Abstracts

Rationale: Tuberous Sclerosis Complex (TSC) is a neurocutaneous disorder characterized by intraventricular tumors and severe epilepsy in up to 90% of patients. TSC is caused by mutations in TSC1 or TSC2, genes that encode the proteins TSC1 (hamartin) and TSC2 (tuberin), and which function as a complex in the regulation of the mTOR pathway. Absence of TSC1 or TSC2 leads to mTOR hyperactivity and formation in the brain of benign tumors known as tubers. Although resection of tubers may result in significant reduction of seizure frequency, it remains unclear which neuronal subtype within cortical tubers or peri-tuberal zones contribute to seizure generation in TSC. To determine how loss of Tsc2 in differentiated excitatory neurons may be involved in epileptogenesis, we examined EEG activity in a mouse model with forebrain-specific NEX-Cre-mediated deletion of the Tsc2 gene in post-mitotic excitatory neurons.  Methods: To assess EEG activity, mouse littermate pups (WT, NEX-Cre+/Tsc2^WT/WT; HT, NEX-Cre+/Tsc2 ^WT/flox; KO, NEX-Tsc2^flox/flox) were implanted with a cortical 3-pin electrode pedestal at postnatal day 10 (P10). EEG signals (filtered at 0.5-200Hz, Natus Nicolet) were collected at P11 through P21. Video-EEG recording sessions lasted a total of 3 hours each day (2x 1.5 hours recording with a 1.5-hour rest interval in the home cage). EEG signals were reviewed visually and processed offline using LabChart v8. After removal of artifacts, power spectra were obtained from EEG traces band passed filtered (0-50Hz) and transformed by the Hann-Cosine method using a 2000 FFT size. To determine seizure threshold, separate cohorts underwent seizure induction with the chemiconvulsant pentylenetetrazole (PTZ; 50 mg/kg b.w.) at P12 or P15. (EEG: n=10 WT; 7 HT, 5 KO; PTZ: P12 n= 9 WT, 9 HT, 8 KO, P15: 5 WT, 6 HT, 6 KO) Results: Tsc2 KO mice exhibited significant growth deficiencies and premature death (lifespan range 11-19 days).HT mice were slightly underweight compared to WT littermates but otherwise thrived and survived past weaning. Spontaneous seizures were evident in KO mice beginning at P11 and recurring until death. Background EEG activity was reduced in amplitude and power in KO mice (p<0.05) and was normal in HT mice compared to WT. No epileptiform activity was detected in WT or HT mice. Epileptiform activity in KO mice appeared initially as isolated interictal spike activity that evolved into frequent polyspike discharges. Whereas exploratory time in KO mice was normal on the first day of recording, subsequent days consisted of long periods of immobility interrupted by brief periods of exploration. Immobility time coincided with epochs of epileptiform activity. Seizure induction with PTZ revealed KO to be more susceptible than WT and HT. At P12 and P15, more KO exhibited PTZ-induced generalized tonic-clonic activity (Stage 6) as measured by a modified Racine scale (87% and 100%, respectively). An age-dependent increase in seizure susceptibility was also observed in WT (20% and 40%) and HT (33% and 66%) at P12 and P15, respectively. Conclusions: Conditional deletion of Tsc2 in differentiated forebrain principal neurons in mice is associated with severe seizures in early postnatal development, significant stunting of growth, and premature death. Electrographically, KO mice exhibit long epochs of spike activity and abnormal background EEG activity compared to HT and WT. KO also express greater vulnerability to PTZ-evoked seizures. These results underscore the importance of Tsc2 in differentiated neurons and validate the NEX-Tsc2 KO mouse as a candidate tool in the study of TSC-associated epilepsy. Funding: Department of Defense grantGrant # W81XWH-12-1-0017