Clocks in the choroid: Localization of PER3 in chicks and humans

Abstract

Purpose : Besides its circulatory role, the choroid is a mediator between vision-induced retinal signals and scleral growth. Diurnal changes in choroidal thickness are a fundamental component in refractive development and also may reflect changes in blood flow. Diurnal thickness changes occur in humans and animals and could be regulated at least in part by an internal clock. Recently, the mRNAs of four clock genes were found to undergo diurnal oscillations in chick choroid (Stone et al., ARVO 2018). We here search for the morphological basis of one of these genes, Per3.

Methods : Choroids of 12-week old chickens were prepared for immunohistochemistry at the time point of highest PER3-mRNA expression (8 am; Stone et al., ARVO 2018) maximize the likelihood of detection. Combined antisera to PER3 (anti-human) and VIP, PGP9.5, and neurofilaments (160/200) were applied, followed by confocal laser scanning microscopy. As a control in chicken, tissues with known clock-activity were studied: hypophysis, cortex, thyroid gland. Additionally, human choroids were screened for PER3-immunoreactivity at similar time point as in animal experiments (from the cornea bank Dept. Ophthalmology, Paracelsus Medical University and meeting the Declaration of Helsinki).

Results : In chicken, PER3-immunoreactivity (PER3-IR) was clearly identified in endocrine cells of the hypophysis and thyroid gland as well as in cortical neurons. In choroid, PER3-IR revealed numerous small round cells of 10 µm diameter, often arranged in clusters within the choroidal stroma. These cells were not co-localized with VIP. Other spindle shaped cells co-expressed VIP/PER3-IR and were therefore identified as intrinsic choroidal neurons (ICNs). In human choroid, numerous small and round to ovoid cells were detected displaying PER3-IR, and these were often closely related to neuronal elements, as seen with the pan-neuronal marker PGP9.5. Intrinsic choroidal neurons as detected with neurofilaments displayed co-localization with PER3.

Conclusions : The immunohistochemical detection of PER3 in distinct cell types including ICNs, along with the circadian rhythm in PER3 mRNA, implies the existence of endogenous choroidal clocks. Future studies are necessary to classify the small PER3+ cells, and to determine the functions driven by these endogenous clocks.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.