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

描述（由申请人提供）：在小鼠中研究神经肽受体在乙醇依赖中的作用的基因靶向研究最近取得了令人着迷的结果。敲除mu阿片受体（MOP）基因可阻断乙醇的饮用和乙醇的操作性反应。促肾上腺皮质激素释放因子受体1 （CRF1）基因敲除可降低基础和酒精戒断条件下的焦虑水平。总的来说，数据表明，阻断这些受体系统可以减少酒精摄入量。这些“传统”基因靶向研究的局限性在于：(1)基因敲除发生得较早，因此代偿机制可能在发育过程中发生；(2)受体的敲除发生在整个动物体内，因此没有提供有关募集神经回路的信息。为了解决这些问题，我们将基于INIA（酒精中毒综合神经科学倡议）关于EA在过度饮酒中的作用的总体假设，在成年动物的扩展杏仁核（EA）中诱导MOP和CRF1受体基因的特异性敲除。首先，我们将利用现有的两种突变小鼠系，一种具有固定的MOP受体基因（最近在我们的实验室中创建），另一种具有固定的CRF1受体基因（合作）。其次，我们将开发一种在EA中表达Cre重组酶的新型转基因小鼠系。为此，我们将使用WFS1 （Wolfram综合征1）基因的BAG启动子，我们最近确定该基因为EA标记基因（Specific Aim 1）。第三，我们将与WFS1-Cre小鼠杂交，产生成年小鼠EA中MOP （Specific Aim 2）和CRF1 （Specific Aim 3）受体基因的条件敲除。这两个条件系将在整个大脑的受体表达、吗啡反应（MOP）和基础行为（Specific Aims 2和3）方面得到充分表征。这两条条件线最终将在过量饮酒的行为模型中得到广泛研究，包括DID和WID模型，以及急性乙醇反应和乙醇戒断（Specific Aim 4）。重要的是，在Specific Aim 1中产生的WFS1-Cre转基因小鼠将代表一种独特的工具，用于条件删除扩展杏仁核中任何其他感兴趣的基因，并将在成瘾研究中普遍有用。

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.