Abstracts

Rationale: Whether individual seizures cause brain cell death in humans or animal models is debated. One hypothesis is that each seizure produces subtle cell loss. The alternate hypothesis is that one event, such as a single, prolonged seizure, is responsible for most, if not all, epilepsy-associated neuron loss. Nonetheless, each theory is supported by published studies. Perforant pathway stimulation causes seizures, significant neuronal injury, and spontaneous epilepsy in rodents. However, the minimum duration of stimulation-induced seizures required to produce brain cell death in na ve rodents, and precisely which hippocampal neurons are the first to die, remain unresolved issues. Methods: Bilateral perforant pathway stimulation, comprised of continuous 2 Hz paired-pulse stimuli with a 40 ms interpulse interval, along with one 10 sec epoch of 20 Hz single pulses each minute, was applied to awake, freely moving Sprague-Dawley rats. Concurrent recording directly from the hippocampal granule cell layer allowed us to monitor hippocampal activity in real-time, and control the duration of stimulation for each animal (10 seconds to 40 minutes), individually. At the end of each stimulation epoch, isoflurane anesthesia was administered to rapidly terminate seizure activity. Seven days after stimulation, animals were perfusion-fixed with 4% paraformaldehyde. 40 m-thick coronal brain sections were mounted on glass slides, dehydrated, stained with FluoroJade-B (FJB), and analyzed with fluorescence microscopy to ascertain neuronal degeneration. Results: Perforant pathway stimulation induced and maintained hippocampal seizures throughout the duration of each stimulation epoch. The minimum stimulation duration that produced FJB-detectable neurodegeneration was 40 minutes. FJB-stained neurons were limited to the dentate hilus of the dorsal hippocampus. Conclusions: These data rebut the hypothesis that a brief, individual seizure causes brain cell death in rats. We did not see any evidence of neurodegeneration produced by stimulation durations of up to and including 30 minutes. Neurons in the dentate hilus are among the first to die after perforant pathway stimulation under urethane anesthesia and are some of the most vulnerable to kainate- and pilocarpine-induced seizures. These data confirm these observations, and demonstrate that hilar neurons are the first neurons to succumb to perforant pathway stimulation-induced seizure activity in awake rats.