A standardized method to assess the endogenous activity and the light-response of the retinal clock in mammals

The bioluminescence reporter PER2::Luciferase (PER2::Luc) provides a powerful tool to study the regulation of biological clocks in explant tissues, including the retinal clock. However, the establishment of a standardized procedure to replicate experimental conditions and to enable meaningful comparisons between findings from different studies is still lacking. In addition, different parameters may affect the retinal circadian bioluminescence signal and its dynamic in in vitro assays. In the present study, we first evaluated the effect of sex and age on the main parameters of the mouse retinal clock. We then examined the impact of medium change on PER2::Luc rhythm and compared two light stimulation protocols of the retinal clock.

In a first set of experiments, retinal explants from both male and female Per2 mice of different ages (1 to 8 months) are cultured and the period, phase, amplitude, and rhythmic power of PER2::Luc oscillations are analyzed. In a second set of experiments, we quantified the effect of a medium change done after 4, 6, 8, 9, or 10 days of culture on the phase and period of retinal explants. Finally, we compared the phase shift and the period change resulting from two methods of light stimulations of retinal explants: the first involved the transfer of the cultured tissues from the Lumicycle into a light stimulation chamber, while the second used a light delivery apparatus embedded in the Lumicycle.

We do not observe any sex-dependent effects on the amplitude, period, phase, and rhythmic power of the in vitro retinal PER2::Luc oscillations in animals aged of 2 to 3 months. The most remarkable effect of age is on the amplitude of PER2::Luc oscillations that significantly decrease from 1 to 4-5 months, whereas the endogenous period and rhythmic power increase slightly until 2 to 3 months and then do not change until 8 months. The phase is not affected by age. We then show that a medium change occurring after 4 days of culture does not alter the phase of PER2::Luc rhythm by comparison with day 0, whereas a medium change done after 6, 8, 9, or 10 days in culture advances the phase and lengthens the period. Finally, we observe that the physical displacement of the culture dishes containing retinal explants, even in complete darkness, induces a strong phase shift of PER2::Luc oscillations.

Our work shows that the retina cultures are particularly sensitive to some aspects of the culture procedure, and it provides an accurate standard protocol to avoid biases due to artifactually induced phase shifts resulting from the medium change or physical displacement.