The method is based on open source software to systematically evaluate the local motor activities for the tracking behavior of adult and the larvae flies from recorded video. This method requires low cost device integration to record and analyze behavior, making it an affordable and effective approach for screening fly locomotion. This technology is employed to identify behavioral modification in a fly model of human disease with local motion impairment, but it is not suitable for treating disease, disabilities, or difficulties.
It is essential to clear the relevant video window during the analysis process, namely the original and the processing video window. To begin, mix reagent A and reagent B of the silica kit at a ratio of one to 10, according to the manufacturer's instructions, to prepare the open field arena for drosophila locomotion tracking. Next, set the HD camera on a tripod, adjusting it so that the camera lens is perpendicular to the surface of the silica arena.
Then open the video with Fiji. Drag the progress bar to the initial frame and tacitly approve. Choose the whole body of the fly using the freehand selection tool.
Click on image, adjust, and brightness and contrast to adjust the white balance until the gray value of the selected area approaches the broad background. Transfer the anesthetized fly to the open field arena. Once the fly recovers from the anesthesia, put the arena dish with the fly under the camera and shake it quickly from side to side to ensure the fly is in motion when the recording begins.
Press the record button on the camera application to start video recording. Upon completion of the recording, press the stop button to terminate the video recording. Convert the recorded videos into AVI format with M-J pagan coding so they can be opened and analyzed using Fiji.
Also set the frames per second of the video to 15 fps for adult flies and 12 fps for larvae. To open the video with Fiji, select the two options, use virtual stack and convert to gray scale, in the pop-up window. Obtain a processing window by using the set active image tool of the animal tracker plugin, and create a tracking area that circles the arena in the original video window, using the oval tool.
Set the filters and the parameters of the two filters for the first blank frame in the processing window. Then select the next frame in the original video window, and choose the filtered surface of the processing window. Once a filtered processing window is selected, use the set threshold tool to turn the tract fly with a red profile, covered in the processing window.
Then use the set blob detector to let the computer recognize the fly with a red profile, covered in the processing window. Set the last frame for the adult fly or larvae, as described in the text manuscript. Use the show blobs tool to present a tracking rectangle in the original video window.
Start the tracking and export the tracking file after the monitoring is completed. Load the track and zone files using the animal tracker and tracking analyzer plugin. Select the desired index using zone settings, and alter the parameter settings.
Calculate the time of the frame interval using the frame rate. Produce the quantitative analysis charts using spreadsheet software and graph pad prism, after being analyzed in the tracking analyzer. Open the track file.
Copy all coordinates to Microsoft Office Excel and split the cells using the space key. To calculate the immobility time per frame interval, select all calculated results and insert a column chart to visually exhibit the immobility time by the margin of the whole column chart. To illustrate the changes in the angle of direction, select all calculated results and insert a scatter diagram.
Adult flies and third Instar larvae, treated with Rotenone, had significant locomotor deficits compared to those of control flies, fed with solvent DMSO. Quantitative analysis of the Rotenone treatment in adult flies showed a significant decrease in the distance traveled and the mean velocity. And a significant increase in the immobility time.
Representative graphs showed fewer peaks for the movement speed per frame interval in flies fed with Rotenone compared to those in the control, indicating the severity of the locomotor activity deficit. The institution-istic immobility column of moved pixels per frame also showed significantly less movement within one minute for Rotenone fed flies, compared to the control flies. Representative graphs illustrating the moving angle of direction changes in Rotenone fed, and control animals also revealed alterations in the direction chosen by flies.
Similar results were observed for the third Instar larvae. The results revealed that the behavioral movement of tracking larvae fed with Rotenone was significantly impaired compared to the control. The most crucial step in the analysis process is forming the first of blank frame, as it evidence whether the software can check effectively.
This technology could allow researchers to delve deeper into the underlying mechanism of multiple defects, such as nerve or muscle injury.
