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  

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.

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361 次观看.  Southeast University. 首先，拿一瓶苍蝇，每七天将它们转移到一个新瓶子中，与它们的生长周期同步。在转移三天后收集从初始瓶子孵化的下一代苍蝇，并将它们放入新瓶子中。接下来，将玻璃管放入一个大烧杯中。倒入双蒸水，煮沸20分钟。然后取出并捆扎玻璃管。用双蒸水冲洗三到五次，然后将玻璃管放入烤箱中晾干。使用移液管，将 4 毫升样品缓冲液和利血平加入 10 毫升小烧杯...