Melatonin Entrains PER2::LUC Bioluminescence Circadian Rhythm in the Mouse Cornea

Abstract

Purpose: Previous studies have reported the presence of a circadian rhythm in PERIOD2::LUCIFERASE (PER2::LUC) bioluminescence in mouse photoreceptors, retina, RPE, and cornea. Melatonin (MLT) modulates many physiological functions in the eye and it is believed to be one of the key circadian signals within the eye. The aim of the present study was to investigate the regulation of the PER2::LUC circadian rhythm in mouse cornea and to determine the role played by MLT.

Methods: Corneas were obtained from PER2::LUC mice and cultured to measure bioluminescence rhythmicity in isolated tissue using a Lumicycle or CCD camera. To determine the time-dependent resetting of the corneal circadian clocks in response to MLT or IIK7 (a melatonin type 2 receptor, MT2, agonist) was added to the cultured corneas at different times of the day. We also defined the location of the MT2 receptor within different corneal layers using immunohistochemistry.

Results: A long-lasting bioluminescence rhythm was recorded from cultured PER2::LUC cornea and PER2::LUC signal was localized to the corneal epithelium and endothelium. MLT administration in the early night delayed the cornea rhythm, whereas administration of MLT at late night to early morning advanced the cornea rhythm. Treatment with IIK7 mimicked the MLT phase-shifting effect. Consistent with these results, MT2 immunoreactivity was localized to the corneal epithelium and endothelium.

Conclusions: Our work demonstrates that MLT entrains the PER2::LUC bioluminescence rhythm in the cornea. Our data indicate that the cornea may represent a model to study the molecular mechanisms by which MLT affects the circadian clock.

Figure 1

The PER2::LUC bioluminescence rhythms in mouse cornea. Bioluminescence obtained from a cultured PER2::LUC mouse cornea was measured with a Lumicycle for 10 days (A). Rhythmic PER2::LUC bioluminescence was observed for more than 80 days when cultures received weekly medium exchange (B). Arrows indicate the time of medium exchanges.

Figure 2

The localization of PER2::LUC to the cornea layers. Cornea obtained from PER2::LUC mice was immunostained with anti-firefly luciferase antibody (A). Specific staining was only observed in epithelium and endothelium layers of cornea (A). Control (i.e., tissue incubated without the primary antibody) did not show any immunoreactivity (B). Images of PER2::LUC bioluminescence obtained with the CCD camera (C). Circadian bioluminescence rhythms in epithelium (D1) and endothelium (D2) were recorded for 3 consecutive days.

Figure 3

Phase response curve of PER2::LUC bioluminescence rhythm to MLT administration. Representative examples of PER2::LUC bioluminescence rhythms in response to vehicle (A) and MLT (B). The arrows indicate when the Vehicle (Veh) or MLT was added to the culture dishes respectively. The value of the phase-shift for each individual cornea rhythm was plotted to create a PRC (C). White circles indicate cultures treated with Veh and black squares indicate culture treated with MLT. Data were divided into six bins at 4-hour intervals for statistical analysis (D) (Tukey test, *P < 0.05). n = 5 to 8 for each bin.

Figure 4

Phase response curve of PER2::LUC bioluminescence rhythm to IIK7 administration: 1 nM of IIK7 induced a significant phase-shift at CT 12 to 16 and CT 20 to 24 (A); 1 μM of IIK7 also induced significant phase-shift at CT 0 to 4 and CT 12 to 24 (B). White circles indicate cultures treated with Veh and black squares indicate culture treated with IIK7. Data were divided to six bins at 4-hour intervals for statistical analysis ([C] for 1 nM and [D] for 1 μM) (Tukey test, *P < 0.05). n = 7 to 17 for each bin.

Figure 5

Immunoreactivity of MT₂ receptor in the cornea. Immunoreactivity of MT₂ was localized to the corneal epithelium and endothelium (A). Control (i.e., tissue incubated with the control peptide antigen) did not show any immunoreactivity (B).