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Michael L. Woodruff, Carter Cornwall, Gordon L. Fain; Bleaching Adaptation in a Mammalian Photoreceptor. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1174. doi: https://doi.org/.
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We developed a method to record from isolated rods whose pigment has been bleached over a much wider range than previously possible, and whose pigment is regenerated with exogenous 11-cis retinal. We have used this method to examine the physiology of bleached and regenerated rods from a mammalian retina.
We made conventional suction-electrode recordings from isolated wild-type mouse rods. In initial experiments, large bleaches produced irreversible desensitization and cell death. Successful recordings could however be obtained over bleaches as great as 90% of the total rhodopsin if (1) we included 0.1% BSA in the perfusing medium, (2) illuminated over a prolonged period with a light intensity that bleached no more that 0.2% of the pigment per second, and (3) waited as long as 60 min after the bleach for the rods to come to steady state. Pigment was regenerated by adding lipid vesicles containing 11-cis retinal. The amount of the bleach was estimated from the previously determined in vitro photosensitivity of mouse rods.
Isolated rods at steady-state after bleaching showed a reduction in circulating current, sensitivity, and time constant of recovery (tau_REC), which all decreased monotonically with the extent of the bleach. Regeneration with 11-cis retinal produced a nearly complete recovery of the amplitude, sensitivity, and waveform of the response. The decrease in sensitivity could be predicted from a simple model in which bleaching light reduced quantum catch and created a photopigment intermediate, probably opsin, which activates the visual cascade in proportion to the amount of bleached pigment and generates an equivalent background light.
Our experiments show that it is possible to recreate the visual cycle in vitro. This advance will make it possible for us to study bleaching adaptation in a mammalian photoreceptor in greater detail than was previously possible, and to study the effect of introducing exogenous chromophore analogues into wild-type photoreceptors.
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