Microfluorometric Measurement of the Formation of All-Trans-Retinol in the Outer Segments of Single Isolated Vertebrate Photoreceptors

The first step in the detection of light by vertebrate photoreceptors is the photoisomerization of the retinyl chromophore of their visual pigment from 11-cis to the all-trans configuration. This initial reaction leads not only to an activated form of the visual pigment, meta II, that initiates reactions of the visual transduction cascade but also to the photochemical destruction of the visual pigment. By a series of reactions termed the visual cycle, native visual pigment is regenerated. These coordinated reactions take place in the photoreceptors themselves as well as the adjacent pigment epithelium and M�ller cells. The critical initial steps in the visual cycle are the release of all-trans- retinal from the photoactivated pigment and its reduction to all-trans- retinol. The goal of this monograph is to describe methods of fluorescence imaging that allow the measurement of changes in the concentration of all-trans- retinol as it is reduced from all-trans- retinal in isolated intact salamander and mouse photoreceptors. The kinetics of all-trans- retinol formation depend on cellular factors that include the visual pigment and photoreceptor cell type, as well as the cytoarchitecture of outer segments. In general, all-trans- retinol forms much faster in cone cells than in rods.