Neurocircuitry Mapping and Genotyping Core
Robert J. Hitzemann, Ph.D.
Oregon Health and Science University
Department of Behavioral Neuroscience
2U01AA013484 27 September 2001 - 31 August 2016
This is a competing renewal application for a UO1 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 funiding 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 (Iancu et al. 2010) and b) the development of a strategy for and implemention 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 UO1. As the HDID and controls are derived from a 8-way inbred strain cross (HItzemann et al. 1994), RNAseq is particularly useful , given that masking oligonucleotide array data is never optimal (see Walter et al. 2007,2009). N = 32/group; previous work (Iancu 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. Ourr working hypothesis is that it is differences between co-expression networks and not the differential expression of individual genes that has the greatest translational value (see e.g. Oti et al. 2008; Zhao et al. 2010).In Aim 3, samples from ethanol exposed macaques (Grant UO1- INIA-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.