Abstracts

Rationale: Patients with early infantile epileptic encephalopathy (EIEE) experience severe seizures and cognitive impairment and are at increased risk for sudden unexpected death in epilepsy (SUDEP). De novo mutations in the voltage-gated sodium channel gene SCN8A, encoding Nav1.6, are linked to EIEE type 13 (EIEE13). We studied a heterozygous knock-in mouse expressing the EIEE13 patient mutation, p.Asn1768Asp (Scn8aN178D/+) to understand mechanisms of hyperexcitability. Methods: Membrane properties were recorded from acute hippocampal slices from mutant and wildtype littermates mice. And isolated transient and persistent sodium current (INa) were recorded from acutely dissociated mutant and WT CA1 and CA3 hippocampal pyramidal neurons. Results: Persistent INa density increased from -19.293.14 pA/pF in WT to -33.455.31 pA/pF in mutant CA1 pyramidal neurons (P=0.030) and increased from -11.502.57 in WT to -23.384.27 pA/pF in mutant CA3 pyramidal neurons (P=0.002). There were no significant changes in transient INa in pyramidal neurons from either region.In acute hippocampal slices from mutant mice we observed hyperexcitability and abnormal action potential (AP) waveforms in CA1 pyramidal neurons, including early afterdepolarization (EAD)-like responses in the late repolarization phase. We investigated the mechanism underlying this EAD-like response using whole-cell current-clamp recording techniques. Application of 50-100 M of the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV) had no effect on the EAD-like responses, suggesting that they were not mediated by NMDA receptors and thus dissimilar to previously described depolarizing after-potentials (DAPs). In contrast, application of 10 M 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-4-thiazolidinecarboxylic acid ethyl ester (SN-6), a reverse mode Na+/Ca2+ exchange inhibitor, completely blocked the EAD-like responses while leaving the AP intact, however, this effect of SN-6 was not completely reversible by washout. These results suggest that increased persistent INa via Nav1.6 may result in higher localized intracellular Na+ concentrations that are sufficient for operation of reverse Na+/Ca2+ exchange and that downstream intracellular Ca2+-mediated events may be responsible for the observed abnormal AP waveforms. To test if Ca2+-mediated spikes contribute to the EAD waveform, we examined the effect of tetrodotoxin (TTX). Application of 500 nM TTX to brain slices completely and reversibly blocked both the AP and the EAD-like response. Conclusions: Taken together, these results suggest that the EAD-like responses in mutant CA1 pyramidal cells may be AP-dependent and Ca2+-mediated, but not Ca2+ spike-mediated events. In conclusion, our work adds to a growing body of evidence suggesting that increased persistent INa density leads to pathology, in this case, a severe EIEE. Our study is the first to demonstrate neuronal hyperexcitability in a mouse model of EIEE13 due to mutation of SCN8A, and thus offers insight into the mechanism of this devastating pediatric disease. The future development of Nav1.6 selective blockers of persistent INa may lead to novel therapeutic treatments for EIEE. Funding: Supported by NIH grants R01-NS-076752 to LLI, U01-NS-090364 to LLI, and R01-NS-034509 to MHM.