Coordinated dynamic gene expression changes in the central nucleus of the amygdala during alcohol withdrawal.

The central nucleus of the amygdala (CeA) is richly innervated with dopaminergic and adrenergic fibers from the ventral tegmental area and brainstem. Functionally, it integrates somatic and emotional information, processing autonomic and affective states, and plays a role in the motivation and stress of alcohol addiction (McBride, 2002). Subsequently, alcohol withdrawal affects all levels of amygdalar function, altering behavioral, emotional, physiological, and neuronal phenotype (Cruz et al., 2012; McBride, 2002; McBride et al., 2010). In part, these effects are thought to arise from changes in gene expression (Koob, in press). However, because alcohol alters CeA function broadly (Clapp et al., 2008), inhibiting glutamate excitation (Obara et al., 2009; Roberto et al., 2004), enhancing c-aminobutyric acid (GABA)-triggered chloride flux (Bajo et al., 2008), and altering intracellular signaling via multiple pathways (Hans-son et al., 2008), characterizing relevant gene expression changes during alcohol withdrawal is a complex undertaking. Improved insight into these expression changes may inform our understanding of the neurobiological substrates of alcohol dependence and compulsive use aimed at limiting the negative experiences of withdrawal and protracted abstinence (Koob, in press). Despite the role of the CeA in mediating alcohol-related consumption and dependence behaviors, only modest gene expression changes have been reported in the region under various exposure paradigms. For instance, in a genome-wide study of alcohol-preferring rats, differential expression was shown in only 23 genes 24 hours after a last operant ethanol (EtOH) exposure period (Rodd et al., 2008). While these included a GABAA receptor subunit (Gabrr2) and a regulator of metabotropic glutamate receptors (Homer2), the limited changes observed were potentially attributable to random chance. In a follow-up study, McBride and colleagues (2010) measured CeA gene expression 1, 6, and 24 hours after an 8-week series of repeated 1-hour binge exposure sessions in alcohol-preferring rats. Here, the CeA showed a more robust response, with expression changes in 402 named genes from 34 Gene Ontology annotation categories including: GABA signaling, second messenger signaling systems, enzyme-linked receptor protein signaling, regulation of protein kinase, and the MAPKKK cascade. Together, these studies suggest that alcohol-induced expression changes in the CeA are likely to be both time- and context-dependent. Consequently, a more complete understanding requires frequent sampling under multiple experimental conditions. Toward this goal, this study examines the temporal patterns of gene expression in the CeA during the first 48 hours of alcohol withdrawal. Recent work in our laboratory has focused on examining withdrawal-induced expression changes on a systems level, providing insight into regulatory dynamics in the dorsal vagal complex (DVC), a reciprocally connected portion of the medulla that supplies afferents to and receives efferents from the CeA (Freeman et al., 2012a,b). Here, we use a similar approach to study the CeA during dependence and withdrawal. By focusing on a limited gene set, representing GABAA, N-methyl-D-aspartic acid (NMDA), and functionally important G-protein-coupled receptor (GPCR) subunits that involve other neuromodulatory signals including corticotropin-releasing hormone, angiotensin-peptide system, metabotropic glutamate receptors, and other neuropeptide systems, as well as downstream signaling components, we gain a dynamic view of the transcriptional contributions to withdrawal following chronic alcohol exposure. With this approach, we discovered coexpressed gene relationships and potential underlying regulatory mechanisms. During withdrawal, 47% of measured transcripts experienced statistically significant expression changes. Often, these changes were withdrawal-specific—their expression levels were not significantly different from control values during chronic exposure. Over the course of withdrawal, we identified 4 significant temporal expression patterns that suggest roles for several transcription factors (TFs) previously implicated in alcoholic liver disease and inflammation in coordinating the amygdala's withdrawal-related expression change, including Cebp, Usf-1, Smad3, Ap-2, and c-Ets.