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

Weiterentwicklung der Behandlung von Schlafstörungen: Ein Drosophila-basierter Screening-Ansatz

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 im Rampenlicht: Überwindung von Herausforderungen bei der Schlafmessung von Drosophila mit dem DAM-System 

Hochdurchsatz-Screening von niedermolekularen Medikamenten für altersbedingte Schlafstörungen mit 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  

Drug-Screening-Methode zur Schlafregulation in einem älteren Drosophila melanogaster-Modell

Nehmen Sie zunächst eine Flasche Fliegen und setzen Sie sie alle sieben Tage in eine neue Flasche um, synchron zu ihrem Wachstumszyklus. Sammle die nächste Generation von Fliegen, die drei Tage nach dem Umfüllen aus der ersten Flasche schlüpfen, und setze sie in eine frische Flasche. Als nächstes legst du das Glasröhrchen in ein großes Becherglas.Mit doppelt destilliertem Wasser füllen und 20 Minuten kochen lassen. Anschließend das Glasröhrchen herausnehmen und bündeln. Spülen Sie es drei- bis fünfmal mit doppelt destilliertem Wasser aus und stellen Sie das Glasröhrchen zum Trocknen in einen Ofen.Geben Sie mit einer Pipette vier Milliliter Probenpuffer und Reserpine in ein 10 Milliliter kleines Becherglas. Für die Negativkontrollgruppe wird Dimethylsulfoxid zugegeben, bis die Konzentration 0,2 % erreicht. Führen Sie vorsichtig ein geeignetes Stück Glasröhrchen in ein kleines Becherglas ein, um das Fließen des Mediums zu erleichtern. Warten Sie, bis das Nährmedium vollständig fest geworden ist.Bevor Sie das Glasröhrchen entfernen, wischen Sie die Außenwand ab, um ein Überwachungsglasröhrchen zu erhalten, um sicherzustellen, dass ein Ende das Nährmedium mit Medikamenten enthält. Anschließend festes Paraffin in einem Becherglas bei 70 Grad Celsius erhitzen, bis es schmilzt. Tauchen Sie das Ende des Glasröhrchens etwa fünf Millimeter in das flüssige Paraffin und entfernen Sie es schnell.Warten Sie, bis das Paraffin erstarrt ist, um das Ende des Glasröhrchens zu versiegeln. Legen Sie die betäubten Fliegen in paraffinversiegelte Glasröhrchen und blockieren Sie das Non-Food-Ende mit einem saugfähigen Wattebausch. Laden Sie die Röhren auf einen Infrarotmonitor, um sie zu überwachen.Richten Sie jede Röhre so aus, dass die Infrarotstrahlen vertikal durch die Mitte des aktiven Bereichs der Fliege verlaufen. Stellen Sie den Monitor in einen Inkubator, der in der Dunkelkammer des Fliegenschlafs untergebracht ist.

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 ...