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

描述（由申请人提供）：在本提案中，我描述了一系列实验，将研究PSD-95在腹侧被盖区（VTA）多巴胺神经元突触传递、突触可塑性和药物诱导的突触可塑性中的作用。这将涉及对PSD-95敲除或野生型小鼠制备的切片制备中的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，在PSD-95敲除小鼠的VTA多巴胺细胞中表达融合GFP的重组PSD-95。在VTA多巴胺神经元中已经描述了多种形式的长期增强（LTP）和长期抑制（LTD），并且已知在体内滥用药物可以引起这些细胞的突触可塑性。然而，人们对这些现象背后的分子机制知之甚少。大量关于海马突触的研究表明，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在药物诱导的行为可塑性方面有改变。然而，PSD-95在VTA多巴胺神经元突触和药物诱导的可塑性中的作用尚未得到研究。我将首先研究PSD-95基因敲除小鼠VTA多巴胺神经元的基础突触传递是否发生改变。然后，我将测试PSD-95敲除小鼠在这些细胞中是否表现出正常的LTP和LTD。接下来，我将确定PSD-95敲除是否改变了VTA多巴胺神经元的药物诱导可塑性，这是对体内给药的反应。最后，我将测试PSD-95基因敲除后突触功能和可塑性的改变是否可以通过在VTA多巴胺神经元中表达重组PSD-95来恢复。这将通过将表达PSD-95的慢病毒与GFP融合，将其立体定向注射到VTA中来完成。这些实验结果将显著增强我们对VTA多巴胺神经元突触可塑性的分子机制以及PSD-95在药物诱导可塑性中的作用的认识。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，从而也可以开发出更好的治疗这种毁灭性疾病的方法。这项研究的目的是阐明在奖赏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.