Nr4a1 and GPR88 as novel gene targets for alcoholism: mouse genetic approaches

描述（由申请人提供）：我们假设Nr4a1和GprBB主要在扩展杏仁核（EA）水平上调节酒精摄入和依赖，并代表治疗酒精中毒的潜在靶点。我们将使用常规和ea条件敲除小鼠来测试这两个候选基因，以及在酒精研究中被认为是有效靶点的已知基因（mu阿片受体）。这些独特的突变系将在行为（目标1）和成像（目标2）水平上进行研究，以表征这三个基因在过量饮酒中的含义。在目的1中，我们将检查我们实验室中可用的常规基因敲除小鼠(i)慢性间歇性暴露于酒精蒸气（CIE）后的酒精退出（ii）随暴露于空气/酒精蒸气（CIE/TBC）导致休闲/过度饮酒（Aim la）的自愿饮酒史。在Aim lb中，我们将通过杂交floxed小鼠和Wfsl-Cre小鼠（均在过去的资助期内产生）来生产和表征具有所有三个基因的ea条件小鼠。在Aim 1c中，我们将检查Aim la中的条件突变系。传统基因敲除小鼠的表型将确定目标基因在酒精摄入中的功能作用，而EA条件突变小鼠的表型将揭示EA电路在这些行为中的含义。在目标2中，我们将使用尖端的DT-MRI和纤维跟踪，并在小鼠中实施FcMRI （coll）。J. Hennig, Freiburg, Germany)在经历过量饮酒史的小鼠中识别结构和连接重塑。实验条件将在野生型小鼠（Aims 2a和d）中进行优化，然后在纵向实验中应用于慢性暴露（Aim 2b）或CIE/TBC范式（Aim 2c）中自愿饮酒条件下具有最强行为表型的突变小鼠（数据来自Aim 1）。Aim 2将从动态解剖学角度扩展我们对基因功能的了解，并为从啮齿动物到人类的转化研究提供一个框架。总之，该提案整合了独特的小鼠遗传工具和成像方法，在分子和系统水平上对酒精依赖的新基因进行功能研究。与INIA合作伙伴的互动涉及材料（小鼠系、aav - shrna）和知识（成像、小鼠和病毒）的共享。

DESCRIPTION (provided by applicant): We hypothesize that Nr4a1 and GprBB regulate alcohol intake and dependence, mainly at the level of the extended amygdala (EA), and represent potential targets to treat alcoholism. We will test this hypothesis using conventional and EA-conditional knockout mice for the two candidate genes, as well as for a known gene considered a valid target in alcohol research (the mu opioid receptor). These unique mutant lines will be studied at behavioral (Aim 1) and imaging (Aim 2) levels to characterize implication of the three genes in excessive alcohol drinking. In Aim 1 we will examine conventional knockout mice available in our laboratory for (i) alcohol withdrawal after chronic intermittent exposure to alcohol vapors (CIE) and (ii) voluntary drinking along a history of exposure to air / alcohol vapors (CIE/TBC) leading to recreational / excessive drinking (Aim la). In Aim lb we will produce and characterize EA-conditional mice for all three genes by crossing floxed mice and Wfsl-Cre mice, all of which were generated in the past funding period. In Aim 1c we will examine conditional mutant lines as in Aim la. Phenotypes in conventional knockout mice will establish a functional role of targeted genes in alcohol intake, and phenotypes in EA-conditional mutant mice will uncover implication of EA circuitry in those behaviors. In Aim 2, we will use cutting-edge DT-MRI and fiber tracking, and implement FcMRI in mice (coll. J. Hennig, Freiburg, Germany) to identify structural and connectivity remodeling in mice undergoing a history of excessive drinking. Experimental conditions will be optimized in wild-type mice (Aims 2a and d), then applied to mutant mice with strongest behavioral phenotype (data from Aim 1) under conditions of chronic exposure (Aim 2b) or voluntary drinking in the CIE/TBC paradigm (Aim 2c) in longitudinal experiments. Aim 2 will expand our knowledge of gene function with a dynamic anatomical dimension and provide a framework for translational studies from rodents to humans. Together, the proposal integrates unique mouse genetic tools and imaging methodology, to functionally study novel genes in alcohol dependence at molecular and system levels. Interactions with INIA partners involve sharing of material (mouse lines, AAV-shRNAs) and knowledge (imaging, mice and viruses).