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.     

Topography of the enteric nervous system in Peyer's patches of the porcine small intestine

The mechanisms of intercommunication between the immune and nervous systems are not fully understood. In the case of the intestine, the enteric nervous system is involved in the regulation of immune responses. It was therefore decided to employ immunohistochemical techniques to investigate the structural organization of the enteric nervous system in Peyer's patches of the porcine small intestine. Using antibodies against various nervous system-specific markers (protein gene product 9.5, neuron-specific enolase, neurofilament 200, S-100 protein and the glial fibrillary acidic protein), an intimate and specific structural association could be demonstrated between enteric nerves and the compartments of Peyer's patches: follicles, interfollicular regions and domes. Peyer's patches have a close topographical relationship to the two submucosal plexuses. Enteric nerves are located around the follicle in the interfollicular area — the so-called traffic area-and in the dome area, which plays an important role in the uptake and presentation of antigens.     

Multiple α2-Adrenoceptor Signalling Pathways Mediate Pigment Aggregation Within Melanophores

It has previously been shown that α₂-adrenoceptors (α₂-ARs) mediate pigment granule (melanosome) aggregation in melanophores of the teleost fish Labrus ossifagus. The present investigation scrutinized the signalling mechanisms of melanosome aggregation induced by sympathetic nerve stimulation or by exogenous addition of α-AR agonists and cAMP analogues. The following was observed: i) nerve-induced melanosome aggregation was associated with a rapid decrease in the cAMP level; ii) noradrenaline or medetomidine (an α₂-AR agonist) caused melanosome aggregation and reduced the cAMP content; iii) RP-S-CI-cAMP, a membrane-permeating inhibitor of protein kinase A induced melanosome aggregation; and iv) B-HT 920 (an α₂-AR agonist) and methoxamine (an α₁-AR agonist) induced melanosome aggregation, although they did not reduce cAMP. It has been suggested that in some teleost species α₁-ARs mediate melanosome aggregation by increasing the level of intracellular calcium. However, we found that the effect of methoxamine in melanophores from Labrus ossifagus could be blocked by yohimbine (an α₂-AR antagonist) but not by equimolar concentration of prazosin (an α₁-AR antagonist). Furthermore, 1 μM ionomycin (a calcium ionophore) did not induce melanosome aggregation. Our findings therefore do not indicate that α₁-ARs and/or an increase in intracellular calcium mediate melanosome aggregation in Labrus ossifagus. Our results suggest that α₂-AR-mediated melanosome aggregation is induced by multiple signalling pathways. One of these involves a reduction in cAMP, but none involves an increase in intracellular calcium.     

Immunohistochemistry of Grandry corpuscles in the oral mucosa of the duck bill: a light- and electron-microscopic study

Grandry corpuscles in the oral mucosa of the upper bill of the duck were immunohistochemically studied using antisera against calcitonin gene-related peptide (CGRP), galanin, methionine-enkephalin, neuropeptide Y (NPY), somatostatin, substance P (SP) and vasoactive intestinal peptide (VIP). Grandry corpuscles in the lamina propria selectively showed only SP-like immunoreactivity. Herbst corpuscles distributed near Grandry corpuscles were negative to all antisera applied. Although immunoreactive products in the Grandry corpuscles were found as granules in the peripheral cytoplasm of the Grandry cell, the axon terminals and satellite cells exhibited no reactivity. In pre-embedding electron-microscopic sections, SP-like immunoreactive products visualized with 3,3-diaminobezidine were localized in the granules of Grandry cells, but no labeling was observed in the cytoplasmic matrix or cell organelles. Electron-immunocytochemical labeling with colloidal gold by the post-embedding method clearly demonstrated that the SP antigen was localized only in the granules. It is presumed that Grandry cells have a secretory function. However, the function and the method of release of the SP contained in the observed granules remains obscure. Some CGRP-, NPY-, SP- and VIP-like-immunoreactive nerve fibers with varicosities associated with blood vessels and nerve fiber bundles of various sizes were observed in the lamina propria, but no such fibers penetrated into the intraepitherial layer. Nerve fibers positive for SP and VIP were also found in the interlobular connective tissue of the palatine glands. Some SP-positive neurons were detected in the vicinity of the palatine glands.     

Vasoactive intestinal polypeptide (VIP) in the pig pancreas: role of VIPergic nerves in control of fluid and bicarbonate secretion

Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas.     

Ultrastructure and innervation of the swimbladder of Tetractenos glaber (Tetraodontidae)

Summary The general structure, ultrastructure and innervation of the swimbladder of the smooth toadfish, Tetractenos glaber, were examined with light-microscopic, fluorescence-histochemical, and transmission electron-microscopic techniques. The structure of the swimbladder is similar to that of other euphysoclists. Fluorescence histochemistry showed adrenergic fibres in both the secretory and resorptive areas of the swimbladder. Transmission electron microscopy revealed two morphologically distinct axon profiles type-I profiles containing many small, flattened vesicles; type-II profiles containing both large, granular vesicles and rounded, small clear vesicles in varying proportions.The gas-gland cells and surrounding muscularis mucosae are innervated by both type-I and type-II fibres. Type-I fibres also innervate pre-rete arteries. The rete- and gas-gland capillaries do not appear to be innervated. Arteries running to the resorptive area are innervated by type-I fibres. Both type-I and type-II profiles make contact with the muscularis mucosae in the resorptive area. Only type-I fibres innervate the radial dilator muscle in the oval sphincter region, whereas only type II fibres innervate the circular muscle of the oval sphincter.Type-I fibres took up -methyl-noradrenaline, and could not be found after pre-treatment with 6-hydroxydopamine. They are, therefore, assumed to be adrenergic. Type-II fibres were tentatively identified, by exclusion, as cholinergic.     

Fine structural and cytochemical study of the innervation of smooth muscle in an amphibian (Bufo marinus) lung before and after denervation

Summary The innervation of the toad (Bufo marinus) lung was studied with transmission electron microscopy and fluorescence techniques, both before and after 12 or 20 days close vagosympathetic denervation. Four cytologically distinct types of neuronal processes were recognised, in relation to the visceral muscles of the lung. These were described as cholinergic, adrenergic, nonadrenergic/non-cholinergic (NANC) and sensory on the basis of the characteristics of their vesicular content and cytochemical reactions. An apparent efferent innervation of visceral smooth muscle was achieved by NANC (50%), cholinergic (25%) and adrenergic (25%) fibres. A few sensory fibres were also present. After denervation only NANC fibres persisted, showing that the cell bodies of these fibres were intrapulmonary. The vascular smooth muscle was supplied by cholinergic, adrenergic and sensory fibres. In the walls of the proximal branches of the pulmonary artery were fibres containing large dense-cored vesicles. These profiles, which were associated with the vasa vasorum, were similar to neurosecretory fibres. After denervation all neural profiles associated with the vasculature had degenerated. The observations suggest that vagal vasodepressor effects in the toad lung are mediated indirectly through relaxation of visceral muscle strands which in their contracted state compress vascular channels.The authors would like to thank Dr. J.R. McLean for technical advice on fluorescence microscopy. This work was supported by a grant from the Australian Research Grants Committee     

Development of innervation to the atrial myocardium of the rabbit

Summary The development of innervation to the atrial myocardium of rabbits from 20th day of gestation to 35 days postnatal was studied ultrastructurally by electron microscopy and by demonstration of catecholamines by histofluorescence. Special attention was directed to the first morphologic appearance of nerve fibers and terminals and the closeness of juxtaposition of terminals with myocardial cells. Adrenergic and cholinergic terminals were identified on the basis of their differential ability to take-up and store the false adrenergic neurotransmitter 5-hydroxydopamine. Adrenergic terminals were first encountered at 20 days of gestation whereas cholinergic terminals could not be positively identified until the 24th day of gestation. Throughout development adrenergic terminals were more numerous than cholinergic, about 71 % of the terminals encountered being adrenergic. Many terminals approach closely (20–30 nm) to the sarcolemma of the muscle cells of the atrium. In many instances adrenergic and cholinergic fibers travel together in the same nerve bundle and are closely apposed without intervening Schwann-cell cytoplasm. Such a relationship could allow peripheral interaction between these fibers in the myocardium.Supported in part by the Kentucky Heart Association, Human Development Studies Program of the University of Kentucky and DHEW Grant 1 RO1 HL 22226-01 HED from the National Heart, Lung and Blood Institute. The technical assistance of Merle Wekstein is appreciated     

Nerves containing substance P,vasoactive intestinal polypeptide,enkephalin or somatostatin in the guinea-pig taenia coli

Summary The guinea-pig taenia coli is rich in peptide-containing nerves. Nerve fibres containing substance P (SP), vasoactive intestinal peptide (VIP), or enkephalin, were numerous in the smooth muscle while somatostatin fibres were very few. Nerve fibres displaying SP or VIP immunoreactivity were numerous in the myenteric plexus. Enkephalin nerve fibres were fairly numerous in the plexus while somatostatin nerve fibres were sparse. Nerve cell bodies containing immunoreactive SP or VIP were regularly seen in the plexus. Delicate varicose elements of the different types of nerve fibres were found to ramify around nerve cell bodies in a manner suggestive of innervation.In the electron microscope the various peptide-storing nerve fibres (i.e., elements containing SP, VIP or enkephalin) were found to contain a varying number of fairly large, electron-opaque vesicles in the varicose swellings. These vesicles represent the storage site of the neuropeptides.The isolated taenia coli responded to electrical nerve stimulation with a contraction. After cholinergic and adrenergic blockade the contractile response was replaced by a relaxation followed by a contraction upon cessation of stimulation. SP contracted the taenia while VIP caused a relaxation. The enkephalins raised the resting tension slightly while somatostatin had no effect. These observations are compatible with a role for SP as an excitatory neurotransmitter and for VIP as an inhibitory one, and with the view that both SP neurones and VIP neurones act as motor neurones. In preparations contracted by SP the electrically induced contractions were reduced in amplitude while the electrically induced relaxations seen after adrenergic and cholinergic blockade were enhanced in amplitude. In preparations relaxed by VIP there was an increased contractile response to electrical stimulation, while in the atropine + guanethidine-treated preparation the electrically induce relaxations were reduced in amplitude. The enkephalins reduced the contractile response to electrical stimulation, while somatostatin induced a very small reduction in the amplitude of such responses. These observations suggest that SP neurones and VIP neurones may play additional roles as interneurones. Somatostatin neurones probably act as interneurones. Enkephalin-containing fibres may serve to modify the release of transmitter from other nerves in the smooth muscle, perhaps through axo-axonal arrangements. Alternatively, the enkephalin nerve fibres in the smooth muscle are afferent elements involved in mediating sensory impulses to the myenteric plexus.