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M. E. Estevez, V. J. Kefalov, M. C. Cornwall; Microspectrophotometric Measurements of Cone Pigment Regeneration Independent of the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3528.
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In rod photoreceptors the regeneration of visual pigment after bleaching (the visual cycle) requires the participation of the retinal pigment epithelium (RPE) which recycles chromophore from all-trans retinol into 11-cis retinal. However, recent biochemical studies have suggested a novel visual cycle for cones that is located entirely within the retina and dependent on Müller cells. To address this possibility we directly measured, by microspectrophotometry, cone pigment regeneration in intact photoreceptors under physiological conditions and in the absence of the RPE.
A photon-counting microspectrophotometer was used to measure visual pigment absorbance spectra from the outer segments of single isolated photoreceptors of tiger salamander retinas that were separated from RPE. One retina from each animal was used to determine the baseline absorbance spectra of dark-adapted cells. The other retina, intact or chopped (i.e. dissociated from Müller cell contact), was exposed to bright light (bleached) and then returned to darkness for two hours prior to spectral measurements. All experiments were designed to mimic the conditions of parallel physiological experiments, presented separately, in order to directly compare cone pigment regeneration and cone sensitivity under identical experimental conditions.
Exposure of dissociated retina to bleach induced a 6-fold decrease in optical density (OD) of red cones, which corresponds to 83% pigment loss. This is consistent with a lack of pigment regeneration in isolated cones. In contrast, exposure of an intact retina to an identical bleach produced only 1.05-fold decrease in OD of red cones (only 5% pigment loss), but a 5-fold decrease in OD of red rods (82% pigment loss). This indicates substantial cone (but not rod) pigment regeneration within the intact retina. Cone pigment regeneration was inhibited by treating the intact retina with a Müller cell-specific inhibitor, alpha-aminoadipic acid (L-α-AAA). Under these conditions, the bleach induced 60% cone pigment loss. In all experiments, including those of retinas treated with L-α-AAA, pigment lost from bleaching was completely restored upon treatment with exogenous 11-cis retinal.
Our results demonstrate the presence of a chromophore recycling mechanism within a rod-dominant retina. This pathway regenerates exclusively cone visual pigment and not rod pigment. This pathway involves Müller cells and operates independently of the RPE.
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