In vivo and in vitro Development of S- and M-cones in Rat Retina

Abstract

Abstract: : Purpose: The factors that control opsin expression and the development of cone types are largely unknown. Since organotypic cultures are well suitable for studying putative regulatory factors under defined experimental conditions, we investigated the developmental regulation of short and middle-wavelength sensitive (SWS and MWS) opsin expression in an organ culture of the rat retina and compared it to cone development in vivo. Methods: Organotypic retinal cultures were prepared using postnatal days (P) 0-2 retinas as described elsewhere (Pinzón-Duarte et al. 2000, Vis. Res. 40), and allowed to develop for 5 to 15 days in vitro. For the in vivo studies, retinas of pigmented rats were collected between P0 and P120. Retinas were immunostained using specific opsin antibodies for S- and M-cones (JH455, JH492, respectively; gift from Dr. J. Nathans). Morphometric analyses were performed on retinal whole mounts and radial sections using confocal microscopy. Results: In vivo, S-cones were already present at birth whereas M-cones were only detected after P4. S-cones reached their maximal density at P10 (∼14600 cones/mm₂), M-cones at P12 (∼12500 cones/mm₂). Thereafter, the number of cones decreased considerably (P60: 980 S-cones/mm₂ ; 6450 M-cones/mm₂). In contrast to former in vitro studies where only S-cone expression has been reported, both SWS- and MWS-opsins were present in our retinal culture system after 5 days in vitro. However, numbers of labeled cones in general were below the in vivo values. The distribution of SWS- and MWS-opsin within the cones was the same in vivo and in vitro. Up to P12, opsin labeling was found over the entire cone surface whereas in later stages, cone opsins were more restricted to the inner and outer segments. Conclusion: Our results show that it is possible to express both SWS and MWS opsins in a rat organotypic retinal culture. Our culture model thus provides a powerful tool for the identification of factors regulating opsin expression and cone patterning.

Keywords: 517 photoreceptors • 560 retinal culture • 564 retinal development