Several enzymes formerly used as markers for a particular type of glial cell have recently been detected in both astrocytes and oligodendrocytes. Carbonic anhydrase, previously regarded as oligodendrocyte- (and myelin-) specific, has now been demonstrated in protoplasmic astrocytes in gray matter of rat forebrain. A second enzyme, glutamine synthetase, served as an astrocyte marker for ten years. However, this enzyme has now been localized to oligodendrocytes in spinal cord and in white matter of brain, as well as to astrocytes in gray matter of brain, where it was first observed. Currently, significant issues in glial cell development in the brain in vivo are still unresolved, and the development of protoplasmic astrocytes, in particular, is most obscure. One possibility is that carbonic anhydrase and glutamine synthetase, at their minor loci (astrocytes and oligodendrocytes, respectively), may be remnants of glial-cell precursors that may not have been detected previously. Alternatively, they may occur in known precursors. In either case immunocytochemical staining of these enzymes will provide new insights concerning glial cell lineage. Antibodies against a third glial-cell enzyme, the Yb subunit of glutathione-S-transferase (""""""""Yb""""""""), will be used to obtain more information concerning the development of astrocytes, in particular, since Yb occurs in astrocytes at all stages in their maturation. Additional new data show another enzyme, the RLM6 isozyme of cytochrome P-450 ('RLM6'), to be in oligodendrocytes.
The specific aims will be as follows. 1) Sections from developing rat brains will be immunostained with antibodies against carbonic anhydrase, glutamine synthetase, and Yb and the stained cells identified by double immunostaining for reliable """"""""markers."""""""" Because oligodendrocytes fail to develop normally in the brain of the myelin-deficient mutant rat, and there is severe gliosis, one might expect certain glial-cell precursors to accumulate during this mutant's 3- to 4-week lifespan. Therefore the developmental immunocytochemical studies will also be performed in sections from the brains of this mutant. 2) Quantitative developmental data will be obtained by performing enzyme assays on bulk-isolated cells. 3) Glial cells will be cultured and immunostained for the above enzymes during development in vitro. 4) Development of RLM6 will be examined in rat brains and in vitro. The findings are expected to help resolve the issues raised above.
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