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. 2024 Apr 22;15:3406. doi: 10.1038/s41467-024-47571-3

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© The Author(s) 2024

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 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. 1 — a Top, an overlaid image of FM1-43⁺ boutons and MAP2⁺ dendrites. Bottom, magnification of boxes and 3D pseudo-color images to show FM1-43 fluorescence (indicative of Pr) of synapses. Punctum height, peak fluorescent intensity. b Plot of functional synapse density (D) against mean Pr ( $\bar{\Pr}$ ) at distal dendritic branches (n = 66, 67 branches from 5, 5 biological repeats). Arrows, the trend towards D^Hi $\bar{\Pr}$ ^Lo or D^Lo $\bar{\Pr}$ ^Hi configuration. Inset, plots of D against 1/Pr (top) and total Pr (ΣPr) against $\bar{\Pr}$ (bottom). Hyperbolic fitting, R² = 0.87, P < 0.0001. Linear regressions, R² = 0.84, 0.0056; P < 0.0001, = 0.25. Blue lines/error bands, fitted curves/95% confidence interval (CI). Biological repeats, individual coverslips of culture. c Shifted Pr distribution (n = 863, 885 boutons from 5, 4 repeats; two-sided Kolmogorov–Smirnov test, ****P < 0.0001). Dashed line indicates where Pr = 0.4. d Left, confocal images to show FM1-43 staining elicited by single AP (1AP) inputs (30AP@0.5 Hz) and burst inputs (6 repetitive trains of 5AP@100 Hz burst) in the same boutons. Black and white arrows, examples of high-Pr and low-Pr boutons. Middle, plot of the release probability upon bursts (Pr_burst) against Pr in the same boutons (n = 458, 763 boutons from 3, 4 repeats). Dashed line, bisector. Right, ΔPr (=Pr_burst–Pr) versus Pr. Curve/error band, mean/SEM. e Plots of synaptic configurations (indicated by D ~ $\bar{\Pr}$ combinations) against ΣPr and ΣPr_burst (in pseudo color) at individual dendritic branches (n = 58, 53 branches from 4, 4 repeats, some branches from b). f Box-whisker plot of ΣPr and ΣPr_burst at dendritic branches under physiological and elevated Mg²⁺ conditions (n = 58, 53 branches from 4, 4 repeats; data from e). Two-sided Kolmogorov–Smirnov tests, P = 0.0930 (NS), ****P < 0.0001. Box borders/line, quartiles/median; whiskers, min and max. Source data are provided as a Source Data file.