Article Figures & Data
Figures
- Figure 1.
a, Left, Schematic representing experimental protocol used to subject flies to orbital motion. DAM monitors were fixed by double-sided adhesive tape on an orbital shaker that rotated the platform in the horizontal plane as shown (120 rpm, unless specified). Right, Horizontal white and black bars represent day and night period under 12:12 h light:dark cycles. Dashed bars represent the duration for which orbital motion was given: OMD (orbital motion during 12 h of daytime), OMN (orbital motion during 12 h of nighttime), and OMC (orbital motion for the entire 24 h cycle). Sleep profile of CS flies exposed to (b) OMD, (c) OMN, and (d) OMC where ● represents treated flies and ○ represents controls. Mean sleep across 3 d averaged across flies (30 min bins) is plotted across time of day with 12 h light phase (white horizontal bar) and 12 h dark phase (black horizontal bar). Sleep levels were also estimated by videography as illustrated by Movie 1. e, Mean sleep during the day (unfilled bars) and night (filled bars) under OMD, OMN, and OMC. Error bars indicate SEM. ANOVA followed by post hoc multiple comparisons Tukey's test revealed a significant increase (p < 0.05) in daytime sleep when exposed to OMD and OMC, whereas OMN has no significant effect (p > 0.05). Nighttime sleep is not significantly altered in any regime. Sample size varied from n = 24 to 32 flies. f, Sleep profiles of male control and OMD flies. g, Daytime sleep of OMD males is higher (p < 0.005) than controls. *p < 0.05. **p < 0.005. ***p < 0.0005.
- Figure 2.
a, Sleep profile of strains OR, w1118, and yw flies exposed to OMD (●), plotted along with controls (○). b, Mean daytime sleep of controls (gray bars) and OMD flies (black bars). ANOVA followed by post hoc comparisons revealed an increase in sleep (p < 0.0005) in CS, OR, w1118, and yw flies exposed to OMD. c, Mean bout length of OR strain flies is significantly increased by OMD during the day and reduced in the subsequent night. d, Activity per waking min (activity counts summed across all waking 1 min bins divided by number of waking bins) of OMD-exposed CS, OR, w1118, and yw flies is comparable to controls (p > 0.05). Daytime sleep latency is smaller for OMD-exposed CS, OR, and yw (p < 0.0005 for OR and yw, p < 0.02 for CS) but remains unchanged in w1118 flies (p = 0.16). Nighttime sleep latency is not affected (p > 0.05) in any of the fly strains. n > 20 flies per strain. e, Mean sleep levels estimated by videography in a longitudinal experiment for 1 h durations during early (ZT1-ZT2) and mid-day (ZT6-ZT7) for 32 flies. Data averaged across 2 d with the same treatment and then across flies. Baseline = no orbital motion; OMD = flies subjected to orbital motion during daytime (ZT0-ZT12). Sleep levels are significantly higher during both morning and mid-day, 1 h sampling windows. All other details same as Figure 1.
- Figure 3.
a, Left, Sleep profiles of CS flies with OMD and controls, when physical disturbance was given at ZT06. Right, Sleep latency after physical disturbance showing OMD flies fall asleep sooner than controls (p < 0.05). b, Left, Sleep profile of flies exposed to OMD from CT0 to CT12, along with light pulse of 500 lux for 10 s on the first day in DD at CT04. Right, Prepulse sleep of OMD flies is significantly higher (p < 0.05) than controls. After light pulse, sleep in OMD flies is not different from controls (p = 0.22). c, Arousal in response to light pulses of varying durations indicates a higher threshold for OMD flies. Data shown as 0 s are the fraction of flies that awoke spontaneously at ZT4 in OMD and control groups and are considered as the baseline response. For each duration, OMD flies were compared with respective controls using the χ2 test of independence. n > 59 flies for each treatment-duration combination. Asterisks indicate significance at p < 0.05.
- Figure 4.
a, Sleep profiles of three sets of CS flies: control (○), OMD (●), and OMD-caf (▴). Left panels, Pretreatment profiles where both OMD and OMD-caf show significant sleep increase (p < 0.05) compared with control. Right panels, While OMD flies (not fed on caffeine) show higher sleep levels than controls, sleep levels of flies fed with caffeine (OMD-caf) fall equal to control levels. b, Mean daytime sleep of control (white bar), OMD (black bar), and OMD-caf (gray bar), showing significant increase (p = 0.02 and p = 0.0001) in sleep in response to OMD before caffeine treatment (pre-caf); whereas following caffeine treatment (caf), OMD flies continue to show increase (p = 0.001) in sleep; however, OMD-caf flies show similar sleep levels as controls (p = 0.99). c, Mean sleep, estimated by videography for 2 h during peak sleep induction (i.e., mid-day ZT6-ZT8; averaged across 3 replicate experiments/trials), for 2 groups: experimental (expt) and controls (ctrl) flies on 3 consecutive days. Error bars indicate SEM. Baseline day = no orbital motion; OMD pre Caf day = all flies subjected to orbital motion during daytime (ZT0-ZT12); OMD Caf day = same as pre Caf day, except that expt flies were transferred to caffeinated food while ctrl were transferred to normal food. On OMD Caf day, caffeine-treated flies exhibit significantly reduced sleep compared with noncaffeinated controls (p < 0.01) as well as their own sleep on the precaffeine day (p < 0.05) (N = 3, n > 16 flies per trial). d, Sleep profiles of OMD and OMHD (between ZT00 and ZT06 only) flies plotted along with controls. Mean sleep of flies showing significant increase (p = 0.0001) in sleep in OMD and OMHD flies during the first half of the day (ZT00-ZT06); whereas in the second half of the day (ZT06-ZT12), OMHD flies sleep significantly lesser (p = 0.0001) than controls and OMD flies. e, Orbital motion for half day results in negative sleep rebound subsequently, as estimated by video records. Mean sleep (across 3 replicate experiments) is plotted during 1 h windows at ZT1-ZT2 and ZT6-ZT7 for 3 consecutive days. Error bars indicate SEM. B1 = Baseline days, no orbital motion; OMD = flies subjected to OMD during daytime (ZT0-ZT12); OMHD = flies subjected to OMD for half day (ZT0-ZT6); N = 3, n = 27-31 flies per trial OMHD flies exhibit significantly reduced sleep at ZT6-ZT7 compared with all other time points except baseline ZT1 (p < 0.007).
- Figure 5.
a, b, Sleep profiles of loss-of-function mutants of (a) period (per0) and Clock (ClkJrk). (b) Mean daytime sleep of per0 and clkjrk flies with OMD is higher than controls (t test, p = 0.0008 for per0 and p = 0.0001 for clkjrk). c, Activity profiles of per0 and clkjrk. d, During daytime, activity counts/waking min of per0 and clkjrk flies subjected to OMD did not differ from their controls (p > 0.05). Other details same as in Figure 1. n ≥ 13.
- Figure 6.
a, Left, Sleep profiles of Orco flies. a, Right, Sleep profiles of Or83bGAL4>UASkir2.1 flies. b, Daytime sleep of Orco and Or83bGAL4>UASkir2.1 flies shows statistically significant increase (p < 0.0001) in response to OMD compared with controls. c, Left, Sleep profile of CS flies in DD (CS-DD) and (right) norpA flies subjected to OMD. d, Daytime sleep of CS flies is significantly greater in response to OMD compared with controls (p < 0.05). Similarly, norpA flies show increase in sleep (p < 0.0001) when exposed to OMD compared with controls. n ≥ 16.
- Figure 7.
a, b, Sleep profiles of (a) nan36a/+ and (b) nandy5/+ flies and trans heterozygotes nan36a/nandy5 where OM was provided for only the first half of the day (ZT0-ZT6). b, Daytime sleep levels of OMHD flies are increased in all the genotypes (p = 1.0 trans-heterozygotes vs nan36a/+ and p = 0.89 trans-heterozygotes vs nandy5/+) in the first half and similar decrease (p = 0.99 trans-heterozygotes vs nan36a/+ and p = 0.05 trans-heterozygotes vs nandy5/+) in the second half compared with controls for all three genotypes. c, Sleep profiles of flies with nan-ablated flies, nanGAL4/UAShid, and controls (nanGAL4 and UAShid). d, Daytime sleep of nanGAL4/UAShid is lower (p < 0.0005 for nanGAL4/+ and p < 0.01for UAShid/+) than controls. e, Sleep profiles of nan silenced by tetanus toxin, nanGAL4/UAStntac (ac, active form), and control nanGAL4/UAStntinac flies (inac, inactive). f, Sleep is significantly lower (p = 0.01) in nanGAL4/UAStnt (ac) than nanGAL4/UAStnt (inac). g, Sleep profile under OMHD of silenced flies nanGAL4/UAStnt and their parental controls (nanGAL4 and UAStnt). h, During the first half of the day, silenced flies sleep less (p = 0.05 for nanGAL4 and p = 0.02 for UAStnt) than controls; whereas in the second half of the day, silenced flies show similar level of rebound sleep as controls (p = 0.30 for nanGAL4 and p = 0.98 for UAStnt). n > 13.
- Figure 8.
a, b, Sleep profiles of control (a) nanGAL4/+ and (b) nanGAL4/UASshibirets flies at 21°C and 28°C. c, Daytime sleep shows a similar increase (p = 0.99, ANOVA followed by Tukey's test) in response to OMD in both the genotypes at 21°C. At 28°C, silenced flies show significant difference (p < 0.0005) in daytime sleep compared with the controls. At 28°C, nighttime sleep is also reduced (p = 0.0009) in silenced flies compared with controls. d, Sleep profiles of control (nanGAL4/+) and nan-activated (nanGAL4/UASdTRPA1) flies. e, Daytime sleep in two genotypes does not differ at 21°C; however, when temperature is increased to 28°C, nanGAL4/UASdTRPA1 flies show an increase (p < 0.0001 for nanGAL4/+ and p = 0.005 for dTRPA1/+) in sleep with respect to the sleep of controls. n > 20 flies per genotype.
- Figure 9.
a, Sleep profiles of control flies with intact antennae and flies in which antennae were removed. b, Daytime sleep is significantly increased (p = 0.04) in controls in response to OMD, whereas daytime sleep of flies with dissected antennae flies is similar (p = 0.58).
- Figure 10.
a, Sleep profiles of silenced flies (JO15GAL4/UAStnt) and their controls (JO15GAL4 and UAStnt). b, Daytime sleep is significantly increased in controls (p = 0.001 for UAStnt, p = 0.03 for JO-15GAL4) as well in silenced flies (and p = 0.007) in response to OMD. c, Sleep profiles of silenced flies (NP1046GAL4/UAStnt) and their controls (NP1046GAL4 and UAStnt). d, In response to OMD, daytime sleep is significantly increased (p = 0.008 for UAStnt, p = 0.003 for NP1046GAL4) in controls, whereas it is similar (p = 1.0) in silenced flies. n > 14.
- Figure 11.
a, Sleep profiles of JO15GAL4, UAStnt, and JO15GAL4/UAStnt. b, In response to OMHD, daytime sleep is increased in the first half of the day (positive values, induced sleep) and is similar in the silenced flies as well as their controls (p = 0.93 for both controls); whereas in the second half of the day, controls JO15GAL4 and UAStnt show sleep rebound (negative values), which is different (JO15GAL4/UAStnt vs -UAStnt, p < 0.0006 and vs -J015GAL4, p = 0.05) from the silenced flies. c, Sleep profiles of 6303GAL4 and 6303GAL4/UAStnt. d, In response to OMHD, daytime sleep is increased in the first half of the day in the silenced flies as well as their controls (6303GAL4/UAStnt vs -UAStnt, p = 0.82 and vs -6303GAL4, p = 0.38; Fig. 8c,d); whereas in the second half of the day, controls 6303GAL4 and UAStnt show sleep rebound, which is different (6303GAL4/UAStnt vs -UAStnt, p = 0.02 and vs -6303GAL4, p = 0.002) from the silenced flies. e, Sleep profiles of NP1046GAL4, UAStnt, and NP1046GAL4/UAStnt (f) in response to OMHD daytime sleep are increased in the first half of the day in case of controls and are different (p = 0.02 for UAStnt and p = 0.004 NP1046GAL4) from silenced flies; whereas in the second half of the day, controls NP1046GAL4 and UAStnt have similar (p > 0.05) sleep as the silenced flies. g, Sleep profiles of control (NP1046GAL4/+ and UASdTRPA1) and temperature-activated (NP1046GAL4/UASdTRPA1) flies. h, Daytime sleep of activated flies in respect to their controls is similar at 21°C; however, when temperature is increased to 28°C, sleep of NP1046GAL4/UASdTRPA1 flies shows increase (p < 0.0001) in sleep with respect to both the controls (ANOVA followed by Tukey's test). n > 14.
- Figure 12.
a, Sleep profiles of experimental (temperature-activated) flies (JO15GAL4/UASdTRPA1) and their controls (JO15GAL4 and UASdTRPA1) at 21°C and 28°C. b, Daytime sleep of experimental flies at 21°C is significantly lower (p < 0.0001) than UASdTRPA1 but is similar (p > 0.05) to JO15GAL4; daytime sleep of experimental flies during activation (28°C) is significantly lower (p < 0.0001) than UASdTRPA1 but is similar (p > 0.05) to JO15GAL4. Daytime sleep of experimental flies at 21°C (after activation) is similar (p > 0.05) to UASdTRPA1 and JO15GAL4. c, Sleep profiles of temperature-activated flies (NP6250GAL4/UASdTRPA1) and their controls (NP6250GAL4 and UASdTRPA1) at 21°C and 28°C. d, Daytime sleep of experimental flies before activation (21°C) is significantly lower (p < 0.0001) than NP6250GAL4 but is similar (p > 0.05) to UASdTRPA1; daytime sleep of experimental flies during activation (28°C) and after activation is similar (p > 0.05) to controls (UASdTRPA1 and NP6250GAL4). e, Sleep profiles of experimental flies (6303GAL4/UASdTRPA1) and their controls (6303GAL4 and UASdTRPA1) at 21°C and 28°C. f, Daytime sleep of activated flies before (21°C), during (29°C), and after activation (21°C) is similar (p > 0.05) to UASdTRPA1 but is greater than 6303GAL4 (p < 0.0005). n > 28.
Movies
- Movie 1.
Representative video illustrates a sleep bout that was identified on a baseline day (when orbital motion was not provided), between ZT6 and ZT7. All flies in the frame were active at the start of the video. Shortly afterward, the first fly became motionless while the other flies continued to be active. This fly remained in a motionless state for ∼13 min before resuming activity toward the end of the video. During this period, the fly did not show any nontranslational movement either, which is consistent with this behavior being sleep.
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