Release from the cone ribbon synapse under bright light conditions can be controlled by the opening of only a few Ca(2+) channels

Abstract

Light hyperpolarizes cone photoreceptors, causing synaptic voltage-gated Ca(2+) channels to open infrequently. To understand neurotransmission under these conditions, we determined the number of L-type Ca(2+) channel openings necessary for vesicle fusion at the cone ribbon synapse. Ca(2+) currents (I(Ca)) were activated in voltage-clamped cones, and excitatory postsynaptic currents (EPSCs) were recorded from horizontal cells in the salamander retina slice preparation. Ca(2+) channel number and single-channel current amplitude were calculated by mean-variance analysis of I(Ca). Two different comparisons-one comparing average numbers of release events to average I(Ca) amplitude and the other involving deconvolution of both EPSCs and simultaneously recorded cone I(Ca)-suggested that fewer than three Ca(2+) channel openings accompanied fusion of each vesicle at the peak of release during the first few milliseconds of stimulation. Opening fewer Ca(2+) channels did not enhance fusion efficiency, suggesting that few unnecessary channel openings occurred during strong depolarization. We simulated release at the cone synapse, using empirically determined synaptic dimensions, vesicle pool size, Ca(2+) dependence of release, Ca(2+) channel number, and Ca(2+) channel properties. The model replicated observations when a barrier was added to slow Ca(2+) diffusion. Consistent with the presence of a diffusion barrier, dialyzing cones with diffusible Ca(2+) buffers did not affect release efficiency. The tight clustering of Ca(2+) channels, along with a high-Ca(2+) affinity release mechanism and diffusion barrier, promotes a linear coupling between Ca(2+) influx and vesicle fusion. This may improve detection of small light decrements when cones are hyperpolarized by bright light.

Fig. 1.

Variance-mean analysis of cone I_Ba tail currents provides an estimate of the number of Ca²⁺ channels per cone terminal. A: overlay of 100 I_Ba tail currents evoked by 2-ms test steps in 2 mM Ba²⁺ and 5 μM BayK8644. Passive membrane properties were subtracted with a P/100 leak subtraction protocol. Inset: overlaid tail currents and region of nonstationary fluctuation analysis. The increase in variance can be seen from the thickening of the family of overlaid traces. B: variance and mean of I_Ba tail currents were plotted and fit with a parabolic equation (see materials and methods). In this example, the fit yielded a single-channel current (I) of −1.29 pA and 1,164 channels (N).

Fig. 2.

Deconvolution of Ca²⁺ channel openings and release rates from the presynaptic I_Ca and excitatory postsynaptic current (EPSC) of a simultaneously voltage-clamped cone and horizontal cell provides an estimate of Ca²⁺ channel cooperativity. In these experiments, we used 1.8 mM Ca²⁺ without BayK8644. A: cone I_Ca overlaid on the EPSC recorded simultaneously from a horizontal cell. B: rate of Ca²⁺ channel openings per millisecond per ribbon obtained from deconvolution of I_Ca (see materials and methods). Deconvolution of the EPSC with the average miniature EPSC (mEPSC) waveform gives the release rate per ribbon. In this example, release rate was normalized for 6 ribbon contacts. C: dividing the release rate per ribbon by the rate of Ca²⁺ channel openings per ribbon gives the number of release events per opening. The reciprocal value provides the number of channel openings per fusion event. In this example, peak efficiency of 0.3 vesicle fusion events per channel opening (3.3 channel openings/fusion event) was reached at 20.9 ms, 4.7 ms after the beginning of the test step.

Fig. 3.

Efficiency of release evoked by activation of I_Ca with a ramp voltage protocol. A: overlay of cone I_Ca evoked by a ramp voltage protocol (−90 to + 60 mV, 0.5 mV/ms) with the EPSC recorded from a simultaneously voltage-clamped horizontal cell. B: deconvolution of I_Ca and the EPSC showed a peak cooperativity of 0.56 release events per opening (1.8 openings/simultaneous fusion event). Traces are plotted as a function of voltage applied during the ramp protocol (V_m).

Fig. 4.

Efficiency of release evoked by activation of I_Ca with a step to −30 mV. A: overlay of cone I_Ca with the EPSC recorded simultaneously from a horizontal cell during a step depolarization from −70 mV to −30 mV (100 ms). V_h, holding potential. B: deconvolution of I_Ca and the EPSC showed a peak efficiency of 0.43 release events per opening (or 2.3 channel openings/fusion event).

Fig. 5.

Effects of changing Ca²⁺ channel open probability with 3 μM nifedipine. A: cone I_Ca overlaid on the simultaneously recorded horizontal cell EPSC evoked by a step from −70 mV to −10 mV (100 ms) applied to the cone in the presence of 3 μM nifedipine. A bright UV flash was applied 100 ms into the step to reduce nifedipine antagonism of Ca²⁺ channels. B: release events per channel opening deconvolved from the EPSC and I_Ca at the beginning of the test step. C: release events per channel opening deconvolved from the EPSC and I_Ca after reduction of nifedipine antagonism with the UV flash.

Fig. 6.

Effects of Ca²⁺ buffering on release efficiency. A, C, and E: cone I_Ca evoked by a step from −70 mV to −10 mV (100 ms) overlaid on the simultaneously recorded horizontal cell EPSC. B, D, and F: number of vesicle fusion events per channel opening obtained by deconvolution of the EPSC and I_Ca, respectively. In A and B, the cone pipette solution contained 0.5 mM EGTA. In C and D, the cone pipette solution contained 1 mM BAPTA. In E and F, we used a gramicidin-perforated patch recording technique to maintain endogenous Ca²⁺ buffering. G: average peak efficiency of release obtained when using a step depolarization to −10 mV and cone Ca²⁺ buffering provided by 5 mM EGTA (n = 18), 0.5 mM EGTA (n = 6), 1 mM BAPTA (n = 6), or endogenous Ca²⁺ buffers (perforated patch, n = 5). H: average peak efficiency of release with these same buffers obtained with a ramp voltage protocol (0.5 mV/ms; 5 mM EGTA, n = 9; 0.5 mM EGTA, n = 6; 1 mM BAPTA, n = 6; perforated patch, n = 5).

Fig. 7.

Simulations of release at the cone ribbon synapse. A: diagram of the geometric arrangements used for the original model without a diffusion barrier. B: release rates (dashed line) and Ca²⁺ channel openings (solid trace) per ribbon predicted by the model using 5 mM EGTA as the intracellular Ca²⁺ buffer in the cone terminal. C: release rates (dashed line) and Ca²⁺ channel openings (solid trace) per ribbon predicted by the model using 1 mM BAPTA as the intracellular Ca²⁺ buffer. D: release events per Ca²⁺ channel opening (5 mM EGTA, 1 mM BAPTA).

Fig. 8.

Effects of changing model parameters on simulations of release. In A–C, we used the exocytotic Ca²⁺ sensor properties found for Mb1 goldfish bipolar cells by Heidelberger et al. (1994). In D–F, we used Ca²⁺ channel properties (maximum open probability = 0.015 and single-channel current = 0.6 pA) reported for heterologously expressed Ca_V1.4 channels by Doering et al. (2005). In G–I, we simulated a 10-fold increase in channel number by lowering Ca²⁺ channel open probability to 0.035. In J–L, we increased the distance between Ca²⁺ channels and release sites by 50 nm. For all 4 of these manipulations, we show the predicted release rates (dashed lines) and Ca²⁺ channel openings (solid traces) per ribbon when using 5 mM EGTA (A, D, G, and J) or 1 mM BAPTA (B, E, H, and K). We also illustrate predicted release events per Ca²⁺ channel opening with both buffers in C, F, I, and L (5 mM EGTA, 1 mM BAPTA).

Fig. 9.

Simulations of release in the presence of a diffusion barrier. A: diagram illustrating the cone ribbon synapse geometry with inclusion of a diffusion barrier that slowed diffusion (1/200). B: release rates (dashed lines) and Ca²⁺ channel openings (solid trace) per ribbon predicted by the model using 5 mM EGTA as the intracellular Ca²⁺ buffer in the cone terminal. C: release rates (dashed lines) and Ca²⁺ channel openings (solid trace) per ribbon predicted using 1 mM BAPTA as the intracellular Ca²⁺ buffer. D: release events per Ca²⁺ channel opening (5 mM EGTA, 1 mM BAPTA).