This program contains four projects devoted to the neurochemistry and cell biology of nervous system development. All studies are to be performed in rats. We propose: (1) to isolate and purify glial precursor cells from the developing and mature rat brain, to follow the differentiation of these cells in culture, and to test the hypothesis that these precursor cells serve as a reservoir for proliferating astrocytes and oligodendrocytes following injury. (2) To extend our studies of cellular localization of the glial-specific enzymes: Yp and Yb isoforms of glutathione-stransferase, carbonic anhydrase, glutamine synthetase and the RLM6 isozyme of cytochrome P-450 in the developing normal and myelin-deficient rat. These studies which will include immunostaining of tissue sections and cell cultures and enzyme assays of bulk isolated cells, are expected to help resolve some current issues in glial cell development, with particular emphasis on protoplasmic astrocytes. (3) To study the expression of a novel 66KD CNS-specific neurofilament protein in rat cerebellum during development and in cultured granule cells. The axonal cytoskeleton of the granule cells will be examined by electron microscopy and immuno-EM in vivo and in vitro. A cDNA probe will be constructed to the 66 KD NF protein and used for in situ hybridization in tissues and in Northern blot analyses of extracted RNAs. (4) To examine the heat-shock (stress) response in the CNS of Lewis rats induced to develop EAE, a disease involving inflammation, demyelination and extensive reactive gliosis; and in glial cells in culture during development and differentiation. Heat-stress mRNA and protein will be analyzed by Northern and Western blots, in situ hybridization, immuno-cytochemistry and in vitro protein synthesis. The methodology for these four studies includes cell isolation, cell culture, immunocytochemistry, enzyme assays, cDNA construction, gene sequencing, RNA isolation, Northern blots, Western blots, protein synthesis, gel electrophoresis, in situ hybridization, subcellular fractionation and polysome analysis. The long range goals of the program are to provide detailed information on some well defined processes of development and differentiation of the nervous system, so that we may better understand how they are perturbed by genetic or environmental influences. The results obtained should also lead to information concerning the generation of reactive astrocytes following injury and of oligodendro- cytes for remyelination.
Showing the most recent 10 out of 54 publications
