RGS6 in mesolimbic circuits as a therapeutic target for alcohol use disorders

项目摘要/摘要 我们的广泛目标是为酒精使用障碍的发病机制提供新的机制洞察力 (AUDS)通过确定G蛋白信号转导调节因子6(RGS6)在中脑边缘环路中作为一种 酒精寻求和奖励行为的关键调节剂(乙醇)。乙醇是最常被滥用的药物 尽管经过几十年的研究，但Etoh寻觅背后的神经生物学机制 行为和依赖还没有完全被理解。因此，几乎没有有效的治疗方法来减少 酒精渴望或戒断症状，禁欲仍然是预防AUDS的唯一方法。 这一建议是基于我们的发现，RGS6促进了小鼠的乙醇寻求和奖励 行为和随后的GWA发现RGS6是一个与人类AUDS连锁的基因。基于我们的 在之前的工作中，我们假设RGS6通过负性调节神经元来促进乙醇寻找行为 G-αI/O偶联受体(GPCRs)与酒精中毒有关。事实上，我们发现RGS6-/-小鼠消耗 免费使用Etoh较少，更不容易受到Etoh奖励、依赖和戒断的影响。 D2Rs或GABRs的拮抗作用和对多巴胺转运体(DAT)活性的抑制 通过G-αI/O偶联GPCRs，部分和完全恢复了RGS6-/-小鼠减少的乙醇消耗， 分别进行了分析。我们的研究结果表明，通过抑制VTA DA神经元中GPCRGDAT I-α的上调， 促进乙醇诱导的多巴胺(DA)神经传递和行为反应。RGS6对此的控制 这一途径代表了一种以前未知的调节GαI强度和持续时间的机制 在VTA神经元中发出信号以滴定DA水平，这些DA水平与乙醇的急性和慢性行为影响有关。 在这里，我们建议检验中央假设，即RGS6调节VTA DA中的G蛋白信号 神经元在促进多巴胺能神经传递中发挥关键作用，多巴胺能神经传递负责乙醇寻找和 奖励行为。目标1将确定中脑边缘RGS6在乙醇寻求和奖励行为中的作用 利用选择性缺失VTA DA神经元中RGS6的小鼠以及RGS6或其G的补救研究 蛋白质缺陷突变体。目标2将确定RGS6在VTA DA神经传递和EtoH中的作用 利用光遗传学在小鼠身上的消耗。我们将使用最先进的细胞类型特定的光遗传学， NAC的神经元集合记录和钙成像，以及一种新的乙醇触发的光发生 确定RGS6如何在真实情况下调节中脑边缘DA神经传递和乙醇消耗的方法 时间在自由移动的老鼠身上。 这些研究意义重大，因为它们将阐明中脑边缘环路中的一条全新的途径， 大脑中一条主要的多巴胺能通路与乙醇行为奖赏和成瘾有关。我们会 解决在理解和治疗AUDS方面取得进展的关键障碍，因为RGS6已经被确定 作为一种人类AUD连锁基因，我们试图增加我们对其在中脑边缘回路中所起作用的基本知识。

Project Summary / Abstract Our broad goal is to provide novel mechanistic insight into the pathogenesis of alcohol use disorders (AUDs) by ascertaining how Regulator of G protein signaling 6 (RGS6) functions in the mesolimbic circuit as a critical modulator of alcohol (EtOH) seeking and reward behaviors. EtOH is the most commonly abused drug worldwide and, despite decades of research, the neurobiological mechanisms underlying EtOH seeking behavior and dependence are not fully understood. As a result, there are few effective therapeutics to reduce alcohol cravings or withdrawal symptomology, and abstinence remains the only way to prevent AUDs. This proposal is based upon our discovery that RGS6 promotes mouse EtOH seeking and reward behaviors and on a subsequent GWAS identifying RGS6 as a gene linked to human AUDs. Based on our previous work, we hypothesized that RGS6 promotes EtOH seeking behavior by negatively regulating neuronal Gαi/o-coupled receptors (GPCRs) implicated in alcoholism. Indeed, we found that RGS6-/- mice consume less EtOH when given free access and are less susceptible to EtOH reward, dependence and withdrawal. Antagonism of D2Rs or GABABRs and inhibition of dopamine transporter (DAT) activity, which is upregulated by Gαi/o-coupled GPCRs, partially and completely restored the reduced EtOH consumption in RGS6-/- mice, respectively. Our findings suggest that by inhibiting GPCR-Gαi-DAT upregulation in VTA DA neurons, RGS6 promotes EtOH-induced dopamine (DA) neurotransmission and behavioral responses. RGS6 control of this pathway would represent a previously unknown mechanism modulating the intensity and duration of Gαi signaling in VTA neurons to titre DA levels responsible for acute and chronic behavioral effects of EtOH. Here we propose to test the central hypothesis that RGS6 regulation of G protein signaling in VTA DA neurons plays a critical role in promoting dopaminergic neurotransmission responsible for EtOH seeking and reward behaviors. Aim 1 will determine the role of mesolimbic RGS6 on EtOH seeking and reward behaviors using mice with selective deletion of RGS6 in VTA DA neurons as well as rescue studies with RGS6 or its G protein-defective mutant. Aim 2 will determine the role of RGS6 in VTA DA neurotransmission and on EtOH consumption in mice using optogenetics. We will employ state of the art cell type-specific optogenetics, neuronal ensemble recordings and Ca2+ imaging in the NAc, as well as a novel EtOH-triggered optogenetic approach to determine how RGS6 modulates mesolimbic DA neurotransmission and EtOH consumption in real time in freely moving mice. These studies are significant as they will illuminate an entirely novel pathway in the mesolimbic circuit, a major dopaminergic pathway in the brain implicated in EtOH behavioral reward and addiction. We will address a critical barrier to progress in the understanding and treatment of AUDs as RGS6 has been identified as a human AUD-linked gene and we seek to increase our basic knowledge of its role in the mesolimbic circuit.