Calcium-induced calcium release in rod photoreceptor terminals boosts synaptic transmission during maintained depolarization

Abstract

We examined the contribution of calcium-induced calcium release (CICR) to synaptic transmission from rod photoreceptor terminals. Whole-cell recording and confocal calcium imaging experiments were conducted on rods with intact synaptic terminals in a retinal slice preparation from salamander. Low concentrations of ryanodine stimulated calcium increases in rod terminals, consistent with the presence of ryanodine receptors. Application of strong depolarizing steps (-70 to -10 mV) exceeding 200 ms or longer in duration evoked a wave of calcium that spread across the synaptic terminals of voltage-clamped rods. This secondary calcium increase was blocked by high concentrations of ryanodine, indicating it was due to CICR. Ryanodine (50 microm) had no significant effect on rod calcium current (I(ca)) although it slightly diminished rod light-evoked voltage responses. Bath application of 50 microm ryanodine strongly inhibited light-evoked currents in horizontal cells. Whether applied extracellularly or delivered into the rod cell through the patch pipette, ryanodine (50 microm) also inhibited excitatory post-synaptic currents (EPSCs) evoked in horizontal cells by depolarizing steps applied to rods. Ryanodine caused a preferential reduction in the later portions of EPSCs evoked by depolarizing steps of 200 ms or longer. These results indicate that CICR enhances calcium increases in rod terminals evoked by sustained depolarization, which in turn acts to boost synaptic exocytosis from rods.

Fig. 1

Bath-applied ryanodine (1 μM) stimulated Ca²⁺ increases in terminals and cell bodies of rods loaded with the calcium-sensitive dye Fluo4. (A) Control image. (B) Image obtained after bath application of ryanodine for 3 min. The images show stacks of confocal sections taken at 1-μm intervals. Image acquisition time was 125 ms per confocal slice. Rod terminals are indicated by arrows; readily detectable increases are visible in the two terminals at right; little increase was observed in the terminal at left. Ryanodine was bath applied for 3 min. Scale bar, 10 μm.

Fig. 2

Changes in [Ca²⁺]_i in the synaptic terminal of a voltage-clamped rod in the retinal slice evoked by depolarizing steps (−70 to −10 mV). Calcium changes were visualized by including a high-affinity calcium-sensitive dye, Oregon Green 488 BAPTA-1 (100 μM; K_d = 0.17 μM), in the patch pipette solution. This single confocal section was cropped to show the terminal, axon and base of the cell body. The top row (A) shows responses to a step of 50 ms; the bottom row (B) shows responses to a step of 500 ms. Image timing is shown diagramatically below each set of images. Image 1 is a control image obtained prior to the step. Image 2 shows the image obtained at the beginning of the test step. For the 50-ms step (A), image 3 was obtained 250 ms after the end of the 50-ms test step whereas for the 500-ms step (B), image 3 was obtained 300 ms into the test step. Image 4 in A and B were both obtained 2.5 s after the beginning of the test step. Arrows point to a local hot spot of Ca²⁺ increase. Image acquisition time: 55 ms. Scale bar, 10 μm.

Fig. 3

Changes in [Ca²⁺]_i in a single confocal slice from a rod terminal in the retinal slice evoked by depolarizing steps (−70 to −10 mV). Calcium changes were visualized by including a low-affinity calcium-sensitive dye, Oregon Green 488 BAPTA-5N (100 μM; K_d = 20 μM), in the patch pipette solution. The images show the terminal, axon and base of the cell soma. The top row (A) shows responses to a step of 50 ms; the bottom row (B) shows responses to a step of 500 ms. Image timing is shown diagramatically below each set of images. Image 1 shows a control image obtained prior to the step. Image 2 shows the image obtained at the beginning of the test step. For the 50-ms step (A), image 3 shows the image beginning 400 ms after termination of the 50-ms test step. For the 500-ms step (B), image 3 shows the last image obtained during the test step. Arrows indicate a local hot spot of Ca²⁺ increase. Image acquisition time: 48 ms. Scale bar, 10 μm.

Fig. 4

Intraterminal calcium levels increase with duration of the test step (−70 to −10 mV). Calcium levels (ΔF/F) were measured using the dye Oregon Green 488 BAPTA 5N at the hot spot in rod terminals. In control conditions, calcium levels (filled squares and solid line, n = 7) increased abruptly when the step was lengthened from 100 to 200 ms, accompanying the spread of calcium through the terminal illustrated in Figs 2 and 3. Introducing ryanodine (25 μM, open squares and dashed line, n = 5) into rods through the patch pipette abolished the abrupt increase in calcium levels measured at the hot spot, in addition to inhibiting the secondary spread evoked by longer steps as shown in Fig. 5.

Fig. 5

Addition of ryanodine (25 μM) to the pipette solution reduced the secondary spread of calcium through the rod terminal. Changes in [Ca²⁺]_i in a single confocal slice from a rod terminal in the retinal slice evoked by depolarizing steps (−70 to −10 mV, 500 ms). The images show the terminal and cell soma. Image timing is shown diagramatically below the images. Image 1 shows a control image obtained prior to the step. Image 2 shows the first image obtained during the test step. Image 3 shows the last image obtained during the 500-ms test step. Arrows indicate a local hot spot of Ca²⁺ increase. Pipette solution contained the low-affinity Ca²⁺ sensitive dye Oregon Green 488 BAPTA-5N (100 μM). Image acquisition time: 48 ms. Scale bar, 10 μm.

Fig. 6

Bath-applied ryanodine (50 μM) strongly inhibited light-evoked currents in a horizontal cell (HC; panels A and B) but produced only a weak inhibition of light-evoked voltage responses in rods (C) and had no effect on rod I_Ca measured using a ramp voltage protocol (D). The three traces from A are superimposed in B to illustrate better ryanodine-induced changes in the waveform of the light-evoked current. For the records in panels B, C and D: solid black trace, control; grey trace, ryanodine; and thin dotted black trace, wash.

Fig. 7

Blocking CICR with high concentrations of ryanodine inhibited EPSCs evoked in horizontal cells by depolarizing steps (−70 to −10 mV, 200 ms) applied to rods. (A) Bath-applied ryanodine (50 μM) inhibited a later component of the EPSC. The EPSC recovered following washout. (B) Introducing ryanodine (50 μM) directly into the rod through the patch pipette also reduced a later component of the EPSC. The control record was obtained immediately after rupturing into the rod and the ryanodine record was obtained 4 min later. (C) EPSCs remained stable for long periods in control recordings in which ryanodine was omitted from the pipette solution. The record on the left was obtained immediately after patch rupture and the records to the right were obtained 15 and 45 min later, respectively. As illustrated by the record obtained 45 min after patch rupture, response rundown was accompanied by a preferential reduction in the early, fast component of the EPSC, not the later portions that were preferentially reduced by ryanodine.

Fig. 8

Intracellular ryanodine (50 μM) slightly reduced EPSC charge transfer for steps ≥ 200 ms, but did not significantly alter the initial component of release from rods. Data were fit with the bi-exponential function A = Amp₁(1 − exp(−t/τ₁) + Amp₂(1 − exp(−t/τ₂) where A = amplitude of normalized EPSC charge transfer and t = step duration. Control (solid line): τ₁ = 19.8 ms, Amp₁ = 0.89, τ₂ = 2.6 s, Amp₂ = 4.3. Ryanodine (dashed line): τ₁ = 17.7 ms, Amp₁ = 0.93, τ₂ = 72.4 s, Amp₂ = 54.2. Control (filled squares): n = 10. Ryanodine (open circles): n = 7.