Figure 4. Responses of Class IV neurons with a knockdown of Ca-α1D and behavioral analysis of the knockdown larvae upon noxious thermal stimulation.

(A–D) Responses of control Class IV neurons (Control, n = 18 and white RNAi ^GL00094, n = 11) and Ca-α1D knockdown neurons (Ca-α1D RNAi ^JF01848, n = 10 and Ca-α1D RNAi ^HMS00294, n = 9) to 1 s irradiation of a 48-mW IR laser. (A) Raster plots of firing (left) and magnitudes of ΔR_peak corresponding to Ca²⁺ transients (right). Trials are sorted in a descending order of the magnitude of ΔR_peak (right bars). Red bar indicates the 1 s irradiation and red raster lines are US. (B) Occurrence rate of dendritic Ca²⁺ transients in each genotype([Control] 72.2 ± 17.6%, [white RNAi GL00094] 81.8 ± 19.4%, [Ca-α1D RNAi ^JF01848] 30.0 ± 24.2%, [Ca-α1D RNAi ^HMS00294] 0.0 ± 0.0%; mean ± 95% confidence interval [Clopper-Pearson method]). (C and D) Histograms of the total US number (C) or the total peak number (D) during the irradiation in the control and the knockdown neurons. The total peak number of firing rate fluctuation is defined as in the text. See also Figure 3A and its legend. (E and F) The distribution of nocifensive escape locomotion latency for wandering third-instar larvae of the wild type and the Ca-α1D mutant that were stimulated with a 47°C probe. The numbers of larvae tested are indicated and data are presented as percentage. (E) The distributions of the wild-type and Ca-α1D mutant larvae (mean latency: [w¹¹¹⁸] 1.78 s, [Ca-α1D ^AR66/X10] 6.12 s; Wilcoxon rank-sum test). (F) The distributions of control larvae and larvae with Class IV neuron-specific white or Ca-α1D knockdown. The Ca-α1D mutant and the Ca-α1D knockdown larvae showed significant delayed nocifensive responses in comparison to the wild type (E) and the controls (Control and white RNAi ^GL00094) (F), respectively. p values in F are those of the test between white RNAi ^GL00094 and each of the Ca-α1D knockdowns (mean latency: [Control] 3.87 s, [white RNAi ^GL00094] 3.39 s, [Ca-α1D RNAi ^JF01848] 5.21 s, [Ca-α1D RNAi ^HMS00294] 5.25 s; Wilcoxon rank-sum test with Bonferroni correction). (G) Percentage of larvae avoiding blue light (457–487 nm; 0.72 mW/mm²; [Control] 59.6 ± 8.0%, [Ca-α1D RNAi ^JF01848] 53.9 ± 10.3%, [Ca-α1D RNAi ^HMS00294] 51.0 ± 8.5%; mean ± 95% confidence interval [Clopper-Pearson method]). We employed GMR-hid to ablate Bolwig’s organs in the photo-avoidance assay. p values are indicated (two-tailed Fisher’s exact test). Sixty-five to 104 larvae were tested for each condition.

DOI: http://dx.doi.org/10.7554/eLife.12959.018

Figure 4—figure supplement 1. Simultaneous recordings of firing responses and dendritic Ca²⁺ transients in fillet preparations of Ca-α1D knockdown larvae upon noxious thermal stimulation.

(A and B) Responses of control Class IV neurons (Control and white RNAi ^GL00094) and Ca-α1D knockdown neurons (Ca-α1D RNAi ^JF01848 and Ca-α1D RNAi ^HMS00294) to 1 s irradiation of a 48-mW IR laser. (A) Time courses of firing rates of the wild type and the Ca-α1D knockdown larvae upon noxious thermal stimulations. (B) The max. firing rate (defined in Methods) of the Control, white RNAi ^GL00094, Ca-α1D RNAi ^JF01848, and Ca-α1D RNAi ^HMS00294 showed normal firing rates during the IR-laser irradiation ([Control] 96.2 ± 5.1 Hz, n = 18 cells; [white RNAi ^GL00094] 100.0 ± 8.4 Hz, n = 11 cells; [Ca-α1D RNAi ^JF01848] 85.5 ± 4.1 Hz, n = 10; [Ca-α1D RNAi ^HMS00294] 82.5 ± 7.7 Hz, n = 9). (C) Quantification of timing of maximum excitation ('Time to Max. Firing Rate' as defined in Methods) of Class IV neuron of each genotype. (Time to Max. Firing Rate: [Control] 47.5 ± 16.8 ms, [white RNAi ^GL00094] 37.8 ± 11.9 ms; [Ca-α1D RNAi ^JF01848] 21.1 ± 3.3 ms; [Ca-α1D RNAi ^HMS00294] 23.5 ± 1.6 ms). Data are presented as mean ± s.e.m (A–C). No statistically significant difference was observed between the Ca-α1D RNAi neurons and wild type or white RNAi ^GL00094 by unpaired t-test with Bonferroni correction (B and C). Genotypes: (Control) w¹¹¹⁸/+; ppk-Gal4/+; 3×[ppk-TNXXL] (attP2)/+ (white RNAi ^GL00094) ppk-Gal4/+; 3×[ppk-TN-XXL] (attP2)/UAS-white RNAi ^GL00094 (Ca-α1D RNAi ^JF01848) ppk-Gal4/+; 3×[ppk-TNXXL] (attP2)/UAS-Ca-α1D RNAi^JF01848 (Ca-α1DRNAi ^HMS00294) ppk-Gal4/+; 3×[ppk-TNXXL] (attP2)/UAS-Ca-α1DRNAi^HMS00294

DOI: http://dx.doi.org/10.7554/eLife.12959.020