Abstracts

Rationale: More effective treatments are needed to treat status epilepticus (SE). NMDA receptors (NMDARs) are activated during SE, cause neuronal death, and may contribute to drug refractoriness. We tested whether NMDAR activation enhanced glutamatergic transmission during SE. Methods: SE was induced in adult rats by injection of pilocaprine. Biochemical and electrophysiological studies were performed 10 min (10 min SE) or 60 min (60 min SE) after the first stage 5 behavioral seizure. The animals were continuously monitored by video-EEG recordings in some experiments. Surface GluA1 subunit expression was determined by a biotinylation or a BS³ assay. AMPA (2.5 μM)-evoked currents were recorded from CA1 pyramidal neurons (PNs) using standard whole-cell patch clamp technique. Organotypic hippocampal slice cultures treated with HKNMDA (10 mM KCl and 10 μM NMDA) for 1 hr were used to test NMDAR regulation of GluA1 subunit surface expression. Results: Cell surface expression of GluA1 subunit-containing AMPARs increased in the hippocampal CA1 region of 60 min SE animals (161 ± 23%, n=5, p < 0.05) but not of 10 min SE animals (130 ± 26%, n=7, p>0.05). This finding was confirmed using a BS³ assay which found reduced intracellular expression of GluA1 subunit in 60 min SE animals. Further, AMPAR-mediated whole cell currents recorded from CA1 pyramidal neurons were also larger in 60 min SE animals compared to controls (20.6 ± 3.8 pA, n=7 vs 12.5 ± 1.5 pA, n=10, p<0.05). MK801 (2 mg/kg) treatment at 10 min SE did not block seizures but prevented the increase in surface expression of GluA1 subunit at 60 min SE. Further, treatment of hippocampal slice cultures with HKNMDA also increased the surface expression of GluA1 subunits (185 ± 35% of controls, n=7, p<0.05) and this enhancement was blocked by co-treatment with MK801. Also, blockade of NR2B but not NR2A subunit-containing NMDARs prevented the increase in GluA1 subunit surface expression. Finally, chelation of calcium by BAPTA-AM also prevented the increase in surface GluA1 subunit expression in HKNMDA-treated slice cultures. We tested whether NMDAR blockade by MK801 conferred diazepam sensitivity to SE animals. MK801 a specific NMDAR antagonist administered in combination with diazepam at 10 min SE terminated seizures in all the animals (n=4) with a median time of 9.36 min. In contrast, MK801 or diazepam given alone were ineffective in shortening the SE. Conclusions: NMDAR dependent mechanisms increase the cell surface expression of GluA1 subunit-containing AMPARs during SE. Blocking this AMPAR plasticity by MK801 may underlie the effectiveness of a MK801-diazepam combination therapy in terminating benzodiazepine refractory SE.