Our protocol is a vision test for mice. It is both robust and easy to replicate. While many mouse vision tests are available, the looming experiment is one of a few methods to test the image forming pathway, which is not a visual reflex pathway.
While many other visual behavior assay require extensive training in animal surgery, the looming technique requires little experimental training. Begin by constructing a rectangular open lid enclosure using aluminum framing and PVC panels to house the mouse during looming visual stimuli presentation. Lay a sheet of paper to cover the entire floor of the enclosure to ensure easy cleanup between trials.
Then, add an opaque shelter in a corner of the enclosure with an entrance facing the center of the arena for easy entrance and exit. Secure a camera with a wide-angle lens to a table mounted stand adjacent to the enclosure for capturing the mouse's behavior. Next, set up a computer monitor on top of the enclosure.
Prepare a looming pattern using experimental software to code for an expanding black circle. Set the stimulus to begin at the visual angle of two degrees and expand to 50 degrees over 250 milliseconds and set the code to repeat the stimulus 10 times with an interval of one second. Begin by selecting a mouse for the experiment as well as three blind mice to ensure response to looming stimulus was truly a visually guided behavior.
Place the healthy mouse in the enclosure and let it freely explore its surroundings for seven to 15 minutes prior to stimulus onset. 10 seconds prior to the stimulation, start the video capture on the camera. Once the mouse is away from the shelter and moving freely in the open arena, start the looming visual stimuli.
Finally, transfer the mouse back to its original cage. Clean the enclosure for the next mouse by spraying the walls and refuge with 70%ethanol and wiping it down and replace the paper floor liner, if soiled. Reposition the refuge to the same initial location if moved during animal transfer and enclosure cleaning.
At the computer, save the video clip for each mouse in AVI format without file compression in order to ensure no data loss during transfer to the analysis software. Then, use analysis software to track the animal's motion around the arena prior to, during, and after stimulus presentation. Calculate the velocity and the distance of the mouse from the refuge.
If the image of the arena is distorted due to the video angle, correct the x and y coordination prior to the calculation. Finally, compare the parameters before and after looming stimulus onset to determine how the mouse responded to the stimuli. Whether by freezing, fleeing, or demonstrating no change in behavior.
Captured video clips were analyzed using a commercial analytics software with a manual tracking function. Using the tracking feature, the mouse's position was identified in each frame of the video before, during, and after the looming stimuli. When flight occurred, the velocity abruptly increased and the distance to the shelter reduced accordingly.
In contrast, the velocity was near zero millimeters per second when mice froze. The velocity was reduced compared to the control before looming and the distance to the dome did not change during this period. It is important to remember to get a baseline of movement prior to the stimulus onset or a quantitative reaction to the stimulus cannot be determined.
It is important to conduct multiple tests for accurate vision evaluation. Pupil light reflex is one commonly used method, which ensures that the mouse has healthy eyes. The mechanism of looming response is not fully understood.
This technique may provide opportunities to explore mechanisms of moving object recognition and fear responses for neuroscientists.
