Name: Drug Screening Method for Sleep Regulation in an Elderly Drosophila melanogaster Model
Uploaded: 2023-10-20T14:15:00
Description: 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

수면 장애 치료의 발전: 초파리 기반 스크리닝 접근법

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.

저자 스포트라이트: DAM 시스템을 사용한 초파리 수면 측정의 과제 극복 

Drosophila melanogaster를 사용하여 나이 관련 수면 장애를 위한 High-throughput 작은 분자 약 검열

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.

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.

Research

JoVE Journal

Behavior

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

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 ...