelvic ganglion neurons from adult rat

Successful culturing of neurons from adult animals has been historically difficult for a relatively long time. In this study, we reported the development of a novel method for the isolation and the culture of major pelvic ganglion (MPG) neurons from adult rat. The cultured cells were identified by neuron morphology and staining with neuronal marker (neurofilament-200, NF-200). The results demonstrate that the new protocol we used was reliable in obtaining a relatively high yield of MPG neurons. Furthermore, it improves the speed and simplicity in neuronal isolation. The viability of neurons can be maintained for about 2 weeks, which should be sufficient for investigating physiological and pathological processes occurring in mature major pelvic ganglia. And this may provide a useful assessment to currently available techniques for the culture of adult neurons.     

Immunohistochemical properties and spinal connections of pelvic autonomic neurons that innervate the rat prostate gland

Autonomic innervation of the prostate gland supplies the acini, and non-vascular and vascular smooth muscle. The activity of each of these tissues is enhanced by sympathetic outflow, whereas the role of the parasympathetic nervous system in this organ is unclear. In the present study, a range of methods was applied in rats to determine the location of autonomic neurons supplying this gland, the immunohistochemical properties of these neurons, the spinal connections made with the postganglionic pathways and the distribution of various axon types within the gland. Injection of the retrograde tracer, FluoroGold, into the ventral gland visualised neurons within the major pelvic ganglion and sympathetic chain. Fluorescence immunohistochemical studies on the labelled pelvic neurons showed that most were noradrenergic (also containing neuropeptide Y, NPY), the others being non-noradrenergic and containing either vasoactive intestinal peptide (VIP) or NPY. Sympathetic dyelabelled neurons were identified by the presence of varicose nerve terminals stained for synaptophysin on their somata following lesion of sacral inputs. Parasympathetic innervation of dye-labelled neurons was identified by continued innervation after hypogastric nerve lesion. Most noradrenergic prostate-projecting neurons were sympathetic, as were many of the non-noradrenergic VIP neurons. Parasympathetic prostate-projecting neurons were largely non-noradrenergic and contained either VIP or NPY. All substances found in retrogradely labelled somata were located in axons within the prostate gland but had slightly different patterns of distribution. The studies have shown that there are a significant number of non-noradrenergic sympathetic prostate-projecting neurons, which contain VIP.     

Colocalization of NO and VIP in neurons of the submucous plexus in the rat intestine

Since very few previous studies have carried out the quantitative analysis for the colocalization of nitric oxide (NO) and vasoactive intestinal peptide (VIP) in the submucous neurons in the rat digestive tract, we applied in vivo treatment of colchicine to enhance the immunoreactivity and examined the colocalization of NO synthase (nNOS) and VIP in neurons of the submucous plexus throughout the rat digestive tract. The density of nNOS-containing neurons in the submucous plexus in the stomach corpus (103±25 cells/cm², n=3) and that in the antrum (157±9 cells/cm², n=3) were significantly lower than those in small and large intestine. However no difference was detected in the cell density among duodenum (1967±188 cells/cm², n=3), jejunum (2640±140 cells/cm², n=3), ileum (2070±42 cells/cm², n=3), proximal colon (2243±138 cells/cm², n=3) and distal colon (2633±376 cells/cm², n=3). The proportion of nNOS-immunoreactive (IR), nNOS/VIP-IR and VIP-IR neurons to the total number of submucous neurons was examined. nNOS/VIP-IR neurons comprised 45–55% of total number of submucous neurons from the duodenum to the proximal colon, however those comprised 66.4±5.1% in the distal colon. The results showed that the dense distribution of nNOS-containing neurons was found in the submucous plexus throughout the small and large intestine, and large population of submucous neurons co-stored nNOS and VIP.     

Arginine vasopressin causes morphological changes suggestive of fluid transport in rat choroid plexus epithelium

Summary The experiments described herein use an in vitro preparation of choroid plexus to demonstrate that it is a vasopressin-responsive organ by morphologic criteria. Choroid plexus from rats was incubated for one hour in graded concentrations of arginine vasopressin (AVP). Within physiologic range of molar concentration, incubation in vasopressin induced a decrease in basal and lateral spaces in choroid plexus epithelial cells as well as an increase in number of dark cells. The number of cells with basal spaces decreased significantly from 82.7±9.2 in control tissue to 19±18 in tissue incubated in 10^-12 M AVP; similarly, the number with lateral cellular spaces decreased from 20±8.8 to 7.6±2.2 cells in 10^-10 M AVP. Dark cells increased in number from 3.8±2.6 in control conditions to 49±4 with 10^-9 M vasopressin. These data suggest important effects of arginine vasopressin in cerebrospinal fluid (CSF) on choroid plexus, compatible with enhanced fluid transport across choroid epithelial cells.     

The distribution of noradrenergic nerves and small,intensely fluorescent (SIF) cells in the cat urinary bladder

Summary Fluorescence and electron microscopy have been used to study the distribution of noradrenergic nerves in the smooth muscle of the cat urinary bladder. Using the former technique, relatively few fluorescent noradrenergic nerves were observed in the body and fundus, while a rich plexus occurred adjacent to muscle cells of the bladder neck. The trigone could not be distinguished neuromorphologically from detrusor muscle in this region. Electron microscopy showed that the majority of noradrenergic terminals in the body and fundus were associated with presumptive cholinergic axons, while in the bladder neck noradrenergic terminals formed typical neuroeffector relationships with individual smooth muscle cells.Numerous ganglia occurred both in the adventitia and among the smooth muscle bundles, particularly in the bladder neck. The majority of the nerve cell bodies were non-fluorescent, although many contained bright orange autofluorescent granules, believed to be lysosomes. A small minority of ganglion cells were associated with fluorescent noradrenergic nerve terminals, thereby providing structural evidence for limited intraganglionic inhibition. In addition, occasional groups of small intensely fluorescent (SIF) cells were observed in some intramural ganglia and these were subsequently identified in the electron microscope. The possibility that these cells may provide a second inhibitory influence on bladder activity was considered.     

Retinal ganglion cells of high cytochrome oxidase activity in the rat

Retinal ganglion cells in the rat were studied using the heavy metal intensified cytochrome oxidase and horseradish peroxidase histochemical methods.The results show that a population of large retinal ganglion cells was consistently observed with the cytochrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birth.These cytochrome oxidase rich ganglion cells appeared to have large somata,3-6 primary dendrites and extensive dendritic arbors,and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP).However,the morphological details of some of the cells revealed by the cytochrome oxidase staining method are frequently better than those shown by the HRP histochemical method.These results suggest that the mitochondrial enzyme cytochrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal genglion cells with high metabolic rate in the rat.     

Three-dimensional observation of the fibroblast-like cells associated with the rat myenteric plexus,with special reference to the interstitial cells of Cajal

Summary An extensive cellular network becomes visible over the myenteric plexus of the rat after removal of the overlying tissues under the scanning electron microscope. The cells are mainly stellate and have many slender processes via which they interconnect. They form a three-dimensional network and are closely associated with the ganglia and nerve bundles, and also extend over the smooth muscle cells. They are considered to correspond to the interstitial cells of Cajal because of their peculiar arrangement and their topography. Transmission electron-microscopic evidence demonstrates that the majority of those cells have features of fibroblasts. Gap junctions and intermediate junctions are observed between these fibroblast-like cells, and also between them and smooth muscle cells. Examination of serial thin sections reveals that single fibroblast-like interstitial cells connect to both circular and longitudinal muscle cells via gap junctions. It is suggested that the network of interstitial cells conducts electrical signals.     

Distribution and origin of neuropeptide Y-immunoreactive fibers in the penis of the rat

Summary The present study investigated the distribution of neuropeptide Y-immunoreactive fibers to the penis of the rat. In the corpora cavernosa penis, a dense plexus of fibers was asociated with arteries, intrinsic cavernosal muscle, and veins including the deep dorsal vein. In the corpus spongiosum, immunoreactive fibers were present around vascular smooth muscle and at the periphery of the acini of the paraurethral glands. Immunohistochemistry of penile neurons identified by retrograde tracer injection into the penis indicates that about 5% of the penile neurons in the pelvic plexus contained the neuropeptide while larger percentages of penile neurons in the sympathetic chains were immunoreactive for neuropeptide Y. Chemical and surgical sympathectomy greatly reduced the neuropeptide Y- and catecholamine-containing fibers in the erectile tissue but had no clear effect on the neuropeptide Y fibers around the paraurethral glands; a tissue that is not innervated by adrenergic fibers. It is concluded that (1) the widespread distribution of neuropeptide Y indicates that it may function in the control of penile blood flow, (2) with the possible exception of the paraurethral glands, the sympathetic chain is the most likely source of neuropeptide Y fibers in both erectile bodies of the penis, and (3) this peptide may play a role in the secretory functions of the paraurethral glands.     

Localization of l-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

Summary The localization of l-glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, was studied in the rat major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex by indirect immunofluorescence technique with a specific antiserum raised in rabbits. GAD immunoreactivity was demonstrated in small cells of these ganglia. The GAD-immunoreactive small cells were 10–20 m in diameter and formed clusters or occured as solitary cells. The principal neurons were non-reactive but they were surrounded by immunoreactive processes. Studies on colocalization of GAD with tyrosine hydroxylase (TH), the rate-limiting enzyme of the catecholamine synthesis, in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex indicated that all GAD-immunoreactive small cells were also labelled with TH. In the major pelvic ganglion all TH-immunoreactive SIF cells were also immunoreactive for GAD. However, in the coeliac-superior mesenteric ganglion complex there occured TH-immunoreactive small cells which showed no immunoreactivity to GAD. It is suggested that the small GAD-immunoreactive cells represent small intensely fluorescent (SIF) cells.