(A) Exemplary AP evoked EPSCs of wildtype hippocampal autaptic neurons (black, n = 23) and syb2 ko neurons (blue, n = 7) or syb2 ko neurons expressing the syb2 mutant variants (polyL, red, n = 13 and polyV, green, n = 12). (B1–B3) Loss of syb2 abolishes evoked release (syb2 ko). Expression of the polyV mutant in syb2ko neurons rescues EPSC amplitude and charge, while the polyL mutant significantly reduces the evoked amplitude and charge compared to control. The time to reach the maximum EPSC amplitude (TTP) is unaffected by the mutants when compared to wt. (C) Whole cell responses from wildtype (black, n = 22), syb2 ko (blue, n = 4), syb2 ko + polyL (red, n = 9) and syb2 ko + polyV (green, n = 11) neurons evoked by application of 500 mM hypertonic sucrose solution. (D) The readily releasable pool charge (RRP, as quantified from hypertonic stimulation shown in C) is fully (polyV) or only partially (polyL) restored to levels of wt cells. (E) Exemplary recordings of mEPSCs activity from syb2 ko neurons expressing syb2, polyL or polyV mutant. (F) The polyL mutant reduces the mEPSC frequency compared to wildtype and ko+syb2 while polyV fully rescues release. (G) Amplitude of the quantal events is not changed in the TMD mutants compared to controls. Data for (E–F) are collected from the following number of cells: wildtype (38), syb2 ko (8), ko+syb2 (52), ko+polyL (23) and ko+polyV (21). ANOVA followed by Kruskal-Wallis post hoc test was performed. (H) Representative confocal images of syb2 ko neurons expressing either syb2 or its mutant variants. To illustrate the synaptic localization of the syb2 signal, neurons were co-stained for the synaptic vesicle marker protein Synaptophysin. (I) Quantification of the immunosignals for syb2 and its mutants. The average fluorescence intensity of syb2 is unchanged for the syb2 variants and reaches the levels observed for wildtype neurons. Note that the syb2 staining is absent in syb2 ko neurons (wt, n = 18; syb2 ko, n = 14; syb2 ko+syb2, n = 16; syb2 ko+polyL, n = 15; syb2ko+polyV, n = 18). (J) The synapse density (determined from the number of synaptophysin positive puncta per 50 µm neurite length) is indistinguishable in wt cells or the syb2ko neurons expressing either syb2 or the mutant variants. Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, one-way analysis of variance versus control.