Generating novel mouse tools to investigate brain region and cell-type specific circadian molecular mechanisms of reward and motivation

 产品说明： 基础和临床研究表明，昼夜节律和成瘾之间存在广泛的双向相互作用。由于环境或遗传干扰而造成的昼夜节律系统紊乱可能会增加成瘾的脆弱性，而长期使用药物会导致昼夜节律紊乱，这种紊乱在戒断期间持续存在，并可能导致复发。虽然这些关系很有趣，但人们对昼夜节律系统与成瘾过渡之间关系的分子机制知之甚少。 动物研究表明，长期暴露于可卡因导致特定昼夜节律基因（即，分子钟的组成部分）在中脑边缘多巴胺奖励系统。 奖赏回路的一个主要会聚区域和调节药物奖赏和动机的关键底物是丘脑核（NAc）。NAc主要由主要表达多巴胺1或2受体（D1+或D2+）的两种特定亚型的中型多刺神经元（MSN）组成。这两种MSNs亚型在可卡因奖赏行为的调节中具有不同的作用，尽管这些差异背后的分子机制仍不清楚。最近的人类遗传学研究已经鉴定了编码昼夜节律转录因子神经元PAS结构域蛋白2（NPAS 2）的基因的变体，该基因与高度共病成瘾障碍的精神障碍相关。我们已经确定了一个新的作用，NPAS 2的可卡因奖励的调节通过活动在D1+ MSN的NAc。R21的目标如下：1）利用CRISPR/Cas9技术来产生分裂Cre小鼠，使我们能够使用交叉遗传学方法来靶向纹状体中的D1+或D2+ MSN; 2）仅在纹状体D1+或D2+ MSN中产生NPAS 2 Q缺陷小鼠，以研究调节奖励和动机的细胞类型特异性分子机制（R33期）。 R33的目的是通过1）研究Npas 2在可卡因条件奖赏的昼夜节律调节中的作用来表征这些小鼠2）NPAS 2 Q介导的NAcR中可卡因诱导的树突可塑性的昼夜节律转录和3）使用共聚焦显微镜、FACs、RNA seq和具有初步ChIPseq数据的综合分析阐明可卡因奖赏的NPAS 2调节的细胞类型特异性分子机制。这些研究将进一步阐明分子钟在成瘾转变中的作用，重要的是，为更广泛的科学界提供新型转基因小鼠，以进一步研究药物奖励和成瘾表型的“直接”和“间接”途径调节的分子机制。这些研究将利用CRISPR/Cas9技术和先进的分子和行为方法来研究成瘾行为昼夜节律调节的新机制。

 DESCRIPTION: Basic and clinical research suggests there are extensive bidirectional interactions between circadian rhythms and addiction. Disruptions to the circadian system, either by environmental or genetic perturbation, may increase the vulnerability to addiction, while chronic drug use leads to circadian disruptions that persist during abstinence and may contribute to relapse. Although these relationships are intriguing, very little is known about the molecular mechanisms underlying the relationship between the circadian system and the transition to addiction. Animal studies have demonstrated that chronic exposure to cocaine leads to alterations in the expression and function of specific circadian genes (i.e., components of the molecular clock) in the mesolimbic dopamine reward system. A major region of convergence for reward circuitry and a key substrate that regulates drug reward and motivation is the nucleus accumbens (NAc). The NAc is comprised of mostly two specific subtypes of medium spiny neurons (MSNs) that predominantly express either dopamine 1 or 2 receptors (D1+ or D2+). These two subtypes of MSNs have distinct roles in the regulation of cocaine reward behaviors, although the molecular mechanisms underlying these differences remain unclear. Recent human genetics studies have identified variants in the gene coding for the circadian transcription factor neuronal PAS domain protein 2 (NPAS2) associated with psychiatric disorders that are highly comorbid with addiction disorders. We have identified a novel role of NPAS2 in the regulation of cocaine reward via activity in D1+ MSNs of the NAc. The R21 aims are as follows: 1) Leverage CRISPR/Cas9 technology to generate split Cre mice that enable us to use intersectional genetics approaches to target D1+ or D2+ MSNs specifically in the striatumR and 2) Generate NPAS2Qdeficient mice exclusively in striatal D1+ or D2+ MSNs to investigate the cell type specific molecular mechanisms regulating reward and motivation (R33 phase). The R33 aims will characterize these mice by 1) investigating the role of Npas2 in the regulation of circadian regulation of cocaine conditioned reward (conditioned place preference) and self-administration 2) NPAS2Q mediated circadian transcription on cocaine-induced dendritic plasticity in the NAcR and 3) elucidating the cell type specific molecular mechanisms of NPAS2 regulation of cocaine reward using confocal microscopy, FACs, RNA seq, and integrative analyses with preliminary ChIPseq data. These studies will further clarify the role of the molecular clock in the transition to addiction, and importantly, provide the broader scientific community with novel transgenic mice to further investigate the molecular mechanisms of 'direct' and 'indirect' pathway regulation of drug reward and addiction phenotypes. These studies will leverage CRISPR/Cas9 technologies and advanced molecular and behavioral approaches to study a novel mechanism of circadian regulation of addiction behaviors.