FIGURE 6 |. Genetic silencing of either DN1a or TPN-II perturbs normal daytime sleep restructuring by cold.

In wild type flies, cold temperature has both acute and persistent effects on daytime activity and sleep. In (A,F,G,I-K) activity and sleep were quantified in 30min bins in 2 consecutive days per condition (B,C: 1 day/condition). Schematics on top illustrate the experimental design. Data plots represent sleep (above) and activity bar graphs (below) and are averages ±SEM across days and across individual flies; filled circles in sleep plots and black dots above activity bars indicate time points that are significantly different between cold (18°C, blue) and 25°C (gray) conditions (p<0.05, paired 2-sided t-test); dark shades indicate lights-off (night), ZT: zeitgeber time. (A) In wild-type animals, cold conditions increase morning sleep and suppresses morning activity (ZT0–3); in contrast, cold reduces both sleep and activity in the evening (ZT6–12); moreover, the onset of evening sleep is advanced (green arrowhead in A). As a result, the net effect of cold is an advancement of daytime sleep. (B) Morning cold (ZT0–3) rapidly suppresses activity and increases sleep; following the return to 25°C sleep and activity quickly return to normal levels. (C) Evening cold (ZT6–12) decreases sleep, so that, together, morning and evening effects recapitulate all day cold conditions (N=31 animals in A, 26 in B,C, and see D for quantifications). (E) A split-Gal4 driver allows selective targeting of DN1as (shown driving GFP; 2-photon z-stack, scale bar =20μm). (F-L) Silencing DN1as or TPN-IIs using selective split-Gal4s perturbs sleep restructuring by cold temperature. (F,I,J) Control genotypes. (N=30 in F, N=61 in I, N=62 in J). (G) Silencing DN1as by expression of tetanus toxin light chain (TNT) partially mimics cold conditions, producing flies that sleep more in the morning even at 25°C, and that fail to restructure their afternoon sleep in response to cold; (N=19 animals). (K) Silencing TPN-II output with TNT also produces flies that fail to restructure afternoon sleep in response to cold (N=52 animals; see H, L for quantifications; In all boxplots, box edges: 25th and 75th percentiles; thick lines: median; whiskers: data range; gray dots: individual data points/flies; *=p<0.05 in paired 2-sided t-test comparing 25°C vs 18°C within genotype or 2-way ANOVAs with a Bonferroni correction for multiple comparisons across genotypes/temperatures; NS= no significant difference).