Molecular basis of excessive alcohol drinking

酒精中毒是一种在世界范围内发病率、死亡率和人类痛苦都很大的疾病，其首要特征是过度饮酒。综合运用行为学、遗传学和分子生物学的研究工具，将检验“两次命中假说”，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，即遗传差异和酒精神经适应性变化的几个基因的表达涉及过度饮酒的特征。这项应用的第一个目标是使用特征良好的、经过基因选择的近交系大鼠IP和INP(分别偏爱和不偏爱)，以及称为重复酒精剥夺效应(RADE)的环境诱导过量饮酒范例，利用mRNA差异显示(DD)完成初步的分子分析，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相关的基因。特别关注的将是延伸的杏仁核，它被认为与酒精中毒的“渴望”方面有关。在最初的DD屏幕中克隆的基因将作为额外的靶点添加到定制的大鼠DNA微阵列中，然后将随着时间的推移和几个大脑区域的表达情况进行分析，以评估重要的全球变化。第二个具体目标是分离杏仁核特异基因，其独特的目的是将其调整为分子工具，用于剖析这一独特的大脑区域的整体作用。目标三将包括分析新开发的INIA小鼠模型的大脑基因表达，该模型将被创建为自我给酒，其程度将在戒断时引发依赖迹象。最初将再次执行DD，随后进行定制DNA微阵列分析，包括添加新的小鼠DD靶标。第四个目的是使用实时定量聚合酶链式反应、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Western blotting和免疫组织化学来验证与过量饮酒相关的后续蛋白质水平变化。关于基因表达谱分析方案和结果的所有信息将通过储存在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，将完成来自AIMS 1-3的已识别基因的子集的功能表征。

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.