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.

项目摘要/摘要 伏隔核（NAC）中的多巴胺（DA）信号对自然奖励的响应增加，IS 被认为是编码环境刺激的激励显着性，这激发了目标指导的行为。 全部 滥用药物（包括酒精）增加了NAC中的DA信号，这意味着该系统在开发中 成瘾。与急性效应相反，从慢性酒精退出期间降低了DA信号传导 接触。有人提出，内源性Kappa阿片受体（KOR）/Dynorphin系统是 慢性酒精给药期间NAC中的上调，可能会导致DA信号通过 抑制性KORS位于DA终端。通过各种异发子对DA释放的调节（包括 Kor）已使用快速扫描循环伏安法（FSCV）在NAC切片中研究了DA终端。 测量DA释放并应用电刺激到激发DA末端。但是，电气 刺激组织会导致刺激场中所有神经元过程的同时激发。这 NAC整合了来自多个大脑区域的输入，导致高水平的神经元异质性 组织。从非药物过程中释放神经递质的人可以单独和局部提供 DA末端释放的调节。因此，可能通过对DA末端释放的药理影响 直接（DA末端的受体）和/或间接（其他末端或细胞上的受体）机制。这个级别 复杂性混淆了Kor对DA终端的影响的分析。在此提案中，我们介绍了一个新颖的模型 通过在NAC中靶向DA神经元刺激的体外研究DA末端功能 并使用FSCV测量释放。我们将以病毒构建的编码注入腹侧对段区域 通道Ropopsin-2（ChR2）的表达诱导DA神经元中的ChR2表达。这将允许选择性 使用蓝光在NAC切片中刺激DA终端。我们建议通过针对 CHR2在Cre th：Cre the型转基因小鼠中使用CRE诱导的病毒构建体专门针对DA神经元的表达。 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身份DA的身份，并证明释放是动作电位 依赖。此外，我们将利用模型的提高灵敏度来衡量Kor的直接行动 DA终端释放。最后，我们将评估慢性后DA终端Kor灵敏度的变化 小鼠乙醇给药。