Figure 6: RIC neurons mediate the inhibition of ASIs by ASHs.

(a) ASI Ca²⁺ responses to the 10 mM Cu²⁺ stimulation in wild-type, ASH silenced, tbh-1 mutant and tbh-1p::tbh-1 rescued worms. (b) The expression of R-GECO1 in RIC neurons driven by tbh-1 promoter and the specific expression of TeTx in ASH neurons realized by the use of the FLP–FRT site-specific recombination system. Scale bar, 20 μm. (c) Permanently or temporarily silencing ASH neurons with TeTx or ArchT had a similar effect on RIC Ca²⁺ responses to the 10 mM CuSO₄ stimulation. (d) ASIs showed an obvious on-response of Cu²⁺-elicited Ca²⁺ signals after blocking or inhibiting RICs. (e) The delay time of Cu²⁺-evoked calcium transients of ASH, RIC and ASI neurons in worms indicated. The delay time was calculated as the time of stimulus application to the time when the single-exponential fitted curve of individual Ca²⁺ on-response trace cuts fitted straight line of basal Ca²⁺ signals before stimulation. (f) The bending magnitude of Cu²⁺-evoked reversals in wild type, mutant and transgenic worms (n=30 assays). 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 a and c–e, and two-way ANOVA test in f. Corrected with Bonferroni t-test for multiple comparisons. Values that differ significantly are indicated (*P≤0.05, **P≤0.01 and ***P≤0.001 compared with the wild-type N2 control).