Phosphatase inhibition prevents the activity‐dependent trafficking of GABA_A receptors during status epilepticus in the young animal

Abstract

SUMMARYObjectives: To determine if the activity-dependent trafficking of c2 subunit-containing c-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. 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 c2 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 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 c2 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 c2 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 c2 subunit-containing GABA A Rs and the mIPSC amplitude. Significance: This study demonstrates that the plasticity of c2 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.

Status epilepticus: Role for etiology in determining response to benzodiazepines

“…Development of benzodiazepine pharmacoresistance during LiPilo-SE in adult animals and animals as young as postnatal day 15 (P15) has been well established 8,11,16,17 . We confirmed this finding in P23 to P25 animals in which seizures of 3 minutes (early SE) or 60 minutes (late SE) in duration were treated with diazepam ranging in dose from 3 mg/kg to 30 mg/kg.…”

“…Animals were monitored for seizures behaviorally and electrographically using video-EEG recordings 11 . For treatment studies, continuous spiking greater than 2 Hz and amplitude more than 2 times the background was considered as the start of SE.…”

“…The antibodies used in these studies were anti-γ2 subunit antibody (clone 10F10-C1-B8, 2–3 µg/ml) 11 , anti-β3 subunit antibody (1:500, Novus), anti-Kv4.2 (1:500, EMD Millipore), and anti-β-actin antibody (1:1000, Sigma-Aldrich).…”

Pathophysiology of status epilepticus

“…It has long been recognized that benzodiazepines lose their potency as status epilepticus continues and that this is partly due to the internalization of synaptic (gammasubunit) containing GABA(A) receptors [35][36][37]. This involves the activation of NMDA receptors and then calcineurin-dependent internalization of the GABA(A) receptors [38,39]. This process points to three specific treatment approaches.…”

Status Epilepticus

“…Increasing the activity of PKC in acutely obtained hippocampal slices from SE-treated animals served to increase the percentage of phosphorylated receptors with a corresponding increase in the surface expression of the receptors and a restoration of the mIPSC amplitude. Other studies have shown that inhibitors of PP2A as well as protein phosphatase 2B (PP2B; calcineurin) was also effective in modulating the activity-dependent trafficking of the GABA A receptors leading to similar restoration of the surface expression of the receptors and a restoration of the mIPSC amplitude (Eckel et al 2015;Joshi et al 2015). Future experiments in vivo are required to determine if activation of protein kinases or inhibition of phosphatases during SE alters the time course or treatment of SE.…”