Figure 4. Glutamatergic DN1ps are necessary for morning and evening differences in rebound.

(A-C) 20 x images of split Gal4 line that labeling presynaptic (A), postynaptic (B) and overlay (C) of Glu⁺ DN1ps (R51H05 AD; R18H11 DBD >SYT GFP; DenMark) co-stained for BRP (blue). (D–F) Averaged activity eductions for female flies during the first 2 days of 12:12 LD. The light-phase is indicated by white bars while the dark-phase is indicated by black bars. Morning and evening anticipation indices are represented in blue and red respectively. (G) Average sleep during the baseline day for Glu⁺ DN1ps ablated (R51H05 AD; R18H11 DBD >hid) (N=30) (green), Gal4 control (R51H05 AD; R18H11 DBD> +) (N=36) and hid control (pBDP split >hid) (N=26) (purple). Sleep per 24 hr is indicated in the bottom right. (H–J) Comparison of sleep lost, baseline sleep, and sleep gain following deprivation at morning and evening timepoints in Glu⁺ DN1p ablated flies. Morning times are matched with evening time points with similar baselines. (H) hid control flies with no ablated neurons (pBDP split >hid) (N=26) exhibit greater rebound in the morning compared to matched evening time point (p<0.0001, paired t-test). (I) Gal4 control flies with no ablated neurons (R51H05 AD; R18H11 DBD> +) (N=19) exhibit greater rebound in the morning compared to matched evening time point (p<0.01, paired t-test) (J) Flies with Glu⁺ DN1ps ablated (R51H05 AD; R18H11 DBD >hid) (N=21) do not exhibit a significant difference in sleep gain between matched morning/evening time points (p>0.09, paired t-test). (K) Comparison of sleep gain at ZT1.5 between flies with Flies with Glu⁺ DN1ps ablated (R51H05 AD; R18H11 DBD >hid) (N=21) and their controls (pBDP split >hid) (N=26) and (R51H05 AD; R18H11 DBD> +) (N=19). R51H05 AD; R18H11 DBD >hid flies exhibit significantly less rebound at ZT1.5 compared to hid control (p<0.05, ANOVA) and a non-significant decrease compared to Gal4 control (p>0.05, ANOVA). Data are means +/- SEM.

Figure 4—figure supplement 1. Standardized time points show similar effects to points with matched baseline sleep (A–B) Rebound sleep heatmaps (above) illustrate average sleep as a function of time of day when rebound occurred (ZT) and minutes after SSD episode.

Missing time points are filled using matlab linear interpolation function. Baseline sleep heatmaps (below) illustrate average sleep during 30 min bins. (A) Gal4 controls with no ablated neurons (R51H05 AD; R18H11 DBD> +) (N=19) display low baseline sleep following lights on and preceding lights off. Immediately following SD flies show high sleep except in the hours preceding lights off. Flies tend to sleep less as rebound time proceeds. (B) Glu⁺ DN1ps ablated (R51H05 AD; R18H11 DBD >hid) (N=14) flies display low baseline sleep following lights on and preceding lights off. Immediately following SD flies show high sleep except in the afternoon and hours preceding lights off. Flies tend to sleep less as rebound time proceeds. (C,D) Comparison of sleep lost, baseline sleep, and sleep gain following deprivation at morning and evening timepoints in clock neuron-ablated flies. Morning times are matched with evening time points with similar baselines. (C) Gal4 control flies with no ablated neurons (R51H05 AD; R18H11 DBD> +) (N=19) exhibit greater rebound in the morning compared to the evening time point (p<0.01, paired t-test) however exhibit greater sleep lost in the evening (p<0.05). (D) Flies with Glu⁺ DN1ps ablated (R51H05 AD; R18H11 DBD >hid) (N=14) do not exhibit a significant difference in sleep gain between the morning and evening time points (p>0.05, paired t-test) however exhibit greater sleep lost in the evening (p<0.05, paired t-test). Data are means +/- SEM.