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

项目概要/摘要 我们的广泛目标是提供新的机制洞察酒精使用障碍的发病机制 通过确定G蛋白信号转导调节因子6（RGS 6）如何在中脑边缘回路中作为一种神经递质发挥作用， 酒精（EtOH）寻求和奖励行为的关键调节剂。乙醇是最常被滥用的药物 尽管经过几十年的研究，乙醇寻求的神经生物学机制仍然存在， 行为和依赖性还没有被完全理解。因此，很少有有效的治疗方法来减少 酒精渴望或戒断症状，而禁欲仍然是预防AUD的唯一方法。 这个提议是基于我们的发现，RGS 6促进小鼠EtOH寻求和奖励 行为和随后的GWAS鉴定RGS 6为与人类AUD相关的基因。基于我们 之前的工作中，我们假设RGS 6通过负调节神经元的神经元，促进乙醇寻求行为， Gαi/o偶联受体（GPCRs）与酒精中毒有关。事实上，我们发现RGS 6-/-小鼠消耗 当给予自由访问时，EtOH较少，并且对EtOH奖励、依赖和戒断较不敏感。 D2 Rs或GABABRs的拮抗作用以及多巴胺转运蛋白（DAT）活性的抑制（上调） 通过Gαi/o偶联的GPCR，部分和完全恢复了RGS 6-/-小鼠中减少的EtOH消耗， 分别我们的研究结果表明，通过抑制VTA DA神经元中GPCR-Gαi-DAT的上调，RGS 6 促进EtOH诱导的多巴胺（DA）神经传递和行为反应。RGS 6控制 途径将代表一种以前未知的调节Gαi强度和持续时间的机制 VTA神经元中的信号传导以滴定负责EtOH的急性和慢性行为效应的DA水平。 在这里，我们提出测试中心假设，RGS 6调节G蛋白信号在腹侧被盖区DA 神经元在促进负责EtOH寻求的多巴胺能神经传递中起关键作用， 奖励行为目的1将确定中脑边缘RGS 6对EtOH寻求和奖励行为的作用 使用VTA DA神经元中选择性缺失RGS 6的小鼠以及RGS 6或其G的拯救研究 蛋白质缺陷突变体目的2将确定RGS 6在腹侧被盖区DA神经传递中的作用和对EtOH的作用。 使用光遗传学在小鼠中的消耗。我们将采用最先进的细胞类型特异性光遗传学， NAc中的神经元系综记录和Ca 2+成像，以及新型EtOH触发的光遗传学 确定RGS 6如何调节真实的脑中中脑边缘DA神经传递和EtOH消耗的方法 自由活动的小鼠。 这些研究意义重大，因为它们将阐明中脑边缘回路中的一个全新途径， 脑中涉及EtOH行为奖励和成瘾的主要多巴胺能通路。我们将 解决了在理解和治疗AUD方面取得进展的关键障碍，因为RGS 6已被确定 作为人类AUD相关基因，我们寻求增加我们对其在中脑边缘回路中作用的基本知识。

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.