Pupillometer

A pupillometer (A-2000 Pupillometer; Neuroptics, Irvine, CA, USA) was used to measure pupillary responses to light stimuli. This system uses two cameras and two independently controlled light sources that allow for independent stimulation and pupil tracking. The mice were dark adapted for 4 hours prior to testing. They were anesthetized with ketamine and xylazine (50 mg/kg and 5 mg/kg body weight, respectively). The light profile consisted of two red-light stimuli (2.0 log W/m²) for 1 second; two ultra-low white-light stimuli (–2.5 log W/m²), two low white-light stimuli (–1.5 log W/m²), two medium white-light stimuli (–0.5 log W/m²), and two high white-light stimuli (0.5 log W/m²), each 0.1 second long; and, finally, a blue-light stimulus (–2.0 log W/m²) for 1 second. Each stimulus had a weighted delay time to allow the pupil size to recover. After each red-light stimulus there was a 60-second delay; after each ultra-low white-light stimulus, a 19.9-second delay; after each low white-light stimulus, a 29.9-second delay; after each medium white-light stimulus a 39.9-second delay; and after the high white-light stimulus, a 49.9-second delay. The maximum size of the pupil after dark adaptation was quantified at about 2 minutes after anesthesia.