Optical Isolation of Dopamine Signals and Regulation by Ethanol

项目摘要/摘要 伏核(NAC)中的多巴胺(DA)信号对自然奖赏和IS的反应增加 认为编码环境刺激的激励突显，从而激励目标导向的行为。全 滥用药物，包括酒精，增加了NAC中的DA信号，暗示这一系统参与了NAC的发育 上瘾的人。与急性效应相反，在慢性酒精戒断过程中，DA信号会减少 曝光。有研究认为，内源性kappa阿片受体(KOR)/强啡肽系统是 在慢性酒精摄入过程中，NAC的表达上调，并可能通过 抑制性KRs位于DA终末。通过各种异源受体(包括 用快速扫描循环伏安法(FSCV)对位于DA端子上的KOR)进行了研究 测定多巴胺的释放和应用电刺激兴奋多巴胺终末。然而，电气 对组织的刺激导致刺激场中所有神经元突起的同时兴奋。这个 NAC整合了来自多个脑区的输入，导致了大脑中神经元高度异质性 组织。来自非DAR能过程的神经递质的释放单独和共同地提供局部 DA终末释放的调节。因此，对DA末端释放的药理作用可能通过 直接(DA终末受体)和/或间接(其他终末或细胞上的受体)机制。这一级别 复杂性的降低混淆了对DA终端KOR效应的分析。在这个方案中，我们引入了一种新的模型 通过光遗传学靶向刺激NAc中的DA神经元来研究DA终末功能 以及使用FSCV测量释放。我们将在腹侧被盖区注射一种编码病毒的构建物 通道视紫红质2(ChR2)的表达诱导DA神经元ChR2的表达这将允许选择性地 蓝光刺激NAC脑片DA终末。我们建议通过目标定位来开发这项技术 利用Cre诱导的病毒构建物在TH：Cre转基因小鼠中特异性地向DA神经元表达ChR2。 我们将使用免疫组织化学和共聚焦显微镜来评估表达的特异性。另外，我们 将表征诱发信号，以验证其作为DA的身份，并证明释放是动作电位 依附的。此外，我们将使用该模型改进的敏感性来衡量KOR对 DA端子释放。最后，我们将评估慢性疾病后DA终末KOR敏感性的变化。 乙醇对小鼠的作用。

Project Summary/Abstract Dopamine (DA) signaling in the nucleus accumbens (NAc) increases in response to natural reward and is thought to encode the incentive salience of environmental stimuli, which motivates goal-directed behaviors. All drugs of abuse, including alcohol, increase DA signaling in the NAc, implicating this system in the development of addiction. In contrast to acute effects, DA signaling is reduced during withdrawal from chronic alcohol exposure. It has been proposed that the endogenous kappa opioid receptor (KOR)/dynorphin system is upregulated in the NAc during chronic alcohol administration, and may contribute to reduced DA signaling via inhibitory KORs located on DA terminals. Modulation of DA release through various heteroreceptors (including KOR) located on DA terminals has been investigated in NAc slices using fast scan cyclic voltammetry (FSCV) to measure DA release and application of electrical stimulation to excite DA terminals. However, electrical stimulation of the tissue results in simultaneous excitation of all neuronal processes in the stimulation field. The NAc integrates inputs from multiple brain regions, resulting in a high level of neuronal heterogeneity in the tissue. Release of neurotransmitters from non-DAergic processes provide, individually and together, local regulation of DA terminal release. Thus, pharmacological effects on DA terminal release may occur through direct (receptors on DA terminals) and/or indirect (receptors on other terminals or cells) mechanisms. This level of complexity confounds analysis of KOR effects on DA terminals. In this proposal we introduce a novel model for in vitro investigations of DA terminal function by optogenetically targeting DA neuron stimulation in the NAc and measuring release using FSCV. We will inject the ventral tegmental area with a viral construct encoding expression of channelrhodopsin-2 (ChR2) to induce ChR2 expression in DA neurons. This will allow selective stimulation of DA terminals in NAc slices using blue light. We propose to develop this technique by targeting ChR2 expression specifically to DA neurons utilizing a Cre-inducible viral construct in TH:Cre transgenic mice. We will assess the specificity of expression using immunohistochemistry and confocal microscopy. Also, we will characterize the evoked signal to validate its identity as DA and demonstrate that release is action potential dependent. Furthermore, we will use the model's improved sensitivity for measures of direct actions of KOR on DA terminal release. Finally, we will assess the changes in DA terminal KOR sensitivity following chronic ethanol administration in mice.