Age-dependent changes in the susceptibility to thiopental anesthesia in mice: Analysis of the relationship to the functional expression of GABA transporter
Highlights
► Thiopental-induced anesthesia is more pronounced in younger mice. ► We examine the contribution of GABA transporter (GAT) to thiopental anesthesia. ► GAT inhibitors enhanced thiopental anesthesia in mice age-dependently. ► The ability of GAT inhibitor to inhibit GABA uptake increased developmentally. ► The mechanism explains the age-dependent contribution of GAT.
Introduction
The potency of various anesthetics changes during development. Susceptibility to barbiturates, used therapeutically for sedation and hypnosis, is higher in childhood, as documented experimentally in young animals (Jondorf et al., 1958, Halevy and Orkin, 1974). Changes in metabolism in the liver during development have been considered to be involved in such susceptibility (Jondorf et al., 1958, Kato et al., 1964). However, metabolism cannot fully explain such developmental changes in susceptibility, since recovery from anesthesia induced by barbiturates results from a redistribution from the brain before metabolism in the liver (Burch and Stanski, 1983).
Although the mechanisms underlying anesthesia have not been fully elucidated, it has been demonstrated that a primary inhibitory neurotransmitter, γ-aminobutyric acid (GABA), is involved in the actions of anesthetics (Rudolph and Antkowiak, 2004, Franks, 2006, Muller et al., 2011). Numerous attempts have been made to determine how the GABAergic system participates in anesthesia, providing evidence that various classes of anesthetic and hypnotic agents including volatile anesthetics, barbiturates, and benzodiazepines act on the GABAA receptor to enhance GABAergic inhibition of neuronal activity in the mammalian nervous system. Therefore, changes in the GABAergic system during development may contribute to the age-dependent susceptibility to barbiturates. Indeed, ontogenic changes in the GABAergic system including GABAA receptor expression have been demonstrated (Coyle and Enna, 1976).
Regulation of the GABAergic system can occur through not only postsynaptic GABAA receptor function but also presynaptic events including GABA synthesis, storage and release (Iversen et al., 2009). The reuptake of GABA by the GABA transporter (GAT) also plays a key role in regulating the synaptic clearance of GABA (Conti et al., 2004). We have previously reported that in a behavioral model in mice, GABA uptake inhibitors potentiated the anesthesia induced by the volatile anesthetics halothane and isoflurane (Sugimura et al., 2002), in association with findings that these anesthetics inhibited [3H]GABA uptake in COS-7 cells transfected with mouse GAT-1 cDNA and in rat brain synaptosomes (Sugimura et al., 2001). These results suggested GAT to be crucial to the anesthesia induced by halothane and isoflurane. On the other hand, we also observed that in younger mice the facilitatory effects of GABA uptake inhibitors were not consistent every time, and preliminary experiments suggested an age-dependent effect of the inhibitors on barbiturate-induced anesthesia (unpublished observation). Based on these results, we hypothesized that GAT could be a candidate for the site that contributes to developmental changes in the susceptibility to anesthesia induced by barbiturates and/or in the sensitivity to GABA uptake inhibitors.
In the present study, we used in vivo and in vitro systems to examine (1) whether changes in GAT function contribute to developmental differences in susceptibility to thiopental-induced anesthesia, (2) whether the potency of GAT inhibitors in facilitating thiopental-induced anesthesia depends on age, and if so (3) what mechanism are involved. It was concluded that GAT is unlikely to participate directly in the age-dependent changes in susceptibility to thiopental, while the efficacy of GABA uptake inhibitors in enhancing thiopental anesthesia is at least partly due to changes in the sensitivity of GAT to the inhibitors.
Section snippets
Chemicals
Drugs used were thiopental sodium salt (Ravonal®, Mitsubishi Tanabe Pharma Co., Osaka, Japan), SKF89976A hydrochloride (Tocris Bioscience, Bristol, UK), NO-711 hydrochloride (Sigma-Aldrich, St. Louis, MO, USA), and diazepam (Wako Pure Chemical Industries, Ltd., Osaka, Japan). [3H]GABA (0.87 TBq/mmol) was obtained from NEN Life Science Products, Inc. (Boston, MA, USA).
Animals
The present study was approved by the Committee of Research Facilities for Laboratory Animal Science in Okayama University, and
Age-dependent effects of thiopental anesthesia
Thiopental-induced anesthesia was examined in terms of onset and duration of LORR in 3-, 5- and 7-week-old ICR mice. Fig. 1A shows the dose–response relationship for the incidence of LORR. Three-week-old mice displayed a higher sensitivity to thiopental than 5- or 7-week-old mice. A dose-dependent increase in the duration but not onset of LORR induced by thiopental was observed at all ages (Fig. 1B, Table 2, and data not shown). Effects of thiopental on the duration of LORR were more pronounced
Discussion
Although numerous attempts have been made to clarify the involvement of the GABAergic system in the mechanisms of anesthesia (Rudolph and Antkowiak, 2004, Franks, 2006, Muller et al., 2011), its relevance to developmental changes in susceptibility to anesthetics has not been fully clarified. We focused on the involvement of GAT, a presynaptic GABA uptake system important for fine-tuning of GABA concentrations at synaptic clefts (Conti et al., 2004), in the actions of the intravenous anesthetic
Acknowledgments
The authors wish to thank B. Ko, R. Hara, T. Machida, Y. Kitagawa, H. Tanimura, and H. Nakama for their help in the initial part of this work. This study was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology (NS and SK).
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