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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. 2 — A Schematic illustrating that loss of GluRA receptors decreases postsynaptic Ca²⁺ influx and may reduce pCaMKII levels at postsynaptic compartments. B Membrane topology of the GluRIIA subunit with the Q615R mutation shown in the pore-forming M2 domain. C Schematics illustrating Ca²⁺ permeability through GluRA receptors and the Ca²⁺ buffer parvalbumin (PV), with the associated reductions in Ca²⁺ observed in postsynaptic compartments. Genotypes: GluRIIA^Q615R (w;GluRIIA^Q615R); G14 > PV (w;G14-GAL4/+;UAS-PV/+); GluRIIA^Q615R + G14 > PV (w;GluRIIA^Q615R,G14-GAL4/GluRIIA^Q615R;UAS-PV/+). D Schematized GluRs and postsynaptic Ca²⁺ levels with representative Ca²⁺ imaging traces. Line scans below are derived from postsynaptic GCaMP6f images of individual spontaneous Ca²⁺ transients in the indicated genotypes: wild type (w;SynapGCaMP6f/+), GluRIIA^−/− (w;GluRIIA^SP16;SynapGCaMP6f/+), GluRIIA^Q615R (w;GluRIIA^Q615R;SynapGCaMP6f/+), G14 > PV (w;G14-GAL4/+;UAS-PV/SynapGCaMP6f), GluRIIA^Q615R + G14 > PV (w;GluRIIA^Q615R,G14-GAL4/GluRIIA^Q615R;UAS-PV/SynapGCaMP6f). E Representative images of NMJ boutons immunostained with anti-GluRIIA, -GluRIIB, -GluRIID, -pCaMKII, -CaMKII, and -DLG antibodies in the indicated genotypes shown in (D) without SynapGCaMP6f expression. Experiments were repeated four times independently with similar results. F Quantification of the normalized changes in fluorescence intensity (ΔF/F) of spontaneous Ca²⁺ transient events at individual boutons in the indicated genotypes in (D) (wild type: n = 12; GluRIIA^−/−, n = 12, p < 0.0001; GluR^Q615R: n = 12, p < 0.0001; G14 > PV: n = 11, p < 0.0001; GluRIIA^Q615R + G14 > PV: n = 12, p < 0.0001). Repeated measures one-way ANOVA with Dunnett’s multiple comparisons test with a significance value of 0.05. p Value adjusted for multiple comparisons. G Quantification of the mean fluorescence intensity of anti-GluRIIA, -GluRIIB, -GluRIID, -pCaMKII, -CaMKII, and -DLG in the indicated genotypes normalized to wild-type values. (wild type: n = 18; GluRIIA^−/−, n = 12, p < 0.0001 for GluRIIA, p < 0.0001 for GluRIIB, p = 0.9490 for GluRIID, p < 0.0001 for pCaMKII, p = 0.9848 for CaMKII, p = 0.0010 for DLG; GluRIIA^Q615R: n = 12, p = 0.6473 for GluRIIA, p > 0.9999 for GluRIIB, p = 0.9889 for GluRIID, p = 0.3169 for pCaMKII, p = 0.9971 for CaMKII, p = 0.6843 for DLG; G14 > PV: n = 11, p = 0.8479 for GluRIIA, p = 0.7851 for GluRIIB, p = 0.6389 for GluRIID, p = 0.0547 for pCaMKII, p = 0.4991 for CaMKII, p = 0.0616 for DLG; GluRIIA^Q615R + G14 > PV: n = 12, p = 0.8829 for GluRIIA, p = 0.4087 for GluRIIB, p > 0.9999 for GluRIID, p = 0.9980 for pCaMKII, p = 0.9965 for CaMKII, p = 0.9903 for DLG). Repeated measures One-way ANOVA with Dunnett’s multiple comparisons test with a significance value of 0.05. p Value adjusted for multiple comparisons. Error bars indicate ±SEM. Asterisks indicate statistical significance using One-way ANOVA: *p < 0.05, **p < 0.01, ****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.