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Tolerance to inhibition by ethanol of N-methyl-d-aspartate-induced depolarization in rat locus coeruleus neurons in vitro

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Abstract

Intracellullar recordings were made in a pontine slice preparation of the rat brain containing the nucleus locus coeruleus. The pressure application of N-methyl-d-aspartate (NMDA) produced reproducible depolarizations of stable amplitude. Superfusion with ethanol (100 mM) for 15 min inhibited the depolarizing response to NMDA; the effect of ethanol was rapidly reversed on washout. When the superfusion time of ethanol (100 mM) was increased to 60 min, its inhibitory effect disappeared after 50 to 60 min. Moreover, after the subsequent washout of ethanol a withdrawal-like increase in the sensitivity to NMDA became evident. Hence, adaptive mechanisms of locus coeruleus neurons during the long-time contact with ethanol may be modelled in an in vitro system.

Introduction

A growing amount of experimental data indicates that the characteristic signs following ethanol consumption are due to changes of certain neurotransmitter receptors and voltage-dependent ion channels (Weight, 1992). A likely target for ethanol effects is the N-methyl-d-aspartate (NMDA) type of excitatory amino acid receptor (Tabakoff and Hoffman, 1996). While the acute application of ethanol inhibits NMDA-induced cationic currents in various neurons, chronic treatment with ethanol leads to an upregulation of NMDA receptor function and binding (Hoffman and Tabakoff, 1994). Such an upregulation contributes both to adaptive changes during ethanol exposure and to withdrawal reactions after its termination.

Noradrenergic neurons of the nucleus locus coeruleus are involved in a number of cognitive and emotional processes (Foote et al., 1983). Moreover, the modulation of the spontaneous discharge of locus coeruleus neurons contributes to physical aspects of opioid (Rasmussen et al., 1990) and probably also ethanol (Engberg and Hajos, 1992a, Engberg and Hajos, 1992b) withdrawal. Under in vivo conditions, acutely applied ethanol inhibits the NMDA-induced increase in the firing of locus coeruleus neurons (Engberg and Hajos, 1992a), whereas following cessation of chronic ethanol treatment an enhanced sensitivity to NMDA develops (Engberg and Hajos, 1992b).

It is difficult to evaluate the interaction between ethanol and NMDA in a slice preparation, because ethanol alters the extracellularly recorded basal firing rate of locus coeruleus neurons by itself (Shefner and Tabakoff, 1985; Fröhlich et al., 1994). Hence, the conclusion that ethanol and NMDA interact in a truly antagonistic manner at the NMDA receptor-channel (Fröhlich et al., 1994) is still controversial. The aim of the present experiments was twofold. Firstly, it was investigated whether intracellular recordings from locus coeruleus neurons confirm an attenuation of the NMDA-induced depolarization by ethanol. Secondly, it was attempted to clarify whether during a longer lasting contact with ethanol the excitatory effect of NMDA declines culminating in a withdrawal-like increase in NMDA-sensitivity after washout.

Section snippets

Brain slice preparation

Mid-pontine slices of the rat brain were prepared and maintained as previously described (Nieber et al., 1995). In brief, male Wistar rats (150–200 g) were anaesthetized with ether and decapitated. Coronal slices about 400 μm thick, containing the caudal part of the locus coeruleus, were prepared in oxygenated medium at 1–4°C with a Lancer vibratome. A single slice was placed in a recording chamber and superfused at a rate of 2 ml/min with medium saturated with 95% O2–5% CO2 and kept at

Results

A total of thirty-one locus coeruleus neurons included in this study had a resting membrane potential of 53.8±1.9 mV. The generation of spontaneous action potentials was prevented by passing continuous hyperpolarizing current via the recording electrode.

When NMDA was applied every 5 min by pressure from a micropipette onto locus coeruleus neurons, depolarizations of a rather slow time-course were evoked (Fig. 1A). These responses had a tendency to slightly decrease over a period of 130 min,

Discussion

The present results confirm the observation that ethanol inhibits NMDA receptor-channels of locus coeruleus neurons (Engberg and Hajos, 1992a; Fröhlich et al., 1994). However, a somewhat higher concentration of ethanol was needed to interact with the depolarizing responses to NMDA than in most other neuronal systems (Weight, 1992). In agreement with this, our previous extracellular study showed an attenuation of the NMDA-evoked increase in the firing rate of locus coeruleus neurons at 10 to 100

Acknowledgements

This work was supported by the Bundesministerium für Bildung, Forschung und Technologie, Biologische und psychosoziale Faktoren von Drogenmißbrauch und Drogenabhängigkeit (01EB9425).

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