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Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome(1,2,3).

Dinday MT, Baraban SC - eNeuro (2015)

Bottom Line: Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurological Surgery, Epilepsy Research Laboratory, University of California San Francisco , San Francisco, California 94143.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Electrophysiology assay to identify drugs that rescue the scn1Lab mutant epilepsy phenotype. a, Representative field electrode recording epochs (5 min in duration) are shown for the “positive” compounds identified in the locomotion assay. All recordings were obtained with an electrode placed in the forebrain of agar-immobilized scn1Lab larvae that was previously tested in the locomotion assay. A suppression of epileptiform electrographic discharge activity was noted in mutants exposed to dimethadione. b, Bar plot showing the mean number of epileptiform events in a 10 min recording epoch for scn1Lab larvae exposed to cytarabine (N = 6), dimethadione (N = 6), theobromine (N = 6), and norfloxacin (N = 6). The mean ± SEM is shown. The fish shown were tested in the locomotion assay first. c, Bar plot showing the total distance traveled before (black bars) and after (white bars) exposure to a test compound; 10 min recording epoch and six fish per drug. The mean ± SEM is shown.
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Figure 3: Electrophysiology assay to identify drugs that rescue the scn1Lab mutant epilepsy phenotype. a, Representative field electrode recording epochs (5 min in duration) are shown for the “positive” compounds identified in the locomotion assay. All recordings were obtained with an electrode placed in the forebrain of agar-immobilized scn1Lab larvae that was previously tested in the locomotion assay. A suppression of epileptiform electrographic discharge activity was noted in mutants exposed to dimethadione. b, Bar plot showing the mean number of epileptiform events in a 10 min recording epoch for scn1Lab larvae exposed to cytarabine (N = 6), dimethadione (N = 6), theobromine (N = 6), and norfloxacin (N = 6). The mean ± SEM is shown. The fish shown were tested in the locomotion assay first. c, Bar plot showing the total distance traveled before (black bars) and after (white bars) exposure to a test compound; 10 min recording epoch and six fish per drug. The mean ± SEM is shown.

Mentions: Extracellular recording electrodes are a reliable, reproducible, and sensitive approach to monitor electroencephalographic activity in agar-immobilized larval zebrafish (Baraban et al., 2005; Baraban, 2013). Field electrodes offer high a signal-to-noise ratio and can be placed, using direct visualization in transparent larvae, into specific CNS structures (i.e., telencephalon or optic tectum). Using a local field electrode, we can efficiently monitor the occurrence of electrographic seizure events in the same zebrafish that were previously tested in the locomotion assay. Based on a positive nontoxic result in two independent locomotion assays, four drugs moved on to electrophysiology testing at concentrations between 500 µm and 1 mm (Fig. 3). Consistent with a “false-positive” classification, spontaneous epileptiform discharge activity was observed for three of these drugs: norfloxacin, theobromine, and cytarabine. Dimethadione, previously shown to inhibit spontaneous epileptiform discharges in thalamocortical slices at concentrations between 1 and 10 mm (Zhang et al., 1996), suppressed burst discharge activity in scn1Lab mutant larvae (Fig. 3a,b). To identify whether any of these four compounds exert nonspecific effects on behavior, they were also tested on freely swimming WT zebrafish larvae (5 dpf) at a concentration of 500 µm. Comparing the total distance moved during a 10 min recording epoch before, and after, the application of a test compound failed to reveal any significant changes in locomotor activity (Fig. 3c).


Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome(1,2,3).

Dinday MT, Baraban SC - eNeuro (2015)

Electrophysiology assay to identify drugs that rescue the scn1Lab mutant epilepsy phenotype. a, Representative field electrode recording epochs (5 min in duration) are shown for the “positive” compounds identified in the locomotion assay. All recordings were obtained with an electrode placed in the forebrain of agar-immobilized scn1Lab larvae that was previously tested in the locomotion assay. A suppression of epileptiform electrographic discharge activity was noted in mutants exposed to dimethadione. b, Bar plot showing the mean number of epileptiform events in a 10 min recording epoch for scn1Lab larvae exposed to cytarabine (N = 6), dimethadione (N = 6), theobromine (N = 6), and norfloxacin (N = 6). The mean ± SEM is shown. The fish shown were tested in the locomotion assay first. c, Bar plot showing the total distance traveled before (black bars) and after (white bars) exposure to a test compound; 10 min recording epoch and six fish per drug. The mean ± SEM is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596025&req=5

Figure 3: Electrophysiology assay to identify drugs that rescue the scn1Lab mutant epilepsy phenotype. a, Representative field electrode recording epochs (5 min in duration) are shown for the “positive” compounds identified in the locomotion assay. All recordings were obtained with an electrode placed in the forebrain of agar-immobilized scn1Lab larvae that was previously tested in the locomotion assay. A suppression of epileptiform electrographic discharge activity was noted in mutants exposed to dimethadione. b, Bar plot showing the mean number of epileptiform events in a 10 min recording epoch for scn1Lab larvae exposed to cytarabine (N = 6), dimethadione (N = 6), theobromine (N = 6), and norfloxacin (N = 6). The mean ± SEM is shown. The fish shown were tested in the locomotion assay first. c, Bar plot showing the total distance traveled before (black bars) and after (white bars) exposure to a test compound; 10 min recording epoch and six fish per drug. The mean ± SEM is shown.
Mentions: Extracellular recording electrodes are a reliable, reproducible, and sensitive approach to monitor electroencephalographic activity in agar-immobilized larval zebrafish (Baraban et al., 2005; Baraban, 2013). Field electrodes offer high a signal-to-noise ratio and can be placed, using direct visualization in transparent larvae, into specific CNS structures (i.e., telencephalon or optic tectum). Using a local field electrode, we can efficiently monitor the occurrence of electrographic seizure events in the same zebrafish that were previously tested in the locomotion assay. Based on a positive nontoxic result in two independent locomotion assays, four drugs moved on to electrophysiology testing at concentrations between 500 µm and 1 mm (Fig. 3). Consistent with a “false-positive” classification, spontaneous epileptiform discharge activity was observed for three of these drugs: norfloxacin, theobromine, and cytarabine. Dimethadione, previously shown to inhibit spontaneous epileptiform discharges in thalamocortical slices at concentrations between 1 and 10 mm (Zhang et al., 1996), suppressed burst discharge activity in scn1Lab mutant larvae (Fig. 3a,b). To identify whether any of these four compounds exert nonspecific effects on behavior, they were also tested on freely swimming WT zebrafish larvae (5 dpf) at a concentration of 500 µm. Comparing the total distance moved during a 10 min recording epoch before, and after, the application of a test compound failed to reveal any significant changes in locomotor activity (Fig. 3c).

Bottom Line: Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurological Surgery, Epilepsy Research Laboratory, University of California San Francisco , San Francisco, California 94143.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus