Nicotinic receptors and alcohol reinforcement

项目总结 本申请是针对PA-13-194：酒精和尼古丁联合成瘾的机制而提交的。 编码α5烟碱型乙酰胆碱受体亚单位的基因ChRNA5的等位基因变异， 增加了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、基因 和行为技术来研究α5*nAChRs的作用，特别是在内侧缰核的那些 (MHb)-α5*nAChRs密集表达的脚间核途径，在调节 酒精的激励特性。在特定的目标I中，我们将评估两种品系的小鼠的饮酒情况 α5*nAChR信号缺陷：α5亚单位敲除小鼠和人源化α5敲除小鼠 NAChR亚单位基因已被转基因，以表达烟草和酒精的主要危险等位基因 人类的依赖性。其次，我们将使用颅内自我刺激(ICSS)程序来评估 酒精对这些突变小鼠品系的奖赏和厌恶作用。我们预测酒精摄入量将是 在α5*nAChR信号缺陷的小鼠中，酒精的厌恶作用减少，增加。具体而言 目的研究酒精对MHB-IPN系统的影响。首先，我们将使用光遗传学耦合 通过电生理记录来检测酒精对兴奋性和抑制性传递的影响 并确定α5*nAChRs在这些效应中的作用。第二，我们将使用Rb 外排实验测定饮酒对α5*nAChRs活性的影响。 我们预测酒精会刺激兴奋性MHb传入IPN--一种“厌恶”信号--这种效应是 α5*nAChR信号通路的缺陷使其减弱。我们进一步预测，长期饮酒会导致 MHB-IPN系统中α5*nAChRs活性降低，可能与酒精的形成有关 饮酒习惯。在具体目标三中，我们将调查mhb-ipn系统和α5*nAChRs的参与 在这个系统中，在规范饮酒方面。首先，我们将使用优雅的依赖于Cre重组酶 化学遗传学(DREADD)方法刺激或抑制MHB-IPN系统中的神经元，或更多 有选择地只有那些表达α5*nAChRs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在MHb中重新表达原本缺失的α5nAChR亚基 或α5KO小鼠的IPN神经元，并检测对这些小鼠饮酒的影响。我们预测 MHB-IPN系统，以及该系统中的α5*nAChRs，在调节酒精摄入方面起着关键作用。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.