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Meeting Abstracts  |   January 2007
Effect of Isoflurane and Other Potent Inhaled Anesthetics on Minimum Alveolar Concentration, Learning, and the Righting Reflex in Mice Engineered to Express α1γ-Aminobutyric Acid Type A Receptors Unresponsive to Isoflurane
Author Affiliations & Notes
  • James M. Sonner, M.D.
    *
  • David F. Werner, B.S.
  • Frank P. Elsen, Ph.D.
  • Yilei Xing, M.D.
    §
  • Mark Liao, B.S.
  • R Adron Harris, Ph.D.
    #
  • Neil L. Harrison, Ph.D.
    **
  • Michael S. Fanselow, Ph.D.
    ††
  • Edmond I. Eger, M.D.
    ‡‡
  • Gregg E. Homanics, Ph.D.
    §§
  • * Associate Professor of Anesthesia and Perioperative Care, § Postdoctoral Fellow, ∥ Research Assistant, ‡‡ Professor of Anesthesia and Perioperative Care, Department of Anesthesia and Perioperative Care, University of California, San Francisco, California. † Graduate Student, §§ Associate Professor of Anesthesiology and Pharmacology, Departments of Anesthesiology and Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania. ‡ Postdoctoral Fellow, ** Professor of Anesthesiology and Pharmacology, Departments of Anesthesiology and Pharmacology, Weill Medical College of Cornell University, New York, New York. # Professor of Molecular Biology, Waggoner Center for Alcohol and Addiction Research, University of Texas, Austin, Texas. †† Professor of Psychology, The Department of Psychology, University of California, Los Angeles, California.
Article Information
Meeting Abstracts   |   January 2007
Effect of Isoflurane and Other Potent Inhaled Anesthetics on Minimum Alveolar Concentration, Learning, and the Righting Reflex in Mice Engineered to Express α1γ-Aminobutyric Acid Type A Receptors Unresponsive to Isoflurane
Anesthesiology 1 2007, Vol.106, 107-113. doi:
Anesthesiology 1 2007, Vol.106, 107-113. doi:
ALL inhaled anesthetics supply two essential elements of anesthesia: immobility and amnesia. A current consensus argues that these elements result from the combined effects of inhaled anesthetics on several ligand-gated and voltage-gated channels.1 The γ-aminobutyric acid type A receptors (GABAARs) have been considered prime candidates as targets of inhaled anesthetic action2 because they are widely distributed in the central nervous system and because many inhaled agents promote their function at clinically relevant concentrations. In fact, enhancement of GABAAR function seems to underlie the production of anesthesia by the intravenous anesthetics propofol and etomidate, which have effects on GABAAR that are similar to those of inhaled anesthetics.3 Many inhaled anesthetics similarly prolong GABAAR-mediated inhibition of spinal motoneurons,4 which may account in part for the immobilizing effect of inhaled anesthetics, and enhance inhibition in the hippocampus,5 which might mediate amnestic effects. However, recent studies suggest that GABAARs might not be important mediators of immobility. For example, although xenon, cyclopropane, and isoflurane differ greatly in their capacity to enhance the GABAAR response to GABA, the intrathecal administration of the GABAAR antagonist, picrotoxin, produces a modest increase in minimum alveolar concentration (MAC) that does not differ among these anesthetics.6 
Our group has developed a strain of knock-in mouse to test the hypothesis that GABAARs containing the α1subunit mediate some or all of the clinically important behavioral effects of inhaled volatile anesthetics such as isoflurane. The α1subunit is the most abundant of the GABAAR α subunits, being present in approximately 40% of all GABAAreceptors in the brain,7 with a relatively ubiquitous distribution throughout the cerebral cortex, thalamus, cerebellum, and hippocampus. The mice were genetically engineered to express two point mutations in the GABRA1 locus that changes serine (S) to histidine (H) at position 270 and leucine (L) to alanine (A) at position 277 in the α1subunit polypeptide. In vitro  , the S270H mutation selectively eliminates GABAAR potentiation by isoflurane and desflurane, but not halothane. Incorporation of the second L277A mutation restores the GABA sensitivity of the mutant receptor to normal.8,9 
If GABAAR-containing α1subunits mediate any of the behavioral effects of the inhaled anesthetics, these knock-in mice should have a reduced sensitivity to isoflurane (but not halothane), relative to wild-type controls. We used three standard behavioral tests: limb withdrawal in response to noxious stimulation (i.e.  , measurement of “MAC”), acquisition of fear conditioning to tone and to context (i.e.  , pavlovian conditioning), and loss of the righting reflex (LORR), measures widely used in studies of anesthetic effects in rodents.
Materials and Methods
Experimental Subjects
Institutional animal care and use committees (University of California, San Francisco, California, and University of Pittsburgh, Pittsburgh, Pennsylvania) approved our studies of male and female mice bred from mice heterozygous for the S270H and L277A mutations, producing wild-type (SL/SL), heterozygous knock-in (SL/HA), and homozygous knock-in (HA/HA) mice. Animals were housed under a 12-h light-and-dark cycle and had continuous access to standard mouse chow and tap water.
Hippocampal Slice Electrophysiology
Brain slices were prepared from adult mice (age range, 28–52 days), and miniature inhibitory postsynaptic currents (mIPSCs) were recorded and analyzed as previously described.10 Interneurons and pyramidal cells in the CA1 stratum radiatum and stratum lacunosum-moleculare layer of the hippocampus were identified using differential interference contrast microscopy.10 The identity of pyramidal cells was then verified by pronounced accommodation of action potential firing, to distinguish them from interneurons also present in the pyramidal cell layer.11 As in a previous study,10 fewer than 10% of the recorded neurons in the CA1 pyramidal cell layer were interneurons. Whole cell patch clamp recordings were obtained with borosilicate glass pipettes that had a resistance of 2–4 MΩ when filled with pipette solution containing 130 mm cesium methanesulfonate (CH3SO3Cs), 8.3 mm sodium methanesulfonate (CH3SO3Na), 1.7 mm NaCl, 1 mm CaCl2, 10 mm EGTA (intracellular free calcium ion concentration: approximately 0.01 μm), 2 mm Mg-ATP, 0.3 mm Na-GTP, and 10 mm HEPES (pH 7.2, osmolarity 295 mOsm). In the recording chamber, brain slices were constantly superfused at a rate of 3.5 ml/min with artificial cerebrospinal fluid containing 117 mm NaCl, 3.6 mm KCl, 25 mm NaHCO3, 1.2 mm NaH2PO4, 1.2 mm MgCl2, 2.5 mm CaCl2, and 11 mm glucose (pH 7.4, osmolarity 305 mOsm). To eliminate action potential–evoked synaptic events, 0.5 μm tetrodotoxin was added to the artificial cerebrospinal fluid. Drugs were applied via  a gravitational glass-syringe/polytetrafluoroethylene-tube system to reduce the loss of volatile anesthetics. A 10-min preapplication of each drug was performed before the 5-min data recording epoch that was used for analysis; this ensured that the anesthetic drugs were at equilibrium throughout the data collection period.12 The mIPSCs were recorded at room temperature for 5 min with an acquisition frequency of 10 kHz and a filter frequency of 2 kHz. The holding potential was −60 mV. Analysis of the mIPSC data traces was performed as described previously.10 Bath application of 20 μm bicuculline blocked all mIPSC activity (n = 3, data not shown). The weighted decay time constant (τdecay) was calculated as τdecay= (Af× tf+ As× ts)/(Af+ As).10 To ensure an adequate recording configuration throughout the experiments, we determined the access resistance before and after each recording using the membrane properties feature of the acquisition software (pClamp 9.0; Axon Instruments, Foster City, CA). Recordings were excluded from the analysis when the difference between membrane and access resistance decreased below a 10-fold value.
Volatile anesthetic solutions were prepared in airtight surgical bags. Each bag contained 100 ml artificial cerebrospinal fluid including 0.5 μm tetrodotoxin plus volatile anesthetic, as described previously. The values obtained for amplitudes and τdecaywere compared to the respective control values in volatile anesthetic–free solution to calculate the volatile anesthetic effect on mIPSCs as percentage change for each experiment, and comparisons between genotypes were made using analysis of variance.10 
Whole-animal Behavioral Studies
Studies of learning and memory applied techniques described previously.13–15 Mice (n = 254) were exposed to a target concentration 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 of isoflurane and was allowed to explore the chamber for 3 min before training began. Mice then received three tone-shock pairs (tone training) consisting of a 30-s tone (90 dB, A-scale, 2,000 Hz) coterminating with a 2-s electric shock (11-Hz bipolar square waves). Ninety seconds separated tone-shock pairs. Animals were returned to their home cages within 60 s after the last shock. The shock currents were 2 mA at 0, 0.1%, 0.2%, 0.3%, and 0.4% isoflurane. At 0.5% and 0.75% isoflurane, we used 3-mA currents. For each test, the anesthetic concentration was calculated as the mean of the concentrations measured in the training chambers before and after training of that set of four mice.
Both context and tone testing took place the day after training. For tone testing, each animal was placed in a special test chamber and, after 3 min of exploration, a tone (90 dB, A-scale, 2,000 Hz) was continuously sounded for 8 min; no shock was administered. Context testing was conducted 1–2 h later. For context testing, the mice were returned to the chambers used to supply the electric shocks, whereas tone testing took place in chambers providing an entirely different environment from that provided by the training chambers. For both context and tone testing, four mice were observed simultaneously, one in each of four separate test chambers, via  a video camera. No personnel were in the tone or context training or testing rooms during training or testing. To score freezing to either tone or context, an observation of one of the four 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.16 The observation periods were 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.16 For each group score at a given isoflurane concentration, the mean freeze score and standard error of the mean (SE) were calculated.
A least-squares linear regression was applied to the raw data for fear to context for 0–0.5% isoflurane. For fear to tone data, a least-squares linear regression was applied to the data for 0–0.75% isoflurane. The concentration (EC50) producing a 50% decrease in freezing scores from control (no isoflurane) and the SE of this concentration were calculated and used to compare freezing to context and freezing to tone for the three genetic groups. In addition, two-way analysis of variance using genotype and anesthetic concentration as factors was performed to determine whether there was a difference in the effect of genotype on freeze scores. A value of P  < 0.05 was regarded as significant for all comparisons.
We measured MAC for desflurane, isoflurane, and halothane in 88 mice (28 SL/SL, 36 SL/HA, and 24 HA/HA) as described previously.17 These mice were a random subset of mice after measurements of fear conditioning had been made. Each mouse was used as a subject for one, two, or three of the test anesthetics but was used only once for a test of a given anesthetic. For each mouse, MAC was calculated as the mean of the greatest inspired concentration that permitted movement in response to tail clamp and the smallest concentration that prevented movement. Genotyping of each test mouse was accomplished after the determinations of learning and memory, and MAC. Differences in MAC between genotypes were determined using analysis of variance.
Adult (8- to 12-week-old) male and female mice (n = 15–18 per genotype) were tested for sensitivity to inhaled anesthetics using the LORR assay as described.18,19 Briefly, mice were placed in individual wire mesh cages in a rotating carousel in a sealed acrylic chamber and anesthetized. Within the chamber, carbon dioxide was maintained at less than 1% atm, and temperature was maintained at 35°± 0.2°C. Chamber atmosphere and anesthetic concentrations were monitored continuously with a Datex Capnomac Ultima device (Datex-Ohmeda, Helsinki, Finland), and fresh oxygen was delivered at a rate of 1.5 l/min. Mice were equilibrated with the desired concentration (% atm) of halothane (Halocarbon Laboratories, River Edge, NJ), isoflurane (Halocarbon Laboratories), or enflurane (Anaquest, Madison, WI) for 15 min, after which they were scored by an observer blind to the genotypes. Scores were quantal; a positive response for LORR occurred when mice were not able to right themselves two times during five revolutions of the carousel at 4 rpm. Mice were allowed to recuperate in oxygen for at least 20 min before being equilibrated with the next anesthetic concentration. The dose–response relation for each anesthetic was analyzed using the Z statistic.20 
Results
Hippocampal Slice Electrophysiology
Miniature inhibitory postsynaptic currents were recorded from interneurons (n = 36) and pyramidal cells (n = 23) in the CA1 subfield of the hippocampus. The cell properties (input resistance, cell capacitance) were not significantly different between genotypes in interneurons and pyramidal cells, and there was also no significant difference between the groups of interneurons and pyramidal cells. In interneurons, the decay phase of mIPSCs recorded in neurons from HA/HA animals was significantly faster compared with mIPSCs recorded in SL/SL and SL/HA animals (figs. 1A and B). In pyramidal cells, we observed a significant faster decay time constant in HA/HA animals compared with SL/SL animals (fig. 1B). However, mIPSC amplitude and frequency were not significantly different between the genotypes (data not shown).
Fig. 1. Example traces of miniature postsynaptic currents (mIPSCs) under control conditions and during application of volatile anesthetics. (  A  ) Average mIPSC traces (from 50 single events) of hippocampal interneurons from the three genotypes (wild-type SL/SL; heterozygous SL/HA; homozygous HA/HA) under control conditions. The current amplitude is normalized to the maximum amplitude to visually compare the decay time of the mIPSCs. (  B  ) Bar diagram of average decay time constants from interneurons and pyramidal cells for all three genotypes. Number of experiments is given in brackets, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL; # significantly different from SL/HA). (  C  ) Effect of 0.16 and 0.31 mm isoflurane on average current traces in all three genotypes. (  D  ) Effect of 0.27 mm halothane on average current traces in all three genotypes. 
Fig. 1. Example traces of miniature postsynaptic currents (mIPSCs) under control conditions and during application of volatile anesthetics. (  A  ) Average mIPSC traces (from 50 single events) of hippocampal interneurons from the three genotypes (wild-type SL/SL; heterozygous SL/HA; homozygous HA/HA) under control conditions. The current amplitude is normalized to the maximum amplitude to visually compare the decay time of the mIPSCs. (  B  ) Bar diagram of average decay time constants from interneurons and pyramidal cells for all three genotypes. Number of experiments is given in brackets, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL; # significantly different from SL/HA). (  C  ) Effect of 0.16 and 0.31 mm isoflurane on average current traces in all three genotypes. (  D  ) Effect of 0.27 mm halothane on average current traces in all three genotypes. 
Fig. 1. Example traces of miniature postsynaptic currents (mIPSCs) under control conditions and during application of volatile anesthetics. (  A  ) Average mIPSC traces (from 50 single events) of hippocampal interneurons from the three genotypes (wild-type SL/SL; heterozygous SL/HA; homozygous HA/HA) under control conditions. The current amplitude is normalized to the maximum amplitude to visually compare the decay time of the mIPSCs. (  B  ) Bar diagram of average decay time constants from interneurons and pyramidal cells for all three genotypes. Number of experiments is given in brackets, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL; # significantly different from SL/HA). (  C  ) Effect of 0.16 and 0.31 mm isoflurane on average current traces in all three genotypes. (  D  ) Effect of 0.27 mm halothane on average current traces in all three genotypes. 
×
Bath application of isoflurane (0.16 and 0.31 mm; 1 MAC corresponds to 0.31 mm) prolonged the decay phase of mIPSCs in SL/SL animals, and this effect was concentration dependent. The effect of isoflurane was significantly reduced in HA/HA animals compared with wild-type animals as shown in example current traces in figure 1C. Halothane application (0.27 mm, the aqueous concentration at 1 MAC) significantly prolonged the decay time of mIPSCs in SL/SL animals and in the mutant genotypes, with no significant differences (fig. 1D). To determine the average effect of the volatiles on the decay time constants, we calculated the percentage change between control conditions and after 10 min of volatile application. The average data are shown in two bar diagrams in figures 2A and B. We found that the effect of isoflurane on mIPSC decay times was reduced in interneurons and pyramidal cells (figs. 2A and B). The amplitudes and frequencies of mIPSCs were not significantly affected by volatile anesthetic application in any of the three genotypes (data not shown).
Fig. 2. Effect of volatile anesthetics on decay time constants of miniature postsynaptic currents in interneurons and pyramidal cells. (  A  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in interneurons for all three genotypes (wild-type SL/SL, heterozygous SL/HA, and homozygous HA/HA). Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). (  B  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in pyramidal cells for all three genotypes. Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). 
Fig. 2. Effect of volatile anesthetics on decay time constants of miniature postsynaptic currents in interneurons and pyramidal cells. (  A  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in interneurons for all three genotypes (wild-type SL/SL, heterozygous SL/HA, and homozygous HA/HA). Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). (  B  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in pyramidal cells for all three genotypes. Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). 
Fig. 2. Effect of volatile anesthetics on decay time constants of miniature postsynaptic currents in interneurons and pyramidal cells. (  A  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in interneurons for all three genotypes (wild-type SL/SL, heterozygous SL/HA, and homozygous HA/HA). Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). (  B  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in pyramidal cells for all three genotypes. Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). 
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Behavioral Studies
Mice were tested for the amnestic effects of inhaled anesthetics using a fear conditioning assay. Control values for fear to context and fear to tone (i.e.  , in the absence of anesthetic) did not differ among the three genotypes of mice. Slopes and intercepts did not differ by genotype among mice conditioned to context or among those conditioned to tone (figs. 3 and 4). Mice of different genotypes did not differ in conditioning to tone (F2,248= 0.11, P  = 0.89) or context (F2,226= 0.16, P  = 0.85). The EC50± SE (in % atm isoflurane) for freezing to tone was 0.34 ± 0.05 for SL/SL mice (n = 72), 0.34 ± 0.04 for SL/HA mice (n = 113 mice), and 0.31 ± 0.06 for HA/HA mice (n = 66). For freezing to context, these values were 0.28 ± 0.05 for SL/SL (n = 64), 0.25 ± 0.04 for SL/HA (n = 101), and 0.24 ± 0.06 for HA/HA (n = 64).
Fig. 3. Fear to context was measured as the percent of time a mouse froze in the presence of the environment in which the mouse had received three foot shocks on the preceding day. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.5% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the slope was −90.4 ± 12.2 and the intercept was 50.3 ± 3.6 (n = 64). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −95.8 ± 11.6 and 48.2 ± 3.4 (n = 101 mice). For HA/HA (homozygous knock-in) mice, these values were −85.1 ± 15.0 and 41.6 ± 4.2 (n = 64). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
Fig. 3. Fear to context was measured as the percent of time a mouse froze in the presence of the environment in which the mouse had received three foot shocks on the preceding day. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.5% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the slope was −90.4 ± 12.2 and the intercept was 50.3 ± 3.6 (n = 64). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −95.8 ± 11.6 and 48.2 ± 3.4 (n = 101 mice). For HA/HA (homozygous knock-in) mice, these values were −85.1 ± 15.0 and 41.6 ± 4.2 (n = 64). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
Fig. 3. Fear to context was measured as the percent of time a mouse froze in the presence of the environment in which the mouse had received three foot shocks on the preceding day. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.5% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the slope was −90.4 ± 12.2 and the intercept was 50.3 ± 3.6 (n = 64). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −95.8 ± 11.6 and 48.2 ± 3.4 (n = 101 mice). For HA/HA (homozygous knock-in) mice, these values were −85.1 ± 15.0 and 41.6 ± 4.2 (n = 64). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
×
Fig. 4. Fear to tone was measured as the percent of time a mouse froze in the presence of the tone supplied previously (the day before). Freezing was tested in an environment that otherwise differed (in shape, texture and shape of ground and walls, smell, lighting) from the environment in which the mouse had been trained. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.75% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the intercept was −105.9 ± 10.9 and the slope was −105.9 ± 11.0, 72.4 ± 4.2 (n = 72). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −109.6 ± 9.0 and 74.6 ± 3.4 (n = 113 mice). For HA/HA (homozygous knock-in) mice, these values were −120.2 ± 17.6 and 74.7 ± 5.4 (n = 66). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
Fig. 4. Fear to tone was measured as the percent of time a mouse froze in the presence of the tone supplied previously (the day before). Freezing was tested in an environment that otherwise differed (in shape, texture and shape of ground and walls, smell, lighting) from the environment in which the mouse had been trained. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.75% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the intercept was −105.9 ± 10.9 and the slope was −105.9 ± 11.0, 72.4 ± 4.2 (n = 72). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −109.6 ± 9.0 and 74.6 ± 3.4 (n = 113 mice). For HA/HA (homozygous knock-in) mice, these values were −120.2 ± 17.6 and 74.7 ± 5.4 (n = 66). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
Fig. 4. Fear to tone was measured as the percent of time a mouse froze in the presence of the tone supplied previously (the day before). Freezing was tested in an environment that otherwise differed (in shape, texture and shape of ground and walls, smell, lighting) from the environment in which the mouse had been trained. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.75% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the intercept was −105.9 ± 10.9 and the slope was −105.9 ± 11.0, 72.4 ± 4.2 (n = 72). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −109.6 ± 9.0 and 74.6 ± 3.4 (n = 113 mice). For HA/HA (homozygous knock-in) mice, these values were −120.2 ± 17.6 and 74.7 ± 5.4 (n = 66). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
×
Mice were tested for the immobilizing effects of inhaled anesthetics in response to a noxious stimulus using the standard tail clamp/withdrawal assay. MAC values did not differ between genotypes for any anesthetic tested (table 1). Using one-way analysis of variance, for isoflurane, F2,48= 0.05 (P  = 0.95); for desflurane, F2,32= 1.32 (P  = 0.29); and for halothane, F2,38= 0.07 (P  = 0.94).
Table 1. Effect of the α1γ-Aminobutyric Acid Type A Receptor Knock-in Mutations on Minimum Alveolar Concentration 
Image not available
Table 1. Effect of the α1γ-Aminobutyric Acid Type A Receptor Knock-in Mutations on Minimum Alveolar Concentration 
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Mice were also tested for the motor ataxic effects of inhaled anesthetics using the standard LORR assay. In this assay, halothane EC50values did not differ between SL/SL (0.68 ± 0.02) and HA/HA (0.69 ± 0.02) mice (fig. 5A). However, differences in the EC50values were observed for isoflurane (0.63 ± 0.02 for SL/SL vs.  0.72 ± 0.02 for HA/HA; P  < 0.01; fig. 5B) and enflurane (1.09 ± 0.03 for SL/SL vs.  1.21 ± 0.03 for HA/HA; P  < 0.001; fig. 5C).
Fig. 5. (  A  ) The concentration of halothane producing loss of righting reflexes did not differ between SL/SL (wild-type) and HA/HA (homozygous knock-in) mice. HA/HA mice inhaling isoflurane (  B  ) or enflurane (  C  ) required more anesthetic than SL/SL mice for loss of righting reflexes (  P  < 0.01 and  P  < 0.001, respectively). 
Fig. 5. (  A  ) The concentration of halothane producing loss of righting reflexes did not differ between SL/SL (wild-type) and HA/HA (homozygous knock-in) mice. HA/HA mice inhaling isoflurane (  B  ) or enflurane (  C  ) required more anesthetic than SL/SL mice for loss of righting reflexes (  P  < 0.01 and  P  < 0.001, respectively). 
Fig. 5. (  A  ) The concentration of halothane producing loss of righting reflexes did not differ between SL/SL (wild-type) and HA/HA (homozygous knock-in) mice. HA/HA mice inhaling isoflurane (  B  ) or enflurane (  C  ) required more anesthetic than SL/SL mice for loss of righting reflexes (  P  < 0.01 and  P  < 0.001, respectively). 
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Discussion
The knock-in mice harboring the HA/HA mutation were designed to enable us to evaluate the significance of the α1subunit to the anesthetic effects of isoflurane. The electrophysiologic recordings from hippocampal neurons show that the decay time constants of mIPSCs in interneurons from HA/HA mice decay significantly faster compared with the mIPSCs of wild-type mice (SL/SL). In addition, whereas isoflurane and halothane prolonged mIPSCs in the wild-type animals, the effects of isoflurane (but not halothane) was significantly reduced in the HA/HA animals. These findings are all consistent with the expression in the hippocampus of mutated α1subunits, and with a role for GABAARs containing α1subunits in generating inhibitory synaptic currents in pyramidal cells and interneurons. The smaller residual effect of isoflurane in the HA/HA animals presumably reflects the activation of a distinct population of isoflurane-sensitive GABAARs at hippocampal synapses, perhaps containing α2and/or α3subunits. At GABAergic synapses onto cerebellar Purkinje cells,21 for example, or thalamocortical relay neurons,22 the IPSC is generated by activation of a more homogeneous population of GABAARs containing α1subunits, and IPSC kinetics are fast (τ= 5–10 ms). The kinetics of hippocampal IPSCs10 are slower and presumably reflect a mixed population of receptors.
The mice harboring the α1subunit HA/HA mutation displayed normal sensitivity to the amnestic effects of isoflurane. This suggests that GABAAreceptors containing the α1subunit do not mediate the capacity of isoflurane to produce amnesia. This is an interesting result, because GABAAreceptors containing the α1subunit make up approximately half of the total receptor population in the rodent brain.7 In addition, our hippocampal electrophysiology shows a decrease in the pharmacologic effect of the anesthetic in vivo  in a structure known to be important for learning, the hippocampus. Clearly, such receptors are not critical to the amnestic effect of isoflurane, as they have been shown to be for benzodiazepines.23 The amygdala is another structure known to be important for the acquisition of fear conditioning, and its circuitry is thought to use GABAARs that contain other subunits, such as α2and α5, that have been implicated in the anxiolytic and amnestic effects of the benzodiazepines.23 Therefore, several factors limit the generalization of our results from the GABAAR-containing α1subunits and isoflurane to other GABAAR subtypes and anesthetics. In addition, the faster decay of IPSCs in the HA/HA mice compared with wild-type mice may also lead to compensation for the HA/HA mutation. Although GABAARs containing the α1subunit may not mediate the capacity of isoflurane to produce amnesia, this may not apply to other inhaled anesthetics.
We also conclude that GABAARs containing the α1subunit do not mediate the capacity of desflurane, isoflurane, or halothane to produce immobility in the face of noxious stimulation (“MAC”). This result is less surprising in the sense that inhaled anesthetics are believed to act at the level of the spinal cord, where the GABAAR α1subunit is not highly expressed, although immobility induced by the intravenous anesthetics propofol and etomidate is reduced by mutations in the GABAAR β3subunit.3 The current results for MAC are also consistent with results from pharmacologic studies that suggest no mediation by GABAAR of the immobility produced by inhaled anesthetics.6,24,25 
The HA/HA mice showed an increase in the EC50for isoflurane and enflurane, but not halothane in the LORR test. Because the GABAAR containing the mutated α1subunit does not respond to isoflurane or enflurane, but retains sensitivity to halothane, this result provides evidence that the circuits involved in the LORR response involve GABAARs containing α1subunits. The LORR response produced by ethanol was not altered by this mutation26 —this difference may reflect the much larger potentiation of GABAAR function by anesthetic concentrations of isoflurane as compared with ethanol. The α1HA/HA mutation produces a 14% change in LORR for isoflurane, suggesting that other GABAAR subtypes or additional anesthetic targets are important in generating LORR.
Taken together, these studies suggest the surprising conclusion that several behavioral actions of isoflurane do not require activation of GABAARs containing α1subunits, despite the ubiquity of this receptor subtype at subsynaptic locations in a variety of cortical and subcortical regions, whereas LORR does indeed require GABAARs containing α1subunits. Recent studies suggest that extrasynaptic GABAARs, which can contain α4, α5, α6, and/or Δ subunits, may be especially sensitive to ethanol and volatile anesthetics,27–30 and these may also prove to be significant for the behavioral actions of these drugs. Mice bearing mutations containing α4, α5, α6, and/or Δ subunits in these receptors that parallel those engineered for the current report for α1subunits will allow an evaluation of this hypothesis.
References
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Fig. 1. Example traces of miniature postsynaptic currents (mIPSCs) under control conditions and during application of volatile anesthetics. (  A  ) Average mIPSC traces (from 50 single events) of hippocampal interneurons from the three genotypes (wild-type SL/SL; heterozygous SL/HA; homozygous HA/HA) under control conditions. The current amplitude is normalized to the maximum amplitude to visually compare the decay time of the mIPSCs. (  B  ) Bar diagram of average decay time constants from interneurons and pyramidal cells for all three genotypes. Number of experiments is given in brackets, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL; # significantly different from SL/HA). (  C  ) Effect of 0.16 and 0.31 mm isoflurane on average current traces in all three genotypes. (  D  ) Effect of 0.27 mm halothane on average current traces in all three genotypes. 
Fig. 1. Example traces of miniature postsynaptic currents (mIPSCs) under control conditions and during application of volatile anesthetics. (  A  ) Average mIPSC traces (from 50 single events) of hippocampal interneurons from the three genotypes (wild-type SL/SL; heterozygous SL/HA; homozygous HA/HA) under control conditions. The current amplitude is normalized to the maximum amplitude to visually compare the decay time of the mIPSCs. (  B  ) Bar diagram of average decay time constants from interneurons and pyramidal cells for all three genotypes. Number of experiments is given in brackets, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL; # significantly different from SL/HA). (  C  ) Effect of 0.16 and 0.31 mm isoflurane on average current traces in all three genotypes. (  D  ) Effect of 0.27 mm halothane on average current traces in all three genotypes. 
Fig. 1. Example traces of miniature postsynaptic currents (mIPSCs) under control conditions and during application of volatile anesthetics. (  A  ) Average mIPSC traces (from 50 single events) of hippocampal interneurons from the three genotypes (wild-type SL/SL; heterozygous SL/HA; homozygous HA/HA) under control conditions. The current amplitude is normalized to the maximum amplitude to visually compare the decay time of the mIPSCs. (  B  ) Bar diagram of average decay time constants from interneurons and pyramidal cells for all three genotypes. Number of experiments is given in brackets, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL; # significantly different from SL/HA). (  C  ) Effect of 0.16 and 0.31 mm isoflurane on average current traces in all three genotypes. (  D  ) Effect of 0.27 mm halothane on average current traces in all three genotypes. 
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Fig. 2. Effect of volatile anesthetics on decay time constants of miniature postsynaptic currents in interneurons and pyramidal cells. (  A  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in interneurons for all three genotypes (wild-type SL/SL, heterozygous SL/HA, and homozygous HA/HA). Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). (  B  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in pyramidal cells for all three genotypes. Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). 
Fig. 2. Effect of volatile anesthetics on decay time constants of miniature postsynaptic currents in interneurons and pyramidal cells. (  A  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in interneurons for all three genotypes (wild-type SL/SL, heterozygous SL/HA, and homozygous HA/HA). Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). (  B  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in pyramidal cells for all three genotypes. Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). 
Fig. 2. Effect of volatile anesthetics on decay time constants of miniature postsynaptic currents in interneurons and pyramidal cells. (  A  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in interneurons for all three genotypes (wild-type SL/SL, heterozygous SL/HA, and homozygous HA/HA). Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). (  B  ) Bar diagram of volatile anesthetic–induced percentage change on decay time constants of miniature postsynaptic currents in pyramidal cells for all three genotypes. Number of experiments is given in parentheses, and significance is indicated at the  P  < 0.05 level (* significantly different from SL/SL). 
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Fig. 3. Fear to context was measured as the percent of time a mouse froze in the presence of the environment in which the mouse had received three foot shocks on the preceding day. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.5% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the slope was −90.4 ± 12.2 and the intercept was 50.3 ± 3.6 (n = 64). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −95.8 ± 11.6 and 48.2 ± 3.4 (n = 101 mice). For HA/HA (homozygous knock-in) mice, these values were −85.1 ± 15.0 and 41.6 ± 4.2 (n = 64). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
Fig. 3. Fear to context was measured as the percent of time a mouse froze in the presence of the environment in which the mouse had received three foot shocks on the preceding day. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.5% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the slope was −90.4 ± 12.2 and the intercept was 50.3 ± 3.6 (n = 64). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −95.8 ± 11.6 and 48.2 ± 3.4 (n = 101 mice). For HA/HA (homozygous knock-in) mice, these values were −85.1 ± 15.0 and 41.6 ± 4.2 (n = 64). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
Fig. 3. Fear to context was measured as the percent of time a mouse froze in the presence of the environment in which the mouse had received three foot shocks on the preceding day. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.5% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the slope was −90.4 ± 12.2 and the intercept was 50.3 ± 3.6 (n = 64). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −95.8 ± 11.6 and 48.2 ± 3.4 (n = 101 mice). For HA/HA (homozygous knock-in) mice, these values were −85.1 ± 15.0 and 41.6 ± 4.2 (n = 64). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
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Fig. 4. Fear to tone was measured as the percent of time a mouse froze in the presence of the tone supplied previously (the day before). Freezing was tested in an environment that otherwise differed (in shape, texture and shape of ground and walls, smell, lighting) from the environment in which the mouse had been trained. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.75% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the intercept was −105.9 ± 10.9 and the slope was −105.9 ± 11.0, 72.4 ± 4.2 (n = 72). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −109.6 ± 9.0 and 74.6 ± 3.4 (n = 113 mice). For HA/HA (homozygous knock-in) mice, these values were −120.2 ± 17.6 and 74.7 ± 5.4 (n = 66). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
Fig. 4. Fear to tone was measured as the percent of time a mouse froze in the presence of the tone supplied previously (the day before). Freezing was tested in an environment that otherwise differed (in shape, texture and shape of ground and walls, smell, lighting) from the environment in which the mouse had been trained. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.75% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the intercept was −105.9 ± 10.9 and the slope was −105.9 ± 11.0, 72.4 ± 4.2 (n = 72). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −109.6 ± 9.0 and 74.6 ± 3.4 (n = 113 mice). For HA/HA (homozygous knock-in) mice, these values were −120.2 ± 17.6 and 74.7 ± 5.4 (n = 66). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
Fig. 4. Fear to tone was measured as the percent of time a mouse froze in the presence of the tone supplied previously (the day before). Freezing was tested in an environment that otherwise differed (in shape, texture and shape of ground and walls, smell, lighting) from the environment in which the mouse had been trained. A greater percentage time spent freezing indicated greater remembrance. Increasing isoflurane concentration from 0 to 0.75% decreased freezing in a rectilinear manner. There was no difference by genotype in the capacity of isoflurane to interfere with fear to context as assessed by slope, intercept, or the effective concentration producing 50% of the maximal response (EC50) of the regression lines. For SL/SL (wild-type) mice, the intercept was −105.9 ± 10.9 and the slope was −105.9 ± 11.0, 72.4 ± 4.2 (n = 72). For SL/HA (heterozygous) mice, the slope and intercept (± SE) were −109.6 ± 9.0 and 74.6 ± 3.4 (n = 113 mice). For HA/HA (homozygous knock-in) mice, these values were −120.2 ± 17.6 and 74.7 ± 5.4 (n = 66). Data for genotypes are displaced slightly on the abscissa to decrease overlapping of error bars. 
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Fig. 5. (  A  ) The concentration of halothane producing loss of righting reflexes did not differ between SL/SL (wild-type) and HA/HA (homozygous knock-in) mice. HA/HA mice inhaling isoflurane (  B  ) or enflurane (  C  ) required more anesthetic than SL/SL mice for loss of righting reflexes (  P  < 0.01 and  P  < 0.001, respectively). 
Fig. 5. (  A  ) The concentration of halothane producing loss of righting reflexes did not differ between SL/SL (wild-type) and HA/HA (homozygous knock-in) mice. HA/HA mice inhaling isoflurane (  B  ) or enflurane (  C  ) required more anesthetic than SL/SL mice for loss of righting reflexes (  P  < 0.01 and  P  < 0.001, respectively). 
Fig. 5. (  A  ) The concentration of halothane producing loss of righting reflexes did not differ between SL/SL (wild-type) and HA/HA (homozygous knock-in) mice. HA/HA mice inhaling isoflurane (  B  ) or enflurane (  C  ) required more anesthetic than SL/SL mice for loss of righting reflexes (  P  < 0.01 and  P  < 0.001, respectively). 
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Table 1. Effect of the α1γ-Aminobutyric Acid Type A Receptor Knock-in Mutations on Minimum Alveolar Concentration 
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Table 1. Effect of the α1γ-Aminobutyric Acid Type A Receptor Knock-in Mutations on Minimum Alveolar Concentration 
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