The neuronal and molecular targets of the inhalational general anesthetic xenon are a matter of debate. The current knowledge is largely based on studies using neurons in culture or heterologous expression systems. In the current study, the authors evaluated for the first time the effect of xenon on synaptic transmission in the basolateral amygdala in an in vitro brain slice preparation of the mouse.
A patch clamp technique was used to evaluate the effects of xenon on N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs), as well as on gamma-aminobutyric acid type A receptor-mediated inhibitory postsynaptic currents. The currents were either evoked upon electrical stimulation (NMDA-eEPSCs, AMPA-eEPSCs) or upon focal, laser-guided photolysis of caged l-glutamate (p-NMDA-Cs, p-AMPA-Cs). In addition, the authors investigated the effects of xenon on miniature EPSCs.
Xenon reversibly reduced basal synaptic transmission but had no effect on gamma-aminobutyric acid type A receptor-mediated inhibitory synaptic transmission. Xenon concentration-dependently diminished NMDA-eEPSCs and p-NMDA-Cs to the same amount. Likewise, xenon-induced reduction of AMPA-eEPSCs and p-AMPA-Cs did not differ. Xenon did not affect the frequency of miniature EPSCs but reduced their amplitude.
In the current study, xenon considerably depressed NMDA and AMPA receptor-mediated synaptic transmission in the basolateral amygdala without affecting inhibitory synaptic transmission. The results provide evidence that the effects of xenon on NMDA- and AMPA-EPSCs are primarily mediated via postsynaptic mechanisms.
THE noble gas xenon is an inhalational anesthetic with analgesic and neuroprotective properties combined with beneficial pharmacokinetic and cardiovascular characteristics.1During the past two decades, the safety and efficacy of xenon anesthesia in human patients has been demonstrated in numerous clinical trials.2–4However, the mechanisms how xenon exerts its anesthetic and analgesic properties have not been clarified yet.
In a simplified manner, general anesthesia can be described as a state with reduced central nervous system excitability. This state can be reached by enhancement of inhibitory or reduction of excitatory neurotransmission, or the combination of both.5
Enhancement of inhibitory γ-aminobutyric acid–mediated (GABAergic) synaptic transmission is considered to be of fundamental importance for the anesthetic mechanism of, e.g. , volatile and intravenous anesthetics.6,7Two studies8,9showed an enhancement of Cl−currents through heterologously expressed recombinant γ-aminobutyric acid type A (GABAA) receptors by xenon. In contrast, other studies10,11demonstrated that xenon exerts no effects on GABAAreceptor–mediated inhibitory postsynaptic currents (GABAA-IPSCs) recorded from cultured hippocampal neurons. Therefore, there is general agreement that effects on GABA receptors most probably do not contribute to the xenon anesthetic state.12
Glutamate is the major excitatory neurotransmitter, and it is generally accepted that xenon reduces glutamatergic current responses. However, it is not yet clear which subtype of glutamate receptors (N -methyl-d-aspartate [NMDA], α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA], or kainic acid receptors) plays the most important role in mediating the xenon anesthetic state.12The first investigations on neuronal mechanisms of xenon postulated an exclusive action on NMDA receptors.10,11Meanwhile, several in vitro studies showed that xenon reduces currents not only through recombinant NMDA receptors,9but also through recombinant non-NMDA receptors.13,14Furthermore, an in vivo study using the model organism Caenorhabditis elegans showed diverse anesthesia-associated behavioral changes, which were induced by xenon via AMPA but not NMDA receptors.15
To our knowledge, there are no data yet available investigating the effect of xenon on synaptic transmission in acute brain slice preparations. Current insights of the neuronal and molecular targets of xenon are instead based on studies using heterologous expression systems or neurons in culture. The aim of the current study was to determine the effects of xenon on inhibitory and excitatory synaptic transmission mediated via native GABAA, NMDA, and AMPA receptors in an acute mice brain slice preparation, which provides a considerable degree of presynaptic and postsynaptic functionality and thus represents a much less artificial model for central nervous system neuronal signaling.
Materials and Methods
The experimental protocols were approved by the Ethical Committee on Animal Care and Use of the Government of Bavaria, Germany. Male C57Bl6 mice, 28–42 days old, were killed by cervical dislocation, and the brains were rapidly removed into ice-cold artificial cerebrospinal fluid (ACSF) containing 125 mm NaCl, 2.5 mm KCl, 25 mm NaHCO3, 2 mm CaCl2, 2 mm MgCl2, 25 mm d-glucose, and 1.25 mm NaH2PO4(all from RBI/Sigma, Deisenhofen, Germany). Saturation with a mixture of 95% O2–5% CO2(carbogen gas) led to a pH of 7.4. Coronal slices (350 μm thick) were prepared using a microtome (HM 650 V; Microm International, Walldorf, Germany). Slices were allowed to recover in a storage chamber (34°C) for at least 1 h before being transferred to the recording chamber.
A platinum ring with nylon filaments was used to fix the slice on the bottom of the recording chamber, which was continuously perfused (2 ml/min) with ACSF. We used infrared-phase contrast-enhanced videomicroscopy (Zeiss, Oberkochen, Germany; for details, see Dodt et al. 16) to visualize the somata of neurons within the basolateral amygdala (BLA). Whole cell patch clamp recordings were performed from principal neurons, which were identified according to their accommodating firing pattern and long-lasting afterhyperpolarizations.17
The patch pipettes were pulled from thin-walled borosilicate glass tubes with inner filament (OD 1.5 mm, ID 1.17 mm, GC150TF-10; Clark Electromedical Instruments, Pangbourne Reading, United Kingdom) and heat polished using a two-step horizontal puller (DMZ-Universal Puller; Zeitz-Instruments, Munich, Germany). Pipettes had a series resistance of 4–6 MΩ when filled with a solution containing 130 mm K-d-gluconate, 5 mm KCl, 0.5 mm EGTA, 2 mm MgCl2, 10 mm HEPES, 5 mm d-glucose, and 20 mm Na2-phosphocreatine (all from RBI/Sigma). Currents were recorded with a switched voltage clamp amplifier (SEC 10 l; NPI Electronic, Tamm, Germany) with switching frequencies of 60–80 kHz (25% duty cycle). Series resistance was monitored continuously and compensated in bridge mode. All patch clamp experiments were performed at room temperature (22°–25°C) to ensure a sufficient oxygenation of the neurons.
Figure 1depicts a scheme of the stimulation and recording conditions. Electrically evoked postsynaptic currents (ePSCs) were induced by square pulse stimuli (6–20 V, 0.05–0.2 ms, interstimulus interval 15 s) delivered via an ultrafine bipolar tungsten electrode (insulated to the tip, 5-μm tip diameter), which was placed to the lateral nucleus of the amygdala. Preceding each tissue stimulation, neuronal input resistance was determined by injecting a current pulse hyperpolarizing by 10 mV for 200 ms.
Photolytically evoked excitatory currents were induced upon focal photolysis of caged l-glutamate.16Thereby, the beam of an ultraviolet laser (355-nm wavelength, frequency-tripled Nd:YVO4, 100-kHz pulse repetition rate; DPSS Lasers, San Jose, CA) was focused by the objective (60×, 0.9 numerical aperture; Olympus, Tokyo, Japan) on a small spot (5 μm in diameter) positioned on a dendrite approximately 10–20 μm from the soma. Laser stimulation was delivered alternating with electrical stimulation in intervals of 15 s. Once a stable whole cell recording had been obtained, “caged glutamate” (γ-α-carboxy-2-nitrobenzyl [γ-CNB]–caged glutamate) was added to the recirculating perfusate (0.25 mm). Caged glutamate had no discernible effect on neurons per se .16Glutamate was released by Q-switching brief laser pulses (3–5 ms; intensity 1–2 mW), applied at regular intervals of 30 s throughout the experiment.
To isolate specific currents, we used d(−)-2-amino-5-phosphonopentanoic acid (AP5; 50 μm), 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (5 μm), 3-amino-propyl(diethoxymethyl)phosphinic acid (CGP35348; 200 μm), and bicuculline methiodide (20 μm). Of these four receptor antagonists, an appropriate cocktail was used, with 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo- benzo[f]quinoxaline-7-sulfonamide omitted for AMPA, AP5 omitted for NMDA, and bicuculline methiodide omitted for GABAAreceptor–mediated responses. The metabotropic GABABreceptors were blocked, because their activation would interfere with either AMPA or NMDA receptor currents and would not allow a detailed analysis of receptor kinetics. For AMPA receptor–mediated currents, holding potential was set to −70 mV, and for NMDA and GABAAreceptor–mediated currents, holding potential was set to −30 and −50 mV, respectively.
Miniature excitatory postsynaptic currents (mEPSCs) were continuously recorded (10 min) at −70 mV in the presence of 1 μm tetrodotoxin, 50 μm AP5, 200 μm CGP35348, and 20 μm bicuculline methiodide. mEPSCs were completely blocked in the presence of 5 μm 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (data not shown).
The whole cell responses were amplified, low-pass filtered (3 kHz), and then digitized (ITC-16 Computer Interface; Instrutech Corp., Port Washington, NY) with a sampling frequency of 9 kHz, and stored to a hard drive (Power Macintosh G3 computer, data acquisition software Pulse version 8.5; Heka Electronic GmbH, Lambrecht, Germany). The obtained data were analyzed with IGOR Pro software (WaveMetrics, Portland, OR). For detection of mEPSCs, the amplitude threshold was defined as the threefold amplitude of the baseline variance (noise level), and the events identified were subsequently verified visually. We then quantified the frequency and peak amplitudes of the detected events.
Under control conditions, the superfusing ACSF was exposed to a mixture of 65% N2–30% O2–5% CO2; for xenon application, the ASCF was gassed with a mixture of 65% xenon–30% O2–5% CO2. In some subsets of experiments, xenon was applied at three lower concentrations using gas mixtures of 30% xenon–35% N2, 18% xenon–47% N2, and 5% xenon–60% N2, respectively (each gas mixture supplemented with 30% O2and 5% CO2). Replacement of nitrogen with xenon did not change the pH of the gassed ACSF. All gas mixtures were purchased from Linde AG (Unterschleissheim, Germany) and applied at a flow rate of 0.3–0.5 l/min to the ACSF reservoir. Oxygen and carbon dioxide concentrations were verified with a calibrated gas monitor (Datex Capnomac Ultima; Duisburg, Germany). Tubing was made of polytetrafluoroethylene (Teflon; VWR International, Darmstadt, Germany) to minimize loss of xenon. Concentration measurements of dissolved xenon were accomplished using Headspace-Gaschromatography (RCC Ltd., Itingen, Switzerland). When gassing the ACSF with the 65% (30%, 18%, or 5%) xenon gas mixture, the revealing mean ± SD (n = 4) concentration of xenon in ACSF was 1.9 ± 0.2 mm (1.1 ± 0.1, 0.6 ± 0.1, or 0.2 ± 0.1 mm). Saturation (> 95%) was achieved within 5 min after the start of gassing. For comparison, other studies describe xenon saturation of 3.9 mm or 2.0 mm when gassing with pure xenon,8,93.4 mm when gassing with 80 vol% xenon,11,13or 3.54 mm when gassing with 84 vol%,14which would result in a calculated mean of approximately 2.3 mm at an assumed 65 vol% xenon saturation. The difference from our obtained concentration of 1.9 mm might be explained by an increased evaporation due to our open-chamber system.
Tetrodotoxin, AP5, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide, bicuculline methiodide, γ-CNB–caged glutamate (all from RBI/Sigma, Deisenhofen, Germany), GYKI52466 (a kind gift from David Leander, Ph.D., Neuroscience Division, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, IN), and CGP35348 (Novartis Laboratories, Basel, Switzerland) were bath applied at known concentrations via the superfusion system.
For statistical evaluation of the xenon effect on the neuronal excitability, we first partitioned the 40-min recording time into equidistant subintervals of 5-min length and then determined the averaged relative amplitudes in each of them. A two factorial multivariate analysis of variance with repeated measures design was applied on the averaged relative amplitudes with interval as a within-subjects factor and kind of receptor as a between-subjects factor. By the analysis of variance, differences between the various intervals were tested by tests with contrasts, whereas differences between the various receptors were tested by post hoc tests. Averaged resting membrane potential, input resistance, and series resistance of neurons as well as averaged decay time constants and charge transfer of the current responses over the three phases—baseline (5–10 min), xenon (20–25 min), and washout (35–40 min)—were tested for significant differences also by a multivariate analysis of variance with repeated-measures design. Differences in the amplitudes and frequency of mEPSCs were tested about significances with one-factorial (one-way) analysis of variance. As a nominal level of significance, we accepted α= 0.05. It was corrected (according to the Bonferroni procedure) for all posteriori tests (tests with contrasts and post hoc tests).
The IC50values and Hill coefficients were assessed on mean normalized data with explicit weighing by SEM according to the four parameter logistic equation %Control = 100/(1 + ([Antagonist]/IC50)Hill Coef). Numerical data are presented as mean ± SEM, with the number of experiments (= neurons) indicated, if not stated otherwise. In graphs where error bars are not shown, they are smaller than the size of the symbol.
All control recordings were made from slices superfused with ACSF, which was saturated with a mixture of 65% N2–30% O2–5% CO2. Despite the reduced oxygen concentration, the recording of stable postsynaptic currents was feasible. In a recent work,18we showed in detail that decreasing the oxygen concentration in the ACSF from 95% to 30% did not produce changes in the resting membrane potential or input resistance (Rin) of neurons in the BLA. Furthermore, neither rise time and decay time constant of electrically evoked NMDA-eEPSCs nor rate and amplitude of miniature AMPA-mEPSCs were changed when reducing oxygen concentration from 95% to 30%.18
For xenon application, the gas mixture of 65% N2–30% O2–5% CO2was changed to a mixture containing 65% xenon–30% O2–5% CO2. Analysis of variance revealed that 1.9 mm xenon did not alter the resting membrane potential, input resistance, or series resistance of neurons in the BLA (table 1).
To evaluate whether xenon reduces overall neuronal excitability, the effect of 1.9 mm xenon on the basal synaptic transmission in the BLA was examined. Compound postsynaptic currents evoked by electrical stimulation (ePSCs) were recorded in the absence of any receptor blockers from neurons visualized by infrared videomicroscopy. Ten to 15 min after xenon application (1.9 mm), the ePSCs recorded from neurons of the BLA were reduced to 80.2 ± 4.2% of control responses (fig. 2A). The averaged relative amplitude in this interval was significantly lower than in the two baseline intervals (tests with contrasts in multivariate analysis of variance, P < 0.05). Upon termination of xenon application, the ePSCs recovered to control level. Application of xenon for longer than 15 min did not result in an additional reduction of ePSCs (data not shown).
To evaluate whether xenon exerts any effects on inhibitory synaptic transmission in the BLA, electrically evoked GABAAreceptor–mediated eIPSCs were recorded in the presence of 50 μm AP5, 5 μm 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulfonamide, and 200 μm CGP35348 at a holding potential of −50 mV. Xenon (1.9 mm) did not affect the amplitudes of GABAA-eIPSCs (fig. 2B). In agreement with other authors,19,20the decay of GABAA-eIPSCs was fitted biexponentially, with two time constants, τdecayfast and τdecayslow. Under xenon, τdecayfast was significantly and reversibly prolonged (table 2). However, charge transfer of GABAA-eIPSCs, which was calculated by integrating the area of the current traces, was not changed (table 2).
N -Methyl-d-aspartate receptor–mediated EPSCs were evoked using either electrical stimulation (NMDA-eEPSCs) or photolytic uncaging of glutamate (p-NMDA-Cs) (fig. 3). Xenon (1.9 mm) reversibly diminished electrically evoked NMDA-eEPSCs recorded from neurons in the BLA to 64.6 ± 5.3% of control (n = 6; fig. 3A). Photolytically evoked p-NMDA-Cs were significantly reduced by 1.9 mm xenon to 72.9 ± 5.1% of control (n = 6; fig. 3B; tests with contrasts in multivariate analysis of variance, P < 0.05). When comparing the degree of reduction between NMDA-eEPSCs and p-NMDA-Cs with Bonferroni post hoc tests in multivariate analysis of variance, we did not find any significant difference. Current decay of NMDA-eEPSCs was fitted biexponentially. Both time constants of NMDA-eEPSCs, τdecayfast and τdecayslow, remained unchanged under 1.9 mm xenon (table 3).
Electrically (AMPA-eEPSCs) or photolytically (p-AMPA-Cs) evoked AMPA receptor–mediated currents were recorded in the BLA (fig. 4). Xenon (1.9 mm) reduced AMPA-eEPSCs to 56.2 ± 4.9% of control responses (n = 6; fig. 4A). p-AMPA-Cs were reduced in the presence of 1.9 mm xenon to 62.1 ± 4.8% of control (n = 6; fig. 4B). In both cases, the averaged relative amplitudes in the third 5-min interval after xenon application was significantly lower than in the first and second baseline intervals (tests with contrasts, P < 0.05). Reduction of the amplitudes of AMPA-eEPSCs and p-AMPA-Cs was not significantly different. Current decays of AMPA-eEPSCs were fitted monoexponentially and were not altered when 1.9 mm xenon was applied (table 3).
In some additional sets of experiments, we evaluated whether the effect of xenon on NMDA and AMPA receptor currents is concentration dependent. Therefore, we used xenon gas mixtures containing 30% xenon–35% N2–30% O2–5% CO2, 18% xenon–47% N2–30% O2–5% CO2, or 5% xenon–60% N2–30% O2–5% CO2. Saturating the ACSF with these gas mixtures revealed a mean ± SD concentration of xenon of 1.1 ± 0.1, 0.6 ± 0.1, or 0.2 ± 0.1 mm, respectively. Under 1.1 mm (0.6 mm) xenon, electrically evoked NMDA-eEPSCs were reversibly diminished to 74.8 ± 5.9% (81.2 ± 4.1%) of control (n = 4 for each concentration; fig. 3A), whereas photolytically evoked p-NMDA-Cs were reduced to 76.8 ± 1.9% (86.3 ± 4.9%) of control (n = 4 for each concentration; fig. 3B). There was no significant difference in the degree of reduction of NMDA-eEPSCs and p-NMDA-Cs under 1.1 or 0.6 mm xenon (fig. 3C). Xenon at 1.1 mm (0.6 mm) reduced electrically evoked AMPA-eEPSCs to 68.3 ± 7.2% (78.0 ± 6.1%) of control responses (n = 4 for each concentration; fig. 4A) and photolytically evoked p-AMPA-Cs to 71.9 ± 3.2% (86.0 ± 5.0%) of control responses (n = 4; fig. 4B). Likewise, there was no significant difference in the degree of reduction of AMPA-eEPSCs and p-AMPA-Cs under 1.1 or 0.6 mm xenon (fig. 4C). Application of 0.2 mm xenon did not result in a significant change of NMDA-eEPSC amplitudes (95.3 ± 4.3% of control, n = 5), p-NMDA-C amplitudes (96.0 ± 3.8% of control, n = 5), AMPA-eEPSC amplitudes (93.1 ± 4.7% of control, n = 6), or p-AMPA-C amplitudes (95.3 ± 2.9% of control, n = 6). Moreover, we did not find a significant difference in the degree of reduction when comparing NMDA-eEPSCs and AMPA-eEPSCs, or p-NMDA-Cs and p-AMPA-Cs under 1.9, 1.1, 0.6, or 0.2 mm xenon, indicating a similar sensitivity of the NMDA and AMPA receptors to xenon.
The calculated IC50values for xenon against the NDMA and AMPA receptor–mediated eEPSCs were 3.5 mm (Hill =−0.9) and 2.3 mm (Hill =−1.0). The IC50values for xenon against p-NMDA-Cs and p-AMPA-Cs were 5.6 mm (Hill =−0.8) and 2.7 mm (Hill =−1.2).
Miniature EPSCs (mEPSCs) were recorded in the presence of 1 μm tetrodotoxin from neurons that also yielded current responses upon electrical stimulation. mEPSCs recorded from neurons in the BLA occurred at a frequency of 8.9 ± 1.3 Hz and had a mean amplitude of 5.6 ± 0.9 pA. Application of xenon (1.9 mm) reduced the mEPSC amplitudes (fig. 5A). Figure 5Bshows the effect of xenon on cumulative distributions of mEPSC amplitudes and interevent intervals. Xenon increased the proportion of mEPSCs with smaller amplitudes, but had no effect on the distribution of interevent intervals. Figure 5Cshows pooled data from five experiments. Xenon reduced the mean mEPSC amplitude to 4.1 ± 0.3 pA, whereas the mean frequency remained unchanged.
In the current study, for the first time, brain slices were used to study effects of xenon on glutamatergic and GABAergic synaptic transmission at the single neuron level. We found that xenon concentration-dependently reduces pharmacologically isolated NMDA and AMPA receptor–mediated synaptic transmission in slices of the BLA of adult mice, while not affecting GABAergic IPSCs. Furthermore, the analysis of receptor currents elicited by photolytic uncaging of glutamate and mEPSCs revealed that the xenon-induced effects are primarily mediated by postsynaptic mechanisms.
Published values of minimum alveolar concentrations of xenon for humans range from 63 to 71 vol%.21,22Using a published23solubility coefficient of 0.0887 for xenon at 37°C, calculated24minimum alveolar concentration equivalents for dissolved xenon range from 2.2 to 2.5 mm. Therefore, with a measured xenon concentration of 1.9 ± 0.3 mm dissolved in ACSF at room temperature, the amount of xenon concentration applied in our experiments lies well within the clinically relevant range. As specific test experiments in the current study and others18demonstrated, supplying brain slices with 30% O2is obviously sufficient to maintain complete neuronal functionality and induced no change in the physiology of the neurons.
Enhancement of GABAAreceptor–mediated inhibitory synaptic transmission is considered to be a crucial mechanism for numerous general anesthetics to reversible depress central nervous system function.6,7,25Under our experimental conditions, we observed virtually no effect of xenon on GABAAreceptor–mediated IPSCs: GABAA-eIPSCs amplitudes remained unchanged, and although τdecayfast of GABAA-eIPSCs was prolonged under xenon, total charge transfer, which determines the strength of inhibition,26,27was not changed by xenon. Enhancement of GABAergic synaptic transmission seems to be critically involved in the anesthetic mechanisms of, e.g. , volatile anesthetics.6,7It has been shown that these agents prolong the decay time constant of GABAAIPSC to 260–500% and consecutively increase charge transfer.28,29In comparison, the prolongation of τdecayfast of GABAA-eIPSCs that was observed under xenon in the current study is considerably small (138%) and not associated with an increased charge transfer. Therefore, a relevant enhancement of GABAergic synaptic transmission by xenon under our conditions seems very unlikely.
These results are in agreement with studies demonstrating that xenon exerts no effects on isolated GABAA-IPSCs evoked from hippocampal autaptic synapses.10,11However, for xenon, two reports8,9describe a xenon-induced increase of Cl−currents through α1β2, α1β2γ2L, or α1β2γ2SGABAAreceptors that were heterologously expressed in Xenopus oocytes9or human embryonic kidney 293 cells.8Although by far the largest population (representing almost half of all GABAAreceptors in the brain) is that containing the α1β2γ2subunit combination,30neither in the current study nor in that by De Sousa et al. 11was the specific subunit composition determined. It has been shown that changes in the β subunit31or the presence of additional Δ or θ subunits32,33substantially affect GABAAreceptor response to neuroactive drugs. In the amygdala, the subunit composition of α2βnγ1predominates.30As such, different subunit compositions might thus explain the different sensitivity of GABAAreceptors to xenon. However, one should consider that the subunit distribution in neurons of brain slices rather resembles the in vivo distribution than in defined heterologous expression systems. Overall, our data support the generally accepted hypothesis12that xenon-induced enhancement of GABAergic inhibition does not crucially account for the xenon anesthetic state in vivo .
Nevertheless, xenon reversibly diminished basal synaptic transmission in the BLA. This might account for the anesthetic properties of xenon, which is in accord with the overall view of the anesthetic state being described as a state with reduced central nervous system excitability. At a holding potential of –70 mV, basal synaptic transmission is governed primarily by NMDA and AMPA receptor activity. Using pharmacologically isolated NMDA- and AMPA-eEPSCs, we analyzed in detail how xenon affects excitatory synaptic transmission.
Xenon induced a reversible reduction of NMDA receptor–mediated eEPSCs, whereas current kinetics remained unchanged. NMDA receptor depression might be a pivotal mechanism for mediating anesthesia and analgesia,34,35and an NMDA receptor–depressing effect has been described for, e.g. , propofol, nitrous oxide, ketamine, and some volatile anesthetics.7A reduced NMDA receptor function was accepted to be the major mechanism for the action of xenon.10–12Doubtlessly, when regarding single synaptic transmission, the NMDA receptor activation plays only a minor role in governing the excitatory signaling. However, frequent stimulation activates NMDA receptors, which might result in synaptic plasticity, and excessive NMDA receptor activation causes excitotoxicity. Hence, antagonizing NMDA receptors might induce acute effects, which on the one hand induce amnesia and on the other hand prevent neurotoxicity.
Whether xenon also affects non-NMDA receptors was considered to be unlikely, mainly because xenon had little effect on AMPA/kainate receptors of dissociated hippocampal neurons forming autaptic synapses.10,11Recently, however, it has been shown that xenon diminishes current responses from heterologously expressed AMPA13and kainate receptors,14as well as AMPA- and kainate-induced current responses from cultivated mice cortical neurons.14Moreover, recent experiments15obtained from C. elegans suggest that the xenon anesthetic state might not be mediated via NMDA but via AMPA receptor depression.
In the current study, we could demonstrate antagonistic properties of xenon against non-NMDA receptor–mediated EPSCs. The generic term non-NMDA receptor comprises AMPA and kainate receptors, with the fast glutamatergic synaptic transmission being primarily mediated via AMPA receptors,36,37as tested also in the current study when non-NMDA-eEPSCs were entirely blocked by the specific AMPA receptor antagonist GYKI52466 (data not shown). Our findings fit well with data from studies13,14demonstrating a sensitivity of agonist-induced AMPA receptor currents to xenon.
We found that xenon concentration-dependently reduces NMDA and AMPA receptor–mediated synaptic currents with similar potency. The dose–response relation for each receptor was calculated by the four-parameter logistic equation. Because of methodologic limitations, xenon concentrations beyond 1.9 mm cannot be tested. Therefore, the respective IC50and Hill values can only be regarded as extrapolated. Furthermore, for calculating the respective IC50and Hill values, it has to be assumed that xenon is a full antagonist. A full antagonism of xenon on excitatory ion channels under normobaric conditions has not been shown yet. However, one cannot exclude that under hyperbaric conditions, NMDA and AMPA receptor currents can be completely blocked by xenon. Although the IC50values for xenon against NMDA receptor–mediated currents are higher than the IC50values for the AMPA receptor–mediated currents, this difference is not statistically significant and might result from the aforementioned limitations. Therefore, we conclude that xenon equipotently antagonizes NMDA and AMPA receptors. Nevertheless, these extrapolated IC50values for each receptor subtype are in the range of the EC50values for xenon calculated from in vivo data.24
Although xenon impairs NMDA- and AMPA-eEPSCs with a similar potency as described for AMPA receptor currents induced in cultured neurons or expression systems, it is still unclear whether xenon acts via presynaptic or postsynaptic mechanisms. The similar influence of xenon on electrically and photolytically evoked currents combined with the analysis of mEPSCs is in favor of a postsynaptic mechanism of xenon on excitatory synaptic transmission. Xenon reduced mEPSC amplitudes with no effect on glutamatergic mEPSC frequency, providing further evidence that xenon-induced suppression of synaptic transmission might not result from a lower frequent synaptic glutamate release. A reduction of the mEPSC amplitude might be due to blocked postsynaptic receptors and/or a reduced size of quantal transmitter release. Although we cannot distinguish between these two mechanisms, a postsynaptic mechanism seems much more likely, because xenon depressed NMDA-eEPSCs and p-NMDA-Cs, and AMPA-eEPSCs and p-AMPA-Cs to the same extent, excluding additional presynaptic mechanisms.
In summary, we showed that clinical relevant concentrations of xenon depress NMDA and AMPA receptor–mediated synaptic transmission in the BLA. Our data provide evidence that this depression might be mainly due to postsynaptic mechanisms. The amygdala, especially the basolateral nucleus, seems to play an important role in anesthetic-induced amnesia,38,39pain processing,40and regulation of sympathetic tone.41Xenon is an anesthetic agent that mediates profound analgesia and amnesia with only moderate cardiovascular side effects.1Regarding these particular properties, the observed depression of excitatory synaptic transmission in the BLA might be crucial for the xenon anesthetic state.
The authors thank Kurt Biedermann, Ph.D. (Chemist, Department of Analytics, RCC Ltd., Itingen, Switzerland), for concentration measurements of dissolved xenon and Alexander Yassouridis, Ph.D. (Biostatistician, Department of Biostatistics, Max-Planck-Institute of Psychiatry, Munich, Germany), for statistical advice.