Genetic Influences Over Nicotine Withdrawal

描述（申请人提供）：根据世界卫生组织的数据，世界上每分钟约有1000万支香烟被售出，每8秒钟就有一人死于烟草使用（WHO，2002）。尼古丁是烟草的主要成瘾成分，与尼古丁戒断相关的情感和躯体症状导致戒烟困难。虽然目前的戒烟疗法是有帮助的，但没有一种疗法可以声称有很高的成功率。因此，非常需要更好地了解尼古丁戒断的行为和生物学因素。 吸烟的持续性受遗传因素的影响，最近发现的尼古丁依赖与CHRNA 5-CHRNA 3-CHRNB 4 nAChR基因簇中的单核苷酸多态性（SNP）之间的相关性突出了这一点。这个应用程序建立在观察，缺乏？5-含有烟碱乙酰胆碱受体（？5* nAChR）消除戒断的躯体体征并减少焦虑样反应。? 5* nAChR在内侧缰核（MHb）和脚间核（IPN）中表达。实验室最近的研究表明，在MHb或IPN中注射nAChR拮抗剂美加明足以使尼古丁处理的小鼠出现戒断症状。 我们将首先问是否rs 16969968 SNP，这导致D398 N氨基酸的变化，可以改变的生物物理和药理学特性？在MHb和IPN中表达的5* nAChR亚型。膜片钳实验将进行异源表达系统的nAChRs包括D398或N398？5个亚基变体。第二，调查的直接作用？MHb和IPN中的5* nAChR，慢病毒载体将用于表达？MHb和IPN的5个基因变异？5无效小鼠或敲低水平的内源性？5在野生型小鼠的MHb和IPN中。来自这些小鼠的MHb和IPN切片将用于膜片钳实验，以确定遗传操作对尼古丁诱导的尖峰放电频率增加的影响。在其他实验中，注射慢病毒的小鼠将暴露于饮用水中的尼古丁六周，以研究美卡明胺引起的戒断。这些研究将确定是否表达？5* nAChR对于尼古丁戒断的表现以及rs 16969968 15 SNP产生的潜在分子变化是否转化为体内不同的戒断症状是必要且充分的。 公共卫生相关性：我们的研究将利用基因工程小鼠和新的遗传技术，并将结合联合收割机行为分析，分子生物学和药理学来研究一个确定的单核苷酸多态性对尼古丁戒断症状的影响。这些研究可能会导致个性化戒烟疗法的发展。

DESCRIPTION (provided by applicant): According to the World Health Organization, about 10 million cigarettes are sold every minute in the world and every eight seconds someone dies from tobacco use (WHO, 2002). Nicotine is the main addictive component of tobacco, and the affective and somatic symptoms associated with nicotine withdrawal contribute to the difficulty in quitting tobacco use. While current therapies for smoking cessation are helpful, none can claim a very high rate of success. Therefore, there is a great need for a better understanding of the behavioral and biological factors underlying nicotine withdrawal. The persistence of cigarette smoking is influenced by genetic factors as highlighted by the recently found correlation between nicotine dependence and single nucleotide polymorphism (SNP) in the CHRNA5-CHRNA3-CHRNB4 nAChR gene cluster. This application builds on the observation that lack of ?5-containing nicotinic acetylcholine receptors (?5* nAChRs) abolishes somatic signs of withdrawal and reduces anxiety-like responses. ?5* nAChRs are expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN). Recent studies from the lab have shown that injection of the nAChR antagonist, mecamylamine, in either the MHb or IPN is sufficient to precipitate withdrawal signs in nicotine-treated mice. We will first ask whether the rs16969968 SNP, which causes a D398N aminoacid change, can alter the biophysical and pharmacological properties of the ?5* nAChR subtypes expressed in MHb and IPN. Patch clamp experiments will be conducted in heterologous expression systems on nAChRs comprising the D398 or N398 ?5 subunit variants. Second, to investigate the direct role of ?5* nAChRs in MHb and IPN, lentiviral vectors will be used to either express ?5 gene variants in the MHb and IPN of ?5 null mice or to knock down the levels of endogenous ?5 in the MHb and IPN of wild type mice. MHb and IPN slices from those mice will be used in patch clamping experiments to determine the effect of the genetic manipulations on nicotine-induced increases in spike firing frequency. In other experiments, lentivirus-injected mice will be exposed to nicotine in the drinking water for six weeks to study mecamylamine-precipitated withdrawal. The studies will determine whether expression of ?5* nAChRs is necessary and sufficient for the manifestation of nicotine withdrawal and whether the potential molecular changes produced by the rs16969968 15 SNP translate into different withdrawal symptoms in vivo. PUBLIC HEALTH RELEVANCE: Our studies will take advantage of genetically engineered mice and novel genetic techniques, and will combine behavioral analysis, molecular biology, and pharmacology to study the influence of an identified single nucleotide polymorphism on the manifestations of nicotine withdrawal. The studies could lead to the development of personalized smoking cessation therapies.