The role of the circadian transcription factor NPAS2 in the nucleus accumbens to regulate cocaine reward

描述(由申请人提供)：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。我们已经确定了昼夜节律转录因子神经元PAS结构域蛋白2(NPAS2)在可卡因奖赏调节中的新作用，可能是通过它与转录因子核因子kappaB(NFκB)的相互作用以及它在NAc的D1+MSN中的选择性表达来实现的。K01的目的如下：1)研究NPAS2在调节不同的可卡因奖赏行为(即条件性位置偏爱和自我给药)中的功能作用，通过表达在NAC的D1+或D2+MSN；2)检测急慢性可卡因暴露对Npas2和Npas2表达的影响。 以及这些转录因子在NAc中的相互作用；以及3)研究NPAS2是否正向调节Nac1+和D2+MSN中的NFκB介导的转录。因此，建议的K01奖将表征这些新的细胞类型特定的分子机制在调节可卡因奖励的NAC中的作用。这些研究还将进一步阐明分子时钟在成瘾转变中的作用。这些研究目标与NIDA和NIH Brain Initiative的战略目标保持一致，通过整合创新的分子和行为方法来确定大脑中特定细胞类型的功能意义。这位候选人的长期职业目标是开发一项独立的研究计划，调查昼夜节律系统在调节奖赏回路中的作用，这是向药物成瘾过渡的基础。应聘者在昼夜节律研究方面有很强的背景，需要额外的培训来发展翻译成瘾研究方面的专业知识，并将先进的分子方法和复杂的行为分析相结合。拟议的K01职业发展奖将确保候选人在下列领域成功地过渡到独立研究人员：1)在基础研究和翻译研究中对成瘾的神经生物学具有概念上的专长；2)在方法上熟练地将先进的分子技术，包括蛋白质免疫沉淀、免疫组织化学和脑细胞类型特异性病毒介导的基因转移与复杂的行为分析相结合，包括可卡因的条件性位置偏爱和小鼠静脉注射可卡因的自我给药；3)将以前的概念和研究能力与新获得的神经生物学技术和方法以及成瘾研究方面的概念专业知识相结合，成为一名独立的调查员。候选人将由一组在成瘾的细胞和分子机制、奖赏神经回路和药物诱导的神经可塑性以及昼夜节律方面拥有专业知识的教员团队指导和建议。个性化的职业发展计划包括与导师和顾问的定期会议、技能培训、正式课程作业、出席会议、正式研究报告以及技术和方法培训。匹兹堡大学和精神病学系为成为一名成功的独立成瘾研究人员提供了必要的基础设施，包括卓越的核心和导师研究设施，个性化的职业发展计划和培训机会，以及该机构和系主任培养有前途的年轻研究人员的长期承诺。

DESCRIPTION (provided by applicant): 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. We have identified a novel role of the circadian transcription factor, neuronal PAS domain protein 2 (NPAS2) in the regulation of cocaine reward, potentially via its interactions with the transcription factor nuclear factor kappa B (NFκB) and its selective expression in D1+ MSNs of the NAc. The aims of the K01 are as follows: 1) to investigate the functional role of NPAS2 in the regulation of different cocaine reward behaviors, (i.e., conditioned place preference and self-administration), via expression in D1+ or D2+ MSNs of the NAc; 2) to examine the effects of acute and chronic cocaine exposure on the expression of Npas2 and NFκB, along with the interactions between these transcription factors in the NAc; and 3) to investigate whether NPAS2 positively regulates NFκB-mediated transcription in D1+ and D2+ MSNs of the NAc. Therefore, the proposed K01 award will characterize the role of these novel cell type specific molecular mechanisms in the NAc that regulate cocaine reward. These studies will also further clarify the role of the molecular clock in the transition to addiction. These research goals are aligned with the strategic goals of NIDA and the NIH BRAIN Initiative to identify the functional significance of specific cell types in the brain by integrating innovative molecular and behavioral approaches. The candidate's long-term career goal is to develop an independent research program investigating the role of the circadian system in the regulation of reward circuitry that underlies the transition to drug addiction. The candidate has a strong background in circadian rhythms research and requires additional training to develop expertise in translational addiction research and the combination of advanced molecular approaches and complex behavioral assays. The proposed K01 Career Development Award will ensure the candidate's successful transition to an independent investigator by providing extensive conceptual and methodological training in the following areas: 1) conceptual expertise on the neurobiology of addiction for basic and translational research; 2) methodological proficiency in the combination of advanced molecular techniques, including protein immunoprecipitation, immunohistochemistry, and brain cell type specific viral-mediated gene transfer, with complex behavioral assays, including cocaine conditioned place preference and intravenous cocaine self-administration in mice; and 3) to integrate previous conceptual and research abilities with newly acquired neurobiological techniques and approaches, and conceptual expertise in addiction research, to become an independent investigator. The candidate will be mentored and advised by a team of faculty with expertise in the cellular and molecular mechanisms of addiction, reward neural circuitry and drug-induced neural plasticity, and circadian rhythms. The individualized career development plan includes a combination of regular meetings with mentors and consultants, grantsmanship skills training, formal coursework, conference attendance, formal research presentations, and technical and methodological training. The University of Pittsburgh and the Department of Psychiatry provides the necessary infrastructure to become a successful independent addiction researcher, including exceptional core and mentor research facilities, individualized career development programs and training opportunities, and a long-standing commitment by the institution and the departmental chair to develop promising young investigators.