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Eduardo C. Solessio, Yumiko Umino; The Steady-state Pupillary Light Reflex In Unrestrained Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4346.
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The pupil controls the amount of light that enters the eye. Thus, in behavioral studies of visual sensitivity using mice, where the precise level of retinal illuminance is important, the attenuating effects of the pupil must be considered. However, measurements of the steady-state pupillary light reflex (PLR) in restrained or sedated mice can be biased by distress or the mydriatic effects of anesthesia and differ from those measured in freely behaving mice. Here we compare the PLR of mice measured in 3 different conditions: a) sedated with ketamine/xylazine (K+X) anesthesia, b) awake and restrained, and c) unrestrained, moving freely inside an enclosure typically used to assess optomotor sensitivity in rodents.
Dark-adapted C57BL/J6 mice were restrained by hand in a light-tight enclosure. Their pupils were visualized with a dissecting microscope retrofitted with an I-R sensitive digital video camera. Light stimulus was provided by a fiber optics ring light guide or a Ganzfeld connected to a custom-built light source. To measure the pupillary light reflex of unrestrained mice we installed the infrared video camera in one corner of the Optomotry enclosure and proceeded to take photographs of the lateral view of mice in various luminance levels. Pupil areas were measured off-line.
Pupils of mice receiving a full dose of K+X (90 mg/kg of ketamine; 9 mg/kg xylazine) were unresponsive to light, while pupils of sedated mice (half dose of K+X) exhibited very weak responses to light. The PLR in awake, gently restrained mice were robust and consistent, but depended strongly on the method used to deliver light. The pupillary response in a Ganzfeld is more sensitive and extends over a wider response range than the PLR measured when light is delivered with a Ring illuminator (EC50 approximately 9 vs 11 log photons sec-1 cm-2 respectively). Next we measured the steady-state PLR of unrestrained, freely behaving mice. Their pupils constricted progressively with luminance, however their PLR was approximately 10-fold less sensitive than that of restrained mice held under Ganzfeld illumination. We will also discuss the impact of the PLR on our measurements of visual sensitivity in mice.
Steady-state PLRs measured in restrained or sedated mice do not accurately represent the PLR of freely behaving mice.
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