The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila
December 14th, 2020
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.
Sleep homeostasis, the increase in sleep following sleep loss, is a defining characteristic of sleep. Sleep deprivation and sleep restriction are thus powerful tools to study sleep regulation and function. This protocol efficiently sleep deprives and sleep restricts flies while minimizing potential confounds.
SNAP deprives flies of greater than 95%of sleep even in flies with high sleep drive. Importantly, agitation of flies with the SNAP does not harm flies and induce a rebound comparable to that obtained with hand deprivation, which is a standard for minimally disruptive sleep deprivation. Visualizing how the SNAP keeps flies awake will help investigators use and optimize this protocol.
Begin by collecting eclosing flies into vials, separating males and females. House the flies in groups of less than 20 and keep them for three to five days in a light and humidity-controlled environment. Prepare an appropriate number of tubes with fly food at one end and seal the end with wax.
Individually place awake behaving flies into the tubes using an aspirator and plug the tubes with a foam stopper. Load the tubes into activity monitors to monitor sleep, making sure that the tubes are placed in the correct orientation. The end of the tube of food should be at the top of the SNAP to ensure that flies do not get pushed into the food.
Place activity monitors in the recording chamber and monitor sleep for at least two full days to estimate baseline sleep. Save locomotor activity counts of flies in one-minute bins from the time of lights on a given day to lights on the previous day using activity recording software. Estimate sleep from the locomotor activity data with custom macros using five minutes of inactivity as the threshold for a bout of sleep.
If sleep is stable over the two baseline days, place activity monitors into the SNAP for overnight sleep deprivation on the third day. Make sure activity monitors are secured in place with monitor holder pins. The monitor cords are plugged in and the monitors oriented correctly.
Once the lights go on after overnight sleep deprivation, unplug activity monitors and take them out of the SNAP immediately. Place the flies in a recording chamber where they will be undisturbed for two days to monitor recovery sleep. For each individual fly, calculate the hourly difference between sleep obtained during sleep deprivation and the corresponding hour during baseline, then sum the hourly differences to calculate total sleep lost.
Next, calculate the hourly difference between sleep obtained during recovery and the corresponding hour during baseline, then sum the hourly differences to calculate total sleep gained. Calculate the average percentage of sleep recovered over 12, 24, and 48 hours of the recovery period for each genotype. Finally, compute the average and maximum daytime sleep bout length on baseline and recovery days for each genotype.
Flies were sleep deprived in the SNAP and recovered sleep during the day. The effectiveness of SNAP in keeping flies awake was demonstrated with the high activity exhibited by flies during sleep deprivation. To quantitatively estimate the effectiveness of sleep deprivation and of recovery, sleep lost during deprivation and then regained in the recovery days was calculated for each individual fly.
Importantly, there was no significant change in baseline sleep between the deprivation day and the baseline day, indicating that sleep is stable in these flies. The SNAP effectively deprived flies of over 98%of their nighttime sleep. Flies recovered approximately 20%of their sleep in the first 12 hours and did not recover additional sleep during the night.
They began to recover sleep the following day and recovered 36%of their sleep over 48 hours. Sleep homeostasis is characterized by both increased sleep duration and by increased sleep depth during the recovery period. Daytime sleep consolidation is commonly used as a readout of sleep depth.
Sleep consolidation can be assessed as the average sleep bout duration over the entire day. However, as sleep pressure is dissipated during recovery, the average sleep bout duration is reduced. Changes in the maximum sleep bout duration can provide a more sensitive metric.
This procedure can be easily modified to minimize sleep loss, thus control for non-specific effects of the stimulus. SNAP can be configured to restrict sleep, thus mimicking chronic sleep loss in humans. It can also be used to measure arousal thresholds.
Sleep deprivation using the SNAP has yielded important insights into sleep function through studies that examine the negative consequences of sleep loss. By identifying manipulations that interfere with the expression of rebound sleep, sleep deprivation with the SNAP has also helped to elucidate homeostatic mechanisms that regulate sleep.
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Overview
This article presents a detailed protocol for sleep deprivation and sleep restriction in Drosophila melanogaster using the Sleep Nullifying Apparatus (SNAP). The SNAP efficiently deprives flies of sleep while minimizing confounding effects, enabling robust analysis of sleep homeostasis and recovery. The method is adaptable for various experimental needs, including sleep restriction and arousal threshold assessment.
Key Study Components
Area of Science
- Neuroscience
- Sleep research
- Behavioral genetics
Background
- Sleep homeostasis is a key feature of sleep, characterized by increased sleep following deprivation.
- Sleep deprivation and restriction are essential tools for studying sleep regulation and function.
- Traditional deprivation methods may introduce confounding physiological or behavioral effects.
- Minimally disruptive techniques are needed to accurately assess sleep function in model organisms.
Purpose of Study
- To describe and validate a protocol for efficient sleep deprivation and restriction in flies using the SNAP.
- To minimize unintended consequences of deprivation stimuli.
- To enable precise measurement of sleep loss, recovery, and homeostatic responses.
Methods Used
- Collection and separation of newly eclosed flies by sex.
- Housing flies in controlled light and humidity conditions.
- Placement of individual flies in food-containing tubes for activity monitoring.
- Baseline sleep monitoring using activity monitors and custom macros (five minutes inactivity threshold).
- Overnight sleep deprivation using the SNAP, which administers a stimulus every 10 seconds to induce negative geotaxis.
- Post-deprivation recovery monitoring and quantitative analysis of sleep loss and rebound.
- Assessment of sleep consolidation and bout duration as measures of sleep depth.
Main Results
- SNAP deprived flies of over 95% (up to 98%) of nighttime sleep, even in high sleep drive genotypes.
- Flies exhibited robust sleep rebound, recovering approximately 20% of lost sleep in the first 12 hours and 36% over 48 hours.
- Baseline sleep remained stable, indicating minimal confounding effects from the deprivation protocol.
- Daytime sleep consolidation and maximum sleep bout duration provided sensitive metrics for sleep depth during recovery.
Conclusions
- The SNAP is an effective and minimally disruptive tool for sleep deprivation and restriction in Drosophila.
- It enables precise quantification of sleep loss and homeostatic rebound.
- The protocol can be adapted for chronic sleep restriction and arousal threshold studies, facilitating deeper insights into sleep regulation mechanisms.
