6-minute intervals thereafter, each vial was gently tapped 3 times on a table and the number of sedated flies (i.e. flies that were noticeably uncoordinated or immobile) in each vial was recorded 30 seconds after the final tap. Ethanol sedation assays were terminated typically at 60–90 minutes or when all flies were immobile. The percentage of non-sedated flies was calculated for each vial at each 6-minute interval, resulting in a sedation time-course for each vial. Sedation time 50 (ST50, time to 50% sedation) values were interpolated from third-order polynomial curve fits (the least complex curve that fit the data well, (R2 = 0.96±0.001, n=1221) using Excel (Microsoft, Redwood, WA, U.S.A.) or Prism 4.03 (GraphPad, San Diego, CA, U.S.A.) from the time-course data for each vial.
