Abstracts

Rationale: Tuberous Sclerosis Complex (TSC) is a neurocutaneous disorder associated with early onset severe epilepsy in up to 90% of patients and results from mutations of the Tsc1 or Tsc2 genes. Hyperactivation of the mTOR pathway leads to formation of cortical lesions which are associated with areas of aberrant neuronal hyperexcitability. These epileptogenic regions are made up of a heterogeneous group of abnormal cells including dysmorphic and cytomegalic neurons as well as immature neurons with glial properties. It is unclear which neuronal subtypes contribute to seizure activity. Throughout brain development mTOR activation occurs across a developmental continuum and the role of post-mitotic mTOR hyperactivation in mature principal neurons has not been explored. Therefore, to parse out the contribution of post-mitotic hyperactive mTOR signaling in seizure generation we used a mouse model of TSC with Cre-mediated deletion of Tsc2 in excitatory neurons of the forebrain driven by the NEX promoter. Methods: Video electroencephalopgraphy (vEEG) activity was recorded from littermate mouse pups [Wild type, (WT) NEX-Cre+/Tsc2WT/WT; Heterozygous, (HT) NEX-Cre+/Tsc2 WT/flox; Knockout (KO), NEX-Tsc2flox/flox] at postnatal day 10 (P10)-P21 with each session lasting 3 hours/day (n=7-9). EEGs were reviewed visually and digitally analyzed using LabChart. Power spectra were calculated from EEG raw data. To determine seizure threshold, separate cohorts underwent seizure induction with the chemiconvulsant pentylenetrazole (PTZ; 50mg/kg) at P12 or P15 (n=12-14). Adult WT and HT mice were implanted at P45 followed by PTZ seizure induction at P49 (n=10-13). Percent time spent in epileptiform activity and seizure frequency, latency, and duration were quantified. Results: Spike bursts were detected as early as P10 in KO mice (30% time spent in epileptiform activity). KO mice exhibited spontaneous generalized tonic-clonic (GTC) seizures (5 seizures/recording session), significantly reduced EEG delta power (p<0.01, WT vs. KO), and premature death (P16). WT and HT mice did not exhibit epileptiform activity or spontaneous seizures. HT mice displayed lower EEG delta power beginning at P14 (p<0.001, WT vs. HT and KO). There were significant differences in the percentage of mice exhibiting GTC seizures after PTZ injection (P12: p<0.001, WT vs. KO; P15: p<0.001, WT vs. KO; p<0.05, WT vs. HT). KO mice did not recover after entering status epilepticus. The latency to GTC seizures was significantly shorter in KO mice at P12 (p<0.01, WT vs. KO) and P15 (p<0.01, WT vs. KO) and in HT mice at P15 (p<0.01, WT vs. HT). At P49, HT mice displayed higher epileptiform activity (% time in epileptiform activity, p<0.05) and higher mortality (p<0.05 WT vs. HT) 1 hour following PTZ-seizure induction compared to WT. Conclusions: Our findings suggest that forebrain specific neuronal deletion of Tsc2 post-mitotically is sufficient to elicit a severe early postnatal epilepsy phenotype in the NEX-Tsc2 KO mouse pups. Furthermore, there appears to be a more subtle phenotype present in the NEX-Tsc2 HT mice. Thus, the NEX-Tsc2 mice provide a novel model of epilepsy associated with Tsc2 deficiency and mTOR pathway dysregulation for investigation of network hyperexcitability, epileptogenesis, and seizures in TSC. Funding: No funding