Autoradiographic localization of acetylcholine receptors in the Schwann cell membrane of the squid nerve fiber.

Intact and slit nerve fibers of the squid Sepioteuthis sepioidea were incubated in a 50-nM solution of [125I] alpha-bungarotoxin in artificial seawater, in the absence and in the presence of D-tubocurarine (10(-4) M). The distribution of the radioactive label was then determined by electron microscope autoradiography. It was found that, in the fibers exposed solely to the radioactive toxin, the label was located mainly at the axon-Schwann cell boundary in the intact nerve fibers or at the axonal edge of the Schwann cell layer in the axon-free nerve fiber sheaths. Label was also present in those regions of the Schwann cell layer rich in intercellular channels. No signs of radioactivity were observed in the nerve fibers exposed to the labeled toxin in the presence of D-tubocurarine. These results indicate that the acetycholine receptors previously found in the Schwann cell plasma membrane are mainly located over the cell surfaces facing the neighboring axon and the adjacent Schwann cells. These findings represent a further advance in the understanding of the relationship between the axon and its satellite Schwann cell.

Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence.

Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.

Calcitonin gene-related peptide is a stimulator of renin secretion.

Calcitonin gene-related peptide (CGRP) was found to stimulate renin secretion in vivo in normal human volunteers. Moreover, CGRP stimulated the release of renin in vitro from isolated rat renal juxtaglomerular cells (half-maximal effective concentration [EC50] 100 nM) concomitant with stimulation of cAMP production (EC50 60 nM). Immunoreactive CGRP was recognized in rat renal cortical nerve fibers, and intact rat CGRP was identified in extracts of the rat renal cortex. Because CGRP containing sensory nerve fibers are seen in the region of the juxtaglomerular apparatus, it would seem that the release of CGRP from these afferent nerves may be involved in the physiological control of renin secretion.

Structural similarities and differences amongst neurofilaments.

Neurofilaments purified from cat, ox, Loligo and Myxicola nerve fibres are composed of different numbers of polypeptides with different molecular weights. Mammalian neurofilaments also differ from those of marine invertebrates by being about 20% larger in diameter. Despite the differences, X-ray diffraction patterns from all the neurofilaments indicate a common alpha-helical content with alpha-helices arranged in coiled-coils. The patterns from Myxicola neurofilaments also indicate a long-range periodicity along the length of these filaments which is of the order of 25.2 nm.

Fluorescent histochemical localisation of quinacrine-positive neurones in the guinea-pig and rabbit atrium.

Quinacrine-fluorescent nerve fibres and nerve cell bodies are described in the right and left atria of the guinea-pig and rabbit. The nerve cells (20 to 35 micrometers in diameter) are found predominantly in the right atrium in both species. The nerve fibres are varicose and innervate both the muscle and many blood vessels. The quinacrine fluorescent neural structures are unaffected by chemical sympathectomy with 6-hydroxydopamine. The distribution of quinacrine-positive nerve fibres and cell bodies are compared to the distribution of adrenergic and acetylcholinesterase-positive nerves in the atrium of both species. Quinacrine fluorescence appears to be selective for non-adrenergic, non-cholinergic nerves and the possibility that it is binding to high contents of ATP discussed.

The regeneration of substance P-containing nerve fibers in the process of burn wound healing in the guinea pig skin.

The regeneration of substance P (SP)-containing nerve fibers in the process of burn wound healing in the guinea pig skin has been studied by immunohistochemistry. SP-like immunoreactivity, which was specifically localized in neural elements of intact skin, was found to disappear in the burn wound including its margin on day 2 post burn. The SP-containing nerve fibers were first detected in periods later than day 2 post burn, and the regeneration seemed to occur in association with regeneration of blood vessels at the wound margins. These nerve fibers gradually increased in number and acquired maximum density on day 14 post burn. In addition, such renewed fibers showed sprouting to form a dense network, which has never been observed in intact skin, in the upper granulation tissue just beneath the regrowing epidermis. Following that peak period, the density of the fibers gradually decreased to less than that of controls. The characteristic process of regeneration of SP-containing nerve fibers, having a peak period of fiber density at least in burn wound healing, appeared similar to that of the regeneration of sympathetic catecholaminergic nerve fibers reported previously.

Ultrastructural pathology of nerve fibers in calcium-induced myelopathy.

Calcium has been proposed as a mediator of nerve fiber degeneration following traumatic injury of the spinal cord. It induces a spongy, necrotizing myelopathy similar in its evolution to that observed in experimental spinal cord trauma. The current study was undertaken to determine the ultrastructural changes in the central nervous system (CNS) nerve fibers associated with calcium-induced myelopathy. A 10% calcium chloride (CaCl2) solution (pH 7.4) was slowly dripped on the dorsal surface of the surgically exposed lower thoracolumbar spinal cord of adult male Sprague-Dawley rats. The posterior and lateral columns of the spinal cords were fixed and processed for electron microscopy. Controls consisted of tissue from normal and sham-operated animals, as well as those receiving equal volumes and osmolarities of sodium chloride (NaCl), magnesium chloride (MgCl2), and potassium chloride (KCl) at the same pH. In the CaCl2 treated animals, spongiosis of increasing severity developed in white matter, as the result of periaxonal, adaxonal and intramyelinic swelling. Vesicular demyelination was consistently observed, beginning within one hour (h) and progressing with increasing severity up to 24-72 h. Axonal changes included pleomorphic spheroids, granular degeneration and intra-axonal calcification. The ultrastructural changes in the nerve fibers provoked by calcium were indistinguishable from those previously reported in experimental spinal cord trauma. These observations strengthen the hypothesis that calcium initiates the nerve fiber degeneration following spinal cord injury.

Biochemical and immunocytochemical characterization of neuropeptide Y in the immature rat ovary.

Neuropeptide Y (NPY)-like immunoreactivity has been found in nerves that innervate the rat ovary. In this study, we used immunohistochemical and biochemical methods to identify NPY in the prepubertal rat ovary. The normal distribution of NPY-containing nerve fibers and the route by which these nerves enter the ovary were analyzed with indirect immunofluorescence techniques. In ovaries with intact nerves, a profuse network of NPY-labeled fibers was observed surrounding blood vessels. Immunoreactive fibers were also seen in the interstitial tissue and coursing between follicles. Occasionally some fibers appeared to enter the follicles. Surgical transection of the superior ovarian nerve had no effect on NPY immunoreactivity; however, transection of the plexus nerve completely eliminated NPY-labeled nerve fibers in all ovarian compartments. The nature of this immunoreactivity was examined in extracts of pooled ovaries that were subjected to reverse phase HPLC and then analyzed by RIA. The major peak of NPY immunoreactivity in each extract eluted at the same time or slightly before synthetic porcine NPY. Two additional peaks of NPY-like immunoreactivity that eluted much earlier than porcine NPY were found in each extract. We conclude that the plexus nerve carries NPY afferents to the ovary and that the ovary contains NPY-like peptides, one of which has a retention time on reverse phase HPLC nearly identical to that of porcine NPY, whereas two others elute with earlier retention times. While the identity and composition of these substances remain to be determined, the presence of peptides that display NPY-like immunoreactivity in the ovary as well as the profuse network of NPY-containing fibers strongly imply a physiological involvement of NPY in the regulation of ovarian function.

Localization of alpha-melanocyte-stimulating hormone in rat brain and pituitary.

The distribution of alpha-melanocyte-stimulating hormone (alpha-MSH) was studied in the rat brain with an immunoperoxidase technique. alpha-MSH-containing cells were found in the arcuate nucleus of the hypothalamus. Cells staining for alpha-MSH were also localized in the intermediate lobe of the pituitary. alpha-MSH-containing nerve fibers extended throughout regions of the hypothalamus, thalamus, and midbrain. Two weeks after hypophysectomy, alpha-MSH-positive cells anf fibers were still present in the brain. These results indicate that alpha-MSH of non-pituitary origin is synthesized and stored by neural structures in the rat brain. The detection of alpha-MSH by radioimmunoassay in the rat brain and pituitary supports these observations.

Serotonergic elements of the mammalian pituitary.

Serotonin [5-hydroxytryptamine (5-HT)] is a constituent of the mammalian pituitary gland. We have used light and electron microscopic immunocytochemistry to locate immunoreactivity in the pituitary glands of mice and bats. In addition, we have compared the distribution of endogenous 5-HT immunoreactivity with that of exogenous [3H]5-HT taken up by elements of the pituitaries and detected by radioautography. 5-HT immunoreactivity was found in neurites in the posterior and intermediate lobes of the glands. These neurites also took up [3H]5-HT. The neural elements reactive with antisera to 5-HT or which labeled with [3H]5-HT differed in their distribution from neurites that labeled with [3H]dopamine or [3H]norepinephrine; moreover, lesioning of catecholaminergic neurites with 6-hydroxydopamine had no effect on the serotonergic structures. 5-HT immunoreactivity was also found in endocrine cells of the anterior lobes of the pituitaries of both species. No 5-HT was detected in neural elements of the anterior lobe, and in fact, no staining with nerve-specific antibodies to neurofilament proteins was found in the anterior pituitary. The ultrastructure of the granules of the 5-HT-immunoreactive endocrine cells suggested that they might be a subset of gonadotrophs, the cell type previously shown to take up exogenous [3H] 5-HT. 5-HT immunoreactivity was found within the granules of these cells. In bats, 5-HT immunoreactivity was present in the anterior pituitaries of active animals when 5-HT levels were high, but was greatly diminished in glands of hibernating animals when the 5-HT content was low. It is concluded that some cells that may be gonadotrophs contain endogenous 5-HT and also have an uptake mechanism for the amine. The function of 5-HT in the anterior pituitary remains to be determined, but 5-HT has previously been shown to inhibit the secretagogue action of LHRH on gonadotrophs.

Calcitonin gene-related peptide in thyroid nerve fibers and C cells: effects on thyroid hormone secretion and response to hypercalcemia.

Calcitonin gene-related peptide (CGRP) in the thyroid has a dual localization to nerve fibers around blood vessels and follicles and to parafollicular (C) cells. CGRP was found to coexist with substance P (SP) in most of the nerve fibers; a few CGRP fibers seemed to lack SP, and a few SP fibers seemed to be devoid of CGRP. In the C cells, CGRP coexisted with calcitonin (CT). Cervical vagotomy (extirpation of the nodose ganglion) eliminated approximately 50% of the CGRP/SP fibers in the thyroid without any overt influence on CGRP/CT in the C cells. Removal of the superior cervical ganglion or chemical sympathectomy (6-hydroxydopamine treatment) affected neither thyroid CGRP/SP nerve fibers nor CGRP/CT-storing C cells. CGRP nerve cell bodies were numerous in the jugular-nodose ganglionic complex (notably in the jugular portion); in many of them, CGRP coexisted with SP. A few scattered CGRP nerve cell bodies also occurred in the laryngeal ganglion, whereas none was found in the thyroid ganglion. Hypercalcemia evoked by vitamin D2 treatment, which is known to degranulate thyroid C cells, reduced the thyroid content of both CGRP and CT. As tested in mice in vivo, CGRP and SP alone or together had no effect on basal or TSH- or isoprenaline-induced thyroid hormone secretion. Vasoactive intestinal peptide-stimulated iodothyronine release, on the other hand, was enhanced by CGRP, but not by SP. SP had no effect on combined vasoactive intestinal peptide-CGRP-stimulated iodothyronine release. These findings suggest that CGRP participates in the control of thyroid hormone secretion and that, like CT, CGRP in the C cells is under control of the serum calcium level.

Immunohistochemical localization of adrenocorticotropin in the rat brain.

ACTH and some of its fragments have been shown to play a role in a variety of adaptative mechanisms. To clearly identify the nervous structures containing ACTH in the rat brain, an immunohistochemical localization of this peptide was conducted at both light and electron microscopic levels. Nervous fibers staining for ACTH were found to be largely distributed throughout regions of hypothalamus, thalamus, and midbrain. Positive fibers could also be occasionally observed in the spinal cord. Immunostained neuronal cell bodies were only detected in the arcuate nucleus. Essentially, the same results were obtained 2 and 8 weeks after hypophysectomy. In animals pretreated with colchicine, the intensity in the staining of cell bodies was markedly increased, making possible the detection of a larger number of cell bodies. At the electron microscopic level, it was demonstrated that ACTH is contained in dense core vesicles present in nervous fibers and endings. These results indicate that ACTH of nonpituitary origin is synthesized in the central nervous system and could probably be considered as a neurotransmitter of still undefined function.

Calcitonin gene-related peptide: occurrence in pancreatic islets in the mouse and the rat and inhibition of insulin secretion in the mouse.

The intrapancreatic cellular distribution and effects on basal and stimulated insulin secretion of the 37-amino-acid polypeptide, calcitonin gene-related peptide (CGRP), were investigated in the mouse. The cellular localization of CGRP was also studied in the rat pancreas. In both species, CGRP was demonstrated in pancreatic islet cells and nerve fibers. Immunocytochemical double staining experiments revealed the CGRP-immunoreactive cells in the mouse to be identical with a majority population of the insulin cells. In the rat, on the other hand, CGRP-immunoreactive cells were identical with somatostatin cells. CGRP-immunoreactive nerve fibers were observed, in both species, running in the exocrine parenchyma, particularly around blood vessels, and they were occasionally seen also within the islets. In in vivo experiments, CGRP was found to inhibit both basal and stimulated insulin secretion in the mouse. Thus, 6 min after the iv injection of CGRP (0.85 nmol/kg), plasma insulin levels were 13 +/- 2 (SE) microU/ml compared to 30 +/- 4 microU/ml in controls (P less than 0.01). At this dose level, CGRP inhibited the insulin secretory response to carbachol, leaving that to glucose unaffected. However, at a higher dose level (4.25 nmol/kg), CGRP inhibited glucose-induced insulin secretion as well. We conclude that CGRP occurs in islet cells and in intrapancreatic nerve fibers of both the mouse and the rat, and inhibits both basal and stimulated insulin secretion in vivo in the mouse.

Peptide histidine isoleucine amide stimulates thyroid hormone secretion and coexists with vasoactive intestinal polypeptide in intrathyroid nerve fibers from laryngeal ganglia.

Peptide histidine isoleucine amide (PHI) and vasoactive intestinal polypeptide (VIP) are fragments of the same precursor molecule, prepro-VIP, and coexistence of the two peptides is, therefore, to be expected. Nerve fibers displaying PHI and VIP immunoreactivity occurred around blood vessels and follicles in the thyroid gland of several species. Sequential staining with antibodies against PHI and VIP revealed coexistence of the two peptides in the same population of nerve cell bodies in ganglia situated along the laryngeal nerves and in intrathyroid nerve fibers. Chemical sympathectomy (6-hydroxydopamine treatment), surgical sympathectomy (removal of the superior cervical ganglia), and unilateral cervical vagotomy (removal of the nodose ganglion) failed to affect the number and distribution of PHI/VIP fibers in the thyroid gland. Taken together, the findings suggest that both the perivascular and interfollicular PHI/VIP fibers originate in laryngeal ganglia. PHI weakly stimulated basal thyroid hormone secretion in mice in vivo, but did not influence the response to TSH or VIP. PHI had no effect on calcitonin secretion in rats. Like VIP, PHI may play a physiological role in the regulation of thyroid hormone secretion.

Retrograde tracing of nerve fibers to the rat middle cerebral artery with true blue: colocalization with different peptides.

The origin of nerve fibers to the rat middle cerebral artery was studied by retrograde tracing with the fluorescent tracer True Blue (TB) in combination with immunocytochemistry to known perivascular peptides. Application of TB to the middle cerebral artery labeled nerve cell bodies in the ipsilateral superior cervical ganglion, the otic ganglion, the sphenopalatine ganglion, the trigeminal ganglion, and the cervical dorsal root ganglion at level C2. A few labeled nerve cell bodies were seen in contralateral ganglia. Judging from the number and intensity of the labeling, the superior cervical ganglion and the trigeminal ganglion and dorsal root ganglion at level C2 contributed most to the innervation. A moderate number of nerve cell bodies were labeled in the sphenopalatine and otic ganglia. The TB-labeled nerve cell bodies were further examined for the presence of neuropeptides. For that purpose antibodies raised against neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP) were used. A considerable portion of the TB-labeled nerve cell bodies in the superior cervical ganglion contained NPY. About half of the labeled nerve cell bodies in the sphenopalatine and otic ganglia contained VIP. In the trigeminal ganglion and in the dorsal root ganglion at level C2, one-third of the TB-labeled nerve cell bodies were CGRP-immunoreactive, while only few nerve cell bodies contained SP. The study provides direct evidence for the origin of cerebrovascular peptidergic nerve fibers and demonstrates that not only ipsilateral but also contralateral ganglia contribute to the innervation of the cerebral circulation.

Nerve fibres containing gastrin-releasing peptide around pial vessels.

Nerve fibres containing immunoreactive gastrin-releasing peptide (GRP) were demonstrated around pial blood vessels of cat, guinea pig, rat, and mouse. A sparse supply was found around spinal cord blood vessels, whereas the choroid plexus seemed to be devoid of GRP fibres. Sympathectomy did not affect the number or distribution of the GRP fibres. The administration of neither GRP nor its closely related analogue, bombesin, contracted or dilated feline pial arteries in vitro.

Nerve fibers containing neuropeptide Y in the cerebrovascular bed: immunocytochemistry, radioimmunoassay, and vasomotor effects.

Perivascular nerve fibers containing neuropeptide Y (NPY)-like immunoreactivity were identified around cerebral blood vessels of human, cat, guinea pig, rat, and mouse. The major cerebral arteries were invested by dense plexuses; veins, small arteries, and arterioles were accompanied by few fibers. Removal of the superior cervical ganglion resulted in a reduction of NPY-like material in pial vessels and dura mater. Pretreatment with 6-hydroxydopamine or reserpine reduced the number of visible NPY fibers and the concentration of NPY in rat cerebral vessels. Sequential immunostaining with antibodies toward dopamine-beta-hydroxylase (DBH) (an enzyme involved in the synthesis of noradrenaline) and NPY revealed an identical localization of DBH and NPY in nerve cell bodies in the superior cervical ganglion and in perivascular fibers of pial blood vessels, suggesting their coexistence. Administration of NPY in vitro resulted in concentration-dependent contractions that were not modified by a sympathectomy. The contractions induced by noradrenaline, 5-hydroxytryptamine, and prostaglandin F2 alpha and the dilator responses to calcitonin gene-related peptide were not modified by NPY in rat cerebral arteries. However, the constrictor response to NPY was reduced by 70% in the presence of the calcium entry blocker nifedipine, and abolished following incubation in a calcium-free buffer. These data suggest an interaction of NPY at a postsynaptic site, which for induction of contraction may open calcium channels in the sarcolemma of cerebral arteries.

Quantitative ultrastructural analysis of enkephalin-, substance P-, and VIP-immunoreactive nerve fibers in the circular muscle of the guinea pig small intestine.

The present work was undertaken to determine what proportion of all nerve fibers in the circular muscle of the guinea pig small intestine contain the neuropeptides enkephalin, substance P, and vasoactive intestinal peptide and in which combinations these peptides occur in the fibers. It was envisaged that such an analysis would provide insights into the chemical identity of excitatory and inhibitory nerve fibers that innervate the muscle. Whole-mount preparations from normal and extrinsically denervated gut were labelled with antiserum to the individual peptides or with combinations of antipeptide antisera and processed for electron microscopy. Reactive and nonreactive vesicle-containing nerve fiber profiles were examined and counted in ultrathin sections. Vesicle-containing nerve fiber profiles immunoreactive for enkephalin, substance P, or vasoactive intestinal peptide had similar morphologies in that they all contained variable proportions of small clear and large granular vesicles. In all samples stained for single peptides or combinations of peptides, a small proportion of immunoreactive profiles approached smooth muscle cells to within 15-20 nm with no intervening basal lamina. A total of 14,694 vesiculated nerve fiber profiles from three control and three extrinsically denervated animals were scored for the presence of immunoreactivity to enkephalin, substance P, vasoactive intestinal peptide, or combinations of these peptides. Analysis of variance showed that the number of profiles labelled for substance P was not different from the number of profiles labelled for vasoactive intestinal peptide and that the number labelled with the substance P and vasoactive intestinal peptide antisera simultaneously were not different from the sum of the numbers obtained with each alone. The number of profiles labelled for substance P plus enkephalin was greater than the number labelled for substance P alone and the number labelled with vasoactive intestinal peptide plus enkephalin was greater than that with vasoactive intestinal peptide alone. Simultaneous labelling for substance P and vasoactive intestinal peptide resulted in immunoreactivity in the same number of profiles as did reaction for all three peptides at the same time. In both cases, about 95% of the profiles were labelled. The results from extrinsically denervated muscle were not different from control circular muscle. These results indicate that nearly all the intrinsic nerve fibers supplying the circular muscle of the guinea pig small intestine contain either substance P or vasoactive intestinal peptide but not both.(ABSTRACT TRUNCATED AT 400 WORDS)

Atlas of serotonin-containing neurons in the optic lobes and brain of the crayfish, Cherax destructor.

An atlas of neurons in the brain of the crayfish Cherax destructor that are immunoreactive to antibodies raised against serotonin has been compiled from whole mount preparations. Neuronal networks of serotonin-containing cells are identified in the optic lobes and protocerebrum, in the deutocerebrum, and in the tritocerebrum. The consistency of the whole-mount technique allows 50 out of a total of about 100 immunoreactive cells to be individually identified according to their neuronal architecture or the location of their cell somata or axons. Apart from six neurons with axons in the oesophageal connectives, all the immunoreactive cells are intrinsic to the optic lobes and brain.

Calcitonin gene-related peptide (CGRP) in the human spinal cord: a light and electron microscopic analysis.

The distribution of CGRP immunoreactivity in the cervical, thoracic, lumbar, and sacral levels of the human spinal cord was mapped at the light microscopic level with the aid of a rabbit-generated antiserum against human calcitonin gene-related peptide (CGRP). CGRP-positive fibers formed a dense plexus in lamina I, II, the reticulated region of lamina V, and the tract of Lissauer at all spinal cord levels. The distribution of fibers showed some variations dependent on the cord level analyzed. At the light microscopic level, intervaricose fiber diameters consistently measured 1.0 micron or less, and two types of CGRP varicosities were observed: a small (1 to 2 microns in diameter), relatively round profile and a larger, (3 to 4 microns in diameter) oval or oblong profile. At the electron microscopic level, immunostained varicosities contained a mixture of round clear vesicles and vesicles that contained dense cores. The CGRP immunoreaction product was often associated with vesicles containing dense cores. The reaction product was also seen associated with clear round vesicles or in the cytoplasmic matrix. Postsynaptic elements included dendritic spines, small and large diameter dendritic shafts and vesicle containing profiles. The presence of CGRP in the superficial dorsal horn of human spinal cord is highly suggestive of a role in primary afferent transmission as postulated in lower vertebrates. This study establishes the distribution of CGRP at four different spinal levels in human cord and will serve as a basis for future studies related to the pathologic conditions affecting sensory systems.