1. Treated and whole brains dissected at Medical University of South Carolina (Marcelo F. Lopez laboratory)
2. Frozen brains sub-dissected at UTHSC (Megan K. Mulligan laboratory)
3. RNA extraction (Qiagen RNeasy kit) at UTHSC (Mulligan and Williams laboratories)
4. Hybridized to Affymetrix Clariom D (aka Affymetrix MTA 1.0 ST) array at UTHSC MRC (Lorne Rose, UTHSC Molecular Resource Center)
5. Initial QC and normalization (COMBAT) at UTHSC (Megan K. Mulligan laboratory)
6. Transcriptome entry and phenotype entry (Arthur Centeno, Megan K. Mulligan, and Robert W. Williams)
Chronic ethanol exposure:
Mice were allowed to self-administer alcohol (15% v/v vs. water) for 2 h a day (5 days a week) 6 weeks prior to treatment in order to establish baseline consumption. Access to 15% alcohol versus water started 30 min prior to the start of the dark cycle. Following establishment of baseline drinking, male mice representative of each strain were separated into groups to be exposed to either weekly cycles of CIE exposure (CIE group) or air control (AIR group) exposure.
Mice assigned to the CIE treatment group were exposed to alcohol vapor for 16 h a day followed by 8 h of withdrawal for 4 days per week. Following the fourth vapor exposure, mice were given a 72-h abstinence/withdrawal period before resuming ethanol intake in the home cage for 5 days. Mice in the AIR control treatment group were similarly treated but exposed only to air in the inhalation chambers. This pattern of CIE or air control exposure followed by 5 days of ethanol self-administration was repeated for four cycles. A fifth cycle of CIE (or air) exposure followed the fourth ethanol intake evaluation period, and brain tissue was collected at multiple time points (8h, 3d, 7d, 14d) after the last cycle ended. Pyrazole (1 mmol/kg) was used to stabilize blood ethanol levels (BEC) and was administered to both CIE and AIR groups. A subset of mice received no pyrazole.
1. Normalization on Affymetrix Expression Console (SST-RMA Gene Level).
2. Batch effect detected (RNA concentration dependent) and corrected in ComBat