Pupillary Light Reflex Measurement Protocol

Pupillary light reflex (PLR) was conducted as previously described [21] (link), [22] (link). Mice were entrained to a 24 h LD cycle then dark-adapted for 1 to 1.5 hours prior to the beginning of the experiment. Measurements were taken between ZT5 and ZT8 (where ZT12 =  time lights off). Pupillary responses were elicited by applying a light stimulus (200 µW/cm²) provided by Xenon arc lamp (Cairn Research Ltd.) filtered with a 480 nm monochromatic interference filter (half bandwidth ≤10 nm) and transmitted to the integrating sphere using a quartz fibre optic. Consensual pupil constriction could then be monitored in the left eye by illuminating it with infrared (>900 nm) and recording it with a CCD camera fitted with a 140 mm lens in a parallel plane to the cornea. The eye was recorded for 2 s with no light stimulus, then for a further 58 s as the light stimulus was applied. Pupil area was measured in images captured from the videotaped records using VirtualDub (web-based software package, VirtualDub.org) and Matlab R2008a (The Mathworks, Cambridge, UK).