DEVELOPMENT OF HYPOTHALAMIC NEURONS IN VITRO

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The objective of this project is to increase our understanding of the role of genetic and epigenetic and epigenetic influences on neuronal growth and development in the mammalian central nervous system. The development of neurons of the hypothalamus, a region of the brain involved in homeostatic regulation, will be followed in tissue culture. Dissociated neurons from the embryonic rat hypothalamus will be cultured in serum- free conditions using a method we have developed, and their growth and development monitored using quantitative morphometric techniques. The expression of specific neuronal phenotypes and of neurotransmitter receptors will be monitored using appropriate monoclonal and monospecific antibodies. Alterations in these parameters as a result of treatment with hormones and growth factors and by different culture substrates will provide evidence for a specific epigenetic influence of these treatments; a stable pattern of gene expression will provide evidence for genetic control. The effect of the culture procedure on neuronal survival and development will also be explored using 3H-thymidine as a marker of neuronal "birthdate". Alterations in neuronal survival or neuronal phenotype as a function of the timing of dissociation will be evaluated. The result of these experiments will be an appreciation of effects of the extracellular environment, including growth factors and hormones, on hypothalamic neuronal development.