To analyze the interdependence of neurons and astroglia during central nervous system development, a rapid method for purifying early postnatal cerebellar neurons and astroglia, and recombining them in vitro, has been developed. The influence of neurons on astroglial shape and proliferation has been evaluated with an in vitro model system previously used to describe the role of cerebellar astroglia in neuronal migration and positioning (Hatten, M. E., and R. K. H. Liem, 1981, J. Cell Biol., 90:622-630; and Hatten, M. E., R. K. H. Liem, and C. A. Mason, 1984, J. Cell Biol., 98:193-204. Cerebellar tissue harvested from C57Bl/6J mouse cerebellum on the third or fourth day postnatal was dissociated into a single cell suspension with trypsin, and enriched glial and neuronal fractions were separated with a step gradient of Percoll. Highly purified astroglial and neuronal fractions resulted from subsequently preplanting the cells on a polylysine-coated culture surface. In the absence of neurons, astroglia, identified by staining with antisera raised against purified glial filament protein, assumed a flattened shape and proliferated rapidly. In the absence of astroglia, cerebellar neurons, identified by staining with antisera raised against the nerve growth factor-inducible large external (NILE) glycoprotein and by electron microscopy, formed cellular reaggregates, had markedly impaired neurite outgrowth, and survived poorly. When purified neurons and isolated astroglia were recombined, astroglial proliferation slowed markedly and the flattened shape expressed in the absence of neurons transformed into highly elongated profiles that resembled embryonic forms of cerebellar astroglia. After longer periods (48-72 h) in the presence of neurons, astroglia had "Bergmann-like" or "astrocyte-like" shapes and neurons commonly associated with them. These results suggest that neurons influence the differentiation of astroglia.

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