In some experiments, oocytes were exposed to GABA (1 or 10 μM) only once for 30 seconds before drug washout (see Figures 1, 2A and 4). In GABA concentration-response curve experiments (Figure 2B and C), each oocyte served as its own control, i.e., multiple concentrations of GABA (300 nM–30 μM) where tested on each cell in a random order with a 5 minute wash-out between GABA applications. In acute ethanol experiments, each oocyte served as its own control and the response to 1 μM GABA before and after 44 mM ethanol pretreatment was compared after a 5 minute wash-out. Specifically, GABA responses were tested before ethanol perfusion and after a 60 second pretreatment with ethanol. Immediately after the ethanol pretreatment 1 μM GABA was again superfused for 30 seconds in the continued presence of 44 mM ethanol. In chronic ethanol experiments, half of the oocytes from an injection group were incubated with ethanol (44 mM or 100 mM) in Oocyte Ringer solution for 16 hours immediately before electrophysiological recording (approximately 2 days after RNA injection).
