The longterm goal of this project is to develop new approaches to the treatment and understanding of neuropsychiatric diseases such as Parkinson's disease and Schizophrenia. The main objective of the present gant is to immortalize fetal rat mesencephalic dopaminergic cells to be used, initially, for transplantation studies in the rat model of hemiparkinsonism. Rat fetal cells from embryonic day-12 to day-16 will be immortalized with retroviral vectors containing myc oncogenes. These cells will first be characterized as their neuronal or glial characteristics using antibodies against neurofilament and glial fibrillary acid proteins. Subsequently, catecholaminergic neurons will be identified by tyrosine hydroxylase immunocytochemistry. Thereafter, the cells will be tested in vitro for their ability to synthesize, release, and take up dopamine (DA). In addition, assays for DA receptors will also be run using homogenates of these immortalized cells. Cells of interest which synthesize and release a significant amount of DA will be transplanted in the brains of rats that have received unilateral injections of 6-hydroxydopamine (6-OHDA) in the nigrostriatal pathway. The effects of these cells on circling behavior will then be assessed. The ability of these cells to survive, make connections with other striatal cells, and then affect the biochemistry of the basal ganglia will then be investigated. These studies will include the use of immunocytochemistry to evaluate cell survival and fiber outgrowth; receptor autoradiography to assess the status of DA receptors after transplantation; and in situ hybridization histochemistry to quantitate the effects of the cells on striatal proenkephalin and protachykinin mRNAs. In subsequent years, the ability of these cells to survive as well as synthesize higher amounts of DA will be potentiated by introducing the TH cDNA into immortalized fetal cells of mixed characteristics (glial/neuronal) and into immortalized DA neurons. The effects of these cells will then be studied at the behavioral, biochemical, and molecular levels using the rat model of hemiparkinsonism induced by 6-OHDA.
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