Twelve-week-old neural cultures were used to examine GABAA subunit mRNA expression following alcohol exposure. In total, 22 neural cell lines derived from 15 donor subjects (13 lines from 8 CTLs, 9 lines from 7 ADs) were used. We cultured 3–4 coverslips per subject, per condition in either neural differentiation media (sham condition) or neural differentiation media supplemented with 50 mM alcohol for 21 days. Neural differentiation media with or without alcohol was replaced daily. Evaporation results in ~18 mM alcohol remaining at 24 hours after treatment (Lieberman et al. 2012). Wells fed with alcohol-containing media were separated in different culture plates to eliminate transfer of alcohol vapor into the sham condition wells. RNA was extracted upon completion of the three-week alcohol exposure using TRIzol reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fischer Scientific, Pittsburgh, PA) and cDNA was synthesized from 2 μg RNA using a High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific).
