The present proposal will use a multidisciplinary approach to further elucidate the physiological events leading to the proestrus surge of prolactin (PRL) and the mechanism by which the PRL surge is coupled to the proestrus surge of luteinizing hormone (LH). Findings from ongoing experiments suggest that the suprachiasmatic nucleus (SCN) and the medial preoptic nucleus (MPN) are both responsible for the PRL and LH surge. It is also apparent that interactions between the two structures generate a signal which directly or indirectly regulates the luteinizing hormone releasing hormone (LHRH) neurons. Initial experiments are designed to explore the effects of SCN or MPN lesions on the ability of progesterone to induce the preovulatory LH and PRL surge. Secondly, we will elucidate the effects of the lesions on the hypothalamic content of LHRH and preproLHRH. We will also explore through Northern blot (RNA) and in situ hybridization the synthetic activity of the LHRH neurons during proestrus as compared to estrus, and evaluate the effects of SCN or MPN lesions on the LHRH synthetic activity. Thereafter we will initiate in vivo studies to explore the sensitivity of the hypothalamic-pituitary axis to estrogen following MPN/SCN lesions. In vitro slice preparation experiments will continue to study the electrophysiological properties of the SCN/MPN neurons. We will use intracellular and patch-clamp recording techniques to determine the synaptic input, the chemosensitivity to the neurotransmitters/neuromodulators (serotonin 5HT), norepinephrine/epinephrine (NE/E), acetylcholine (Ach), dopamine and Beta-endorphin (Beta-end)), and the effects of estrogen on membrane excitability of SCN/MPN neurons. Intracellular staining with procion yellow (PY) will be done in conjunction with immunocyto-chemistry to identify the morphology and cytochemistry of these neurons. Finally, we will use the sagittal slice preparation to study the electrophysiological interaction between the MPN and the SCN to elucidate the basic mechanism by which the two structures initiate the signal leading to the proestrus surge of LH and PRL.
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