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

 描述：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。环境或遗传干扰对昼夜节律系统的干扰可能会增加成瘾的易感性，而长期使用药物会导致昼夜节律紊乱，在戒酒期间持续存在，并可能导致复发。尽管这些关系很耐人寻味，但人们对昼夜节律系统和成瘾转变之间的关系的分子机制知之甚少。动物研究表明，长期接触可卡因会导致中脑边缘多巴胺奖赏系统中特定的昼夜节律基因(即分子时钟的组成部分)的表达和功能发生变化。伏隔核(NAC)是奖赏回路的一个主要汇聚区，也是调节药物奖赏和动机的关键底物。NAC主要由两种特定亚型的中棘神经元(MSN)组成，它们主要表达多巴胺1或2受体(D1或D2)。这两种亚型的MSN在可卡因奖赏行为的调节中扮演着不同的角色，尽管这些差异背后的分子机制尚不清楚。最近的人类遗传学研究发现，昼夜节律转录因子神经元PAS结构域蛋白2(NPAS2)的基因编码变异与精神障碍有关，精神障碍与成瘾障碍高度共存。我们已经确定了NPAS2通过在NAC的D1MSN中的活动来调节可卡因奖励的一个新的作用。R21的目标如下：1)利用CRISPR/Cas9技术产生分裂的Cre小鼠，使我们能够使用交叉遗传学方法来靶向纹状体区的D1MSN或D2MSN；以及2)仅在纹状体D1MSN或D2MSN中产生NPAS2Q缺陷小鼠，以研究调节奖赏和动机的细胞类型特定的分子机制(R33期)。R33 AIMS将通过1)研究Npas2在可卡因条件性奖赏(条件性位置偏爱)和自我给药的昼夜调节中的作用2)NPAS2Q介导的可卡因诱导的树突状可塑性的昼夜转录和3)利用共聚焦显微镜、流式细胞仪、RNA序列以及与初步ChIPseq数据的综合分析来阐明NPAS2调节可卡因奖赏的细胞类型特定的分子机制。这些研究将进一步阐明分子时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的转基因小鼠，以进一步研究药物奖赏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的昼夜调节的新机制。

 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.