The contribution of electrophysiology to knowledge of the acute and chronic effects of ethanol

Pharmacology & Therapeutics 84 (1999) 333–353

Associate editor: H.J. Little

The contribution of electrophysiology to knowledge of the acute and chronic effects of ethanol Hilary J. Little* Drug Dependence Unit, Department of Psychology, Durham University, South Road, Durham, DH1 3LE, UK

Abstract This review describes the effects of ethanol on the components of neuronal transmission and the relationship of such effects to the behavioural actions of ethanol. The concentrations of ethanol with acute actions on voltage-sensitive ion channels are first described, then the actions of ethanol on ligand-gated ion channels, including those controlled by cholinergic receptors, 5-hydroxytryptamine receptors, the various excitatory amino acid receptors, and g-aminobutyric acid receptors. Acute effects of ethanol are then described on brain areas thought to be involved in arousal and attention, the reinforcing effects of ethanol, the production of euphoria, the actions of ethanol on motor control, and the amnesic effects of ethanol; the acute effects of ethanol demonstrated by EEG studies are also discussed. Chronic effects of alcohol on neuronal transmission are described in the context of the various components of the ethanol withdrawal syndrome, withdrawal hyperexcitability, dysphoria and anhedonia, withdrawal anxiety, craving, and relapse drinking. Electrophysiological studies on the genetic influences on the effects of ethanol are discussed, particularly the acute actions of ethanol and electrophysiological differences reported in individuals predisposed to alcoholism. The conclusion notes the concentration of studies on the classical transmitters, with relative neglect of the effects of ethanol on peptides and on neuronal interactions between brain areas and integrated patterns of neuronal activity. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Ethanol; Electrophysiology; Ion channel; Neurotransmitter Abbreviations: ALDH, aldehyde dehydrogenase; AMPA, (R,S)-a-amino-3-hydroxy-5-methyl-4-isoxole; BK channels/currents, large conductance, calciumactivated potassium channels/currents; EPSP, excitatory postsynaptic potential; GABA, g-aminobutyric acid; 5-HT, 5-hydroxytryptamine; IPSP, inhibitory postsynaptic potential; LTP, long-term potentiation; mGluR, glutamate metabotropic receptor; NMDA, N-methyl-D-aspartate; NP, ethanol nonpreferring; P, ethanol preferring; REM, rapid eye movement; VTA, ventral tegmental area.

Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Voltage-sensitive ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Sodium channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Calcium channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Potassium channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Ligand-gated ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Cholinergic receptor-mediated responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Glutamate-activated channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. 5-Hydroxytryptamine receptor-mediated responses . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. g-Aminobutyric acid and glycine receptor-mediated transmission . . . . . . . . . . . . . . 3.5. Other ligand-activated responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Neuronal effects of ethanol; relationships to behavioural actions . . . . . . . . . . . . . . . . . . . . 4.1. Effects of ethanol on arousal and attention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Reinforcing effects of ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Production of euphoria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Motor control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Amnesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: 144-191-374-7768; fax: 144-191-3747774. E-mail address: [email protected] (H.J. Little) 0163-7258/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S0163-7258(99)00 0 4 0 - 6

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5.

6.

7.

4.6. Electroencephalogram studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The ethanol withdrawal syndrome, and beyond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Withdrawal hyperexcitability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Dysphoria and anhedonia during withdrawal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Withdrawal anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Craving and relapse drinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic influences on the effects of ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Genetic differences in the acute actions of ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Electrophysiological differences in individuals predisposed to alcoholism . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The predominant value of electrophysiological techniques in determining the changes in CNS activity caused by ethanol lies in the fact that functional changes can be measured. Other techniques provide information about alterations produced by ethanol in receptor density, mRNA levels, neurotransmitter concentrations, and other measures, while behavioural studies provide information about the results of such changes. Electrophysiology, however, combines analytic capability with functional information. Use of brain slices in electrophysiology combines the analytical advantages of studying living neurones, and the ability to control the external environment and apply exact concentrations of drugs, with the opportunity to examine changes in neuronal function. The patch-clamp technique, which can be used on dissociated cells or brain slices, takes this a step further in enabling measurement of individual ion conductances and the ability to alter the internal milieu of the cells. In vivo recording provides more information in one sense than brain slice methods, as it involves recordings of the functional activity of neurones in their natural state with intact afferent input, but does not permit such detailed analysis as the in vitro studies. In addition, the widespread use of general anaesthetics in vivo can cause major problems in the interpretation of results. Anaesthetics affect a wide variety of different transmitter systems (Little, 1996), and the effects of ethanol in such studies are measured against these background changes. Ethanol is a general anaesthetic, albeit not a very useful one from the practical point of view. Recording from implanted electrodes in conscious animals avoids this problem, but this is at present feasible only in certain brain areas that are more accessible and contain neurones of sufficient size to permit stable recordings. The development of more sophisticated technology that enables recordings from more difficult locations and multi-unit, as well as single-unit, recordings is a valuable advance and one that is likely to provide important information in the future. At the other end of the spectrum, recordings from oocytes in which different combinations of receptor subunits can be expressed and the functional activity recorded provide detailed information about the importance of subunit composition and amino acid sequence in ion channel function. The studies covered in this review have been primarily

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limited to electrophysiological studies carried out on mammalian neurones and oocytes. The effects of ethanol demonstrated by electrophysiological techniques have been considered in relation to its acute behavioural properties and to the development of dependence on ethanol. There is evidence that alcoholism has a genetic component, and the genetic influences on the actions of ethanol that have been demonstrated by electrophysiology are discussed at the end of the review. Perhaps the most important aspect of the electrophysiological studies on the actions of ethanol is the concentration of ethanol at which effects are produced. The plasma concentrations of ethanol that have been associated with various behaviour patterns are illustrated in Table 1. Brain concentrations of ethanol, however, do not exactly parallel those in plasma. Nurmi et al. (1994) demonstrated that brain ethanol concentrations after intraperitoneal administration of 1 g/kg ethanol (Fig. 1) rose more quickly than plasma concentrations, with a higher and earlier maximum (at approximately 5 min after injection), followed by a fast decline (probably due to redistribution to less well-perfused organs), which then slowed over the next 30 min to then follow the blood concentrations in an approximately parallel fashion. Concentrations of ethanol of approximately 5–20 mM are found in the body during mild intoxication (i.e., mood changes, anxiolysis, excitation, impaired cognition), while sedation, anxiolysis, and motor incoordination are associated with 20–50 mM ethanol. Ethanol concentrations higher than 50–100 mM cause general anaesthesia and risk of fatalities. Deitrich

Table 1 Plasma ethanol concentrations at which the behavioural effects of ethanol have been reported in humans and laboratory rodents Effect Humans Alterations in mood Impaired attention; increased accident risk Ataxia Intoxication Loss of consciousness Death Rodents Sedation Ataxia Loss of righting reflex

mM

mg%

5 11 20 44 50 111

22 50 90 200 227 500

22 27 66

100 122 300

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Fig. 1. Ethanol concentrations in brain and plasma measured at 1-min intervals after intraperitoneal administration of 1 g/kg ethanol in rats of the alcohol-preferring (AA; top diagram) and alcohol avoiding (ANA; middle diagram) lines and the outbred Wistar strain (lower diagram). The open circles indicate the concentrations of ethanol, mean 6 SEM in tail blood and the vertical lines the whole brain concentrations. Reproduced from Nurmi et al. (1994), with permission of the authors and the copyright holder, Elsevier, New York.

and Harris (1996) contribute a valuable discussion on concentrations and distribution of ethanol. The in vivo doses of ethanol that can produce behavioural changes may be lower than has often been thought, as both behavioural and electrophysiological changes have been reported after doses as low as 0.125–0.25 g/kg ethanol. It is often stated that ethanol has to be over a certain (unspecified) level in the brain before behavioural effects are produced that are worth consideration, and the unfortunate concept of “pharmacologically relevant” doses or concentrations is employed. However, whilst the contribution of taste and smell in influencing ethanol consumption is by no means negligible, the concept of “pharmacologically relevant” concentrations has, in fact, no scientific basis. It would be valid only if all of the effects of ethanol on central neurones, and the concentrations at which they are produced, are known, which is obviously not the case. The ap-

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plication of this concept can lead to disregard of effects of low ethanol concentrations that may cause subtle, but important, changes in behaviour. Another important aspect is the occurrence of behavioural excitation at low doses of ethanol (Pohorecky, 1977). The dose-response relationship for ethanol, therefore, is not a simple one, and the effects of different concentrations in vitro need to be interpreted in this context. The effects of ethanol in recordings have in some cases been shown to have a biphasic pattern; for example, the action of ethanol on electrographic seizures produced by electrical stimulation was found to be biphasic, with potentiation of epileptiform activity at 10 mM and depression at 60–300 mM (Cohen, S. M. et al., 1993). The locomotor activating effect of ethanol has been blocked by dopamine antagonists and attributed to activation of mesolimbic dopamine pathways (Strombom et al., 1977; Broadbent et al., 1995). The activation of locomotor activity by ethanol, however, does vary between rodent strains and species (Frye & Breese, 1981; Pohorecky, 1977). It is notable that a similar behaviour pattern is also seen with low doses of all general anaesthetic agents, with hyperexcitability at low doses, approximately one-third of the full general anaesthetic dose, followed at higher doses by sedation and anaesthesia. Ethanol has many different pharmacological actions, as described in Sections 2–4, and these are likely to be produced by actions at different sites. It has very selective effects on certain target sites within neurones, while other components of neuronal transmission are affected only by ethanol concentrations in the lethal range. Recent studies have demonstrated clearly defined locations for the actions of ethanol within, for example, the N-methyl-D-aspartate (NMDA) receptor complex (Peoples & Weight, 1995), and future work may demonstrate that these are equivalent to the receptor binding sites described for other drugs. However, specific binding sites, such as are well established for most pharmacological compounds, have not been clearly identified for ethanol. The word “nonspecific” has been used to describe the effects of ethanol, and is often misinterpreted to mean that ethanol would have the same effects at all sites, which clearly is not the case, as the studies below illustrate. The word “nonspecific,” however, has a pharmacological meaning, to indicate a lack of specific receptor binding sites. It is apparent from the electrophysiological studies described in Sections 2–4 that ethanol has selective actions on different sites within neurones. However, the origins of this selectivity of action are as yet uncertain. Alcohol, along with other general anaesthetic agents, was originally suggested to act on the lipid bilayer of the cell membrane (Meyer, 1899; Overton, 1901; see also Lipnick, 1989). These early theories derived from the high correlation between lipid solubility and general anaesthetic potency and the studies were primarily concerned with the mechanism of the general anaesthetic actions of drugs (reviewed by Little, 1996). Much of this work studied the effects of high ethanol

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concentrations on the fluidity of artificial membranes, which are very different from biological membranes in chemical composition, structure, and bond formation with exogenous compounds. Although comparatively low ethanol concentrations can affect the lipids of neuronal membranes, for example, increased fluidity of synaptosomal membranes with 25 mM ethanol was demonstrated by Chin and Goldstein (1977). Work suggesting direct actions of ethanol on proteins was at first largely on nonmammalian models, and also concentrated largely on the general anaesthetic effects of high ethanol concentrations. That ethanol, and other compounds with no apparent receptor binding sites, must act on either the lipid bilayer or the proteins in cell membranes, however, is an oversimplification, and both the interactions between lipids and proteins and the existence of hydrophobic areas within proteins need to be considered. More recent approaches, which take into account the structure of both the lipids and the proteins in biological membranes and the functional integration between their components, have contributed valuable results. The alcohols have been suggested to affect ligand-gated ion channels by acting on a hydrophobic area within the receptor protein (Li et al., 1994; Peoples & Weight, 1995). This would explain the correlation between their anaesthetic potency and lipid solubility and the “cut-off” phenomenon, in which the longer-chain alcohols do not have greater potency, despite their greater lipid solubility. From the point of this review, however, it must be remembered that these studies have been aimed at elucidation of the mechanisms of the general anaesthetic actions of the alcohols and have investigated the effects of high concentrations of ethanol. Li et al. (1994) saw 50% inhibition of NMDA receptor-mediated ion conductance with 130 mM ethanol, and 45% inhibition of ATP-gated ion channel conductance was seen with 100 mM ethanol (Peoples & Weight, 1995). Whether or not corresponding mechanisms are involved in the actions of ethanol at the lower concentrations that produce its range of behavioural actions remains to be seen. Other aspects that are of relevance are the role of the lipid-protein interface and the demonstration that the effects of ethanol vary with the position of the lipids within the bilayer. An important result that illustrates the difference between the effects of ethanol on membrane fluidity and those of temperature was that 25 mM ethanol increased fluidity of the exofacial leaflet of synaptosomal membranes, but had no effect on the cytofacial leaflet (Wood & Schoeder, 1992). This work demonstrates that measurement of bulk fluidity, even in biological membranes, does not give a true indication of the effects produced by ethanol. Comparatively few studies have been carried out on the lipid-protein interface, although effects of ethanol have been demonstrated (Slater et al., 1993; Ho et al., 1994), and studies on this interface for other neuronal proteins that are known to be sensitive to ethanol would be of interest.

The molecular target site(s) at which the synaptic actions of ethanol are produced, although of considerable scientific interest, is perhaps not the most vital problem, or even the most difficult to solve, for the electrophysiologist. The real challenge lies in making connections between the effects of ethanol on neuronal transmission and its behavioural actions. As this review describes, ethanol causes a number of selective changes in the components of synaptic transmission and many more have yet to be identified. However, comparatively little is known about which of its synaptic actions are responsible for its various behavioural effects. In this review, I have attempted to make correlations between the neuronal and behavioural properties of ethanol, but it is fully appreciated that this is at best a preliminary comparison and is limited in many cases by our lack of knowledge of the neuronal pathways and synaptic changes that are responsible for behaviour in the nonintoxicated state. I hope, however, that my attempts will provide an incentive for electrophysiologists, and encourage consideration of whole brain systems, as well as the discrete components of synapses. 2. Voltage-sensitive ion channels The effects of ethanol on ion channels are quite selective, as some types of channel are affected by comparatively low concentrations, while others are little altered until lethal levels are reached. 2.1. Sodium channels Ethanol, at concentrations relevant to its behavioural actions, does not affect Na1/K1 spike generation. Only very high concentrations affect voltage-sensitive sodium channels; Frenkel et al. (1997) demonstrated an ethanol EC50 of 1.03 M for the blockade of sodium channels from human cerebral cortex incorporated into lipid bilayers. 2.2. Calcium channels Depression of voltage-activated calcium conductances has been reported with behaviourally relevant concentrations of ethanol, but the relationship between ethanol concentration and effect varies with the tissue and the particular currents involved. Oakes and Pozos (1982) found evidence for decreases in calcium currents in dorsal root ganglion cells with ethanol at 11–110 mM, but concentrations below 11 mM increased spike duration, an effect suggested to be due to increased calcium conductance. A time-dependent biphasic effect of ethanol on L-channels was suggested by the patch-clamp study of McArdle et al. (1992), who found that 42 mM ethanol caused a small increase in the current and probability of opening, followed by a decrease in both these parameters. Influx of calcium via N and P/Q channel subtypes was decreased by ethanol at 10–100 mM (Solem et al., 1997). At concentrations of 30 mM and above, ethanol was found by Twombly and co-workers (1990) to decrease transient cal-

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cium currents in cultured neuroblastoma cells, while 100 mM and above decreased the long duration currents. Ethanol concentrations of 5–50 mM, however, were shown by Mullikin-Kilpatrick and Treistman (1994) to block calcium currents in PC12 cells, identified as being of the L-subtype. Wang et al. (1991a, 1991b) demonstrated blockade of both transient and long-duration calcium channels in neurohypophyseal terminals, suggested to account for the decrease in vasopressin release; the long duration channels were significantly affected by 10 mM ethanol. The effect of ethanol was shown to be on the gating characteristics of the channels (Wang et al., 1994). 2.3. Potassium channels Voltage-sensitive potassium channels are very insensitive to ethanol; a study of 10 different types of such channels expressed in oocytes showed some variability in the sensitivity at 200 mM ethanol and above (Anantharam et al., 1992). Potassium-specific inward rectification in locus coeruleus neurones was increased by ethanol, 40–200 mM, but this effect was attributed to increases in extracellular potassium concentrations (Osmanovic & Shefner, 1994). The conductance of calcium-dependent potassium channels underlying after-hyperpolarisations in hippocampal CA1 cells was reported to be increased by ethanol, 5–20 mM (Carlen et al., 1982), but other authors failed to find this effect. Siggins et al. (1987) saw no changes or decreases in after-hyperpolarisations in CA1 cells at ethanol concentrations between 11 and 150 mM. The differences may have been due to the method of application of ethanol, as Carlen et al. (1982) used microdrop application and Siggins et al. (1987) used superperfusion of submerged tissue slices. Large conductance, calcium-activated potassium channels (BK channels) in neurohypophyseal cell terminal showed increased activation with low concentrations (10 mM and above) of ethanol (Dopico et al., 1996). Activation of these channels (decreasing neuronal firing) may contribute to the decreased release of vasopressin and oxytocin and to the sedative actions of ethanol. Activation of large conductance calcium-activated channels with ethanol, 30 mM, was also seen in GH3 pituitary tumour cells (Jakab et al., 1997). A comparison of two cloned potassium channels expressed in oocytes showed that ethanol activated BK currents, but inhibited Shaw2 potassium channels. However, ethanol had less effect in these BK currents than those in neuronal membranes (Chu & Treistman, 1997). BK channels cloned from mouse brain (mslo channels) also showed activation by ethanol, with an EC50 of 24 mM (Dopico et al., 1997). Important effects of both acute and chronic ethanol have been demonstrated on the mesolimbic system, but the ion conductances that underlie these changes have not been elucidated. As potassium channels are involved in control of the spontaneous activity in dopamine neurones in this system (Silva & Bunney, 1988; Grace & Onn, 1991), it is possible that these may be targets for the actions of ethanol.

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3. Ligand-gated ion channels 3.1. Cholinergic receptor-mediated responses Much attention has been paid recently to co-dependence on both ethanol and nicotine, as around 90% of alcoholics are also dependent on nicotine. Behavioural studies in rodents have provided evidence for acute interactions between ethanol and nicotinic receptors. The locomotor activating effects of ethanol were found by Blomqvist et al. (1992) to be prevented by mecamylamine, a nicotinic antagonist that enters the brain. As the effect of ethanol in raising extracellular dopamine in the nucleus accumbens was also prevented by mecamylamine (Blomqvist et al., 1993), the mesolimbic dopamine pathway was suggested to be an important site of interaction between ethanol and nicotine. As this pathway is also thought to play an important role in dependence and in the reinforcing actions of drugs (see Section 4.2), these results may be relevant to the extensive co-dependence on ethanol and nicotine seen in humans. In addition, the depressant effects of ethanol on muscarinic transmission may be involved in the amnesic actions of ethanol; facilitation of memory at low doses has been reported (Parker et al., 1981). In view of the substantial clinical and behavioural evidence linking ethanol and nicotine, it is surprising that the effects of ethanol on cholinergic receptor responses have not been more widely studied by electrophysiological techniques. In halothane-anaesthetised rats, Covernton and Connelly (1997) demonstrated differences in the ethanol sensitivity of nicotinic receptor subtypes expressed in oocytes. The a3b4-subunit combination was the most sensitive to ethanol, with variable changes at low concentrations of ethanol (1–30 mM) and consistent potentiation at high concentrations (100–300 mM), while the a3b2-, a4-1b2-, and a4-1b4subunit combinations also showed potentiation of responses at the higher ethanol concentrations. The a7 homomeric receptor was insensitive to the low concentrations of ethanol and showed variable responses to the high concentrations, although Yu et al. (1996) found the chick a7 receptor to be inhibited by 5–100 mM ethanol, with an ED50 of 33 mM. At 100 mM, ethanol depressed the response of locus coeruleus neurones to nicotine (Froehlich et al., 1994). Synergism between ethanol and nicotine, applied by pressure injection, in cerebellar Purkinje cells was attributed to involvement of nicotinic acetylcholine receptors in the actions of ethanol (Freund & Palmer, 1997). Mancillas et al. (1986) demonstrated potentiation of the excitatory acetylcholine responses of hippocampal CA1 neurones in vivo with 1.5 g/kg ethanol, with no change in glutamate responses. At low concentrations (11–22 mM), ethanol enhanced the effects of muscarinic agonists and slow muscarinic inhibitory postsynaptic potentials (IPSPs) in hippocampal CA1 and CA3 cells (Madamba et al., 1995). Higher concentrations of ethanol depress certain cholinergic responses. Calcium-dependent chloride currents elicited in oocytes by muscarinic M1 receptor activation were de-

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creased by concentrations of ethanol in the higher range, 50–150 mM (Sanna et al., 1994), with an IC50 of 78 mM (Minami et al., 1997b). 3.2. Glutamate-activated channels Blockade of neuronal responses mediated by the NMDA subtype of glutamate receptor has been found to occur at lower concentrations of ethanol than effects on other glutamate receptor-mediated responses. Lovinger et al. (1989) found an inhibitory action of ethanol on NMDA responses between 5 and 50 mM, while responses to kainate or quisqualate were affected only slightly by these concentrations. A similar difference in sensitivity was also found in the spinal cord, with less effect of ethanol on neonatal neurones (Wong et al., 1997), although hippocampal slices from immature rats showed greater sensitivity to the depressant effects of ethanol on NMDA receptor-mediated excitatory postsynaptic potentials (EPSPs) than tissues from mature animals (Swartzwelder et al., 1995). Gruol (1992) found that ethanol, 22 and 44 mM, increased the inhibitory component of the responses of cultured cerebellar Purkinje neurones to quisqualate. The presence of magnesium was shown to increase the actions of ethanol on NMDA receptor-mediated responses in hippocampal CA1 pyramidal cells, reducing the IC50 from 107 mM to 47 mM; the IC50 for AMPA kainate responses in this study was over 170 mM (Martin et al., 1991; Morrisett et al., 1991). The patch-clamp study of Lima-Landman and Albuquerque (1989) demonstrated that very low concentrations of ethanol, 1.74–8.65 mM, increased the probability of NMDAactivated channel opening in cultured rat hippocampal cells, but did not affect channel open time, while high concentrations, 86.5–174 mM, decreased both the probability of channel opening and the mean open time. However, considerable regional and local variations occur in the actions of ethanol on NMDA receptors; for example, NMDA receptor responses in the lateral septum in vivo were insensitive to ethanol (Yang et al., 1996). Neiber et al. (1998) found no selectivity in the actions of ethanol on responses of locus coeruleus neurones to NMDA and to AMPA. All NMDA receptor subunit combinations have been found to be affected by ethanol, but N1-NR2C and NR1/NR2D channels are less sensitive to ethanol than the NR1-NR2A or NR1-NR2B combinations, with the N2B subunit being particularly sensitive (Kuner et al., 1993; Masood et al., 1994; Dildy-Mayfield & Harris, 1995; Chu et al., 1995; Buller et al., 1995; Yang et al., 1996). Glycinesensitive and insensitive components to the effect of ethanol, and increased glycine-sensitive desensitisation, have been described (Buller et al., 1995). Homomeric assemblies of subunit splice variants expressed in oocytes exhibited similar sensitivity to ethanol to that seen in neurones, with the NR1-LL variant being more sensitive than the NR1-SS assembly (Kolchine et al., 1993). Higher concentrations of ethanol (50–100 mM) were re-

quired to decrease responses mediated by AMPA/kainate receptors in hippocampal slices (ethanol 25–100 mM; Martin et al., 1995) or expressed in oocytes (ethanol 50–100 mM; Dildy-Mayfield & Harris, 1992a). Responses to low concentrations of kainate were more sensitive to ethanol than responses to high concentrations (Dildy-Mayfield & Harris, 1992b). All GluR subunits appear to be sensitive to ethanol, particularly GluR3 and GluR6 (Dildy-Mayfield & Harris, 1992a). Raised calcium concentrations increased this effect of ethanol, possibly via increased protein kinase activity. Metabotropic receptor responses have been less investigated, but Netzeband and Gruol (1995) studied effects of ethanol on the firing rate changes produced by metabotropic receptor agonists in cultured cerebellar Purkinje neurones suggested to involve glutamate metabotropic receptor (mGluR)1 subunits. Ethanol, 66 mM, but not 33 mM, increased the duration of response to 1-aminocyclopentane1,3-dicarboxylic acid, a metabotropic glutamate receptor agonist, but inhibited burst activity induced by quisqualate, which acts on the AMPA subtype of glutamate receptor. Minami et al. (1998) showed that ethanol inhibited mGLuR5-induced calcium-dependent chloride currents in oocytes, but had less effect on the currents induced by glutamate via mGluR1 receptors. The sensitivity of NMDA receptor-mediated responses to ethanol is well established, but wide regional variations occur. Identification of subunit selectivity may explain some of the sensitivity differences. The action is likely to contribute to the amnesic effects of ethanol, and possibly the anxiolytic actions. Effects of higher concentrations of ethanol on AMPA/kainate receptor-mediated responses are likely to be involved in the sedative and general anaesthetic actions of ethanol. However, little attention appears to have been paid to the possibility of potentiation of excitatory amino acid responses by very low ethanol concentrations, which might contribute to the excitant effects seen in vivo after low doses of ethanol. 3.3. 5-Hydroxytryptamine receptor-mediated responses Ethanol, between 25 and 100 mM, potentiates the effects of 5-hydroxytryptamine (5-HT) at the 5-HT3 subtype of receptor (Lovinger, 1991; Lovinger & White, 1991) in neuroblastoma and nodose ganglion cells, the effect being dependent on the concentration of 5-HT present. The variability in the effects of ethanol seen in these studies has been suggested to be due to different channel states rather than to subunit composition or other factors (Lovinger & Zhou, 1994), and the mechanisms involved include increased channel activation and decreased deactivation and desensitisation (Zhou et al., 1998). Ethanol, 50–150 mM, inhibits the 5HT1C receptor activation of calcium-dependent chloride currents in oocytes (Frye et al., 1991; Sanna et al., 1994). The depressant action of 5-HT on after-hyperpolarisations in hippocampal neurones, thought to be mediated by 5-HT1A and 5-HT4 recep-

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tors, showed a slowly developing potentiation with ethanol, 30 mM (Lau & Frye, 1996). Calcium-dependent chloride currents induced in oocytes by activation of 5-HT2A receptors were found to be inhibited by ethanol, with an IC50 of 41 mM (Minami et al., 1997a). Antagonists at 5-HT3 receptors are used therapeutically as anti-emetic agents, so by deduction, the actions of ethanol in potentiating 5-HT3 receptor-mediated responses could mediate the nausea and vomiting caused by relatively high doses of ethanol. The reinforcing properties of ethanol may involve effects on 5-HT transmission, as 5-HT reuptake inhibitors decrease voluntary consumption of ethanol (Sellers et al., 1992). It is possible that the interaction demonstrated in ventral tegmental neurones by Brodie et al. (1995), when they reported potentiation of the excitant effects of ethanol by 5-HT, is involved in the reinforcing actions of ethanol. 3.4. g-Aminobutyric acid and glycine receptor-mediated transmission Although there is widespread belief that potentiation of the chloride conductance increases elicited by g-aminobutyric acid (GABA) plays an important part in the behavioural effects of ethanol, particularly the anxiolytic actions, examination of the evidence, particularly that from electrophysiological studies that measure functional changes, shows that this effect of ethanol is very variable. The factors that determine whether or not potentiation of GABAA receptor-mediated transmission by ethanol is observed are still not completely elucidated, but certain aspects are now considered to be important, as described below. It is notable that the variability (and frequent elusiveness) of potentiation of GABAA-mediated inhibition by ethanol is not seen in neurochemical studies, for example, those that demonstrated potentiation of the effects of GABA on synaptosomal chloride flux (Ticku et al., 1986), and the reason for this difference is not clear. Responses to applied GABA- and GABAA-mediated inhibition were found to be increased in neurones of the cerebral cortex (Nestoros, 1980) in spinal cord by 20–50 mM ethanol (Celentano et al., 1988), in hippocampus by ethanol at 70 mM and above (Takada et al., 1989; Procter et al., 1992), and in dorsal root ganglion by 30–300 mM ethanol (Nishio & Narahashi, 1990). In contrast, many authors failed to see potentiation of the effects of GABA by ethanol. For example, on hippocampal neurones, Gage and Robertson (1985) saw no effect with 10–200 mM ethanol; Carlen et al. (1982) no effect with ethanol concentrations between 5 and 20 mM, although some potentiation was seen with 100 mM ethanol; and Siggins et al. (1987) no effect with ethanol 22–80 mM. Inhibitory currents in hippocampal cells appear to be less affected by ethanol than in other regions. Procter et al. (1992) demonstrated potentiation of GABAA receptormediated EPSPs in cerebral cortex, but not hippocampus. In an important set of studies, potentiation by ethanol of

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GABA responses, recorded in culture, was seen in only certain hippocampal, cortical, and spinal neurones (Aguayo & Pancetti, 1994). Wafford and Whiting (1992) suggested that the potentiation of GABA occurred only when the g2L variant subunit was present in the GABA receptor complex. Celentano et al. (1988) found potentiation of GABA responses in cultured spinal cord neurones was seen only after repeat applications of ethanol, and Nishio and Narahashi (1990) reported potentiation by ethanol only of the initial GABA response of dorsal root ganglion cells, prior to desensitisation. The factors that affect the appearance, or otherwise, of GABAA potentiation by ethanol, therefore, include the brain region, the species, the receptor subunit composition, the duration of application of ethanol, and the state of phosphorylation of the GABA receptor. In addition, the importance of synaptic connections in identification of the actions of ethanol was illustrated by the demonstration that ethanol markedly potentiated the responses to GABA of cerebellar Purkinje cells in vivo, only when such depression was enhanced by noradrenergic input or by b-adrenergic agonists (Lin et al., 1991; Freund & Palmer, 1996). Evidence from in vivo recordings has suggested that the depression of locus coeruleus firing by ethanol reduces the tonic enhancement of the GABA-mediated inhibition of cerebellar Purkinje cells (Harris & Sinclair, 1984). Other studies indicated the effects of ethanol may involve GABAB receptors, as blockade of GABAB-mediated inhibition revealed an effect of ethanol, 22–66 mM, in potentiating GABAA inhibition in hippocampal pyramidal cells (Wan et al., 1996). Allan and Harris (1991), however, found that GABAB receptor activation was necessary for detection of the potentiation by ethanol of GABA-induced flux through chloride channels expressed in Xenopus oocytes. In contrast to the effects on the GABAA subtype of receptor, potentiation has not been seen when the effects of ethanol have been investigated on other subtypes of GABA receptors. Responses mediated by the GABAB subtype do not appear to be greatly affected by concentrations of ethanol that cause behavioural changes. GABAB receptor-mediated responses in hippocampal cells were found to be unaltered by 10–100 mM ethanol (Frye et al., 1991; Frye & Fincher, 1996). Currents through GABA receptors formed of rho receptor subunits expressed in oocytes were inhibited by ethanol, 10–100 mM (Mihic & Harris, 1996). Glycine-elicited chloride currents have also been reported to be potentiated by GABA. Aguayo and Pancetti (1994) demonstrated potentiation of glycine-activated currents in cultured mouse hippocampal and spinal neurones over a wide range of concentrations (1–450 mM) of ethanol. A voltage-clamp study on chick spinal cord neurones showed ethanol (20–50 mM) increased the actions of both GABA and glycine (Celentano et al., 1988). Both the a1 and a2 glycine subunits showed such potentiation with ethanol (10–200 mM) when expressed in oocytes, but homomeric a1 receptors were more sensitive to ethanol than a2 recep-

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tors (Harris et al., 1995; Mascia et al., 1996). However, strychnine-sensitive chloride currents in primary cultures of septal cholinergic neurones were unaffected by ethanol at 1–100 mM (Kumamoto & Murata, 1996). The anxiolytic actions of ethanol are seen at low doses, but the uncertainty in the interactions between ethanol and GABA makes it difficult to draw conclusions about the role in these of potentiation of GABAA transmission by ethanol, although it is likely that increased GABAA receptor-activated chloride conductance in sensitive neurones could contribute to the anxiolysis, and possibly to the reinforcing effects of ethanol. The sensitivity of certain areas of the brain, such as medial septum, to this action of ethanol may be of relevance in the anxiolytic and the amnesic actions of ethanol. The effects of higher concentrations of ethanol are likely to play a major role in the production of sedation. 3.5. Other ligand-activated responses Neuronal transmission mediated by the receptor systems, particularly GABA and glutamate transmission described in Sections 2–4, has been extensively studied by electrophysiological techniques. However, there is a very large number of other neurotransmitters in the CNS that play vitally important roles in the control of behaviour, even if they do not have such a ubiquitous distribution in the brain as GABA and glutamate. These have received very little attention, particularly from electrophysiologists, but also from researchers using other types of technique. The inhibitory responses of hippocampal CA1 cells to somatostatin were also reported by Mancillas et al. (1986) to be potentiated following 1.5 g/kg ethanol, while responses to GABA were not thought to be altered. Hyperpolarisation of locus coeruleus cells elicited by noradrenaline was unaffected by ethanol at 100 mM (Neiber et al., 1998). Recordings of field EPSPs in hippocampal CA1 pyramidal cells provided no evidence of effects due to changes in adenosine concentrations with ethanol, 20 mM and 100 mM (Diao & Dunwiddie, 1996). High concentrations of ethanol (IC50 68 mM) inhibited the ATP-activated currents in dorsal root ganglion cells via the neuronal P2X purinergic receptor (Li et al., 1994, 1998). 4. Neuronal effects of ethanol; relationships to behavioural actions The effects of ethanol on neuronal activity within brain regions has received somewhat less attention than its effects on discrete ion channels, but results have demonstrated important changes in neuronal firing that can be related to certain of the behavioural actions of ethanol. 4.1. Effects of ethanol on arousal and attention As described at the beginning of this review, ethanol has an array of effects on arousal, with, in certain circum-

stances, stimulation at lower doses and, more universally, sedation at high doses. The mechanism(s) underlying the general anaesthetic properties of ethanol and other agents has been discussed in an earlier review (Little, 1996). The locus coeruleus plays a role in attention processes; reported effects of ethanol on neurones in this area have included both excitation and inhibition of activity. Sensoryevoked responses in the locus coeruleus in response to applied sensory stimuli were demonstrated in rats anaesthetised with chloral hydrate or halothane to be reduced in magnitude and in temporal reliability by ethanol doses from 0.5 to 3 g/kg, while spontaneous firing and antidromic activation of neurones were unaffected (Aston-Jones et al., 1982). In a slice preparation of the locus coeruleus, however, Shefner and Tabakoff (1985) found that the effect of ethanol depended on the spontaneous firing rate of the neurones prior to addition of ethanol, at concentrations relevant to the behavioural effects of ethanol. Below 10 nM, ethanol depressed, had no effect on, or increased the firing rate, while higher concentrations resulted in decreased firing. Froehlich et al. (1994) found increased firing of action potentials in vitro with ethanol, 100 mM, but depression of responses to NMDA with 10–100 mM ethanol. Neiber et al. (1998), however, did not see increased firing or effects of the lower concentrations of ethanol on responses to NMDA. Depression of activity of locus coeruleus neurones by very low concentrations of ethanol (0.5–10 mM) was demonstrated by Palmer et al. (1992) in ocular grafts of these neurones. Low concentrations of ethanol (1 mM for cerebellar cells and 10 mM for hippocampal neurones) caused excitation of co-grafted hippocampal and cerebellar neurones, while these neurones were depressed by higher concentrations of ethanol. The excitations were demonstrated to be dependent on the locus coeruleus cell depression, illustrating the importance in the effects of ethanol of the afferent input to cells. Ethanol increases the activity of dopaminergic neurones in the substantia nigra, an effect that may underlie the locomotor stimulant effect of low doses of ethanol. Doses of ethanol that would produce motor stimulation and ataxia (0.5–2 g/kg) increased the firing rate of dopamine neurones in the substantia nigra in unanaesthetised rats, but only depressant actions of ethanol were seen in anaesthetised animals (Mereu et al., 1984), suggesting that results obtained from dopaminergic neurones in anaesthetised animals may not provide a true indication of the actions of ethanol in these neurones during the behavioural effects of this drug. 4.2. Reinforcing effects of ethanol “Reinforcing” effects of drugs are those that strengthen the relationship between stimulus and response and, therefore, will increase the probability of occurrence of the behaviour that preceded the effects of the drug (Skinner, 1938; White & Milner, 1992). This term is often used synonymously with the “rewarding” actions of drugs, but there are

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important differences between these attributes (White, 1989). The concept of reward implies “pleasure” and includes euphoric actions (discussed in Section 4.3). Both these and the reinforcing properties of drugs have important roles in the development of dependence. There is considerable evidence implicating the mesolimbic dopamine system in the reinforcing properties of drugs, including ethanol, which is, for example, self-administered by rodents into the ventral tegmental area (VTA), where the cell bodies of this system are located. It should be recognised, however, that the mesolimbic dopamine system is also involved in responses to aversive experiences, and the role in reinforcement has been questioned (Salamone et al., 1997). Effects of ethanol on endogenous opiate transmission (see Section 4.3) are also likely to be involved in reinforcement. Very low doses of ethanol (ED50 162 mg/kg i.v.) increase the firing of ventral tegmental dopaminergic neurones in unanaesthetised, but not anaesthetised, rats (Mereu et al., 1984; Gessa et al., 1985). This area was considerably more sensitive to ethanol than neurones in the substantia nigra, as recorded in the same study (see above). In isolated slices of the VTA, ethanol, 20 mM and above, was shown to have a direct action in stimulation of firing, seen when synaptic transmission was blocked (Brodie et al., 1990). No depression of activity was seen even at ethanol concentrations up to 320 mM. The excitant action, seen in 75% of dopaminergic neurones in the VTA, has been shown to involve increased time-dependent inward rectification (Brodie & Appel, 1998), and later studies showed that this effect of ethanol was increased by co-application of 5-HT (Brodie et al., 1995). The effect of general anaesthesia in converting the activating effects of ethanol on the VTA into depressant action explains the results of Criado et al. (1994), who reported that ethanol, 1.2 g/kg, decreased the VTA facilitation of the activity of hippocampal cells. These results illustrate the problems of interpretation of the effects of ethanol, with its range of properties, including general anaesthesia, against a background of the actions of another anaesthetic agent.

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nucleus accumbens core region in vitro was prevented by naloxone. This effect was thought to be due to a naloxonesensitive action of ethanol on glutamate release (Nei et al., 1994). An in vivo study, however, showed that the depression by ethanol (1.2–1.4 g/kg) of activation of nucleus accumbens neurones from the amygdala was not affected by naloxone in conscious, freely moving animals (Criado et al., 1997). At reinforcing concentrations, ethanol, therefore, would stimulate VTA neurones, but also has depressant actions within the nucleus accumbens, some of which involve endogenous opiate transmission. 4.4. Motor control The effects of ethanol in vivo on motor control are well established and are of great social importance in the major contribution of ethanol to the accident rate. The results described here demonstrate that ethanol, at concentrations that have ataxic effects in vivo, has direct effects in depressing spinal reflexes and altering cerebellar function. Many of the early electrophysiological studies on ethanol investigated these effects and were reviewed by Kalant and Woo (1981). Depression of the monosynaptic reflex in spinal cord neurones was produced by moderate ethanol concentrations (22–108 mM) measured in blood during in vivo recordings (Lathers & Smith, 1976). Studies on the cerebellum in vivo demonstrated that low doses of ethanol, 0.25–1 g/kg, increased the rate of firing of Purkinje cells (Sinclair & Lo, 1986). Inhibition of these cells due to release of GABA from adjacent neurones was decreased by the same dose range of ethanol. Increased firing rates were also seen in cerebellar slices with low concentrations (9–17 mM) of ethanol, while depression of firing occurred at concentrations above this range (George & Chin, 1984). Urrutia and Gruol (1992) demonstrated that current-evoked spiking in cultured cerebellar Purkinje cells was enhanced by 22 mM ethanol, although this concentration depressed corresponding activity in hippocampal neurones; at 44 mM, ethanol decreased activity in both types of cells. Excitant effects of ethanol on cerebellar interneurones were reported by Freund et al. (1993).

4.3. Production of euphoria The euphoria produced by ethanol, primarily at low doses, is one of the main reasons for its social use, along with the anxiolytic actions. The euphorigenic actions may involve effects on endogenous opiate systems and on dopamine transmission. Comparatively little electrophysiological work has been carried out on the effects of ethanol on endogenous opiate responses, which is surprising, as one of the most important advances in therapeutic treatment for alcohol dependence is naltrexone (Volpicelli et al., 1992). This opiate receptor antagonist has been shown to decrease relapse drinking in alcoholics, and is thought to act by decreasing the euphoric, rewarding, and/or reinforcing effects of alcohol. Nei et al. (1993) demonstrated that the effect of ethanol, 22–66 mM, in reducing glutamatergic EPSPs in cells in the

4.5. Amnesia The amnesic actions of ethanol are seen both after acute administration and following chronic high consumption. Long-term potentiation (LTP) is a form of synaptic plasticity thought to play an important role in memory and learning. Ethanol decreases hippocampal LTP, an effect originally shown with a high concentration of 100 mM (Sinclair & Lo, 1986), but then demonstrated to occur with concentrations as low as 5 mM when the EPSP slope was used as a measure of potentiation (Blitzer et al., 1990). The greater sensitivity of LTP to ethanol in immature rats (Swartzwelder et al., 1995) may be important in effects of alcohol on adolescents. The effects of ethanol on LTP appear to be due to its depression of NMDA receptor-mediated responses (Morrisett & Swartzwelder, 1993).

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Single unit recording from the dorsal hippocampus, made under general anaesthesia, demonstrated excitatory effects at low doses of ethanol (0.1 and 0.2 g/kg, i.v.), while slightly higher doses (0.4 and 0.8 g/kg, i.v.) showed mixed effects, with depressant actions predominating at the 0.8 g/kg dose, with parallel effects on the cortical EEG (Grupp, 1980). An additional type of action on hippocampal dentate granule cells was demonstrated by Fu et al. (1992), who found that 50 mM ethanol increased gap junctional resistance, thus decreasing electrotonic coupling. Hippocampal theta rhythm is a regular firing pattern seen in field potentials, which is driven from the medial septum. Theta rhythm has been reported to be suppressed by ethanol, at doses of 0.75 g/kg and above (Kaheinin et al., 1988). Depression of theta rhythm, particularly the high-frequency component, was demonstrated with low doses of ethanol (0.75–1 g/kg) in conjunction with deficits in working memory in rats (Givens, 1995). This author also found increases in relative theta power with very low doses of ethanol, 0.25 and 0.5 g/kg. 4.6. Electroencephalogram studies Early studies on EEG in conscious animals and humans demonstrated that low doses, causing behavioural arousal and euphoria, resulted in desynchronisation of the EEG, reduced mean amplitude, and increases in theta and alpha activity (Lucas et al., 1986; Ehlers et al., 1989; Cohen, H. L. et al., 1993a). Doses of alcohol that caused depression of activity increased synchronisation of the EEG, decreased frequency, and increased mean voltage. In young male volunteers, 1 g/kg ethanol increased EEG power in the theta and beta bands (Stenberg et al., 1994), but in conscious rats, 0.75 g/kg ethanol decreased EEG power over all frequencies (Ehlers et al., 1992a, 1992b, 1992c). The EEG changes have been found to outlast the presence of ethanol in the blood, both in rodents (Young et al., 1982) and in humans (Lehtinen et al., 1981). Ghosh et al. (1991) found no acute effect of ethanol vapour on EEG, despite decreases in rapid eye movement (REM) sleep, in rats, but Djik et al. (1992) demonstrated enhanced delta and theta range frequency during non-REM and REM sleep in humans after ethanol, 0.6 g/kg; these changes continued into the second night after the ethanol. Enhanced delta frequency EEG power was also seen in middle-aged men, together with increases in alpha and band power densities, 6 hr after an afternoon dose of ethanol, 0.55 g/kg (Landolt et al., 1996). Early work on evoked potentials was reviewed by Kalant and Woo (1981). These studies showed the later component of cerebrocortical-evoked responses in humans was the most affected by ethanol, suggesting actions on the indirect input via thalamus and reticular formation, rather than the direct input via the primary sensory pathways. Evidence from brain stem-evoked potentials in conscious rats also indicated greater effects of ethanol on later components of the potentials, for example, with blood ethanol levels of 22–33 mM after 2–3 g/kg ethanol (Squires et al., 1978; Chu et al.,

1978). Single unit-evoked potentials elicited in the somatosensory cortex in conscious monkeys by light touch, however, were unaffected by intravenous doses of 0.1–2.5 g/kg of ethanol (Collins & Roppolo, 1980). The early peaks of visual-evoked potentials in humans have been found to be less affected by ethanol than the later peaks (Rohrbaugh et al., 1987; Colrain et al., 1993), suggesting a selective effect on conscious processing rather than automatic stimulus evaluation. The P3 component, elicited by infrequent stimuli that attract attention, is particularly sensitive to alcohol, showing increases in latency and decreases in amplitude (e.g., Wall & Ehlers, 1995). Increased latencies and reduced amplitude of the auditory N1 and P2 waves and the N2b and P3b complex have been reported in human studies (Pfefferbaum et al., 1979; Teo & Ferguson, 1986) or example at alcohol breath concentrations of 0.05 and 0.08 (Krull et al., 1993), although Hetzler et al. (1982) demonstrated increases in P2, with depression and increased latency of other components, in visual cortex-evoked potentials in rats after ethanol, 1.5 and 2.5 g/kg. In conscious rats, ethanol, 0.75 g/kg, increased latency and increased amplitude of the N1 and P2 component responses evoked by auditory stimuli (Ehlers et al., 1992a, 1992b, 1992c). Evidence has been obtained of selective effects of ethanol on responses elicited by unattended, rather than attended, stimuli that may have important relevance to human ability to avoid accidents (Campbell & Lowick, 1987). Jaaskelainen et al. (1995) demonstrated decreases in mismatch negativity response to a deviant auditory stimulus within a train in social drinkers after ethanol, 0.5 g/kg (blood concentrations 1–2 mM), suggesting deficits in automatic processing of unattended stimuli that may be important in driving. Potentials associated with attentional functions remained unchanged in this study. The effects of alcohol (0.8 g/kg) on human evoked potentials on discrimination of a target auditory stimulus was found to depend on the ease of discrimination, with reduction in the N1 and P3 amplitudes only when discrimination was easy (Campbell et al., 1984). Heavy social drinkers exhibited shorter P2 latencies and decreased P3 amplitudes in a word identification task (Nichols & Martin, 1996) and had longer P3 latencies in a simulated driving task (Nichols & Martin, 1993). Lehtokoski et al. (1998) showed that ethanol, 0.55 g/kg, in social drinkers decreased the N1 amplitude (automatic change detection) and increased the latency of the N2b and mismatch negativity (allocation of attentional resources to stimulus processing) of auditory evoked potentials; the P1 amplitude was increased only for attended stimuli. Naltrexone blocked the effects of ethanol on the P1 and N2b components, but increased its effect on mismatch negativity latency.

5. The ethanol withdrawal syndrome, and beyond Dependence on any drug is diagnosed by certain criteria, not all of which are easy to model in animals. These criteria

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include progressively increasing consumption, unsuccessful attempts to reduce intake, continued excessive use of the drug despite adverse effects, and production of a physiological withdrawal syndrome. Methods used in rodents to model alcohol consumption in humans include voluntary choice between alcohol and water and operant self-administration of alcohol. Other methods of chronic administration of alcohol involve repeated intragastric administration, inhalation of alcohol vapour, and consumption via a liquid diet. In comparison with the study of other aspects of dependence, production of the acute withdrawal syndrome is relatively straightforward, and the signs in rodents closely resemble the symptoms in humans, consisting of hyperexcitability, tremor, convulsions, and anxiety-related behaviour. The effects of ethanol withdrawal in humans, however, are not confined to symptoms of hyperexcitability. Dysphoria, anhedonia, and craving for alcohol are all seen, and anxiety and craving, in particular, continue far beyond the acute phase of withdrawal hyperexcitability, often for months or years. Electrophysiological studies have provided considerable information about the mechanisms underlying the neuronal hyperexcitability seen in the time period immediately following cessation of alcohol consumption, and recently, these techniques have been applied to study of the more prolonged neuronal changes that may be involved in the propensity of alcoholics to repeatedly return to excess drinking, despite having maintained abstinence through the acute withdrawal phase. The use of different routes of administration of alcohol, both voluntary and involuntary, and of a wide range of durations of treatment, from days to years, makes comparison of work on this topic from different laboratories very difficult. It is not possible to compare the degree of “dependence” with the different models, since there is no consensus on how this can be estimated, i.e., amount of alcohol consumed, propensity to self-administer ethanol, or production of a withdrawal syndrome. Studies of withdrawal hyperexcitabil-

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ity in vitro, however, have been useful, although the time courses are likely to be different from those in vivo, owing to differences in the amount of metabolism of alcohol in vivo and in tissue preparations. The importance of the interval between cessation of alcohol intake and the various physiological measurements is often neglected (see Section 5). A question frequently asked (by referees, editors, and audiences!) concerns the blood concentrations of ethanol during chronic administration in animals by the oral route, such as via the drinking fluid or a liquid diet. However, such blood concentration measurements are not as informative as might at first be thought, particularly when samples are taken at only one or two times a day. The lack of parallelism between the blood and brain concentration of ethanol has been discussed in Section 1. In addition to this, the pattern of drinking of the animals results in marked circadian variation in concentrations, and this has been elegantly illustrated by the results of Jelic et al. (1998). These authors measured the blood concentrations, every few hours over 24 hr, in two mouse strains (CBA and TO) undergoing oral chronic ethanol administration via a liquid diet (Fig. 2). The blood concentrations varied from less than 10 mM to over 100 mM, and the two strains showed different patterns of intake, with the maximal concentration in CBA mice being seen at 19.00 hr and that in the TO strain at 09.00 hr (ethanol consumption values were 11–19 g/kg/day for the CBA mice and 18– 22 g/kg/day for the TO strain). 5.1. Withdrawal hyperexcitability Epileptiform patterns with desynchronised activity and spiking are seen in EEG recordings during withdrawal from chronic alcohol consumption. Early work by Walker and Zornetzer (1974) and Hunter and Walker (1978) showed this to originate in subcortical regions, the posterior thalamus, reticular formation, and amygdala, then to spread to

Fig. 2. Plasma concentration of ethanol measured at intervals over 24 hr during consumption of a liquid diet containing ethanol. The right-hand diagram illustrates the concentrations in CBA strain mice and the left-hand diagram, the concentrations in mice of the TO strain. Measurements were made after 7 days’ consumption of the liquid diet. Reproduced from Jelic et al. (1998), with permission of the authors and the copyright holder, Springer-Verlag, Heidelberg.

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the cerebral cortex. During withdrawal from repeated gastric intubation of ethanol, recordings from the inferior colliculus and the pontine reticular formation showed increased activity just prior to audiogenic seizures, although evidence was not found that these areas played a major role in the origin of withdrawal hyperexcitability (Faingold & Riaz, 1994; Chakravarty & Faingold, 1998). Veatch and Gonzalez (1996), using EEG recordings, found evidence of increased spiking activity during ethanol withdrawal that was apparent first in the hippocampus, followed by changes in the amygdala and motor and visual cortices. The severity of this activity in area CA1 of the hippocampus was related to the duration of chronic ethanol exposure, while that in hippocampal area CA3 was increased by repeated cycles of withdrawal. Gonzalez and Sun (1982) showed, using single unit recordings in vivo, significantly lowered thresholds for activation of rat hippocampal CA1 cells by medial septal stimulation, between 6 hr and 8 hr after the animals were withdrawn from chronic ethanol treatment. Evoked potentials have shown increases in latency and decreases in amplitude during withdrawal after up to 5 weeks of ethanol administration to experimental animals (Beglieter et al., 1972: Beirley et al., 1980; Bogart et al., 1991). After 1 month of ethanol vapour treatment, rats showed reductions in N1 and P2 amplitudes in response to auditory stimuli, seen at 24 hr and 2 weeks’ withdrawal (Ehlers & Chaplin, 1991). Two months of ethanol treatment in rats resulted in withdrawal in increased latency and reduced amplitude of the N1 wave of the visual-evoked responses recorded in anaesthetised rats that persisted for at least 1 week after cessation of the ethanol consumption (Kjellstrom et al., 1994). Measurements of evoked potentials during withdrawal in alcoholics have demonstrated increases in the latency of N2 and P3 components and decreases in amplitude of the N1 and P3 components (Porjesz & Begleiter, 1987; Parsons et al., 1990). An increase in amplitude of the auditory-evoked N1 and P2 components, however, was found to be predictive of the severity of withdrawal in alcoholics (Juckel et al., 1994). The amplitude of N1 and P2 appeared to differentiate alcoholics with and without a history of withdrawal seizures (Noldy & Carlen, 1991). More detailed electrophysiological studies have shown that increases in excitatory amino acid transmission play a major role in ethanol withdrawal hyperexcitability and the production of convulsions. The majority of these studies have used hippocampal neurones, as the hippocampus is known to play an important role in seizure activity. Burst firing in hippocampal CA1 neurones after chronic ethanol treatment in vivo, rarely seen in slices from control animals, was prevented by an antagonist of AMPA/kainate receptors, but not by an NMDA receptor antagonist (Shindou et al., 1994). Both AMPA/kainate receptor-mediated and NMDA receptor-mediated transmission were increased in hippocampal slices during withdrawal from chronic ethanol consumption by rats, as illustrated in Fig. 3a (Molleman & Little, 1995a; Whittington et al., 1995).

Muscarinic receptor function in the rat hippocampal CA1 area was reported to be impaired after chronic ethanol treatment, with decreased cholinergic facilitation of population spikes, but cholinergic inhibition of the field EPSPs and inhibition of post-spike after-hyperpolarisations were unaffected (Rothberg et al., 1993; Frye et al., 1995). Measurements of voltage-sensitive calcium currents have demonstrated increased conductance during the acute withdrawal phase, which is likely to contribute to withdrawal hyperexcitability. Shindou et al. (1994) reported an increase in the plateau component of the calcium spike in hippocampal neurones in vitro. Huang and McArdle (1993) showed 10 days of chronic ethanol consumption increased whole cell calcium currents in hippocampal neurones of long sleep, but not short sleep, mice; channel gating properties were unchanged, but the subtype(s) of calcium channel involved was not determined. Calcium currents in the dentate gyrus in hippocampal slices from withdrawal seizure-

Fig. 3. Synaptic potentials in CA1 neurones in hippocampal slices prepared after chronic ethanol administration. The time scale indicates the withdrawal time, i.e., the time from cessation of ethanol consumption. (a) Fast EPSP mediated by AMPA/kainate receptors. Modified from Molleman and Little (1995a). (b) Fast IPSPs mediated by GABAA receptors. Reproduced from Whittington et al. (1995), with permission of the copyright holder, Macmillan Press Limited, Basingstoke.

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prone mice, after cessation of 2- to 5-hr ethanol perfusion, showed increases in high voltage-activated currents, thought to be of the N or P subtype of calcium channel (Perez-Velazquez et al., 1994). This change was not apparent in slices from withdrawal seizure-resistant animals. Whittington et al. (1995) demonstrated a dihydropyridine-sensitive component in fast NMDA receptor-mediated EPSPs during withdrawal from chronic ethanol administration in vivo, that was not seen in control preparations. Evidence from behavioural and other methods has suggested that withdrawal hyperexcitability and convulsions may be due to decreases in GABAA transmission, but electrophysiological studies investigating functional changes during withdrawal have not consistently supported this hypothesis. Much of the behavioural evidence is derived from studies demonstrating decreases in withdrawal tremor and convulsions with drugs that increase GABAA transmission, such as the benzodiazepines, but these drugs prevent convulsions due to many different underlying mechanisms, so demonstration of an anticonvulsant action tells us little about the neuronal changes that cause the convulsions. IPSPs recorded in hippocampal CA1 and CA3 neurones, after a schedule of chronic ethanol treatment in vivo, which produced both behavioural and electrophysiological signs of withdrawal, did not demonstrate any decreases in either GABAA- or GABAB receptor-mediated inhibition (Whittington et al., 1995; Frye & Fincher, 1996). GABAA inhibition in hippocampal CA1 cells was actually increased, rather than decreased, at 2 hr after withdrawal and was unchanged at later times, 4–7 hr after slice preparation, when hyperexcitability was observed in field potentials (Fig. 3b). This hyperexcitability was prevented by dihydropyridine calcium channel antagonists and by NMDA antagonists (Whittington & Little, 1990; Whittington et al., 1992; Ripley & Little, 1995; Bailey et al., 1998). Recordings from neurones acutely isolated from the medial septum/diagonal band area at the end of chronic ethanol administration showed no changes in any aspect of GABAA transmission (Frye et al., 1996). However, regional differences may occur, since Ibbotson et al. (1997) found reduced inhibitory transmission in cerebrocortical cells after chronic ethanol consumption. Repeated withdrawal from chronic ethanol intake results in kindling, a phenomenon in which the thresholds for convulsions are lowered, in a prolonged, possibly permanent, fashion (Ballenger & Post, 1978; Becker, 1994), and this effect may contribute to alcohol dependence. There is evidence that repeated withdrawal may have more effect on GABA transmission than a single withdrawal episode, as Kang et al. (1996) reported a prolonged decrease in GABAmediated inhibition in hippocampal slices after intermittent chronic ethanol administration. The results of Veatch and Gonzalez (1996) described above demonstrated localised changes in activity in areas of the hippocampus following repeated ethanol withdrawal. The evidence above suggests that increased excitatory amino acid transmission (NMDA and AMPA/kainate recep-

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tor-mediated), increased calcium channel conductance, and possibly decreased GABA transmission make a major contribution to the production of withdrawal convulsions. Other aspects of the alcohol withdrawal syndrome, however, may have different underlying mechanisms. 5.2. Dysphoria and anhedonia during withdrawal The dysphoria that characterises the aversive affects of alcohol withdrawal has been suggested to be due to changes in the mesolimbic dopamine system. Anhedonia is also characteristic of the acute phase of ethanol withdrawal and may also involve this dopamine system. Alterations in 5-HT transmission, although less studied with electrophysiological techniques, may also contribute to the affective components of alcohol withdrawal. Changes in firing of dopaminergic neurones in the VTA during withdrawal from chronic ethanol treatment have been recorded in vivo, but the results were not consistent and the recording conditions appear to have considerable influence. Under halothane anaesthesia, decreases in the spontaneous firing rates were found, but no differences were seen in the number of spontaneously active cells (Diana et al., 1992). Shen and Chiodo (1993), however, recording under chloral hydrate anaesthesia, found a significant decrease in the number of actively firing VTA neurones, but no decrease in firing rates, and concluded that depolarisation block had occurred. Diana et al. (1995), recording under neuromuscular blockade, found “drastic reductions” in the firing rates, but no change in the number of spontaneously firing cells; they concluded that depolarisation block was not involved in the changes. Comparison of VTA activity during ethanol withdrawal under chloral hydrate anaesthesia and under neuromuscular blockade demonstrated decreases in firing rate in both conditions, but no evidence of depolarisation block. A study using ventral tegmental slices demonstrated decreases in firing rate and changes in firing pattern in dopaminergic neurones during acute ethanol withdrawal hyperexcitability (Molleman & Little, 1995b). No changes were found in 5-HT1A/5-HT4 receptor-mediated after-hyperpolarisations in hippocampal slices prepared during chronic ethanol administration (Lau & Frye, 1996). 5.3. Withdrawal anxiety Anxiety is common both during the acute phase of withdrawal from alcohol and in alcoholics who have been abstinent for some time. Certain electrophysiological studies have shown prolonged neuronal changes that might be related to this phenomenon. Durand and Carlen (1984) found the amplitude and duration of IPSPs in hippocampal slices to be decreased 3 weeks after withdrawal from an ethanol liquid diet. Both GABA-mediated inhibition and the after-hyperpolarisation that is considered to be due to calcium-dependent potassium conductance were decreased. Later experiments from the same group (Reynolds et al., 1990) failed to show any

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change in IPSPs in hippocampal slices prepared 24 hr after withdrawal from 15 weeks of ethanol treatment. Abraham et al. (1984) found evidence of decreased excitability in hippocampal granule cells, but increased responses of CA1 cells, after 20 weeks ethanol treatment followed by 8 weeks withdrawal. The persistence of these changes indicates that they may be related to the anxiety that is seen during prolonged abstinence rather than to behavioural changes during the acute stages of withdrawal. Durand and Carlen (1984) found decreased calciumdependent potassium currents in rat hippocampal slices measured 3 weeks after ethanol withdrawal. A later study from this group (Reynolds et al., 1990) used a slightly different chronic ethanol treatment (lower doses, 3.8 g/kg/day instead of 10–15 g/kg/day, given for 15 rather than 20 weeks). In this case, the duration of the after-hyperpolarisations in hippocampal CA1 pyramidal cells, measured 16–24 hr after the last dose of ethanol treatment, was significantly increased. The corresponding potentials in dentate gyrus granule cells of the hippocampus were unchanged by the chronic treatment. No changes were found in GABAA-mediated inhibitory synaptic potentials in this study. 5.4. Craving and relapse drinking One of the major problems in the treatment of alcoholics is the propensity to relapse drinking. This is a particularly vital aspect, perhaps the most important in the understanding of alcohol dependence, since alcoholics frequently go through the acute withdrawal syndrome and remain abstinent for weeks or months, then relapse back into excess drinking. Such relapse has been reported, for example, in 74–90% (McKenna et al., 1993) and in 80% (Naranjo & Kadlec, 1991) of alcoholics during the 1–2 years following therapeutic treatment. Prolonged effects of excessive alcohol consumption, including craving, anxiety, and sleep disturbances, are seen in alcoholics after months or even years of abstinence, as described in Sections 5.1–5.3, many electrophysiological studies have investigated the acute phase of the withdrawal syndrome that occurs immediately after cessation of alcohol intake, but there has been little interest until recently in more prolonged neuronal changes that may contribute to relapse drinking. Functional changes have been reported in dopaminergic neurones at intervals after withdrawal from ethanol. In recordings in vivo from rats under local anaesthetic and neuromuscular blockade, Diana et al. (1996) reported that electrophysiological changes in VTA neurones were seen 24 hr after the end of the behavioural withdrawal signs. Decreases in neuronal firing lasted for 72 hr after cessation of the ethanol intake, while the withdrawal signs ceased at 48 hr. Studies on dopaminergic ventral tegmental neurones in midbrain slices prepared at intervals after cessation of chronic ethanol administration have shown that changes occur within the VTA. The firing rates of dopaminergic neurones in this location were still decreased both 24 hr and 6 days after etha-

nol withdrawal, when no withdrawal hyperexcitability was evident, but these returned to normal by 2 months withdrawal (Bailey & Little, 1997; Bailey et al., 1997, 1998). EEG recordings in alcoholics who had been abstinent for 5 years showed more uniform distribution of theta amplitudes than matched controls (Pollock et al., 1992). EEG differences may possibly reflect the tendency of alcoholics to relapse. Bauer (1994) demonstrated greater high-frequency EEG activity (b power, measured within 17 days of drinking cessation), compared with either abstaining alcoholics or controls, in alcoholics who relapsed within 3 months of withdrawal. Although the difference was small, it was not considered to be due to alcohol family history or personality disorder (despite the fact that both these variables have been associated with this difference; Bauer & Hesselbrock, 1993a). Prolonged changes in evoked potentials, lasting beyond the acute behavioural withdrawal phase, may indicate differences in responses of alcoholics to stimuli, for example, the P3 component, which is considered to reflect responses to novel or important stimuli. Porjesz and Begleiter (1987) found decreases in the P3 component in alcoholics that were still present after 4 months abstinence. Glenn et al. (1994) demonstrated lower amplitudes of the N1A, N2A, and P3A components, in both relapsing alcoholics and successful abstainers, that persisted for 14 months after initial abstinence. The latter changes were not attributable to genetic predisposition (see Section 6), as equivalent proportions of controls and alcoholics had close relatives with alcohol dependence. Alcoholics, after a minimum of 14 days of abstinence, showed increased N2 and P3 latencies and reduced P3 amplitude in response to an auditory task, but in a visual task, only a reduced P3 amplitude was seen compared with nonalcoholics (Kathmann et al., 1996). Elderly alcoholics were shown by Biggins et al. (1995) to have increased latency of P3A and P3B responses to novel, rare nontarget stimuli. These changes were more in evidence in the visual than in the auditory modality, and were thought to occur independently. However, not all studies have demonstrated this difference in abstinent alcoholics. Brain stem-evoked potentials in alcoholics showed differences at 1 month’s abstinence, particularly in the peak V latency and the III-V and I-V intervals, that gradually recovered, although not completely, over the next year (Cadaveira et al., 1994).

6. Genetic influences on the effects of ethanol The importance of genetic influences in the development of alcohol dependence has been established by evidence from both clinical and experimental studies. The former have demonstrated significantly higher rates of alcoholism in relatives of alcoholics, with increasing concordance with closer genetic relationship. The importance of the D2 dopamine receptor gene in dependence on drugs, and in other psychiatric problems, is still controversial (Noble, 1993), but identification of individuals at high risk of alco-

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holism, owing to a positive family history of the disorder, has enabled both mechanistic and therapeutic developments. Strains of rats and mice vary considerably in their willingness to drink alcohol, in the sensitivity to the acute effects of alcohol, and in the severity of the withdrawal syndrome. These variations have been utilised in the breeding of selected lines, for example, of rats that drink either almost entirely dilute alcohol or almost all water, when given a choice between drinking the two fluids (George & Goldberg, 1989). The development of techniques for identifying the genes responsible for these differences has led to considerable advances in this area in recent years. 6.1. Genetic differences in the acute actions of ethanol The ethanol preferring (P) line of rats showed greater slow-wave EEG activity and lower-peak theta frequency than the NP (ethanol nonpreferring) line after vehicle administration (Morzorati et al., 1988; Robledo et al., 1994). Presentation of alcohol to P and NP rats during a selfadministration paradigm resulted in an early increase in EEG power in P rats, compared with presentation of saccharin or baseline values, while the NP line showed a corresponding change later in time. The differences in theta rhythm between P and NP lines have been attributed to differences in burst firing in neurones in the medial septal area (Breen & Morzorati, 1996). However, the difference in theta activity in hippocampus of alcohol nonpreferring (NP) rats and alcohol prefering (P) rats was not seen in the high alcohol drinking (HAD) and low alcohol drinking (LAD) rat lines (Morzorati et al., 1994). Event-related potentials after alcohol administration in the two pairs of lines reflected the smaller responses of the P line to alcohol (Morzorati et al., 1991) and qualitative differences. Doses of 0.5 and 1 g/ kg ethanol decreased the cortical N1 component in NP rats, but increased the hippocampal N1 component in P rats (Ehlers & Chaplin, 1991). Some of the differential sensitivity of neurones to the effects of ethanol on GABA responses is due to genetic differences. Aguayo et al. (1994) found cultured mouse neurones were considerably more sensitive to potentiation of GABA by ethanol (effects seen with 0.5 mM and above) than cultured rat neurones (potentiation seen only with ethanol 200 mM and above). Differential sensitivity of medial septal/ diagonal band neurones from different rat strains was studied by Frye et al. (1994), who demonstrated that potentiation of GABA responses with ethanol, 3–300 mM, was seen in neurones from Fischer 344, ACI, and Wistar Kyoto strains, but not in Sprague Dawley or high alcohol sensitive (HAS) rats. Alcohol sensitivity varied within neurones from each strain and was not correlated with alcohol preference. The genetic influence on the actions of alcohol on the cerebellum was shown in the study of Spuhler et al. (1982) on the sensitivity of cerebellar Purkinje cells to alcohol in 8 inbred strains of mice, measured under urethane anaesthesia. Sensitivity of ethanol-sensitive (HAS) and -insensitive (LAS)

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rat lines to ethanol-induced loss of righting reflex was found to be reflected in greater sensitivity and slower development of acute tolerance of the HAS line to the depressant effect of ethanol on the firing rate of cerebellar Purkinje cells (Palmer et al., 1992; Pearson et al., 1997). The in vitro effects of ethanol in exciting ventral tegmental neurones and the potentiation of this effect by 5-HT did not differ between the alcohol-avoiding F334 and the alcohol-preferring Lewis rats (Brodie et al., 1995). However, the study of Minabe et al. (1995) in anaesthetised rats showed that the number of spontaneously active neurones in the ventral tegmentum and the substantia nigra pars compacta was significantly lower in Lewis than in F344 rats, while more burst firing activity was seen in the Lewis rats. Effects of alcohol on the EEG was found in early studies to be dependent on the patterns prior to ethanol consumption. Subjects with low-voltage alpha activity showed large increases in amplitude after alcohol, while those in which activity was desynchronised, with low proportion of alpha frequency or alternatively pronounced alpha activity, demonstrated little change after alcohol (Propping, 1980). The genetic influence is illustrated by the similarity of effects of alcohol on EEG in monozygotic twins, which is far greater than that in dizygotic twins; alcohol increased this similarity (Sorbel et al., 1996). 6.2. Electrophysiological differences in individuals predisposed to alcoholism EEGs in men with a positive family history of alcoholism have been reported to show more fast alpha frequency activity than matched controls with negative family history (Ehlers & Shuckit, 1991; Bauer & Hesselbrock, 1993a), not all studies have demonstrated such differences (Cohen et al., 1991). The latter authors demonstrated that alcohol consumption elicited different EEG patterns, with greater increases in slow alpha activity, in men at high risk for development of alcoholism; the difference was attributed to genetic factors (Cohen, H. L. et al., 1993b). Slow alpha activity has been reported to predict two risk factors (drinking to get “high” and age at first drink) in nonalcoholic young men (Deckel et al., 1995). This was suggested to indicate anterior brain dysfunction that might involve the prefrontal cortex; the sample included high sensation seeking and antisocial personality diagnoses. The combination of antisocial personality diagnosis and positive family history of alcoholism was associated with more fast alpha activity in right frontal lobe EEG (Bauer & Hesselbrock, 1993b). A preliminary prospective study, however, showed that in high risk men, a smaller alpha frequency EEG response to alcohol was related to the development of alcohol dependence 10 years later (Volavka et al., 1996). The EEG of sons of alcoholics has been demonstrated to be more highly “organised” than controls, an effect independent of the difference in alpha activity, but the interpretation of this is not yet clear (Ehlers et al., 1996). Asian individuals

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with the inactive aldehyde dehydrogenase (ALDH) encoded by the ALDH2*2 allele showed a lower slow wave alpha EEG response to alcohol than the ALDH2*1/2*1 genotype, who did not show the flushing response to alcohol (Wall et al., 1993). Family history of alcoholism has been demonstrated to be associated with differences in evoked potentials, particularly the P3 component (see Section 5.4), which was found to be reduced in amplitude in children of alcoholics who had no personal exposure to alcohol (Begleiter et al., 1984; Berman et al., 1993). This difference does not appear to continue into adulthood (Hill et al., 1995), although Bauer et al. (1994) reported that in young adults performing visual, but not auditory, tasks the effects of a distracter on P3 latency correlated with family history of alcoholism and with antisocial personality diagnosis. Keenan et al. (1997) found longer auditory P3 latencies, but no difference in P3 amplitude, in alcoholics, which was not related to length of sobriety or family history. An association between the reduced P3 component and the possession of the DRD2 dopamine receptor A1 allele was reported in children with positive alcoholic family history (Hill et al., 1998), but Noble et al. (1994) found an association between long P3 latency (but not amplitude) and possession of the A1 allele in boys with and without a positive family history of alcoholism. Greater P3 components of auditory-evoked responses after alcohol ingestion were found to be associated with the ALDH2*1/2*2 genotype and deficiency of aldehyde dehydrogenase, considered to reflect a reduced risk of alcohol dependence (Wall & Ehlers, 1995).

7. Conclusions Electrophysiological studies on voltage-sensitive and ligand-operated ion channels have provided a large amount of valuable information concerning the acute pharmacological effects of alcohol, especially when these have utilised molecular biology techniques to investigate the role of different receptor subunits, although, as illustrated by the divergent results on the effects of ethanol on GABAA transmission, this has not always been in agreement with information obtained using neurochemical techniques. The effects of alcohol on different brain areas have been less investigated, and there is a perceptible gap in our knowledge of the effects of alcohol on integrated neuronal activity, both within and between different brain regions. One of the most notable aspects of the acute electrophysiological studies described in this review is the frequent lack of correlation of the synaptic studies with the relative importance of the sites of action of ethanol, both to its acute behavioural actions and to the development of alcohol dependence. For the ligand-gated channels, the concentration of work has been very much on classical sites, the GABAand NMDA-activated channels, with some recent interest in 5-HT3 receptor responses, but there has been comparatively little investigation of the effects of ethanol on other ligand-

gated ion channels. Many behavioural studies have demonstrated the importance of endogenous opiates in voluntary consumption of alcohol by both rodents and humans, but only a small number of electrophysiological investigations have provided information about the functional interactions of alcohol with opiate transmission. In addition, the distribution of interest does not reflect the acute sensitivity of channels to alcohol. The work of Mancillas et al. (1986), for example, demonstrated the sensitivity of somatostatin responses to low doses of alcohol, but few studies have continued this line of investigation. The biphasic, or multiphasic, effects of alcohol on behaviour have, in a small number of electrophysiological studies, been demonstrated to be reflected in the actions of alcohol on neurones. The results obtained from these studies suggest that different patterns of action may be seen at very low concentrations of ethanol, which often have not been considered in electrophysiological experiments. The complexity of the synaptic actions of ethanol have been particularly illustrated by the work of Palmer and co-workers, and more information about such aspects would be of considerable interest. It is clear from this review that electrophysiological techniques can be usefully applied to the study of effects of chronic alcohol intake and the mechanisms that may underlie dependence on alcohol. The neuronal changes that result in the symptoms of alcohol withdrawal are now relatively well understood, and information about genetic differences in the mechanism of action of alcohol is rapidly advancing. However, the application of electrophysiological methods to the study of more subtle behavioural changes that lead to relapse drinking and loss of control of alcohol consumption in alcoholics remains a major challenge to researchers in this area. References Abraham, W. C., Rogers, C. J., & Hunter, B. E. (1984). Chronic ethanolinduced decreases in the responses of dentate granule cells to perforant path input in the rat. Exp Brain Res 54, 406–414. Aguayo, L. G., & Pancetti, F. C. (1994). Ethanol modulation of the gamma-aminobutyric acid-activated and glycine-activated Cl current in cultured mouse neurones. J Pharmacol Exp Ther 270, 61–69. Aguayo, L. G., Pancetti, F. C., Klein, R. L., & Harris, R. A. (1994). Differential effects of GABAergic ligands in mouse and rat hippocampal neurones. Brain Res 647, 97–105. Allan, A. M., & Harris, R. A. (1991). Ethanol-induced changes in chloride flux are mediated by both GABAA and GABAB receptors. Alcohol Clin Exp Res 15, 233–236. Anantharam, V., Bayley, H., Wilson, A., & Treistman, S. N. (1992). Differential effects of ethanol on electrical properties of various potassium channels expressed in oocytes. Mol Pharmacol 42, 499–505. Aston-Jones, G., Foote, S. I., & Bloom, F. E. (1982). Low doses of ethanol disrupt sensory responses of brain noradrenergic neurones. Nature 296, 857–860. Bailey, C. P., & Little, H. J. (1997). Prolonged alterations in VTA neuronal function after chronic ethanol intake. Pharmacologist 39, 379. Bailey, C. P., Andrews, N., Davis, B., McKnight, A. T., Hughes, J., & Little, H. J. (1997). Prolonged changes in monoamines and metabolites in the ventral tegmental area after chronic ethanol treatment. Br J Pharmacol 122, 337P. Bailey, C., Manley, S. J., Wonnacott, S., Molleman, A., Watson, W. P., &

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