Figure 2. Octopamine is required to activate salt inducible kinase 3 (SIK3)-regulated K⁺ buffering program in glia.

(A) Representative images of peripheral nerves showing aberrant HDAC4 localization in octopamine synthesis and transport mutants. Grayscale images show HDAC4 staining; glial nuclei are outlined in red. Scale bars 15 μm. (B) Quantification of nucleo:cytoplasmic ratio of HDAC4 for genotypes in (A). n ≥ 20. Data are presented as fold changes relative to Repo>HDAC4. One-way ANOVA with Tukey's multiple comparisons; ***, p<0.001; ****, p<0.0001. (C) Quantification of number of nerve swellings per animal in HDAC4 overexpressing control larvae (Repo>HDAC4) and octopamine mutants. n ≥ 20 larvae per genotype. One-way ANOVA with Tukey's multiple comparisons; ***, p<0.001. ****, p<0.0001. (D) Quantification of HDAC4 localization in octopamine synthesis mutants, mutants with glial expression of LexA RNAi as control or PKA-C1 RNAi. Octopamine synthesis mutants (tdc2^R054) exhibit HDAC4 accumulation in glial nuclei; this nuclear localization is rescued by abolishing protein kinase A (PKA) catalytic activity in glia (Repo>PKA-C1 RNAi). One-way ANOVA with Tukey's multiple comparisons; ****, p<0.0001. (E) Representative images of nerves demonstrating the effect of 30 mg/ml octopamine or 30 mg/ml tyramine on octopamine synthesis mutant glia in an ex vivo assay. Scale bars 15 μm. (F) Quantification of HDAC4 nucleo:cytoplasmic ratio for genotypes in (D). Octopamine (30 mg/ml), but not TA (30 mg/ml) or KCl (500 mM), suppresses HDAC4 nuclear localization in glia. n ≥ 15. One-way ANOVA with Tukey's multiple comparisons; ***, p<0.001; NS = not significant, p>0.05. (G) Quantification of number of nerve swellings per animal in larvae with glial expression of LexA RNAi as control (Repo>), Octβ1R RNAi, PKA-C1 RNAi, or co-expression of Octβ1R RNAi and PKA-C1 RNAi. n ≥ 20. Student's t test; ****, p<0.0001. Data are mean ± SEM.