SEX STEROID ACTIVATION OF OPIOID CIRCUITS IN THE CNS

描述(申请者摘要)：我们的长期目标是了解 性腺(性)类固醇调节G蛋白偶联受体 神经传递是荷尔蒙激活行为的基础。在这些人中 研究最多的是下丘脑中的类固醇反应回路 规范女性生殖行为，前凸。初步结果 提示雌激素和黄体酮对类阿片有不同的作用。 受体(MOR)和阿片受体样孤儿受体(OFQR)。现在 该提案将研究雌激素和黄体酮转换的机制 从抑制到抑制的下丘脑回路的功能平衡 通过依次激活MOR和OFQR来促进前凸。 具体地说，拟议的实验将检验一个普遍的假设：雌激素。 增加MOR的传递和黄体酮减弱抑制的MOR 激活并增加OFQR的传递，导致前凸。这个 提案分为三个具体目标。第一，初步研究 表明雌激素作用于细胞内受体，导致内源性阿片类药物 释放导致MOR激活。因为有两种雌激素受体， ER-α和ER-β，一个根本的问题是哪个受体介导 雌激素诱导的效应。在ER-Alpha“敲除”(Erko-Alpha)小鼠中， 免疫细胞化学将被用来监测MOR的激活。就地 杂交技术将用于监测OFQR基因的表达水平。这些实验将 确定特定的雌激素受体在MOR激活和 OFQR的表达，可能为阿片类药物的类固醇调节提供线索 其他回路中的受体。其次，类固醇可能使阿片受体解偶联。 从细胞内信号级联改变生理平衡 MOR和OFQR。[35S]-GTPgS绑定将用于确定临时和 性激素对MOR和ORQR偶联G蛋白的部位特异性调节。 这将为阿片受体的功能耦合提供一种测量方法。 第三，几种内源性阿片肽激活MOR，包括脑啡肽， β-内啡肽和内吗啡。只有一种内源性配体被描述过 对于OFQR，孤儿FQ/伤害素，但OFQR也可能有几个配体。至 确定哪些内源性阿片类药物调节前凸，被动 免疫中和和行为分析将检查OFQR是否只是 由孤儿FQ/伤害素激活，以及MOR是否主要激活 通过内吗啡或其他内源性阿片类药物。这样的实验将在功能上 定义类固醇和阿片类药物调节前凸的相互作用，以及 演示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.