Figure 6. Active signal propagation in the dendrites of c4da.

(A) Representative images and curves showing the responses (ΔF/F₀) of the soma (red arrowhead), the homolateral dendrite (position p1: yellow arrowhead) and contralateral dendrite (position p2: white arrowhead) of c4da-wt, nimodipine-treated c4da-wt, c4da-Ca-α1Dⁱ and Ca-α1D^X10/AR66 to the stimuli (4 mN, 60 μm probe). The asterisk indicated the force application point. Scale bar: 50 μm. C4da-wt: ppk-gal4/+; ppk-cd4-tdtom/20×uas-ivs-gcamp6s. Ca-α1Dⁱ: ppk-gal4/20×uas-ivs-gcamp6s; ppk-cd4-tdtom/ uas-Ca-α1Dⁱ. Ca-α1D^X10/AR66: Ca-α1D^X10/ Ca-α1D^AR66; ppk-gal4/20×uas-ivs-gcamp6s. (B) Statistical quantification of calcium increases in the homolateral dendrites in response to the stimuli (4 mN, 60 μm probe). The genotypes were the same as those shown in panel (A). Mann Whitney U test was used for the comparison between c4da-wt and the mutants. Kruskal Wallis test was used for the comparison among the groups with drug treatment. ***: p<0.001. **: p<0.01. ns: no significance. (C) and (D) The responses (ΔF/F₀) of nimodipine treated c4da-wt to the proximal and distal stimuli from the probes of different sizes. (C): 60 μm probe stimuli. (D) : 30 μm probe stimuli. Kruskal Wallis test was used. ***: p<0.001. **: p<0.01. *: p<0.05. ns: no significance. (E) and (F) The responses (ΔF/F₀) of c4da-wt (n=10 cells) treated with 2.5 μM nimodipine to the proximal (E) and distal (F) stimuli delivered using the probes of two different sizes. Solid lines: 30 μm probe. Dashed lines: 60 μm probe. (G) The responses (ΔF/F₀) of c4da-Ca-α1Dⁱ and c4da-Ca-α1D^X10/AR66 to the stimuli applied at different dendritic regions (proximal and distal) and delivered using the probes of two different sizes (60 μm: 4 mN, 30 μm: 1.5 mN). (H) The behavioral responses of the c4da-Ca-α1Dⁱ larvae to mechanical poking using the probes of two different sizes. wt: w1118. ppk-gal4: ppk-gal4; +/+. uas-Ca-α1Dⁱ: +/+; uas-Ca-α1Dⁱ. Ca-α1Dⁱ: ppk-gal4/+; uas-Ca-α1Dⁱ /+. Ca-α1D^X10/AR66: Ca-α1D^X10/ Ca-α1D^AR66; +/+. uas-Ca-α1Dⁱ larvae: 30 μm probe (n=61 larvae from three experiments), 60 μm probe (n=61 larvae from three experiments). Ca-α1Dⁱ larvae: 30 μm probe (n=56 larvae from three experiments), 60 μm probe (n=60 larvae from three experiments). Ca-α1D^X10/AR66 larvae: 30 μm probe (n=43 larvae from three experiments), 60 μm probe (n=47 larvae from three experiments). One-way ANOVA test was used for the comparison among different genotypes. ***: p<0.001. ns: no significance. In panels (B), (C), (D), (G) and (H), data were presented as mean ± std. In panels (B), (C), (D) and (G), the numbers of cells were indicated below the scattered data points. In panels (E) and (F), data were presented as mean ± sem. In panels (C), (D), (E), (F), (G) and (H), the corresponding data from c4da-wt were provided for comparison.

Figure 6—figure supplement 1. Statistics quantification of the calcium increase in the contralateral dendrites in response to the force stimuli.

C4da-wt: ppk-gal4/+; ppk-cd4-tdtom/20×uas-ivs-gcamp6s. Scrambledⁱ: ppk-gal4/20×uas-ivs-gcamp6s, ppk-cd4-tdtom/uas-Scrambledⁱ. Ca-α1Dⁱ: ppk-gal4/20×uas-ivs-gcamp6s; ppk-cd4-tdtom/uas-Ca-α1Dⁱ. Ca-α1D^X10/AR66: Ca-α1D^X10/Ca-α1D^AR66; ppk-gal4/20×uas-ivs-gcamp6s. The force stimuli (4 mN) were applied using a 60 μm probe. The numbers of cells were indicated below the scattered data points. Mann Whitney U test was used for the comparison between c4da-wt and the mutants. Kruskal Wallis test was used for the comparison among the groups with drug treatment. *: p<0.05. **: p<0.01. **: p<0.001. ns: no significance.

Figure 6—figure supplement 2. The expression and localization of Ca-α1D in vivo.

(A) The expression level of Ca-α1D in wild-type and Ca-α1Dⁱ was examined using RT-PCR. Because Ca-α1D expressed in various types of neurons and the number of c4da in the whole animal was small, we knocked down the expression of Ca-α1D in the whole animal using tub-gal4. Using RT-PCR, we found that the expression level of Ca-α1D was significantly reduced in the Ca-α1Dⁱ strain. The expression of rp49 was used as the reference for normalization. Note that the same strain was also used as a knockdown mutant in other functional studies (Terada et al., 2016; Basak et al., 2021). (B) The expression pattern of Ca-α1D analyzed based on the FlyCellAtlas (Li et al., 2022) single-cell RNA-sequencing datasets We found that Ca-α1D mainly expressed in excitable cells, such as neurons (red square) and muscle cells (blue square). In particular, it expressed in the multidendritic neurons that specifically expressed ppk1 (black arrowhead). (C) To confirm the expression of Ca-α1D in c4da, we examined the Mi{ET1}Ca-α1D^MB06807 line. However, no significant signal was observed. Control: ppk-cd4-tdgfp/+; uas-cd4-tdtom/+. Ca-α1D^MB06807: Mi{ET1}Ca-α1D^MB06807/ppk-cd4-tdgfp; uas-cd4-tdtom/+. n=30 cells for each genotype. (D) We generated a transgenic strain that expressed mCherry-Ca-α1D under the endogenous promoter of Ca-α1D. However, no signal was observed (n=12 cells). Control: ppk-cd4-tdgfp/+; +/+. mcherry-Ca-α1D: ppk-cd4-tdgfp/+;mcherry-Ca-α1D. (E) We used a previously reported antibody (see Materials and Methods) to examine the localization of Ca-α1D. However, no signal was observed. (F) We generated a transgenic strain to express mCherry-Ca-α1D under the control of the ppk-gal4 strain. However, no signal was observed (n=12 cells). Control: ppk-cd4-tdgfp/+; ppk-gal4/+. uas-mcherry-Ca-α1D: ppk-cd4-tdgfp/+; 10×uas-mcherry-Ca-α1D/ppk-gal4. In panel C, D and F, scale bar: 30 μm. In panel (E), scale bar: 100 μm. In panel (A, C, D and F), data were presented as mean ± std. Student’s t test was used for statistical analysis. *: p<0.05; ns: no significance.