Janet R. Keast

Choline acetyltransferase- and peptide immunoreactivity of submucous neurons in the small intestine of the guinea-pig

SummaryThe peptides cholecystokinin (CCK), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP), and the synthesizing enzyme for acetylcholine, choline acetyltransferase (ChAT) were localized immunohistochemically in nerve cell bodies of the submucous ganglia in the small intestine of the guinea-pig. VIP-like immunoreactivity was found in 45% of submucous neurons. ChAT immunoreactivity was observed in a separate group of nerve cells, which made up 54% of the total population. There were three subsets of neurons immunoreactive for ChAT: (1) ChAT neurons that also contained immunoreactivity for each of the peptides CCK, SOM and NPY, representing 29% of all submucous neurons; (2) ChAT neurons that also contained SP-like immunoreactivity, representing 11% of all submucous neurons, and (3) ChAT cells that did not contain any detectable amount of the peptides that were localized in this study.

Origins of Peptide and Norepinephrine Nerves in the Mucosa of the Guinea Pig Small Intestine

Norepinephrine, acetylcholine, and certain peptides are contained in mucosal nerves and have potent effects on transepithelial water and electrolyte fluxes. It is difficult to ascribe roles for these nerves as their sources are unknown. The present studies were undertaken to determine the origins of nerve fibers that are found in the mucosa of the guinea pig small intestine and which contain one of the following substances: vasoactive intestinal peptide, substance P, somatostatin, neuropeptide Y, cholecystokinin, or norepinephrine. Nerve fiber origins were ascertained by making lesions to sever pathways through which the nerves could reach the mucosa. The lesioning operations were homotopic autotransplants of short (2 cm) segments of intestine; myectomies, in which a 5-10-mm length of intestine was stripped of longitudinal muscle and myenteric plexus; and extrinsic denervation, in which nerves reaching the intestine through the mesentery were severed. The results of these studies, considered along with previously published work, led to the upcoming conclusions. Nerve fibers in the mucosa showing immunoreactivity for vasoactive intestinal peptide, somatostatin, cholecystokinin, and neuropeptide Y arise from cell bodies in the overlying submucous plexus. Substance P fibers arise in part from the overlying submucous plexus and in part from the overlying myenteric plexus. Mucosal norepinephrine fibers arise from extrinsic sympathetic ganglia. Enkephalin, gastrin-releasing peptide, and 5-hydroxytryptamine, which are in some enteric nerves, are not found in submucous nerve cells and few, if any, fibers containing these substances supply the mucosa. Thus, the mucosa receives a dense nerve supply, much of which arises locally from submucous ganglia.

A possible neural source of nitric oxide in the rat penis

NADPH diaphorase staining was used to indicate the presence of nitric oxide synthase (NOS) in whole mounts of rat major pelvic ganglion (MPG) and sections of rat penis. Many stained neurons were observed in the MPG and were distributed in a manner identical to that of retrogradely labelled penile neurons described previously. Staining was also observed within many axons of the penile (cavernous) nerve and in varicose terminals associated with various tissues of the penis. The results suggest that many, if not all, penile neurons of the MPG contain NOS and that a neural source of NO within the penis is likely.

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

SummaryIn 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.

Visualization and immunohistochemical characterization of sympathetic and parasympathetic neurons in the male rat major pelvic ganglion

Pelvic ganglia contain a mixture of sympathetic and parasympathetic neurons which are innervated by either lumbar (sympathetic) or sacral (parasympathetic) preganglionic axons, respectively. However, until recently no stain for these axon terminals has been available. In the present study of the male rat major pelvic ganglion, a ganglion which supplies axons to the lower urinary and digestive tracts and internal reproductive organs, the total population of preganglionic axon terminals was immunostained using an antiserum against synaptophysin, a protein associated with all small synaptic vesicles (such as the presumptive cholinergic vesicles present in all preganglionic terminals). Selective bilateral lesions of either the hypogastric or pelvic nerves, which carry the sympathetic and parasympathetic preganglionic axons, respectively, were carried out and three to seven days later ganglia were examined immunohistochemically for the distribution of residual synaptophysin-positive terminals. Neurons remaining innervated following hypogastric nerve lesion were therefore classified as parasympathetic and those innervated after pelvic nerve section were defined as sympathetic. These two cell groups are present in approximately equal proportions. Double-staining immunofluorescence to identify which transmitters or peptides are present in either sympathetic or parasympathetic neurons showed that the majority (approximately 75%) of sympathetic neurons are presumed to be noradrenergic (i.e. contain tyrosine hydroxylase immunoreactivity) and are also immunoreactive for neuropeptide Y; the remainder contain vasoactive intestinal peptide-immunoreactivity but not tyrosine hydroxylase and may be cholinergic. Parasympathetic neurons were virtually all non-noradrenergic (tyrosine hydroxylase negative) and were also of two histochemical types, with some neurons containing neuropeptide Y- and others containing vasoactive intestinal peptide-immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical evidence for the presence of calcium-binding proteins in enteric neurons

SummaryImmunoreactivity for vitamin D-dependent calcium-binding protein (CaBP) has been localized in nerve cell bodies and nerve fibres in the gastrointestinal tracts of guinea-pig, rat and man. CaBP immunoreactivity was found in a high proportion of nerve cell bodies of the myenteric plexus, particularly in the small intestine. It was also found in submucous neurons of the small and large intestines. Immunoreactive nerve fibres were numerous in the myenteric ganglia, and were also common in the submucous ganglia and in the intestinal mucosa. Immunoreactive fibres were rare in the circular and longitudinal muscle coats. In the myenteric ganglia of the guinea-pig small intestine the immunoreactivity is restricted to one class of nerve cell bodies, type-II neurons of Dogiel, which display calcium action potentials in their cell bodies. These neurons were also immunoreactive with antibodies to spot 35 protein, a calcium-binding protein from the cerebellum. From the distribution of their terminals and the electrophysiological properties of these neurons it is suggested they might be sensory neurons, or perhaps interneurons. The discovery of CaBP in restricted sub-groups of enteric neurons may provide an important key for the analysis of their functions.

Testosterone has potent, selective effects on the morphology of pelvic autonomic neurons which control the bladder, lower bowel and internal reproductive organs of the male rat

Although gonadal steroids are important determinants of the development and activity of various neuronal circuits in the brain and spinal cord, their function has been relatively poorly studied in the peripheral nervous system. In the present work, the effects of pre- and postpubertal castration were examined on the morphology of autonomic neurons that supply pelvic visceral organs in male rats. These neurons were identified by peripheral injection of fluorescent retrograde tracers and, in the major pelvic ganglion, were further classified as sympathetic or parasympathetic by means of immunostaining for tyrosine hydroxylase. Sizes of ganglion cell somata were indicated by areas of nucleated profiles in cryosections. The results show that, irrespective of whether castration was carried out at two or seven weeks-of-age, noradrenergic pelvic neurons that supply the vas deferens, prostate gland, urinary bladder or colon achieved only approximately 60% of the size of those in unoperated controls. In contrast, cholinergic pelvic neurons were unaffected by castration unless they supplied reproductive targets. Pre- and paravertebral sympathetic neurons that supplied the pelvic viscera were only slightly smaller following castration or were unchanged, depending on their target. All effects of castration were prevented by testosterone replacement following surgery. Androgen receptor-immunoreactivity was particularly prevalent in the nuclei of some pelvic ganglion neurons. The studies suggest that circulating androgens are essential for the maturation and maintenance of the structure of select groups of autonomic neurons that supply the viscera. The presence of androgen receptor immunoreactivity in many of these neurons indicates that direct neuronal effects of androgens are possible. However this does not exclude other less direct mechanisms of steroid action on neurons, such as by an effect on target organs, neurotrophic factor release or peripheral vascular supply. These studies point to the androgenic steroids as potentially important determinants of autonomic reflex function.

Plasticity of pelvic autonomic ganglia and urogenital innervation

Pelvic ganglia contain a mixture of sympathetic and parasympathetic neurons and provide most of the motor innervation of the urogenital organs. They show a remarkable sensitivity to androgens and estrogens, which impacts on their development into sexually dimorphic structures and provide an array of mechanisms by which plasticity of these neurons can occur during puberty and adulthood. The structure of pelvic ganglia varies widely among species, ranging from rodents, which have a pair of large ganglia, to humans, in whom pelvic ganglion neurons are distributed in a large, complex plexus. This plexus is frequently injured during pelvic surgical procedures, yet strategies for its repair have yet to be developed. Advances in this area will come from a better understanding of the effects of injury on the cellular signaling process in pelvic neurons and also the role of neurotrophic factors during development, maintenance, and repair of these axons.

Glutamate and aspartate immunoreactivity in dorsal root ganglion cells supplying visceral and somatic targets and evidence for peripheral axonal transport.

Glutamate (Glu) is released by primary sensory neurons at their central synapses, although immunohistochemical studies have shown that only a proportion of these cell bodies are Glu‐immunoreactive. Antisera raised against Glu or aspartate (Asp) were used to investigate whether neurons that store high levels of these substances have a unique neuropeptide content or target projection. In male rats, many lumbar and sacral dorsal root ganglion cells and their associated glia show high levels of Glu or Asp immunoreactivity, and fewer than half of these also express substance P or calcitonin gene‐related peptide. Conversely, only a minority of peptide‐containing neurons store high levels of excitatory amino acids. When neurons that were labelled retrogradely from somatic (skin, gastrocnemius muscle) or visceral (bladder, rectum) targets were immunostained for peptides or amino acids, there was some variation in the peptide expression of their sensory nerve supply, but there was very little or no difference in the prevalence of Glu‐ or Asp‐immunoreactive neurons. In vitro studies on isolated lumbar dorsal root ganglia showed that, after crushing nerve roots, Glu and Asp were transported in both central and peripheral directions, similar to substance P. These studies showed that primary afferent neurons store different levels of Glu and Asp in their somata but that this is not correlated with their target tissue or peptide content. This suggests that both visceral and somatic sensory neurons may vary considerably in the way they release, store, or metabolise amino acids. Peripheral and central transport of amino acids suggests that, in some neurons, reuptake at the synapse may need to be supplemented by amino acids that are produced or accumulated in the soma. J. Comp. Neurol. 424:577–587, 2000.

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

SummaryThe 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.