A large-scale mutagenesis screen to identify seizure-resistant zebrafish

Abstract

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 locomotion-tracking software. Electrographic activity was monitored by using a field-recording electrode placed in the optic tectum of agar-immobilized 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.

FIG. 1

Electrographic seizure activity in immature zebrafish. A: Representative gap-free field recording from an electrode placed in the optic tectum of an agar-immobilized zebrafish larvae at 3 and 7 days postfertilization (dpf). Fish were exposed to 15 mM PTZ for 40 min. B: Individual burst-discharge waveforms from zebrafish exposed to 15 mM PTZ at 3 and 7 dpf. For comparison, a representative field recording from an electrode placed in CA3 stratum pyramidale mouse hippocampus maintained in vitro and perfused with artificial cerebrospinal fluid containing 1.5 mM PTZ is shown at right. Note the similarities in burst-discharge patterns between 7-dpf zebrafish larvae and mouse.

FIG. 2

A screen for seizure-resistant zebrafish. A: Sample tectal recording in agar-embedded zebrafish larvae (7 dpf) exposed to 15 mM PTZ for 10 h. Flat EEG, a lack of heart beat, and death were noted several hours later. Traces are clipped by the data-acquisition program. Arrow, Initiation of status-like electrographic seizure activity. B: Plot of responsive WT zebrafish larvae at various time points during prolonged exposure to PTZ; fish are freely swimming in a Petri dish, 30–50 fish per plate. Three different PTZ concentrations are shown. Note that 15 mM PTZ resulted in clonus-like convulsions and fatalities starting at ~15 h of continuous drug exposure, confirmed by visual observation. Seizure resistant (SR) mutants were identified as a dish containing 20–30% responsive fish at 16–19 h PTZ exposure. C: Summary of PTZ survival assay data for six mutant strains. SR assays were performed in duplicate on all positive mutants (first and second PTZ) and then, in triplicate, on all outcrossed zebrafish (OX first, second, third). Results from each of these assays, presented as a percentage of zebrafish responsive per clutch, are shown for six putants. All other fish exhibited <5% responsiveness (not shown). Mendelian ratio of 25% responsiveness was taken as a “positive hit,” with a 5% margin of error (dashed lines in plot).

FIG. 3

Behavioral characterization of mutant s334. A: Sample locomotion plots from 2-min recording epochs obtained in embryo medium (A1) and 10 min after exposure to medium containing 15 mM pentylenetetrazole (PTZ) (A2). Sibling WT fish exhibited many clonus-like convulsions; confirmed by simultaneous monitoring of a live video feed. Putant s334 fish exhibited only stage II whirlpool-like circling behavior and were identified based on observation of their behavior in response to PTZ. Plots of the percentage of time spent moving during the 2-min recording epoch are shown at right (A3); n = 22 siblings; n = 8 s334 putants. Data are presented as mean ± SEM. *p < 0.001 with a Student’s t test. Note that baseline movement is fairly minimal, and PTZ exposure significantly increases this measurement for sibling WT zebrafish (A3, top plot) but not putant s334 fish (A3, bottom plot). B: WT sibling and s334 putant fish are indistinguishable in their appearance at 7 dpf. Fish shown here were first sorted by using locomotion tracking. Scale bar, 1 mm. C: Box plot illustrating the absence of stage III convulsions in putant s334 zebrafish (s334) and siblings (sib) sorted by using locomotion tracking. In this plot, the median value is illustrated as a vertical line in the box. Inner quartiles are shown as the edges of the box, and the outer quartiles, as lines extending from the box. Outliers are shown as black circles.

FIG. 4

Electrophysiologic characterization of mutant s334. A: Schematic showing the recording configuration for agar-immobilized zebrafish; a stimulating electrode was placed on one eye, and a recording electrode was placed in contralateral optic tectum (top). Representative input–output responses for stimulations at 0.7, 1.4, and 2.1 mV in wild-type larvae are shown below. B: Input–output plot for field EPSP responses obtained in a representative WT zebrafish during perfusion with normal bathing medium (blue circles) and 40 min after perfusion with medium containing 1 mM kynurenate (red circles). C: Stimulation-evoked tectal response from an s334 putant zebrafish and WT sibling embedded in agar. Fish were bathed in normal medium. Stimulation artifacts were clipped (open circle). D: Sample field recordings from the same fish as in C. Fish were exposed to 15 mM PTZ for 45 min. Note the presence of interictal and ictal-like discharge in WT fish, as expected (red arrows and inset) and the absence of such discharge in s334 putant (inset). E: Box plot illustrating the frequency of large-duration ictal-like discharges in PTZ-exposed zebrafish. Note the complete absence of ictal-like activity (putant = 0) in potential s334 homozygote mutants. Values as described in Fig. 3C.

FIG. 5

Additional electrophysiologic characterization of mutant s334. Tectal field recordings from sibling WT and putant s334 zebrafish exposed to 4-aminopyridine. Note the absence of large-duration ictal-like discharge activity in 334 putant fish (arrows in WT).