okadaic-acid and Status-Epilepticus

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.. 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.. 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.. 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.

Topics: Animals; Animals, Newborn; Anticonvulsants; Cells, Cultured; Diazepam; Disease Models, Animal; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Hippocampus; Immunosuppressive Agents; In Vitro Techniques; N-Methylaspartate; Neurons; Okadaic Acid; Organ Culture Techniques; Phosphoric Monoester Hydrolases; Pilocarpine; Protein Transport; Rats; Rats, Sprague-Dawley; Receptors, GABA; Status Epilepticus; Tacrolimus

Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM kinase II has not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long-lasting decrease in CaM kinase II activity in the hippocampal neuronal culture model of low Mg2+-induced spontaneous recurrent epileptiform discharges (SREDs). We show here that the decrease in CaM kinase II activity associated with SREDs in hippocampal cultures involves a Ca2+/N-methyl-d-aspartate (NMDA) receptor-dependent mechanism. Low Mg2+-induced SREDs result in a significant decrease in Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptide autocamtide-2. Reduction of extracellular Ca2+ levels (0.2 mM in treatment solution) or the addition of dl-2-amino-5-phosphonovaleric acid (APV) 25 microM blocked the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. Antagonists of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid receptor or L-type voltage sensitive Ca2+ channel had no effect on the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. The results of this study demonstrate that the decrease in CaM kinase II activity associated with this model of epileptogenesis involves a selective Ca2+/NMDA receptor-dependent mechanism and may contribute to the production and maintenance of SREDs in this model.

Topics: Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cells, Cultured; Cellulose; Electrophysiology; Enzyme Inhibitors; Epilepsy; Hippocampus; Immunohistochemistry; Magnesium Deficiency; Neurons; Okadaic Acid; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Recurrence; Status Epilepticus

The development of symptomatic epilepsy is a model of long-term plasticity changes in the central nervous system. The rat pilocarpine model of epilepsy was utilized to study persistent alterations in calcium/calmodulin-dependent kinase II (CaM kinase II) activity associated with epileptogenesis. CaM kinase II-dependent substrate phosphorylation and autophosphorylation were significantly inhibited for up to 6 weeks following epileptogenesis in both the cortex and hippocampus, but not in the cerebellum. The net decrease in CaM kinase II autophosphorylation and substrate phosphorylation was shown to be due to decreased kinase activity and not due to increased phosphatase activity. The inhibition in CaM kinase II activity and the development of epilepsy were blocked by pretreating seizure rats with MK-801 indicating that the long-lasting decrease in CaM kinase II activity was dependent on N-methyl-D-aspartate receptor activation. In addition, the inhibition of CaM kinase II activity was associated in time and regional localization with the development of spontaneous recurrent seizure activity. The decrease in enzyme activity was not attributed to a decrease in the alpha or beta kinase subunit protein expression level. Thus, the significant inhibition of the enzyme occurred without changes in kinase protein expression, suggesting a long-lasting, post-translational modification of the enzyme. This is the first published report of a persistent, post-translational alteration of CaM kinase II activity in a model of epilepsy characterized by spontaneous recurrent seizure activity.

Topics: Animals; Brain; Calcium-Calmodulin-Dependent Protein Kinases; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Intercellular Signaling Peptides and Proteins; Isoenzymes; Male; Okadaic Acid; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Pilocarpine; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Secondary Prevention; Seizures; Status Epilepticus; Time Factors