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. 2022 Nov 30;29(6):1683–1697. doi: 10.1038/s41380-022-01882-3

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Rat hippocampal neurons cotransfected with GFP-tagged human Shank2 constructs and a shShank2 vector, were stained with antibodies against GFP as well as PSD-95. Confocal and 2D STED imaging was performed on GFP-Shank2 clusters (a) or on PSD-95 clusters colocalising with Shank2 (e; areas of colocalisation are indicated in white, left panel). Single clusters of Shank2/PSD-95 observed in confocal mode were resolved into subclusters in super-resolution mode. Scale bar: 1 µm. Quantitative analysis was performed on 30 clusters from 10 neurons per each condition, obtained from three independent experiments. b The size of clusters in the confocal mode did not show any difference between WT Shank2 and the two mutant variants. c The size of Shank2 subclusters is significantly increased when expressing the G643R mutant compared to WT and L1800W. d Number of subclusters was significantly higher in clusters positive for WT Shank2 compared to both mutant variants. f The PSD-95 cluster size (confocal mode) was not significantly altered among the variants. g The size of PSD-95 subclusters was increased when expressing the G643R mutant compared to WT and L1800W. h Number of PSD-95 subclusters per cluster was significantly reduced for both mutant variants. **, ***, ****: significantly different, p < 0.01, 0.001, 0.0001 respectively; Kruskal–Wallis test, followed by Dunn’s multiple comparison test. i Miniature excitatory postsynaptic currents (mEPSCs) recorded in primary cultured hippocampal neurons cotransfected with shShank2 vector and human Shank2 construct. j Cumulative probability of mEPSC inter-event intervals was significantly altered for both variants. ****: p < 0.0001; Kolmogorov–Smirnov test. k The mean amplitude of mEPSCs was significantly larger in p.G643R variant cells compared to wild type cells. *p < 0.05; one-way ANOVA with Dunnett’s multiple comparison test.