Calcium dependence of neurotransmitter release at a high fidelity synapse.

Abdelmoneim Eshra,Stefan Hallermann,Hartmut Schmidt,Jens Eilers

doi:10.7554/elife.70408

Abstract

The Ca2+-dependence of the priming, fusion, and replenishment of synaptic vesicles are fundamental parameters controlling neurotransmitter release and synaptic plasticity. Despite intense efforts, these important steps in the synaptic vesicles' cycle remain poorly understood due to the technical challenge in disentangling vesicle priming, fusion, and replenishment. Here, we investigated the Ca2+-sensitivity of these steps at mossy fiber synapses in the rodent cerebellum, which are characterized by fast vesicle replenishment mediating high-frequency signaling. We found that the basal free Ca2+ concentration (<200 nM) critically controls action potential-evoked release, indicating a high-affinity Ca2+ sensor for vesicle priming. Ca2+ uncaging experiments revealed a surprisingly shallow and non-saturating relationship between release rate and intracellular Ca2+ concentration up to 50 μM. The rate of vesicle replenishment during sustained elevated intracellular Ca2+ concentration exhibited little Ca2+-dependence. Finally, quantitative mechanistic release schemes with five Ca2+ binding steps incorporating rapid vesicle replenishment via parallel or sequential vesicle pools could explain our data. We thus show that co-existing high- and low-affinity Ca2+ sensors mediate priming, fusion, and replenishment of synaptic vesicles at a high-fidelity synapse.

Highlights

During chemical synaptic transmission Ca2+ ions diffuse through voltage-gated Ca2+ channels, bind to Ca2+ sensors, and thereby trigger the fusion of neurotransmitter-filled vesicles (Südhof, 2012)
We investigated the Ca2+-sensitivity of these steps at 7 cerebellar mossy fiber synapses, which are characterized by fast vesicle recruitment 8 mediating high-frequency signaling
Our results show the co-existence of Ca2+ sensors with high- and low-affinities that cover a large range of intracellular Ca2+ concentrations and mediate fast signaling at this synapse

Summary

Introduction

During chemical synaptic transmission Ca2+ ions diffuse through voltage-gated Ca2+ channels, bind to Ca2+ sensors, and thereby trigger the fusion of neurotransmitter-filled vesicles (Südhof, 2012). The technical development of caged Ca2+ compounds (Kaplan and Ellis-Davies, 1988) allows to experimentally elevate the Ca2+ concentration homogenously by photolysis and the direct measurement of the Ca2+-sensitivity of vesicle fusion (reviewed by Neher, 1998; Kochubey et al, 2011) First experiments with this technique at retinal bipolar cells of goldfish found a very low sensitivity of the release sensors with a half saturation at ∼100 μM Ca2+ concentration and a fourth to fifth order relationship between Ca2+ concentration and neurotransmitter release (Heidelberger et al, 1994), similar to previous estimates at the squid giant synapse (Adler et al, 1991; Llinás et al, 1992). Analysis at an inhibitory central synapse revealed a high-affinity Ca2+ sensor and in addition a profoundly Ca2+-dependent priming step (Sakaba, 2008)

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