Optical Isolation of Dopamine Signals and Regulation by Ethanol

项目总结/摘要 多巴胺（DA）信号在丘脑核（NAc）中的增加是对自然奖励的反应， 被认为编码环境刺激的激励显著性，从而激发目标导向行为。 所有 滥用药物，包括酒精，增加了NAc中的DA信号，暗示该系统参与了发育。 上瘾的人与急性效应相反，DA信号在慢性酒精戒断期间减少 exposure.已经提出内源性κ阿片受体（KOR）/强啡肽系统是 在慢性酒精给药期间NAc上调，并可能通过以下途径导致DA信号转导减少： 抑制性KOR位于DA末端。通过各种异源受体（包括 用快速扫描循环伏安法（FSCV）研究了位于多巴胺（DA）末端的KOR 以测量DA释放和施加电刺激以激发DA末端。然而，电 组织的刺激导致刺激场中所有神经元过程的同时激发。的 NAc整合了来自多个脑区的输入，导致了大脑中高水平的神经元异质性。 组织.从非DA能过程中释放的神经递质单独和共同提供了局部的 DA末端释放的调节。因此，对DA末端释放的药理学作用可能通过以下途径发生： 直接（DA末端上的受体）和/或间接（其它末端或细胞上的受体）机制。这个水平 的复杂性混淆了KOR对DA末端的影响的分析。在这个建议中，我们引入了一个新的模型， 用于通过光遗传学靶向NAc中的DA神经元刺激来体外研究DA末端功能 并使用FSCV测量释放。我们会在腹侧被盖区注射一种病毒， 通道视紫红质-2（ChR 2）的表达以诱导DA神经元中ChR 2的表达。这将允许选择性 使用蓝光刺激NAc切片中的DA末端。我们建议通过瞄准 在TH：Cre转基因小鼠中利用Cre诱导的病毒构建体特异性地向DA神经元表达ChR 2。 我们将使用免疫组织化学和共聚焦显微镜评估表达的特异性。另外我们 将描述诱发信号的特征，以验证其作为DA的身份，并证明释放是动作电位 依赖。此外，我们将使用该模型的改进灵敏度的措施，直接行动的KOR对 DA终端释放最后，我们将评估DA终末KOR敏感性的变化， 乙醇给药小鼠。

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.