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Comparative Study
. 2005 Oct 1;568(Pt 1):83-95.
doi: 10.1113/jphysiol.2005.091942. Epub 2005 Jul 1.

Opsin activation of transduction in the rods of dark-reared Rpe65 knockout mice

Jie Fan  1 , Michael L Woodruff, Marianne C Cilluffo, Rosalie K Crouch, Gordon L Fain
Affiliations
Comparative Study

Opsin activation of transduction in the rods of dark-reared Rpe65 knockout mice

Jie Fan et al. J Physiol. .

Abstract

Rpe65 knockout mice (Rpe65-/-) are unable to synthesize the visual pigment chromophore 11-cis retinal; however, if these animals are reared in complete darkness, the rod photoreceptors accumulate a small amount of 9-cis retinal and its corresponding visual pigment isorhodopsin. Suction-electrode recording of single rods from dark-reared Rpe65-/- mice showed that the rods were about 400 times less sensitive than wild-type control rods and that the maximum responses were much smaller in amplitude. Spectral sensitivity measurements indicated that Rpe65-/- rod responses were generated by isorhodopsin rather than rhodopsin. Sensitivity and pigment concentration were compared in the same mice by measuring light responses from rods of one eye and pigment concentration from the retina of the other eye. Retinas had 11-35% of the normal pigment level, but the rods were of the order of 20-30 times less sensitive than could be accounted for by the loss in quantum catch. This extra desensitization must be caused by opsin-dependent activation of the visual cascade, which leads to a state equivalent to light adaptation in the dark-adapted rod. By comparing the sensitivity of dark-reared Rpe65-/- rods to that produced in normal rods by background light, we estimate that Rpe65-/- opsin is of the order of 2.5x10(-5) as efficient in activating transduction as photoactivated rhodopsin (Rh*) in WT mice. Dark-reared Rpe65-/- rods are less desensitized than rods from cyclic light-reared Rpe65-/- mice, have about 50% more photocurrent and degenerate at a slower rate. Retinas sectioned after 9 months in darkness show a larger number of photoreceptor nuclei in dark-reared animals than in cyclic light-reared animals, though both have fewer nuclei than in cyclic light-reared wild-type retinas. Both also have shorter outer segments and a lower free-Ca2+ concentration. These experiments provide the first quantitative measurement of opsin activation in physiologically responding mammalian rods.

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Figures

Figure 1
Figure 1. Rods from dark-reared Rpe65−/− mice are less sensitive than wild-type mouse rods but more sensitive than rods from Rpe65−/− mice raised in cyclic light
A, wild-type mouse rod current responses to 20 ms flashes of 500 nm light at intensities of 2.1, 7.9, 21, 69, 220 and 590 photons μm−2. Traces are global means from 10 rods, and for each rod from 10 to 12 presentations for the dimmest light, 4–10 for lights of intermediate intensity, and 2–5 for the brightest flashes. B, Rpe65−/− mouse rod currents to 20 ms flashes but at intensities of 220, 590, 2140, 7940, 26300 and 85100 photons μm−2. Responses are global means from 42 rods and 5–12 presentations per rod at each flash intensity. C, peak amplitude as function of flash intensity for rods from WT (•), dark-reared Rpe65−/− (○) and light-cycle-reared Rpe65−/− mice (□). WT data averaged from 21 rods, dark-reared Rpe65−/− from 37. The Rpe65−/− cyclic light-reared data are replotted from Woodruff et al. (2003).
Figure 2
Figure 2. Spectral sensitivity of dark-reared Rpe65−/− rods follows isorhodopsin absorption spectrum
Mean relative absorption of pigment from one retina each of 9 Rpe65−/− mice (□) was fitted with isorhodopsin nomogram calculated as in Lamb (1995) for a λmax of 487 nm. Mean relative absorption from one retina of a WT mice (○) was fitted with a rhodopsin nomogram for a λmax of 503 nm. Mean dark-adapted sensitivities of 7 Rpe65−/− rods each measured at five wavelengths (▪) were normalized by dividing by the sensitivity at 480 nm, and the result was then multiplied by the value of relative sensitivity at 480 nm predicted from the nomogram for isorhodopsin (0.986).
Figure 3
Figure 3. Dependence of sensitivity of rods in Rpe65−/− mice on pigment concentration
Mean dark-adapted sensitivity (SFM) from 3 to 7 Rpe65−/− photoreceptors for each of 12 animals (total of 63 rods from all animals). Eight animals were placed in darkness for a period of 15 weeks (□) or 37 weeks (▴) beginning 3 weeks after birth, and 4 were kept in darkness from birth for 20 weeks (▪). Sensitivities were corrected for the difference in quantum efficiency between isorhodopsin and rhodopsin (0.22 versus 0.67) and normalized to mean dark-adapted sensitivity of WT animals (SFWT). From each Rpe65−/− retina, pigment concentration was measured and normalized to pigment concentration in WT after adjusting for the loss of photoreceptors in Rpe65−/− retinas and the smaller length of the rod outer segments compared with wild-type. This fraction (Φ) was then subtracted from unity to give the fraction of pigment lost. Dashed curve is loss in sensitivity expected from loss in quantum catch alone. Continuous curve is fit to eqn (5). See text.
Figure 4
Figure 4. Comparison of adaptation by background light in WT rods with desensitization by opsin activation in Rpe65−/− rods
A, suction-electrode responses to 20 ms, 500 nm flashes from single WT rod, in darkness (left-most traces) and in presence of background light of 500 nm at intensities of 311, 939 and 2900 photons μm−2 s−1. Each trace is the mean of 5–12 presentations. B, mean peak response amplitude plotted as function of flash intensity for 21 dark-adapted WT rods (•), taken from Fig. 1C and light-adapted WT rods (open symbols) at background intensities (increasing for curves from left to right) of 27 (□), 101 (○), 311 (▴), 939 (⋄), and 2900 (⊥) photons μm−2 s−1. Also shown are responses of 37 dark-adapted Rpe65−/− rods replotted from Fig. 1C (▪) after adjusting light intensities for loss in quantum catch (see text). Error bars give s.e.m.s. C, mean small-amplitude responses normalized to peak amplitude from 10 dark-adapted WT rods (thick line), 36 dark-adapted, dark-reared Rpe65−/− rods (medium line), and 19 light-adapted WT rods (thin line). Actual peak amplitudes and flash intensities for three responses (dark-adapted WT, dark-adapted Rpe65−/−, light-adapted WT): 3.6 pA, 15 photons μm−2 0.48 pA, 1150 photons μm−2; 0.71 pA, 135 photons μm−2.
Figure 5
Figure 5. Weber-Fechner curve for WT mouse rods
Mean sensitivity of from 4 to 12 WT rods in presence of background light normalized to dark-adapted WT sensitivity and plotted as function of background intensity (intensity values as in legend to Fig. 4B). Curve is eqn (2) with I0 set to 50 photons μm−2 s−1.
Figure 6
Figure 6. Retinal histology of WT and Rpe65−/− mice
Light micrographs from age-matched 11-month-old cyclic light-reared WT (A), cyclic light-reared Rpe65−/− mice (B), and Rpe65−/− mice reared in dark for 9 months (C). Micrographs were taken from the same region in each eye 250 μm superior to the optic nerve head. Scale bar, 25 μm.
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