Influence of ASD-Shank2 SNVs on excitatory glutamatergic synapses. A Representative images of hippocampal neurons transfected with EGFP, Shank2-wildtype or ASD-associated Shank2 SNVs, S557N, V717F or L1722P, cultured without (left) or with (right) 10 μM zinc supplementation from DIV 9 till DIV 16 (transfected neurons shown in green), and immuno-stained with VGluT1 (Alexa Fluor 594, shown in red) and Homer (Alexa Fluor 647, shown in blue). Scale bar is 20 μm. B Quantification of VGluT1 and Homer co-localised puncta density per 10 µm length of dendrite in hippocampal neurons transfected with EGFP, Shank2-wildtype or ASD-associated Shank2 SNVs, S557N, V717F or L1722P (above) and in transfected hippocampal neurons supplemented with 10 µM zinc from days in vitro (DIV) 9 till DIV 16 (below). C Cumulative probability graph of miniature excitatory post-synaptic current (mEPSC) inter-event interval in neurons expressing EGFP, Shank2-wildtype or ASD-associated Shank2 SNVs, S557N, V717F or L1722P. D Cumulative probability plot of mEPSC inter-event interval in control EGFP- and zinc treated EGFP-expressing hippocampal neurons. E Cumulative probability plot of mEPSC inter-event interval in control and zinc-treated Shank2-wildtype-expressing hippocampal neurons. (F–H) Cumulative probability plots of mEPSC inter-event intervals in S557N, V717F and L1722P-expressing neurons cultured in the presence of absence of zinc. Example mEPSC traces are shown for each Shank2 variant. I, J Bar graphs of mEPSC amplitudes from EGFP, Shank2WT, S557N, V717F, and L1722P-expressing neurons grown in control (I) and zinc supplemented (J) media. Data were statistically analysed using two-way ANOVA with Tukey’s multiple comparisons test. NS not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.