Robert W Peoples PhD

1. INTRODUCTION

Alcohol is arguably the oldest drug known to man, its use dating back at least 10,000 yr to the dawn of human civilization (1). Although illicit drug use often receives more attention in contemporary society, alcohol abuse exacts a devastating toll: In the United States at present, over 7% of the population meet diagnostic criteria for alcohol abuse or alcoholism (2), over 28% of children under 18 yr of age are exposed to alcohol abuse or dependence in the home (3), and the overall economic cost to society of alcohol abuse has been estimated at $ 185 billion (4). Despite intensive research since the latter part of the previous century, it is clear that the biological actions that are responsible for the characteristic effects of ethyl alcohol, or ethanol, on human physiology and behavior are still incompletely understood. Because of the simple chemical structure of ethanol (it differs from water only by two methylene groups) and its low potency (it produces most of its biological effects at millimolar concentrations), alcohol undoubtedly interacts with multiple sites in the central nervous system. The biological effects of alcohol almost certainly reflect its concerted actions at a number of these sites. Of the many possible targets of alcohol actions, neurotransmitter receptors, and in particular, neurotrans-mitter-gated receptor-ion channels are currently believed to be among the most important (5). Because the neurotransmitter glutamate mediates the majority of fast excitatory neurotransmission in the central nervous system via actions on glutamate-gated receptor-ion channels (6), effects of alcohol on these ion channels could profoundly alter central nervous system function.

In addition to postsynaptic effects on glutamate receptors, alcohol could also influence glutamater-gic neurotransmission presynaptically by altering release of glutamate or its clearance from the synap-tic cleft. Few studies have addressed this, however (7-9), and results of studies in brain slices suggest that presynaptic effects of alcohol on glutamatergic transmission are likely to be of lesser physiological importance relative to postsynaptic effects (10,11). The content of this chapter will be restricted to the effects of acute exposure to alcohol on pharmacologically isolated or recombinant glutamate receptors. Although effects of repeated or chronic exposure to alcohol on glutamate receptors are of great interest because of their relevance to human alcohol abuse and alcoholism, this topic is addressed in Chapters 24-26. For a more detailed discussion of the effects of alcohol on glutamatergic synaptic transmission, including studies performed prior to glutamate receptor cloning, the reader is referred to the review by Weight (12).

2. ACTIONS OF ALCOHOL ON NMDA RECEPTORS

Glutamate-gated membrane ion channels are broadly divided into V-methyl-d-aspartate (NMDA) receptors and non-NMDA receptors [(6); see Chapters 1 and 2].) NMDA receptor-ion channels are involved in nervous system excitability, cognitive function, forms of neural plasticity believed to

From: Contemporary Clinical Neuroscience: Glutamate and Addiction Edited by: Barbara H. Herman et al. © Humana Press Inc., Totowa, NJ

Fig. 1. Ethanol inhibits NMDA receptors at physiologically relevant concentrations. Traces are current activated by 25 ||M NMDA and 10 ||M glycine and its inhibition by 50 mM ethanol (EtOH) in a rat hippocampal neuron. The bars over the traces correspond to the duration of agonist and ethanol application. (Data from ref. 24.)

underlie learning and memory, and motor coordination (13-16), all of which have obvious relevance to the intoxicating effects of alcohol. Perhaps the first evidence of an effect of alcohol on NMDA receptors was the finding that ethanol, as well as the alcohols methanol, 1-propanol, and 1-butanol, inhibited NMDA-evoked 22Na+ efflux from rat striatal slices (17). The first direct evidence for alcohol inhibition of NMDA receptors in neurons was reported in a study in mouse hippocampal neurons in culture, in which alcohols from methanol to isopentanol inhibited NMDA-, kainate-, and quisqualate-activated ion current (18). Other studies in the same year also demonstrated ethanol inhibition of NMDA receptor single-channel currents (19) and ethanol inhibition of NMDA-stimulated 45Ca2+ uptake (20,21), cyclic GMP production (21,22), and neurotransmitter release (23). Importantly, ethanol inhibits NMDA receptors at physiologically relevant concentrations (Fig. 1). Although the potency of ethanol may vary depending on experimental conditions, the large number of studies to date that have reported inhibition of NMDA receptor-mediated responses by concentrations of ethanol in the intoxicating range in many different tissues and preparations using various experimental techniques testifies to the robustness of this effect.

2.1. Effects of Alcohol on NMDA Receptor Subunits

V-methyl-d-aspartate receptors are heteromeric assemblies containing NR1 subunits, of which there are eight variants due to alternate RNA splicing of three cassettes (N1, C1, and C2), and NR2 subunits, of which there are four subtypes, NR2A-NR2D (6). Because the distribution of these sub-units varies among brain regions (25), any differences in ethanol sensitivity among subunits could result in brain region-specific effects of ethanol (26-28). Such differences in ethanol sensitivity among NR1 subunit splice variants and NR2 subunits have been observed in some, but not all, studies. In Xenopus laevis oocytes expressing recombinant NMDA receptor subunits, ethanol inhibition of NMDA receptors was greatest when the NR1 subunit contained the N1, C1, and C2 cassettes, appeared to decrease in NR1 subunits lacking either the N1 or C1 cassettes and was lowest in NR1 subunits containing only the C2 cassette (29). Interestingly, these differences in ethanol potency were not observed when calcium in the extracellular bathing solution was replaced with barium. In a later study, ethanol sensitivity of native NMDA receptors in rat striatal neurons or recombinant NMDA

Fig. 1. Ethanol inhibits NMDA receptors at physiologically relevant concentrations. Traces are current activated by 25 ||M NMDA and 10 ||M glycine and its inhibition by 50 mM ethanol (EtOH) in a rat hippocampal neuron. The bars over the traces correspond to the duration of agonist and ethanol application. (Data from ref. 24.)

Nmda Receptor Alcohol

Fig. 2. Ethanol inhibition of NMDA receptors is not competitive with NMDA. The graph plots the percentage of current activated by 250 |lM NMDA as a function of NMDA concentration in the absence (filled circles) and presence (open circles) of 100 mM ethanol. Solutions of NMDA also contained 10 |M glycine. Each data point is the mean ± S.E. of six to seven neurons. The curves shown are the best fits of the data to the equation y = Emax/[1 + (X/EC50)", where x and y are concentration and response, respectively, EC50 is the half-maximal concentration, n is the slope factor (Hill coefficient), and Emax is the maximal response. Ethanol decreased the maximal response of the NMDA concentration-response curve without changing the EC50 of NMDA. (Data from ref. 40.)

NMDA(ijM)

Fig. 2. Ethanol inhibition of NMDA receptors is not competitive with NMDA. The graph plots the percentage of current activated by 250 |lM NMDA as a function of NMDA concentration in the absence (filled circles) and presence (open circles) of 100 mM ethanol. Solutions of NMDA also contained 10 |M glycine. Each data point is the mean ± S.E. of six to seven neurons. The curves shown are the best fits of the data to the equation y = Emax/[1 + (X/EC50)", where x and y are concentration and response, respectively, EC50 is the half-maximal concentration, n is the slope factor (Hill coefficient), and Emax is the maximal response. Ethanol decreased the maximal response of the NMDA concentration-response curve without changing the EC50 of NMDA. (Data from ref. 40.)

receptors in transfected cells did not differ depending on the presence or absence of the NR1 N-termi-nal cassette (30). In some studies, the NR2 subunit type was reported to alter ethanol sensitivity of NMDA receptors, with NR2A and NR2B subunit-containing receptors generally being the most sensitive to ethanol inhibition (31-36), whereas other studies reported little if any influence of the NR2 subunit on ethanol sensitivity (30, 37-39). If there is any consensus on this point at present, it is that NR2A and NR2B subunits under some conditions confer the highest ethanol sensitivity, with NR2B being perhaps the most sensitive, whereas NR2C and NR2D subunits are less sensitive. The contribution of NR1 splice variants to ethanol sensitivity in neurons under normal physiological conditions is probably of lesser importance. It should be appreciated, however, that any differences in ethanol sensitivity among NMDA receptor subunits are subtle at best. Thus, brain region-specific ethanol sensitivity of NMDA receptors is most probably not attributable to simple differences in ethanol sensitivity of subunit combinations, but instead is more likely to arise primarily from factors such as phosphorylation state or intracellular modulatory proteins (see below). Differences in regional expression of NMDA receptor subunits may still be important, however, in that modulation of ethanol sensitivity by phosphorylation or intracellular proteins may differ among subunits.

2.2. Mechanisms of Alcohol Action on NMDA Receptors

Studies designed to identify the mechanism of alcohol action on the NMDA receptor have been inconclusive to date. The agonist-binding site of the NMDA receptor is clearly not the site of ethanol action, based on observations of noncompetitive inhibition in experiments using electrophysiological recording of NMDA-activated current in hippocampal neurons (40) (Fig. 2), NMDA-evoked release of [3H]norepinephrine from cerebral cortical slices (23,41) or of [3H]dopamine from striatal slices (42), NMDA-stimulated Ca2+ influx in cerebellar granule cells (43) or dissociated whole brain cells (44), and NMDA-activated current in Xenopus oocytes injected with rat hippocampal mRNA (28). In addition, radiolabeled ligand-binding experiments in membranes from mouse cerebral cortex or hippocampus indicate that ethanol does not alter the binding affinity of [3H]l-glutamate or of the NMDA

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