Progressive NKCC1-Dependent Neuronal Chloride Accumulation during Neonatal Seizures

Seizures induce excitatory shifts in the reversal potential for GABA(A)-receptor-mediated responses, which may contribute to the intractability of electro-encephalographic seizures and preclude the efficacy of widely used GABAergic anticonvulsants such as phenobarbital. We now report that, in intact hippocampi prepared from neonatal rats and transgenic mice expressing Clomeleon, recurrent seizures progressively increase the intracellular chloride concentration ([Cl-](i)) assayed by Clomeleon imaging and invert the net effect of GABA(A) receptor activation from inhibition to excitation assayed by the frequency of action potentials and intracellular Ca2+ transients. These changes correlate with increasing frequency of seizure-like events and reduction in phenobarbital efficacy. The Na+-K+-2Cl(-) (NKCC1) cotransporter blocker bumetanide inhibited seizure-induced neuronal Cl- accumulation and the consequent facilitation of recurrent seizures. Our results demonstrate a novel mechanism by which seizure activity leads to [Cl-](i) accumulation, thereby increasing the probability of subsequent seizures. This provides a potential mechanism for the early crescendo phase of neonatal seizures.