Synaptotagmin-7 is required for synchronous but not asynchronous facilitation of glutamate release at cortical boutons

Short-term synaptic plasticity and the kinetics of neurotransmitter release vary widely across synapses, but population measurements obscure the mechanisms that generate this diversity. While the Ca ²⁺ sensor Synaptotagmin-7 (Syt7) has been implicated in facilitation, vesicle replenishment and asynchronous vesicle exocytosis, its precise contributions to these processes remain debated. We used quantal-resolution imaging to measure synchronous and asynchronous glutamate release at individual cortical boutons in wild type and Syt7 ^-/- neurons. Stratifying boutons by release efficacy and applying failure-based analysis to isolate trials where the first action potential evoked no release allowed us to separate facilitation from vesicle depletion. Syt7 deletion selectively eliminated activity-dependent facilitation of synchronous release but left facilitation of asynchronous release intact, although its overall magnitude was reduced. We further show that synchronous and asynchronous events arise from functionally distinct vesicle populations. These findings demonstrate that activity-dependent facilitation of synchronous and asynchronous exocytosis are mechanistically separable, enabling synapses to independently tune distinct temporal components of neurotransmission.