Spatially non-overlapping Ca signals drive distinct forms of neurotransmission.

Calcium (Ca) signaling is tightly regulated within a presynaptic bouton. Here, we visualize Ca signals within hippocampal presynaptic boutons using GCaMP8s tagged to synaptobrevin, a synaptic vesicle protein. We identify evoked presynaptic Ca transients (ePreCTs) that derive from synchronized voltage-gated Ca channel openings, spontaneous presynaptic Ca transients (sPreCTs) that originate from ryanodine sensitive Ca stores, and a baseline Ca signal that arises from stochastic voltage-gated Ca channel openings. We find that baseline Ca, but not sPreCTs, contributes to spontaneous glutamate release. We employ photobleaching as a use-dependent tool to probe nano-organization of Ca signals and observe that all three occur in non-overlapping domains within the synapse at near-resting conditions. However, increased depolarization induces intermixing of these Ca domains via both local and non-local synaptic vesicle turnover. Our findings reveal nanosegregation of Ca signals within a presynaptic terminal that derive from multiple sources and in turn drive specific modes of neurotransmission.