The structure of (+)-FFA (A), (-)-FFA (B), (+)-norFFA (C) and (−)-norFFA (D)

Behavioral (A) and electrophysiological (B, C) antiepileptic activity of valproate (VPA), (±) fenfluramine [(±)-FFA], topiramate (TPM), stiripentol (STP), cannabidiol (CBD), clobazam (CLB), levetiracetam (LEV), carbamazepine (CBZ), phenytoin (PHT), and lamotrigine (LTG) in the zebrafish scn1Lab^−/− mutant model. (A) Locomotor activity of larvae pre-exposed to antiepileptic drugs (AEDs) for 22 h. Data were assessed over the total tracking period of 10 min and expressed as cm/100 s. Results were pooled from 3–4 independent experiments, with 207 larvae for each of the VHC-treated groups, and 22–31 larvae for each AED-treated group. (B, C) Noninvasive local field potential (LFP) recordings from the optic tectum of larvae pre-exposed to antiepileptic drugs (AEDs) for 22 h. Epileptiform discharges are quantified by the cumulative duration (mean ± SEM) (B) and frequency (mean ± SEM) (C) of events per 10-min recording. With 72 larvae for the VHC-treated group, 10–15 larvae for each AED-treated group. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparison test (locomotor assay), Kruskal–Wallis testing with Dunn’s multiple comparisons (LFP measurements). Significance levels: ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001. WT wide type, VHC vehicle

Behavioral (A, D, G) and electrophysiological (B, C, E, F, H, I) antiepileptic activity of combination treatment (colored in blue) of valproate (VPA) with stiripentol (STP) (A–C), (±) fenfluramine [(±)-FFA] (D–F), and cannabidiol (CBD) (G–I) in the zebrafish scn1Lab^−/− mutant model. (A, D, G) Locomotor activity of larvae pre-exposed to antiepileptic drugs (AEDs) for 22 h. Data were assessed over the total tracking period of 10 min and expressed as cm/100 s. Results were pooled from 3–4 independent experiments, with 120 larvae for each of the VHC-treated groups, and 20–30 larvae for each AED-treated group. (B, C, E, F, H, I) Noninvasive local field potential (LFP) recordings from the optic tectum of larvae pre-exposed to antiepileptic drugs (AEDs) for 22 h. Epileptiform discharges are quantified by the cumulative duration (mean ± SEM) (B, E, H) and frequency (mean ± SEM) (C, F, I) of events per 10-min recording. With 23 larvae for the VHC-treated group, 12–14 larvae for each AED-treated group. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparison test (locomotor assay), Kruskal–Wallis testing with Dunn’s multiple comparisons (LFP measurements). Significance levels: ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001. WT wild type, VHC vehicle

The total uptake of (+)-FFA (A), (−)-FFA (B), (+)-norFFA (C) and (−)-norFFA (D) in the larvae head after different exposure times (30 min, 4 h and 22 h). The concentration in the head of larvae pre-exposed to (+)-FFA (A), (−)-FFA (B), (+)-norFFA (C) and (−)-norFFA (D) at their respective MTCs for 30 min, 4 h and 22 h, separately, and total uptake of compound = compound concentration/head weight. With 5 larvae heads for each sample, and 5 replicates for a total of 25 larvae heads per treatment. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparison test. Significance levels: ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001. WT wild type, VHC vehicle, FFA fenfluramine, norFFA norfenfluramine

Behavioral antiepileptic activity of (+)-FFA (A), (−)-FFA (B), (+)-norFFA (C) and (−)-norFFA (D) in the zebrafish scn1Lab^−/− mutant model, and ( ±)-FFA (colored in blue, A–D), used as a positive control. (A–D) Locomotor activity of larvae pre-exposed to different concentration of enantiomers of FFA and norFFA for 22 h. Data were assessed over the total tracking period of 10 min and expressed as cm/100 s. Results were pooled from 3–4 independent experiments, with 67–75 larvae for each VHC-treated group, and 21–28 larvae for each compound-treated group. Statistical analysis: one-way ANOVA with Dunnett’s multiple comparison test. Significance levels: ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001. WT wild type, VHC vehicle, FFA fenfluramine, norFFA norfenfluramine

Representative local field potential recordings. Ten-min noninvasive local field potential (LFP) recordings from the optic tectum of larvae pre-exposed to (+)-FFA, (−)-FFA, (+)-norFFA and (−)-norFFA for 22 h. WT wild type, VHC vehicle, FFA fenfluramine, norFFA norfenfluramine

Electrophysiological antiepileptic activity of (+)-FFA (A, E), (−)-FFA (B, F), (+)-norFFA (C, G) and (−)-norFFA (D, H) in the scn1Lab^−/− mutant model, and ( ±)-FFA (colored in blue, A–H) used as a positive control. (A–H) Noninvasive local field potential (LFP) recordings from the optic tectum of larvae pre-exposed to (+)-FFA, (−)-FFA, (+)-norFFA and (−)-norFFA for 22 h. Epileptiform discharges are quantified by the cumulative duration (mean ± SEM) (A–D) and frequency (mean ± SEM) (E–H) of events per 10-min recording. With 27–45 larvae for each VHC-treated group, and 10–16 larvae for each compound-treated group. Statistical analysis: Kruskal–Wallis testing with Dunn’s multiple comparisons. Significance levels: ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001. WT wild type, VHC vehicle, FFA fenfluramine, norFFA norfenfluramine