Experimental (nSybG4>Nkt.IR; tubGAL80ts or repoG4>Nkt.IR; tubGAL80ts) and control flies were reared at 23°C and LD 12:12 and then maintained in the same conditions as adults for 4 days. A 30°C step-up in temperature was used to inactivate GAL80ts and turn on GAL4 activity. Although only 2 days are shown in panels A and E, sleep was significantly decreased on each of the 4 days at 30°C in experimental flies compared to controls. A) Four-day sleep curves for nSybG4>Nkt.IR; tubGAL80ts and controls. B) Average day and night sleep amounts for experimental and control populations. C) Day and night sleep average and maximum bout lengths. D) Average bout numbers for day and night sleep. n = 12-15 flies for all genotypes. E) Four-day sleep curves for repoG4>Nkt.IR; tubGAL80ts and controls. F) Average day and night sleep amounts for experimental and control populations. G) Day and night sleep average and maximum bout lengths. H) Average bout numbers for day and night sleep. For both neuronal and glial knockdown, similar results were obtained in two independent biological replicates. n = 11-16 flies for all genotypes. ***, p < 0.001, **, p < 0.01 and *, p < 0.05 compared to controls. See also Figures S1, S2 and S4.