MICE harboring an N265M point mutation in the β3
subunit of the γ-aminobutyric acid type A (GABAA
) receptor are useful tools for investigating the role of this subunit in general anesthesia.1,2
Recently, the effects of several volatile anesthetics have been examined in these mice to answer the question of whether the β3
subunit is involved in mediating amnesia, hypnosis, or immobility.3–5
Conclusions drawn from these behavioral studies are based on the assumption that, on the molecular level, the N265M mutation significantly attenuates the effects of volatile anesthetics at β3
receptors. However, experimental evidence supporting this assumption is limited.
Siegwart et al.6
reported that enflurane augments GABA-evoked currents in wild-type but significantly less in mutated α2
recombinant receptors expressed in human embryonic kidney 293 cells. In this work, effects of enflurane were evaluated in the presence of 3 μm GABA in wild-type and 30 μm GABA in mutant receptors corresponding to EC5
values. However, these findings do not predict how the mutation affects GABAA
receptor-mediated synaptic transmission. At intact central synapses, the GABA concentration within the synaptic cleft rises into the millimolar range.7
Furthermore, effects of volatile anesthetics at GABAA
receptors substantially depend on the concentration of GABA. In the case that this concentration is approximately 1 mm, volatile anesthetics not only enhance GABAA
receptor function but also induce receptor blockade.8,9
Hapfelmeier et al.10
and Neumahr et al.11
studied the effects of isoflurane and sevoflurane on currents evoked by 1 mm GABA at recombinant α1
receptors. Using an ultrafast application method, they discovered that anesthetic-induced blockade of GABAA
receptors results in a reduced peak current. At an intact central synapse, receptor blockade by volatile anesthetics decreases inhibitory postsynaptic current (IPSC) peak amplitudes, whereas potentiation of GABAA
receptor function manifests as a prolongation of current decays. The latter effect increases GABAA
receptor-mediated synaptic currents, whereas the former effect decreases synaptic currents. Because the potentiating action dominates, the overall effect of volatile anesthetics is enhancement of GABAA
receptor function. Banks and Pearce9
have carefully analyzed these dual actions. The concentration-response curves revealed a dissociation of the effects on the amplitude and time course of IPSCs, suggesting the involvement of distinct molecular mechanisms. This conclusion is corroborated by the recent finding that a point mutation in the α subunit of the GABAA
receptor abolishes the enhancing but not blocking effect of isoflurane in a recombinant system.12
Taken together, these observations raise the question of whether the β3
(N265M) mutation exclusively modulates the potentiating action or both the blocking and potentiating actions of volatile anesthetics. So far, experimental data demonstrating how volatile anesthetics modulate GABAergic synaptic transmission at intact synapses in preparations derived from β3
(N265M) mutant mice are absent. In the case that the mutation exclusively alters anesthetic-induced prolongation of IPSC decay times, while leaving the blocking action unchanged, the mutation should turn the overall potentiating effect of volatile anesthetics into a blocking one. This hypothetical mechanism would greatly complicate the interpretation of data originating from comparative behavioral studies on wild-type and β3
(N265M) mutant mice.3–5
In the current work, we first explore the effects of enflurane on GABAA
receptor-mediated IPSCs recorded from neurons in cultured neocortical brain slices, derived from wild-type and β3
(N265M) mutant mice. Enflurane was chosen for this investigation because its dual actions on GABAA
receptors have been characterized in great detail previously.9,13
In a previous study, the action of enflurane on spontaneous action potential firing of neocortical neurons in brain slices from wild-type and β3
(N265M) mutant mice has been examined only for one single small concentration.3
Therefore, here we compare the effects of this volatile anesthetic over a variety of concentrations to test whether β3
receptors are involved in the depressant effects of volatile anesthetics on neocortical neurons reported recently.14
Materials and Methods
Mice of both sexes homozygous for an asparagine to methionine point mutation at position 265 of the GABAA
subunit (N265M) and homozygous wild-type controls on the same genetic background as described previously (statistically 87.5% 129/SvJ, 12.5% 129/Sv) were used for this study.3
All procedures were approved by the animal care committee (Eberhard-Karls-University, Tuebingen, Germany) and were in accordance with German law on animal experimentation.
Organotypic Slice Cultures
Neocortical slice cultures were prepared from 2- to 5-day-old mice as described by Gähwiler et al.15,16
In brief, for the preparation of somatosensory cortex, animals were deeply anesthetized with halothane and decapitated. Cortical hemispheres were aseptically removed and stored in ice-cold Gey solution. After removal of the meninges, 300-μm-thick coronal slices were cut. Slices were transferred onto clean glass coverslips and embedded in a plasma clot. The coverslips were transferred into plastic tubes (Nunc, Roskilde, Denmark) containing 750 μl nutrition medium and incubated in a roller drum at 37°C. After 1 day in culture, antimitotics were added. The suspension and the antimitotics were renewed twice a week. Cultures were used after 2 weeks in vitro
Preparation and Application of Test Solutions
Test solutions were prepared by dissolving enflurane in artificial cerebrospinal fluid (ACSF) to yield the desired concentration as described previously.13
A closed, air-free system was used to prevent evaporation of the anesthetic.
Enflurane (Abbott, Wiesbaden, Germany) was applied via bath perfusion using syringe pumps (ZAK, Marktheidenfeld, Germany), connected to the experimental chamber via Teflon tubing (Lee, Frankfurt, Germany). The flow rate was approximately 1 ml/min. When switching from ACSF to drug-containing solutions, the medium in the experimental chamber was replaced by at least 95% within 2 min. Effects of the anesthetic were stable approximately 5 min later. To ensure steady state conditions, recordings during anesthetic treatment were performed 10 min after commencing the change of the perfusate.
Extracellular network recordings were performed in a recording chamber mounted on an inverted microscope. Slices were perfused with ACSF consisting of 120 mm NaCl, 3.3 mm KCl, 1.13 mm NaH2PO4, 26 mm NaHCO3, 1.8 mm CaCl2, and 11 mm glucose. ACSF was bubbled with 95% oxygen and 5% carbon dioxide. ACSF-filled glass electrodes with a resistance of approximately 3–5 MΩ were positioned on the surface of the slices and advanced into the tissue until extracellular spikes exceeding 100 μV in amplitude were visible. All experiments were conducted at 34°–36°C. Data were acquired on a personal computer with the Digidata 1200 AD/DA interface and Axoscope 9 software (Axon Instruments, Foster City, CA).
Whole cell patch clamp recordings were performed with an EPC 7 amplifier (List, Darmstadt, Germany) at room temperature. Extracellular medium consisted of 120 mm NaCl, 3.3 mm KCl, 1.13 mm NaH2PO4, 1 mm MgCl2, 26 mm NaHCO3, 1.8 mm CaCl2, and 11 mm glucose. To block glutamatergic currents, 6-cyano-7nitroquinoxaline-2,3-dione and dl-2-amino-5-phosphonopentanoic acid (50 μm each) were added to the extracellular medium. Cells in neocortical slices were identified as pyramidal neurons according to their morphologic appearance on a television monitor using infrared illumination and a 40× water immersion objective. Patch pipettes were fabricated from borosilicate glass using a P-2000 laser puller (Sutter Instruments, Novato, CA), fire polished, and coated with Sylgard (Dow Corning, Seneffe, Belgium) to reduce electrode capacitance. When filled with recording solution containing 145 mm CsCl, 1 mm MgCl2, 5 mm EGTA, 10 mm HEPES, and 4 mm ATP at pH 7.2, patch pipettes had a resistance of 1.5–3.5 MΩ. Neurons were held at −70 mV. IPSCs were sampled at 10 kHz with the Digidata 1200 AD/DA interface and Clampex 9.0 software (Axon Instruments) for a recording period of 180 s.
Extracellular recorded spikes were counted offline using custom routines in OriginPro7 (OriginLab Corporation, Northampton, MA). The mean of spikes occurring during a recording period of 180 s was used as the average spike rate.
Equation (Uncited)Image Tools
Inhibitory postsynaptic current data were analyzed off-line using self-written programs in OriginPro7. Spontaneous events were counted using an automated event detection algorithm. Events were discarded if the next event occurred during the decay tail (“stacked” events). Decay phases were best fit by two exponential components, using a biexponential equation in the form
where I(t) is the current amplitude at any given time t; c is the baseline current; τfast and τslow are the fast and slow time constants of current decay, respectively; and Af and As are the estimated fast and slow intercepts of the components at time zero.17
Drug-induced changes in the net charge transferred during GABAA
receptor-mediated synaptic events were estimated by calculating the area under the curve of averaged IPSCs. Furthermore, the frequency and amplitudes of IPSCs were evaluated in the absence and presence of the anesthetic.
We used the Student t test for statistical testing. P values less than 0.05 were considered significant. All results are given as mean ± SEM.
Spontaneous Inhibitory Postsynaptic Currents from Wild-type and β3(N265M) Mutant Mice Do Not Differ under Control Conditions
We sampled spontaneous IPSCs from pyramidal cells in cultured brain slices derived from the somatosensory cortex of wild-type and β3
(N265M) mutant mice by performing whole cell patch clamp recordings in the voltage clamp mode. Spontaneous IPSCs were characterized by frequency of occurrence, amplitude, the rapid and slow phases of current decay, and net charge transfer. IPSC characteristics as obtained in the absence of enflurane are summarized in figure 1
. The frequency of occurrence was 2.01 ± 0.14 Hz (n = 44) in the wild-type slices and 1.91 ± 0.16 Hz (n = 33) in the β3
(N265M) mutant slices. These mean values were not statistically different. Similarly, peak amplitudes of averaged IPSCs did not differ between slices from wild-type (61.34 ± 3.25 pA, n = 43) and β3
(N265M) mutant mice (60.15 ± 3.79 pA, n = 32). Current decays were fitted by two time constants (τfast and τslow) using a biexponential model. The fast component of the decay time was 10.81 ± 0.63 ms (n = 41, 73.5 ± 2.2% of amplitude) in the wild-type and 10.17 ± 0.60 ms (n = 33, 75.7 ± 3.4% of amplitude) in β3
(N265M) mutant slices. Regarding the slow phase of the current decay, the β3
(N265M) mutant showed smaller values (30.92 ± 2.55 ms, n = 32) than the wild type (37.65 ± 2.76 ms, n = 42). However, this difference did not reach statistical significance. Previous investigators have reported similar values for neocortical pyramidal neurons.18
Finally, we calculated the area under the curve as a measure of charge transfer. These values were also not different between wild-type (1.28 ± 0.09 pC, n = 41) and β3
(N265M) mutant (1.20 ± 0.16 pC, n = 34) mice. In summary, IPSC parameters did not differ between wild-type and β3
(N265M) mutant mice in the absence of enflurane.
Different Actions of Enflurane on Spontaneous Inhibitory Postsynaptic Currents in Wild-type and β3(N265M) Mutant Neurons at Concentrations Below 1 MAC
Representative recordings of spontaneous IPSCs in slices from wild-type mice, performed in the absence and presence of enflurane (0.6 mm), are shown in figure 2A
. The anesthetic reduced IPSC amplitudes and prolonged current decays. In figures 2B–D
, averaged IPSCs are displayed. The current decay was prolonged by 0.3 mm enflurane, and the amplitude was slightly decreased (fig. 2C
). Both effects were stronger in the presence of 0.6 mm enflurane (fig. 2D
In figure 2E
, representative traces obtained from experiments on β3
(N265M) mutant slices are presented. Enflurane at 0.3 mm did not attenuate the amplitude of the averaged IPSC, and the current decay was only marginally prolonged (figs. 2F–G
). The latter effect was enhanced at 0.6 mm enflurane but still less pronounced compared with the IPSCs monitored in slices from wild-type mice (fig. 2H
). Even at this high concentration, enflurane did not reduce the amplitude of the mean IPSC in the β3
summarizes the effects of enflurane on IPSCs monitored in wild-type and β3
(N265M) mutant slices. The effects of the anesthetic on the amplitudes of averaged IPSCs are displayed in figure 3A
: At 0.3 mm, enflurane did not change the IPSC amplitudes significantly, but there was a trend to smaller values in the wild type (50.08 ± 3.97 pA, n = 14) compared with mutant (63.72 ± 6.24 pA, n = 9). At 0.6 mm enflurane, however, the amplitude of the averaged IPSC was significantly reduced in the wild type (37.15 ± 2.64 pA, n = 13), whereas no such effect could be observed in the β3
(N265M) mutant (66.59 ± 6.35 pA, n = 7). Figure 3B
shows the actions of enflurane on the rapid phase of the current decay (τfast). This parameter was prolonged by enflurane in a concentration dependent manner in the wild type (0.3 mm enflurane 17.42 ± 1.07 ms, n = 14; 0.6 mm enflurane 23.49 ± 2.26 ms, n = 12) but not in the β3
(N265M) mutant (0.3 mm enflurane 9.59 ± 0.42 ms, n = 8; 0.6 mm enflurane 12.36 ± 0.96 ms, n = 8). Prolongation of τfast in the wild-type was significantly different from the β3
(N265M) mutant and from control conditions. The corresponding effects of enflurane on the slow phase of the current decay (τslow) are presented in figure 3C
. Again, enflurane led to longer decay times in the wild type but not in the β3
(N265M) mutant. In the wild type, enflurane (0.3 mm) increased τslow to 66.14 ± 3.87 ms (n = 14), an effect that was further enhanced at 0.6 mm enflurane to 117.45 ± 12.86 ms (n = 12). In contrast, in the β3
(N265M) mutant, there was only a minor prolongation to 37.16 ± 4.66 ms (n = 8) at 0.3 mm enflurane and to 35.74 ± 3.74 ms (n = 8) at 0.6 mm. Again, the effect of enflurane on the wild-type preparation was significantly different from β3
(N265M) mutant preparations and from control conditions.
To draw conclusions about the overall effect of enflurane on IPSCs, we calculated the net charge transferred by averaged IPSCs (fig. 3D
). In wild-type preparations, this parameter was increased by enflurane in a concentration-dependent manner (0.3 mm enflurane 2.05 ± 0.15 pC, n = 13; 0.6 mm enflurane 2.62 ± 0.12 pC, n = 12). In the β3
(N265M) mutant, however, only a small and nonsignificant effect was observed (0.3 mm enflurane 1.06 ± 0.11 pC, n = 9; 0.6 mm enflurane 1.48 ± 0.14 pC, n = 7). The net charge transferred during IPSCs as determined in the presence of enflurane in wild-type slices was significantly different from control conditions as well as from the charge transferred in β3
(N265M) mutant slices in the presence of the anesthetic.
The frequency of spontaneous IPSCs was decreased by enflurane in a concentration-dependent manner in both preparations (table 1
), but no significant difference was observed between wild-type and β3
(N265M) mutant slices.
At Sedative and Hypnotic Concentrations Enflurane Exerts Stronger Depression of Spontaneous Action Potential Firing in Wild-type Than in β3(N265M) Mutant Neocortical Slices
The effects of enflurane on spontaneous action potential firing of neocortical neurons were investigated. As in our previous studies, spontaneous neuronal activity was reinforced by removing Mg2+
ions from the extracellular solution.19
Under control conditions, action potential firing occurred typically in bursts (episode of ongoing activity). In slices from wild-type mice, episodes of ongoing activity showed a frequency of 0.16 ± 0.01 Hz (n = 51). In β3
(N265M) mutant cultures, this frequency was 0.15 ± 0.01 Hz (n = 53). These mean values were not statistically different and lie in the range of previously reported values.19
Mean action potential rates were slightly higher in slices from wild-type than in β3
(N265M) mutant preparations (13.62 ± 2.03 vs.
9.93 ± 0.86 Hz, n = 65/64), but this did not reach statistical significance. In summary, activity patterns monitored in slices from wild-type and β3
(N265M) mutant mice did not differ under control conditions.
Enflurane depressed spontaneous action potential firing in a concentration-dependent manner in cultures from both wild-type and β3
(N265M) mutant mice (fig. 4
). At concentrations of 0.2 and 0.4 mm, enflurane depressed action potential firing in the wild type to a significantly greater extent than in the β3
(N265M) mutant (0.2 mm enflurane: WT 37.75 ± 4.06%, n = 25, MU 13.45 ± 10.4%, n = 18; 0.4 mm enflurane: WT 55.53 ± 4.08%, n = 20, MU 19.44 ± 8.13%, n = 15). However, at higher concentrations, enflurane was similarly effective in slices from wild-type and β3
(N265M) mutant mice.
Characteristics of Spontaneous IPSCs under Drug-free Conditions
In recombinant α2
receptors expressed in human embryonic kidney 293 cells, the β3
(N265M) mutation may alter channel gating, agonist binding, or both. Support for the latter conclusion is derived from the finding that EC50
values for activation of α2
receptors by GABA were 47 μm in wild-type and 122 μm in α2
This prompts the question of whether the change in GABA efficacy, introduced by the mutation, is mirrored in the time course of IPSCs. In our study, IPSCs from wild-type and β3
(N265M) mutant mice did not differ under drug-free conditions regarding frequency, amplitude, current decay, and charge transfer (fig. 1
). This observation is based on 44 recordings in neocortical slices prepared from wild-type and 33 recordings in slices from β3
(N265M) mutant mice. By monitoring from a single neuron, 200–300 IPSCs were sampled and used for further analysis. The finding that under drug-free conditions IPSCs did not differ in slices from wild-type and mutant mice implies that GABAA
receptors involved in these IPSCs were exposed to almost saturating agonist concentrations.7
At first glance, this explanation seems to be at variance with the data published by Liao et al.4
These authors report that in the absence of anesthetics, β3
(N265M) mutant mice display less freezing in a pavlovian fear conditioning paradigm compared with wild-type mice. One of a number of possibilities to explain this difference is that extrasynaptic GABAA
receptors in the hippocampus, cerebellum, or neocortex may be playing an important role in such a behavioral outcome. Because of their location, these extrasynaptic receptors are exposed to GABA concentrations much lower than in the synaptic cleft,7,20
possibly close to the EC50
values cited above.
Actions of Enflurane on Spontaneous IPSCs
When recording miniature IPSCs from hippocampal pyramidal cells, Banks and Pearce9
observed that at concentrations close to 1 MAC-immobility (0.58 mm) enflurane depressed IPSC amplitudes by 40% and increased the time constant of current decay by 3.5-fold. These findings are consistent with results obtained with cerebellar Purkinje cells, which express almost exclusively GABAA
receptors composed of α1
In the latter study, enflurane (0.64 mm) attenuated amplitudes of miniature and spontaneous IPSCs by 55% and increased current decay times by 5.1-fold. This indicates that these GABAA
receptors are also sensitive to enflurane and that the blocking effects are not subtype specific.
In the current work, effects of enflurane on spontaneous IPSCs recorded from neocortical neurons in brain slices prepared from wild-type mice were comparable with the results mentioned above. At 0.6 mm, IPSC amplitudes were decreased by 39%, and decay times were prolonged by 117% (τfast) and 211% (τslow), respectively.
How is this blocking and potentiating action of enflurane altered by the β3
(N265M) mutation? The results summarized in figure 3
indicate that the mutation eliminates both effects. This stands in contrast to the S270H mutation, which was introduced in the α1
subunit of recombinant α1
receptors, which exclusively affects the potentiating action of the volatile anesthetic isoflurane, leaving anesthetic-induced receptor blockade unaltered.12
Because the β3
(N265M) mutation abolished both (i.e.
, the prolongation of current decay and the blocking action of enflurane), the different effects of the anesthetic on spontaneous action potential activity monitored in slices from wild-type and β3
(N265M) mutant mice can be explained as follows: The weaker depression by enflurane in the β3
(N265M) mutant at concentrations between MAC-awake and MAC-immobility (fig. 4
) does not involve blockade of mutated GABAA
receptors but is produced by the lack of an effect of enflurane on mutated β3
receptors in this concentration range.
How can we explain that the β3(N265M) mutation abolishes both the blocking and potentiating effects of enflurane? One possibility is that both potentiation and inhibition arise from occupation of a molecular site that is disrupted by the mutation. Another possibility is that the mutation is distant from the enflurane binding site but produces a conformational change, thereby affecting both the prolonging and blocking action of the anesthetic.
The finding that the effects of enflurane on IPSCs were largely abolished by the β3
(N265M) mutation indicates that a major fraction of IPSCs represented currents carried by β3
receptors. This was unexpected, because only approximately 15–20% of all GABAA
receptors in the neocortex contain β3
However, neocortical pyramidal cells are innervated by different classes of GABAergic interneurons.22
Furthermore, specific synaptic contacts, as defined by the identity of the presynaptic cell, frequently show specific GABAA
receptor subtypes. For example, α1
subunits, which most commonly coassemble with β2
subunits, are enriched at synapses between fast spiking basket cells and pyramidal cells.23
In contrast, α2
subunits, which frequently coassemble with β3
subunits, are enriched at synapses between cholecystokinin-positive basket cells and pyramidal cells. It seems unlikely that under our recording conditions (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N
-methyl-d-aspartate receptors were blocked to isolate GABAA
receptor-mediated events) fast spiking basket cells were active, because these neurons exhibit a rather negative membrane resting potential.24
Therefore, IPSCs monitored in the course of our experiments may predominantly originate from a yet unidentified subpopulation of interneurons, namely those displaying the most positive membrane resting potential.
At 0.6 mm, we observed that enflurane decreased the frequency of spontaneous IPSCs by approximately 30%. This effect probably results from a decrease in spontaneous action potential firing of presynaptic neurons, because at the concentrations tested in this study, the anesthetic does not depress action potential-independent GABA release.9
Because GABAergic interneurons express GABAA
receptors, these may also have been the mediators of enflurane-induced depression of presynaptic activity.25
Interestingly, attenuation of presynaptic activity by enflurane did not differ between wild-type and β3
(N265M) mutant mice. This finding is consistent with the observation that β3
subunits are mostly expressed in pyramidal cells and not in GABAergic interneurons.26
Are β3-Containing GABAA Receptors in the Neocortex Relevant Targets for Volatile Anesthetics?
By combining in vivo
and in vitro
recordings, Hentschke et al.14
have recently identified the neocortex as a major target of subhypnotic concentrations of enflurane. The authors showed that concentration-dependent depression of spontaneous action potential firing of neurons in the somatosensory cortex caused by enflurane is almost identical in vivo
and in isolated cortical brain slices, indicating a minor contribution of subcortical structures. In the same study, GABAA
receptors, located on cortical pyramidal cells, were identified as a prime target of the anesthetic.
Here we report that at clinically relevant concentrations between MAC-awake and MAC-immobility, enflurane depresses spontaneous action potential firing in cortical slices from β3(N265M) mutant mice to a lesser extent than in slices from wild-type mice, suggesting that β3-containing GABAA receptors significantly contribute to this effect.
However, β3-containing GABAA receptors are not the exclusive molecular target of enflurane, because at concentrations exceeding MAC-immobility, full suppression of neuronal activity also occurred in slices from β3(N265M) mutant mice.
How do these findings on cortical neurons in vitro
relate to the observation that enflurane concentrations causing loss of righting reflexes do not differ in wild-type and β3
(N265M) mutant mice?5
The latter result clearly indicates that ablation of righting reflexes does not involve effects of enflurane on neocortical neurons. Righting reflexes are mediated by brainstem centers including the vestibular nuclei in the medulla and pontine reticular nuclei, integrating mechanoreceptor input into motor commands. In fact, focal application of barbiturates into the brainstem mesopontine tegmentum ablates the righting reflex.27,28
Furthermore, discrete injections of GABAA
receptor agonists in the tuberomammillary nucleus produce loss of righting reflexes.29
Therefore, enflurane possibly abolishes righting reflexes via
these routes and not via
depressing neocortical neurons. In addition, the data discussed so far suggest that the molecular targets by which enflurane depresses cortical neurons and abolishes righting reflexes are distinct. Assuming that righting reflexes involve anesthetic actions in brainstem nuclei, but not in neocortex, enflurane-sensitive ion channels in the neocortex and brainstem should be different molecular entities. What channels are abundant in only one of these brain regions? Strychnine-sensitive glycine receptors are expressed in the brainstem, but not in neocortical networks.30
Glycine and GABAA
receptors are equally sensitive to volatile anesthetics.31
Therefore, we speculate that enflurane ablates righting reflexes largely via
molecular targets located in subcortical structures. These may include glycine receptors, non-β3
receptors, and possibly yet unidentified molecular targets.
Taken together, our results provide further evidence for the hypothesis that the sedative and hypnotic properties of anesthetic agents are related to drug actions in different parts of the brain.2
While sedation involves targets in the neocortex, hypnosis, as defined by the absence of righting reflexes, seems to be mediated predominantly by subcortical structures.
The authors thank Claudia Holt (Technical Assistant, Eberhard-Karls-University, Tuebingen, Germany) for excellent technical assistance.
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© 2006 American Society of Anesthesiologists, Inc.