Evidence that GAD65 mediates increased GABA synthesis during intense neuronal activity in vivo

Abstract

In this study we tested the hypothesis that the 65-kDa isoform of glutamate decarboxylase (GAD65) mediates activity-dependent GABA synthesis as invoked by seizures in anesthetized rats. GABA synthesis was measured following acute GABA-transaminase inhibition by gabaculine using spatially localized 1H NMR spectroscopy before and after bicuculline-induced seizures. Experiments were conducted with animals pre-treated with vigabatrin 24 h earlier in order to reduce GAD67 protein and also with non-treated controls. GAD isoform content was quantified by immunoblotting. GABA was higher in vigabatrin-treated rats compared to non-treated controls. In vigabatrin-treated animals, GABA synthesis was 28% lower compared to controls [p < 0.05; vigabatrin-treated, 0.043 ± 0.011 μmol/(g min); non-treated, 0.060 ± 0.014 μmol/(g min)] and GAD67 was 60% lower. No difference between groups was observed for GAD65. Seizures increased GABA synthesis in both control [174%; control, 0.060 ± 0.014 μmol/(g min) vs. seizures, 0.105 ± 0.043 μmol/(g min)] and vigabatrin-treated rats [214%; control, 0.043 ± 0.011 μmol/(g min); seizures, 0.092 ± 0.018 μmol/(g min)]. GAD67 could account for at least half of basal GABA synthesis but only 20% of the two-fold increase observed in vigabatrin-treated rats during seizures. The seizure-induced activation of GAD65 in control cortex occurs concomitantly with a 2.3-fold increase in inorganic phosphate, known to be a potent activator of apoGAD65in vitro. Our results are consistent with a major role for GAD65 in activity-dependent GABA synthesis. © 2006 International Society for Neurochemistry.