Abstract P001 P001 is a continuation and expansion of currently funded Hitzemann PARC project. Dr. Richards Leads the project as Dr Hitzemann (Co-I) transitions to retirement (1/1/2023). Dr. Ozburn (Co-I) provides resident expertise in transcriptome analysis and testing of nominated ethanol consumption treatment targets using a number of techniques. Previously published results from this project17,18, along with monkey transcriptome and epigenetic findings (P003, P004), converged to focus the PARC application on the tetrapartite synapse. Use of the heterogeneous stock-collaborative cross (HS-CC) mice continues as a unique aspect of P001. Advantages of using the HS-CC, which capture 90% of mouse genetic diversity, are described in Hitzemann et al.39. Residual individual variation in ethanol consumption that persists after selective breeding for high preference is a new key focus. S.A.1. expands our analysis of the ethanol preference risk transcriptome from a network perspective. Three key regions of the addiction circuit: prelimbic cortex (PL), nucleus accumbens core (NAcC), and central nucleus of the amygdala (CeA), will be studied in short-term selectively bred high preference (HP) and low preference (LP) lines from HS-CC founders. Further, because RNA-Seq from bulk tissue is a blunt instrument for annotating the cell types associated with specific genes and network modules, single cell RNA- Seq (scRNA-Seq) will be used for this purpose. Cognitive flexibility, identified in our PARC macaque studies as a predictor of later ethanol consumption (see P004), will be measured in the HP and LP lines for P001 in C001. Brains from these animals will be shared with P004 to track whether changes in the extracellular matrix (ECM), a prominent feature of the tetrapartite synapse, are related to differences in risk for ethanol consumption and in cognitive flexibility. S.A.2. is a new focus on transcriptional features associated with residual individual variation in chronic ethanol consumption among members of the HP line. Transcriptional analysis will be completed (P001) for the PL, NAcC and CeA, and integrated with analysis of DNA methylation in the PL and NAcC in P003 for tissues from the same subset of higher and lower ethanol-consuming HP mice. Prior to ethanol drinking, all individuals will be cognitively tested, so that cognitive flexibility can be examined as a predictor phenotype for the entire distribution of mice, as well as for the extreme tails included in transcriptome and DNA methylation analyses. S.A.3. derives from our current project and P003 indicating a role for the primary cilium in ethanol preference. We will measure primary cilia number and length in mice with differential risk for ethanol consumption (ethanol-naïve HP and LP mice), and after different amounts of chronic ethanol intake (residual variation in HP). This aim also will study the importance of receptors and signaling molecules found only in the primary cilium on ethanol consu...