We found down-regulation of Btn1a1 which has previously demonstrated an ability to be critical for milk lipid secretion

At the 8-hour time point, mice are in acute withdrawal based on hypothalamic-pituitary-adrenocortical axis elevation, but they do not display seizures because they are a relatively seizure-resistant strain. A multi-level analysis approach was utilized which included differential expression, network analysis, cell-type specificity, and time-series clustering analyses. These approaches include computational algorithms that enhance the analysis of gene coexpression networks existing in diverse expression datasets. In addition, since the brain transcriptome is organized into gene modules associated with major cell classes and specific synaptic and cellular functions, we classified sets of genes based upon subcellular localization. Neurons and glial cells are characterized by unique transcriptional signatures and these signatures can be identified reliably from analysis of complex brain tissue without isolating homogeneous populations of cells. Classifying significantly regulated genes into cell type-specific signatures improves the quality of inference and potentially leads to refined hypotheses. A time-series analysis was performed to identify clusters of genes with similar expression patterns. Each brain region displayed distinct clusters overlapping with WGCNA modules enriched in differentially expressed and cell type-specific genes. In the AMY, a cluster was identified in which all genes were included with modules enriched with differentially expressed, and enriched with astrocyte and microglia genes. The expression of this cluster of genes was significantly increased at 0- and 8-hours, but returned to baseline at 120-hours. IPA analysis indicated that this cluster was enriched in "Inflammatory Disease"-related genes. Interestingly, B2m is known to be important for immune responses and has been shown to be alcohol responsive in multiple studies. Blednov et al. demonstrated that deletion of B2m reduced ethanol consumption in the limited access two-bottle choice test for ethanol intake, supporting a hypothesis that genes within this cluster may play a role in mediating voluntary drinking. In addition, the innate immune cytokine Cd74 was a member of this cluster. The expression of this gene is rapidly induced by alcohol and has been linked to the progression of cytokine responses during alcohol withdrawal which is consistent with changes observed 8 hours post-treatment. In NAC, a cluster of genes overlapped with modules enriched with microglia and oligodendrocytes. Similar to the AMY, IPA analysis indicated that this cluster was enriched in "Inflammatory Disease"-related genes. Slc1a2 is highly expressed in microglia and has been linked to neurodegenerative disorders as well as drug dependence. This gene is differentially expressed in human alcoholic post-mortem brain and drugs acting on this target alter motivation to drink in dependent animals. In addition, genetic variation in glutamate transporters confers risk-taking behavior in alcoholics. Together, these findings support a role for Slc1a2 in alcohol intake and dependence. As in the AMY, the cytokine Cd74 was also identified in NAC, suggesting that it may have a role in innate immune responses in multiple brain regions. Other genes in this cluster that are differentially expressed in mouse models and human alcoholics include Htra1 and Il17rc. Tsc22d3 and Gata-2 are alcohol-responsive members of this gene set but are not members of inflammatory response pathways. Tsc22d3 functions as a transcriptional regulator and is differentially expressed in human alcoholics. Tsc22d3 may be associated with neuroplastic changes in response to drugs of abuse, including ethanol in mouse striatum. In contrast to the glial signature of many of the clusters, one PFC gene cluster was highly overlapping with a WGCNA module enriched with differentially expressed neuronal genes. Mean t-value distributions indicated that the magnitude and direction of change were clearly different in the PFC compared to the other brain regions, showing both a significant down- and up-regulation of genes at the 0- and 8-hour time points. These data suggest that chronic ethanol vs withdrawal changes in neuronal gene regulation may reflect greater transcriptional control in PFC than in the enriched cell types in AMY and NAC. The most prominent pathway identified by IPA analysis indicated that this cluster was enriched in "Neurological Disease"-related genes. Bdnf has a well-documented role in synaptic plasticity and addiction. We recently demonstrated that Bdnf is significantly down-regulated in homogenized medial PFC tissue as well as in purified synaptoneurosome preparations. Reductions of Bdnf levels and knockdown of Bdnf expression increase ethanol-drinking behavior.