Nanoscale Distribution of Presynaptic Ca²⁺ Channels and Its Impact on Vesicular Release during Development

Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca²⁺ channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Ca_v2.1 channels, local [Ca²⁺] imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca²⁺ buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca²⁺ sensors for vesicular release are located at the perimeter of VGCC clusters (<30nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course.