We propose to investigate the molecular basis of the impaired ability of hypothalamic dopaminergic neurons of the aged brain to synthesize L-dihydroxyphenylalanine (DOPA), to release dopamine, and to respond to prolactin stimulation. We shall investigate the hypothesis that this impaired ability is a consequence of a deficiency of exposed prolactin receptors (PR) exists in the plasma membranes of aged dopaminergic neurons compared to young dopaminergic neurons and that such a deficiency is a result of altered fludity of the lipid membranes of the neurons. To do so, the number of exposed PR in the aging hypothalamus will be evaluated using radiolabelled monoclonal antibodies to prolactin, and the effects of alterations of lipid membrane fluidity on the available number of PR will be investigated. Lipid membrane fluidity will be changed by modifying the cholesterol-to-phospholipid ratio of the membranes. The consequences of such modifications on the responses of aging neurons to prolactin stimulation will be investigated by examining the intracellular events involved in the synthesis of dopamine, the in vitro synthesis (in organ culture) of tyrosine hydroxylase (TH), the transformation of TH from an inactive form through phosphorylation to an active form, the specific activity of TH per mole of the enzyme, the synthesis of DOPA, and the release of dopamine into hypophysial portal blood. Monoclonal antibodies against TH and against PR as well as affinity-purified, monospecific polyclonal antibodies against prolactin will be utilized in these stuides. These proteins will be used in blocking experiments as well as experiments in which isolation and/or quantification of TH and PR are undertaken. The activity of TH in vivo will be assessed by quantifying the rate of synthesis of DOPA in the median eminence. The rate of release of dopamine by hypothalamic dopaminergic neurons will be quantified by determining the rate of release of dopamine into hypophysial portal blood. Using these various approaches, we propose to ascertain the extent to which down-regulation of PR in the hypothalamus of the aging brain is involved in the reduced capacity of the aged brain to synthesize DOPA and release dopamine.
NIH ExPorter
NIH ExPorter