Ethanol Remodeling of Striatal Microcircuits

描述(由申请人提供)：这项研究提案调查了酒精暴露如何诱导大脑回路重构，从而导致持续的酒精消费。这个项目将重点放在大脑中一个强烈控制习惯的区域--纹状体。习惯形成I依赖于通过大麻素1型受体(CB1)的内源性大麻素信号，CB1是一种突触前终端蛋白，介导持久形式的突触可塑性。纹状体CB1的大部分定位于抑制性微回路终末，它有效地控制着该脑区的功能输出。有趣的是，现在有几条证据表明乙醇对内源性大麻素系统有直接影响。尽管纹状体对习惯的控制与酗酒有关，但在这方面的数据很少。这项建议试图了解急性和慢性乙醇暴露如何重塑纹状体抑制微电路，以促进习惯性的乙醇消费。这将通过三个具体的实验目标进行调查。1)确定乙醇对特定纹状体抑制性微回路突触GABA能传递的急性影响。这一目标将通过将急性小鼠脑片暴露在乙醇中，并从光遗传学角度评估对特定纹状体抑制性微回路突触的强度和可塑性的影响来实现。这些变化背后的分子机制将通过评估乙醇和内源性大麻素系统之间的相互作用来检验。2)评估黑暗中饮酒(DID)对纹状体抑制微回路突触GABA能传递的慢性影响。DID范式对人类条件下长期的、狂欢的乙醇消费进行了建模。为了检测DID对小鼠纹状体抑制性微环路突触的持久变化，将制作脑片，并评估乙醇对特定微环路突触的有效性和可塑性的变化。我们将测试酒精诱导的内源性大麻素系统的扰动如何引起抑制性微电路的这些不适应的稳态反应。3)在体光遗传学研究中，确定纹状体抑制微电路在DID所致乙醇消耗量增加中的作用。为了实现这一目标，在体内使用光遗传学方法，在DID范例中的饮酒行为期间，将以毫秒的精度在线可逆地抑制包含抑制微电路的特定细胞类型。在DID期间的这种操作对DID后乙醇消费行为的贡献将通过两瓶(乙醇和水)选择试验来评估。这些研究的结果应该会为我们了解乙醇的增强效应是如何通过大脑回路特异性改变而介导的，提供重大进展。在我的研究生和博士后培训中，我获得了必要的神经解剖学和电生理技术方面的培训，这些技术是有效执行这项研究计划所必需的。在我师从Ariel Y.Deutch博士的研究生工作中，我接受了广泛的神经解剖学和行为药理学技术培训，包括立体定向微量注射神经束示踪剂和毒素、成像质谱学、免疫组织化学和运动控制的行为测量。通过这次训练，我获得了一种对大脑回路进行概念化的专家能力。在大卫·M·洛文格博士的博士后培训中，我已经精通评估突触功能的电生理分析。此外，在我的立体定位手术能力的帮助下，我在实验室中率先使用光遗传学来确定突触特异性变化对电路重塑的贡献。在该提案的指导阶段，研究将在NIAAA的我的导师David M.Lovinger博士的实验室进行。安德鲁·霍姆斯博士将担任我的共同导师。洛文格博士是乙醇和突触可塑性研究的权威，在体外和体内电生理记录方面拥有丰富的经验。霍姆斯博士是一名行为神经科学和乙醇研究专家，他将监督我在评估动物行为方面的持续培训。在NIAAA的Veronica Alvarez博士和Geroge Kuros博士的共同协作下，执行研究提案所需的所有实验工具和研究指导都将随时可用。最后，NIAAA可用于发展我的教学、指导、管理、拨款撰写和负责任的研究技能的资源创造了一个支持性的环境，在这个环境中我可以很容易地完成我提出的培训计划。

DESCRIPTION (provided by applicant): The research proposal investigates how ethanol exposure induces brain circuit remodeling to result in persistent ethanol consumption. This project will focus on a brain region that strongly controls habits, the striatum. Habit formation i dependent upon endocannabinoid signaling through the cannabinoid type 1 receptor (CB1), a presynaptic terminal protein that mediates lasting forms of synaptic plasticity. The majority of striatal CB1 is localized to inhibitory microcircuit terminals, which potently control the functionl output of this brain region. Interestingly, several lines of evidence now indicate a direct effect f ethanol on the endocannabinoid system. Despite the relevance of striatal control of habits to alcoholism, a paucity of data exists in this regard. This proposal seeks to understand how acute and chronic ethanol exposure remodels the striatal inhibitory microcircuitry to promote habitual ethanol consumption. This will be investigated through three specific experimental aims. 1) Determine the acute effects of ethanol on GABAergic transmission at specific striatal inhibitory microcircuit synapses. This aim will be accomplished by exposing acute mouse brain slices to ethanol and optogenetically assessing effects on the strength and plasticity of specific striatal inhibitory microcircuit synapses. The molecular mechanism underlying these changes will be examined by assessing the interaction between ethanol and the endocannabinoid system. 2) Assess the chronic effects of ethanol drinking in the dark (DID) on GABAergic transmission at synapses of the striatal inhibitory microcircuitry. The DID paradigm models long-term, binge-like ethanol consumption present in the human condition. To examine DID-induced lasting changes to striatal inhibitory microcircuit synapses of mice, brain slices will be made and the ethanol-induced changes to efficacy and plasticity of specific microcircuit synapses will be assessed. We will test how these maladaptive homeostatic responses of the inhibitory microcircuitry arise from ethanol-induced perturbation of the endocannabinoid system. 3) Determine the contribution of the striatal inhibitory microcircuitry to DID-induced increased ethanol consumption using in vivo optogenetics. To accomplish this aim, a specific cell type comprising the inhibitory microcircuitry will be reversibly inhibited on-line, with millisecond precision, durng drinking behavior in the DID paradigm using an optogenetics approach in vivo. The contribution of this manipulation during DID to post-DID ethanol consumatory behavior will be assessed using a two-bottle (ethanol and water) choice assay. The results of these studies should provide significant advances in our knowledge of how the reinforcing effects of ethanol are mediated by brain circuit-specific alterations. In my graduate and postdoctoral training, I gained the necessary training in neuroanatomical and electrophysiological techniques necessary to effectively execute this research proposal. In my graduate work under Dr. Ariel Y. Deutch, I trained extensively in neuroanatomical and behavioral pharmacology techniques including stereotaxic microinfusion of neuronal tract tracers and toxins, imaging mass spectrometry, immunohistochemistry, and behavioral measures of motor control. Through this training, I gained an expert ability to conceptualize brain circuitry. In my postdoctoral training under Dr. David M. Lovinger, I have become proficient in electrophysiological analyses assessing synaptic function. Moreover, I have pioneered the use of optogenetics in the lab, aided by my stereotaxic surgery abilities, for determining the contribution of synapse-specific changes to circuit remodeling. During the mentored phase of the proposal, research will be conducted at NIAAA in the laboratory of Dr. David M. Lovinger, my mentor. Dr. Andrew Holmes will serve as my co-mentor. Dr. Lovinger is an authority in ethanol and synaptic plasticity research and has extensive experience in in vitro and in vivo electrophysiological recording. Dr. Holmes, a behavioral neuroscience and ethanol research expert, will oversee my continued training in assessing animal behavior. With the combined collaboration of Dr. Veronica Alvarez and Dr. Geroge Kunos, both at NIAAA, all experimental tools and research guidance necessary for executing the research proposal will be readily accessible. Finally, the resources available at NIAAA for developing my teaching, mentorship, managerial, grant writing, and responsible conduct of research skills create a supportive environment in which to readily accomplish my proposed training plan.