Drosophila strains and fly husbandry
All flies for experiments were maintained at 25 °C in a 12 h–12 h light–dark cycle, except for the temperature-sensitive Gal80 experiment in which Gal80 flies were kept at 18 °C until the experiment and activated Gal4 at 30 °C for 2 days. For the gene switch experiment, 500 μM mifepristone (RU486, sigma, catalogue no. M8046) was added to sucrose/agar food. For the RU486 control food, the same amount of 80% ethanol was added to sucrose/agar food. The following Drosophila stocks were used in this study: CantonS (Sehgal laboratory stock), HmlΔ-LexA LexAop-mCherry (J. Shim), HmlΔ-Gal4 UAS-EGFP (BL30139, BL30140), Srp-Hemo-mCherry (BL78358, BL78359, BL78362, BL78363), 9-137-Gal4 (Sehgal laboratory stock), NP2222-Gal4 (Sehgal laboratory stock), C584-Gal4 (Sehgal laboratory stock), TH-Gal4 (BL8848), R23E10-Gal4 (BL49032), R85G01-LexA (BL54285), R54C07-LexA (BL61562), Srp-Gal4 (L. Waltzer), Srp-Hemo-Gal4DBD (I. Evans), Srp-Hemo-Gal4AD (I. Evans), eater-dsRed (U. Banerjee), Ppn-Gal4 (BL77733), NP6293-Gal4 (Sehgal laboratory stock), moody-Gal4 (Sehgal laboratory stock), Repo-GeneSwitch (Sehgal laboratory stock), UAS-CD4::GRASP (BL58755), eater1 (BL68388), UAS-eater (BL36325), eater RNAi (BL25863, V4301), UAS-mCD8GFP (BL5137), UAS-mCD8::RFP, LexAop-mCD8::GFP (BL58754) UAS-LD::GFP (M. Welte), UAS-GFP::LSD2 (M. Welte), UAS-Sirt1 (BL44216), GLaz-GFSTF (BL60526), UAS-h.MEGF11(BL78460), MZ0709-Gal4 (M. Freeman), Alrm-Gal4 (M. Freeman), Repo-Gal4 (L. Griffith), Repo-LexA (M. Freeman), put RNAi (BL35195), Lsd-1 RNAi (V30884), NimC3 RNAi (V22920), GLaz RNAi (V4806), Lsd-2 RNAi (V40734), UAS-PvrDN (BL58431), Nplp2 RNAi (BL54041), NimC1 RNAi (BL25787), NimC4 RNAi (BL61866), NimB1 RNAi (BL55937), NimC2 RNAi (BL25960), Smox RNAi (BL41670), PGRP-LC RNAi (BL33383), drpr RNAi (BL67034), NimB4 RNAi (BL55963), Apoltp RNAi (BL 51937), NimA RNAi (V104204), Col4a1 RNAi (BL44520), NimB2 RNAi (BL62289), NimB5 RNAi (V15758), babo RNAi (BL25933), Karl RNAi (V9446), LpR2 RNAi (BL31150), apolpp RNAi (BL28946), Acsl RNAi (V3222), LpR1 RNAi (BL27249), crq RNAi (BL40831), NimB3 RNAi (V330502).
Sleep recording
For the fly sleep recording, mated 5- to 7-day-old flies were loaded into glass tubes containing 5% sucrose with 2% agarose. At least 2 days after loading into the monitors, sleep was analysed for 3 days. Single beam monitors were used for RNAi screening and the sleep deprivation experiment, but other sleep recordings were performed with multibeam monitors. Sleep was defined as failure of the fly to cross the red beam in the monitor for 5 or more minutes, analysis of data was performed with an in-house built code as described previously56.
Sleep deprivation
We used mechanical sleep deprivation. To achieve deprivation, flies in single beam monitors were fixed to a vortex machine and shaken randomly for 2 s every 20 s over a 12-h period (from ZT12–ZT24). To estimate rebound sleep, 6 h of sleep after sleep deprivation was compared with sleep on the pre-deprivation day at the same ZT time (ZT0–ZT6). Analysis of per cent sleep gain was as described previously57. In short, 6 h of daytime sleep on the day before deprivation was subtracted from the 6 h after deprivation (sleep gain). Similarly, sleep loss was calculated by subtracting sleep during deprivation to night-time sleep a day before the deprivation (sleep loss). Percentage of sleep gain was calculated by amount of sleep gain relative to amount of sleep loss.
Fly tissue clearing and haemocyte quantification
For visualizing circulating haemocytes, we optimized two different protocols58,59. Heads of female HmlΔ-LexA LexAop-mCheery flies were cut with a micro-scissor and fixed with 4% paraformaldehyde for 4 h at room temperature with rotation. After fixation, heads were incubated with 100% methanol at 4 °C overnight. Methanol was removed the following day and heads were incubated with BABB solution (2:1 ratio of benzyl benzoate and benzyl alcohol) for at least 6 h. After removing the BABB solution, heads were mounted on glass slides with VECTASHIELD solution without 4′,6-diamidino-2-phenylindole (DAPI). Imaging was performed immediately after mounting and a 408-nm excitation laser was used for auto-fluorescent signals. For thermogenetic experiments where wake or sleep neuronal populations were manipulated, flies were maintained at 18 °C until TrpA1 was activated for 1 day at 30 °C. Following this, they were processed as above. For haemocyte counts, we used the three-dimensional (3D) object counter in ImageJ software.
Immunohistochemistry
Brains were dissected from female flies, fixed in a 4% paraformaldehyde solution and washed three times using 0.4% PBS TritonX-100. After three washes, samples were blocked using 10% normal goat serum for 30 min at 25 °C. Samples were incubated with the desired primary antibodies overnight at 4 °C and then washed three times using 0.4% PBS TritonX-100 (PBST). Samples were incubated with secondary antibodies (Life Tech, catalogue nos. A32723, A32740, A32742, A32731 and A21236) diluted 1:250 for 2 h. Samples were then washed three times with 0.4% PBST. After washing, samples were rinsed and kept in VECTASHIELD until they were mounted on glass slides. For haemocytes count, we did not remove air sacs to maximize the number of haemocytes. The following primary antibodies were used: α-NimC1 (a gift from I. Ando; 1:100), α-brp (Developmental Studies Hybridoma Bank (DSHB), catalogue no. nc82; 1:100), α-Repo (DSHB, catalogue no. 8D12; 1:100), α-cleaved dcp1 (Cell Signaling, catalogue no. 9578S; 1:100), Oil-Red O (Sigma, catalogue no. O9755) and BODIPY 493/503 (Fisher, catalogue no. D3922, 1:1,000). Images were obtained with a Leica Stellaris STED confocal microscope. For haemocyte counts, we used the 3D object counter in ImageJ software.
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