The intracellular calcium concentration ([Ca²⁺]) plays a crucial role in triggering neurotransmitter release and regulating short-term plasticity (STP). High [Ca²⁺] concentrations within microdomains initiate transmitter release, while global increases in [Ca²⁺] influence short-term facilitation by accelerating vesicle recruitment. The calyx of Held synapse is used to study these processes, providing quantitative dose-response curves and kinetic information. The study reveals that [Ca²⁺] has two distinct roles: accelerating "molecular priming" (vesicle docking and machinery buildup) and promoting tight coupling between releasable vesicles and Ca²⁺ channels. This coupling is essential for vesicle sensitivity to short [Ca²⁺] transients during action potentials. The robustness of the release machinery is highlighted, with changes in synaptic properties often not reflecting a one-to-one relationship with molecular perturbations. The calyx of Held also demonstrates that vesicle docking and priming are enhanced by globally increased [Ca²⁺], contributing to short-term depression during sustained activity. The study further explores the mechanisms of facilitation, synchrony, and spontaneous release, emphasizing the complex interplay between vesicle exocytosis and vesicle pool availability. The calyx of Held's heterogeneous vesicle pool and its release kinetics are discussed, along with the role of [Ca²⁺]-dependent recruitment in short-term depression. The molecular mechanisms underlying vesicle heterogeneity and the rate-limiting steps for AP-induced release are also examined, suggesting that molecular priming and positional priming may play key roles.The intracellular calcium concentration ([Ca²⁺]) plays a crucial role in triggering neurotransmitter release and regulating short-term plasticity (STP). High [Ca²⁺] concentrations within microdomains initiate transmitter release, while global increases in [Ca²⁺] influence short-term facilitation by accelerating vesicle recruitment. The calyx of Held synapse is used to study these processes, providing quantitative dose-response curves and kinetic information. The study reveals that [Ca²⁺] has two distinct roles: accelerating "molecular priming" (vesicle docking and machinery buildup) and promoting tight coupling between releasable vesicles and Ca²⁺ channels. This coupling is essential for vesicle sensitivity to short [Ca²⁺] transients during action potentials. The robustness of the release machinery is highlighted, with changes in synaptic properties often not reflecting a one-to-one relationship with molecular perturbations. The calyx of Held also demonstrates that vesicle docking and priming are enhanced by globally increased [Ca²⁺], contributing to short-term depression during sustained activity. The study further explores the mechanisms of facilitation, synchrony, and spontaneous release, emphasizing the complex interplay between vesicle exocytosis and vesicle pool availability. The calyx of Held's heterogeneous vesicle pool and its release kinetics are discussed, along with the role of [Ca²⁺]-dependent recruitment in short-term depression. The molecular mechanisms underlying vesicle heterogeneity and the rate-limiting steps for AP-induced release are also examined, suggesting that molecular priming and positional priming may play key roles.