Spontaneous vesicle release is not tightly coupled to voltage-gated calcium channel-mediated Ca²⁺ influx and is triggered by a Ca²⁺ sensor other than synaptotagmin-2 at the juvenile mice calyx of held synapses

It is well known that voltage-gated calcium channels (VGCCs)-mediated Ca²⁺ influx triggers evoked synaptic vesicle release. However, the mechanisms of Ca²⁺ regulation of spontaneous miniature vesicle release (mini) remain poorly understood. Here we show that blocking VGCCs at the juvenile mice (C57BL/6) calyx of Held synapse failed to cause an immediate change in minis. Instead, it resulted in a significant reduction (~40%) of mini frequency several minutes after the blockage. By recordingVGCCactivity and single vesicle fusion events directly at the presynaptic terminal, we found that minis did not couple to VGCC-mediated Ca²⁺entry, arguing for a lack of direct correlation between mini and transient Ca²⁺ influx. Moreover, mini frequencies displayed a lower apparent Ca²⁺ cooperativity than those of evoked release. In agreement with this observation, abrogation of the Ca²⁺ sensor synaptotagmin-2 had no effect on apparent Ca²⁺cooperativity of minis. Together, our study provides the first direct evidence that spontaneous minis are not mediated by transient Ca²⁺ signals through VGCCs and are triggered by a Ca²⁺-sensing mechanism that is different from the evoked release at these microdomain VGCC–vesicle coupled synapses.