Figure 3

stj^RNAi and stol^RNAi have different effects on axonal Ca_v2 channel abundance and AP shape. (A–C) Projection views (10 optical sections) of DLM MN axons with endogenously tagged Ca_v2^GFP channels, with reported normal function⁵³. (A) As compared to control (top panel) stj^RNAi (middle panel) decreases Ca_v2^GFP label in DLM MN axons (encircled by dotted white line), whereas stol^RNAi (bottom panel) increases labeling intensity. (B) Quantification of mean gray of Ca_v2^GFP puncta in confocal sections reveals a ~ 20% significant decrease in stj^RNAi (light gray bar, **p = 0.006, n = 9) but a ~ 20% significant increase in stol^RNAi (dark gray bar, **p = 0.001, n = 10) as compared to control (white bar, N = 9). Error bars represent SD; statistics, one-way ANOVA, LSD post-hoc test. (C) Pupal MN5 action potentials (APs) were recorded in 1.8 (left) and 5 mM Ca²⁺ saline (right) and elicited by somatic square pulse current injection. APs showed a Ca²⁺ shoulder (C, top, left trace, see arrow) that was abolished by the VGCC blocker Cd²⁺ (500 µM; C, top, gray trace) and broadened in high Ca²⁺(C, top, right trace). APs were smaller and narrower in stj^RNAi (C, second row, left trace) as compared to control (C, top, left trace) and were neither narrowed by Cd²⁺ (C, second row, gray trace) nor broadened in 5 mM Ca²⁺ (C, second row, right trace). stol^RNAi often caused a double peak (C, third row, left trace) and APs were broadened in high Ca²⁺ (C, third row, right trace), but abolished in Cd²⁺ (C, third row, gray trace). stj^RNAistol^RNAi resulted in APs that were as small as with stj^RNAi but slightly broader, both in 1.8 (C, bottom, left trace) and in 5 mM Ca²⁺ (right trace). Cd²⁺ caused a small narrowing effect (C, bottom, gray trace). (D) Cadmium reduced AP half width significantly more in control (*p = 0.012) than in stj^RNAi (***p < 0.001) but significantly less than with stol^RNAi (*p = 0.016). In stj^RNAistol^RNAi the Cd²⁺ effect was small, but significantly larger as compared to stj^RNAi (*p = 0.016). Diagrams show single data points and medians (Kruskal–Wallis ANOVA, Dunn’s post-hoc test).