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3-Containing GABAA Receptors Limits the Immobilizing Actions of GABAergic AnestheticsExperimental Anesthesiology Section, Department of Anesthesiology, Eberhard-Karls-University, Tuebingen, Germany (C.G., B.A.); RJ Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, California (R.J.); Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland (U.R.); and Laboratory of Genetic Neuropharmacology, McLean Hospital, Department of Psychiatry, Harvard Medical School, Belmont, Massachusetts (U.R.)
Received May 1, 2007; accepted June 21, 2007
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Abstract |
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3(N265M) knock-in mice. In the wild type, etomidate prolonged decay times of GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) and concomitantly reduced the frequency of action potential-dependent IPSCs. Etomidate prolonged the decay time of GABAA receptors at all tested concentrations. At concentrations greater than 1.0 µM, anesthetic-induced decrease of GABA release via modulation of presynaptic GABAA receptors and enhancement of postsynaptic GABAA receptor-function compensated for each other. The results suggest that the limited immobilizing efficacy of these agents is probably due to a presynaptic mechanism and that GABAergic agents with a specificity for post-versus presynaptic receptors would probably have much stronger immobilizing actions, pointing out novel avenues for drug development.
; Smith and Thwaites, 1999; Watson and Shah, 2000; Rudolph and Antkowiak, 2004; Grasshoff et al., 2006a). In clinical practice, these agents are routinely used for providing hypnosis and amnesia but rarely are administered to achieve immobility. This difference in efficacy reported in vivo was also observed in neuronal networks from spinal ventral horn interneurons in vitro (Grasshoff and Antkowiak, 2004). Ventral horn interneurons control the timing and excitability of motoneurons, the output structure of the spinal cord (Kiehn, 2006). Immobility, defined as the anesthetic-induced ablation of spontaneous and evoked movements in response to a noxious stimulus, is primarily mediated by molecular targets in the spinal cord (Collins et al., 1995; Campagna et al., 2003). However, the reasons for the low effectiveness of GABAergic anesthetics in producing immobility are not known.
To explain the low immobilizing capacity of intravenous GABAergic anesthetics, we hypothesized that besides depressing neuronal excitability in the ventral horn of the spinal cord, these agents concomitantly reduce GABA release. Because anesthetics acting predominantly by modulation of GABAA receptor function require GABA to be present at its binding site (Orser et al., 1994; Banks and Pearce, 1999), a decrease in GABA release can be expected to abate the efficacy of such therapeutics in reducing neuronal excitability. To answer the question of whether this mechanism accounts for the limited immobilizing properties of GABAergic anesthetics, the effects of etomidate on neuronal network activity were investigated in organotypic spinal slices. We quantified the impact of the anesthetic on the time course of GABAA receptor-mediated synaptic events, on GABA release onto neurons in the ventral horn, and on the activity of the intact spinal network. Previous work on 3(N265M) knock-in mice has demonstrated that etomidate causes immobility measured as the loss of hind limb withdrawal reflexes predominantly via GABAA receptors harboring 3 protein subunits (Jurd et al., 2003; Rudolph and Antkowiak, 2004). To find out which of the effects of etomidate examined in spinal slices involves these receptor subtypes, recordings were carried out not only in slices derived from wild type but also in organotypic spinal slices prepared from 3(N265M) mutant mice. Our results suggest that intravenous GABAergic anesthetics like etomidate depress excitatory and GABAergic interneurons in the ventral horn of the spinal cord. The modulation of presynaptic 3-containing GABAA receptors reduces the release of GABA and limits thereby the efficacy of etomidate to cause profound overall inhibition in the spinal cord.
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Materials and Methods |
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3 subunit (N265M) on the same genetic background (Jurd et al., 2003). All procedures were approved by the animal care committee (Eberhard-Karls-University, Tuebingen, Germany) and were in accordance with the German law on animal experimentation.
Spinal Slice Cultures. Preparation of spinal cord slices from embryos obtained from either pregnant wild-type or mutant mice (E 15) was performed according to the method originally described by Braschler and coworkers (1989) and as reported previously (Grasshoff and Antkowiak, 2004). In brief, embryos were rapidly decapitated and placed in ice-cold Gey's balanced salt solution consisting of 1.5 mM CaCl2, 5 mM KCl, 0.22 mM KH2PO4, 11 mM MgCl2, 0.3 mM MgSO4, 137 mM NaCl, 0.7 mM NaHCO3, and 33 mM D-glucose (all from Sigma, Taufkirchen, Germany). Spinal columns were freed from inner organs and limbs and were cut transversely into 300 µm thick slices using a vibratome. The slices were placed on a coverslip and embedded in a plasma clot consisting of 13 µl of heparin-treated chicken plasma (Sigma) and coagulated by 13 µl of a thrombin solution (Sigma). The coverslips were inserted into plastic tubes containing 0.75 ml of nutrient fluid including 10 nM neuronal growth factor (Sigma) and initially incubated in an atmosphere of 95% oxygen/5% carbon dioxide at 36.0°C for 1 to 2 h. Nutrient fluid (100 ml) consisted of 25 ml of horse serum (Invitrogen, Karlsruhe, Germany), 25 ml of Hanks' balanced salt solution (Sigma), and 50 ml of basal medium Eagle (Sigma). The roller tube technique described by Gähwiler (1981) was used to maintain the tissue. After 1 day in culture, antimitotics (10 µM 5-fluoro-2-deoxyuridine, 10 µM cytosine--D-arabino-furanoside, and 10 µM uridine; all from Sigma) were added to reduce proliferation of glial cells. The slices were used after 14 days in vitro for whole-cell patch-clamp and extracellular recordings.
Extracellular Recordings. The spinal cord slices were continuously perfused with an artificial cerebrospinal fluid (ACSF) consisting of 120 mM NaCl, 3.3 mM KCl, 1.13 mM NaH2PO4, 26 mM NaHCO3, 1.8 mM CaCl2, and 11 mM D-glucose. The ACSF was bubbled with 95% oxygen/5% carbon dioxide. Glass electrodes filled with ACSF (resistance, approximately 2–5 M
) were used for recording extracellular signals, which were bandpass-filtered (passband 200-5000 Hz) to isolate action potential activity. The electrodes were advanced into the tissue until extracellular single or multiunit spike activity (usually exceeding 100 µV in amplitude) could be clearly identified. Signals were digitized on a personal computer at 10 kHz via a Digidata 1200 analog-to-digital/digital-to-analog interface and Axoscope (Molecular Devices, Sunnyvale, CA).
Whole-Cell Voltage-Clamp Recordings. Whole-cell voltage-clamp recordings were performed on visually identified ventral horn interneurons at room temperature (20–24°C). The cultures were perfused with ACSF as specified above, with the addition of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 µM), DL-2-amino-5-phosphonopentanoic acid (APV, 50 µM), and 1 µM strychnine (all from Sigma). Signals were acquired with an EPC 7 amplifier (List Medical, Darmstadt, Germany), low pass-filtered at 3 kHz, and digitized on a personal computer at 10 kHz via a Digidata 1200 interface and Clampex (Axon Instruments). Patch pipettes were pulled from borosilicate capillaries (1.5 mm outside diameter) and coated with Sylgard. Pipette resistances were between 1.5 and 2.5 M after fire-polishing. The pipette solution contained 121 mM CsCl, 24 mM CsOH, 10 mM HEPES, 5 mM EGTA, 1 mM MgCl2, and 2 mM ATP, adjusted to pH 7.3 with 1 M NaOH (all from Sigma).
Preparation and Application of Test Solutions. Test solutions were prepared by dissolving etomidate (Etomidat-Lipuro; Braun, Melsungen, Germany) in ACSF equilibrated with 95% oxygen/5% carbon dioxide. Etomidate was administered via the bath perfusion using gas-tight syringe pumps (ZAK Medicine Technique, Marktheidenfeld, Germany), which were connected to the experimental chamber via Teflon tubing (Lee, Frankfurt, Germany). The flow rate was approximately 1 ml/min. To ensure steady-state conditions, recordings during anesthetic treatment were carried out 10 to 15 min after starting the perfusate change.
Data Analysis. Data were analyzed with in-house software written in OriginPro version 7 (OriginLab Corp, Northampton, MA) and MATLAB version 6.5 (The MathWorks Inc., Natick, MA). IPSC current decays were fitted with monoexponential functions. Data analysis of extracellular recordings was performed as described previously (Grasshoff and Antkowiak, 2004). After close inspection of the raw data, action potentials were detected by setting a threshold well above baseline noise. The mean firing rate was obtained from single or multiunit activity; it is defined as the number of detected action potentials divided by the recording time of 180 s. The natural firing mode of spinal ventral horn neurons in culture consisted of bursts of action potentials separated by silent periods. Comparative statistics were performed with a two-tailed Student's t test for paired data. Unless otherwise stated, results are given as mean ± S.E.M. Concentration-response curves were fitted by Hill equations, as described previously (Antkowiak and Helfrich, 1998). Estimated EC50 values were derived from these fits.
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3 Subunits on Etomidate Effects on Spinal Network Activity. It has been reported that the suppression of noxious-evoked movements in response to etomidate is completely abolished in mice harboring a point mutation (N265M) in the second transmembrane region of the 3 subunit of GABAA receptors (Jurd et al., 2003). Because the ablation of spontaneous or stimulus-induced movements by general anesthetics is known to be primarily spinally mediated (Collins et al., 1995; Campagna et al., 2003), this leads to the hypothesis that the depression of spinal neurons by etomidate is largely mediated by GABAA receptors containing a 3 subunit. We tested the hypothesis by comparing the effects of etomidate on spinal network activity in cultured slices from wild-type and 3(N265M) knock-in mice. Network activity was measured by extracellular recordings of ventral horn interneurons, which were visually identified in the slices. Concentration-dependent effects of etomidate on average discharge rates of spinal neurons are summarized in Fig. 1. Figure 1A displays original recordings under control conditions and in the presence of 0.75 µM etomidate measured in slices from wild-type and 3(N265M) mutant mice. In Fig. 1B, concentration-response relationships were fitted by Hill equations. In wild-type animals, the estimated EC50 value is 0.39 ± 0.04 µM and thereby within the relevant range for general anesthesia in rats (Dickinson et al., 2003) and swine (Johnson et al., 2003). It is remarkable that full depression of spontaneous activity could not be achieved. The concentration-response curve of etomidate showed an upper limit close to 60%. Etomidate displayed no depressant effects on network activity in organotypic slices from 3(N265M) mutant mice within the clinically relevant range. The deletion of etomidate's depressant effects by the 3(N265M) point mutation strongly supported our hypothesis that the depression of spinal neurons is predominantly mediated by GABAA receptors containing a 3 subunit. At higher concentrations, etomidate diminished network activity slightly in slices from the mutant mice (Fig. 1B) but was still less effective compared with the lowest concentration (0.25 µM) tested in the wild type.
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3 knock-in mice are shown in Fig. 3A. In slices obtained from wild-type mice, etomidate prolongs half-decay times and concurrently reduces the number and amplitudes of GABAergic sIPSCs. In slices from 3 knock-in mice, 1.5 and 2.5 µM etomidate induce only a minimal increase in half-decay times, whereas it did not affect amplitudes or frequencies of GABAergic sIPSCs. Figure 3B demonstrates a concentration-dependent increase of half-decay times resembling the effects of etomidate on mIPSCs. In contrast to the actions on mIPSCs, etomidate diminishes amplitudes and reduces frequencies of spontaneous GABAergic IPSCs. This result suggests that etomidate decreases the presynaptic release of GABA.
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3 subunit by comparing the effects of 1.5 and 2.5 µM etomidate on sIPSCs in cultured spinal slices of wild-type and 3 knock-in mice. Figure 3C summarizes the results from the corresponding experiments. Although 2.5 µM etomidate induces a 3-fold prolongation of the half-decay time of GABAergic IPSCs in the wild type, it exerts only a negligible prolonging effect in the mutant, consistent with the hypothesis that GABAA receptors mediating postsynaptic effects of etomidate contain a 3 subunit. Regarding presynaptic effects of etomidate, 2.5 µM etomidate neither reduces amplitudes of GABAergic sIPSCs nor depresses the IPSC rate in the mutant, whereas the anesthetic at a concentration of 2.5 µM diminished the amplitude by half and exerted a 60% depression of IPSC rate in the wild type. From these results, we deduce that not only postsynaptic but also presynaptic GABAA receptors mediating spinal effects of etomidate incorporate a 3 subunit. Effects of Etomidate on Total Charge Transfer of GABAergic sIPSCs in Ventral Horn Interneurons. The results so far indicate that the limited efficacy of etomidate in depressing spinal neurons can be explained by concomitant effects on pre- and postsynaptic GABAA receptors exerting opposing effects on network activity. Although the prolonging of half-decay times by etomidate increases the charge transfer, the reduction of IPSC amplitudes and IPSC rates decreases the impact of etomidate on charge transfer. Assuming that the changes in total charge transfer of GABAA receptors reflect the effects of etomidate on the network level, we expect a limitation in total charge transfer at concentrations greater than 1.5 µM. The increase in total charge transfer of spontaneous GABAergic IPSCs was calculated as the product of the integral of averaged spontaneous IPSCs with the frequency of the events. Figure 4 displays the effects of etomidate on the total charge transfer. The curve demonstrates a concentration-dependent increase in total charge transfer and a ceiling starting at a concentration of 1.5 µM etomidate, thereby mirroring the run of the concentration-response curve measured from extracellular recordings of spinal network activity as depicted in Fig. 1.
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Discussion |
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3(N265M) mutant mice. The striking result is that the effects of etomidate on action potential activity of ventral horn neurons saturated at concentrations exceeding 1.0 µM, which are known to cause immobility. As displayed in Fig. 1, the concentration-response curve plateaued to an uppermost limit of approximately 60% depression of the firing rate, thereby resembling the concentration-response curve of another GABAergic anesthetic, propofol, in the ventral horn of the spinal cord (Grasshoff and Antkowiak, 2004). In sharp contrast to intravenous GABAergic drugs, volatile anesthetics like sevoflurane, isoflurane, or enflurane do not display a limitation in their effectiveness in depressing ventral horn neurons (Grasshoff and Antkowiak, 2004, 2006). Compared with intravenous anesthetics, volatile anesthetics are known to affect, besides GABAA receptors, a multitude of molecular targets in the spinal cord (Cheng and Kendig, 2000, 2002, 2003; Wong et al., 2001; Campagna et al., 2003). Although the experimental conditions in vitro are different from the in vivo measurement of immobility, our results reflect closely those obtained from studies in humans in whom intravenous GABAergic anesthetics were far less effective in depressing involuntary movements compared with volatile anesthetics (Ashworth and Smith, 1998; Smith and Thwaites, 1999; Watson and Shah, 2000). However, in clinical study settings, immobility can also be achieved with intravenous anesthetics at concentrations approximately 5-fold higher than those required for hypnosis (Smith et al., 1994). At these high concentrations, intravenous anesthetics like propofol frequently require the application of ephedrine to keep the blood pressure stable. The capability of GABAergic anesthetics to produce immobility, despite their limited efficacy in reducing neuronal excitability in the spinal cord, can be explained by the involvement of supraspinal sites. It has been demonstrated previously that local administration of pentobarbital, another anesthetic acting predominantly via GABAA receptors (Zeller et al., 2007), into the mesopontine tegmental anesthesia locus can cause deep anesthesia (Devor and Zalkind, 2001; Sukhotinsky et al., 2005). This result, together with findings of the present study, allows the conclusion that the immobilizing properties of GABAergic anesthetics are mediated in part via the ventral horn of the spinal cord and, in part, via supraspinal structures, including the mesopontine tegmental anesthesia locus. In summary, these results lead to the conclusion that general anesthetics acting via positive modulation of GABAA receptors display a restricted capacity in depressing the excitability of neurons in the ventral horn of the spinal cord, leading to the question of which mechanism is responsible for this limitation. Depression of GABA Release Is Limiting the Efficacy of Etomidate in the Ventral Horn. Observing that GABAergic anesthetics display a limited efficacy in reducing spinal network activity, we hypothesized that besides depressing neuronal excitability, these agents simultaneously inhibit GABA release in the ventral horn of the spinal cord via presynaptic GABAA receptors. The results of the current study support this hypothesis because etomidate reduces action potential-dependent GABA release in a concentration-dependent manner (Fig. 3B). Anesthetic-induced depression of GABA release onto ventral horn neurons, caused by a presynaptic mechanism of action, is expected to increase the excitability of these cells. In contrast, anesthetic actions mediated via postsynaptic GABAA receptors decreases the excitability of ventral horn neurons. Thus, pre- and postsynaptic actions of etomidate affect neurons in the ventral horn in opposing ways. As demonstrated by the experiments on the total charge transfer the pre- and postsynaptic actions of etomidate largely balance out between 1 and 5 µM, thereby explaining the limited efficacy of etomidate in reducing spinal network activity.
Involvement of 3-Containing GABAA Receptors in the Actions of Etomidate. The ability of the intravenous anesthetics etomidate and propofol to modulate GABAA receptors is uniquely dependent on the identity of the subunit in the receptor complex (Siegwart et al., 2002; Jurd et al., 2003). Receptors containing 2 or 3 but not 1 subunits are highly susceptible to both anesthetics. Moreover, point mutations in the subunits have dramatic effects on the sensitivity of GABAA receptors to anesthetics (Belelli et al., 1997; Mihic et al., 1997) For example, the 3(N265M) mutation was shown to largely abolish the modulatory effects of etomidate at GABAA receptors (Siegwart et al., 2002). Subsequent work on 3(N265M) knock-in mice showed that the immobilizing properties of etomidate almost exclusively depend on 3 subunits (Jurd et al., 2003). It is most striking that etomidate failed to cause loss of hind limb withdrawal reflexes in 3 knock-in mice, even when delivered at very high concentrations close to the lethal dose. In contrast, a similar mutation in the 2 subunit did not alter the ability of etomidate to ablate motor reflexes (Reynolds et al., 2003).
In the present study, we characterized the effects of etomidate in spinal slices derived from wild-type and mutant mice. Within a range of concentrations commonly assumed to produce immobility in vivo (1.0–1.5 µM) (Dickinson et al., 2003), effects of etomidate at GABAA receptors and on action potential firing were almost completely abolished by the 3(N265M) mutation. Because the mutation also abolished the ability of etomidate to ablate the hind limb withdrawal reflex in knock-in mice (Jurd et al., 2003), our results clearly support the idea that the ventral horn of the spinal cord presents a major contribution to etomidate-induced immobility. In addition, the current results suggest that the majority of GABAA receptors in the ventral horn harbor 3 subunits, a finding that is quite different in the brain, where 3 subunits are present in only 20 to 30% of all GABAA receptors (Benke et al., 1994). As mentioned above, etomidate exerts opposing actions on spinal network activity via pre- and postsynaptic GABAA receptors in the ventral horn, raising the question of which of these two groups of receptors is able to be modulated by etomidate. Because not only the prolonging effect on IPSCs but also the reduction of the frequency of spontaneous IPSCs was completely abolished by the 3(N265M) mutation, it is clear that both functional groups of GABAA receptors express 3 subunits. Thus, intravenous GABAergic anesthetics like etomidate and propofol, acting via 3-containing GABAA receptors, act on both excitatory and inhibitory neurons in the ventral horn. This opposing effect therefore limits the effectiveness of etomidate to cause profound overall inhibition in the spinal cord and thus limits the immobilizing capability of this intravenous GABAergic anesthetic.
Future Prospects for the Design of Intravenous Anesthetics to Produce Immobility. In summary, the results provide one explanation of why intravenous GABAergic anesthetics are poor immobilizers. We demonstrate that etomidate has the ability to simultaneously depress not only excitatory neurons in the ventral horn of the spinal cord but also GABAergic interneurons, thus reducing the release of GABA in the same spinal region. Because general anesthetics potentiate the effect of GABA at GABAA receptors, they require the neurotransmitter to exert their depressant effects. This action counteracts the depressant effects on spinal network activity and causes thereby a limitation of the anesthetic efficacy. Furthermore, the experiments using 3(N265M) mutant mice revealed that the release of GABA in the ventral horn is regulated by GABAA receptors incorporating a 3 subunit. However, the localization of these functionally presynaptic receptors is unclear. They can be present either at the presynaptic terminal or at presynaptic inhibitory neurons. Apart from these presynaptic GABAA receptors, those mediating the depressant anesthetic effects possess a 3 subunit as well. Thus, GABAergic anesthetics in current clinical use act unspecifically at both pre- and postsynaptic receptors in the spinal cord. Immunohistochemical experiments have demonstrated that GABAA receptors located on spinal interneurons harbor different types of 
subunits (Bohlhalter et al., 1996). Electrophysiological experiments using cultured spinal slices from -knock-in mice revealed first evidence that these GABAA receptors are located on excitatory and inhibitory interneurons (Grasshoff et al., 2006b), opening the opportunity to develop subtype-selective agents that can be used clinically as potent muscle relaxants or immobilizers in the future.
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ABBREVIATIONS: ACSF, artificial cerebrospinal fluid, APV, DL-2-amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; IPSC, inhibitory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; sIPSC, spontaneous inhibitory postsynaptic current.
Address correspondence to: Dr. Christian Grasshoff, Experimental Anesthesiology Section, Department of Anesthesiology and Intensive Care, Eberhard-Karls-University, Schaffhausenstr. 113, D-72072 Tuebingen. E-mail: christian.grasshoff{at}uni-tuebingen.de
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) or 
). For each concentration, the mean value and standard error were obtained from 8 to 12 cells. The curve represents a Hill fit to the data (R2 = 0.966). The half-maximal effect (EC50) occurred at 0.39 ± 0.04 µM, and the maximal depression of action potential activity was 65.6 ± 2.5% in wild-type mice. The clinically relevant concentration range is marked by a gray vertical bar.
