Only a Few Ca2+ Channel Openings Are Required to Stimulate Exocytosis at the Cone Ribbon Synapse

Abstract

Purpose: : To determine the minimum number of L-type Ca²⁺ channel openings necessary for fusion of a single vesicle at the cone ribbon synapse.

Methods: : Synaptic release was evoked by depolarizing voltage-clamped cones while recording excitatory post-synaptic currents (EPSCs) simultaneously from OFF bipolar or horizontal cells in the salamander retina slice preparation.

Results: : Comparisons of EPSCs, cone Ca²⁺ currents (I_Ca), and single Ca²⁺ channel amplitude determined from variance-mean analysis of cone I_Ca suggested that ≤4 Ca²⁺ channel openings accompany fusion of each vesicle during the first few ms of depolarization. We refined this estimate by deconvolving the rate of Ca²⁺ channel openings from cone I_Ca and the rate of vesicle release from simultaneously-recorded EPSCs, finding that ≤3 channel openings are needed for vesicle fusion. Reducing channel open probability by use of weaker voltage steps or treatment with nifedipine did not alter efficiency suggesting few excess channel openings occur during strong depolarization. We simulated release at cone synapses using empirically-determined values for channel number, synaptic dimensions, vesicle pool size, Ca²⁺ -dependence of release, and Ca²⁺ channel properties. Computer simulations replicated experimental data if we assumed that the spread of Ca²⁺ is restricted by diffusion barriers or fast endogenous buffers. Consistent with diffusion barriers or immobile buffers, release efficiency was not significantly altered by introduction of different diffusible Ca²⁺ buffers into the cone terminal (0.5 mM EGTA, 5 mM EGTA, 1 mM BAPTA).

Conclusions: : Our results suggest that when cones are hyperpolarized (e.g., in bright light), there is an efficient coupling between Ca²⁺ channel opening and vesicle fusion at the cone synapse. Restricting the spread of Ca²⁺ with diffusion barriers (e.g., arciform density) and buffers prevents promiscuous fusion of multiple vesicles by a single channel opening. Tight coupling between Ca²⁺ channel opening and fusion may promote detection of small contrast changes in bright light conditions. By contrast, release rates in darkness are governed by Ca²⁺-dependent delivery of vesicles down the ribbon resulting in a looser coupling between channel openings and fusion that helps to reduce synaptic noise.

Keywords: synapse • retina: distal (photoreceptors, horizontal cells, bipolar cells) • photoreceptors