Abstracts

Rationale: Tuberous sclerosis complex (TSC), an autosomal dominant disorder due to inactivating mutations in TSC1 or TSC2, frequently manifests with a constellation of neurological deficits including early life epilepsy, mental retardation and autism. The mechanism of epileptogenesis and refractory epilepsy in TSC is poorly understood. Our recent study shows that NKCC1 is overexpressed relative to KCC2, and depolarizing GABA_ARs are present in biopsy samples from human TSC patients (Talos et al., 2012). The present work is a follow up study in a TSC mouse model for purposes of preclinical trail. Methods: We used the Tsc1cc Nes-rtTA++ mouse model that can uniquely replicate development of giant cells with multi-organelle dysfunction including severe clinical seizures (Goto et al., 2011), to investigate the functional changes of GABA_ARs. Spontaneous and evoked field potential activity was recorded using extracellular recordings in ex vivo neocortical slices removed at P28-P40 and compared among groups. We also used gramicidin perforated patch clamp recordings to examine the reversal potential of GABA evoked currents. Western blot was performed to test NKCC1 and KCC2 protein expression in TSC mutant mice and wild-type controls. Results: We first demonstrated epileptiform activity in neocortical slices from TSC1 mutant. Spontaneous epileptiform discharges in layer V were detected in 5 of 12 neocortical slices from TSC mutant mice, but 0 of 15 slices from wild-type mice. In slices from TSC mutant mice, evoked field potentials were often associated with polyphasic activity and/or bursts of action potentials, suggesting an overall hyperexcitability in cortical slices of TSC mutant mice. The polarity of neurotransmission mediated by GABA_ARs is dependent on the transmembrane Cl- gradient mainly regulated by the NKCC1 and the KCC2. Quantitative western blot analysis from cortical samples demonstrated a significant upregulation of NKCC1 expression (p<0.05) and low KCC2 expression (p<0.05) in TSC mutant compared to wild-type controls, suggesting a compromised Cl- homeostasis. These changes can be seen as early as P7. To further examine if increased expression of NKCC1 reversed the polarity of GABA_AR mediated inhibition in TSC mutant mice, we measured reversal potentials of GABA evoked currents (E_GABA) in layer V dysplastic pyramidal cells. The mean E_GABA in TSC mutant mice (-52.7±3.1mV, n=7) was significantly higher than wild-type controls (-69.2±5.2mV, n=6, p<0.05), confirming that GABA_AR mediated synaptic transmission may be excitatory in TSC mutant mice. Conclusions: These results suggest an important role of GABA_AR mediated excitation in tonic seizure activity in TSC mice, which may represent a new therapeutic target for seizure control in TSC. In addition, these data suggest that this mouse model will provide a suitable substrate for preclinical testing of bumetanide to determine its efficacy in increasing the efficacy of conventional GABA agonist anticonvulsant drugs.