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.
The purpose of the proposed research is to understand what genes are associated with animal models of excessive ethanol consumption. Detecting these genes and probably more importantly their associated gene networks may lead to new therapeutic targets for the treatment of alcoholism.
|Hitzemann, Robert; Bottomly, Daniel; Iancu, Ovidiu et al. (2014) The genetics of gene expression in complex mouse crosses as a tool to study the molecular underpinnings of behavior traits. Mamm Genome 25:12-22|
|Hitzemann, Robert; Darakjian, Priscila; Walter, Nikki et al. (2014) Introduction to sequencing the brain transcriptome. Int Rev Neurobiol 116:1-19|
|Hitzemann, R; Bottomly, D; Darakjian, P et al. (2013) Genes, behavior and next-generation RNA sequencing. Genes Brain Behav 12:1-12|
|Iancu, Ovidiu D; Oberbeck, Denesa; Darakjian, Priscila et al. (2013) Selection for drinking in the dark alters brain gene coexpression networks. Alcohol Clin Exp Res 37:1295-303|
|Crabbe, John C; Kendler, Kenneth S; Hitzemann, Robert J (2013) Modeling the diagnostic criteria for alcohol dependence with genetic animal models. Curr Top Behav Neurosci 13:187-221|
|Fei, Suzanne S; Wilmarth, Phillip A; Hitzemann, Robert J et al. (2011) Protein database and quantitative analysis considerations when integrating genetics and proteomics to compare mouse strains. J Proteome Res 10:2905-12|
|Bottomly, Daniel; Walter, Nicole A R; Hunter, Jessica Ezzell et al. (2011) Evaluating gene expression in C57BL/6J and DBA/2J mouse striatum using RNA-Seq and microarrays. PLoS One 6:e17820|
|Kaur, Simranjit; Ryabinin, Andrey E (2010) Ghrelin receptor antagonism decreases alcohol consumption and activation of perioculomotor urocortin-containing neurons. Alcohol Clin Exp Res 34:1525-34|
|Iancu, Ovidiu D; Darakjian, Priscila; Walter, Nicole A R et al. (2010) Genetic diversity and striatal gene networks: focus on the heterogeneous stock-collaborative cross (HS-CC) mouse. BMC Genomics 11:585|
|Phillips, Tamara J; Reed, Cheryl; Burkhart-Kasch, Sue et al. (2010) A method for mapping intralocus interactions influencing excessive alcohol drinking. Mamm Genome 21:39-51|
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