Pharmacological inhibition of HDAC4 suppresses neuronal hyperexcitability and seizure susceptibility of SIK3 mutants. (A) Quantification of nerve swellings in wild-type and SIK3Δ48 mutant larvae raised on control or TSA-containing (10 µM) diet. TSA, a pan-HDAC inhibitor, completely rescues the nerve defects in SIK3 mutants. n = 20. Two-way ANOVA with Tukey’s multiple comparisons; ****, P < 0.0001. (B) Representative physiological traces recorded from larval NMJs. Spontaneous EJPs (asterisks) caused by loss of SIK3 from glia are suppressed in larvae fed TSA-containing diet. These events do not occur in control larvae (Repo>) that display only mEJPs (arrowheads). (C) Quantification of frequency of spontaneous EJPs for genotypes in B. Events with amplitudes ≥4 mV were considered spontaneous EJPs, and the spontaneous EJP frequencies were averaged per genotype per condition. Control diet: n = 9 for Repo>, n = 9 for Repo>SIK3 RNAi; TSA-rich diet: n = 9 for Repo>, n = 8 for Repo>SIK3 RNAi. Two-way ANOVA with Tukey’s multiple comparisons; **, P < 0.01. (D) Representative EJP traces recorded from larval NMJs. TSA suppresses the supernumerary evoked events in SIK3 RNAi-expressing larvae following a stimulus without impacting the evoked response of controls. Control diet: n = 8 for Repo>, n = 10 for Repo>SIK3 RNAi; TSA-rich diet: n = 8 for Repo>, n = 8 for Repo>SIK3 RNAi. (E) Time course of vortex-induced seizure behaviors in control and SIK3 RNAi-expressing flies fed control or TSA-containing diet as adults. n ≥ 5 groups of 10 flies per genotype for each time point. Two-tailed Student’s t test; **, P < 0.01. Data are mean ± SEM.