Fig. 2 ∣. High-speed trans-segmental imaging of sensory-evoked calcium activity in behaving GFAP-GCaMP6f mice.

a, Schematic of the experimental approach. A laminectomy was performed over the lumbar spinal cord of GFAP-GCaMP6f mice (N = 4). The wearable macroscope was mounted above it. Pinch stimuli of different intensities and set duration (1.5 s ± 0.5 s) were delivered to the awake animal’s proximal tail. Running speed was recorded by placing the animal on a spherical treadmill under focal restraint (for open field data, see Extended Data Fig. 10 and Supplementary Video 8); scale bar (left inset), 1,400 μm. b, Average intensity projection image from a time-lapse recording taken at a focal depth of ~50 μm below the pia overlaid with ~10 μm × 10 μm ROIs. Only ROIs with at least 20% (yellow) and 40% ΔF/F (orange) in response to an innocuous pinch (p < 200 g) are shown. Innocuous stimuli evoked only sparse astrocyte activity on the left (L) and right (R) sides of the spinal cord and across anterior (A) and posterior (P) lumbar spinal segments (Supplementary Video 6). The calcium transients of two example ROIs are shown above the activity map (for all transients, see Supplementary Fig. 2a-c (left column)). c, Heat map of innocuous tail pinch-evoked activity for the example recording shown in b. Each row depicts the percentage of active ROIs (>20% ΔF/F) 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. d, Activity map from the same animal and focal depth as in b for a noxious pinch trial (p > 500 g). Noxious stimuli evoked widespread, bilateral astrocyte excitation across spinal segments (Supplementary Video 7). Two example ROI calcium transients are shown above the activity map (for all transients, see Supplementary Fig. 2a-c (right column)). e, Heat map of noxious tail pinch-evoked astrocyte activity for the recording shown in d. Each row depicts the percentage of active ROIs (>20% ΔF/F) across a given mediolateral (Y) position. Pressure stimulus amplitude, locomotor activity and average calcium signal across the FOV are shown above the activity heat map. f, Population data showing the percentage of active ROIs with at least 20 or 40% ΔF/F for innocuous and noxious stimulus trials. g, Population data showing the average calcium transient onset latency for noxious pinch trials across all, anterior or posterior regions (Methods); NS, not significant. h,i, Population data showing the individual ROI and average calcium response amplitude (h) and duration (i) for noxious pinch trials. j,k, Population data showing the average calcium transient amplitude (j) and duration (k) on the left and right sides of the spinal cord for noxious pinch trials. The data in f are from nine innocuous and eight noxious pinch trials in four mice. The data in h and i (left) are from 67,696 ROIs with ΔF/F of >20%, eight recordings and four mice. The data in g, h and i (right) and j and k are from eight recordings and four mice. Paired two-sided t-tests determined P values (g: 0.5953, 0.8364 and 0.4787; j: 0.2956; k: 0.5852), 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.