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A pure neuronal culture grown in a defined serum-free environment has been developed and characterized. Insulin was the only hormone found to enhance the growth of neurons obtained from embryonic chicken brains during the early proliferative stage, a time when many neurons survived without the addition of any growth factors to the culture. With increasing embryonic age, there was an increase in the number of neurons requiring transferrin. By the time neurons reached a postmitotic state in older brains, they were completely dependent on both insulin and transferrin for survival and growth. Because this culture is free of glial cells and serum, it provides an effective basis for investigating molecular mechanisms underlying neuronal development.Current understanding of neuronal development has progressed slowly due to the complexity of the developing brain, in which astrocytes, oligodendrocytes, neurons, and their precursors interact in an ever-changing chemical environment. This complexity has been a major obstacle to defining the stages and characterizing the biochemical changes through which neurons progress during development. Our approach towards unravelling the developmental process has been to isolate, culture, and study a population composed of a single cell type as it proliferates and differentiates. This investigation defines the requirements for neuronal growth in vitro and the changes occurring in these requirements as the neurons develop in vivo.We have studied 6-to 9-embryonic day (ED) chicken brains because they are composed of a nearly pure population of neuronal cells that during this period progress in vivo from a dividing to a postmitotic state (1-3). The majority of neuronal cultures previously described (4, 5) contain serum. Its ill-defined and variable composition and its ability to promote the proliferation of glial cells, which produce neuronal growth factors (6), have prevented investigators from defining the molecular growth requirements of neurons. The only previous attempt to culture chicken brain cells in a defined medium, after an exposure to serum, did not delineate the factors critical for neuronal survival and, in addition, did not characterize the cell types of the culture (7).In this study, we have investigated the essential growth requirements for pure neuronal populations obtained from embryos of various ages. We found these requirements to change with development. The neuronal cultures were characterized by immunocytochemical and biochemical means and were found to be essentially free of glial cells. This eliminated the possibility of the factors acting on, or via, glial cells and increases the effectiveness of this culture system for studying neuronal development. MATERIALS AND METHODSCell Culture Preparation. Neuronal cultures were prepared from 6-, 7-, 8-, or 9-ED brains. After the brains had been dissected from the chicken embryos and the meninges had been stripped off, the tissue was put in 1:1 mixture of Dulbecco-Vogt modified Eagle's medium and Ham's F-12 medium...

Myelin basic protein (MBP) is one of the most important myelin components. Based on our previous studies, we hypothesized that neurons might have regulatory effects on the production of MBP by oligodendrocytes, and we conducted studies designed to verify this hypothesis. Oligodendroglia-rich cultures from total brain of neonatal rats or mice and pure cultures of embryonic rats or chicks were prepared. Cultures of mouse fibroblasts and astrocytes were prepared as well. We show here that MBP production by oligodendrocytes was greatly enhanced by treatment with either pure neurons, rat neuronal conditioned medium, or chick neuronal conditioned medium, while chemically defined, hormonally supplemented medium or medium conditioned by astrocytes and fibroblasts had no effect on MBP expression. We conclude that the production of MBP by oligodendrocytes is regulated by a nonspecies specific soluble neuronal factor. The conservation of this phenomenon from avian to rodent species implies its critical role in myelination and suggests its potential application as a treatment in demyelination.

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