The overall goal of this procedure is to assess muscle performance and function in zebrafish using touch-evoked response and swimming assays. This method can help answer questions in the field of neuro-muscular research such as identifying impaired muscle performance or neurodegenerative defects in zebrafish diseased models. The main advantage of this technique is that it provides an automated high throughput method to assess muscle performance in zebrafish diseased models.
To perform the assay, place a petri dish filled with approximately 25 milliliters of embryo medium onto an illuminated, temperature controlled stage set to approximately 28 degrees Celsius. Mount the high speed camera above the dish. Launch the video recording software and set the capture speed to 1, 000 frames per second to ensure fast swimming speeds can be captured.
Position the embryo in the center of the petri dish clearly visible in the field of view. Press Record and then deliver a mechanosensory stimulus by gently touching the embryo with a blunt needle on top of the head. Stop the recording once the embryo has swam out of the field of view or returned to rest.
Repeat the testing of the same larvae mainly to habituation or promote muscle weakness in some diseased models resulting in reduced response to the tactile stimulus, therefore, each embryo should only be tested once. To quantify the swimming behavior, launch the software and select the single larvae locomotion with our Background Subtraction Module to open the AVI video file. Select the freehand or polygon tool from the Menu bar and select the region of the movie that encompasses both the original position of the fish and the area to which it swims whilst excluding the probe used to deliver the mechanosensory stimulus.
Click on Experiment in the Menu bar and select Execute. At the prompt, save the raw data analysis file in a PHR format to the desired location. Next, click Start to begin the analysis.
Once the fish has swam out of the field of view or the video clip has ended, conclude the analysis by clicking Stop under the Experiment Menu and a window displaying the results will appear. Scroll to the right of the window to obtain the maximum acceleration value. Export the data by clicking the Export Instantaneous Results button under the Results dropdown menu.
Select the appropriate Raw Data Analysis file and click to open. This will save a text file in the destination folder that can be opened in a spreadsheet program. Place the test larvae into a 48 well plate, one per well.
Next, fill the wells with water to just below the top of the well ensuring there are no bubbles. Store the plates at 28 degrees Celsius for one hour. Place the plate into a recording chamber equipped with an infrared digital camera that can detect larvae in the dark.
Circadian rhythms and external environmental stimuli may significantly affect zebrafish swimming behavior. The time of day and lighting conditions needs to be standardized and water temperature needs to be tightly regulated. Launch the software and select the Tracking Module.
Under File, click on Generate New Protocol and edit the number of wells used for the experiment which in this example is 48. Next, click on Parameters and select Protocol Parameters then Time to set the experiment duration and integration period to 10 minutes. Also in Protocol Parameters, click Options and ensure the numeroscope check box is selected.
To set the recording areas, highlight the entire grid and double click on one of the wells. Click on the Draw Areas button and draw around the top left, top right and bottom left wells and click Build. The software will then determine the position of each well.
At this point, also draw in the scale bar and click on Apply to Group. Once completed, click on the Draw Areas button. Next, select the color of the fish which in this case is black and slide the Detection Threshold bar to a level where only the fish movements are highlighted with no background.
Enter Movement Thresholds for detection of inactivity and small, enlarged movements. In this example, in an inactivity threshold of six millimeters per second and an activity burst threshold of 30 millimeters per second was used. Click on the Parameters Menu then Light Driving settings and set the light intensity of the chamber to be at 0%Close the recording chamber door and begin video recording.
The experiment will be completed in 10 minutes as indicated by the timer on the screen. Once completed, click the Experiment drop down Menu and select Stop. A dialog box with the results will be displayed.
To examine the results using Excel, click on Open Containing folder and open the file that appears in the resulting folder. Finally, the video can be replayed to review whether the locomotion values recorded accurately depict the swimming movements of the fish. This can be achieved by comparing the movement observed in the video file to the locomotion profile generated by the software.
Snapshot images of a zebrafish embryo taken during a touch-evoked assay show the typical movement of an individual over the first 0.2 seconds after application of the stimulus. Here, the figure shows an acceleration profile for the first 0.2 seconds of the burst swimming escape response in wild type versus myopathic individuals. Acceleration was seen to peak in both strains in this time window and peak maximum acceleration is proportional to the force generating capacity of the skeletal muscle.
Maximum acceleration values were averaged for the wild type and myopathic strains. The myopathic fish showed a significant decrease in maximum acceleration indicating reduced muscle function. 10 minute locomotion assays of embryos recorded the movement patterns and types of both wild type and myopathic embryos and generated diagrammatic representations of swimming movements.
Periods of slow movement represented by green lines and fast movement represented by red lines were mapped as well as periods of inactivity represented by black lines. Wild type individuals showed high activity levels with relatively no inactive periods in contrast to the myopathic individuals which were less active over the test period. This was reflected in significant differences in the average numbers of movements and distance traveled by the wild type versus myopathic fish.
Once mastered, the touch-evoked response assay can be performed in 15 minutes for 15 fish and the locomotion assays can be completed in approximately 10 minutes for up to 48 fish. While attempting this procedure, it is important to carefully handle embryos as this may affect their activity. Following this procedure, other techniques such as immunolabeling or electron microscopy can be performed to answer additional questions related to muscle pathology.
After watching this video, you should have a good understanding of how to measure muscle performance in early zebrafish development.
