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. 2022 Dec 10;13:7656. doi: 10.1038/s41467-022-35417-9

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© The Author(s) 2022

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — A Schematic depicting cleavage of the synaptic vesicle SNARE complex by BONT-C expression (w;OK319-Gal4/+;UAS-BoNT-C/+), preventing all vesicle fusion. Representative electrophysiological traces and quantifications are shown for the indicated genotype, confirming no evoked or miniature transmission at NMJs silenced by BoNT-C. Quantification of average mEPSP amplitude (B), EPSP amplitude (C), and quantal content (D) values in the indicated genotypes (wild type: n = 8; OK319 > BoNT-C, n = 8, p < 0.0001 for mEPSP, p < 0.0001 for EPSP, unpaired, two-tailed t-test). B Schematic and postsynaptic Ca²⁺ imaging using a new SynapGCaMP8f indicator in wild type (w;SynapGCaMP8f/+) and BoNT-C silenced (+OK319 > BoNT-C: w;OK319-GAL4/+;SynapGCaMP8f/UAS-BoNT-C) NMJs. Quantification of transients, confirming no synaptic Ca²⁺ events in postsynaptic compartments following BoNT-C silencing. (wild type: n = 8; OK319 > BoNT-C, n = 8, p < 0.0001, unpaired, two-tailed t-test, with a significance value of 0.05). C Representative images of NMJs immunostained for pCaMKII, GluRIIA, and CaMKII in wild type, BoNT-C silenced, and GluRIIA mutants with BoNT-C silencing (w;OK319-GAL4,GluRIIA^SP16/GluRIIA^SP16;UAS-BoNT-C/+). Experiments were repeated three times independently with similar results. D Quantification of mean fluorescence intensity of the indicated antibodies (wild type: n = 12; OK319 > BoNT-C, n = 12, p = 0.1373 for pCaMKII, p = 0.3770 for GluRIIA, p = 0.9974 for CaMKII; OK319 > BoNT-C + GluRIIA^−/−: n = 10, p < 0.0001 for pCaMKII, p < 0.0001 for GluRIIA, p = 0.9835 for CaMKII). Repeated measures one-way ANOVA with Dunnett’s multiple comparisons test with a significance value of 0.05. p Value adjusted for multiple comparisons. Note that pCaMKII levels are unchanged in BoNT-C silenced NMJs, while pCaMKII is abolished by loss of GluRIIA. Error bars indicate ±SEM. Asterisks indicate statistical significance using t-test or one-way ANOVA: ****p < 0.0001; ns, not significant. n values indicate biologically independent NMJs. Absolute values for normalized data are summarized in Table S1. Source data are provided as a Source Data file.