Abstracts

RATIONALE: Hypoxic encephalopathy is the most common cause of neonatal seizures and can be associated with long-term neurocognitive deficits and epilepsy. In our perinatal rodent model, hypoxia-induced seizures result in an increase in hippocampal network excitability and increase long-term seizure susceptibility. We have previously reported a decrease in GABAA IPSC[ssquote]s in hippocampal CA1 observed at 96 hours after hypoxia. As the phosphatase calcineurin (CaN) can regulate GABAA receptor (GABAAR) function, the goal of this study was to examine the role of CaN in hypoxia-induced hyperexcitability. We evaluated CaN expression in hippocampus following hypoxia induced seizures. We examined the role of CaN in hypoxia-induced hyperexcitability by evaluating the effect of the CaN inhibitor FK-506 on the altered IPSC[ssquote]s in hippocampal slices in vitro and on seizure incidence in the in vivo animal model.
METHODS: Rat pups at postnatal day (P) 10-11 were placed to 4-7% of O2 for 15 min for induction of seizures. For western blot analysis, blots from whole cell hippocampal protein samples (removed at 24 hours post hypoxic seizure or from littermate controls) were incubated with the CaN A antibody. For electrophysiology, hippocampal slices were removed at 1 hour post hypoxia-induced seizures. Whole-cell patch-clamp studies were made in CA1 pyramidal neurons from hypoxia-treated and age-matched control rats. Spontaneous ISPCs (sIPSCs) mediated by GABAAR were recorded as index for GABAergic neurotransmission. IPSC frequency was compared at baseline and after addition of CNQX (20[mu]M), APV(50[mu]M), or FK-506 (10[mu]M). To study the effects of FK-506 in vivo, FK-506 (10 mg/kg i.p.) was administered 30 min prior to hypoxia and seizure frequency was compared to that of hypoxic vehicle-treated controls.
RESULTS: Western blots showed that CaN A protein was significantly increased 24 hour following hypoxia (p[lt]0.04). Both the frequency and amplitude of CA1 pyramidal neuronal sIPSCs mediated by GABAARs were significantly decreased as early as 1 hour after hypoxia (n=5) compared to control slices (n=5, p[lt]0.01). Subsequent application of FK-506 in slices from hypoxic rats significantly reversed this decrease in sIPSCs (n=4, p[lt]0.001). Similarly, application of CNQX and APV also reversed this decrease (n=5, p[lt]0.001). Finally, rats treated with FK-506 prior to hypoxia (n=12) exhibited significantly fewer hypoxia-induced seizures compared to vehicle treated hypoxic control littermates (n=11) (p[lt]0.0001).
CONCLUSIONS: These data support the hypothesis that early increases in CaN activity may play a role in the impaired GABAAR signaling seen in area CA1 after hypoxia-induced seizures, and may contribute to hypoxia-induced neuronal hyperexcitability. The action of FK-506 to reverse hypoxia-induced IPSC downregulation was similar to the effect of glutamate receptor blockade, suggesting that CaN may be activated by glutamate receptors after hypoxic seizures. The anticonvulsant effect of FK-506 in our rodent model indicates that FK-506-like agents may have clinical potential in the treatment of refractory perinatal seizures.
[Supported by: NINDS RO1 NS31718-10 (FEJ)]