Nicotinic receptors and alcohol reinforcement

项目摘要 本申请是根据PA-13-194：酒精和尼古丁共同成瘾的机制提交的。 编码α5烟碱乙酰胆碱受体（nAChR）亚单位的基因CHRNA 5的等位基因变异， 增加对酒精和烟草依赖的脆弱性。在这里，我们将使用尖端的分子，遗传 和行为技术来研究α5* nAChRs的作用，特别是内侧缰核中的作用。 （MHb）-脚间核（IPN）途径，其中α5* nAChR密集表达，在调节 酒精的动机特性在具体目标I中，我们将评估两种小鼠的饮酒情况， 缺乏α5* nAChR信号传导：α5亚基敲除小鼠和“人源化”敲入小鼠，其中α5亚基敲除小鼠和“人源化”敲入小鼠的α5* nAChR信号传导缺失。 nAChR亚基基因已被遗传修饰，以表达烟草和酒精的主要风险等位基因 人类的依赖。其次，我们将使用颅内自刺激（ICSS）程序来评估 酒精在这些突变小鼠品系中的奖励和厌恶作用。我们预测，酒精摄入量将 在α5* nAChR信号传导缺陷的小鼠中，酒精的抑制作用增加，而酒精的抑制作用减少。在特定 目的二，考察乙醇对MHb-IPN体系的影响。首先，我们将使用光遗传学结合 用电生理记录来检查酒精对兴奋性和抑制性传递的影响 在MHb-IPN突触，并确定α5* nAChR在这些影响中的作用。第二，我们将使用铷 外排试验，以确定饮酒对MHb-IPN系统中α5* nAChR活性的影响。 我们预测，酒精刺激兴奋性MHb输入到IPN -一个“厌恶”信号-这种效应是 通过缺乏α5* nAChR信号传导而减弱。我们进一步预测，长期饮酒会导致 MHb-IPN系统中α5* nAChRs的活性降低，这可能有助于酒精的发展 饮酒习惯在具体目标III中，我们将研究MHb-IPN系统的参与，以及α5* nAChRs 在这个系统中，在调节饮酒方面。首先，我们将使用一个优雅的Cre重组酶依赖 化学遗传学（DREADD）方法来刺激或抑制MHb-IPN系统中的神经元，或更多 选择性地仅那些表达α5* nAChR的MHb-IPN神经元，并检查对饮酒的影响。 其次，我们将使用病毒介导的基因转移来重新表达MHb中原本缺失的α5 nAChR亚基 或IPN神经元的α5基因敲除小鼠，并检查对这些小鼠饮酒的影响。我们预测 MHb-IPN系统和该系统中的α5* nAChR在调节酒精摄入中起关键作用。这 创新的研究计划可能会对酒精依赖的机制产生新的见解， 支持开发全新类别的治疗剂。

PROJECT SUMMARY This application is submitted in response to PA-13-194: Mechanisms of alcohol and nicotine co-addiction. Allelic variation in CHRNA5, the gene encoding the α5 nicotinic acetylcholine receptor (nAChR) subunit, increases vulnerability to alcohol and tobacco dependence. Here, we will use cutting-edge molecular, genetic and behavioral techniques to investigate the role for α5* nAChRs, particularly those in the medial habenula (MHb)-interpeduncular nucleus (IPN) pathway where α5* nAChRs are densely expressed, in regulating the motivational properties of alcohol. In Specific Aim I, we will assess alcohol drinking in two lines of mice with deficient α5* nAChR signaling: α5 subunit knockout mice and “humanized” knock-in mice in which the α5 nAChR subunit gene has been genetically modified to express the major risk allele for tobacco and alcohol dependence in humans. Second, we will use the intracranial self-stimulation (ICSS) procedure to assess the rewarding and aversive effects of alcohol in these lines of mutant mice. We predict that alcohol intake will be increased, and aversive effects of alcohol decreased, in mice with deficient α5* nAChR signaling. In Specific Aim II, we will investigate the effects of alcohol on the MHb-IPN system. First, we will use optogenetics coupled with electrophysiological recordings to examine the effects of alcohol on excitatory and inhibitory transmission at the MHb-IPN synapse and determine the role for α5* nAChRs in these effects. Second, we will use rubidium efflux assays to determine the effects of alcohol drinking on the activity of α5* nAChRs in the MHb-IPN system. We predict that alcohol stimulates excitatory MHb inputs to IPN – an “aversion” signal – and that this effect is attenuated by deficient α5* nAChR signaling. We further predict that prolonged alcohol intake results in diminished activity of α5* nAChRs in MHb-IPN system, which may contribute to the development of the alcohol drinking habit. In Specific Aim III, we will investigate the involvement of the MHb-IPN system, and α5* nAChRs in this system, in regulating alcohol drinking. First, we will use an elegant Cre recombinase-dependent chemogenetics (DREADD) approach to stimulate or inhibit neurons in the MHb-IPN system, or more selectively only those MHb-IPN neurons that express α5* nAChRs, and examine effects on alcohol drinking. Second, we will use virus-mediated gene transfer to re-express otherwise absent α5 nAChR subunits in MHb or IPN neurons of the α5 KO mice and examine the effects on alcohol drinking in these mice. We predict that the MHb-IPN system, and α5* nAChRs in this system, play a key role in regulating alcohol intake. This innovative program of research may yield novel insights into the mechanisms of alcohol dependence that supports development of entirely new classes of therapeutic agents.