Anti-OPN4 polyclonal antibody

Purpose Intraocular pressure (IOP) is known to have a strong circadian rhythm, yet how light/dark cycles entrain this rhythm is unknown. The purpose of this study was to assess whether, like the retina, the mammalian ciliary body and IOP clocks have an intrinsic ability to entrain to light/dark cycles. Methods Iris-ciliary body complexes were obtained from period2:luciferase (PER2::LUC) mice and cultured to measure bioluminescence rhythmicity. Pairs of the iris-ciliary body complex were exposed to antiphasic 9:15 h light/dark cycle in vitro. After 4 days of exposure to light/dark cycles, bioluminescence was recorded to establish their circadian phases. In addition, pairs of the iris-ciliary body complex co-cultured with the retinas or corneas of wild-type mice were also investigated. The IOP circadian changes of free-running Opn4(-/-); rd1/rd1 mice whose behavior was antiphasic to wild-type were measured by a rebound tonometry, and compared with wild-type mice. Opn3, Opn4, and Opn5 mRNA expression in the iris-ciliary body were analyzed using RT-PCR. Results The iris/ciliary body complex expressed Opn3, Opn4, and Opn5 mRNA; however, unlike in retina and cornea, neither the iris-CB complex nor the co-cultured complex was directly entrained by light-dark cycle in vitro. The diurnal IOP change of Opn4(-/-); rd1/rd1 mice showed an antiphasic pattern to wild-type mice and their rhythms followed the whole-animal behavioral rhythm. Conclusions Despite expressing mRNA for several non-visual opsins, circadian rhythms of the iris-ciliary body complex of mice do not entrain directly to light-dark cycles ex vivo. Unlike retina, the iris/ciliary body clocks of blind mice remain synchronized to the organismal behavioral rhythm rather than local light-dark cycles. These results suggest that IOP rhythm entrainment is mediated by a systemic rather than local signal in mice.

PURPOSE. The purpose of this study was to evaluate a chromatic pupillometry protocol for specific functional assessment of rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) in dogs. METHODS. Chromatic pupillometry was tested and compared in 37 dogs in different stages of primary loss of rod, cone, and combined rod/cone and optic nerve function, and in 5 wildtype (WT) dogs. Eyes were stimulated with 1-s flashes of dim (1 cd/m(2)) and bright (400 cd/m(2)) blue light (for scotopic conditions) or bright red (400 cd/m(2)) light with 25-cd/m(2) blue background (for photopic conditions). Canine retinal melanopsin/Opn4 was cloned, and its expression was evaluated using real-time quantitative reverse transcription-PCR and immunohistochemistry. RESULTS. Mean+/-SD percentage of pupil constriction amplitudes induced by scotopic dim blue (scDB), scotopic bright blue (scBB), and photopic bright red (phBR) lights in WT dogs were 21.3%+/-10.6%, 50.0%+/-17.5%, and 19.4%+/-7.4%, respectively. Melanopsin-mediated responses to scBB persisted for several minutes (7.7+/-4.6 min) after stimulus offset. In dogs with inherited retinal degeneration, loss of rod function resulted in absent scDB responses, followed by decreased phBR responses with disease progression and loss of cone function. Primary loss of cone function abolished phBR responses but preserved those responses to blue light (scDB and scBB). Although melanopsin/Opn4 expression was diminished with retinal degeneration, melanopsin-expressing ipRGCs were identified for the first time in both WT and degenerated canine retinas. CONCLUSIONS. Pupil responses elicited by light stimuli of different colors and intensities allowed differential functional assessment of canine rods, cones, and ipRGCs. Chromatic pupillometry offers an effective tool for diagnosing retinal and optic nerve diseases.