The influence of manipulations to alter ambient GABA concentrations on the hypnotic and immobilizing actions produced by sevoflurane, propofol, and midazolam
Highlights
► Ambient GABA is an important determinant of responses to propofol and midazolam. ► Ambient GABA minimally alters behavioral responses to sevoflurane. ► Sevoflurane (0.23 mM) minimally alters discharge properties of cortical neurons.
Introduction
The GABAergic system in the central nervous system (CNS) is a key target of general anesthetics (Mihic et al., 1997, Sonner et al., 2003, Rudolph and Antkowiak, 2004, Hemmings et al., 2005, Franks, 2006). Two types of GABAergic inhibition are known; a phasic form (phasic inhibition) regulating neural excitability via the activation of postsynaptic GABAA receptors by intermittent GABA release from presynaptic terminals, and a persistent tonic form (tonic inhibition) generated by continuous activation of extrasynaptic GABAA receptors by low concentrations of ambient GABA (Brickley et al., 1996). Growing evidence suggests that tonic inhibition mediated by extrasynaptic GABAA receptors might contribute to the actions of intravenous anesthetics such as propofol (Bai et al., 2001, Bieda and MacIver, 2004). These extrasynaptic GABAA receptors have different pharmacological and kinetic properties compared with synaptic GABAA receptors, as a result of the distinct subunit compositions (Glykys and Mody, 2007). Given that extrasynaptic GABAA receptors respond to low ambient levels of GABA, manipulations of ambient GABA concentrations may affect cellular and behavioral responses to general anesthetics.
Two manipulations studied were 1) the genetic absence of glutamate decarboxylase (GAD) 65 gene (GAD65−/−), and 2) the pharmacological manipulation of GABA uptake using GABA transporter inhibitor. GAD is the only synthetic enzyme responsible for the conversion of l-glutamic acid to GABA. The brain contains two forms of GAD, which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular locations, and interaction with the GAD cofactor pyridoxal phosphate (Erlander et al., 1991). The 67-KDa isoform (GAD67) is found mainly in the cell body, whereas GAD65 is localized to the nerve terminal and is reversibly bound to the membrane of synaptic vesicles (Namchuk et al., 1997). GAD65−/− mice remain viable without apparent anatomical deficits and postsynaptic GABAA receptor density is unchanged (Kash et al., 1997), although the survival rate of GAD65−/− mice was slightly reduced with age, largely due to spontaneous seizures (Stork et al., 2000). As a result of reduced GABAergic tone, GAD65−/− mice appear to show increased anxiety levels (Kash et al., 1999, Kubo et al., 2009a), different sensitivity to pentobarbital (Kash et al., 1999), and hyperalgesia to thermal, but not chemical, stimulation (Kubo et al., 2009b). On the other hand, inhibition of GABA uptake and/or metabolism is a strategy for enhancing ambient GABA concentrations. GABA is cleared from the synaptic cleft by specific, high-affinity, sodium- and chloride-dependent transporters, which are thought to be located on presynaptic terminals and surrounding glial cells, i.e., four distinct GABA transporters, GAT-1, GAT-2, GAT-3 and BGT-1 (Borden, 1996). NO-711, a potent and selective GAT-1 inhibitor, was used because GAT-1 is responsible for the majority of neuronal GABA transport.
We have reported that sevoflurane enhances GABAergic inhibition (Nishikawa and MacIver, 2001, Nishikawa and Harrison, 2003, Nishikawa et al., 2005), suggesting that GABAA receptor is one of the plausible molecular targets. In addition, several targets have been also proposed for inhalational general anesthetics; glycine receptors (Mascia et al., 1996), two-pore-domain potassium channels (Sirois et al., 2000), NMDA receptors (Sonner et al., 2003), HCN channels (Chen et al., 2005), and some subtypes of sodium channels (Wu et al., 2004), whereas a specific point mutation in GABAA receptor is critical for propofol and etomidate (Jurd et al., 2003). These data suggest that the relative contributions of GABAergic inhibition to in vivo anesthetic actions are different between sevoflurane and intravenous anesthetics. We first tested the hypothesis that genetic and pharmacological manipulations to alter ambient GABA concentrations would affect loss of righting reflex (LORR), a surrogate measure of hypnosis, and loss of tail-pinch withdrawal reflex (LTWR), a measure of immobilization, produced by sevoflurane, propofol, and midazolam. We then studied the influence of these manipulations on in vitro sevoflurane actions on membrane properties of frontal cortical layer V neurons using patch-clamp methods. The present study provides evidence that genetic and pharmacological manipulations to alter ambient GABA concentrations (tonic conductance) affect the response to propofol and midazolam, but minimally affect the actions of sevoflurane.
Section snippets
Mice
All animal procedures and protocols used in this study were approved by the Animal Care Committee of Gunma University Graduate School of Medicine (protocol # 05-71) and performed through NIH guidelines for the care and use of laboratory animals. All efforts were made to minimize animal suffering and to reduce the number of animals used.
The generation of glutamate decarboxylase 65 (GAD65) knockout mice used in the present study was described by Yanagawa et al. (1999) and Yamamoto et al. (2004).
GABA levels in the brain and the spinal cord are reduced in GAD65−/− mice
We measured the GABA content in the whole brain and spinal cord in GAD65−/− mice at 12–16-weeks old. GAD65−/− mice showed a significant reduction in GABA levels in the brain (76.7% of WT, P < 0.001, n = 6 each, Fig. 1A) and the spinal cord (68.5% of WT, P < 0.001, n = 5 for WT and n = 6 for GAD65−/− mice, Fig. 1B). Although a compensatory mechanism involving the balance between inhibitory and glutamatergic excitatory neurotransmission might have been expected, the difference in glutamate and
Discussion
GAD65−/− mice appeared to show normal sensitivity to sevoflurane despite a 20–30% reduction in GABAergic inhibitory tone, whereas they showed reduced sensitivity to propofol and midazolam. In contrast, enhanced GABA concentrations by NO-711 prolonged the duration of LORR and LTWR by propofol and midazolam, but not sevoflurane. Sevoflurane enhanced tonic inhibition of layer V cortical neurons; however, these effects were not strong enough to alter discharge properties of cortical neurons. These
Conclusions
The present study provides in vivo evidence that genetic and pharmacological manipulations to alter ambient GABA concentrations have significant effects on the hypnotic and immobilizing actions of propofol and midazolam, whereas these manipulations minimally alter cellular and behavioral responses to sevoflurane.
Acknowledgments
This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to K.N. [#20390412], K.K. [#21890032], and Y.Y. [#20019010, 22300105]. This work was also supported by a Grant-in-Aid from the Japan Medical Association, Tokyo, Japan to K.N., JST, CREST and Takeda Science Foundation to Y.Y. The authors thank the staff at Institute of Experimental Animal Research, Gunma University Graduate School of Medicine, for
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