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与 在调节阿片类药物依赖和戒断方面的作用。在过去10 多年来，Nestler、Aghajanian、Alreja和Duman博士的实验室 进行了高度整合的生化和电生理学研究， 研究，以探讨潜在的分子和细胞机制 阿片耐受性，依赖性和戒断在这些神经元， 将这些发现与阿片戒断的行为表现联系起来。 一个主要的发现是，慢性阿片类药物管理上调 LC中cAMP途径在受体和受体之间的几个主要步骤 生理反应，腺苷酸环化酶、蛋白质 激酶A（PKA）和蛋白激酶的几种底物，包括 酪氨酸羟化酶和CREB（cAMP反应元件结合蛋白）， 主要cAMP调节转录因子。现在有几条证据 支持这样的观点，即cAMP途径的上调有助于 LC神经元表现出阿片耐受、依赖和戒断。 拟议的研究将进一步表征cAMP的作用， 阿片类药物作用的途径。一个主要目标是确定具体的 腺苷酸环化酶的亚型和PKA的特定亚基， 通过吗啡处理上调LC，并研究 这种上调发生的分子机制。我们有 证实了特异性腺苷酸环化酶和PKA亚单位的上调 在蛋白质和mRNA水平，这表明这些适应可能 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， 并非所有这些吗啡诱导的适应都是通过 CREB的变化我们将进一步寻求CREB在阿片类药物中的作用， 通过使用病毒介导的基因转移的作用，其中CREB或特异性 CREB抑制剂在LC神经元中过表达。我们将利用小说 转基因小鼠，目前在转基因核心的发展，其中 CREB或CREB抑制剂在LC中以诱导型过度表达。 时尚.第二个主要目标是探索阿片耐受的机制 在LC神经元中。我们将重点关注GRKs（G蛋白受体）的作用 激酶）和RGS蛋白（G蛋白信号传导的调节剂）作为新的 受体-G蛋白解偶联的机制可能与耐受有关。 总之，所提出的分子、细胞和行为研究有望 来增进我们对长期适应的理解， 阿片类药物诱导LC神经元。

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.