Many gonadal-steroid dependent neuroendocrine events and behaviors are thought to be mediated by steroid hormones acting through preoptic, hypothalamic and limbic system neurons which contain steroid-specific receptor proteins. The experiments in this proposal are designed to reveal, on a cell-by-cell basis (IA) the extrahypothalamic axonal connections of gonadal steroid hormone-concentrating neurons by using steroid autoradiography combined with retrograde neuroanatomical tracing. These experiments will reveal exactly how steroid concentrating neurons fit in neuroendocrinologically of behaviorally relevant neural circuits. Other experiments (IB) are designed to detect the chemical content, neurotransmitter or neuropeptide-related, of gonadal steroid hormone-concentrating neurons, also on a cell-by-cell basis, using steroid autoradioagraphy combined with immunocytochemistry. These neurons could be part of the mechanism by which gonadal steroid hormones influence neurotransmitter or neuropeptide regulation of hypothalamic releasing hormones, which subsequently effect anterior pituitary secretion. Experiments (II) designed to examine the neuroendocrine properties of intrinsic neurons of the POA or medial basal hypothalamus (MBH) will identify steroid-concentrating capacity, or neurotransmitter or neuropeptide content, of neurons that survive surgical isolation of islands of POA or MBH. The physiologic importance of the intrinsic hypothalamic neurons may be considerable; cell-by-cell analysis of their properties has yet to be carried out. The action of steroid hormones on the genome of cells that contain specific steroid hormone receptors is the best documented mechanism by which steroids influence cells. Experiments (III) designed to optimize the use of in situ hybridization with cDNA probes on cells of the brain will be carried out using probes to 1) prooxytocin; 2) provasopressin and 3) tyrosine hydroxylase. Then, these procedures will be used in a paradigm designed to demonstrate qualitatively and quantitatively the effects of physiological state or steroid hormones on mRNA levels of hypothalamic neurons, detected in situ with the cDNA probes. Limited attempts (IV) to analyze the estradiol receptor (E-R) content of hypothalamic neurons with immunocytochemical methods using primary antibodies to the E-R will be carried out. The long term goal of all these experiments is to reveal, on a cell-by-cell basis, the characteristics of gonadal.
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