Abstracts

Perinatal hypoxia increases hippocampal network excitability and long term seizure susceptibility (Jensen et al., 1992). We previously showed that maturational differences in AMPA subtypes of glutamate receptors play a role hypoxia-induced epileptogenesis (Sanchez et al., 2000). In addition, hypoxia increases calcineurin (CaN) activity and expression within 24 h (Dai et al., 2002). NMDA subtypes play a major role in epileptogenesis and are developmentally regulated via CaN, with CaN inducing a switch from NR2B to NR2A (Shi et al., 2000). We used a rodent model of perinatal hypoxia to determine whether early life seizures induced alterations in NMDA receptor subunit expression and function.
Rats at postnatal day 10-11 were placed in 4-7% O[sub]2 [/sub]for 15 min to induce seizures. 24 h after seizures, hippocampal slices were prepared and incubated at room temperature for 1 hr before recording. Patch-clamp recordings in whole-cell configuration and bath application of the NMDA receptor antagonists Ro25-6981 and D-AP5 were used to compare the seizure-induced alterations in NMDA currents in pyramidal cells of hippocampal slices from hypoxic and normal rats. Membrane protein from hippocampal homogenates was prepared 24 h after seizures, and NMDAR subunit expression was analyzed by Western blotting and immunodetected with NR2A, NR2B, or NR1 antibodies.
NMDA (200[micro]M) evoked large responses in hippocampal pyramidal neurons from control rats (-138.5[plusmn]24 pA, n=25) and in rats at 24 h post hypoxic seizures (-115.8[plusmn]19.6 pA, n=16, p[gt]0.05). Ro25-6981, a highly selective and potent NR2B receptor antagonist (Fischer et al., 1997) markedly inhibited NMDA responses from control rats by 62% (37.8[plusmn]4.4% of the control current, n=9, p[lt]0.0001). In contrast, Ro25-6981 had little effect on NMDA responses in slices from rats 24 hrs post hypoxia (88[plusmn]4.6% of the control current, n=5, p[gt]0.05). D-AP5 (50[micro]M) completely blocked the residues of the Ro25-6981-resistant component of the NMDA responses in these slices from rats 24h post hypoxia (n=4). The lack of effect of Ro25-6981 indicates that NR2B subunits contribute minimally to NMDA currents in rats after hypoxia. Western blot analysis showed a significant decrease (32 [plusmn] 7%, n=8) in NR2B subunit protein in samples from rats 24 h post hypoxic seizure compared to control rats (n=7, p[lt]0.05). There were no changes in NR2A and NR1 expression 10[plusmn]14% (n=4) and 1[plusmn]12% (n=4), respectively compared to control rats (n=4, p[gt]0.05).
NR2B receptor function and expression is downregulated in hippocampus at 24 h post hypoxia-induced seizures, coincident with the time window in which we observed increased CaN activity and function. These data suggest seizures may modify NR2 expression via CaN. Future studies will be required to determine whether these changes are proepileptogenic or compensatory in the immature brain.
[Supported by: NINDS RO1 NS31718 (FEJ)]