Fig. 3: Distinct dynamics of astrocytic Ca²⁺ and cAMP.

a–d Simultaneous imaging of GCaMP and Pink Flamindo with repetitive [3 s × 10, 7 s inter-stimulus interval] (a) and continuous [30 s] (b) PS. The peak amplitude of Pink Flamindo differs substantially between the two PS paradigms, whereas GCaMP shows similar peak amplitudes (n = 4–7 sessions, 4–6 mice) (c). Durations of astrocytic Ca²⁺ and cAMP responses to 30-s PS are distinct (d) (n = 6 sessions, 4 mice). e–k 120-s PS of NAergic fibers. The long-time stimulation of NAergic fibers consists of 10-s PS and 2-s imaging that repeats 12 times (e). GCaMP signals show faster response and shorter duration (f, g), and Pink Flamindo signals exhibit slower increase and long-lasting duration (f, h). The dynamics of extracellular NA similarly long with that of astrocytic cAMP (i). Quantification of time to peak (j, n = 4–8 sessions, 4–6 mice) and duration (k, n = 4–8 sessions, 4–6 mice). Scale bar: 20 μm. l–n GCaMP and Pink Flamindo responses classified into active cells and less active cells (l). Red and green circles indicate active and less active cells, respectively. Combinatorial classification of GCaMP and Pink Flamindo responsiveness (m). Correlation of GCaMP signal changes and Pink Flamindo signal changes (n, n = 249 cells, 7 mice, Pearson correlation; R² = 0.32, slope = 0.557). Scale bar: 100 μm. Analysis was performed from average of individual sessions in a–k. l–n were analyzed from individual cell responses. Box plots: box range, 25–50–75% quatile; square, mean; whiskers, max–min. Student’s t-test with Welch’s correction: c, d; one-way ANOVA with Turkey’s test: c; paired t-test: j, k; *p < 0.05, **p < 0.01, ***p < 0.001.