Name: a simple technique to assay locomotor activity in drosophila
Uploaded: 2023-02-24T14:00:00
Description: Watch this Scientific Journal Video about A Simple Technique to Assay Locomotor Activity in Drosophila at JoVE.com

This work was supported by a special launch fund from Soochow University and the National Science Foundation of China (NSFC) (82171414). We thank Prof. Chunfeng Liu&#39;s lab members for their discussion and comments.

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.
<ol>
	<li>Mix reagent A and reagent B at a ratio of 1:10, according to the manufacturer&#39;s instructions for the silica kit (see Table of Materials). Ensure that sodium bicarbonate is added t...

<ol>
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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...

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 neuropathies5,6. Drosophila is now used to study multiple human diseases, neurodevelopment, and neurodegenerative diseases such as Parkinson&#39;s disease and amyotrophic lateral sclerosis7,8. Detecting the motor ability of the disease models is crucial for understanding the pathogenic progress and may provide a phenotypic correlation to the molecular mechanisms underlying the disease process.
Recently, commercially available software tools and cost-effective programs have been developed for Drosophila locomotor detection strategies, such as high-throughput testing in grouped flies9,10 and measuring locomotion in real-time11,12. One such conventional approach is rapid interactive negative geotaxis (RING), also called the climbing assay, which includes multiple channels that allow for a large fly population with the same gender and age to be contained, reducing variation while data collecting9,13. Another pre-testing method for analyzing locomotor behavior is TriKinetics Drosophila activity monitor (DAM), a device that uses multiple beams to detect fly activity movement within a thin glass tube14. The device records position continuously, which represents automated locomotion by calculating the beam-crossings to study the activity and circadian rhythm of flies over a longer period of time15. Although these methods have been widely used in analyzing behavioral defects in fruit flies to determine changes in behavioral locomotion, they always require special testing equipment or complex analysis processes, and restrict their application in some models with a limited, simple device. Animal-tracing group-based strategies for testing the adult Drosophila, such as FlyGrAM11 and the Drosophila island assay10, implement social recruitment and individual tracking in a predefined area. Nevertheless, social individual restriction in defied areas might have a negative effect on identifications in the images, caused by the collision or overlapping of flies. Even though some open-source materials-based methods, such as TRex16, MARGO12, and FlyPi17, have an emergency, they can fast-track trace the flies with flexible usage in behavioral testing. These testing approaches are associated with elaborate experimental apparatus installations, special software requirements, or professional computer languages. For larvae, measuring the total distance traveled across the number of grid border lines per unit of time18, or rough counting the body wall contractions for individuals manually19, are the predominant methods for assessing their locomotor ability. Due to the lack of precision in equipment or devices and analysis methods, some behavioral locomotion of larvae might escape detection, making it difficult to accurately assess behavioral movement, especially fine movement15.
The present developed method utilizes the AnimalTracker application programming interface (API), compatible with the Fiji (ImageJ) image processing program, to systematically evaluate the locomotor activity of both adult and larval flies by analyzing their tracking behavior from high-definition (HD) videos. Fiji is an open-source software ImageJ distribution that can combine robust software libraries with numerous scripting languages, resulting in rapid prototyping of image processing algorithms, making it popular among biologists for its image analysis capabilities20. In the current approach, Fiji&#39;s integration into the AnimalTracker API is exploited to develop a unique Drosophila behavioral assay with personalized algorithm insertion, and provides a useful step for detailed documentation and tutorials to support robust analytical capabilities of locomotor behavior (Figure 1). To circumvent the complication of objective identifications in the images caused by the collision or overlapping of flies, each arena is restricted to hosting only one fly. Upon assessing the tracking precision of the approach, it was implemented to trace and quantify the locomotor movements of Drosophila that were administered with the toxic drug rotenone, which is generally used for animal models of Parkinson&#39;s disease, ultimately discovering locomotion impairment in the drug treatment21. This methodology, which employs open-source and free software, does not necessitate high-cost instrumentation, and can precisely and reproducibly analyze Drosophila behavioral locomotion.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal tracker</td>
			<td>Hungarian Brain Research Program</td>
			<td>version: 1.7</td>
			<td>pfficial website: http://animaltracker.elte.hu/main/downloads</td>
		</tr>
		<tr>
			<td>Camera software</td>
			<td>Microsoft</td>
			<td>version: 2021.105.10.0</td>
			<td>built-in windows 10 system</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>DELL</td>
			<td>Vostro-14-5480</td>
			<td>a comupter running win 10 system is available</td>
		</tr>
		<tr>
			<td>Drosophila carbon dioxide anesthesia workstation</td>
			<td>Wu han Yihong technology</td>
			<td>#YHDFPCO2-018</td>
			<td>official website: http://www.yhkjwh.com/</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td>Fiji team</td>
			<td>version: 1.53v</td>
			<td>official website: https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Format factory software</td>
			<td>Pcfreetime</td>
			<td>version: X64 5.4.5</td>
			<td>official website: http://www.pcfreetime.com/formatfactory/CN/index.html</td>
		</tr>
		<tr>
			<td>Graph pad prism</td>
			<td>GraphPad Software</td>
			<td>version: 8.0.2</td>
			<td>official website: https://www.graphpad-prism.cn</td>
		</tr>
		<tr>
			<td>Hight definition camera</td>
			<td>TTQ</td>
			<td>Jingwang2 (HD1080P F1.6 6-60mm)</td>
			<td>official website: http://www.ttq100.com/product_show.php?id=35</td>
		</tr>
		<tr>
			<td>Office software</td>
			<td>Microsoft</td>
			<td>version: office 2019</td>
			<td>official website: https://www.microsoftstore.com.cn/software/office</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Bkman</td>
			<td>110301003</td>
			<td>size: 60 mm</td>
		</tr>
		<tr>
			<td>Silica gel</td>
			<td>DOW</td>
			<td>SYLGARD 184 Silicone Elastomer Kit</td>
			<td>Mix well according to the instructions</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Macklin</td>
			<td>#144-55-8</td>
			<td>Mix well with silica gel</td>
		</tr>
	</tbody>
</table>

a simple technique to assay locomotor activity in drosophila

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.

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</st...

Watch this Scientific Journal Video about A Simple Technique to Assay Locomotor Activity in Drosophila at JoVE.com

A Simple Technique to Assay Locomotor Activity in Drosophila

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.

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

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