Extended Data Fig. 10 ∣. High-speed trans-segmental imaging of sensory-evoked calcium activity in freely behaving GFAP-GCaMP6f mice.

a,b, Average intensity projection images from a time-lapse recording in a freely moving GFAP-GaMP6f mouse (Supplementary Video 8) taken at ~50 μm focal depth below the pia overlaid with ~10 μm × 10 μm ROIs. Only active ROIs above the indicated ΔF/F thresholds in response to an innocuous tail pinch/touch (p < 200 g) (a) or a noxious tail pinch (>500 g) (b) are shown. Like in focally restrained mice (Fig. 2), innocuous tail pinch/touch evoked only sparse activity across spinal segments, while noxious pinch resulted in widespread, bilateral astrocyte excitation. c, Innocuous tail pinch/touch and noxious tail pinch-evoked calcium activity (bottom) for the example recording shown in a–b. Each row depicts the percent of active ROIs across a given mediolateral (Y) position. The corresponding pressure stimulus, locomotor activity, and average calcium transient across the FOV are shown above the activity heat map. Innocuous tail touch/pinch and noxious pinch onsets are indicated by gray vertical lines. d, Population data showing the percent of active ROIs for innocuous and noxious stimulus trials. e, Population data showing the average calcium transient onset latency for noxious pinch trials across all, anterior, or posterior regions (Methods). f,g, Population data showing the individual ROI and average calcium response amplitude (f) and duration (g) for noxious pinch trials. Only 0.4% of ROIs had a transient longer than 6 s. h,i, Population data showing the average calcium transient amplitude (h) and duration (i) on the left and right sides of the spinal cord for noxious pinch trials. The data in d–i are from 11 innocuous and 8 noxious pinch trials in 2 mice. The data in f and g (left) are from 124,928 ROIs with ΔF/F > 20%, 8 recordings, and 2 mice. Paired two-sided t-tests determined P values (e: 0.2431, 0.1231, 0.2131; h: 0.3521; i: 0.5216), and all bar plots are presented as mean ± s.e.m. The box and whisker plots mark the median and the 25th and 75th percentiles, and the whiskers cover the minimum and maximum of the data.