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

1. Experimental preparation
<ol>
	<li>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.</li>
	<li>House flies of a single sex in groups of &#60;20. 
	NOTE: Housing flies in a socially enriched environment (groups of &#62;50) modulates sleep drive6,13 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 &#60;20 avoids this potential confound.</li>
	<li>Keep flies in vials for 3-5 days in a light and humidity controlled environment. 
	&#8203;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.</li>
</ol>
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.
<ol>
	<li>Prepare an appropriate number of tubes with fly food at one end. 
	NOTE: Diet and metabolism are known to influence sleep33,34, hence it is particularly important to place flies on the same food on which they were reared.</li>
	<li>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.</li>
	<li>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. 
	&#8203;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.</li>
</ol>
3. Recording sleep
<ol>
	<li>Load flies in tubes into activity monitors to monitor sleep. 
	&#8203;NOTE: The SNAP rocks monitors back and forth from -60&#176; to +60&#176; every ~10 s. The monitors are held at -60&#176; for ~5.9s ; it takes ~2.9 s for the tray holding the monitors to move from -60&#176; to +60&#176; and ~1 s to move back from +60&#176; to -60&#176;. The cycle length can be altered as needed by adjusting the voltage supplied to the motor.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Place activity monitors in the recording chamber to monitor sleep.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Estimate sleep from the locomotor activity data with custom macros using 5 min of inactivity as the threshold for a bout of sleep35. 
	&#8203;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.</li>
</ol>
4. Sleep deprivation and recovery
<ol>
	<li>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).</li>
	<li>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 times8,32,37,38, but sleep has to be stable to reliably evaluate rebound sleep. Sleep is stable when the difference in sleep between baseline days is &#177; 100 min.</li>
	<li>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 &#34;up&#34; position. Resetting the tubes is important to ensure that all tubes have the full range of motion at the beginning of each cycle.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#177; 100 min of sleep calculated prior to the initiation of sleep deprivation as candidates for inclusion. On occasion, an individual fly&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

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...

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 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 memory6,7,8,9,10,11,12, structural plasticity13,14,15, visual attention16, recovery from neuronal injury17,18, mating and aggressive behaviors19,20, cell proliferation21, and responses to oxidative stress22,23 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 homeostat8,9,23,24,25,26,27,28,29. 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 states30,31.
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 &#62;98% of night-time sleep, even in flies with high sleep drive8,32. 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.

<table><tbody><tr><td>&lt;strong&gt;Locomotor activity tubes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Fisher Tissue Prep Wax</td><td>Thermo Fisher</td><td>13404-122</td><td>Wax used for sealing tubes</td></tr><tr><td>Glass tubes</td><td>Wale Apparatus</td><td>244050</td><td>We cut 5mm diameter Pyrex glass&amp;nbsp;tubes into 65mm long tubes&amp;nbsp;to record sleep. Pre-cut tubes&amp;nbsp;can also be purchased.</td></tr><tr><td>Nutri Fly Bloomington Formulation fly food</td><td>Genesee Scientific</td><td>66-113</td><td>Labs might use their own fly food recipe. It is important that sleep be recorded on the same food that flies were reared in.</td></tr><tr><td>Rotary glass cutting tool</td><td>Dremel Multi Pro</td><td>395</td><td>Used to cut 65mm long glass tubes&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Monitoring Sleep&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DAM System and DAMFileScan software</td><td>Trikinetics</td><td></td><td>Software used to acquire data from DAM monitors and save the acquired data in an appropriate format</td></tr><tr><td>Data acquisition computer</td><td>Lenovo</td><td>Idea Centre AIO3</td><td>A equivalent computer from any manufacturer can substitute</td></tr><tr><td>Drosophila Activity Monitors</td><td>Trikinetics</td><td>DAM2</td><td>These monitors are used to record flies&#039; locomotor activity</td></tr><tr><td>Environment Monitor</td><td>Trikinetics</td><td>DEnM</td><td>Not essential, but an easy way to monitor environmental conditions in the chamber where sleep is recorded</td></tr><tr><td>Light Controller</td><td>Trikinetics</td><td>LC4</td><td>A convenient way to control the timing of when the SNAP is turned on and off</td></tr><tr><td>Power Supply Interface Unit for DAM</td><td>Trikinetics</td><td>PSIU-9</td><td>Required for data acquisition computers to record Trikinetics locomotor acitvity data</td></tr><tr><td>RJ11 connector</td><td>7001-64PC</td><td>Multicomp</td><td>DAM monitors accept RJ11 jacks</td></tr><tr><td>Splitters</td><td>Trikinetics</td><td>SPLT5</td><td>Used to connect upto 5 DAM monitors</td></tr><tr><td>Telephone cable wire</td><td>Radioshack</td><td>278-367</td><td>Phone cables to acquire data from DAM monitors</td></tr><tr><td>&lt;strong&gt;Sleep Deprivation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Power supply</td><td>Gw INSTEK</td><td>GPS-30300</td><td>Power supply for the SNAP</td></tr><tr><td>Sleep Nullifying Apparatus</td><td>Washington University School of Medicine machine shop</td><td></td><td></td></tr></tbody></table>

the sleep nullifying apparatus: a highly efficient method of sleep depriving drosophila

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 &#62;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.

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<sup class="xref...

Watch this Scientific Journal Video about The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila at JoVE.com

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

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.

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

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal ...

the-sleep-nullifying-apparatus-highly-efficient-method-sleep

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

3.3K Views.  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.

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

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Quantitative Measurement of the Immune Response and Sleep in Drosophila

A complex interaction between the immune response and host behavior has been described in a wide range of species. Excess sleep, in particular, is known to occur as a response to infection in mammals 1 and has also recently been described in Drosophila melanogaster2. It is generally accepted that sleep is beneficial to the host during an infection and that it is important for the maintenance of a robust immune system3,4. However, experimental evidence that supports this hypothesis is limited4, and the function of excess sleep during an immune response remains unclear. We have used a multidisciplinary approach to address this complex problem, and have conducted studies in the simple genetic model system, the fruitfly Drosophila melanogaster. We use a standard assay for measuring locomotor behavior and sleep in flies, and demonstrate how this assay is used to measure behavior in flies infected with a pathogenic strain of bacteria. This assay is also useful for monitoring the duration of survival in individual flies during an infection. Additional measures of immune function include the ability of flies to clear an infection and the activation of NF&kappa;B, a key transcription factor that is central to the innate immune response in Drosophila. Both survival outcome and bacterial clearance during infection together are indicators of resistance and tolerance to infection. Resistance refers to the ability of flies to clear an infection, while tolerance is defined as the ability of the host to limit damage from an infection and thereby survive despite high levels of pathogen within the system5. Real-time monitoring of NF&kappa;B activity during infection provides insight into a molecular mechanism of survival during infection. The use of Drosophila in these straightforward assays facilitates the genetic and molecular analyses of sleep and the immune response and how these two complex systems are reciprocally influenced.

Quantitative Measurement of the Immune Response and Sleep in Drosophila

To understand a link between the immune response and behavior, we describe a method to measure locomotor behavior in Drosophila during bacterial infection as well as the ability of flies to mount an immune response by monitoring survival, bacterial load, and real-time activity of a key regulator of innate immunity, NF&kappa;B.

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

Immunology and Infection

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

Behavior

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr&#160;to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but effective method to observe and document the behavior or morphology of flies. Flies were placed in a translucent observation chamber c.a. &#216;15 x 5mm (no food inside) or &#216;15 x 12 mm (with an 8 mm-high piece of food inside). After covering with an ultraviolet (UV)/clear filter with high light transmittance, the chamber was placed under a 5-50x zoom stereo microscope, and mini light-emitting diode (LED) video lights were placed on both sides of the microscope to illuminate the chamber to obtain uniform, soft, bright, and nearly shadow-free light. Then, a compact digital camera with 3-5x optical zoom, which can record 1080 P high-definition or higher resolution video (at a frame rate of &#8805;30 fps), was connected to the eyepiece of microscope through a bracket, and photographs or videos were taken through the eyepiece. By adjusting the zoom knob of the zoom stereo microscope, it was very easy to track the flies and take panoramic or detailed close-up images as needed, while the camera recorded everything under the microscope. Because the flies can stay at any position in the chamber, they can be observed and recorded from all directions. The photographs or videos taken are of good image quality. This method can be used both for scientific research and teaching.

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but ...

Advancing Sleep Disorder Treatments: A Drosophila-Based Screening Approach

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders characterized by shortened sleep duration and fragmented patterns. These sleep disruptions also correlate with an increased risk of various illnesses in the elderly, including diabetes, cardiovascular diseases, and psychological disorders. Unfortunately, existing drugs for sleep disorders are associated with significant side effects such as cognitive impairment and addiction. Consequently, the development of new, safer, and more effective sleep disorder medications is urgently needed. However, the high cost and lengthy experimental duration of current drug screening methods remain limiting factors.
This protocol describes a cost-effective and high-throughput screening method that utilizes Drosophila melanogaster, a species with a highly conserved sleep regulation mechanism compared to mammals, making it an ideal model for studying sleep disorders in the elderly. By administering various small compounds to aged flies, we can assess their effects on sleep disorders. The sleep behaviors of these flies are recorded using an infrared monitoring device and analyzed with the open-source data package Sleep and Circadian Analysis MATLAB Program 2020 (SCAMP2020). This protocol offers a low-cost, reproducible, and efficient screening approach for sleep regulation. Fruit flies, due to their short life cycle, low husbandry cost, and ease of handling, serve as excellent subjects for this method. As an illustration, Reserpine, one of the tested drugs, demonstrated the ability to promote sleep duration in elderly flies, highlighting the effectiveness of this protocol.

Author Spotlight: Overcoming Challenges in Drosophila Sleep Measurement Using DAM System 

Presented is a protocol for high-throughput drug screening to improve sleep by monitoring the sleep behavior of fruit flies in an elderly Drosophila model.

Sleep, an essential component of health and overall well-being, often presents challenges for older individuals who frequently experience sleep disorders ...

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant environmental parameters that synchronize animal circadian clocks. In Drosophila circadian photo-responses are mediated by a solely blue light photoreceptor named CRYPTOCHROME (CRY). Upon photoreception, CRY changes its conformation and initiates the proteosomal dependent degradation of TIMELESS (TIM). TIM is an important pacemaker protein, thus degradation of TIM will reset the circadian clock. Under constant light conditions (LL), wild type flies quickly become arrhythmic because of the constant degradation of TIM, while flies bearing defects with circadian photo-responses will still be rhythmic. Thus LL triggered arrhythmicity has been used for screening of components in circadian light input pathways. A brief short light pulse in the night can also dramatically shift phases of circadian rhythms. As expected, this phase shift response is reduced in flied with defects in circadian photoresponse. Thus analyzing locomotion behavior rhythmicity under LL or phase changes after short light pulse in constant darkness (DD) are two major methods to study circadian photoresponse. Here we describe how to design and analyze LL and phase response experiments. LL arrhythmicity is suitable for screening light input pathways mutants, whereas phase response validates the results and provide further information for light sensitivity.

Drosophila locomotor activity is a robust and quantitative measurement of circadian photo-responses. We describe protocols for designing behavior experiments for circadian photo-responses and analyzing the data. Studying the circadian photo-responses is important for dissecting the neuronal and molecular mechanisms of light entrainment.

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant ...

Biology

Drosophila Activity Monitor (DAM): A Method to Measure Locomotor Activity in Flies

Source: Chiu, J. C., et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/2157/" target="_blank">Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila</a>. J. Vis. Exp. (2010).
This video describes the Drosophila activity monitor (DAM) system used to track locomotor activity. Researchers use activity data collected from the DAM to study circadian rhythms in fruit flies. The featured protocol clip shows how to load flies in the device and record activity data for circadian experiments.

Source: Chiu, J. C., et al. Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila. J. Vis. Exp. (2010).
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The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Summary

Explore More Videos

Chapters in this video

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Quantitative Measurement of the Immune Response and Sleep in Drosophila

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

The FlyBar: Administering Alcohol to Flies

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

Drosophila Activity Monitor (DAM): A Method to Measure Locomotor Activity in Flies