Developmental regulation of calcium-dependent feedback in Xenopus rods

Abstract

The kinetics of activation and inactivation in the phototransduction pathway of developing Xenopus rods were studied. The gain of the activation steps in transduction (amplification) increased and photoresponses became more rapid as the rods matured from the larval to the adult stage. The time to peak was significantly shorter in adults (1.3 s) than tadpoles (2 s). Moreover, adult rods recovered twice as fast from saturating flashes than did larval rods without changes of the dominant time constant (2.5 s). Guanylate cyclase (GC) activity, determined using IBMX steps, increased in adult rods from approximately 1.1 s(-1) to 3.7 s(-1) 5 s after a saturating flash delivering 6,000 photoisomerizations. In larval rods, it increased from 1.8 s(-1) to 4.0 s(-1) 9 s after an equivalent flash. However, the ratio of amplification to the measured dark phosphodiesterase activity was constant. Guanylate cyclase-activating protein (GCAP1) levels and normalized Na+/Ca2+, K+ exchanger currents were increased in adults compared with tadpoles. Together, these results are consistent with the acceleration of the recovery phase in adult rods via developmental regulation of calcium homeostasis. Despite these large changes, the single photon response amplitude was approximately 0.6 pA throughout development. Reduction of calcium feedback with BAPTA increased adult single photon response amplitudes threefold and reduced its cutoff frequency to that observed with tadpole rods. Linear mathematical modeling suggests that calcium-dependent feedback can account for the observed differences in the power spectra of larval and adult rods. We conclude that larval Xenopus maximize sensitivity at the expense of slower response kinetics while adults maximize response kinetics at the expense of sensitivity.

Figure 1.

Light responses of larval and adult Xenopus rod photoreceptors. (A) Isolated rod photoreceptors from a stage 48 tadpole (left) and froglet (right) in the recording chamber following retinal dissociation. Typically rod outer segments (ROS) of froglet rods were three times the length of ROS in young tadpole rods. ROS diameters of both young tadpole and froglet rods used for this study had reached mature dimensions of 6–7 μm. Bar, 10 μm. (B) Photocurrents recorded from a tadpole rod using the suction electrode technique. Photocurrents were elicited by a series of 520-nm flashes, 20 ms in duration of the intensities 0.32, 1.1, 4.9, 12.0, 54.0, 123.7, 514.3 hυ/μm². Recordings of dim flashes are averages of eight responses, recordings of saturating flashes are averages of two responses. (C) Photocurrents recorded from a froglet rod stimulated as in B. Relative to tadpole rods, the saturating current tripled in magnitude and the responses were faster at onset and recovery. (D) Dependence of saturating current on rod length. The plot shows averaged values grouped according to stage of development: young tadpoles corresponding to stages 48–50, limbbud tadpoles corresponding to stages 54–56, and froglets. Double error bars indicate SEM. On average, froglet ROS were three times as long as ROS of young tadpoles. The saturating currents grew in the same proportion, suggesting that the density of the currents (0.3 pA/μm) does not change with development. Current density was estimated from the slope of the line interpolating the data and passing through the origin (R² > 0.99).

Figure 2.

Intensity–response curves of larval and adult Xenopus rod photoreceptors. (A) Responses are plotted as a function of flash intensity for tadpoles (black circles, n = 6) and froglets (gray triangles, n = 8). The amplitude of the responses of froglet rods were three times larger than the responses of tadpole rods, similar to the saturating currents. The gray circles represent the scaled (i.e., multiplied by three) responses of tadpole rods. (B) The squared value of the mean responses (black) was compared with the ensemble variance (gray) for young tadpole and froglet rods (16–25 flash repetitions). Flash intensity was 0.32 hυ/μm². Applying Poisson statistics to these examples, the estimated mean number of photoisomerizations per flash (R*) was 1.1 for the tadpole rod, and 2.5 for the froglet rod. The estimated values of the collecting areas were 3.8 and 8.6 μm² for the tadpole and froglet rods, respectively. (C) Tadpole and froglet rod collecting areas plotted as a function of ROS length. On average, the collecting areas grew in proportion to ROS length and triple in value, indicating that the optical density per unit of ROS length (pigment density) does not vary with stage of development. (D) Peak responses from A plotted as a function of photoisomerizations per flash (R*). On average, the response of both tadpole and froglet rods to single photoisomerizations (arrow) did not differ (∼0.6 pA). At low light intensities (0–10 R*), the responses from tadpole and froglet rods were the same.

Figure 3.

Time course of Xenopus rod responses to single photoisomerizations. (A) Averages of single photon responses from nine tadpole rods (black circles) and eight froglet rods (gray squares). On average, the population responses had the same amplitude of ∼0.6 pA, however the time-to-peak in tadpole rods (2 s) was considerably longer than that of froglet rods (1.3 s). In addition, the responses of froglet rods recover at a faster rate than those of tadpole rods. Error bars represent SEM and are shown every fourth data point for clarity. (B) Average single-photon responses normalized relative to the saturating currents. The normalized responses of tadpole rods (black circles) grew at a faster rate and reached higher amplitudes than those of froglet rods (gray squares). The solid black lines represent the fits of the activation theory of Pugh and Lamb (1993) to the rising phase of the normalized responses. The amplification of the tadpole rods was 1.6 times greater than in froglet rods, a difference that cannot be explained in terms of their respective ROS volumes. (C) Responses of froglet rods (gray squares) were scaled ×1.6 to match the amplification of the tadpole rods (black circles). The recovery of the froglet rod responses was significantly faster than that of the tadpole rod, suggesting that the shutoff mechanisms strengthen as the rod develops.

Figure 4.

Responses of Xenopus rods to saturating flashes. (A) Responses of tadpole (black) and froglet (gray) rods to saturating flashes producing ∼6 × 10³ photoisomerizations were recorded. Responses of froglet rods recovered faster from saturation than tadpole rods. Responses were normalized relative to their respective saturating currents. (B) Time to 50% recovery from saturation as a function of the number of photoisomerizations (R*) elicited by a saturating flash was measured. The recovery time of tadpole rods (black circles, n = 9) increased with flash intensity. Two linear segments were used to fit the data. The dominant time constant value of 2.5 s was estimated from the slope of the shallow segment. In response to flashes producing the same number of photoisomerizations, froglet rods (triangles, n = 8) recovered approximately twice as fast as tadpole rods. Recovery time of froglet rods increased with intensity at the same rate as that of tadpole rods but more intense flashes (15×) were required to produce similar recovery times. Displacement of the tadpole data by 2.7 ln units brought the two curves to a close match (gray circles). However, only a 0.5 ln units shift along the abscissa can be accounted for by the 1.6-fold increase in amplification in the tadpole rods. Therefore, the remaining ∼10-fold difference must result from a stronger shutoff mechanism present in froglet rods.

Figure 5.

Guanylate cyclase activity in developing Xenopus rods. (A) Overlay shows currents recorded from a froglet rod in response to brief applications of IBMX at various instances (periods) after stimulation with a saturating flash. Black trace is the control response to 6,000 photoisomerizations. The gray traces are a succession of responses with IBMX applied 1, 5, and 9 s after the flash. Pulse application of IBMX lasted 2 s, concentration was 0.5 mM. (B) Guanylate cyclase activity during the response to a saturating flash was estimated. In froglet rods (triangles, n = 5), resting GC activity (α'_dark) was ∼1.1 s⁻¹ and rose rapidly to reach a peak value of 3.8 s⁻¹, 5 s after the flash. In tadpole rods (circles, n = 6), α'_dark was 1.6× as active (1.8 s⁻¹), and increased gradually in response to the flash, requiring 9 s to reach its peak activity of ∼4 s⁻¹. (C) Relative guanylate cyclase activity (α'/α'_dark) of froglet rods (triangles) increased 3.5-fold in 5 s. α'/α'_dark in tadpole rods (circles) grew at a slower rate, increasing only 1.4-fold 5 s after the flash and reaching a peak 2.2-fold increase 9 s after the flash. In that same time (9 s after the flash), α'/α'_dark in the froglet rods had already recovered halfway to its resting value. (D and E) The time course of the recovery phase of α'/ α'_dark matched closely the recovery of the photocurrents in both tadpole and froglet rods. The shorter time to onset of recovery of the froglet rod response correlates with the fast increase in α'/α'_dark activity, while the slower onset of recovery in tadpole rods corresponds to a comparatively sluggish change in α'/α'_dark activity. These results suggest that feedback activity mediated by GC strengthens during development to speed up the recovery phase of the responses.

Figure 6.

GCAP1 immunolabeling in dark-adapted Xenopus retina. (A) In larval retinas (stage 48), anti-GCAP1 labeling was found mainly in cone photoreceptors with very weak labeling of rod outer segments. (B) In froglet retinas, anti-GCAP1 labeling was found in both cone and rod photoreceptors, although labeling was significantly stronger in cones than in rods. Note that GCAP1 staining in cones also increased as the retinas matured. In (C) control tadpole and (D) froglet retinas probed with secondary antibody, fluorescence levels can be hardly detected. All confocal images were processed identically.

Figure 7.

Calcium buffering in developing Xenopus rods. Population average single-photon responses from young tadpole rods (n = 6) and froglet rods (n = 6) loaded with BAPTA-AM were compared with control responses without BAPTA-AM. (A) The recovery of the single-photon response of tadpole rods with BAPTA-AM slowed down relative to tadpole control response. The single-photon response amplitude increased 1.6-fold with BAPTA-AM. (B) The single-photon response of froglet rods was greatly altered by BAPTA-AM. The onset of the response with BAPTA was coincident with the control response; however, the peak amplitude of the responses with BAPTA in the rods increased by almost threefold over the control amplitude. The recovery phase overshoots the baseline, the product of delayed activation of GC.

Figure 8.

Power spectral density of Xenopus rod single-photon responses in control conditions and when intracellular Ca²⁺ changes were buffered with BAPTA-AM. (A) Population average power spectra were computed from single-photon responses of froglet (squares) and tadpole (circles) rods in control solution and froglet (upward triangle) and tadpole (downward triangle) rods loaded with BAPTA-AM. Data were fit with piecewise-linear approximations. Arrows indicate approximate cutoff frequencies. Under both conditions, the tadpole rod spectra overlap closely over the entire frequency range. Above 0.3 Hz, the froglet rod spectra obtained in control and BAPTA-containing conditions also overlap one another. However, <0.3 Hz the spectrum of the BAPTA-loaded rods continues to gain power, while the control froglet spectrum diverges to superimpose on the low frequency power density of the froglet responses <∼0.15 Hz. (B) Population average power spectra of single photon responses were normalized relative to the square of their respective saturating current values. Spectra of tadpole rods (control and BAPTA) and froglet rod responses with BAPTA overlap closely. The spectrum of control froglet rod responses has lower power levels at frequencies <0.15 Hz. Data fit with model predictions (see appendix ).

Figure 9.

Measured and predicted power spectral densities of Xenopus rod single-photon responses. Power spectra of froglet rod responses with BAPTA (triangles) and under control conditions (squares) are compared with predictions from a linear model. Three parameters in the feedback stage of the model were varied independently to determine their influence on the shape of the spectrum. (A) the gain (H_f); (B) the cutoff frequency (β_f); and (C) the buffering capacity (ΔB_Ca), which is inversely related to both gain and cutoff frequency of the feedback loop (Eqs. A3–A5). Values assigned to the model parameters are indicated along with the respective functions.