JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

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

Published: December 14th, 2020

DOI:

10.3791/62105

1Department of Neuroscience, Washington University School of Medicine

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 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.

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 animals1,2,3,4,5.

Sleep in the fly has many similarities with human sleep, including a robust homeostatic response to sleep loss4,5. Numerous studies of sleep in the fly have u....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 drive6,

    Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Sleep in Drosophila was independently characterized in 2000, by two groups4,5. 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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by NIH grants 5R01NS051305-14 and 5R01NS076980-08 to PJS.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Nath, R. D., et al. The Jellyfish Cassiopea Exhibits a Sleep-like State. Current Biology. 27 (19), 2984-2990 (2017).
  2. Vorster, A. P., Krishnan, H. C., Cirelli, C., Lyons, L. C. Characterization of sleep in Aplysia californica. Sleep. 37 (9), 1453-1463 (2014).
  3. Zhdanova, I. V., Wang, S. Y., Leclair, O. U., Danilova, N. P. Melatonin promotes sleep-like state in zebrafish. Brain Research. 903 (1-2), 263-268 (2001).
  4. Shaw, P. J., Cirelli, C., Greenspan, R. J., Tononi, G. Correlates of sleep and waking in Drosophila melanogaster. Science. 287 (5459), 1834-1837 (2000).
  5. Hendricks, J. C., et al. Rest in Drosophila is a sleep-like state. Neuron. 25 (1), 129-138 (2000).
  6. Ganguly-Fitzgerald, I., Donlea, J., Shaw, P. J. Waking experience affects sleep need in Drosophila. Science. 313 (5794), 1775-1781 (2006).
  7. Seugnet, L., Suzuki, Y., Vine, L., Gottschalk, L., Shaw, P. J. D1 receptor activation in the mushroom bodies rescues sleep-loss-induced learning impairments in Drosophila. Current Biology. 18 (15), 1110-1117 (2008).
  8. Donlea, J. M., Thimgan, M. S., Suzuki, Y., Gottschalk, L., Shaw, P. J. Inducing sleep by remote control facilitates memory consolidation in Drosophila. Science. 332 (6037), 1571-1576 (2011).
  9. Seidner, G., et al. Identification of Neurons with a Privileged Role in Sleep Homeostasis in Drosophila melanogaster. Current Biology. 25 (22), 2928-2938 (2015).
  10. Li, X., Yu, F., Guo, A. Sleep deprivation specifically impairs short-term olfactory memory in Drosophila. Sleep. 32 (11), 1417-1424 (2009).
  11. Melnattur, K., et al. A conserved role for sleep in supporting Spatial Learning in Drosophila. Sleep. , 197 (2020).
  12. Seugnet, L., Suzuki, Y., Donlea, J. M., Gottschalk, L., Shaw, P. J. Sleep deprivation during early-adult development results in long-lasting learning deficits in adult Drosophila. Sleep. 34 (2), 137-146 (2011).
  13. Donlea, J. M., Ramanan, N., Shaw, P. J. Use-dependent plasticity in clock neurons regulates sleep need in Drosophila. Science. 324 (5923), 105-108 (2009).
  14. Bushey, D., Tononi, G., Cirelli, C. Sleep and synaptic homeostasis: structural evidence in Drosophila. Science. 332 (6037), 1576-1581 (2011).
  15. Huang, S., Piao, C., Beuschel, C. B., Götz, T., Sigrist, S. J. Presynaptic Active Zone Plasticity Encodes Sleep Need in Drosophila. Current Biology. 30 (6), 1077-1091 (2020).
  16. Kirszenblat, L., et al. Sleep regulates visual selective attention in Drosophila. Journal of Experimental Biology. 221, (2018).
  17. Singh, P., Donlea, J. M. Bidirectional Regulation of Sleep and Synapse Pruning after Neural Injury. Current Biology. 30 (6), 1063-1076 (2020).
  18. Stanhope, B. A., Jaggard, J. B., Gratton, M., Brown, E. B., Keene, A. C. Sleep Regulates Glial Plasticity and Expression of the Engulfment Receptor Draper Following Neural Injury. Current Biology. 30 (6), 1092-1101 (2020).
  19. Kayser, M. S., Yue, Z., Sehgal, A. A critical period of sleep for development of courtship circuitry and behavior in Drosophila. Science. 344 (6181), 269-274 (2014).
  20. Kayser, M. S., Mainwaring, B., Yue, Z., Sehgal, A. Sleep deprivation suppresses aggression in Drosophila. Elife. 4, 07643 (2015).
  21. Szuperak, M., et al. A sleep state in Drosophila larvae required for neural stem cell proliferation. Elife. 7, (2018).
  22. Vaccaro, A., et al. Sleep Loss Can Cause Death through Accumulation of Reactive Oxygen Species in the Gut. Cell. 181 (6), 1307-1328 (2020).
  23. Kempf, A., Song, S. M., Talbot, C. B., Miesenböck, G. A potassium channel β-subunit couples mitochondrial electron transport to sleep. Nature. 568 (7751), 230-234 (2019).
  24. Donlea, J. M., Pimentel, D., Miesenböck, G. Neuronal machinery of sleep homeostasis in Drosophila. Neuron. 81 (4), 860-872 (2014).
  25. Liu, S., Liu, Q., Tabuchi, M., Wu, M. N. Sleep Drive Is Encoded by Neural Plastic Changes in a Dedicated Circuit. Cell. 165 (6), 1347-1360 (2016).
  26. Pimentel, D., et al. Operation of a homeostatic sleep switch. Nature. 536 (7616), 333-337 (2016).
  27. Sitaraman, D., et al. Propagation of Homeostatic Sleep Signals by Segregated Synaptic Microcircuits of the Drosophila Mushroom Body. Current Biology. 25 (22), 2915-2927 (2015).
  28. Foltenyi, K., Greenspan, R. J., Newport, J. W. Activation of EGFR and ERK by rhomboid signaling regulates the consolidation and maintenance of sleep in Drosophila. Nature Neuroscience. 10 (9), 1160-1167 (2007).
  29. Seugnet, L., et al. Notch signaling modulates sleep homeostasis and learning after sleep deprivation in Drosophila. Current Biology. 21 (10), 835-840 (2011).
  30. Seugnet, L., Galvin, J. E., Suzuki, Y., Gottschalk, L., Shaw, P. J. Persistent short-term memory defects following sleep deprivation in a Drosophila model of Parkinson disease. Sleep. 32 (8), 984-992 (2009).
  31. Tabuchi, M., et al. Sleep interacts with aβ to modulate intrinsic neuronal excitability. Current Biology. 25 (6), 702-712 (2015).
  32. Melnattur, K., Zhang, B., Shaw, P. J. Disrupting flight increases sleep and identifies a novel sleep-promoting pathway in Drosophila. Science Advances. 6 (19), 2166 (2020).
  33. Thimgan, M. S., Suzuki, Y., Seugnet, L., Gottschalk, L., Shaw, P. J. The perilipin homologue, lipid storage droplet 2, regulates sleep homeostasis and prevents learning impairments following sleep loss. PLOS Biology. 8 (8), (2010).
  34. Keene, A. C., et al. Clock and cycle limit starvation-induced sleep loss in Drosophila. Current Biology. 20 (13), 1209-1215 (2010).
  35. Shaw, P. J., Tononi, G., Greenspan, R. J., Robinson, D. F. Stress response genes protect against lethal effects of sleep deprivation in Drosophila. Nature. 417 (6886), 287-291 (2002).
  36. Andretic, R., Shaw, P. J. Essentials of sleep recordings in Drosophila: moving beyond sleep time. Methods Enzymol. 393, 759-772 (2005).
  37. Seugnet, L., et al. Identifying sleep regulatory genes using a Drosophila model of insomnia. Journal of Neuroscience. 29 (22), 7148-7157 (2009).
  38. Bushey, D., Huber, R., Tononi, G., Cirelli, C. Drosophila Hyperkinetic mutants have reduced sleep and impaired memory. Journal of Neuroscience. 27 (20), 5384-5393 (2007).
  39. Geissmann, Q., et al. Ethoscopes: An open platform for high-throughput ethomics. PLOS Biology. 15 (10), 2003026 (2017).
  40. Faville, R., Kottler, B., Goodhill, G. J., Shaw, P. J., van Swinderen, B. How deeply does your mutant sleep? Probing arousal to better understand sleep defects in Drosophila. Scientific Reports. 5, 8454 (2015).
  41. Huber, R., et al. Sleep homeostasis in Drosophila melanogaster. Sleep. 27 (4), 628-639 (2004).
  42. Klose, M., Shaw, P. Sleep-drive reprograms clock neuronal identity through CREB-binding protein induced PDFR expression. bioRxiv. , (2019).
  43. Dissel, S., et al. Sleep restores behavioral plasticity to Drosophila mutants. Current Biology. 25 (10), 1270-1281 (2015).
  44. Gerstner, J. R., Vanderheyden, W. M., Shaw, P. J., Landry, C. F., Yin, J. C. Fatty-acid binding proteins modulate sleep and enhance long-term memory consolidation in Drosophila. PLoS One. 6 (1), 15890 (2011).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved