The stimulation-induced decrease in pericyte calcium fluorescence is impaired after spreading depolarization. (A) The fluorescence intensity of GCaMP6s expressed by pericytes and vascular smooth muscle cells was extracted and normalized to a 15-s pre-stimulation baseline. The minimum values are shown for vascular smooth muscle cells located on penetrating arterioles (PA, brown) and pericyte cell bodies on first (pink), second (yellow), or third (blue) order capillaries before and 20 min after spreading depolarization for six animals. Vascular smooth muscle cells and pericytes located from penetrating arterioles to second order capillaries responded to stimulation, and the decrease in calcium was larger for pericytes located on first order capillaries. After spreading depolarization, somatosensory stimulation did not induce any change in calcium fluorescence compared to baseline at any vessel branch. (B–E) Representative examples of the fluorescence intensity of GCaMP6s expressed in pericytes normalized to a 15-s pre-stimulation baseline. The dashed line indicates the baseline and the pink square the 15-s 2-Hz whisker stimulation period. Data are shown for vascular smooth muscle cells located on penetrating arterioles (B) and pericyte cell bodies on first (C), second (D), or third (E) order capillaries before (left) and 20 min after spreading depolarization (right). A blue circle marks the minimum value during stimulation. Data represent the mean ± SEM. **P < 0.01, *P < 0.001 two-way ANOVA; ^###P < 0.0001, ^##P < 0.001, ^#P < 0.01 compared to baseline with post hoc Sidak test. In A, the plus symbol indicates < 0.05, two-tailed t-test, comparing values between penetrating arterioles and first order capillaries before spreading depolarization. ns = non-significant; SD = spreading depolarization.