Figure 1. Cortical astrocyte Ca²⁺ event rate and SWA are negatively correlated across behavioral states.

(A) Experimental in vivo imaging and electrophysiology setup. Mice expressing astrocytic GCaMP6f were head-fixed on a horizontal treadmill to record astrocyte Ca²⁺, LFP, EMG, and locomotion. (B) Left: example image of GCaMP6F expression in L2/3 V1 astrocytes in an awake, head-fixed mouse. Right: example of LFP, EMG, locomotion, and astrocyte Ca²⁺ event data using this experimental setup. (C) Left: AQuA (Wang et al., 2019) detects astrocyte Ca²⁺ events in 3 min GCaMP time series. Right: representative spatiotemporal plot of AQuA-identified Ca²⁺ events displaying event time and duration on the x-axis, and the unidimensional (y) spatial extent on the y-axis. Events are color-coded by behavioral state (wake=blue, NREM sleep=pink). Inset demonstrates that event count throughout paper is quantified using event onset. (D) Ca²⁺ event rate and SWA are negatively correlated (Pearson’s correlation, p<0.0005). Each point represents a single 2 min bin (for all data in this figure except panel I, n = 4 mice, 19 hr). (E) Example of Ca²⁺ event rate (bottom row) across three behavioral states: NREM sleep, locomotory wake, and stationary wake. (F) Across animals, Ca²⁺ event rate is lowest during sleep, higher during stationary wake, and highest during locomotory wake, while (G) the inverse is true for SWA (for F and G, rank sum test, data are represented as median, 25th and 75th percentile). (H) Ca²⁺ event rate and SWA are negatively correlated within each behavioral period: sleep (red, Pearson’s correlation, p<0.0005) and stationary wake (cyan, Pearson’s correlation, p<0.0005) (I), but Ca²⁺ events in IP₃R2 KO mice are less correlated with SWA in sleep (Pearson’s correlation, p<0.0005) and stationary wake (Pearson’s correlation, p>0.05, n = 5 mice, 22 hr).

Figure 1—figure supplement 1. PCA reveals that behavioral states can be characterized by unique properties of astrocyte Ca²⁺ events.

(A) Individual quantification of Ca²⁺ event size (top left), duration (top right), and amplitude (bottom) across sleep, stationary wake, and locomotory wake reveal significant differences in size and duration between locomotory wake and the other two states, but no difference between sleep and stationary wake (rank sum test). Data are represented as median, 25th and 75th percentile (n = 4 mice, 19 hr, 1089 locomotory wake periods, 443 stationary wake periods, 285 sleep periods). (B) Top: Weight matrix extracted from PCA, incorporating 20 Ca²⁺ event properties obtained with AQuA (n = 4 mice, 19 hr, 87,189 events). PC 1 largely represents spatial features, PC 2 represents temporal features, and PC 3 represents features associated with amplitude. Bottom: Percent of variance explained by each PC. (C) Cumulative distribution of the score for each PC for sleep, stationary wake, and locomotory wake events demonstrates significant differences among all three states (two-sample Kolmogorov-Smirnov test).