Abstracts

Rationale: Hippocampal granule cells generated in the weeks before and after an epileptogenic brain injury can integrate abnormally into the dentate gyrus, potentially mediating temporal lobe epileptogenesis. This study tests the hypothesis that these abnormal cells promote epileptogenesis following status epilepticus (SE)-induced brain injury. Specifically, we examined whether ablating developing dentate granule cells following SE would decrease seizure frequency and attenuate cellular abnormalities within the dentate gyrus. Methods: NesCreER^T2:DTr^fl/wt:GFP^+/- transgenic mice were used for this study. Tamoxifen was given once per week for five weeks beginning at P21 to induce the expression of the diphtheria toxin receptor (DTr) in Nestin expressing progenitor cells, making these cells vulnerable to diphtheria toxin (DT) induced apoptosis. DTr is continuously expressed in recombined progenitor cells, as well as their progeny, making it possible to ablate specific cell lineages at any later time point by treating the animals with DT. At P56 (8 weeks), the mice underwent pilocarpine-induced SE. Beginning two days after SE, DT (or saline control) was given once per day for five days to ablate progenitor cells and developing dentate granule cells. At P105-112 (7 to 8 weeks after SE), mice were implanted with cortical electrodes for 24/7 video-EEG monitoring. Mice were monitored for 2-4 weeks and were then sacrificed for histological studies. Results: The NesCreER^T2:DTr^fl/wt:GFP^+/- transgenic mouse model used here was highly effective at inducing DTr expression in abnormal granule cells. Prox1 immunolabeling of hippocampal granule cells revealed co-expression of DTr in 81.0±2.2% of hilar ectopic cells in pilocarpine-treated NesCreER^T2:DTr^fl/wt:GFP^+/- mice (n=15) 2-3 months after SE. DT administration resulted in a significant ablation of DTr expressing cells within the hilar region (DT, n=4, 5.6±1.9 cells/hilar section; saline, n=16, 27.1±4.3 cells/hilar section; SEM; p=0.008, Mann-Whitney). Importantly, this methodology effectively reduced the number of Prox1 immunoreactive cells ectopically located in the hilus (DT, n=4, 11.1±5.2 cells/hilar section; saline, n=16, 32.6±17.1 cells/hilar section; SEM; p=0.012, Mann-Whitney). Conclusions: Aberrant integration of granule cells born in the weeks before and after SE is hypothesized to contribute to temporal lobe epileptogenesis. The approach used here allows us to simultaneously ablate neural progenitors present after the insult as well as granule cells born in the weeks prior to the insult. This methodology was highly effective at reducing the number of ectopic granule cells; a population implicated in epileptogenesis. 24/7 video-EEG monitoring is currently ongoing to determine if DT treatment affects seizure frequency following pilocarpine-induced SE. Histological studies are also being conducted to determine if DT treatment reduces cellular abnormalities associated with epilepsy. Together, the methods provide a powerful new approach for assessing the impact of abnormal granule cells on temporal lobe epileptogenesis.