Figure 4.

Light-induced Ca²⁺ hyperactivity depends on total light dose, not the pulse energy. (A) Schematic representation of the experiment design is shown. Spontaneous Ca²⁺ transients were recorded during a 2-min control period to establish a baseline activity (p1; 920 nm, 90 fs, 0.5 Hz). Then, during 10 min, three different protocols were applied: 1) the cells were either imaged as in the preceding figures (with the same hyperactivity scenario as before), or 2) the laser was shuttered and the cells allowed to recover from the previous recording. In a third variant, 3) we disrupted the laser mode-locking to expose the cells to continuous-wave (CW) radiation with the same average power $\overline{P}$ as during pulsed excitation (see Materials and Methods and Fig. S6). The respective impact of these different protocols on the astrocytic Ca²⁺ activity was read out in another 2-min recording (with the same parameters as p1). (B) Raster plots illustrate the outcome of the three different protocols. (C) Cumulative Ca²⁺ activity traces in the astrocyte processes during p1 and p3 are shown. (D) Statistical analysis of data was shown in (C). Shown are the activity increases for all conditions between the beginning of p1 (0–60 s) and end of p3 (660–720 s; one-sided nonparametric Wilcoxon Mann-Whitney two-sample rank test). Of note, the Ca²⁺ activity in the processes during p3 is indistinguishable between exposures to pulsed versus CW light, both of which are significantly higher than with the laser shuttered. All experiments started from initial activity levels during p1. n.s, not shown for clarity. (E) Shown are somatic ΔF/F₀ traces for the three scenarios and statistical analysis in (F). For CW illumination, somatic activity is significantly higher in p3 compared to either no or pulsed excitation during p2, respectively. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, n.s., not significant. To see this figure in color, go online.