Abstracts

Rationale: Although benzodiazepines and other GABAA receptors allosteric modulators are frequently used to treat human neonatal seizures, their efficacy may derive primarily from actions on subcortical structures. Side effects of benzodiazepines in non-seizing human neonates include myoclonus, seizures and abnormal movements. Excitatory actions of GABA in developing cortical neurons may underlie both these side effects and the reduced cortical anticonvulsant activity of benzodiazepines. We used neocortical organotypic slice cultures to study: 1) spontaneous cortical epileptiform activity during early development; 2) developmental changes in [Cl-]i and 3) whether diazepam’s anticonvulsant effect correlated with neuronal [Cl-]i and the predicted directions of GABA-gated Cl- currents in this cortical preparation.Methods: Field potential recordings were used to measure epileptiform activity in neocortical organotypic slices from wild type mice. Cl- changes during development were assessed by multi-photon imaging of neurons in murine slice cultures transgenically expressing a Cl--sensitive fluorophore (Clomeleon). Clinically relevant concentrations of diazepam were used to test the anticonvulsant effectiveness at ages corresponding to premature neonates through early infancy.Results: 1) Neocortical organotypic slices at days in vitro 5 (DIV5) exhibited spontaneous epileptiform activity. 2) Epileptiform event duration decreased with age. 3) There was a progressive decrease in [Cl-]i over the same age range. 4) Diazepam was ineffective in decreasing epileptiform activity at DIV5-6, but progressively more effective at older ages through DIV15. 5) Interestingly, at DIV5-6, diazepam worsened epileptiform activity in 50% of the slices.Conclusions: The neocortical organotypic slice is a useful model to study spontaneous epileptiform activity. Decreasing [Cl-]i during development corresponds to increasing efficacy of diazepam. At the earliest ages, diazepam worsened epileptiform activity in 50% of the cases, providing a potential explanation for the reports of seizures and myoclonus induction by benzodiazepines in premature human neonates, as well as the limited electrographic efficacy of these drugs for the treatment of neonatal seizures.