Fig. 5: Mechanisms underlying the frequency-dependence of dendritic spike modulation.

a Astrocyte expressing GCaMP5g (Supplementary Fig. 4) and tdTomato (red, scale bar: 20 µm; region of interest: dotted while line, sparing the cell body and major branches). Quantification of Ca²⁺ signals by the fluorescence intensity ratio (R, GCaMP5g / tdTomato) and its changes (ΔR) over its resting value (R₀). Comparison of ΔR/R0 during alveus stimulation with the baseline period before stimulation (see methods section for panels a–c). b Percentage of astrocytes with Ca²⁺ signaling increase of >5% was significantly larger with 10 Hz (blue) stimulation than with 40 Hz (73.1 % vs. 9.52 %, two-sided Fisher’s exact test, p = 0.0000022, n = 19 out of 26 cells and 2 out of 25 cells). c Change of ΔR/R0 during alveus stimulation across all cells (10 Hz vs 40 Hz: 1.07 ± 0.008 vs. 1.02 ± 0.007, n = 26 and 25, p = 0.0000046, two-sided Mann–Whitney U-test). d, e ZD7288 (10 µM) prevented the modulation of dendritic spiking by alveus stimulation while the effect of direct CBR stimulation by WIN55 (1 µM) was preserved. d Threshold stimulus. Left panel: 10 Hz (0.47 ± 0.05 µA vs. 0.49 ± 0.06 µA, n = 8, p = 0.33, two-sided paired Student’s t test) and WIN55 (0.48 ± 0.05 µA vs. 0.38 ± 0.04 µA, n = 10, p = 0.025, two-sided paired Student’s t test). Right panel: change during treatment (solid circles, left panel) relative to baseline (open circles, left panel), Kruskal-Wallis test with post-hoc two-sided Mann–Whitney U-test. e Slow component. Left panel: 10 Hz (14.75 ± 2.87 mV vs. 14.27 ± 3.05 mV, n = 8, p = 0.52, two-sided paired Student’s t test) and WIN55 (16.24 ± 2.21 mV vs. 18.79 ± 2.41 mV, n = 10, p = 0.0010, two-sided paired Student’s t test). Right panel: change during treatment (solid circles, left panel) relative to baseline (open circles, left panel), Kruskal-Wallis test with post-hoc two-sided Mann–Whitney U-test. Right panels of (d and e). The white control bar with 10 Hz alveus stimulation represents control data from Fig. 4d, e. f Ca²⁺ events in astrocytes expressing GCaMP6f (f, upper left panel, scale bar 10 µm) detected using AQuA⁷³ (lower left panel: sample events in orange, yellow and red; right panel: corresponding traces, scale bars: 0.5 ΔF/F and 1 min). g Change in Ca²⁺ event frequency during alveus stimulation (f_10Hz / f_baseline − 1) across all cells. Control (gray) vs. ZD7288 (yellow, ZD, 10 µM): 1.44 ± 0.56 vs. −0.34 ± 0.15 (p = 0.00067, n = 8 and 6 cells from 4 and 3 mice, two-sided Mann–Whitney U-test). Two-sided one sample Wilcoxon Signed Rank tests: p = 0.008 for control and p = 0.13 for ZD (“Method” section for panels f, g). Data are expressed as mean ± s.e.m. in the legend and displayed in box plots. The box indicates the 25th and 75th, the whiskers the 5th and 95th percentiles, the horizontal line in the box the median and the mean is represented by a filled circle. Supplementary Table 2 for further information. Throughout panels *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.