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Featured ArticleResearch Articles, Behavioral/Cognitive

Mechanosensory Stimulation via Nanchung Expressing Neurons Can Induce Daytime Sleep in Drosophila

Shahnaz Rahman Lone, Sheetal Potdar, Archana Venkataraman, Nisha Sharma, Rutvij Kulkarni, Sushma Rao, Sukriti Mishra, Vasu Sheeba and Vijay Kumar Sharma
Journal of Neuroscience 10 November 2021, 41 (45) 9403-9418; DOI: https://doi.org/10.1523/JNEUROSCI.0400-21.2021
Shahnaz Rahman Lone
1Chronobiology Laboratory, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore, Karnataka 560064, India
2Department of Zoology, Central University of Punjab, Bathinda, Punjab 151001, India
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Sheetal Potdar
3Chronobiology and Behavioral Neurogenetics Laboratory, Neuroscience Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore, 560064, Karnataka India
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Archana Venkataraman
1Chronobiology Laboratory, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore, Karnataka 560064, India
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Nisha Sharma
2Department of Zoology, Central University of Punjab, Bathinda, Punjab 151001, India
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Rutvij Kulkarni
3Chronobiology and Behavioral Neurogenetics Laboratory, Neuroscience Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore, 560064, Karnataka India
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Sushma Rao
3Chronobiology and Behavioral Neurogenetics Laboratory, Neuroscience Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore, 560064, Karnataka India
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Sukriti Mishra
2Department of Zoology, Central University of Punjab, Bathinda, Punjab 151001, India
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Vasu Sheeba
3Chronobiology and Behavioral Neurogenetics Laboratory, Neuroscience Unit, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore, 560064, Karnataka India
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  • ORCID record for Vasu Sheeba
Vijay Kumar Sharma
1Chronobiology Laboratory, Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore, Karnataka 560064, India
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Abstract

The neuronal and genetic bases of sleep, a phenomenon considered crucial for well-being of organisms, has been under investigation using the model organism Drosophila melanogaster. Although sleep is a state where sensory threshold for arousal is greater, it is known that certain kinds of repetitive sensory stimuli, such as rocking, can indeed promote sleep in humans. Here we report that orbital motion-aided mechanosensory stimulation promotes sleep of male and female Drosophila, independent of the circadian clock, but controlled by the homeostatic system. Mechanosensory receptor nanchung (Nan)-expressing neurons in the chordotonal organs mediate this sleep induction: flies in which these neurons are either silenced or ablated display significantly reduced sleep induction on mechanosensory stimulation. Transient activation of the Nan-expressing neurons also enhances sleep levels, confirming the role of these neurons in sleep induction. We also reveal that certain regions of the antennal mechanosensory and motor center in the brain are involved in conveying information from the mechanosensory structures to the sleep centers. Thus, we show, for the first time, that a circadian clock-independent pathway originating from peripherally distributed mechanosensors can promote daytime sleep of flies Drosophila melanogaster.

SIGNIFICANCE STATEMENT Our tendency to fall asleep in moving vehicles or the practice of rocking infants to sleep suggests that slow rhythmic movement can induce sleep, although we do not understand the mechanistic basis of this phenomenon. We find that gentle orbital motion can induce behavioral quiescence even in flies, a highly genetically tractable system for sleep studies. We demonstrate that this is indeed true sleep based on its rapid reversibility by sensory stimulation, enhanced arousal threshold, and homeostatic control. Furthermore, we demonstrate that mechanosensory neurons expressing a TRPV channel nanchung, located in the antennae and chordotonal organs, mediate orbital motion-induced sleep by communicating with antennal mechanosensory motor centers, which in turn may project to sleep centers in the brain.

  • chordotonal organs
  • mechanosensation
  • nanchung
  • orbital motion
  • sleep
  • sleep rebound

Introduction

The phenomenon of sleep is exhibited by organisms across the animal kingdom, yet, our understanding regarding functions of sleep is still inadequate (Saper et al., 2010; Shaw et al., 2013). Animals shut down many vital behaviors, such as feeding and reproduction, and are more vulnerable to predators during sleep, yet the sleeping state constitutes one-half to one-third of their lifespan (Allada and Siegel, 2008; Siegel, 2009; Shaw et al., 2013). Circadian clocks determine the timing of sleep, while its quality (intensity) and quantity (duration) are determined by the homeostatic system (for review, see Shaw et al., 2013). Studies on organisms, including fruit flies, worms, zebrafish, and other model organisms, have revealed that many aspects of sleep are genetically determined (for review, see Allada and Siegel, 2008; Crocker and Sehgal, 2010; Potdar and Sheeba, 2013; Shaw et al., 2013). In flies, there is also evidence to suggest that environmental stimuli, like light, temperature, social cues, physiological states, such as age, mating status, sex, etc., can modulate quality and quantity of sleep (Donlea et al., 2009; Lone and Sharma, 2012; Lone et al., 2016; Parisky et al., 2016; Lamaze et al., 2017). Over the last two decades, we have begun to understand the role of circadian circuitry in modulating sleep and to appreciate the contributions of homeostatic processes and their interaction with the circadian circuitry using the fly (Parisky et al., 2008; Shang et al., 2008, 2013; Sheeba et al., 2008; Donlea et al., 2011; Guo et al., 2016; Liu et al., 2016; Yadlapalli et al., 2018).

In humans, walking during daytime has been shown to influence sleep duration and quality of nighttime sleep (Morita et al., 2011). In addition to the conventional prescription of somnolence-inducing drugs, nonpharmacological aids using cognitive behavioral therapy, which include relaxation techniques that can reduce hyperarousal in patients, have been used to treat insomnia (Siebern et al., 2012). Additionally, transcranial magnetic stimulation (Massimini et al., 2007), transcranial direct current stimulation (Marshall et al., 2006; Reato et al., 2013), open-loop audiovisual stimulation (Tang et al., 2016), and acoustic stimulation (Bellesi et al., 2014) have been shown to aid sleep in mammals. Although it is common knowledge that infants in cradles, adults in rocking chairs, and passengers in moving vehicles fall asleep readily, yet the mechanisms underlying motion-induced sleep remain unclear. Human subjects on a linearly accelerated swing made a faster transition to sleep, exhibiting increased number of rapid eye movements compared with those who stayed stationary (Woodward et al., 1990). A more recent study, which examined daytime sleep (afternoon nap) in human subjects, demonstrated that gentle rocking movements enabled participants to quickly transition from waking to sleep and enabled them to experience longer duration non-rapid eye movement Stage 2 sleep (Bayer et al., 2011). Yet another study on human volunteers (Perrault et al., 2019) showed that sleep latency is reduced by nighttime rocking, and there are fewer arousals compared with the night when volunteers were not subjected to rocking. Increase in slow oscillations associated with consolidated sleep and memory formation was also reported by the study (Perrault et al., 2019). Similarly, the stimulation of vestibular and proprioceptive sensory inputs of mice were also seen to elicit a sustained boosting of slow-wave oscillations and increased density of sleep spindles, both of which are indicators of deep sleep (Bayer et al., 2011; Kompotis et al., 2019). The authors hypothesized that rocking-mediated sensory inputs may directly or indirectly impact sleep centers in the brain; however, the underlying neuronal mechanisms remain elusive. Here we provide evidence for mechanosensory stimulation-induced sleep, in fruit flies (Drosophila melanogaster). We show that this sleep induction is reversible and independent of the circadian clock but regulated by homeostatic mechanisms and mediated by the mechanosensory receptor nanchung (Nan) expressing neurons located in the antennae and chordotonal organs.

Materials and Methods

Flies were collected, sexed, and maintained as virgins in a temperature- and humidity-controlled room (25 ± 1°C temperature and 75% relative humidity) under 12:12 h light/dark (LD) cycles, at a density of 30 flies per vial. In most of our experiments, light intensity of 500 lux was used during the light phase of the LD cycles. Four-day-old flies were loaded into glass tubes (65 mm length × 5 mm dia) with 5% sucrose-agar at one end and cotton at other end by anesthetizing them using carbon dioxide. Flies were recorded with the help of Drosophila Activity Monitors (DAM5) (TriKinetics). Two pairs of infrared emitters and sensors aligned roughly to the center of the glass tube, making a pair of cross beams, which picks up any locomotion of flies as they cross the middle part of the glass tube. Activity was recorded in 1 min bin, and any 5 min of continuous quiescence was considered as sleep (Hendricks et al., 2000; Shaw et al., 2000), estimated using a sliding time window. In the majority of experiments, the monitors were then placed on VDRL orbital shakers kept plugged inside incubators. In a few cases, an incubator shaker (Innova 42, Eppendorf) was used, where experimental monitors were placed roughly in the center of the shaking platform with orbit radius of 1.9 cm, while controls were placed on the fixed shelf inside the same incubator. Orbital motion of 120 rpm was used in most experiments (unless specified otherwise). Activity monitors were fixed on the shaking platforms (1 feet × 1 feet) using double-sided adhesive tape, attached at the bottom of the monitor.

To plot sleep and activity profiles, data pooled in 30 min bins and averaged across 3 d were used. For most of the experiments, flies were given OM during daytime and were left undisturbed on the shaker during night. The experiment described in Figure 1 is an exception; here OMD flies were removed from the shaker at Zeitgeber Time 12 (ZT12) and were placed along with controls, whereas OMN flies were placed on the shaker at ZT12. At ZT00, OMD flies were placed on the shaker, whereas OMN flies were removed and placed along with controls. OMC flies remained on the shaker for the duration of 24 h. For perturbation experiments (see Fig. 2a), at ZT04 or ZT06, experimental flies and control flies were subjected to perturbation by physically shaking the monitors. For light pulse exposure under DD (see Fig. 2b), light of 500 lux was switched on for 10 s without causing any disturbance to the flies. For arousal experiments (see Fig. 3c), control and experimental flies were entrained under 20 lux intensity and light pulses (250 lux) of different duration (0, 20, 40, or 60 s) were given to the flies at ZT4. Separate experiments were performed for each duration with 59-64 flies for each treatment. For all cases, the number of flies that woke up (within 10 min, i.e., equal to two sleep episodes) in response to the light pulses were counted and used to calculate arousal response. To analyze the effect of caffeine on OMD-induced sleep (see Fig. 4), a final concentration 1 mg/ml of fly medium was used. Sleep change (Δ sleep) was estimated by subtracting mean daytime or nighttime sleep of controls from daytime or nighttime sleep of each OMD fly. For Figure 8d, e, where OMD is not used, sleep change was calculated as change in sleep of GAL4>dTRPA1 with respect to each parental control.

Videography experimental methods

Fly handling and locomotor assay setup

Canton S flies of both sexes were collected on the day of emergence using CO2 anesthesia and housed in vials containing standard cornmeal medium for 2-3 d until the start of the assay. On the third day after emergence, ∼96 female flies were separated under CO2 anesthesia and transferred into 5 mm locomotor tubes containing 5% sucrose agar. These tubes were distributed equally among two Drosophila activity monitors and a clay tube holder. One monitor and the clay tube holder were placed in the tray of an orbital shaker, which was placed inside a Percival incubator maintaining a 12:12 LD cycle and an ambient temperature of 25°C. The other monitor was placed on a shelf within the same incubator. One day of acclimatization was provided, and data were collected from the fifth day after emergence. Depending on the experiment, recordings lasted 3-4 d with continuous data being collected via the DAM system and video data being collected during specific time windows. Owing to logistic limitations, only one set of flies could be video recorded at any time. Hence, a longitudinal design was used for all experiments whereby the same set of flies was subjected to different treatments on different days.

Video recording setup

To verify the patterns observed using the DAM setup, we video recorded fly activity in locomotor tubes. The video recording setup consisted of a clay tube holder into which 32 locomotor tubes could be placed. This holder was placed in the tray of the orbital shaker and video recorded using an iPhone 4S mobile phone as a webcam. iVCam software was used to capture videos directly onto the computer at a rate of 15 fps. The phone was raised above the tube holder using a metal frame attached to the shaker tray. As the phone camera underwent orbital motion along with the locomotor tubes, relative motion between the two was (largely) eliminated and videos could be recorded with a mostly stationary background (which is essential for video tracking). These videos were tracked using Ctrax software (Branson et al., 2009), and any errors in tracking were corrected using the FixErrors GUI provided along with it.

Data processing

The tracked data consisted of fly positions in every frame of the video. Despite correcting for relative motion between the camera and locomotor tubes, we observed slight periodic movement of the background in our videos. To minimize the resultant noise in measurement of fly positions, we marked dots at different points on the clay holder and tracked their movement. In each frame, the centroid of these points was calculated and fly positions were redefined with this centroid as the origin. Thus, by defining fly positions relative to the moving centroid, any movement of a fly's position because of movement of the background was eliminated. These corrected, frame-wise data were used to obtain mean fly positions in every second of the video.

Sleep measurements

A fly was considered to be inactive in a given second if its position changed by less than half its body length over that second. If inactivity lasted for 300 consecutive seconds or longer, then the entire bout was considered to be a potential sleep bout. If displacement of the fly over this bout was also less than half its body length, then the bout was considered to reflect sleep. Each such bout occurring over the course of the video was identified, and total sleep was calculated for each individual.

To test whether effects of orbital motion are under homeostatic control

We provided three treatments over 3 d: First day (B) baseline reference, when no orbital motion was provided. Second day (O) orbital motion provided during the light phase of the day (12 h). Third day (OB) orbital motion provided only during the first half of the light phase (6 h). On each of these days, videos were recorded between ZT1 and ZT2 and between ZT6 and ZT7. This experiment was repeated 3 times with a fourth day being included in two replicates. As no orbital motion was provided on this day, it served as a second control day (B2) that was used to test the effect of age on sleep levels.

Effect of caffeine during orbital motion

To test the effects of caffeine, the following treatments were provided over 3 d: First day (B) baseline reference, when no orbital motion was provided. Second day (O1) orbital motion provided during the light phase of the day (12 h). Third day (O2) orbital motion provided during light phase of the day (12 h). On the third day, half of all flies were transferred into fresh food tubes containing sucrose agar with 1 mg/ml caffeine while the rest were transferred into sucrose agar tubes. On each day, videos were recorded between ZT6 and ZT8. This experiment was also performed 3 times.

Experimental design and statistical analysis

Each experiment or assay included all possible control genotypes or experimental regime with age-matched individuals. Unless stated otherwise, data collected over a period of 3 d were averaged and analyzed using t test or, if multiple groups and factors were involved, subjected to ANOVAs followed by post hoc multiple comparisons using Tukey's HSD test. For videography experiments, at each time point in a given replicate experiment, total sleep was measured for all flies and averaged. The average values were compared using a repeated-measures ANOVA with time point as the repeated measure, followed by multiple comparisons using Tukey's HSD. These analyses were performed using Statistica version 7 software. For arousal experiments (see Fig. 3c), we performed χ2 tests of independence for each stimulus strength to test whether arousal response was dependent on treatment. More details are provided along with results at appropriate sections.

Results

Orbital motion promotes rest across multiple fly strains

Recent studies showed that gentle rocking movements decreased the latency by which a daytime nap could be induced and also altered sleep quality in human subjects (Bayer et al., 2011). Nighttime sleep quality and subsequent behavioral attributes could be enhanced by rocking in human volunteers (Perrault et al., 2019). Studies on mice have also suggested that proprioceptive and auditory stimulations of specific types can induce sleep (Bayer et al., 2011; Kompotis et al., 2019). Based on these studies of mammals including humans where rocking motion was found to induce daytime sleep, we set out to examine the effect of sustained orbital motion on flies. Here we report, for the first time, that gentle orbital motion (Fig. 1a) induces a resting state in D. melanogaster. The earliest studies that established that flies experience bona fide sleep demonstrated that 5 min of continuous inactivity, as detected by the DAM (see Materials and Methods), can be classified as a sleep bout (Hendricks et al., 2000; Shaw et al., 2000). Female Canton-S (CS) flies showed higher levels of sleep, when provided orbital motion either during daytime (OMD), or continuously throughout day and night (OMC) compared with controls, which were not exposed to orbital motion but remained in the same incubator (Fig. 1b–d). In males also, OMD caused a significantly higher daytime sleep compared with controls (p < 0.0001; t test, Fig. 1f,g). Daytime sleep of females is generally significantly lower than males in many fly strains (Huber et al., 2004; Andretic and Shaw, 2005); consequently, daytime sleep induction by orbital motion is more conspicuous and consistent. Therefore, for the most part, we report results on the phenomenon of orbital motion-aided enhancement of daytime sleep of females, unless specified otherwise. Orbital motion of either 80 or 120 rpm showed significant effect of treatment (control or OMD) (treatment, F(1,141) = 25.12, p < 0.0001; speed, F(2,141) = 0.008, p = 0.99; ANOVA). Post hoc comparisons suggest significant impact of 80 rpm (p = 0.03) and 120 rpm (p = 0.007); however, 600 rpm could not increase sleep (p = 0.22) (data not shown). Speeds <80 rpm were not technically feasible. We found that seven different positions on the shaker platform are equally (p < 0.05 for comparisons between OMD and controls) capable of sleep induction compared with the controls, suggesting that small differences in centripetal or centrifugal forces do not impair this phenomenon (data not shown).

Figure 1.
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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.

To uncover any possible strain-specific effect, we examined three other strains of D. melanogaster, Oregon-R (OR), w1118, and yellow white (yw), and found that they all show increase in daytime sleep because of OMD compared with controls (p = 0.0001 for Oregon-R (OR), w1118, and p = 0.0002 for yw; ANOVA followed by Tukey's HSD for post hoc comparisons; Fig. 2a,b). Increase in daytime sleep could impact nighttime sleep. However, we do not observe any decrease in nighttime sleep in Canton-S flies, although there is an appreciable impact on nighttime sleep in Oregon-R. Next, we analyzed sleep consolidation and found that mean sleep bout length increased during daytime (p = 0.0003; Fig. 2c) in response to OMD in case of Oregon-R flies, whereas nighttime bout length is reduced (p = 0.03, ANOVA followed by Tukey's test; Fig. 2c). For all strains, we verified that the orbital motion-induced sleep is not because of a general decline in activity since activity counts per waking minute (daytime) for OMD flies is not different from controls (p > 0.05, ANOVA followed by Tukey's test; Fig. 2d). We also observed that orbital motion during daytime induces flies to fall asleep quickly after lights-ON, with a statistically significant decrease in daytime sleep latency for most of the strains (p = 0.0002 for OR and p = 0.0003 for yw; p = 0.01 for CS; for w1118 p = 0.16; Fig. 2d), whereas nighttime sleep latency was unaffected (Fig. 2d). Thus, we detect a robust phenotype of daytime sleepiness because of OMD that is over and above baseline sleep across four fly strains. By video recording female CS flies (Movie 1) in a longitudinal experiment, we confirmed that, when subjected to OM, they indeed undergo prolonged periods of rest, consistent with conventional definition of fly sleep (Shaw et al., 2000; Hendricks et al., 2000). Sleep enrichment was detected both during ZT1-ZT2 (p < 0.00001) and ZT6-ZT7 (p = 0.0005; Wilcoxon matched pair test; Fig. 2e) compared with baseline day. Further, we confirmed that this is not because of age-dependent increase in activity as the day following 2 d of OM shows a reduction in daytime sleep with levels similar to baseline days 1 and 2 (data not shown). Thus, an independent method corroborates our finding that orbital motion induces inactivity akin to sleep.

Figure 2.
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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.

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.

Orbital motion-mediated quiescence is “true” sleep with properties of reversibility and homeostatic control

Since it is possible that the quiescence induced in flies experiencing orbital motion is merely inactivity because of inability to locomote, we examined whether OMD induced inactivity meets the accepted criteria for sleep in flies: first its reversibility and second its homeostatic control (Campbell and Tobler, 1984; Hendricks et al., 2000; Shaw et al., 2000). To examine reversibility of OMD-induced sleep, both OMD and control groups of flies were subjected to manual physical disturbance by shaking monitors for a few seconds during the middle of the day, which is also the peak of daytime sleep in flies, that is, at ZT06 (by convention, ZT00 is lights-ON under a 12:12 h LD cycle) (Fig. 3a). Physical disturbance awoke flies in both groups (∼52% and ∼60%), suggesting that this state of quiescence is reversible. Importantly, flies that experienced OMD fell asleep sooner after physical perturbation compared with controls (sleep latency, p = 0.0001, t test; Fig. 3a, right), suggesting that OMD-induced sleep drive has a potent effect on flies even after a recent experience of physical disturbance. It also suggests that orbital motion is effective in promoting sleep at times other than ZT00. We also tested this directly by initiating OMD at ZT04 instead of at ZT00, and similar results were observed for sleep levels and latency (p = 0.001; data not shown).

Figure 3.
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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.

Next, we tested whether orbital motion could induce sleep in the absence of light by first subjecting flies to OMD under LD 12:12 for 4 d following which they were placed in constant darkness and flies continued to receive OMD for 12 h of subjective day. We found that OMD-treated flies exhibited higher sleep levels even during “subjective daytime” (Fig. 3b, right; prepulse sleep). When exposed to a brief (10 s) light pulse of 500 lux, 4 h after onset of OMD (Circadian Time 04 [CT04]) (Fig. 3b, left, arrowhead), a similar fraction of OMD flies awoke as controls (54% of controls and 60% of OMD flies), which further confirmed that orbital motion-induced quiescence is reversible. Significant effects were detected for both treatment (OMD/control) and light perturbation (treatment, F(1,116) = 11.05, p = 0.001; perturbation, F(1,116) = 4.78, p = 0.03, ANOVA). Before administration of light pulse, OMD flies exhibited significantly higher daytime sleep compared with controls (p = 0.03; Fig. 3b, right); however, following light pulse, this difference was not statistically significant (p = 0.22; Fig. 3b, right). This may be because of overall reduction in sleep in both controls and treated flies due to the light pulse's impact on both circadian and sleep parameters. To observe whether increase in sleep is associated with higher arousal threshold, we first entrained flies under dim light:dark cycles (20:0 lux) and subjected them to relatively high intensity (250 lux) light pulses of different durations at ZT4. We do not detect any significant difference in the fraction of flies that awoke spontaneously at ZT4 (χ2df=1 = 1.6958, n = 128, p = 0.19). The number of flies that awoke in response to the 20 and 40 s pulses were significantly lower (χ2df=1 = 4.6436, n = 124, p = 0.03 and χ2df=1 = 4.3476, n = 125, p = 0.03, respectively; Fig. 3c) in OMD group compared with the control group. This fraction was similar when 60 s pulse was applied (χ2df=1 = 2.0089, n = 122, p = 0.15). Thus, we find that flies subject to OMD are less likely to be aroused with lower intensity stimulations.

Like humans, flies are also susceptible to the arousal-promoting effects of caffeine (Shaw et al., 2000). We asked whether the OMD is reversible by caffeine. Here, of three groups of flies, two received OMD while the third group served as control. Stage (before and after caffeine) and treatment (with or without caffeine) and their interaction were all found to be statistically significant factors (stage F(1,112) = 12.70, p = 0.0005; treatment F(2,112) = 11.77, p < 0.0001; stage × treatment F(2,112) = 9.93, p = 0.0001, ANOVA). On the day before caffeine treatment (pre-caf), both sets of OMD-exposed flies slept significantly more than controls (p = 0.02 and p = 0.0001; Fig. 4a, left, b). On the next day, immediately after lights-ON, one of the OMD-exposed groups was fed caffeinated food (1 mg/ml, OMD-caf), while the other two groups (OMD and controls) were transferred to fresh food medium without caffeine (Fig. 4a, right). Flies that received caffeine along with OMD slept significantly less during daytime compared with those exposed only to OMD (p = 0.001) but not less than the controls (p = 0.99) (Fig. 4b). Expectedly, nighttime sleep of flies fed with caffeine during the day was also lower than the other groups possibly because of the persistence of the arousal-promoting effect of caffeine into the night. This result was verified using videography in three replicate experiments where flies exhibiting OM-induced sleep were divided into two groups and given food containing either 1 mg/ml caffeine or no caffeine. Following subsequent OM, caffeinated flies showed significantly lower sleep compared with controls experiencing only OMD (p = 0.0025), despite showing comparable sleep levels before caffeine treatment (p = 0.825) (Fig. 4c). This suggests that orbital motion-induced sleep can be blocked by pharmacological intervention, demonstrating reversibility of sleep induced by orbital motion and indicating that inactivity is not because of the orbital motion preventing locomotion of flies.

Figure 4.
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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).

To examine whether orbital motion-induced sleep can be modulated by homeostatic pathways, we divided flies into three groups: the first group was subjected to orbital motion for 12 h during daytime (ZT00-ZT12, OMD), the second group only during the first half of the daytime (ZT00-ZT06, orbital motion during half day [OMHD]) and the third group served as control. There is significant effect of treatment (OMD/OMHD/Control) and time of day (first half/second half) (treatment F(2,30) = 32.59, p = 0.0; time of day F(1,30) = 17.0, p = 0.0002; time of day × treatment F(2,30) = 80.31, p < 0.0001, ANOVA followed by the Tukey's test). During the first half of the day, there was a statistically significant increase in sleep in both groups exposed to orbital motion (OMD and OMHD; p = 0.0001 for both groups, Fig. 4d, compared with controls). However, during the second half of the day, the OMHD group of flies slept significantly less than OMD and even lower compared with controls (p = 0.0001; Fig. 4d, right). This result was also confirmed by longitudinal videography experiments where we find that, after being subjected to orbital motion for half a day, flies experience negative sleep rebound as demonstrated by reduced sleep levels (in the subsequent 1 h window after OMD) relative to sleep on baseline day (Fig. 4e; p = 0.0067). This is possibly because of accelerated decline in sleep drive that is believed to occur via homeostatic processes (Hendricks et al., 2000; Shaw et al., 2000). Together, we demonstrate that several signature features of sleep are conserved in flies exposed to OMD, suggesting that OMD-mediated quiescence is indeed “true” sleep.

Orbital motion induced sleep is independent of circadian clock genes period and clock

Circadian clocks are known to influence the timing of sleep (Borbély, 1982; Borbély et al., 2016). Moreover, since we saw that the behavior of OMD-induced sleep persists even under constant darkness during subjective day (Fig. 3b), we asked whether the circadian clock is required for this sleep induction. We subjected flies carrying loss-of-function mutation for the period (per0) and clock (ClkJrk) genes to OMD (Fig. 5a) and found that mutant flies exposed to orbital motion continued to show a statistically significant increase in daytime sleep (p = 0.0008 for per0 and p = 0.0001 for clkjrk; t test; Fig. 5b) compared with controls, while their activity counts per waking minute did not differ (Fig. 5c,d). This suggests that core circadian clock genes period and Clock are not necessary for OMD-induced sleep.

Figure 5.
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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.

Negligible role of olfaction and vision in OMD sleep

Orbital motion could, in principle, affect sensory systems, such as olfaction and vision, which in turn may affect sleep circuitry. A previous study on rats has shown that olfactory stimulation can induce slow wave activity (Fontanini and Bower, 2006), while cutaneous stimulation in cats resulted in synchronized brain activity akin to sleep (Pompeiano and Swett, 1962). To test for such effects, we subjected mutants defective in olfactory and visual inputs to OMD. We tested null mutants of Orco (Or83bo), which are defective in their olfactory ability for most odors (Larsson et al., 2004) and found that mutant flies subjected to OMD show increased (p < 0.0001, t test; Fig. 6a, left, b) daytime sleep compared with controls. Further, when we silenced the Or83b-expressing olfactory neurons by expressing potassium channels (UASkir2.1), flies exposed to OMD showed increased sleep (p < 0.0001, t test; Fig. 6a, right, b) compared with controls, which suggests that olfactory signals do not play any role in mechanically stimulated sleep induction.

Figure 6.
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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.

To examine the role of vision, we subjected CS flies to LD cycles for 3 d and then transferred them to DD along with orbital motion for the first 12 h under darkness and estimated sleep levels during subjective day. We found a statistically significant increase (p < 0.05, t test; Fig. 6c, left, d) in daytime sleep in OMD flies compared with controls even under darkness, which suggests that visual cues are not needed for sleep induction. We also found that norpA mutants, known to have defective vision (Bloomquist et al., 1988), also showed sleep induction when exposed to OMD (p < 0.0001, t test; Fig. 6c, right, d). Together, these results suggest that orbital motion-aided sleep induction can occur even under suboptimal performance of sensory modalities, such as olfaction and vision.

Mechanosensory signals via nanchung expressing neurons mediate orbital motion aided sleep induction

The mechanosensory system helps organisms integrate mechanical information to guide their locomotor activity (Tuthill and Wilson, 2016). Since orbital motion may transduce mechanosensory signals to the sleep circuit, we examined possible roles of various mechanosensory signaling pathways in OMD-induced sleep. Fruit flies D. melanogaster have the ability to sense and respond to touch via specialized mechanosensory centers, the chordotonal organs (Kim et al., 2003), which have been also shown to relay temperature and vibration signals to circadian clocks via yet unknown mechanisms (Sehadova et al., 2009; Simoni et al., 2014). Additionally, mechanosensory neurons present on the legs help flies to avoid aversive conditions by inducing walking behavior (Ramdya et al., 2015). The chordotonal organs express mechanoreceptor Nanchung (Nan), which on the basis of sequence similarity is predicted to be an ion channel subunit, similar to vanilloid-receptor-related (TRPV) channels, and is activated by forces ranging from osmotic to mechanical pressures (Kung, 2005). These channels also respond to changes in gravity, sound, and humidity levels (Kamikouchi et al., 2009).

We found that flies carrying loss-of-function mutation in nan, trans-heterozygotes nandy5/nan36a as well as heterozygotes nandy5/+ and nan36a/+ (Kim et al., 2003) have no impairment in OMD-induced increased sleep (data not shown). With the same mutants subjected to orbital motion for only the first half of the day (OMHD) (Fig. 7a,b), we see significant impact of genotype and treatment (genotype F(2,76) = 5.15, p = 0.007; treatment F(1,76) = 64.76, p < 0.0001; ANOVA followed by Tukey's test). Flies show a similar increase in sleep in the first half (p = 1.0 trans-heterozygotes vs nan36a/+ and p = 0.89 trans-heterozygotes vs nandy5/+); and negative rebound in the second half (p = 0.99 trans-heterozygotes vs nan36a/+ and p = 0.05 trans-heterozygotes vs nandy5/+), suggesting that reduced nan levels do not hamper sleep induction by orbital motion. Orbital motion is likely to be transduced to higher-order brain centers through mechanosensory pathways. Therefore, we targeted the chordotonal organs, which are important mechanosensory structures in flies, using nanGAL4 driver by expressing the pro-apotoptic gene hid (nanGAL4>UAShid). When we compared sleep change (average sleep of controls with sleep of flies receiving OMD = Δ sleep), there is significant effect of genotype and treatment (genotype F(2,106) = 6.69, p = 0.001; treatment F(1,106) = 22.40, p < 0.0001, ANOVA). Ablated nanGAL4>UAShid flies showed a significantly reduced response to OMD compared with their parental controls (p < 0.0005 for nanGAL4/+ and p < 0.01for UAShid/+; Fig. 7c,d). In a separate experiment, we blocked synaptic signaling in nan neurons using an active form of tetanus toxin (nanGAL4>UAStnt(ac)) along with controls expressing an inactive form (nanGAL4>UAStnt(inac)). Sleep induction in nanGAL4>UAStnt(ac) and Δ sleep is significantly less than nanGAL4>UAStnt(inac) (p = 0.01, t test; Fig. 7e,f). These results suggest that orbital motion induces sleep via sensory input provided by Nan-expressing neurons. We have found similar results when nan-expressing neurons were silenced by UASdORCKC1. Sleep is increased in nanGAL4<UASdORKNC flies subjected to OMD (p = 0.04); however, in nanGAL4<UASdORKC flies, there was no significant change in sleep when flies were subjected to OMD (p = 0.76; data not shown). To rule out the possibility of ablation and silencing by nanGAL4 causing locomotor defects, we subjected nanGAL4>UAStnt (ac) and their controls to light pulse during subjective day (CT4). We found that similar number of flies wake up in silenced (43%) and their parental groups (59% and 47%), suggesting that simply silencing these neurons does not affect their ability to respond to wake-promoting cues. When we subjected them to OMHD, there is significant impact of the genotype, treatment, and interaction between them (genotype F(2,146) = 6.03, p = 0.003; treatment F(1,146) = 30.28, p < 0.0001; genotype × treatment F(2,146) = 3.66, p = 0.02; Fig. 7g,h). Silenced (nanGAL4>UAStnt (ac)) flies slept less compared with controls (p = 0.05 for nanGAL4 and p = 0.02 for UAStnt); however, when released from orbital motion treatment in the second half of the day, nanGAL4>UAStnt (ac) flies exhibited negative sleep rebound similar to parental controls (p = 0.30 for nanGAL4 and p = 0.98 for UAStnt), suggesting that, despite the fact that we do not detect enhanced sleep by our methods, the sleep circuits in these flies are experiencing some form of sleep signals during the first half of the day, followed by release in sleep pressure during the second half and hence exhibit sleep rebound. Since nanGAL4>UAStnt (ac) flies exhibit activity and sleep similar to the controls during the morning and evening transitions, it does not appear to be because of any impairment in their ability to sleep after their release from OMHD or sleep in general.

Figure 7.
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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.

Transient silencing and activation of nan neurons have opposite effects

Since our results thus far suggest that nan neurons transduce mechanosensory stimuli produced by orbital motion to sleep centers, we asked whether such effects can be brought about by transient modifications of this circuit. To begin with, we silenced Nan neurons transiently by overexpressing UASshibirets (Fig. 8a–c). There is significant impact of the temperature, time of day, and genotype (time of day F(1,166) = 33.87, p < 0.0001; temperature F(1,166) = 18.68, p < 0.0001; genotype F(1,166) = 12.35, p = 0.0005, ANOVA). At lower temperature of 21°C, sleep induction because of OMD (Δ sleep = difference in sleep levels from same genotype not receiving OMD) in nanGAL4/UASshibirets is similar to controls (during daytime, p = 0.99; Fig. 8a–c). When we increased temperature to 28°C, silenced flies (nanGAL4/UASshibirets) do not show any increase in sleep; however, there are significant differences (p = 0.0009) between silenced flies and their Gal4 controls. Next, we asked whether transient activation of Nan neurons alone can induce sleep in the absence of orbital motion (Fig. 8d,e). We expressed temperature-gated cation channel dTRPA1 under the nanGAL4 driver. There is significant impact of the temperature, time of day, and genotype (time of day F(1,188) = 315.48, p < 0.0001; temperature F(1,188) = 6.35 p = 0.012; genotype F(1,188) = 28.99, p < 0.0001, time of day × temperature F(1,188) = 6.84, p = 0.009, time of day × genotype F(1,188) = 6.45, p = 0.01, temperature × genotype F(1,188) = 11.61, p = 0.0008, ANOVA). We found that, at subthreshold temperatures (21°C), nanGAL4/dTRPA1 flies sleep as much as nanGAL4/+ and dTRPA1/+ controls (sleep difference of nanGAL4/dTRPA1 compared with both parental controls p > 0.05; Fig. 8d,e). However, when we increased temperature to 28°C (activation temperature of dTRPA1) (Viswanath et al., 2003), and Nan neurons are expected to enhance firing frequency (Hamada et al., 2008; Tang and Riegel, 2013), sleep level is significantly higher in nanGAL4/dTRPA1 flies compared with nanGAL4/+ (p < 0.0001) and dTRPA1/+ (p = 0.005) controls (Fig. 8d,e), which shows that electrical activity of Nan neurons can contribute to orbital motion-induced sleep.

Figure 8.
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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.

Antennal Mechanosensory and Motor Center (AMMC) neurons are critical for OMD phenotype

A recent study on human subjects showed that acoustic inputs by way of brief auditory tones (0.5-4 Hz) induced slow wave activity with features very similar to natural sleep (Tononi et al., 2010). In D. melanogaster, antennae house ∼500 mechanosensory cells (Kamikouchi et al., 2006). We examined the role of mechanosensory cues received by the antennae by surgically removing both the antennae 4-day-old of CS flies (CS ant-) and left them undisturbed for 6 d to ensure complete loss of neurons and ablation. Subsequently, we found that antennae-less flies do not exhibit sleep induction because of OMD compared with controls with intact antennae (p = 0.04 for controls and p = 0.58 for CS ant-; Fig. 9a,b). These results suggest that mechanosensory signals from the antennae are necessary for this sleep phenotype.

Figure 9.
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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).

The Johnston's organ (JO) at the antennal base detects mechanosensory cues (Göpfert and Robert, 2003; Tauber and Eberl, 2003; Kamikouchi et al., 2006; Albert et al., 2007; Nadrowski et al., 2011); in addition, there are mechanosensory neurons present on the legs of flies (Tuthill and Wilson, 2016). The axons of JO neuron subgroups signal to specific zones of AMMC and wedge areas of the brain (Kamikouchi et al., 2006). We silenced neurons that innervate specific subregions of the AMMC by expressing tetanus toxin. We used JO-15GAL4 to target regions A and B, and found that sleep induction under OMD is unaffected (p = 0.001 for UAStnt, p = 0.03 for JO-15GAL4 and p = 0.007 for JO-15GAL4/UAStnt-; Fig. 10a,b). Additionally, we silenced region B alone using NP1046GAL4 and found that flies do not show an increase in sleep in response to OMD (p = 1.0; Fig. 10c,d), whereas parental controls show increase in sleep (p = 0.008 for UAStnt, p = 0.003 for NP1046GAL4). Thus, it appears that region A is not critical for sleep induction, whereas neurons in the area B are important for OMD phenotype.

Figure 10.
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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.

Next, we used OMHD regime to examine differences in sleep homeostasis when these brain regions are silenced (Fig. 11). In case of JO15GAL4/UAStnt (silenced area A), there is significant effect of the genotype and regimen (genotype F(2,88) = 7.67, p = 0.0006; regimen F(1,88) = 150.0, p < 0.0001; genotype × regimen F(2,88) = 4.11, p = 0.01, ANOVA). Sleep induction occurred during the first half of the day, similar to parental controls (p = 0.93 for both controls; Fig. 11a,b). However, interestingly, no negative sleep rebound was elicited in silenced flies, whereas controls do show the expected negative rebound (JO15GAL4/UAStnt vs -UAStnt, p < 0.0006 and vs -J015GAL4, p = 0.05). We verified these results by another driver line 6303GAL4 (Fig. 11c,d), which also targets area A, and find significant impact of the genotype and regimen (genotype F(2,115) = 8.64, p = 0.0005; regimen F(1,88) = 31.41, p < 0.0001, ANOVA). Here also, sleep induction occurred during the first half of the day, similar to parental controls (6303GAL4/UAStnt vs -UAStnt, p = 0.82 and vs -6303GAL4, p = 0.38; Fig. 11c,d). Unlike parental genotypes, silenced 6303GAL4/UAStnt do not show negative rebound (6303GAL4/UAStnt vs -UAStnt, p = 0.02 and vs -6303GAL4, p = 0.002). On the other hand, when flies with silenced area B (NP1046GAL4/UAStnt) were subjected to OMHD (Fig. 11e,f), there is significant effect of the regimen and interaction genotype × regimen (regimen F(1,97) = 127.04, p < 0.0001; genotype × regimen F(2,97) = 7.19, p = 0.001, ANOVA). Silenced flies do not show sleep enrichment in the first half of the day, whereas parental controls do show sleep induction (p = 0.02 UAStnt; p = 0.004 -NP1046GAL4). Despite this, during the second half of the day, silenced flies show similar levels of negative sleep rebound as controls (p = 0.92 UAStnt; p = 0.73 -NP1046GAL4; Fig. 11e,f). We also transiently activated AMMC subregions by dTRPA1 and found that there is significant impact of the genotype, temperature, and interaction between genotype × temperature (genotype F(2,261) = 52.25, p < 0.0001; temperature F(2,261) = 19.64, p < 0.0001, genotype × temperature F(4,261) = 35.93, p < 0.0001, ANOVA). Post hoc analysis suggests that there is increase (p < 0.0001 compared with both controls) in daytime sleep when area B is activated (NP1046GAL4/UASdTrpA1; Fig. 11g,h), whereas no such change is seen with JO15GAL4 and 6303GAL4 drivers with common target of region A and 6250GAL4 targeting C and E common with 6303GAL4 (Fig. 12), thus suggesting the importance of these neurons in sleep. Together, our results suggest that the area B of the AMMC is important for sleep enrichment in response to OMD, whereas area A sends signals to the homeostatic circuitry.

Figure 11.
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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.
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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.

Discussion

We find that gentle-movement-induced sleep that humans experience can be replicated in several fly strains, and we attempted to decipher the neuronal circuits involved. We designed an experimental paradigm involving exposure to orbital motion (Fig. 1a) and found that it promotes daytime sleep of fly strains without compromising their overall locomotor activity levels (Fig. 2) and that this phenotype is especially evident in females, which otherwise exhibit significantly lesser and lighter daytime sleep (van Alphen et al., 2013). Our results are consistent with previous studies that showed that rocking can induce sleep in humans and mice (Bayer et al., 2011; Kompotis et al., 2019; Perrault et al., 2019). We showed that the orbital motion-induced quiescence is reversible (Fig. 3), circadian clock-independent (Fig. 5), homeostatically modulated (Fig. 4d), and mediated by mechanosensory Nan neurons present in the chordotonal organs (Fig. 7). The fact that activation of Nan neurons alone causes sleep induction in the absence of orbital motion suggests that mechanosensory cues activate the chordotonal Nan neurons to promote sleep. Nan+ cells are present in tactile bristles in the femur, wing margin bristles (Li et al., 2016), and predominantly in second segment of antennae and are considered to be key components of fly mechanosensory system (Walker et al., 2000; Kim et al., 2003; Effertz et al., 2011; Yan et al., 2013). Our results suggest that they mediate mechanosensory stimulation-mediated sleep induction (Fig. 8).

JO in the antennae is the largest mechanosensory organ in D. melanogaster and helps in the processing of hearing, touch, vestibular sensing, and proprioception. JO neurons terminate in the AMMC. Here we find that the antennae and other neurons typically involved in detection of auditory signals are also involved in OMD sleep. We further found that neurons originating in the JO and terminating in the AMMC are critical for the OMD phenotype. Thus, it appears that signals from the antennae are processed in the AMMC before being sent to sleep centers. Our studies point to the importance of at least two areas of JO (A and B) in inducing sleep by mechanosensation, each having distinct roles: homeostatic control and sleep induction, respectively. Differences in the responses elicited by silencing area B by the two drivers JO15GAL4 and NP1046GAL4 are not surprising since the areas under these drivers also give differential responses to sound frequencies (Patella and Wilson, 2018).

Antennal displacements activate mechanosensory JO neurons that are housed within the antenna (Göpfert and Robert, 2002). JO neurons project through the antennal nerve into distinct areas of AMMC in the central brain (Kamikouchi et al., 2006). The AMMC sends projection neurons to the wedge area which have proximal branches in the protocerebral bridge from where they extend into fan-shaped and ellipsoid body (Homberg, 1994; Franconville et al., 2018; Okubo et al., 2020) known to be important for sleep (Donlea et al., 2011; Liu et al., 2016). Wedge cells also project to lateral accessory lobes believed to facilitate communication between central complex and motor centers (Namiki and Kanzaki, 2016). Thus, we propose a pathway by which the stimuli received from the antennae, and other structures including bristles on the wing and legs reach the AMMC, which in turn convey this information via the wedge area to sleep centers which can induce sleep while informing the homeostatic centers of the dissipating sleep drive.

Curiously, flies carrying two different alleles of nan mutations do not exhibit any defects in OMD-induced sleep or OMHD-induced negative rebound (Fig. 7a,b). This is in contrast to the clear defects seen when the nan-expressing neurons were specifically manipulated using the GAL4-UAS means, by various approaches: ablation, silencing, or activation (Fig. 7c–h). This may be because of compensatory processes early in development in case of nan mutants or because of yet unknown mechanosensory proteins present within these neurons, which perhaps enable nan mutants to perform various functions, including the response to OMD. Future studies may reveal molecular and biophysical mechanisms involving ion channels other than Nanchung that underlie this phenomenon.

Most sensory stimuli are perceived by GPCRs, except touch, vibration, and pressure, which are sensed by mechanosensory receptors (Kung, 2005). Mechanosensory neurons respond to such cues by converting them into receptor potentials (Gong et al., 2004), and many organisms have evolved the ability to respond to ligands, which changes the magnitude of such receptor potentials. In our studies, we observe a graded nature of the sleep induction with increasing speeds, which is probably because mechanosensory neurons are activated at the relatively lower speeds leading to sleep induction, whereas at higher speeds other mechanosensors/mechanosensory neurons signal arousal. We speculate that the levels of activation of the mechanosensory neurons targeted by some of the drivers we used are within the range for sleep induction and do not activate arousal centers. Further, while daytime orbital motion gives a clear increase in sleep, nighttime motion also increases sleep, although the difference from controls is not as large. This is likely because flies already sleep for a large portion of the night and, hence, they encounter a ceiling effect that does not allow for much further increase because of orbital motion. Recently, an independent study by Öztürk-Çolak et al. (2020) also reported that mechanosensory stimulation via vibration could induce sleep. Thus, our independent approaches indicate that sleep induction occurs because of stimulation of mechanosensory receptors, that sleep is deeper during orbital motion/when vibrational stimuli are present, and that chordotonal organs are involved. Our studies go on to suggest that TRPV channel nanchung-expressing neurons are involved, and that potentially the circuit involves important sensory hubs in the AMMC region which may project to higher order sleep centers in the brain.

We reason that differences in the type of motion and intensity of mechanical stimulation invoke widely different responses. Rapid to-and-fro motion of ∼1000 rpm would cause sleep deprivation (Huber et al., 2004), whereas slow orbital motion of 80-120 rpm would result in sleep induction. A similar feature of no-impact followed by sleep inducing effect, followed by sleep disrupting effect of sensory stimulation was also hypothesized to explain the effect of increased acoustic stimulation in humans (Bellesi et al., 2014). Stimulation above a certain intensity threshold can effectively induce sleep, whereas stimulation below the threshold is ineffective and stimulation well past the threshold can be disruptive. Since increased sensory threshold is a cardinal feature of the sleeping state, the ability of repetitive low-amplitude sensory input to induce sleep in an awake animal is counterintuitive. However, recent studies in mammals, including humans (Bayer et al., 2011; Kompotis et al., 2019; Perrault et al., 2019), showed that rocking can enhance the propensity to enter into deep-sleep state. The authors hypothesize that certain types of low-grade sensory stimuli can potentially increase sleep propensity by impinging on brain regions that receive inputs regarding a relaxed or low-stress state or by stimulating sleep centers in the hypothalamus or brain stem region. Alternatively, the authors propose that rocking enhances the degree of synchrony in neural activity in thalamocortical regions, which in turn are associated with deep sleep (Bayer et al., 2011).

Our data suggest that, like humans and mice, flies can respond to the mechanosensory cues that result in sleep enrichment, suggesting the conserved role of mechanosensation in sleep. We find that orbital motion increased consolidation of fly sleep as suggested by the differential arousal threshold. This sleep is also under homeostatic control as demonstrated by negative rebound. Previous studies have suggested that, in mammals, rocking enhances the degree of synchrony in neural activity in thalamocortical regions, which in turn is associated with deep sleep (Bayer et al., 2011). In nature, where many environmental factors change rapidly, animals constantly need to distinguish different sensory cues and integrate information to make decisions that determine their survival. Mechanosensation is an important and fast means to adjust to unpredictable environmental features (Tuthill and Wilson, 2016). In addition, mechanical stimuli are perceived through proprioceptors by insects that execute many complex behaviors, such as running, foraging, courting, fighting, etc. It is possible that, in nature, continuous repetitive mechanical stimulation is indicative of constancy or lack of a need to be aroused and therefore is conducive for sleep.

Nonpharmacological intervention to alleviate sleep disorders is highly desirable since pharmacological agents often have off-target effects and lingering effects that persist into the wakeful state. The fly model for sleep has been particularly useful in unraveling genetic components underlying sleep regulation as well as the neuronal pathways involved, including cellular and molecular components (for review, see Donelson and Sanyal, 2015). Our finding of a pathway of sensory stimulation that can alleviate sleep levels in this model organism should enable future studies that suggest efficient therapeutic measures to treat sleep defects in a wide range of conditions, including circadian sleep phase syndromes, neurodegenerative conditions with associated sleep loss, metabolic syndrome, and so on.

Footnotes

  • This work was supported by Science and Engineering Research Board CRG/2019/006802 to V.S.; Ramanujan fellowship SB/S2/RJN-005/2016 and EMR/2017/001625 to S.R.L.; Department of Biotechnology, Government of India consumable Grant BT/INF/22/SP27679/2018 to V.S.; Jawaharlal Nehru Center for Advanced Scientific Research, Bangalore intramural funding to V.S. and V.K.S.; and Council for Scientific and Industrial Research CSIR for funding to N.S. and S.M. We thank three anonymous reviewers of this journal for comments on improving a previous version of this paper; Martin Göpfert and other members of the fly community for fly lines; and Manishi Srivastava for assistance with some preliminary experiments.

  • The authors declare no competing financial interests.

  • Correspondence should be addressed to Vasu Sheeba at sheeba{at}jncasr.ac.in or Shahnaz Rahman Lone at lonesr{at}gmail.com

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The Journal of Neuroscience: 41 (45)
Journal of Neuroscience
Vol. 41, Issue 45
10 Nov 2021
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Mechanosensory Stimulation via Nanchung Expressing Neurons Can Induce Daytime Sleep in Drosophila
Shahnaz Rahman Lone, Sheetal Potdar, Archana Venkataraman, Nisha Sharma, Rutvij Kulkarni, Sushma Rao, Sukriti Mishra, Vasu Sheeba, Vijay Kumar Sharma
Journal of Neuroscience 10 November 2021, 41 (45) 9403-9418; DOI: 10.1523/JNEUROSCI.0400-21.2021

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Mechanosensory Stimulation via Nanchung Expressing Neurons Can Induce Daytime Sleep in Drosophila
Shahnaz Rahman Lone, Sheetal Potdar, Archana Venkataraman, Nisha Sharma, Rutvij Kulkarni, Sushma Rao, Sukriti Mishra, Vasu Sheeba, Vijay Kumar Sharma
Journal of Neuroscience 10 November 2021, 41 (45) 9403-9418; DOI: 10.1523/JNEUROSCI.0400-21.2021
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Keywords

  • chordotonal organs
  • mechanosensation
  • nanchung
  • orbital motion
  • sleep
  • sleep rebound

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