The proposed studies will test the hypothesis that high circulating prolactin (PRL) levels inhibit cyclic LH surges by an action on the brain. High PRL may inhibit LH surges by any of the following mechanisms. 1. Hyperprolactinemia could abolish the specific neurotransmitter signal(s) required for cyclic LH release. We will examine the pattern of turnover of changes in norepinephrine, dopamine and serotonin in discrete brain regions before and during LH surges. 2. Hyperprolactinemia could disrupt the rhythm of the """"""""biological clock"""""""" upon which all cyclic physiological functions, including LH surges, depend. We will examine the circadian rhythms in serotonin turnover, and Alpha- and Beta-adrenergic serotonergic receptors, particularly in the suprachiasmatic nucleus, because these are indices of the biological clock which may directly or indirectly affect LH release. 3. Hyperprolactinemia could affect LHRH-containing neurons to alter the amount and/or the kinetics of LHRH release. We will study LHRH release kinetics from synaptosomes prepared from the median eminence or from medial basal hypothalamic slices. The following methods will be used. A microdissection technique allows for the removal of discrete nuclei, fiber pathways and terminal projection fields from rat brain. We will estimate norepinephrine and dopamine turnover by measuring neurotransmitter concentrations before and after synthesis inhibition. Serotonin turnover will be estimated by assessment of 5-hydroxyindoleacetic acid/serotonin ratios and 5HT accumulation after inhibition of metabolism. High pressure liquid chromatography with electrochemical detection allows for quantitation of all of these compounds within samples from individual rats. Neurotransmitter receptors will be localized and quantitated using topical quantitative autoradiography. The ability of depolarizing stimuli to evoke LHRH release from median eminence synaptosomes or medial basal hypothalamic slices will be examined. The animal model to be used is the estadiol-treated ovariectomized rat. Our previous work demonstrates that elevated PRL can abolish cyclic LH release depending upon the estradiol treatment paradigm. This animal model allows us to investigate the direct neural effects of hyperprolactinemia on cyclic LH release in the absence of confounding ovarian effects.
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