Opioid Receptors in Excitatory Synapses

描述（由申请人提供）：长期阿片类药物滥用损害神经元发育并导致成瘾者认知缺陷。除了作用于抑制性gaba能突触外，我们最近的研究发现mu-阿片受体（MOR）还可以调节树突棘的稳定性，而树突棘主要是兴奋性谷氨酸能突触。由于缺乏AMPA受体亚基的转基因小鼠吗啡耐受性和依赖性下降，因此这种树突棘稳定性的变化可能有助于阿片成瘾。本项目的总体目标是阐明参与阿片调节树突棘的细胞内信号通路。基于我们和其他人之前的研究，我们提出了以下中心假设模型：兴奋性突触突触后阿片受体的激活抑制蛋白激酶和Rho gtpase的活性。特别是，Rho GTPase Rac 1的抑制，通过改变肌动蛋白细胞骨架，导致随后的树突和棘的崩溃。结合遗传、活体成像和电生理技术，将在培养的游离神经元中测试该模型。我们将通过追求三个具体目标来检验这一模式。1. 我们将进一步描述MORs及其内化在树突棘突触后调节中的作用。受体内化和脊柱可塑性都被认为是药物成瘾和耐受性发展的重要因素。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，会引起树突棘浓度依赖性的双相变化。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 GTPase介导树突棘的阿片调节。这一目标将确定吗啡如何通过改变肌动蛋白细胞骨架诱导脊柱可塑性。3. 我们将鉴定和确定介导树突棘阿片调节的蛋白激酶。这一目的将确定MOR与Rac1之间的信号通路。通过完成这些具体目标，我们将确定介导吗啡诱导的脊柱塌陷的三个主要信号传导步骤：介导吗啡作用的受体；介导吗啡反应的蛋白激酶；以及与肌动蛋白相互作用的蛋白质对吗啡的作用起作用。这些信号步骤的确定将提供负责阿片类药物诱导的兴奋性突触变化的信号通路的主要框架，从而将在分子和细胞水平上提供有关阿片类药物滥用的新信息。

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.