Abstracts

Rationale: Patients with Dravet Syndrome (DS), a severe pediatric epileptic encephalopathy, display a high incidence of sudden unexpected death in epilepsy (SUDEP) as well as developmental delay at all milestones. Seizures are pharmacoresistant and the mechanisms underlying SUDEP are unclear. DS results primarily from de novo mutations in the voltage-gated sodium channel gene SCN1A, leading to haploinsufficiency, with a subset of patients displaying inherited homozygous loss-of-function mutations in SCN1B, encoding the sodium channel β1/β1B subunits. One of the critical events during brain development is the maturation of inhibitory GABAergic signaling, occurring by the second postnatal week in mice, which relies on the establishment of the neuronal chloride gradient through the action of the cation-chloride co-transporters KCC2 and NKCC1. We proposed previously that the Scn1b null mutation delays developmental maturation of GABAergic inhibition in central neurons in mice. Here, we confirm delayed GABAergic maturation, and demonstrate that treatment with the NKCC1 inhibitor bumetanide delays the occurrence of SUDEP in Scn1b null mice without prevention of seizures. Methods: Gramicidin perforated patch clamp recordings were performed from brain slices of P14-18 Scn1b null and WT mice to measure the reversal potential of GABAA receptor-mediated currents. Western blotting of forebrain membranes from P5-17 Scn1b null and WT mice was performed to measure protein levels of KCC2 and NKCC1. Bumetanide was administered subcutaneously at 0.2 mg/kg to litters of mouse pups from Scn1b+/- matings twice daily beginning at P0-P1. Results: The mean reversal potential for GABA in slices from Scn1b null neurons (-53.1 ± 4.03 mV, n=7) was significantly more depolarized than that in slices from age-matched WT littermates (-82.7 ± 4.6 mV, n=6, P < 0.01). However, total protein expression of the cation-chloride transporters KCC2 or NKCC1 was not different between genotypes at any of ages tested, whether prior to or after seizure onset in the DS mice (Scn1b WT vs null; age P5 (n=8 WT, n=6 null), P10 (n=7 WT and null) or P16-17 (n=9 WT, n=8 null)). Twice daily administration of the NKCC1 inhibitor bumetanide beginning at P0-P1 resulted in a significant delay in the occurrence of SUDEP in Scn1b null mice, with survival ranging from P22 through P46 (n=7), compared to untreated Scn1b null mice, which have a 100% incidence of SUDEP beginning at P17 with rare survival past P21. Interestingly, treatment with bumetanide did not prevent the occurrence of seizures in Scn1b null mice. No adverse drug effects were observed in WT (n=6) or Scn1b+/- (n=11) littermates. Conclusions: We propose that bumetanide may be therapeutically effective in some DS patients to reduce SUDEP risk. Funding: Supported by NIH R37NS076752 to LLI.