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

Abstract

Summary Objectives To determine if the activity-dependent trafficking of γ2 subunit-containing γ-aminobutyric acid type A receptors (GABAARs) 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. Methods The efficacy and potency of diazepam 0.2-10 mg/kg administered 3 or 60 min 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 γ2 subunit-containing GABAARs was assessed using a biotinylation assay, and GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs) were recorded using whole-cell patch-clamp recording techniques from dentate granule cells in hippocampal slices acutely obtained 60 min 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. Results Diazepam terminated seizures of 3 min but not 60 min duration, even at the highest dose. In the SE-treated slices, the surface expression of γ2 subunit-containing GABAARs was reduced and the amplitude of the mIPSCs was diminished. Inhibition of protein phosphatases prevented the activity-induced reduction of the γ2 subunit-containing GABAARs in organotypic slice cultures. Furthermore, treatment of SE-treated slices with FK506 or OA restored the surface expression of the γ2 subunit-containing GABAARs and the mIPSC amplitude. Significance This study demonstrates that the plasticity of γ2 subunit-containing GABAARs 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 GABAARs during SE can be targeted to develop novel adjunctive therapy for the treatment of benzodiazepine-refractory SE.