Molecular basis of excessive alcohol drinking

过量饮酒的分子基础

• 批准号: 6533683
• 负责人: SUSAN E. BERGESON
• 金额: $ 22.23万
• 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/6533683
• 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.

酗酒是一种在全世界范围内发病率、死亡率和人类痛苦相当高的疾病，其首要特征是过量饮酒。行为、遗传和分子工具的整合将被用来检验“双重打击假说”，即遗传和环境因素都会导致过量饮酒。基于发现的分子方法将检验扩展的假设，即遗传差异和几个基因表达的酒精神经适应性变化与过度饮酒特征有关。该应用的第一个目标是使用经过充分表征的、基因选择的大鼠近交系 iP 和 iNP（分别是偏好和非偏好），以及称为 RADE（重复酒精剥夺效应）的环境诱导过度饮酒范例，以使用 mRNA 差异显示 (DD) 完成初始分子分析，并分离和鉴定与过度饮酒表型背后的遗传倾向和酒精特异性神经适应有关的基因。特别关注的是扩展的杏仁核，它被认为与酗酒的“渴望”方面有关。在最初的 DD 筛选中克隆的基因将作为额外的靶标添加到定制的大鼠 DNA 微阵列上，然后随着时间的推移以及多个大脑区域的表达情况进行分析，以评估重要的全局变化。第二个具体目标是分离杏仁核特定基因，其独特目的是将其用作分子工具来剖析这个独特大脑区域的整体作用。目标三将包括分析新开发的 INIA 小鼠模型大脑中的基因表达，该模型将被创建为自我饮酒，达到在戒断时引发依赖迹象的程度。首次执行 DD 后将进行定制 DNA 微阵列分析，包括添加的新小鼠 DD 靶标。第四个目标是使用实时 PCR、Northern 和 RPA 分析以及原位杂交来验证表达。在可行的情况下，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相关的随后的蛋白质水平变化。有关基因表达谱的方案和结果的所有信息将通过存放在 MGI 数据库 (http://www.informatice.jax.org) 以及新创建的 INIA 网站中免费共享。最后，将完成目标 1-3 中已识别基因子集的功能表征。