The mammalian brain is composed of a variety of cell types which are characterized by unique functions, as well as complex and specific spatial and temporal interactions with other cells. This complexity is reflected in the 3-4 fold higher level of transcription of the genome in brain than in other organs. Recombinant DNA technology and a combination of in situ hybridization and immunohistochemistry techniques now make it possible to study the complex functions of individual brain cells. By constructing and using recombinant DNA libraries to DNAs isolated from neonate and adult rat cerebellum, it will be possible to precisely quantitate the changes in RNA transcription patterns which take place during development, and to determine the extent to which the genes expressed are unique to this brain area. The differential gene expression of individual cell types will then be analyzed by in situ hybridization. Using this technique, the different cell types in cerebellum will be analyzed throughout development for their content of specific messenger RNAs (mRNAs) so as to quantitate the relative abundance of these mRNAs in the different cell types, and to determine whether the mRNAs of an individual cell type fluctuate more dramatically in response to developmental signals than can be quantitated at the tissue level. The induction of differentiation-specific mRNAs in individual cell types will be precisely correlated with developmental events such as cell division, migration, and the development of specific synaptic inputs. As it is possible that the individual cell types themselves may be more biochemically heterogeneous than is suggested by their similar morphology, in situ hybridization with recombinant DNA probes for mRNAs encoding putative neurotransmitter enzymes will determine whether or not a single cell type such as the cerebellar Purkinje cell is biochemically heterogeneous. These techniques will also be used to determine the transcriptional changes which accompany synaptic transmission. The cerebellar Purkinje cell, with its unique and well characterized circuitry and biochemistry, will again be used as a model to determine the effects that its specific synaptic inputs have on gene expression. Completion of these specific aims will provide the answers to several fundamental questions in neurobiology, as well as significantly advance our understanding of abnormal brain development. They also significantly contribute to our understanding of events underlying neuronal malfunction and loss.
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