Immunohistochemical localization of myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase in flattened membrane expansions produced by cultured oligodendrocytes

Abstract Oligodendrocytes, the cells responsible for myelin sheath formation in the central nervous system, were isolated from primary dissociated mixed glial cultures prepared from newborn mouse forebrain, and further cultured in a serum-free defined culture medium. Single and double indirect immunofluorescence using antibodies against the myelin glycolipids, galactocerebroside and sulfatide, and the myelin proteins, myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase, was used to investigate the composition of the flat membrane extensions produced by some oligodendrocytes in culture. Galactocerebroside and sulfatide were both expressed on the external surface of the plasma membrane of oligodendrocyte cell bodies and processes and also the membrane expansions. Neither myelin basic protein nor 2',3'-cyclic nucleotide 3'-phosphohydrolase were expressed on the external surface of oligodendrocytes. Myelin basic protein could be localized to the cell body and the membrane expansions but not the major and fine processes. The localization of these myelin components suggests that the expansions have characteristics of the mature myelin membrane. 2',3'-Cyclic nucleotide 3'-phosphohydrolase was found to be localized in the cell body, and in total contrast to myelin basic protein, in the major processes and the fine interconnecting processes, but not the membrane expansions. In some of the cells 2',3'-cyclic nucleotide 3'-phosphohydrolase was present at the outer extremities of the flat membrane sheets, giving the appearance of an extending growth region. Our results thus clearly show that 2',3'-cyclic nucleotide 3'-phosphohydrolase is localized within oligodendrocytes in discrete regions of plasma membranes and suggest that this protein has a possible role in the early stages of myelin formation.