Fig. 6: Calcium dynamics in single and two clustered spines during t-LTP induction.

a Single 2P images of a spine and dendrite from a L5 pyramidal neuron loaded with Alexa Fluor 594 (shown in a) and Fluo-4 (shown in b). Red ellipses and blue polygons indicate the ROIs selected for analysis. b Two-photon calcium signal images before (left panels) and after (right panels) a +7 ms pre–post pairing. The 1st, 2nd, and 40th repetitions of the pairing protocol are shown. Change in calcium fluorescence from baseline (ΔF) is color coded. Only positive changes in fluorescence are shown. White ellipses and polygons indicate the ROIs selected for analysis. c Average and individual \(\frac{{\Delta {G}}}{{R}}\) traces from spine ROIs for each of the 40 repetitions during the experiment depicted in a, b. Dotted line is the time when pairing occurred. d, e Population averages of \(\frac{{\Delta {G}}}{{R}}\) measured in d spines and e dendrites before the pairing performed in one spine (left panels; P = 0.28 and 0.13, respectively, n = 7 spines and dendrites, from six neurons, and four mice) and two spines (middle panels; P < 0.0001 and P = 0.006, respectively, n = 12 spines and 6 dendrites, from four neurons, and four mice, one-way repeated-measures ANOVA). Right panels show \(\frac{{\Delta {G}}}{{R}}\) population averages for one (black lines) and two spines (green lines). f, g As in a, b, but for two clustered spines. h Average and individual \(\frac{{\Delta {G}}}{{R}}\) traces from ROIs (sp1 versus sp2) from each of the 40 repetitions during the pre–post pairing in the experiment depicted in f, g. Dotted line is the time when pairing occurred. i, j Population averages of \(\frac{{\Delta {G}}}{{R}}\) measured in i spines and j dendrites after the pairing in one spine (left panels; P < 0.0001 and P = 0.03, respectively, n = 7 spines and dendrites) and two spines (middle panels; P < 0.0001 and P = 0.0001, respectively, n = 12 spines and 6 dendrites, one-way repeated-measures ANOVA). Right panels show \(\frac{{\Delta {G}}}{{R}}\) population averages in one spine (black lines) and two spines (green lines). Shaded area represents SEM. n.s. not significant; *P < 0.05; **P < 0.01; ***P < 0.001, post hoc Dunnet’s test. sp spine.