To further investigate the potential role of mini-w in ethanol sedation assays, we determined if RNAi-mediated knockdown of mini-w in the nervous system via elav-Gal4 (Olofsson and Page, 2005) altered ethanol sensitivity in this paradigm. In all experiments, the elav-Gal4 and UAS-white-RNAi transposons themselves contained the mini-w marker (Figure S2). Nervous system expression of two w RNAi transgenes (v30033 and v30034) led to eye color phenotypes indistinguishable from w1118 null flies (Figure S2I–M), demonstrating that expression of v30033 and v30034 substantially inactivates mini-w. ST50s from ethanol sedation assays were not significantly different in w1118 nulls and red-eyed control flies expressing mini-w (elav-Gal4/+, v30033/+ and v30034/+; Figure 4B). Ethanol sensitivity in white-eyed flies expressing w RNAi in the nervous system (elav-Gal4;v30033 and elav-Gal4/v30034) was significantly increased compared to the elav-Gal4/+ control, but not compared to the v30033/+ or v30034/+ controls (Figure 4B), indicating that knockdown of mini-w has a negligible effect. Internal ethanol concentrations were comparable in all control and w knockdown strains tested (Figure 4C). Additionally, the development of rapid tolerance was observed in all control and w knockdown groups
