Inactivation of mu opioid and CRF1 receptor genes in the extended amygdala

描述（由申请人提供）：最近在小鼠中研究神经肽受体在乙醇依赖性中的作用的基因靶向产生了令人着迷的结果。敲除μ阿片受体（MOP）基因可阻断饮酒和对乙醇的操作性反应。敲除促肾上腺皮质激素释放因子受体1（CRF 1）基因可降低基础和酒精戒断条件下的焦虑水平。总之，数据表明，这些受体系统的封锁减少酒精摄入量。这些“常规”基因靶向研究的局限性在于（i）基因敲除发生较早，因此在发育期间可能发生代偿机制，以及（ii）受体敲除发生在整个动物中，因此未提供关于募集的神经回路的信息。为了解决这些问题，我们将诱导MOP和CRF 1受体基因的敲除，特别是在成年动物的扩展杏仁核（EA）的基础上，INIA（酒精中毒综合神经科学倡议）的整体假设，EA在过度饮酒的作用。首先，我们将利用两个现有的突变小鼠品系，一个具有floxed MOP受体基因（我们实验室最近创建的），另一个具有floxed CRF 1受体基因（合作）。第二，我们将开发一种新的转基因小鼠品系表达Cre重组酶在EA。为此，我们将使用一个BAG启动子WFS 1（Wolfram syndrom 1）基因，我们最近确定为EA标记基因（特异性目的1）。第三，我们将用WFS 1-Cre小鼠繁殖floxed小鼠，以产生成年小鼠EA中MOP（Specific Aim 2）和CRF 1（Specific Aim 3）受体基因的条件性敲除。将对两个条件性细胞系的整个脑中的受体表达、吗啡反应（MOP）和基础行为（特定目标2和3）进行充分表征。最终将在过量饮酒的行为模型（包括DID和WID模型）以及急性乙醇反应和乙醇戒断（具体目标4）中广泛研究这两种条件性品系。重要的是，在特异性目的1中产生的WFS 1-Cre转基因小鼠将代表一种独特的工具，用于条件性缺失延伸杏仁核中的任何其他感兴趣的基因，并且通常可用于成瘾研究。

DESCRIPTION (provided by applicant): Gene targeting in mice to study the role of neuropeptide receptors in ethanol dependence has recently yielded fascinating results. Knockout of the mu opioid receptor (MOP) gene blocks ethanol drinking and operant responding for ethanol. Knockout of the Corticotropin Releasing Factor receptor 1 (CRF1) gene reduces levels of anxiety under basal and alcohol withdrawal conditions. Altogether data demonstrate that blockade of these receptor systems reduce alcohol intake. Limitations of these "conventional" gene targeting studies are that (i) gene knockout occurs early, therefore compensatory mechanisms could take place during development, and (ii) knockout of the receptors occurs throughout the entire animal, therefore no information on the recruited neurocircuitry is provided. To address these issues, we will induce the knockout of MOP and CRF1 receptor genes specifically in the extended amygdala (EA) of adult animals, based on the overall hypothesis of INIA (Integrative Neuroscience Initiative on Alcoholism) regarding the role of the EA in excessive alcohol consumption. First, we will take advantage of two existing mutant mouse lines, one with a floxed MOP receptor gene (recently created in our laboratory), and another with a floxed CRF1 receptor gene (collaboration). Second, we will develop a novel transgenic mouse line expressing Cre recombinase in the EA. To do this, we will use a BAG promoter for the WFS1 (Wolfram syndrom 1) gene, that we have recently identified as an EA marker gene (Specific Aim 1). Third, we will breed floxed mice with the WFS1-Cre mouse to produce the conditional knockout of MOP (Specific Aim 2) and CRF1 (Specific Aim 3) receptor genes in the EA of adult mice. The two conditional lines will be fully characterized for receptor expression throughout the brain, for morphine responses (MOP) and for basal behaviors (Specific Aims 2 and 3). The two conditional lines will finally be extensively studied in behavioral models of excessive alcohol drinking, including the DID and WID models, as well as for acute ethanol responses and ethanol withdrawal (Specific Aim 4). Importantly, the WFS1-Cre transgenic mice generated in Specific Aim 1 will represent a unique tool for the conditional deletion of any other gene of interest in the extended amygdala, and will be generally useful in addiction research.