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Journal of Physiology (1992), 449, pp 279-293 With 9 figures Printed in Great Britain

ENHANCEMENT OF y-AMINOBUTYRIC ACID-ACTIVATED C1CURRENTS IN CULTURED RAT HIPPOCAMPAL NEURONES BY THREE VOLATILE ANAESTHETICS

BY MATHEW V. JONES*, PENELOPE A. BROOKSt AND NEIL L. HARRISONt From the Department of Anesthesia and Critical Care, The University of Chicago, Chicago, IL 60637, USA, the *Department of Pharmacological and Physiological Sciences, The University of Chicago, Chicago, IL 60637, USA and the tDepartment of Physiology, The Royal Free Hospital School of Medicine, London (Received 22 March 1991) SUMMARY

1. The effects of the volatile anaesthetics enflurane, halothane and isoflurane on yaminobutyric acid (GABA)A receptor-mediated chloride currents were studied in cultured rat hippocampal neurones. Transient current responses were obtained by brief pressure application of GABA to the cell body of neurones under voltage clamp. 2. All three anaesthetics increased the peak amplitude and duration of current responses to brief applications of GABA. These effects were fully reversible, and did not involve alterations in the reversal potential for GABA responses. 3. The experimental concentrations of anaesthetics were measured directly using gas chromatography. The enhancement of GABA currents increased with increasing anaesthetic concentration. Clinically effective concentrations of anaesthetics (between 1 and 1-5 times MAC (minimum alveolar concentration) produced significant enhancement of GABA currents. 4. These results demonstrate that the changes in the time course of synaptic inhibition reported in the presence of the volatile anaesthetics are likely to result from modification of the function of postsynaptic GABAA receptor-channel complexes. These findings also support the hypothesis that GABAA receptor complexes serve as common molecular target sites for a variety of structurally diverse anaesthetic molecules. INTRODUCTION

Most theories of anaesthesia have related to the hydrophobic nature of neuronal lipid membranes and have highlighted the ease with which many anaesthetics dissolve in octanol and other 'membrane-like' substances (reviewed by Seeman, 1972; Roth, 1979). However, it was recently suggested that the striking correlation between lipid solubility and anaesthetic potency might implicate hydrophobic t To whom correspondence should be addressed. MS 9253 10

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regions of neuronal membrane protein as sites of action for general anaesthetics (Franks & Lieb, 1984). General anaesthetics have been reported to directly hyperpolarize neuronal membranes (Madison & Nicoll, 1982), alter neuronal firing patterns (Fujiwara, Higashi, Nishi, Shimaji, Sugita & Yoshimura, 1988) and modify synaptic transmission (Maclver & Roth, 1988). Synaptic inhibition in the rat and human brain is usually mediated by yaminobutyric acid (GABA) (McCormick, 1989). Eccles, Schmidt & Willis, (1963) noted that presynaptic (GABA-mediated), but not postsynaptic (glycine-mediated), inhibition of spinal motoneurones was prolonged by many general anaesthetics, and Nicoll (1972) suggested that enhancement of synaptic inhibition mediated by GABA might be a common mode of anaesthetic action. Inhibition in hippocampus (Nicoll, Eccles, Oshima & Rubia, 1975; Gage & Robertson, 1985) and olfactory cortex (Scholfield, 1980) is prolonged by many soluble anaesthetic agents such as barbiturates and steroidal anaesthetics. The mammalian central nervous system contains two major classes of GABA receptors. The 'classical' GABAA receptor is now known to be a family of heterogeneous multi-subunit complexes (Schofield et al. 1987; Shivers et al. 1989) containing an integral Cl--selective ion channel. GABAA receptors mediate the fast inhibitory postsynaptic potential (IPSP) in many brain regions, while GABAB receptors (Hill & Bowery, 1981) mediate a slower 'late' IPSP (Dutar & Nicoll, 1988; McCormick, 1989). The GABAA receptors are modulated in an allosteric manner (Olsen, 1982) by sedative and anaesthetic drugs such as the barbiturates (Barker & Ransom, 1978; Segal & Barker, 1984; Akaike, Hattori, Inomata & Oomura, 1985) and steroidal anaesthetics (Harrison, Majewska, Harrington & Barker, 1987). Volatile anaesthetic interactions with inhibition and GABA have received less attention than the actions of the soluble anaesthetics. However, a recent study in the anaesthetized rat (Pearce, Stringer & Lothman, 1989) reported that the time course of inhibition of CAl neurones was greatly prolonged by volatile anaesthetics, relative to urethane-anaesthetized controls. In sensory neurones, it was reported that GABAactivated chloride currents were either increased or decreased by volatile anaesthetics (Nakahiro, Yeh, Brunner & Narahashi, 1989). Moody, Suzdak, Paul & Skolnick (1988) reported effects of volatile anaesthetics on muscimol-stimulated and picrotoxin-sensitive uptake of radiolabelled Cl- into synaptoneurosomes; however, the anaesthetic concentrations employed in the study were several times greater than those that produce anaesthesia. Another study using radioligand binding techniques (Huidobro-Toro, Bleck, Allan & Harris, 1987) provided more compelling evidence for an interaction of alcohols and some volatile anaesthetics with GABAA receptors. We have studied the effects of the three volatile anaesthetics, enflurane, halothane, and isoflurane, on chloride current responses to exogenously applied GABA in cultured central neurones. Marked increases in the amplitude and duration of chloride current were observed in the presence of each anaesthetic, at concentrations known to induce surgical anaesthesia in man. Some of this work has appeared previously in abstract form (Brooks & Barker, 1990; Jones, Brooks & Harrison, 1990; Brooks, Jones, Barker & Harrison, 1990).

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METHODS

Cell culture and electrophysiology Neuronal cultures were prepared from the hippocampi of 19-20 day rat embryos using methods similar to those previously described (Huettner & Baughmann, 1986). Donor mother rats were killed by exposure to a rapidly rising concentration of diethyl ether, followed by cervical dislocation. Embryo rats were removed and rapidly decapitated. the brains removed and the hippocampi dissected out. Following enzymatic treatment with papain and mechanical trituration, neurones were plated onto culture dishes coated with poly-D-lysine. The cultures were maintained in Eagle's minimal essential medium supplemented with 2 mM-glutamine and 5 % heat-inactivated horse serum. Cultures were used for recording 2-5 weeks after plating. Recordings were made at 25 0C, using the whole-cell patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981), the methods being identical to those we have previously described (Lambert & Harrison, 1990). The solution in the recording pipette contained (in mM): potassium gluconate, 145; MgCl2, 2; N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulphonic acid) (HEPES), 5; CaCl2, 0-1; ethyleneglycol-bis-(/-aminoethyl ether)-lN-N-'-N\'-tetraacetic acid (EGTA), 11; K2ATP, 5; adjusted to pH 7-2 with KOH. Responses to GABA recorded using this solution did not exhibit significant 'run-down' over recording periods up to 3 h (Lambert & Harrison, 1990). Neurones were continuously superfused at 2 ml/min with extracellular solution containing (in mM): NaCl, 145; KCl, 3; CaCl2. 15; MgCl2, 1; HEPES 10; D-glucose, 6; adjusted to pH 7-4 with NaOH. Tetrodotoxin (200 nm) was added to the extracellular medium. GABA was applied to the cell body of the neurone under study by pressure ejection (< 70 kPa, 5-100 ms, 0 05-01 Hz) from blunt-tipped micropipettes containing between 10-50 /aM-GABA. Micropipettes containing 10 ,uM-GABA were always used in constructing concentration-effect plots. Current responses to GABA were digitized using an analog-to-digital converter (TL-125-1 interface; Axon Instruments, Foster City, CA, USA), low-pass filtered at 3 kHz and stored on a 80386-20 MHz computer (Zeos, St Paul, MN, USA) for later analysis. Acquisition and analysis were performed using software modified from original software written by Dr Aaron Fox ('AXOBASIC'; Axon Instruments). Measurement of anaesthetic concentration All anaesthetics were applied to the preparation via the extracellular medium. The anaesthetic solutions were prepared by adding liquid anaesthetics to extracellular medium under air-tight conditions. A standard sample was obtained by sampling the anaesthetic solution after allowing sufficient time for equilibration. Experimental samples were withdrawn directly from the bath during electrophysiological recordings. Standard and experimental samples were quick-frozen immediately and treated identically. All samples were handled using septum-capped autosampler vials and Hamilton Gastight syringes. Samples were injected (90 °C) onto a 5% SE-30 packed column (Ohio Specialty Chemical; 6 ft long; 100 °C) and detected by flame ionization (100 °C). N2 was used as carrier, at a flow rate of 10-20 ml/min. Peak heights were used to quantify concentrations. Experimental concentrations were determined by comparison with peak heights from standard samples. Equilibration of the anaesthetic concentration in the recording chamber required approximately 10 min (e.g. for enflurane, Fig. 1). Column retention times for all three anaesthetics were between 0 5 and 2 min. Criteria for acceptable experiments Neurones used in experiments had input resistance ) 200 MQ, resting membrane potentials more negative than -40 mV (mean+S.E.M., -55-1 + 1-3 mV), and gave current responses to GABA applications < 100 ms in duration. Peak amplitude of GABA current (IGABA)' half-decay time (Ti), and charge transfer during the GABA response (QGABA) (the latter calculated by numerical integration of the entire GABA response) were determined by computer-assisted analysis for each individual GABA response. Ten successive control responses were used to calculate baseline values for these parameters. Experiments were rejected if IGABA' 2l, or baseline membrane current failed to recover after removal of anaesthetic. Calculation of MAC equivalents One MAC is defined as the minimum alveolar concentration of anaesthetic required to produce immobility (in man) in response to a noxious stimulus in 50% of trials (Eger, Saidman & Brandstater, 1965). MAC equivalents for the volatile anaesthetics are calculated from published 10-2

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MAC values (see e.g. Quasha, Eger & Tinker, 1980), using published solubility coefficients (Allott, Steward, Flook & Mapleson, 1973). To calculate MAC equivalents at 25 °C, published values of temperature coefficients of solubility were used (Steward, Allott, Cowles & Mapleson, 1973). MAC equivalents at 25 °C of the anaesthetics used here are: enflurane, 0-46 mM; halothane, 0-32 mM; isoflurane, 0-51 mM. 1-0

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Fig. 1. Anaesthetic equilibration with the experimental medium. An example of the time course of enflurane concentration in the experimental medium. After beginning perfusion of a solution containing 10 mM-enflurane, there is a rapid increase in enflurane concentration in the bath, which reaches a steady-state level at around 0-8 mM, 10 min after beginning perfusion. After returning the perfusion to control medium, the bath concentration of enflurane declines. Ten minutes after beginning wash-out, enflurane concentration has declined to unmeasurably low levels. Each filled circle represents the (mean+standard deviation) of four injections, from a single bath sample, onto a gas chromatography column. The inset shows the separation of enflurane (left peak) and halothane (right peak) by gas chromatography, from a solution containing 1 mm of each. Retention time (in min) is printed above each peak.

Quantitative analysis Due to the volatile nature of the anaesthetics, it is not technically possible to deliver defined anaesthetic concentrations reproducibly in these experiments. In order to quantify the alterations in GABA-activated current, a number of experiments were performed with each anaesthetic using brief pulses from a micropipette containing 10 ,/im-GABA. Frequently several anaesthetic concentrations were tested on the same neurone. The data from these comparable experiments were pooled and are presented here as concentration-response plots, in which the percentage increase in IGABAqt1, or QGABA is plotted against log [anaesthetic].

Source8 of anaesthetic8 and other chemicals Enflurane and isoflurane were obtained from Anaquest, (Madison, WI, USA); halothane was obtained from Halocarbon Laboratories, (N. Augusta, SC, USA). Unless specified, all other chemicals used were obtained from Sigma Chemical Co. (St Louis, MO, USA).

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RESULTS

As we have previously described for this system (Lambert & Harrison, 1990), brief application of GABA to neurones held at -40 mV in voltage clamp elicited transient outward current responses (IGABA) when the patch pipette solution contained B

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