Neurocircuitry Mapping and Genotyping Core

描述（由申请人提供）：这是一份名为“神经回路映射和基因分型核心”的U01资助的竞争性续期申请；该申请是作为NIAAA赞助的“酒精中毒综合神经科学倡议(INIA)-West”（G. Koob， PI）的成员提交的。该申请延续了当前资助期对研究和核心活动的关注。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（加权基因协方差网络分析，WGCNA）进行中大型样本研究（lancu等人，2010年）；b)制定定量RNAseq策略并实施（Bottomly等人，2011年；附录a）。有了这些工具，我们建议1)在重复的高夜间饮酒（HDID）小鼠系和HS/NPT对照动物中直接测序转录组（~ 25,000,000 75 bp reads/sample）， 2)在适当的对照组中完成慢性间歇乙醇（CIE）过程的HDID动物测序转录组。本分析所需的组织将由克拉布U01提供。由于HDID和对照来自8位自交系杂交株（Hitzemann et al. 1994）， RNAseq特别有用，因为屏蔽寡核苷酸阵列数据从来都不是最佳的（见Walter et al. 2007,2009）。N = 32/组；先前的工作（lancu et al. 2010）表明，这种规模的样本足以进行拟议的分析。采用激光捕获显微解剖法（LCM）采集样品；重点分析的区域将是杏仁核中央核（CeA）、边缘下皮层（IL）、边缘前皮层（PL）。枕皮质（OC）将被用作控制区。目标1侧重于酗酒，而目标2侧重于慢性乙醇暴露如何影响有限获取2瓶选择范式下的乙醇消费。我们的工作假设是，共同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Oti et al. 2008; Zhao et al. 2010）。在目标3中，将对暴露于乙醇的猕猴（Grant U01- INlA-Stress）的样本进行测序。来自CeA和皮质区25和32的数据将与特定目的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.