Zebrafish have recently emerged as an attractive in vivo model for epilepsy. Seven-day-old zebrafish larvae exposed to the GABAA antagonist pentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-like behavior, and epileptiform electrographic activity. A previous study showed that 12 out of 13 antiepileptic drugs (AEDs) suppressed PTZ-mediated increases in larval movement, indicating the potential utility of zebrafish as a high-throughput in vivo model for AED discovery. However, a question remained as to whether an AED-induced decrease in locomotion is truly indicative of anticonvulsant activity, as some drugs may impair larval movement through other mechanisms such as general toxicity or sedation. We therefore carried out a study in PTZ-treated zebrafish larvae, to directly compare the ability of AEDs to inhibit seizure-like behavioral manifestations with their capacity to suppress epileptiform electrographic activity. We re-tested the 13 AEDs of which 12 were previously reported to inhibit convulsions in the larval movement tracking assay, administering concentrations that did not, on their own, impair locomotion. In parallel, we carried out open-field recordings on larval brains after treatment with each AED. For the majority of AEDs we obtained the same response in both the behavioral and electrographic assays. Overall our data correlate well with those reported in the literature for acute rodent PTZ tests, indicating that the larval zebrafish brain is more discriminatory than previously thought in its response to AEDs with different modes of action. Our results underscore the validity of using the zebrafish larval locomotor assay as a rapid first-pass screening tool in assessing the anticonvulsant and/or proconvulsant activity of compounds, but also highlight the importance of performing adequate validation when using in vivo models.

Turmeric, obtained from the rhizomes of Curcuma longa, is used in South Asia as a traditional medicine for the treatment of epilepsy. To date, in vivo studies on the anticonvulsant activity of turmeric have focused on its principal curcuminoid, curcumin. However, poor absorption and rapid metabolism have limited the therapeutic application of curcumin in humans. To explore the therapeutic potential of turmeric for epilepsy further, we analyzed its anticonvulsant activity in a larval zebrafish seizure assay. Initial experiments revealed that the anticonvulsant activity of turmeric in zebrafish larvae cannot be explained solely by the effects of curcumin. Zebrafish bioassay-guided fractionation of turmeric identified bisabolene sesquiterpenoids as additional anticonvulsants that inhibit PTZ-induced seizures in both zebrafish and mice. Here, we present the first report of the anticonvulsant properties of bisabolene sesquiterpenoids and provide evidence which warrants further investigation toward the mechanistic understanding of their neuromodulatory activity.

In view of the clinical need for new anti-epileptic drugs (AEDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used for traditional epilepsy treatment in the Congo, identifying 4 crude plant extracts that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of an anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, an bis-indole alkaloid with known inhibitory activity at glycogen synthase kinase (GSK)-3, a well known protein kinase. Target validation experiments with partial loss of function of GSK-3 via antisense knockdown in zebrafish showed anticvonvulant activity against PTZ-induced seizures. Subsequently, indirubin, together with the more potent and GSK-3 specific derivative 6-bromoindirubin-3'-oxime (BIOacetoxime) and the structurally unrelated GSK-3β inhibitor 2-methyl-5-[3-[4-(methylsulfinyl)phenyl]-5-benzofuranyl]-1,3,4-oxadiazole (TCS2002), were tested in zebrafish and rodent seizure assays. All 3 compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. All 3 compounds also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz psychomotor refractory seizure mouse model. Moreover, BIOacetoxime, the most potent GSK-3 inhibitor was also able to exert anticonvulsant actions in 6-Hz fully kindled mice. Our findings provide the first evidence for anticonvulsant activity of selective GSK-3 inhibitors, thereby implicating GSK-3 as a potential new drug target for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of anti-epileptic medicinal plant extracts as an effective strategy for the discovery of new AEDs with novel mechanisms of action.

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