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. 2020 Aug 26;11:4276. doi: 10.1038/s41467-020-17861-7

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© The Author(s) 2020

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 Experimental protocol for t-LTP induction in two clustered dendritic spines (<5 µm apart). b Representative experiment for t-LTP induction (pre–post pairing of +7 ms) in two clustered spines. Traces correspond to the average of 10 uEPSPs recorded in the soma and generated by 2P uncaging before (control, black trace) and after t-LTP induction (red trace) over indicated spines (red dots). c Time course of uEPSP amplitude, neck length, and spine head volume (P < 0.001, P < 0.001, P = 0.47, respectively, n = 8 spine pairs, one-way repeated-measures ANOVA) changes following STDP induction (+7 ms) in two clustered spines. n.s. not significant; **P < 0.01, ***P < 0.001, post hoc Dunnet’s test. d Experimental protocol for t-LTP induction (+7 ms) in two distributed spines (>5 μm apart). e t-LTP induction (+7 ms) in two nearly simultaneously activated spines is dependent on interspine distance. The estimated value of λ represents the boundary between clustered and distributed spines. f Same as b, but for two distributed spines (>5 μm apart). g Time course of uEPSP amplitude, neck length and spine head volume (P = 0.45, 0.07, and 0.64, respectively, one-way repeated-measures ANOVA) following STDP induction (+7 ms) in two distributed spines. n.s. not significant, post hoc Dunnet’s test. h Changes in uEPSP amplitude, neck length, and head volume for individual (1 sp), clustered (<5 μm) and distributed (>5 μm) spines (2 sp) 15–25 min after t-LTP induction (+7 ms) (clustered: uEPSP = 121.73 ± 7.92%, P = 0.02, n = 8 spine pairs; neck length = 74.33 ± 6.35%, P = 0.0039; spine head volume = 107.92 ± 3.69%, P = 0.039; distributed: uEPSP = 100.03 ± 6.18%, P = 0.95, n = 12 spine pairs; neck length = 95.88 ± 4.87%, P = 0.28; spine head volume = 103.71 ± 2.61%, P = 0.11, two-sided Wilcoxon test; *P < 0.05, **P < 0.01). Shaded area and error bars represent SEM. Lines, bars, and dots in c, g, h: uEPSP = black, neck length (NL) = red, and head volume (HV) = blue.