Effect of acute ethanol on medidal prefronal cortex across BXD genetic mapping panel and progenitors

In order to elucidate the molecular mechanisms underlying individual variation in sensitivity to ethanol we profiled the prefrontal cortex transcriptomes of two inbred strains that exhibit divergent responses to acute ethanol, the C57BL6/J (B6) and DBA/2J (D2) strains, as well as 27 members of the BXD recombinant inbred panel, which was derived from a B6 x D2 cross. With this dataset we were able to identify several gene co-expression networks that were robustly altered by acute ethanol across the BXD panel. These ethanol-responsive gene-enriched networks were heavily populated by genes regulating synaptic transmission and neuroplasticity, and showed strong genetic linkage to discreet chromosomal loci. Network-based measurements of node importance identified several hub genes as established regulators of ethanol response phenotypes, while other hubs represent novel candidate modulators of ethanol responses. Animals were injected intraperitoneally (IP) with saline or 1.8 g/kg of ethanol. As part of a parallel study of ethanol induced anxiolysis, all mice underwent behavioral testing that included 15 minutes of restraint in a 50 mL conical tube followed by 10 minutes in a light-dark chamber. Mice were killed by cervical dislocation four hours following IP injection. Immediately thereafter, brains were extracted and chilled for 1 minute in iced phosphate buffer before being microdissected into 8 constituent regions, including the medial prefrontal cortex. Samples were randomly assigned to batch groups prior to total RNA extraction, cRNA synthesis and hybridization. Each microarray represent a pooling of 4-5 animals.为阐明个体对乙醇敏感性差异的分子机制，我们对两种对急性乙醇反应截然不同的近交系小鼠——C57BL/6J（B6）与DBA/2J（D2）品系——以及由B6×D2杂交衍生的BXD重组近交系群体的27个成员的前额叶皮层转录组进行了谱分析。 依托该数据集，我们成功鉴定出多个在BXD群体中经急性乙醇处理后发生显著且稳定改变的基因共表达网络。这些富集乙醇响应基因的网络中，大量基因参与突触传递与神经可塑性调控，且与特定染色体位点存在强遗传连锁关联。基于网络节点重要性的评分分析发现，多个枢纽基因已被证实为乙醇响应表型的调控因子，其余枢纽则代表了全新的乙醇响应候选调节物。 实验过程中，小鼠经腹腔注射（intraperitoneally, IP）生理盐水或1.8 g/kg乙醇。作为乙醇诱导抗焦虑平行研究的一部分，所有小鼠均接受行为学测试：先在50 mL锥形管中束缚15分钟，随后置于明暗箱中适应10分钟。腹腔注射4小时后，通过颈椎脱臼法处死小鼠。随即取出脑组织，在冰磷酸盐缓冲液中冷却1分钟，随后经显微解剖分为8个脑区，其中包括内侧前额叶皮层。在总RNA提取、cRNA合成与微阵列（microarray）杂交前，样本被随机分配至不同批次组。每一份微阵列样本均混合了4-5只小鼠的组织。