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 Ca(2+) influx triggers evoked synaptic vesicle release. However, the mechanisms of Ca(2+) 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 recording VGCC activity and single vesicle fusion events directly at the presynaptic terminal, we found that minis did not couple to VGCC-mediated Ca(2+) entry, arguing for a lack of direct correlation between mini and transient Ca(2+) influx. Moreover, mini frequencies displayed a lower apparent Ca(2+) cooperativity than those of evoked release. In agreement with this observation, abrogation of the Ca(2+) sensor synaptotagmin-2 had no effect on apparent Ca(2+) cooperativity of minis. Together, our study provides the first direct evidence that spontaneous minis are not mediated by transient Ca(2+) signals through VGCCs and are triggered by a Ca(2+)-sensing mechanism that is different from the evoked release at these microdomain VGCC-vesicle coupled synapses.