Optical Isolation of Dopamine Signals and Regulation by Ethanol

多巴胺信号的光学隔离和乙醇的调节

• 批准号: 8718204
• 负责人: James Melchior
• 金额: $ 4.27万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8718204
• 关键词: Action Potentials; Acute; Address; Affect; Affective; Agonist; Air; Alcohol abuse; Alcohol consumption; Alcohol dependence; Alcohols; Anhedonia; Area; Back; Bathing; Behavior; Brain; Brain region; Breathing; Buffers; Cells; Chronic; Confocal Microscopy; Corpus striatum structure; Data; Development; Disease; Dopamine; Dynorphins; Electric Stimulation; Electrical Stimulation of the Brain; Ethanol; Excision; Goals; Health; Heterogeneity; Human; Immunohistochemistry; In Vitro; Incentives; Investigation; Light; Measures; Mediating; Methods; Modeling; Mus; Negative Reinforcements; Neuromodulator; Neurons; Neurotransmitters; Nucleus Accumbens; Opioid Receptor; Optics; Prevalence; Prevention; Process; Progressive Disease; Receptor Activation; Receptor Inhibition; Regulation; Rewards; Rodent; Scanning; Shapes; Signal Pathway; Signal Transduction; Slice; Specificity; Stimulus; System; Techniques; Testing; Tetrodotoxin; Tissues; Transgenic Mice; Tyrosine; Tyrosine 3-Monooxygenase; United States; Up-Regulation; Ventral Tegmental Area; Viral; Virus; Withdrawal; Withdrawal Symptom; addiction; alcohol exposure; base; calmodulin-dependent protein kinase II; cell type; dopaminergic neuron; drug of abuse; economic cost; improved; in vitro Model; inhibitor/antagonist; insight; mouse model; neurotransmitter release; novel; optogenetics; postsynaptic; promoter; receptor; receptor sensitivity; recombinase; response; uptake; vapor; vesicular monoamine transporter

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.

项目概要/摘要 伏隔核 (NAc) 中的多巴胺 (DA) 信号随着自然奖赏的反应而增加，并且 被认为编码环境刺激的激励显着性，从而激发目标导向的行为。 全部 包括酒精在内的滥用药物会增加 NAc 中的 DA 信号传导，表明该系统与发育有关 的成瘾。与急性效应相反，长期饮酒戒断期间 DA 信号减弱 接触。有人提出，内源性κ阿片受体（KOR）/强啡肽系统是 长期饮酒期间 NAc 上调，并可能通过以下途径减少 DA 信号传导： 抑制性 KOR 位于 DA 终端。通过各种异源受体（包括 使用快速扫描循环伏安法 (FSCV) 在 NAc 切片中研究了位于 DA 端子上的 KOR） 测量 DA 释放和应用电刺激来激发 DA 终端。然而，电 对组织的刺激导致刺激场中所有神经元过程的同时兴奋。这 NAc 整合来自多个大脑区域的输入，导致神经元的高度异质性 组织。从非DAergic过程中释放的神经递质单独或一起提供局部 DA末端释放的调节。因此，对 DA 末端释放的药理作用可能通过 直接（DA 终端上的受体）和/或间接（其他终端或细胞上的受体）机制。这个级别 复杂性混淆了 KOR 对 DA 终端影响的分析。在这个提案中，我们引入了一种新颖的模型 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。我们将用编码的病毒构建体注射到腹侧被盖区 表达通道视紫红质 2 (ChR2) 以诱导 DA 神经元中 ChR2 的表达。这将允许选择性 使用蓝光刺激 NAc 切片中的 DA 末端。我们建议通过瞄准来开发这项技术 在 TH:Cre 转基因小鼠中，利用 Cre 诱导型病毒构建体，ChR2 特异地针对 DA 神经元进行表达。 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 依赖。此外，我们将利用该模型改进的灵敏度来衡量 KOR 对 DA 终端释放。最后，我们将评估慢性病后 DA 末端 KOR 敏感性的变化 对小鼠进行乙醇给药。