The overall goal of this assay is to measure voluntary ambulation in mouse models of muscular dystrophy. This method can help answer key questions in the muscular dystrophy field about the progression of muscle weakness in mouse models of muscular dystrophy, and the effectiveness of potential therapeutics. The main advantage of this technique is that it provides a non-invasive and reproducible measurement of animal activity that can be easily adapted to many different research settings.
In a quiet temperature controlled room at a consistent time of day, place a camera on a wire mesh above the activity chamber to allow recording across the full arena and clean the chamber with disinfectant. Immediately prior to the activity recording allow the mouse to grip the pole bar of a digital force gauge and gently pull back until the pole bar is released. Record the peak tension in Newtons for each trial.
With one minute of rest between trials. Immediately following the grip strength assay, place the mouse in the chamber and start the video recording, allowing the mouse to explore freely. After six minutes stop the recording, and return the mouse to it's home cage.
For tracking analysis of the video data, first open the recording in the appropriate video editing software, and set the speed to two times. Export the decimated video in mp4 file format for tracking analysis, and open the video in the appropriate video analysis program. To set the calibration for the video recording, use the line tool to draw a line along one side of the chamber.
Right click on the line, select calibrate measure, and enter the actual size of the chamber side in centimeters. To begin semi-automated tracking of the mouse position click on the move cursor. Starting at the initial frame in the video, right click on the point to be tracked.
The tracking point will be marked with a blue circle. Use the right arrow on the keyboard to advance the frame. The tracking point should automatically move based on the position of the tail base.
If the tracking position is not aligned with the point of interest in a given frame, manually align the blue circle to the tracking position. When the entire video has been tracked, save the video and the tracing overlay and select export to spreadsheet to export the positional data of the tracing. Consistent with the results reported from similar assays, dystrophin null mdx mice challenged with an exercise protocol immediately before testing, demonstrate significantly less voluntary movement in a six minute open field test, than do wild type animals.
Indeed, following the mild exertion of a grip strength challenge, the mdx mice typically remained still for the first few minutes of the ambulation protocol, with a modest increase in movement by minutes four to five. When comparing wild type and mdx animals without an exercise challenge however, no differences in the distance traveled are detected. Mdx mice transgenic for utrophin expression are indistinguishable from wild type animals with no significant differences in the total distance traveled, or at any of the commulative time points observed.
To determine the effect of decreasing the sampling rate, the total distance traveled from one minute of full frame rate video was measured, and compared to the distance measurements derived from down sampled versions of the data set. Reducing the frame rate by half decreased the measured distance by 5%of the original distance calculated. The accuracy of the measured distance drops off sharply after this point, although even highly decimated data sets still closely approximate the path of the animal, highlighting the importance of considering the frame rate and signal processing in assessing animal activity, and demonstrating that high spacial resolution can be retained in down sampled video data.
After watching this video you should have a good understanding of how to record and analyze mouse ambulation in an open field setting. Once mastered, this analysis can be completed in 15 to 30 minutes if performed properly. This low cost and non-invasive assay allows researchers to examine motor activity in mouse models of muscular dystrophy, and can serve as an informative first step for further functional measurements.
