drug screening method for sleep regulation in an elderly drosophila melanogaster model

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

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.

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

Watch this Scientific Journal Video about Drug Screening Method for Sleep Regulation in an Elderly Drosophila melanogaster Model at JoVE.com

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

노인 초파리 멜라노가스터 모델의 수면 조절을 위한 약물 스크리닝 방법

시작하려면 파리 한 병을 가져다가 성장주기에 맞춰 7 일마다 새 병으로 옮깁니다. 옮긴 지 3일 후에 첫 번째 병에서 부화하는 다음 세대의 파리를 수집하여 새 병에 넣습니다. 그런 다음 유리관을 큰 비커에 넣습니다.이중 증류수를 채우고 20분 동안 끓입니다. 그런 다음 유리관을 제거하고 묶습니다. 이중 증류수로 3-5회 헹구고 유리관을 오븐에 넣어 건조시킵니다.피펫을 사용하여 4mL의 샘플 완충액과 레저핀을 10mL의 작은 비커에 넣습니다. 음성 대조군의 경우 농도가 0.2%가 될 때까지 디메틸설폭사이드를 첨가적절한 길이의 유리관을 작은 비커에 조심스럽게 삽입하여 매체의 흐름을 용이하게 합니다. 배양 배지가 완전히 응고될 때까지 기다리십시오.유리관을 제거하기 전에 외벽을 닦아 모니터링 유리관을 확보하여 한쪽 끝에 약물이 들어 있는 배양 배지가 들어 있는지 확인합니다. 그런 다음 비커에 고체 파라핀을 섭씨 70도에서 녹을 때까지 가열합니다. 유리관의 끝을 액체 파라핀에 약 5mm 담갔다가 빠르게 제거합니다.파라핀이 응고되어 유리관의 음식 끝을 밀봉할 때까지 기다립니다. 마취된 파리를 파라핀으로 밀봉된 유리관에 넣고 흡수성 면봉으로 비식품 끝을 막습니다. 튜브를 적외선 모니터에 로드하여 계속 감시합니다.적외선이 파리 활성 범위의 중심을 수직으로 통과하도록 각 튜브를 정렬합니다. 파리 수면 암실에 있는 인큐베이터 안에 모니터를 설정합니다.

drug-screening-method-for-sleep-regulation-an-elderly-drosophila

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Behavior

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

373 조회수.  Southeast University. 시작하려면 파리 한 병을 가져다가 성장주기에 맞춰 7 일마다 새 병으로 옮깁니다. 옮긴 지 3일 후에 첫 번째 병에서 부화하는 다음 세대의 파리를 수집하여 새 병에 넣습니다. 그런 다음 유리관을 큰 비커에 넣습니다.이중 증류수를 채우고 20분 동안 끓입니다. 그런 다음 유리관을 제거하고 묶습니다. 이중 증류수로 3-5회 헹구고 유리관을 오븐에 넣어 건조시킵니다....

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

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.

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 ...

연구

행동분석학

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.