Expression of Novel Opsins and Intrinsic Light Responses in the Mammalian Retinal Ganglion Cell Line RGC-5. Presence of OPN5 in the Rat Retina Paula S. Nieto, Diego J. Valdez, Victoria A. Acosta-Rodrı´guez, Mario E. Guido* CIQUIBIC (CONICET)- Departamento de Quı ´mica Biolo ´ gica, Facultad de Ciencias Quı ´micas, Universidad Nacional de Co ´ rdoba, Co ´ rdoba, Argentina Abstract The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca 2+ mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca 2+ levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca 2+ mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell. Citation: Nieto PS, Valdez DJ, Acosta-Rodrı ´guez VA, Guido ME (2011) Expression of Novel Opsins and Intrinsic Light Responses in the Mammalian Retinal Ganglion Cell Line RGC-5. Presence of OPN5 in the Rat Retina. PLoS ONE 6(10): e26417. doi:10.1371/journal.pone.0026417 Editor: Stuart E. Dryer, University of Houston, United States of America Received April 8, 2011; Accepted September 26, 2011; Published October 17, 2011 Copyright: ß 2011 Nieto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Agencia Nacional de Promocio ´ n Cientı ´fica y Te ´cnica (FONCyT, PICT 2004 No 967 and PICT 2006 No 898), Consejo Nacional de Investigaciones Cientı ´ficas y Tecnolo ´ gicas de la Repu ´ blica Argentina (CONICET), Secretarı ´a de Ciencia y Te ´cnologı ´a de la Universidad Nacional de Co ´ rdoba (SeCyT-UNC), Fundacio ´ n Florencio Fiorini and Ministry of Sciences and Technology of Co ´ rdoba. MEG is a fellow of the John Simon Guggenheim Memorial Foundation, 2009. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mguido@fcq.unc.edu.ar Introduction Retinal ganglion cells (RGCs) play a key role in the circadian system of all vertebrates, being responsible for synchronizing central pacemakers to the environmental illumination conditions that coordinate the temporal organization of behavior and physiology. RGCs send visual and photic information to the brain through the axons forming the optic nerve and projecting to areas in the central nervous system involved in image- and non-image-forming tasks [1– 5]. In vertebrates, a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin (Opn4) [6–8] are responsible for transducing information about ambient lighting conditions to brain areas involved in non-image- forming tasks (entrainment of the circadian clock, pupillary light reflexes and suppression of melatonin synthesis) [1,2,7,9–11]. These ipRGCs may have evolved from a common ancestor with rhabdomeric photoreceptors of invertebrates [6,12–19]. Our recent observations support the idea that the phototransduction cascade operating in primary cultures of chicken RGCs is closely related to that taking place in rhabdomeric cells, involving a phosphoinositide cascade [18,20]. In addition, cells of the inner retina may express other photopigments and photoisomerases such as encephalopsin/ panopsin (OPN3), neuropsin (OPN5), peropsin, retinal G protein coupled receptor (RGR), vertebrate ancient (VA) opsin and cone opsins [4,21–28]. Though their exact function in the retina is unknown, these photopigments may cooperate with classical opsins in the process of photon capture and chromophore regeneration, or be directly involved in tasks requiring precise photic responsiveness. A clonal rat retinal cell line named RGC-5 displays RGC characteristics based on expression of specific markers such as Thy-1, Brn-3c, Neuritin, NMDA and GABAb receptors, sensitiv- ity to glutamate excitotoxicity and neurotrophin withdrawal [29– 31]. These cells constitute a widely used model for studying physiological and pathophysiological processes in retinal cells. We recently demonstrated that proliferating RGC-5 cells express different clock genes such as Per1, Clock and Bmal1, while serum stimulation induced the expression of the immediate-early gene (IEG) proteins c-Fos and PER1 [29]. IEGs encode for, among others, the inducible transcription factors of the Fos and Jun families; they are rapid and transiently induced in response to diverse physiological stimuli, acting as cellular messengers in the coupling of extracellular signals to long-term cellular changes [32]. In the retina, light stimulation selectively induces IEG expression in RGCs of mammals and birds [33–37] and in neurons of the central pacemaker, the suprachiasmatic nucleus (SCN) [38–42]. In the present work we investigated the expression of different classical and non-visual opsins and potential intrinsic light responsiveness in RGC-5 cells. To this end, we first examined PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26417