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The rat 1B1075 mRNA encodes a 533-residue novel chromogranin/secretogranin-like acidic protein that contains an apparent secretion signal, several pairs of tandem basic residues, and internally repeated sequence elements. 1B1075 transcripts are detected, by blotting and in situ hybridization, at the highest levels in the neocortex, hippocampus, cerebellar cortex, selected pontine and diencephalic nuclei, and presumptive pituitary corticotrophs, at lower levels in specific nuclei in most other brain regions, but in none of several other tissues. Utilizing antisera to several nonoverlapping synthetic peptide fragments of the predicted protein sequence, we detect a brain- and pituitary-specific 57-kDa protein in cellular processes and fiber tracts, generally consistent with axonal transport from the cell bodies identified by in situ hybridization. Ultrastructural studies demonstrate that this protein is a component of intraneuronal vesicles in axons and vesicle-like structures in dendrites. Based on these data, we suggest the name Secretogranin III for the 1B1075 gene product. In related collaborative studies, a mouse deleted for the 1B1075-homologous gene has been produced that should allow assessment of its physiological role. more...

High-molecular-weight forms of clathrin light chains LCa and LCb contain inserted sequences and are expressed in brain tissue but have not been observed in peripheral tissues. Monoclonal antibodies specific for the high-molecular-weight form of LCb and all forms of LCa were used to analyze their expression in different species and different neuronal cell types. High-molecular-weight light chains were found in bovine, rat, mouse, chicken, and human brain, indicating a conserved pattern of expression. Neuron-specific expression of the high-molecular-weight light chains was suggested by analysis of human brain gray matter and white matter. The former contained a higher proportion of light chains with insertion sequences. Immunohistochemical analysis localized the high-molecular-weight form of LCb to synapses and neuronal perikarya, but not to glial cells. Immunofluorescent labeling of cultured chicken dorsal root ganglia confirmed expression in neurons but not Schwann cells. These results indicate that the high-molecular-weight forms of clathrin light chains are restricted in expression and found in neuronal cells. more...

The bombesin-like peptides are a family of structurally related amidated peptide ligands that are known to have a variety of potent pharmacological actions on various cells, including neurons in the rat brain. Two mammalian representatives of the bombesin family of peptides have been identified, gastrin-releasing peptide (GRP) and neuromedin B (NMB). Previously, we cloned the rat preproGRP gene and determined the locations of neurons expressing this gene using in situ hybridization. In this study, we describe the structure and sequence of the rat preproNMB gene, and the first detailed cellular localization of preproNMB mRNA in rat brain using in situ hybridization. Nucleotide sequence analysis of cDNA and genomic clones reveals a 117 amino acid precursor whose overall structure is similar to that described for human preproNMB. Sequence similarity between the rat NMB and GRP genes is observed only over a limited 10 amino acid sequence encoding the carboxy termini of the GRP and NMB peptides, the region shown to be necessary and sufficient for high-affinity receptor binding. In situ hybridization studies performed with cRNA probes specific for NMB or GRP mRNA show that the distribution of cells expressing either mRNA in brain is very distinct. NMB mRNA is found most prominently in the olfactory bulb, dentate gyrus, and dorsal root ganglion. In contrast, the highest levels of GRP mRNA are observed in the forebrain (isocortex and hippocampal formation). This heterogeneity of mRNA distribution for these peptides suggests that these 2 structurally related peptides may have very distinct functions as neuropeptides in the rat nervous system. more...

This communication describes the cellular and ultrastructural localization in the central nervous system of P-glycoprotein (P-GP) recognized by a murine monoclonal antibody, MRK 16. At the ultrastructural level P-GP was strictly confined to the luminal surface of the endothelial cells which comprise the capillary vessels of the brain and the spinal cord. No P-GP was found in the endothelial cells of other organs. Our findings may be useful as a means to define the blood-brain barrier, and they imply that the blood-brain barrier is anatomically characterized by the presence of intercellular tight junctions between continuous nonfenestrated endothelial cells. more...

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system. more...

The confocal scanning laser microscope (CSLM) offers improved optical resolution and contrast, high photometric precision, and the ability to make optical sections. These benefits were explored for use in quantitative analysis of immunofluorescence-labeled axon terminals. Guidelines were obtained for adjustments of the CSLM parameters. In the present applications, bleaching of the fluorescence did not represent a serious obstacle to analysis with the CSLM. A method was developed to distinguish the background fluorescence from the specific fluorescence labeling. This procedure made way for the development of automated quantification of immunolabeled axon terminals. The automated procedures substantially reduced the man-hour expenditure for analysis and provided highly reproducible quantifications compared with manual methods. The increased resolution and contrast of the CSLM allowed measurements of the fluorescence signal strength of individual axon terminals. The CSLM also allowed detection of co-localized neurochemicals in axon terminals. more...

Eight MAbs have been developed against chordin and designated as At2-At9. It is shown that all antibodies are directed against identical, spatially overlapping or closely positioned epitopes of chordin. The chordin molecule has repetitive sites wherein epitopes for the eight MAbs are located. This site lies within a proteinase-resistant fragment of chordin, presumably a glycopeptide, of molecular mass between 2 and 10 kDa. Fluorescence staining of cryostat sections from stellate sturgeon with the use of At5 (indirect Coons' method) has revealed a positive reaction with notochord cells and sheath and with the spinal cord. No significant reaction with cartilage, muscle and kidney was detected. more...

A procedure is presented which modifies the Sternberger peroxidase--antiperoxidase (PAP) technique in order to visualize additional amounts of immunodeposits representing the antigen substance (SP) in 5-micrometer paraffin tissue sections of rat spinal cord. For increased sensitivity, the new procedure utilizes a "double bridge" and diaminobenzidine in low pH buffer. The modifications have made possible the visualization of immunoreactive beaded processes and punctate bodies, which were then traced to determine patterns of SP circuitry. Using the modified PAP procedure, the greatest number of immunoreactive processes appeared in the dorsal horn, where some punctate bodies and varicose processes could be seen adjacent to the myelinated afferent fiber bundles that penetrate the substantia gelatinosa as dorsal root collaterals. Additional immunoreactive processes and punctate bodies coursed through the myelinated afferent fiber bundles that penetrate the dorsolateral white matter, and extend into the intermediolateral gray region. Substance P was also identified within immunoreactive processes found in Rexed's laminae V and VI, as well as the central canal region, the dorsal gray commissure, and the ventral gray and white commissures. Since the modifications improved the visualization of SP-containing processes in sparsely populated regions of the spinal cord, especially the ventral horn, they may be useful in demonstrating other antigens that normally occur in small quantities within tissues. more...

The molecular organization of surface galactose residues in glycoproteins of the intact myelin sheath was investigated using the enzymatic membrane probe, galactose oxidase. Rat spinal cords treated under physiological conditions with this nonpermanent probe were labeled specifically in galactose residues by reduction with tritiated sodium borohydride. The enzymatically modified proteins from isolated myelin were analyzed electrophoretically and their specific radioactivities determined. Results indicated tritium label associated with a surprising variety of high molecular weight proteins. The most extensively labeled peak corresponded to the major myelin glycoprotein as indicated by the coincidence of tritium label with that of [14C]fucose used as an internal marker for the glycoproteins. The radioactivity associated with this protein was 1.1 to 2.7 times higher after treatment with galactose oxidase when compared to reduction in the absence of the enzyme and 1.4 to 4.8 times higher when oxidized and reduced after prior treatment with neuraminidase. The results suggest a complex heterogeneity of minor glycoproteins associated with isolated myelin. It is concluded that from this complexity of glycoproteins, a major glycoprotein is at least partially localized on the external surface of either the intact myelin sheath or the closely associated oligodendroglial plasma membrane. Such a localization of this glycoprotein and the probable localization of the other glycoproteins enhances their potential role in specific interactions in the process of mpyelination or myelin maintenance. more...

The lactoperoxidase-catalyzed iodination technique was utilized to incorporate radioactive iodine into membrane proteins which lie on the outer surface of the myelin sheath. An intact, myelinated nerve bundle, the dorsal column of the cat spinal cord, was employed. The enzymatically iodinated proteins were identified by polyacrylamide gel electrophoresis, and the specific radioactivity was determined. Results indicated that several high molecular weight proteins were predominantly labeled by the nonpenetrating lactopreoxidase. Proteolipid protein was also labeled, although to a lesser extent; basic protein was not labeled under these conditions. The data suggest that several high molecular weight proteins are exposed on the outer surface of the myelin sheath. Proteolipid protein is at least partially exposed on the outer surface, although it could be present at both membrane surfaces. Evidence is presented which suggests that the basic protein is located at the inner surface of the membrane, corresponding to the major dense line of myelin. more...

1. We determined the number of beta-receptors in the whole spinal cord of the adult rat and in the cervical, thoracal, and lumbal/sacral parts. 2. The undivided spinal cord contains 47 +/- 10 fmol/mg beta-receptors (KD = 2066 +/- 982 pmol/liter), and the cervical part of the spinal cord contains 53 +/- 8 fmol/mg protein (KD = 3224 +/- 1775 pmol/liter). The thoracal part shows 40 +/- 1 fmol/mg protein (KD = 3229 +/- 104 pmol/liter), and the lumbal/sacral spinal cord contains 48 +/- 8 fmol/mg protein (KD = 3610 +/- 1610 pmol/liter). 3. Competitive inhibition studies with l-practolol, dl-atenolol, and ICI 118,551 were performed and we calculated by a computer program in the whole spinal cord the following ratio of beta-receptor subtypes: 80 +/- 5% Beta 1-receptors and 20 +/- 5% beta 2-receptors. 4. The basal and (-)-isoproterenol- and NaF-stimulated activity of adenylate cyclase was highest in the cervical part of the spinal cord and equally distributed between the thoracal and the lumbal/sacral parts. 5. The whole synaptosomal protein of the cervical part of the spinal cord contained 132 +/- 20 fmol, the thoracal part 117 +/- 3 fmol, and the lumbal/sacral part 133 +/- 22 fmol. more...

The contractile protein, actin, is a major component of the central nervous system (CNS). Its accumulation at presynaptic and postsynaptic sites has been suggested by biochemical and ultrastructural studies. The present work examines directly the distribution of actin within the CNS by immunocytochemical means. Sections of rat CNS were stained with an antiserum against chicken gizzard actin, using a peroxidase-antiperoxidase technique. In Vibratome sections, reaction product appeared widely distributed in the perikaryal cytoplasm of neurons, in axons and dendrites, and in astrocytes and blood vessels. In paraffin sections, however, much of the cytoplasmic reaction was not observed. Instead, the reaction product was concentrated in processes within the neuropil and in discrete densities about the perimeters of neurons. This latter distribution, observed in areas in which large axosomatic synapses are known to occur, was consistent with a concentration at synaptic sites. Immunocytochemistry performed at the ultrastructural level, using a pre-embedding technique, revealed a diffuse cytoplasmic staining, with increased accumulation under the neuronal plasma membrane, particularly at postsynaptic densities. more...

During screening of a rat spinal cord lambda gt11 cDNA library with poly- and monoclonal antibodies against the postsynaptic glycine receptor a cDNA was isolated which covers an open reading frame encoding a protein of calculated mol. wt 94 kd. Sequence analysis identified a novel type of neuron-specific protein (named neuraxin) which is characterized by an unusual amino acid composition, 12 central heptadecarepeats and putative protein and/or membrane interaction sites. The gene encoding neuraxin appears to be unique in the haploid rat genome and conserved in higher vertebrates. Northern blot and in situ hybridization revealed neuraxin mRNA to be expressed throughout the rodent central nervous system (CNS). In spinal cord, neuraxin transcripts were abundant in motoneurons which also expressed glycine receptor subunit mRNA. A bacterial fusion protein containing approximately 90% of the neuraxin sequence was found to specifically bind tubulin. Polyclonal neuraxin antibodies cross-reacted with microtubule-associated protein 5 (MAP5), and a monoclonal antibody against MAP5 recognized the neuraxin fusion construct. Based on these data we suggest that neuraxin is related to MAP5 and may be implicated in neuronal membrane-microtubule interactions. more...

The immunohistochemical localization of two myelin specific proteins-basic protein (BP) and proteolipid protein (PLP)-was compared during the process of myelination. Although both proteins were present in oligodendrocytes, (i) neither protein was observed in oligodendrocytes not already closely associated with nerve fibers exhibiting a fluorescent coating; (ii) in any discrete anatomical area oligodendrocytes were positive for BP before PLP was visible; and (iii) as myelination progressed, immunoreactivity for BP in oligodendrocytes appeared to decrease and simultaneously PLP immunofluorescence became visible in this cell type. During the period of active myelination, fibers exhibited a distinct varicose appearance. As myelination progressed, the myelin sheath increased in thickness and these varicosities became less prominent, eventually completely disappearing. Therefore, the nature and the appearance of varicosities can be used as an index of the relative stage of maturation of myelin in an individual fiber. In general, PLP appeared in fibers at a later stage of maturation than did BP based on the above criteria. However, in a relatively small number of fine fibers PLP was observed at a very early stage. In fully mature myelin, very large fibers were frequently more intensely fluorescent for BP than PLP, whereas fine myelinated fibers were more intensely stained for PLP. These observations are consistent with the following interpretations. (i) Substantial differentiation of oligodendrocytes occurs prior to appearance of either of these proteins by immunofluorescence. (ii) BP is added to the myelin sheath prior to PLP and there appears to be a shift in priority of synthesis from BP to PLP in individual oligodendrocytes during the process of myelination. (iii) Very small fibers often contain low concentrations of BP relative to PLP, and conversely, very large fibers may contain a high concentration of BP relative to PLP. Thus, the relative concentration of these proteins in myelin appears not to be constant but may vary as a function of the size of the myelinated fiber. more...

Infection of athymic (nu/nu) mice with Theiler's murine encephalomyelitis virus results in an acute encephalitis which resembles poliomyelitis. Immunohistochemistry and in situ hybridization were used to delineate the presence of viral proteins and RNA in the nervous systems of nude mice infected with the Daniels strain of Theiler's virus. This system permits the analysis of a viral infection in the absence of an effective immune response. By immunohistochemistry, viral antigen was found in the processes and cell bodies of neurons and glial cells. Besides the presence of viral antigen in these cell types, by in situ hybridization, Theiler's virus RNA was also found in cells associated with vascular endothelium in the brains and spinal cords of these infected mice. Theiler's virus RNA-positive endothelial cells were observed not only near the primary lesions but also away from demonstrable lesions in normal-appearing regions in the central nervous system. Earlier work had suggested an intra-axonal dissemination for this virus (M. C. Dal Canto and H. L. Lipton, Am. J. Pathol. 106:20-29, 1982). Our findings are consistent with this model but also suggest an additional mechanism for virus spread within the central nervous system, i.e., by infecting vascular cells and crossing the blood-brain barrier. Lastly, after Theiler's murine encephalomyelitis virus infection, not only glial cells but also endothelial cells express major histocompatibility complex class II (la) antigen on their surface (M. Rodriguez, M. L. Pierce, and E. A. Howie, J. Immunol. 138:3438-3442, 1987). Our demonstration of Theiler's virus-infected endotheliumlike cells may explain interactions of virus products in stimulating antigen presentation. more...

A recent publication described autopsy samples from five brains in which positive staining for iron was observed as surrounding demyelinated plaques. Because this has not been previously reported, autopsy material from 13 patients who were being followed up at the Multiple Sclerosis (MS) Clinic, University Hospital, The University of Western Ontario, London, Ontario, was reviewed. A total of 32 paraffin-embedded blocks containing demyelinated plaques of varying age were sectioned and stained using a standard acid ferrocyanide technique (Peris' test) for iron. Microscopic examination failed to detect the presence of significant positive staining either within or surrounding the areas of demyelination. Positive histochemical staining for iron identifies hemosiderin, the presence of which suggests either iron overload or remote hemorrhage. Support for these two processes in the pathogenesis of MS could not be provided from our material. more...

Substance P and 5-hydroxytryptamine containing presynaptic boutons on presumed alpha-motoneurons were studied in the chicken ventral horn using the indirect antibody peroxidase-antiperoxidase technique in conjunction with monoclonal antibodies to 5-hydroxytryptamine and substance P both at light and electron microscopic levels. At the light microscopic level, substance P immunoreactivity was observed as large dot-like structures in close apposition to the soma and dendrites of presumed alpha-motoneurons. Individual immunoreactive structures were separated from each other. On the other hand, 5-hydroxytryptamine immunoreactivity was observed as many small dot- or fiber-like structures outlining the soma and dendrites of large neurons in the ventral horn. Electron microscopically, 5-hydroxytryptamine immunoreactive presynaptic boutons were identified as the type which contained elongated dense cored vesicles concomitant with flattened clear vesicles, while the substance P immunoreactivity was observed in presynaptic boutons which contained spherical dense cored vesicles (diameter range 60-110 nm) together with spherical clear vesicles. Both types of dense cored vesicles were often adjacent to the presynaptic membrane of synaptic specialization. These immunocytochemical results suggest that alpha-motoneurons may be directly modulated by substance P and 5-hydroxytryptamine in the chicken. more...

1. Effects of taurine on primary afferent terminals in the frog spinal cord were examined by a sucrose-gap method applied to a dorsal root (9th or 10th segment). 2. In a normal Ringer solution, taurine (1 mM, applied for 5s at a rate of 0.04 ml s-1, 0.2 mumol) caused a hyperpolarization, but a higher concentration (10 mM, applied at the same rate, 2.0 mumol) caused a biphasic response consisting of a hyperpolarization followed by a slow onset depolarization. A similar biphasic response could also be observed in tetrodotoxin-treated preparations. 3. When the concentration of extracellular Mg2+ was increased up to 9.0 mM, the depolarizing response to taurine was augmented. The rate of the augmentation was dependent upon the extracellular Mg2+ concentration. 4. The depolarizing effect was selectively antagonized by bicuculline in concentrations (10-30 microM) that had no significant antagonizing action on gamma-aminobutyric acid (GABA)-induced depolarization. On the other hand the hyperpolarizing effect of taurine was selectively reduced by strychnine (0.1 microM) which had no antagonizing effect on responses to glycine. 5. These results suggest that in the frog spinal cord there are at least two subtypes of taurine receptor whose pharmacological profiles resemble GABA and glycine receptors in the mammalian central nervous system, and whose sensitivity may be modulated by extracellular Mg2+. more...

The cold non-depolymerizable fractions obtained during the standard procedure for the isolation of microtubules from ox brain stem-cerebral hemispheres and spinal cord have been studied. The cerebral-hemisphere preparation was composed of 10-nm filaments but also contained large amounts of membranes. The polypeptide content included tubulin, microtubule-associated proteins and minor proteins corresponding to the neurofilament triplet of proteins of mol.wt. 210 000, 160 000 and 70 000 respectively. The brain-stem preparation contained more 10-nm filaments than membranes. The polypeptide content consisted of the neurofilament triplet (35%), tubulin (30%) and minor proteins. In contrast, the spinal-cord preparation was mainly composed of 10-nm filaments, free of membranes and containing essentially the neurofilament protein triplet (64%). These filaments appeared very similar to the peripheral-nervous-system neurofilaments described by several authors. Since the best neurofilament from the central nervous system often contained less than 15% of the neurofilament protein triplet, our spinal-cord preparation is an improvement on the usual neurofilament preparation. This simple and rapid method gave large amounts of 10-nm filaments (100 mg per 100 g of spinal cord) characterized by the absence of membranous material, a low content of tubulin and the 50 000-mol.wt.-protein component, and a high content of neurofilament peptides. Thus, the presence of tubulin in 10-nm filament preparations seems to be related to the contaminant membranous material and not to be linked to the interaction in vitro of tubulin or microtubules with neurofilaments, as has been suggested previously. more...

One of the polypeptides (H1) of the rabbit nervous system occurs in an altered form (H2) in some rabbits. The electrophoretic mobility of H2 on sodium dodecyl sulfate-polyacrylamide gels is about 6% greater than that of H1, suggesting that the two polypeptides differ in molecular weight by about 10,000. The alteration is genetically determined since (i) rabbit phenotypes corresponding to all possible genotypes (H1H1, H1H2, H2H2) were present in an outbred population of rabbits, (ii) the frequency of the phenotype corresponding to the heterozygous genotype (H1H2) was smaller in partially inbred rabbit populations than in outbred populations, and (iii) all of the individuals examined from two partially inbred strains (WH/J and X/J) were of the phenotype that would be expected if they were homozygous (H2H2) for the rare allele of the gene. Preliminary evidence indicates that this polymorphic polypeptide is most abundant in the white matter of the nervous systems of several mammalian species, and is distributed with buffers of low ionic strength and centrifuged at 100,000 Xg. more...

To study the distribution of glycine immunoreactive neurons in the spinal cord and brain, antisera were raised against glycine conjugated to protein carriers. High-titer rabbit glycine antiserum was purified by affinity chromatography. Testing against other amino acids and peptides with immuno dot blots and ELISA assays showed little apparent cross-reaction with glutamate, aspartate, glutamine, taurine, and 17 other amino acids and related compounds. Similarly, the antiserum showed little apparent recognition of glycine when glycine was incorporated into peptides. A slight cross-reactivity with GABA, beta-alanine, and cysteine was found. Immunocytochemical labeling of tissue sections could be blocked with glycine conjugated to a heterologous carrier protein but not by other amino acids conjugated to that protein. Immunocytochemistry at the light microscope level with immunofluorescence and silver-intensified colloidal gold revealed a wide distribution of glycine-like immunoreactivity throughout all laminae of the rat spinal cord and in all segments studied from the cervical, thoracic, lumbar, and sacral cord. Immunoreactive boutons were found terminating on both cell bodies and on dendrites. Ultrastructural analysis with postembedding colloidal gold immunocytochemistry demonstrated large numbers of immunoreactive boutons making symmetrical type synapses with neuronal perikarya, including motor neurons, and with proximal and distal dendrites. Presynaptic glycine immunoreactive boutons were found in both ventral and dorsal horn. Immunoreactivity was concentrated over regions rich in vesicles, and over mitochondria in immunoreactive boutons, but not over mitochondria in postsynaptic dendrites. Glycine-immunoreactive perikarya were identified both in the dorsal horn and in the ventral horn. Myelinated and unmyelinated glycine-immunoreactive axons were noted both in the gray and white matter of the cord. The density of immunoreactive axons varied in the white matter, with the greatest number of immunoreactive axons found in the white matter adjacent to the gray matter in lateral and ventral white. Significantly fewer immunoreactive axons were found in the white matter of the dorsal columns. Myelin sheaths around axons were unlabeled. The distribution of glycine-immunoreactive boutons correlated well with the distribution of glycine receptor immunoreactivity on postsynaptic elements of the spinal cord, tested with different monoclonal antisera against strychnine-purified glycine receptor. Glycine receptor immunoreactivity was found throughout the gray matter of both rat and primate.(ABSTRACT TRUNCATED AT 400 WORDS) more...

In the present study we have employed immunoperoxidase techniques to investigate the distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the spinal cord and sensory ganglia of the cat. The spinal distribution of VIP-containing neuronal processes was also compared with that of substance P (SP), somatostatin (SOM), and cholecystokinin-8 (CCK) at lumbar, sacral, and coccygeal levels. At sacral levels, VIP was found to be contained in small and medium-sized primary sensory neurons and in dorsal rootlets. Deafferentation, by either ganglionectomy or dorsal rhizotomy, resulted in a nearly complete loss of VIP immunoreactivity in the spinal cord. The spinal distribution of VIP fibers and terminals was most dense and extensive in sacral segments. Forming a thin shell around the dorsal horn, collaterals, apparently originating from Lissauer's tract, projected either medially or laterally through lamina I. Laterally, many VIP axons terminated in lateral laminae V to VII. Others projected further through the neck of the dorsal horn to medial lamina V and the gray matter near the central canal. Medially, VIP axons descended through lamina I to expand into terminal fields in the posterior commissure and medial lamina V. At the ultrastructural level, VIP-like immunoreactivity was found in dense core vesicles within axonal enlargements containing both large dense core and smaller clear round vesicles. Synaptic connections were infrequently observed but, when encountered, were of the simple axodendritic type. The spinal distribution of VIP-containing fibers was remarkably similar to that reported for pelvic nerve visceral afferents, both in termination patterns within the spinal gray matter and in localization to the sacral cord. The density of SP-, SOM-, and CCK-containing fibers and terminals was constant at all levels examined (L4 to Co4). In marked contrast, the distribution of VIP fibers, much like that of pelvic nerve afferents, was mostly confined to sacral segments. Thus, although SP, SOM, and CCK may be contained within a population of sacral visceral afferents, they must be common to afferent systems in other segments as well. VIP, however, appears to be preferentially contained within pelvic visceral afferent fibers confined mostly to sacral segments. more...

The protein C anticoagulant system is mediated by thrombin and is highly accelerated by thrombomodulin. We studied the distribution of thrombomodulin antigen (TM Ag) in the rabbit using an affinity-purified antibody raised in a goat against rabbit thrombomodulin. The preservation of TM Ag was highly dependent on immediate fixation of the surface on which it is located. TM Ag was found on the endothelium of the entire vasculature, whereas it was absent from all connective tissue, smooth and striated muscle, secretory epithelia, cartilage, bone, neural tissue, and all parenchyma examined. A new finding was the presence of TM Ag on nonvascular surfaces of body cavities (the mesothelia of pleura, pericardium, and peritoneum, the synovial membrane, and the arachnoid enveloping the central nervous system). By use of a functional assay, TM activity was recovered in buffered saline/detergent solution which was either injected into the intraperitoneal cavity of rabbits in vivo or incubated with the surface of the arachnoid in vitro. These findings extend the importance of anticoagulant mechanisms to the systems of slowly circulating fluids, in which they might be required for maintenance of the flow, and to mesothelial cavities, in which they could be necessary for preventing adherence between the surfaces, in conditions associated with pathological exudation. more...

The existence of bombesin/gastrin-releasing peptide-like immunoreactivity (BN-GRP-LI) in rat sensory ganglia and spinal cord was confirmed using immunocytochemistry, gel filtration chromatography, and high performance liquid chromatography combined with radioimmunoassay. Immunohistochemical studies showed that in the spinal sensory ganglia of the rat about 5% of the neurons exhibited BN-GRP-LI, whereas about 20% of the neurons exhibited substance P-like immunoreactivity (SP-LI). The two immunoreactivities were found in different cells, but both were located in small ganglion cells. In the posterior horn of the spinal cord, BN-GRP-LI and SP-LI were located in the superficial layers, and this distribution was different from that of Met5-enkephalin-like immunoreactivity. The results are in agreement with the concept that there is a primary sensory pathway from the sensory ganglia to the spinal cord which contains BN-GRP-LI and that these neurons are separate from those containing substance P. In extracts prepared from spinal ganglia, two molecular weight forms of BN-GRP-LI were found using gel filtration chromatography. The high molecular weight form coeluted with porcine GRP and the low molecular weight form was smaller than bombesin. The low molecular weight BN-GRP-LI extracted from spinal cord was more hydrophilic than bombesin or ranatensin. more...

Norepinephrine, dopamine, and 5-hydroxytryptamine concentrations were determined in the central nervous systems of genetically epilepsy-prone rats (GEPR) and in control rats. Norepinephrine concentrations were abnormal in all major areas of the central nervous system of the GEPR, with decrements existing in the telencephalon, hypothalamus-thalamus, midbrain, pons-medulla and spinal cord. An increment in the concentration of this neurotransmitter existed in the cerebellum. Dopamine concentrations were normal in all areas of the GEPR brain. Abnormalities in 5-hydroxytryptamine concentrations were also present in the GEPR. They were exclusively decrements and occurred in the telencephalon, hypothalamus-thalamus, midbrain, and pons medulla. Concentrations of this neurotransmitter were normal in the cerebellum and spinal cord. Coupled with our earlier pharmacologic data, these observations support our concept that noradrenergic and/or 5-hydroxytryptaminergic decrements are etiologically important in seizure susceptibility in the GEPR. The lack of abnormalities in brain dopamine concentrations strengthens our hypothesis that dopaminergic transmission does not regulate seizure susceptibility in this model. more...

Gangliosides were isolated from whole spinal cords and cord myelin of human, cat, and rabbit by a revised methodology. The method included the sequential application of DEAE-Sephadex column chromatography, base treatment, Sephadex G-50 column chromatography, and finally Iatrobeads column chromatography. The human whole spinal cord was found to contain about one-tenth of the ganglioside concentration as in cerebral gray matter and about one-third of that in cerebral white matter. Low levels of gangliosides were also found in cat and rabbit whole cords. Only N-acetyl neuraminic acid could be detected in the ganglioside fractions of all three species. The whole cords also possessed unique ganglioside patterns when compared with the patterns of cerebral tissues. The most prominent and consistent features were the reduced concentration of Gd1a and increased amounts of Gm3 and Gd3. Human, but not cat and rabbit, spinal cord also contained Gm4 as one of the major gangliosides. Myelin prepared from the spinal cords of all three species also contained gangliosides. The amounts were only about half of those in the respective cerebral white matter myelin. The cord myelin ganglioside pattern was generally similar to the cerebral white matter myelin within the same species. Gm1 was the most abundant ganglioside in the cord myelin. Gm4 was found to be highly enriched only in myelin prepared from human sources. more...

Complete serial sectioning of the medulla oblongata in monkey, cat, guinea pig, and japanese dancing mouse and incubation for somatostatin-immunoreaction was carried out. Numerous regions of the medulla oblongata such as the nucleus reticularis gigantocellularis, nucleus cuneatus et gracillis, nucleus raphe magnus, nucleus tractus solitarius, nucleus vestibularis, and parts of the oliva contain dense networks of somatostatin-immunoreactive nerve fibers. Cell bodies were seen in the nucleus reticularis medullae oblongatae. In the spinal cord the sections from each segment were analyzed, showing the highest concentrations of somatostatinergic fibers in the substantia gelantinosa of the columna dorsalis. Cell bodies were seen in the zona intermedia centralis, especially in the upper cervical segments. Many positive fibers were also seen in the entire zona intermedia and the columna ventralis. Especially prominent was the immunoreactivity in the zona intermediolateralis of the thoracic segments and the columna ventralis of the lower lumbar and sacral segments. more...

A protein of molecular size 180 kilodaltons is associated with 10-nanometer filaments in neurons and is immunologically distinct from smaller putative neurofilament subunits and from 10-nanometer filament proteins in nonneuronal cells, such as myotubes and fibroblasts. Neurons do not contain vimentin, the major filament protein in many other cells, including the nonneuronal cells in cultures of neural tissue. more...

This study surveyed the coexistence of serotonin-like immunoreactivity (5-HT-IR) with substance P-like immunoreactivity (SP-IR) in fibers and terminals within various portions of the spinal cord of the rat. A previously characterized technique of immunofluorescent double labeling was used to stain 5-HT-IR red and SP-IR green, and a search was then made for single fibers that fluoresced both colors. These were found to be most common in the ventral horn, wherein 99% of 5-HT-IR fibers were also immunoreactive for SP. Coexistence of these substances was very rare in the superficial dorsal horn: fewer than 3% of 5-HT-IR fibers were immunoreactive for SP. In the region surrounding the central canal and in the intermediolateral cell column (IML), over half of all 5-HT-IR fibers and terminals were doubly labeled. In the white matter, doubly labeled fibers were most common in the ventral funiculus and somewhat less common in the lateral funiculus. They were rare in the dorsal columns. It is concluded that the coexistence of 5-HT and SP in nerve fibers and terminals is associated with somatic and sympathetic autonomic motoneurons. The role of the coexistence of 5-HT and SP in somatosensation, including pain, is unclear. more...

A new panel of greater than 300 monoclonal antibodies (mAbs) was prepared to the high, middle, and low Mr rat neurofilament (NF) subunits (NF-H, NF-M and NF-L, respectively). NF proteins were purified both from native, i.e., phosphorylated rat NFs and from enzymatically dephosphorylated rat NFs. The resulting mAbs were used to biochemically and immunochemically distinguish and characterize distinct and differentially phosphorylated isoforms of NF subunits. By immunoblot, all mAbs specific for NF-L and some mAbs specific for NF-M detected their specific NF subunit regardless of whether or not the NFs had been treated with alkaline phosphatase, and such antibodies were termed "phosphate-independent" or P[ind] mAbs. The other mAbs were specific for NF-M, NF-H, or for both NF-M and NF-H, and they recognized epitopes in the COOH termini of these subunits. Significantly, the latter mAbs could discriminate different isoforms of NF-M and NF-H, depending on the phosphorylation state of each variant. Such mAbs were assigned to one of 4 distinct categories on the basis of their performance in immunoblots of progressively dephosphorylated rat NF samples and by immunohistochemistry of various adult rat nervous tissues: (1) P[-] mAbs preferentially stained neuronal perikarya and dendrites, and they recognized only extensively dephosphorylated (and nonphosphorylated) NF-H; (2) P[+] mAbs stained axons more strongly than perikarya, and primarily blotted phosphorylated, but not nonphosphorylated, forms of NF-H and NF-M; (3) P[++] mAbs stained axons almost to the exclusion of perikarya, and in blots recognized only the extensively phosphorylated forms of NF-H and NF-M (i.e., subunits subjected to limited enzymatic dephosphorylation); (4) P[ ] mAbs also predominantly stained axons, but the briefest alkaline phosphatase treatment abolished the NF-M and NF-H immunobands produced by these mAbs. Two-dimensional gel analysis and immunoblotting of total proteins from adult rat dorsal root ganglion verified mAb specificity in situ, and showed that differentially phosphorylated isoforms of NF-M and NF-H occur in vivo. This provided additional evidence that mAbs can detect all 4 phosphorylation-dependent endogenous isoelectric variants of NF-H and NF-M.(ABSTRACT TRUNCATED AT 400 WORDS) more...

Antisera raised to scrapie-associated fibril (SAF) proteins were used to detect scrapie-specific polypeptides in three different non-purified brain preparations: a synaptosomal-mitochondrial fraction, 20% brain homogenate and 20% brain homogenate extracted with Sarkosyl. The concentration of SAF proteins in the preparations was greater than the quantity of SAF as detected by negative stain electron microscopy. This suggests that not all of the protein exists in the form of SAF. An immunologically reactive 33K to 35K protein was detected in both normal and scrapie brain preparations. This protein was susceptible to complete proteinase K (PK) digestion in normal brain preparations and it is suggested that scrapie infection is responsible for post-translational modifications which confer PK resistance in scrapie preparations. These modifications may also play a role in the antigenic differences seen in a variety of scrapie agents. SAF-specific proteins were also detected in the spinal cords and spleens from scrapie-affected animals. Detergent extraction of material followed by PK treatment and Western blot analysis is a highly specific and sensitive method for the detection of SAF proteins. This procedure could be applied to human neurological diseases of unknown aetiology. more...

GABA- and glycine-containing neuronal elements were visualized in the rat spinal cord by immunohistochemistry employing antisera raised against conjugated GABA and glycine. GABA- and glycine-like immunoreactivities were localized in axon terminals and neuronal perikarya. GABA- and glycine-like immunoreactive terminals were in synaptic contact with perikarya and dendrites of most spinal neurons. Single spinal motoneurons appeared to be in contact with GABA- or glycine-like immunoreactive terminals. These findings suggest that these neurons are regulated postsynaptically by both GABA and glycine. more...

Vesicle-associated membrane protein (VAMP) 1 is a 120-amino acid protein which co-purifies with cholinergic synaptic vesicles from the marine ray Torpedo californica. We used the Torpedo gene to isolate two independent classes of VAMP cDNA clones from rat brain. Nucleotide sequence analysis of the cDNAs predicts proteins which are 84 and 75% homologous to Torpedo VAMP-1. The amino-terminal 24-28 amino acid residues which comprise the proline-rich head are only about 50% homologous between the different VAMPs, yet the proline-rich character is maintained. The 69 amino acids which comprise the hydrophilic core are highly homologous to Torpedo VAMP-1, with only 2 amino acid substitutions in rat VAMP-1 and 6 in rat VAMP-2. The carboxyl-terminal 23 amino acids of all of the VAMP proteins maintain the hydrophobic character necessary to serve as a membrane anchor. Both VAMP transcripts are expressed differentially in the rat central nervous system. Whereas VAMP-2 is more highly expressed in the whole brain, VAMP-1 is expressed at a higher level in the spinal cord. more...

We studied the distribution of sugar residues in the oligosaccharide chains of complex carbohydrates in tissue sections of rat spinal cord, brainstem, and sensory ganglia using twelve lectin-horseradish peroxidase conjugates. Glycoconjugates containing terminal galactose residues were localized apparently in the Golgi apparatus in a population of predominantly small B-type neurons in spinal and trigeminal ganglia. Large A-type neurons rarely showed reactivity with galactose-binding lectins. A cells stained for glycoconjugates with N-glycosidically linked oligosaccharides and glycogen. The central and peripheral processes of the small neurons, mostly unmyelinated C fibers in sensory roots and spinal nerves, contained an abundance of glycoconjugates with terminal alpha-galactose residues. The central projections and terminals of small to medium-sized primary sensory neurons in the spinal and trigeminal ganglia were visualized in Lissauer's tract and the substantia gelatinosa in the spinal cord, and in the spinal trigeminal tract and the nucleus trigeminus in the lower medulla with lectins specific for terminal alpha-galactose residues. In addition, fibers of the solitary system and the area postrema were reactive with these lectins. The peripheral and central nervous system elements with affinity for galactopyranosyl-specific lectins correspond in distribution with neuroanatomical regions thought to be involved in the transmission and relay of somatic and visceral afferent inputs such as pain and temperature. Such specific localization of a glycosubstance to a distinct subpopulation of neurons and their peripheral and central processes suggests that the particular glycoconjugate may be of physiological significance. more...

Antisera generated to substance P-Gly (SP-G) and substance P-Gly-Lys (SP-G-K), the likely unamidated COOH-terminally extended forms of substance P, were used to quantify and localize substance P precursor forms in hamster brain stem and spinal cord. The precursor determinant SP-G-K was liberated from larger heterogeneous forms by mild trypsinization of tissue extracts and was converted into the second precursor determinant, SP-G, by subsequent treatment with carboxypeptidase B. The basal levels of SP-G-K in brain stem and spinal cord were approximately equal to 0.5 pg/mg of tissue and rose 43- to 64-fold after trypsinization. Basal levels of SP-G were comparable to those of SP-G-K and rose 10- to 29-fold after combined enzyme treatments. Immunohistochemical labeling of axons and somata with anti-SP-G-K increased dramatically after trypsinization. This labeling was eliminated by preadsorption with authentic SP-G-K but not substance P or SP-G. Gel-permeation chromatography revealed SP-G-K-like immunoreactivity in fractions corresponding to considerably higher molecular weight than mature substance P. Collectively, these results support the hypothesis that substance P is synthesized from larger precursors and demonstrate that extended precursor forms are normally present in the axons and somata of neural systems that synthesize substance P. more...

In the nervous tissue of miniature pig, type Göttingen, three fucosyl-containing gangliosides are major gangliosides. All three were found in dorsal-root ganglia and spinal cord, but only two of them in the forebrain. The concentrations of these gangliosides were increased in chloroquine intoxication. Their chemical structures were determined by component analysis, partial acid and enzymatic degradation and assay of the partially methylated sugars after hydrolysis, reduction and acetylation. Mass spectrometric analysis of the intact gangliosides as permethylated, permethylated-reduced and permethylated-reduced-silylated derivatives confirmed the following structures of the three gangliosides. (See Formula in text). more...

The interaction between noradrenergic and serotonergic mechanisms on the central regulation of blood pressure in the rat was studied, using central experimental hypertension produced by chemical lesions of the locus coeruleus (LC) with 6-hydroxydopamine (6-OHDA). The hypertension (LC-hypertension) was blocked by pretreatment with 6-OHDA intraventricularlly administered and also with desipramine. An increase of serotonin (5-HT) turnover in the cortex and especially in the spinal cord was revealed in the hypertension. Intraperitoneally administered para-chlorophenylalanine elevated blood pressure and reduced 5-HT and 5-hydroxyindole acetic acid content in the cortex and especially in the spinal cord, indicating a decrease 5-HT turnover. Intraventricularlly administered 5, 6-dihydroxytryptamine (5, 6-DHT) resulted in only bradycardia but not an elevation in blood pressure and did not block the development of LC hypertension, and lowered 5-HT content in the cortex and especially in the spinal cord. Intraspinally administrated 5, 6-DHT evoked mild but significant elevation in blood pressure but not in heart rate, and lowered 5-HT content only in the spinal cord. Furthermore, the disappearance of the LC was histologically observed in the rats with LC hypertension. The results of these experiments demonstrate that (1) LC hypertension is probably due to a specific lesion of the LC with 6-OHDA, (2) LC hypertension is accompanied by an increase of 5-HT turnover in the cortex and especially in the spinal cord, which may participate in a depressor function in LC hypertension, (3) ablation of bulbospinal serotonergic neurons produce mild elevation in blood pressure. (4) the balance between the activity of central noradrenerfic and serotonergic neurons may play a role in maintaining normotension, and their unbalance may induce hypertension. more...

Substance P- and cholecystokinin-like immunoreactivities have been shown to coexist in some, but not all, dorsal root ganglion cell bodies of the rat. Quantitative immunofluorescence techniques were used in the present study to describe densities of substance P- and cholecystokinin-like immunoreactive varicosities in several spinal cord nuclei. By combining simultaneous immunofluorescent techniques on one tissue section with computerized image processing, coexistence of substance P- and cholecystokinin-like immunoreactivity in varicosities was also quantified. By comparing spinal cord regions between normal and unilateral dorsal rhizotomy affected animals, densities of substance P- and cholecystokinin-like immunoreactive varicosities associated with primary afferent neurons were established. To determine the densities of immunoreactive varicosities that were related to unmyelinated primary afferent fibers, data were compared between normal animals and those treated neonatally with capsaicin. Four major observations were made: (1) Substance P- and cholecystokinin-like immunoreactivity coexist in populations of varicosities in sensory and autonomic regions of rat spinal segment L6. (2) Within the superficial laminae of the dorsal horn, varicosities containing both substance P- and cholecystokinin-like immunoreactivity and cholecystokinin-like immunoreactivity alone are of primary afferent neuron origin, but those containing only substance P-like immunoreactivity are most likely of spinal or descending neuronal origin. (3) Capsaicin-insensitive cholecystokinin-like immunoreactive varicosities were present predominantly in lamina I. These data suggest some cholecystokinin-like immunoreactive varicosities are associated with myelinated primary afferent neurons. (4) Primary afferent fibers containing substance P- and cholecystokinin-like immunoreactivity project to intermediate gray regions of the rat spinal cord. A large proportion of these fibers are capsaicin sensitive, suggesting that they are unmyelinated. more...

In this paper we describe a 66-kD protein that co-purifies with intermediate filaments from rat optic nerve and spinal cord but can be separated further by ion-exchange chromatography. This protein is distinct from the 68-kD neurofilament subunit protein as judged by isoelectric focusing, immunoblotting, peptide mapping, and tests of polymerization competence. This protein is avidly recognized by the monoclonal anti-intermediate filament antigen antibody, previously demonstrated to recognize a common antigenic determinant in all five known classes of intermediate filaments. Also, when isolated this protein binds to various intermediate filament subunit proteins, which suggests an in vivo interaction with the intermediate filament cytoskeleton, and it appears to be axonally transported in the rat optic nerve. Because of this ability to bind to intermediate filaments in situ and in vitro we have named this protein alpha-internexin. A possible functional role for the protein in organizing filament assembly and distribution is discussed. more...

Affinity-purified goat anti-rabbit immunoglobulin G (GAR) was conjugated with (3H)-propionyl succinimidate and used to localize substance P (SP), enkephalin (ENK), and serotonin immunoreactive sites in the spinal dorsal horn and medulla of the rat and cat. Autoradiographic localization was demonstrated on paraffin, frozen, Vibratome, and 2 micron plastic sections. The latter were obtained from radiolabeled Vibratome sections that were embedded in epoxy resin. The distribution of SP, ENK, and serotonin demonstrated by radioimmunocytochemistry was comparable to that observed on semiadjacent sections using peroxidase-antiperoxidase (PAP) immunocytochemistry. The autoradiograms, however, were generated using primary antibody concentrations up to five times more dilute than concentrations used for the PAP procedure. Indirect radioimmunocytochemistry using a (3H) anti-immunoglobulin G second antibody can be used to localize a variety of monoclonal and polyclonal antisera. It is quantifiable at the light microscopic level and can be potentially used with peroxidase histochemistry to double label immunoreactive structures at the ultrastructural level. more...

As part of a series of studies in which we are attempting to determine the roles of the lateral spinal (LSn) and lateral cervical (LCn) nuclei in somatic sensation, we have examined the fibers and terminals within these nuclei in the rat using the indirect immunofluorescence technique. Eleven antisera were used. Within the LSn, antisera against dynorphin 1-8 (DYN), substance P (SP), and Met-enkephalin (ENK) produced labeling of a large number of processes in all segmental levels examined. Processes labeled with these antisera frequently apposed the cell bodies and dendrites of LSn neurons. Antisera against somatostatin (SOM) and FMRF-NH2 (FMRF) labeled smaller numbers of processes within the LSn. Few, if any, processes in the LSn were labeled using antisera against serotonin, cholecystokinin octapeptide, oxytocin, neurotensin, corticotrophin-releasing factor, and vasoactive intestinal polypeptide. In contrast to the LSn, the LCn contained virtually no labeled processes irrespective of the antiserum employed. An area was found adjacent to the LCn in the medial portion of the dorsal lateral funiculus (DLf) of C2 that resembled the LSn in several of its anatomical characteristics: like the LSn, the medial portion of the C2 DLf contained small multipolar neurons; it was similar to the LSn in its medial-lateral extent; and following staining with each antiserum, the LSn and the medial DLf of C2 contained a similar number of labeled processes. The peptide-containing area in the medial DLf of C2 was found to be continuous with the LSn. We therefore propose that this region is a rostral extension of the LSn.(ABSTRACT TRUNCATED AT 250 WORDS) more...

An antiserum was generated against a synthetic peptide corresponding to amino acids 95-117 of bovine proenkephalin, and a sensitive radioimmunoassay was developed. Comparison of the reactivities of the synthetic peptide, its specific cleavage products, and other synthetic peptides showed that the important immunological determinant was contained within residues 101-109 of bovine proenkephalin (-Gly-Gly-Glu-Val-Leu-Gly-Lys-Arg-Tyr-). Radioimmunoassay of fractions after gel filtration of bovine adrenal medullary chromaffin granule lysate showed three pools of immunoreactivity: pool 1 (Mr 20,000-30,000), pool 2 (Mr 10,000-20,000), and pool 3 (Mr approximately 5,000). Further characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that the antiserum recognized 27-, 20.5-, 16.5-, and 5.6-kilodalton enkephalin-containing proteins. The radioimmunoassay was also used to detect proenkephalin-like material in extracts of rat adrenal and regions of rat brain and spinal cord following gel filtration. Immunoreactivity from the rat adrenal chromatographed predominantly as high molecular weight material (Mr 31,500-43,500), whereas material in regions of rat brain showed a broader molecular weight distribution (Mr 4,000-43,500). This indicated differences in the processing of proenkephalin between rat adrenal and brain tissue. Differences were also seen in the molecular weight profile of immunoreactivity in different brain regions, most noticeable in the case of striatum and hypothalamus, suggesting regional differences in processing. Based on quantitation of higher molecular weight immunoreactive proenkephalin-like material and free Met-enkephalin immunoreactivity in different brain regions, it was apparent that extensive processing of proenkephalin occurs in brain. We concluded that antisera against proenkephalin-(95-117) recognize a wide range of intermediates in the processing of proenkephalin in both bovine adrenal medulla and rat adrenal, brain, and spinal cord, making it a useful tool for further studies concerned with the expression and post-translational processing of proenkephalin. more...