(A) Fluorescent micrograph (merged channels) of the vulva region, showing red (mCherry) and green (GCaMP6), expressed in AS MNs (with the use of the Q system), and GFP, expressed in vulva muscles. Angle between vulva and the two flanking AS6 and AS7 neurons is indicated. (B, C) Representative analysis of time traces of Ca2+ signals (ΔF/F) in AS6 and AS7, as well as the angle defined by the vulva and the two neurons during crawling (B, 15 s, forward; C, 20 s, reverse). (D, E) Cross-correlation analysis (mean ± SEM) of single periods of the body wave (5 s each) for each of the AS6 and AS7 GCaMP6 signals with the vulva angle, as well as for the two Ca2+ signals, during forward (D) or backward (E) locomotion. (F) Comparison of the mean cross-correlation peaks (during the 2 s centered on the peak) of the fluorescence of AS6 or AS7 and the neuron/vulva angle, or between AS6 and AS7 neurons, for forward or reverse locomotion. (G) Comparison of the peak Ca2+ signals (mean ± SEM) in AS6 and AS7, during forward or reverse locomotion, respectively. See also Figure 5—video 1. Number of animals is indicated in D-G. Statistical test: ANOVA with Tukey’s post-hoc test.