Zebrafish offer a valuable neural regeneration model because of their ability to recover from spinal cord injury. This method describes swim endurance and swim behavior as readouts of functional spinal cord repair. This technique provides reliable and quantifiable assessment of swim endurance and swim behavior in adult zebrafish.
These methods are equally applicable to studies of neuromuscular and musculoskeletal development, disease, and regeneration. To assess swim endurance, open the flow velocity control software. Click on the box labeled Experiment and uncheck Uswim and Uwater.
Then change the flow speed in the Uwater box on the bottom left to adjust water current velocities. To begin an automated protocol, click on the Start logging box. In the dialogue window that opens, choose Automated from the dropdown list.
To open a previously saved protocol file, click on the file icon next to the protocol file. Next, set up the output file by clicking on the file icon next to the log file. In the file explorer window that opens, name the output file and save it in the desired location.
Then, set up a split lap timer window to ensure simultaneous access to the flow velocity control software and timer windows on the computer screen. Next, set up a fish collection tank to house the exhausted fish after their removal from the swim tunnel. Fill the collection tank and a long polyvinyl chloride tube with zebrafish system water.
Place one end of the prefilled tube in the collection tank and another in the buffer tank, ensuring that water can freely flow from the buffer tank into the collection tank. Clamp the upper end of the tube with a binder clip to prevent water flow, and use the binder clip to control the outflow of water as needed. Close the swim tunnel with the swim endurance lid before placing one group of fish inside the swim tunnel.
To acclimate the fish to the swim tunnel and flow direction, start the split lap timer and adjust the current velocity to zero centimeters per second for the first five minutes, nine centimeters per second for the next five minutes, and 10 centimeters per second for the following five minutes. Following the acclimation, open the protocol and start the automated flow velocity control program, which will increase the water current velocity by two centimeters per second every minute. Monitor the fish for exhaustion.
Exhausted fish will be pushed toward the back end of the swim tunnel. To ensure a fish is exhausted, gently tap the back end of the tunnel or create a shadow over that area to stimulate the fish to swim. Exhausted fish did not respond to the startle stimulus and lay flat at the back end of the tunnel.
When a fish is exhausted, unclamp the fish collection tube. Open the swim tunnel window and collect the fish in the collection tank. Record the time at exhaustion using the split lap timer.
After all the fish are exhausted and collected in the collection tank, click on the Emergency Stop button on the flow velocity control software and stop the timer. To capture movies for the swim behavior assay, place a group of fish in the swim tunnel and close the tunnel using a standard, fully-enclosed lid. Then, open a new recording window and name the file.
Do not click Record yet. Before beginning a new experiment, place a paper towel or piece of fabric on the side of the swim tunnel to ensure all behaviors are due to fish swimming, and not due to a startle response caused by movement in the environment. After ensuring that the water is calm and no ripples are moving across the frame, click on Record in the camera software window to start recording the movie file.
Then click on Start in the flow velocity control software to begin the protocol, which will continue uninterrupted. Watch the movie to ensure that no frames are dropped, there are no bubbles in the field-of-view, and all fish are recorded. Once the movie recording is completed, click on Emergency Stop to end the flow velocity control protocol and check that the data output file is saved automatically.
Then, close the recording window to save the movie file. After the recording, remove the lid. Carefully retrieve the fish and return them to their tank.
To analyze the captured movies, open the tracking_v2. ijm script in Fiji and click on Run to begin the program. In the popup window, choose the folder containing the swim behavior movies to track and click on Open.
Look for frame one of the first movie, a dialogue box, and the region-of-interest, or ROI manager, that will come up. Follow the directions given in the dialogue box and create an ROI at the bottom of the swim tunnel chamber. Then click on OK and ensure that no black corners are seen.
The threshold window will open along with an edited thresholded frame one. Change the color scheme from black and white to red and adjust the max value until frame one only shows the fish in red and nothing else. Record the threshold and click OK in the dialogue box.
For aligning, assembling, and acquire descriptive statistics, open the SwimBehavior_v7-3. R script in RStudio and click on Source in the top right corner of the script section. In a new window that opens, choose the folder containing the _raw.
csv files generated by Fiji and click Open. The program will run automatically. In the popup dialogue box, confirm the number of fish in each movie.
Click on Yes if the given numbers are correct or no if the numbers are incorrect. Once the files are aligned, check for a newly-generated _aligned. csv file.
After ensuring that the program combines the data, runs statistics, and plots output graphs, check for the analysis files generated in a new folder labeled Results within the parent folder containing the _raw. csv and _aligned. csv files.
Swim endurance assessed at two, four, and six weeks post-injury showed a 60%loss of swim endurance capacity at two weeks. The regenerating fish gradually regained swim endurance at four and six weeks post-injury. In the swim behavior assay, controls swam steadily in the front part of the swim tunnel chamber corresponding to an elevated Y position.
In contrast, at two weeks post-injury, injured fish could not maintain steady swim capacity against the current. Consequently, their swim tracks are more irregular, with an overall decrease in Y position. Y position increased at four and six weeks post-injury, indicating that regenerating animals gradually regained their ability to swim.
Furthermore, relative to uninjured controls, lesioned animals at two weeks post-injury were markedly less active, stalled in the rear quadrant of the swim tunnel, and lost their ability to swim against low-current velocities. Consistent with their innate ability to achieve functional recovery, lesioned animals gradually normalized swim behavior parameters at four and six weeks post-injury. The use of the flow velocity control software in this protocol is optional.
The alternative is to manually control the water current motor. The assays described in this study can be used to pre-screen for neural, muscular, or skeletal phenotypes. The tissue of interest can then be harvested for histological or molecular examination.
Our lab and others have used this protocol to identify genes that are required for innate neural repair and factors that are sufficient to enhance neural regeneration.
