Propofol and Isourane Enhancement of Tonic Gamma-Aminobutyric Acid Type A Current in Cardiac Vagal Neurons in the Nucleus Ambiguus

Xin Wang, PhD, MD

BACKGROUND: General anesthesia with propofol and isoflurane induces alterations of the cardiovascular system, including hypotension and changes in heart rate. The preganglionic cardiac vagal neurons (CVNs) are one of the major central components controlling heart rate and autonomic regulation. In this study, we examined whether propofol and isoflurane act on phasic or tonic -aminobutyric acid type A (GABAA) receptor-mediated inhibition in CVNs. METHODS: CVNs were identified in vitro by retrograde fluorescent labeling. Phasic and tonic GABA currents in CVNs were examined using the whole cell patchclamp technique. RESULTS: Propofol (10 M) increased the membrane holding currents by 63 13% and prolonged the decay time of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) from 42.3 2.8 ms in control to 61.8 4.5 ms. Isoflurane, at concentrations of 100, 300, and 500 M, decreased GABAergic mIPSCs frequency by 26.0 16%, 64.6 10.4%, and 70.5 9.8%, prolonged the decay time of GABAergic mIPSCs from 47.9 7.3 to 64.5 8.1 ms, 70.3 10.4 ms, and 66.8 8.1 ms, and increased the membrane holding currents by 32.8 12.8%, 42.7 10%, and 39.9 3%, respectively. The GABAergic antagonist gabazine (25 M) blocked GABAergic mIPSCs, but failed to alter the enhanced holding potential induced by propofol and isoflurane. In contrast, the channel blocker of GABAA receptors, picrotoxin (100 M), reversed the propofol and isoflurane-evoked increase in membrane holding current. CONCLUSION: The results demonstrate that the general anesthetics propofol and isoflurane enhance both phasic and tonic GABAA receptor-mediated inhibition of CVNs.
(Anesth Analg 2009;108:1428)

amma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. Synaptic release of GABA activates postsynaptic ionotropic GABAA receptors and evokes hyperpolarizing inhibitory postsynaptic currents (IPSCs or phasic inhibition).13 GABAA receptors are the most abundant inhibitory neurotransmitter receptors in the mammalian brain and are widespread in somatic, dendritic, and axonal regions of neuronal membrane, some of which are extrasynaptic and distant from sites of neurotransmitter release.1 The activation of extrasynaptic GABAA receptors by ambient GABA produces
From the Department of Pharmacology and Physiology, and Department of Anesthesiology and Critical Care Medicine, The George Washington University, Washington DC. Accepted for publication June 26, 2008. Supported by a National Scientist Development Award from the American Heart Association. Address correspondence and reprint requests to Xin Wang, PhD, MD, Department of Pharmacology and Physiology, and Department of Anesthesiology and Critical Care Medicine, The George Washington University, 2300 Eye Street NW, Ross Hall 654, Washington, DC, 20037. Address e-mail to xinwang@gwu.edu. Copyright 2008 International Anesthesia Research Society
DOI: 10.1213/ane.0b013e31818d8b79

persistent tonic currents, which can produce inhibitory chloride currents that are more than three times larger than that carried by IPSCs when integrated.4 Both volatile and IV general anesthetics can allosterically enhance GABA-evoked chloride currents mediated by the GABAA receptors. Propofol, a widely used clinical IV anesthetic, enhances both tonic and phasic GABA currents in the nucleus tractus solitarius5 and hippocampal neurons.6,7 This effect may be mediated by acting on and GABAA receptors.8 The volatile anesthetic isoflurane prolongs GABAA decay time but reduces the frequency or amplitude of IPSCs. Several reports have shown a diverse set of isoflurane-induced actions on GABAergic IPSCs in various neurons.9 11 Its action on tonic GABAA-mediated conductance showed different results with a selective enhancement in murine hippocampal neurons,12 but had no effect on the nucleus tractus solitarius13 and rat hippocampal pyramidal neurons.14 General anesthetics directly affect the circulation system and induce alterations in hemodynamic homeostasis, including hypotension and variable changes in heart rate depending on the depth of
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anesthesia. However, little is known about the mechanisms by which general anesthetics modulate the central autonomic control of cardiovascular function in the brainstem. The function of parasympathetic cardiac vagal neurons (CVNs) in the nucleus ambiguus in the brainstem is essential for the control of heart rate. CVNs are intrinsically silent and their activity is regulated by the activation and modulation of inhibitory and excitatory synaptic inputs,15,16 as well as an intrinsic persistent GABAergic tonic current.17 In this study, we tested whether propofol and isoflurane act on GABAA receptors in CVNs in the nucleus ambiguus because multiple GABAA receptors are present in CVNs within the nucleus ambiguus,17 each of which could be a target of anesthetics and could result in changes in both phasic and tonic inhibition of CVNs, eliciting the cardiovascular consequence of anesthetics.

receptors to be recorded as an inward current at a holding voltage of 80 mV. Voltage-clamp recordings were made with Axopatch 200B and pClamp 8 software (Axon Instruments, Union City, CA). All tonic and synaptic activity in CVNs was recorded at 80 mV. No more than one experiment was performed in each slice of tissue.

Focal Drug Application to Isolate GABAergic Synaptic Inputs to CVNs

Focal drug application was performed using a PV830 Pneumatic PicoPump pressure delivery system (WPI, Sarasota, FL). GABAergic neurotransmission to CVNs was isolated by continuous focal application of d-2-amino-5-phosphonovalerate (AP-5, 50 M), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 M), and strychnine (1 M) to block N-methyl, d-aspartate (NMDA), non-NMDA, and glycinergic receptors, respectively. Tetrodotoxin (1 M) was used to block synaptic events induced by action potential. Tonic GABAergic currents were examined by selectively blocking GABA synaptic events with picrotoxin. All drugs were obtained from Sigma.

METHODS
All animal procedures were performed with the approval of the Animal Care and Use Committee of The George Washington University in accordance with the recommendations of the panel on euthanasia of the American Veterinary Medical Association and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. SpragueDawley rat pups (postnatal days 17; Hilltop, Scottsdale, PA) were anesthetized and exposed to hypothermia to slow the heart. The heart was exposed by a right thoracotomy and the retrograde fluorescent tracer X-rhodamine-5-(and 6)-isothiocyanate (Molecular Probes, Eugene, OR) was injected into the fat pads at the base of the heart. After a 24 48-h recovery, pups were anesthetized with isoflurane, killed by cervical dislocation, and the hindbrain was rapidly removed and placed in cold physiological saline solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 5 mM glucose, 10 mM HEPES, bubbled with 100% O2, pH 7.4). Medullar slices (500 600 m) that included CVNs were made and the tissue was placed in a recording chamber that allowed perfusion (3 mL/min) with artificial cerebrospinal fluid (125 mM NaCl, 3 mM KCl, 2 mM CaCl2, 26 mM NaHCO3, 5 mM glucose, 5 mM HEPES, equilibrated with 95% O25% CO2, pH 7.357.4). Parasympathetic CVNs in the nucleus ambiguus were identified by the presence of the fluorescent tracer, as described previously15. Neurons containing fluorescent tracer were identified by superimposing the fluorescent and infrared images on a video monitor (Sony, Tokyo, Japan). Patch pipettes (2.54.5 M) were visually guided to the surface of individual CVNs using differential interference optics and infrared illumination (Zeiss, Oberkochen, Germany). Patch pipettes were filled with a solution containing 150 mM KCl, 4 mM MgCl2, 2 mM EGTA, 2 mM Na-ATP, and 10 mM HEPES, pH 7.4. This pipette solution causes the Cl current induced by the activation of GABA
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Application of IV Anesthetics Propofol and Inhaled Anesthetic Isolfurane

Propofol, at a clinically relevant concentration (10 M),18 was added into the perfusion for 10 min after isolated GABAergic postsynaptic inhibitory currents. Gabazine was subsequently applied to block GABA synaptic events for 510 min before picrotoxin was administrated for 10 min. A 12-mM stock of saturated isoflurane solution was allowed to equilibrate overnight in a sealed volumetric flask at room temperature. For each exposure, this saturated isoflurane solution was diluted into artificial cerebrospinal fluid immediately before experiments and was perfused for 10 min at a flow rate 3.0 mL/min at 25C. The concentrations of isoflurane used in the study were 10, 50, 100, 300, and 500 M. These concentrations were chosen based on the minimum alveolar concentration equivalent aqueous EC50 concentration for isoflurane of 270 M (in human) and 310 M (in rat), after adjusting for room temperature and expressed as free aqueous-phase concentration in saline.19 21 This experimental setup used a sealed glass syringe and Teflon tubing to prevent loss of isoflurane from the apparatus; in addition, the actual isoflurane concentrations applied to the tissue was assessed. To determine any difference between calculated and actual isoflurane concentrations, bath samples (100 L) from the recording chamber were collected from the calculated 1 mM concentration at 5, 10, and 15 min after exposure. Samples were extracted into heptane (500 L) in which halothane (50 M) was added as an internal standard. Gas chromatography/ mass spectrometry was used to accurately measure isoflurane concentrations of the samples taken from the bath.
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Statistical Analysis
Analysis of tonic and spontaneous synaptic currents was performed using Clampfit (Version 8.2, Axon instruments) and MiniAnalysis (Version 5.6.12, Synaptosoft) with minimal acceptable amplitude at 6 9 pA. Results are presented as means sem. Data within groups using different treatments were analyzed by one-way analysis of variance for repeated measures followed by Dunnetts post-tests. P 0.05 was considered to be statistically significant. Statistical analysis was performed using Graphpad Prism 5.0 (Graphpad Software, San Diego, CA) and Microcal Origin 6.1 (OriginLabs Corp., Northhampton, MA).

(Fig. 3B). Gabazine (25 M) did not significantly change the holding current in CVNs but did abolish the GABAergic IPSCs (Fig. 3A). Application of picrotoxin (100 M) reversed the isoflurane-induced increase in holding current to a level which was not significantly different compared with control values (Fig. 3A).

DISCUSSION
This study demonstrates that both the volatile anesthetic isoflurane and IV anesthetic propofol act on GABAA receptor-mediated phasic and tonic inhibition in brainstem parasympathetic cardioinhibitiory vagal neurons in the nucleus ambiguus. Consistent with previous studies, the IV drug propofol22 and volatile anesthetic isoflurane11 prolong the decay time of GABAergic mIPSCs, and these were likely caused by a decrease in GABAA receptor desensitization and deactivation. This study confirmed and advanced these results and found both propofol and isoflurane also augment tonic GABAergic holding current in CVNs. Tonic activation of GABAA receptors results in neurodepression by inhibition of intrinsic neuronal excitability. The mechanisms responsible for the anesthetic-induced increase in tonic inhibition in CVNs could involve three critical aspects: 1) increase of concentration of ambient GABA in extrasynaptic regions; 2) modulating the affinity of GABAA receptors thereby increasing the sensitivity of CVNs to ambient GABA; and 3) direct activation and modulation of GABAA receptors function. The concentration of GABA in the extracellular space reflects the number and activity of GABArelease elements and the action of GABA transporters. Application of the GABA transporter blocker, NO-711, did not change the tonic current in CVNs, suggesting that factors other than GABA transporters determine ambient GABA concentration.17 Although propofol had been found to increase GABA release in rat cortical synaptosomes23 and granule cells of the dentate gyrus,24 neither propofol nor isoflurane enhanced the frequency of GABAergic IPSCs in brainstem CVNs.11,22 In addition, isoflurane inhibited GABA IPSCs frequency in murine hippocampal neurons12 and CVNs,11 and a high concentration of propofol also decreased the frequency of GABAergic IPSCs in CVNs.22 Therefore, it seems likely that propofol and isoflurane enhance tonic currents through mechanisms other than an increase in ambient GABA concentration. The pharmacological modulation of tonic inhibition depends on the intrinsic properties of GABAA receptors, specifically subunit composition and subcellular distribution as well as GABAA receptor kinetics. The 122 and 332 are densely expressed in the brainstem and the 2 subunit exhibits a widespread extrasynaptic localization.25 The subunit containing GABAA receptors have a very high affinity for GABA and are particularly resistant to desensitization; however, there is no evidence of the subunit expression
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RESULTS
Spontaneous mIPSCs were examined to characterize the anesthetic modulation of phasic GABAergic currents in cardiac parasympathetic neurons in the nucleus ambiguus. Under control conditions, GABAergic mIPSCs occurred at a frequency of 1.38 0.2 Hz, amplitude of 37.6 6.5 pA, rise time of 8.9 0.9 ms, and decay phase of 47.9 7.3 ms. Application of propofol (10 M) induced a 63 13% increase in the membrane holding currents in CVNs (Figs. 1A and B). The holding currents were enhanced from an average control of 160.9 41 pA to 235.25 53 pA (P 0.003, n 8, Fig. 1B). The decay time constant of GABAergic mIPSCs was also prolonged from 42.3 2.8 ms in control to 61.8 4.5 ms at a dose of 10 M (P 0.03, n 8, Fig. 1C). Propofol evoked no significant change in GABA mIPSCs amplitude or frequency (data not shown). The GABAA antagonist gabazine (25 M) blocked the GABAergic mIPSCs events, but failed to alter the enhanced holding potential (P 0.01, compared with control, Figs. 1A and B). In contrast, picrotoxin (100 M) reversed the propofol-evoked increase in membrane holding current and decreased the holding currents beyond control values (P 0.01, compared with control, n 8, Figs. 1A and B). Because the effect of isoflurane on tonic GABA currents were more complex,12,14 we studied isoflurane in a series of doses from 10 to 500 M. Isoflurane altered GABA mIPSCs at concentrations of 100 M and larger. Isoflurane decreased the frequency of GABAergic mIPSCs by 26.0 6.4% (P 0.04, n 7), 64.6 10.4% (P 0.03, n 6), and 70.5 9.8% (P 0.009, n 6) at 100, 300, and 500 M, respectively (Figs. 2A and B). At the same concentrations, isoflurane also prolonged the decay time of GABAergic mIPSCs from an average control of 47.9 7.3 ms to 64.5 8.1 ms (P 0.04, n 7), 70.3 10.4 ms (P 0.02, n 6), and 66.8 8.1 ms (P 0.02, n 6), respectively (Figs. 2C and D). Application of isoflurane increased the membrane holding currents by 32.8 12.8% (P 0.03, n 7), 42.7 10% (P 0.004, n 6), and 39.9 3% (P 0.02, n 6) at doses of 100, 300, and 500 M, respectively
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Figure 1. Propofol increased membrane holding currents and prolonged the decay time of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in cardiac vagal neurons. (A) A typical example showing that propofol induced an increase in the membrane holding current; gabazine blocked synaptic events but not the increase of the holding currents, whereas picrotoxin decreased the holding currents beyond control values. (B) Summary data of membrane holding current changes induced by propofol, gabazine, and picrotoxin from eight cardiac vagal neurons. Data are presented as mean sed, *P 0.05, **P 0.01 compared with controls. (C) Propofol evoked prolongation of GABAergic mIPSCs decay time (ms) are illustrated as a typical example (left) and summary data (right).

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phase. Isoflurane dose-dependently inhibited the frequency of GABAergic mIPSCs at concentrations of 100, 300, and 500 M. (A) A typical example showing isoflurane inhibition of GABAergic frequency. (B) Bar diagram of average percentage changes of -aminobutyric acid (GABA) mIPSCs frequency from cardiac vagal neurons (CVNs) for three doses of isoflurane. At the same concentrations, isoflurane also prolonged the decay time of GABAergic mIPSCs. (C) A typical example using 300 M isoflurane and (D) summary data for different doses of isoflurane. Number of experiments is given in brackets. Data are presented as mean sed, *P 0.05, **P 0.01 compared with controls.

Figure 2. Isoflurane induced changes in GABAergic miniature inhibitory postsynaptic currents (mIPSCs) frequency and decay

in the brainstem. Given the rapid changes that occur with propofol and isoflurane, it seems unlikely these changes in GABAergic function are due to increased sensitivity of GABA receptors in CVNs to ambient GABA. Based on Macdonalds comprehensive kinetic models of ligand-gated ion channels,26 28 desensitization and deactivation kinetics can play an important role in anesthetic modulation of GABAergic function. For example, propofol has been shown to slow deactivation and reduce desensitization in some, but not all, GABA isoforms.8 Anesthetics that enhance GABAA receptor function act by prolonging IPSCs and increasing the proportion of time that the channel spends in the open state. Consistent with a previous study,22 propofol
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slowed the rate of deactivation kinetics by prolonging GABAergic IPSCs decay time constant in the CVNs, which results in an increase of the GABA-induced charge transfer. Propofol has been found to act on and GABAA receptors,8 and the presence of a 3 subunit is necessary for propofol modulation of GABA receptors and tonic inhibition in hippocampal pyramidal neurons.29 31 It is likely that propofol facilitation of tonic current may be mediated by multiple GABAA receptors in CVNs containing 3 and 2 subunits and that changes of GABA function with propofol and isoflurane occur via reductions in GABAA receptor desensitization and prolongation of deactivation kinetics. The IV anesthetic propofol and inhaled anesthetic isoflurane bind to different transmembrane segments
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3. Isoflurane increased -aminobutyric acid (GABA) tonic currents in cardiac vagal neurons. (A) Representative trace showing a typical example of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) and baseline current changes induced by isoflurane at 300 M. In the presence of isoflurane membrane, holding currents were significantly increased. Gabazine abolished GABAergic synaptic events, but did not change the holding currents. Picrotoxin (100 M) reversed the isoflurane-induced increase in holding currents. (B) Bar diagram showing summary data of percentage changes of membrane holding currents induced by various doses of isoflurane. Significance is denoted as *P 0.05, ***P 0.001 compared with controls.

Figure

of GABAA receptors.32 Although and subunits are critical targets for the propofol action on GABAA receptors, subunits (1, 2, and 3) are the common sites for volatile anesthetics, including isoflurane.33 Localization of the different subunits indicates the 1 and 3 subunit-containing GABAA receptors are densely expressed, whereas 2, 4, 5, and 6 are relatively weakly expressed in the brainstem.25 Further studies would be required to test which specific subunits are responsible for the isoflurane-induced increase in tonic GABA currents in CVNs. It is likely that propofol and isoflurane directly activate GABAA receptors at sites distinct from the GABA binding site,34 40 and this is supported by the lack of gabazine antagonism of the increased tonic currents. Picrotoxin acts not at the GABA recognition site but perhaps within the ion-channel pore and prevents ion flow. Our previous study demonstrated picrotoxin not only blocked GABAergic IPSCs, but also decreased tonic holding currents in CVNs.17 The present study shows that the propofol and isoflurane increased membrane holding currents are reversed by picrotoxin but not gabazine. This suggests that the enhancement of GABAergic receptor function observed with propofol and isoflurane occurs by an action on the GABA receptor independent of GABA binding site that is blocked by gabazine. Furthermore, the GABA receptor subunits specifically sensitive to picrotoxin play a role in mediating propofol and
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isoflurane-induced increase in the persistent tonic currents in CVNs. In conclusion, CVNs are sensitive to both propofol and isoflurane at clinically relevant concentrations. Propofol and isoflurane increased not only the decay time of phasic GABAergic mIPSCs, but also enhanced tonic current. In addition, isoflurane also decreased the frequency of GABAergic mIPSCs in parasympathetic cardiac neurons. Increase in tonic inhibition and prolongation of GABAergic mIPSCs decay kinetics in CVNs would result in a decrease of firing in CVNs, thereby evoking a tachycardia with propofol and isoflurane; however, a decrease in the frequency of GABA mIPSCs with isoflurane would disinhibit CVNs and minimize the decreased parasympathetic cardiac activity, resulting in less tachycardia with isoflurane. This differential modulation of GABAA receptors in CVNs are likely responsible for the differential alterations in heart rate and baroreflex during clinical anesthesia with propofol and isoflurane. ACKNOWLEDGMENTS The author would like to thank Dr. Tarek Z Deeb for his thoughtful comments and constructive suggestions for editing the manuscript. REFERENCES
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