Fig. 1. Sustained functional hyperemia escalates and is associated with delayed astrocyte Ca²⁺ transients.

a Cartoon and timeline of the awake mouse, acute cranial window (skull + dura removal), 2-photon imaging experiment in barrel cortex. b Time series images showing dilation of a Rhodamine (Rhod)-B-dextran labeled penetrating arteriole (magenta, median filtered image) and Ca²⁺ responses (cyan) of a GCaMP6s-expressing astrocyte in response to 30 s whisker stimulation at 3, 20 and 40 s in an Aldh1l1-CreERT2 × RCL-GCaMP6s mouse (representative of n = 9 mice). Time stamps refer to stimulation onset as 0 s. c Traces of arteriole dilation (black) to 5 and 30 s whisker stimulation. N = 28 trials (T) of 12 penetrating arterioles (PA) from 9 mice. d Summary data of peak arteriole dilation to 1, 5, and 30 s whisker stimulation. Friedman test (one-sided). N = 7 PA from 6 mice. e Summary of response onset (calculated from each trial as 3 × SD above baseline) for dilation and astrocyte Ca²⁺ from 13 PA of 9 mice. Arteriole dilation T = 39, astrocyte endfoot Ca²⁺:T = 30 astrocyte process Ca²⁺: T = 27. Mixed effect analysis (two-sided) with Holm–Sidak’s multiple comparison. f Astrocyte endfoot (dark green) and astrocyte arbor (light green) Ca²⁺ traces to 30 s whisker stimulation. g Time series images of GcaMP6s expressing vascular smooth muscle cells (VSMC)(yellow) within a penetrating arteriole loaded with Rhod-B-Dextran (magenta). Each image is an average projection of 4 raw images. h VSMC Ca²⁺ trace during 30 s whisker stimulation. N = 30 PA from 11 mice. All average trace and summary dot plot data show mean ± SEM. For further statistical details see Supplementary Table 1. Source data are provided as a Source Data file.