Molecular basis of excessive alcohol drinking

酗酒是全世界发病率、死亡率和人类痛苦都很高的一种疾病，其首要特征是过度饮酒。将使用行为、遗传和分子工具的综合手段来检验“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一个扩展的假设，即遗传差异和酒精神经适应变化的几个基因的表达与过量饮酒的特征有关。本应用程序的第一个目标是使用具有良好特征的遗传选择的自交系大鼠，iP和iNP（分别为偏好和非偏好），以及一种称为RADE（重复酒精剥夺效应）的过度饮酒环境诱导范式。利用mRNA差异显示（DD）完成初步分子分析，分离并鉴定与过度饮酒表型相关的遗传倾向和酒精特异性神经适应相关的基因。我们将特别关注扩展的杏仁核，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目标添加到定制的大鼠DNA微阵列上，然后随着时间的推移和几个大脑区域的表达将被描绘出来，以评估重要的全球变化。第二个具体目标是分离杏仁核特定基因，并将其作为分子工具来解剖这一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，该模型将用于自我管理酒精，在一定程度上将引发戒断后的依赖迹象。DD将再次初始执行，然后进行自定义DNA微阵列分析，包括添加的新小鼠DD目标。第四个目标是使用Real-time PCR， Northern和RPA分析以及原位杂交来验证表达。在可行的情况下，Western blotting和免疫组织化学将用于验证与过量饮酒分析相关的蛋白质水平变化。所有关于基因表达谱的协议和结果的信息将通过沉积在MGI数据库（http:/www.informatice.jax.org）以及新创建的INIA网站上免费共享。最后，将完成Aims 1-3中已鉴定基因子集的功能表征。

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.