(283a) A High-Throughput, Multi-Dimensional Investigation of the Brain's Transcriptomic Response to Alcohol Withdrawal

Alcohol withdrawal is a highly complex, dynamic process that occurs during the transition from an alcohol dependent state to a non-dependent state. It is characterized by a wide array of physiological and emotional symptoms that suggest a loss of neural control in response to the cessation of alcohol intake. In order to understand the role and function of the brain in producing these symptoms, we examined two specific brain regions during the response to alcohol withdrawal: the nucleus tractus solitarius (NTS) for its role in homeostatic regulation, and the central nucleus of the amygdala (CeA) for its role in emotional regulation.

To investigate the dynamic response to alcohol withdrawal in the NTS and the CeA, we performed a multi-dimensional, high-throughput analysis of the transcriptome. Adult, male rats were fed either an alcohol-containing liquid diet or a calorically-matched control diet. In order to distinguish the effects of withdrawal from the effects of chronic alcohol intake, we studied rat triplets: an alcohol-adapted rat withdrawn from alcohol, an uninterrupted alcohol-adapted rat, and an alcohol-naïve control rat. We also studied the dynamic progression of alcohol withdrawal by varying the length of withdrawal before brain sample collection, including five time points within the first 48 hours of withdrawal. We collected 203 samples that included all combinations of both brain regions, all three alcohol states, and 5 time points with at least 5 replicates per condition. For each of the 203 samples, we measured gene expression data for 192 transcripts with Fluidigm's BioMark high-throughput quantitative reverse transcription PCR platform.

Our multi-dimensional systems level analysis of this transcriptomic data revealed that the NTS and the CeA exhibit distinct patterns of gene expression that are not apparent when considering a small number of genes, as is typical in conventional approaches; the transcriptomic profiles were more highly correlated by brain region than by alcohol treatment or time point following withdrawal, indicating distinct molecular phenotypes for each brain region. However, the intraregional changes resulting from chronic dependence and withdrawal were widespread and at the expected levels of less than 2-fold in all genes considered. This indicates that the deregulation apparent in alcohol withdrawal does not originate from a single gene or pathway, but rather from the concerted regulation of many genes and pathways in the context of the specific regional molecular phenotype. These gene expression patterns were altered with the progression of alcohol withdrawal and differed between the two brain regions. These results provide a new systems-level view of multifaceted and regionally specific regulation in the brain as it is affected by chronic alcohol intake and subsequent withdrawal from that adapted state.