Abstracts

Rationale: Dravet syndrome (DS) is a rare, severe and refractory epilepsy syndrome that starts within the first year of life. In 80% of the cases a mutation is present in the SCN1A-gene (neural sodium channel, type 1, subunit α) (Dravet, C. Epilepsia 52, 3–9 (2011)). In a recent clinical study, add-on treatment with fenfluramine led to seizure-freedom in 70% of the DS children (Ceulemans, B. et al. Epilepsia 53, 1131–9 (2012)). Fenfluramine (FA) is a potent 5-hydroxytryptamine (5-HT, serotonin) releaser that activates multiple 5-HTsubtype receptors. We recently confirmed the efficacy of FA as an anti-epileptiform compound in an antisense knockdown zebrafish model of DS (Zhang, Y. et al. PLoS One 10, e0125898 (2015)). In the present study we used zebrafish larvae with a homozygous mutation in the SCN1A orthologue gene (scn1Lab) that recapitulate DS well (Baraban, S. C., Dinday, M. T. & Hortopan, G. A. Nat. Commun. 4, 2410 (2013)) and examined which 5-HTsubtype receptors i) are potentially interesting targets for DS therapeutics; and ii) are involved in the anti-epileptic activity of FA.Methods: A series of highly specific 5-HT-agonists and -antagonists was applied in zebrafish larvae assays, including an automated behavioral tracking (locomotor activity); followed by forebrain local field potential (LFP) recording, measuring epileptiform discharges.Results: Our results show that treatment with FA or some functional analogs (5-HT1D-, 5-HT1E-, 5-HT2A-, 5-HT2C- and 5-HT7-agonist) significantly decreased epileptiform locomotor activity in homozygous scn1Lab-/- mutants (One-way ANOVA; p<0.001 vs. vehicle-treated (control)). Moreover, most of these compounds did not decrease the locomotor activity in age-matched wildtype zebrafish larvae, pointing to a selective effect on scn1Lab-/- mutants. By recording LFPs we confirmed the anti-epileptiform activity of FA and the 5-HT1D-, 5-HT2C-, and especially the 5-HT2A-agonist (Mann-Whitney test; p<0.05 vs. vehicle-treated (control)). Combinatorial treatment of FA and 5-HT-antagonists highlighted the 5-HT1D, 5-HT2A and 5-HT2C receptors in the anti-epileptiform activity of FA.Conclusions: In conclusion by using highly specific 5-HT-agonists and -antagonists in homozygous scn1Lab-/- mutants, that recapitulate Dravet syndrome well; we were able to identify 5-HTsubtype receptors that appear to be involved in the mechanism-of-action of fenfluramine; though other as of yet unknown mechanisms cannot be ruled out. These findings might also open new avenues in the field of anti-epileptic drug discovery for efficient and safe DS treatment.