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Abstract

Introduction

Protocol

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Behavior

High-Throughput Small Molecule Drug Screening For Age-Related Sleep Disorders Using Drosophila melanogaster

Published: October 20th, 2023

DOI:

10.3791/65787

1School of Life Science and Technology, Key Laboratory of Developmental Genes and Human Disease, Southeast University

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 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.

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 ....

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This protocol uses the 30-day-old w1118 flies from the Bloomington Drosophila Stock Center (BDSC_3605, see Table of Materials).

1. Preparation of the aged fruit flies

  1. Food preparation
    1. Prepare standard corn starch culture medium by mixing 50 g/L cornflakes, 110 g/L sugar, 5 g/L agar, and 25 g/L yeast. Heat the cornflakes and yeast with water to gelatinize, and then fully dissolve all the substances.
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Reserpine is a small-molecule inhibitor of the vesicular monoamine transporter (VMAT), which inhibits the reuptake of monoamines into presynaptic vesicles, leading to increased sleep33. The sleep-promoting effects of Reserpine were examined in 30-day-old flies, with the control group being fed solely with the solvent dimethyl sulfoxide (DMSO). In the Reserpine group, older flies exhibited significantly increased sleep during both the day and night compared to the DMSO group. F.......

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The described method is suitable for rapidly screening small and medium-sized sleep drugs. Currently, most mainstream high-throughput drug screening methods are based on biochemical and cellular levels. For example, the structure and properties of the receptor are examined to search for specific ligands that can bind to it22. Another approach involves analyzing the binding mode and strength of molecular fragments of selected drugs using Nuclear Magnetic Resonance (NMR) with mass spectrometry

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We thank Prof. Junhai Han's lab members for their discussion and comments. This work was supported by the National Natural Science Foundation of China 32170970 to Y.T and the "Cyanine Blue Project" of Jiangsu Province to Z.C.Z.

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Name Company Catalog Number Comments
Ager BIOFROXX 8211KG001
Artificial Climate Box PRANDT PRX-1000A official website:https://www.nbplt17.com/PLTXBS-Products-20643427/
DAM2 Drosophila Activity Monitor TriKineics DAM2 official website:https://www.trikinetics.com/
DAM2system TriKineics version:v3.03 official website:https://www.trikinetics.com/
DAMFileScan TriKineics version:1.0.7.0 official website:https://www.trikinetics.com/
Dimethyl Sulfoxide SIGMA 276855
Drosophila Activity Monitoring Incubator Tritech Research DT2-CIRC-TK official website:https://www.tritechresearch.com/DT2-CIRC-TK.html
Drosophila Bottles Biologix 51-17720 official website:http://biologixgroup.com/goods.php?id=48
Drosophila: w1118 Bloomington Drosophila Stock Center  BDSC_3605
Excel Microsoft version:Excel 2016 official website:https://www.microsoftstore.com.cn/software/office/excel
Glass tubes TriKinetics PPT5x65 official website:https://www.trikinetics.com/
MATLABR2022b MathWorks version:9.13.0.2049777 official website:https://ww2.mathworks.cn/products/matlab.html
Prism GraphPad Version:Prism 8.0.1 official website:https://www.graphpad.com/features
Reserpine MACKLIN R817202-1g
Saccharose SIGMA 1245GR500
SCAMP Vecsey Lab N/A official website:https://academics.skidmore.edu/blogs/cvecsey/

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