Our long term goal is to understand how gonadal (sex) steroids regulate G protein-coupled receptor mediated neurotransmission that underlies hormonal activation of behavior. Among the best studied steroid-responsive circuits are those in the hypothalamus that regulate the female reproductive behavior, lordosis. Preliminary results suggest that estrogen and progesterone have differential effects on mu-opioid receptors (MOR) and opioid receptor-like orphanin receptor (OFQR). The present proposal will examine mechanisms through which estrogen and progesterone shift the functional balance of a hypothalamic circuit from inhibition to facilitation of lordosis by sequentially activating MOR and then OFQR. Specifically, the proposed experiments will test a general hypothesis: estrogen increases transmission at MOR and progesterone attenuates the inhibitory MOR activation and increases transmission at OFQR resulting in lordosis. The proposal is divided into three specific aims. First, preliminary studies indicate estrogen acts at intracellular receptors to cause endogenous opioid release resulting in MOR activation. Because there are two estrogen receptors, ER-alpha and ER-beta, a fundamental question is which receptor mediates estrogen-induced effects. In the ER-alpha 'knock-out' (ERKO-alpha) mouse, immunocytochemistry will be used to monitor MOR activation. In situ hybridization will be used to monitor OFQR mRNA levels. These experiments will determine the role of specific estrogen receptors underlying MOR activation and OFQR expression and may provide clues about steroid regulation of opioid receptors in other circuits. Second, steroids may uncouple opioid receptors from intracellular signaling cascades altering the physiologic balance between MOR and OFQR. [35S]-GTPgS binding will be used to determine the temporal and site-specific sex steroid regulation of MOR and ORQR coupling to G proteins. This will provide a measure of functional coupling of the opioid receptors. Third, several endogenous opioid peptides activate MOR, including: enkephalins, beta-endorphin and endomorphins. Only one endogenous ligand has been described for OFQR, orphanin FQ/nociceptin, but OFQR may also have several ligands. To determine which endogenous opioids regulate lordosis, passive immunoneutralization and behavioral analysis will examine whether OFQR is only activated by orphanin FQ/nociceptin, and whether MOR is principally activated by endomorphins or other endogenous opioids. Such experiments will functionally define the interaction of steroids and opioids modulating lordosis, and demonstrate a category of analyses that compare patterns of receptor activation and behavioral output of specific circuits.
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