Spatially non-overlapping Ca²⁺ signals drive distinct forms of neurotransmission

Calcium (Ca²⁺) signaling is tightly regulated within a presynaptic bouton. Here, we visualize Ca²⁺ signals within hippocampal presynaptic boutons using GCaMP8s tagged to synaptobrevin, a synaptic vesicle protein. We identify evoked presynaptic Ca²⁺ transients (ePreCTs) that derive from synchronized voltage-gated Ca²⁺ channel openings, spontaneous presynaptic Ca²⁺ transients (sPreCTs) that originate from ryanodine sensitive Ca²⁺ stores, and a baseline Ca²⁺ signal that arises from stochastic voltage-gated Ca²⁺ channel openings. We find that baseline Ca²⁺, but not sPreCTs, contributes to spontaneous glutamate release. We employ photobleaching as a use-dependent tool to probe nano-organization of Ca²⁺ 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 Ca²⁺ domains via both local and non-local synaptic vesicle turnover. Our findings reveal nanosegregation of Ca²⁺ signals within a presynaptic terminal that derive from multiple sources and in turn drive specific modes of neurotransmission.