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. 2020 May 18;11:2464. doi: 10.1038/s41467-020-16315-4

Fig. 4. PostSynTagMA photoconversion with back-propagating action potentials (bAPs).

Fig. 4

2P image stacks of postSynTagMA expressing CA1 pyramidal neurons were taken before and after 15 pairings of trains of bAPs with photoconverting violet light (395 nm, 16 mW mm−2, 500 ms duration, 1 s delay). Synaptic transmission was blocked. a R/G ratios of individual synapses before (R0/G0) vs after (R1/G1) photoconversion. Note the variability in photoconversion within conditions. Dotted black line is the unity line. Magenta: 50 bAPs. Blue: 3 bAPs. Green: 0 bAP. b Three different metrics vs. PSD size (linear regression lines with 95% confidence intervals, see Supplementary Fig. 6a-c for regressions). ΔR is positively correlated with PSD size (0 bAPs: rs = 0.11, p = 0.03; 3 bAPs: rs = 0.23, p < 0.0001; 50 bAPs: rs = 0.34, p < 0.0001). Normalizing ΔR by (G0 + G1) removes the correlation with PSD size (0 bAP: rs = −0.07, p = 0.164; 3 bAPs: rs = −0.03, p = 0.583; 50 bAPs: rs = 0.004 p = 0.876). rs is the correlation coefficient (Spearman’s rho). c Histograms of photoconversion ΔR/(G0 + G1) for each condition (0 bAP: median = 0.139, mean = 0.164; 3 bAPs: median = 0.327, mean = 0.406; 50 bAPs: median = 0.984, mean = 1.452). Values above 2 are binned. Right bar graph: ΔR/(G0 + G1) vs the number of bAPs (data are median and interquartile range; Kruskal-Wallis test followed by Dunn’s multiple comparsions: 0 bAP vs. 3 or 50 bAPs ****p < 0.0001, see Supplementary Fig. 6c for individual points). 0 bAP: n = 356 synapses, 1 neuron; 3 bAPs: n = 472 synapses, 1 neuron; 50 bAPs: n = 2587 synapses, 3 neurons. d Example green, red and merged images of SynTagMA-labeled synapses photoconverted with 0, 3 or 50 bAPs. 3D plots show the positions of individual synapses; markers are colored according to the calculated photoconversion value (ΔR/(G0 + G1)). Scale bar: 1 µm.