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

Avançando nos tratamentos de distúrbios do sono: uma abordagem de triagem baseada em Drosophila

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.

Autor em destaque: Superando desafios na medição do sono de Drosophila usando o sistema DAM 

Triagem de drogas de moléculas pequenas de alto rendimento para distúrbios do sono relacionados à idade usando 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.

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  

Método de Triagem de Fármacos para Regulação do Sono em um Modelo Idoso de Drosophila melanogaster

Para começar, pegue uma garrafa de moscas, transfira-as para uma nova garrafa a cada sete dias em sincronia com seu ciclo de crescimento. Colete a próxima geração de moscas que eclodem da garrafa inicial três dias após a transferência e coloque-as em uma garrafa fresca. Em seguida, coloque o tubo de vidro em um copo grande.Encha com água bidestilada e ferva por 20 minutos. Em seguida, retire e embrulhe o tubo de vidro. Enxágue de três a cinco vezes com água bidestilada e coloque o tubo de vidro em um forno para secar.Usando uma pipeta, adicione quatro mililitros de tampão de amostra e reserpina em um copo pequeno de 10 mililitros. Para o grupo controle negativo, adicionar dimetilsulfóxido até que a concentração atinja 0,2%Inserir cuidadosamente um comprimento apropriado de tubo de vidro em um copo pequeno para facilitar o fluxo do meio. Espere que o meio de cultura se solidifique totalmente.Antes de remover o tubo de vidro, limpe a parede externa para adquirir um tubo de vidro de monitoramento, garantindo que uma das extremidades contenha o meio de cultura com drogas. Em seguida, aqueça a parafina sólida em um copo a 70 graus Celsius até derreter. Mergulhe a extremidade do tubo de vidro na parafina líquida por cerca de cinco milímetros e remova-o rapidamente.Espere a parafina solidificar para selar a extremidade alimentar do tubo de vidro. Coloque as moscas anestesiadas em tubos de vidro selados com parafina e bloqueie a extremidade não alimentar com uma bola de algodão absorvente. Coloque os tubos em um monitor infravermelho para vigiá-los.Alinhe cada tubo para que os raios infravermelhos percorram verticalmente o centro da faixa ativa das moscas. Coloque o monitor dentro de uma incubadora alojada na câmara escura de sono das moscas.

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 ...