Abstracts

Hypoxic encephalopathy is the most common cause of neonatal seizures. We have established a rodent model that mimics the acute seizures and chronic pro-epileptogenic effects of neonatal hypoxia. We have previously shown enhanced LTP and kindling in CA1 neurons in hippocampal slices removed from P10 rats following hypoxic seizures. Furthermore, we observed increases in PKA, PKC and CamK II activity at 1 hour following hypoxic seizures. Kinase activity has been demonstrated to induce changes in AMPA receptor (AMPAR) phosphorylation in cultured neurons and hippocampal slices. These changes can regulate excitatory synaptic transmission by modulating AMPAR channel properties and receptor trafficking. We hypothesized that increased kinase activity may lead to increased AMPAR phosphorylation and activity, which in turn might underlie the hyperexcitability observed in our model of hypoxic seizures., Rat pups were subjected to hypoxia (15 minutes at 4 [ndash] 7% O[sub]2[/sub]) on postnatal day (P) 10. Protein extracts were prepared from hippocampal tissue obtained at 1, 6, 12, 24 and 48 hours post hypoxia. PKA, PKC and CamK II kinase activity was assayed using ELISA assays. Immunoblot analysis was performed using phospho-specific antibodies against serine 831 and 845 epitope of AMPAR subunit GluR1. The whole-cell patch clamp technique was used to record spontaneous and evoked EPSCs in CA1 pyramidal neurons in hippocampal slices. The pipette solution contained 110 mM Cs, 10 mM TEA and 5 mM QX-314 to block voltage-sensitive K⁺ and Na⁺ currents. The slices were superfused with ACSF containing picrotoxin (30 [micro]M) and DL-AP5 (50[micro]M) to block GABA and NMDA receptor currents, respectively. Schaffer collaterals were stimulated by a bipolar electrode in CA3., CamK II activity was increased at 1 hour (1.7 fold, [italic]p[/italic] [lt] 0.025) and 6 hours (1.2 fold, [italic]p[/italic] [lt] 0.05), but not at 24 hours, following hypoxia-induced seizures compared to control. Similarly, PKA (8.6 fold, [italic]p[/italic] = 0.006) and PKC (10 fold, [italic]p[/italic] = 0.037) activity was increased in animals 1 hour after hypoxia. The phosphorylation of GluR1 receptors at serine 831 (3.0 fold, [italic]p[/italic] [lt] 0.05) and serine 845 (3.5 fold, [italic]p[/italic] [lt] 0.05) was increased for 24 hours before returning to baseline 48 hours after hypoxia. Consistent with increased phosphorylation, both amplitude and frequency of spontaneous and evoked AMPAR-mediated EPSCs appeared to be increased in hippocampal slices harvested one hour after hypoxia ([italic]n[/italic] = 4)., Our data suggest that hypoxia-induced seizures induce a transient increase in kinase activity resulting in increased phosphorylation of AMPAR subunit GluR1 and increased activity of synaptic AMPARs. These changes in AMPARs may underlie increased hippocampal excitability, epileptiform activity and kindling following hypoxic seizures., (Supported by NS 31718.)