Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading.

Lack of neurite growth in optic nerve explants in vitro has been suggested to be due to nonpermissive substrate properties of higher vertebrate central nervous system (CNS) white matter. We have searched for surface components in CNS white matter, which would prevent neurite growth. CNS, but not peripheral nervous system (PNS) myelin fractions from rat and chick were highly nonpermissive substrates in vitro. We have used an in vitro spreading assay with 3T3 cells to quantify substrate qualities of membrane fractions and of isolated membrane proteins reconstituted in artificial lipid vesicles. CNS myelin nonpermissiveness was abolished by treatment with proteases and was not associated with myelin lipid. Nonpermissive proteins were found to be membrane bound and yielded highly nonpermissive substrates upon reconstitution into liposomes. Size fractionation of myelin protein by SDS-PAGE revealed two highly nonpermissive minor protein fractions of Mr 35 and 250-kD. Removal of 35- and of 250-kD protein fractions yielded a CNS myelin protein fraction with permissive substrate properties. Supplementation of permissive membrane protein fractions (PNS, liver) with low amounts of 35- or of 250-kD CNS myelin protein was sufficient to generate highly nonpermissive substrates. Inhibitory 35- and 250-kD proteins were found to be enriched in CNS white matter and were found in optic nerve cell cultures which contained highly nonpermissive, differentiated oligodendrocytes. The data presented demonstrate the existence of membrane proteins with potent nonpermissive substrate properties. Distribution and properties suggest that these proteins might play a crucial inhibitory role during development and regeneration in CNS white matter.

A phosphatidylinositol-linked peanut agglutinin-binding glycoprotein in central nervous system myelin and on oligodendrocytes.

Here we report the isolation and initial biochemical characterization of a 120-kD peanut agglutinin-binding glycoprotein from the adult human central nervous system (CNS), which is anchored to membranes through a phosphatidylinositol linkage. Myelin incubated with phosphatidylinositol-specific phospholipase C released the protein as a soluble polypeptide of 105 kD, which was isolated with peanut agglutinin-agarose affinity chromatography. The protein was found to be highly glycosylated. The protein appears to be confined to the CNS, where its developmental expression is region specific and parallels myelination. It is in greater quantity in white matter than in gray matter and it is in isolated human CNS myelin. Furthermore, ovine oligodendrocytes in culture contain the protein on their surfaces and release it into the supernatant as a soluble 105-kD form. We call this protein the oligodendrocyte-myelin protein.

Phorbol ester receptors: autoradiographic identification in the developing rat.

Autoradiography with 3H-labeled phorbol dibutyrate was used for the light microscopic detection of phorbol ester receptors in rat fetuses. In 15- and 18-day fetuses, as well as in adult rats, receptors were found to be concentrated in the central nervous system. The localization of receptors in the ventral marginal zone of the fetal neural tube, the lens of the eye, and other sites suggests a role for phorbol ester receptors in cellular process extension and cell-cell interaction.

Localization of FGF receptor mRNA in the adult rat central nervous system by in situ hybridization.

Fibroblast growth factor receptor (FGF-R) mRNA expression was examined in the adult rat CNS. Northern blot analysis showed a distinct 4.3 kb transcript in various CNS regions. In situ hybridization revealed widely distributed, but specific, populations of cells that express FGF-R mRNA. The most intense hybridization signals were observed in the hippocampus and in the pontine cholinergic neurons. The limbic system and brainstem nuclei, including motor nuclei, showed robust labeling. Cerebellar granule cells and spinal cord neurons were positive for FGF-R mRNA. The distribution of FGF-R mRNA differed significantly from that of NGF receptor mRNA; particularly, no hybridization signal was detected in basal forebrain cholinergic neurons. These results strongly suggest that FGF or FGF-like molecules may exert effects on specific neuronal populations in the mature CNS.

Laminin-like antigen in rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment.

Using several antibodies against rat or human laminin and an avidin-biotin immunocytochemical protocol, laminin-like immunoreactivity was detectable in the rat nervous system in expected locations, i.e., associated with blood vessels and reactive astrocytes. However, laminin staining was also abundantly present within neuronal cell bodies in most parts of the developing and adult rat CNS. Medial septum neuronal immunoreactivity was lost after septo-hippocampal disconnection, but could be preserved or even restored by intraventricular administration of nerve growth factor. Thus, at least for medial septum neurons, this laminin-like molecule can be accumulated or produced independent of direct hippocampal (target) contact. It remains to be determined whether CNS neuronal "laminin" processes activities similar to those found for laminin in vitro.

Non-specificity of anti-carbonic anhydrase C antibody as a marker in human neurooncology.

Because the presence of carbonic anhydrase C (CA C) has been demonstrated in the oligodendrocytes of the mouse, rat and man, anti-CA C serum has been considered to be a possible specific marker for these cells. In order to determine its value in human neurooncology, specimens from 110 human tumors from the central and peripheral nervous systems as well as from five cases of cerebral infarction and two of multiple sclerosis were tested immunohistochemically by the peroxidase-antiperoxidase method with anti-CA C serum. Reactive astrocytes, oligodendrocytes in the neural parenchyma surrounding tumors, and neurons included in areas of neoplasia showed CA C immunopositivity. In 92% of the astrocytomas and 56% of the glioblastomas variable numbers of tumor cells were positive. Some tumor cells positive for glial fibrillary acidic protein in ependymomas and astroblastomas were also CA C-positive. Schwannomas (86%), neurofibromas (100%) and meningiomas (86%) showed CA C positivity of the tumor cells, as did choroid plexus papillomas and gangliogliomas. However, all the medulloblastomas, neuroblastomas, central neurocytomas or melanomas tested in this study were entirely CA C-negative. In some examples of squamous cell carcinoma, leiomyoma, leiomyosarcoma and fibrous histiocytoma, CA C-positive neoplastic cells were also demonstrated. Our findings indicate that since various types of neoplastic and reactive cells express CA C positivity, the anti-CA C serum cannot be used as a specific marker for any tumor in human neurooncology.

Cyclo(His-Pro): its distribution, origin and function in the human.

Cyclo(His-Pro), or histidyl-proline diketopiperazine, is a cyclic dipeptide endogenous to blood, cerebrospinal fluid (CSF), semen, brain, spinal cord, and gastrointestinal tract of humans. Although a part of cyclo(His-Pro) clearly appears to be derived from the limited proteolysis of thyrotropin-releasing hormone by Pyroglutamate aminopeptidase, the biosynthetic origin of the remainder of the peptide can only be speculated. The levels of this peptide in blood and CSF fluctuate in health and disease in a manner appropriate for a physiologically active endogenous molecule.

Central serotonergic receptors: evidence for heterogeneity and characterization by ligand-binding.

Evidence has accumulated which suggests that receptors for serotonin exist in multiple forms both in the central nervous system and the periphery. This has come from the use of a variety of techniques and a number of different tissues. In the central nervous system the ligand-binding technique has proven particularly useful for characterizing different types of serotonin receptors, and two major classes of central serotonin receptors have been proposed on the basis of studies using this procedure. The first group (5-HT1 receptors) is defined by the high-affinity binding of 3H-serotonin in the brain, and the second (5-HT2 receptors) is defined by the high-affinity binding of 3H-spiperone in the frontal cortex. The 5-HT1 sites have themselves been shown to be a heterogeneous group, and recent studies suggest that it is possible to synthesize tryptamine analogues which can discriminate between the different types of 5-HT1 receptors. Such studies suggest the possibility of designing new selective serotonin agonists and antagonists for the study of the effects of specific receptor subtypes on behavioral and physiological activities.

Substance P-like immunoreactivity is present in the central nervous system of Limulus polyphemus.

The distribution of substance P-like immunoreactivity (substance P-li) in the central nervous system of Limulus polyphemus was studied by using indirect immunocytochemical techniques. Six bilaterally symmetrical pairs of cell clusters in the circumesophageal connectives and the subesophageal mass contain substance P-li. Two of those pairs are the source of a system of efferent fibers that is involved in the expression of circadian rhythms of photosensitivity by the lateral eye. Substance P-li-containing cells were also observed scattered along the length of the circumesophageal connectives, which contain abundant stained fibers and some terminals. Substance P-li fibers leave through the ventral and dorsal nerves of the posterior circumesophageal ring. The neuropil of the subesophageal mass contains an abundance of stained terminals. Immunoreactive fibers can be seen throughout the length of the two longitudinal connectives of the ventral cord, in discrete fiber tracts in the lateral edges of the interganglionic connectives, and in the dorsal and ventral nerves of abdominal ganglia 1-4. Each of these ganglia contains three pairs of substance P-immunoreactive cell body clusters: an anterolateral, a medial longitudinal, and a medial posterior cluster. Substance P-li fibers entering through the ventral (posterior) nerves form very distinctive fascicles in each side of the ganglia, giving off fibers throughout their length. The neuropil is filled with immunoreactive terminals distributed homogeneously. The anterolateral clusters of the abdominal ganglia may be involved in cardioregulation. The six pairs of clusters in the posterior circumesophageal ring, and perhaps some of those in the abdominal ganglia, are believed to constitute a neurosecretory system, projecting to multiple targets throughout the organism. This system is postulated to modulate various sensory inputs and motor activity, and could be driven by a circadian clock, as well as by other systems responsible for integrated organismic responses.

Expression of the L11 neuropeptide gene in the Aplysia central nervous system.

Neuron L11 in the abdominal ganglion of Aplysia californica is thought to be both cholinergic and peptidergic. In previous studies, we isolated a cDNA clone encoding the precursor for an L11 secreted protein(s) by differentially screening an abdominal ganglion cDNA library. We now report the isolation of genomic clones encoding the L11 cDNA sequences. Analysis of these clones reveals that the gene is present in a single copy per haploid genome. RNA blotting and cDNA cloning demonstrate that the L11 gene is expressed not only in the abdominal ganglion but in the head ganglia as well. To define the positions of cells expressing this gene and to follow their processes, we raised antibodies to synthetic peptides defined by the cDNA sequence. Histochemistry revealed about 100 neurons containing immunoreactive material. These cells arborize in the neuropil and are distributed throughout the central nervous system, representing about 0.5% of the Aplysia central neurons. In addition, cells in the abdominal ganglion send processes to the mantle floor at the base of the gill via the genital and branchial nerves. Our data suggest that this network of cells expresses the single L11 peptide gene.

Glial fibrillary acidic (GFA) protein in vertebrates: immunofluorescence and immunoblotting study with monoclonal and polyclonal antibodies.

We report a comparative immunofluorescence and immunoblotting study of GFA protein, the subunit of glial filaments, in nonmammalian vertebrates. The study was conducted with polyclonal antibodies raised to human and shark antigen and with monoclonal antibodies isolated from mice immunized with chicken and bovine antigen. With the exception of cyclostomes, glial filaments appeared remarkably conserved in vertebrate phylogeny, both with respect to the molecular weight and immunoreactivity of their protein subunit. In most species, the antibodies decorated a single band in brain, spinal cord, and optic nerve extracts by the immunoblotting procedure. This band had the same molecular weight in the different CNS regions. With the exception of the turtle, species differences in the molecular weight of the band were not greater than those observed among mammalian vertebrates (human, bovine, and rat). However, there were some exceptional findings in fish. In goldfish and trout brain and spinal cord extracts, the antibodies decorated with the same intensity two bands. In accordance with previous immunofluorescence findings, goldfish optic nerve extracts were negative by the immunoblotting procedure. In four fishes (sea bass, tautog, trout, and scup), optic nerves reacted with the antibodies. However, the band decorated by the antibodies was higher in molecular weight than that obtained from brain and spinal cord extracts. Glial fibers were demonstrated by immunofluorescence in the brain, spinal cord, optic nerve, and retina of most species studied. In amphibia immunofluorescent structures were comparatively few, probably accounting for the negative results by immunoblotting. A comparative immunohistological study of the cerebellum showed the presence of perpendicular glial fibers in the molecular layer of most species examined. Birds and amphibia were different in this respect. Bergmann glia in chicken were GFA negative. In the frog and the toad, immunofluorescent fibers in the molecular layer of the cerebellum were haphazardly oriented. Ependymal radial glia was GFA-negative in the cerebellum of subavian vertebrates. Antisera raised in rabbit to shark GFA protein reacted with the same bovine GFA fragments recognized by polyclonal and monoclonal antibodies raised to human and bovine antigens, respectively, i.e., 30-kDa N-bromosuccinimide fragment (tryptophan cleavage); 35-kDa 2-nitro-5-thiocyanobenzoic acid fragment (cysteine cleavage); 18-kDa cyanogen bromide fragment (methionine cleavage). Conversely, the chicken GFA monoclonal antibodies selected for this study only reacted with noncleaved protein.

Wheat germ agglutinin-apoHRP gold: a new retrograde tracer for light- and electron-microscopic single- and double-label studies.

In this report, we describe a new colloidal-gold-labelled retrograde tracer, wheat germ agglutinin (WGA) conjugated to enzymatically inactive horseradish peroxidase (apoHRP). This protein gold complex (WGAapoHRP-Au) is a sensitive marker for retrograde tracing of the projections of CNS neurons at the light-microscopic (LM) level when a silver-enhancement procedure is used to detect the gold in the tracer. For electron-microscopic (EM) analysis, the silver-enhanced sections undergo a further gold-toning step. This protects against rapid oxidation and dissolution of the silver precipitate during the osmication procedure. A major advantage of WGAapoHRP-Au is that it can be used in a variety of multiple-labelling studies. When the retrograde transport of the new tracer is combined with that of the fluorescent dye, True Blue, neurons that have bifurcating axons can be readily demonstrated. Simultaneous immunofluorescent detection of the cytochemistry of the double-retrogradely labelled neurons is also possible. In contrast to a WGA-HRP gold complex, the new complex has no enzymatic activity. Thus HRP-based techniques (e.g., anterograde transport of WGA-HRP or peroxidase-antiperoxidase immunocytochemistry) can be performed on tissue that contains retrogradely labelled neurons marked with WGAapoHRP-Au without having to pretreat tissue so as to destroy endogenous HRP enzyme activity. At the EM level, the gold is readily distinguished from DAB immunoreaction product. This makes both LM and EM double-labelling studies possible. The great sensitivity of the new tracer, its compatibility with a variety of aldehyde fixatives, its ease of detection, and the fact that it can be simultaneously used with several fluorescent and HRP-based immunocytochemical and tracing techniques make WGAapoHRP-Au a valuable tool for LM and EM characterization of CNS cytochemistry and connectivity.

Central somatostatin systems revealed with monoclonal antibodies.

The distribution of central neurons displaying somatostatin immunoreactivity was studied using three monoclonal antibodies to cyclic somatostatin. The sensitive ABC immunoperoxidase technique was employed. A large number of positive cell groups including many previously undescribed populations were detected throughout the brain and spinal cord. Telencephalic somatostatin neurons included periglomerular cells in the olfactory bulb, mitral cells in the accessory olfactory bulb, and multipolar cells in the anterior olfactory nuclei, neocortex, amygdala, hippocampus, lateral septum, striatum, and nucleus accumbens. Within the hypothalamus, positive neurons were found in the periventricular, suprachiasmatic, and arcuate nuclei, and throughout the anterior and lateral hypothalamus. The entopeduncular nucleus and zona incerta contained many positive neurons, and the lateral habenula had a dense terminal field suggesting a pallidohabenula somatostatin pathway. Somatostatin neurons were also found in association with many sensory systems. Positive cells were present in the superior and inferior colliculi, the ventral cochlear nuclei, the ventral nucleus of the lateral lemniscus, nucleus cuneatus, nucleus gracilus, and the substantia gelatinosa. Various cerebellar circuits also displayed somatostatin immunoreactivity. Golgi cells throughout the cerebellar cortex were intensely stained, and some Purkinje cells in the paraflocculus also showed a positive reaction. Positive fibers were present in the granular layer and large varicose fibers were present in the inferior cerebellar peduncle. Many nuclei known to project to the cerebellum, including the nucleus reticularis tegmenti pontis, the medial accessory inferior olive, the nucleus prepositus hypoglossi, and many areas of the reticular formation contained positive neurons. These studies demonstrate that these new monoclonal antibodies are of great value for the study of central somatostatin systems. Previously described somatostatin systems are readily detected with these antibodies, and in addition, many otherwise unrecognized somatostatin cell groups have been discovered.

Organization of atriopeptin-like immunoreactive neurons in the central nervous system of the rat.

The atrial natriuretic peptide, atriopeptin, is a circulating hormone that plays an important role in the regulation of fluid and electrolyte homeostasis. Several recent studies have shown that atriopeptin-like immunoreactivity is present within the central nervous system as well as peripheral tissues. In the present report, we describe in detail the organization of atriopeptin-like immunoreactive (APir) perikarya and fibers in the central nervous system of the rat. The most prominent collection of APir perikarya was found in the hypothalamus, adjacent to the anteroventral tip of the third ventricle. Additional groups of APir perikarya were observed along the wall of the third ventricle and in the paraventricular and arcuate nuclei. Separate, smaller groups with distinctive morphology were seen in the lateral hypothalamic area, in the supra-mammillary, medial, and lateral mammillary nuclei, medial habenular nucleus, bed nucleus of the stria terminalis, and the central nucleus of the amygdala. In the pons and brain-stem, APir neurons were observed in the pedunculopontine and laterodorsal tegmental nuclei, as well as in the ventral tegmental area, Barrington's nucleus, the parabrachial nucleus, and the nucleus of the solitary tract. The densest terminal fields of APir fibers were found in the paraventricular nucleus of the hypothalamus, the bed nucleus of the stria terminalis, the median eminence, and the interpeduncular nucleus. The presence of atriopeptin immunoreactivity within the central nervous system suggests that atriopeptin may function as a central neuromediator. Potential functions of this candidate neuromediator deduced from its anatomical distribution are discussed, including the possibility that atriopeptin may function as both a central neuromediator and a systemic hormone in the regulation of the cardiovascular system.

The distribution of serotonin in the CNS of an elasmobranch fish: immunocytochemical and biochemical studies in the Atlantic stingray, Dasyatis sabina.

The distribution of serotonin (5HT) in the brain of the Atlantic stingray was studied with peroxidase-antiperoxidase immunocytochemistry and high-pressure liquid chromatography. The regional concentrations of 5HT determined for this stingray fell within the range of values previously reported for fishes. A consistent trend in vertebrates for the hypothalamus and midbrain to have the highest concentrations and the cerebellum the lowest was confirmed in stingrays. Neuronal cell bodies and processes exhibiting 5HT-like immunoreactivity were distributed in variable densities throughout the neuraxis. Ten groups of 5HT cells were described: (I) spinal cord, (II-IV) rhombencephalon, (V, VI) mesencephalon, (VII, VIII) prosencephalon, (IX) pituitary, and (X) retina. There were three noteworthy features of the 5HT system in the Atlantic stingray: (1) 5HT cells were demonstrated in virtually every location in which 5HT-containing cells have been described or alluded to in the previous literature. The demonstration of immunopositive cells in the spinal cord, the retina, and the pars distalis of the pituitary suggests that 5HT may be an intrinsic neurotransmitter (or hormone) in these regions. (2) The distribution of 5HT cells in the brainstem shared many similarities with that in other vertebrates. However, there were many 5HT cells outside of the raphe nuclei, in the lateral tegmentum. It appears that the hypothesis that "lateralization" of the 5HT system is an advanced evolutionary trend cannot be supported. (3) 5HT fibers and terminals were more widely distributed in the Atlantic stingray brain than has been reported for other nonmammalian vertebrates on the basis of histofluorescence. It appears that this feature of the 5HT system arose early in phylogeny, and that the use of immunohistochemistry might reveal a more general occurrence of widespread 5HT fibers and terminals.

Histochemical labeling of beta-adrenergic receptors in the mouse central nervous system by 9-amino-acridin propranolol.

A new fluorescent beta-adrenergic antagonist, 9-amino-acridin propranolol (9-AAP), was administered intravenously to living mice. In the cerebral cortex, the highest concentration of 9-AAP was noted in the hippocampal formation, where it distinctly labeled the hippocampal pyramidal cell layer and the granule cell layer of the dentate gryus. High uptake occurred in the pyramidal cell layer of the piriform cortex. In the neocortex, fluorescence was less dense and more diffuse but confined to the basal layers. A similar pattern was observed in the basal layers of the cingulate cortex, but an additional high-density dotted fluorescence labeled its layer II. In the cerebellar cortex, 9-AAP was localized within the Purkinje cell layer. In the spinal cord, the highest density of fluorescence was observed in the nuclear collections of alpha-motoneurons. The findings were similar to those observed in the central nervous system of the rat and support the reproducibility of the method. 9-AAP may be used in vivo as a fluorescent probe to map out the central beta-adrenergic receptor system.

Expression of the rat amylin (IAPP/DAP) gene.

We have used the polymerase chain reaction with mixed sequence primers to generate a probe for rat amylin and have used this to detect expression in various rat tissues. Amylin mRNA is found in greatest concentrations in the pancreas where a single mRNA species can be detected giving a hybridisation signal intensity approximately 10% that of insulin mRNA. When the beta cell population was depleted with streptozotocin, both amylin and insulin mRNAs were reduced to a similar extent. Consistent with its supposed role in the control of carbohydrate metabolism, amylin mRNA was also found in the stomach. Unlike the related peptide, CGRP, amylin mRNA is not present in the thyroid and is not widely distributed in the central nervous system. The only nervous tissue in which it could be detected was the dorsal root ganglion. Surprisingly, amylin mRNA was also found in the lung though only at very low levels.

Selection of antibodies for the immunofluorescent localisation of cyclic GMP in the central nervous system.

Radioimmunoassay techniques have been used to determine why only a small number of antibodies against cyclic GMP are able to show immunofluorescent staining in the central nervous system. However, neither the titre, avidity nor specificity as determined in liquid-phase radioimmunoassay employing [3H]cyclic GMP, have been able to predict whether a given antibody will show specific staining, or account for the different sites of immunofluorescent localisation observed with these antibodies. These observations are discussed in terms of the populations of antibody molecules employed in the radioimmunoassay and immunofluorescent procedures.

Myelin-associated glycoprotein (MAG) distribution in human central nervous tissue studied immunocytochemically with monoclonal antibody.

Recent biochemical data show that myelin-associated glycoprotein (MAG) is the antigen for a monoclonal antibody found in sera of patients with IgM paraproteinemia and neuropathy (Braun et al. 1982). Immunoreactivity of this antibody with CNS has not been described. To study this, monoclonal anti-MAG was used in the avidin-biotin-peroxidase complex method (Hsu et al. 1981) to immunostain paraffin and epon sections of human CNS. Well characterized polyclonal MAG antiserum (Quarles et al. 1981) was employed in comparison tests. In paraffin sections of developing CNS, both monoclonal and polyclonal MAG antisera stained oligodendroglia and myelin. In adult CNS, periaxonal regions of myelin sheaths were immunostained in paraffin sections and semithin epon sections treated with monoclonal and polyclonal anti-MAG. In electron-microscopic experiments that included milder pretreatment of epon thin sections and more precise reaction product localization, entire thickness of myelin sheaths were immunostained. Thus, in electron micrographs, monoclonal and polyclonal anti-MAG immunoreactivity also have the same localization. In other electron-microscopic experiments, the same reaction product localization was observed with antiserum to myelin basic protein (MBP), a known constituent of compact myelin. Thus, results with this monoclonal anti-MAG provide important new evidence to support the localization of MAG in compact CNS myelin. Our data also suggest that monoclonal antibodies against MAG will be useful in studies of the pathogenesis of multiple sclerosis and other demyelinating diseases.

Glial fibrillary acidic protein (GFAP): the major protein of glial intermediate filaments in differentiated astrocytes.

The glial fibrillary acidic protein (GFA protein or GFAP) is the major protein constituent of glial intermediate filaments in differentiated fibrous and protoplasmic astrocytes of the central nervous system. Proteins having similar molecular weights, isoelectric points, and immunoreactivity with GFAP have been found in cells of neural crest and ectodermal origin. A putative function ascribed to glial filaments is its role as a component of the cytoskeleton in defining and maintaining the shape of the astrocyte. Since 1980, over 350 reports have utilized antisera to GFAP for immunochemical and immunocytochemical studies.