We thank Prof. Junhai Han&#39;s lab members for their discussion and comments. This work was supported by the National Natural Science Foundation of China 32170970 to Y.T and the &#34;Cyanine Blue Project&#34; of Jiangsu Province to Z.C.Z.

This protocol uses the 30-day-old w1118 flies from the Bloomington Drosophila Stock Center (BDSC_3605, see Table of Materials).
1. Preparation of the aged fruit flies
<ol>
	<li>Food preparation
	<ol>
		<li>Prepare standard corn starch culture medium by mixing 50 g/L cornflakes, 110 g/L sugar, 5 g/L agar, and 25 g/L yeast. Heat the cornflakes and yeast with water to gelatinize, and then fully dissolve all the substances.</li>
		<l...

Advancing Sleep Disorder Treatments: A &lt;em&gt;Drosophila&lt;/em&gt;-Based Screening Approach

<ol>
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H., Sehgal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+screen+in+adult+Drosophila+identifies+VMAT+as+a+regulator+of+sleep.">Small-molecule screen in adult Drosophila identifies VMAT as a regulator of sleep.</a> Journal of Neuroscience. 33 (19), 8534-8464 (2013).</li><li>Jin, X., Gu, P., Han, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+Drosophila+sleep+deprivation+using+single-chip+board.">Protocol for Drosophila sleep deprivation using single-chip board.</a> STAR Protocols. 2 (4), 100827 (2021).</li><li>Kashyap, A., Singh, P. K., Silakari, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+on+fragment+based+drug+design+approach+for+drug+discovery.">Counting on fragment based drug design approach for drug discovery.</a> Current Topics in Medicinal Chemistry. 18 (27), 2284-2293 (2018).</li><li>Qi, W., Ding, D., Salvi, R. 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A., Shaposhnikov, M. V., Moskalev, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronobiotics+KL001+and+KS15+extend+lifespan+and+modify+circadian+rhythms+of+Drosophila+melanogaster.">Chronobiotics KL001 and KS15 extend lifespan and modify circadian rhythms of Drosophila melanogaster.</a> Clocks Sleep. 3 (3), 429-441 (2021).</li><li>Cavas, M., Beltran, D., Navarro, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioural+effects+of+dimethyl+sulfoxide+(DMSO):+changes+in+sleep+architecture+in+rats.">Behavioural effects of dimethyl sulfoxide (DMSO): changes in sleep architecture in rats.</a> Toxicology Letters. 157 (3), 221-232 (2005).</li><li>Pfeiffenberger, C., Lear, B. C., Keegan, K. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ethomics+in+large+groups+of+Drosophila.">High-throughput ethomics in large groups of Drosophila.</a> Nature Methods. 6 (6), 451-457 (2009).</li><li>Kabra, M., Robie, A. A., Rivera-Alba, M., Branson, S., Branson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JAABA:+interactive+machine+learning+for+automatic+annotation+of+animal+behavior.">JAABA: interactive machine learning for automatic annotation of animal behavior.</a> Nature Methods. 10 (1), 64-67 (2013).</li><li>Donelson, N. 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The described method is suitable for rapidly screening small and medium-sized sleep drugs. Currently, most mainstream high-throughput drug screening methods are based on biochemical and cellular levels. For example, the structure and properties of the receptor are examined to search for specific ligands that can bind to it22. Another approach involves analyzing the binding mode and strength of molecular fragments of selected drugs using Nuclear Magnetic Resonance (NMR) with mass spectrometry<sup c...

Sleep, one of the essential behaviors necessary for human survival, is characterized by two main states: rapid eye movement (REM) sleep and non-rapid eye movement (NREM) sleep1. NREM sleep comprises three stages: N1 (the transition between wakefulness and sleep), N2 (light sleep), and N3 (deep sleep, slow wave sleep), representing the progression from wakefulness to deep sleep1. Sleep plays a crucial role in both physical and mental health2. However, aging reduces total sleep duration, sleep efficiency, slow-wave sleep percentage, and REM sleep percentage in adults3. Older individuals tend to spend more time in light sleep compared to slow-wave sleep, making them more sensitive to nocturnal awakenings. As the number of awakenings increases, average sleep time decreases, resulting in a fragmented sleep pattern in the elderly, which may be associated with excessive excitation of Hcrt neurons in mice4. Additionally, age-related declines in circadian mechanisms contribute to an earlier shift in sleep duration5,6. In combination with physical illness, psychological stress, environmental factors, and medication use, these factors make older adults more susceptible to sleep disorders, such as insomnia, REM sleep behavior disorder, narcolepsy, periodic leg movements, restless legs syndrome, and sleep-disordered breathing7,8.
Epidemiological studies have shown that sleep disorders are closely linked to chronic diseases in the elderly9, including depression10, cardiovascular disease11, and dementia12. Addressing sleep disorders plays a crucial role in improving and treating chronic diseases and enhancing the quality of life for older adults. Currently, patients primarily rely on drugs such as benzodiazepines, non-benzodiazepines, and melatonin receptor agonists to enhance sleep quality13. However, benzodiazepines can lead to downregulation of receptors and dependence after long-term use, causing severe withdrawal symptoms upon discontinuation14,15. Non-benzodiazepine drugs also carry risks, including dementia16, fractures17, and cancer18. The commonly used melatonin receptor agonist, ramelteon, reduces sleep latency but does not increase sleep duration and has hepatic function-related concerns due to extensive first-pass elimination19. Agomelatine, a melatonin receptor agonist and serotonin receptor antagonist, improves depression-related insomnia but also poses a risk of liver damage20. Consequently, there is an urgent need for safer drugs to treat or alleviate sleep disorders. However, current drug screening strategies, based on molecular and cellular experiments combined with automated systems and computer analysis, are expensive and time-consuming21. Structure-based drug design strategies, relying on receptor structure and properties, require a clear understanding of receptor three-dimensional structure and lack predictive capabilities for drug effects22.
In 2000, based on the sleep criteria proposed by Campbell and Tobler in 198423, researchers established simple animal models to study sleep24, including Drosophila melanogaster, which exhibited sleep-like states25,26. Despite anatomical differences between Drosophila and humans, many neurochemical components and signaling pathways regulating sleep in Drosophila are conserved in mammalian sleep, facilitating the study of human neurological diseases27,28. Drosophila is also extensively used in circadian rhythm studies, despite differences in core oscillators between flies and mammals29,30,31. Therefore, Drosophila serves as a valuable model organism for studying sleep behavior and conducting sleep-related drug screening.
This study proposes a cost-effective and simple phenotype-based approach for screening small-molecule drugs to treat sleep disorders using aged flies. Sleep regulation in Drosophila is highly conserved25, and the decline in sleep observed with age may be reversible through drug administration. Thus, this sleep phenotype-based screening method can intuitively reflect drug efficacy. We feed the flies with a mixture of the drug under investigation and food, monitor and record sleep behavior using the Drosophila Activity Monitor (DAM)32, and analyze the acquired data using the open-source SCAMP2020 data package in MATLAB (Figure1). Statistical analysis is performed using statistics and graphing software (see Table of Materials). As an example, we demonstrate the effectiveness of this protocol by presenting experimental data on Reserpine, a small-molecule inhibitor of the vesicular monoamine transporter reported to increase sleep33. This protocol provides a valuable approach to identify drugs for treating age-related sleep problems.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ager</td>
			<td>BIOFROXX</td>
			<td>8211KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Climate Box</td>
			<td>PRANDT</td>
			<td>PRX-1000A</td>
			<td>official website:https://www.nbplt17.com/PLTXBS-Products-20643427/</td>
		</tr>
		<tr>
			<td>DAM2 Drosophila Activity Monitor</td>
			<td>TriKineics</td>
			<td>DAM2</td>
			<td>official website:https://www.trikinetics.com/</td>
		</tr>
		<tr>
			<td>DAM2system</td>
			<td>TriKineics</td>
			<td>version:v3.03</td>
			<td>official website:https://www.trikinetics.com/</td>
		</tr>
		<tr>
			<td>DAMFileScan</td>
			<td>TriKineics</td>
			<td>version:1.0.7.0</td>
			<td>official website:https://www.trikinetics.com/</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>SIGMA</td>
			<td>276855</td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Activity Monitoring Incubator</td>
			<td>Tritech Research</td>
			<td>DT2-CIRC-TK</td>
			<td>official website:https://www.tritechresearch.com/DT2-CIRC-TK.html</td>
		</tr>
		<tr>
			<td>Drosophila Bottles</td>
			<td>Biologix</td>
			<td>51-17720</td>
			<td>official website:http://biologixgroup.com/goods.php?id=48</td>
		</tr>
		<tr>
			<td>Drosophila: w1118</td>
			<td>Bloomington Drosophila Stock Center&#160;</td>
			<td>BDSC_3605</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>version:Excel 2016</td>
			<td>official website:https://www.microsoftstore.com.cn/software/office/excel</td>
		</tr>
		<tr>
			<td>Glass tubes</td>
			<td>TriKinetics</td>
			<td>PPT5x65</td>
			<td>official website:https://www.trikinetics.com/</td>
		</tr>
		<tr>
			<td>MATLABR2022b</td>
			<td>MathWorks</td>
			<td>version:9.13.0.2049777</td>
			<td>official website:https://ww2.mathworks.cn/products/matlab.html</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td>Version:Prism 8.0.1</td>
			<td>official website:https://www.graphpad.com/features</td>
		</tr>
		<tr>
			<td>Reserpine</td>
			<td>MACKLIN</td>
			<td>R817202-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>SIGMA</td>
			<td>1245GR500</td>
			<td></td>
		</tr>
		<tr>
			<td>SCAMP</td>
			<td>Vecsey Lab</td>
			<td>N/A</td>
			<td>official website:https://academics.skidmore.edu/blogs/cvecsey/</td>
		</tr>
	</tbody>
</table>

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 

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

Reserpine is a small-molecule inhibitor of the vesicular monoamine transporter (VMAT), which inhibits the reuptake of monoamines into presynaptic vesicles, leading to increased sleep33. The sleep-promoting effects of Reserpine were examined in 30-day-old flies, with the control group being fed solely with the solvent dimethyl sulfoxide (DMSO). In the Reserpine group, older flies exhibited significantly increased sleep during both the day and night compared to the DMSO group. F...

Watch this Scientific Journal Video about Overcoming Challenges in Drosophila Sleep Measurement Using DAM System at JoVE.com

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 ...

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Drug Screening Method for Sleep Regulation in an Elderly Drosophila melanogaster Model

Drug Screening Method for Sleep Regulation in an Elderly Drosophila melanogaster Model  

To begin, take a bottle of flies, transfer them into a new bottle every seven days in sync with their growth cycle. Collect the next generation of flies that hatch from the initial bottle three days after transferring, and place them into a fresh bottle. Next, place the glass tube into a large beaker.Fill it with double distilled water and boil for 20 minutes. Then remove and bundle the glass tube. Rinse it three to five times with double distilled water, and place the glass tube in an oven to dry.Using a pipette, add four milliliters of sample buffer and reserpine into a 10 milliliter small beaker. For the negative control group, add dimethyl sulfoxide until the concentration reaches 0.2%Carefully insert an appropriate length of glass tube into a small beaker to facilitate the flow of the medium. Wait for the culture medium to fully solidify.Before removing the glass tube, wipe the outer wall to acquire a monitoring glass tube ensuring that one end contains the culture medium with drugs. Then heat solid paraffin in a beaker at 70 degrees Celsius until it melts. Immerse the glass tube's end in the liquid paraffin for about five millimeters and quickly remove it.Wait for the paraffin to solidify to seal the food end of the glass tube. Put the anesthetized flies into paraffin sealed glass tubes and block the non-food end with an absorbent cotton ball. Load the tubes onto an infrared monitor to keep watch over them.Align each tube so the infrared rays run vertically through the center of the flies active range. Set the monitor inside an incubator housed in the flies sleep darkroom.

Analyzing the Effect of Reserpine on the Sleep Behavior of Drosophila melanogaster Using SCAMP2020 Data Package in MATLAB

Analyzing the Effect of Reserpine on the Sleep Behavior of Drosophila melanogaster Using SCAMP2020 Data Package in MATLAB  

To begin, monitor the sleep behaviors of flies using an infrared monitoring device and import the collected data in TXT format into the DAM file scan 107 software for scanning. Set the start time of the segmentation data to either 0800 or 0801 on the third morning after initiating the monitors to obtain one or 30 minute bin length data, respectively. Set the termination time to 8:00 AM on the third day post the set starting time.Adjust the bin length option to one minute and alter the output file type to channel files. Rename the file and generate the output. Now launch the SCAMP 2020 software package in MATLAB.Double click on Vecsey Sleep and Circadian Analysis MATLAB program. Added it sub-folder titled Vecsey SCAMP Scripts into the path. Locate the SCAMP.m file in that folder and run it. In the subsequent popup window sequentially select the folder named one minute and 30 minute. Then select a monitor and click on load individual C plots to preview.Inspect the image that appears and deselect the corresponding channel of any dead flies. Rename each channel in every monitor based on the corresponding drug to be tested. Select all monitors and click Analyze selected data for the analysis.Select the default option and then click on analyze for chosen bin feature. Check the export data box. Click on graph 30 minute data types for all days for selected groups, and export all data to generate the output of the results.Select the S30 file from the CSV file and locate the corresponding mean value and standard error data for each monitor. Save this data in Excel to make some adjustments before posting it into GraphPad Prism to formulate a sleep status diagram. Afterwards, locate the file named S-T-D-U-R and compute the average values of daytime, nighttime, and total sleep for each fly over three days.Paste data into GraphPad Prism to draw graphs. The sleep patterns of Reserpine and Dimethyl Sulfoxide on female and male flies over three consecutive days are shown. Quantitative analysis of the sleep behavior demonstrates a significant increase in sleep time in aged females fed Reserpine.The experiment on male flies also demonstrates a positive correlation between Reserpine concentration and the promotion of sleep.

1.4K Views.  Southeast University.  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.

Advancing Sleep Disorder Treatments: A Drosophila-Based Screening Approach

Summary