Article Figures & Data
Figures
- Figure 1.
Physiological response properties of WT and Rh+/− rods. A, Representative light-evoked flash families from WT (left, black) and Rh+/− (right, gray) rods. Patch-clamp recordings were made in whole-cell mode [membrane potential (Vm) = −40 mV]. Twenty millisecond light flashes given at time 0 yielded flash strengths ranging from 1 to 800 R*/rod, increasing by a factor of 2. Recordings are representative of data collected from several experiments. B, Intensity–response plot (mean ± SEM) of WT rods (black; n = 7)) and Rh+/− rods (red; n = 7)). The sensitivity (I1/2) was derived from the best fitting Hill equation, I1/2 = 15.6 ± 0.4 and 29.8 ± 0.6, respectively. C, Microspectrophotometric measurements of rhodopsin from WT rods (black) and Rh+/− rods (red). Optical density (visual pigment content) was measured from outer segments of 12 WT rods (OD = 0.219 ± 0.03, mean ± SEM) and 14 Rh+/− rods (OD = 0.106 ± 0.012, mean ± SEM) showing a reduction in half of pigment concentration in Rh+/− mice (***p < 0.0001; see Materials and Methods). D, Western blots for rhodopsin showing that concentration in Rh+/− rods (1.5 ± 0.3 a.u., normalized over β-actin content.) compared with WT rods (3.7 ± 0.4 a.u., normalized over β-actin content) is reduced by half (***p < 0.001).
- Figure 2.
Discrete noise events in rod photoreceptors in complete darkness. A, Average normalized dim-flash responses recorded with the suction electrode technique from WT (black) and Rh +/− (gray) rods. Responses were derived from 10 and 11 rods (<25% of maximum response amplitude), and the time-to-peak values were 246 ± 15.1 and 237.5 ± 18.8 ms, respectively (mean ± SEM). B, Average normalized dim-flash responses recorded with the suction electrode technique from R+/+;GCAPs−/− (black) and Rh+/−;GCAPs−/− rods (gray), N = 36 and N = 46, respectively, and time-to-peak values were 427.9 ± 30.3 and 432.3 ± 36.8 ms, respectively. C, Representative recordings of outer-segment membrane currents recorded with the suction electrode technique from dark-adapted Rh+/+;GCAPs−/−. Rods were first stimulated with a saturating light flash to demonstrate viability of the cell and then placed in darkness. Discrete noise events (*) were distinguished by “match filtering” the dark-noise recordings with the derived dim-flash response (see Materials and Methods). D, Representative recordings of outer-segment membrane currents recorded with the suction electrode technique from Rh+/−;GCAPs−/−. Same as in C.
- Figure 3.
Reduced PDE6 expression in PDEAB+/− rods. A, Representative immunoblots for PDE6 β and γ subunit expression in PDE6AB+/+, PDE6AB+/−, and PDE6AB−/− retinas. Examples of protein expression in 6 retinas (2 for each well) per genotype. B, Quantification of PDE6 β subunit expression in WT (red), PDE6AB+/− (light red), and PDE6AB−/− (pink) rods, normalized to the β-actin loading control. The expression of the β subunit was significantly decreased in both PDE6AB+/− rods (70% of the PDE6AB+/+, **p < 0.01, N = 3) and PDE6AB−/− rods (>1% of the PDE6AB+/+, ***p < 0.001, N = 3). C, Quantification of the PDE6 γ subunit in WT (brown), PDEAB+/− (light brown), and PDEAB−/− (red) rods, normalized to the β-actin loading control. The expression of the γ subunit was significantly decreased in both PDE6AB+/− (73% compared with PDE6AB+/+, **p < 0.01, N = 3) and PDE6AB−/− (>1% compared with PDE6AB+/+, ***p < 0.001, N = 3).
- Figure 4.
Effect of transduction protein concentration on physiological properties of rods. A, Representative light-evoked responses from WT rods (C57BL/6J, black), Tr+/− rods (blue), and PDE6AB+/− rods (red). Patch-clamp recordings were made in whole-cell mode [membrane potential (Vm) = −40 mV]. Twenty millisecond light flashes given at time 0 yielded flash strengths ranging from 4 to 550 R*/rod, increasing by a factor of 2. Reduction in expression of transducin or PDE yielded no marked change in response waveforms compared with C57BL/6J rods. B, Intensity–response plot (mean ± SEM) of WT rods (black, n = 10), Tr+/− rods (blue, n = 6), and PDE+/− (red, n = 11). The sensitivity (I1/2) was derived from the best fitting Hill equation, I1/2 = 19.8 ± 1.8, 23.2 ± 3.6, and 20.2 ± 3.2, respectively. Reduction in rod transducin corresponded with a small, albeit nonsignificant, decrease in flash sensitivity (p = 0.47). Reduction in PDE resulted in no significant change in flash sensitivity (p = 0.94). C, Averaged single-photon responses from WT (black), Tr+/− (blue), and PDE+/− (red) rods, time-to-peak 222.5 ± 0.5, 245. ± 19.8, and 232. ± 32.8 ms, respectively. Reduction of neither transducin nor PDE resulted in a significant change in response kinetics.
- Figure 5.
Effects of reduction in PDE and transducin expression on continuous noise. A, Representative recordings of membrane currents from a WT littermate rod (black) and a Tr+/− rod (blue). Whole-cell patch-clamp recordings were done in complete darkness, followed by a saturating light flash (6750 R*/rod) to close all of the CNG channels. B, Continuous noise power spectra from WT (black, n = 6) and Tr+/− (blue, n = 6) rods (mean ± SEM). Spectra were constructed by fast Fourier transformation of the cellular dark noise, derived by subtracting the instrumental noise (saturated response) from dark noise recordings. C, Cumulative power of dark noise between 0.1 and 10 Hz. No statistical difference between WT (black) and Tr+/− (blue) was observed. D, Representative recording of membrane currents from a WT littermate (black) and PDE+/− rod (red). Whole-cell patch-clamp recordings were done in complete darkness, followed by a saturating light flash (6750 R*/rod) to close all the CNG channels. E, Continuous noise power spectra from WT (black, n = 10) and PDE+/− (red, n = 10) rods (mean ± SEM). Spectra were constructed by fast Fourier transformation of the cellular dark noise, derived by subtracting the instrumental noise (saturated response) from dark noise recordings. F, Cumulative power of dark noise between 0.1 and 10 Hz (mean ± SEM). A statistically significant (p = 0.02) approximately twofold difference in dark noise between WT (black) and PDE+/− (red) was observed.
Tables
Rods Time Events Discrete rate Rh+/+;GCAPs−/− 10 5052 s 68 0.013 s−1 Rh+/−;GCAPs−/− 11 5965 s 39 0.006 s−1 Collected data of discrete noise events recorded in total darkness from dark-adapted Rh+/+;GCAPs−/−; and Rh+/−;GCAPs−/− rods.
Total continuous noise Continuous noise 0.1–1 Hz Continuous noise 5–10 Hz Tr+/+ 0.24 ± 0.08 (N = 6) 0.1 ± 0.04 (N = 6) 0.0014 ± 0.007 (N = 5) Tr+/− 0.21 ± 0.03 (N = 6) 0.09 ± 0.03 (N = 6) 0.007 ± 0.001 (N = 6) PDE6+/+ 0.42 ± 0.04 (N = 10) 0.33 ± 0.07 (N = 11) 0.029 ± 0.003 (N = 11) PDE6+/− 0.75 ± 0.05* (N = 6) 0.65 ± 0.1** (N = 6) 0.013 ± 0.001** (N = 6) The power of the cellular dark noise (pA2) was calculated as the frequency integral of total power in darkness subtracted by the light-isolated instrumental noise between 0.1 and 10 Hz, 0.1 and 1 Hz, and 5 and 10 Hz.
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