Abstracts

Rationale: Neonatal seizures are refractory to current drugs and can result in chronic epilepsy and long-term cognitive deficits. The mammalian Target of Rapamycin (mTOR), a key regulator of protein translation has been implicated in epileptogenesis in adult models of epilepsy (J Neurosci 29:6964-6972; J Neurosci 29:8259-8269). We hypothesized that the mTOR Complex 1 (mTORC1) kinase activity is developmentally enhanced during the neonatal period, coincident with heightened synaptic plasticity and increased seizure susceptibility and that post-seizure activation of the mTORC1 pathway may be critically involved in creating epileptic networks in the immature brain. We assessed the developmental pattern of the mTORC1 activity in rat hippocampus and cortex and tested the efficacy of mTORC1 inhibitor rapamycin in attenuating long-term increases in neuronal excitability and seizure susceptibility in a rat model of neonatal seizures. Methods: The levels of mTOR and its activity readout phospho-p70S6 (Thr389) kinase (p70S6K) from postnatal day (P)3 to adulthood were quantified by western blot. Hypoxic seizures (HS) were induced by graded global hypoxia at P10. Control and HS rats were treated with rapamycin (at 3 mg/kg i.p.) or vehicle, 24h before and 1h after exposure to hypoxia. Differences in susceptibility to kainic acid (2 mg/kg, i.p.) were evaluated at 72h post-HS. Spontaneous AMPA receptor-mediated EPSCs (sEPSCs) in CA1 pyramidal neurons were recorded in ex vivo hippocampal slices removed 48h post-HS. Results: mTOR protein expression is significantly lower during the neonatal period (P10/11) compared to adulthood (69 6.1% of adult, n=5, p<0.05 in the hippocampus, and 54 5.2% of adult, n=5, p<0.001 in the cortex). In contrast, mTORC1 activity, assessed by p70S6K phosphorylation levels, is significantly higher at P10/11, relative to adult (43.8 12.3 fold increase, n=5, p<0.05 in the hippocampus and 70.7 8.4 fold, n=7, p<0.001 in the cortex), suggesting that in developing brain mTORC1 pathway may be critically involved in epileptogenesis. Indeed, mTORC1 inhibitor rapamycin blocked the post-HS increased seizure susceptibility, as demonstrated by significantly longer latency to onset of first behavioral seizure after kainate administration at P13 (control vehicle: 21.73 1.61 min, n=13; HS vehicle: 16.35 0.51 min, n=10; HS rapamycin: 23.24 2.21 min, n=11; p=0.021). In addition, rapamycin treatment reversed the subacute increases in sEPSCs amplitude in the CA1 pyramidal neurons in ex vivo hippocampal slices removed 48h post-HS (control vehicle: 100 6.27%, n=6; HS vehicle: 137.62 3.39% of control, n=6; HS rapamycin: 107.93 4.64% of control, n=6; p<0.001).