Fig. 2

c-fos expression is higher in larvae following combined treatment with clonidine and caffeine than following caffeine alone. a qRT-PCR on groups of ~ 20 larvae (n = 4 and n = 5 biological replicates per condition) reveals that larvae treated with caffeine had a significant, 71-fold increase in c-fos expression compared to water-treated larvae (*p < 0.05, two tailed Wilcoxon rank sum test performed on the “dCt” metric, see Fig. S3a). b c-fos expression of larvae soaked in clonidine before and during caffeine exposure was significantly higher by 47% than in larvae exposed to caffeine alone (n = 6 biological replicates per condition, **p < 0.01, see Fig. S3b). c The relative c-fos expression induced by different combinations of vehicle, clonidine and caffeine is positively, linearly correlated (R2 = 0.985) with the total rebound sleep induced by these drugs. qRT-PCR was performed on groups of 37 larvae (see Fig. S3c). Note that c plots together the results of two separate experiments; in both experiments there were four groups of larvae each treated with one of the four combinations of clonidine, caffeine, DMSO and water, but in one experiment c-fos expression was measured after drug treatment, and in the other rebound sleep was measured (sleep data is per Fig. 1a and b). Each square in a–c is the mean of three technical replicates