Zebrafish (Danio rerio) assays provide a versatile pharmacological platform to test compounds on a wide range of behaviors in a whole organism. A major challenge lies in the lack of knowledge about the bioavailability and pharmacodynamic effects of bioactive compounds in this model organism. Here, we employed a combined methodology of LC-ESI-MS/MS analytics and targeted metabolomics with behavioral experiments to evaluate the
anticonvulsant and potentially toxic effects of the angular dihydropyranocoumarin
pteryxin (PTX) in comparison to the
antiepileptic drug sodium valproate (VPN) in zebrafish larvae. PTX occurs in different Apiaceae plants traditionally used in Europe to treat
epilepsy but has not been investigated so far. To compare potency and efficacy, the uptake of PTX and VPN into zebrafish larvae was quantified as larvae whole-body concentrations together with
amino acids and
neurotransmitters as proxy pharmacodynamic readout. The
convulsant agent
pentylenetetrazole (PTZ) acutely reduced the levels of most metabolites, including
acetylcholine and
serotonin. Conversely, PTX strongly reduced neutral
essential amino acids in a LAT1 (SLCA5)-independent manner, but, similarly to VPN specifically increased the levels of
serotonin,
acetylcholine, and
choline, but also
ethanolamine. PTX dose and time-dependent manner inhibited PTZ-induced seizure-like movements resulting in a ~70% efficacy after 1 h at 20 µM (the equivalent of 4.28 ± 0.28 µg/g in larvae whole-body). VPN treated for 1 h with 5 mM (the equivalent of 18.17 ± 0.40 µg/g in larvae whole-body) showed a ~80% efficacy. Unexpectedly, PTX (1-20 µM) showed significantly higher bioavailability than VPN (0.1-5 mM) in immersed zebrafish larvae, possibly because VPN in the medium dissociated partially to the readily bioavailable
valproic acid. The anticonvulsive effect of PTX was confirmed by local field potential (LFP) recordings. Noteworthy, both substances specifically increased and restored whole-body
acetylcholine,
choline, and
serotonin levels in control and PTZ-treated zebrafish larvae, indicative of
vagus nerve stimulation (VNS), which is an adjunctive therapeutic strategy to treat
refractory epilepsy in humans. Our study demonstrates the utility of targeted metabolomics in zebrafish assays and shows that VPN and PTX pharmacologically act on the autonomous nervous system by activating parasympathetic
neurotransmitters.