Role of Central Amygdala Astrocyte Plasticity in Ethanol Dependence

项目摘要/摘要 酒精使用障碍(AUD)影响着1600多万美国人，导致数百万可预防的死亡，以及 造成巨大的经济和社会负担。因此，更好地了解 AUD的神经生物学机制将为开发改进的治疗方法提供洞察力 战略。酒精依赖是以酒精摄入量过多为特征的AUD的特征， 躯体戒断症状和消极情绪。杏仁中央核(CEA)是大脑的关键部位 与酒精依赖有关的结构。例如，在CEA中，突触传递是失调的 在慢性酒精暴露和CEA活性增加后，饮用和戒酒需要CEA活性 依赖期间的症状。越来越多的证据表明，星形胶质细胞是 然而，我们对星形胶质细胞在突触传递和行为中的作用缺乏基本的了解。 对酒精的依赖。两个特别重要的问题是：1)长期接触酒精会对 星形胶质细胞-神经元的相互作用和2)星形胶质细胞在调节酒精后果中是否具有因果作用 依赖和退缩。拟议的研究将通过测试 主要假说认为慢性酒精暴露改变了CEA和 星形细胞GABA转运体GAT3介导乙醇戒断和依赖升级的乙醇摄取。 该实验方法利用慢性间歇性乙醇(CIE)蒸汽吸入暴露的大鼠模型 酒精饮酒结合超分辨率共聚焦显微镜和三维星形胶质细胞 形态分析。最重要的假设将在以下两个具体目标中得到检验。AIM 1将测试 慢性酒精暴露调节CEA中星形胶质细胞-神经元相互作用的假说。星形细胞- 在CIE暴露后，将评估突触的接近程度，以确定 星形胶质细胞的可塑性。星形胶质细胞-突触的接近程度也将在酒精访问后进行评估 戒断以确定自愿饮酒如何调节依赖和非依赖性星形胶质细胞的可塑性 依赖的老鼠。目标2将验证CEA中星形细胞GAT3调节依赖升级的假设 酒精摄入和躯体戒断。一种吗啡反义寡核苷酸策略将用于 在CEA中敲除GAT3以确定GAT3是否是升级的乙醇表达所必需的 酒精依赖期间的摄取和躯体戒断。相比之下，病毒过度表达的策略将是 用于增加星形细胞GAT3在CEA中的表达，以确定GAT3是否足以调节 酒精摄入量和戒断量增加。GAT3调控对星形胶质细胞可塑性的影响 也要接受评估。总之，这些研究将为星形胶质细胞在乙醇中的作用提供新的见解。 依赖和促进我们对潜在的神经底物的理解，这些潜在的神经底物可以被干预 AUD的治疗。

PROJECT SUMMARY/ABSTRACT Alcohol use disorder (AUD) affects over 16 million Americans, contributes to millions of preventable deaths, and causes enormous financial and societal burdens. Therefore, gaining a better understanding of the neurobiological mechanisms underlying AUD would provide insight for developing improved therapeutic strategies. Alcohol (ethanol) dependence is a hallmark of AUD that is characterized by excessive alcohol intake, somatic withdrawal symptoms, and negative mood. The central nucleus of the amygdala (CeA) is a key brain structure involved in ethanol dependence. For example, synaptic transmission is dysregulated in the CeA following chronic ethanol exposure and CeA activity is required for escalated ethanol drinking and withdrawal symptoms during dependence. Accumulating evidence suggests that astroglial cells are essential regulators of synaptic transmission and behavior, however, we lack a basic understanding regarding the role of astrocytes in in ethanol dependence. Two particularly important questions are 1) how does chronic ethanol exposure affect astrocyte-neuron interactions and 2) do astrocytes have a causal role in regulating the consequences of ethanol dependence and withdrawal. The proposed studies will begin to address these questions by testing the overarching hypothesis that chronic ethanol exposure alters astrocyte-synapse proximity in the CeA and the astrocytic GABA transporter, GAT3, mediates ethanol withdrawal and dependence-escalated ethanol intake. The experimental approach utilizes a rat model of chronic intermittent ethanol (CIE) exposure by vapor inhalation and ethanol drinking in combination with super resolution confocal microscopy and three-dimensional astrocyte morphology analysis. The overarching hypothesis will be tested in the following two specific aims. Aim 1 will test the hypothesis that chronic ethanol exposure modulates astrocyte-neuron interactions in the CeA. Astrocyte- synapse proximity will be assessed following CIE exposure to determine the temporal properties of changes in astrocyte plasticity. Astrocyte-synapse proximity will also be assessed following ethanol access during withdrawal to determine how voluntary ethanol drinking modulates astrocyte plasticity in dependent and non- dependent rats. Aim 2 will test the hypothesis that astrocytic GAT3 in the CeA regulates dependence-escalated ethanol intake and somatic withdrawal. A morpholino antisense oligonucleotide strategy will be used to knockdown GAT3 in the CeA to determine whether GAT3 is necessary for the expression of escalated ethanol intake and somatic withdrawal during ethanol dependence. In contrast, a viral overexpression strategy will be used to increase astrocytic GAT3 expression in the CeA to determine whether GAT3 is sufficient to modulate escalated ethanol intake and withdrawal. The consequences of GAT3 manipulation on astrocyte plasticity will also be assessed. Together, these studies will provide novel insight into the role of astrocytes in ethanol dependence and advance our understanding of potential neural substrates amenable to intervention for the treatment of AUD.