Abstracts

Rationale: Prolonged seizures or status epilepticus can produce transient hypoxia, and also predispose to later seizures. Hypoxia Inducible Factor-1 alpha (HIF-1α), which promotes cell survival after hypoxia, might alter inhibition and precipitate or protect against subsequent seizure activity. Whether HIF-1α is stimulated by prolonged seizures or contributes to GABA_A receptor (GABAR) dysfunction, has not been directly tested. Both protein kinase C (PKC) and the Ca²⁺/calmodulin dependent phosphatase, calcineurin (CaN), can modulate GABAR phosphorylation and bind to RACK1; CaN also promotes HIF-1α expression by blocking RACK1 dimerization. We hypothesized that prolonged seizure activity might predispose to further seizures by causing hypoxia and triggering hypoxia-induced GABAR dysfunction. Methods: To test for possible interactions between HIF-1α and GABAR regulation, we used the lithium/pilocarpine status epilepticus model and performed immunohistochemistry to determine if prolonged chemically-induced seizures cause time-dependent accumulation of HIF-1α in rat brain regions associated with seizure activity, and whether HIF co-localized with RACK-1, CaN or other proteins associated with GABAR regulation. Male Sprague-Dawley rats (PN33-40) received lithium (3 mEq/kg IP), followed 18 h later by scopolamine methylbromide (2 mg/kg IP) and 30 min later by pilocarpine (60 mg/kg IP). Seizure activity was observed for 1 h and stopped with diazepam (10 mg/kg IP). Control rats were injected with vehicle (X 3), then diazepam. Immediately after seizure or after 24 h recovery, rats were perfused under isoflurane anesthesia. Brains were paraffin thin sectioned (10 µm) and analyzed by immunofluorescence. Results: Immediately after 1 h seizure, the protein expression of GABAR (β2/3 subunit), RACK1 and HIF-1α was increased compared to controls and decreased after 24 h recovery. Preliminary confocal experiments suggested co-localization of HIF-1α and RACK1 in controls which decreased immediately after seizures and returned to baseline after 24 h; hippocampal CaN immunostaining shifted from a dendritic to somatic localization in both pyramidal and dentate granule cell regions. Conclusions: Prolonged lithium-pilocarpine-induced seizures altered HIF-1α-related signaling components, which may contribute to altered neuronal inhibition associated with status epilepticus.