Abstracts

Rationale: Hypoxic encephalopathy is the most common cause of neonatal seizures frequently and results in the development of epilepsy and cognitive impairment. In our rat model of hypoxia-induced neonatal seizures, we have observed enhanced AMPAR-mediated spontaneous and miniature EPSCs in hippocampal CA1 neurons (Zhou et al., AES abstract 2007:IW49), elevated activity of protein kinases PKC, PKA and CaMKII and concomitant post-translational modification of AMPAR GluR1/2 subunits (Rakhade et al., AES abstract 2007:IW39). The perinatal hippocampus contains many AMPAR-silent synapses. We hypothesized that seizures decrease the proportion of silent synapses, and alter the expression of long term potentiation (LTP) in the developing brain. Methods: Rat pups were subjected to hypoxia (15 minutes at 4-7% O₂) on postnatal day (P)10. Ex vivo hippocampal slices were prepared at 1, or 24 and 48 hours after hypoxic seizures in vivo. The whole-cell patch clamp technique was used to record GluR-mediated spontaneous (s) and evoked (e)EPSCs in CA1 pyramidal neurons and measure sEPSC frequency and eEPSC failure rates at -60mV compared to +40mV. The minimal stimulation method was used to stimulate Schaffer collaterals to record eEPSCs. To record EPSCs, the pipette solution contained Cs, TEA and QX-314. Picrotoxin (30 µM) was added in ACSF. LTP was induced using tetanic stimulation (100 Hz) and fEPSP slope was evaluated at 24-48 hours (hr) after seizures. Results: One hour (hr) following hypoxic seizures, hippocampal CA1 neurons exhibited significant increases in GluR-mediated sEPSC frequency at -60 mV. The ratio of sEPSC frequency at -60/+40 mV was 1.31 ± 0.3 (n = 6) in slices removed at 1 hr from rats experiencing hypoxic seizures, compared to 0.26 ± 0.06 (n = 4; p = 0.02) in the normoxia group. When minimal stimulation of Schaffer collaterals was used to evoke EPSCs, the eEPSC failure rates in slices removed 1 hr following hypoxic seizures were not significantly different at -60 and +40 mV (91.6 ± 6.5% at -60 mV and 67.8 ± 10.4% at +40mV, n = 4, p = 0.111). In contrast, the eEPSC failure rates in normoxic control slices were significantly increased at -60 mV compared to +40 mV (100 ± 0% at -60mV, 63.3 ± 10.8% at +40mV, n = 4, p = 0.04), consistent with the presence of silent synapses in control slices. Furthermore, at 48hr following hypoxic seizures, fEPSP amplitude and slope were decreased, compared to normoxia. In addition, LTP was reduced at 48hrs in post-hypoxic seizure group (35% of pre-tetanus fEPSP slope), compared to normoxia group (55%). Conclusions: Our data suggest that hypoxia-induced seizures can cause an early decrease in AMPA-lacking silent synapses in hippocampal CA1 neurons in slices prepared following hypoxic seizures at P10. In addition, LTP was diminished compared to slices from normal litter mates. These results suggest that hypoxic seizures not only induce early and rapid alterations in synaptic function, but also change or occlude the expression of LTP during brain development, which may in part contribute to the long-term cognitive deficits observed in this neonatal seizure model.