LAUSR

BACKGROUND Locomotor assays in zebrafish have emerged as a screening test in early drug discovery for antiseizure compounds. However, parameters differ considerably between published studies, which may explain some discrepant results with (candidate) antiseizure medications. NEW METHOD We optimized a locomotor-based seizure assay in zebrafish with pentylenetetrazol (PTZ) as the pharmacological proconvulsant to generate a therapeutic window in which proconvulsant-treated zebrafish larvae could be discriminated from a non-treated control. To generate a reliable control, exposure time and concentration of valproate (VPA, anticonvulsant) was optimized. RESULTS Wells with one or three larvae show a similar PTZ dose-dependent increase in locomotion with less variability in motility for the latter. Zebrafish immersed in 10 mM PTZ showed a significant increase in movement with a sustained effect, without any indication of toxicity. Animals treated with 3 mM VPA showed the strongest reduction of PTZ-induced movement without toxicity. The decrease in PTZ-induced locomotion was greater after 18 h versus 2 h. COMPARISON WITH EXISTING METHOD(S) For the larval zebrafish PTZ-induced seizure model, varying experimental parameters have been reported in literature. Our results show that PTZ is often used at toxic concentrations, and we provide instead reliable conditions to quantify convulsant behaviour using an infrared-beam motility assay. CONCLUSIONS We recommend using three zebrafish larvae per well to quantify locomotion in 96-multiwell plates. Larvae should preferably be exposed to 10 mM PTZ for 1 h, consisting of 30 min acclimation and 30 min subsequent recording. As positive control for anticonvulsant activity, we recommend exposure to 3 mM VPA for 18 h before administration of PTZ.