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*Department of Anesthesia and Perioperative Care, University of California, San Francisco, California; Institute of Pharmacology and Toxicology, University of Zurich, Zurich, Switzerland; and Waggoner Center for Alcohol and Addiction Research, University of Texas, Austin, Texas
Address correspondence and reprint requests to Dr. Edmond I Eger II, Department of Anesthesia, S-455, University of California, San Francisco, CA 94143-0464. Address e-mail to egere{at}anesthesia.ucsf.edu.
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Abstract |
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1ß2
2 GABAA receptors whereas isoflurane provides a large enhancement. Consistent with previous work on 1ß22 GABAA receptors, we found in Xenopus oocytes that 5 MAC cyclopropane enhanced the effect of GABA on 1ß22 GABAA receptors by only 76%, and by a nearly identical enhancement in 1ß32, and 6ß32 receptors. In contrast, a much smaller concentration of isoflurane (1 MAC) produced a 160% to 310% enhancement in these receptors. If, relative to isoflurane, cyclopropane minimally increases GABA-induced chloride currents at any GABAA receptor subtype, the present data for MAC are consistent with the notion that GABAA receptors do not mediate the immobility produced by inhaled anesthetics. |
Introduction |
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The N265M point mutation also affected the potency of enflurane and halothane, a finding consistent with a previous report that recombinant 2ß3(N265M)2 GABAA receptors are significantly less sensitive to enflurane compared with wild-type receptors (5). Although this mutation did not change the concentrations of these anesthetics required to abolish the righting reflex, suppression of the hindlimb withdrawal reflex required 16% more enflurane and 21% more halothane in mutant compared with wild-type mice (1). These findings suggested that ß3-containing GABAA receptors might have a major role in mediating the immobilization produced by propofol and etomidate, and a minor role in the immobilization produced by enflurane and halothane.
We sought to further explore this implication in the present study. In particular, we examined whether the mutation could be used to assess the extent to which the subpopulation of ß3-containing GABAA receptors mediates MAC (the minimum alveolar concentration of inhaled anesthetic required to eliminate movement in response to noxious stimulation in 50% of subjects) for two inhaled anesthetics, isoflurane and cyclopropane. Bearing in mind that no in vitro data have determined the degree to which the ß3(N265M) point mutation affects the action of isoflurane and cyclopropane, we hypothesized that if the ß3-containing GABAA receptors mediate the reported increase in the duration of the "withdrawal reflex" or MAC for enflurane and halothane (1), then the increase in MAC should be less for an anesthetic that has a minimal effect on the 1ß22 GABAA receptor in vitro [e.g., cyclopropane (6)], and possibly other GABAA receptors, than for an anesthetic that has a much greater effect at the same receptor subtype in vitro [e.g., isoflurane (6)]. Recent studies indicate that the 1ß22 GABAA receptor, which is by far the most abundant GABAA receptor subtype, likely mediates the sedative but not the anesthetic action at least of etomidate (1,7), and it is currently unknown whether cyclopropane might modulate the activity of some GABAA receptor subtype(s) in a manner that might contribute to immobility.
The target(s) mediating the amnesic action of inhaled anesthetics (e.g., isoflurane) is also unknown. In the present study, we therefore explored the potential involvement of ß3-containing GABAA receptors in the capacity of inhaled anesthetics to depress learning and memory by comparing the actions of isoflurane in conditioning paradigms in ß3(N265M) mice and wild-type mice. Furthermore, we wanted to quantify the contribution of ß3-containing GABAA receptors to the immobilizing action of isoflurane and cyclopropane. We assumed that, as for the 1ß22 GABAA receptor (see above), other GABAA receptor subtypes would be sensitive to isoflurane but relatively insensitive to cyclopropane. Thus, we expected that the MAC for isoflurane, but not the MAC for cyclopropane, would be increased in ß3(N265M) mice.
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Methods |
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Three studies were performed. First, using a Pavlovian approach, we determined whether the mutant mice were resistant to isoflurane’s capacity to impair learning and memory. Second, we determined whether the MAC values for isoflurane and cyclopropane differed for mutant versus wild-type mice. Third, we assessed the effects of cyclopropane and isoflurane on GABA-induced currents through 1ß22, 1ß32, and 6ß32 GABAA receptors expressed in Xenopus laevis oocytes.
Studies of learning and memory applied techniques described previously (9–11). Mice were exposed to target concentrations of isoflurane (confirmed with gas chromatography) for 30 min before training. Each animal was then rapidly transferred to a training chamber containing the target concentration and was allowed to explore the chamber for 3 min before training began. Animals then received 3 tone-shock pairs consisting of a 30-s tone (90 dB, A-scale, 2000 Hz) co-terminating with a 2-s electric shock (11-Hz bipolar square waves); shock pairs were 90 s apart. Animals were returned to their home cages within 60 s after the last shock. The shock currents were 1 or 2 mA at 0%, 0.1%, and 0.2% isoflurane, and 2 mA at 0.3% and 0.4% isoflurane. At all larger concentrations of isoflurane, we used 3-mA currents.
Tone testing took place the day immediately after training in chambers providing an entirely different environment from that provided by the training chambers. We then assessed freezing to context. For tone testing, each animal was placed in the test chamber, and, after 3 min of exploration, a tone (90 dB, A-scale, 2000 Hz) was continuously sounded for 8 min; shocks were not administered. Four animals were observed simultaneously, one in each of four separate test chambers, via a video camera. Two hours after the tone test, each mouse was tested for fear conditioning to context by an 8-min observation in the identical chamber in which it was trained the previous day. Again, four animals were observed simultaneously, one in each of the four chambers, via a video camera. No personnel were in the tone or context testing rooms during testing. To score freezing to either tone or context, an observation of 1 of the 4 animals was made every 2 s. Therefore, each animal was scored once every 8 s. Behavior was judged as freezing if there was no visible movement except for breathing (12). The observation periods were also video recorded for scoring by a blinded observer.
The percentage of time an animal froze during the 8-min observation periods was calculated as the number of observations judged to be freezing divided by the total number of observations in 8 min, i.e., 60 observations (12). For each group score at a given isoflurane concentration, the mean and standard error of the mean (sem) were calculated. Nonlinear regression was performed to calculate a 50% effective dose, Hill coefficient (n), and the maximal or starting value of the dose-response curve (A) for fear conditioning according to the equation:
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A Student’s t-test was applied to comparisons of results for wild-type versus mutant mice. A value of P < 0.05 was regarded as significant for all comparisons.
MAC (the minimum alveolar concentration of isoflurane or cyclopropane that produced immobility in 50% of mice in response to a tail clamp) was determined in groups of 4–8 mice using techniques previously described (13,14). Rectal temperatures were monitored and maintained between 35.9° and 38.7°C (range for all mice). Anesthetic concentrations were monitored with an infrared analyzer (as above). The initial anesthetic concentration was imposed for a 40-min equilibration period. A small initial target concentration was chosen to ensure response to tail clamp. We then increased the anesthetic concentration in steps of 15%–20% of the preceding concentration, holding each step for a minimum of 20 (cyclopropane) to 30 (isoflurane) min before again applying the tail clamp. This continued until all mice ceased moving in response to the tail clamp. At the end of each step (after application of the tail clamp), a sample of gas was taken for anesthetic analysis by gas chromatography.
The gas chromatograph was calibrated with secondary standards from tanks containing isoflurane or from concentrations obtained by serial dilution from 100% cyclopropane. For the tests of learning and memory, for each group, the isoflurane concentration was calculated as the mean and standard error (se) of the concentrations measured in the cylinders and in the training chambers before and after training of that group. For the studies of MAC, the MAC for each mouse was calculated as the mean of the concentrations that bracketed movement-nonmovement. The group MAC and se were calculated from these individual values. Differences between wild-type and mutant mice were assessed with a Student’s t-test. We accepted P < 0.05 as significant.
Isolation, injection, and electrophysiological studies were conducted as previously described (6) with small differences detailed below. The cDNAs encoding the GABAA subunits were generously provided by Drs. M. H. Akabas (rat 1 and 2s), P. J. Whiting (human ß2 and ß3), and N. Iwata (human 6). After linearization, the cDNA encoding the wild-type and mutant subunits was used as a template for the synthesis in vitro of 5'-capped RNA (mCAP RNA Capping Kit; Stratagene, La Jolla, CA). X. laevis oocytes were manually isolated and then injected into the cytoplasm with 30 nL of diethyl pyrocarbonate-treated water containing 16 ng of cRNA encoding GABAA subunits. The ratio was /ß/ 3:3:10 in nanograms/oocyte. The injected oocytes were kept at 13°C in incubation media. Recordings were conducted 5–6 days after injection. The oocytes were perfused with ND96 buffer (2 mL/min) during the electrophysiological studies (perfusion buffer composition was [in millimolar]: 96 NaCl, 1 CaCl2, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.5). The maximal response to GABA (determined with application of 3 mM GABA) allowed the subsequent determination of the GABA concentration equivalent to 5% effective concentration (EC5). A washout of 5 min was observed between all GABA applications, except after 3 mM GABA (15 min). A saturated solution was obtained by bubbling 100% cyclopropane through perfusate solution in a closed vial for at least 10 min. The resulting cyclopropane concentration that perfused the oocyte is equivalent to 5 MAC (6). Similarly, we produced a perfusate containing 1 MAC of isoflurane. After 2 applications of EC5 GABA, cyclopropane or isoflurane was preapplied for 1 min and then immediately coapplied with GABA for 30 s. EC5 GABA was applied again. The coexpression of the subunit was determined by coapplication of GABA and 10 µM Zn++ (the presence of the subunit greatly decreases the inhibition produced by Zn++).
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Results |
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In the absence of isoflurane (control), mutant mice did not freeze as much as wild-type mice. Percentages of time spent freezing for context and for tone in wild-type mice were 73% ± 4% and 77% ± 4% (n = 22) versus 48% ± 5% and 36% ± 4% (n = 36; P < 0.01 for context and P < 0.001 for tone). The nature of and the reasons for this difference in reactivity have not been investigated in detail. Nonetheless, the EC50 values for context did not differ significantly between wild-type and mutant mice (0.19% ± 0.037% isoflurane for the wild-type; n = 83; 0.28% ± 0.044% for the mutant; n = 134; P = 0.14; Fig. 1). Similarly, the EC50 values for tone did not differ (0.46% ± 0.043% versus 0.68% ± 0.13%; P = 0.17; Fig. 2).
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The isoflurane MAC for mutant mice (1.93% ± 0.03%; mean ± se; n = 14) was 17.0% larger than MAC for wild-type mice (1.65 ± 0.04; n = 14; P < 0.001). Similarly, the cyclopropane MAC for mutant mice (27.6% ± 0.55%; n = 16) was 13.6% larger than MAC for wild-type mice (24.3 ± 0.46; n = 8; P < 0.01). Ten of the 16 mutant mice died before completion of the study with cyclopropane (i.e., at the highest step of the study). The MAC of the 6 surviving mice was 27.2% ± 0.41%, a value 11.9% larger than that for the wild-type mice (P < 0.05). For the mice that died, we assumed that movement would not have occurred at the next step, 15% more than the step at which movement had occurred. Thus, the MAC we calculated for cyclopropane in mutant mice probably underestimated the true MAC.
A saturated solution of cyclopropane (100% cyclopropane, equivalent to approximately 5 MAC) increased the current induced by EC5 GABA by 76% ± 4% in 1ß22S (n = 5), 74% ± 3% in 1ß32S (n = 5), and 66% ± 9% in 6ß32S (n = 6). A solution of 1 MAC isoflurane (0.29 mM) produced an enhancement of 234% ± 21% in 1ß22S (n = 5), 308% ± 18% in 1ß32S (n = 5), and 162% ± 14% in 6ß32S (n = 5) on the responses induced by EC5 GABA. All values are the mean ± se.
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Discussion |
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The finding of a nonsignificant trend to greater average EC50 values for the mutant mice presents one limitation of this study. Another limitation is the finding that the control (no anesthetic) freeze scores were smaller for the mutant mice. That is, although the present work finds no difference in the effect of isoflurane on learning and memory for wild-type versus mutant mice, we cannot exclude the possibility that there is a difference (and thus an effect of mutation on anesthetic potency).
Had we found a difference in tone or context conditioning, we would have measured the effect of the point mutation on the depression of learning and memory produced by cyclopropane. Our rationale would have been that if the ß3-containing GABAA receptors mediate part or all of the capacity of inhaled anesthetics to depress learning and memory, the mutation should have had less influence on the effect of cyclopropane than of isoflurane because cyclopropane has a far smaller capacity to enhance the in vitro action of GABA on GABAA receptors, based on previous in vitro studies at the 1ß22 and 2ß1 GABAA receptors (6) and the studies of 1ß32S and 6ß32S in the present report. 1-Containing GABAA receptors mediate the anterograde amnesic action of diazepam (15), suggesting that the most abundant receptor subtype, 1ß22, which represents approximately 50% of GABAA receptors in the brain, mediates this action. The present study finds that the depressant effects of isoflurane on learning and memory do not significantly differ between ß3(N265M) mice and wild-type mice indicating no role of ß3-containing GABAA receptors in this effect. These effects of isoflurane might be mediated by ß1- or ß2-containing GABAA receptors or other target receptors or ion channels.
The reasoning of a potential differential sensitivity of GABAA receptors for isoflurane and cyclopropane also applied to our study of MAC in the wild-type versus the ß3(N265M) mutant mice. Consistent with the reported effects of halothane and enflurane (1), we found that the mutant mice had a 17% larger MAC for isoflurane than did the wild-type mice (P < 0.001). If the ß3-containing GABAA receptors mediate the capacity of inhaled anesthetics to produce immobility, then we anticipated a smaller effect would be seen with cyclopropane. However, the effect of cyclopropane on MAC was comparable to that for isoflurane; MAC for cyclopropane in the mutant mice was 13.6% larger than MAC in wild-type mice (P < 0.01). We probably underestimated this larger MAC because, as noted in the results, 10 of the 16 mutant mice given cyclopropane died before the response at the largest concentration could be tested.
Some of the results for MAC and learning and memory are unexpected and we speculate on their explanation.
a) Our assumption that ß3-containing wild-type GABAA receptors are much more sensitive to isoflurane than to cyclopropane was based, in part, on published in vitro data obtained for the 1ß22 GABAA receptor, which is the most abundant GABAA receptor subtype but does not mediate the immobilizing effects of etomidate or propofol, and on data obtained for the 2ß1 GABAA receptor (6). The complete subunit composition of the GABAA receptor that mediates the immobilizing response of etomidate and propofol is currently unknown. In addition to the ß3 subunit, it likely contains an as yet undefined subunit(s) and likely either a or a 
subunit. In particular, for ß3-containing GABAA receptors (or subsets thereof), cyclopropane might have a higher affinity or potency than at the 1ß22 GABAA receptor. In this case, there may be no or only a minor difference in sensitivity of the receptor subtype mediating the immobilizing effects to isoflurane and cyclopropane. However, our results for 1ß32S and 6ß32S receptors (see next paragraph) suggest that the GABAA receptor containing the ß3 subunit is not necessarily more sensitive than other GABAA receptors.
To expand on the previous findings in recombinant 1ß22 receptors, we examined whether cyclopropane enhanced GABA-induced chloride currents at 1ß32 and 6ß32 receptors expressed in oocytes and found small, similar effects of 5 MAC cyclopropane. However, the 1ß32 subunit combination is rare in vivo and the 6ß32 subunit combination is restricted to the cerebellum, and thus is not involved in the immobilizing action of general anesthetics. Further experiments are needed to unequivocally clarify the effects of cyclopropane on all potentially relevant ß3-containing GABAA receptor subtype combinations.
Consistent with the possibility that specific and as yet undefined GABAA receptor subtypes may be sensitive to cyclopropane, the sensitivity of GABAA receptors to ethanol is determined by the type of ß, , or subunits. GABAA receptors containing—in addition to 4ß or 6ß—the subunit instead of the 2 subunit are 3 times more sensitive to ethanol, and receptors containing the ß3 subunit (i.e., in 4ß3 or 6ß3) are almost 10 times more sensitive to ethanol than receptors containing the ß2 subunit (in the presence of a subunit) (16). These ß3- and -containing receptors, which may be found at extrasynaptic locations but not the synaptic 2-containing receptors, may be primary targets for ethanol at concentrations achieved by social drinking and also for general anesthetics (16). It is an interesting and speculative hypothesis that the GABAA receptor subtypes likely mediating ethanol action might also mediate part of the action of volatile anesthetics such as isoflurane and potentially cyclopropane.
b) In 5(H105R) point mutated mice, the expression of the extrasynaptic 5-containing GABAA receptors is reduced in the hippocampus, resulting in facilitation of trace fear conditioning (17). In the case of the ß3-containing GABAA receptor, in Western blot and immunohistochemistry analyses, the expression levels of 1, 2, 3, ß2/3, and 2 subunits in the ß3(N265M) mice were indistinguishable from those in wild-type receptors (1). In vitro data suggest that the ß3(N265M) point mutation decreases the GABA sensitivity of 2ß32 GABAA receptors from an EC50 of 47 ± 5 µM for the wild-type receptor to an EC50 of 122 ± 10 µM (5). We currently do not know exactly what the relevance of this difference is in vivo. If the ß3-containing GABAA receptors are less sensitive to GABA, this might explain at least in part the differential freezing response to context and tone of ß3(N265M) and wild-type mice in the Pavlovian test described in this report. Thus, similar to the 5(H105R) mouse line, the ß3(N265M) mouse line displays a drug-independent phenotype which future studies of these mice will assess further. We currently cannot exclude the possibility that the sensitivity to the as yet unknown target(s) mediating the immobilizing action of cyclopropane is increased in ß3(N265M) mice, in which case the increase in MAC for cyclopropane would be independent of GABAA receptors.
c) As mentioned in the Introduction, the ß3(N265M) point mutation does not completely abolish enflurane’s action in vitro (5), and isoflurane’s and cyclopropane’s actions at this point mutation have not been studied in vitro. Potentially, point mutations in the subunits, which have been predicted to line binding cavities for halothane and isoflurane within GABAA receptors (18), would have a more profound effect (e.g., on isoflurane action) than observed in the ß3(N265M) mice.
We demonstrated previously that intrathecal administration of the GABAA receptor blocker picrotoxin did not increase the MAC of isoflurane more than the MAC of cyclopropane (19,20). In addition, other evidence suggests that GABAA receptors are not crucial to the immobilizing effects of inhaled anesthetics (reviewed in Ref. 19). Notwithstanding the aforementioned assumptions made in this study, if ß3-containing GABAA receptors were the only or the major targets mediating the immobilizing response to isoflurane and cyclopropane, we would have expected increases parallel to the observations made on etomidate and propofol which were unable to immobilize the ß3(N265M) mice (1). Because this is clearly not the case, our present finding that the MACs for isoflurane and cyclopropane are increased only partially in ß3(N265M) mice is consistent with targets other than ß3-containing GABAA receptors having a major role in mediating the immobilizing actions of these drugs.
In summary, we suggest that although the ß3(N265M) point mutation markedly influences the potency of propofol and etomidate, and thus that ß3-containing GABAA receptors are a likely site of action for these injected anesthetics (1), ß3-containing GABAA receptors are of essentially no or of limited importance as mediators of the amnesia or immobility produced by inhaled anesthetics.
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Footnotes |
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EIE is a paid consultant to Baxter Healthcare Corp.
Accepted for publication December 8, 2004.
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