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NEUROPHARMACOLOGY
C.V. Starr Laboratory for Molecular Neuropharmacology, Department of Anesthesiology, Weill Cornell Medical College, New York, New York (F.J., M.Y., P.A.G., N.L.H.); and Departments of Anesthesiology and Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania (D.C., G.E.H.)
Received November 21, 2007; accepted December 18, 2007.
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Abstract |
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4-subunit knockout (Gabra4–/–) mice, although 250 µM isoflurane enhanced synaptic inhibition in VB neurons from both wild-type and Gabra4–/– mice. These data indicate an obligatory requirement for 4-subunit expression in the generation of the isoflurane-activated current. In addition, isoflurane directly activated 4β2
GABAA-Rs expressed in human embryonic kidney 293 cells, and it was more potent at 4β2 than at 1β2
2 receptors (the presumptive extrasynaptic and synaptic GABAA-R subtypes in VB neurons). We conclude that the extrasynaptic GABAA-Rs of thalamocortical neurons are sensitive to low concentrations of isoflurane. In view of the crucial role of the thalamus in sensory processing, sleep, and cognition, the modulation of these extrasynaptic GABAA-Rs by isoflurane may contribute to the sedation and hypnosis associated with low doses of this anesthetic agent.
). In all cases, the dose of general anesthetic required to produce amnesia, sedation, and hypnosis is much lower than that required to produce immobility. For example, the commonly used inhaled anesthetic, isoflurane, prevents voluntary response to spoken commands in human subjects (Dwyer et al., 1992) at concentrations that are only 30 to 40% of the levels required for immobility in response to a painful stimulus.
In the waking state, the thalamus is involved in the processing and relay of sensory information to the cortex, and it is also important in the maintenance of high-frequency synchronous oscillatory activity that is associated with attention (Steriade et al., 1993; von Krosigk et al., 1993; Steriade, 2000; Ribary, 2005). During slow-wave sleep, however, relay neurons in the thalamus are hyperpolarized and generate 1 to 3 Hz (delta wave) activity that entrains neurons in the cortex (Steriade, 2000; Steriade, 2003).
Therefore, the thalamo-corticothalamic loop has long been identified as a key target for anesthetic action to induce sedation and hypnosis (Angel, 1993; Alkire et al., 2000; White and Alkire, 2003; Alkire and Miller, 2005). In vivo, the firing rate of thalamocortical relay cells is strongly suppressed by isoflurane (Detsch et al., 1999), and these in vivo inhibitory effects of isoflurane can be reversed by local application of the GABAA receptor (GABAA-R) antagonist bicuculline (Vahle-Hinz et al., 2001), indicating that GABAA-Rs contribute significantly to the effects of the anesthetic on the firing of thalamic neurons, although other mechanisms involving K+ conductances have also been proposed to contribute to the anesthetic effects in the thalamus (Ries and Puil, 1999a).
In addition to the "classic" GABAA-Rs that mediate synaptic inhibition, an additional population of GABAA-Rs has recently been demonstrated to occur at extrasynaptic sites in the central nervous system, and a population of such receptors exists on thalamocortical relay neurons (Belelli et al., 2005; Cope et al., 2005; Jia et al., 2005; Bright et al., 2007). These extrasynaptic GABAA-Rs (consisting mainly of 4, β2, and subunits) are persistently activated by low concentrations of GABA and have distinct pharmacological properties that differentiate them from the synaptic GABAA-Rs in relay neurons of the thalamus (which consist mainly of 1, β2, and 2 subunits) (Jia et al., 2005). GABA-mediated tonic inhibition in thalamic neurons requires expression of the GABAA-R 4 subunit, because tonic currents are absent in thalamic relay neurons from GABAA-R 4-subunit knockout (Gabra4–/–) mice (Chandra et al., 2006). The inhibitory function of these extrasynaptic GABAA-Rs has been shown to be enhanced by the novel hypnotic gaboxadol and by the i.v. anesthetic etomidate (Belelli et al., 2005; Cope et al., 2005; Jia et al., 2005).
In the present study, we investigated the actions of isoflurane on thalamocortical neurons in the mouse ventrobasal (VB) thalamus. We found that isoflurane, at clinically relevant concentrations (25–250 µM) (Franks and Lieb, 1994), evoked sustained currents in VB neurons. These sustained currents were blocked by gabazine, a selective antagonist of GABAA-Rs. In addition, we examined the actions of isoflurane on GABAA-Rs expressed in human embryonic kidney (HEK)293 cells, using subunit compositions chosen to resemble synaptic and extrasynaptic GABAA-Rs found in the thalamus. We found that isoflurane directly activated the"extrasynaptic" 4β2 receptors and showed greater potency at 4β2 receptors than at the "synaptic" 1β22s receptors. The isoflurane-activated current was absent in VB neurons from Gabra4–/– mice, which do not express extrasynaptic GABAA-Rs (Chandra et al., 2006). Our data suggest that isoflurane is a potent modulator of extrasynaptic GABAA-Rs in VB neurons.
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Materials and Methods |
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Slices were perfused with carbogenated artificial cerebrospinal fluid, which contained the following: 124 mM NaCl, 2.5 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3, 1.25 mM NaH2PO4, and 10 mM glucose. Whole-cell patch clamp recordings from visually identified thalamic neurons were performed using an Axopatch 200A amplifier (Molecular Devices, Sunnyvale, CA) at room temperature (20–22°C) as described previously (Jia et al., 2005). The intracellular solution for voltage-clamp recordings contained the following: 140 mM CsCl, 4 mM NaCl, 1 mM MgCl2, 10 mM HEPES, 0.05 mM EGTA, 2 mM ATP-Mg, and 0.4 mM GTP-Mg; pH was adjusted to 7.2 with CsOH. Intracellular solution for current-clamp recordings contained the following: 130 mM K+-gluconate, 5 mM NaCl, 2 mM MgCl2, 10 mM HEPES, 0.5 mM EGTA, 2 mM ATP-K+, and 0.3 mM GTP-Na+; pH was adjusted to 7.25 with KOH. Spontaneous inhibitory postsynaptic currents (IPSCs) were recorded at –60 mV and isolated by bath application of 2 to 5 mM kynurenic acid. Access resistance was monitored throughout the recording period; cells were included for analysis only if the series resistance was less than 20 M
and the change in series resistance was less than 20% over the course of the experiment. Data were analyzed as described previously (Jia et al., 2005); very briefly, off-line analysis was performed using MiniAnalysis 5.5 (Synaptosoft, Decatur, GA), SigmaPlot 6.0 (SPSS Inc., Chicago, IL), and Excel 2000 (Microsoft, Redmond, WA). The holding current shift was measured as the difference in the holding current before and during drug application. IPSCs were detected and analyzed using MiniAnalysis as described previously (Jia et al., 2005). Unless otherwise indicated, averaged data are expressed as mean ± S.E.M. Statistical significance was assessed using Student's t test or one-way ANOVA with Dunnett's test, and p < 0.05 was considered statistically significant.
Recordings from HEK293 Cells Expressing Recombinant GABAA-Rs. The cDNAs encoding the human 1, mouse 4, rat β2, human 2s, and human subunits were subcloned into the pcDNA3.1 expression vector and transiently expressed in HEK293 cells (American Type Culture Collection, Rockville, MD), as described in Jia et al. (2005). Ligand-gated currents were recorded at room temperature (voltage-clamped at –60 mV) using an Axopatch 200 amplifier (Molecular Devices). The extracellular solution contained the following: 145 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 1 mM MgCl2, 6 mM D-glucose, and 10 mM HEPES; pH was adjusted to 7.4 with NaOH. The intracellular solution used to fill patch pipettes contained the following: 145 mM N-methyl-D-glucamine hydrochloride, 0.1 mM CaCl2, 5 mM ATP-K, 1.1 mM EGTA, 2 mM MgCl2, and 5 mM HEPES; pH was adjusted to 7.2 with KOH. GABA and/or isoflurane were applied rapidly (
50-ms exchange time) to the cell via a multichannel motor-driven solution exchange device (Rapid Solution Changer RSC-100; Molecular Kinetics, Pullman, WA).
Concentration-response amplitude data were analyzed as described previously (Jia et al., 2005); concentration-response data for each individual cell were fitted (using a sum of least-squares method) to a Hill equation of the form as follows: I = Imax x [agonist]nH/([agonist]nH + EC50nH), where I is the peak current, Imax is the maximal whole-cell current amplitude, [agonist] is the agonist concentration, EC50 is the agonist concentration eliciting a half-maximal current response, and nH is the Hill coefficient. Peak current in response to an EC20 concentration of GABA alone (IEC20) was defined as the control response, and isoflurane was then preapplied for 20 s before coapplication with GABA(EC20) to ensure that isoflurane had reached equilibrium with the receptors. Isoflurane-induced potentiation was calculated as the percentage increase in peak current relative to control. Currents elicited directly during preapplication of isoflurane were measured and normalized to IEC20 to quantify the direct activation of GABAA receptors by isoflurane in the absence of GABA. Statistical significance was assessed using a one-way ANOVA with a Dunnett's multiple comparison post-test. Data are presented as mean ± S.E.M.
Drugs and Preparation of Volatile Anesthetic Solutions. GABA, gabazine [4-[6-imino-3-(4-methoxyphenyl) pyridazin-1-yl] butanoic acid hydrobromide], and kynurenic acid (4-oxo-1H-quinoline-2-carboxylic acid) were purchased from Sigma-Aldrich (St. Louis, MO). Isoflurane [2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane] was obtained from Abbott Laboratories (North Chicago, IL). A stock solution of 10 mM GABA was prepared daily. Isoflurane solutions were prepared by injection of liquid anesthetic with a gas-tight syringe (Hamilton, Reno, NV) into i.v. solution bags containing 100 ml of extracellular solution and were used within 2 h (Krasowski and Harrison, 2000). Isoflurane solutions were applied to the brain slice preparations via perfusion through polytetrafluoroethylene tubing; the drug-containing solution was applied to the HEK cells locally using a rapid solution changer. We have previously shown that losses of anesthetic to the air and the tubing using this approach are less than 5% (Krasowski and Harrison, 2000).
Generation and Use of 4-Subunit Knockout Mice. The 4-subunit gene knockout (KO) mice were generated as described previously (Chandra et al., 2006). All knockout (Gabra4–/–) and wild-type (Gabra4+/+) littermates used were age-matched and were on the same genetic background (129 x 1/S1 x C57BL/6J hybrid; F2–F6 generations). Those conducting the experiments were blind to genotype in all studies.
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; Cope et al., 2005; Jia et al., 2005; Chandra et al., 2006; Bright et al., 2007). Isoflurane is able to directly activate GABAA-Rs (Yang et al., 1992; see also Fig. 5), as well as potentiate the action of GABA at these receptors (Jones and Harrison, 1993; see also Fig. 5), and so for the sake of simplicity, we will refer to the sustained current recorded from neurons in brain slices as the "tonic current" or "isoflurane-activated current."
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We then performed experiments using a voltage-ramp protocol to compare the reversal potential of isoflurane-activated currents (EIso) to the Cl– equilibrium potential (ECl). Currents induced by a slow ramp voltage command (+40 to –40 mV, 10 s) were recorded before and after the perfusion of 250 µM isoflurane, and EIso was calculated from the subtracted traces as shown in Fig. 2B; EIso was 5 ± 1 mV (n = 6), which was very close to the predicted ECl (2.1 mV), calculated using the Nernst equation. These results support our interpretation that isoflurane-induced currents arise via the enhancement of a chloride conductance mediated by GABAA-Rs.
Isoflurane Prolongs the Decay Time of IPSCs. We also examined the effects of isoflurane on synaptic GABAA-Rs in the thalamus. In thalamic VB neurons, synaptic GABAA-Rs consist primarily of 1, β2, and 2 subunits (Jia et al., 2005). Spontaneous IPSCs were readily observed in VB neurons and were completely blocked by gabazine (Fig. 2A). Isoflurane (25–250 µM) had no effect on either the amplitude or frequency of these IPSCs (Fig. 3). Low concentrations of isoflurane (25 µM) had no effect on the decay time of IPSCs (% change: 4 ± 4%, n = 10), but higher concentrations of isoflurane significantly increased IPSC decay time (85 µM, 28 ± 6%, n = 10; 250 µM, 133 ± 12%, n = 10), and these findings are completely consistent with observations made in hippocampal neurons (Jones and Harrison, 1993; Banks and Pearce, 1999; Nishikawa and MacIver, 2001; Caraiscos et al., 2004; Verbny et al., 2005).
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). Therefore, to facilitate measurements of firing rate, we depolarized the membrane potential to approximately –60 mV by constant current injection. At this membrane potential, VB neurons were generally silent but displayed sustained action potential (AP) firing in response to injection of depolarizing current. The amplitude of the current step (500-ms duration) was adjusted to induce 10 APs (Fig. 4A), corresponding to a firing frequency of 20 Hz, and we then compared the numbers of APs evoked by depolarizing current steps before and after isoflurane application. Isoflurane (25 µM) significantly reduced the number of evoked APs, from 9.5 ± 1.0 to 7.5 ± 1.0% (p < 0.01, n = 6). In addition to the effect on spike firing, isoflurane (25 µM) also decreased the membrane input resistance (Rm) to 90 ± 2% of control (p < 0.01, n = 6).
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Isoflurane Directly Activates GABAA 4β2 Receptors Expressed in HEK293 Cells. The GABAA-Rs activated at inhibitory synapses onto VB neurons are thought to be of the 1β22 subtype (Zhang et al., 1997; Huntsman and Huguenard, 2000), whereas the extrasynaptic receptors are thought to be 4β2 (Belelli et al., 2005; Jia et al., 2005; Chandra et al., 2006). To compare the isoflurane sensitivity of these proposed synaptic and extrasynaptic GABAA-R subtypes, we tested the effect of isoflurane on 1β22s and 4β2 GABAA-Rs expressed in HEK293 cells. Concentration-response curves for GABA (data not shown) revealed that 4β2 receptors (EC50 2.1 ± 0.1 µM, n = 43) are more sensitive to GABA than 1β22s receptors (EC50 22.7 ± 2.1 µM, n = 20; p < 0.001), but the maximal GABA current was smaller in 4β2 receptors than in 1β22s receptors (223 ± 16 and 1741 ± 193 pA, respectively), all consistent with earlier observations (Jia et al., 2005).
We then examined the modulation of these recombinant GABAA-R subtypes over a range of concentrations of isoflurane (25–800 µM). Isoflurane was applied for 20 s to a HEK cell held under a voltage clamp before coapplication with GABA (EC20) for another 20 s. Typical recordings from both 1β22 and 4β2 receptors are shown in Fig. 5, A and B (two different HEK cells for each subtype). At all of the concentrations tested, isoflurane enhanced the amplitude of the GABA-evoked response to a greater degree in 4β2 receptors than in 1β22 receptors (Fig. 5C).
In the presence of isoflurane, the peak current observed in response to GABA is actually the sum of two components, IDirect (i.e., the current induced by isoflurane alone, due to direct receptor activation) and IGABAEC20+ISO (i.e., the isoflurane-potentiated GABA-evoked current). In a more detailed analysis, we compared these two currents in 1β22 and 4β2 receptors. As shown in Fig. 6A, isoflurane, over the entire range of concentrations tested (25–800 µM), directly activated 4β2 receptors in the absence of GABA, whereas in 1β22s receptors, only higher concentrations of isoflurane (
200 µM) were able to induce direct currents. In addition, isoflurane (at all of the concentrations tested) activated proportionally larger direct currents (IDirect/IGABAEC20) in 4β2 receptors than in 1β22s receptors. At the same time, isoflurane was more potent as a modulator (in terms of potentiation of the response to GABA) at 4β2 receptors than at 1β22s receptors (Fig. 6B). Note that these data illustrate the potentiating effect of isoflurane, calculated by subtracting IDirect (Fig. 6A) from the total current (Fig. 5C).
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10 pA), we decided to test the possibility that the currents were influenced by the change of solutions and the associated movement artifacts. Therefore, we used the same protocol but omitted the isoflurane. Under these test conditions, when switching from one stream of saline to another, there was no significant enhancement of the GABA response (1β22s:0 ± 10%, n = 5; 4β2: –5 ± 5%, n = 7) or direct current response to the solution exchange (1β22s:2 ± 1%, n = 5; 4β2: –4 ± 2%, n = 7).
Isoflurane-Evoked Currents Are Absent in VB Neurons from Gabra4–/– Mice. In a final set of experiments, we used a knockout mouse strain to determine whether extrasynaptic GABAA-Rs are required for the generation of isoflurane-induced currents in the thalamus. We have previously demonstrated that extrasynaptic GABAA-Rs are absent in thalamic relay neurons from Gabra4–/– mice (Chandra et al., 2006). No significant isoflurane-evoked currents were detected in VB neurons from Gabra4–/– mice (25 µM: 0 ± 1 pA, n = 10; 85 µM: 0 ± 1 pA, n = 12; 250 µM: 1 ± 1 pA, n = 11). In contrast, wild-type (WT) neurons showed measurable isoflurane-evoked currents (25 µM: 8 ± 2 pA, n = 8; 85 µM: 17 ± 3 pA, n = 8; 250 µM: 44 ± 4 pA, n = 9), which were comparable to isoflurane-evoked currents recorded from standard C57BL/6 mice (Fig. 7). This difference between the genotypes was highly significant (p < 0.001 at all three concentrations of isoflurane). In contrast, we found that the modulation of IPSCs in VB neurons by isoflurane was similar in wild-type and 4-subunit knockout mice. As shown in Fig. 7C, isoflurane had a comparable effect on the decay time of IPSCs recorded in VB neurons from wild-type and Gabra4–/– mice. IPSC decay times in the absence or presence of isoflurane (at the specified concentration) were (WT versus KO) as follows: control, 15 ± 1 versus 14 ± 1 ms; 25 µM, 16 ± 2 versus 15 ± 1 ms; 85 µM, 19 ± 1 versus 19 ± 2 ms; 250 µM, and 33 ± 3 versus 31 ± 3 ms. These results are consistent with the hypothesis that extrasynaptic (and not synaptic) GABAA-Rs mediate the isoflurane-evoked tonic current in thalamic neurons.
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; Krasowski et al., 1998; Banks and Pearce, 1999; Krasowski and Harrison, 2000; Li and Pearce, 2000; Nishikawa and MacIver, 2001; Nishikawa and Harrison, 2003; Hemmings et al., 2005). In the present study, we found that isoflurane (25–250 µM) induced sustained currents in thalamic relay neurons and that this was completely dependent on the presence of extrasynaptic GABAA-Rs (4β2 subtype). Recordings from 4β2 receptors expressed in HEK293 cells demonstrated that isoflurane not only potentiated the GABA response but it also directly activated these receptors in the absence of GABA. Therefore, the sustained current evoked by isoflurane in thalamic relay neurons probably results from the sum of the direct activation of extrasynaptic GABAA-Rs and the enhancement of the action of ambient GABA on these receptors.
General anesthesia in mammals is a complex phenomenon, involving a combination of desirable effects such as amnesia, hypnosis, and immobility (Campagna et al., 2003). Although immobilization is commonly used as a measure of anesthetic potency (Hemmings et al., 2005), 3-fold lower concentrations are required for sedation and hypnosis (Dwyer et al., 1992). GABAA-Rs may not be involved in the immobilizing action of inhaled anesthetics, which seems to occur at the level of the spinal cord, and is resistant to GABA antagonists (Sonner et al., 2003), but there is strong evidence that GABAA-Rs are involved in the sedation and hypnosis induced by many general anesthetics (reviewed by Hemmings et al., 2005).
The thalamus relays sensory information to the appropriate modality-specific areas in the sensory cortex and also participates in the transitions between waking and sleep states (Saper et al., 2001; Steriade, 2003). The thalamus is therefore a potential target for the sedative and hypnotic actions of volatile anesthetics, and it has been previously argued that the thalamus is a principal site of drug action for generating the anesthetized state (Angel, 1993). A variety of studies support the idea that volatile anesthetics modulate thalamic function. At the whole-brain level, positron emission tomography imaging demonstrates that the volatile anesthetics isoflurane and halothane both produce a particularly large decrease in glucose utilization in the thalamus (White and Alkire, 2003). Consistent with a decrease in metabolic activity, the firing rate of thalamocortical relay cells in vivo is strongly suppressed by anesthetic concentrations of isoflurane (Detsch et al., 1999).
AK+ conductance mechanism has been implicated in the inhibitory actions of isoflurane in the thalamus in vitro (Ries and Puil, 1999a,b), but in other studies, the effect of isoflurane to inhibit spike firing induced by a mechanical stimulus in the thalamus in vivo is blocked by local application of the GABAA-R antagonist bicuculline (Vahle-Hinz et al., 2001). In the present study, we demonstrate that tonic currents activated by isoflurane in thalamic neurons in vitro are mediated by GABAA-Rs, because the isoflurane-induced currents were totally blocked by a specific GABAA-R antagonist, gabazine. In addition, the reversal potential of isoflurane-induced currents (EIso) is close to the calculated equilibrium potential for chloride ions (ECl) in our experiments and far from K+ equilibrium potential (EK).
Extrasynaptic GABAA-Rs and tonic inhibition have been observed in the thalamus, hippocampus, dentate gyrus, cortex, and cerebellum (Brickley et al., 1996; Nusser and Mody, 2002; Jia et al., 2005; Keros and Hablitz, 2005). The presence of a low concentration of GABA in the extracellular space leads to persistent activation of the extrasynaptic GABAA-Rs, and the resulting tonic inhibition regulates the excitability of individual neurons and the activity of neural networks (Mody and Pearce, 2004; Semyanov et al., 2004; Farrant and Nusser, 2005; Jia et al., 2007). In the thalamus, subunit containing extrasynaptic GABAA-Rs appear to be a common target for a variety of sedative and hypnotic agents, including gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) (Belelli et al., 2005; Cope et al., 2005; Jia et al., 2005) and tetrahydrodeoxycorticosterone (Cope et al., 2005). Several laboratories also report that extrasynaptic GABAA-Rs containing the subunit are sensitive to low concentrations of ethanol (Mody et al., 2007; Olsen et al., 2007; Santhakumar et al., 2007; Smith and Gong, 2007; but see: Borghese and Harris, 2007; Botta et al., 2007).
It is noteworthy that not all extrasynaptic GABAA-Rs contain subunits; in hippocampal CA1 pyramidal neurons, for example, one population of extrasynaptic GABAA-Rs contains the 5 subunit (Caraiscos et al., 2004). Like extrasynaptic receptors containing subunits, 5 subunit containing GABAA-Rs are sensitive to a spectrum of anesthetic agents. At the same concentration of isoflurane as used in the present study, Caraiscos et al. (2004) observed that 25 µM isoflurane potentiated the tonic current in cultured hippocampal pyramidal neurons obtained from wild-type, but not 5-subunit knockout, mice. In addition, the i.v. anesthetic etomidate was shown to potentiate tonic currents recorded in cultured hippocampal pyramidal neurons expressing the GABAA-R 5 subunit. In this case, the amnestic, and not sedative-hypnotic, effect of the drug is dependent on 5-subunit expression (Cheng et al., 2006). These studies suggest that extrasynaptic GABAA-Rs are a common molecular target for central nervous system depressants (Orser, 2006).
In the thalamus, extrasynaptic GABAA-Rs that contain the subunit also seem to require inclusion of the GABAA-R 4 subunit because tonic currents are absent in relay neurons from GABAA-R 4-subunit knockout (Gabra4–/–) mice (Chandra et al., 2006). Our experiments on thalamic relay neurons from Gabra4–/– mice suggest that this population of extrasynaptic GABAA-Rs exclusively mediates the sustained currents elicited by low concentrations of isoflurane, although the prolongation of synaptic currents by isoflurane (85 µM) is independent of 4-subunit expression, because IPSCs were potentiated to a similar extent by isoflurane in wild-type and 4-subunit knockout mice. We conclude that the 4 subunit containing GABAA-Rs provides the molecular basis for the isoflurane-induced current and that this current is completely independent of synaptic GABAA-Rs.
We examined the pharmacological properties of heterologously expressed GABAA-Rs and configured in order to resemble natively expressed extrasynaptic (4β2) receptors, which are more sensitive to GABA than 1β22s receptors (Brown et al., 2002; Storustovu and Ebert, 2006). We found that isoflurane is a more potent modulator at 4β2 GABAA-Rs (which are analogous to native extrasynaptic receptors) than at 1β22 receptors (which are analogous to native synaptic receptors). One interesting finding is that isoflurane, at a concentration as low as 25 µM, directly activates 4β2 receptors in the absence of GABA. In contrast, only high concentrations of isoflurane (200 µM) are able to induce significant direct currents in 1β22s receptors, consistent with earlier observations (Krasowski et al., 1998; Krasowski and Harrison, 2000; Raines et al., 2003).
In conclusion, we show that extrasynaptic GABAA-Rs in the thalamus are potently activated by isoflurane. Because the thalamus plays a critically important role in both sensory processing and sleep regulation, we suggest that the sedative and hypnotic actions of isoflurane at subanesthetic concentrations may be mediated in part through this population of receptors.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GABAA-R(s), GABAA receptor(s); VB, ventrobasal; HEK, human embryonic kidney; IPSC, inhibitory postsynaptic current; KO, knockout; EIso, isoflurane-activated current; ECl, Cl– equilibrium potential; AP, action potential; Rm, membrane input resistance; WT, wild-type.
Address correspondence to: Dr. Neil Harrison, Department of Anesthesiology, Weill Cornell Medical College, 1300 York Avenue, Room A-1050, New York, NY 10065. E-mail: neh2001{at}med.cornell.edu
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References |
|---|
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Angel A (1993) Central neuronal pathways and the process of anaesthesia. Br J Anaesth 71: 148–163.
Alkire MT, Haier RJ, and Fallon JH (2000) Toward a unified theory of narcosis: brain imaging evidence for a thalamocortical switch as the neurophysiologic basis of anesthetic-induced unconsciousness. Conscious Cogn 9: 370–386.[CrossRef][Medline]
Alkire MT and Miller J (2005) General anesthesia and the neural correlates of consciousness. Prog Brain Res 150: 229–244.[Medline]
Banks MI and Pearce RA (1999) Dual actions of volatile anesthetics on GABAA IPSCs: dissociation of blocking and prolonging effects. Anesthesiology 90: 120–134.[CrossRef][Medline]
Belelli D, Peden DR, Rosahl TW, Wafford KA, and Lambert JJ (2005) Extrasynaptic GABAA receptors of thalamocortical neurons: a molecular target for hypnotics. J Neurosci 25: 11513–11520.
Borghese CM and Harris RA (2007) Studies of ethanol actions on recombinant -containing -aminobutyric acid type A receptors yield contradictory results. Alcohol 41: 155–162.[CrossRef][Medline]
Botta P, Radcliffe RA, Carta M, Mameli M, Daly E, Floyd KL, Deitrich RA, and Valenzuela CF (2007) Modulation of GABAA receptors in cerebellar granule neurons by ethanol: a review of genetic and electrophysiological studies. Alcohol 41: 187–199.[CrossRef][Medline]
Brickley SG, Cull-Candy SG, and Farrant M (1996) Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J Physiol 497: 753–759.
Bright DP, Aller MI, and Brickley SG (2007) Synaptic release generates a tonic GABAA receptor-mediated conductance that modulates burst precision in thalamic relay neurons. J Neurosci 27: 2560–2569.
Brown N, Kerby J, Bonnert TP, Whiting PJ, and Wafford KA (2002) Pharmacological characterization of a novel cell line expressing human 4β3 GABAA receptors. Br J Pharmacol 136: 965–974.[CrossRef][Medline]
Campagna JA, Miller KW, and Forman SA (2003) Mechanisms of actions of inhaled anesthetics. N Engl J Med 348: 2110–2124.
Caraiscos VB, Newell JG, You-Ten KE, Elliott EM, Rosahl TW, Wafford KA, MacDonald JF, and Orser BA (2004) Selective enhancement of tonic GABAergic inhibition in murine hippocampal neurons by low concentrations of the volatile anesthetic isoflurane. J Neurosci 24: 8454–8458.
Chandra D, Jia F, Liang J, Peng Z, Suryanarayanan A, Werner DF, Spigelman I, Houser CR, Olsen RW, Harrison NL, et al. (2006) GABAA receptor 4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol. Proc Natl Acad Sci U S A 103: 15230–15235.
Cheng VY, Martin LJ, Elliott EM, Kim JH, Mount HT, Taverna FA, Roder JC, Macdonald JF, Bhambri A, Collinson N, et al. (2006) 5 GABAA receptors mediate the amnestic but not sedative-hypnotic effects of the general anesthetic etomidate. J Neurosci 26: 3713–3720.
Cope DW, Hughes SW, and Crunelli V (2005) GABAA receptor-mediated tonic inhibition in thalamic neurons. J Neurosci 25: 11553–11563.
Detsch O, Vahle-Hinz C, Kochs E, Siemers M, and Bromm B (1999) Isoflurane induces dose-dependent changes of thalamic somatosensory information transfer. Brain Res 829: 77–89.[CrossRef][Medline]
Dwyer R, Bennett HL, Eger EI 2nd, and Heilbron D (1992) Effects of isoflurane and nitrous oxide in subanesthetic concentrations on memory and responsiveness in volunteers. Anesthesiology 77: 888–898.[CrossRef][Medline]
Farrant M and Nusser Z (2005) Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors. Nat Rev Neurosci 6: 215–229.[CrossRef][Medline]
Franks NP and Lieb WR (1994) Molecular and cellular mechanisms of general anaesthesia. Nature 367: 607–614.[CrossRef][Medline]
Hemmings HC Jr, Akabas MH, Goldstein PA, Trudell JR, Orser BA, and Harrison NL (2005) Emerging molecular mechanisms of general anesthetic action. Trends Pharmacol Sci 26: 503–510.[CrossRef][Medline]
Huntsman MM and Huguenard JR (2000) Nucleus-specific differences in GABAA-receptor-mediated inhibition are enhanced during thalamic development. J Neurophysiol 83: 350–358.
Jia F, Pignataro L, and Harrison NL (2007) GABAA receptors in the thalamus: 4 subunit expression and alcohol sensitivity. Alcohol 41: 177–185.[CrossRef][Medline]
Jia F, Pignataro L, Schofield CM, Yue M, Harrison NL, and Goldstein PA (2005) An extrasynaptic GABAA receptor mediates tonic inhibition in thalamic VB neurons. J Neurophysiol 94: 4491–4501.
Jones MV and Harrison NL (1993) Effects of volatile anesthetics on the kinetics of inhibitory postsynaptic currents in cultured rat hippocampal neurons. J Neurophysiol 70: 1339–1349.
Keros S and Hablitz JJ (2005) Subtype-specific GABA transporter antagonists synergistically modulate phasic and tonic GABAA conductances in rat neocortex. J Neurophysiol 94: 2073–2085.
Krasowski MD and Harrison NL (2000) The actions of ether, alcohol and alkane general anaesthetics on GABAA and glycine receptors and the effects of TM2 and TM3 mutations. Br J Pharmacol 129: 731–743.[CrossRef][Medline]
Krasowski MD, Koltchine VV, Rick CE, Ye Q, Finn SE, and Harrison NL (1998) Propofol and other intravenous anesthetics have sites of action on the -aminobutyric acid type A receptor distinct from that for isoflurane. Mol Pharmacol 53: 530–538.
Li X and Pearce RA (2000) Effects of halothane on GABAA receptor kinetics: evidence for slowed agonist unbinding. J Neurosci 20: 899–907.
Llinás R and Jahnsen H (1982) Electrophysiology of mammalian thalamic neurones in vitro. Nature 297: 406–408.[CrossRef][Medline]
Mody I, Glykys J, and Wei W (2007) A new meaning for "Gin & Tonic": tonic inhibition as the target for ethanol action in the brain. Alcohol 41: 145–153.[CrossRef][Medline]
Mody I and Pearce RA (2004) Diversity of inhibitory neurotransmission through GABAA receptors. Trends Neurosci 27: 569–575.[CrossRef][Medline]
Nishikawa K and Harrison NL (2003) The actions of sevoflurane and desflurane on the -aminobutyric acid receptor type A: effects of TM2 mutations in the and β subunits. Anesthesiology 99: 678–684.[CrossRef][Medline]
Nishikawa K and MacIver MB (2001) Agent-selective effects of volatile anesthetics on GABAA receptor-mediated synaptic inhibition in hippocampal interneurons. Anesthesiology 94: 340–347.[CrossRef][Medline]
Nusser Z and Mody I (2002) Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. J Neurophysiol 87: 2624–2628.
Olsen RW, Hanchar HJ, Meera P, and Wallner M (2007) GABAA receptor subtypes: the "one glass of wine" receptors. Alcohol 41: 201–209.[CrossRef][Medline]
Orser BA (2006) Extrasynaptic GABAA receptors are critical targets for sedative-hypnotic drugs. J Clin Sleep Med 2: S12–S18.[Medline]
Raines DE, Claycomb RJ, and Forman SA (2003) Modulation of GABAA receptor function by nonhalogenated alkane anesthetics: the effects on agonist enhancement, direct activation, and inhibition. Anesth Analg 96: 112–118.
Ribary U (2005) Dynamics of thalamo-cortical network oscillations and human perception. Prog Brain Res 150: 127–142.[Medline]
Ries CR and Puil E (1999a) Ionic mechanism of isoflurane's actions on thalamocortical neurons. J Neurophysiol 81: 1802–1809.
Ries CR and Puil E (1999b) Mechanism of anesthesia revealed by shunting actions of isoflurane on thalamocortical neurons. J Neurophysiol 81: 1795–1801.
Santhakumar V, Wallner M, and Otis TS (2007) Ethanol acts directly on extrasynaptic subtypes of GABAA receptors to increase tonic inhibition. Alcohol 41: 211–221.[CrossRef][Medline]
Saper CB, Chou TC, and Scammell TE (2001) The sleep switch: hypothalamic control of sleep and wakefulness. Trends Neurosci 24: 726–731.[CrossRef][Medline]
Semyanov A, Walker MC, Kullmann DM, and Silver RA (2004) Tonically active GABAA receptors: modulating gain and maintaining the tone. Trends Neurosci 27: 262–269.[CrossRef][Medline]
Smith SS and Gong QH (2007) Ethanol effects on GABA-gated current in a model of increased 4β GABAA receptor expression depend on time course and preexposure to low concentrations of the drug. Alcohol 41: 223–231.[CrossRef][Medline]
Sonner JM, Zhang Y, Stabernack C, Abaigar W, Xing Y, and Laster MJ (2003) GABAA receptor blockade antagonizes the immobilizing action of propofol but not ketamine or isoflurane in a dose-related manner. Anesth Analg 96: 706–712.
Steriade M (2000) Corticothalamic resonance, states of vigilance and mentation. Neuroscience 101: 243–276.[CrossRef][Medline]
Steriade M (2003) The corticothalamic system in sleep. Front Biosci 8: d878–d899.[Medline]
Steriade M, McCormick DA, and Sejnowski TJ (1993) Thalamocortical oscillations in the sleeping and aroused brain. Science 262: 679–685.
Storustovu SI and Ebert B (2006) Pharmacological characterization of agonists at -containing GABAA receptors: Functional selectivity for extrasynaptic receptors is dependent on the absence of 2. J Pharmacol Exp Ther 316: 1351–1359.
Vahle-Hinz C, Detsch O, Siemers M, Kochs E, and Bromm B (2001) Local GABAA receptor blockade reverses isoflurane's suppressive effects on thalamic neurons in vivo. Anesth Analg 92: 1578–1584.
Verbny YI, Merriam EB, and Banks MI (2005) Modulation of -aminobutyric acid type A receptor-mediated spontaneous inhibitory postsynaptic currents in auditory cortex by midazolam and isoflurane. Anesthesiology 102: 962–969.[CrossRef][Medline]
von Krosigk M, Bal T, and McCormick DA (1993) Cellular mechanisms of a synchronized oscillation in the thalamus. Science 261: 361–364.
White NS and Alkire MT (2003) Impaired thalamocortical connectivity in humans during general-anesthetic-induced unconsciousness. Neuroimage 19: 402–411.[CrossRef][Medline]
Yang J, Isenberg KE, and Zorumski CF (1992) Volatile anesthetics gate a chloride current in postnatal rat hippocampal neurons. FASEB J 6: 914–918.[Abstract]
Zhang SJ, Huguenard JR, and Prince DA (1997) GABAA receptor-mediated Cl– currents in rat thalamic reticular and relay neurons. J Neurophysiol 78: 2280–2286.
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