Supplementary Figure 9: TRPV1–mediated activation and ablation of Hcrt neurons affects sleep–wake behaviors.

(a) A representative example of Tg(hcrt:TRPV1–RFPT) and Tg(hcrt:EGFP) co–expression in a 5 d.p.f. larva detected using anti–EGFP (green) and anti–RFPT (magenta) IHC. Images show a maximum intensity projection of a 50 μm confocal z–stack. Scale bars, 20 μm. (b) RFPT–positive and EGFP–positive cell counts in Tg(hcrt:TRPV1–RFPT);Tg(hcrt:EGFP) larvae (n = 8) and control Tg(hcrt:EGFP) larvae (n = 2). TRPV1–RFPT was expressed in 85% of EGFP–positive Hcrt neurons, and EGFP was expressed in 95% of TRPV1–RFPT–positive neurons. Thus, the Tg(hcrt:TRPV1–RFPT) line shows specific and nearly comprehensive expression of the transgene in Hcrt neurons. (c,d) Behavioral phenotypes during activation of Hcrt neurons with 1 μM Csn in Tg(hcrt:TRPV1–RFPT) larvae (n = 44) compared to their WT siblings (n = 44). (e,f) Behavioral phenotypes following ablation of Hcrt neurons with 10 μM Csn in Tg(hcrt:TRPV1–RFPT) larvae (n = 52) compared to their WT siblings (n = 43). Box plots indicate median (solid black line), 25^th and 75^th percentiles (box) and data range (whiskers). *P<0.05, **P<0.01, ***P<0.001 for transgenic larvae compared to their WT siblings by the Wilcoxon rank–sum test. (g,h) Representative images of QRFP neurons detected by anti–EGFP IHC (green), and c–fos expression detected by FISH (magenta), in Tg(hcrt:TRPV1–RFPT) larval brains after incubation 10 μM Csn for 20 minutes. Sagittal images of a 3 d.p.f. larval brain (g, scale bar, 100 μm) and a higher magnification view of the boxed area (h, scale bar, 20 μm) are oriented with rostral at left. Images are maximum intensity projections of 40 µm confocal z–stacks. A gamma correction was uniformly applied across all images to visualize cells with lower signal.