2.4. Video and Image Analysis
The tail movement was analyzed using an open-source software, Kinovea (www.kinovea.org, France). The software facilitates dynamic analysis for a variety of applications. After importing a tail movement video recorded at a speed of 160 frames per second (fps) using the camera mounted on the microscope, the tail tip position could be efficiently tracked over the duration of the video. The software output a data file that can be used to calculate the RD and TBF of the movement tracked. Tracking was monitored to ensure that the software was correctly identifying the tail position to ensure accuracy. If required, the tracking position could be manually adjusted using the convenient point tracking tool. The RD value was taken to be the time between the beginning and end of tail movement. The start of movement coincided with the start of electric stimulation with the end of movement varying between fish. The TBF was calculated as the ratio of the number of complete cycles to the RD. Any small flick movement patterns were excluded to keep tail analysis methods consistent with previous methods of behavioral screening in the field[20], [28]. To be counted as a part of a cycle, a tail movement had to pass the designated threshold of \(\pm 0.25mm\) relative to the axial centerline of the TR. A full cycle is composed of four threshold passes.