Neurocircuitry Mapping and Genotyping Core

描述（由申请人提供）：这是一个竞争性的更新申请U 01赠款题为“神经电路映射和基因分型核心”;该申请是作为一个成员提交的NIAAA赞助的“综合神经科学倡议对酒精中毒（INIA）-西（G。Koob，PI）。该申请继续将当前资助期的重点放在研究和核心活动上。当前资助期的关键核心活动是a）掌握使用加权基因协方差网络分析（WGCNA）进行中到大样本量（lancu et al. 2010）和B）制定定量RNAseq的策略和实施（Bottomly et al，2011;附录A）。有了这些工具，我们建议1）直接测序重复的黑暗中高饮酒（HDID）小鼠系和HS/NPT对照动物的转录组（约25，000，000个75 bp读数/样品），2）测序已完成慢性间歇性乙醇（CIE）程序的HDID动物的转录组和适当的对照组。该分析所需的组织将由Crabbe U 01提供。由于HDID和对照来源于8向近交系杂交（Hitzemann et al.1994），RNAseq特别有用，因为掩蔽寡核苷酸阵列数据从来不是最佳的（参见Walter et al.2007，2009）。N = 32/组;先前的工作（lancu et al. 2010）表明，该样本量足以进行拟定分析。通过激光捕获显微解剖（LCM）收集样品;用于分析的区域优先级将是杏仁核的中央核（CeA）>边缘下皮层（IL）>边缘前皮层（PL）。枕叶皮质（OC）将用作对照区域。目标1关注酗酒，而目标2关注慢性乙醇暴露如何影响有限获取2瓶选择范式中的乙醇消费。我们的工作假设是共表达网络之间的差异而不是单个基因的差异表达具有最大的翻译价值（参见例如Oti et al. 2008; Zhao et al. 2010）。在目标3中，将对来自乙醇暴露的猕猴（Grant U 01- INlA-Stress）的样品进行测序。将CeA和皮质区25和32的数据与特定目标1和2中获得的结果进行比较。

DESCRIPTION (provided by applicant): This is a competing renewal application for a U01 grant entitled "Neurocircuitry Mapping and Genotyping Core"; the application is submitted as a member of the NIAAA sponsored "Integrative Neuroscience Initiative on Alcoholism (INIA)-West (G. Koob, PI). The application continues the focus of the current funding period on both research and core activities. Key core activities of the current funding period were a) the mastery of the use of the Weighted Gene Co-variance Network Analysis (WGCNA) for moderate to large sample sizes (lancu et al. 2010) and b) the development of a strategy for and implementation of quantitative RNAseq (Bottomly et al, 2011; Appendix A). With these tools in hand, we propose 1) to directly sequence the transcriptome ( ~ 25,000,000 75 bp reads/sample) in both replicate High Drinking in the Dark (HDID) mouse lines and in the HS/NPT control animals and 2) to sequence the transcriptome HDID animals that have completed the chronic intermittent ethanol (CIE) procedure with the appropriate control groups. The tissues needed for this analysis will be provided by the Crabbe U01. As the HDID and controls are derived from a 8- way inbred strain cross (Hitzemann et al. 1994), RNAseq is particularity useful, given that masking oligonucleotide array data is never optimal (see Walter et al. 2007,2009). N = 32/group; previous work (lancu et al. 2010) has illustrated that samples of this size are adequate for the proposed analyses. Samples are collected by laser capture micro-dissection (LCM); the regional priority for analysis will be the central nucleus of the amygdala (CeA) > the infralimbic cortex (IL) > the prelimbic cortex (PL). The occipital cortex (OC) will be used as a control region. Aim 1 focuses on binge drinking whereas aim 2 focuses on how chronic ethanol exposure affects ethanol consumption in limited access 2-bottle choice paradigm. Our working hypothesis is that differences between co-expression networks and not the differential expression of individual genes have the greatest translational value (see e.g. Oti et al. 2008; Zhao et al. 2010). In Aim 3, samples from ethanol exposed macaques (Grant U01- INlA-Stress) will be sequenced. Data from the CeA and cortical areas 25 and 32 will be compared to the results obtained in specific aims 1 and 2.