The hippocampus is especially vulnerable to seizure-induced damage and excitotoxic neuronal injury. This study examined the time course of neuronal death in relationship to seizure duration and the pharmacological mechanisms underlying seizure-induced cell death using low magnesium (Mg 2+ ) induced continuous high frequency epileptiform discharges (in vitro status epilepticus) in hippocampal neuronal cultures. Neuronal death was assessed using cell morphology and Fluorescein diacetate-Propidium iodide staining. Effects of low Mg 2+ and various receptor antagonists on spike frequency were assessed using patch clamp electrophysiology. We observed a linear and timedependent increase in neuronal death with increasing durations of status epilepticus. This cell death was dependent upon extracellular calcium that entered primarily through the N-methyl-D-aspartate (NMDA) glutamate receptor channel subtype. Neuronal death was significantly decreased by coincubation with the NMDA receptor antagonists and was also inhibited by reduction of extracellular calcium (Ca 2+ ) during status epilepticus. In contrast, neuronal death from in vitro status epilepticus was not significantly prevented by inhibition of other glutamate receptor subtypes or voltage-gated Ca 2+ channels. Interestingly this NMDA-Ca 2+ dependent neuronal death was much more gradual in onset compared to cell death from excitotoxic glutamate exposure. The results provide evidence that in vitro status epilepticus results in increased activation of the NMDA-Ca 2+ transduction pathway leading to neuronal death in a time dependent fashion. The results also indicate that there is a significant window of opportunity during the initial time of continuous seizure activity to be able to intervene, protect neurons and decrease the high morbidity and mortality associated with status epilepticus. KeywordsLow Mg 2+ model of status epilepticus; Neuronal death; NMDA-Ca 2+ pathway

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SummaryIt is established that the majority but not all of the seizure-induced cell death is associated with status epilepticus while spontaneous recurrent seizures associated with epilepsy do not cause neuronal death. Extracellular effects and compensatory changes in brain physiology complicate assessment of neuronal death in vivo as the result of seizures. In this study we utilized a well-characterized in vitro hippocampal neuronal culture model of both continuous high-frequency epileptiform discharges (status epilepticus) and spontaneous recurrent epileptiform discharges (acquired epilepsy) to investigate the direct effects of continuous and episodic electrographic epileptiform discharges on cell death in a carefully controlled extracellular environment. The results from this study indicate that continuous high-frequency epileptiform discharges can cause neuronal death in a time-dependent manner. Episodic epileptiform seizure activity occurring for the life of the neurons in culture was not associated with increased neuronal cell death. Our data confirm observations from clinical and some animal studies that spontaneous recurrent seizures do not initiate cell death. The hippocampal neuronal culture model provides a powerful in vitro tool for carefully evaluating the effects of seizure activity alone on neuronal viability in the absence of various confounding factors and may provide new insights into the development of novel therapeutic agents to prevent neuronal injury during status epilepticus.

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