Circadian Pacemakers and the Cone Paraboloid

Abstract

Abstract: : Purpose: Retinal photoreceptors are known to possess circadian oscillators. The present study aimed to characterize the dynamic structural rearrangements of organelles within cone photoreceptors in lizard retinas as a function of circadian rhythm and to identify potential pacemaker structures. Methods: Lizards (Anolis carolinensis, adult) were maintained with cyclic 12 hour light/dark (LD) cycles for at least 15 days in ventilated chambers equipped with an externally controlled light source. Eyes were harvested at 6–hour intervals in cohorts of mice (n = 4) using the time of light onset as the "zero" timepoint (zeitgeber time (ZT) = 0) and were prepared for frozen or plastic sections. An additional group of animals was placed in constant light (LL) for an additional 15 days and eyes were similarly harvested. The circadian morphological dynamics of photoreceptor organelles and of melatonin immunoreactivity were evaluated by histological and immunohistochemical methods. Periodic Acid Schiff’s (PAS) staining was performed to evaluate presence of glycogen. In vitro reverse hemolytic plaque assay (RHPA) was employed to detect circadian alterations in melatonin secretion from individual cone photoreceptors. Results: PAS staining showed that the cone paraboloid was detectable only at ZT12 under LD cycles or at ZT0, the opposite timepoint, under LL conditions. Photoreceptor melatonin immunoreactivity was localized within paraboloids, showing strong intensity at midnight (ZT18) and weak intensity at midday (ZT6). RHPA assay showed that individual photoreceptor cones can act as circadian oscillators when isolated in vitro. Conclusions: Paraboloids appear to be circadian oscillators in lizard cone photoreceptors. Cones undergo rhythmic structural circadian changes and these changes can be shifted by altering the LD cycle. Further, isolated cones cultured in vitro can rhythmically produce melatonin in a circadian fashion.

Keywords: circadian rhythms • photoreceptors • microscopy: light/fluorescence/immunohistochemistry