Abstracts

Rationale: Inhibition is essential for the prevention of runaway excitation within neural networks as well as critical for diverse neural coding. Effective inhibition depends upon the capacity for neurons to tightly regulate (Cl-)i. Mature neurons maintain low levels of (Cl-)i via the K+-Cl- cotransporter KCC2, a cation chloride co-transporter (CCCs). During development, immature neurons express low levels of KCC2 with concurrent elevated levels of (Cl-)i that support chloride efflux during GABAergic synaptic transmission. This period of heightened excitability during perinatal development is critical for circuit formation and synaptic plasticity however it also increases the susceptibility for early life seizures such as in hypoxic-ischemic encephalopathy (HIE). Developmentally, KCC2 expression undergoes a robust increase of approximately 200% from P7 to P10 in CD1 pups. Ischemia-induced seizures in the CD-1 mouse model of ischemic seizures, are associated with phenobarbital (PB)-resistant seizures at P7 and PB-responsive seizures at P10.The model provides an excellent paradigm to test the ability of CLP290, a proposed KCC2 functional enhancer, to restore KCC2 mediated inhibition and rescue PB-refractoriness at P7. Methods: P7 or P10 CD-1 mouse pups of both sexes underwent unilateral carotid ligation without transection, followed by 2h vEEG recording to quantify seizure burdens and PB-efficacy. Pups were randomly assigned to either the ligate-only, CLP290 post, or pre-treatment groups. The post-treatment groups received a single injection immediately after ligation, while the pre-treatment groups received an injection 4h before ligation as well as one immediately after ligation. Pups received intraperitoneal (I.P.) injections of 5, 10, or 20 mg/kg of CLP290 in HPCD. CLP290 is a carbamate pro-drug of CLP257 that crosses the blood brain barrier. To investigate differences in anti-seizure efficacy between CLP257 and CLP290, 10mg/kg of CLP257 in HPCD was administered to a post and pre-treatment group. After 1h of baseline vEEG recording, all treatment groups received a loading dose of PB (25 mg/kg I.P. in saline) at 1h. The selective KCC2 antagonist VU 0463271 in 5% DMSO was substituted for PB in a subgroup of ligate-only pups. 24h after ligation, brains were harvested and processed for western blot analyses to investigate KCC2 expression levels. Results: The KCC2 enhancer CLP290 (pre-treatment group; 10mg/kg; IP) significantly rescued PB-resistance (p=0.004) at P7. Western blot analysis also showed rescue of the ischemia-induced KCC2 degradation when compared to the non-treated group. The KCC2 phosphorylation site S940, which is associated with the membrane insertion and stability of KCC2 as well as an increased chloride extrusion capacity, was dephosphorylated following ischemic seizures. CLP 290 rescued KCC2 S940 phosphorylation in treated P7 pups. As expected CLP257 pre-treatment failed to rescue PB-resistance. Further, the selective KCC2 antagonist VU 0463271 increased the seizure burden, supporting the role of KCC2 in neonatal seizure susceptibility. Conclusions: These data indicate that therapies which specifically increase KCC2 function maybe a sound approach for refractory seizures associated with compromised KCC2 function. Funding: Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Number R01HD090884 (SDK)