Head-fixed pupillometry was recorded as described by Gao et al 2020 (link) (Gao et al., 2020 (link)). Briefly, mice were first acclimated to a custom-built head fixation device for 5 days, 30 min per day. A monochromatic CMOS camera equipped with a macro zoom lens (MVL7000, ThorLabs) and an infrared LED lamp (LIU850A, ThorLabs) were used to collect images from the left pupil at 5 frames per second. The start of camera recording and the delivery of mild aversive stimulus were triggered by TTL signal controlled by Clampex. 5 consecutive air puffs (100 ms, 60 psi) or tail shocks (1s, ~250 μA) were started 5 min after the baseline recording, with the interval between stimulation range from 30-60s. The 29G tubing for air puff was placed 2mm under the tip of the snout so that the air puff won’t cause eye blink of the mouse. The recorded videos were subsequently analyzed using Bonsai 2.4, and changes in pupil size (area) were plotted.
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