Cell-surface antigen distinguishes sensory and autonomic peripheral neurones from central neurones.
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Biomedical subjects
Publications and source records attributed to R Mirsky.
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We have produced a monoclonal antibody that reacts with all classes of intermediate filaments in immunofluorescence assays, including glial filaments in astrocytes, neurofilaments in axons, tonofilaments in epithelial PtK2 cells and intermediate filaments in fibroblasts. It also binds to Z lines in skeletal muscle. In SDS-polyacrylamide gels, the antibody binds to most and perhaps all of the major intermediate filament proteins that have been previously defined, including glial fibrillary acidic protein, the three vertebrate neurofilament proteins (the "neurofilament triplet"), vimentin, desmin, several cytokeratins and the neurofilament proteins of squid and the marine worm Myxicola. In addition, the antibody binds to a protein with an approximate molecular weight of 66,000 that may be a component of all intermediate filaments. These findings suggest that all vertebrate and invertebrate intermediate filament proteins share a common antigenic determinant and raise the possibility that all intermediate filaments contain a 66,000 molecular weight protein.
The complex nervous networks found throughout the mammalian gut--the enteric nervous system--are histologically, ultrastructurally, and, to some extent, functionally--similar to the central nervous system. The glial cells of the small enteric ganglia are generally classified as Schwann or satellite cells, since they are found in the peripheral nervous system, possess nuclei which ultrastructurally resemble those of Schwann cells and are derived from the neural crest. However, it has been argued that these cells resemble astrocytes of the central nervous system with respect to gross and fine structure, and their relationship with the enteric neurones and their processes. In immunohistochemical studies of these cells, both in frozen sections of gut wall and in tissue culture preparations of the enteric plexuses, we found evidence that the enteric glial cells are rich in glial fibrillary acidic protein (GFAP), a protein associated with the 100 A glial intermediate filaments, and hitherto believed to be specific to astrocytes of the central nervous system only.
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We have used antibodies to identify Schwann cells and oligodendrocytes and to study the expression of myelin-specific glycolipids and proteins in these cells isolated from perinatal rats. Our findings suggest that only Schwann cells which have been induced to myelinate make detectable amounts of galactocerebroside (GC), sulfatide, myelin basic protein (BP), or the major peripheral myelin glycoprotein (P0). When rat Schwann cells were cultured, they stopped making detectable amounts of these myelin molecules, even when the cells were associated with neurites in short-term explant cultures of dorsal root ganglion. In contrast, oligodendrocytes in dissociated cell cultures of neonatal optic nerve, corpus callosum, or cerebellum continued to make GC, sulfatide and BP for many weeks, even in the absence of neurons. These findings suggest that while rat Schwann cells require a continuing signal from appropriate axons to make detectable amounts of myelin-specific glycolipids and proteins, oligodendrocytes do not. Schwann cells and oligodendrocytes also displayed very different morphologies in vitro which appeared to reflect their known differences in myelinating properties in vivo. Since these characteristic morphologies are maintained when Schwann cells and oligodendrocytes were grown together in mixed cultures and in the absence of neurons, we concluded that they are intrinsic properties of these two different myelin-forming cells.
We have used 4 cell-type-specific markers to identify individual glial and neuronal cells in dissociated cell cultures of neonatal rat sciatic nerve, dorsal root ganglia (DRG), optic nerve, cerebellum, corpus callosum, cerebral cortex and leptomeninges. Schwann cells were identified with antibodies against rat neural antigen-1 (Ran-1), neurons with tetanus toxin, astrocytes with antibody against the glial fibrillary acidic protein (GFAP) and oligodendrocytes with antibody against galactocerebroside. All of these ligands react with cell surface molecules except for anti-GFAP antibody which binds to intracellular glial filaments. Using two-fluorochrome immunofluorescence we have studied the distribution of various glycoproteins and glycolipids on these 4 major neural cell types in short-term cultures. We have found that (1) although Ran-1 is expressed on glial and neuronal tumours, it was not found on normal astrocytes, oligodendrocytes or neurons; (2) Thy-1 was present on fibroblasts and some neurons but not on the majority of leptomeningeal cells or on oligodendrocytes or astrocytes in short-term cultures (however, it was expressed on some astrocytes in longer term cultures); (3) the 'large external transformation sensitive' (LETS) protein could be detected on fibroblasts and leptomeningeal cells but not on neurons or glial cells; (4) GM1 was present on all neurons, most oligodendrocytes and approx. 50% of other cell types; sulfatide and GM3 were only detectable on oligodendrocytes, while globoside was only found on some neurons. In addition, we were able to identify putative microglial cells by the presence of cell surface receptors for IgG and by their phagocytic activity; they did not express and of the cell-type-specific defining markers.
gamma-Aminobutyric acid (GABA) is an inhibitory neurotransmitter in the peripheral nervous system of certain invertebrates and is thought to be a major transmitter in the vertebrate central nervous system. In this report we present evidence that GABA may also be a neurotransmitter in the vertebrate peripheral autonomic nervous system. We have used light and electron microscopic autoradiography to analyse high-affinity uptake of 3H-GABA into the myenteric plexus of the guinea pig taenia coli, both in situ and in a tissue culture preparation. In the isolated myenteric plexus, we have measured the specific activity of glutamic acid decarboxylase (GAD; EC 4.1.1.15), the enzyme responsible for conversion of glutamic acid to GABA in GABAergic neurones, and assessed the ability of this tissue to accumulate 3H-GABA newly synthesised from 3H-glutamic acid. Furthermore, we have measured the levels of endogenous GABA in strips of taenia coli containing the myenteric plexus.
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Several cationic anaesthetics and barbiturates including bupivacaine, lignocaine and pentobarbital reversibly inhibit myoblast fusion in rat primary muscle cultures. The same drugs also cause disruption of myotube structure, which is normally complete within 2 h; fusion of myoblasts and reformation of myotubes takes place on removal of the drugs. The disruption of myotubes caused by tertiary amine anaesthetics observed in vitro appears to mimic muscle fibre disruption caused by the same drugs in vivo. The effects of temperature, culture age, medium constitution and calcium flux on anaesthetic-induced myotube breakdown have been examined.
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The distribution of three cell surface markers on dissociated cultures of the rat dorsal root ganglion has been investigated using indirect immunofluorescence. Tetanus toxin binds to a nondividing class of cells with neuronal morphology. Ran-1 is expressed by Schwann cells. Thy-1 is expressed both by fibroblasts and by all the neurones. These assignments have been confirmed by detection of any two of these markers in a single culture using antisera conjugated to different fluorochromes. Approximately 20% of the nonneuronal cells in these cultures were negative for all three markers.
Surface cultures of 15-19 day old fetal mouse brain that do not initially express the Thy 1 antigen develop the antigen between 4 and 11 days in culture. The antigen can be detected by immunofluorescence of live cells on the culture plate on two morphologically distinct types of cells. Both neuronal-looking cells and a more general category of cells which includes fibroblastic cells can express the Thy 1 antigen. The antigen is not expressed on certain cells of the glial type. This method enables one to distinguish between different types of brain cells in culture both morphologically and immunologically at the same time.
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