Abstracts

Zebrafish represent an important model organism in genetics and neurodevelopment. Despite burgeoning interest, very little effort has been directed toward using zebrafish to study neurological disorders. Although seizures induced in rodents provided insights into epileptogenesis, many questions remain unanswered. To address this issue, we propose to establish a simple vertebrate model of induced seizures. Here, we hypothesize that zebrafish larvae which are small and can be produced in large numbers, can exhibit complex seizure activity. To highlight the usefulness of such a model we describe behavioral, electrophysiological and molecular changes that occur in zebrafish exposed to a common convulsant agent. Experiments were performed on zebrafish ([italic]Danio rerio[/italic]) larvae of the TL strain. To induce seizures, zebrafish larvae at 7 day post-fertilization (d.p.f.) were exposed to pentylenetetrazole (PTZ). For behavioral studies, zebrafish were monitored using locomotion tracking software (EthoVision 3.0). For electrophysiology studies, zebrafish were immobilized in agar. A field electrode containing 2 mM NaCl was placed in the tectum or forebrain. Electrical activity was monitored using a patch-clamp amplifier in current-clamp mode. For molecular studies, c-fos mRNA levels were examined by RT-PCR and whole-mount in situ hybridization. PTZ elicited seizure-like behavior in all zebrafish tested ([italic]n[/italic] = 34). At 15 mM PTZ, rapid clonus-like movements followed by loss of posture were consistently observed (10 min). Video tracking analysis of this behavior indicated an average distance traveled (in cm) of 19.0 [plusmn] 1.3 and a [quot]movement[quot] score (in %) of 1.71 [plusmn] 0.22. These scores were significantly greater (p [lt] 0.0001, ANOVA) than those obtained at baseline (Ringer[apos]s solution): 6.7 [plusmn] 1.0 cm and 0.11 [plusmn] 0.05 %, respectively. PTZ-induced electrographic seizure activity could be simultaneously recorded in forebrain and tectum. Activity consisted of ictal- and interictal-like components and was highly synchronized between the two brain regions. RT-PCR analysis of c-fos expression at various time-points following PTZ exposure indicated a clear increase in expression. Findings were confirmed using in situ hybridization which showed significant c-fos mRNA in forebrain, tectum and cerebellum. Here we demonstrate that zebrafish represent a valuable model for studying mechanisms of seizure generation and propagation. Analysis of behavioral, electrophysiological and molecular changes that occur with exposure to a common convulsant agent is but one step in our efforts to use zebrafish in the field of epilepsy research. Future studies incorporating forward-genetic and chemical screening, as well as application of morpholino or transgenic technologies will, no doubt, lead to a better understanding of epilepsy. (Supported by EFA (Holden Targeted Investigation Program), NIH and Klingenstein Fund.)