␥-Aminobutyric acid, which is synthesized by two isoforms of glutamate decarboxylase (GAD), inhibits the transfer of nociceptive signals from primary afferent fibers to the central nervous system. However, the roles of a 65-kDa isoform of GAD (GAD65)-mediated GABA in nociceptive processing are less clear. This study tested whether partial reductions in GABAergic inhibitory tone by GAD65 gene knockout [GAD65(Ϫ/Ϫ)] would contribute to the regulation of pain threshold in mice. Experiments were performed on male wild-type (WT) mice and GAD65(Ϫ/Ϫ) mice. Acute nociception and inflammatory pain tests were compared between WT mice and GAD65(Ϫ/Ϫ) mice. GABA A receptor-mediated inhibitory postsynaptic currents were also examined by use of the whole-cell patch-clamp method in somatosensory cortical neurons in brain slices. In the hot plate test, which reflects supraspinal sensory integration, a significant reduction in the latency was observed for GAD65(Ϫ/Ϫ) mice. Intraperitoneal administration of the GABAethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (C 21 H 22 N 2 O 3 ⅐HCl; NO-711), dose-dependently prolonged the latency in both genotypes, suggesting that GABA concentration contributes to acute thermal nociception. However, there was no genotype difference in responses to the tailimmersion test or the von Frey test, indicating that spinal reflex and mechanical nociception are kept intact in GAD65(Ϫ/Ϫ) mice. There was no genotype difference in responses to chemical inflammatory nociception (formalin test and carrageenan test). Although properties of the phasic component of inhibitory postsynaptic currents were similar in both genotypes, tonic inhibition was significantly reduced in GAD65(Ϫ/Ϫ) mice. These results support the hypothesis that GAD65-mediated GABA synthesis plays relatively small but significant roles in nociceptive processing via supraspinal mechanisms.

GABA is synthesized by two isoforms of glutamate decarboxylase (GAD), GAD65, and GAD67. However, the relative contributions of GAD65-mediated GABA synthesis to the in vivo actions of anesthetics remain unknown. To address this issue, we used mice deficient in the 65-kDa isoform of GAD and tested the hypothesis that partial reduction of GABA content in GAD65-deficient mice [GAD65(Ϫ/Ϫ)] would contribute to hypnotic and immobilizing actions of the anesthetics. The open field test, loss of righting reflex (LORR), loss of tail-pinch withdrawal response (LTWR), and locomotor activity were compared between wild-type (WT) mice and GAD65(Ϫ/Ϫ) mice. Effects of general anesthetics on both phasic and tonic GABAergic currents were examined using the patch-clamp method in frontal cortex pyramidal neurons in brain slices. The duration of propofol (100 mg/kg i.p.)-induced LORR and the duration of propofol (150 mg/kg i.p.)-induced LTWR in GAD65(Ϫ/Ϫ) mice were significantly reduced compared with WT mice. In contrast, no difference was seen for ketamine. Preinjection of the GABA transporter 1 inhibitor, NO-711 (C 21 H 22 N 2 O 3 ⅐ HCl) (0.75 mg/kg i.p.), reinstated diminished actions of propofol in GAD65(Ϫ/Ϫ) mice. Cortical pyramidal neurons in GAD65(Ϫ/Ϫ) mice had smaller tonic conductances, and propofol-induced enhancement of tonic inhibition was smaller than in WT mice, suggesting that genotype differences in GAD65-mediated GABAergic inhibitory tone may be, at least in part, a cellular basis underlying behavioral differences. In conclusion, GAD65(Ϫ/Ϫ) mice show a diminished response to propofol, but not ketamine, indicating that GAD65-mediated GABA synthesis plays an important role in hypnotic and immobilizing actions of propofol.