MOLECULAR AND CELLULAR BASIS OF OPIATE ACTION IN THE LOCUS COERULEUS

蓝斑(LC)的去甲肾上腺素能神经元已被牵连 在调节身体鸦片类药物依赖和戒断方面。在过去的10年中 多年来，内斯特勒、阿加贾尼安、阿雷贾和杜曼博士的实验室 进行了高度集成的生化和电生理 研究潜在的分子和细胞机制 这些神经元对阿片类药物的耐受、依赖和戒断 将这些发现与阿片类药物戒断的行为表现联系起来。 一个主要的发现是，长期服用阿片类药物会上调 LC中受体和受体之间几个主要步骤的cAMP通路 生理反应，腺酰环化酶、蛋白质升高 蛋白激酶A(PKA)和几种蛋白激酶底物，包括 酪氨酸羟基酶和cAMP反应元件结合蛋白 主要的cAMP调节转录因子。现在有几条证据 支持这样的观点，即cAMP途径的这种上调有助于 LC神经元表现出对阿片类药物的耐受、依赖和戒断。 拟议的研究将进一步确定营地的作用。 阿片类药物作用的途径。一个主要目标是确定特定的 腺酰环化酶的亚型和PKA的特定亚基是 吗啡处理后LC的上调，并探讨其作用机制。 这种上调发生的分子机制。我们有 特异性腺酰环化酶和PKA亚基表达上调 在蛋白质和信使核糖核酸水平上，表明这些适应可能 至少部分发生在基因表达水平上。的确， 初步调查提供了直接证据表明，一些人，但 并不是所有这些吗啡诱导的适应都是通过 CREB中的变化。我们将进一步推动CREB在阿片类药物中的作用 使用病毒介导的基因转移的作用，其中CREB或特定的 CREB抑制因子在LC神经元中高表达。我们将利用小说 转基因小鼠，目前正在转基因核心中开发，其中 CREB或CREB抑制物在LC中高表达 时尚。第二个主要目标是探索阿片类药物耐受的机制。 在LC神经元中。我们将重点介绍GRKs(G蛋白受体)的作用 激酶)和RGS蛋白(G蛋白信号调节因子)作为新的 受体-G蛋白解偶联的机制可能与耐受有关。 总之，拟议的分子、细胞和行为研究有望 以促进我们对长期适应的理解 阿片类药物对LC神经元有诱导作用。

The noradrenergic neurons of the locus coeruleus (LC) have been implicated in mediating physical opiate dependence and withdrawal. Over the last 10 years, the laboratories of Drs. Nestler, Aghajanian, Alreja, and Duman have carried out highly integrated biochemical and electrophysiological studies to investigate the molecular and cellular mechanisms underlying opiate tolerance, dependence, and withdrawal in these neurons and to relate these findings to behavioral manifestations of opiate withdrawal. One major finding is that chronic opiate administration up regulates the cAMP pathway in the LC at several major steps between receptor and physiological response, with increases seen for adenylyl cyclase, protein kinase A (PKA), and several substrates for the protein kinase, including tyrosine hydroxylase and CREB (cAMP-response element binding protein), a major cAMP-regulated transcription factor. Several lines of evidence now support the view that this upregulation of the cAMP pathway contributes to opiate tolerance, dependence, and withdrawal exhibited by LC neurons. The proposed studies will further characterize the role of the cAMP pathway in opiate action. One major aim is to identify the specific subtypes of adenylyl cyclase and specific subunits of PKA that are upregulated in the LC by morphine treatment and to investigate the molecular mechanisms by which this upregulation occurs. We have demonstrated upregulation of specific adenylyl cyclases and PKA subunits at the protein and mRNA levels, suggesting that these adaptations may occur, at least in part, at the level of gene expression. Indeed, preliminary investigations have provided direct evidence that some, but not all, of these morphine-induced adaptations are mediated via alterations in CREB. We will further pursue a role for CREB in opiate action by use of viral-mediated gene transfer where CREB or a specific inhibitor of CREB are over expressed in LC neurons. We will utilize novel transgenic mice, currently in development in the Transgenic Core, in which CREB or the CREB inhibitor are over expressed in the LC in an inducible fashion. A second major aim is to explore mechanisms for opiate tolerance in LC neurons. We will focus on a role for GRKs (G protein receptor kinases) and for RGS proteins (regulators of G protein signaling) as novel mechanisms of receptor-G protein uncoupling possibly related to tolerance. Together, the proposed molecular, cellular, and behavioral studies promise to advance our understanding of the long-term adaptations that chronic opiates induce in LC neurons.