of 24 h prior to replenishment. We measured the ethanol concentration at 0, 12, and 24 h after media replenishment (Fig. 5B) and found the half-life of 75 mM ethanol in solution is ~7 h (Fig. 5C). Interestingly, the sister well without ethanol application would be “contaminated” with a low concentration of ethanol, peaking at 12 h (~10 mM) (Fig. 5B). To determine the impact of CIE exposure on synaptic transmission, we recorded mIPSCs from CIE and sister-cultured AD-iNs in the same culture plate (Fig. 5B) (Note: we normally culture control and treatment groups in the same plate to reduce batch effects in in vitro experiments). In order to bypass any homeostatic scaling changes in synaptic transmission (Lovinger & Abrahao, 2018), N40 or D40 AD-iNs subjected to 10-day CIE were incubated in 40 mM ethanol for the entire electrophysiological recording periods, while recordings in control neurons cultured in sister wells were done without ethanol (Fig. 5A). We observed no difference for control AD-iNs in terms of average mIPSC frequency (Fig. 5C) and amplitude (Fig. 5D). Interestingly, CIE AD-iNs showed an opposite phenotype to that observed with acute ethanol application (Fig. 3). The frequency of mIPSCs was significantly increased in D40
