Whole-Body Imaging of Neural and Muscle Activity during Behavior in Hydra vulgaris: Effect of Osmolarity on Contraction Bursts

Effect of experimental conditions on contractions and locomotion behavior. Data from mounted preparations in A–C and from 1-h freely moving Hydra in D–F. A, Upper images, Changes in body length during longitudinal contractions. Lower images, Foot detachment. Scale bar, 500 μm. Number of contractions (B) and foot detachments (C) were counted. D, Upper images depict changes in body length during longitudinal contractions. Lower images depict foot detachment followed by locomotion. Scale bar, 1 mm. Number of contractions (E) and foot detachment/locomotion (F) were counted. The four conditions used were food (Food), osmolarity (Osmo), size (Size), and temperature (Temp). Control medium (ctr). Error bars shown as mean ± SEM, with symbol marks denoting data points from individual Hydra (N = 9–16 for B, C; N = 15–30 for E, F). Tukey’s multiple comparisons tests were performed following one-way ANOVA for osmolarity experiment, and Student’s t test was performed for others: ns ≥ 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Effect of experimental conditions on ectoderm and endoderm muscle activity. A, upper images, Measurements of contractions in Hydra expressing GCaMP6s in ectoderm muscle. Lower images, contractions in Hydra expressing GCaMP6s in endoderm muscle. Scale bar, 500 μm. B, Schematic summarizing steps to detect peaks of CB pulses from raw traces extracted from 2-h calcium imaging movies. RP1 pulses were not present in muscle activity. C–H, Each type of response was analyzed with four variables: (C) ectoderm CB pulse number; (D) endoderm CB pulse number; (E) ectoderm CB total time; (F) endoderm CB total time; (G) ectoderm CB total time; (H) endoderm CB total time. The four conditions used were food (Food), osmolarity (Osmo), size (Size), and temperature (Temp). Control medium (ctr). Error bars are shown as the mean ± SEM, with symbol marks denoting data points from individual Hydra (N = 3–6). Tukey’s multiple comparisons tests were performed following one-way ANOVA for osmolarity experiment, and Student’s t test was performed for others: ns ≥ 0.05; *p < 0.05.

Effect of experimental conditions on neuronal activity. A, upper images, Activation of CB neurons. Lower images, Activation of RP1 neurons. Scale bar, 500 μm. B, Schematic summarizing steps to detect peaks of CB and RP1 pulses from raw traces extracted from 2-h calcium imaging. C–H, Analysis of parameters: (C) CB pulse number; (D) RP1 pulse number; (E) CB total time; (F) RP1 total time; (G) CB pulse frequency; (H) RP1 pulse frequency. The four conditions used were food (Food), osmolarity (Osmo), size (Size), and temperature (Temp). Control medium (ctr). Error bars are shown as the mean ± SEM, with symbol marks denoting data points from individual Hydra (N = 3–8). Tukey’s multiple comparisons tests were performed following one-way ANOVA for osmolarity experiment, and Student’s t test was performed for others: ns ≥ 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.