Rod Photoreceptors Avoid Saturation in Bright Light by the Movement of the G Protein Transducin

Rod responses in bright continuous light

We recorded rod-mediated signals from isolated, whole mouse retina to maximize the signal-to-noise ratio of the recording. To eliminate signals from cones and other retinal cells, we used retinae from Gnat2^−/− mice lacking the gene for cone transducin (Ronning et al., 2018), and we perfused the retina with 40 μm DL-2-amino-4-phosphonobutyric acid to block bipolar-cell responses and 100 µm BaCl₂ to eliminate currents from Müller glia (see Materials and Methods) (Vinberg et al., 2014). We first recorded dark-adapted (DA) responses to a series of flashes with light of increasing intensity, which are given in the first column of Figure 1. We then recorded responses to a similar family of flashes immediately after turning on a background light (indicated as time 0 min) and at various times during continuous background-light exposure. The background light was provided by an LED source with its peak at 560 nm (see Materials and Methods). Light intensities are expressed as equivalent photons at the λ_max of the rod pigment, designated as ϕ. The first row shows results at a background intensity of 1.3 × 10⁴ ϕ µm⁻² s⁻¹, which we estimate initially to produce ∼5000 bleached rhodopsin molecules (Rh*) per rod per second and is near the intensity we and others have reported to produce saturation in single rods (Mendez et al., 2001; Morshedian et al., 2018). The following three rows show similar experiments at brighter backgrounds. At time 0, beginning immediately after turning on the background, we recorded a response-intensity series and could detect small responses to incremental flashes with maximum amplitudes of 15–25 µV for each of the background intensities we used, ∼2%–3% of the DA maximum amplitude. Since in suction-electrode recordings from the rod outer segment the maximum dark current are typically 15–20 pA (Field and Rieke, 2002; Gross et al., 2012; Morshedian et al., 2018), responses recorded from single rods at time 0 would have been <1 pA in amplitude and difficult to detect, explaining why rods have previously been assumed to be completely saturated. When, however, the background light was left on for many minutes, the maximum amplitude of the response grew and at 90 min could become as large as 100 µV, or >10% of the maximum amplitude recorded before background illumination.

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Figure 1.

Representative trans-retinal (ERG) recordings of isolated rod responses to flashes of 505 nm light in DA Gnat2^−/− mouse retinae, immediately (0 min), 30 min, and 90 min after the onset of a 560 nm background light. Background light intensity is expressed in ϕ µm⁻² s⁻¹, which are photons effective at the λ_max of mouse rhodopsin at 503 nm (see Fig. 5). The 505 nm flashes (in ϕ µm⁻²) were as follows: 0.80, 4.8, 19, 72, 2.5 × 10², 7.7 × 10², and 2.3 × 10³ (DA before 1.3 × 10⁴ and 3.6 × 10⁴ ϕ µm⁻² s⁻¹ background); 1.2, 6.5, 53, 2.5 × 10², and 7.1 × 10² (DA before 1.3 × 10⁵ and 3.6 × 10⁵ ϕ µm⁻² s⁻¹ background); 1.6 × 102, 9.6 × 10², 3.8 × 10³, 1.4 × 10⁴, 4.9 × 10⁴, 1.5 × 10⁵, and 4.6 × 10⁵ (1.3 × 10⁴ ϕ µm⁻² s⁻¹ background); 1.6 × 10³, 9.6 × 10³, 3.8 × 10⁴, 1.4 × 10⁵, 4.9 × 10⁵, 1.5 × 10⁶, 4.5 × 10⁶ (3.6 × 10⁴ ϕ µm⁻² s⁻¹ background); 4.7 × 10³, 2.1 × 10⁴, 7.5 × 10⁴, 3.0 × 10⁵, 1.2 × 10⁶, 3.4 × 10⁶ (1.3 × 10⁵ and 3.6 × 10⁵ ϕ µm⁻² s⁻¹ background).

To confirm that light-evoked responses could be recorded from single rods, we made suction-electrode recordings under similar conditions. Figure 2A shows averaged DA responses from rods recorded before the presentation of a background of 10⁶ ϕ µm⁻² s⁻¹, even brighter than the brightest background used in Figure 1. We then turned on the illumination and continued recording from these same rods for the times indicated in Figure 2B. At each time, we gave the same flash (6.1 × 10⁶ ϕ µm⁻²), which was the brightest our suction-electrode photostimulator could deliver. Initially, responses were very small but gradually grew over 30 min and averaged nearly 0.5 pA, or ∼3% of the average amplitude in darkness. This value is approximately the same as the percent amplitude we recorded after 30 min in the brightest background of Figure 1. We believe this amplitude to be a lower limit because the waveform of the response suggests that even larger responses could have been evoked by brighter flashes, and because mouse rods recorded with suction electrodes typically lose some of their circulating current with time during a recording.

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Figure 2.

Single-cell suction electrode recordings from WT mouse rods. A, Average responses to 505 nm flashes recorded from 16 DA mouse rods. Flashes were 1.6, 4.6, 21, 51, 190, 540, 1300, 2200, and 5100 ϕ µm⁻². B, Responses in the presence of 565 nm background of 1.0 × 10⁶ ϕ µm⁻² s⁻¹. Traces are average responses from 30 flashes of 6.1 × 10⁶ ϕ µm⁻² recorded from 6 rods. C, Mean flash responses from 11 rods first exposed for 60 min to 565 nm background light of 1.0 × 10⁶ ϕ µm⁻² s⁻¹, then allowed to reach steady state after another 60 min in darkness. Flashes were 9.4 × 10⁴, 1.9 × 10⁵, 4.1 × 10⁵, 6.7 × 10⁵, and 1.36 × 10⁶ ϕ µm⁻². D, Response-intensity functions from cells of C. Data were fitted with saturating exponential relations of R = R_max[1 – e^(–kϕ)], where R_max is the maximum response amplitude in pA, k is a constant in ϕ⁻¹ µm², and ϕ is the number of effective photons in the flash per square micron (Lamb et al., 1981). The best fitting parameters for DA rods were R_max = 12.4 pA and k = 5.1 × 10⁻³ ϕ⁻¹ µm²; and for rods after illumination, R_max = 0.93 pA and k; = 7.1 × 10⁻⁶ ϕ⁻¹ µm².

Changes in sensitivity and maximum amplitude

To provide a more quantitative description of the time course of the change in rod responsivity during prolonged background exposure, we have plotted response-intensity curves in Figure 3A, B for the background intensity of 1.3 × 10⁵ ϕ µm⁻² s⁻¹ from Figure 1. The curves in Figure 3B show responses in the presence of the background on an expanded ordinate. All of the data have been fitted to a Michaelis-Menten curve, as follows: (3) where R is the response amplitude in µV, R_max is the best-fitting maximum amplitude of R, ϕ is the number of incident photons in the flash per square micron, and ϕ_½ is the best-fitting value of ϕ at R = ½R_max (half-saturation constant). The values of the best-fitting parameters are given in the legend to Figure 3. These data show that, from 15 to 90 min, ϕ_½ varied within a narrow range from ∼5 × 10⁴ to 2 × 10⁵ ϕ µm⁻², at first decreasing at 30 and 45 min and then increasing again at 60-90 min, perhaps reflecting a slow loss in sensitivity. During these small changes in the half-saturation constant, R_max monotonically increased, reaching its maximum value at ∼75 min.

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Figure 3.

Response amplitude and sensitivity of rods in background light. A, Mean response-intensity relations recorded from Gnat2^−/− mouse retinae, DA and every 15 min after onset of a background of 1.3 × 10⁵ ϕ µm⁻² s⁻¹ (n = 6 for each condition). The data were fitted with Equation 3, with the parameters as follows: DA, R_max = 746 µV and ϕ_1/2 = 26.8 ϕ µm⁻²; 0 min, R_max = 18.4 µV and ϕ_1/2 = 9.85 × 10⁵ ϕ µm⁻²; 15 min, R_max = 26.5 µV and ϕ_1/2 = 2.02 × 10⁵ ϕ µm⁻²; 30 min, R_max = 33.1 µV and ϕ_1/2 = 4.45 × 10⁴ ϕ µm⁻²; 45 min, R_max = 43.0 µV and ϕ_1/2 = 4.93 × 10⁴ ϕ µm⁻²; 60 min, R_max = 53.4 µV and ϕ_1/2 = 8.71 × 10⁴ ϕ µm⁻²; 75 min, R_max = 74.7 µV and ϕ_1/2 = 1.37 × 10⁵ ϕ µm⁻²; 90 min, R_max = 72.5 µV and ϕ_1/2 = 1.55 × 10⁵ ϕ µm⁻². B, Data in background light from A, with the ordinate rescaled to 10% that in A. C, Maximal response amplitude (R_max) to a bright flash plotted as a function of time in the presence of background light. Flashes were 4.6 × 10⁵ ϕ µm⁻² for the 1.3 × 10⁴ ϕ µm⁻² s⁻¹ background, 4.5 × 10⁶ ϕ µm⁻² for the 3.6 × 10⁴ ϕ µm⁻² s⁻¹ background, and 3.4 × 10⁶ ϕ µm⁻² for the 1.3 × 10⁵ and 3.6 × 10⁵ ϕ µm⁻² s⁻¹ backgrounds. D, Sensitivity normalized to DA sensitivity plotted and as a function of time in background light. C, D, n = 6 for each condition.

In Figure 3C, D, we plot changes in the maximum amplitude and sensitivity for all four background light intensities from Figure 1. Because in some measurements (e.g., the 0 min data in Fig. 3B), we could not accurately determine R_max with even the brightest flash from our photostimulator, we estimated flash sensitivity as response amplitude per incident light at the illumination necessary to give a response that was 10% of the largest voltage we could measure for any set of data at a given time and background intensity. We chose the value of 10% because it was large enough to measure accurately yet still within the near-linear range of the response-intensity curve. The data in Figure 3C confirmed the results of Figure 3B, showing that R_max grew monotonically during background light exposure. The time-dependent increase in R_max was substantially greater for the brighter backgrounds than for the dimmest. Similarly, rod sensitivities ( increased between 0 and 45 min of background exposure for all but the dimmest light intensities, plateauing between 60 and 90 min (Fig. 3D).

These data pose two questions. (1) How can sensitivity and R_max both increase from 0 to 45 min in the presence of constant background light, and why is the extent of increase smaller for the dimmest light than for the brighter intensities? (2) How can sensitivity between 60 and 90 min remain nearly constant, even in the brightest background? We can calculate the fraction of pigment bleached in these experiments from the photosensitivity equation as follows: (4) where F is the fraction bleached, ϕ is the light intensity in incident photons µm⁻² s⁻¹ at the λ_max of the photopigment, and P is the photosensitivity for mouse rhodopsin of 5.7 × 10⁻⁹ µm² (Woodruff et al., 2004; Nymark et al., 2012). The fraction of pigment remaining is 1 – F. After a 90 min exposure to 3.6 × 10⁵ photons µm⁻² s⁻¹, Equation 2 predicts that a single rod in the absence of regeneration would have ∼2 × 10⁻⁵ of its normal complement of rhodopsin, or ∼1200 rhodopsin molecules. Since a single mouse rod has ∼800 disks (Nickell et al., 2007), there would be on average 1.5 rhodopsin molecules per disk. In experiments not shown, we have exposed rods for as long as 4 h at this intensity and at 10⁶ incident photons µm⁻² s⁻¹, and rods continue to respond much as in Figure 1. Clearly, some mechanism must exist under these experimental conditions for regenerating rhodopsin (Tikidji-Hamburyan et al., 2017).

Role of transducin translocation

The intensity dependences of the increase in response amplitude and sensitivity appear similar to those required for the light-induced translocation of transducin from the rod outer segment to the inner segment (Sokolov et al., 2002; Lobanova et al., 2007). Moreover, for the three brighter backgrounds in Figure 3D, sensitivity increased by a factor of ∼10 and R_max by a factor of ∼5, consistent with a small fraction of transducin still remaining in the outer segment after translocation is complete (Sokolov et al., 2002). A reduction of outer-segment transducin concentration would decrease the gain of phototransduction. This decrease in gain could allow rods to avoid saturation by decreasing activation of the cGMP phosphodiesterase, which would increase the free concentration of cyclic GMP and augment the circulating current and R_max. The increase in R_max could in turn explain most of the increase in sensitivity.

To explore a possible role of transducin translocation in changes in rod sensitivity and response amplitude in background light, we used the A3C⁺ mouse (Majumder et al., 2013). In this animal, the normal Gnat1 gene for the transducin α subunit Gα_t was substituted with A3C⁺-Gnat1, which introduces an additional, artificial S-palmitoylation site on Gα_t. This third palmitoylation site increases the affinity of Gα_t for outer-segment disk membranes and impedes Gα_t from dissociating during light stimulation. As a result, only about half as much transducin moves to the inner segment in the A3C⁺ mice compared with control animals during continuous light exposure (Majumder et al., 2013).

The results in Figure 4 show that this reduction in transducin translocation, though partial and incomplete, has nevertheless a significant effect on the changes in sensitivity and R_max. Figure 4A shows responses recorded from a Gnat1^−/−;Gnat2^−/−;A3C⁺ retina, first in darkness and then after exposure to 3.1 × 10⁵ ϕ µm⁻² s⁻¹. Responses before presentation of the background were somewhat smaller than in a WT retina (Fig. 1), perhaps in part because the Gnat1^−/−;Gnat2^−/−;A3C⁺ mouse has only 80% of the transducin as the Gnat1^+/+;Gnat2^−/− mouse, and perhaps also because Gnat1^−/−;Gnat2^−/−;A3C⁺ mice are known to undergo slow degeneration (Majumder et al., 2013). The sensitivity and waveform of the responses were, however, nearly unaltered. Responses in the presence of the background light were almost undetectable immediately after turning on the light and continued to be smaller, even after correction for the difference of current in darkness, growing much more slowly in amplitude than in a Gnat2^−/− retina.

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Figure 4.

Recordings as in Figure 1 from Gnat2^−/− and Gnat1^−/−Gnat2^−/−A3C⁺ mice. A, Representative responses from a Gnat1^−/−Gnat2^−/−A3C⁺ retina DA and at indicated times after onset of a 560 nm background light of 3.1 × 10⁵ ϕ µm⁻² s⁻¹. B, Maximum response amplitude (R_max) to a flash stimulus recorded every 2 min after the onset of background light of 3.1 × 10⁵ ϕ µm⁻² s⁻¹ in Gnat1^−/−Gnat2^−/−A3C⁺ (n = 4, black) and Gnat2^−/− (n = 9, red) retinae. Flashes were 6.1 × 107 ϕ µm⁻². C, Mean flash sensitivities of Gnat1^−/−Gnat2^−/−A3C⁺ (n = 7) and Gnat2^−/− (n = 9) retinae plotted as a function of time in the presence of a background light of 3.1 × 10⁵ ϕ µm⁻² s⁻¹.

In Figure 4B, C, we compare the mean changes in the values R_max and sensitivity for Gnat1^−/−;Gnat2^−/−;A3C⁺ (black symbols) and Gnat2^−/− retinae (red symbols). Nearly all of the increases we observed in Figure 3 have been greatly reduced. Although increases could still be observed, they were smaller than we might have expected, given that nearly half of transducin continues to translocate in these animals. If we had prevented all of the transducin from moving, we think it possible that rods would have remained completely unresponsive in bright backgrounds.

Rhodopsin bleaching

To investigate possible mechanisms of rhodopsin regeneration, we first measured its concentration directly with MSP during exposure to the various background lights we used in previous experiments. The measuring beam was focused onto a group of rod outer segments lying on their sides, as described previously (Nymark et al., 2012). The background exposure was initiated and then briefly turned off at set times so that the MSP measurements could be made. The total light exposure produced by the MSP measurement itself was small and did not affect the concentration of rhodopsin we were attempting to measure (see Materials and Methods).

Figure 5A shows measurements of rhodopsin OD (noisy traces) taken at various times during exposure to 6.7 × 10⁴ ϕ µm⁻² s⁻¹. The data have been fitted with pigment absorbance curves (Govardovskii et al., 2000) calculated for a λ_max of 503 nm. From these fits, we extracted values of relative peak absorbance at each of the times the measurements were made. These values are given in Figure 5B for three different background light intensities of 2.6 × 10⁴, 6.7 × 10⁴, and 6.0 × 10⁵ ϕ µm⁻² s⁻¹. The straight lines through the data give the value of 1 – F calculated from Equation 4. The fraction of rhodopsin remaining (equal to the relative OD in the limit of low rhodopsin concentration) agreed almost perfectly with the fraction remaining calculated from the photosensitivity of the rhodopsin, the intensity of exposure, and time. There was no evidence of rhodopsin regeneration within the resolution of the MSP measurement.

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Figure 5.

MSP measurements of OD of WT mouse rods during pigment bleaching. A, Examples of absorbance spectra recorded first in DA retina and then after exposure to 570 nm light of 6.7 × 10⁴ ϕ µm⁻² s⁻¹ for 5, 10, 15, 20, 30, 40, 60, 90, and 120 min. Spectra were fitted with rhodopsin templates (Govardovskii et al., 2000) with λ_max = 503 nm. B, Bleaching of rhodopsin expressed as normalized OD at 500 nm from recordings as in A for background intensities of 2.6 × 10⁴ ϕ µm⁻² s⁻¹ (red squares, n = 3), 6.7 × 10⁴ ϕ µm⁻² s⁻¹ (blue circles, n = 5), and 6.0 × 10⁵ ϕ µm⁻² s⁻¹ (green triangles, n = 3). Lines indicate fraction of pigment remaining (1 – F) calculated from Equation 4.

Rhodopsin regeneration in bright backgrounds

The data in Figure 5 show that no significant regeneration of rhodopsin occurs for bleaches reducing the rhodopsin concentration to within ∼0.02 (or 2%) of the DA concentration, but the experiments in Figures 1–3 indicate that some pigment must nevertheless be re-forming in bright background light. To explore this apparent discrepancy, we have measured the changes in sensitivity produced by bleaching and used these measurements to estimate the rhodopsin concentration. We adopted a protocol we have previously used to measure the effect of rhodopsin bleaching for isolated mouse rods in the absence of RPE or exogenous sources of 11-cis retinal (Nymark et al., 2012; Pahlberg et al., 2017). In those earlier experiments, we exposed mouse rods to light calculated to bleach a predetermined fraction of rhodopsin from Equation 4, and we then turned the light off and waited a period of 45-60 min to allow the sensitivity of the photoreceptors to reach steady state. We showed that the relative sensitivity of the rods at steady state after a bleach is well described by the following: (5) where S_F is the sensitivity at steady state after bleaching, is the sensitivity in the dark before the bleach, F is the fraction of rhodopsin bleached, and k is a constant. This equation takes into account the decrease in sensitivity produced by reduction in the concentration of rhodopsin (the decrease in quantum catch), together with light adaptation produced by activation of phototransduction by bleached pigment (Jones et al., 1996). Our thought was to make similar measurements on our preparation but with light calculated to bleach larger values of F than previously. On the assumption that rods will continue to behave according to Equation 5, we hoped to use the change in sensitivity to estimate the actual fraction of rhodopsin remaining in the rod, including any regeneration that may have occurred.

The results of these experiments are given in Figure 6. The Gnat2^−/− retinae were exposed to steady light of intensity 3.6 × 10⁵ ϕ µm⁻² s⁻¹ as in Figures 1 and 3, but for the times indicated in Figure 6. At the cessation of each exposure, we waited 45-60 min for the rods to reach steady state and then measured the sensitivity, in a manner identical to our previous work for single rods (Nymark et al., 2012; Pahlberg et al., 2017). Sokolov et al. (2002) showed that movement of transducin back into the outer segment is much slower than translocation outward, having a time for half-completion of 2.5 h (see also Zhang et al., 2011). Over the time course of these experiments, the great majority of transducin moving to the inner segment will have remained there.

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Figure 6.

Sensitivity and rhodopsin concentration after long exposures to bright light. A, Responses were recorded as in Figure 1 first in darkness (DA, n = 24). Retinae were then exposed for a variable duration (as indicated in the figure) to 560 nm light of 3.6 × 10⁵ ϕ µm⁻² s⁻¹ and returned to darkness for an additional 45-60 min to allow the rods to come to steady state. Data from bleached conditions are averages from 3 retinae for each condition and give response amplitude as a function of flash strength. B, Sensitivity from A, normalized to DA sensitivity. Dashed red line is Equation 5, with k = 70 and 1 – F calculated from Equation 4. Black line is also Equation 5, but with 1 – F taken from its value inferred from the black line in C. Arrow points to the sensitivity where the two lines diverge at ∼0.8%-1% of DA rhodopsin concentration. C, Inferred concentration of rhodopsin (1 – F). Dashed red line is 1 – F from Equation 4, assuming no regeneration. Black line is 1 – F calculated from the numerical solution to Equations 2a and 2b, with the constants k_d = 2.0 × 10⁻⁴ s⁻¹ and k_r = 1.8 × 10⁻¹¹ µm². Arrow points to the value of 1 – F where the two curves diverge.

The changes in sensitivity and response amplitude are given in Figure 6A for light exposures ranging from 2 to 180 min (3 h). In Figure 6B, we show the relative sensitivity at each of the times at which measurements were made. The dashed red line is Equation 5 with Equation 4 substituted for F to account for the loss of pigment. For changes of relative sensitivity up to ∼10⁻⁴, this line gave a good fit to the data much as in previous work, although the value of the constant k (70) was somewhat larger in our whole-retina preparation than for single rods recorded with suction electrodes (35, Nymark et al., 2012; 24, Pahlberg et al., 2017). Beyond a relative sensitivity of 10⁻⁴, the data were better fit by a curve for which the fraction of rhodopsin remaining was no longer given by Equation 4 but was larger than predicted by this equation. This altered unbleached fraction is shown in Figure 6C. The dashed red line again gives the prediction of Equation 4 for the fraction of pigment remaining (1 – F), and the black continuous line is the concentration of rhodopsin required to fit the sensitivity measurements in Figure 6B for long exposures to the background light.

On the assumption that the rod continues to behave according to Equation 5 for longer background exposures, the difference between the red dashed and black continuous lines in Figure 6C gives the amount of additional rhodopsin present in the rods, which must have been formed by some process of regeneration. As we show in Figure 7, this regeneration seems not to occur in darkness but only in the presence of illumination. We therefore modeled this process by allowing bleached rhodopsin either to re-form rhodopsin with a light-dependent rate constant k_r, or to decay into free opsin and atRAL with a rate constant k_d (see Eqs. 2a and 2b). The fraction of rhodopsin calculated by the model is then given as the black continuous line in Figure 6C. The value of k_r that we obtained to the best fit of our data was 1.8 × 10⁻¹¹ µm², which is ∼0.3% of the photosensitivity of rhodopsin (Woodruff et al., 2004). These data indicate that the amount of additional pigment that is formed is small and can only be detected when the calculated fraction of pigment drops to <∼1%. From that point onward, some process produces enough additional rhodopsin to keep the pigment concentration nearly constant, so that sensitivity never drops below 10⁻⁵ to 10⁻⁴ of that in darkness.

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Figure 7.

Mechanism of regeneration. A, Response-intensity relations recorded before, during, and after the retinae were exposed to a 60 min, 560 nm background of 3.6 × 10⁵ ϕ µm⁻² s⁻¹. Responses are means from 5 retinae. B, Smaller responses from A, replotted on an expanded scale. C, Sensitivity during or after background exposure normalized to DA sensitivity (n = 5). Dashed vertical line at 0 min indicates when background was turned off. D, HPLC analysis of retinoid content in WT mouse retinae in dark (0 min) and after exposure to a 560 nm bleaching light of 1.0 × 10⁶ ϕ µm⁻² s⁻¹ (DA, n = 4; 30 min, n = 3; 60 min, n = 7, 90 min, n = 5). Retinoid levels normalized to those in darkness are given for 11-cis retinal (black), 9-cis retinal (red), and atRAL (blue). Open symbols represent sum of 9-cis and 11-cis retinal. Dotted horizontal line indicates estimate of 11-cis retinal in DA cones. The first data point for 9-cis retinal (0 min, DA) is uncertain because it is close to the detection limit of the instrument.

Similar data were obtained from single rods recorded with suction electrodes (Fig. 2C,D). Rods were exposed for 60 min to a 565 nm background light delivering 10⁶ ϕ µm⁻² s⁻¹. For this intensity and duration, Equation 4 predicts that the fraction of pigment remaining should have been ∼10⁻⁹, or a single molecule of rhodopsin every 15-20 rods. Relative sensitivity decreases to ∼10⁻⁵, much as for the 180 min exposure to the 3.6 × 10⁵ ϕ µm⁻² s⁻¹ background in Figure 6. These experiments confirm that regeneration can maintain the rhodopsin concentration at 0.1%-1% that in darkness, sufficient to allow the rods to continue to respond to bright, maintained illumination at least for several hours and perhaps indefinitely. And since the experiments of Figure 2C, D were done with suction-electrode recording on single rods, this process of regeneration must be occurring within the rod itself.

Possible mechanisms of regeneration

How does this regeneration occur? All of the experiments in Figures 1–4 were done either on single photoreceptors or with isolated retina. Every effort was made to remove all of the RPE when preparing the retina for recording. The background lights were chosen to be between 560 and 570 nm. At these wavelengths, there should be little activation of isomerization from retinal condensed with PE to form the retinyl-lipid, N-retinylidene-PE (N-ret-PE) (Kaylor et al., 2017) or retinal G-protein-coupled receptor opsin (Morshedian et al., 2019). The protocols of our experiments seem therefore to have eliminated the principal known mechanisms of 11-cis retinal chromophore regeneration.

Since, however, the amount of additional rhodopsin required in Figure 6C is quite small, it seemed to us possible that some residual process might supply chromophore to the rods. To see whether this mechanism operates in darkness, we exposed retinae to a background light of 3.6 × 10⁵ ϕ µm⁻² s⁻¹ for 60 min and then measured sensitivity as a function of time in darkness. These data are shown in Figure 7A, B. The black circles give the response-intensity values just before turning the background off, and the other curves show responses in darkness from 2-90 min. These curves seem to indicate that very little change in sensitivity occurs after the light is turned off.

We quantified these changes from Figure 7A, B in the same way as for Figure 3D and plotted the relative sensitivity S_F/ as a function of time in Figure 7C. The dotted vertical line indicates the time when the background light was extinguished. Sensitivity increased by a factor of ∼2 within the first 2 min, probably at least in part reflecting the removal of light adaptation after turning off the illumination. From 2 until 90 min, the sensitivity was nearly constant, varying by no more than a factor of 1.5. These data provide no evidence of any process in darkness that regenerates a significant amount of rhodopsin.

Another possibility is that visual pigment is regenerated by reversed photoisomerization of atRAL in the light. To investigate this possibility, we illuminated mouse retinas with a 560 nm background at an intensity of 10⁶ ϕ µm⁻² s⁻¹. At 30, 60, and 90 min, the retinas were removed and the retinoid content was evaluated with HPLC, which has greater sensitivity for small changes in retinoid species. We measured levels of both 11-cis and 9-cis retinals because both can form visual pigments (see, e.g., Hurley et al., 1977). The amount of 11-cis retinal was between 0.02 and 0.03 of that in darkness at all three time points of background exposure. The sum of the 11-cis and 9-cis retinal amounts varied from 0.04 to 0.06 of the amount of 11-cis retinal in the dark. Because these values did not change greatly with time, they may reflect a steady-state during the bright continuous illumination used in this experiment. Cones in mouse make up only ∼3% of the total photoreceptor population (Carter-Dawson and LaVail, 1979) and have outer-segment volumes ∼0.4 that of rods (Nikonov et al., 2006). The fraction of chromophore coming from the cones is therefore unlikely to have exceeded 1%, which we have indicated with the dashed horizontal line in Figure 7D. Most of the additional 11-cis and 9-cis retinals in Figure 7D are likely to have been produced by isomerization of atRAL released by bleaching of rhodopsin. This regeneration of chromophore could have originated from one or more of a number of mechanisms, including N-ret-PE isomerization, photoisomerization of unbound atRAL (see, e.g., Kropf and Hubbard, 1970), or reversal of one of the intermediates of rhodopsin bleaching, such as meta I, meta II, or meta III. We review these possibilities and their possible physiological significance below.