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

Abstract

It is well known that voltage-gated calcium channels (VGCCs)-mediated Ca2ϩ influx triggers evoked synaptic vesicle release. However, the mechanisms of Ca 2ϩ 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 (ϳ40%) 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 2ϩ entry, arguing for a lack of direct correlation between mini and transient Ca 2ϩ influx. Moreover, mini frequencies displayed a lower apparent Ca 2ϩ cooperativity than those of evoked release. In agreement with this observation, abrogation of the Ca 2ϩ sensor synaptotagmin-2 had no effect on apparent Ca 2ϩ cooperativity of minis. Together, our study provides the first direct evidence that spontaneous minis are not mediated by transient Ca 2ϩ signals through VGCCs and are triggered by a Ca 2ϩ -sensing mechanism that is different from the evoked release at these microdomain VGCC-vesicle coupled synapses.

Calcium dependence of spontaneous neurotransmitter release

“…At neocortical excitatory and inhibitory synapses, the dependence of spontaneous release on [Ca 2+ ] o was similarly low ( n = 0.45–0.63) (Groffen et al, ; Vyleta & Smith, ; Williams et al, ). Likewise, at the calyx of Held, a large central synapse, n for spontaneous release is 0.43 (Dai, Chen, Tian, & Sun, ). Overall spontaneous release is [Ca 2+ ] o dependent, albeit with a weaker concentration dependence than evoked release.…”

“…While a majority of groups reported that VACCs do not trigger mEPSCs at conventional central synapses in culture and acute brain slices, others report the contrary (Dai et al, ; Ermolyuk et al, ; Xu, Jun, Pang, Ok‐Ho, & Südhof, ). One hypothesis to explain the discrepancy is that Cd 2+ may enter the cell and increase [Ca 2+ ] i via off‐target mechanisms (Ermolyuk et al, ), leading to vesicle depletion so that block of VACCs has no detectable impact on synaptic transmission.…”

Sr2+ has low efficiency in regulating spontaneous release at the Calyx of Held synapses

“…Many factors regulate the resting intraterminal Ca 2+ ‐concentration which largely contributes to the Ca 2+ ‐dependent minis (Dai et al, ; Ermolyuk et al, ; Pang & Sudhof, ; Sun et al, ; Vyleta & Smith, ). The major contributing factors include the stochastic Ca 2+ ‐channel opening, Ca 2+ release from internal Ca 2+ ‐stores and Ca 2+ ‐sensing receptor (CaSR, a Ca 2+ ‐sensing G‐protein coupled receptors) (Coulombe, Faure, Robin, & Ruat, ; Dai et al, ; Ermolyuk et al, ; Friel & Chiel, ; Lou, Scheuss, & Schneggenburger, ; Pang & Sudhof, ; Sun et al, ; Vyleta & Smith, ). We first observed the divalent cationic influx when extracellular Ca 2+ was switched to Sr 2+ .…”

“…Single current from VGCCs was recorded in the presence of 1 μM tetrodotoxin (TTX), 20 mM TEA–Cl as described in detail previously (Dai, Chen, Tian, & Sun, ). Changing Ca 2+ ‐concentrations was always balanced by adjusting the concentration of NaCl to keep the osmolarity.…”

“…Single Ca 2+ /Sr 2+ currents were recorded by EPC‐10 amplifier (HEKA) with sampling frequency of 20 kHz and online low‐pass filtering at 5 kHz. The Clampfit software (Molecular Device) was used for offline analyses of single channel signals, including low‐pass filtering at 300 Hz and identifying single channel open events (Dai et al, ). We recorded the single VGCC activities in On‐Cell mode with HEKA 10 and set the holding voltage of the pipette to 0 mV (same as the reference potential in the bath solution), to make the membrane potential as resting potential and recorded the spontaneous openings of single VGCCs at rest state.…”