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© 1999 International Anesthesia Research Society
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Abstract |
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Halothane affects synaptic transmission in the rat hippocampus with a 50% effective dose (ED50) correlating with clinical figures for minimum alveolar anesthetic concentration (MAC). Halothane dose-dependently suppresses glutamate receptor-mediated excitatory postsynaptic potentials (EPSPs) in the rat hippocampus. It also inhibits voltage-gated Na+ channels. The anticonvulsant lamotrigine acts as a Na+ channel antagonist and inhibits glutamate release after Na+ channel activation. Given their known similar sites of action, the combination of halothane and lamotrigine may alter the inhibition produced by either drug alone. Extracellular recordings of field EPSPs were obtained from the dentate gyrus in the presence of 100 µM picrotoxin (to block GABAA receptors). Stimulation at 30 Hz (200 ms, pulse duration 0.1 ms, six pulses) allowed us to investigate use-dependent block (UDB). Once a stable equilibrium was established, halothane and lamotrigine were administered via the perfusate, and recordings were collected. Both halothane (n = 12) and lamotrigine (n = 6) exhibited reversible inhibition of the EPSP (ED50 0.28 mM [1.2%] and 100 µM, respectively) at low-frequency stimulation. Slices (n = 6) exposed to halothane 0.2 mM (0.75%), then to lamotrigine, showed reduced sensitivity compared with lamotrigine alone. Halothane 0.2 mM potentiated the control UDB (Pulse 6: 31% ± 11% control versus 20.5% ± 2.5% halothane 0.75%; P < 0.05; n = 6). Lamotrigine had no effect on control UDB. The combination (n = 6) did not alter UDB effects compared with controls or lamotrigine alone. Halothane may reduce the effect of lamotrigine on glutamate release, either at the receptor or via effects at the inactivated Na+ channel. The site of interaction requires further examination.
Implications: The general and local anesthetic drugs halothane and lamotrigine act at both the glutamate receptor and the Na+ channels and, in our experiments, independently inhibited synaptic transmission at low-frequency stimulation. Although halothane potentiated control use-dependent block, lamotrigine had no effect. Halothane attenuated the inhibitory dose-dependent effects of lamotrigine on synaptic transmission at a low frequency. The clinical importance of this interaction in patients presenting for anesthesia remains unanswered.
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Introduction |
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Synaptic transmission is affected by volatile anesthetics administered at clinically relevant concentrations (1). Although the precise sites and mechanisms involved have yet to be elucidated, there is general agreement that these anesthetics depress excitatory transmission and enhance synaptic inhibition as a result of their actions via the neurotransmitters glutamate (2–4) and GABA (5,6), respectively. Halothane has a consistently inhibitory action on glutamatergic excitation, and it affects both glutamate ionotropic receptor subtypes (N-methyl-D-aspartate [NMDA] and non-NMDA) (7). The action of halothane on metabotropic glutamate receptors is less well known. The depression of excitatory transmission may be due to reduced transmitter release or to anesthetics reducing the quantal content of motorneuron excitatory postsynaptic potentials (EPSP) but not affecting miniature EPSP amplitude (8).
Glutamate release is affected by drugs other than anesthetics. Both neuronal Ca2+ channel blockers and certain Na+ channel blockers may attenuate glutamate release (9,10). The anticonvulsant lamotrigine (3,5-diamino-6[2,3-dichlorophenyl]-1,2,4-triazine) is a known Na+ channel blocker (11). Studies suggest that lamotrigine acts primarily at voltage-gated Na+ channels, although there is some debate as to whether its principal action occurs at the fast or slow inactivated channels (12,13). In intact brain slices, lamotrigine acts at the voltage-gated Na+ channels that are responsible for the action potential upstroke in a voltage- and use-dependent manner (13). Although lamotrigine (10–300 µM) seems not to affect basal glutamate release, it profoundly inhibits glutamate release after Na+ channel stimulation by veratrine (14).
Given the known effects of lamotrigine and of halothane on both Na+ channels and glutamate release, we examined the effects of lamotrigine on nerve volley amplitude (presynaptic spike), field EPSP, and use-dependent block. The combined effects of halothane and lamotrigine on synaptic mechanisms may alter the inhibition produced by either drug alone. A demonstrated interaction may have clinical application, given the increasing use of this anticonvulsant in clinical practice.
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Methods |
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After receiving institutional animal investigation committee approval, all experiments were performed on transverse slices of the hippocampus of the male Wistar rat (weight 50–100 g) by standard methods (17). Brains were rapidly removed after decapitation and placed in cold oxygenated (95% O2, 5% CO2) artificial cerebrospinal fluid (concentrations [in mM]: NaCl 120, KCl 2.5, NaH2PO4 1.25, NaHCO3 26, MgSO4 2.0, CaCl2 1.5, D-glucose 10). Slices were cut at a thickness of 350 µm using a Camden vibroslice (Campden Inst. Ltd., Loughborough, Leicester, UK) and transferred to a storage container containing oxygenated medium at room temperature. The slices were then transferred as required to a recording chamber for submerged slices and continuously perfused at a rate of 5–7 mL/min with oxygenated medium at 30–32°C for at least 1 h. The perfusate for all experimental slices contained picrotoxin (100 µM) to block GABAA receptor-mediated synaptic transmission.
Halothane was delivered using a Tec 3 series vaporizer (Cyprane Ltd., Keighley, England) in the gas supply line. The oxygen/carbon dioxide mixture passed through the vaporizer at a constant flow rate (2 L/min) and was humidified and bubbled through a reservoir containing the artificial cerebrospinal fluid solution. The bath perfusate was recirculated using a roller pump to provide a maximally stable system for the application of volatile anesthetics. The vaporizer was calibrated by relating the millimolar gas concentration in the saline perfusate to the percentage of gas delivered by the vaporizer to the gas line. This was performed by adding 10-L samples from the tissue bath to 25 mL of n-hexane in a volumetric flask. The mixture was shaken well, and duplicate samples were transferred to vials for analysis. Samples were analyzed using a gas chromatograph with electron capture detector and autosampler. The column used was 30 m long with an internal diameter of 0.32 mm and film thickness of 4.0 µm. The carrier gas was nitrogen; injector temperature was 280°C, detector temperature was 300°C; initial oven temperature was 80°C for 1 min; rate was 10°C/min to 140°C; total run time was 7 min; and the injection was splitless.
The O2/CO2/halothane mixture was allowed to equilibrate for at least 15 min before measurements were recorded. A second reservoir filled with artificial cerebrospinal fluid and continuously oxygenated was maintained for washout of the slice to allow examination of electrical recovery after drug application.
A glass stimulating electrode was placed in the outer two thirds of the molecular layer of the dentate gyrus to stimulate the presynaptic afferent fibers entering the hippocampal formation in the medial perforant pathway. Stimulation was performed with a stimulator every 20 s (intensity 5–15 V, duration 0.1 ms), evoking both nerve volley (presynaptic spike) and the field EPSP in the dentate granule cell dendrites of 30%–50% maximal amplitude EPSP. The medial perforant path was distinguished by the presence of characteristic paired pulse depression at short interstimulus intervals (20–50 ms) (16,17). High-frequency stimulation was performed by applying a single train of 30-Hz frequency (200 ms duration; each pulse 0.1 ms) at the same intensity as the low-frequency stimulation. The recording electrode was placed in the middle third of the dentate molecular layer to record paired population granule cell dendritic postsynaptic responses to medial perforant pathway stimulation. Evoked responses were amplified using a microelectrode amplifier and were displayed on standard storage oscilloscope.
Drugs used were halothane (2-bromo-2-chloro-1,1,1-trifluoroethane; May & Baker, Dagenham, England) and lamotrigine (Wellcome Foundation, London, England). All other chemicals were obtained from Sigma Chemical Co. (Poole, Dorset, UK). Lamotrigine was dissolved in 0.1 M HCl and was diluted with the perfusate to the desired concentrations for the experiments. Lamotrigine was bath-applied to the slices as part of the perfusate during recording for at least 20 min at each dose examined. Halothane application has been described. Data were recorded before drug application, during drug application, and, in some cases, after washout with artificial cerebrospinal fluid.
All currents recorded were filtered at 10-kHz bandpass, digitized on-line (Lab PC+; National Instruments, Austin, TX) and analyzed off-line using the Strathclyde electrophysiology software (J. Dempster, University of Strathclyde, Glascow, UK). EPSP amplitudes were measured as peak negativity from baseline. EPSP slopes were also measured, as amplitude may be reduced by the presence of population spikes and noise. There were no significant differences between the results obtained with either slope or amplitude. Drug effects are expressed as a percentage of control (effect measure/control measure x 100), and summarized results are expressed as mean ± SEM. For high-frequency stimulation, data are given as a percentage of the slope of the first impulse in the train. Differences among the control, lamotrigine, and lamotrigine with halothane groups were determined by using analysis of variance by ranks. Pairwise comparisons were performed by using paired two-tailed Student’s t-tests. Data are reported as mean ± SEM, and P < 0.05 was taken to indicate statistical significance.
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Results |
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The low-frequency evoked EPSP amplitude showed a dose-dependent reduction when exposed to stepwise increases in halothane concentration (Fig. 1C), with a 50% effective concentration (EC50) of 1.2% (0.28 mM) (n = 12). Nerve volley amplitude also responded dose-dependently (Fig. 1B), with the maximal reduction in amplitude being 61% ± 9% at a halothane concentration of 4% (0.72 mM). Representative examples of an EPSP and nerve volley are illustrated in Figure 1A.
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Lamotrigine applied to the slice preparation in concentrations ranging from 100 nM to 100 µM also demonstrated a dose-dependent effect. At 100 µM, the nerve volley amplitude was reduced to 52.6% ± 19% of control, and the EPSP was reduced to 50% ± 12% of its control value (n = 6). Increasing the lamotrigine concentration to 250 µM caused no further reduction in EPSP amplitude (n = 3). When the hippocampal preparation was exposed to halothane 0.75% (0.2 mM), and thereafter to stepwise increases in lamotrigine (100 nM to 100 µM), the inhibition in both nerve volley and EPSP amplitude was attenuated (Fig 2). The maximal inhibition of nerve volley amplitude for the combination was 73% ± 17% of control. The effect of the combination on the EPSP amplitude was more profound: 101% ± 10% and 88% ± 9% of control values for 50 µM and 100 µM lamotrigine, respectively. This was significantly different from the values for lamotrigine alone (P < 0.05).
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) in increasing doses from 0.1 to 100 µM reduced the NV amplitude compared with control (*P < 0.05). The combination of lamotrigine and halothane 0.75% (0.2 mM; 
) attenuated the inhibition caused by lamotrigine alone. The inhibitory effect of halothane before lamotrigine application has been corrected to 100% (first point). There was no significant difference from control at any point. At 100 nM lamotrigine only, the combination with halothane differed significantly from lamotrigine alone ($P < 0.05). B, Lamotrigine (Use-dependent inhibition was examined using a high-frequency train (30 Hz, 200-ms duration, six stimuli) of an intensity similar to low-frequency stimulation. The results of such stimulation in the absence of drug manipulation (n = 12) are given in Figure 3A with a representation of a typical waveform. Regardless of the dose administered, lamotrigine did not alter control use-dependent block. At 100 µM, the sixth pulse in the train was 46% ± 12% of the first pulse, which was not significantly different from the control value (31% ± 11%; n = 6) (Fig. 3B). Halothane potentiated control use-dependent block. A significant effect was seen at all concentrations (P < 0.05), with the maximal effect at concentrations of 3% (0.64 mM). This effect was not linearly dose-dependent. Halothane 3% (0.64 mM) resulted in Pulse 6 being reduced to 6% ± 2% of the first pulse in the train from a control of 31% ± 11% (n = 6) (Fig. 3C).
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) had no effect on use-dependent block compared with control (
). Data for Pulse 6 are illustrated and are given as mean ± SEM (n = 6). C, Halothane (Examination of use-dependent inhibition in preparations exposed to halothane 0.75% (0.2 mM) and to increasing doses of lamotrigine showed a pattern largely similar to that obtained when lamotrigine was used alone. Data for lamotrigine 10 µM are given in Figure 4A. At no point was there a significant difference between the curves (n = 6). At lamotrigine concentrations of 100 µM, there was a potentiation of the second pulse in the train to 132% ± 34%, compared with the first pulse. When combined with halothane 0.75% (0.2 mM), the second pulse was 74% ± 13% (P < 0.05). Although the use-dependent inhibition remained greater for the combined drug group compared with the control and lamotrigine alone groups, at no subsequent point was it significant (Pulse 6: 46% ± 11% vs 22% ± 6% for the lamotrigine and combined drug groups, respectively; P = 0.15).
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) did not exhibit use-dependent block different from that of controls (
) and to lamotrigine 10 µM ( |
Discussion |
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The demonstrated effects of halothane and lamotrigine administered alone on tonic EPSP amplitude confirm previous work (1,8). The precise site of action of halothane remains unspecified, but halothane has known effects on glutamate release (1). Whether this is primarily due to presynaptic or to postsynaptic actions is controversial (2,4). MacIver et al. (4) examined the effects of volatile anesthetics on paired pulse facilitation at Schaffer collateral fiber glutamate synapses in CA1 pyramidal neurons. Although they demonstrated a concentration-dependent depression of glutamate-mediated excitatory synaptic transmission, the increase in facilitation was consistent with a presynaptic action. Perouansky et al. (2) confirmed halothane suppression of glutamatergic excitatory postsynaptic currents, but they thought that the predominant action was presynaptic because of halothane’s voltage insensitivity, lack of action on kinetics, and equal depression of NMDA and non-NMDA current amplitudes. Although some authors have demonstrated that the NMDA receptors are 3–4 times more sensitive to the action of halothane than the non-NMDA receptors (7), others have shown AMPA-induced currents to be more sensitive (18). We demonstrated larger decreases in EPSP amplitude for any given halothane concentration compared with the amplitude decrease for the nerve volley. This may indicate that halothane affects transmitter release and postsynaptic events, as well as nerve volley conduction.
Lamotrigine showed a less profound, but similarly dose-dependent, reduction in both nerve volley and EPSP amplitude. The EC50 of 100 µM (at an estimated resting membrane potential of -70 mV) [previous work in our laboratory has shown the resting membrane potential for granule cells to be -68 ± 4 mV (15)] is less than that demonstrated by Xie et al. (13) for currents at holding potentials of -90 mV, but it compares more favorably with the data described by Kuo and Lu (12), who reported an approximate ED50 of 70 µM at a holding potential of -80 mV. Both groups of investigators demonstrated that the tonic inhibition produced by lamotrigine was voltage-dependent.
Halothane demonstrated use-dependent inhibition of EPSP amplitude at 30-Hz stimulation frequency, in agreement with previous work (19). Lamotrigine did not exhibit use-dependent effects at 30 Hz. Previous work confirms the absence of use-dependent inhibition with lamotrigine, regardless of dose, at stimulation frequencies of 11–50 Hz and of short duration (3.5 ms) (13). However, significant inhibition of current amplitudes was observed for pulses of longer duration (20 ms). Our stimulation protocol (six pulses, 200 ms, pulse duration 0.1 ms) produced shorter pulses than those demonstrating frequency-dependent blockade in the work of Xie et al. (13). Because use-dependent inhibition is believed to occur due to either selective interaction of a drug with the open state of a channel or drug interaction with a channel conformation, this duration difference was thought to indicate lamotrigine’s interaction with the slow inactivated conformation of the Na+ channel. Kuo and Lu (12) suggested that lamotrigine bound preferentially to the inactivated state of the Na+ channel but that the kinetics of recovery indicated binding to the fast inactivated state.
The effects of lamotrigine in the presence of halothane 0.75% (0.2 mM) on tonic and high-frequency EPSP amplitude are not easily resolved because they differ depending on the frequency of stimulation. During low-frequency stimulation, the combination of drugs shows a much reduced effect on the EPSP compared with lamotrigine alone (0.2 µM only). Both drugs affect glutamate release, but lamotrigine seems to primarily affect glutamate release after Na+ channel activation by veratrine (14). Lamotrigine’s effects on glutamate release after electrical Na+ channel activation are less clear. Compared with carbamazepine and oxcarbazepine, lamotrigine has similar potency in inhibiting glutamate, [3H]-GABA, and [3H]-dopamine release stimulated by veratrine (10). Although the effect of lamotrigine on electrically stimulated glutamate release was not investigated, carbamazepine and oxcarbazepine are 5–7 times less potent inhibitors of electrically mediated transmitter release. The inhibition of glutamate release by halothane may prevent the less potent lamotrigine from affecting glutamate release to the same extent as when administered alone.
Halothane and lamotrigine have known effects at the Na+ channel. Although the effects of volatile anesthetics at ion channels has been discounted as a meaningful mechanism at clinically useful concentrations (20), more recent work has suggested that the concentration of volatile anesthetic required to inhibit voltage-dependent Na+ currents is highly dependent on the resting membrane potential (21). Cells examined at holding potentials of -60 mV require minimum alveolar anesthetic concentrations to produce 50% reduction in Na+ currents, whereas cells held at -120 mV require 3–5 times as much volatile anesthetic. Rehberg et al. (21) suggested two possible mechanisms by which anesthetics suppress central nervous system Na+ channel currents. They may exert a potential-independent suppression of resting/open Na+ channels or may produce a hyperpolarizing shift in the voltage dependence of Na+ channel inactivation. The modulated receptor hypothesis of drug interaction, in which different channel states have different affinities for drug binding, best describes the volatile anesthetic modification of Na+ channels, binding preferentially to the inactivated channel. The action of lamotrigine at inactivated Na+ channels may result in a degree of competition with halothane at those channels and may explain the change from lamotrigine alone (Figure 2).
Another possible explanation for the effects of the combination of drugs is that halothane and other volatile anesthetics hyperpolarize the cell membrane, thus reducing the resting membrane potential (22). As previously discussed, the action of lamotrigine in reducing EPSP amplitudes is voltage-dependent; therefore, the hyperpolarizing effect of halothane would shift lamotrigine’s dose-response curve to the right by this mechanism alone. However, the hyperpolarization caused by halothane (1–4 mM) is on the order of 4–5 mV; therefore, this mechanism only partly explains the effect of the combination of lamotrigine and halothane.
The clinical implications of this interaction require further exploration. Does the administration of halothane to a patient receiving lamotrigine for the control of epilepsy present a greater risk for perioperative convulsions? The brain concentrations of lamotrigine that are clinically effective range from 10 to 40 µM, much lower than the ED50 in our experiments. Interestingly, these concentrations are often increased to 70 µM in the presence of other anticonvulsants. The lack of effect of the combination of drugs on use-dependent block suggests that halothane does not exert its usual effect. This may indicate that higher halothane concentrations are necessary to produce the same effect.
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Acknowledgments |
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This research was supported by grants from the Mater Foundation.
We thank Dr. Mary Lehane and Professor J. J. A. Heffron, Laboratory Director, Analytical Biochemistry and Toxicology Laboratory, University College Cork, for their help in analyzing halothane samples for gas chromatography.
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Footnotes |
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The results were presented in part at the 73rd International Anesthesia Research Society annual meeting, Los Angeles, CA, March 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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- Pocock G, Richards CD. Excitatory and inhibitory synaptic mechanisms in anaesthesia. Anaesth 1993;71:134–47.
- Perouansky M, Baranov D, Salman M, Yaari Y. Effects of halothane on glutamate receptor-mediated excitatory postsynaptic currents: a patch-clamp study in adult mouse hippocampal slices. Anesthesiology 1995;83:109–19.[Web of Science][Medline]
- Perouansky M, Kirson ED, Yaari Y. Halothane blocks synaptic excitation of inhibitory interneurons. Anesthesiology 1996;85:1431–8.[Web of Science][Medline]
- MacIver MB, Mikulec AA, Amagasu SM, Monroe FA. Volatile anesthetics depress glutamate transmission via presynaptic actions. Anesthesiology 1996;85:823–34.[Web of Science][Medline]
- MacDonald RL, Olsen RW. GABAA receptor channels. Ann Neurosci 1994;17:569–602.
- Tanelian DL, Kosek P, Mody I, MacIver MB. The role of the GABAA receptor/chloride channel complex in anesthesia. Anesthesiology 1993;78:757–76.[Web of Science][Medline]
- Narimatsu E, Tsai YC, Gerhold TD, et al. A comparison of the effect of halothane on N-methyl-D-aspartate and non–N-methyl-D-aspartate receptor-mediated excitatory synaptic transmission in the hippocampus. Anesth Analg 1996;82:843–7.[Abstract]
-
Weakly JN. Effects of barbiturates on "quantal" synaptic transmission in spinal motoneurons. J Physiol 1969;204:63–77.
[Abstract/Free Full Text] - Wu G, Kim HK, Zornow MH. Transient brain ischaemia in rabbits: the effect of omega-conopeptide MVIIC on hippocampal excitatory amino acids. Brain Res 1995;692:118–22.[Web of Science][Medline]
-
Waldmeier PC, Baumann PA, Wicki P, et al. Similar potency of carbamazepine, oxcarbazepine and lamotrigine in inhibiting the release of glutamate and other neurotransmitters. Neurology 1995;45:1907–13.
[Abstract/Free Full Text] - Leach MJ, Baxter MG, Critchley MA. Neurochemical and behavioral aspects of lamotrigine. Epilepsia 1991;32 (Suppl 2):S4–8.
- Kuo CC, Lu L. Characterization of lamotrigine inhibition of Na+ channels in rat hippocampal neurones. Br J Pharmacol 1997;121:1231–8.[Web of Science][Medline]
- Xie X, Lancaster B, Peakman T, Garthwaite J. Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurones. Pflugers Arch 1995;430:437–46.[Web of Science][Medline]
- Leach MJ, Marden CM, Miller AA. Pharmacological studies of lamotrigine, a novel potential antiepileptic drug. II. Neurochemical studies on the mechanism of action. Epilepsia 1986;27:490–7.[Web of Science][Medline]
- O’Connor JJ, Rowan MJ, Anwyl R. Long lasting enhancement of NMDA receptor-mediated synaptic transmission by metabotropic glutamate receptor activation. Nature 1994;367:557–9.[Medline]
- McNaughton BL. Evidence for two physiologically distinct perforant pathways to the fascia dentata. Brain Res 1980;199:1–19.[Web of Science][Medline]
- O’Leary DM, Cassidy EM, O’Connor JJ. Group II and III metabotropic receptors modulate paired pulse depression in the rat dentate gyrus in vitro. Eur J Pharmacol 1997;340:35–44.[Web of Science][Medline]
- Kirson ED, Yarri Y, Perouansky M. Presynaptic and postsynaptic actions of halothane at glutamatergic synapses in the mouse hippocampus. Br J Pharmacol 1998;124:1607–14.[Web of Science][Medline]
- Strichartz G. Use dependent conduction block produced by volatile general anesthetic agents. Acta Anaesthesiol Scand 1980;24:402–6.[Web of Science][Medline]
- Franks NP, Lieb WR. Molecular and cellular mechanisms of general anesthesia. Nature 1994;367:607–13.[Medline]
- Rehberg B, Xiao Y-H, Duch DS. Central nervous system sodium channels are significantly suppressed at clinical concentrations of volatile anesthetics. Anesthesiology 1996;84:1223–33.[Web of Science][Medline]
-
Nicoll RA, Madison DV. General anesthetics hyperpolarize neurons in the vertebrate central nervous system. Science 1982;217:1055–7.
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