Abstracts

Rationale: Hippocampal granule cells with aberrant neuronal connections are hypothesized to contribute to the development of temporal lobe epilepsy. To test this hypothesis, we have developed a transgenic mouse model of epilepsy in which PTEN is deleted from hippocampal granule cells by crossing loxP flanked PTEN mutants to a Gli1-CreERT2 driver line. Treatment of these animals with tamoxifen at P14 leads to the selective deletion of PTEN from hippocampal granule cells and cortical glial cells. PTEN KO granule cells in these animals develop abnormally, exhibiting basal dendrites and mossy fiber axon sprouting. PTEN KO cortical glial cells appear morphologically normal. Animals develop spontaneous seizures around 8 weeks of age. We have hypothesized that abnormal granule cells drive seizures in this model; however, it is conceivable that PTEN KO glial cells contribute to this phenotype. Therefore, to localize the source of seizures, dual channel EEG recordings were conducted from hippocampus and overlying neocortex. Methods: At P14, Gli1-CreERT2 hemizygous, PTENfl/fl, GFP+/- reporter mice received a subcutaneous injection of 250 mg/kg of tamoxifen. At 6 weeks of age, mice were wirelessly monitored 24/7 by video/EEG via electrodes placed in neocortex and hippocampus. Following continuous EEG monitoring, mice were sacrificed for histological studies. Triple immunostaining of brain sections was conducted for GFP, PTEN, and NeuN to assess neuronal morphology and determine the percentage of PTEN KO cells in cortex and dentate gyrus. ZnT-3 antibodies were used to reveal sprouted mossy fiber axons, and antibodies to phospho-S6 were used to assess activation of the mTOR pathway. Sections were analyzed using laser confocal microscopy. Following continuous EEG monitoring, mice were sacrificed and the percentage of newly-generated granule cells lacking PTEN was determined using confocal microscopy. Mossy fiber axonal sprouting, basal dendrites, and ps6 expression were all assessed in control and epileptic animals. Results: PTEN knock out animals exhibited spontaneous seizures within 6 weeks after tamoxifen treatment, while no seizures were observed in littermate controls. Epileptic animals exhibited a significant increase in pS6 expression, indicative of mTOR signaling, and a significant increase in mossy fiber sprouting, demonstrating the formation of recurrent excitatory circuits. Dual lead recordings to monitor hippocampus and cortex revealed robust seizure activity in hippocampus, with no spread of seizure activity to overlying cortex at earlier time points. As the epileptic syndrome progressed in these animals, hippocampal seizure activity was associated with epileptiform bursting in cortex. Conclusions: The present findings strongly suggest that seizures in the Gli1-CreERT2::PTEN model of epilepsy originate from hippocampus. This implies that epileptogenesis is driven in these animals by aberrantly integrated PTEN KO granule cells. Aberrantly integrated granule cells are common in temporal lobe epilepsy, and the current results support a pro-epileptogenic role for these neurons.