Fig. 2: Clamping astrocyte Ca²⁺ reduces the late phase of functional hyperemia.

a Cartoon of astrocyte Ca²⁺ extrusion tool: a high-affinity plasma membrane Ca²⁺ ATPase hPMCA2w/b (CalEx). b Viral vector strategy to express CalEx (Top Left) in astrocyte (or control virus- Bottom Left) plus GCaMP6f targeting astrocytes (Top Right) or neurons (Bottom Right). c Timeline from viral vector intracortical injection to the imaging experiment. d Representative post hoc immunofluorescence of CalEx expression against the fused Hemagglutinin (HA) reporter completed in 7 CalEx and 5 Control mice. e Representative image of gfaABC₁D-GCaMP6f expressing astrocytes (cyan, median filtered) and Rhodamine (Rhod)-B-Dextran loaded vasculature (magenta, median filtered) during startle in layer 2/3 of the barrel cortex in a control mouse (similar in 5 more mice) (channels were merged from separate acquisitions as startle experiments were performed before vascular dye loading). f Average arteriole diameter traces in CalEx and Control for 5 s (Left) and 30 s (Right) whisker stimulation. g Left: summary data showing peak arteriole dilation of the last 5 s of the stimulation period. Right: net Area Under the Curve (AUC: 40 s from stimulation onset) in the four conditions. Control, 5 s stim: n = 26 penetrating arterioles (PA), Control 30 s stim: n = 28 PA from 11 mice; CalEx, 5 s and 30 s stim: n = 23 PA from 10 mice. h Evoked astrocyte endfoot Ca²⁺ event occurrence. Black (control) and purple (CalEx) slices are events, white is no event detected. An event is >3 standard deviation of baseline. i Summary time series data of astrocyte endfoot Ca²⁺ in CalEx vs control for both 5 s (Left) and 30 s (Right) whisker stimulation. j Left: Summary bar graph of peak astrocyte endfoot Ca²⁺ signal. Right: net AUC (stimulation + 10 s) data in the four conditions. Control, 5s stim: n = 51 trials, penetrating arterioles (PA), Control 30 s stim: n = 57 trials, 18 PA from 6 mice; CalEx, 5 s stim: n = 36 trials, and 30 s stim: n = 38 trials 12 PA from 5 mice. k–m Same as h–j but for astrocyte arbor Ca²⁺. Control, 5 s stim: n = 48 trials, 16 PA, Control 30 s stim: n = 54 trials, 17 PA from 6 mice; CalEx, 5 s stim: n = 36 trials, and 30 s stim: n = 37 trials 12 PA from 5 mice. n AAV9.hSyn.GCaMP6f expressing neurons (cyan) and Rhodamine-B-Dextran loaded vasculature (magenta, median filtered image) in layer 2/3 of the barrel cortex. Representative example of 10 experiments. o Average neuronal Ca²⁺ traces in CalEx and Control for 5 s (Left) and 30 s (Right) whisker stimulation. p Left: summary data showing peak neuronal Ca²⁺ during the stimulation period. Right: net AUC of the stimulation period in the four conditions. Control, 5 s stim: n = 30 trials, 10 PA; Control 30 s stim: n = 30 trials, 10 PA from 5 mice; CalEx, 5 s stim: n = 32 trials, and 30 s stim: n = 38 trials 11 PA from 5 mice. All average trace and summary dot plot data show mean ± SEM. All statistical tests are Two-way ANOVA with Tukey’s multiple comparisons (two-sided). For further statistical details see Supplementary Table 2. Source data are provided as a Source Data file.