Ca2+imaging with G-CaMP2. (A – C) Double immunofluorescence analysis of hippocampal CA1 region from tetO-G-CaMP2:Tg2 (Ctrl) mice using antibodies to G-CaMP2 and S100B. Scale bar, 50 μm. (D – F) Confocal images of double immunofluorescence analysis of hippocampal CA1 of Tg1:tetO-G-CaMP2:Tg2 (TTg) mice using antibodies to G-CaMP2 and lacZ. Scale bar, 100 μm. (G – J) Two-photon images of control (Ctrl) and TTg astrocytes with G-CaMP2 fluorescence. Magenta areas are regions of interest (ROIs) in processes that showed changes in fluorescence over time. Green areas indicate soma. (G) and (H) are the same Ctrl astrocyte and (I) and (J) are the same TTg astrocyte, but under no-puff (G) and (I) and 500-ms puff (H) and (J) conditions. Scale bar, 10 μm. (K – N) Ca2+ traces in the ROIs indicated by arrows and the numbers in (G – J). Red traces in (L) and (N) show changes in the fluorescence induced by 500-ms puff application of Alexa594 with DHPG. Vertical dotted lines in red indicate the onset of puff stimulation. Red asterisks indicate peaks of each Ca2+ event. Vertical and horizontal ranges of gray bands below the asterisks represent the range from the baseline + 1SD to + 2SD and the event duration, respectively. Peaks whose duration could not be identified were excluded from the events. Vertical and horizontal scale bars, respectively, indicate 20% (ΔF/F 0) and 20 s. (O, P) Mean (±SEM) duration and mean areas under the curve, respectively, of individual events under no puff, 100-ms puff, and 500-ms puff conditions in Ctrl and TTg groups. A significant increase upon stimulation was observed only in the Ctrl group. *P < 0.05, **P < 0.01, ***P < 0.001, with Bonferroni’s correction for multiple comparisons.