The long-term goal of the proposal is to understand the cellular actions of estrogen and morphine on hypothalamic neurosecretory neurons which control anterior pituitary secretions. The application is based upon an ongoing NIDA- funded research program regarding the electrophysiology of hypothalamic neurosecretory neurons. The Principal Investigator is now seeking to increase the power of his techniques by including whole-cell patch recordings. This will greatly expand the candidate's research capabilities and is in keeping with his career development plans. The proposal has a training and research component. The award would allow the applicant to increase his time in the laboratory and to receive training in vital new biological techniques. The research component of the application seeks to further our understanding of the role of estrogens in controlling neurosecretory neurons of the hypothalamus. Since estrogen appears to decrease the number of functional mu opioid receptors in a manner similar to chronic morphine treatment, the research focuses on the interaction of and the common cellular components affected by both agents. Hypothalamic slices will be prepared from ovariectomized female guinea pigs which have been treated with morphine or placebo. The first series of experiments will study the interaction of chronic morphine in vivo and acute estrogen in vitro in altering the mu opioid mediated membrane hyperpolarization of identified arcuate (ARC) neurons by measuring the shift in the mu opioid dose-response curve in the two groups of animals.Cells from morphine- treated or placebo- treated, ovariectomized (oil-treated) guinea pigs will be tested with mu opioid agonists before and after perfusion with 17 Beta-estradiol. The time- course and site of estrogen's rapid actions to decrease the potency of the mu opioid response will also be determined. Finally, the effects of chronic morphine on a heterologous receptor response (GABA-B) which is coupled to the inwardly rectifying K channel and affected by estrogen will be measured. Other experiments will study the effects of mu opioid agonists on supraoptic (SON) vasopressin and oxytocin magnocellular neurons by ascertaining the specific K and Ca conductances affected by mu opioid agonists and characterize the interaction of estrogen and chronic morphine on SON neurons.A third series of experiments will investigate the cellular mechanism by which estrogen and morphine decrease the potency of mu opioid agonists to hyperpolarize ARC (SON) neurons by measuring the expression of G-alpha-i and G-alpha-o mRNA using in situ hybridization and immunocytochemistry to identify the cells. These experiments will be followed by whole cell patch recording from ARC (SON) neurons to study the role of G-proteins in the estrogen- mediated attenuation of the mu opioid response.
