Figure 1. Spotty Ca²⁺ signals in rat hippocampal astrocyte-neuron co-cultures.

a. Images of astrocytes expressing Lck-GCaMP3 in co-cultures. Left: basal fluorescence of Lck-GCaMP3 for an astrocyte before any spotty Ca²⁺ signals. Right: a maximum projection image of a 300 frame video. Eight regions of interest are shown (as 1–8). The intensity profiles of these eight ROIs are shown in b. c. Still frames between 141 and 240 s and between 171 and 270 s from the graph in panel a for ROI 4 and ROI 6. The time between images is 1 s. d. Images of spotty Ca²⁺ signals for ROI 1 and ROI 3. A graph on the right shows the full width of half maxima (FWHM) of the events (5.0 ± 0.6 μm, n = 10 sites). e. Intensity profiles of the six spotty Ca²⁺ signals observed by Fluo-4 Ca²⁺ indicator with total internal reflection fluorescence (TIRF) microscopy. f. Images of spotty Ca²⁺ signals visualized by TIRF microscopy. Spotty Ca²⁺ signals detected by TIRF occurred with a frequency of 1.8 events/min (n = 46 sites from 27 cells), lasted 0.6 ± 0.07 s (n = 408 events) and displayed peak dF/F values of ~100% (1.0 ± 0.03, n = 427). The graph on the right shows the FWHM of the events (5.1 ± 0.4 μm, n = 19 sites). g. Spotty Ca²⁺ signals imaged with Lck-GCaMP3 were reduced by ~95% in Ca²⁺ free conditions (Supplementary Video 2, n = 155 sites from 14 cells). Vertical lines are s.e.m.