There is unequivocal evidence of inherited deficiencies in human central biogenic monoamine neurotransmission which are the direct result of genetic defects in tetrahydrobiopterin (BH4) metabolism. This evidence lends support to the theory of abnormal function within monoaminergic neurons as expressed in current hypotheses of the biology of schizophrenia and affective illness. Our understanding of the metabolism of BH4, the essential cofactor for tyrosine and tryptophan hydroxylase activity within monoaminergic neurons, is limited by the difficulty of biochemical studies using intact animals. The present proposal outlines a research plan designed to study BH4 metabolism within two anatomically defined populations of intact dopamine (DA) containing neurons derived from the embryonic mouse hypothalamus and mesencephalon and maintained in tissue culture. These studies will combine immunohistochemical, high performance liquid chromatographic and radionuclide tracer techniques with intact and cell-free preparations of theses cultures to investigate the cellular localization and metabolism of BH4. In addition, a series of experiments are proposed which are intended to determine the effect of chronically decreased BH4 content on DA levels and synthesis. Known inhibitors of BH4 synthesis will be tested for their capacity to acutely inhibit BH4 synthesis. Long term incubation with inhibitor(s) will then be undertaken, and BH4 content monitored. The functional impact of decreased intracellular BH4 content on DA levels and biosynthesis will be examined, as will the capacity of exogenous BH4 added to the incubation medium to reinstate DA function. It is hoped that these studies will help elucidate possible differences between DA neurons with respect to BH4 metabolism, and will also lead to the development of an intact cell system for the study of the interaction between DA biosynthesis and BH4 metabolism in DA neurons.
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