Acute coexpression of a truncated syntaxin1A produces resistance. (A) The syntaxin1A protein shown with 3 HABC, H3 (with amino acid sequence), and transmembrane domains. Upstream activation sequence (UAS)-driven constructs with human influenza hemagglutinin (HA) epitope tags were created to drive overexpression of full-length wild-type syntaxin1A (syx^FL) and syx²²⁷, a mutant truncated in a similar position to the md130 mutation that confers resistance to isoflurane in nematodes.¹⁰  (B) Western blot of syntaxin1A protein expression in flies expressing the UAS element alone (UAS-syx²²⁷/+, left) and UAS and Gal4 elements together (UAS-syx²²⁷/+; Actin-Gal4/+, right). Endogenous syntaxin1A protein is shown in red (black arrowhead) and the truncated protein as labeled with HA antibody is shown in green (green arrowhead). (C) ED50 values for flies expressing syx²²⁷ in all neurons (orange, Elav-Gal4/+; UAS-syx²²⁷, n = 40) for responsiveness and baseline locomotion endpoints compared to genetic control (grey, Gal4 Control, Elav-Gal4/+, n = 40). Error bars show ± standard error of the estimate. Extra sum-of-squares F test, **P < 0.01, ***P < 0.001. (D) Schematic of Gal80^TS experimental protocol to express syx²²⁷ transiently in adult flies. Flies are housed at 25° then moved to 32° overnight (more than12 h) to remove Gal80^TS suppression of Gal4 expression. (E) Western blot of endogenous syntaxin1A protein (red) and HA tag (green) to verify the heat treatment of flies induced expression of syx²²⁷. Flies expressing the UAS element alone (UAS-syx²²⁷/+) and UAS and Gal4-Gal80^TS elements together (Elav-Gal4/+; UAS-syx²²⁷/+; tubulin-Gal80^TS/+) were left at room temperature (first two lanes) or heated overnight. HA protein expression is only present after flies have been heated. (F) Responsiveness and locomotion ED50 values for heated flies (top) expressing syx²²⁷ in all neurons (yellow, Elav-Gal4/+; UAS-syx²²⁷, n = 40) following removal of GAL80^TS suppression compared to Gal4 (grey, Gal4 Control, Elav-Gal4/+, n = 40) and UAS (black, UAS Control, UAS-syx²²⁷/+; tubulin-Gal80^TS/+, n = 40) genetic controls. Bottom is a nonheated control. Error bars show standard error of the estimate. Extra sum-of-squares F test, *P < 0.05, ***P < 0.001. (G) Recovery from isoflurane anesthesia (mean proportion responding) in flies expressing syx²²⁷ in all neurons (orange, Elav-Gal4/+; UAS-syx²²⁷, n = 68) compared with Gal4 (grey, Gal4 Control, Elav-Gal4/+, n = 68) and UAS (black, UAS Control, UAS-syx²²⁷/+, n = 68) genetic controls. Error bars show ± SD. Asterisks indicate the first stimulus where responses of Elav-Gal4 > syx²²⁷ flies were significantly different from control flies. Kruskal–Wallis corrected for multiple comparisons (Dunn). ****P < 0.001. Time points on the x axis show discrete stimulus events every 10 min. Data points show reaction proportion for each genotype at each stimulus event. (H) Triangles in G indicate stimulus at which response is no longer statistically significantly different from baseline. (I) Mean time taken to first initiate locomotion following anesthesia induction. Error bars show ± SD. One-way ANOVA (between-subjects), n.s., not significant.