Fig. 1: Two distinct Ca²⁺ waves at astrocytic fine processes are characterized for the sleep and awake states.

A A schematic drawing depicting the experimental setup combining two-photon imaging of astrocytic Ca²⁺ signaling in the barrel cortex with in vivo whole-cell recordings in single neurons and local field recordings in awake behaving GCaMP6f transgenic mice. B Representative fluorescence photomicrographs of the barrel cortex displaying the expression of GCaMP6f (left panel), SR101-loaded astrocytes (middle panel), and merged images (right panel) illustrating that some of the GCaMP6f-expressing astrocytes were colabeled with SR101-loaded astrocytes. Scale bar = 10 µm. C Representative recordings of EEG and EMG and power spectrum analyses during sleep and in awake mice (blue as sleep; red as arousal). Upper traces are representative ECoG traces from whisker stimulation. Awake states were shown as no apparent evoked ECoG response and strong EMG activity. D Representative fluorescence changes showing that whisker stimulation induced small Ca²⁺ increases during sleep, scale bar = 10 µm. E The analyses of fluorescence changes corresponding to GCaMP6f F/F₀ traces at the fine processes, which are marked as circles in (D), showing the higher amplitude response to the stimulation in awake mice. Note the significant increase in the peak in the awake state. F–J Statistical data show the comparison of whisker stimulation-induced changes in Ca²⁺ transients by measuring ΔF/F₀ (F), latency (G), rise slope (H), decay (I), and duration (J), confirming the differential firing dynamics during sleep and awake states (the data are shown as the mean ± SD, **paired t-test, P < 0.001; n.s not significant, P = 0.161. The dots represent the mean value in each mouse, n = 6 mice in each group).