Dravet syndrome (DS) is a catastrophic pediatric epilepsy with severe intellectual disability, impaired social development and persistent drug-resistant seizures. One of its primary monogenic causes are mutations in Nav1.1 (SCN1A), a voltage-gated sodium channel. Here we characterise zebrafish Nav1.1 (scn1Lab) mutants originally identified in a chemical mutagenesis screen. Mutants exhibit spontaneous abnormal electrographic activity, hyperactivity and convulsive behaviors. Although scn1Lab expression is reduced, microarray analysis is remarkable for the small fraction of differentially expressed genes (~3%) and lack of compensatory expression changes in other scn subunits. Ketogenic diet, diazepam, valproate, potassium bromide and stiripentol attenuate mutant seizure activity; seven other antiepileptic drugs have no effect. A phenotype-based screen of 320 compounds identifies a US Food and Drug Administration-approved compound (clemizole) that inhibits convulsive behaviors and electrographic seizures. This approach represents a new direction in modeling pediatric epilepsy and could be used to identify novel therapeutics for any monogenic epilepsy disorder.
Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate salient phenotypes associated with DS, including seizures, early fatality, and resistance to antiepileptic drugs. To discover new drug candidates for the treatment of DS, we screened a chemical library of ∼1000 compounds and identified 4 compounds that rescued the behavioral seizure component, including 1 compound (dimethadione) that suppressed associated electrographic seizure activity. Fenfluramine, but not huperzine A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were also identified. We conclude that the combination of behavioral and electrophysiological assays provide a convenient, sensitive, and rapid basis for phenotype-based drug screening in zebrafish mimicking a genetic form of epilepsy.
Summary:Purpose: Zebrafish are a vertebrate organism ideally suited to mutagenesis screening strategies. Although a genetic basis for seizure susceptibility and epilepsy is well established, no efforts have been made to study seizure resistance. Here we describe a novel strategy to isolate seizure-resistant zebrafish mutants from a large-scale mutagenesis screen.Methods: Seizures were induced with pentylenetetrazole (PTZ). Zebrafish were analyzed between 3 and 7 days postfertilization (dpf). Genome mutations were induced in founders by using N-ethyl-nitrosourea (ENU). Seizure behavior was monitored by using a high-speed camera and quantified by locomotiontracking software. Electrographic activity was monitored by using a field-recording electrode placed in the optic tectum of agarimmobilized zebrafish.Results: Short-term PTZ exposure elicited a burst-suppression seizure pattern in 3-dpf zebrafish and more complex activity consisting of interictal-and ictal-like discharges at 7 dpf. Prolonged exposure to PTZ induced status epilepticus-like seizure activity and fatality in wild-type zebrafish larvae. With a PTZ survival assay at 6-7 dpf, we identified six zebrafish mutants in a forward-genetic screen covering nearly 2,000 F 2 families. One mutant (s334) also was shown to exhibit reduced behavioral activity on short-term PTZ exposure and an inability to generate long-duration ictal-like discharge.Conclusions: Zebrafish offers a powerful tool for the identification and study of a genetic basis for seizure resistance.
Despite a long tradition of using rats and mice to model epilepsy, several aspects of rodent biology limit their use in large-scale genetic and therapeutic drug screening programs. Neuroscientists interested in vertebrate development and diseases have recently turned to zebrafish (Danio rerio) to overcome these limitations. Zebrafish can be studied at all stages of development and several methods are available for the manipulation of genes in zebrafish. In addition, developing zebrafish larvae can efficiently equilibrate drugs placed in the bathing medium. Taking advantage of these features and adapting electrophysiological recording methods to an agar-immobilized zebrafish preparation, we describe here our efforts to model seizure disorders in zebrafish. We also describe the initial results of a large-scale mutagenesis screen to identify gene mutation(s) that confer seizure resistance. Although the adaptation of zebrafish to epilepsy research is in its early stages, these studies highlight the rapid progress that can be made using this simple vertebrate species.
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