A Simple Technique to Assay Locomotor Activity in Drosophila

Summary

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Abstract

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

Introduction

Drosophila melanogaster provides an excellent model organism for investigating cellular and molecular functions in neuronal disease models created by gene modification¹, drug treatment², and senescence³. The high conservation of biological pathways, physical properties, and disease-associated homolog genes between humans and Drosophila makes the fruit fly an ideal mimic from the molecular to the behavioral level⁴. In many disease models, behavioral deficiency is an important index, providing a helpful model for various human neuropathies^5....

Protocol

W¹¹¹⁸ adult flies and third instar larvae were used for the present study.

1. Experimental preparation

NOTE: An open-field arena for Drosophila locomotion tracking is made withacolorless and odorless silica gel.

1. Mix reagent A and reagent B at a ratio of 1:10, according to the manufacturer's instructions for the silica kit (see Table of Materials). Ensure that sodium bicarbonate is added to the mixture by stirring until the color changes to white. Transfer the mixture to a clean Petri dish and place it in an oven at 40 °C for dryin....

Results

In the present study, locomotor deficits in adult flies and third instar larvae treated with rotenone were examined and compared in their motor activity to that of a control fly fed with the drug solvent dimethyl sulfoxide (DMSO). Treatment with rotenone in Drosophila has been shown to cause dopaminergic neuron loss in the brain²² and lead to significant locomotor deficits²³. As shown in Figure 11 and Figure 12

Discussion

We have designed a method, based on the open-source material AnimalTracker API compatible with the Fiji image processing program, that can enable researchers to systematically evaluate locomotor activity by tracking both adult and individual larval flies. AnimalTracke is a tool written in Java that can be easily integrated into existing databases or other tools to facilitate the analysis of application-designed animal-tracking behavior²⁴. Upon a frame-by-frame analysis by a softw.......

Materials

Name	Company	Catalog Number	Comments
Animal tracker	Hungarian Brain Research Program	version: 1.7	pfficial website: http://animaltracker.elte.hu/main/downloads
Camera software	Microsoft	version: 2021.105.10.0	built-in windows 10 system
Computer	DELL	Vostro-14-5480	a comupter running win 10 system is available
Drosophila carbon dioxide anesthesia workstation	Wu han Yihong technology	#YHDFPCO2-018	official website: http://www.yhkjwh.com/
Fiji software	Fiji team	version: 1.53v	official website: https://fiji.sc/
Format factory software	Pcfreetime	version: X64 5.4.5	official website: http://www.pcfreetime.com/formatfactory/CN/index.html
Graph pad prism	GraphPad Software	version: 8.0.2	official website: https://www.graphpad-prism.cn
Hight definition camera	TTQ	Jingwang2 (HD1080P F1.6 6-60mm)	official website: http://www.ttq100.com/product_show.php?id=35
Office software	Microsoft	version: office 2019	official website: https://www.microsoftstore.com.cn/software/office
Petri dish	Bkman	110301003	size: 60 mm
Silica gel	DOW	SYLGARD 184 Silicone Elastomer Kit	Mix well according to the instructions
Sodium bicarbonate	Macklin	#144-55-8	Mix well with silica gel