Close association of peptidergic nerves with lymphocytes in canine and monkey ileal villi.

Intimate association of peptidergic nerves with lymphocytes of canine and monkey ileal villi was demonstrated by immunohistochemistry and transmission electron microscopy. A swollen, presumably terminal, portion of nerves containing large cored vesicles and small clear vesicles was in direct contact with a lymphocyte. The apposing membranes of the nerve and lymphocyte were thickened and darkened, being separated by a narrow uniform space. The lymphocyte-associated nerves contained immunoreactivity for substance P(SP), calcitonin gene-related peptide (CGRP) or vasoactive intestinal polypeptide (VIP), localized in large-cored vessels. These result support the hypothesis that peptidergic nerves may play a regulatory role in mucosal immune responses.     

Ultrastructural localization of VIP-like immunoreactivity in large dense-core vesicles of ‘cholinergic-type’ nerve terminals in cat exocrine glands

Exocrine glands of the cat were analysed with the peroxidase-antiperoxidase method and routine electron microscopy. Vasoactive intestinal polypeptide (VIP)-like immunoreactivity was observed in certain nerve endings in the submandibular salivary gland, lacrimal gland and Harderian gland. The distribution of the VIP immunoreactive nerve fibres agreed well with earlier light microscopic findings. At the electron microscopic level electron-dense precipitates representing VIP-like immunoreactivity were seen in so-called large dense-core vesicles (median diameter about 990A?) in nerve fibres and varicosities also containing many small (‘immunonegative’) agranular vesicles. In conventional electron microscopy, the small agranular vesicles outnumbered the large dense-core vesicles by about 9 to 1. Immunoreactive fibres and varicosities could be seen close to the secretory acini (distance less than 400A?) and more distant (1500A?or more) to e.g. demilunes, ducts and blood vessels of the glands. The number and distribution of immunoreactive nerve fibres were not affected by sympathectomy. Furthermore, no typical ‘p-type’ bouton profiles, which are dominated by large opaque vesicles (dia. 800–2000A?), could be seen in the ultrastructural analysis of conventional preparations of the glands.The morphological features of the VIP immunoreactive nerve endings could not be distinguished from those often described as representing cholinergic fibres. These findings are in agreement with earlier suggestions of a possible coexistence of acetylcholine and VIP in neurons innervating exocrine glands and indicate possible functions for VIP in the roles of these nerves in evoking vasodilation and exocrine secretion.     

Light and electron microscopic demonstration of neuropeptide Y-like immunoreactive nerves in human cardiac muscle

Summary Neuropeptide Y (NPY)-like immunoreactive nerves were demonstrated in human cardiac muscle. The atrial specimens were obtained from open-heart surgery. The PAP method was applied for immunocytochemistry for light and electron microscopy. A dense, extensive network of NPY-like immunoreactive nerve fibres was seen between cardiac muscle cells and around blood vessels. In electron microscope PAP precipitates were localized in large dense-cored vesicles of 80–120 nm in size in separate nerve terminals or in the terminals situated in the nerve bundles. Close contacts were observed between NPY nerves and muscle cells and blood vessels.The possible functional role of NPY innervation in the human heart is discussed.     

Ultrastructural localization of VIP-immunoreactivity in canine distal oesophagus

Summary The localization of vasoactive intestinal polypeptide (VIP) immunoreactivity in canine distal oesophagus was studied using different fixation and embedding procedures and labelling with protein A-gold. The retention of immunoreactivity and the preservation of ultrastructure was best after fixation with a mixture of 0.1% glutaraldehyde and 4% paraformaldehyde, and embedding in LR White resin using the cold-cured method. VIP immunoreactivity was localized exclusively over large granular vesicles in the myenteric plexus and in nerves of the circular muscle. Most varicose profiles in circular muscle were labelled, but some large granular vesicles in the same profiles which contained labelled vesicles as well as some varicose profiles with large granular vesicles were unlabelled. From these data it was uncertain whether unlabelled large granular vesicles contained VIP or other neuropeptides. A striking finding was the dense and close innervation of the interstitial cells of Cajal with nerve containing VIP-labelled large granular vesicles. This finding is consistent with earlier suggestions that VIP-immunoreactive nerves may innervate these cells which are in gap junction contact with smooth muscle and that this arrangement may be involved in the transmission of non-adrenergic, non-cholinergic nerve activity in distal oesophagus.     

人皮肤经络线上P物质和VIP免疫反应神经纤维与肥大细胞关系的光镜和电镜研究

本研究在已发现神经与肥大细胞之间有突触样联接关系的的基础上,检查了与肥大细胞形成联接的神经纤维终末内是否含有P物质和血管活性肠肽(VIP)。证明:光镜下P物质免疫反应阳性神经纤维与肥大细胞直接接触;将光镜看到的目的物用原位包埋法转到电镜下,可见P物质免疫反应轴索与肥大细胞紧密并靠,两者之间距离不超过20nm。含P物质免疫反应囊泡的轴突末梢在近肥大细胞处形成膨大,有的膨大部直接与肥大细胞接触,形成突触样联接。证实了前人关于与肥大细胞形成联接的轴突末梢内含有P物质的推断。此外,在光镜下证明的P物质和VIP免疫反应神经纤维在皮肤的分布与前人的报道一致,本实验并观察了它们在电镜下的结构特点。     

GRP (gastrin-releasing peptide)-containing nerves in the rat stomach and stress-induced depletion of their synaptic vesicles. An electron microscope study.

Gastrin-releasing peptide (GRP)-containing nerves are extremely numerous in the mucosa of the gastric body. Our previous study demonstrated that depletion of GRP from the nerves occurred in close relation to ulceration in the stomach. The present study deals with the ultrastructure of the GRP-immunoreactive nerves under normal conditions and its changes induced by conditions of stress. The immunoreactivity for GRP was recognized selectively in large cored vesicles in the swollen axoplasm of the nerves. The same axoplasm further revealed immunonegative small clear vesicles which were believed to contain acetylcholine. In materials where the GRP-immunoreactive nerves markedly decreased in number, both large and small synaptic vesicles were depleted from the nerves. These findings suggest that GRP and acetylcholine coexist in single nerves in the oxyntic mucosa, and that by nerve stimulation, they are coreleased into the lamina propria.     

A light and electron microscopical study of enkephalin-immunoreactive structures in the rat neostriatum after removal of the nigrostriatal dopaminergic pathway.

The pattern of enkephalin immunoreactivity was examined in the adult rat neostriatum, at various times after unilateral removal of the nigrostriatal dopamine input by 6-hydroxydopamine injection into the medial forebrain bundle. Animals were examined 12 days, 26 days or 13 months after the lesion. Enkephalin-immunoreactive synaptic boutons (n = 1018) in the control and the dopamine-depleted neostriatum were analysed in the electron microscope. The area of enkephalin-immunoreactive synaptic bouton profiles was significantly larger in the dopamine-depleted neostriatum and this increase was maximal in rats in which the lesion had been made 26 days or 13 months previously (50% increase). The synaptic specializations of these enkephalin-immunoreactive boutons were significantly longer in the neostriatum from the injected side. Dendritic shafts were the principal postsynaptic target of these boutons (67%) but dendritic spines (18%), perikarya (6.5%) and unidentifiable small dendrites or spines (8.5%) were also contacted. The proportions of enkephalin-immunoreactive boutons on the different postsynaptic targets were not altered by the 6-hydroxydopamine lesion. The increase in enkephalin immunoreactivity observed in the dopamine-depleted neostriatum in previous studies may be explained by the increase in the size of enkephalin-immunoreactive synaptic boutons found in the present ultrastructural investigation. The observations do not rule out the possibility that there is also an increase in the number of immunoreactive synaptic boutons, due to, for example, sprouting of the existing enkephalin-containing fibres.     

大鼠神经垂体内后叶加压素神经分泌末梢的GABA能神经支配的免疫电镜证实

为了探讨神经垂体内后叶加压素（ＶＰ）释放的γ－氨基丁酸（ＧＡＢＡ）能神经调控，本研究用包埋前ＡＢＣ法结合免疫电镜双标技术，研究了大鼠神经垂体内ＶＰ能神经和ＧＡＢＡ能神经的超微结构分布及其相互联系。先用ＤＡＢ法显示ＧＡＢＡ免疫反应，然后用钼酸铵－ＴＭＢ法显示ＶＰ免疫反应，再用ＤＡＢ－氯化钴稳定后作免疫电镜包埋。电镜观察发现：在神经垂体内ＧＡＢＡ样免疫反应产物呈电子密度高的颗粒状沉淀，定位于神经末梢内的小清亮羹泡周围和线粒体膜上；ＶＰ样免疫反应产物呈电子密度高的不规则形块状或针状散在于神经分泌末梢内。ＧＡＢＡ样神经末梢分布于神经分泌末梢之间或紧贴毛细血管和垂体细胞，可与神经分泌末梢紧密接触甚至形成突触。ＶＰ样神经分泌末梢内含小透亮囊泡和大颗粒囊泡，并可与ＧＡＢＡ样轴突末梢形成突触。在这种情况下，ＧＡＢＡ样轴突为突触前成分，突触前、后膜呈对称性，突触间隙宽度小于２０ｎｍ，内含电子致密物质。以上结果证实，在大鼠神经垂体内ＶＰ释放受到ＧＡＢＡ神经的直接突触调控。     

Alterations in the distributory pattern of vasoactive intestinal polypeptide immunoreactivity in the ischemic intestines of dogs

Alterations in the distribution of vasoactive intestinal polypeptide (VIP) in normal and ischemic small intestines of dogs were studied by using conventional transmission electron microscope, and immunohistochemistry for light and electron microscopy. At the light microscopic level, immunoreactivity was evident in the intestinal ganglionic cells of control segments. At the electron microscopic level using a pre-embedding method, the entire cytoplasm of the ganglionic cells in the control segments was filled with VIP immunoreactive products, while the post-embedding experiment showed positive reactions only within the VIP granules and Golgi vesicles. After 30 min of ischemia, immunoreactivity was greatly decreased in the ganglionic cells and a large amount of VIP immunoreactive product appeared in the striated border of epithelial cells and in nerve fibers of the subepithelial layer. These results suggest that intestinal ischemia might lead to the release of VIP, which seems to bind to the microvillus membrane of epithelial cells. The relationship between the changes in VIP distribution and its protecting mechanisms of ischemic damage is discussed.     

Age-related changes of enkephalinergic innervation of human sympathetic neurons

The age-related changes in the distribution of enkephalin-immunoreactive (ENK-IR) nerve fibers in relation to the principal neurons were studied in 23 human sympathetic ganglia from patients aged between 22 and 98 years. There were no age-related changes in the diameter of nerve cells and the packing density of the neurons did not change. There was an age-related decrease in the proportion of neurons innervated by enkephalin-containing fibers. In all age groups the neurons innervated by ENK-IR nerve fibers were larger than those neurons which were not surrounded by ENK-IR nerves. The pigment material in the enkephalin-innervated neurons was non-osmiophilic. The small neurons containing dark neuromelanin type of age pigment were not innervated by ENK-IR nerves. It is speculated that ENK-IR preganglionic nerves play a role in maintaining neuronal integrity of sympathetic neurons.     

Sympathetic nerve activation decreases the release of vasoactive intestinal polypeptide from the feline intestine

The splanchnic nerves to the small intestine were stimulated in anaesthetized cats. Activation of the sympathetic nerves caused vasoconstriction, increased net fluid absorption and decreased release of vasoactive intestinal polypeptide (VIP) in the small intestine. In the colon, parasympathetic nerve stimulation elicited vasodilatation and increased release of VIP. Additional stimulation of the sympathetic lumbar colonic nerves decreased the colonic blood flow and inhibited the release of VIP. These effects of the stimulation of the lumbar colonic nerves were blocked by phentolamine. It is concluded that, in the feline intestine, sympathetic nerve stimulation presynaptically decreased the release of VIP via an alpha-adrenergic mechanism.     

Peptide containing nerves in the ureter of the guinea-pig and cat

We report the presence of two regulatory peptides, substance P and vasoactive intestinal polypeptide (VIP), in the ureter and their localisation by both light- and electron-microscopy to autonomic nerves. VIP- and substance P-like immunoreactive nerves showed, in general, a similar anatomical distribution in the various layers of the ureter. Immunoreactive nerves were observed running along the smooth muscle coat, parallel to muscle bundles, around blood vessels and in the submucosa, particularly beneath the epithelium. In addition, scattered VIP-like immunoreactive ganglion cells and nerve fibres were seen in adventitial ganglia around the most distal part of the ureter and ureter-bladder junction in the cat. The guinea-pig ureter contained principally substance P-like immunoreactivity, whereas the cat ureter possessed mainly VIP-like material.The distribution of adrenergic and cholinergic nerves was compared with those containing peptides. Peptide-containing nerves had a more extensive distribution than adrenergic ones, which were mainly associated with blood vessels; however, cholinergic nerves were often localised in the same areas as those possessing peptides. Conventional electron microscopy revealed that separate p-type (peptidergic) and cholinergic nerve terminals were frequently present in the same nerve bundles, although in the cat ureter some 50% of the p-type profiles contained a mixed population of vesicles, characteristic of both cholinergic and p-type nerves. VIP- and substance P-like immunoreactivity were also localised at the ultrastructural level by means of a gold-labelled goat-antirabbit serum.     

Properties of nerve endings with small granular vesicles in the distal colon and rectum of the guinea-pig

The appearance and distribution of nerve endings (varicosities) containing small granular vesicles have been studied in the distal colon and rectum of the guinea-pig with the electron microscope. Two types of varicosity were recognised. They were distinguished by differences in their synaptic vesicles and in their distribution in the layers of the gut wall. The first type resembled noradrenergic nerves in having predominantly (92%) small vesicles and few (8%) large granular vesicles (90 nm diameter). This type was common in the plexuses and at the medial-adventitial border of arteries and arterioles but was sparsely distributed in the muscle coat. The second type had a lower proportion of small vesicles (69%) and a higher proportion (31%) of large granular vesicles (132 nm diameter). This type was absent in Auerbach's plexus, well represented in the muscle coat and Meissner's plexus and not associated with blood vessels. The first type was labelled with 5-hydroxydopamine, a specific marker for noradrenergic nerves, and disappeared after extrinsic denervation. a procedure which causes degeneration of noradrenergic nerves in the gut. The second type was unaffected by 5-hydroxydopamine and extrinsic denervation.It is concluded that the two types of small granular vesicle-containing varicosities belong to different axons and that the first type is noradrenergic. The second type of nerve axon has not been described in the gut before and is intrinsic to it. From the distribution and numbers of these axons in the circular muscle it would seem that they play an important role in gut motility.     

Serotonergic terminals in the neural sheath of the blowfly nervous system: electron microscopical immunocytochemistry and 5,7-dihydroxytryptamine labelling

With serotonin immunocytochemistry we have demonstrated an extensive plexus of immunoreactive varicose fibres in the neural sheath of the nervous system of the blowfly, Calliphora. These fibres are located in the neural sheath of the following regions: the maxillary-labial and labrofrontal nerves of the cerebral ganglia, the cervical connective, the dorsal surface of the thoracicoabdominal ganglia, two pairs of prothoracic nerves and the median abdominal nerve. We identified the serotonin-immunoreactive neural processes in the electron microscope by means of the peroxidase-antiperoxidase method. Immunoreactivity was seen in large granular vesicles (ca 100 nm), on membranes of smaller (ca 60 nm) and larger (ca 100 nm) agranular vesicles, along the inner surface of the axolemma, along neurotubules and outer membranes of mitochondria. By conventional electron microscopy we found numerous varicose neural processes in the neural sheath of some of the above regions. These varicosities are of at least two types. One type corresponds to the serotonin-immunoreactive profiles. A second type contains large granular vesicles (ca 200 nm) of variable electron density. 5,7-Dihydroxytryptamine injected into the head capsule labelled varicosities in the neural sheath, corresponding to the ones identified with serotonin immunocytochemistry. The electron-dense labelling was seen in flattened vesicles within these varicosities. We propose that the serotonin-immunoreactive fibers in the neural sheath constitute neurohemal regions for the release of serotonin into the circulation. The finding of another morphological type of varicose fibers in the neural sheath suggests the presence of further putative neurohormones in these regions.     

Pharmacological and ultrastructural studies on the electron dense cores of the vesicles that accumulate in noradrenergic axons constricted in vitro.

The noradrenaline storage vesicles that accumulate in axons proximal to ligatures applied to cat hypogastric nerves incubatedin vitro have been examined in the electron microscope with different fixation and staining procedures after exposure of the nerves to a variety of drugs which alter their noradrenaline content. After glutaraldehyde/osmium tetroxide fixation and lead citrate staining the granules possess electron-dense cores irrespective of their noradrenaline content. After fixation with Palade's osmium tetroxide or glutaraldehyde/dichromate followed by lead citrate staining, dense cores are not seen after treatment with noradrenaline-depleting agents but reappear after staining additionally with uranyl acetate.It is concluded that the matrix of axonal (large) noradrenaline storage vesicles contains some material in addition to catecholamines and this possibility is discussed in connection with the relationship between large and small dense-cored vesicles seen in sympathetic nerve endings.     

Study of the efferent connections of the enkephalinergic magnocellular dorsal nucleus in the guinea-pig hypothalamus using lesions, retrograde tracing and immunohistochemistry: evidence for a projection to the lateral septum

The efferents of enkephalin-immunoreactive neurons in the magnocellular dorsal nucleus of the guinea-pig were studied using different neuroanatomical methods and indirect immunocytochemical technique. Following unilateral implantation of the fluorescent dye 4',6-diamidino-2-phenylindole in the lateral septal nucleus, retrogradely-labeled perikarya were found in the magnocellular dorsal nucleus. These labeled perikarya reacted with antiserum against enkephalin, demonstrating that enkephalin-immunoreactive neurons in the magnocellular dorsal nucleus project to the lateral septal nucleus. In other experiments, complete bilateral lesions were produced in the magnocellular dorsal nucleus by electrocoagulation. Enkephalin-immunoreactive nerve fibers and terminals were totally depleted in the lateral septal nucleus. This confirms that septal enkephalin-immunoreactive terminals originate in the magnocellular dorsal nucleus and further suggests that this nucleus is the source of all the enkephalin-immunoreactive material found in the septum. Experiments utilizing two different fluorescent dyes, 4',6-diamidino-2-phenylindole and propidium iodide, injected in each side of the lateral septal nucleus, respectively, demonstrated that the magnocellular dorsal nucleus gives off axon collaterals to both sides of the septum, since double-labeling of individual cell bodies was detected in the nucleus. By relating this finding to the results obtained after unilateral destruction of the nucleus, which caused an incomplete loss of enkephalin- immunoreactive material in the lateral septal nucleus ipsilaterally, it is suggested that the enkephalinergic hypothalamo-septal pathway contains unbranching neurons projecting ipsilaterally and branching neurons distributing fibers ipsilaterally and contralaterally. Lesion experiments, and experiments based on the retrograde axonal transport of horseradish peroxidase after intravenous injections, demonstrated that the magnocellular dorsal nucleus contributes neither to the tubero-infundibular nor to the hypothalamo-neurohypophyseal tracts. The lateral septal nucleus receives numerous aminergic and peptidergic projections, indicating the potential importance of this region in physiological and behavioral events. In the guinea-pig, the well-demarcated enkephalinergic pathway demonstrated in this study provides a convenient model for the experimental study of the enkephalinergic innervation of the lateral septal nucleus.     

Ultrastructural morphometric analysis of enkephalin-immunoreactive terminals in the ventrocaudal periaqueductal gray: analysis of their relationship to periaqueductal gray-raphe magnus projection neurons

The periaqueductal gray plays an important role in the descending modulation of nociception. While the importance of endogenous opioids to periaqueductal gray circuits that modulate nociception is supported by many studies, the ultrastructural relationships between enkephalin-immunoreactive axon terminals and the surrounding periaqueductal gray neuropil have not been quantitated in the rat. Further, the possible interaction between enkephalin-immunoreactive axon terminals and periaqueductal gray neurons that project to the rostroventral medulla has not been described. The present study utilized electron microscopic immunocytochemistry to quantitate the normal neuronal associations of enkephalin-immunoreactive terminals in the caudal periaqueductal gray of the rat. A primary focus of this analysis was to ascertain whether any interaction exists between enkephalin-immunoreactive axon terminals and periaqueductal gray neurons that were retrogradely-labeled from the nucleus raphe magnus and adjacent medullary reticular nuclei. We examined the ventrolateral periaqueductal gray and the ventral periaqueductal gray immediately subjacent to the aqueduct and found that both the average terminal diameters and the volume fractions of enkephalin-immunoreactive terminals were very similar. In these two regions, most terminals were observed to be in close apposition to either two or three dendrites that were neither retrogradely-labeled nor enkephalin-immunoreactive, although axonal and perikaryal associations were also observed. In the ventrolateral periaqueductal gray, 22% of all enkephalin-immunoreactive terminals were adjacent to periaqueductal gray-nucleus raphe magnus and periaqueductal gray-reticular nucleus projection neurons. In the periaqueductal gray subjacent to the aqueduct, 32% of all enkephalin-immunoreactive terminals were adjacent to periaqueductal gray-nucleus raphe magnus and periaqueductal gray-reticular nucleus projection neurons. Symmetrical synapses with these retrogradely-labeled neurons were formed by 5.5% of enkephalin-immunoreactive terminals in the ventrolateral periaqueductal gray, and by 4.3% of enkephalin-immunoreactive terminals located subjacent to the aqueduct. We also noted that enkephalin-immunoreactive terminals formed symmetrical synapses with non-retrogradely-labeled, enkephalin-immunoreactive dendrites in the periaqueductal gray. Direct opioid input onto putative excitatory periaqueductal gray output neurons that are hypothesized to modulate nociception was an unexpected finding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural simultaneous localization of vasoactive intestinal polypeptide immunoreactivity and the acetylcholine-like cation in rat endocrine pancreas

The ultrastructural co-localization of vasoactive intestinal polypeptide (VIP) and acetylcholine (ACh)-like cation was demonstrated in the rat endocrine pancreas. Immunoperoxidase cytochemical procedure for the detection of VIP was followed by ionic fixation of ACh by silicotungstic acid. All the VIP-immunoreactive nerve endings displayed punctiform precipitates revealing ACh-like cations in the small clear vesicles. Almost all the VIP-immunoreactive nerve terminals were in contact with endocrine A, B or D cells, but not in the close vicinity of blood vessels. Nerve endings devoid of VIP immunoreactivity but containing ACh-like cations were seen either in contact with endocrine cells or in close vicinity to blood vessels. These data suggest that VIP might modulate, concomitantly with ACh, the hormonal secretion of the endocrine pancreas, although the possibility of the neurosecretion of ACh and VIP into the blood vessels should not be excluded. ACh might also control some function of the endothelial cells of blood vessels.     

Coexistence of vasoactive intestinal peptide and enkephalins in the adrenal chromaffin granules of the frog

The chromaffin cells of the frog adrenal gland were studied at the electron microscopic level by means of the immunoperoxidase technique. Using specific antibodies against Met-enkephalin, Leu-enkephalin and vasoactive intestinal peptide (VIP), the coexistence of the three neuropeptides in the chromaffin cells was demonstrated. Furthermore, all three peptides were co-located in the 200-400 nm dense-core vesicles which also stained for dopamine-beta-hydroxylase. These results, which suggest concomitant release of catecholamines and neuropeptides by the chromaffin cells, support the concept that VIP is involved in the stimulation of adrenal steroid secretion during stress conditions.