Fig. 3: Ratiometric imaging of touch-receptor neurons in unrestrained animals.

A Schematic of touch receptor neurons in C. elegans and behavioral response to gentle mechanical stimulus, and an assay plate with piezo. B Touch receptor neuron PLM labeled with GCaMP8f and mKate2 (cyan, red, respectively). The dashed line shows the worm’s outline. Scale bar corresponds to 50 µm. C Speed response (mean ± s.e.m.) to light touch stimulation (gray bar). Statistics performed with two-tailed paired t-test, ****p = 1.8e-14 (D) Neuronal response (mean ± s.e.m.) to light touch stimulation (gray bar). Statistics as in (C), ****p = 2.5e-11. E Neuronal response to light touch stimuli (gray bar) as in D, for trials with reversal (orange) ****p = 3.1e-04, and forward response (green) **** p = 2.8e-08 within 2 s after stimulus onset. Statistics as in (C). F Boxplot showing mean poststimulus GCaMP8f fluorescence signal, color as in (E). For forward responding worms, N = 15 worms, n = 41 traces, with 1–5 repetitions per animal. For reversal responding worms, N = 12 worms, n = 20 traces, with 1–3 repetitions per animal. The box spans the first to third quartiles, with a line marking the median. Error bars extend to the furthest point within 1.5 times the interquartile range. Outliers are excluded. A mixed linear regression analysis with a z-test used for significance testing revealed that poststimulus GCaMP8f fluorescence signal was significantly influenced by reversals (**p = 0.0032).