Co2 pads

Ontogeny experiments were conducted as previously described (Chakravarti Dilley et al., 2020 ). Briefly, day one males and females (young adults) were compared to day 4–5 males and females (mature adults) of the same genotype. For day 4–5 flies, newly eclosed males and females were collected and aged in group housing on standard food at 25° on a 12 h:12 h LD cycle, and flipped onto new food every 2–3 days. For day 1 flies, newly eclosed males and females were housed until loading into the sleep experiment. Flies were anesthetized on CO2 pads (Genesee Scientific Cat #59–114) and loaded into individual glass tubes (containing 5% sucrose and 2% agar food) for monitoring locomotor activity in the Drosophila Activity Monitoring (DAM) system (Trikinetics, Waltham, MA). Data collection began at ZT0 the following day, at least 16 h after CO2 exposure. Activity was measured in 1 min bins and sleep was identified as 5 min of consolidated inactivity (Shaw et al., 2000 (link); Hendricks et al., 2000 (link)). Data was processed using custom MATLAB script.

Male flies were collected at 1 to 3 days old and aged in group housing. Flies were then anesthetized on CO₂ pads (Genesee Scientific, catalog no. 59-114) and loaded into glass tubes containing 5% sucrose and 2% agar and RU486/rapamycin or EtOH as described above. All data collection began at ZT0 at least 24 hours following CO₂ anesthesia. Locomotor activity was monitored using the DAM system (Trikinetics, Waltham, MA). Activity was measured in 1-min bins, and sleep was defined as 5 min of consolidated inactivity (99 (link)). Data were processed with a custom R script using Rethomics package (100 (link)). Activity index was calculated as the average number of beam breaks per minute of wake time. For all experiments, the first day of data following loading was discarded. For starvation-induced sleep loss experiments, flies were monitored for baseline sleep and then transferred at ZT0 (lights on) into tubes containing only 2% agar and RU486/rapamycin or EtOH. Sleep loss for each fly was calculated as the difference between baseline sleep duration during the first 12 hours (ZT0 to ZT12) of the day preceding starvation and sleep duration during the first 12 hours immediately following transfer into tubes without food.

Adult female flies were collected 2 to 3 days after eclosion and aged in group housing on standard food at 25°C on a 12-hour:12-hour LD cycle (unless otherwise noted). Flies aged 5 to 7 days were anesthetized on CO₂ pads (Genesee Scientific, catalog no. 59-114) and loaded into individual glass tubes (with 5% sucrose and 2% agar) for monitoring locomotor activity in the Drosophila Activity Monitoring (DAM) system (TriKinetics, Waltham, MA) or MultiBeam Activity Monitors (Trikinetics, Waltham, MA) as denoted in figure legends. All sleep experiments were loaded between ZT5-ZT10. Data collection began at ZT0 at least 24 hours following CO₂ anesthesia. Activity was measured in 1-min bins, and sleep was defined as 5 min of consolidated inactivity (31 (link), 32 (link)). Data processing was performed using PySolo (73 (link)). Mechanical sleep deprivation was performed using a Trikinetics vortexer mounting plate. Monitors were shaken for 2 s randomly within every 20-s window for 12 hours during the night. Rebound sleep was calculated as the difference between baseline sleep duration for 12 hours during the day preceding deprivation and rebound sleep duration for 12 hour during the day immediately following nighttime deprivation.