Xenon Attenuates Excitatory Synaptic Transmission in the Rodent Prefrontal Cortex and Spinal Cord Dorsal Horn
Haseneder, Rainer M.D.*; Kratzer, Stephan M.S.†; Kochs, Eberhard M.D.‡; Mattusch, Corinna R.A.§; Eder, Matthias Ph.D.∥; Rammes, Gerhard Ph.D.#
Background: The molecular mechanisms of the inhalational anesthetic xenon are not yet fully understood. Recently, the authors showed that xenon reduces both N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor–mediated synaptic transmission in a brain slice preparation of the amygdala. In the current study, the authors examined the effects of xenon on synaptic transmission in the prefrontal cortex and the spinal cord dorsal horn (substantia gelatinosa).
Methods: In rodent brain or spinal cord slice preparations, the authors used patch clamp technique to investigate the impact of xenon on NMDA and AMPA receptor–mediated excitatory postsynaptic currents, as well as on γ-aminobutyric acid type A receptor–mediated inhibitory postsynaptic currents. The currents were either evoked upon electrical stimulation (NMDA-eEPSCs and AMPA-eEPSCs) or upon photolysis of caged L-glutamate (p-NMDA-Cs and p-AMPA-Cs). In addition, the authors investigated the effects of xenon on AMPA receptor–mediated miniature excitatory postsynaptic currents.
Results: In both central nervous system regions, xenon had virtually no effect on inhibitory postsynaptic currents. In the prefrontal cortex (spinal cord), xenon reversibly reduced NMDA-eEPSCs to approximately 58% (72%) and AMPA-eEPSCs to approximately 67% (65%) of control. There was no difference in the xenon-induced reduction of NMDA-eEPSCs and p-NMDA-Cs, or AMPA-eEPSCs and p-AMPA-Cs. Xenon did not affect the frequency of miniature excitatory postsynaptic currents but reduced their amplitude.
Conclusions: In the current study, the authors found that xenon depresses NMDA and AMPA receptor–mediated synaptic transmission in the prefrontal cortex and the substantia gelatinosa without affecting γ-aminobutyric acid type A receptor–mediated synaptic transmission. These results provide evidence that the effects of xenon are primarily due to postsynaptic mechanisms.
THE inhalational anesthetic xenon combines profound anesthetic and analgesic properties with a low side effect profile. A number of clinical trials during the past years have evaluated the safety and efficacy of xenon anesthesia in human patients.1,2
However, the detailed mechanisms by which xenon exerts its anesthetic and analgesic properties are still a matter of debate.3
Anesthesia, when simplistically described as a status with reduced neuronal activity, might emerge from an enhancement of inhibitory or reduction of excitatory central nervous system (CNS) signaling or a combination of both.
Current knowledge speaks in favor of the excitatory rather than the inhibitory transmitter system to transduce the molecular mechanisms of xenon. Studies using heterologously expressed N
-methyl-d-aspartate (NMDA) receptors4
or neurons in culture5,6
brought evidence that the NMDA receptor might be a major target for xenon action. However, meanwhile, several studies provide evidence for non-NMDA receptors to play an important role in the xenon anesthetic state. Current flow through heterologously expressed non-NMDA receptors of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate type are reduced in the presence of xenon,7,8
as are non-NMDA receptor–mediated currents recorded from cultured cortical neurons.8
In addition, the two-pore domain–related potassium channel type 1 has been shown to be activated by xenon,9
possibly contributing to the xenon anesthetic state. Consistent with these in vitro
findings, in vivo
studies using the model organism Caenorhabditis elegans10
or human volunteers11,12
provide evidence that mechanisms beyond an exclusive NMDA receptor antagonism play a role in mediating the xenon anesthetic state.
In a recently published study,13
we demonstrated that xenon depresses both NMDA and AMPA receptor–mediated excitatory while not affecting γ-aminobutyric acid type A (GABAA
) receptor–mediated inhibitory synaptic transmission in an acute murine brain slice preparation of a subcortical brain structure, the basolateral amygdala. Because the basolateral amygdala is critically involved in anesthetic-induced amnesia,14,15
addictive behavior, and the formation of negative emotions such as fear (review: Sah et al.16
), these results may explain the amnestic and memory-ablating properties of xenon. However, detailed investigations on the neuronal mechanisms of xenon action in other CNS areas are missing.
In the current study, we therefore determined the impact of xenon on GABAA
, NMDA, and AMPA receptor–mediated synaptic transmission in two distinct CNS areas that might be critically involved in producing the hypnotic and analgesic effects of xenon. The prefrontal cortex (PFC) was chosen as a cortical brain area being critically involved in higher brain functions such as consciousness (review: Bodovitz17
), whereas the substantia gelatinosa (SG) of the spinal cord dorsal horn, as the first site of synaptic integration of nociceptive inputs,18,19
is involved in mechanisms of pain processing.
Materials and Methods
The experimental protocols were approved by the Ethical Committee on Animal Care and Use of the Government of Bavaria, Germany. For the experiments in the PFC, 28- to 42-day-old male C57Bl6 mice were killed by cervical dislocation, and the brains were rapidly removed and placed in 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 NaH2
(all from RBI/Sigma, Deisenhofen, Germany). Saturation with a mixture of 95% O2
(carbogen gas) led to a pH of 7.4. Sagittal slices (350 μm thick) were prepared using a microtome (HM 650 V; Microm International, Walldorf, Germany). For the experiments in the SG of the spinal cord dorsal horn, 14- to 28-day-old male Wistar rats were killed by cervical dislocation, and coronary slices of lumbar spinal cord were obtained as described previously.20
All 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 slices on the bottom of the recording chamber, which was continuously perfused (2 ml/min) with ACSF. We used infrared videomicroscopy and the gradient contrast system (Zeiss, Oberkochen, Germany; for details, see Dodt et al.21
) to visualize the neurons within layer V of the PFC or within the SG. The SG was clearly identifiable as a translucent band across the superficial dorsal horn.
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 an open tip 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 ameliorate oxygenation of the neurons.
Electrically evoked postsynaptic currents (ePSCs) were elicited by square pulse stimuli (6–100 V, 0.05–0.5 ms, interstimulus interval 15 s) delivered via a bipolar tungsten electrode, which was placed either in layer V near the apical dendrite of the recorded neuron (5-μm electrode tip diameter) or in the dorsal root entry zone of the spinal cord slices (50-μm electrode tip diameter). Preceding each tissue stimulation, neuronal input resistance was determined by injecting a current pulse hyperpolarizing by 10 mV for 200 ms. In the PFC, in a subset of experiments, paired-pulse stimulation was performed by delivering the same stimulus at 50-ms interpulse intervals. The paired-pulse ratio was determined by dividing the amplitude of the second current response by the amplitude of the first one.
Photolytically evoked excitatory currents were induced upon focal photolysis of caged L-glutamate.21
For this purpose, 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. When a stable whole cell recording had been obtained, γ-α-carboxy-2-nitrobenzyl caged glutamate at a final concentration of 0.25 mm was added to the recirculating perfusate. Caged glutamate had no discernible effect on neurons per se
Glutamate 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 (NBQX; 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 NBQX omitted for AMPA, AP5 omitted for NMDA, and bicuculline methiodide omitted for GABAA receptor–mediated responses. For the experiments in the spinal cord, strychnine at a final concentration of 2 μm was added to the ACSF, and for recordings of NMDA receptor–mediated currents in the spinal cord, MgCl2 was reduced to 0.5 mm. For AMPA receptor–mediated currents, the holding potential was set to −70 mV, and for NMDA and GABAA receptor–mediated currents, the 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 NBQX (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
; for xenon application, the ASCF was gassed with a mixture of 65% xenon–30% O2
. In a subset of experiments, xenon was applied at a lower concentration using a gas mixture of 30% xenon–35% N2
and 5% CO2
. Replacement of N2
by xenon did not change the pH of the gassed ACSF. We have shown repeatedly13,22
that it is feasible to obtain stable synaptic responses from brain slice preparations for at least 40 min under these conditions with relatively low oxygen concentration. 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 gas chromatography (RCC Ltd., Itingen, Switzerland). When gassing the ACSF with the 65% (30%) xenon gas mixture, the concentration of xenon in ACSF was 1.9 ± 0.2 mm (1.1 ± 0.1 mm) (mean ± SD; n = 4). For comparison, other studies describe xenon saturation of 3.9 mm 23
or 2.0 mm 4
when gassing with pure xenon, 3.4 mm when gassing with 80 vol% xenon,6,7
or 3.54 mm when gassing with 84 vol%,8
which 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, NBQX, bicuculline methiodide, strychnine, γ-α-carboxy-2-nitrobenzyl caged glutamate (all from RBI/Sigma), and CGP35348 (Novartis Laboratories, Basel, Switzerland) were bath applied at known concentrations via the superfusion system.
For all statistical evaluations, SPSS Statistics version 16 (SPSS GmbH Software, Munich, Germany) was used. The 40-min recording time of ePSCs, electrically evoked inhibitory postsynaptic currents (eIPSCs), electrically evoked excitatory postsynaptic currents (eEPSCs), and photolytically evoked currents was first partitioned in equidistant subintervals of 5 min, and then the averaged relative amplitudes in each of them were determined. A two-factorial multivariate analysis of variance (MANOVA) 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. Differences between the various intervals were tested by tests with contrasts, and differences between the various receptors and stimulation methods were tested by post hoc tests. MANOVA was also performed for testing differences in the amplitudes and frequency of mEPSCs between the control and xenon recordings. As the nominal level of significance, we accepted α = 0.05. It was corrected (according to Bonferroni procedure) for all a posteriori tests (tests with contrasts and post hoc tests). 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.
In a first set of experiments, the effect of 1.9 mm xenon on compound synaptic transmission in the PFC or SG was examined. Compound postsynaptic currents upon electrical stimulation (ePSCs) were recorded from neurons in the PFC (layer V) or the SG. Ten to 15 min after xenon application, the ePSCs recorded from the PFC and SG were reduced to 70.1 ± 10.3% and 74.3 ± 4.2%, respectively (figs. 1A and B
). The averaged relative amplitudes in this interval were significantly lower than in the two control intervals (tests with contrasts in MANOVA, PFC: P
= 0.022, SG: P
= 0.003), with no significant difference between PFC and SG (Wilks multivariate test, P
= 0.661). Upon termination of xenon application, the ePSCs nearly reversed to control level. Application of xenon for a longer period (up to 25 min) did not result in an additional reduction of current responses (data not shown). Neither resting membrane potential (PFC: control −71.3 ± 0.9 mV, xenon −71.1 ± 0.2 mV; SG: control −50.6 ± 2.2 mV, xenon −52.8 ± 4.7 mV) nor input resistance (PFC: control 272.2 ± 26.7 MΩ, xenon 294.3 ± 29.9 MΩ; SG: control 823.7 ± 117.8 MΩ, xenon 789.9 ± 117.0 MΩ) of the neurons were changed in the presence of 1.9 mm xenon.
From neurons in the PFC and SG, electrically evoked GABAA
receptor–mediated eIPSCs (GABAA
-eIPSCs) were recorded in the presence of 50 μm AP5, 5 μm NBQX, and 200 μm CGP35348 at a holding potential of −50 mV. Xenon, 1.9 mm, did not affect the amplitudes of GABAA
-eIPSCs (figs. 1C and D
) in the PFC or in the SG. The decay of the GABAA
-eIPSCs was fitted biexponentially, with two time constants, τdecay
fast and τdecay
slow, and total charge transfer was calculated by integrating the area under the GABAA
-eIPSCs. In both CNS regions, we did not detect an apparent influence of xenon on either of these parameters (table 1
In further sets of experiments, the impact of xenon on NMDA and AMPA receptor–mediated excitatory synaptic transmission in the PFC (fig. 2
) and SG (fig. 3
) was investigated.
-methyl-d-aspartate receptor–mediated current responses were evoked using either electrical stimulation (NMDA-eEPSCs) or photolytic uncaging of glutamate (p-NMDA-Cs). Xenon, 1.9 mm, reversibly diminished NMDA-eEPSCs recorded from neurons in the PFC (SG) to 57.7 ± 4.4% (71.9 ± 3.4%) of control (PFC: n = 6, SG: n = 5; tests with contrasts in MANOVA, PFC: P
= 0.008, SG: P
= 0.004; figs. 2A and 3A
). Photolytically evoked p-NMDA-Cs were reduced by xenon to 58.6 ± 3.6% (PFC; n = 6) and 71.5 ± 5.6% (SG; n = 5) of control (tests with contrasts in MANOVA, PFC: P
= 0.001, SG: P
= 0.002; figs. 2A and 3A
). There was no significant difference in the degree of xenon-induced reduction when comparing NMDA-eEPSCs with p-NMDA-Cs in the PFC or in the SG (Bonferroni post hoc
tests in MANOVA, PFC: P
= 0.951, SG: P
= 0.937). Moreover, when comparing the impact of xenon on NMDA receptor–mediated currents in the PFC with that in the SG, we did not see a significant difference (Wilks multivariate test, NMDA-eEPSC: P
= 0.166, p-NMDA-Cs: P
= 0.104). The current decay of NMDA-eEPSCs was fitted biexponentially, with two time constants, τdecay
fast and τdecay
slow. In both CNS regions, a clear influence of xenon on deactivation kinetics of NMDA-eEPSCs was not detectable (table 2
Electrically (AMPA-eEPSCs) and photolytically (p-AMPA-Cs) evoked AMPA receptor–mediated currents were recorded in the PFC and SG. Xenon reduced the amplitudes of AMPA-eEPSCs to 66.9 ± 4.1% (PFC; n = 6; fig. 2B
) and 64.5 ± 6.5% (SG; n = 5; fig. 3B
). p-AMPA-Cs were reduced in the presence of xenon to 63.7 ± 2.9 (PFC; n = 5; fig. 2B
) and 58.2 ± 3.1% (SG; n = 5; fig. 3B
) of control. Reduction of the amplitudes of AMPA-eEPSCs and p-AMPA-Cs was not significantly different in the PFC or in the SG (Bonferroni post hoc
tests in MANOVA, PFC: P
= 0.826, SG: P
= 0.209). Comparison of the xenon effect on AMPA receptor–mediated current responses in the PFC with that in the SG revealed no statistical significant difference (Wilks multivariate test, AMPA-eEPSC: P
= 0.452, p-AMPA-Cs: P
= 0.640). We did not see a conspicuous influence of 1.9 mm xenon on current decay kinetics of AMPA-eEPSCs (table 2
In a subset of experiments, we tested whether a lower concentration of xenon (1.1 mm), which most likely corresponds to minimum alveolar concentration (MAC)awake
, affected NMDA and AMPA receptor–mediated current responses in the PFC. Xenon, 1.1 mm, reversibly diminished NMDA-eEPSCs to 76.5 ± 3.7% of control (n = 4; tests with contrasts in MANOVA, P
< 0.05) with no significant difference from the reduction of p-NMDA-Cs caused by 1.1. mm xenon (78.0 ± 4.1%; n = 4; fig. 2A
, triangles). Likewise, 1.1 mm xenon reduced the amplitudes of AMPA-eEPSCs (83.3 ± 3.4%; n = 4) and p-AMPA-Cs (79.9 ± 4.2%; n = 4) to a similar extent (fig. 2B
Miniature excitatory postsynaptic currents were recorded in the presence of 1 μm tetrodotoxin (fig. 4
). mEPSCs recorded from neurons in the PFC (SG) occurred at a frequency of 9.0 ± 0.7 (4.9 ± 2.1) Hz and had a mean amplitude of 4.2 ± 0.3 (5.7 ± 0.4) pA. Application of 1.9 mm xenon reduced the mEPSC amplitudes in the PFC (fig. 4A
) and SG (fig. 4E
). Figures 4B and F
show the effect of xenon on cumulative distributions of mEPSC amplitudes and interevent intervals. Xenon, 1.9 mm, increased the proportion of mEPSCs having smaller amplitudes but had no effect on the distribution of interevent intervals. Figures 4C and G
show pooled data from five experiments performed in the PFC and five experiments performed in the SG. Xenon reduced the mean mEPSC amplitude to 3.7 ± 0.1 (5.1 ± 0.2) pA, whereas the mean frequency remained unchanged.
In an additional set of experiments, AMPA-eEPSCs upon paired-pulse stimulation were recorded from PFC neurons. The paired-pulse ratio did not change when 1.9 mm xenon was applied (P
= 0.111, n = 7; fig. 4D
Recently, we demonstrated that the inhalational anesthetic xenon depresses excitatory while not affecting inhibitory synaptic transmission in the murine basolateral amygdala.13
Because the effects of an anesthetic on the cellular level may vary depending on the local network under consideration,24,25
we now investigated the effect of xenon in two additional CNS regions. In the current study, we show that in acute rodent slice preparations of the cortex and the spinal cord, both NMDA and AMPA receptor–mediated synaptic transmission are depressed by xenon, whereas GABAA
receptor–mediated inhibitory synaptic transmission remains unchanged.
Based on published MAC values of xenon for humans (63–71 vol%)26,27
and a solubility coefficient of 0.0887 for xenon at 37°C,28
the calculation of MAC equivalents of dissolved xenon reveals a value of 2.2–2.5 mm. Therefore, with a measured xenon concentration of 1.9 ± 0.3 mm dissolved in ACSF at room temperature, the xenon concentration applied during most of our experiments lies close to MACimmobility
, whereas the lower concentration of 1.1 mm, which was applied in a subset of experiments, corresponds closely to MACawake
The results provided in the current study further support the hypothesis that xenon does not act via
an enhancement of γ-aminobutyric acid (GABA)–mediated synaptic transmission. For intravenous30,31
and volatile anesthetics,32
as well as for nitrous oxide,23,33
a substantial potentiating effect on GABA-mediated currents has been reported, and for intravenous and volatile anesthetics, there is growing evidence that they mediate their anesthetic properties, at least in part, via
an enhancement of inhibitory synaptic transmission (reviews: Campagna et al.
Rudolph and Antkowiak35
). In the current study, we observed no effect of xenon on the amplitudes of GABAA
-eIPSCs recorded from native neurons in the PFC or in the SG. A slight effect of xenon on GABAA
-eIPSC deactivation time constants, and total charge transfer, which determines the strength of inhibition,36,37
cannot be completely excluded by our data. However, we certainly can exclude a pronounced effect, as it has been described, e.g.
, for volatile anesthetics, prolonging IPSC time constants to 260–500%.32,38
Our results are in accord with studies5,6
showing that xenon exerts no effects on GABAA
-IPSCs evoked from cultured hippocampal neurons, but contrasts with two studies describing a xenon-induced increase of Cl−
currents through GABAA
receptors, which were heterologously expressed in Xenopus
or human embryonic kidney 293 cells.23
In the latter studies, the xenon-induced potentiation occurred only at nonsaturating GABA concentrations, which are below the range that occurs synaptically,36
thus possibly explaining the discrepancy with our data. However, the possibility, that extrasynaptic GABAA
receptors, which are activated at subsaturating GABA concentrations (reviews: e.g.
, Walker and Semyanov,39
Hemmings et al.40
) and mediate a tonic neuronal inhibition, are affected by xenon, cannot be ruled out by our data.
In both PFC and SG, compound synaptic transmission was reduced under xenon. For a further detailed investigation of the underlying mechanisms, we used pharmacologic isolation to separately investigate the effect of xenon on NMDA and AMPA receptor–mediated synaptic transmission.
In both CNS regions, we observed a pronounced xenon-induced reduction of NMDA receptor–mediated currents without apparent change of current decay kinetics. An NMDA receptor–depressing effect has been described for, e.g.
, propofol, nitrous oxide, ketamine, and also some volatile anesthetics (review: Rudolph and Antkowiak35
), and a depressant effect on the NMDA receptor has been suggested as the main mechanism for the xenon anesthetic state.5
NMDA receptor–mediated currents recorded from amygdalar neurons in a brain slice preparation,13
from neurons in culture,5,6
and from heterologously expressed NMDA receptors4
were reported to be reduced by xenon. In the current study, in the PFC as well as in the SG, photolytically evoked NMDA receptor currents, which are generated beyond the influence of the presynaptic terminal, were depressed by xenon to the same amount as were electrically evoked NMDA receptor–mediated currents. This finding provides strong evidence for a mainly postsynaptic mechanism of xenon on NMDA receptors in the PFC and SG.
Similar to our recently published results in the basolateral amygdala, also AMPA receptor–mediated currents in the PFC and SG were reduced by xenon. These results further support the hypothesis that the xenon anesthetic state is not only due to an NMDA receptor depression, as has been suggested initially.5
In fact, evidence for a participation of the AMPA receptor in mediating the xenon anesthetic state derives not only from in vitro
but also from in vivo
studies using the model organism C. elegans 10
or human volunteers.11,41
Because AMPA receptor currents upon electrical stimulation and upon photolytic uncaging of L-glutamate were reduced to the same amount, we propose a postsynaptic mechanism of xenon action, both in the PFC and in the SG. The analysis of the effect of xenon on AMPA receptor–mediated mEPSCs in the PFC and SG, as well the analysis of the xenon effect on the paired-pulse ratio of AMPA-eEPSCs in the PFC, further support this hypothesis. Xenon reduced mEPSC amplitudes while not affecting mEPSC frequency, which speaks, according to the classic interpretation of mEPSC analysis,42
in favor of a postsynaptic mechanism of xenon action. Changes in paired-pulse ratio and neurotransmitter release are inversely correlated.43,44
Therefore, our findings of decreased AMPA-eEPSCs with unchanged paired-pulse ratio suggest that xenon inhibits excitatory synaptic transmission without changing the probability of presynaptic neurotransmitter release.
The analysis of current kinetics of GABAA, NMDA, and AMPA receptor–mediated currents revealed considerably faster current responses recorded from SG neurons compared with neurons in the PFC. Possible explanations for these discrepancies might be species differences and/or the approximately threefold higher input resistance of the SG neurons, which reflects a smaller cell size and thus a less pronounced current distortion typically produced by space clamp deficits.
When comparing the amount of reduction of NMDA and AMPA receptor–mediated currents in the PFC and the SG, we did not see a difference between the two CNS regions. Furthermore, when comparing the results of the current study with the recently published results of the xenon effect in the basolateral amygdala,13
no significant difference occurred in terms of the impact of xenon on NMDA or AMPA receptor–mediated currents recorded in the PFC, SG, or amygdala (Wilks multivariate test, P
> 0.05). We therefore hypothesize an equipotent xenon action on the cortical, subcortical, and spinal levels.
The PFC is essentially involved in higher cognitive functions such as working memory,45–47
level of wakefulness,49–51
Therefore, the observed xenon action in PFC neurons might account for the hypnotic properties of xenon, in fact, at concentrations close to MACimmobility
and close to MACawake
. Moreover, because layer V pyramidal neurons in the PFC represent the major output neurons of the PFC,54
a xenon-induced disruption of excitatory synaptic transmission to these cells might also affect further important subcortical processes regulated by the PFC.
Current knowledge regarding the action of xenon on the spinal cord level is limited to few studies. It has been shown that xenon depresses spinal cord dorsal horn neuronal activity in vivo
slows ventral root potentials and spinal monosynaptic reflexes in vitro
and attenuates long-term potentiation of C-fiber evoked potentials.58
However, precise insights into the mechanisms of xenon action on dorsal horn level are missing. In our study, we showed that xenon depresses both NMDA and AMPA receptor–mediated synaptic transmission to SG neurons, presumably via
postsynaptic mechanisms. Nociceptive signals transmitted by thin (Aδ) and unmyelinated (C) primary afferent fibers are conveyed to neurons in the superficial laminae (SG) of the spinal cord dorsal horn as the first site of their synaptic integration,18,19
and inhibition of SG neuron activity might be a key mechanism of antinociception.59
Therefore, the observed xenon effect on NMDA and AMPA receptor–mediated synaptic transmission might crucially account for the profound analgesic60,61
properties of xenon. In addition, another endpoint of general anesthesia, immobility, is largely produced by anesthetic action on the spinal cord level.62–65
It is still a matter of debate whether depression of ventral or dorsal horn neuronal activity contributes more to immobility.66,67
Although accumulating evidence suggests the ventral horn as the primary site,67,68
the involvement of dorsal horn depression to produce immobility cannot be ruled out.66,69
Hence, the depression of synaptic transmission to SG neurons observed in our study might also account for the immobilizing properties of xenon.
In summary, we have shown that a clinical relevant concentration of xenon depresses NMDA and AMPA receptor–mediated synaptic transmission in the PFC and the SG, while not affecting GABA-mediated synaptic transmission. Our data provide evidence that the depression might be mainly due to postsynaptic mechanisms. According to the primary functions of the investigated CNS areas, the observed depression of excitatory synaptic transmission might account for the hypnotic and analgesic properties of the general anesthetic xenon.
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, Head of the Research Group Biostatistics, Max Planck Institute of Psychiatry, Munich, Germany), for statistical advice.
1. Rossaint R, Reyle-Hahn M, Schulte Am Esch J, Scholz J, Scherpereel P, Vallet B, Giunta F, Del Turco M, Erdmann W, Tenbrinck R, Hammerle AF, Nagele P: Multicenter randomized comparison of the efficacy and safety of xenon and isoflurane in patients undergoing elective surgery. Anesthesiology 2003; 98:6–13
2. Wappler F, Rossaint R, Baumert J, Scholz J, Tonner PH, van Aken H, Berendes E, Klein J, Gommers D, Hammerle A, Franke A, Hofmann T, Schulte Esch J: Multicenter randomized comparison of xenon and isoflurane on left ventricular function in patients undergoing elective surgery. Anesthesiology 2007; 106:463–71
3. Preckel B, Weber NC, Sanders RD, Maze M, Schlack W: Molecular mechanisms transducing the anesthetic, analgesic, and organ-protective actions of xenon. Anesthesiology 2006; 105:187–97
4. Yamakura T, Harris RA: Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels: Comparison with isoflurane and ethanol. Anesthesiology 2000; 93:1095–101
5. Franks NP, Dickinson R, de Sousa SL, Hall AC, Lieb WR: How does xenon produce anaesthesia? Nature 1998; 396:324
6. de Sousa SL, Dickinson R, Lieb WR, Franks NP: Contrasting synaptic actions of the inhalational general anesthetics isoflurane and xenon. Anesthesiology 2000; 92:1055–66
7. Plested AJ, Wildman SS, Lieb WR, Franks NP: Determinants of the sensitivity of AMPA receptors to xenon. Anesthesiology 2004; 100:347–58
8. Dinse A, Fohr KJ, Georgieff M, Beyer C, Bulling A, Weigt HU: Xenon reduces glutamate-, AMPA-, and kainate-induced membrane currents in cortical neurones. Br J Anaesth 2005; 94:479–85
9. Gruss M, Bushell TJ, Bright DP, Lieb WR, Mathie A, Franks NP: Two-pore-domain K+ channels are a novel target for the anesthetic gases xenon, nitrous oxide, and cyclopropane. Mol Pharmacol 2004; 65:443–52
10. Nagele P, Metz LB, Crowder CM: Xenon acts by inhibition of non-N-methyl-D-aspartate receptor-mediated glutamatergic neurotransmission in Caenorhabditis elegans
. Anesthesiology 2005; 103:508–13
11. Rex S, Schaefer W, Meyer PH, Rossaint R, Boy C, Setani K, Bull U, Baumert JH: Positron emission tomography study of regional cerebral metabolism during general anesthesia with xenon in humans. Anesthesiology 2006; 105:936–43
12. Laitio RM, Kaisti KK, Laangsjo JW, Aalto S, Salmi E, Maksimow A, Aantaa R, Oikonen V, Sipila H, Parkkola R, Scheinin H: Effects of xenon anesthesia on cerebral blood flow in humans: A positron emission tomography study. Anesthesiology 2007; 106:1128–33
13. Haseneder R, Kratzer S, Kochs E, Eckle VS, Zieglgansberger W, Rammes G: Xenon reduces N
-methyl-d-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic transmission in the amygdala. Anesthesiology 2008; 109:998–1006
14. Dutton RC, Maurer AJ, Sonner JM, Fanselow MS, Laster MJ, Eger EI: The concentration of isoflurane required to suppress learning depends on the type of learning. Anesthesiology 2001; 94:514–9
15. Alkire MT, Nathan SV: Does the amygdala mediate anesthetic-induced amnesia? Basolateral amygdala lesions block sevoflurane-induced amnesia. Anesthesiology 2005; 102:754–60
16. Sah P, Faber ES, Lopez De Armentia M, Power J: The amygdaloid complex: Anatomy and physiology. Physiol Rev 2003; 83:803–34
17. Bodovitz S: The neural correlate of consciousness. J Theor Biol 2008; 254:594–8
18. Yoshimura M, Jessell TM: Primary afferent-evoked synaptic responses and slow potential generation in rat substantia gelatinosa neurons in vitro
. J Neurophysiol 1989; 62:96–108
19. Schneider SP, Perl ER: Comparison of primary afferent and glutamate excitation of neurons in the mammalian spinal dorsal horn. J Neurosci 1988; 8:2062–73
20. Haseneder R, Kurz J, Dodt HU, Kochs E, Zieglgansberger W, Scheller M, Rammes G, Hapfelmeier G: Isoflurane reduces glutamatergic transmission in neurons in the spinal cord superficial dorsal horn: Evidence for a presynaptic site of an analgesic action. Anesth Analg 2004; 98:1718–23
21. Dodt HU, Eder M, Schierloh A, Zieglgansberger W: Infrared-guided laser stimulation of neurons in brain slices. Sci STKE 2002; 2002:L2
22. Ranft A, Kurz J, Becker K, Dodt HU, Zieglgansberger W, Rammes G, Kochs E, Eder M: Nitrous oxide (N2O) pre- and postsynaptically attenuates NMDA receptor-mediated neurotransmission in the amygdala. Neuropharmacology 2007; 52:716–23
23. Hapfelmeier G, Zieglgansberger W, Haseneder R, Schneck H, Kochs E: Nitrous oxide and xenon increase the efficacy of GABA at recombinant mammalian GABAA
receptors. Anesth Analg 2000; 91:1542–9
24. Franks NP, Lieb WR: Molecular and cellular mechanisms of general anaesthesia. Nature 1994; 367:607–14
25. Antkowiak B: How do general anaesthetics work? Naturwissenschaften 2001; 88:201–13
26. Cullen SC, Eger EI, Cullen BF, Gregory P: Observations on the anesthetic effect of the combination of xenon and halothane. Anesthesiology 1969; 31:305–9
27. Nakata Y, Goto T, Ishiguro Y, Terui K, Kawakami H, Santo M, Niimi Y, Morita S: Minimum alveolar concentration (MAC) of xenon with sevoflurane in humans. Anesthesiology 2001; 94:611–4
28. Weathersby PK, Homer LD: Solubility of inert gases in biological fluids and tissues: A review. Undersea Biomed Res 1980; 7:277–96
29. Goto T, Nakata Y, Ishiguro Y, Niimi Y, Suwa K, Morita S: Minimum alveolar concentration-awake of xenon alone and in combination with isoflurane or sevoflurane. Anesthesiology 2000; 93:1188–93
30. Holland KD, Canney DJ, Rothman SM, Ferrendelli JA, Covey DF: Physiological modulation of the GABA receptor by convulsant and anticonvulsant barbiturates in cultured rat hippocampal neurons. Brain Res 1990; 516:147–50
31. Sanna E, Mascia MP, Klein RL, Whiting PJ, Biggio G, Harris RA: Actions of the general anesthetic propofol on recombinant human GABA(A) receptors: Influence of receptor subunits. J Pharmacol Exp Ther 1995; 274:353–60
32. Nishikawa K, MacIver MB: Agent-selective effects of volatile anesthetics on GABAA
receptor–mediated synaptic inhibition in hippocampal interneurons. Anesthesiology 2001; 94:340–7
33. Dzoljic M, Van Duijn B: Nitrous oxide-induced enhancement of gamma-aminobutyric acidA
-mediated chloride currents in acutely dissociated hippocampal neurons. Anesthesiology 1998; 88:473–80
34. Campagna JA, Miller KW, Forman SA: Mechanisms of actions of inhaled anesthetics. N Engl J Med 2003; 348:2110–24
35. Rudolph U, Antkowiak B: Molecular and neuronal substrates for general anaesthetics. Nat Rev Neurosci 2004; 5:709–20
36. Mody I, De Koninck Y, Otis TS, Soltesz I: Bridging the cleft at GABA synapses in the brain. Trends Neurosci 1994; 17:517–25
37. Hentschke H, Schwarz C, Antkowiak B: Neocortex is the major target of sedative concentrations of volatile anaesthetics: Strong depression of firing rates and increase of GABAA receptor-mediated inhibition. Eur J Neurosci 2005; 21:93–102
38. Banks MI, Pearce RA: Dual actions of volatile anesthetics on GABAA
IPSCs: Dissociation of blocking and prolonging effects. Anesthesiology 1999; 90:120–34
39. Walker MC, Semyanov A: Regulation of excitability by extrasynaptic GABA(A) receptors. Results Probl Cell Differ 2008; 44:29–48
40. Hemmings HC Jr, Akabas MH, Goldstein PA, Trudell JR, Orser BA, Harrison NL: Emerging molecular mechanisms of general anesthetic action. Trends Pharmacol Sci 2005; 26:503–10
41. Laitio RM, Kaisti KK, Laangsjo JW, Aalto S, Salmi E, Maksimow A, Aantaa R, Oikonen V, Sipila H, Parkkola R, Scheinin H: Effects of xenon anesthesia on cerebral blood flow in humans: A positron emission tomography study. Anesthesiology 2007; 106:1128–33
42. del Castillo J, Katz B: The effect of magnesium on the activity of motor nerve endings. J Physiol 1954; 124:553–9
43. Mennerick S, Zorumski CF: Paired-pulse modulation of fast excitatory synaptic currents in microcultures of rat hippocampal neurons. J Physiol 1995; 488(pt 1):85–101
44. Kim J, Alger BE: Random response fluctuations lead to spurious paired-pulse facilitation. J Neurosci 2001; 21:9608–18
45. Bodner M, Shafi M, Zhou YD, Fuster JM: Patterned firing of parietal cells in a haptic working memory task. Eur J Neurosci 2005; 21:2538–46
46. Baddeley A: Working memory: Looking back and looking forward. Nat Rev Neurosci 2003; 4:829–39
47. Goldman-Rakic PS: Cellular basis of working memory. Neuron 1995; 14:477–85
48. Dias R, Robbins TW, Roberts AC: Dissociation in prefrontal cortex of affective and attentional shifts. Nature 1996; 380:69–72
49. Thomas M, Sing H, Belenky G, Holcomb H, Mayberg H, Dannals R, Wagner H, Thorne D, Popp K, Rowland L, Welsh A, Balwinski S, Redmond D: Neural basis of alertness and cognitive performance impairments during sleepiness, I: Effects of 24 h of sleep deprivation on waking human regional brain activity. J Sleep Res 2000; 9:335–52
50. Yamasaki H, LaBar KS, McCarthy G: Dissociable prefrontal brain systems for attention and emotion. Proc Natl Acad Sci U S A 2002; 99:11447–51
51. Lambe EK, Olausson P, Horst NK, Taylor JR, Aghajanian GK: Hypocretin and nicotine excite the same thalamocortical synapses in prefrontal cortex: Correlation with improved attention in rat. J Neurosci 2005; 25:5225–9
52. Lau HC, Passingham RE: Relative blindsight in normal observers and the neural correlate of visual consciousness. Proc Natl Acad Sci U S A 2006; 103:18763–8
53. Freitas da Rocha R, Pereira A Jr, Bezerra Coutinho FA: N-methyl-D-aspartate channel and consciousness: From signal coincidence detection to quantum computing. Prog Neurobiol 2001; 64:555–73
54. Sesack SR, Deutch AY, Roth RH, Bunney BS: Topographical organization of the efferent projections of the medial prefrontal cortex in the rat: An anterograde tract-tracing study with Phaseolus vulgaris leucoagglutinin. J Comp Neurol 1989; 290:213–42
55. Utsumi J, Adachi T, Miyazaki Y, Kurata J, Shibata M, Murakawa M, Arai T, Mori K: The effect of xenon on spinal dorsal horn neurons: A comparison with nitrous oxide. Anesth Analg 1997; 84:1372–6
56. Miyazaki Y, Adachi T, Utsumi J, Shichino T, Segawa H: Xenon has greater inhibitory effects on spinal dorsal horn neurons than nitrous oxide in spinal cord transected cats. Anesth Analg 1999; 88:893–7
57. Watanabe I, Takenoshita M, Sawada T, Uchida I, Mashimo T: Xenon suppresses nociceptive reflex in newborn rat spinal cord in vitro
; comparison with nitrous oxide. Eur J Pharmacol 2004; 496:71–6
58. Benrath J, Kempf C, Georgieff M, Sandkühler J: Xenon blocks the induction of synaptic long-term potentiation in pain pathways in the rat spinal cord in vivo
. Anesth Analg 2007; 104:106–11
59. Moore KA, Baba H, Woolf CJ: Synaptic transmission and plasticity in the superficial dorsal horn. Prog Brain Res 2000; 129:63–80
60. Nakata Y, Goto T, Saito H, Ishiguro Y, Terui K, Kawakami H, Tsuruta Y, Niimi Y, Morita S: Plasma concentration of fentanyl with xenon to block somatic and hemodynamic responses to surgical incision. Anesthesiology 2000; 92:1043–8
61. Lachmann B, Armbruster S, Schairer W, Landstra M, Trouwborst A, Van Daal GJ, Kusuma A, Erdmann W: Safety and efficacy of xenon in routine use as an inhalational anaesthetic. Lancet 1990; 335:1413–5
62. Antognini JF, Schwartz K: Exaggerated anesthetic requirements in the preferentially anesthetized brain. Anesthesiology 1993; 79:1244–9
63. Rampil IJ: Anesthetic potency is not altered after hypothermic spinal cord transection in rats. Anesthesiology 1994; 80:606–10
64. Rampil IJ, King BS: Volatile anesthetics depress spinal motor neurons. Anesthesiology 1996; 85:129–34
65. Antognini JF, Carstens E, Atherley R: Does the immobilizing effect of thiopental in brain exceed that of halothane? Anesthesiology 2002; 96:980–6
66. Sonner JM, Antognini JF, Dutton RC, Flood P, Gray AT, Harris RA, Homanics GE, Kendig J, Orser B, Raines DE, Rampil IJ, Trudell J, Vissel B, Eger EI: Inhaled anesthetics and immobility: Mechanisms, mysteries, and minimum alveolar anesthetic concentration. Anesth Analg 2003; 97:718–40
67. Kim J, Yao A, Atherley R, Carstens E, Jinks SL, Antognini JF: Neurons in the ventral spinal cord are more depressed by isoflurane, halothane, and propofol than are neurons in the dorsal spinal cord. Anesth Analg 2007; 105:1020–6
68. Antognini JF, Carstens E: Increasing isoflurane from 0.9 to 1.1 minimum alveolar concentration minimally affects dorsal horn cell responses to noxious stimulation. Anesthesiology 1999; 90:208–14
69. Mitsuyo T, Dutton RC, Antognini JF, Carstens E: The differential effects of halothane and isoflurane on windup of dorsal horn neurons selected in unanesthetized decerebrated rats. Anesth Analg 2006; 103:753–60
This article has been cited 1 time(s).
Molecular PainXenon inhibits excitatory but not inhibitory transmission in rat spinal cord dorsal horn neuronsMolecular Pain
© 2009 American Society of Anesthesiologists, Inc.
Publication of an advertisement in Anesthesiology Online does not constitute endorsement by the American Society of Anesthesiologists, Inc. or Lippincott Williams & Wilkins, Inc. of the product or service being advertised.