cry mutation increases sleep duration selectively under short photoperiods.

(A)Daily sleep duration in male circadian clock gene mutants per0, tim0, clkjrk, cyc0, and cryb compared to wild-type (WT) males under 4L20D, 8L16D, 12L12D, 16L8D, 20L4D (12L12D, n = 86, 63, 91, 14, 91, 53 flies; 8L16D, n = 21, 31, 31, 10, 32, 31 flies; 12L12D, n = 31, 14, 32, 14, 27, 32 flies; 16L8D, n = 36, 30, 33, 17, 36, 30 flies; 20L4D, n = 42, 55, 83, 26, 91, 89 flies).(B)Average sleep traces of cryb and WT male flies under 4L20D. (C-F) The daily sleep duration (C), waking activity (D), sleep bout number (E), average sleep bout length (F) for cryb and WT female and male flies under 4L20D (n = 56, 88, 58, 91 flies). For (B), statistical differences between cryb and WT is determined by paired two-tailed t-test, *P < 0.001. For A and C-E, statistical differences is determined by two-tailed Student’s t-test, ***P < 0.001. Error bars represent standard error of the mean (SEM). ZT, Zeitgeber Time (ZT0 is the time of lights on).

CRY functions in GABAergic neurons and promotes wakefulness via GABA signaling.

(A, B) Daily sleep duration of male flies with cry knocked down in GABAergic neurons by VGATGAL4 (A) (n = 72, 74, 96, 49, 54 flies) or Gad1GAL4 (B) (n = 34, 31, 31, 25, 23 flies) monitored under 4L20D.(C) Daily sleep duration of cry knocked down in GABAergic neurons by Gad1GAL4 monitored under 12L12D (n = 20, 19, 30, 26, 32, 20 flies). For A-C, One-way ANOVA with Bonferroni multiple comparison test: compared to GAL4 control, ***P < 0.001; compared to UAS control, #P < 0.05, ###P < 0.001.(D) Daily sleep duration of male WT and cryb flies fed with EOS or NipA under 4L20D (n = 31, 32, 30, 30, 25, 32 flies). Two-tailed Student’s t-test: compared to WT, ###P < 0.001; compared to vehicle control, *P < 0.05, ***P < 0.001.(E) Daily sleep duration of cry RNAi and control flies fed with NipA under 4L20D (n = 72, 74, 95, 49, 54, 23, 24, 26, 15, 24 flies). For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, ***P < 0.001; compared to UAS control, ###P < 0.001. For comparing to vehicle control, two-tailed Student’s t-test was used, $$$P < 0.001.(F) Daily sleep duration of male flies with cry and gad1 knocked down in GABAergic neurons monitored under 4L20D (n = 72, 73, 24, 33, 45, 33, 27 flies). For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, **P < 0.01, ***P < 0.001; compared to UAS control, ###P < 0.001. For comparing to RNAi control, two-tailed Student’s t-test was used: compared to UAScryRNAi/GAL4 control, $$$P < 0.001; compared to UASGad1RNAi/GAL4 control, &&&P < 0.001.(G) Daily sleep duration of male flies with cry and VGAT knocked down in GABAergic neurons monitored under 4L20D (n = 72, 73, 46, 41, 45, 66, 34 flies). For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, ***P < 0.001; compared to UAS control, #P < 0.05, ### P < 0.001. For comparing to RNAi control, two-tailed Student’s t-test was used: compared to UAScryRNAi/GAL4 control, $$$P < 0.001; compared to UASVGATRNAi/GAL4 background, not significant. (H) Daily sleep duration of cry mutant flies with VGAT knocked down in GABAergic neurons monitored under 4L20D (n = 72, 59, 20, 23, 69, 22 flies). For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, **P < 0.01, ***P < 0.001; compared to UAS control, #P < 0.05, ###P < 0.001. For comparison between mutant vs. control, two-tailed Student’s t-test was used: compared to WT background, &&&P < 0.001; compared to UVGATRNAi/GAL4, not significant. Error bars represent SEM; G4, GAL4; U, UAS; NS, not significant.

GABA-A receptor mediates the effects of CRY on sleep/wakefulness.

(A, C, D) Daily sleep duration of male WT and cry mutant flies fed with THIP (A) (n = 28, 29, 16, 19 flies), SKF-97541 (C) (n = 25, 21, 23, 18 flies) or CBZ (D) (n = 28, 25, 31, 29 flies) under 4L20D. Two-tailed Student’s t-test: compared to WT, ###P < 0.001; compared to vehicle control, ***P < 0.001.(B, E) Sleep profile of male WT and cry mutant flies fed with THIP (B) or CBZ (E) under 4L20D in A and D, respectively. White box indicates light period while black box indicates dark period. (F, H) Daily sleep duration of male cry RNAi and control flies fed with THIP (F) (n = 72, 74, 95, 49, 54, 23, 30, 27,27, 20 flies) or CBZ (H) (n = 72, 74, 95, 49, 54, 18, 22, 26, 30, 24 flies) under 4L20D. For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, ***P < 0.001; compared to UAS control, ##P < 0.01, ###P < 0.001. For comparing to vehicle control, two-tailed Student’s t-test was used, $$$P < 0.001. (G, I) Sleep profile of male cry RNAi and control flies fed with THIP (G) or CBZ (I) under 4L20D in F and H, respectively. White box indicates light period while black box indicates dark period. (J) Daily sleep duration of male rdlMD-RR flies with cry knocked down in GABAergic neurons under 4L20D, along with relevant controls (n = 72, 73, 66, 44, 45, 33, 45, 54, 38, 33 flies). For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, ***P < 0.001; compared to UAS control, ###P < 0.001. For comparing to WT or UAScryRNAi/GAL4 control, two-tailed Student’s t-test was used: compared to WT, &P < 0.05; compared to UAScryRNAi/GAL4 control, $$$P < 0.001. (K, L) Sleep profile of male rdlMD-RR flies with cry knocked down in GABAergic neurons using cryRNAi-1 (K) or cryRNAi-2 (L) under 4L20D in J. Error bars represent SEM; G4, GAL4; U, UAS.

CRY increases the neural activity of the l-LNvs to promote wakefulness.

(A-D) Brains from male WT and cry mutant flies dissected at ZT1, 7, 13, 19 under 4L20D (A) or 12L12D (C) are immunostained with GABA (green) and PDF (red) antisera, and representative l-LNvs are displayed. Merged signal is shown as yellow. Bar graphs represent normalized GABA intensity in the l-LNvs under 4L20D (B) (n = 26-56 cells) and 12L12D (D) (n = 32-128 cells). The average value of the control group at ZT1 is set to 1. (E) Representative live image of the l-LNvs expressing GCaMP6m and tdTomato using PdfGAL4.Brain samples are dissected at the indicated time points under 4L20D. (F) Quantification of GCaMP6m signal intensity normalized to that of tdTomato (n = 25-54 cells). Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001. (G) Daily sleep duration of male flies expressing Kir2.1 in PDF neuron using PdfGAL4 and controls, monitored under 4L20D (n = 32, 42, 30 flies). One-way ANOVA with Bonferroni multiple comparison test: compared to GAL4 control, *P < 0.05; compared to UAS control, ###P < 0.001. (H) Daily sleep duration of male cry mutant flies expressing TrpA1 in the l-LNvs using c929GAL4 and relevant controls, monitored under 4L20D and 29℃ to activate TrpA1 (n = 42, 42, 38, 35, 45, 37 flies). For comparison with UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, ***P < 0.001; compared to UAS control, ###P < 0.001. For comparison between mutant vs. control, two-tailed Student’s t-test was used: &P < 0.05, &&&P < 0.001. (I) Daily sleep duration of cry mutant flies expressing NachBac in the l-LNvs using c929GAL4 and relevant controls, monitored under 4L20D (n = 45, 30, 32, 48, 55, 48 flies). For comparison with UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, *P < 0.05, ***P < 0.001; compared to UAS control, ###P < 0.001. For comparison between mutant vs. control, two-tailed Student’s t-test was used: &P < 0.05, &&&P < 0.001. (J, K) Daily sleep duration of male Pdf01-cryb (J) (n = 58, 91, 89, 31 flies) and Pdfrhan5304;cryb (K) (n = 58, 91, 31, 31 flies) mutants along with relevant controls, monitored under 4L20D. Two-tailed Student’s t-test: compared to WT background, ***P < 0.001; compared to Pdf01or Pdfrhan5304, ###P < 0.001; compared to cryb, &&P < 0.01, &&&P < 0.001. The scale bar represents 15 µm. Error bars represent SEM. G4, GAL4; U, UAS.

CRY acts in the s-LNvs to inhibit their neural activity and promote wakefulness.

(A) Brains of male flies expressing nls-GFP driven by Gad1GAL4 maintained under 4L20D and immunostained with PDF antisera. Representative l-LNv and s-LNv are displayed. (B) Quantification of GFP signal intensity of PDF neuron (n = 20, 49, 43 cells). Two-tailed Student’s t test: ***P < 0.001. (C) Daily sleep duration of flies with cry knocked down in the s-LNvs using R6GAL4 and relevant controls, monitored under 4L20D (n = 45, 60, 63, 34, 39 flies). (D-G) Brains from male WT and cry mutant flies dissected at ZT1, 7, 13, 19 under 4L20D (D) or 12L12D (F) are immunostained with GABA (green) and PDF (red) antisera, and representative s-LNvs are displayed. Merged signal is shown as yellow. Bar graphs represent normalized GABA intensity in the s-LNvs under 4L20D (E) (n = 20-53 cells) and 12L12D (G) (n = 29-67 cells). The average value of the control group at ZT1 is set to 1. (H) Representative live image of the s-LNvs expressing GCaMP6m and tdTomato using PdfGAL4. Brain samples are dissected at the indicated time points under 4L20D. (I) Quantification of GCaMP6m signal intensity normalized to that of tdTomato (n = 29-47 cells). Two-tailed Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001. (J) Daily sleep duration of male flies expressing TrpA1 in the s-LNvs using R6GAL4 and relevant controls, monitored under 4L20D and 29℃ to activate TrpA1 (n = 45, 54, 29, 22, 56, 22 flies). (K) Daily sleep duration of male cry mutant flies expressing TrpA1 in the s-LNvs using R6GAL4 and relevant controls, monitored under 4L20D and 29℃ to activate TrpA1 (n = 33, 42, 23, 35, 36, 20 flies). (L) Daily sleep duration of male cry mutant flies over-expressing HK in the s-LNvs using R6GAL4 and relevant controls, monitored under 4L20D (n = 24, 29, 23, 30, 37, 22 flies). For comparison with UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, *P < 0.05, ***P < 0.001; compared to UAS control, ##P < 0.01, ###P < 0.001. For comparison between mutant vs. control, two-tailed Student’s t-test was used: compared to WT background, &&P < 0.01, &&&P < 0.001. The scale bar represents 15 µm. Error bars represent SEM. G4, GAL4; U, UAS; NS, not significant.

s-LNvs release GABA onto the l-LNvs.

(A) l-LNvs of male flies expressing syt-GFP in the s-LNvs using R6GAL4 maintained under 4L20D and immunostained with PDF antisera. (B) l-LNvs of male flies expressing trans-Tango in the s-LNvs using R6GAL4 maintained under 4L20D and immunostained with HA and PDF antisera. (C) Brains from male flies with VGAT (top) or Gad1 (bottom) knocked down in the s-LNvs using R6GAL4 and controls maintained under 4L20D and immunostained with GABA (green) and PDF (red) antisera. Representative l-LNvs are displayed. Merged signal is shown as yellow. (D, E) Bar graphs represent normalized GABA intensity in the l-LNvs of flies with VGAT (D) (n = 29, 37 cells) or Gad1 (E) (n = 37, 51 cells) knocked down in the s-LNvs using R6GAL4, monitored under 4L20D. The average value of the control group is set to 1. Two-tailed Student’s t-test: **P < 0.01, ***P < 0.001. (F) Daily sleep duration of male cry mutant flies with VGAT knocked down in the s-LNvs using R6GAL4 and relevant controls, monitored under 4L20D (n = 31, 31, 32, 29, 43, 19 flies). For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, *P < 0.05, **P < 0.01; compared to UAS control, ###P < 0.01. For comparison between mutant vs. control, two-tailed Student’s t-test was used: compared to WT background, &&&P < 0.001. (G) Sleep profile of male cry mutant flies with VGAT knocked down in the s-LNvs using R6GAL4 and relevant controls in F, monitored under 4L20D. (H) Daily sleep duration of male cry mutant flies with Gad1 knocked down in the s-LNvs using R6GAL4 and relevant controls, monitored under 4L20D (n = 59, 59, 53, 60, 62, 45 flies). For comparison between RNAi flies vs. UAS/GAL4 controls, one-way ANOVA with Bonferroni multiple comparison test was used: compared to GAL4 control, *P < 0.05, **P < 0.01, ***P < 0.001; compared to UAS control, #P < 0.05, ##P < 0.01. For comparison between mutant vs. control, two-tailed Student’s t-test was used: compared to WT background, &&&P < 0.001. (I) Sleep profile of male cry mutant flies with Gad1 knocked down in the s-LNvs using R6GAL4 and relevant controls in H, monitored under 4L20D.The scale bar represents 15 µm. Error bars represent SEM. G4, GAL4; U, UAS; NS, not significant.

Short photoperiod reduces GABA level at the l-LNvs and increases sleep duration.

(A-D) Brains from male WT flies dissected at ZT1, 7, 13, 19 under 4L20D or 12L12D are immunostained with GABA (green) and PDF (red) antisera. Representative l-LNvs (A) and s-LNvs (C) are displayed. Merged signal is shown as yellow. Bar graphs represent normalized GABA intensity in the l-LNvs (B) (n = 28-55 cells) and s-LNvs (D) (n = 21-59 cells). The average value of the control group at ZT1 is set to 1. Student’s t-test: ***P < 0.001. (E-I) The daily sleep duration (E), sleep profile (F), sleep bout number (G), average sleep bout length (H) and waking activity (I) of male WT flies under 4L20D and 12L12D (n = 91, 32 flies). Two-tailed Student’s t-test: ***P < 0.001. The scale bar represents 15 µm. Error bars represent SEM. (J) A model demonstrating how CRY regulates the GABAergic s-LNv/l-LNv circuitry under short vs. longer photoperiods to promote arousal.

cry knock-out mutation lengthens sleep duration under 4L20D.

Daily sleep duration of male WT flies and cry knock-out flies, monitored under 4L20D (n = 53, 35, 47). Student’s t-test: *P < 0.05. Error bars represent SEM.

CRY does not regulate sleep duration via TIM.

Daily sleep duration of male WT flies and flies mutant for cry and/or tim, monitored under 4L20D (n = 58, 63, 91, 33 flies). Two-tailed Student’s t-test: compared to WT background, ***P < 0.001; compared to tim0, ###P < 0.001. Error bars represent SEM.

Screening for anatomical substrates that mediate the effects of CRY on sleep/wakefulness.

(A) Difference in sleep duration between male cry RNAi flies compared to GAL4 and UAS controls under 4L20D (n = 20-92). (B) Plots of relative mRNA abundance for cry determined by qRT-PCR in whole-head extracts of cryRNAi and control flies under 4L20D (n = 4). The value of the GAL4 control group was set to 1. Mann-Whitney test: compared to GAL4 control, *P < 0.05; compared to UAS control, #P < 0.05. G4, GAL4; U, UAS.

CRY promotes wakefulness via GABA signaling.

(A, B) Sleep profile of male flies with cry knocked down in GABAergic neurons by VGATGAL4 (A) or Gad1GAL4 (B) monitored under 4L20D. (C, D) Sleep profile of male WT and cry mutant flies fed with NipA (D) or EOS (E) under 4L20D in Fig 2D. White box indicates light period while black box indicates dark period. (E) Sleep profile of cry RNAi and control flies fed with NipA under 4L20D in Fig 2E. (F) Sleep profile of male flies with cry and gad1 knocked down in GABAergic neurons monitored under 4L20D in Fig 2F. (G) Sleep profile of male flies with cry and VGAT knocked down in GABAergic neurons monitored under 4L20D in Fig 2G. (H) Sleep profile of cry mutant flies with VGAT knocked down in GABAergic neurons monitored under 4L20D in Fig 2H. (I, J) Plots of relative mRNA abundance for Gad1 (J) and VGAT (K) determined by qRT-PCR in whole-head extracts of RNAi and control flies under 4L20D (n = 6). The value of the GAL4 control group was set to 1. Mann-Whitney test: compared to GAL4 control, **P < 0.01; compared to UAS control, ##P < 0.01. (K) Daily sleep duration of male flies with cry and gfp knocked down in GABAergic neurons by Gad1GAL4 monitored under 4L20D (n = 72, 26, 29, 20, 30, 31, 68, 32, 31, 29, 29). One-way ANOVA with Bonferroni multiple comparison test: compared to GAL4 control, ***P < 0.001; compared to UAS control, #P < 0.05, ###P < 0.001. Error bars represent SEM; G4, GAL4; U, UAS; NS, not significant.

GABAergic neurons project to and form synapses with the l-LNvs.

(A) l-LNvs of flies expressing syt-GFP in GABAergic neurons using Gad1Gal4 maintained under 4L20D and immunostained with PDF antisera. (B) l-LNvs of flies expressing Pre-mGRASP in GABAergic neurons using Gad1Gal4 and t-GRASP in the l-LNvs using PdfLexA maintained under 4L20D. Brains are immunostained with PDF antisera. (C) l-LNvs of flies expressing trans-Tango in GABAergic neurons using Gad1Gal4 maintained under 4L20D and immunostained with HA and PDF antisera. G4, GAL4; U, UAS; LexAop, LexA operator.

CRY promote wakefulness by impinging on the excitability of l-LNv and PDF signaling.

(A) Sleep profile of male flies expressing Kir2.1 in PDF neuron using PdfGAL4 and controls, monitored under 4L20D in Fig 4G. White box indicates light period while black box indicates dark period. (B) Sleep profile of male cry mutant flies expressing TrpA1 in the l-LNvs using c929GAL4 and relevant controls in Fig 4H, monitored under 4L20D and 29℃ to activate TrpA1. (C) Sleep profile of cry mutant flies expressing NachBac in the l-LNvs using c929GAL4 and relevant controls in Fig 4I, monitored under 4L20D. (D, E) Daily sleep duration of male Pdf01-cryb (D) and Pdfrhan5304; cryb (E) mutants along with relevant controls in Fig 4J and K, respectively, monitored under 4L20D.

cry expression in the s-LNvs is necessary but not sufficient for maintaining normal sleep/wakefulness.

(A) Sleep profile of cry knocked down in s-LNvs by R6GAL4 monitored under 4L20D in Fig 5C. White box indicates light period while black box indicates dark period. (B) Daily sleep duration of cry knocked down in PDF-GABAergic neurons using Gad1GAL4 and PdfGAL80, monitored under 4L20D (n =101, 26, 29, 27, 31, 23, 32, 73, 63). One-way ANOVA with Bonferroni multiple comparison test: compared to GAL4 control, ***P < 0.001; compared to UAS control, #P < 0.05, ###P < 0.001. Error bars represent SEM. G4, GAL4; G80, GAL80; U, UAS.

s-LNvs appear to be GABAergic neurons.

(A) Brains from male flies with VGAT knocked down in the s-LNvs using R6GAL4 and control maintained under 4L20D and immunostained with GABA (green) and PDF (red) antisera. Representative s-LNvs are displayed. Merged signal is shown as yellow. (B) Bar graphs represent normalized GABA intensity in the s-LNvs with VGAT knocked down using R6GAL4 (n = 25, 32 cells). The average value of the control group is set to 1. Two-tailed Student’s t-test, ***P < 0.001. The scale bar represents 15 µm. Error bars represent SEM. G4, GAL4; U, UAS.

CRY promotes wakefulness by impinging on the excitability of the s-LNvs.

(A) Sleep profile of male flies expressing TrpA1 in the s-LNvs using R6GAL4 and relevant controls in Fig 5J, monitored under 4L20D and 29℃ to activate TrpA1. White box indicates light period while black box indicates dark period. (B) Sleep profile of male cry mutant flies expressing TrpA1 in the s-LNvs using R6GAL4 and relevant controls in Fig 5K, monitored under 4L20D and 29℃ to activate TrpA1. (C) Sleep profile of male cry mutant flies over-expressing HK in the s-LNvs using R6GAL4 and relevant controls in Fig 5L, monitored under 4L20D. Error bars represent SEM. G4, GAL4; G80, GAL80; U, UAS.