Abstracts

RATIONALE: Genetic factors can play a major role in the development of epilepsy. With recent progress in human genetics it is now clear that mutations resulting in ion channel dysfunction, neurotransmitter receptor alterations, or brain malformations predispose an individual to epilepsy. To more rapidly identify additional epilepsy-related gene mutations, we propose a large-scale mutagenesis screening strategy in zebrafish. However, before mutagenesis screening can occur, we must determine whether (i) seizures can be reliably elicited in zebrafish and (ii) seizure activity in zebrafish resembles that observed in higher vertebrates.
METHODS: For behavioral seizure scoring studies, zebrafish larvae (danio rerio, 7 days old) were placed in a 98-well tissue culture plate (1 fish/well). Zebrafish were videotaped using a Sony digital camera and scored by an investigator blind to the status of the fish. For electrophysiological seizure studies, zebrafish were immobilized in agar and placed on the stage of an Olympus upright microscope. A field electrode containing 2 mM NaCl (2-5 M[OMEGA]) was placed in the tectum under visual control. Seizure activity was induced by adding pentylenetetrazol (PTZ, 2.5-15 mM), a common convulsant agent, to the bathing medium.
RESULTS: Wild-type zebrafish were exposed to PTZ for up to 24 hours (n = 490). During PTZ exposure, zebrafish exhibited three distinct stages of seizure-like behavior: Stage I, increased swimming movement; Stage II, rapid circling; and Stage III: tonic paralysis-type convulsions. Seizure-like behaviors were elicited in a concentration-dependent manner. At a concentration of 15 mM PTZ, Stage III seizure activity and fatality was observed in nearly all fish. Bath application of 15 mM PTZ (a concentration shown to reliably evoke behavioral seizures) elicited spontaneous epileptiform-like discharge activity in all electrophysiological trials (n = 32): amplitude = 0.7 [plusminus] 0.2 mV; duration = 659 [plusminus] 46 ms; frequency = 5.3 [plusminus] 0.7 Hz. Epileptiform discharge developed gradually, and was stable for the duration of the recording period (45 - 180 min). Activity was abolished by bath application of tetrodotoxin (1-5 [mu]M; Na channel blocker) or kynurenic acid (2.5 mM; glutamate receptor antagonist). Epileptiform discharge was suppressed by valproate (5 mM; n = 4) and ethosuximide (5-10 mM; n = 4) but was insensitive to carbamazepine (100-200 [mu]M; n = 4). This anticonvulsant profile is similar to that reported for PTZ-evoked seizures in rodents [Epilepsy Res. 9:1-10, 1991].
CONCLUSIONS: Here we demonstrate, for the first time, that seizures in zebrafish are similar to those observed in higher vertebrates. These findings suggest that large-scale zebrafish mutagenesis screening studies may reveal important epilepsy-related gene mutations.
Support: Sandler Family Supporting Foundation & UCSF Innovation in Basic Science Award