Abstracts

Rationale: Neonatal seizures are refractory to conventional anticonvulsants and can result in chronic epilepsy and long-term cognitive deficits. Neonatal period is characterized by enhanced activity-dependent plasticity including homeostatic plasticity through which neurons use the neuroplasticity machinery to maintain stable neural activity. Recent studies have indicated polo-like kinase 2 (PLK2) as part of this novel molecular mechanism required in downward scaling of synaptic strength in response to elevated neuronal activity in vitro. As seizures are a naturally occurring source of neuronal overactivation, we hypothesized that PLK2-mediated homeostatic plasticity may provide endogenous protection against neonatal seizures-induced potentiation of synaptic strength. Methods: Hypoxic seizures (HS) were induced by global hypoxia in P10 rats. Control and HS rats were treated with BI-2536 (30 mg/kg i.p.), MG132 (5 mg/kg i.p.), or vehicle, 30 min before exposure to hypoxia. Ex vivo hippocampal slices were prepared at 1, 12, 24, 48, 72, 96, 120, 168, 240-480h after HS. Whole-cell patch clamp recordings in CA1 pyramidal neurons were made to examine intrinsic membrane properties (IMP) and spontaneous AMPAR EPSCs (sEPSCs). Real-time PCR and Immunochemistry were used to examine the PLK2 mRNA and protein expression. Results: Although developmental maturation was apparent, the IMPs including resting membrane potential, input resistance, input-output function were not significantly different between post-HS groups and controls at different time points (all p>0.05, n=6-12), providing little evidence for intrinsic homeostatic modulation following HS. In contrast to IMPs, dynamic changes of AMPAR function were found at different time points after HS. Compared to controls, AMPA sEPSC amplitude was significant higher at post-HS 1h (178.9 7.8% of control, n=12, p<0.001) and 12h (162.8 7.0% of control, n=9, p<0.001). Enhancement of AMPA sEPSC amplitude gradually reduced at post-HS 24-72h (24h: 137.7 5.1% of control; 48h: 130.8 6.2% of control, n=11-12, p<0.01) and then kept at relatively constant level until post-HS12-13 days (5-7d: 131.6 5.7% of control; 13-20d: 153.9 8.3% of control, n=8-9, p<0.01), suggesting a possible self-compensative mechanism following HS. PLK2 mRNA significantly increased at 1, 6 and 24 h after HS (mRNA fold change at 1h: 1.18 0.03; 6h: 1.70 0.18; 24h: 1.24 0.04; n=4, p<0.05). In addition, significant increase of neuronal PLK2 protein expression was detected at 24-72 h post-HS. In vivo application of the PLK2 inhibitor BI-2536 blocked the subacute down-regulation of AMPAR function (sEPSC amplitude at post-HS 18-24h: 173.9 11.1% of control; at 48h: 169.3 8.8% of control, n=6-9, p<0.001). Furthermore, the PLK2 breakdown inhibitor MG132 resulted in full recovery from HS induced AMPAR function enhancement (sEPSC amplitude at post-HS 24h: 96.9 8.4% of control, n=6, p>0.05). Conclusions: These results suggest an important role of PLK2 mediated homeostatic compensation in preventing early life HS-induced potentiation of synaptic strength in vivo.