Electron microscopic immunocytochemical study of somatostatin neurons in the periventricular nucleus of the rat hypothalamus with special reference to their relationships with homologous neuronal processes

The neurons containing somatostatin in the rat periventricular nucleus were studied by using a modified electron microscopic immunocytochemical technique that improves both the penetration of immunoreagents into unembedded immunostained tissues and the preservation of ultrastructural morphology. Inside perikarya and dendrites, immunostaining was not only associated with neurosecretory granules but also with ribosomes and saccules of the cis face of the Golgi apparatus. In the axonal profiles found in this region the labeling was observed both on neurosecretory granule cores and on the limiting membrane of small synaptic-like vesicles. Throughout the periventricular nucleus, both non-synaptic and synaptic relationships were shown between labeled neurons. Non-synaptic relationships mainly consisted of direct apposition of the membranes of neighboring neurons by dendrosomatic, somasomatic or dendrodendritic contacts. These labeled perikarya and dendrites were also synaptically contacted by labeled axonal endings containing numerous aggregated synaptic-like vesicles. The physiological significance of the synaptic and non-synaptic relationships between somatostatinergic neurons is discussed in terms of possible synchronization between homologous neurons of the somatostatin neuroendocrine system and control of these neurons by a central ultra-short loop feedback mechanism.     

Immunohistochemical localization of protein-O-carboxylmethyltransferase in rat brain neurons

The distribution of the enzyme protein-O-carboxylmethyltransferase (EC 2.1.1.24) has been investigated in the rat brain using both immunohistochemical and biochemical techniques. The enzyme, which carboxylmethylates free aspartic and glutamic acid residues of protein substrates, was localized in neurons, but not other cell types throughout the brain. The highest immunoreactivity was detected throughout the cortex, followed by the hippocampus, the corpus striatum, the thalamus and the amygdala. Immunoreactive cells were detected in other brain regions but were not as prominent as those regions listed above. The distribution of immunoreactivity in the hippocampus was most striking, with considerable labelling of the pyramidal and granule cells in all regions. Numerous pyramidal cells were labelled in the cerebral cortex, with some ascending processes exhibiting immunoreactivity. The corpus striatum was uniformly labelled, suggesting that the enzyme was not localized to any specific neurotransmitter system. The antisera employed in this study was generated against purified bovine brain protein-O-carboxylmethyltransferase and Western immunoblot analysis showed cross reactivity against both rat brain and human erythrocyte forms of the enzyme. Enzyme activity and methyl acceptor protein capacity were examined in 1.5 mm coronal sections of rat brain. The regions with highest enzyme activities were found in cross-sections containing cortex and corpus striatum or cortex and hippocampus. The lowest enzyme activities were noted in slices of brainstem and cerebellum, areas exhibiting low amounts of immunoreactive protein-O-carboxylmethyltransferase. Methyl acceptor protein capacity was highest in slices of cortex and corpus striatum, cortex and hippocampus and was lowest in slices of brainstem and cerebellum. These results demonstrate that protein-O-carboxylmethyltransferase has an unique neuronal pattern of distribution in the rodent central nervous system, and suggest that the carboxylmethylation of proteins may be of functional significance in these neurons.     

The olfactory marker protein in the olfactory system of the mouse during development

The olfactory marker protein, a protein specific to the olfactory sensory neurons, has been studied in mouse during embryogenesis and in the postnatal period up to 30 days, with the unlabeled antibody enzyme method of immunohistochemistry. Olfactory neurons, which are morphologically detectable in 10-day-old embryos, do not contain olfactory marker protein. The protein is present in the olfactory neuroepithelium at embryonic day 14 and its appearance coincides with the establishment of sensory synapses in the olfactory bulb. Neurons containing the protein increase in number up to 30 days after birth. At 15 days of embryonic life, immunostaining was observed in sensory axons at the rostral tip of the olfactory bulb, and by embryonic day 17 a plexus of stained fibres has covered the bulbar surface. Between embryonic day 15 and postnatal day 1, olfactory axons have been observed to reach the mitral cell layer. In the vomeronasal system the olfactory marker protein is present at later stages and both the receptors' perikarya and their axons and axon terminals in the accessory olfactory bulb show a lower level of staining than the olfactory system proper.This study of the olfactory marker protein has allowed us to correlate its appearance with significant developmental phenomena.     

Ultrastructural study of presumptive pro-opiocortin producing neurons in the rat hypothalamus

In order to analyse secretory mechanisms in presumptive pro-opiocortin neurons, an ultrastructural study of their perikarya was performed in the hypothalamic arcuate nucleus of adult rats intraventricularly injected with colchicine. This study was carried out both in tissue fixed with aldehydes alone and in tissue fixed with aldehydes and osmium tetroxide.In tissue not exposed to osmium tetroxide, pro-opiocortin neurons can be adequately identified by immunostaining in semi-thin sections with both ACTH 17–39 and β-endorphin antisera. But in adjacent ultra-thin sections of the same neurons, their ultra-structural aspect cannot be studied in detail since, without fixation in osmium tetroxide. the cellular membranes are not preserved. Among the organelles, only lysosomes and elementary granules can be unambiguously identified in the cytoplasm; the latter are selectively immunostained with ACTH antiserum.In semi-thin sections of tissue fixed with aldehydes and then with osmium tetroxide, the pro-opiocortin neurons can be stained only with ACTH 17–39 antiserum and then very poorly when compared with aldehyde-fixed tissue. A detailed ultra-structural study of the presumptive pro-opiocortin perikarya was possible in adjacent ultra-thin sections stained with uranyl acetate and lead citrate. They have the ultrastructural characteristics of active secretory neurons: their cytoplasm is filled with many organelles; it contains an abundant rough endoplasmic reticulum, numerous lysosomes and secretory granules probably accumulated in perikarya by the colchicine-induced blockage of the axonal flow. The Golgi complexes are conspicuously enlarged. They are probably the source of lysosomes and granules. Images of crinophagy and autophagic vacuoles suggest an autoregulatory mechanism in pro-opiocortin neurons to reabsorb organelles accumulated by colchicine.These results show that neurons containing pro-opiocortin have organelles necessary for a synthesis of neuropeptides and their packaging in granules. This supports the hypothesis that the presence of pro-opiocortin-derived peptides in brain is, at least partly, due to their neural synthesis by hypothalamic secretory neurons.     

Light and electron microscopic immunocytochemical localization of vasoactive intestinal polypeptide VIP-like activity in the rat small intestine

Vasoactive intestinal polypeptide nerve processes and cell bodies were identified by electron microscopic immunocytochemistry in the rat small intestine. Labeled nerve processes were numerous in the inner circular smooth muscle coat and mainly in the mucosa, but were absent in the longitudinal muscle layer. Submucosal blood vessels were often surrounded by immunoreactive vasoactive intestinal polypeptide positive nerves, in close associations (distance less than 40 mn) to blood vessel basement membranes and to smooth muscle cells. In the ganglia of the myenteric and submucous plexuses, labeled fibers surrounded unstained neural cell bodies. The synaptic vesicles of vasoactive intestinal polypeptide positive terminals were 35-40 nm in diameter and some dense core vesicles (80-120 nm in diameter) were also observed in the same profiles. These observations suggest that vasoactive intestinal polypeptide nerves may participate in regulating smooth muscle activity and local blood flow in the small intestine.     

Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. I. Morphological properties and immunocytochemical localization of transmitter candidates

We have developed procedures for dissociating neurons from the myenteric plexus of the small intestine of newborn rats and for growing those neurons in cell cultures for up to 3 months. Neurons in these cultures retain many of the differentiated properties of myenteric neurons in vivo. This is the first of a series of 3 papers describing those properties. In this paper, we describe the morphology of cultured neurons that we have observed with light and electron microscopy; we also describe the patterns of straining observed when immunocytochemical techniques were used to localize neurotransmitter candidates in the cultured neurons. Intracellular injections of a fluorescent dye, Lucifer yellow, revealed that many of the cultured neurons had morphologies similar to those of myenteric neurons in vivo. When thin sections of cultures were viewed in an electron microscope, many neurons were observed to have numerous small (40-60 nm), clear synaptic vesicles and/or large (80-150 nm), opaque-cored (p-type) vesicles. Synaptic profiles were most often observed on neuronal somata. Neurons containing immunoreactive serotonin, substance P, somatostatin, enkephalin, bombesin and gastrin/cholecystokinin were observed in about the same proportions as they occur in the intact myenteric plexus. Neurons containing immunoreactive vasoactive intestinal polypeptide were found in higher numbers than reported in vivo. Neurons containing immunoreactive neurotensin, secretin and glutamate decarboxylase were not observed. An antiserum directed against choline acetyltransferase stained 40-50% of the neurons. We conclude that myenteric neurons continue to express much of their normal differentiated properties even when they are removed from the gut, dissociated into a suspension of single cells and grown in culture. Such cultures will be useful for correlating the morphological, biophysical, pharmacological and synaptic properties of individual myenteric neurons and for testing the ability of altered environmental conditions to change those properties.     

Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat

The localization and distribution of somatostatin (growth hormone release-inhibiting hormone; somatotropin release-inhibiting factor) have been studied with the indirect immunofluorescence technique of Coons and collaborators and the immunoperoxidase method of Sternberger and coworkers using specific and well-characterized antibodies to somatostatin, providing semiquantitative, detailed maps of somatostatin-immunoreactive cell profiles and fibers. Our results demonstrate a widespread occurrence of somatostatin-positive nerve cell bodies and fibers throughout the central nervous system of adult, normal or colchicine-treated, albino rats. The somatostatin cell bodies varied in size from below 10 micron up to 40 micron in diameter and could have only a few or multiple processes. Dense populations of cell somata were present in many major areas including neocortex, piriform cortex, hippocampus, amygdaloid complex, nucleus caudatus, nucleus accumbens, anterior periventricular hypothalamic area, ventromedial hypothalamic nucleus, nucleus arcuatus, medial to and within the lateral lemniscus, pontine reticular nuclei, nucleus cochlearis dorsalis and immediately dorsal to the nucleus tractus solitarii. Extensive networks of nerve fibers of varying densities were also found in most areas and nuclei of the central nervous system. Both varicose fibers as well as dot- or "dust-like" structures were seen. Areas with dense or very dense networks included nucleus accumbens, nucleus caudatus, nucleus amygdaloideus centralis, most parts of the hypothalamus, nucleus parabrachialis, nucleus tractus solitarii, nucleus ambiguus, nucleus tractus spinalis nervi trigemini and the dorsal horn of the spinal cord. One exception is the cerebellum which only contained few somatostatin-positive cell bodies and nerve fibers. It should be noted that somatostatin-positive cell bodies and fibers did not always conform to the boundaries of the classical neuroanatomical nuclei, but could often be found in areas between these well-established nuclei or occupying, in varying concentrations, only parts of such nuclei. It was difficult to identify with certainty somatostatin-immunoreactive axons in the animals studied. Some pathways could, however, be demonstrated, but further experimental studies are necessary to elucidate the exact projections of the somatostatin-immunoreactive neurons in the rat central nervous system.     

Light and electron microscopic localization of acetylcholinesterase activity in the rat renal nerves

Acetylcholinesterase activity is shown in the renal nerves of the rat with the technique of Karnovsky and Roots. By light microscopy, the acetylcholinesterase-positive nerves are seen in association with blood vessels, including the glomerular arterioles, and occasionally with renal tubules. By electron microscopy the precipitate appears extracellularly around axons and varicosities. DFP inhibits the deposition of precipitate. Previous demonstration by serial section electron microscopy in the rat revealed that all nerves around the glomerular arterioles contain small dense-cored vesicles characteristic of adrenergic nerves, indicating that the acetylcholinesterase-positive nerves demonstrated here are likely to be adrenergic nerves containing acetylcholinesterase.     

Distribution and subcellular localization of calmodulin in adult and developing brain tissue

The distribution and subcellular localization of calmodulin in adult and developing cerebellum was studied in rats by immunocytochemistry. Calmodulin immunoreactivity was found both in neurons and in glial cells. Within neurons the staining was particularly intense in the cell nucleus and in dendrites, the cytoplasm of the cell body was more lightly stained than the nucleus, and light immunoreactivity was observed in axons. Electron microscopic analysis confirmed the association of calmodulin with the nuclear chromatin, while the nucleolus remained unstained. The reaction product was also found overlying the membranes of several organelles, in postsynaptic densities and decorating both dendritic and axonal microtubules. In developing Purkinje cells, calmodulin immunoreactivity was found as early as 5 days after birth. During the initial phases of dendritic development (5-10 days post-natal), the reaction product was associated with the organelles of the apical cone, while little or no staining was observed in the elongating dendrites or in the cell nucleus. Later in development, calmodulin was found in primary and secondary dendrites, and by 20 days after birth immunoreactivity appeared in the cell nucleus, and in the postsynaptic densities of immature spines located in dendrites. The presence of calmodulin in the apical cone suggests the possibility that this protein may participate in the regulation of microtubule formation during the initial stages of dendritic development. Its presence in dendrites at later stages (during the period of synaptogenesis) may indicate that it also participates in the formation of synapses between the parallel fibres and dendritic spines.     

Serotonin immunoreactive nerve fibers and terminals in the rat pituitary--light- and electron-microscopic studies

Serotonin-immunoreactive nerve fibers were demonstrated to form a plexus in the intermediate lobe of rat pituitary. Such fibers were sporadic in the posterior lobe where they occurred exclusively in the zone adjacent to the intermediate lobe, and in the anterior lobe. With the aid of electron-microscopic immunocytochemistry, labeled fibers were shown to constitute the majority of neuronal processes found in the intercellular region of the intermediate lobe. They were frequently seen to establish synaptoid contacts with endocrine cells.     

Light and electron microscopic immunocytochemical localization of glutamine synthetase in the superficial pineal gland of the rat.

Glutamine synthetase (L-glutamate:ammonia ligase; EC 6.3.1.2), an enzyme catalysing the ATP-dependent formation of glutamine from glutamate and ammonia, was detected immunocytochemically only in glial (interstitial) cells of the superficial pineal gland of the rat. The results show the important role of pineal glial cells in the metabolism of the presumptive neurotransmitters, glutamate and gamma-aminobutyric acid (GABA) as well as in detoxification of ammonia.     

Correlation between met-enkephalin and substance P immunoreactivity in the primate globus pallidus

Using immunohistochemical techniques, the distribution of enkephalin and substance P immunoreactive fibers and terminals was studied in the globus pallidus of the non-human primate. In the external segment of the globus pallidus, enkephalin immunoreactivity was very dense while only sparse to moderate substance P staining was observed. Enkephalin immunoreactivity in the inner portion of the internal segment was moderate while such fibers were sparse in the outer portion of the internal segment. Substance P immunoreactivity was dense throughout the internal segment of the globus pallidus.The pattern of enkephalin and substance P immunoreactivity in the globus pallidus of the non-human primate as reported in the present study is of interest with regard to pallidal efferents. The pallidosubthalmic projection, arising from the external segment of the globus pallidus, is likely to be strongly influenced by the very dense network of enkephalin immunoreactive fibers and terminals in this region. Conversely, substance P immunoreactive elements are sparse in the external segment, and are, therefore, unlikely to influence significantly activity carried by the pallidosubthalmic projection. Since the inner portion of the internal segment contains moderate enkephalin immunoreactivity and dense substance P immunoreactivity, the information carried by the lenticular fasiculus may be modulated by both of these putative transmitters. On the other hand, based on the densities of immunoreactivity, the ansa lenticularis, which arises from the outer portion of the internal segment, is likely to be under greater influence of the dense substance P projection to this area.     

The effects of capsaicin pre-treatment on the responses of single neurones to sensory stimuli in the trigeminal nucleus caudalis of the rat: Evidence against a role for substance P as the neurotransmitter serving thermal nociception

Rats were systemically pre-treated with capsaicin either on the first day of life or at an age of 1 month. Both treatments were found to deplete substance P levels in the trigeminal nucleus caudalis (55.6% and 57.9% depletions, respectively). Extracellular single neurone recordings in the trigeminal nucleus caudalis revealed that neither type of capsaicin treatment greatly altered the proportions of neurones responding to non-noxious or noxious mechanical stimulation of the face. However, the proportion of mechanically-nociceptive neurones also responding to noxious thermal stimulation was greatly reduced in neonatally-treated, but not adult-treated rats.As both methods of capsaicin treatment caused similar depletions of substance P, it is concluded that this peptide may not be the neurotransmitter of afferent fibres to the trigeminal nucleus caudalis signalling thermal nociception.     

Oxytocin/vasopressin-like immunoreactivity is present in the nervous system of hydra

Nerve cells have been found in hydra, which react with antisera to oxytocin, vasopressin and mesotocin. These nerve cells have a high density in the ectoderm of basal disk and tentacles and lower density in the ectoderm of peduncle, gastric region and hypostome. A very small number of nerve cells occur also in the endoderm of foot, gastric region and hypostome. By using a technique for simultaneous visualisation of nerve cells reacting with antisera to oxytocin and vasopressin, it can be shown that these nerve cells belong to a single population. In agreement with this, the staining of the nerve cells can be abolished by absorbing each antiserum with either oxytocin, vasopressin, [Lys⁸]vasopressin, vasotocin, mesotocin or isotocin, indicating that the antigenic determinant of hydra cross-reacts with those antibody subpopulations, which recognize common portions (sequence 1–2, 5–7, 9) of the oxytocin/vasopressin-like peptides. With radioimmunoassays that are specific for either oxytocin or vasopressin, only very low amounts of immunoreactivity were measured. In addition, the dilution curves in these assays were not parallel to the standards, indicating that the antigenic determinant of hydra is not oxytocin or vasopressin.The presence of oxytocin/vasopressin-like material in coelenterates, shows that this family of peptides is of great antiquity.     

An immunohistochemical study of the projections of somatostatin-containing neurons in the guinea-pig intestine

Somatostatin-like immunoreactivity was localized in nerves in whole mount preparations of the separated layers of the guinea-pig intestine. The directions in which the neurons project were determined by examining the accumulation of somatostatin-like immunoreactivity after axonal flow was interrupted. In some experiments this was done by crushing or cutting the nerves in isolated preparations which were then maintained in oxygenated Krebs solution for 3–5 h. In other experiments, the nerves were cut in vivo and the animals allowed to survive for 4–8 days before the intestine was examined.Somatostatin immunoreactive nerve cell bodies were found in both the myenteric plexus, where they represented 4.7% of the total population of neurons, and in the submucous plexus, where they formed 17.4% of the total population. The axons of the somatostatin-containing neurons in the submucosa are not polarized while those of the somatostatin-containing neurons in the myenteric plexus of the small intestine project in the anal direction for 8–12 mm to form pericellular baskets around other enteric neurons, some of which are reactive for somatostatin.It is postulated that somatostatin-containing neurons in the myenteric plexus are interneurons in a descending nerve pathway, possibly the one involved in the descending inhibitory reflex of peristalsis.     

Localization of chick retinal visinin-like immunoreactivity in the rat forebrain and diencephalon

The present study is an examination, using an indirect immunofluorescence method, of the distribution of visinin, a 24,000 dalton peptide, in the rat forebrain and diencephalon. Immunoreactive structures were localized in the neuronal elements showing an uneven distribution. Immunoreactive neurons were found in the olfactory bulb, anterior olfactory nucleus, cerebral cortex, amygdaloid complex, ventral portion of the nucleus caudatus putamen, septal area, nucleus accumbens, nucleus paratenialis, nucleus rhomboideus, nucleus reuniens, nucleus paraventricularis hypothalami, nucleus supraopticus, nucleus anterior hypothalami, preoptic area, hypothalamic periventricular nucleus, nucleus mammillaris medialis, medial habenular nucleus, zona incerta, nucleus lateralis thalami, nucleus tractus optici and gyrus dentatus. Immunoreactive fibers were observed in the above areas, particularly near the labelled cells, forming fiber plexuses of varying density. In addition, dense plexuses were also seen in the globus pallidus, anteroventral nucleus of the thalamus, substantia nigra and hippocampus. In the former three structures, no labelled cells were present and in the latter, a few scattered neurons were found, indicating that these fibers originate from extrinsic sources.     

Changes in the cytoplasmic distribution of microtubule-associated protein 2 during the differentiation of cultured cerebellar granule cells

The distribution of microtubule-associated protein 2 in cultured cerebellar granule cell neurons was followed by immunohistochemical staining with specific antibodies. During differentiation in vitro, the neurites of these cells pass through a series of developmental stages. At first the emergent processes contain only trace levels of microtubule-associated protein 2 which is most concentrated in and near the cell body. When the neurites are between two and five cell diameters long they exhibit both microtubule-associated protein 2 and tubulin, apparently evenly distributed, throughout their length. Subsequently microtubule-associated protein 2 is limited to an initial, usually varicose portion of the neurite whereas its long distal extension contains abundant tubulin but is apparently devoid of microtubule-associated protein 2. Thus microtubule-associated protein 2 and tubulin are not necessarily co-distributed with a single neuronal process. In both morphological appearance and in the different distributions of microtubule-associated protein 2 and tubulin they contain, these processes show a mixture of axonal and dendritic properties. Since these same cells do not develop their characteristic dendritic arborizations, our results suggest that when removed from the developing brain, cerebellar granule neurons achieve part but not all of their normal morphological and cytoskeletal differentiation.     

Astrocytic cerebellar cell clones synthesize the beta' isoforms of the beta-tubulin protein family

We have analysed the isotubulin pattern of three astrocytic cell clones, derived from spontaneously established permanent cell cultures originating from 8-day postnatal mice cerebellar explants, in comparison with that of primary astroglial cultures from embryonic brain and cerebellum. These astrocytic clones, which may represent the different astroglial cell types of mouse cerebellum, did not produce the alpha- and beta-acidic isoforms, these being found only in cells of neuronal lineage. However, the three astrocytic clones, but not the primary astroglial cultures, did synthesize the beta'-tubulin isoforms; in addition quantitative analysis of the beta' proteins showed a positive correlation between the ability of the cells to extend processes and their synthesis of the beta' isoforms. These data suggest that the presence of beta'-tubulin is not specific for neuronal cells but may be related to the ability of cells from the nervous system to extend processes.     

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.