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While rods, cones, and intrinsically photosensitive melanopsin-containing ganglion cells (ipRGCs) all drive light entrainment of the master circadian pacemaker of the suprachiasmatic nucleus, recent studies have proposed that entrainment of the mouse retinal clock is exclusively mediated by a UV-sensitive photopigment, neuropsin (OPN5). Here, we report that the retinal circadian clock can be phase shifted by short duration and relatively low-irradiance monochromatic light in the visible part of the spectrum, up to 520 nm. Phase shifts exhibit a classical photon dose-response curve. Comparing the response of mouse models that specifically lack middle-wavelength (MW) cones, melanopsin, and/or rods, we found that only the absence of rods prevented light-induced phase shifts of the retinal clock, whereas light-induced phase shifts of locomotor activity are normal. In a “rod-only” mouse model, phase shifting response of the retinal clock to light is conserved. At shorter UV wavelengths, our results also reveal additional recruitment of short-wavelength (SW) cones and/or OPN5. These findings suggest a primary role of rod photoreceptors in the light response of the retinal clock in mammals., Author summary The mammalian retina contains a circadian clock that plays a crucial role in adapting retinal physiology and visual function to light/dark changes. In addition, the retina coordinates rhythmic behavior and physiology by providing visual input to the master hypothalamic clock in the suprachiasmatic nucleus through a network of retinal photoreceptor cells involving rods, cones, and intrinsically photosensitive melanopsin-containing ganglion cells (ipRGCs). In contrast, recent studies argue that none of these photoreceptors are involved in light responses of the retinal clock and propose that photoresponses are exclusively mediated by the UV-sensitive photopigment neuropsin (OPN5). Our study demonstrates that rods are required to phase shift the retinal clock, while melanopsin and middle-wavelength (MW) cones influence the intrinsic period of the clock.

The mammalian retina contains an endogenous pacemaker regulating retinal physiology and participate to the sybchronization of the temporal phase of the central clock of the SCN to environmental time. This entrainment process involves rods, cones and melanopsin-containing retinal ganglion cells. In contrast with the SCN, the role of these photoreceptors in the light response of the retinal clock is still controversial. While recent studies suggest that none of them is involved in light response of the retinal clock, others support a role for melanopsin. My project aims to dissect the role of these different photoreceptors in wild-type, Per2Luc and/or photoreceptor-deficient mice using in vitro/ex vivo approaches. Monochromatic lights targeting different photoreceptors were applied to retinal explants of Per2Luc or melanopsin-, MW cones- or rods-deficient mice. Our results demonstrate that rods are required for the light-induced phase shift of the retinal clock in the visible spectrum and suggest an additional contribution of SW cones and/or neuropsin in the UV. As the retinal clock is composed of several clocks and in order to determine their response to light, the photic induction of Per1-Per2 and C-Fos genes was analyzed in isolated retinal layers from wild-type and photoreceptor-deficient mice. In mice without melanopsin or MW cones, Per1-Per2 induction by light is abolished in all layers, suggesting a role for these photoreceptors. In summary, our results propose a differential contribution of the retinal photoreceptors as a function of the response recorded (phase shift of PER2::Luc or induction of clock genes by light), La rétine contient une horloge endogène régulant différentes fonctions rythmiques et participe à la synchronisation de l'horloge centrale du SCN au temps géophysique. Cette synchronisation met en jeu les bâtonnets, les cônes et les cellules à melanopsine. Contrairement au SCN, le rôle des photorécepteurs dans la réponse à la lumière de l'horloge rétinienne est controversé. Nos travaux et ceux d'autres équipes soutiennent un rôle de la melanopsine alors que 2 études récentes suggèrent que seule la neuropsine est impliquée. Mon projet vise à disséquer le rôle des différents photorécepteurs par des approches in vitro/ex vivo chez des souris sauvages, Per2Luc et/ou déficientes en photorécepteurs. Des lumières monochromatiques ciblant différents photorécepteurs ont été appliquées à des explants rétiniens en culture de souris Per2Luc ou déficientes en melanopsine, cônes MW ou bâtonnets. Nos résultats montrent un rôle des bâtonnets dans le décalage de phase de l'horloge rétinienne par la lumière dans le spectre visible et une contribution additionnelle des cônes SW et/ou de la neuropsine dans l'UV. L'horloge rétinienne étant composée de plusieurs horloges et afin de déterminer leur réponse à la lumière, l'induction photique des gènes Per1-Per2 et C-Fos a été analysée dans les couches rétiniennes isolées chez des souris sauvages et déficientes en photorécepteurs. Chez les souris sans melanopsine ou cônes MW, l'induction de Per1-Per2 est abolie dans toutes les couches, suggérant un rôle de ces photorécepteurs. En résumé, nos résultats indiquent un rôle différentiel des photorécepteurs en fonction de la réponse mesurée (décalage de phase de PER2::Luc et induction des gènes de l'horloge par la lumière)

Marie-Paule Felder-Schmittbuhl,1,* Hugo Calligaro,2 Ouria Dkhissi-Benyahya2,* 1Institute of Cellular and Integratives Neurosciences, UPR3212, CNRS, Université de Strasbourg, Strasbourg, 2University of Lyon, Stem Cell and Brain Research Institute, INSERM U1208, Bron, France *These authors contributed equally to this work Abstract: The mammalian retina contains an extraordinary diversity of cell types that are highly organized into precise circuits to perceive and process visual information in a dynamic manner and transmit it to the brain. Above this builds up another level of complex dynamic, orchestrated by a circadian clock located within the retina, which allows retinal physiology, and hence visual function, to adapt to daily changes in light intensity. The mammalian retina is a remarkable model of circadian clock because it harbors photoreception, self-sustained oscillator function, and physiological outputs within the same tissue. However, the location of the retinal clock in mammals has been a matter of long debate. Current data have shown that clock properties are widely distributed among retinal cells and that the retina is composed of a network of circadian clocks located within distinct cellular layers. Nevertheless, the identity of the major pacemaker, if any, still warrants identification. In addition, the retina coordinates rhythmic behavior by providing visual input to the master hypothalamic circadian clock in the suprachiasmatic nuclei (SCN). This light entrainment of the SCN to the light/dark cycle involves a network of retinal photoreceptor cells: rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs). Although it was considered that these photoreceptors synchronized both retinal and SCN clocks, new data challenge this view, suggesting that none of these photoreceptors is involved in photic entrainment of the retinal clock. Because circadian organization is a ubiquitous feature of the retina and controls fundamental processes, the coherence from cell to tissue is critical for circadian functions, and disruption of retinal clock organization or its response to light can potentially have a major impact on retinal pathophysiology and vision. Keywords: retina, clock gene, circadian, ipRGC, photoreceptor, light