Fig. 3

Synchronization between different classes of CS neurons. a Photomicrography of the injection sites of the retrograde tracers (cholera toxin subunit B conjugated with Alexa 594 in red and FluoroGold in green) into the contralateral dorsal horn as well as the intermediate zone and ventral horn of cervical segment C4 in one experiment (left) and two-photon microscopy images of a coronal slice showing FG-(green) and ChT-(red) labeled neurons (center), as well as L5 neurons positive to GCaMP6f (right). The field of view corresponds to the area indicated in Fig. 1. b (Top) representative traces of simultaneously imaged CS DH- (green) and IVH-projecting (red) neurons indicated by the arrows in a. Black traces represent the time derivative of the fluorescence signals. Colored dots indicate the frames (events) in which d(DF/F)/dt remained above the threshold (2 times above the SD of the calcium signal). (Bottom) Zoomed-in depictions of the fluorescence traces indicated in a. The shaded areas indicate peri-event intervals triggered by one event occurred in a CS neuron (black dots). Notice that most of the events (dots) occurring during the peri-event interval belong to other CS neurons of the same class (i.e. DH- or IVH-projecting). Thus, for each calcium event occurring in a certain cell, the number of events occurring in a 200-ms or 600-ms peri-event interval (shaded area) of all the simultaneously recorded cells was computed. c Color activity maps showing the number of co-activation events (colored scale) between different classes of CS neurons (DH-DH, IVH-IVH and DH-IVH) imaged simultaneously in the same experiment d, synchronized events per bin (median, 25th, 75th, 10th and 90th percentiles for all pairs of neurons imaged in 8 experiments) occurring in CS neurons triggered by events in other CS neurons of the same class (between DH-projecting DH-DH; between IVH-projecting IVH-IVH) or in CS neurons of different class (DH-IVH; IVH-DH) computed for 200 ms peri-event interval (*P < 0.05, Kruskal–Wallis ANOVA, post hoc Dunn’s test). e the same as C but for the events occurred in 600 ms peri event intervals. (*P < 0.05, Kruskal–Wallis ANOVA, post hoc Dunn’s test). f, g Fraction of synchronized events (mean ± SE) occurring in CS neurons of the same class (DH-DH and VH-VH) or in CS neurons of different class (DH-IVZ and IVZ-DH) computed for 200 (e) and 600 ms (f) peri-event intervals (*P < 0.05, One-way ANOVA, post hoc Tukey test)