Figure 1. Stimulation-evoked Ca2+ signals in astrocytic territories of adult mice monitored by two-photon GCaMP5E imaging. A, Viral (rAAV) transduction of GCaMP5E driven by GFAP promoter in adult mouse hippocampus. Immunofluorescence with GFP antibodies (green) shows robust GCaMP5E expression in GFAP-immunopositive (red) CA1 astrocytes. The vascular endothelium is revealed with CD31 antibodies (blue). or, Stratum oriens; pyr, stratum pyramidale; rad, stratum radiatum. Scale bars: 100 and 10 μm (boxed motif expanded in inset). B, Schematic drawing of electrode placements in the hippocampal slice and fEPSPs recorded during a 20 Hz stimulation train expanded in the bottom trace. C, SD image of fluorescence intensities from a time-lapse recording at 1 Hz frame rate. The positions of GCaMP5E fluorescent astrocytic somata (1–8) and electrodes are indicated. Rec, recording; Stim, stimulation. Scale bar, 20 μm. D, Activity histogram (ΔF/F) of astrocytic somata 1–8. Red bar indicates electrical Scc stimulation. E, Single GCaMP5E fluorescence traces from somata of astrocytes 1–8 during activation. F, Amplitudes of the somatic Ca2+ signals in response to 10 s stimulation with different frequencies (top) and at 20 Hz stimulation with different durations (bottom). G, TTX abolished stimulation-evoked astrocytic Ca2+ responses (paired t test, n = 11 cells, 4 slices from 3 animals). H, SD image of fluorescence intensities from rAAV–GFAP–GCaMP5E-transduced hippocampal striatum radiatum astrocyte (4 Hz frame rate), in which soma (S), process (P), and endfeet (E) are indicated. Scale bar, 10 μm. I, Latency, rise rate, amplitude, and duration of stimulation-evoked (20 Hz, 10 s) GCaMP5E fluorescence responses in astrocytic somata, processes, and endfeet (abbreviations as above). P-S lag indicates the time between the first response occurring in fine process and the somatic response. This lag analysis was performed by one-sample t test, n = 16 cells with paired observations. Other comparisons were done by Kruskal–Wallis test with Dunn's post hoc test, n = 23 (somata), 99 (processes), and 26 (endfeet), 7 slices from 3 animals. J, As in A but with transduction of Lck-GCaMP5E instead of GCaMP5E. Scale bars: low magnification, 50 μm; high magnification, 10 μm. K, As in H but with rAAV–GFAP–Lck-GCaMP5E. L, As in I but with rAAV–GFAP–Lck-GCaMP5E, n = 21 (somata), 106 (processes), and 32 (endfeet), 8 slices from 2 animals; n = 18 cells for the lag analysis. Values are mean ± SEM. *p < 0.05, **p < 0.001.