Figure 4. AQuA resolves astrocytic Ca²⁺ propagation directionality across scales.

(a) Representative in vivo GCaMP6f images during a burst period (top) and inter-burst period (bottom) with overlaid AQuA-detected events. (b) Population Ca²⁺ events represented as percentage of the imaging field active as a function of time. Burst periods (pink) are defined by Ca²⁺ activity >1% of the field of view and >10% of the maximum number of event onsets. (c) In vivo Ca²⁺ events propagate with specific directionality. Top: representative propagative event from the burst in panel a. The propagation direction (change of centroid relative to its original location) for each frame is overlaid on the event (right). Bottom: Total propagation distance versus event size for all events within bursts (n=6 mice, 66 bursts, 14,967 events for all data in this figure). (d) Event propagation direction from all events over the entire field in the burst in e. Arrow length indicates propagation distance. (e) To test consistency of subregional directionality during bursts, sixteen 96×96μm tiles are overlaid on images. (f) Top: All events within highlighted tile in d (red square) for five burst periods, color-coded by propagation direction (top). Bottom: Event propagation direction distributions (P=posterior; A=anterior; M=medial; L=lateral). (g) Cumulative distribution of percentage of bursts with events (within individual tiles/regions) propagating in the same direction in actual (solid) and simulated (dashed) data (one-tailed Wilcoxon rank sum, ***p=3.77e-15) (h) Two representative maps of population burst propagation direction with each event color-coded by onset time relative to the beginning of the burst, demonstrating variability of burst size. (i) Burst propagation direction calculated from onset maps in h (n=66 bursts). Event locations from the first 20% of the frames after burst onset are averaged together to determine burst origin. Event locations from 20% of the last frames after burst onset are averaged together and the difference between this and the origin determines burst propagation distance. Red arrow denotes average of all bursts.