Role of PSD-95 in Synaptic and Drug Induced Plasticity in Dopamine Neurons

描述（由申请人提供）：在此提议中，我描述了一系列实验，将调查PSD-95在腹侧被盖区（VTA）多巴胺神经元中的突触传递、突触可塑性和药物诱导的突触可塑性中的作用。这将涉及在从PSD-95敲除或野生型小鼠制备的切片制备物中从VTA多巴胺神经元进行全细胞膜片钳记录。我还将进行分子“拯救”实验，这将需要使用病毒介导的基因转移，以表达融合到GFP的重组PSD-95在VTA多巴胺细胞从PSD-95敲除小鼠。在腹侧被盖区多巴胺神经元中已经描述了多种形式的长时程增强（LTP）和长时程抑制（LTD），并且已知滥用药物的体内给药可以在这些细胞中引起突触可塑性。然而，人们对这些现象背后的分子机制知之甚少。大量研究表明，突触后支架蛋白PSD-95在兴奋性突触功能和可塑性中起着重要作用。此外，PSD-95基因敲除小鼠最近被证明具有药物诱导的行为可塑性的改变。然而，PSD-95在腹侧被盖区多巴胺神经元中的突触和药物诱导的可塑性中的作用尚未被研究。我将首先调查是否基础突触传递的PSD-95基因敲除小鼠腹侧被盖区多巴胺神经元的改变。然后，我将测试PSD-95敲除小鼠是否在这些细胞中表现出正常的LTP和LTD。接下来，我将确定是否PSD-95敲除改变了药物诱导的可塑性，在腹侧被盖区多巴胺神经元在体内管理以前的滥用药物的特点。最后，我将测试是否观察到的突触功能和可塑性的PSD-95敲除的改变可以通过重组PSD- 95在腹侧被盖区多巴胺神经元的表达来挽救。这将使用表达与GFP融合的PSD-95的慢病毒来实现，所述慢病毒将立体定位地注射到VTA中。这些实验的结果应显着提高我们的知识的基础上的VTA多巴胺神经元的突触可塑性的分子机制，以及PSD-95在药物诱导的可塑性的作用。通过进一步了解大脑的奖赏回路是如何工作的，我们可以更好地了解导致成瘾的大脑机制，从而为这种毁灭性的疾病开发更好的治疗方法。这项资助申请中提出的研究旨在阐明奖励回路中发生的一些药物诱导的分子变化，这些变化是定义成瘾的持久行为改变的基础。

DESCRIPTION (provided by applicant): In this proposal, I describe a series of experiments that will investigate the role of PSD-95 in synaptic transmission, synaptic plasticity and drug-induced synaptic plasticity in dopamine neurons in the ventral tegmental area (VTA). This will involve making whole-cell patch clamp recordings from VTA dopamine neurons in a slice preparation prepared from PSD-95 knockout or wildtype mice. I will also perform molecular "rescue" experiments that will entail using viral-mediated gene transfer to express recombinant PSD-95 fused to GFP in VTA dopamine cells from PSD-95 knockout mice. Multiple forms of long-term potentiation (LTP) and long-term depression (LTD) have been described in VTA dopamine neurons and it is known that in vivo administration of drugs of abuse can elicit synaptic plasticity in these cells. Yet little is known about the molecular mechanisms underlying these phenomena. An extensive body of work primarily on hippocampal synapses suggests that the postsynaptic scaffolding protein, PSD-95, plays an important role in excitatory synaptic function and plasticity. Additionally, PSD-95 knockout mice have recently been shown to have alterations in drug-induced behavioral plasticity. The role of PSD-95 in synaptic and drug induced plasticity in VTA dopamine neurons, however, has not been examined. I will first investigate whether basal synaptic transmission is altered in VTA dopamine neurons from PSD-95 knockout mice. I will then test whether PSD-95 knockout mice exhibit normal LTP and LTD in these cells. Next I will determine if PSD-95 knockouts have altered drug-induced plasticity in VTA dopamine neurons in response to in vivo administration of previously characterized drugs of abuse. Finally, I will test whether observed alterations in synaptic function and plasticity in the PSD-95 knockout can be rescued by expression of recombinant PSD- 95 in VTA dopamine neurons. This will be accomplished using a lentivirus expressing PSD-95 fused to GFP that will be stereotaxically injected into the VTA. The results of these experiments should significantly enhance our knowledge of the molecular mechanisms underlying synaptic plasticity in VTA dopamine neurons as well as the role of PSD-95 in drug-induced plasticity. By furthering our understanding of how the reward circuitry of the brain works we can better understand the brain mechanisms that lead to addiction and thereby also develop better treatments for this devastating illness. The research proposed in this grant application is aimed at elucidating some of the drug-induced molecular changes that occur in the reward circuitry, which underlie the lasting behavioral modifications that define addiction.