Looming Test

The looming test apparatus was made of acrylic and consisted of a refuge alley (40 × 10 × 30 cm³) connected to a circular open area (diameter, 50 cm; height, 30 cm). The floor was frosted white, the refuge walls were black, and the open area wall was transparent. A trigger area (diameter, 25 cm) was defined by a conceptual concentric ring in the center of the open area. Individual mice were placed in the center of the open area and their movements were recorded for no more than 15 min. Mice were given a 5-min habituation period to explore the apparatus and recover from any handling stress, during which time the trigger was not active. Following this, the looming stimuli trigger was automatically activated and triggered when specific mouse behavioral parameters fulfilled predefined conditions (movement within the trigger area at a speed < 0.15 m/s). The recording ended 2 min after looming stimuli were triggered. Looming stimuli were presented on an overhead LCD monitor (42 inches, refresh rate 60 Hz, AOC) displaying a gray background. The monitor was elevated 460 mm above the floor of the arena. Looming stimuli were generated using MatLab with Psychtoolbox-3. The looming stimulus was a dark disk that expanded from 2° to 40° in 300 ms and maintained this size for 50 ms before disappearing and was repeated 15 times at 30-ms intervals. In the negative control group, no visual stimulus was presented; that is, the trigger was not active.