Abstracts

Rationale: Abnormal granule cells are hypothesized to mediate epileptogenesis by impairing the “dentate gate”, a property of the hippocampal circuit that normally limits excess excitation. Previously, we developed a mouse model in which the mTOR inhibitor PTEN is deleted from a subset of granule cells. PTEN knockout cells integrate abnormally, and animals become epileptic, supporting the hypothesis that abnormal granule cells are epileptogenic. Here, we conducted anatomical and physiological studies of PTEN knockout granule cells to elucidate how these cells contribute to epilepsy. Both male and female animals were examined. Findings provide new insights into mechanisms that may underlie temporal lobe epileptogenesis. Methods: Gli1-CreERT2; PTEN flox/flox mice and litternate controls on a C57BL/6 background were treated with tamoxifen on postnatal day 21 to induce PTEN deletion from a subset of hippocampal granule cells. Between 2-6 months of age, acute hippocampal slices were prepared for field potential recording, whole cell recording and single cell morphology studies. Cells from both male and female mice were recorded to identify sex-specific effects. Results: PTEN knockout granule cells showed altered intrinsic excitability, evident as a tendency to fire in bursts. PTEN knockout granule cells also exhibited increased frequency of spontaneous excitatory synaptic currents (sEPSC’s) and decreased frequency of inhibitory currents (sIPSC’s), further indicative of a shift towards hyperexcitability. Morphological studies of PTEN knockout granule cells revealed larger dendritic trees, more dendritic branches and an impairment of dendrite self-avoidance. Finally, analysis of cells from male and female animals demonstrated that both female control and female knockout cells receive more sEPSC’s than corresponding male cells -- but also receive more sIPSC’s – resulting in statistically equivalent EPSC/IPSC ratios. Consistent with this latter observation, extracellularly evoked responses in hippocampal slices were similar between male and female knockouts; albeit both groups of knockouts were abnormal relative to controls. Conclusions: Together, these studies reveal a host of physiological and morphological changes among PTEN knockout cells likely to underlie epileptogenic activity. Comparisons between male and female animals reveal sec-specific differences in inputs, but no interaction between sex and genotype. Funding: This work was supported by the National Institute of Neurological Disorders and Stroke (SCD, Award Numbers R01NS065020 and R01NS062806). VRS received a Fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Brazil. His work, under the mentorship of SD at CCHMC, was part of his PhD Program at the University of São Paulo, Brazil.