Molecular basis of excessive alcohol drinking

过量饮酒的分子基础

• 批准号: 6655002
• 负责人: SUSAN E. BERGESON
• 金额: $ 21.94万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-27 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6655002
• 关键词: alcoholic beverage consumption; alcoholism /alcohol abuse; amygdala; animal genetic material tag; animal tissue; behavioral /social science research tag; cooperative study; craving; drug /alcohol abstinence; gene environment interaction; gene expression; genetic polymorphism; genetic susceptibility; green fluorescent proteins; immunocytochemistry; in situ hybridization; microarray technology; neurophysiology; nucleus accumbens; polymerase chain reaction; western blottings

Alcoholism, a disease of considerable morbidity, mortality and human suffering worldwide, is first and foremost characterized by excessive alcohol drinking. An integration of behavioral, genetic and molecular tools will be used to test the "Two-Hit hypothesis" that both genetic and environmental factors contribute to excessive alcohol intake. Discovery-based molecular methods will test the expanded hypothesis that genetic differences and alcohol neuroadaptive changes in the expression of several genes are involved in excessive alcohol drinking traits. The first objective of this application is to use the well characterized, genetically selected inbred strains of rats, iP and iNP (preferring and non-preferring respectively), and an environmentally induced paradigm for excessive drinking termed RADE (Repeated Alcohol Deprivation Effect), to complete initial molecular analysis using mRNA differential display (DD) and isolate and identify genes involved in both the genetic propensity and alcohol specific neuroadaptation underlying the excessive drinking phenotype. Of particular focus will be the extended amygdala, posited to be involved in "craving" aspects of alcoholism. Genes cloned in the initial DD screen will be added as additional targets on a custom rat DNA microarray and expression will then be profiled over time and across several brain regions to assess important global changes. The second specific aim is to isolate an amygdala specific gene with the distinct purpose of adapting it for use as a molecular tool to dissect the overall role of this unique brain region. Aim three will include profiling gene expression in brain from a newly developed INIA mouse model that will be created to self-administer alcohol to the extent that will elicit signs of dependence upon withdrawal. DD will again be initially performed followed by custom DNA microarray analysis including the added new mouse DD targets. The fourth aim provides validation of expression using Real-time PCR, Northern and RPA analysis, and in situ hybridization. Where feasible, Western blotting and immunohistochemistry will be used to verify consequent protein level changes relevant to excessive alcohol drinking analysis. All information on the protocol and results of the genetic expression profiling will be freely shared by deposition in the MGI data base (http:/www.informatice.jax.org) as well as a newly created INIA website. Finally, functional characterization of a subset of the identified genes from Aims 1-3 will be completed.

酒精中毒是一种在全球范围内发生相当大的发病率，死亡率和人类苦难的疾病，首先是饮酒过量的最重要疾病。行为，遗传和分子工具的整合将用于检验“两次打击的假设”，即遗传和环境因素都会导致过度酒精摄入量。基于发现的分子方法将检验扩大的假设，即遗传差异和酒精神经适应性在几种基因表达中的变化涉及过度饮酒性状。该应用的第一个目标是使用大鼠，IP和INP（分别偏爱和非偏爱的大鼠）特征性，遗传选择的植物菌株（分别和非偏爱），以及对过度饮酒的环境诱导的范式（反复的酒精剥夺效应）（重复的酒精剥夺效应），以使用MRNA差异显示（DD）和鉴定的属性（DD）和鉴定的属性（DD），并既有分子分析（DD），并构成素养（DD）的素养（DD），过多的饮酒表型。特别关注的是扩展的杏仁核，被认为参与了酒精中毒的“渴望”方面。在初始DD筛选中克隆的基因将作为自定义大鼠DNA微阵列上的其他目标添加，然后随着时间的推移和几个大脑区域进行表达，以评估重要的全球变化。第二个具体目的是隔离杏仁核特异性基因，其独特目的是将其适应作为分子工具，以剖析该独特的大脑区域的整体作用。 AIM三将包括来自新开发的Inia小鼠模型中大脑中基因表达的分析，该模型将被创建为自助酒精，以至于会引起依赖戒断的迹象。 DD最初将再次执行，然后进行自定义DNA微阵列分析，包括添加的新鼠标DD目标。第四目的是使用实时PCR，Northern和RPA分析以及原位杂交的表达验证。在可行的地方，将使用蛋白质印迹和免疫组织化学来验证与过量饮酒分析有关的蛋白质水平的变化。关于遗传表达分析的协议和结果的所有信息将由MGI数据库（http：/www.informatice.jax.org）以及新创建的INIA网站中的沉积自由共享。最后，将完成AIMS 1-3的鉴定基因子集的功能表征。