Fig. 5-S2

Chemogenetic stimulation of NPVF neurons promotes sleep.

(A) TRPV1-TagRFP-T expression in NPVF neurons of a 5-dpf Tg(npvf:EGFP);Tg(npvf:KalTA4);Tg(UAS:TRPV1-TagRFP-T) animal shown using IHC for EGFP and TagRFP-T in a 1.1 μm thick image stack. Locomotor activity and sleep in 6–8-dpf Tg(npvf:KalTA4) and Tg(npvf:KalTA4);Tg(UAS:TRPV1-TagRFP-T) siblings treated with DMSO vehicle (B–E) or 2 μM Csn (H–K) starting at 100-hpf until the end of the experiment. Mean ± SEM for one representative experiment (B,C,H,I) or 4 (D,E) or 5 (J,K) pooled experiments are shown. White and black bars under behavioral traces indicate day (14 h) and night (10 h), respectively. (F) Following 48 hr of treatment with 2 μM Csn, 100% more c-fos expression is observed in NPVF neurons of 6-dpf Tg(npvf:EGFP);Tg(npvf:KalTA4);Tg(UAS:TRPV1-TagRFP-T) animals than their Tg(npvf:EGFP);Tg(npvf:KalTA4) siblings, shown using EGFP IHC and c-fos FISH in a representative 2.6 µm thick image stack, and quantified in (G). Yellow and white arrowheads indicate NPVF neurons with and without bright c-fos puncta, respectively. (L) npvf ISH in a 2.4 µm thick image stack of animals treated with 2 μM Csn for 48 h. There was no significant difference in the number of EGFP-positive neurons between Tg(npvf:EGFP);Tg(npvf:KalTA4) and Tg(npvf:EGFP);Tg(npvf:KalTA4);Tg(UAS:TRPV1-TagRFP-T) siblings, assayed using EGFP IHC. Data presented as mean ± SEM. n = number of animals. n.s. = not significant. **p<0.01,***p<0.005 by Student’s t-test. Scale: 20 μm (A), 5 μm (F,L).