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>
		<li>When the medium cools to 50-60 &#176;C, add 6 mL/L of propionic acid and promptly pack them into culture bottles.</li>
	</ol>
	</li>
	<li>Fly rearing and preparation of aged flies
	<ol>
		<li>Breed the fly strain w1118 in bottles containing a standard cornstarch culture medium and put bottles in a constant temperature incubator at 25&#176; C, 68% relative humidity, 500-1000 lux lighting conditions, and a 12 h:12 h light: dark cycle.</li>
		<li>Transfer flies into a new bottle every 7 days according to the growth cycle of flies, keeping the age of individuals in the same bottle consistent.</li>
		<li>Collect the new batch of flies that hatch from the original bottle 3 days after transferring them and put them into a new bottle. Following the principle of changing the bottle every 7 days, they will be cultured until around 30 days old.</li>
	</ol>
	</li>
</ol>
2. Preparation of medicinal food and glass tubes for monitoring
NOTE: The procedure for glass tube preparation follows the work of Jin et al. with modifications34.
<ol>
	<li>Cleaning and drying of glass tubes
	<ol>
		<li>Place the glass tube (5 mm in diameter x 65 mm in length, see Table of Materials) into a large beaker, soak it, and boil it with double distilled water for 20 min. Repeat 3 times.</li>
		<li>Remove and bundle the glass tube, rinse the inside with double distilled water 3-5 times, and place it in an oven for drying.</li>
	</ol>
	</li>
	<li>Preparation of simple culture medium (100 mL)
	<ol>
		<li>Dissolve 1.5 g agar and 5 g sucrose in double distilled water, heat, and concentrate to 100 mL.</li>
		<li>Add 600 &#181;L propionic acid when the medium cools to about 70 &#176;C, preventing it from solidifying using a constant temperature water bath.</li>
		<li>Add approximately 4 mL of simple medium and Reserpine (see Table of Materials) into a 10 mL small beaker until the drug reaches 20 &#181;M or 50 &#181;M. Add dimethyl sulfoxide (DMSO) to the concentration of 0.2% in the negative control group.</li>
	</ol>
	</li>
	<li>Preparation of the glass tubes containing medicine
	<ol>
		<li>To facilitate the flow of the medium, carefully insert a suitable length of glass tube into a small beaker. The medium will naturally enter the glass tube due to atmospheric pressure.</li>
		<li>Pull out the glass tube when the culture medium is completely solidified and wipe the outer wall to obtain a monitoring glass tube with a culture medium containing drugs at one end.</li>
		<li>Heat the solid paraffin in a beaker until it melts at 70 &#176;C, put the end of the glass tube close to the food into the paraffin liquid for about 5 mm, and quickly remove it. Wait for the paraffin to solidify to seal the food end of the glass tube.</li>
	</ol>
	</li>
</ol>
3. Experimental design and fly treatment
<ol>
	<li>Design the experiment for the fly treatment following Table 1.</li>
</ol>
4. Drosophila assembly and sleep monitoring
NOTE: The procedure for Drosophila assembly follows the work of Jin et al.34 with modifications.
<ol>
	<li>Anesthetize flies with CO2 gas, put them into paraffin-sealed glass tubes (one per tube), and block the non-food end with an absorbent cotton ball to prevent flies from escaping and ensure air circulation.</li>
	<li>Load tubes onto the infrared monitor for monitoring them.
	<ol>
		<li>Assemble the glass tubes containing flies onto an infrared monitor in the same direction, and record the monitor number and hole number corresponding to each drug.</li>
		<li>Adjust the alignment of each tube, and make the infrared rays pass vertically through the center of the fly&#39;s activity range.</li>
		<li>Place the monitor inside a 25 &#176;C incubator located in the fly sleep darkroom, following the specified settings: 25 &#176;C temperature, Zeitgeber 12 (ZT12) (equivalent to local time 08:00 p.m.), and ZT24 (equivalent to local time 08:00 a.m.). This setup ensures that the flies experience alternating periods of 12 h of light and darkness. 
		NOTE: Try not to open the door until monitoring data collection is complete to maintain a stable environment in the incubator during monitoring.</li>
		<li>Start monitoring using the DAM2 system (see Table of Materials).</li>
		<li>Once the monitoring is complete, download the collected data in .txt format from the system.</li>
	</ol>
	</li>
</ol>
5. Data processing
NOTE: The data processing using the DAM system, DAMFileScan107, and SCAMP was performed according to the instructions on their official websites (see Table of Materials).
<ol>
	<li>Import the above txt file into DAMFileScan107 software for scanning and divide it as needed to obtain sleep data.
	<ol>
		<li>Set the starting time of segmentation data to 8:01 (1 min segmentation) or 8:00 (30 min segmentation) on the third morning after starting the monitors, and the termination time is 8:00 a.m. three days after the starting time (Figure 2A1). 
		NOTE: Flies must adapt to the monitoring environment for at least one day. So, one can set the split data start time to 8 a.m. on the third day after the monitor begins.</li>
		<li>Split the data at intervals of 1 min and 30 min. Change the option &#34;Bin Length&#34; to 1 minute, change the option &#34;Output File Type&#34; to Channel files, rename, and output. The 30 min data segmentation method is the same as above (Figure 2A2-5). 
		NOTE: When performing data segmentation at 1 min and 30 min intervals, the final renaming of the two files should be consistent; otherwise, it may be unreadable during subsequent Matlab processing. If necessary, the file name can be changed after output to facilitate differentiation.</li>
	</ol>
	</li>
	<li>Data processing using SCAMP2020
	<ol>
		<li>Open the program package SCAMP2020 in Matlab, and double-click on Vecsey Sleep and Circadian Analysis MATLAB Program (SCAMP) (Figure 2B).</li>
		<li>Add its subfolder &#34;Vecsey SCAMP Scripts&#34; to the path, find the file &#34;scamp.m&#34; in that folder, and run it. In the following pop-up window, select the process 1 min and 30 min folders in sequence (Figure 2C,D).</li>
		<li>Select a monitor, click on Load individual See Plots to preview (Figure 3A1), and check the image that appears. Uncheck the corresponding channel of dead flies (Figure 3A2, Figure 3B).</li>
		<li>Repeat the above steps to check all monitors.</li>
		<li>Rename each channel in each monitor based on the corresponding drug to be tested (Figure 3A3), select all monitors, and click on ANALYZE Selected Data for analysis (Figure 3A4).</li>
		<li>Default to the selected option, click on Analyze for Chosen Bin, check Export Data, and finally click on GRAPH 30 min Data Types for All Days for Selected Groups and EXPORT All Data to output the results (Figure 3C).</li>
	</ol>
	</li>
	<li>Select the file named s30 from the CSV file, find the corresponding mean value and standard error data for each monitor, back up it to Excel for modification and adjustment, and paste it into GraphPad Prism (see Table of Materials) to draw a sleep status diagram (Figure 4A,B).</li>
	<li>Find the file named &#34;stdur&#34; and calculate the average values of daytime, nighttime, and total sleep for each fly within three days (Figure 4A,C). Paste the data into the Prism software to complete the difference test and draw a graph.</li>
</ol>

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

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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. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+positional+tracking+for+long-term+analysis+of+Drosophila+sleep+and+locomotion+using+the+&amp;#34;tracker&amp;#34;+program.">High-resolution positional tracking for long-term analysis of Drosophila sleep and locomotion using the &amp;#34;tracker&amp;#34; program.</a> Plos One. 7 (5), e37250 (2012).</li><li>Cichewicz, K., Hirsh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ShinyR-DAM:+a+program+analyzing+Drosophila+activity,+sleep+and+circadian+rhythms.">ShinyR-DAM: a program analyzing Drosophila activity, sleep and circadian rhythms.</a> Communications Biology. 1, 25 (2018).</li></ol>

The authors declare no competing interests.

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

Drug screening techniques include high-throughput screening, which uses automation for drug discovery, structure-based designs, and find receptor binding molecules based on the structure, and a fragment-based design, which uses nuclear magnetic resonance and mass spectrometry to optimize molecular fragments and identify the compounds. The DAM system measures fly sleep by detecting infrared beam bricks. However, this method can overestimate sleep if the flies remain static at one end of the tube, or if the fly is not highly mobile.Furthermore, the order of any tested drug may impact the fly fruit intake, potentially skewing experimental results. Therefore, our experiment will incorporate various stretches to mitigate these factors and enhance accuracy. Compared with video analysis based methods, our method offers high-throughput capabilities, reduce the cost, and simplify operation.So process of experimental data is also relatively straightforward, where the resulting graph visually represents the drug's effects.

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 ...

high-throughput-small-molecule-drug-screening-for-age-related-sleep

Research

JoVE Journal

Behavior

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.

2.2K 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