All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Indiana University School of Medicine (Indianapolis, IN) and are in accordance with the guidelines of the Institutional Animal Care and Use Committee of the National Institute on Drug Abuse, National Institutes of Health, and the Guide for the Care and Use of Laboratory Animals [65]. Two-month-old C57BL/6 mice (~20 g) were purchased from Harlan, Inc. (Indianapolis, IN). Upon arrival, breeder mice were individually housed and acclimated for at least one week before mating began. The mice were maintained on a reverse 12 h light-dark cycle (lights on: 19:00 - 07:00) and provided with laboratory chow and water ad libitum. Two females were placed with one male for two hours between 08:00 and 10:00. When a vaginal plug was detected after the mating period, it was designated as embryonic day 0 (E0). On E8.25 at 15:00, dams were sacrificed using CO2 gas. The embryos were treated at this stage, which is the beginning of neurulation. The window of 46 hrs treatment covered the stages of the formation of the major organs, neural specification and patterning. These stages are known to be vulnerable to alcohol [66].
