# Circadian Rhythms in Murine Ocular Tissues Including Sclera Are Affected by Neurobasal A Medium Preincubation and Mouse Strain, but Not Sex

## TL;DR

This study shows that mouse strain and culturing conditions affect circadian rhythms in eye tissues, including the sclera, but not sex.

## Contribution

The discovery of a novel circadian oscillator in the sclera and the effect of Neurobasal A medium and mouse strain on ocular rhythms.

## Key findings

- Scraped posterior eye cups (PECs) exhibit circadian rhythms, indicating a novel oscillator in the sclera.
- RjOrl:SWISS mice show higher amplitude rhythms in ocular tissues compared to C57BL/6J mice.
- Neurobasal A medium preincubation alters rhythms in ocular tissues, but no sex differences were observed.

## Abstract

Our understanding of ocular clocks has been profoundly advanced by the development of real-time recording of bioluminescence of PER2-luciferase (PER2::LUC) knock-in mouse explants. However, the effect of sex, mouse strain, and culturing conditions on ocular clocks remains unknown. Here, we studied the role these variables play on PER2::LUC bioluminescence rhythms of ocular tissues: retinas, corneas, and posterior eye cups (PECs). We also tested the hypothesis that the sclera contains a circadian oscillator by using scraped PECs as a proxy.

Retinas, corneas, and intact and scraped PECs were obtained from male and female PER2::LUC knock-in mice maintained on either a pigmented C57BL/6J or albino RjOrl:SWISS background. PER2::LUC bioluminescence rhythms in ocular tissues were measured using a Lumicycle.

We compared PER2::LUC bioluminescence rhythms between ocular tissues and found that all ocular tissues oscillated, including the scraped PECs, which were previously not known to oscillate. The rhythms in scraped PECs had lower amplitudes, longer periods, and distinct acrophases compared with other ocular tissues. Immunolabeling revealed the presence of the protein product of the clock gene BMAL1 in the sclera. Ocular tissues of RjOrl:SWISS mice oscillated with higher amplitudes compared with the ones of C57BL/6J, with corneal rhythms being most affected by mouse strain. A 24-hour preincubation with Neurobasal A medium affected rhythms of ocular tissues, whereas sex differences were not detected for these rhythms.

We discovered a novel oscillator in the sclera. PER2::LUC bioluminescence rhythms in murine ocular tissues are affected by Neurobasal A medium preincubation mouse strain but not sex.