Abstracts

Rationale: Integration of morphologically abnormal hippocampal granule cells is hypothesized to contribute to the development of temporal lobe epilepsy. Deletion of PTEN, an inhibitor of the mTOR pathway, has been shown to induce granule cell abnormalities similar to those in an epileptic brain. These mice also exhibit dentate hyperexcitability and develop spontaneous seizures. However, it is unknown whether abnormal PTEN knockout (KO) granule cells directly drive circuit abnormalities, or whether they induce secondary changes among initially normal cells, which in turn mediate abnormal responses. To distinguish between these possibilities, we used an optogenetic strategy to selectively silence PTEN KO cells in hippocampal slices. If PTEN KO cells drive circuit abnormalities, then silencing these neurons should restore normal circuit behavior. Methods: PTEN KO mice (Gli1-CreERT2 hemizygous x PTEN FL/FL) were bred to mice containing a Cre-dependent Archaerhodopsin (Arch) construct. Tamoxifen (250 mg/kg on post-natal day (p) 14 or 21; n=7 or 10, respectively) was injected to induce Arch expression in postnatally-generated PTEN KO cells. Delaying tamoxifen treatment (P14 vs. P21) in this model reduces the severity of the epilepsy phenotype. Arch control mice (PTEN wt/wt; n= 5) and PTEN KO mice without Arch (n=6) were injected with tamoxifen to serve as controls. Once mice reached adulthood (9-23 weeks old), acute hippocampal slices were prepared to record from the granule cell layer during perforant path stimulation. Recordings were performed in baseline conditions, with exposure to a 589nm laser to inhibit Arch expressing cells, and during a recovery phase without light. Results: PTEN knockout slices required significantly less current stimulation to elicit a population spike in the dentate granule layer compare to controls (p < 0.001) under normal conditions. However, when slices were exposed to 589 nm laser, Arch-expressing PTEN KO slices exhibited a significant increase in threshold current (p < 0.001). In p21 Arch PTEN knockout mice, light exposure restored threshold to control levels. Light stimulation of Arch PTEN KO slices also prevented the occurrence of secondary population spikes, a pathological response evident in multiple epilepsy models. Light exposure did not alter fEPSP threshold in any groups, and Arch negative slices were unresponsive to light in all cases. Conclusions: This study demonstrates that pathological physiological responses in PTEN KO mice are mediated by PTEN KO cells, evident by the ability of Arch-mediated silencing of KO cells to restore normal network activity. Further, findings suggest the preservation of a “normal” dentate circuit, which can be revealed by silencing the overlying abnormal circuit. Whether this normal circuit continues to be preserved at later disease stages, and whether downstream circuits develop abnormalities remains to be determined. However, the findings indicate that abnormal PTEN KO granule cells actively drive dentate circuit hyperexcitability for months after disease onset. Funding: This work was supported by the Cincinnati Children’s Hospital Research Foundation and the National Institute of Neurological Disorders and Stroke (SCD, Award Numbers R01NS065020 and R01NS062806; CLL F32NS083239).