Bleaching of mouse rods: microspectrophotometry and suction-electrode recording

When a substantial fraction of rhodopsin in a rod photoreceptor is exposed to bright light, the rod is desensitized by a process known as bleaching adaptation. Experiments on isolated photoreceptors in amphibians have revealed many of the features of bleaching adaptation, but such experiments have not so far been possible in mammals. We now describe a method for making microspectrophotometric measurements of pigment concentration and suction-electrode recording of electrical responses over a wide range of bleaching exposures from isolated mouse rods or pieces of mouse retina. We show that if pigment is bleached at a low rate in the presence of bovine serum albumin (BSA), and intermediate photoproducts are allowed to decay, mouse rods are stably desensitized; subsequent treatment with exogenous 11-cis retinal results in pigment regeneration and substantial recovery of sensitivity to the dark-adapted value. Stably bleached wild-type (WT) rods show a decrease in circulating current and acceleration of the time course of decay, much as in steady background light; similar effects are seen in guanylyl cyclase-activating protein knockout (GCAPs(-/-)) rods, indicating that regulation of guanylyl cyclase is not necessary for at least a part of the adaptation produced by bleaching. Our experiments demonstrate that in mammalian rods, as in amphibian rods, steady-state desensitization after bleaching is produced by two components: (1) a reduction in the probability of photon absorption produced by a decrease in rhodopsin concentration; and (2) an equivalent background light whose intensity is proportional to the fraction of bleached pigment, and which adapts the rod like real background light. These two mechanisms together fully account for the ‘log-linear' relationship in mammalian retina between sensitivity and per cent bleach, which can be measured in the steady state following exposure to bright light. Our methods will now make possible an examination of bleaching adaptation and pigment regeneration in mouse animal lines with mutations or other alterations in the proteins of transduction.