Alcohol and Adenosine-Mediated Glutamate Signaling in Neuro-Glial Interaction

 描述（由申请人提供）：本提案的目的是研究腺苷调节的谷氨酸信号传导的分子机制，这是内侧前额叶皮层（mPFC）-纹状体回路和乙醇寻求行为的重要组成部分。我们将利用 一个独特的和高度集成的实验方法，其中包括新的转基因小鼠和光遗传学，这将有助于阐明腺苷信号在酒精使用障碍中的作用。在之前的资助期间，我们的研究表明，ENT 1（平衡核苷转运蛋白1型; Slc 29 a1），乙醇敏感的腺苷转运蛋白，调节纹状体中的腺苷和谷氨酸信号传导，并在神经元-胶质细胞相互作用中发挥关键作用，以调节乙醇寻求行为。我们最近的研究结果表明，在背内侧纹状体（DMS）中ENT 1的遗传和药理学抑制通过低腺苷A2 A受体功能促进目标导向行为，这有助于在操作性条件反射实验中增加乙醇寻求行为。然而，目前还不清楚星形胶质细胞或神经元ENT 1是否调节腺苷水平和腺苷受体介导的信号在mPFC-DMS电路。我们的主要假设是星形胶质细胞ENT 1调节的腺苷信号在皮质-纹状体回路的谷氨酸神经传递和乙醇消耗中起着重要作用。为了研究这个假设，我们提出了三个目标。首先，我们将研究是否诱导表达ENT 1调节腺苷和谷氨酸信号在皮质纹状体电路。使用最近产生的四环素依赖性诱导ENT 1转基因小鼠，我们将研究是否过度表达ENT 1改变腺苷和谷氨酸信号在纹状体和前额叶皮层以及乙醇寻求行为。在这个目标中，我们将利用CAG（通用启动子）驱动的rtTA（反向四环素控制的反式激活因子）表达小鼠来检查ENT 1在脑中的过表达。我们将研究是否ENT 1依赖信号在两个不同的纹状体区域（NAc和DMS）和mPFC改变乙醇寻求行为。其次，使用星形胶质细胞或神经元特异性ENT 1过表达小鼠，我们将研究星形胶质细胞或神经元ENT 1是否调节乙醇寻求行为。使用星形胶质细胞（GFAP）或神经元特异性（CamK 2a）rtTA表达小鼠，我们计划研究星形胶质细胞或神经元特异性ENT 1是否调节目标导向和乙醇寻求行为。此外，我们将研究腺苷受体的激动剂或拮抗剂是否调节乙醇寻求行为。第三，我们将使用电路特异性光遗传学研究乙醇寻求行为。我们的初步研究表明，抑制含A2 AR的纹状体回路中的DMS促进乙醇寻求行为的操作室。为了检查DMS中纹状体苍白球神经元的光遗传学调节是否调节乙醇寻求行为，我们将在A2 AR启动子的控制下使用激光刺激的通道视紫红质（ChR 2）和盐视紫红质（eNpHR 3）。这些研究将阐明神经元-胶质细胞相互作用和皮质-纹状体回路中腺苷调节谷氨酸信号传导的分子机制及其在乙醇寻求行为中的作用。

 DESCRIPTION (provided by applicant): The purpose of this proposal is to study molecular mechanisms of adenosine-regulated glutamate signaling, which is an essential component of the medial prefrontal cortex (mPFC)-striatal circuit and ethanol seeking behaviors. We will utilize a unique and highly integrated experimental approach, which includes novel transgenic mice and optogenetics, which will contribute to elucidating the role of adenosine signaling in alcohol use disorders. During the previous funding period, our studies revealed that ENT1 (equilibrative nucleoside transporter type 1; Slc29a1), an ethanol-sensitive adenosine transporter, regulates adenosine and glutamate signaling in the striatum and plays a pivotal role in neuron-glial interaction to regulate ethanol-seeking behaviors. Our recent findings demonstrated that genetic and pharmacological inhibition of ENT1 in the dorsomedial striatum (DMS) promote goal- directed behaviors through hypo-adenosine A2A receptor function, which contributes to increased ethanol seeking behaviors in an operant conditioning experiment. However, it is unclear whether astrocytic or neuronal ENT1 regulates adenosine levels and adenosine receptor-mediated signaling in the mPFC-DMS circuits. Our main hypothesis is that astrocytic ENT1-regulated adenosine signaling plays an essential role in glutamate neurotransmission in the cortico-striatal circuits and ethanol consumption. To investigate this hypothesis, we propose three aims. First, we will examine whether inducible expression of ENT1 regulates adenosine and glutamate signaling in the cortico-striatal circuits. Using recently generated tetracyline-dependent inducible ENT1 transgenic mice, we will examine whether overexpression of ENT1 alters adenosine and glutamate signaling in the striatum and prefrontal cortex as well as ethanol-seeking behaviors. In this aim, we will utilize CAG (universal promoter) driven rtTA (reverse tetracycline controlled transactivator) expressing mice to examine overexpression of ENT1 in the brain. We will investigate whether ENT1-dependent signaling in the two distinct striatal regions (NAc and DMS) and mPFC alters ethanol-seeking behaviors. Second, using astrocyte or neuron-specific ENT1 overexpressing mice, we will investigate whether astrocyte or neuronal ENT1 regulates ethanol-seeking behaviors. Using astrocyte (Gfap) or neuron-specific (CamK2a) rtTA expressing mice, we plan to investigate whether astrocyte- or neuron-specific ENT1 regulates goal-directed and ethanol seeking behaviors. In addition, we will examine whether agonists or antagonists of adenosine receptors regulate ethanol-seeking behaviors. Third, we will investigate ethanol-seeking behaviors using circuit-specific optogenetics. Our preliminary study suggests that inhibition of the A2AR-containing striatopallidal circuit in the DMS promotes ethanol-seeking behavior in an operant chamber. To examine whether optogenetic regulation of striatopallidal neurons in the DMS regulates ethanol-seeking behaviors, we will use laser-stimulated channelrhodopsin (ChR2) and halorhodopsin (eNpHR3) under the control of the A2AR promoter. These studies will elucidate molecular mechanisms underlying adenosine-regulated glutamate signaling in both neuron-glial interactions and cortico-striatal circuits and their role in ethanol-seeking behaviors.