Estrogen has profound effects on the structure and function of the CNS. Recent evidence that estrogen has neuroprotective effects and enhances spine density in the hippocampus have made the study of estrogenic activation of the CNS an interesting and timely topic. Much of our understanding of estrogen's actions in the brain is derived from studying steroid responsive cells and circuits that regulate reproductive behaviors. The present proposal is focused on the interaction of two transmitter systems, cholecystokinin (CCK) and endogenous opioid peptides (EOP) in a classic steroid sensitive system that regulates lordosis, the limbic-hypothalamic circuit. These transmitters are highly regulated by estrogen, and both alter reproductive behavior. Our working hypothesis for this proposal is that estrogen alters the interaction among the EOP and CCK systems. Preliminary results suggest that delta opioid receptors mediate estrogen-induced CCK expression, and mu opioid receptors mediate CCK release. We propose five experiments to test parts of this general hypothesis. In the first experiment, we will determine the dependence of estrogen-induced CCK release on increased CCK mRNA expression. The second experiment, using receptor and GTPgammaS binding, will characterize the functional coupling of mu- and delta-opioid receptors in response to estrogen stimulation. The third experiment, using colocalization studies, will provide morphological evidence for direct versus indirect opioid effects on CCK cells. The fourth experiment, using microdialysis and behavioral testing, will provide direct evidence of a functional interaction of CCK and EOP. Finally, in experiment five, using both in vitro and in vivo assays, we will test the interaction of opioids at the CCK receptor and vice versa. These experiments will not only tell us about the actions of steroids in specific circuits, but they will also provide important information about the interactions of neuropeptide circuits regulating behavior.
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