SEX STEROID ACTIVATION OF OPIOID CIRCUITS IN THE CNS

描述（申请人的摘要）：我们的长期目标是了解如何 性腺（性）类固醇调节G蛋白偶联受体介导的 神经传递是荷尔蒙激活行为的基础。中 研究得最好的类固醇反应回路是下丘脑中的那些， 调节女性生殖行为脊柱前凸初步结果 提示雌激素和孕激素对μ-阿片类药物具有不同作用 受体（莫尔）和阿片样受体样受体（OFQR）。本 一项提案将研究雌激素和孕激素转移的机制 下丘脑回路从抑制到 通过依次激活莫尔和OFQR促进脊柱前凸。 具体来说，拟议的实验将测试一个普遍的假设：雌激素 增加莫尔的传递，孕酮减弱抑制性莫尔 激活并增加OFQR的传输，导致脊柱前凸。的 建议分为三个具体目标。第一，初步研究 表明雌激素作用于细胞内受体引起内源性阿片样物质 释放导致莫尔激活。因为有两种雌激素受体， ER-α和ER-β，一个基本的问题是哪个受体介导 雌激素诱导的作用。在ER-α“敲除”（ERKO-α）小鼠中， 免疫细胞化学将用于监测莫尔活化。原位 杂交将用于监测OFQR mRNA水平。这些实验将 确定特定雌激素受体在莫尔激活中的作用， OFQR的表达，并可能提供线索类固醇调节阿片类药物 其他电路中的受体。其次，类固醇可能会使阿片受体解偶联 从细胞内信号级联改变之间的生理平衡 莫尔和OFQR。[35 S]-GTPgS结合将用于确定时间和 莫尔和ORQR与G蛋白偶联位点特异性类固醇调节。 这将提供阿片受体的功能偶联的量度。 第三，几种内源性阿片肽激活莫尔，包括：脑啡肽， β-内啡肽和内啡肽。只有一个内源性配体已被描述 对于OFQR，在FQ/伤害感受素中是一种配体，但OFQR也可能有几个配体。到 确定哪些内源性阿片类药物调节脊柱前凸，被动 免疫中和和行为分析将检查OFQR是否仅 FQ/痛敏肽中的MOR激活，以及莫尔是否主要被激活 由内吗啡肽或其他内源性阿片类药物引起。这些实验将在功能上 定义类固醇和阿片类药物调节脊柱前凸的相互作用，以及 展示了一类比较受体激活模式的分析， 和特定电路的行为输出。

DESCRIPTION (applicant's abstract): 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.