Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture.

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.

Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair.

Peripheral nerve injury results in short-term and long-term changes in both neurons and glia. In the present study, immunohistological and immunoblot analyses were used to examine the expression of the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) within different parts of a functionally linked neuromuscular system extending from skeletal muscle to the spinal cord after peripheral nerve injury. Histological samples were taken from 3 to 150 d after crushing or transecting the sciatic nerve in adult chickens and mice. In unperturbed tissues, both N-CAM and Ng-CAM were found on nonmyelinated axons, and to a lesser extent on Schwann cells and myelinated axons. Only N-CAM was found on muscles. After denervation, the following changes were observed: The amount of N-CAM in muscle fibers increased transiently on the surface and in the cytoplasm, and in interstitial spaces between fibers. Restoration of normal N-CAM levels in muscle was dependent on reinnervation; in a chronically denervated state, N-CAM levels remained high. After crushing or cutting the nerve, the amount of both CAMs increased in the area surrounding the lesion, and the predominant form of N-CAM changed from a discrete Mr 140,000 component to the polydisperse high molecular weight embryonic form. Anti-N-CAM antibodies stained neurites, Schwann cells, and the perineurium of the regenerating sciatic nerve. Anti-Ng-CAM antibodies labeled neurites, Schwann cells and the endoneurial tubes in the distal stump. Changes in CAM distribution were observed in dorsal root ganglia and in the spinal cord only after the nerve was cut. The fibers within affected dorsal root ganglia were more intensely labeled for both CAMs, and the motor neurons in the ventral horn of the spinal cord of the affected segments were stained more intensely in a ring pattern by anti-N-CAM and anti-Ng-CAM than their counterparts on the side contralateral to the lesion. Taken together with the previous studies (Rieger, F., M. Grumet, and G. M. Edelman, J. Cell Biol. 101:285-293), these data suggest that local signals between neurons and glia may regulate CAM expression in the spinal cord and nerve during regeneration, and that activity may regulate N-CAM expression in muscle. Correlations of the present observations are made here with established events of nerve degeneration and suggest a number of roles for the CAMs in regenerative events.(ABSTRACT TRUNCATED AT 400 WORDS)

Norepinephrine and dopamine: assay by mass fragmentography in the picomole range.

Gas chromatography-mass spectrometry makes possible the simultaneous measurement of norepinephrine and dopamine in concentrations of 0.1-milligram tissue samples. Specificity of the assay is confirmed both by the retention time of the compound and by the mass to charge ratio of the fragments recorded. The sensitivity is of the order of 0.5 picomole, and linearity of the response is maintained up to at least 200 picomoles.

Cloning of complementary DNA for GAP-43, a neuronal growth-related protein.

GAP-43 is one of a small subset of cellular proteins selectively transported by a neuron to its terminals. Its enrichment in growth cones and its increased levels in developing or regenerating neurons suggest that it has an important role in neurite growth. A complementary DNA (cDNA) that encodes rat GAP-43 has been isolated to study its structural characteristics and regulation. The predicted molecular size is 24 kilodaltons, although its migration in SDS-polyacrylamide gels is anomalously retarded. Expression of GAP-43 is limited to the nervous system, where its levels are highest during periods of neurite outgrowth. Nerve growth factor or adenosine 3',5'-monophosphate induction of neurites from PC12 cells is accompanied by increased GAP-43 expression. GAP-43 RNA is easily detectable, although at diminished levels, in the adult rat nervous system. This regulation of GAP-43 is concordant with a role in growth-related processes of the neuron, processes that may continue in the mature animal.

Dopamine-sensitive adenyl cyclase: possible role in synaptic transmission.

An adenyl cyclase activated by low concentrations of dopamine has been found in the mammalian superior cervical sympathetic ganglion. The existence of this enzyme may account for the increased amount of adenosine 3',5' monophosphate associated with synaptic activity in the ganglion. The results suggest that the physiological effects of dopamine in the ganglion, and possibly elsewhere in the nervous system, may be mediated by stimulating the synthesis of adenosine 3',5'-monophosphate.

Differences in neuronal lipid composition between superior cervical ganglia and nodose ganglia of the rat.

The lipid content and composition of rat superior cervical ganglia containing sympathetic motor neurons and nodose ganglia containing parasympathetic sensory neurons were studied for the first time to elucidate the mechanism of the different effects of exogenous gangliosides on these neurons in the culture medium. The ganglioside content of the superior cervical ganglia was almost 3-times that of the nodose ganglia. Although both ganglia contained GM3, GD3, GD1b and GT1b as major gangliosides, the nodose ganglia additionally contained a significant amount of sialosyllactoneotetraosylceramide LM1 (10% of total sialic acids). Contrasting with nodose ganglia, vagus fiber and dorsal root ganglia of rats, superior cervical ganglia had a higher content of sulfatide than galactosylceramide. The phospholipid content was lower in superior cervical ganglia than in nodose ganglia. Superior cervical ganglia contained less ethanolamine plasmalogen and more phosphatidylcholine than nodose ganglia. Sphingomyelin in superior cervical ganglia contained mainly medium-chain fatty acids, while that in nodose ganglia contained mainly longer-chain fatty acids. Differences in the fatty acid composition of glycerophospholipids were also observed. The results indicate that the properties of neuronal cell membranes from superior cervical ganglia and nodose ganglia are quite different, and that the differences may reflect the physiological roles of these ganglia.

Slow component-a of axonal transport, nerve myo-inositol, and aldose reductase inhibition in streptozocin-diabetic rats.

This study measured sugars and polyols, weight/unit length, and slow component-a of axonal transport (SCa) in dorsal root afferents of the sciatic nerves of control rats and rats with streptozocin (STZ)-induced diabetes of 4-wk duration. The effects of two treatments--aldose reductase inhibition [Statil ("Statil" is a trademark; the property of Imperial Chemical Industries PLC.) ICI 128436 at 25 mg/kg/day, p.o.] and myo-inositol supplementation (650 mg/kg/day, p.o.)--were studied in control and diabetic groups. Inclusion of untreated controls and diabetics gave a total of six groups for the study. The treatments were begun on the day after injection of STZ and were maintained throughout the protocol. The sciatic nerves of the diabetic (untreated) rats showed accumulation of sorbitol and fructose, depletion of myo-inositol, and an 8% increase in weight/unit length. All of these abnormalities were prevented by treatment with Statil. Treatment of diabetic rats with myo-inositol prevented its depletion in the sciatic nerve, but did not affect the accumulation of sorbitol and fructose nor the increase in weight/unit length. Neither treatment exerted any apparent effect on body weight, blood glucose, nerve weight, or nerve sugars and polyols in the control rats. The diabetic rats showed a retardation of the wave of transported-labeled protein (shown as increased leftward skewness of the wave) and a reduction in mean transport velocity (calculated as the mean velocity for all segments contributing to the transport wave: 0.96 +/- 0.09 mm/day in diabetics versus 1.15 +/- 0.07 mm/day in controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunofluorescence comparisons of anti-actin specificity.

The abilities of antibody populations against brain actin and two immunogenic forms of cardiac actin to react with sarcomeric muscle actin and cytoplasmic non-muscle actin were tested by indirect immunofluorescence, by using isolated skeletal muscle myofibrils and cultured non-neuronal dorsal root ganglion cells as the test systems. All three antibody preparations stained the I-bands of myofibrils, a result that demonstrated the presence of antigenic determinants shared among skeletal, cardiac, and brain actins. However, although antibodies against cytoplasmic brain actin stained the stress fibers of cultured cells, those against glutaraldehyde cross-linked cardiac actin did not, a result that implies that cardiac actin possesses determinants common to sarcomeric actins but not present on cytoplasmic actin. Finally, antibodies against SDS-treated cardiac actin readily stained the stress fibers of cultured cells, in contrast to those against glutaraldehyde cross-linked cardiac actin, a result that suggests that the state of the original immunogen can affect the actin type specificity of the resulting antibody population.

The distribution of serum albumin in rat peripheral nerve.

To clarify the nature of the blood-nerve barrier (BNB) in excluding serum proteins from peripheral nerve endoneurium, we studied the ultrastructural distribution of albumin in peripheral nerve using light and electron microscopic immunocytochemistry. We found that serum albumin is commonly found within the endoneurium, but that it is not normally found intraaxonally. After subperineurial injection of labeled albumin, the albumin redistributed within intrafascicular clefts towards the perivascular spaces and the perineurium. These results suggest that, contrary to previous evidence, axons are exposed to serum proteins in normal nerves, and implies that they may be subject to interaction with abnormal proteins or altered serum proteins without any disruption of the BNB.

Abnormalities in spinal neurons and dorsal root ganglion cells in Tangier disease presenting with a syringomyelia-like syndrome.

A woman with homozygous Tangier disease had progressive syringomyelia-like neuropathy. She died with cardiac failure at age 61. A sural nerve biopsy taken at age 60 had shown lipid storage in Schwann and interstitial cells, and a pronounced loss of unmyelinated fibers. The neurons of the L5 spinal ganglion and, to a lesser extent, all neurons of the sacral spinal cord, contained large lipid inclusions which in electron micrographs differed from those in Schwann and satellite cells. There was no storage material in glial cells. The neuronal inclusions were membrane-bound and consisted of electron-dense and electron-lucent components. There was evidence of neuronal death in the spinal ganglion, and a diameter histogram showed that small cytons had preferentially been lost. The inclusions probably were secondary lysosomes or residual bodies, and resembled giant lipofuscin granules. Nevertheless, they were uncolored and displayed weak autofluorescence as compared to the aging pigment in control ganglia. It is tentatively suggested that the syringomyelia-like neuropathy in Tangier disease represents a lysosomal storage disorder preferentially affecting small dorsal root ganglion cells.

The distribution and origin of a novel brain peptide, neuropeptide Y, in the spinal cord of several mammals.

The distribution of neuropeptide Y [NPY]-immunoreactive material was examined in the spinal cord and dorsal root ganglia of rat, guinea-pig, cat, marmoset, and horse. Considerable concentrations of NPY and similar distribution patterns of immunoreactive nerve fibres were found in the spinal cord of all species investigated. The dorsal root ganglia of the cat and the horse contained numerous immunoreactive nerve fibres, but in these species, as in the other three studied [rat, guinea-pig, marmoset], no positively stained cell bodies were found. Neuropeptide Y-immunoreactive nerves were observed at all levels of the spinal cord, being most concentrated in the dorsal horn. In the rat, guinea-pig, and marmoset, there was a marked increase of NPY-immunoreactive fibres in the lumbosacral regions of the spinal cord, and this was reflected by a considerable increase of extractable NPY. Estimations of NPY-immunoreactive material in the various regions of the rat spinal cord were as follows: cervical, 13.8 +/- 1.0; thoracic, 21.1 +/- 2.5; lumbar, 16.3 +/- 2.9; sacral, 92.4 +/- 8.5 pmol/gm wet weight of tissue +/- SEM. In the ventral portion of the guinea-pig spinal cord they were as follows: cervical, 7.1 +/- 1.2; thoracic, 8.2 +/- 3.6; lumbar, 22.6 +/- 7.0; sacral, 36.7 +/- 9.5 pmol/gm wet weight of tissue +/- SEM. Analysis of spinal cord extracts by reverse phase high performance liquid chromatography [HPLC] demonstrated that NPY-immunoreactive material elutes in the position of pure NPY standard. No changes in the concentration and distribution of the NPY-like material in the rat spinal cord were observed following a variety of surgical and pharmacological manipulations, including cervical rhizotomy, sciatic nerve section and ligation, and local application of capsaicin [50 mM] to one sciatic nerve. It is therefore suggested that most of the NPY-immunoreactive material in the spinal cord is derived either from intrinsic nerve cell bodies or from supraspinal tracts.

Cerebral arterial innervation by nerve fibers containing calcitonin gene-related peptide (CGRP): I. Distribution and origin of CGRP perivascular innervation in the rat.

The origin, density and distribution of calcitonin gene-related peptide (CGRP) immunoreactivity in cerebral perivascular nerves and the trigeminal ganglion of rats were examined in this study. CGRP immunoreactive axons were abundant on the walls of the rostral circulation of the major cerebral arteries in the circle of Willis. The fibers form a grid- or meshwork of longitudinal and circumferential axons studded with numerous varicose swellings. The density of CGRP fibers was particularly high at the bifurcation of major arteries. A few CGRP fibers cross the midline to innervate arteries on the contralateral side of the arterial tree. The arteries of the caudal circulation were sparsely innervated by CGRP fibers. In the trigeminal ganglion, about 30% of the ganglion cells had CGRP immunoreactivity. The cell size of most (75%) of CGRP neurons was less than 30 micron in diameter. There was no significant difference in staining density between small and large CGRP neurons. Unilateral transection of the maxillary and mandibular divisions of the trigeminal nerve caused a substantial decrease of CGRP immunoreactivity in the ipsilateral dorsal two-thirds of the trigeminal nucleus and cervical spinal cord but did not noticeably change the diameter of the vascular lumen or the densities of CGRP fibers in the walls of the cerebral arteries. In contrast, unilateral transection that included the ophthalmic division eliminated CGRP fibers on the ipsilateral cerebral arteries and eliminated CGRP immunoreactivity throughout the trigeminal nucleus in the brainstem and rostral cervical cord. In addition, these lesions caused a significant reduction in the diameter of the denervated arteries. The present study demonstrates that CGRP, a putative neurotransmitter/neuromodulator, is especially abundant in the rostral cerebral circulation and is derived from the ipsilateral ophthalmic division of the trigeminal nerve. In addition, the loss of CGRP perivascular nerves is associated with a reduction of the arterial lumen. This suggests that CGRP is a strong candidate as a nerve-derived trophic factor at trigeminal terminals and provides additional evidence that CGRP is a component in the trigeminovascular system influencing vascular diameter.

Ontogeny of peptide- and amine-containing neurones in motor, sensory, and autonomic regions of rat and human spinal cord, dorsal root ganglia, and rat skin.

The developmental patterns of neurofilament triplet proteins, peptide and amine immunoreactivities were compared in motor (ventral spinal cord), sensory (dorsal spinal cord, dorsal root ganglia, epidermis), and autonomic (intermediolateral cell columns, dermis) regions in the rat and human. In the rat, neurofilament triplet proteins first appeared in motoneurones (embryonic day 13). In the youngest human fetuses studied (6 weeks), immunoreactivity was present throughout the spinal cord. Peptides and amines occurred later. Calcitonin gene-related peptide, galanin, somatostatin, neuropeptide Y and its C-flanking peptide (CPON) were the first to appear localized to motoneurones (embryonic days 15-17 rat; fetal weeks 6-14 human). Numbers of immunoreactive motoneurones decreased toward birth, but immunoreactive fibers increased in the ventral horn with enkephalin, thyrotrophin-releasing hormone, and the monoaminergic markers 5-hydroxytryptamine and tyrosine hydroxylase (all presumably of supraspinal origin) the last to appear perinatally. In the dorsal horn, particularly in the rat, a transient expression of substance P-, somatostatin-, and neuropeptide Y/CPON-immunoreactive cells was detected (embryonic days 15-17). A pronounced increase of calcitonin gene-related peptide-, galanin-, somatostatin- and substance P- immunoreactive fibers was found perinatally in both species. This coincided with an increased detection of cells in the dorsal root ganglia containing these peptides and the earliest appearance of calcitonin gene-related peptide-, somatostatin-, and substance P-immunoreactive fibers in the rat epidermis. Few antigens were localized to the intermediolateral cell columns before embryonic day 20 (rat), fetal week 20 (human), with thyrotrophin-releasing hormone-, 5-hydroxytryptamine-, tyrosine hydroxylase-, and vasoactive intestinal polypeptide-immunoreactive nerves appearing perinatally. In the rat dermis, tyrosine hydroxylase-immunoreactive fibers (sympathetic fibers) and fibers immunoreactive for neuropeptide Y/CPON and vasoactive intestinal polypeptide were detected from postnatal day 1. In conclusion, 1) peptide and amine immunoreactivity develops in motor before sensory or autonomic regions, 2) many peptide-containing cells are transient in fetal life, and 3) central terminals of dorsal root ganglion cells express peptides before terminals in the skin.

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies.

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.

Vasoactive intestinal polypeptide and substance P in primary afferent pathways to the sacral spinal cord of the cat.

An analysis of vasoactive intestinal polypeptide immunoreactivity (VIP-IR) and substance P-IR in the cat spinal cord has revealed marked differences in the distribution of the two peptides. While substance P-IR was located at all levels of the cord, VIP-IR was most prominent in the sacral segments in Lissauer's tract and lamina I on the lateral edge of the dorsal horn. VIP-IR was also present in the sacral cord in (1) laminae V, VII, and X, (2) a thin band on the medial side of the dorsal horn, (3) the dorsal commissure, (4) the lateral band of the sacral parasympathetic nucleus, and (5) in a few animals in Onuf's nucleus. In other segments of the spinal cord VIP-IR was much less prominent but was present in Lissauer's tract and laminae I, II, and X. Substance P-IR was more uniformly distributed at all segmental levels in laminae I-III, V, VII, and X and in the dorsal commissure. In ventrolateral lamina I of the sacral spinal cord both VIP-IR and substance P-IR exhibited a distinctive periodic pattern in the rostrocaudal axis. The peptides were associated with bundles of dorsoventrally oriented axons and varicosities spaced at approximately 210-micron intervals center to center along the length of the spinal cord. The bundles in lamina I continued into lamina V where they further divided into smaller bundles that extended medially through laminae V and VII. The most prominent bundles of VIP axons passed ventrally from lateral laminae V and VII to enter lamina X and the ventral part of the dorsal gray commissure. On the other hand the majority of substance P axons in lamina V turned dorsally to join with axons on the medial side of the dorsal horn and to pass into the dorsal part of the dorsal gray commissure. Rostrocaudal VIP axons were present not only in Lissauer's tract but also in dorsolateral lamina I, in the lateral funiculus and in the ependymal cell layer of the central canal. Following unilateral transection of the sacral dorsal roots (2 weeks-22 months) the density of VIP axons and terminals was markedly reduced in ipsilateral Lissauer's tract and lateral laminae I and V; however, no change was detected in lamina X. Sacral deafferentation reduced substance P-IR in the dorsal gray commissure and in lateral laminae I and V.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunohistochemical distribution of substance P, serotonin, and methionine enkephalin in sexually dimorphic nuclei of the rat lumbar spinal cord.

The purpose of the present study was to identify chemically some potential inputs to lumbar motoneurons of the rat in the spinal nucleus of the bulbocavernosus, ventral motor pool, dorsolateral nucleus, and retrodorsolateral nucleus. Substance P-like immunoreactivity and serotonin-like immunoreactivity were found in all four motor nuclei, with dense immunoreactive profiles surrounding motoneurons and their processes. Enkephalin-like immunoreactivity was restricted to the sexually dimorphic nuclei, the spinal nucleus of the bulbocavernosus, and the dorsolateral nucleus. Within the spinal nucleus of the bulbocavernosus, enkephalin-like immunoreactive profiles were apposed to the processes of motoneurons but not their somata. In contrast, enkephalin-like immunoreactivity surrounded motoneuron somata in the medial part but not the lateral part of the dorsolateral nucleus, in the location of motoneurons projecting to the ischiocavernosus muscle. Moreover, the density of serotonin-like immunoreactivity was also greater in the medial part of the dorsolateral nucleus. On the basis of the chemo-architecture and the connections of the dorsolateral nucleus, we suggest the division of this motor column into a medial part composed of ischiocavernosus motoneurons surrounded by enkephalin- and serotonin-like immunoreactivity and a lateral part that contains neurons that project to the sphincter urethrae muscle. Total spinal transection severely depleted both serotonin-like and substance P-like material in the lumbar ventral horn. No changes in the distribution of enkephalin-like immunoreactivity were observed following this lesion. It is therefore suggested that in the ventral horn, substance P- and serotonin-like material are derived from supraspinal tracts, whereas enkephalin-like material is derived from intrinsic nerve cell bodies of the spinal cord.

Nerve growth factor receptor immunoreactivity in the neuronal perikarya of human sensory and sympathetic nerve ganglia.

We examined immunohistochemically the dorsal root ganglia, sympathetic ganglia, spinal cord, ventral and dorsal roots, and sciatic nerves obtained at autopsy from adult humans, using a monoclonal antibody against the human nerve growth factor receptor. We observed labelling in a granular pattern in the neuronal perikarya of dorsal root and sympathetic nerve ganglia. Ventral horn cells and axons were not labelled.

The Thy-1 glycoprotein on nerve cells in culture: electron-microscopic analysis and biochemical characterization.

The distribution and some biochemical properties of the Thy-1 glycoprotein, an antigen common to brain and thymus-derived lymphocytes, were analyzed in primary nerve cell cultures from mouse embryos. The Thy-1 antigen was detected on the plasma membrane of most neurons and fibroblast-like cells and on some astrocytes. The labelling pattern was homogeneous, except on some neuronal cell bodies where the distribution varied from a few patches to a homogeneous labelling. The Thy-l molecules immunoprecipitated from either nerve cell cultures or from fibroblast-like cells from the meninges had similar molecular weights and isoelectric points. In contrast, Thy-1 from thymocytes was more heterogeneous in molecular weight and in isoelectric point range.

Monoclonal antibody to neural cell surface protein: identification of a glycoprotein family of restricted cellular localization.

A monoclonal antibody, designated anti-NSP-4 (anti-Neural cell Surface Protein-4), was obtained from a hybridoma generated by fusing rat myeloma cells with splenocytes of a rat immunized with membranes from the cerebella of weaver mutant mice. This antibody reacted with several high-molecular weight polypeptides in extracts prepared from the newborn and adult CNS of wild-type mice. The main NSP-4-reactive bands from neonatal cerebellum and spinal cord migrated with apparent molecular weights of 220,000 and 140,000. Major bands of 160,000 and of 175,000, 160,000 and 140,000 molecular weight were revealed in the adult cerebellum and spinal cord, respectively. Reaction of the antibodies with concanavalin A-binding proteins demonstrated the glycoprotein nature of the antigen. Cell types expressing NSP-4 antigen were determined using indirect immunofluorescence on monolayer cultures of early postnatal mouse cerebellar and dorsal root ganglion cells and on sections of developing and adult mouse cerebellum. In cerebellar cultures, the antibody reacted with the surface membrane of a subpopulation of astrocytes and of a small subset of neurones. In dorsal root ganglion cultures, anti-NSP-4 antibodies were highly specific for a subclass of small neurones. Staining for NSP-4 in sections of adult cerebellum was confined to the granular layer where the antibody seemed to label astroglia. In the developing cerebellum, NSP-4 staining outlined cell bodies of neuroblasts and migrating granule cells in the external granular layer. Post-migratory granule cells and Purkinje cells were negative. As in the adult, the labeled structures in the internal granular layer were probably astrocytes. Our results on the in vivo and in vitro localization of NSP-4 show its expression by subclasses of neurones and astrocytes in the cerebellum and by a subclass of neurones in cultures from the peripheral nervous system. The developmentally-regulated changes in the molecular weight forms of the NSP-4 antigen together with the shift in its cellular localization during cerebellar ontogeny suggest a functional significance for this antigen in developmental processes.

Distribution of calcitonin gene-related peptide in the rat peripheral nervous system with reference to its coexistence with substance P.

This immunocytochemical study, using a double-staining method, showed that calcitonin gene-related peptide-like immunoreactive structures are widely distributed in the peripheral nervous system and that many of them coexist with substance P-like immunoreactive structures in single sensory ganglion cells. Neurons positive for calcitonin gene-related peptide but negative for substance P were detected in sensory ganglia. These cells were large (about 30-45 micron in diameter); these primary sensory neurons containing calcitonin gene-related peptide can probably act independently of substance P. There were neurons containing calcitonin gene-related peptide without substance P in the pterygopalatine ganglion, although these cells were less numerous than in the sensory ganglia. In consecutive sections, calcitonin gene-related peptide-like structures occurred in thyroid parafollicular cells, which also contain calcitonin. This suggested that messenger RNA for producing calcitonin gene-related peptide is also present in the thyroid, and like calcitonin, calcitonin gene-related peptide may have a peripheral physiological role.