CIE exposure was performed for a period of 10 days and initiated at DIV40. Briefly, primary glial culture (passage 2) medium was exchanged for Neurobasal™ medium containing B27 and l-glutamine, and this medium was exchanged daily. Conditioned glial medium was used for the CIE exposure paradigm and supplemented with 10 ng/mL of BDNF, NT3, GDNF, and 75 mM ethanol (control plates were fed with identical medium but without ethanol). Ethanol (200 Proof, Decon Laboratories, catalog# 2716) was used. The iNs were fed with the above medium every 24 h for the duration of 10 days. Following the 10-day CIE exposure, iNs were subjected to functional analysis. Evaporation of ethanol following 24-h medium changes in unsealed culture dishes is expected to produce a gradual reduction in ethanol concentration each day. Thus, the daily replenishment of ethanol followed by a gradual loss due to evaporation in unsealed culture dishes mimics a pattern of ethanol exposure more similar to human drinkers (Lieberman, Levine, Kranzler, Abreu, & Covault, 2012). Evaporation of ethanol was examined in 6-well culture dishes with culture media by measurement of
