Abstract
Abstract
The zebrafish has emerged as a new model system in behavioral neurobiology research. There are numerous assays available to analyze locomotor behavior, but most of these assays require sophisticated equipment and proprietary software. A freely available plugin, wrMTrck, originally developed to analyze locomotor response in Caenorhabditis elegans, has been used in this study to measure locomotion in zebrafish.
T
Concise Workflow
Zebrafish embryos were harvested and raised under standard conditions at 28.5°C. 18 The 4 days post-fertilization larvae were divided into three groups namely control (untreated) group and 5 μg/mL MPTP- and 10 μg/mL MPTP-treated groups.19,20 The MPTP treatment was performed for 24 h at the corresponding concentration.
Camera Setup and Image Acquisition
A 5 dpf experimental larva was placed individually in the 80 × 15 mm Petri dish and allowed to acclimatize for 3–5 min. The Nikon D5300 18–15 mm f/3.5–5.6G video camera was placed in a custom-made locomotor assay chamber (LAC) with the dimensions 32 × 36 × 25 cm. The Petri dish was kept on a laptop screen, which served as a light source (Fig. 1A and Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/zeb). Subsequently, video was captured for 60 s and the video was uploaded onto an Asus Windows 8 (32-bit operating system) computer. The video file was converted into multiple image files (3 frames/sec) using Free Studio v.6.6.28.831. Hence, the 60 s video gives 180 frames or image files. Of these 180 files, first and last 60 image files were omitted from further downstream analysis.
Schematic workflow to analyse locomotor behavior in zebrafish.
Analysis of Image Files
Figure 1A depicts workflow and the results obtained from the analysis of the image files using ImageJ and the plugin wrMTrck. 21
• The image files (.jpg) were imported using the ImageJ File>Import>Image sequence.
• The image sequence was converted into grayscale using Image>Type >8 bit.
• To clear the unwanted background, the region of interest was set using the oval selection tool and the region was cleared using edit>clear outside.
• The gray level was normalized using Plugins>Stack Deflicker.
• To highlight the fry from the stack, Image>Stacks>Z Project was done.
• The image calculator function was used to calculate the difference between two stacks by Process>Image calculator.
• The processed image sequence was converted into binary image sequence using Image>Adjust>Threshold.
• The wrMTrck plugin was run using Plugins>wrMTrck with the following optimized conditions and settings (Supplementary Table S1).
• The wrMTrck output gives the number of objects, number of frames, number of tracks, total length, total distance, average speed, maximum speed, and so on in pixels.
• The length of the Petri dish was measured in pixels using the straight line tool (Analyze>Measure).
• The actual Petri dish was measured using a standard ruler to get the length in millimeters (mm).
• Dividing the actual Petri dish length (in mm) by the Petri dish length in pixels gave the conversion factor.
• By multiplying the wrMTrck output value by this conversion factor gave the results in mm.
• The data obtained were analyzed using GraphPad Prism 7. One-way analysis of variance (ANOVA) and Tukey's post hoc tests were performed.
Conclusion
To validate this method, we treated the zebrafish fry with MPTP and analyzed the locomotor behavior using wrMTrck. Expectedly, the MPTP-treated fry showed locomotor impairment compared with the untreated siblings (Fig. 1B). To substantiate further, the acquired image files were also subjected to ImageJ Macro analysis and the locomotor trajectories were drawn and they were found to be identical to the wrMTrck-derived trajectories 22 (Fig. 1C). We also analyzed the image files using Flote v2.1 software to calculate the differences in the number of swim events in the control and MPTP-treated larvae (Supplementary Fig. S2 and Supplementary Video V1 files). 3
From these results, we conclude that we have successfully optimized the wrMTrck to analyze locomotor behavior in 5 dpf zebrafish larvae. This method can be easily adopted successfully in all laboratory conditions.
Footnotes
Acknowledgment
The authors thank the Department of Biotechnology (DBT), Government of India, for funding.
Disclosure Statement
No competing financial interests exist.
References
Supplementary Material
Please find the following supplemental material available below.
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