The gating property of subdued channels is dependent on heat, voltage and Ca2+, and the expression in Class IV neuron is required for larval heat avoidance behavior (Jang et al., 2015). Considering the results of the Cl− ion direction (Figure 2B–C), the possibility of subdued channels contributing to inhibitory currents is low, although an effect of the Subdued channels on neuronal activity cannot be completely excluded. (see below). (A–B) Avoidance behavior of wild-type and subdued mutant larvae expressing TN-XXL in response to thermal stimulation (46°C and 50°C). The onsets of avoidance behavior were delayed at both temperatures, and the fraction of no response was increased in mutant larvae at 46°C. (A) The distribution of response latency (Wilcoxon rank sum test). NR, no response group. (B) Percentage of larvae responding within 5 s with 95% Clopper-Pearson confidence intervals (Fisher’s exact test). (C–F) Responses of wild-type and subdued mutant neurons expressing TN-XXL with different IR-laser power (30, 38 and 44 mW). The IR laser was focused onto the proximal dendritic arbors in filet preparations for 1 s (upper red line in C). (C) Raster plots of firing (left) and magnitudes of the ΔRpeak corresponding to dendritic Ca2+ transients (right). Trials are sorted by descending order of the magnitude of the ΔRpeak. Red raster lines indicate USs. (D) Spike numbers decreased in subdued mutant neurons only at low IR-laser power (30 mW; boxplot; Student’s t-test). (E–F) subdued mutant neurons did not significantly change the number of USs (E; boxplot, Wilcoxon rank sum test) or the amplitudes of the dendritic Ca2+ transients (F; mean ± s.e.m., Student’s t-test). (G–H) Cl− dynamics of subdued mutant neurons expressing SuperClomeleon. The IR laser (30 and 46 mW) was focused onto the proximal dendritic arbors in whole-mount preparations for 1 s (red-dashed boxes in G). (G) Time courses of the FRET ratio of SuperClomeleon at somata (left) and distal dendrites (right) in subdued mutant neurons. The ratio increased upon IR irradiation. Light blue lines indicate each of the Cl− changes, and dark lines represent the averaged amplitudes. (H) Amplitudes of the ΔRpeak for SuperClomeleon increased with IR-laser power in subdued mutant neurons (mean ± s.e.m., Student’s t-test), but they were not significantly different from the values obtained for wild-type neurons in Figure 2C. Furthermore, there were no changes in physiological activities in subdued knockdown neurons (data not shown). These results suggested that Subdued channels can contribute to membrane excitation in Class IV neurons, although their function may be rather limited; in any case, the channels were not a source of inhibitory currents. *p<0.05, **p<0.01, ***p<0.001.