EEG analysis of anti-seizure efficacy of VA in the brains of PTZ-treated zebrafish larvae (A) EEG signals corresponding to PTZ-induced seizure-like activity were recorded in the brains of zebrafish larvae pre-treated with 0, 0.01, 0.1, and 1 μM VA or with 1000 μM VPA for comparison. The total seizure count (a) and cumulative duration of seizures (b) recorded during EEG analyses are shown. We examined 6 to 9 fish in each treatment group for EEG analysis. The results are expressed as the mean ± standard error of the mean (S.E.M.). A one-way analysis of variance (ANOVA) was conducted for statistical analysis, and the significance of the results was confirmed. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; (B) mRNA expression analysis of c-fos in 5 dpf zebrafish larvae using qRT-PCR. Data are presented as mean ± standard error of the mean (S.E.M.). Statistical significance was determined using Student’s t-test. ** p < 0.01, *** p < 0.001; (C) Lateral views of bright-field images of zebrafish embryos treated with VA at various concentrations that showed anti-seizure efficacy. Scale bar = 400 µm. The embryos at 1 dpf (a–e) and larvae at 5 dpf (a’–e’) showed no observable morphological abnormalities. The experiment was conducted with 24 to 54 fish in each treatment group.

EEG analysis of the anti-seizure efficacy of VA in the brains of adult zebrafish administered PTZ. The collected data were represented graphically, depicting the total number of seizures (A) and cumulative duration (B). The experiment was conducted with 6 to 9 fish in each treatment group. The results are presented as mean ± standard error of the mean (S.E.M.). A one-way analysis of variance (ANOVA) was employed for the statistical analysis, and the significance of the findings was subsequently ascertained. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Analysis of validamycin A’s efficacy in mitigating locomotive changes in adult zebrafish via measurements of angular velocity. (A) The red lines and arrows indicate the locations of the zebrafish and abrupt changes in movement direction during a 1-min video recording in a 90 mm Petri dish. (B) Angular velocity was calculated. The sample size for each experimental group ranged from 12 to 26 fish. The collected data are presented as the mean ± standard error of the mean (S.E.M.). Student’s t-test was utilized for the statistical analysis to determine the significance of the observed differences. * p < 0.05, ** p < 0.01.

Quantification of the therapeutic dosage of validamycin A. (A) The half-lethal dose (LD50) was evaluated by determining the survival rate following oral administration of VA to zebrafish at doses of 0, 7000, and 14,000 mg/kg (mpk); (B) Half-effective dose (ED50) was determined after administering varying concentrations of VA—0, 0.75, 7.5, 75, 150, 300, 600, and 1200 mpk—alongside 220 mpk of PTZ to the fish. The ED50 was measured based on the presence or absence of epileptiform discharges. The therapeutic index was calculated by dividing the LD50 by the ED50. For VA, the therapeutic index was determined to be above 34.2, indicating a high safety margin for the drug. Each experimental group comprised 8–10 fish.