The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Summary

Sleep deprivation is a powerful tool to investigate sleep function and regulation. We describe a protocol to sleep deprive Drosophila using the Sleep Nullifying Apparatus, and to determine the extent of rebound sleep induced by deprivation.

Abstract

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal kingdom. As a consequence, sleep deprivation and sleep restriction are powerful tools that are commonly used to provide insight into sleep function. Nonetheless, sleep deprivation experiments are inherently problematic in that the deprivation stimulus itself may be the cause of observed changes in physiology and behavior. Accordingly, successful sleep deprivation techniques should keep animals awake and, ideally, result in a robust sleep rebound without also inducing a large number of unintended consequences. Here, we describe a sleep deprivation technique for Drosophila melanogaster. The Sleep Nullifying Apparatus (SNAP) administers a stimulus every 10s to induce negative geotaxis. Although the stimulus is predictable, the SNAP effectively prevents >95% of nighttime sleep even in flies with high sleep drive. Importantly, the subsequent homeostatic response is very similar to that achieved using hand-deprivation. The timing and spacing of the stimuli can be modified to minimize sleep loss and thus examine non-specific effects of the stimulus on physiology and behavior. The SNAP can also be used for sleep restriction and to assess arousal thresholds. The SNAP is a powerful sleep disruption technique that can be used to better understand sleep function.

Introduction

Sleep is near universal in animals, yet its function remains unclear. Sleep homeostasis, the compensatory increase in sleep following sleep deprivation, is a defining property of sleep, that has been used to characterize sleep states in a number of animals^1,2,3,4,5.

Sleep in the fly has many similarities with human sleep, including a robust homeostatic response to sleep loss^4,5. Numerous studies of sleep in the fly have used sleep deprivation both to infer sleep function by examining the adverse consequences that accrue from extended waking, and to understand sleep regulation by determining the neurobiological mechanisms controlling the homeostatic regulation of sleep. Thus sleep deprived flies were shown to exhibit impairments in learning and memory^{6,7,8,9,10,11,12}, structural plasticity^13,14,15, visual attention¹⁶, recovery from neuronal injury^17,18, mating and aggressive behaviors^19,20, cell proliferation²¹, and responses to oxidative stress^22,23 to name a few. Further, investigations into the neurobiological mechanisms controlling rebound sleep have yielded critical insights into the neuronal machinery that constitutes the sleep homeostat^{8,9,23,24,25,26,27,28,29}. Finally, in addition to revealing fundamental insights into sleep function in healthy animals, sleep deprivation studies have also informed insights into sleep function in diseased states^30,31.

While sleep deprivation is undeniably a powerful tool, with any sleep deprivation experiment, it is important to distinguish phenotypes that result from extended waking, from those induced by the stimulus used to keep the animal awake. Sleep deprivation by hand deprivation or gentle handling, is generally regarded as setting the standard for minimally disruptive sleep deprivation. Here we describe a protocol for sleep depriving flies using the Sleep Nullifying Apparatus (SNAP). The SNAP is a device that delivers a mechanical stimulus to flies every 10s, keeping flies awake by inducing negative geotaxis (Figure 1). The SNAP efficiently deprives flies of >98% of night-time sleep, even in flies with high sleep drive^8,32. The SNAP has been calibrated on bang sensitive flies, agitation of flies in the SNAP does not harm flies; sleep deprivation with the SNAP induces a rebound comparable with that obtained by hand deprivation⁷. The SNAP is thus a robust method to sleep deprive flies while controlling for the effects of the arousing stimulus.

Protocol

1. Experimental preparation

1. Collect flies as they eclose into vials, separating male and female flies.
   NOTE: Sleep experiments are commonly conducted with female flies. It is important to collect virgin females. Mated females will lay eggs that hatch into larvae complicating the analysis of the data.
2. House flies of a single sex in groups of <20.
   NOTE: Housing flies in a socially enriched environment (groups of >50) modulates sleep drive^6,13 potentially confounding measurements of rebound sleep. Further, following social enrichment, sleep will decline over a few days⁶. Thus, baseline sleep is not stable complicating analysis of rebound sleep. Keeping flies in groups of <20 avoids this potential confound.
3. Keep flies in vials for 3-5 days in a light and humidity controlled environment.
   ​NOTE: Age and maturity of flies strongly influence sleep. Sleep is high in one day old flies and stabilizes by 3-5 days of age⁴. Flies are typically maintained on a 12 h light: 12 h dark schedule at 50% humidity.

2. Preparation of tubes for sleep recording

NOTE: Sleep is monitored using locomotor activity monitors. A monitor can hold 32 flies housed individually in 5 mm diameter tubes. Typically, genotypes are analyzed in groups of 16 or 32 flies.

1. Prepare an appropriate number of tubes with fly food at one end.
   NOTE: Diet and metabolism are known to influence sleep^33,34, hence it is particularly important to place flies on the same food on which they were reared.
2. Seal the end of the tubes with wax.
   NOTE: Sleep deprivation and rebound is a five day experiment, and food can dry out if not properly sealed. In properly sealed tubes, food can be maintained for 10 days or more. Thus, it is critical to ensure that the ends of the tubes are sealed well. Flies can also get stuck to wet food, however. Thus, it helps to make tubes 1-2 days before the start of the experiment.
3. Individually place wake, behaving flies into 65 mm long glass tubes for sleep recording using an aspirator and plug the end of the tubes with a foam stopper.
   ​NOTE: Flies are never re-exposed to CO₂ anesthesia when placing flies into tubes for sleep recording. The aspirator is made from rubber tubing with one end covered with cheesecloth and inserted into a 1 mL pipette tip.

3. Recording sleep

1. Load flies in tubes into activity monitors to monitor sleep.
   ​NOTE: The SNAP rocks monitors back and forth from -60° to +60° every ~10 s. The monitors are held at -60° for ~5.9s ; it takes ~2.9 s for the tray holding the monitors to move from -60° to +60° and ~1 s to move back from +60° to -60°. The cycle length can be altered as needed by adjusting the voltage supplied to the motor.
   1. Take care to ensure that tubes are placed in activity monitors in the correct orientation. In the correct orientation, the end of the tube with food is at the top of the SNAP to ensure that flies do not get pushed into the food. In addition, the end with food is on the side of the monitor with the sleep recording jack. This allows activity monitors to be oriented correctly in the SNAP for efficient sleep deprivation while simultaneously monitoring activity.
2. Place activity monitors in the recording chamber to monitor sleep.
3. Monitor sleep for at least two full days to estimate baseline sleep.
   NOTE: The day flies are loaded into activity monitors is typically excluded as an adaptation day to allow flies to adapt to being housed in tubes. Baseline sleep is recorded for at least two full days (48hrs) beginning with the morning following the day flies are loaded.
4. Save locomotor activity counts of flies in 1 min bins from the time of lights on a given day to lights on the previous day using activity recording software (e.g., from 8 AM to 8 AM).
5. Estimate sleep from the locomotor activity data with custom macros using 5 min of inactivity as the threshold for a bout of sleep³⁵.
   ​NOTE: A number of sleep metrics are computed from the locomotor activity counts. These include sleep in min/h over 24 h, total sleep time in 24 h, average and maximum daytime and nighttime sleep bout lengths³⁶.

4. Sleep deprivation and recovery

1. As flies can be sleep deprived for variable lengths of time (e.g., 12 h, 24 h and 36 h) and recovery sleep can also be evaluated at various intervals (e.g., 6 h, 12 h, 24 h and 48 h), determine the duration of recovery by experimental need. Sleep recovery can be visualized using a sleep gain/loss plot or by examining percent sleep recovered over a predetermined interval (e.g., 6 h).
2. If sleep is stable over the two baseline days, on the third day, place activity monitors into the SNAP for overnight sleep deprivation.
   NOTE: Flies will exhibit a robust sleep rebound over a range of sleep times^8,32,37,38, but sleep has to be stable to reliably evaluate rebound sleep. Sleep is stable when the difference in sleep between baseline days is ± 100 min.
3. Make sure activity monitors are secured in place with monitor holder pins, monitor cords plugged in, and monitors oriented correctly with the end with food at the back, and plastic barriers in front (Figure 1).
   NOTE: The SNAP is designed so the cam rotates once every 10 s (Figure 1). The plastic insert resets the tubes by pushing the tubes back when the apparatus is in the "up" position. Resetting the tubes is important to ensure that all tubes have the full range of motion at the beginning of each cycle.
4. Unplug activity monitors, and take monitors out of the SNAP immediately upon lights on following overnight sleep deprivation.
   NOTE: It is critical that sleep deprivation is terminated, and flies are placed in recovery immediately upon lights on following 12 h of overnight sleep deprivation. Even a 20-30 min delay in placing flies into recovery can interfere with the extent of rebound sleep.
5. Place flies in a recording chamber where they will be undisturbed for two days (48 h) to monitor recovery sleep.
   NOTE: If the recording chamber is being used for other experiments, extra care must be taken to avoid stimulating recovering flies.
6. Calculate the amount of sleep lost. For each individual fly, calculate the hourly difference between sleep obtained during sleep deprivation and the corresponding hour during baseline; sum the hourly differences to calculate total sleep lost.
7. Calculate the amount of sleep recovered. For each individual fly, calculate the hourly difference between sleep obtained during recovery and the corresponding hour during baseline; sum the hourly differences to calculate total sleep gained.
   NOTE: Whether a fly is actually sleep deprived is empirical. Thus, the experimenter should examine percent sleep lost. If the fly has not lost a sufficient amount of sleep it can be excluded from the analysis. Although this might be required for other sleep deprivation approaches, it is rarely if ever required for the SNAP. More commonly, sleep may not be stable in a given fly prior to the initiation of sleep deprivation. If sleep is not stable, homeostasis cannot be calculated. We accept a maximum difference of ± 100 min of sleep calculated prior to the initiation of sleep deprivation as candidates for inclusion. On occasion, an individual fly's sleep is distributed unevenly across the 24 h day (e.g., some individuals may obtain 60-70% of their sleep quota during the day and thus only lose a small proportion of their 24 h sleep quota when deprived for 12 h at night). These flies can be evaluated separately.
8. Calculate the average percentage of sleep recovered (relative to baseline) over 12 h, 24 h and 48 h of the recovery period for each genotype.
9. From sleep data, compute the average and maximum daytime sleep bout length on baseline, and recovery days for each genotype.
   NOTE: Rebound sleep in flies is characterized by increased sleep amount, and increased sleep depth in the recovery days. Sleep consolidation is used as measure of sleep depth. Arousal thresholds could also be used as a measure of sleep depth.

Results

Canton S (Cs) was used as a wild-type strain. Flies were maintained on a 12 h light: 12 h dark schedule, and sleep deprived for 12 hours overnight. Inspection of the sleep profiles of Cs flies on the baseline day (bs), sleep deprivation day (sd), and two recovery days (rec1 and rec2) (Figure 2A) suggests that flies were effectively sleep deprived in the SNAP, and recovered sleep during the day consistent with observed reports in the literature

Discussion

Sleep in Drosophila was independently characterized in 2000, by two groups^4,5. In these pioneering studies, flies were deprived of sleep by gentle handling (i.e., hand deprivation) and shown to exhibit a robust homeostatic response to overnight sleep deprivation. Importantly, with any sleep deprivation experiment it is crucial to control for potential confounding effects of the method used to keep the animal awake. Hand deprivation studies set the benchm...

Materials

Name	Company	Catalog Number	Comments
Locomotor activity tubes
Fisher Tissue Prep Wax	Thermo Fisher	13404-122	Wax used for sealing tubes
Glass tubes	Wale Apparatus	244050	We cut 5mm diameter Pyrex glass tubes into 65mm long tubes to record sleep. Pre-cut tubes can also be purchased.
Nutri Fly Bloomington Formulation fly food	Genesee Scientific	66-113	Labs might use their own fly food recipe. It is important that sleep be recorded on the same food that flies were reared in.
Rotary glass cutting tool	Dremel Multi Pro	395	Used to cut 65mm long glass tubes
Monitoring Sleep
DAM System and DAMFileScan software	Trikinetics		Software used to acquire data from DAM monitors and save the acquired data in an appropriate format
Data acquisition computer	Lenovo	Idea Centre AIO3	A equivalent computer from any manufacturer can substitute
Drosophila Activity Monitors	Trikinetics	DAM2	These monitors are used to record flies' locomotor activity
Environment Monitor	Trikinetics	DEnM	Not essential, but an easy way to monitor environmental conditions in the chamber where sleep is recorded
Light Controller	Trikinetics	LC4	A convenient way to control the timing of when the SNAP is turned on and off
Power Supply Interface Unit for DAM	Trikinetics	PSIU-9	Required for data acquisition computers to record Trikinetics locomotor acitvity data
RJ11 connector	7001-64PC	Multicomp	DAM monitors accept RJ11 jacks
Splitters	Trikinetics	SPLT5	Used to connect upto 5 DAM monitors
Telephone cable wire	Radioshack	278-367	Phone cables to acquire data from DAM monitors
Sleep Deprivation
Power supply	Gw INSTEK	GPS-30300	Power supply for the SNAP
Sleep Nullifying Apparatus	Washington University School of Medicine machine shop