title: Phosphatase inhibition prevents the activity-dependent trafficking of GABAA receptors during status epilepticus in the young animal.
abstract: OBJECTIVE
  To determine if the activity-dependent trafficking of γ2 subunit-containing γ-aminobutyric acid type A receptors (GABAA 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 γ2 subunit-containing GABAA Rs was assessed using a biotinylation assay, and GABAA 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 γ2 subunit-containing GABAA Rs was reduced and the amplitude of the mIPSCs was diminished. Inhibition of protein phosphatases prevented the activity-induced reduction of the γ2 subunit-containing GABAA Rs in organotypic slice cultures. Furthermore, treatment of SE-treated slices with FK506 or OA restored the surface expression of the γ2 subunit-containing GABAA Rs and the mIPSC amplitude.
  
  
  CONCLUSIONS
  This study demonstrates that the plasticity of γ2 subunit-containing GABAA 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 GABAA Rs during SE can be targeted to develop novel adjunctive therapy for the treatment of benzodiazepine-refractory SE.
authors.affiliation: Department of Neurology, University of Virginia Health System, Charlottesville, Virginia, U.S.A.
authors.forename: Suchitra
authors.initials: S
authors.lastname: Joshi
authors.affiliation: Department of Neurology, University of Virginia Health System, Charlottesville, Virginia, U.S.A.
authors.forename: Karthik
authors.initials: K
authors.lastname: Rajasekaran
authors.affiliation: Department of Neurology, University of Virginia Health System, Charlottesville, Virginia, U.S.A.
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Hawk
authors.affiliation: Department of Neurology, University of Virginia Health System, Charlottesville, Virginia, U.S.A.
authors.forename: Jasmit
authors.initials: J
authors.lastname: Brar
authors.affiliation: Department of Pediatrics, University of Virginia Health System, Charlottesville, Virginia, U.S.A.
authors.forename: Brittany M
authors.initials: BM
authors.lastname: Ross
authors.affiliation: Department of Neurology, University of Virginia Health System, Charlottesville, Virginia, U.S.A.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Tran
authors.affiliation: Department of Neurology, University of Virginia Health System, Charlottesville, Virginia, U.S.A.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Chester
authors.affiliation: Department of Neurology, University of Virginia Health System, Charlottesville, Virginia, U.S.A.|Department of Pediatrics, University of Virginia Health System, Charlottesville, Virginia, U.S.A.
authors.forename: Howard P
authors.initials: HP
authors.lastname: Goodkin
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000927:Anticonvulsants / Q000494:pharmacology / Q000627:therapeutic use; D002478:Cells, Cultured; D003975:Diazepam / Q000494:pharmacology / Q000627:therapeutic use; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects; D007166:Immunosuppressive Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects; D019319:Okadaic Acid / Q000494:pharmacology; D009924:Organ Culture Techniques; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010862:Pilocarpine / Q000633:toxicity; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D018079:Receptors, GABA / Q000378:metabolism*; D013226:Status Epilepticus / Q000139:chemically induced / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D016559:Tacrolimus / Q000494:pharmacology
chemical_list: D000927:Anticonvulsants; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D007166:Immunosuppressive Agents; D018079:Receptors, GABA; D010862:Pilocarpine; D019319:Okadaic Acid; D016202:N-Methylaspartate; D010744:Phosphoric Monoester Hydrolases; D003975:Diazepam; D016559:Tacrolimus
keywords: Benzodiazepine; FK506; Okadaic acid; Phosphatase