Abstracts

Studies from humans, rodents, and fruit flies have identified dozens of single gene mutations that result in heritable forms of epilepsy or increased seizure susceptibility. Although this genetic basis for epilepsy and seizure susceptibility is well established, there have been no efforts to identify gene mutations conferring seizure resistance. Large-scale mutagenesis screening in simple vertebrates offers a powerful approach to further studies of the genetic basis of epilepsy. Here we describe the initial results of the first forward-genetic screen to identify seizure resistant zebrafish mutants., Approximately 5,000 F1 adult fish were generated from matings between ENU-treated males and wild-type females. From these founders 1,896 F2 families were generated and [sim]500,000 F3 larvae were used for screening. Using a behavioral assay, we isolated several seizure-resistant (SR) familes based on their ability to survive prolonged exposure to the convulsant pentylenetetrazol (PTZ). All SR mutants were further characterized by examining resistance to another convulsant (pilocarpine), tracking behavioral responses, and recording electrical activity. In addition, mutants were genetically mapped by bulked-segregant analysis, and recombination analysis with microsatellite markers was used for fine-mapping., Six seizure-resistant mutant families were identified in a forward-genetic screen over a four-year period. One mutant, s198, was also shown to be resistant in a pilocarpine survival assay, suggesting multi-convulsant resistance. Three of the mutants exhibited reduced behavioral activity upon acute PTZ exposure (s139, s333 & s387), and one of these also showed decreased c-Fos expression by RT-PCR (s333). Electrophysiology data on mutant s334 revealed an inability to generate long-duration ictal-like discharge in response to PTZ or 4-aminopyridine. Mutant s198 was randomly chosen for mapping studies. Analysis of 192 microsatellite markers, evenly distributed throughout the zebrafish genome, placed the mutated gene on linkage group 24. Recombination analysis with 658 meioses localized the mutation within a 1.1-centimorgan region (approximately 800 kb)., In conclusion, SR zebrafish mutants were successfully identified in a large-scale forward-genetic screen. Mutants were isolated with multi-convulsant resistance, reduced seizure behaviors, and no ictal-like discharge. Linkage studies on one mutant, s198, have been completed. Further identification and characterization of the defective genes in these mutants will present a more complete understanding of the genetics of seizures, and provide a unique opportunity for developing a genetically-based cure for epilepsy., (Supported by Epilepsy Foundation of America/Milken Family (M.R.T.) and Klingenstein Fund (S.C.B.).)