Fig. 4: Pyramidal cell activity increases astrocytic Ca²⁺ signaling and promotes dendritic spiking via CBRs and NMDAR co-agonist supply.

a Axonal simulation in the alveus (A) retrogradely activates CA1 pyramidal cells (SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SLM, stratum lacunosum-moleculare). b Example astrocyte (whole-cell patch clamp, 40 µM Alexa Fluor 594 and 400 µM Fluo-4, scale bar: 20 µm). Left panel: Alexa Fluor 594. Right panels, top to bottom: Fluo-4, average of 20 frames during baseline, alveus stimulation and alveus stimulation in the presence of the CBR inverse agonists AM251 (5 µM). Quantification of Ca²⁺ signals by the fluorescence intensity ratio (R, Fluo-4 / Alexa). Overall changes (ΔR) relative to resting value (R₀) during alveus stimulation compared to the baseline period before stimulation (see methods section for panels b, c). c 69.2% of the astrocytes showed an increase of at least 5% (left panel, 9 out of 13 cells). In the responders, the increase was blocked by AM251 (5 µM; 1.31 ± 0.07 vs 1.05 ± 0.03, n = 9, p = 0.016, two-sided paired Student’s t test). d Left panel: the threshold stimulus of dendritic spikes was decreased by alveus stimulation at 10 Hz (0.33 ± 0.03 µA vs. 0.26 ± 0.03 µA, n = 8, p = 0.000071, repeated measures ANOVA and post-hoc Fisher’s LSD) and 20 Hz (0.38 ± 0.03 µA vs. 0.34 ± 0.03 µA, n = 9, p = 0.031, Friedman test and post-hoc two-sided Wilcoxon signed-rank test) but not 4 Hz (0.55 ± 0.03 µA vs. 0.55 ± 0.04 µA, n = 5, p = 1, two-sided paired Student’s t test, separate set of experiments) and 40 Hz (0.33 ± 0.03 µA vs. 0.32 ± 0.04 µA, n = 8, p = 0.31, repeated measures ANOVA and post-hoc Fisher’s LSD). Open circles: baseline. Filled circles: alveus stimulation. Right panel: change relative to baseline (one-way ANOVA with post-hoc Fisher’s LSD). e Left panel: corresponding changes of the slow component (10 Hz: 13.70 ± 2.47 mV vs. 15.74 ± 2.29 mV, n = 8, p = 0.000051, repeated measures ANOVA with post-hoc Fisher’s LSD; 20 Hz: 9.75 ± 1.37 mV vs. 11.08 ± 1.81 mV, n = 8, p = 0.022, repeated measures ANOVA with post-hoc Fisher’s LSD; 4 Hz: 17.97 ± 2.98 mV vs. 18.61 ± 2.94 mV, n = 5, p = 0.23, two-sided paired Student’s t test, separate set of experiments; 40 Hz: 13.70 ± 2.47 mV vs. 14.00 ± 2.39 mV, n = 8, p = 0.41, repeated measures ANOVA with post-hoc Fisher’s LSD). Open circles: baseline. Filled circles: alveus stimulation. Right panel: change relative to baseline (Kruskal-Wallis test with post-hoc two-sided Mann–Whitney U-tests). f In the presence of AM251 (5 µM, yellow), no effect of alveus stimulation (20 Hz) on dendritic spikes (threshold stimulus: 0.36 ± 0.06 µA vs. 0.35 ± 0.05 µA, n = 7, p = 0.54, two-sided paired Student’s t test; slow component amplitude: 14.53 ± 2.21 mV vs. 14.61 ± 2.27 mV, n = 7, p = 0.85, two-sided paired Student’s t test). g Presence of D-amino acid oxidase (DAOO, 0.17 U/ml, red) also prevent the effect of alveus stimulation (10 Hz) (threshold stimulus: 0.36 ± 0.06 µA vs. 0.37 ± 0.07 µA, n = 8, p = 0.73, two-sided paired Student’s t test; slow component: 19.58 ± 3.21 mV vs. 18.60 ± 2.46 mV, n = 8, p = 0.27, two-sided paired Student’s t test). Data are expressed and displayed as mean ± s.e.m. or 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.