Bleaching of mouse rods: microspectrophotometry and suction‐electrode recording

Abstract

Key points• When photoreceptors in vertebrate retina are exposed to bright light, a significant proportion of the photopigment in the rods can be bleached.• Bleaching produces a desensitization of the visual system that recovers slowly as pigment is slowly regenerated, by a process known as dark adaptation.• Experiments on isolated amphibian rods have revealed some of the features of bleach-induced desensitization, but such experiments have not so far been possible on mammals.• We now describe an improved method that makes possible the first direct measurements of pigment concentration and rod photoreceptor responses over a wide range of bleaching exposures from isolated cells or pieces of intact mammalian retina.• Our experiments reveal important features of mammalian bleaching adaptation and will now make possible future studies from mouse animal lines containing genetically altered photoreceptor proteins.Abstract 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 S. Nymark and R. Frederiksen contributed equally to the work. bright light. Our methods will now make possible an examination of bleaching adaptation and pigment regeneration in mouse animal lines with mutations or other al...

Modulation of Mouse Rod Photoreceptor Responses by Grb14 Protein

“…2D). Responses in WT rods decayed more rapidly after bleaching, as we have previously documented (16). A similar effect was observed for Grb14 Ϫ/Ϫ rods.…”

“…2 and Table 1. Bleaching decreased maximum response amplitude and sensitivity in WT and Grb14 Ϫ/Ϫ rods, both by reducing the amount of pigment available to absorb photons and by producing adaptation similar to that produced by background light (16). The effect of bleaching was similar for rods of the two genetic backgrounds.…”

“…16). After waiting 45 min to 1 h for the circulating current and sensitivity of the rod to come to steady state and for any protein translocation to occur, we recorded rod responses from WT (Fig.…”

“…At medium flash intensities, 5-10 responses were averaged, and the interflash interval was increased to 10 s. At bright flash intensities above saturation for the rods, only 3-5 responses were averaged, and the interflash interval was increased to 15-20 s. A 500-nm light was also used for steps of light and for bleaching. The amount of bleaching was determined as in previous experiments (16) The time in saturation (T sat ) was measured as the time from the beginning of the flash to the time at which the mean circulating current recovered to 25% of its dark-adapted value. The value of D was then calculated rod by rod (see Table 1) or from mean values (see Fig.…”

Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells

“…Second, our recordings of A3C rod photocurrents reveal that little desensitization/adaptation is observed using the translocation protocol, which leads to a residual 35% bleached visual pigment (Materials and Methods and SI Text). It is well known that, after visual pigment bleaching in both amphibian and mammalian rods, the sensitivity of phototransduction is reduced because of the sum of the loss of quantum catch and residual phototransduction activity produced by free opsin (24,35). The relatively minor desensitization of A3C rods using the translocation protocol (Fig.…”