Activation of rat locus coeruleus neuron GABA_A receptors by propofol and its potentiation by pentobarbital or alphaxalone

Abstract

The action of propofol on the rat locus coeruleus was examined using intracellular recording from in vitro brain slice preparations. Concentrations of propofol between 3 and 300 μM were tested. At 100 μM, propofol completely inhibited the firing of all neurons tested (n=34); this was associated with a 5.7-mV hyperpolarization (range 0–16 mV, n=33) and a 35.6% reduction in input resistance (range 7.3–66.1%, n=33). The propofol-induced responses were not affected by 2-hydroxysaclofen (50 μM) or BaCl₂ (300 μM), but were completely blocked by bicuculline methiodide (100 μM) or picrotoxin (100 μM), indicating that propofol acts on GABA_A receptors. As assessed by inhibition of the spontaneous firing rate, propofol was 5.6-fold more potent than GABA (γ-aminobutyric acid). Potentiation of the propofol effect by other general anesthetics or other drugs was also investigated. When pentobarbital (100 μM) was tested alone on locus coeruleus cells, no change in membrane potential or input resistance was seen and there was only a 20.3±7.2% (n=8) inhibition of firing rate; however, in combination with 30 μM propofol, it caused a 6.1-fold greater increase in membrane hyperpolarization and a 9.7-fold greater reduction in input resistance than 30 μM propofol alone. A relatively low concentration of alphaxalone (10 μM), when tested alone, had little effect on the membrane potential or input resistance and only produced a 46.0±8.9% (n=8) inhibition of firing rate; however, in combination with 30 μM propofol, it caused a 9.3-fold greater hyperpolarization and an 8.6-fold greater reduction in input resistance compared with 30 μM propofol alone. In contrast, diazepam caused no potentiation of either propofol- or GABA-induced responses. Our data also indicate that locus coeruleus neuron GABA_A receptors possess distinctive pharmacologic characteristics, such as blocking of the propofol effects by zinc and insensitivity to diazepam and the direct action of pentobarbital. On the basis of these pharmacologic properties, we suggest that locus coeruleus neuron GABA_A receptors do not contain the γ subunit.

Introduction

Propofol (2,6-diisopropylphenol) is an intravenous general anesthetic and hypnotic that is structurally unrelated to other anesthetics. In recent years, propofol has been used extensively in clinical anesthesia because of its clinical benefits of a rapid onset, clear emergence, and lack of cumulative effects (for reviews, see Fulton and Sorkin, 1995, Tagliente, 1997). Although considerable information is available on the pharmacokinetic and pharmacodynamic properties of propofol (e.g., Langley and Heel, 1988, Dundee and Clarke, 1989), its cellular mechanism of action on the central nervous system has not yet been entirely elucidated. Propofol is reported not only to potentiate γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission Collins, 1988, Albertson et al., 1991, but also to directly activate the GABA_A receptor Peduto et al., 1991, Hara et al., 1993. However, as far as anesthesia is concerned, GABA is not suitable for clinical use because it cannot cross the blood-brain barrier. The GABA_A receptor–chloride ionophore complex is believed to be a multisubunit protein that contains binding sites for a variety of compounds. For example, GABA, barbiturates, steroids, and benzodiazepines have been suggested to bind to the GABA_A receptor complex and enhance the inhibitory GABA_A response via allosteric modulation of receptor function (Olsen and Tobin, 1990). The efficacy and potency of these drugs as GABA potentiators Shingai et al., 1991, Horne et al., 1993 or as direct GABA_A receptor activators Valeyev et al., 1993, Adodra and Hales, 1995 may be subunit-dependent. For example, it has been shown that the potentiation of GABA responses by benzodiazepines depends on the presence of a γ subunit Pritchett et al., 1989, Ymer et al., 1990, Knoflach et al., 1991. The aims of the present investigation were therefore to provide information on (1) the cellular mechanism of action of propofol on locus coeruleus neurons, (2) the possible correlation of the in vitro dose–response results for propofol with its clinically relevant concentrations, (3) the potency of propofol compared with GABA, (4) potentiating effects of pentobarbital, alphaxalone (a steroid type of general anesthetic), or diazepam on the propofol-evoked responses, and (5) whether locus coeruleus neuron GABA_A receptors contain a γ subunit.

The locus coeruleus brain slice preparation was used in the present investigation for a number of reasons. Firstly, the locus coeruleus contains the greatest concentration of noradrenergic cell bodies in the central nervous system, projections from this small pontine nucleus being responsible for more than half of the noradrenaline terminals in the brain (Amaral and Sinnamon, 1977). Since locus coeruleus neurons have extensive projections to many central nervous system areas, changes in the activity of these neurons would be expected to cause widespread effects. Secondly, using autoradiography, GABA receptors have been demonstrated to be present in the locus coeruleus (Palacios et al., 1981) and binding studies have shown the presence of both GABA_A and GABA_B receptors in the terminal fields of locus coeruleus neurons (Suzdark and Gianutsos, 1985). Neurochemical studies indicate that almost half of the afferent terminals in the locus coeruleus can take up radiolabeled GABA (Iversen and Schon, 1973). Thus, the locus coeruleus provides an excellent site for studying the actions of GABA agonists. Thirdly, functional studies have demonstrated that the locus coeruleus is one of the major neural sites involved in sedation Correa-Sales et al., 1992, Pertovaara et al., 1994 and, more importantly, it is the primary origin of descending noradrenergic analgesic fibers. Electrophysiological studies have provided strong evidence for a role of the locus coeruleus in modulating analgesia (for reviews, see Proudfit, 1988, Jones, 1991, Lipp, 1991).