Abstracts

Rationale: mTOR pathway mutations are associated with autism, cognitive dysfunction and epilepsy. In animal models, loss of the mTOR pathway negative regulator PTEN from hippocampal dentate granule cells leads to dentate hyperexcitability and spontaneous seizures. To better elucidate the process of epileptogenesis, we used a conditional, inducible mouse model system to introduce variable numbers of PTEN knockout granule cells into the hippocampus. Using this system, we generated mice representing a linear increase in the percentage or “load” of PTEN KO cells. We then queried whether epileptogenic changes in the animals followed a corresponding linear pattern, or whether changes occurred through a series of stepwise events. Methods: Gli1-CreERT2, PTEN flox/flox mice were treated with tamoxifen at 2 or 3 weeks-of-age to selectively delete PTEN from granule cell progenitors, generating 22 animals in which the percentage of PTEN KO cells ranged from 0.1-22%. Control mice lacking KO cells were also included (n=13). Mice were implanted with either cortical ECoG electrodes or a hippocampal depth electrode for 24/7 video-seizure monitoring. A second cohort of Gli1-CreERT2, PTEN flox/flox, Archaerhodopsin flox/flox (Arch) mice were also generated to facilitate optogenetic silencing of KO cells. Acute hippocampal slices were prepared from adult (9-23 weeks old) control Arch-expressing (n=5), Arch-PTEN KO (n=17, 3.6-38% KO), and PTEN KO (no Arch, n=6) mice. Evoked responses were recorded from the granule cell layer during perforant path stimulation under baseline conditions, during inhibition of Arch expressing cells, and during a recovery phase. Results: Seizure phenotype progressed in stepwise fashion following a linear increase in KO cell load, with epileptiform spikes and focal (hippocampal) seizures appearing at KO cell loads above 3%, while cortical seizures didn’t appear until loads reached 15% (R=0.889, p<0.000001, Spearman correlation on data ranked by epilepsy phenotype). At the circuit level, KO slices, regardless of cell load, exhibited reduced population spike thresholds and multiple population spikes following perforant path stimulation. On the other hand, the incidence and amplitude of secondary spikes was positively correlated with KO cell load (R=0.60, p<0.01; coastline analysis of line length). Optogenetic experiments revealed that silencing PTEN KO cells consistently restored normal circuit behavior in mice with low KO cell loads (versus control, p=0.6), but failed to fully do so in high load animals (versus control, p<0.01). Conclusions: Together, these findings reveal that the incremental accumulation of abnormal, PTEN KO granule cells can drive stepwise changes in circuit physiology and epilepsy phenotype. Stepwise events suggest that secondary changes are an important component of progression in this epilepsy model. To test this possibility, KO cells were optogenetically silenced to remove them from the circuit. The failure to fully restore normal circuit behavior in high KO slices supports the conclusion that secondary changes contribute to the pathology in these animals. Funding: This work was supported by the National Institute of Neurological Disorders and Stroke (SCD, Awards R01NS065020 and R01NS062806; CLL, F32NS083239).