Figure 2: ASIs inhibit ASH Cu²⁺-elicited Ca²⁺ response and worm avoidance.

(a) Expression of TeTx in ASIs and R-GECO1 in ASHs. Scale bar, 20 μm. ASH (b) and ASI (c) Ca²⁺ responses to a 10-mM Cu²⁺ stimulation after blocking or during optogenetic inhibition of neurotransmission in ASIs. ‘On’ and ‘Off’ indicate Ca²⁺ signals to the presentation and removal of the Cu²⁺ stimulation, respectively. For calculating the ratio of ΔF/F₀, the ΔF and F₀ for the on-response are defined as the fluorescence changes during the application of CuSO₄ and the average fluorescence intensity of 5 s before the Cu²⁺ stimulation, and the ΔF and F₀ for off-response are defined as the initial fluorescence changes 5 s after the removal of CuSO₄ and the average fluorescence intensity of the last 5 s of the on-response, respectively. Grey shading denotes the period of Cu²⁺ application. For ArchT optogenetic inhibition, continuous green light irradiation was applied during the entire recording, ATR, all-trans-retinal; similarly herein after. The ASI (d) and ASH (e) Ca²⁺ signals in response to the 10 mM CuSO₄ stimulation following chemogenetic activation of ASIs. Neurons that specifically expressed TRPV1 were activated by exogenous capsaicin (100 μM). The F₀ of the on-response was defined as the average fluorescence intensity from 0 to 5 s. Light grey and dark grey shading denote the span of applications of capsaicin and capsaicin plus Cu²⁺, respectively; similarly herein after. Comparison of Cu²⁺ sensitivity tested by the quadrant assay (f,g, n≥8 assays) and kinematics of the Cu²⁺-evoked reversal in the dry drop test (h–j, n=30 assays) after genetically blocking ASHs with TeTx or chemogenetically exciting ASIs with TRPV1 plus 10 μM capsaicin. All data are expressed as means±s.e.m. The number on each bar indicates the number of independent tests for each genotype. One-way analysis of variance (ANOVA) test in b–e and h,i, and two-way ANOVA test in f,g,j. Corrected with Bonferroni t-test. Statistical significance was indicated as: *P≤0.05, **P≤0.01 and ***P≤0.001.