Abstracts

Rationale: Loss-of-function mutations in voltage-gated sodium channels cause epilepsy in patients and in a transgenic mouse model. Antiepileptic drugs that affect voltage-gated sodium channels also exacerbate seizures in some epilepsy syndromes. Detailed understanding of this paradoxical effect can lead to better treatments for epilepsy. To this end, we have developed an in vitro model to investigate network mechanisms of seizure generation following partial blockade of voltage-gated sodium channels. Methods: We have maintained organotypic cultures of p4-p7 rat hippocampus for 3 to 4 weeks using a modified interface culture method. Field recordings were carried out in an interface chamber with a tungsten microelectrode. For recording, organotypics were perfused with culture medium containing 20, 50, 100, and 500 nM concentration of tetrodotoxin (TTX), a voltage-gated sodium channel antagonist. Results: Organotypic cultures are hyperexcitable due to a higher probability of cell-to-cell connections, likely a result of in vitro axon sprouting. When recorded in culture medium, hippocampal organotypic cultures exhibit epileptiform activity after two weeks in vitro. Activity is completely abolished if 500 nM of TTX is added to recording/culture medium. However, a paradoxical effect occurs during perfusion with lower concentrations of TTX. After 30 minutes of perfusion with low TTX, slice activity is reduced; however, between 30 and 90 minutes of TTX perfusion, an increase in frequency and amplitude of epileptiform bursts can be observed, followed by gradual cessation of activity. Conclusions: Perfusion with low concentrations of TTX results in accumulation of TTX in the hippocampus culture, due to extremely high affinity of TTX-sodium channel binding. The accumulation of TTX leads to a gradual increase in the number of inactivated voltage-gated sodium channels in the culture. At a critical threshold, a brief period of synchronous firing commences, which is terminated with further accumulation of TTX. We have created a computer simulation of this process with a network where the probability of cell-to-cell transmission was gradually reduced, and achieved similar results. Seizure activity due to partial inactivation of voltage-gated sodium channels in our in vitro model mimics seizures due to loss-of-function sodium channel mutations or toxic doses of drugs such as phenytoin. Our model will permit detailed study of the network mechanisms of such seizures.