Opioid Receptors in Excitatory Synapses

描述（由申请人提供）：慢性阿片类药物滥用损害神经元发育，并导致成瘾者的认知缺陷。除了作用于抑制性GABA能突触外，我们最近的研究发现μ阿片受体（莫尔）还可以调节主要是兴奋性GABA能突触的树突棘的稳定性。由于吗啡耐受性和依赖性已被报道在缺乏AMPA受体亚单位的转基因小鼠中减少，这种树突棘稳定性的变化可能有助于阿片成瘾。这个项目的总体目标是阐明参与阿片类调节树突棘的细胞内信号通路。基于我们和其他人的研究，我们提出了以下中心假设模型：兴奋性突触中突触后阿片受体的激活抑制了蛋白激酶和Rho GTP酶的活性。特别地，Rac 1（一种Rho GT酶）的抑制通过改变肌动蛋白细胞骨架而引起树突状突起和棘的随后的塌陷。结合遗传，活体成像和电生理技术将被用来测试这个模型在培养的解离神经元。我们将通过追求三个具体目标来测试这个模型。1.我们将进一步描述MORs及其内化在树突棘突触后调制中的作用。已经提出受体内化和脊柱可塑性对于药物成瘾和耐受性发展是重要的。阐明这两个关键细胞事件之间的机制联系可能会为我们对阿片类药物成瘾的理解提供新的线索。我们还将确定为什么一些临床相关的阿片类药物，如美沙酮，导致树突棘的浓度依赖性双相变化。临床相关阿片类药物的剂量反应研究可能会在未来潜在地减少这些阿片类药物的成瘾倾向。2.我们将鉴定和确定Rho GTdR介导阿片样物质对树突棘的调节。这一目标将确定吗啡如何通过改变肌动蛋白细胞骨架来诱导脊柱的可塑性。3.我们将鉴定并确定介导阿片类调节树突棘的蛋白激酶。该目标将确定莫尔和Rac 1之间的信令链路。通过完成这些特定的目标，我们将确定介导吗啡诱导的脊髓塌陷的三个主要信号步骤：介导吗啡效应的受体;介导吗啡反应的蛋白激酶;以及有助于吗啡效应的肌动蛋白相互作用蛋白。这些信号步骤的确定将提供一个主要的框架，负责阿片类药物诱导的兴奋性突触的变化的信号通路，从而将提供新的信息阿片类药物滥用在分子和细胞水平。

DESCRIPTION (provided by applicant): Chronic opiate abuse impairs neuronal development and causes cognitive deficits in addicts. In addition to acting on inhibitory GABAergic synapses, our recent study revealed that mu-opioid receptors (MOR) could regulate the stability of the dendritic spines, which are mainly excitatory glutamatergic synapses. Since morphine tolerance and dependence have been reported to decrease in transgenic mice lacking AMPA receptor subunits, changes in the stability of such dendritic spines may contribute to opiate addiction. The overall objective of this proposed project is to clarify the intracellular signaling pathways that are involved in opioid regulation of dendritic spines. Based on our and other's previous studies, we proposed the following central hypothetical model: Activation of the postsynaptic opioid receptors in excitatory synapses inhibits the activities of protein kinases and Rho GTPases. Particularly, the inhibition of Rac 1, a Rho GTPase, causes the subsequent collapse of dendritic protrusions and spines by altering actin cytoskeleton. Combined genetic, living imaging and electrophysiological techniques will be used to test this model in cultured dissociated neurons. We will test this model by pursuing three specific aims. 1. We will further characterize the roles of MORs and their internalization in postsynaptic modulation of dendritic spines. Both receptor internalization and spine plasticity have been proposed to be important for drug addiction and tolerance development. The elucidation of the mechanistic links between these two critical cellular events may shed new light on our understanding of opiate addiction. We will also determine why some clinical relevant opiates, such as methadone, cause concentration-dependent biphasic changes in dendritic spines. The dosage-response studies of clinical relevant opiates may potentially reduce addictive liability of these opiates in the future. 2. We will identify and determine the Rho GTPase that mediates opioid modulation of dendritic spines. This aim will determine how morphine induces plasticity of spines by altering the actin cytoskeleton. 3. We will identify and determine the protein kinase(s) that mediates opioid modulation of dendritic spines. This aim will determine the signaling link between MOR and Rac1. By completing these specific aims, we will have determined three major signaling steps that mediate morphine-induced collapse of spines: the receptor that mediates morphine's effect; the protein kinase(s) that mediates the morphine response; and the actin-interacting proteins that contribute to morphine's effect. The determination of these signaling steps will provide a major framework of the signaling pathway that is responsible for opioid-induced changes in the excitatory synapses, and thus will provide new information about opiate abuse at a molecular and cellular level.