Genetic Influences Over Nicotine Withdrawal

描述（由申请人提供）：据世界卫生组织称，世界上每分钟售出约1 000万支香烟，每8秒钟就有人死于烟草使用（卫生组织，2002年）。尼古丁是烟草的主要成瘾性成分，与尼古丁戒断相关的情感和身体症状导致戒烟困难。虽然目前的戒烟疗法是有帮助的，但没有一种可以声称成功率很高。因此，有必要更好地了解尼古丁戒断背后的行为和生物学因素。吸烟的持久性受到遗传因素的影响，最近发现尼古丁依赖与CHRNA5-CHRNA3-CHRNB4 nAChR基因簇的单核苷酸多态性（SNP）相关。这个应用程序建立在缺乏？含烟碱乙酰胆碱受体(？5* nAChRs)消除躯体戒断症状，减少焦虑样反应。？5* nAChRs表达于内侧束（MHb）和束间核（IPN）。最近来自实验室的研究表明，在MHb或IPN中注射nAChR拮抗剂甲胺，足以在尼古丁治疗的小鼠中沉淀戒断症状。我们首先要问，引起D398N氨基酸变化的rs16969968 SNP是否会改变？5* nAChR亚型在MHb和IPN中表达。膜片钳实验将在含有D398或N398 ？5个亚基变体。第二，调查的直接作用？5* nAChRs在MHb和IPN中，慢病毒载体将被用来表达？MHb和IPN中的5个基因变异？5只小鼠或敲低内源性？5在野生型小鼠MHb和IPN中表达。来自这些小鼠的MHb和IPN切片将用于贴片夹紧实验，以确定基因操作对尼古丁诱导的spike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在饮用水中的尼古丁中六周，以研究甲胺沉淀戒断。这些研究将确定？5* nAChRs对于尼古丁戒断的表现是否必要和充分，以及rs16969968 15 SNP产生的潜在分子变化是否在体内转化为不同的戒断症状。

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.