Fig. 4: Asynchronous release is elevated in parallel with short-term facilitation in low release efficacy synapses.

a Analysis of the relationship between short-term plasticity, synchronicity and efficacy of glutamate release among synaptic outputs of single neurons. Paired-pulse stimulation paradigm and synaptic responses recorded in 2 representative boutons. As in Fig. 1, horizontal green lines show the amplitude of SF-iGluSnFR signal corresponding to the release of a single vesicle quanta (q). Vertical dashed lines depict action potential (AP) timings. Grey bars, 10 s intersweep intervals. Blue and red circles mark synchronous and asynchronous release events, respectively. b–d The relationships between: (b) asynchronous release fraction n_A/n_T and the overall release efficacy n_T, (c) paired-pulse ratio (PPR) and n_T, and (d) n_A/n_T and PPR in (i) a represenrtative cell (n = 137 boutons) and (ii, iii) across all recorded cells (N = 20 cells from 7 culture preparations, each recorded neuron contained at least 50 boutons). (i) Vertical dashed lines mark the subdivision of boutons either according to n_T (T1, T2 and T3, as in Fig. 3) or according to PPR (Depressing/Facilitating). Horizontal dashed lines depict either the average fraction of asynchronous release or the average PPR. Black dots with error bars depict average values within each group (mean ± SEM). (ii) Bar and dot summary plots (mean ± SEM), data from individual cells are connected by dashed lines. (iii) Average values in each group among all recorded cells (mean ± SEM). *** p < 0.001, repeated measures ANOVA (b and c) and two-tailed paired t-test (d) (for exact p values, all pairwise multiple comparisons, and further details of statistical analysis see SourceData.xlsx file).