Figure 1. Astroglial Ca²⁺ Signals Change with Vigilance State and Time of Day.

(A) Representative images from one mouse showing microinjection of AAV2/5 GfaABC₁D-GCaMP6f in cortex results in selective expression of GCaMP6f in GFAP⁺ astrocytes (top), but not in NeuN⁺ neurons (bottom). GCaMP6f labeling (green) is native whereas astrocytes (GFAP; magenta) and neurons (NeuN; magenta) are immunolabeled. 40x objective; scale bar = 20 μm. (B) Cartoon showing dual EEG & EMG recording and astroglial Ca²⁺ imaging in the frontal cortex through a cranial window. (C) Maximum projection ΔF/F images of Ca²⁺ imaging in astrocytes during wake, NREMS, and REMS from Video S1 using the miniature microscope and Inscopix Data Processing Software. The time incorporated for each image corresponds to the traces shown in D (REMS: 50 s; wake: 28 s; NREMS: 48 s). (D) Representative Ca²⁺-induced fluorescent trace from one ROI from Video S1, EEG, EEG power spectrogram, and EMG in each vigilance state. (E) Mean (± s.e.m.) ΔF/F during wake (W), NREMS (NR), and REMS (R) at ZT6 (Friedman). (F) Number of astroglial Ca²⁺ events per 4-s vigilance state epoch expressed as means ± s.e.m. at ZT6 (Friedman). (G) Distribution of Ca²⁺ event amplitudes organized by smallest (low deciles) to largest (high deciles) values during wake, NREMS, and REMS and boxplot comparisons at ZT6. Black squares in boxplots denote means (wake = 4.84; NREMS = 2.71; REMS = 2.30; Friedman). (H) Heatmaps show fluorescence traces from 110 regions of interest (ROI) from a representative mouse during the 12 s before and after transitions between vigilance states. Line plots are mean ΔF/F (± s.e.m.) during state transitions under baseline conditions for n = 6 mice. Vertical, dashed lines indicate time of state transition. (I) Mean ΔF/F during NREMS and baseline NREMS EEG delta (δ) power (i.e. NREM SWA; 0.5 – 4 Hz) at ZT0, 6, and 12. NREM δ power is expressed as a percentage of the last 4 h of the light period. Values are means ± s.e.m. ^a, different from ZT0; ^b, different from ZT6 (linear mixed-effects model). Group means are from n = 586 individual ROIs from 6 mice. *, vigilance state differences. p < 0.05. See also Figure S1 and Video S1.