All data are expressed as means ± SEM. For datasets with two groups, statistical analysis was performed with two-tailed t test to compare between control and test conditions. For datasets with greater than two groups, one-way ANOVA followed by Tukey post hoc test was used to compare groups. Statistical analysis was performed with a confidence level of 95%, and results with P < 0.05 were considered statistically significant.

The average rate of spH-labeled exocytosis was calculated by measuring the slope of a line fit to the cumulative events plot for each cell. The baseline rate was measured by fitting a line to the first 120 s of the experiment. For measurements of agonist-evoked exocytosis, the cursors were set at least 120 s after agonist application and spanned 200 s. For the Ca2+-free exocytotic rate analysis, the cursors for the fit were set immediately after agonist application to obtain the store-release contribution to the release rate. No difference was seen in the exocytosis rates between Orai1fl/fl nestin-Cre and Orai1fl/fl GFAP-Cre astrocytes, and therefore, the results from these cells were pooled into an “Orai1 KO” group.

spH event kinetics were measured with a line scan [12-pixel length (3.24 μm)] through the center of individual events. The fluorescence values were calculated as ∆F/F0, where F0 was the average of the baseline fluorescence for 5 s (25 time points) before the event. The half-width of each event was calculated using the Peak Analyzer function of OriginLab as the width of the curve at half of its maximum peak value.

The frequency and amplitude of Ca2+ fluctuations in GCaMP6f-expressing astrocytes in slices were analyzed using ImageJ and OriginLab. ROIs were drawn manually on the 2D maximum intensity projection of the time series (~540 s) in each experiment around the soma, the primary proximal processes coming off the cell body, and the distal tertiary processes (fig. S7A). The numbers and average sizes of the ROIs drawn around the soma, proximal processes, and distal processes were not statistically significantly different between WT and Orai1fl/fl GFAP-Cre cells. Background was measured in a region of the imaging field not containing any GCamP6f expression. The background-subtracted mean intensity of each ROI was measured at each time point using the ImageJ Multi Measure plugin, and F0 was calculated by averaging the first 20 time points for each ROI. The intensity of the GCamP6f fluctuations at each time point was calculated as ∆F/F0 for each ROI, and frequency and amplitude of the fluctuations were measured using Peak Analyzer function of OriginLab. A moving baseline with asymmetric least squares smoothing was used, and peaks were determined from the local maxima that exceeded a threshold of 10% over the local baseline. The frequency and amplitude for all the peaks in each ROI was averaged and calculated for each compartment (soma, proximal processes, and distal processes) in each cell. Analysis was done over a 3-min duration immediately before and at least 90 s after the administration of thrombin. Cells were excluded from analysis if they had noticeable z-drift or x-y drift during the experiment.

IPSCs were analyzed using MiniAnalysis software (Synaptosoft Inc.). Events were analyzed over a 5-min duration immediately before and 3 min after the administration of thrombin. The threshold for detection of events was set at 20 pA.

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