Abstracts

We have previously shown that seizures in the immature brain, and this is associated with immediate ([lt]1h post seizure) increases in hippocampal hyperexcitability and long term seizure susceptibility (J Neurophysiol 1998, 79:73-81). Epileptogenic sequela of seizures in the immature brain (P10 rat) are likely to be due to alterations in multiple neurotransmitter systems. AMPA receptor current activity is increased within 1 h after seizures. AMPA mediated activation of calcineurin within 1 h after seizures leads to a decrease in GABAergic inhibition. We have recently reported a seizure induced downregulation of the NR2B subunit at 24 h following seizures at P10 (Epilepsia 2003, 44:18-19). In the present study, we examined the time course of alterations in NMDAR function and expression. Postnatal day (P) 10 rats were placed to 4-7% of O[sub]2 [/sub](15 min). Whole-cell recordings were made in pyramidal cells of hippocampal slices removed 1h after hypoxia or from control rats. Single stimulus shocks were delivered through a bipolar electrode to evoke NMDAR-mediated EPSCs (eEPSCs). We used the NR2B specific antagonist Ro25-6981 to evaluate NR2B mediated currents. NR2B expression levels as well as phosphorylation state of the serine (pSer) residue were analyzed by Western blot with anti-pSer or anti-NR2B antibodies. We have previously shown that NMDA responses were negatively regulated by Ro25-6981 (1 [micro]M) in control rats but not at 24h post hypoxia-induced seizures in P10 rats (Epilepsia 2003, 44:18-19). In contrast, at 1 h after hypoxia-induced seizures, there were no differences in NMDA (200[micro]M) induced responses in CA1 pyramidal neurons from slices from hypoxic vs control P10 rats. Furthermore, NMDA responses were significantly inhibited by Ro25-6981 in both groups (70% decrease in control rats vs 77% in hypoxic rats). In addition, Ro25-6981 showed similar inhibition of the eEPSCs mediated by NMDARs, suggesting only synaptic NMDARs are involved in this study (65% decrease in control rats). Unlike 24h post hypoxic seizures, NR2B expression was not altered at 1 h compared to control rats. However, there was a significant decrease (23% of control, p[lt]0.05) in NR2B phosphorylation as measured by pSer labeling. Our data indicate that seizure induced alterations of overall levels of NR2B subunit in the P10 rat brain are not immediate and become apparent by 24h. However, NR2B phosphorylation does change as early as 1h, indicating that seizure-induced receptor dephosphorylation may precede membrane protein decreases. Future studies are required to determine the specific NR2B site involved or whether NR2B subunit dephosphorylation results in subunit removal from the membrane. Furthermore, it is not yet clear whether the seizure-induced decreases in NR2B represent a compensatory response to suppress excitability or in fact contribute to epileptogenesis in this model of neonatal seizures. (Supported by EF/AES fellowship (W.D.); NS31718 (FEJ).)