Abstracts

RATIONALE: Genetic factors are known to play a significant role in the development of epilepsy. With recent progress in human and mouse genetics, it is now clear that mutations can predispose an individual to seizure activity. Identification of gene mutations that confer enhanced seizure susceptibility or a frank epileptic phenotype has greatly increased our understanding of epilepsy. However, mutations that would confer seizure resistance remain undiscovered. To more efficiently identify seizure resistant mutations, we have developed a forward genetic screening strategy in zebrafish. Here we describe our early efforts to characterize pharmacologically-induced seizures in zebrafish larvae and, report our initial identification of several resistant mutants.
METHODS: For induction of seizures, zebrafish larvae (Danio rerio) were exposed to a common convulsant agent (pentylenetetrazole, PTZ). For electrophysiologic seizure studies, zebrafish larvae were immobilized in agar and placed on the stage of an upright microscope. A field recording electrode containing 2 mM NaCl (2-5 M[Omega]) was placed in the tectum under visual control. Electrical activity was monitored using a patch-clamp amplifier in current-clamp mode. In all experiments, PTZ was directly added to the Ringer[ssquote]s bathing medium at a saturating concentration. Wild-type and ENU-mutagenized zebrafish larvae (6 to 8 days old) were used in all studies.
RESULTS: High frequency interictal-like electrographic seizure activity (burst duration, ~650 msec; burst amplitude ~1 mV) was observed during the first 15 to 30 min of PTZ exposure in all electrophysiological trials. After 45 min of PTZ exposure interictal- and ictal-like electrographic seizure activity (burst duration, ~5 sec; burst amplitude, ~6 mV) was consistently observed (n = 49). Electrographic seizure-like activity was abolished by the addition of valproic acid, ethosuximide, tetrodotoxin, kynurenic acid or CNQX/APV to the bathing medium. During PTZ exposure, freely moving wild-type fish were observed to undergo three distinct stages of seizure-like behavior, as described previously (Baraban et al. 2001; Epilepsia 42:3.017). Wild-type zebrafish exposed to PTZ exhibited [dsquote]status-like[dsquote] seizure activity and [gt] 98% of all wild-type fish died within 24 hr of PTZ exposure. Similar seizure-like behaviors were observed in zebrafish mutants exposed to PTZ. To date, we have identified twelve families that carry seizure-resistance mutations.
CONCLUSIONS: Here we demonstrate that a common convulsant agent (PTZ) can be used to reliably induce seizures in zebrafish. These PTZ-induced seizures appear to have a behavioral, electrophysiological and pharmacological profile that is identical to that previously reported in rodents. Further, the initial identification of seizure resistant zebrafish mutants using our forward genetic screening strategy demonstrates that this is a powerful system to discover genes that may prevent, rather than ameliorate, epilepsy.
[Supported by: Epilepsy Foundation of American (Holden Targeted Investigation Program) and the National Institutes of Health.]