Phosphatase inhibition prevents the activity-dependent trafficking of GABAA receptors during status epilepticus in the young animal

ObjectivesTo determine if the activity-dependent trafficking of 2 subunit-containing -aminobutyric acid type A receptors (GABA(A)Rs) that has been observed in older animals and posited to contribute to benzodiazepine pharmacoresistance during status epilepticus (SE) is age-dependent, and to evaluate whether blockade of protein phosphatases can inhibit or reverse the activity-dependent plasticity of these receptors. MethodsThe efficacy and potency of diazepam 0.2-10mg/kg administered 3 or 60min after the onset of a lithium/pilocarpine-induced seizure in postnatal day 15-16 rats was evaluated using video-electroencephalography (EEG) recordings. The surface expression of 2 subunit-containing GABA(A)Rs was assessed using a biotinylation assay, and GABA(A)R-mediated miniature inhibitory postsynaptic currents (mIPSCs) were recorded using whole-cell patch-clamp recording techniques from dentate granule cells in hippocampal slices acutely obtained 60min after seizure onset (SE-treated). The effect of the protein phosphatase inhibitors FK506 and okadaic acid (OA) on the surface expression of these receptors was determined in organotypic slice cultures exposed to high potassium and N-methyl-d-aspartate (NMDA) or in SE-treated slices. ResultsDiazepam terminated seizures of 3min but not 60min duration, even at the highest dose. In the SE-treated slices, the surface expression of 2 subunit-containing GABA(A)Rs was reduced and the amplitude of the mIPSCs was diminished. Inhibition of protein phosphatases prevented the activity-induced reduction of the 2 subunit-containing GABA(A)Rs in organotypic slice cultures. Furthermore, treatment of SE-treated slices with FK506 or OA restored the surface expression of the 2 subunit-containing GABA(A)Rs and the mIPSC amplitude. SignificanceThis study demonstrates that the plasticity of 2 subunit-containing GABA(A)Rs associated with the development of benzodiazepine resistance in young and adult animals is similar. The findings of this study suggest that the mechanisms regulating the activity-dependent trafficking of GABA(A)Rs during SE can be targeted to develop novel adjunctive therapy for the treatment of benzodiazepine-refractory SE.