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