Because the synaptic inhibition in the human brain is largely mediated by gamma-aminobutyric acid (GABA), the GABA receptor is of primary interest for the study of the working mechanism of general anesthetics. This article examines the interaction between this type of ion channel and nitrous oxide (N2O).
Patch clamp recording techniques were applied to investigate the effects of N2O on GABA(A) receptor channels in a whole-cell configuration at room temperature. Acutely dissociated rat hippocampal cells from the CA1 region were used. Rapid application of the agonist muscimol and anesthetics (N2O, pentobarbital, and ethanol) was accomplished using a Y tube application system. Peak chloride (Cl-) currents were measured.
Short-term application of muscimol (5-30 microM) with dissolved N2O (80%, approximately 22.5 mM) increased the Cl- current (approximately 140%) compared with muscimol alone. This effect is comparable with results the authors obtained with ethanol (800 mM) and pentobarbital (100 microM). Prolonged exposure (9 min) to N2O further increased Cl- currents by an additional 50%. Concentrations of N2O lower than 12 mM did not show an enhancement of this current, whereas application of N2O alone did not result in any Cl- conductance.
These results indicate that N2O can enhance GABA(A) channel-mediated Cl- currents by modulating the effect of the specific GABA(A) agonist; it is not active by itself.
ALTHOUGH the working mechanism of anesthetic agents has been a subject of scientific investigations since the 19th century, only recently, with advances in cell electrophysiology, have investigations on the actions of anesthetics at the single cell level been possible. The strong correlation between anesthetic potency and oil solubility does provide a framework within which a hypothesis of the working mechanism of anesthetics must fit. It gives no clues, however, about the exact site(s) of action. Because not only lipids but also proteins have hydrophobic sites, recent research is directed toward exploring the effects of anesthetic agents on proteins controlling the excitation and inhibition of neurons. One of the proteins playing a central role in modulation of neuronal excitability is the gamma-aminobutyric acid (GABA) receptor. Electrophysiologic and pharmacologic studies have identified two primary receptor subtypes, GABAAand GABAB. Whereas the GABABreceptor is less well studied in the central nervous system, modulation of the GABAAreceptor has been extensively explored. Activation of the GABAAreceptor leads to an increase in chloride (Cl sup -) conductance through an ion channel associated with the receptor complex. The GABAAreceptor-mediated Cl sup - conductance has been shown to be enhanced by many different anesthetic agents, such as ethanol, [3,4]benzodiazepines, barbiturates, steroid anesthetics, and propofol. [6,7]The effects of volatile anesthetics have been studied less frequently, probably because of technical difficulties with their administration, although reports of enhancement of GABA-activated Cl sup - currents do exist. [8–10]
Although there are reports on the effect of nitrous oxide (N sub 2 O) on glutamate-gated ion channels and nicotinic receptors, the direct effects of N2O on GABAAreceptor functioning have not been described. The possibility of modification of this important system by N2O, however, has indirectly been indicated by several authors. Modification of N2O analgesia by benzodiazepine receptors, reversal of its anxiolytic effects by benzodiazepine receptor antagonists, [14,15]and development of cross-tolerance between ethanol and N2O suggest an involvement of the GABAergic system in the action of N2O. The hypothesis that N2O modulates the GABAAreceptor function is investigated in this study.
Materials and Methods
This study was approved by the Animal Care and Use Committee at University of Amsterdam. CA1 pyramidal neurons were isolated according to a modified version of the method developed by Kay and Wong. Adult male Wistar rats ([nearly =] 10 weeks old, [nearly =] 200–300 g) were decapitated under ether anesthesia. The hippocampus was dissected and cut into 500-micro meter slices, from which 1-mm2tissue blocks from the CA1 area were dissected and incubated for 90 min at 32 [degree sign] Celsius in oxygenated dissociation solution containing (in mM): 120 NaCl, 20 piperazone-N,N'-bis [2-ethanesulfonic acid], 5 KCl, 1 CaCl2, 1 MgCl2, 25 D-glucose, and 1 mg/ml bovine trypsin (trypsin 1–300, ICN; Nutritional Biochemicals, Cleveland, OH); a pH of 7.0 was set with NaOH. After incubation, the tissue was washed once with dissociation solution without enzyme and kept at room temperature and oxygenated. Before every experiment, one tissue block was triturated in 0.75 ml extracellular medium (composition described subsequently) through a Pasteur pipette. The cell suspension was brought into a perfusion chamber mounted on an inverted microscope. After settling on the bottom, fusiform neurons with a bright and smooth appearance and no visible organelles were selected for recording. Only one cell per tissue block was used for the experiment. These criteria have proved successful for electrophysiologic measurements. 
Patch Clamp and Solutions
Standard patch clamp technique was used to measure GABAAreceptor-mediated Cl sup - currents under whole-cell voltage-clamp conditions with a computer-controlled amplifier (APC-8; Medical Systems Corp., Greenvale, NY). Data acquisition and analysis and control of the amplifier were performed with pClamp 6.0 in combination with a Digidata 1200 A/D converter (Axon Instruments Inc., Foster City, CA). After giga-seal formation and cell membrane rupture, resulting in the whole-cell configuration, the membrane capacitance (usually between 8 and 10 pF) and series resistance were compensated. The dial readout was taken as a measure of the membrane capacitance and was used to normalize for different cell sizes afterward. Holding potential in all experiments was -70 mV. Before application of an agonist, a hyperpolarizing step of -20 mV was applied during 150 ms to determine a nonspecific linear leak conductance. This conductance was used for off-line correction of measured currents for nonspecific linear leak. All measurements were done at room temperature (20–22 [degree sign] Celsius) using patch pipettes of 2–3 M Omega resistance, as measured in the extracellular solution. The pipette solution contained (in mM): 100 CsF, 20 tetraethyl-ammonium chloride, 20 phosphocreatine, 50 U/ml phosphocreatine kinase, 10 EGTA, 10 HEPES, 2 MgCl2, 0.5 CaCl2, 2 Mg-ATP, 0.1 Na-GTP, 0.1 leupeptin; pH was set at 7.3 with CsOH. The chamber was continuously perfused (4 ml/min) with an extracellular medium containing (in mM): 110 NaCl, 10 HEPES, 5 KCl, 25 tetraethyl-ammonium chloride, 5 CaCl2, 5 4-aminopyridine, 1 MgCl sub 2, 25 D-glucose, and 0.001 tetrodotoxin; pH was adjusted to 7.4 with NaOH. Addition of N2O to this solution did not change the pH. Unless specified otherwise, all chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
GABAAreceptor-mediated Cl sup - currents were evoked by application of the selective agonist muscimol using a modified Y tube application system. The agonist and other drugs were dissolved in extracellular solution and continuously supplied by controlled pressure through silicon tubes with an inner diameter of 100 micro meter. Three of these tubes, with different solutions, were positioned within a wider second (glass) pipette with a conic shape and a tip diameter of 100 micro meter. This outer pipette was held under negative pressure to remove agonist and drug-containing solutions before it was able to flow into the bath. During application (500 ms), regulated pressure was added to one of the inner tubes, thereby ejecting the solution from this specific tube into the bath. The pressure was regulated using a pico injector (PLI-100; Medical Systems Corp.) and timed by pClamp. Fast and reproducible applications resulted from a close position of the cell to the application system (<100 micro meter) and continuous flow of the extracellular solution (complete application within 70 ms and complete removal within 300 ms). To ensure that a stable Cl sup - current was achieved, several preapplications (usually 3 or 4) were given before every experiment. All agonist applications with or without additional drugs were performed through this Y tube application system. Only during prolonged exposure to N2O was a drug dissolved in the extracellular fluid. A period of 1 min was allowed for recovery between every application. At the end of experiments, muscimol was applied and the current was compared with initial currents ensuring that the system remained stable during the experiment.
Quantification of Nitrous Oxide in the Recording Chamber and Application Tubing
To assess the accuracy and effectiveness of application of N sub 2 O to the cell, before the experiments, evaluation of the measurement setup was performed. The dissolved gas concentrations were measured with a mass spectrometer (QP 9000; CASE, Biggin Hill, UK) at room temperature. Preparation of different gas mixtures for the calibration of the mass spectrometer was done by a gas mixture pump (Digamix M; Wosthoff, Bochum, Germany). The gas mixture was monitored using a Datex Capnomac Ultima (Datex Instrumentarium Corp., Helsinki, Finland). The calibration was performed in a tonometer (IL 237 Tonometer; Instrumentation Laboratory, Inc., Ijsselstein, The Netherlands), ensuring a complete equilibration between the solution and the supplied gas. The solution in the tonometer was the extracellular solution used in the experiments on cells. The inset in Figure 1shows the linear correlation between the dissolved gas concentration measured by the spectrometer and the N2O concentration as measured by Datex Capnoma Ultima. Once calibrated, the mass spectrometer was used to measure the dissolved N2O concentrations in the perfusion chamber, later to be used for electrophysiologic measurements. The best control of N2O concentration is obtained by continuous perfusion with N sub 2 O-saturated extracellular medium. Figure 1shows the time course of N2O concentration in the perfusion chamber if the perfusion flow is set at 4 ml/min. Once the extracellular fluid was saturated with N2O, it remained saturated for hours when stored in polyethylene tubing (internal diameter 0.86 mm, outer diameter 1.27 mm; Portex Ltd., Hythe, Kent, UK). Because of this, a short-term application with an application pipette, as described earlier, is possible. Once the fluid is exposed to air, in the perfusion chamber, a desaturation occurs as shown in Figure 1. To allow for rapid change of extracellular fluids, a small measurement chamber (0.2 ml) was used in N2O concentration-response experiments.
Calculation of Minimum Alveolar Concentration
The minimum alveolar concentration value for N2O is thought to be [nearly =] 150%(vol/vol) for rodents. Taking into account that this value was established at 37 [degree sign] Celsius, the dissolved N2O at 1 minimum alveolar concentration can be calculated as 27.4 mM. The extracellular solution, in most of our experiments, was saturated with 80% N2O, and using published solubility coefficients the amount of dissolved N2O at 20 [degree sign] Celsius was calculated to be 22.5 mM. Lower concentrations of N2O were obtained by mixing it with oxygen. Oxygen concentration between 20% and 100% did not affect the muscimol induced Cl sup - current.
Analyses of the modulation on the muscimol-activated peak Cl sup - currents were performed using a paired t test. Differences in which the probability value was < 0.05 were considered significant and are indicated by asterisks in the figures. Data obtained during prolonged N2O exposition were analyzed by analysis of variance. Student's t test was used to compare the effects of exposure time on Cl sup - current. Comparison between different modulators of GABAAreceptor was performed by analysis of variance. Results are expressed as the mean +/- SEM with the number of measured cells (n), given in parentheses.
To establish the correct muscimol concentration range to be used for the study of effects of anesthetics on the GABAAreceptor, the concentration-response relationship between muscimol and Cl sup - current was investigated. Figure 2(A) shows an example of the effect of three different concentrations of muscimol on the Cl sup - currents of one hippocampal neuron. During exposure to muscimol concentrations of 10 and 40 micro Meter, a sustained inward current could be observed. Analysis of the current-voltage relation and the reversal potential of these currents showed that the current was mainly carried by chloride ions (data not shown). Higher concentrations of this agonist (see the example of 75 micro Meter in Figure 2(A)) produced a transient current. The decay of the current, at these concentrations, is caused by a combination of desensitization and decrease in Cl sup - gradient.  Figure 2(B) shows a dose-response curve for muscimol concentrations ranging between 0 and 200 micro Meter. Maximum current is induced with concentrations > 100 micro Meter, with a 50% increase as compared to the maximum current (EC50) occurring at [nearly =] 40 micro Meter. Because of the increased desensitization with higher concentrations of muscimol, the rest of the study was performed with concentrations of muscimol less than EC50.
Short-term Exposure to Nitrous Oxide
To study the effects of short-term exposure to N2O on the muscimol-induced Cl sup - currents, the cells were alternately exposed to 10 micro Meter muscimol and 10 micro Meter muscimol with N sub 2 O (Figure 3). An increase of [nearly =] 30% in Cl sup - current is shown in this example. The use of low concentrations of the agonist in the Y tube system, in combination with a high flow of the agonist-free extracellular solution, produced a fast increase and decrease of the current. These currents remained stable during the 500-ms application period (Figure 2). Figure 4shows the effects on the Cl sup - current of short-term N2O exposure at different concentrations of muscimol. This graph shows that short-term administration of N2O with muscimol increases the current to values [nearly =] 40% higher than the application of muscimol without dissolved N2O. Studying the effects of N2O on higher concentrations of muscimol is difficult because of desensitization, but a few cells that had stable currents showed no increase when N2O was applied with the agonist (data not shown). Further, Figure 4shows that in this setting no Cl sup - current could be elicited by N2O without an agonist.
Prolonged Exposure to Nitrous Oxide
To measure the effect of prolonged exposure to N2O, the Cl sup - current was measured before and after the extracellular solution was replaced by an extracellular solution containing dissolved N2O. During a 9-min period, at 3-min intervals, muscimol was applied to the cell after which the extracellular solution was changed again to the one without N2O. Again, at 3-min intervals, muscimol was applied and the Cl sup - currents measured. The data from these experiments, as depicted in Figure 5(A), show that prolonged exposure to N2O further increases the Cl sup - current by [nearly =] 50%(SEM = 22%, n = 9). Exposure up to 9 min did not further increase the Cl sup - current. Recovery when the N2O-containing fluid was replaced by normal extracellular fluid was slow and lasted several minutes.
Nitrous Oxide Concentration-Response
The concentration-response relation for the N2O-augmented Cl sup - current is illustrated in Figure 5(B); all responses are normalized to the peak amplitude of the current induced by 10 micro Meter muscimol without N2O. Because prolonged exposure to N2O was needed to obtain maximal increase of muscimol-induced Cl sup - current, the concentration-response relation was measured by exposing the cells to different concentrations of N2O in the extracellular fluid. A marked increase in Cl sup - current can be observed with concentrations of N2O of >or= to 40%(vol/vol).
Effects of Other GABA sub A Receptor Modulators and Antagonist on the Muscimol-induced Cl sup - Current
Cl sup - currents induced by 10 micro Meter muscimol alone or in combination with either 800 mM ethanol (4.2 vol%) or 100 micro Meter pentobarbital (23 micro gram/ml) were studied (Figure 6). For comparison, data obtained by short-term exposure to a combination of N sub 2 O and muscimol are included. The anesthetics ethanol and pentobarbital induced similar increases in Cl sup - current. Combination of the competitive GABAAantagonist bicuculline (20 micro Meter) with muscimol resulted in a marked decrease of the Cl sup - currents, indicating the GABAAspecificity of the measured responses.
Although GABA binds to both GABAAand GABABreceptors, the chloride ion-selective channel GABAAis seen as the target protein of many anesthetic agents. Inhalational anesthetic agents show a plethora of observable phenomena, and, from a clinical point of view, it is unlikely that they all can be explained through a modification of one single receptor protein. Many reports on modification of other ligand-gated ion channels by inhalational anesthetic agents point toward a nonspecificity on the receptor level. Inhalational anesthetic agents are, however, used largely because they induce loss of consciousness. This effect may be entirely explained by a modulation of GABAAreceptors, as has been learned through the study of benzodiazepines. 
In this work, we studied the effects of short- and long-term application of N2O on GABAAreceptor channels. To avoid interference caused by a network of cells, the experiments were performed on acutely isolated hippocampal neurons. In addition to a naturally present cell-to-cell variation, the procedure of isolation (and especially the use of trypsin) caused an additional variation in quality of cell surfaces. The intercell variation in muscimol-induced Cl sup - currents, as observed most clearly at high muscimol concentrations (Figure 2(B)), is likely to be explained by this variation in quality. To avoid these problems and desensitization, low concentrations of muscimol were used. Figure 4shows the moderate increase in Cl sup - currents that can be obtained with a short-term application of muscimol combined with N2O, and Figure 3shows an example of the currents in such an experiment. The ability to return to control values after application of N2O was considered essential as an additional control that a stable current was present. The observed increase of muscimol-induced current by short-term application of N2O could not be explained by a modification of Cl sup - gradients or other muscimol-activated conductances because the reversal potential (approximately -35 mV) remained the same and currents continued to reverse as a single phase in the presence of N2O (data not shown). Complex gating behavior of the GABAAreceptor does not allow further elucidation of the possible cause of the observed phenomena at this level of research. The possibility of modulation, however, seems to be dependent on the subtype structure of different subunits forming the GABAAreceptor. [25,26]Because some subtypes have possible phosphorylation sites, it has been argued that phosphorylation plays an essential role in the modulation of GABAAreceptor function. [25,27]In addition, it has been shown that subtype composition and the possibility of phosphorylation play an important role in the sensitivity to anesthetic agents. [28–31]If phosphorylation plays a role in the modulation of the GABAAreceptor, the process is supposed to be relatively slow. The time course of a GABA-induced Cl sup - current decrease, by enhancement of phosphorylation, was measured to be several minutes. [25,32]Considering this finding, a 500-ms exposure to N2O, as in our short-term experiments, may not have been long enough to show the modulation to its full extent. Our experiments that apply prolonged exposure to N2O by exchange of extracellular fluid show a further increase in Cl sup - conductance, indicating that in our experiments a slow process is involved as well. Although the nature of this slow process may be found in changes of phosphorylation processes and thus be of relevance to our understanding of interaction between N2O and the GABAAreceptor, one should keep the possibility of an artificial derangement in mind. The process of patch clamping, using suction, introduces contents of pipette solution into the cell, causing dialysis of the intracellular side. This may dilute some cytoplasmic biochemicals, giving rise to changes in intracellular processes. Using a gramicidin perforated patch, further insight into the intracellular modulation of Cl sup - conducting channels, under more physiologic conditions, can be expected.
We compared the increase as induced by N2O with other agents known to modulate the GABA-mediated Cl sup - current. Figure 6shows that the sensitivity of the rat hippocampal neurons to ethanol and pentobarbital in our experiments is in agreement with earlier reports. [3,34]
This study demonstrates the facilitatory effect of N2O on the GABAAreceptor. The effects reported resemble those of other inhalational anesthetic agents, strongly suggesting that these agents, despite their chemical differences, exert similar actions on this important receptor.
The authors thank E. Branderhorst and Prof. C. Ince for help with the mass spectrometry and L. van Urk and A. van der Wardt for technical assistance in the preparation of isolated cells.