Molecular basis of excessive alcohol drinking

酒精中毒是一种在全世界造成相当高的发病率、死亡率和人类痛苦的疾病，其首要特征是过度饮酒。将使用行为、遗传和分子工具的整合来检验“两次打击假说”，即遗传和环境因素都有助于过量饮酒。基于发现的分子方法将测试扩展的假设，即遗传差异和酒精神经适应性变化的几个基因的表达参与过量饮酒的特点。本申请的第一个目的是使用良好表征的、遗传选择的大鼠近交系iP和iNP（分别为偏好和非偏好），以及环境诱导的过度饮酒范例，称为拉德（重复酒精去乙酰化作用），使用mRNA差异显示（DD）完成初始分子分析并分离和鉴定涉及遗传倾向和酒精特异性神经适应的基因，这些基因是过度饮酒表型的基础。特别关注的是扩展的杏仁核，它被认为与酗酒的“渴望”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，然后将随着时间的推移和在几个大脑区域中进行表达分析，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性基因，其独特目的是将其用作分子工具来剖析这一独特大脑区域的整体作用。第三个目标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管理酒精的程度，这将引起戒断后的依赖迹象。最初将再次进行DD，然后进行定制DNA微阵列分析，包括添加的新小鼠DD靶标。第四个目的是使用实时PCR、北方和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蛋白质水平变化。关于遗传表达谱分析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网站中而自由共享。最后，将完成目标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.