Syt1’s release inhibitory function synchronizes release during high-frequency stimulation. Working model for the effect of Syt1’s release inhibitory and vesicle recruitment functions on release coming from rest (First evoked release; left column) and during high-frequency stimulation (right column), split between global Ca2+ induced release in the interpulse-interval (i.e. 75 ms), and peak Ca2+ induced release right after the AP (i.e. 25 ms). With vesicles not subject to Syt1’s release inhibitory function in green (unclamped), and clamped vesicles in red. (a,c,e,g) First evoked release after a period of rest during peak Ca2+ in WT synapses at 32 °C (a), in WT synapses at 22 °C (c), in Syt1 9Pro synapses at 32 °C with a shift towards unclamped vesicles due to impaired Syt1 release inhibition (e), and in Syt1 3K synapses at 32 °C with a decreased number of available vesicles due to impaired constitutive vesicle recruitment (g). (b,d,f,h) Late-train release during high-frequency stimulation, due to global Ca2+ (left), and peak Ca2+ (right), in WT synapses at 32 °C (b) where rapid transition of newly primed vesicles to the clamped (red) state suppresses asynchronous release due to global Ca2+ (b, left), preserving vesicles for synchronous release during peak Ca2+ (b, right). In WT synapses at 22 °C (d), where a slower on-rate of release inhibition leaves vesicles unclamped causing asynchronous release (d, left), and leaving insufficient vesicles for a synchronous response (d, right). In Syt1 9Pro synapses at 32 °C (f) where impaired release inhibition leaves vesicles unclamped (f, left), leaving too few vesicles for synchronous release (f, right). And in Syt1 3 K synapses at 32 °C (h) where global Ca2+ drives increased Ca2+-dependent recruitment, increasing the available pool for synchronous release (h, right). In all panels insets show representations of the post-synaptic response based on the depicted fusion events, with synchronous responses in dark grey, asynchronous responses in light grey, and the baseline represented by the dashed line.