摘要:

AbstractTwo overlapping cosmids containing the 5′ end of human choline acetyltransferase (ChAT) gene have been cloned. Using heterologous probes, we localized two alternative first exons homologous to rodent ChAT exons R and M (Misawa et al.: J Biol Chem 267:20392–20399,1992). The sequence of rodent exon N was not conserved in the human gene. Northern blot analysis of mRNA purified from the human neuroepithelioma cell lines LA‐N2 and MC‐I‐XC revealed that both exons R and M were transcribed in mRNA species of 6.0 and 2.5 kb. Only the 6‐kb species was detected with both R‐ and M‐specific probes in the neuroepithelioma cell line CHP126. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis suggested that the major mRNA species in MC‐I‐XC and CHP126 cells contained the proximal part of exon M spliced to exon 1, which contains the alternative ACG initiation codon. RT‐PCR also allowed the characterization of a mRNA species containing exon R spliced to exon 1, but no species containing both exon R and the distal part of exon M could be detected. RT‐PCR was also used to evidence an alternative exon (tentatively numbered exon 8) in the coding seque

ISSN:0360-4012    

DOI:10.1002/jnr.490400402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractMicrotubule associated proteins (MAPs) interact with tubulin to modulate neurite stability and growth during development. The phosphorylated form of one of these MAPs, MAP1B (MAP1B‐P) is hypothesized to be of particular importance for the regulation of neurite outgrowth. To investigate the mechanisms by which MAP1B and MAP1B‐P contribute to this regulation, we used a new antibody against an isoform of MAP1B‐P to determine its pattern of expression during neuronal development in vitro. We examined cultured hippocampal neurons because these provide a well‐established system to evaluate the development of axons and dendrites. MAP1B, MAP1B‐P and MAP2 colocalized to the cell bodies and minor processes during the first 24 hours of culture, but MAP1B‐P also extended well into the growth cones. As neurite outgrowth and differentiation proceeded, MAP1B and MAP1B‐P became localized to the cell bodies and axons, and MAP2 to the cell bodies and dendrites. After 3 days, MAP1B‐P declined in the cell body and was segregated to the distal axon; MAP1B remained in the cell body, but was also concentrated in the distal axon. Over 5–9 days in culture, MAP1B‐P levels decreased and became undetectable; MAP1B levels decreased later (19–23 days). MAP2 levels, however, remained high through the entire culture period in cell bodies and dendrites. These results are consistent with the hypothesis that MAP1B‐P plays an important role in the initiation and elongation of axons by regulating the dynamics of microtubules near the growth cone: MAP1B‐P expression is greatest during the period of active neurite extension, is particularly prominent in growth cones where axon outgrowth is most active, and decreases along with the decline in active axon extensio

ISSN:0360-4012    

DOI:10.1002/jnr.490400403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractIn the choroid plexus carbonic anhydrase II (CAII) supports the transport of bicarbonate ions, sodium ions, and water from blood to the CSF, and in the myelin sheath CAII supports compaction of myelin by stimulating cotransport of ions and water out from between the myelin membranes. In view of the latter, it is surprising that mutant mice deficient in CAII (Car‐2n) have compact myelin. Since myelin basic protein also takes part in myelin compaction, we bred double CAII‐deficient, myelin‐deficient (MId) mutant mice, in which the adults would have some compact myelin sheaths and a partial deficiency in myelin basic protein, with a view to examining oligodendrocytes and myelin sheaths in the double mutant. Like the parent MId strain, the double mutants displayed tremors and seizures; however, the onset of seizures was delayed significantly in the double mutants, and the lifespan increased by several months. Like the brains of Car‐2n mutants, those of double mutants (MldCar‐2n) were deficient in mRNA and protein for CAII and showed upregulation of a different isozyme, CAIV. In the double mutants, oligodendrocytes were reduced in number, and the myelin sheaths and oligodendrocytes were swollen. The partial protection against seizures, which CAII deficiency conferred, suggests that acidosis in the central nervous system (CNS) of the Car‐2n and MldCar‐2n mice, due to absence of CAII from the choroid plexus, may downregulate the activity of NMDA receptors. The swelling of myelin and oligodendrocytes only in the MldCar‐2n suggests that CAIV activity is not sufficient for maintaining compact myelin when the myelin is partly deficient in myelin basic protein and severely deficient in CAII. The findings provide the first direct evidence for a role of carbonic anhydrase II in the compaction of myelin membranes. © 1995

ISSN:0360-4012    

DOI:10.1002/jnr.490400404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractBecause of the importance of collagens in mediating cell‐substrate interactions and the association of collagens with neural recognition molecules in the peripheral nervous system, the ability of neural recognition molecules to modify the substrate properties of collagens, in particular collagen type I, for cell adhesion was determined. Two cell lines, the N2A neuroblastoma and PC12 pheochromocytoma, were investigated for their capacity to adhere to different collagen types in the absence or presence of several neural recognition molecules. Adhesion of N2A or PC12 cells and membrane vesicles from PC12 cells to collagen type I was reduced when the collagen had been preincubated prior to its application as substrate with the extracellular domain of myelin‐associated glycoprotein (s‐MAG) or, as control, fibroblast tenascin‐C (F‐tenascin). In mixture with other collagen types, s‐MAG was only able to reduce the adhesiveness of collagen types III and V, but not of collagen types II and IV. F‐tenascin reduced the adhesiveness of all collagen types tested. In contrast to F‐tenascin, s‐MAG had to be present during fibrillogenesis to exert its effect, indicating that it must be coassembled into the collagen fibril to block the cell binding site. Cell adhesion to collagen type I was dependent on Mg2+or Mn2+and inhibited by a monoclonal antibody to the α1integrin subunit. The combined observations indicate that s‐MAG and F‐tenascin interfere with cell binding, most probably by modifying the integrin binding site, and that the two molecules act by different mechanisms, both leading to reduction of adhesion.

ISSN:0360-4012    

DOI:10.1002/jnr.490400405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractRegional astrocyte cultures were obtained by dissecting and dissociating medial and lateral sectors of the midbrain from 14‐day Swiss mouse embryos. Once confluent, these cultures were tested by glial fibrillary acidic protein (GFAP) immunocytochemistry to confirm their astrocyte composition and for 2′‐3′ cyclic nucleotide 3′‐phosphohydrolase (CNPase) and microtubule‐associated protein 2 (MAP2) immunocytochemistry to rule out oligodendroglial and neuronal components, respectively. In confluent astrocyte cultures from either sector, virtually all cells were GFAP‐positive elements, most of which were flat cells accompanied by smaller numbers of flat cells with processes. Confluent astrocyte cultures, derived from medial (M) or lateral (L) sectors, were used as substrata for culturing dissociated cells from medial (m) or lateral (1) sectors of 14‐day embryonic midbrains. Fixed cocultures (LI, Lm, Mm, MI) were stained with an anti‐MAP2antibody to verify neuronal aggregation and neuritic morphology. In spite of the morphological constancy of glia substrata at plating, MAP2‐positive cells in cocultures showed differences in the aggregation of somata and in the length, caliber, and branching of neurites. These differences, which depend mostly on the sector of origin of astrocytes, suggest that the substrata may differ in adhesiveness and/or growth‐promoting vs. growth‐interfering properties. Together with evidence for sectorial heterogeneity in brainstem radial glia, the present results raise the possibility that cultured astrocytes have properties that reflect the roles played by their parent radial glia in the developing brai

ISSN:0360-4012    

DOI:10.1002/jnr.490400406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractMicroglia were successfully cultured from human brain tissue from normal and neurologically diseased cases obtained 3.5–10 hours postmortem. Final cell preparations were more than 99% pure as judged by latex bead phagocytosis, expression of microglial phenotypic markers, and absence of astrocytic markers. The expression of complement genes C1qB, C3, and C4 as well as genes for interleukin‐(IL‐)1α, IL‐1β, IL‐6, tumor necrosis factor (TNF)α, IL‐1 receptor antagonist, and transforming growth factor β, but not inducible nitric oxide synthase, by these cells was detected by polymerase chain reaction (PCR) analysis. The pattern of gene expression was evaluated following stimulation of the cells with lipopolysaccharide, phorbol myristate acetate, γ interferon, and β amyloid peptide. There was considerable variation in gene response to these activating agents. However, it was of interest that β‐amyloid peptide (1–40) increased the expression of IL‐1β mRNA in these cells. The number of cases in this study was too small to permit evaluation of microglial response according to the disease state, but the results demonstrate the potential for such studies in the futur

ISSN:0360-4012    

DOI:10.1002/jnr.490400407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractK‐252a treatment produced a 30–50% increase in the uptake of radioactive calcium by PC12 cells within 3–4 minutes. The increase in uptake was partially blocked by inhibitors of voltage‐operated calcium channels, such as nifedipine, but not by inhibitors of receptor‐operated calcium channels, such as nickel or suramin. Introduction of phosphatase 2A into the cells completely blocked the effect of K‐252a. Longterm treatment of the cells with either K‐252a or with nerve growth factor blocked the subsequent actions of either K‐252a or nerve growth factor on calcium uptake, but neither altered the subsequent action of the L‐channel agonist Bay K 8644 on calcium uptake. Calcium uptake was not stimulated by K‐252a in PC12nnr, cells that have little or no high‐affinity nerve growth factor receptors; cells expressing increased levels of high‐affinity nerve growth factor receptors showed a response to K‐252a comparable to that seen in parent PC12. The data suggest that the increased uptake of radioactive calcium produced by K‐252a is mediated by a mechanism very similar to that serving the increased calcium uptake produced by nerve growth factor. © 1995 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain

ISSN:0360-4012    

DOI:10.1002/jnr.490400408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractTransection of the hindlimb dorsal column fibers leads to a partial deafferentation of the neurons of the nucleus gracilis, the first relay of the ascending dorsal column‐medial lemniscal (DC‐ML) neural network. In response to the deafferentation, a synaptic renewal cycle is initiated and the neurons of the nucleus gracilis atrophy. The present study examines the molecular changes that occur in synaptic relays of the ascending DC‐ML following a hindlimb dorsal column transection. Rats were sacrificed 0.5, 1, 24, or 72 hours postlesion. Steady‐state levels of mRNA coding for c‐fos were significantly elevated only at 24 hours postlesion in caudal dorsal medulla, which contains the nucleus gracilis. The increased c‐fos is neuronal in origin since there is an increased level of c‐fos immunoreactivity in both the cluster neurons and the interneurons of the nucleus gracilis. In the third relay of the DC‐ML system, the somatomotor cortex, levels of c‐fos mRNA were significantly decreased 72 hours postlesion. These data indicate that lesions of the hindlimb dorsal column fibers have transneuronal effects on gene expression that extend to at least the third synaptic relay in the DC‐ML system. © 1995 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the Un

ISSN:0360-4012    

DOI:10.1002/jnr.490400409

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractMonoclonal antibody BM88 identifies a neuron‐specific antigen (BM88 antigen) present in the central and peripheral nervous system of the pig (Patsavoudi et al.: Neuroscience 30:463–478, 1989; J Neurochem 56:782–788, 1991). We have previously shown that the antigen is also expressed by cultured neurons derived from newborn rat brain. In the present study we have used the monoclonal antibody BM88 and a specific polyclonal antibody in order to identify the nature of the cross‐reactive antigen in rat brain and to investigate its expression and cellular localization in the developing and adult rat nervous system. Western blot analysis and immunocytochemistry revealed that the rat BM88 antigen displays very similar biochemical properties with its porcine homologue. It is a neuron‐specific integral membrane protein, apparently not glycosylated, consisting of two 23 kD polypeptide chains. Immunoperoxidase staining demonstrated that the BM88 antigen is widely distributed in the brain of 19‐day‐old rat embryos. At this stage, immunoreactivity was particularly prominent in differentiated cellular areas and developing fiber tracts of the embryonic rat brain, but was also present in the neuroepithelium. A similar wide distribution of the BM88 antigen was observed in the adult rat brain. Here, immunoreactivity was detected in the neuropil and neuronal perikarya. Immunocytochemical analysis of the expression of the BM88 antigen during postnatal development of the cerebellar cortex showed that this molecule is particularly concentrated in the Purkinje cells between postnatal days 10 to 15; their somata and developing dendrites were distinctly immunopositive during this period. An agedependent increase in the expression of the BM88 antigen both in brain and in the cerebellum was noted. Electron microscopy confirmed the presence of the BM88 reaction product within the perikarya, axons and dendrites of labeled neurons in the adult brain. The BM88 reaction product was preferentially associated with the limiting membrane of mitochondria, endoplasmic reticulum and small electron‐lucent vesicles, but was also present in the plasma membrane, especially at the level of synaptic densities. Our results show that the BM88 antigen participates in an activity common to all or most neurons, and demonstrate that the expression of this antigen is elevated upon neuronal differentiation and maturation. © 1995

ISSN:0360-4012    

DOI:10.1002/jnr.490400410

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractOligodendrocyte precursor cells that develop into myelin‐forming cells of the central nervous system (CNS) were cultured from newborn rat brain to study how they proliferate and differentiate in normal conditioning medium, and their cell development was characterized by scanning electron microscopy (SEM) observation and immunocytochemical studies.We have identified A2B5‐negative pre‐O2A progenitor cells (so‐called “type‐1” oligodendrocytes) in the secondary cultures on the astrocyte feeder layer. These cells are very small (diameter: 3.5 μm), round, and glossy, and develop into the process‐bearing O2A progenitor cells (called “type‐2” oligodendrocytes), which also express myelin basic protein (MBP) both in the cell body and in their cell processes. Finally, they develop into mature oligodendrocytes (called “type‐3” oligodendrocytes). After MBP expression is elicited in these cells and MBP accumulates in the cell process in the area in contact with the axon, these cells are capable of forming the myelin sheath. Therefore, we examined the mechanism of myelin‐sheath formation of “type‐3” oligodendrocytes using video time‐lapse movies, and demonstrated that these cells initially sent out processes to search for axons several times before the onset of myelination. Then thick filopodia extended towards the axon, and at the same time, the axonal part of neuron moved forward. Finally the ruffling lamellipodial parts wrapped up the axon similarly to a transverse wave with the secured thick filopodial process on the axon acting as scaffolding. These results suggest that our experimental systems are useful in studying normal oligodendrocyte development and their cellular biochemistry, as well as investigating the mechanism of myelin formation by ol

ISSN:0360-4012    

DOI:10.1002/jnr.490400411

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY