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

Fig. 2. Temporal resolution of preSynTagMA photoconversion.

Fig. 2

a A spatial light modulator was used to illuminate parts of the axonal arbor (405 nm, 50 mW cm−2, 100 ms) at different times relative to a brief tetanic stimulation (5 APs). After each trial, new images were acquired. ‘Exact’ & ‘200 ms’ timing share the same color code (cyan) as we used ‘exact’ timing in sypCaMPARI experiments and a 200 ms delay in preSynTagMA experiments. b Ratio of red to green fluorescence (R/G0) from sypCaMPARI boutons illuminated at different times relative to the electrical stimulation. Trial 1 shows the effect of illumination without stimulation. Line color code as in a, n = 6 neurons. c Activity-dependent photoconversion (ΔR/G0) versus delay from start of stimulation to violet light from the same experiments in b. The gray box indicates the time window for efficient photoconversion of sypCaMPARI. d Neurons expressing preSynTagMA (synaptophysin-CaMPARI2 (F391W_ L398V)) were stimulated as in a–c. Note the greatly reduced increase in R/G0 with violet light alone (Trial 1). n = 6 neurons. e Left axis: activity-dependent photoconversion (ΔR/ G0) versus delay of preSynTagMA expressing neurons (circles). n = 12 neurons for the before, 200 ms, and 2 s conditions and n = 6 neurons for the 500 ms, 1 s, 5 s and 10 s conditions. Right axis: photoconversion of CaMPARI2(F391W_L398V) after 50 bAPs and 100 ms violet light (triangles). n = 5 neurons. f Cultured rat hippocampal neuron expressing preSynTagMA, trial # 3 from d. Boxes indicate regions where photoconversion light was applied with different delays (color code as in a). All data are presented as mean ± SEM. In e, a Kruskal-Wallis test followed by the Benjamini-Hochberg FDR method was used. Timing conditions inside the gray box were not significantly different while the timing conditions outside the gray box were significantly different from those within the box (p < 0.001). Scale bars: 50 µm (a), 25 µm (f). Cultured neurons were 14–18 days old, expressing sypCaMPARI or preSynTagMA for 8–16 days.