Enflurane Actions on Spinal Cords from Mice That Lack the β³Subunit of the GABA^AReceptor

A foundation colony was established at Stanford University with six breeding pairs of mice heterozygous (+/−) for the mutant gene incapable of expressing the GABA^Areceptor β³subunit. These breeding pairs were the F6–F7 generations of the mutant line originated at the University of Pittsburgh 3and were derived on a mixed C57GL/6J X 129/Sv/SvJ genetic background. Null mutants (−/−) were identified phenotypically at birth by pink eye color. Wild-type and heterozygous mice have black eyes at birth. All mice were genotyped retrospectively using Southern blot techniques. For the most part, controls were littermates of the nulls; to increase the numbers of homozygous wild types (+/+) in the series, some control experiments were performed using offspring of +/+ pairs.

Protocols were approved by the Institutional Animal Care and Use Committee at Stanford University. Experiments were performed on 1–6-day-old mice, using methods similar to those previously described. 7,8After anesthesia with halothane and decapitation, spinal cords were removed. For extracellular recording, the intact cord from midthoracic through sacral regions was mounted in a superfusion bath and arranged to permit stimulation of a dorsal root and recording from either a ventral root (pEPSP and sVRP) or an adjacent dorsal root (DRP). Stimulus duration was 0.2 ms. Stimulus intensity was adjusted in each experiment to minimize or eliminate the monosynaptic reflex compound action potential from the pEPSP. Intensity was nominally 9 V for the other potentials, which is supramaximal for sVRP and DRP; this intensity is sufficient to recruit small-diameter primary afferent fibers in the dorsal root. 9Isolated spinal cords were perfused at 4 ml/min with artificial cerebrospinal fluid (ACSF) at 27–28°C equilibrated with 95% O²–5% CO², pH 7.3–7.4. The pre-equilibrated ACSF was delivered from glass syringes mounted on an infusion pump set to a constant rate of approximately 4 ml/min. ACSF was of the following composition: 123 mm NaCl, 5 mm KCl, 1.2 mm NaH²PO⁴, 1.3 mm MgSO⁴, 26 mm NaHCO³, 2 mm CaCl², and 30 mm glucose. Single stimuli were delivered to the dorsal root at a constant frequency of 1/50 s throughout the experiment. Responses were digitized, averaged in groups of five, and stored for later analysis.

For patch clamp studies in spinal cord slices, 350-μm-thick slices were sectioned from the lumbar region on a Vibratome (Technical Products International, St. Louis, MO) and removed to oxygenated ACSF at room temperature for a 1-h recovery period. Individual slices were transferred to a chamber constantly superfused with oxygenated ACSF. All experiments were conducted at room temperature. Cells in the spinal cord slice were visualized on a closed-circuit television monitor using infrared illumination and a 40× water immersion objective. Studies were conducted in the large cell bodies in the ventral horn, most commonly seen in the ventral lateral or ventral medial area. In separate studies, these cells were identified as motor neurons by fluorescent labeling with Evans blue dye injected into the hind limb the day before animals were killed, as previously described. 10Patch pipettes were pulled on a Flaming-Brown pipette puller (Sutter Instruments, Novato, CA) and filled with a solution of the following composition: 15 mm NaCl, 110 mm K gluconate, 10 mm HEPES, 2 mm MgCl², 11 mm EGTA, 1 mm CaCl², 2 mm MgATP, pH adjusted with KOH to 7.3. Pipettes typically had a tip resistance of 3–8 MΩ. Motor neuron responses were evoked by electrical stimulation of the dorsal horn via  a concentric bipolar platinum electrode with tip diameter of 0.025 mm, using square wave stimuli 0.1 ms in duration, 1–20 V nominal intensity, with a frequency of 0.03–0.1 Hz. Excitatory postsynaptic currents were measured individually or analyzed statistically by averaging groups of 5–10. In separate experiments, responses were evoked by direct pressure application of glutamate from a pipette positioned near the cell body (Picospritzer, General Valve Division of Parker Hannefin Corporation, Fairfield, NJ). Pressure pulse was 9 psi, and the duration of the pulse was 200 ms. Glutamate concentration in the pipette was 5 mm. We found that these parameters consistently gave a reproducible inward current of good amplitude. Glutamate applications at 1-min intervals produced stable responses over the course of each experiment; receptor desensitization was not observed at this rate of application. Holding potential was −60 mV. The membrane potential value was not corrected for the junction potential, which was −13 mV. Experiments were conducted on a single cell in each slice.

Enflurane was prepared as a saturated stock solution and diluted to the desired concentration in ACSF immediately before use. Concentrations were measured by gas chromatography of samples from the perfusion chamber. Concentrations given with reference to MAC are based on adult rat MAC; MAC for volatile anesthetics in 1-week-old rats is approximately 20% higher than in adults, and a similar age-dependent potency difference probably occurs in mice. 11,12 

In the whole cord studies, effects of enflurane, bicuculline, and strychnine on ventral root responses were measured as area under the curve at the data point corresponding to 30-min exposure and normalized to control values. pEPSP area encompassed the first 100 ms of the response, and SVRP the first 10 s. Effects on the dorsal root potential were measured as amplitudes of the early transient peak (latency determined in each experiment) and of the late steady state response 1 s after the stimulus. Significance of differences between cords from +/+ and −/− animals were tested by Student t  test for unpaired data. Enflurane application was made first alone and then repeated in the presence of the antagonist in the same preparation, using the antagonist-modified response as the new control to control for the effect of the antagonist itself. The Student t  test with paired data was used to analyze these results. In the patch clamp studies, enflurane was applied for 10 min. Enflurane effects were measured by taking the average of responses during the last 5 min after application compared with the average of responses in the 5 min immediately before the start of application. Enflurane effects were expressed as mean ± SEM, and differences among groups were tested with analysis of variance with the Dunnett test for multiple comparisons.

Enflurane exerted identical depressant effects on evoked responses in spinal cords from wild-type mice and mice lacking the β³subunit of the GABA^Areceptor.

Examples of the actions of enflurane on the pEPSP, sVRP, and DRP in cords from −/− mice are shown in figure 2. Comparisons of enflurane actions on these responses between +/+ and −/− animals are shown graphically in figure 3. Enflurane concentrations for pEPSP and DRP were approximately 0.7 MAC, and for the more sensitive sVRP enflurane concentrations were approximately 0.18 MAC. Enflurane depressant effects on all three responses were similar in extent; there were no significant differences in sensitivity between +/+ and −/− cords in either the ventral root responses (figs. 3A and B) or amplitudes of the early peak and late steady state DRP components (figs. 3C and D;P > 0.05).

The population-evoked responses in intact spinal cord reveal all the actions of an anesthetic on each of the pathways that generate them (fig. 1) but do not allow the possibility of discriminating among actions at different points along the pathway, e.g. , direct actions on motor neurons versus  actions mediated by modulatory interneurons. To probe further for enflurane actions on motor neurons themselves that might be altered by the mutation, we conducted patch clamp studies on both synaptically evoked currents and glutamate-evoked currents in visually identified motor neurons during patch clamp conditions.

Short latency currents evoked in motor neurons by stimulation in the dorsal root entry zone are the single-cell counterpart of the pEPSP in intact cord. Enflurane at approximately 1 MAC depressed synaptically evoked currents in cords from both +/+ and −/− mice. Examples are shown in figures 4A and B. As is the case with the pEPSP in intact cord, there were no differences in enflurane depressant effects on either amplitude or area of synaptically evoked currents between +/+ and −/− cords (figs. 4C–4F).

Brief pulses of glutamate were applied in the presence of tetrodotoxin (300 nm) to block impulse activity. Enflurane depressed these currents to a similar extent in both +/+ and −/− animals (fig. 5A). Data are shown in table 1. Figure 5also shows examples of the interaction between GABA^Areceptors and enflurane depression (fig. 5B).

Similarity of the actions of enflurane on cords from +/+ and −/− mice led us to probe for evidence that GABA receptors were, in fact, fewer in mice with the β³subunit deletion. Two strategies were used: direct activation of GABA^Areceptors by agonists, and block of GABA^Areceptors by antagonists.

In intact spinal cord, activation of GABA receptors will depress the pEPSP by acting directly on the circuit from primary afferents to motor neurons (fig. 1). The GABA^Aagonist muscimol (1 μm) depressed the pEPSP much more in cords from +/+ than −/− mice (fig. 6A). Muscimol depressed the response in +/+ cords to 20.7 ± 3.80% of control versus  76.3 ± 3.63% in −/− cords (n = 5, P < 0.0001). This result clearly suggests that the expression of GABA^Areceptors is markedly reduced in mice with the deletion. A limited series of experiments was conducted in which currents in motor neurons were evoked by direct GABA application. Examples from +/+ and −/− mice are shown in figure 6B. Although there are GABA receptors on motor neurons in −/− mice, the currents evoked appear to be much smaller than in wild types. Because current amplitudes vary from neuron to neuron, no quantitative conclusions can be drawn from these experiments.

Examples of bicuculline actions on VRPs are shown in figures 7A and Band on the DRPs in figure 7C. The results are shown quantitatively in graphs for the VRPs in figures 8A and Band for two components of the DRP in figure 8C and D.

In intact rodent spinal cord, GABA^Areceptor–mediated inhibitory tone is substantial; treatment with the GABA^Areceptor antagonists bicuculline (figs. 7A and B) or picrotoxin (data not shown) elevates VRPs and, at high concentrations, spontaneous waves of activity appear. Bicuculline (1 μm) increased the area of the pEPSP and sVRP in spinal cords from both null mutants (−/−) and wild-type (+/+) animals (P < 0.01;figs. 8A and B). There were no differences between −/− and +/+ in extent of bicuculline elevation of EPSP area (P > 0.05;fig. 8A); bicuculline increased sVRP area significantly (P < 0.05) more in −/− than in +/+ cords (fig. 8B). Inspection of the records suggests that this apparent difference is caused by greater distortion of the baseline by the sporadic unsynchronized activity induced by bicuculline in wild-type animals. A typical example of the actions of bicuculline in this respect is shown in figure 7B.

The DRP is mediated, in part, by GABA^Areceptors on the primary afferent nerve terminals. The GABA^Areceptor antagonist bicuculline (10 μm) depressed the amplitude of the early peak of the DRP in both −/− and +/+ cords (fig. 7C). Visually, the effects of bicuculline on the DRP resembled those of the mutation (fig. 7C), decreasing the amplitude of the fast-rising early component and leaving a relatively slow-rising late component insensitive to bicuculline. Bicuculline depressed the early peak of the DRP to a significantly (P < 0.05) greater extent in cords from +/+ than −/− mice (fig. 8C). Bicuculline elevated the amplitude of the late steady state component of this response to a similar extent (P > 0.05) in cords from +/+ and −/− animals (fig. 8D).

We tested the part played by GABA^Areceptors in the depressant actions of enflurane by examining the extent to which bicuculline, by blocking these receptors, could attenuate the actions of enflurane.

In intact cords, a protocol was designed to expose each spinal cord to enflurane alone and to enflurane in the presence of bicuculline, with controls for the effect of bicuculline alone (fig. 9). After an initial 30-min control period, preparations were exposed to a sequence of enflurane (30 min), wash (1 h), bicuculline (30 min), enflurane plus bicuculline (30 min), and wash with bicuculline (1 h). Controls for enflurane effects alone were the initial controls, and for enflurane with bicuculline were new controls at the end of bicuculline treatment. Bicuculline significantly (P < 0.05) attenuated the depressant effects of enflurane on both pEPSP and sVRP in cords from +/+ mice but not in cords from −/− mice (fig. 9).

After the control period, bicuculline (20 μm) was applied, followed by enflurane. Examples for +/+ and −/− cells are shown in figure 5B. Because of the limited time over which cells could be held for each experiment, it was not possible to compare the actions of enflurane alone and in the presence of bicuculline in the same cell, as was performed in intact cords. Comparisons among the groups of cells exposed separately to enflurane or to enflurane plus bicuculline are shown in table 1. Bicuculline significantly (P < 0.05) attenuated the depressant effects of enflurane on glutamate-evoked currents in motor neurons from +/+ but not −/− cells (table 1).

Because enflurane MAC is elevated in the mutants, we had predicted that enflurane would appear less potent in depressing motor neuron responses in spinal cords from −/− than +/+ mice. This was not the case, there being no difference between the groups. Experiments were conducted to test the hypothesis that the equal enflurane sensitivity of normal and mutant cords was caused by changes in other receptors that would compensate for the loss of GABA^Areceptors containing the β³subunit, both to restore excitability toward more normal levels and to serve as substitute targets for enflurane actions.

A logical substitute for missing GABA^Areceptors is the related chloride channel, the glycine receptor, which plays an important inhibitory role in spinal cord. To test for developmental upregulation of glycine receptors, we examined the actions of the glycine antagonist strychnine on the pEPSP. Strychnine (400 nm) increased the area of the pEPSP significantly more (P < 0.05) in −/− than in +/+ cords, suggesting that glycinergic inhibition is, in fact, increased in −/− cords (fig. 10A). To test whether the depressant effects of enflurane were equalized by the increase in glycine receptors, we probed the interaction between glycine receptors and enflurane potency in a protocol similar to that used for GABA^Areceptor–enflurane interactions. If compensatory overexpression of the glycine receptor is responsible for equalizing the actions of enflurane, then strychnine is predicted to attenuate enflurane-depressant effects more in −/− than in +/+ cords. However, although there was a tendency for strychnine to reduce the apparent potency of enflurane in depressing the pEPSP, this was not significant (P > 0.05) in either +/+ or −/− cords, and there were no differences between the two populations in this respect (figs. 10B and C).

In the presence of bicuculline to block GABA^Areceptors, glutamate-evoked currents in mutant mouse spinal motor neurons are significantly (P < 0.05) more sensitive to enflurane than those of wild-type animals (table 1). This result suggests some change in glutamate receptors, perhaps a reduction in number, or in some cellular component that acts on glutamate receptors and may indirectly mediate an anesthetic effect.

Behavioral studies, in which enflurane MAC requirement was increased in mice lacking the β³subunit of the GABA^Areceptor, 4led to the prediction that spinal cord sensitivity to enflurane would be decreased in β³−/− mice. Contrary to the prediction, enflurane exerted similar depressant effects in cords from −/− and +/+ mice. Behaviorally, the increased MAC in −/− mice is not dramatic, approximately 26% for enflurane and less for halothane. 4Given the steepness of the dose–response curve for MAC, this is not a large difference and certainly does not support a dominant role for GABA^Areceptors containing the β³subunit in determining MAC. It is possible that the source of the increased enflurane requirement in −/− mice is supraspinal rather than at the spinal level. MAC is determined predominantly in spinal cord but with supraspinal influences. 6In isolated spinal cord and spinal cord slices, supraspinal modulation is removed. The results of the current study thus suggest that the increase in enflurane requirement observed in intact −/− animals may be the result of a difference in supraspinal enflurane actions. Deficits in GABA-mediated inhibition have been observed in some supraspinal nuclei in −/− mice. 13 

The GABA^Aagonist muscimol exerted much greater depressant effects on the pEPSP in wild-type mice than in mutants, a strong indication that there are fewer GABA^Areceptors in the circuit that mediates this response in −/− mice. The comparatively small currents evoked by GABA in motor neurons of −/− mice are also suggestive of fewer receptors, although quantitative conclusions cannot be drawn from the limited number of experiments. However, the results with the antagonist bicuculline on ventral root potentials suggested equal tonic GABA-mediated inhibition in both populations. There are several possible explanations for the discrepancy. First, GABA^Areceptors of different subunit composition may be expressed in mutant animals. However, it is not likely that differences in bicuculline sensitivity among GABA^Areceptors of different subunit composition account for equal bicuculline sensitivity. GABA^Areceptors are, by definition, sensitive to bicuculline. The type of β subunit present in recombinant receptors expressed in nonneural cells does not greatly influence receptor pharmacology. 14–16Second, and somewhat more likely, at concentrations greater than those used in the current study, bicuculline also blocks glycine receptors in rat motor neurons. 17Because glycine receptors appear to be present in greater numbers in −/− mice, it is possible that the increased population of glycine receptors might be more sensitive to bicuculline than in normal animals. Finally, there is a difference in source of the agonist and location of the receptors between the muscimol and bicuculline experiments. Muscimol directly acts on the monosynaptic reflex circuit to depress the evoked response, whereas tonic GABA inhibition is caused by GABA release from interneurons. Muscimol will activate all accessible receptors, whereas the effects of bicuculline will be predominantly on receptors located at synapses. With respect to the DRP, the results with bicuculline clearly show a decrease in the early bicuculline-sensitive component of the response in −/− animals. This is in agreement with the finding that there are fewer GABA^Areceptors on dorsal root ganglion cells in −/− mice than in wild-type mice. 18Dorsal root ganglion cells are the cell bodies of the primary afferent sensory neurons whose terminals generate the DRP.

Although the correlation between anesthetic potency and enhancement of GABA^Areceptor function is excellent, 19it is not uniform. Some fluorinated alkanols abolish movement in response to a noxious stimulus 20but do not act to increase current through GABA^Areceptors. 21The inert gas xenon is a general anesthetic agent but has little effect on GABA^Areceptors. 22Thus, although actions on GABA^Areceptors may be important for many inhalation agents, they are not essential for some, and for others, actions on other receptors may also contribute to MAC. In a recent study, intrathecal application of picrotoxin increased isoflurane MAC by a maximum of 43%. 23 

We have previously shown in wild-type mice that enflurane depresses glutamate-evoked currents by direct actions, independent of actions on either GABA^Aor glycine receptors. 8In the present studies, blockade of the GABA^Areceptor by bicuculline attenuated but did not block the depressant actions of enflurane on VRPs and on glutamate-evoked motor neuron currents in spinal cords from +/+ mice. This result suggests that GABA^Areceptors contribute to anesthetic actions in these mice but do not account for all of them. In −/− mice, there was no attenuation of the depressant actions of enflurane by bicuculline, suggesting little or no contribution of GABA^Areceptors to anesthetic actions in these animals.

The results with muscimol provide further evidence that GABA^Areceptors are not of dominant importance in determining spinal actions of anesthetics. Muscimol, which activates GABA^Areceptors, was clearly less effective in depressing a ventral root response in −/− animals. Muscimol is not an ideal control for a putative anesthetic action, since volatile anesthetic agents are thought to act by enhancing the effectiveness of GABA rather than by directly gating the receptor. However, it is probable that an anesthetic that exerted its effects predominantly by enhancing GABA^Areceptor activity should have also been less effective in these animals. Because the effects of enflurane alone were similar in the two populations, this result suggests that GABA^Areceptors are not dominant contributors to the effects of enflurane. Furthermore, loss of bicuculline attenuation coupled with identical enflurane actions suggest the molecular basis of spinal anesthetic sensitivity is shifted to another target than GABA^Areceptors in mice that lack β³subunits.

The exaggerated effect of strychnine in cords from −/− mice suggests that glycinergic inhibition is more prominent in these animals than in wild-type controls, as a result of developmental compensation leading to overexpression of this other inhibitory chloride channel. It is plausible to suggest that glycine receptors could, in part, substitute for GABA^Areceptors. Glycine and GABA play similar inhibitory roles in spinal cord and are coreleased from the same interneuronal terminals onto motor neurons. 17Tonic inhibition to rat spinal cord motor neurons is predominantly mediated by glycine rather than by GABA^Areceptors, although the latter also play a part. 24Glycine receptors as well as GABA^Areceptors are subject to anesthetic enhancement. 2However, block of the glycine receptor with strychnine did not significantly attenuate enflurane-depressant effects on the pEPSP in either +/+ or −/− cords, although there was a tendency in that direction. At least for this response, glycine receptors appear to play a relatively small role in anesthetic depressant actions, and there is no evidence that they become the substitute target for enflurane in −/− cords. Studies in rats using intrathecal application of strychnine suggest that, as is the case for a GABA antagonist, MAC cannot be increased by more than approximately 40%; furthermore, there was a plateau effect for both picrotoxin and strychnine such that intrathecal administration of maximally effective amounts of both drugs together increased MAC by a maximum of 47%. 23 

The depressant actions of enflurane were identical in +/+ and −/− mice, yet the role of the GABA^Areceptor in enflurane actions is less in the −/− mice. A decrease in the role of the GABA^Areceptor in enflurane actions, combined with identical enflurane effects, forces the conclusion that the molecular basis for anesthetic depression has shifted as a result of the mutation. Some other receptor(s) or ion channels play a greater role in anesthetic depressant effects in −/− than in wild-type mice. The proportions of molecular substrates in the mix responsible for anesthetic actions have changed. We have shown that the substitute target is not the glycine receptor, although glycine inhibition is increased. Other possibilities include compensatory downregulation of excitatory glutamate receptors to maintain a balance between excitation and inhibition. In addition to changes in both excitatory and inhibitory amino acid–mediated neurotransmission in the mutant mice, there also may be changes in other less well-studied potential anesthetic targets, including ion channels modulated by second messenger-linked receptors or the poorly understood class of voltage-independent baseline potassium channels. Figure 11illustrates this hypothetical shift in the basis for enflurane depression of spinal neurotransmission.

This study represents an early stage in the developing strategy of using genetically engineered animals to probe for target sites of anesthetic action. The results show that a volatile anesthetic agent may have a number of different target sites. Compensatory changes in expression of other receptors certainly occurred during development of this global knockout, and a shift in the contribution of these other receptors to the sum of anesthetic effects has also occurred. These changes were not a predictable result of the mutation. These studies illustrate some of the problems in interpreting results from such animals, a situation that will certainly improve as conditional knockouts and other types of mutations become available. They also illustrate the importance of testing hypotheses of anesthetic mechanisms in intact circuitry where all potential molecular targets are present, in addition to conducting detailed molecular studies on isolated receptors.

The authors thank the members of Program Project Group GM47818 for helpful discussion, Diane Gong (E. I. Eger’s laboratory at the University of California, San Francisco, CA) for measuring enflurane concentrations, and Carolyn Ferguson and JoAnn Steinmiller (University of Pittsburgh, Pittsburgh, PA) for mouse genotype analysis.