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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.

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.

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.

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.

The peroxidase-anti-peroxidase (PAP) method was used to determine the topography and cellular localization of glial fibrillary acidic protein (GFAP), myelin basic protein (MBP) and carbonic anhydrase II (CAII) in the central nervous system (CNS), dorsal root ganglia and dorsal and ventral spinal nerve roots of the sheep. Parallel studies of mouse brain provided comparative data. Several fixatives were compared for their relative merits in preserving marker protein expression: GFAP was well preserved irrespective of the fixative employed; MBP was best preserved in formal sublimate and CAII was best preserved in Carnoy's fluid. In sheep, GFAP expression was seen in protoplasmic and fibrous astrocytes, Bergmann glial cells, a proportion of ependymal cells, amphicytes of spinal ganglia and in a proportion of presumed Schwann cells of dorsal and ventral spinal nerve roots. MBP expression was seen in mature and developing myelin sheaths of the central nervous system and in the cytoplasm of sparse myelinating oligodendroglia of the sub-cortical white matter of the cerebrum. CAII expression was seen in choroid plexus epithelium in all ages of sheep studied and, in a young lamb and an adult sheep, in glia and neuropil of ventral horn grey matter of the spinal cord and in the cytoplasm of white matter glia, presumed fibrous astrocytes, throughout the CNS. Compared with sheep brain, mouse brain showed the following differences in marker protein localization. GFAP was weakly expressed by protoplasmic astrocytes and not expressed in ependyma, oligodendroglia expressing intracytoplasmic MBP were frequent and widespread in neonatal mouse brain, CAII was expressed in myelin and oligodendroglia.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), free glutamate (Glu) and aspartate (Asp) was studied in the neuraxis of rats 48 h after complete transection of brain stem at the intercollicular level. NAA remained unchanged above the transection level but decreased in the neur-axis segments caudal to the lesion. Glu was strongly depressed both in the segments rostral to the transection and in the ones caudal to it. No significant change was found for NAAG and Asp. These findings are consistent with the hypothesis that NAA may be involved in neurotransmission processes and provide further evidence for Glu as transmitter of descending and ascending pathways. In addition, the data obtained do not suggest a direct metabolic or functional relationship between NAA and NAAG nor between these N-acetylderivatives and free Glu and Asp.

The Wobbler mouse (wr) exhibits the loss of motoneurons especially in the cervical spinal cord, and thus has been studied as a model for human motoneuron diseases. Wobbler mice selected at various ages and stages during the disease process show increased levels of thyrotropin releasing hormone and substance P in spinal cord and brainstem (medulla). Enkephalins (methionine and leucine) also increase in the spinal cord and brainstem. Somatostatin increases in hypothalamus, perhaps accounting partly for the small size of this mutant mouse via its effect on growth hormone.

The object of the present study was to examine the effects of temperature, oxidation, and pH on in vitro relative recovery of catecholamine and indoleamine neurotransmitters and their metabolites using microdialysis probes. Relative recovery of norepinephrine (NE), dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA), homovanillic acid (HVA), and 5-hydroxytryptamine (5HT) increased with temperature from 0 to 46 degrees C. For each compound, the increase in the amount recovered with increasing temperature was different. The stability of norepinephrine and dopamine was not affected at any temperature using deoxygenated calibration standard solutions containing ascorbic acid but was greatly reduced when exposed to ambient air without antioxidant treatment; catecholamine metabolites and the indole compounds were less affected. No change for in vitro relative recovery was observed by varying the pH of the perfusing solution from 6 to 8. Thus, temperature control in probe calibration as well as analyte stability using antioxidant treatment are important in reducing the error when estimating extracellular concentrations of neurotransmitter and metabolites.

The purpose of this paper is to illustrate the advantages of immunocytochemical studies of spinal neuropeptides in human pathology and in animal experiments. The distribution of neuropeptides in the gray matter of the human spinal cord is summarized. Data obtained on selected pathological cases are able to determine the origin of certain peptidergic afferents to the spinal cord and suggest that the early disappearance of substance P-positive fibers in the motoneuronal columns plays a role in the pathogenesis of amyotrophic lateral sclerosis. In rats, peptide immunocytochemistry is useful for assessing the plasticity of spinal neurons in a model of chronic pain, in chemically induced degeneration of the gray matter and in a model of spinal cord trauma. Finally, a newly developed culture system of adult rat and human dorsal root ganglia demonstrates that the phenotypic expression of neuropeptides can be modulated experimentally, which offers new perspectives for studying the neurobiological role of these mediators and for the tentative repair of spinal lesions by autografts.

Thin layer chromatography of lysosulfatide showed anomalous Rf-values in contrast with such lysosphingolipids as glucopsychosine and galactopsychosine with neutral, acidic, and alkaline developing solvents. This was thought to be due to the presence of oppositely charged sulfate and amino groups in the lysosulfatide. In the negative mode of fast atom bombardment mass spectrometry, the lysosulfatide showed the pseudo molecular ion (M-H)- peak at m/z 540 and sulfate ion peak at m/z 97, whereas in the positive mode, it showed not only the pseudo molecular ion (M+H)+ peak at m/z 542, but also the major peaks of protonated psychosine at m/z 462 and fragment ions of dehydrated sphingosine at m/z 282 and 264, 13C-NMR signals of all carbons of lysosulfatide were determined by using distortionless enhancement by polarization transfer. The difference in chemical shifts of ring carbons of galactose residue between lysosulfatide and galactopsychosine was largest at C-3 (downfield shift), thereby indicating the location of the sulfate group to be at C-3 of galactose. This conclusion is supported by the 1H-NMR spectra of the lysosulfatide and galactopsychosine. Thus, the chemical structure of lysosulfatide was confirmed by fast atom bombardment mass spectrometry and 13C- and 1H-NMR spectroscopy. Furthermore, 13C-NMR signals of C-1 to C-5 of the sphingosine moiety showed significantly different chemical shifts between the lysosulfatide and galactopsychosine. These differences suggested that C-1 to C-5 of sphingosine might be influenced by intramolecular or intermolecular interaction between the sulfate group of the galactose residue and the amino group of sphingosine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A method is presented for the separation and detection of the myelin marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase on isoelectric focusing gels and by immunoblotting. The gel staining procedure is a modification of a method used to demonstrate enzyme activity on blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis. The results show that immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase can be separated under equilibrium conditions on isoelectric focusing gels with an expanded alkaline pH range after solubilization in a mixture of nonionic/zwitterionic detergents and urea. Enzymatically active 2',3'-cyclic nucleotide 3'-phosphodiesterase focused as two closely spaced bands at pIapp 8.1 and 8.8, respectively, while 2',3'-cyclic nucleotide 3'-phosphodiesterase immunoreactivity was detected as four distinct bands at pIapp 4.2, 7.4, 8.8, and 9.3 and a diffuse band at pIapp 7.9-8.2. By two-dimensional separation these five bands showed molecular weights of about 43-47 kDa, i.e., corresponding to reported values for immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase. Since enzyme activity is associated with only two of the bands, nonspecific and artifactual banding due to, e.g., detergent micelle formation, is unlikely.

The regional brain and spinal cord concentrations of calcitonin gene-related peptide (CGRP) were measured in age-matched (22-23-week-old) spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. The highest concentration of CGRP in the WKY rats was in the spinal cord (172 +/- 9 pmol/g), followed by the medulla oblongata/pons (88 +/- 5 pmol/g). The relative order of distribution in the remaining regions was: hypothalamus (12.6 +/- 0.8 pmol/g) = striatum greater than thalamus greater than midbrain = hippocampus greater than cortex (2.1 +/- 0.3 pmol/g). The concentration of CGRP in the cerebellum was at the level of the assay's sensitivity (0.5 pmol/g). The relative order of distribution in the SHR strain was essentially the same. However, in comparison with the WKY rats, the SHR had significantly lower levels of CGRP in the hippocampus (-47%), striatum (-49%) and medulla oblongata/pons (-24%), and in the spinal cord (-24%). In younger age-matched (16-17-week-old) rats, the spinal cord and medulla oblongata/pons concentrations of CGRP were also lower in SHR than in WKY rats. CGRP is a putative neurotransmitter which, when administered centrally or peripherally, has potent cardiovascular effects. The reduced levels of this peptide may be an important factor in the cardiovascular and/or behavioural abnormalities of the SHR strain.

Cross-reaction of a rat monoclonal antibody (BTP-1) against seventeen substance P analogues was studied. The antibody was of IgG type and related to the carboxyl terminal of substance P, especially methionyl in the terminal, but did not depend on the strength of antagonistic effects of these analogues. It did not show cross-reaction with the following nine peptides: glucagon, endorphin, angiotensin I, II, leucine-enkephalin, methionine-enkephalin, bradykinin, oxytocin and dernorthin, indicating its high specificity to substance P. By means of immuno-enzyme histochemical method, it was shown that stained nerve fibers were located in the gelaliternous substance of Rolando, interpeduncular nucleus, substantia nigra and nerve cell bodies in the vestibular nucleus, lateral tegmental nucleus of mesencephalon and ventral region of third ventricle.

Human beta-calcitonin gene-related peptide (beta-hCGRP) was isolated and purified from spinal cord. The complete characterization of this material was based on definitive mass analysis by fast atom bombardment mass spectrometry together with gas phase sequencing. Combining these data we have characterized the structure of beta-hCGRP including the C-terminal sequence, the presence of the S-S bridge and of phenylalanineamide as the C-terminal amino acid, and fully confirmed the amino acid sequence predicted from the nucleotide analysis.

The lipid specificities of two related integral membrane proteins of central nervous system myelin, the proteolipid (PLP) and DM-20 proteins, which differ only by the deletion of a polar stretch of 35 contiguous amino acid residues, were studied with spin-labeled lipids after reconstitution into dimyristoyl-phosphatidylcholine. The selectivity in populating lipid association sites at the protein interface and in modulating the lipid exchange between protein and bulk lipid sites was quantitated by the relative association constants and the off-rate constants for exchange, respectively, for both proteins. The sequence deleted in DM-20 (residues 116-150 of PLP) is found to play a major role in determining the lipid selectivity for the parent PLP protein.

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.

We analyzed the effects of myelin basic protein (MBP)-specific encephalitogenic T line cells in neonatal syngeneic recipients before and after formation of central nervous system myelin. Lewis rat pups (postnatal days 0, 1, 2, 4, 8) were injected intraperitoneally with MBP-specific T cell line in doses which cause clinical and histologic changes of experimental autoimmune encephalomyelitis (EAE) in adult rats. We correlated the susceptibility to transferred EAE with the developmental appearance of MBP as demonstrated by immunohistochemical staining. MBP was barely demonstrable until postnatal day 2 but became definitely apparent on postnatal day 4. All newborn rats that were injected on postnatal day 0, 1, 2 failed to develop any apparent clinical signs, but histologically some recipients displayed slight inflammatory cell infiltration of the meninges and subpial lesions limited to the lower spinal cord. In striking contrast, rats that were injected on postnatal day 4 or 8, developed clinical signs and, in particular, many of the older recipients became moribund. Histologically, these animals displayed marked inflammatory cell infiltrations and white matter destruction within the spinal cord. Clinical and histologic severity clearly increased with the recipient's age. Histologically, there were some differences between adult rat EAE and newborn rat EAE. In contrast to adult rat EAE which displayed grey and white matter involvement with marked perivascular mononuclear cell infiltrations, newborn rat EAE typically showed very severe changes with edema formation selectively in the white matter and the cellular infiltrates were dominated by polymorphonuclear cells and macrophages. Newborn T line-mediated EAE thus strikingly resembles hyperacute EAE induced in immunocompromised (irradiated) recipients.

A serotonergic input to the lateral cervical nucleus of cats and monkeys (Aotus trivirgatus) was demonstrated with immunohistochemical methods. In both species, the lateral cervical nucleus was found to contain a network of serotonin-immunoreactive fibers. However, the density of labeled fibers was greater in the monkeys than in the cats. Most labeled fibers were thin and had irregularly spaced varicosities. Electron microscopic examination showed that labeled varicosities were in apposition with dendrites, neuronal somata and unlabeled terminals, but synapses were rare. The results demonstrate that the lateral cervical nucleus receives a serotonergic innervation, as is the case with other somatosensory relay structures such as the spinal dorsal horn and the dorsal column nuclei. The presence of a serotonergic innervation suggests that the transmission of somatosensory information through the lateral cervical nucleus is modulated by a descending pathway. However, its effect on the response properties of neurons in the lateral cervical nucleus is unknown.

Calcitonin gene-related peptide (CGRP)-like immunoreactivity was localized immunocytochemically in the large motoneurons in the ventral horn of rat spinal cord. Using fluorescence double-labelling substance P (SP)-immunoreactive nerve fibres were found to surround both the CGRP-positive and negative motoneurons, whereas enkephalin (ENK)-immunoreactive fibres surrounded mainly CGRP-negative cells. All CGRP-like immunoreactive motoneurons were also choline acetyltransferase (ChAT)- and acetylcholinesterase (AChE)-positive. On the other hand a large population of ChAT- and AChE-positive motoneurons were devoid of CGRP-immunoreactivity. It is probable that CGRP/ChAT/AChE-positive cells surrounded by SP-positive fibres have different functions in motoric nervous system than the CGRP-negative ChAT/AChE-positive cells, which are surrounded by ENK-immunoreactive fibres.

We concurrently measured, by radioimmunoassay, levels of substance P (SP), somatostatin (SST), methionine-enkephalin (Met-Enk), cholecystokinin (CCK), peptide hystidyl-isoleucine (PHI), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) in the ventral and dorsal gray matter at each segment of the cervical, thoracic, lumbar, and sacral spinal cord, obtained within 6 hours of death from 4 subjects (ages 17 to 55) with no neurologic disease. Levels (pmol/g gray matter) of SP, SST, and Met-Enk throughout and PHI, VIP, and NPY in lumbar and sacral cord were significantly higher in dorsal than in ventral gray matter. PHI, VIP, and NPY were significantly higher in lumbar and especially sacral cord than in cervical and thoracic segments. In rats, a postmortem delay of up to 8 hours did not affect SP, Met-Enk, PHI, or NPY and decreased SST, CCK, and VIP levels. Thus, there is a characteristic profile of neuropeptide distribution in gray matter, which emphasizes the neurochemical heterogeneity along the rostrocaudal and dorsoventral extent of normal human spinal cord.

The extraocular muscle nuclei in one case of Werdnig-Hoffmann disease were examined immunocytochemically using antibodies against phosphorylated neurofilament (pNF) and ubiquitin (UBQ). The oculomotor and trochlear nuclei showed several chromatolytic ballooned neurons. All ballooned neurons contained epitopes of pNF and UBQ. pNF were present mainly in the periphery of the cell in a ring-like shape and were occasionally seen in the center of some cells. On the other hand, the structures stained by the antibody to UBQ were small vesicles or granules and most of them were aggregated in the center of the cell. These distribution patterns of pNF and UBQ may be unique in Werdnig-Hoffmann disease, since similar patterns were reported in other types of neurons of Werdnig-Hoffmann disease but were not seen in two other motor neuron diseases: classical amyotrophic lateral sclerosis, and familial amyotrophic lateral sclerosis with posterior column and spinocerebellar tract involvement.

Changes in the tissue content of water, Na+, K+, and Mg2+ were measured in spinal cord samples from anesthetized rats subjected to impact trauma (T9 spinal segment) of varying severity: 25 g-cm (low injury), 100 g-cm (high injury). Laminectomized animals served as controls. Rats with high injury were killed at 15 min, 60 min, 4 hr, 24 hr, 3 days, or 7 days after trauma. Those with low injury were killed at 24-hr postinjury. In all groups, spinal cord tissue was rapidly removed (less than 30 sec), frozen in liquid nitrogen, and dissected into the injured segment and adjacent two caudal and rostral segments. Water content was determined as wet weight/dry weight ratios, and cations were measured by atomic absorption spectrophotometry. High injury resulted in statistically significant increases in water content as early as 15 min after trauma. There were concomitant increases in Na+ and decreases in K+ that were apparent at 15 min and were statistically significant by 60-min posttrauma. The increases in water content and Na+ were present to a similar degree in the 4-hr, 24-hr, 3-day, and 7-day groups. In contrast, tissue Mg2+ was reduced and significantly decreased at 4 hr and 24 hr; Mg2+ levels had recovered partially by 3 days and completely by 7 days. Whereas changes in the tissue content of water, Na+, and K+ occurred at 24 hr regardless of the injury severity, changes in total Mg2+ were correlated with the degree of injury. These data are consistent with the conclusion that edema formation (and associated Na+ and K+ changes) after spinal cord trauma may be an epiphenomenon and does not significantly contribute to injury progression. In contrast, reductions in Mg2+ content may represent an important factor in the development of irreversible tissue damage.

A specific radioimmunoassay was developed for metorphamide, an endogenous, amidated opioid octapeptide, originally isolated from bovine brain and human pheochromocytoma tissues. The radioimmunoassay was used to determine the concentration of immunoreactive metorphamide in extracts from dissected regions of rat and guinea pig brain. Radioimmunoassay interfacing with Sephadex gel filtration and reverse phase high performance liquid chromatography confirmed that the immunoreactive substance measured corresponded to authentic metorphamide. Metorphamide was found to be widely distributed in brain regions from both species. However, the concentrations of immunoreactive metorphamide in regions from guinea pig brain were up to 5 times higher than the concentrations of immunoreactive metorphamide in rat brain regions. The results suggest that metorphamide is a specific processing product from proenkephalin in rodent brain.

Immunohistofluorescence studies of the rat central nervous system with antibodies to Phe-Met-Arg-Phe-NH2 (molluskan cardioexcitatory peptide) revealed a widespread neuronal system in the brain, spinal cord, and posterior pituitary. Immunoreactive axons and cell bodies were mainly located in cortical, limbic, and hypothalamic areas. Immunostaining of serial sections of the brain and pituitary showed that the Phe-Met-Arg-Phe-NH2 immunoreactive neurons were different from neurons labeled by antibodies to either Met-enkephalin or the putative Met-enkephalin precursor Tyr-Gly-Gly-Phe-Met-Arg-Phe, which is structurally related to Phe-Met-Arg-Phe-NH2. Control staining by antiserum absorption and radioimmunoassay indicated that the antibodies that caused the specific immunofluorescence recognized peptides with an amidated Arg-Phe sequence at the carboxyl terminus.

A detailed account of the distribution of immunoreactive substance P-containing structures in the rat central nervous system is presented, from results obtained by applying an indirect immunofluorescent technique. High densities of substance P-containing nerve terminals were present in sensory nuclei and other non-sensory structures such as thalamus, hypothalamus and extrapyramidal system. Substance P-reactive neuron cell bodies were present in spinal root ganglia, nucleus habenulae medialis, nucleus interpeduncularis, caudoputamen and globus pallidus. Most of the neocortex and the cerebellar cortices had no substance P-positive elements. The results support the hypothesis that substance P may be a widespread neurotransmitter in the central nervous system.

An immunohistochemically derived morphological description of a diverse population of rat lamina VII and X intraspinal 5HT neurons is provided. These bipolar or multipolar neurons occur most frequently in lamina X, dorsal or dorsolateral to the central canal, in thoracolumbar, sacral, and coccygeal spinal segments. These 5HT intraspinal neurons are found in normal rat spinal cords as well as in spinal cords that have been hemisected or transected 60 days prior to serotonin immunostaining. Therefore, 5HT intraspinal neurons are the probable source of the biochemically detectable 5HT that remains in the spinal cord distal to a spinal transection. In the rat, serotonin intraspinal neurons are most often associated with spinal autonomic nuclei but it is unknown if they are preganglionic in nature.

Tryptamine levels have been determined in mouse brain regions and spinal cord and in rat spinal cord. They were; caudate nucleus 2.5 ng X g-1, hypothalamus less than 0.5 ng X g-1, hippocampus less than 0.7 ng X g-1, olfactory bulb less than 0.7 ng X g-1, olfactory tubercles less than 0.6 ng X g-1, brain stem less than 0.4 ng X g-1, cerebellum less than 1.0 ng X g-1, and the "rest" 0.9 ng X g-1. The mouse whole brain was found to have 0.5 ng X g-1, the mouse spinal cord 0.3 ng X g-1, and the rat spinal cord 0.3 ng X g-1. These concentrations increased rapidly to 22.8 ng X g-1, 14.2 ng X g-1, and 6.6 ng X g-1 respectively at 1 hr after 200 mg X kg-1 pargyline. The turnover rates and half lives of tryptamine in the mouse brain and spinal cord and rat spinal cord were estimated to be 0.14 nmol X g-1 X h-1 and 0.9 min; 0.054 nmol X g-1 X h-1 and 1.5 min and 0.04 nmol X g-1 X h-1 and 1.6 min respectively. The aromatic L-aminoacid decarboxylase inhibitors NSD 1034 and NSD 1055 reduced synthesis of tryptamine in controls and pargyline pretreated animals. Tryptophan increased the concentrations of mouse striatal tryptamine and 5-hydroxytryptamine and brain stem 5-hydroxyindole acetic acid. p-Chlorophenylalanine reduced formation of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid but did not change that of tryptamine.

Following spinal cord injury in rats there was a time-dependent change of vascular permeability as reflected by extravasation of 125I-labelled serum albumin. The change of vascular permeability correlated with tissue calcium and water accumulation suggesting that cord exposure to plasma calcium as a consequence of vascular injury may contribute to the progressive post-traumatic cord necrosis.

The structures of two biologically different myelin proteins, A1 from the central nervous system and P2 from the peripheral nervous system, were investigated. Both proteins were isolated from nerve tissues. Conformational changes in the homogeneous proteins were examined in aqueous solutions by means of circular dichroism measurements. The secondary structures of both proteins proved to be very stable between pH 2.5 and pH 11.7. Unlike the P2 protein, the A1 protein is stable up to pH 13 without detectable conformational changes. The stereochemistry of the polypeptide chains of both proteins is markedly different in the presence of urea. While the value of theta222 for the A1 protein changes linearly with increasing urea concentration, a sigmoidal curve was obtained for the P2 protein. The observed differences in the dichroic properties of the basic myelin proteins A1 and P2 indicate the possibility of further structure - function correlations.

The distribution of glucose-1,6-bisphosphate (G16P2) synthase was measured in more than 70 regions of mouse brain, and nine layers of monkey retina. Activities in gray areas varied as much as 10-fold, in a hierarchical manner, from highest in telencephalon, especially the limbic system, to lowest in cerebellum, medulla, and spinal cord. The synthase levels were significantly correlated among different regions with G16P2 itself, as well as with previously published levels of a brain specific IMP-dependent G16P2 phosphatase. In contrast, neither G16P2 nor either its synthase or phosphatase correlated positively with phosphoglucomutase, and in all regions the G16P2 levels greatly exceeded requirements for activation of this mutase. This strengthens the view that G16P2 has some function besides serving as coenzyme for phosphoglucomutase. However, attempts to correlate the "G16P2 system," as defined by the three coordinately related elements, synthase, phosphatase, and G16P2, with other enzymes of carbohydrate metabolism, or with regional data of Sokoloff et al. [J. Neurochem. 28, 897-916 (1977)] for glucose consumption, were unsuccessful. This leaves open the possibility that brain G16P2 might serve as a phosphate donor for specific nonmetabolic effector proteins.

The effects of vitamin B-12 deficiency on lipids and fatty acids of spinal cord myelin were studied in control and vitamin B-12-deficient fruit bats. Very low plasma and brain vitamin B-12 concentrations were present in animals fed the vitamin B-12-free, all fruit diet. Myelin was isolated from the spinal cord of control and vitamin B-12-deficient animals (n = 3 pools) by means of a flotation method in a discontinuous sucrose gradient. The molar concentration of cholineglycerophosphatide was significantly lower in the deficient bats than in the controls. The molar cholesterol: phospholipid ratio in the deficient bats was also significantly lower. The odd-chain fatty acid 15:1 was not detected in cholineglycerophosphatide from the controls, but comprised 1.4% of the total fatty acids in the deficient group. The odd-chain fatty acids 15:0, 17:1 and 19:0 were present in slightly higher amounts in cholineglycerophosphatide of deficient bats. Lipid and fatty acid differences were related to the vitamin B-12-requiring methionine synthetase and methylmalonyl CoA mutase reactions.

The regional distribution of substance P in postmortem brain and spinal cord of 5 cases of schizophrenia were investigated using the immunohistochemical PAP and ABC technique. Substance P immunoreactive fibers were primarily found in Substantia nigra, nucleus tractus spinalis n. trigemini, nucleus tractus solitarii, nucleus spinalis n. vestibuli, nucleus ambiguous, Central gray matter of the midbrain, globus pallidus and cornua posteriora spinales. The 5 cases showed no differences to the controls. These results agree with the finding reported abroad that the brain tissues were measured by radioimmunoassays. It was also discussed on study method of substance P in Central nervous system.

We attempted to clarify the organ distribution of human and murine proteinase-resistant prion protein (PrPCJD) in Creutzfeldt-Jakob disease (CJD), and to measure the concentration of PrPCJD, using a semi-quantitative Western blot analysis. Human PrPCJD was restricted to the central nervous system, whereas murine PrPCJD was present in the central nervous system and in the lymphoreticular system at the end stage of CJD. PrPCJD concentration in the central nervous system of mice was almost identical to that of humans. The minimum wet weight of an organ with a positive reaction was 0.3 mg for brain, 1 to 3 mg for spleen, 3 mg for spinal cord, 3 mg for lymph node, 10 mg for thymus and 10 to 30 mg for intestine of the CJD-infected mice. There were no immunoreactions in purified PrPCJD fractions from 300 mg of spleen, lymph node, liver or peripheral nervous systems of humans, nor in 300 mg of liver, lung or kidney of CJD-infected mice. Within the limits of our method, the distribution of murine PrPCJD differed from that of human PrPCJD. Antibodies on the Western blot membrane from murine spleen PrPCJD fractions stained the kuru plaques in the CJD-infected mouse brain. Therefore, PrPCJD in the murine spleen probably shares the epitopes of the antigen in the murine kuru plaques. Although the immunological detection of PrPCJD does have limits of sensitivity, PrPCJD concentrations did correlate with infectivity titres in scrapie-infected or CJD-infected mice.

In spinal cords from seven amyotrophic lateral sclerosis (ALS) patients and four controls, we found no difference in thyrotropin-releasing hormone (TRH) concentration relative to protein content, but there was a reduction per tissue wet weight in ALS. Immunohistochemical localization of TRH in ALS cord was unaltered. Histidyl proline diketopiperazine (HisPro-DKP), a possible metabolite of TRH, was significantly elevated per protein content in ALS. CSF levels of TRH and HisPro-DKP were unchanged. These findings suggest that TRH neurons are not primarily affected in ALS, but TRH and tissue protein are lost together as the disease progresses.

The cellular localization and regional distribution of "insulin-like immunoreactivity" and "C-peptide immunoreactivity" were studied in brains of fetuses and newborns from normoglycaemic and diabetic mothers using immunohistochemical techniques. It is shown that "insulin-like material" and "C-peptide-like material" occur in the same nerve cells. No differences in regard to the localization of both polypeptides exist between abort material from normoglycaemic and diabetic mothers.

Light and electron immunocytochemistry using antibodies recognising a class II major histocompatibility complex antigen, fibrin, fibronectin, albumin and factor VIII related antigen has been used to stain sections of spinal cord from normal guinea pigs and those with chronic relapsing experimental allergic encephalomyelitis (CREAE). It was found that class II MHC antigens, fibrin and fibronectin were present in normal blood vessels and at high levels in lesions from animals at all stages of the disease. The possible immunological roles of these antigens suggest their participation in the initiation and maintenance of disease state.

This review examines, in a condensed manner, many of the major achievements related to spinal cord injury research during the last quarter century. Most of the advances have been made within the past 10 years. They include such basic and clinical tools as evoked potentials, regional and local spinal blood flows, neurophysiologic monitoring systems, and methods that detail the morphology and contents of cord tissue. Much of the experimentation conducted within the last 25 years has provided a better understanding and clinical therapeutic approach to the injured spinal cord than at any time before. Such work has exposed significant aspects in the biochemistry and vascular mechanics associated with trauma to the cord. A growing and intriguing area of spinal injury research lies in probing the factors related to neuronal plasticity and regeneration of the cord tissue. This review also examines the role of experimental animal models as well as the clinical and experimental therapies available for acute and chronic spinal cord injury.

There is considerable evidence that somatostatin is released from nerve terminals throughout the central nervous system in response to presynaptic stimulation, thus suggesting a neuromodulator role for the peptide. We here report the partial characterization of immunoreactive somatostatin released from rat nervous system in vitro (hypothalamus, spinal cord and hypothalamic, cortical, thalamic and striatal synaptosomes). Serial dilutions of released somatostatin immunoreactivity showed parallelism with dilutions of synthetic somatostatin standard. Somatostatin immunoreactivity released from all tissue areas coeluted with synthetic tetradecapeptide on Sephadex G-25 (fine grade) gel chromatography; more than 85% of this immunoreactivity bound to Sepharose-anti-somatostatin-serum immunoaffinity columns. In addition, immunoreactive material released from hypothalamus, spinal cord and hypothalamic and cortical synaptosomes inhibited somatotropin (growth hormone, 'STH', 'GH') release from perifused anterior pituitary in a dose-related manner, indicating biological similarity to synthetic somatostatin.

Myelin proteins were quantitated in whole tissue and isolated 'myelin fractions' from spinal cord, brainstem and hemispheres of 'shaking pups', a mutation in Springer-Spaniel dogs characterized by hypomyelination of the CNS. The amount of myelin basic protein (MBP) in the brainstem of affected 4-week-old pups was 2.6% of that in age-matched controls, while the levels of 2'3'-cyclic nucleotide phosphodiesterase (CNP) and myelin-associated glycoprotein (MAG) were 10% and 15% of the control levels, respectively. Similar results were obtained in the spinal cord and hemispheres, and the amounts of these proteins in the mutant pups did not change substantially between 4 and 16 weeks of age. The amount of the 21 kDa MBP compared to the 18 kDa MBP was relatively increased in the shaking pups, suggesting that the small amount of myelin formed was immature. The yields of myelin fractions from the mutant pups were very low; e.g., the yield from the brainstems of 4-week-old mutants was only 2.4% of that from age-matched controls and the yield did not increase by 16 weeks. The isolated myelin fractions contained very little MBP (less than 0.5% of total protein) or proteolipid protein, indicating that they were a very immature form of myelin or consisted largely of non-myelin contaminants. MAG in the 'myelin fractions' from the mutant brainstems were 9-15 fold higher and CNP levels were 2-3 fold higher than those in whole homogenates, suggesting that the isolated fractions were enriched in oligodendroglia-derived membranes. Overall, the biochemical results are consistent with a severe hypomyelination of the CNS in which a small amount of immature myelin is formed.

In 4 monkeys of the species Macaca fascicularis (2 animals) and Papio anubis (2 animals) the three dorsal column nuclei and the lateral cervical nucleus have been investigated immunocytochemically with antiserum against gamma-aminobutyric acid (GABA). Light microscopic studies demonstrated the presence of GABA-positive cells in the gracile nucleus, the internal cuneate nucleus and the lateral cervical nucleus but not in the external cuneate nucleus. Although labeled cells seemed fairly evenly spread in the nuclei there was an increased amount between the clusters of the internal cuneate nucleus and in the border zone between the gracile and the cuneate nucleus. Electron microscopical investigation showed GABA labeling in fairly small neurons with relatively large cell nuclei and low somatic bouton covering. GABA-positive terminals with rounded synaptic vesicles were present in all the investigated nuclei also the external cuneate one. No apparent difference in number of such boutons in the different nuclei or parts of the nuclei was found. GABA-positive boutons mostly synapsed with dendrites but in the dorsal column nuclei also with other larger boutons. Axosomatic contacts between labeled terminals and neuronal perikarya were more common in the lateral cervical nucleus than in the dorsal column nuclei. The results from the different nuclei in the monkey were compared with the results of similar investigations in the cat. It is concluded that there are important species differences especially on the light microscopical level in the lateral cervical nucleus. Thus the GABA labeled cells is rather evenly spread over the nucleus in the monkey whereas in the cat they are concentrated to the ventromedial region.

The intermediate filament protein, glial fibrillary acidic protein (GFAP), is widely used as a cell-specific marker molecule for immunocytochemical identification of astrocyte lineages in cell culture, in tissues during development, and in tissues undergoing pathological changes. This study demonstrates that a reaction pattern of two commercially available polyclonal anti-GFAP antibodies shows extensive similarity to the pattern of reactivity obtained with monoclonal antibodies to neurofilaments in the PNS and spinal cord of human embryos and foetuses, at 5 to 12 weeks of gestation. The polyclonal antibodies to GFAP labelled populations of neurons and their processes in the PNS and in the spinal cord. Monoclonal antibodies to GFAP only labelled glial cells in the spinal cord. Neurofilament adsorption of one of the anti-GFAP antisera abolished the neurofilament-like reaction pattern, while the structures also labelled with monoclonal antibodies to GFAP remained immunostained. The results presented may question previously published data obtained with these and possibly other polyclonal anti-GFAP antibodies.

Norepinephrine (NE) stimulated cyclic adenosine 3',5'-monophosphate (cyclic AMP) accumulation in regional spinal cord and cortical tissue slices from postnatal rats demonstrated distinct developmental patterns. NE-concentration-response studies using a 10-min incubation period demonstrated minimal NE-stimulated cyclic AMP accumulation in whole spinal cord at PD 1-5 with maximal sensitivity on PD 12. Thereafter, sensitivity decreased to adult values at PD 30. Sensitivity changes were reflected in alterations in maximal response only since NE EC50s all averaged 10(-6) M. This agrees with no change in calculated Kd for NE with increased Vmax to PD 12 and a reduction thereafter. Studies on regional spinal cord and cerebral cortical cyclic AMP accumulation indicated peak NE sensitivity in cervical and thoracic cord occurred at PD 10, in lumbar cord at PD 15, and in cerebral cortex at PD 20. The fact that inhibition of phosphodiesterase (PPDE) produced the same percentage alteration in peak accumulation in spinal cord slices regardless of postnatal age suggests that PPDE is not the primary determinant of the ontogenic changes. The results indicate that the postnatal development of spinal NE receptors may be reflected in an increase in the responsiveness of the cyclic AMP system to NE.

Intact neurofilaments were isolated from bovine spinal cord white matter, washed by sedimentation in 0.1 M-NaCl, and extracted with 8 M-urea. Solubilized neurofilament triplet proteins of molecular weights approximately 68,000 (P68), 150,000 (P150), and 200,000 (P200) were purified by preparative electrophoresis, using an LKB 7900 Uniphor apparatus. The method provides for an enhanced yield of purified protein and has markedly reduced admixture of electrophoresed protein with acrylamide and associated protein contaminants. Amino acid compositions of the purified neurofilament triplet proteins are reported and compared.