alcoholism

Alcoholism: A Systems Approach From Molecular Physiologyto Addictive Behavior

RAINER SPANAGEL

Department of Psychopharmacology, Central Institute of Mental Health, University of Heidelberg,Mannheim, Germany

I. Introduction 650A. Alcohol use from an evolutionary and sociocultural perspective 650B. The dark side of alcohol use and abuse 650C. The pros of alcohol consumption 651D. An integrative systems approach towards alcohol addiction 652

II. Primary Targets of Alcohol 653A. Towards the identification of specific alcohol-sensitive sites on receptors and ion channels 653B. Receptor composition determines sensitivity to ethanol 655C. What are the functional consequences of the primary alcohol targets? 656D. Drug discrimination to study the psychotropic effects of ethanol 656

III. Neurochemical Systems and Signaling Pathways Involved in the Action of Alcohol 657A. The mesolimbic dopamine system and modulatory neurochemical systems: actions of alcohol 657B. Acquisition of alcohol reinforcement is mediated by mesolimbic DA neurons 659C. Are endogenous opioids and endocannabinoids involved in mediating the rewarding and

pleasurable effects induced by alcohol? 662D. Signaling pathways involved in alcohol reinforcement 663

IV. Gene Transcription and Epigenetic Effects Mediated by Alcohol 666A. Gene transcription induced by ethanol 666B. Epigenetic effects induced by ethanol 668

V. Synaptic and Cellular Effects Mediated by Alcohol 670VI. Neuronal Network Effects Induced by Alcohol 671

A. Multielectrode recording to reveal neuronal network activity underlying alcohol-related behavior 671B. Human brain imaging to identify the neuroanatomical and neurochemical substrates of

addictive behavior 672C. Animal brain imaging to identify the neuroanatomical and neurochemical substrates of addictive

behavior 675VII. Behavioral Effects Induced by Alcohol: From Controlled Drinking to Alcoholism 676

A. An animal model to study different phases of alcohol consumption 676B. An animal model to study alcohol-seeking behavior 677

VIII. Comorbidity, Genetic, and Environmental Factors That Contribute to Alcohol Use and AddictiveBehavior 678

A. Anxiety and alcohol drinking/addictive behavior 678B. Depression and alcohol drinking/addictive behavior 679C. Gene environment interactions and alcohol drinking/addictive behavior 680

IX. Treatment Aspects 682A. Preclinical medication developments for the treatment of craving and relapse 682B. Translational approach in medication development and new clinical trials 685C. Individualized pharmacotherapy for alcoholism 690

X. Summary and a Perspective of Systems-Oriented Alcohol Research 690A. A retrospective view of neurobiological alcohol research 690B. A summary of the present review 691C. A perspective of systems-oriented alcohol research 692

Spanagel R. Alcoholism: A Systems Approach From Molecular Physiology to Addictive Behavior. Physiol Rev 89:649705, 2009; doi:10.1152/physrev.00013.2008.Alcohol consumption is an integral part of daily life in manysocieties. The benefits associated with the production, sale, and use of alcoholic beverages come at an enormouscost to these societies. The World Health Organization ranks alcohol as one of the primary causes of the global

Physiol Rev 89: 649705, 2009;doi:10.1152/physrev.00013.2008.

www.prv.org 6490031-9333/09 $18.00 Copyright 2009 the American Physiological Society

burden of disease in industrialized countries. Alcohol-related diseases, especially alcoholism, are the result ofcumulative responses to alcohol exposure, the genetic make-up of an individual, and the environmental perturba-tions over time. This complex gene environment interaction, which has to be seen in a life-span perspective, leadsto a large heterogeneity among alcohol-dependent patients, in terms of both the symptom dimensions and theseverity of this disorder. Therefore, a reductionistic approach is not very practical if a better understanding of thepathological processes leading to an addictive behavior is to be achieved. Instead, a systems-oriented perspective inwhich the interactions and dynamics of all endogenous and environmental factors involved are centrally integrated,will lead to further progress in alcohol research. This review adheres to a systems biology perspective such that theinteraction of alcohol with primary and secondary targets within the brain is described in relation to the behavioralconsequences. As a result of the interaction of alcohol with these targets, alterations in gene expression and synapticplasticity take place that lead to long-lasting alteration in neuronal network activity. As a subsequent consequence,alcohol-seeking responses ensue that can finally lead via complex environmental interactions to an addictivebehavior.

I. INTRODUCTION

A. Alcohol Use From an Evolutionary andSociocultural Perspective

A conventional evolutionary perspective is that psy-choactive drug use in humans is a novel feature of ourenvironment and of cultural developments (338). How-ever, given the fact that the evolution of animals pro-ceeded in a world rich in drugs, a novel theory favors theconcept that drug and alcohol intake by mammals andother species has always been an everyday occurrence(123, 479).1 Thus occasional and even chronic intake ofalcohol through sugar-rich plant products susceptible tofermentation, such as nectar, sap, and fruit, might be abehavioral feature that has been shaped over millions ofyears from the fruit fly to numerous mammals includingprimates and humans. This current theory is best exem-plified by a very recent discovery in a primary tropicalrainforest in West Malaysia, where pentailed tree shrews(Ptilocercus lowii) consume intoxicating amounts of al-cohol on a daily basis (531). Pentailed tree shrews aremammals closely resembling modern primates early an-cestors who lived more than 50 million years ago, andtheir major daily food source is the nectar from the ber-tam palm Eugeissona tristis. This indigenous plant bearsflowers that actively produce, by means of a number ofhitherto unknown yeast species, alcohol in concentra-tions up to 3.8%, which is comparable to that of beer. Inthis million-year-old ecosystem, the pentailed tree shrewhas adapted to a daily intake of intoxicating amounts ofalcohol, most probably by means of metabolic tolerance,without suffering from any obvious negative conse-quences (531). In conclusion, this new discovery favorsthe hypothesis that from an evolutionary perspective al-cohol intake behavior has been shaped over millions of

years and should be considered as being part of ournormal behavioral repertoire, embedded today in tradi-tional and sociocultural contexts.

The great majority of Western modern society regu-larly consumes alcohol. The main reasons for the con-sumption of alcohol are that it can produce positive moodstates and has stress-relieving effects. Thus alcohol is adaily incentive and, in addition to coffee and tea, alcoholicbeverages are the most important commodities world-wide. In fact, Europeans spend 100 billion euros onalcoholic beverages annually, which is reflected by thehigh rate of alcohol consumption per capita of 10 liters ofpure ethanol per year. Luxemburg has the highest level ofconsumption worldwide at more than 13 liters per year. Incomparison, the alcohol consumption per capita in NorthAmerica in the last decade averaged 8.5 liters per year(Fig. 1).

B. The Dark Side of Alcohol Use and Abuse

Consuming and abusing these huge amounts of alco-hol clearly also has a dark side, with enormous health andsocioeconomic impacts on the world population. Thus in1020% of consumers, chronic alcohol use and abusecontributes to a multiplicity of medical complicationsincluding damage to organs and immune functions. Al-though most body organs are affected by alcohol intoxi-cation and chronic alcohol use, severe alcohol-induceddiseases are most notable in the liver, pancreas, andbrain. Alcohol-induced brain damage is a particular prob-lem during pregnancy, resulting in fetal alcohol syn-drome, which represents the most common form of ac-quired mental disability, affecting up to 7/1,000 infants(340). During adolescence, the consequences of alcoholdrinking, especially of binge drinking, on organ dysfunc-tion and damage are largely unknown despite the fact thatby 2007 binge drinking among adolescents had reached aprevalence rate of 30% in various European countries.

New research programs have been recently launched,in particular by the National Institute of Alcohol and Alco-holism (NIAAA), to gain a better understanding of binge

1 The terms alcohol and ethanol are used interchangeably through-out this review. However, the term ethanol is mostly used in the contextof a specific effect, e.g., a specific pharmacological effect.

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drinking during adolescence (www.niaaa.nih.gov). Initial re-sults clearly indicate the negative consequences of suchbehavior. Thus the young adolescent brain displays highersensitivity to alcohol-induced brain damage and cognitiveimpairment than the adult brain, in humans as well as inrodents (104, 449, 469, 529). Furthermore, the onset of alco-hol use during adolescence leads to a higher susceptibility tostress-induced alcohol consumption (444, 147) and a greaterrisk of developing alcohol addiction in adulthood (167).

Alcohol use and abuse affects all social and ethnicgroups; in almost every family in Western societies therewill be someone who has suffered, directly or indirectly,from alcohol abuse. In an estimate of the factors respon-sible for the global burden of disease, alcohol contributesto 3.2% of all deaths worldwide (530). Moreover, withregard to the world population, the percentage of the totaldisability-adjusted life years (DALYs; calculated by addingthe years of life lost due to premature mortality and theyears of life lost due to living with disability) resultingfrom chronic alcohol abuse has been estimated to be ashigh as 4% (compared to 2.2% for AIDS). Alcohol use andabuse not only entails deleterious consequences to thephysical and psychological health of the afflicted individ-uals (345), but also serious societal and economic falloutin the form of criminality, decreased productivity, andincreased healthcare costs. As a consequence, on a world-wide scale, 10% of an industrialized nations gross do-mestic product is spent in connection with alcohol useand abuse.

Alcohol abuse has a high comorbidity with otherpsychiatric disorders (238, 481). People who suffer fromanxiety disorders and depression often use alcohol as akind of self-medication (see sect. VII), but in most casesthe driving force of alcohol abuse is the development of

an addictive behavior. Addiction is defined as a syndromein which alcohol or drug use pervades all life activities ofthe user.2 Life becomes governed by the drug, and theaddicted patient can lose social compatibility (e.g., loss ofpartner and friends, loss of job, crime). Behavioral char-acteristics of this syndrome include compulsive drug use,craving, and chronic relapses that can occur even afteryears of abstinence. The diagnostic criteria for alcoholaddiction (in DSM-IV termed as alcohol dependence) ac-cording to this definition are listed in Table 1.

C. The Pros of Alcohol Consumption

Despite the enormous negative health and socioeco-nomic impact of alcohol use and abuse on the worldpopulation, light-to-moderate alcohol consumption alsohas several beneficial human health effects. These includereduced risk of coronary heart disease, type 2 diabetes,and some types of cancer (187). A substantial proportionof the benefit of moderate drinking is due to the pureethanol component of alcoholic beverages; however, dif-ferences in the beneficial effects of various alcoholic bev-erages may occur (98). In particular, red wine contains ahigh number of polyphenols, such as resveratrol that canincrease the function of the endogenous antioxidant sys-tem (27). Although research continues on resveratrol, the

2 Note that the term dependence is avoided in this review. Addic-tion is a pathological behavioral syndrome that has to be strictly sepa-rated from physical dependence. Transient neuroadaptive processesunderlie physical dependence to alcohol, whereas persistent changeswithin specific neuronal systems underlie addictive behavior. To avoidany confusion between clinicians, psychologists, and preclinicians, theterm dependence should refer to a state of physical dependence.

FIG. 1. Alcohol consumption per capita in liters of pure ethanol.

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concentration in wine seems too low to account for theso-called French Paradox, which is the observation thatthe French suffer a relatively low incidence of coronaryheart disease despite a diet relatively rich in saturatedfats. Very recently, another group of polyphenols, knownas procyanides, has been identified. Tests of 165 winesdemonstrated that the greatest concentrations are foundin European red wines from certain areas, which corre-lates with the longevity in those regions, such as south-western France (99).

D. An Integrative Systems Approach TowardsAlcohol Addiction

Taking into consideration all the pros and cons ofalcohol and drug use, it is an ongoing challenge for allcountries and governmental regulations to find a balancedway in which alcohol and other psychoactive drugs maybe embedded into our daily life. In this context, it isimportant to have a solid understanding of how alcoholacts to induce its effects and, even more importantly, tounderstand the pathological mechanisms leading to ad-diction.

Over the last 20 years, great progress has been madein alcohol pharmacology. Today we have a solid under-standing of how alcohol acts in the brain to induce itsacute behavioral effects. Despite the generally held viewthat alcohol is an unspecific pharmacological agent, re-cent molecular pharmacology studies demonstrated thatalcohol has only a few known primary targets. These arethe N-methyl-D-aspartate (NMDA), -aminobutyric acid A(GABAA), glycine, 5-hydroxytryptamine-3 (5-HT3), andneuronal nicotinic acetylcholine (nACh) receptors, aswell as L-type Ca2 channels and G protein-activatedinwardly rectifying K channels (507). Following the firsthit of alcohol on specific targets in the brain, a second

wave of indirect effects on a variety of neurotransmitter/neuropeptide systems is initiated (507), leading to thetypical acute behavioral effects of alcohol, ranging fromdisinhibition to sedation and even hypnosis, with increas-ing concentrations of alcohol.

It should be emphasized that alcohol can also exert avariety of actions and behavioral effects via its metabolicproducts. Thus acetaldehyde, which is the first productgenerated during alcohol metabolism, can affect the ac-tivity of different neurotransmitter systems and, subse-quently, can contribute to the behavioral effects of alco-hol (381). Nonoxidative alcohol metabolites, such as fattyacid ethyl esters, exert powerful effects on intracellularCa2 homeostasis (368) and therefore may also be impor-tant in mediating, at least in part, the actions of ethanol.

Multiple signaling pathways activated by alcohol andpossibly by its metabolites lead to alterations in geneexpression (114, 408). As a consequence of repeated al-cohol intake, more or less long-lasting cellular and neu-rophysiological changes that trigger alcohol-seeking be-havior become apparent in the brain reinforcement sys-tem. Whether or not this behavioral response transformsinto an addictive behavior finally depends on the geneticmake-up of an individual, as well as on numerous envi-ronmental factors (Fig. 2).

Addictive behavior is, therefore, the result of cumu-lative responses to alcohol exposure, the genetic make-upof an individual, and environmental perturbations overtime. The complex gene environment interaction leadsto a large clinical heterogeneity, in terms of both thesymptom dimensions and the severity of the disorder.Having highlighted this complex interaction, it is obviousthat a reductionistic approach has certain limitations inachieving a better understanding of the pathological pro-cesses leading to an addictive behavior. Instead, a per-spective of systems-oriented biomedicine, in which all

TABLE 1. Diagnostic guidelines: DSM-IV criteria for alcohol dependence

Criteria for Alcohol Dependence

A definite diagnosis of alcohol addiction should be made by three or more of the following seven criteria, occurring at any time in the same12-month period:

1. Tolerance2. Withdrawal3. Alcohol is often taken in larger amounts or over a longer period than was intended4. There is a persistent desire or there are unsuccessful efforts to cut down or control alcohol use5. A great deal of time is spent in activities necessary to obtain alcohol, use alcohol, or recover from its effects6. Important social, occupational, or recreational activities are given up or reduced because of alcohol use7. Alcohol use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have

been caused or exacerbated by the alcohol (e.g., continued drinking despite recognition that an ulcer was made worse by alcoholconsumption)

Diagnostic guidelines/criteria for alcohol dependence are from Diagnostic and Statistical Manual of Mental Disorders (4th ed.) (DSM-IV).Washington, DC: American Psychiatric Association, 1994. Similar diagnostic guidelines have been developed by the World Health Organization(ICD-10). Note: DSM-IV is currently undergoing revision with publication of DSM-V planned in 2011. There is an ongoing discussion whethertolerance should be further included and whether a more quantitative measure such as the frequency of engaging in a harmful drinking pattern mightnot be a more practical approach for early diagnosis and intervention (207 and several commentaries in the same issue).

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interactions and dynamics of all endogenous and environ-mental factors involved are centrally integrated (Fig. 2), issuggested to lead to further progress (5).

II. PRIMARY TARGETS OF ALCOHOL

How does alcohol affect the functions of the centralnervous system (CNS)? It is only recently that a shift fromthe so-called lipid theory (the primary targets of ethanolare membrane lipids) to the protein theory (the primarytargets of ethanol are membrane proteins, especially re-ceptors) has taken place (363). Into the 1990s, differentlipid theories postulated that alcohol acted via some per-turbation of the membrane lipids of CNS neurons. Inparticular, effects on membrane fluidity and disorderingof the bulk lipid phase of membranes were originally anattractive hypothesis of alcohol action because it pro-vided a possible mechanism by which alcohol could affectmembrane proteins, such as ion channels, via an action onmembrane lipids.

There are, however, clear limitations to this hypoth-esis. First, the effects of alcohol on membrane disorderare generally measurable only at alcohol levels well abovethe pharmacological range [500 mg/dl blood alcohollevels (BALs); these levels are close to the LD50 of ethanolin humans].3 Significant effects of membrane disorderingon protein function are even more difficult to envision at

pharmacologically relevant alcohol concentrations. Forexample, at very high intoxicating BALs associated withloss of consciousness (300 mg/dl), there would only be1 alcohol molecule per 200 lipid molecules (363). Sec-ond, membrane effects induced by alcohol concentrationsexceeding the pharmacological range can be mimicked byan increase in temperature of just a few tenths of a degreeCelsius (363), which clearly does not produce behavioralsigns of alcohol intoxication or appreciably alter the func-tion of membrane proteins such as neurotransmitter-gated ion channels. Therefore, the reported effects ofalcohol on membrane fluidity and organization seem to bea purely biophysical phenomenon with no relevance tothe pharmacological CNS effects of alcohol. Taking evenmore refinements of the lipid theory into consideration(363), it remains very unlikely that membrane lipids arethe primary targets of alcohol.

A. Towards the Identification of Specific Alcohol-Sensitive Sites on Receptors and Ion Channels

The protein theory predicts that alcohol acts specif-ically on membrane proteins such as receptors and ionchannels. The main reason for a shift towards the proteintheory originates from findings that alcohol, at concentra-tions in the 1020 mM range, directly interferes with thefunction of several ion channels and receptors.4 In a keypublication, David Lovinger et al. (283) demonstrated that

3 For historical reasons, blood alcohol concentrations are calcu-lated as g/kg blood plasma given in percent. Since the specific weight ofplasma is 1.23, a BAL of 500 mg/dl corresponds to 4.06.

4 For reference, a low intoxicating BAL of 50 mg/dl is equivalent toan ethanol concentration of 10.6 mM.

FIG. 2. This scheme shows a systems approach to-wards a better understanding of the acute and chroniceffects of alcohol. This review follows exactly this ap-proach. Thus sections II and III describe the primary andsecondary targets of alcohol including signaling trans-duction. Section IV discusses effects on gene transcrip-tion along with epigenetic effects. Synaptic and cellulareffects are summarized in section V. Section VI describesneuroimaging and anatomical work leading to an under-standing of the neuronal networks underlying the actionof alcohol. Finally, sections VII and VIII describe behav-ioral responses and their interaction with environmentaleffects such as stress. Note, although pharmacokineticsof ethanol also determine the behavioral response toacute and chronic ethanol exposure, this review does notfocus on the pharmacokinetic aspects.

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NMDA function was inhibited by ethanol in a concentra-tion-dependent manner over the range of 550 mM, arange that also produces intoxication. The amplitude ofthe NMDA-activated current was reduced 61% by 50 mMethanol. What is more, the potency for inhibition of theNMDA-activated current by several alcohols is linearlyrelated to their intoxicating potency. This suggests thatethanol-induced inhibition of responses to NMDA recep-tor activation may contribute to the neural and cognitiveimpairments associated with intoxication (283). But howcan ethanol directly interfere with NMDA receptor func-tion?

The NMDA receptor is a ligand-gated ion channelwith a heteromeric assembly of NR1, NR2 (A-D), and NR3

subunits. The NR1 subunit is crucial for channel function,the NR2 subunits contain the glutamate-binding site, andthe NR3 subunits have some modulatory function onchannel activity, especially under pathological conditions.Electrophysiological studies show that ethanol interactswith domains that influence channel activity (536), sug-gesting that residues within transmembrane (TM) do-mains may be involved. In the search for a possible bind-ing site of alcohol at the NMDA receptor, several site-directed mutagenesis studies have been performed andputative binding sites in TM3 and -4 of the NR1 and NR2Asubunits, respectively, identified (389, 390, 409, 450)(Fig. 3). In particular, a substitution of alanine for aphenylalanine residue in the TM3 of the NR1 subunit

FIG. 3. Site-directed mutagenesis reveals sites of actionof ethanol on the NMDA receptor. Exchanges on aminoacids (AA) and their consequences on ethanol inhibition ofNMDA currents are indicated. Residues in the TM3 and TM4domains of the NR1 subunit were identified that either en-hanced (green) or reduced (red) ethanol inhibition of NMDAcurrents. In particular, substitution of TM3 alanine for phe-nylalanine (F639A) strongly reduced ethanol inhibition, andthis effect was reversed by replacing TM4 glycine with tryp-tophane (G822W). (Figure kindly provided by J. J. Wood-ward and C. T. Smothers.)

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strongly reduced the ethanol sensitivity of recombinantNMDA receptors (409).

Besides the NMDA receptor, other receptors or ionchannels expressed within the CNS also have putativealcohol-binding sites. In particular, the function of GABAAreceptors is enhanced by ethanol. The GABAA receptor/chloride channel complex is a pentameric ligand-gatedion channel and the major inhibitory neurotransmitterreceptor in the mammalian brain. Several subunits havebeen identified, with the majority of GABAA receptorsbeing composed of -, -, -, and -subunits (23). With theuse of different receptor constructs, a region in the TMdomains of the / subunits of the GABAA receptor wasidentified which is involved in the action of ethanol (318),where it can potentially bind to a water-filled proteincavity between the second and third TM segments ofthese receptor subunits. In addition to its effects onGABAA receptors, ethanol also directly affects glycinereceptors. Thus there is considerable evidence to indicatethat ethanol acts on specific residues in the TM domains(318) as well as on the extracellular domain of glycinereceptors, and the net effect on receptor function is thesummation of positive and negative modulatory effects ofethanol on different ethanol-sensitive binding sites (103).Furthermore, ethanol potentiates neuronal nACh (336)and 5-HT3 receptor function (282, 289). The 5-HT3 recep-tor mediates fast synaptic transmission at postsynapticsites and regulates neurotransmitter release presynapti-cally, and its alcohol sensitivity has been consistentlyshown in different in vitro preparations (308).

Non-ligand ion channels also constitute a primarytarget of ethanol. Thus ethanol inhibits dihydropyridine-sensitive L-type Ca2 channels, and single-channel re-cordings suggest that the effects of ethanol on gating areconsistent with the interaction of a single drug moleculewith a single target site, possibly the L-channel itself(522). In addition, ethanol opens G protein-activated in-wardly rectifying K channels (GIRKs) (246, 269). Selec-tive enhancement of GIRK2 function by intoxicating con-centrations of ethanol was demonstrated for homomericand heteromeric channels, and a region of 43 amino acidsin the carboxy (COOH) terminus has been identified thatis critical for the action of ethanol on these channels (246,269).

B. Receptor Composition Determines Sensitivityto Ethanol

These primary inhibitory and facilitatory actions ofethanol on ion channels and receptors depend on a num-ber of variables, in particular the ethanol concentrationand the subunit composition of a particular channel orreceptor. For example, ethanols action on GABAA recep-tors strongly depends on the subunit composition. While

most subunit compositions of GABAA receptors displayresponses to ethanol only at high concentrations (60mM), it has been found that very low concentrations (13mM) of ethanol do alter the activity of GABAA receptorscontaining subunits. These GABA receptors are exclu-sively associated with 4/6 subunits and the 3 subunitin vivo. Moreover, in 4 subunit combinations, recep-tors containing the 3 subunit have been found to bealmost 10 times more sensitive than receptors containingthe 2 subunit, suggesting that the 3 subunit also con-stitutes an ethanol-sensitive site (519). However, mousemodels in which either the 3 subunit was geneticallydeleted or knock-in mice that carry a single point muta-tion5 in the subunit do not differ in their acute responseto ethanol when compared with wild-type animals (424).These findings suggest that extrasynaptic subunit-containing GABAA receptors (without a prominent role ofthe associated 3 subunit), but not their synaptic subunit-containing counterparts, are primary targets forethanol.

The subunit composition of glycine receptors andother receptors is also critical in the response to ethanol.Thus 1-containing glycine receptors appear to be moresensitive to low concentrations of ethanol than 2-con-taining receptors (317). Furthermore, ethanol concentra-tions lower than 100 mM are known to potentiate only24, 44, 22, and 42 subtypes of nACh receptors.In contrast, 32 and 34 subtypes are not affected bythese ethanol concentrations, while 7 receptor functionis inhibited (178). Higher ethanol concentrations are lessselective and potentiate almost all nACh receptors. As aresult of the differential distribution of the aforemen-tioned receptors as well as their subunits throughout thebrain (e.g., 5-HT3 and neuronal nACh receptors are pri-marily expressed in the cerebral cortex and some limbicregions, while the NR1/NR2B subtype of NMDA receptoris primarily expressed in forebrain regions), ethanol af-fects some brain regions more than others.

It is not yet possible directly to measure by means ofbiophysical methods the binding of an ethanol moleculeto these receptors or ion channels due to the fact thatethanol is a small molecule with low binding energy andis only efficient in the mid-millimolar range. These phar-macological characteristics preclude a direct assessmentof an ethanol protein-binding site. However, with thediscovery of the LUSH protein in the fruit fly Drosophilamelanogaster, it became possible to model how TM res-idues can form a specific protein-binding pocket for eth-anol. The high-resolution crystal structures of LUSH incomplex with a series of short-chain alcohols were ob-tained by David Joness team in 2003 (254). LUSHs struc-

5 N265M: the in vivo action of general anesthetics is stronglyattenuated by this point mutation (227).

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ture reveals a specific alcohol-binding site. LUSH exists ina partially molten globule state. The presence of ethanolat pharmacologically relevant concentrations 50 mMshifts the conformational equilibrium to a more compactstate (65), demonstrating that ethanol induces a confor-mational change of the binding protein, an importantrequirement for a functional binding site. A group ofamino acids form a network of concerted hydrogen bondsbetween the protein, and the ethanol molecules provide astructural motif to increase alcohol-binding affinity at thissite. This motif seems to be conserved in a number ofmammalian ligand-gated ion channels, and it is thereforesuggested that the alcohol-binding site in LUSH repre-sents a general model for putative alcohol-binding sites inproteins such as the NMDA or GABAA receptors.

Finally, it should be noted that alcohol is an importantodor signal in the sensory spectrum of fruit flies, and wild-type flies have an active olfactory avoidance mechanism toprevent attraction to concentrated alcohol whereas lushmutant flies are abnormally attracted to high concentrationsof ethanol, propanol, and butanol but have normal chemo-sensory responses to other odorants (244). The ability offruit flies to detect ethanol is important for chemotaxistowards food sources. However, adult flies are also sus-ceptible to intoxication and death in high ethanol envi-ronments (76), in a range similar to that observed inhumans, making them an ideal animal model for the studyof alcohol intoxication (329). In conclusion, there is aselective advantage in the ability of fruit flies to avoidenvironments with dangerously high alcohol concentra-tions, and LUSH is required for this response.

C. What Are the Functional Consequences of thePrimary Alcohol Targets?

Taken together, over the last 20 years it has beendemonstrated that ethanol acts directly on membranereceptors and ion channels. This favors the protein the-ory, and the current view commonly held is that ethanolhas only a few known primary targets that include NMDA,GABAA, 5-HT3, and nACh receptors, as well as L-typeCa2 channels and GIRKs, where concentrations as lowas 1 mM produce alterations in the function of thesereceptors and ion channels.

Although more structural information about the pu-tative alcohol-binding sites on proteins such as the NMDAreceptor continues to be acquired, the functional impactof these binding sites is still to be discovered. Advanceswill only be achieved by novel knock-in models such asthose already described for the GABAA receptor (227), inwhich the wild-type receptor subunits are replaced bythose containing alcohol-insensitive or -hypersensitivesites. In the meantime, we have to be content with the useof either knockout mice or specific pharmacological in-

terventions in combination with an appropriate behav-ioral test for acute alcohol intoxication. A commonly usedprocedure is the loss of righting reflex (LORR), a behav-ioral test that probes the relevance of a particular recep-tor in alcohol intoxication. In this test, either a rat ormouse is injected with a high dose of ethanol (34 g/kgintraperitoneally) and upon becoming ataxic is consid-ered to have lost the righting reflex. The animal is thenplaced on its back and LORR duration is calculated as thetime that elapses until the animal is able to right itself.Although the LORR provides a reliable measure of CNSsensitivity in response to alcohol, it can be only used fora behavioral readout of the effects of hypnotic alcoholconcentrations of at least 50 mM, which corresponds toBALs above 250 mg/dl. However, as stated above, most ofthe putative membrane protein-binding sites for alcoholare sensitive to much lower concentrations of ethanol;thus how is it possible to investigate whether alcoholbinding to these targets has any psychotropic effects?

D. Drug Discrimination to Study the PsychotropicEffects of Ethanol

Drug discrimination studies with ethanol as a train-ing drug provide a valuable tool to study the psychotropiceffects during alcohol exposure. Drug discriminationstudies can be conducted in humans as well as in labora-tory animals and have been used for more than 30 years tounderstand whether a specific binding site on a protein ismediating an ethanol-like interoceptive stimulus; the numer-ous studies are well archived under www.dd-database.org.During a discrimination test the experimentor asks: Do youfeel like having alcohol? In fact, the discriminative ethanolstimulus very much corresponds to the subjective effectsexperienced by social drinkers and can already be de-tected by BALs of 30 mg/dl (214).

As shown in Figure 4, animals can be trained in anoperant task to discriminate ethanol from saline and, sub-sequently, in a so-called substitution/generalization test, aspecific pharmacological agent (e.g., an NMDA receptorblocker such as memantine or ketamine) is applied to testwhether this compound produces an ethanol-like stimulus.It is important in animal drug discrimination studies thatself-administered ethanol can substitute for investigator-ad-ministered ethanol, as this demonstrates that the psycho-tropic effects of self-administered ethanol are similar tothose produced by investigator-administered ethanol (288).Moreover, healthy social drinkers undergoing a computer-assisted intravenous alcohol self-infusion paradigm experi-enced a similar alcohol effect as with drinking (549), sug-gesting that irrespective of the route of administration sim-ilar psychotropic effects of alcohol are achieved.

Substitution studies have shown that a complete sub-stitution for ethanol is exerted by NMDA receptor antag-

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onists and certain GABA-mimetic drugs acting throughdifferent sites within the GABAA receptor complex (193,251). Thus it has been consistently shown in mice, rats,and monkeys that noncompetitive antagonists at the

NMDA receptor, such as dizocilpine (MK-801), phencyc-lidine (PCP), ketamine, or memantine, which all act as anion channel blocker, generalize to the ethanol cue whilecompetitive NMDA antagonists have often shown onlypartial substitution for ethanol (92, 166, 198, 209, 443).Moreover, it has been demonstrated that ketamine pro-duced dose-related ethanol-like subjective effects in de-toxified alcoholics (255), suggesting that, at least in part,NMDA receptors mediate the subjective effects of ethanolin humans. Furthermore, the ethanol stimulus effect maybe increased (i.e., stronger recognition) by drugs acting atnicotinic cholinergic receptors and 5-HT3 receptor ago-nists (251). Finally, depending on the training dose ofethanol, different receptors are involved in mediating thediscriminative stimulus properties of the drug (165).

In conclusion, the ethanol stimulus is composed ofseveral components, with the NMDA receptor and GABAAreceptor complex being of particular importance. Thisdemonstrates that the primary sites of alcohols action donot simply induce intoxication but also mediate subjec-tive effects. Therefore, an understanding of the receptormechanisms that mediate the discriminative stimulus ef-fects of alcohol can be used to develop medications aimedat decreasing the subjective effects induced by alcohol.

III. NEUROCHEMICAL SYSTEMS ANDSIGNALING PATHWAYS INVOLVEDIN THE ACTION OF ALCOHOL

The first hit of alcohol on specific targets in the brainleads to the typical acute subjective effects comprising thediscriminative stimulus properties of this drug, and associ-ated with these psychotropic effects, the intoxication signalranging from disinhibition to sedation and even hypnosisoccurs with increasing concentrations of alcohol. Followingthis first hit of alcohol, a second wave of indirect effects ona variety of neurotransmitter/neuropeptide systems is initi-ated (507); it is believed that this second wave, which mainlyinvolves monoamines, opioids, and endocannabinoids, iscrucial for the initiation of alcohol reinforcement and re-ward.

A. The Mesolimbic Dopamine System andModulatory Neurochemical Systems:Actions of Alcohol

The brain regions that play an important role in me-diating the reinforcing effects of drugs of abuse, includingalcohol, have been identified by a variety of neurophar-macological studies that include lesion, microinjection,and microdialysis experiments. However, the ground-breaking work was performed in 1954 by Olds and Millner(347). Their electrical brain stimulation experimentsmade it apparent that the brain must have some special-

FIG. 4. Drug/ethanol discrimination is widely recognized as one ofthe major methods for studying the psychotropic effects of drugs. Indrug discrimination studies, effects of drugs serve as discriminativestimuli that indicate how reinforcers (e.g., food pellets) can be obtained.For example, animals can be trained to press one of two levers to obtainfood after receiving an ethanol injection (here the red active lever is onthe right side, pressing the white lever has no consequences; 1.0 g/kg ipas training dose), and to press the other lever to obtain food afterinjection of vehicle (saline; here the red active lever is on the left side,pressing the white lever has no consequences). Once the discriminationhas been learned, the animal will press the appropriate lever accordingto whether it has received ethanol or saline; accuracy in most experi-ments is very good (90% or more correct). Trained subjects can then beused 1) to determine an ethanol dose-response curve (left bottom panel;note: a dose of 0.5 g/kg already produces 60% response accuracy, mean-ing that some animals already recognize the ethanol stimulus) and 2) todetermine whether a test substance (e.g., an NMDA receptor antagonistsuch as memantine or ketamine; right bottom panel) is identified asbeing like or unlike the ethanol training dose. This is the so-calledgeneralization, or substitution, test (476).

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ized brain sites for reinforcement and reward functions.In these experiments brain sites were identified whereelectrical stimulation was rewarding in the sense that arat will stimulate itself in these places frequently andregularly for long periods of time if permitted to do so (foran illustration of this technique, see Ref. 425). Drugs ofabuse lead to an increase in sensitivity of the animal to theelectrical stimulation. However, only oral self-administra-tion of ethanol and not experimenter-administered etha-nol facilitates rewarding electrical brain stimulation(328). The midbrain dopamine (DA) system, in particular,is sensitive to electrical self-stimulation and has beencharacterized as a neurochemical substrate of reinforce-ment (433, 533, 534). Midbrain A10 DA neurons involvedin the initiation of reinforcement processes originate inthe ventral tegmental area (VTA) and project to structuresclosely associated with the limbic system, most promi-nently the nucleus accumbens (NAC) shell region as wellas the prefrontal cortex (PFC). Activation of the midbrainDA system by all kinds of reinforcers has been demon-strated in animals and humans. For example, by means ofneuroimaging methods in humans (see sect. VIB), it hasbeen shown that social attractiveness (230), sex and or-gasm (155, 202), even classical music (but only in musi-cians; Ref. 51) can induce enhanced activity in the NAC.Also, a variety of drugs abused by humans, includingalcohol, leads to enhanced mesolimbic DAergic activity,preferentially in the NAC shell region (115, 213, 379). Inthe following text, animal studies are described that ex-amine the relationship between alcohol and midbrain DA.

Various techniques have indicated that the mesolim-bic DAergic system is activated when alcohol is adminis-tered to laboratory animals. The VTA, in particular, hasbeen implicated in the effects of alcohol. Thus, followingthe key publication by Gessa et al. (157), which showedthat low systemic doses of ethanol produce a dose-depen-dent increase in the firing rate of DAergic neurons, later itwas consistently shown that alcohol stimulates DA trans-mission in the mesolimbic pathway (115). With the use ofmicrodialysis, it was found that acute administration ofalcohol results in preferential release of DA from the NACshell region (379). It is suggested that the manner bywhich acute alcohol administration increases extracellu-lar DA within the NAC is via changes in GABAergic feed-back into the VTA. Alcohol may decrease the activity ofthese GABAergic neurons, which subsequently leads to adisinhibition of mesolimbic DA neurons (467). This sug-gested mode of action is supported by the observationthat DA levels within the NAC remained elevated aftersystemic alcohol administration, whereas somatoden-dritic release in the VTA had already declined, implyingthat alcohol also has local effects in the NAC (247). Sincelocal infusion of a DA-reuptake inhibitor through the di-alysis probe into the NAC elevated DA levels therein and,in parallel, decreased DA levels in the VTA (247), it is

suggested that elevating DA levels in the NAC activates along-loop negative GABAergic feedback system to theVTA, which regulates DA cell body neuronal activity (228,247, 286, 467). In recent studies it has finally been dem-onstrated that the NAC is the primary hot spot for the DAreleasing properties of ethanol but that a secondary effectoccurs in the VTA as well (136, 278) (Fig. 5).

However, DAergic activity is regulated not only via along-loop negative GABAergic feedback system andGABAergic interneurons within the VTA but also by a vari-ety of other systems. Glutamatergic activity in particularalso seems to control the mesolimbic DAergic pathway(148, 286). Glutamatergic projections from the PFC, bednucleus of the stria terminalis, laterodorsal tegmentalnucleus, and lateral hypothalamus feed into the VTA(350). In addition, glutamatergic projections from thePFC, hippocampus, amygdala, and paraventricular nu-cleus feed into the NAC, and glutamate release from anyone of these projection terminals can act on ionotropicglutamate receptors in the NAC shell to induce DA release(44, 205, 361). In addition, glutamatergic neurons withinthe VTA have recently been identified (537), which mightalso influence DAergic activity via different glutamatereceptors. Microdialysis studies have revealed biphasiceffects of ethanol on glutamate release within the NAC.Thus, at low doses, ethanol may elevate extracellularglutamate levels in the NAC, whereas at higher doses itreduces glutamate overflow (148, 324). Whether this ef-fect of alcohol on glutamatergic transmission within themesolimbic DA system is of relevance for the activity ofDA A10 neurons is less clear. For instance, infusion of anNMDA receptor antagonist into the VTA did not affect the

FIG. 5. Similar to all other drugs of abuse, ethanol stimulatesdopamine (DA) release preferentially in the nucleus accumbens (NAC)shell region, and it is suggested that this neurochemical event is involvedin the initiation of alcohol reinforcement. Although multiple neurotrans-mitter and neuropeptide systems are involved in the initiation of thisneurochemical event, the disinhibition of GABAergic neurons appears tobe one major contributory mechanism. [Modified from Spanagel andWeiss (467).]

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DA-enhancing effects of ethanol (135). This is surprisingin light of the fact that several other drugs of abuse act viaglutamatergic input on the activity of midbrain DA neu-rons (148, 234) and, as such, clearly requires further re-search.

The dorsal raphe nucleus 5-HT system also modu-lates the DAergic activity of the VTA and the NAC (333).This 5-HT effect is mainly mediated via the 5-HT3 receptor(284). Blockade of 5-HT3 receptors therefore selectivelyprevents both ethanol-induced DA release in the NAC (71)and the somatodentritic release of DA in the VTA (69),whereas activation of 5-HT3 receptors increases DA re-lease within the VTA of Wistar and alcohol-preferring Prats (275). 5-HT3 receptor-mediated effects on DA releasemay be due to a mixed primary action of ethanol on thisreceptor and a secondary effect of ethanol-induced sero-tonin release.

Neuronal nACh receptors are also a primary target ofethanol and are known to modulate the release of DA. ThenACh receptor antagonist mecamylamine given systemi-cally blocks the DA-releasing properties of ethanol (49).Furthermore, blockade of nAch receptors within the VTAalso inhibits the stimulating effects of conditioned etha-nol cues on DA neurons (279). This suggests that the nAchreceptor-mediated acetylcholine/DA interaction may rep-resent an important neurochemical access point of con-ditioned alcohol reinforcement. Moreover, this neuro-chemical interaction points to the synergistic effects ofalcohol and nicotine in terms of reinforcement processesand provides a neurochemical correlate for the fact thatalcohol drinking is strongly associated with smoking(272).

There also seems to be an interesting link betweenthe acetylcholine/DA interaction and neuropeptides in-volved in feeding behavior such as ghrelin. Centrally ad-ministered ghrelin has DA-stimulating properties (218,219) which appear to be mediated via central nAch recep-tors, suggesting that ghrelin activates cholinergic inputinto DA neurons. There is cholinergic input from thelaterodorsal tegmental area to the VTA, and growth hor-mone secretagogue receptors (GHSR-1A), the functionalghrelin receptor, are expressed in both areas (219). It hasbeen demonstrated that local administration of ghrelininto the VTA or the laterodorsal tegmental area enhancesDA release in the NAC (219), suggesting that ghrelin maystimulate the mesolimbic DAergic system via activation ofGHSR-1A in the VTA and laterodorsal tegmental area.Although a direct link between ethanol, ghrelin, and DAhas not yet been investigated, it is known that ghrelinregulates not only energy balance and feeding behaviorbut is also likely to be directly involved in drug (105, 487)and alcohol reinforcement (428). It is currently unknownwhether other neuropeptides involved in feeding behavioralso modulate the action of ethanol on DAergic neurons.Such neuropeptides may include orexin A and B, which

are synthesized exclusively in neurons of the lateral hy-pothalamus (417) and are activated in response to naturaland drug reinforcers (176) including alcohol (262, 428). Inaddition, stimuli conditioned to alcohol availability alsoactivate hypothalamic orexin neurons (110). Since thereis a lateral hypothalamic orexin projection to both theVTA (139) and the NAC (21), it is probable that ethanolhas an access point to the mesolimbic reinforcementsystem via these neuropeptides.

Finally, glycine receptors also modulate the DArelease properties of A10 neurons since they are aprimary target of ethanol. Thus reversed microdialysisof the competitive glycine receptor antagonist strych-nine into the NAC decreases accumbal extracellular DAlevels, whereas reversed microdialysis of the agonistglycine increases DA levels in the NAC (326). Further-more, local perfusion of strychnine not only decreasesaccumbal DA levels per se, but also completely preventsan increase in accumbal DA levels following administra-tion of ethanol (327).

In summary, systemic alcohol has multiple actionsaffecting the NAC, the VTA, and their afferents, i.e., thereare multiple neurochemical points of access to DAergicA10 neurons. Most of these neurochemical access pointsrepresent primary targets of alcohol. Note that the activityof A10 neurons is also modulated by endocannabinoidsand endogenous opioid systems (these modulatory mech-anisms will be discussed in section IIIC). However, themost important questions remain unanswered: 1) Whatare the behavioral consequences of the activation andmodulation of DAergic A10 neurons by alcohol, and 2) arealcohol reinforcement and reward and conditioned re-sponses closely linked to DAergic activity?

B. Acquisition of Alcohol Reinforcement IsMediated by Mesolimbic DA Neurons

Alcohol-induced activation of mesolimbic A10 neu-rons appears to be associated with the reinforcing prop-erties of alcohol, since rats will directly self-administeralcohol into the VTA (149). In a more detailed study, Roddet al. (402) demonstrated that rats will self-administerethanol directly into the posterior but not into the anteriorVTA. Coadministration of the DA D2/3 agonist quinpiroleinto the VTA at a concentration that activates DA D2autoreceptors and thereby reduces the firing rates of VTADA neurons was shown to prevent the acquisition ofself-administration behavior into the posterior VTA. Thiseffect was restored by the withdrawal of quinpirole or theinfusion of the DA D2 antagonist sulpiride into the VTA(402). The results of this study indicate that alcohol isreinforcing within the posterior VTA and suggest thatactivation of VTA DA neurons is involved in this process(402).

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Numerous pharmacological studies have further in-vestigated the role of midbrain DA in alcohol reinforce-ment, but the results have been conflicting. Although6-hydroxy-DA-induced lesions do not affect the mainte-nance of alcohol self-administration (212, 241, 287, 386),they substantially reduce the acquisition of alcohol drink-ing (212, 386). These findings indicate that the acquisitionand maintenance of primary alcohol reinforcement maybe mediated by different neuronal mechanisms and thatfunctional midbrain DA neurons are not necessarily re-quired to maintain alcohol self-administration. However,postsynaptic changes in DA receptor signaling appear tobe involved in the maintenance of voluntary alcohol in-take since DA D1 and D2 receptor knockout mice displayaltered alcohol consumption (102). In particular, operantalcohol self-administration behavior is markedly reducedin DA D2 receptor-deficient mice (373, 396). Quantitativetrait locus (QTL) analysis using recombinant inbredmouse strains localized a QTL for alcohol preference atthe location of the DA D2 receptor on mouse chromsome9 (484). Furthermore, D1, D2, and D3 receptor agonistsand antagonists are capable of modulating ethanol con-sumption in common stock rats (91, 369, 412) as well as inalcohol-preferring rats (125, 307, 489).6

DA measurements in different alcohol-preferring ratstrains have also produced conflicting results. Alcoholself-administration has been shown to produce a consid-erably greater relative stimulation of mesolimbic DA re-lease in alcohol-preferring P-rats than in control Wistarrats (31, 231, 524). In contrast to these findings, a similardose-dependent increase in mesolimbic DA release inFinish alcohol-preferring AA rats and corresponding alco-hol-avoiding ANA rats (454) has been reported by Kiian-maa et al. (242). Furthermore, in a well-designed experi-ment by the same authors (343), a group of AA rats drank10% ethanol voluntarily in a limited access paradigmwhile a yoked group of AA rats and a yoked group of ANArats received the same amount of ethanol intragastricallyby intubation. Subsequently, the different animal groupsunderwent in vivo microdialysis. Then, DA release wasmonitored in the NAC after intraperitoneal challenge of 1g/kg ethanol. The AA and the ANA rats that receivedethanol noncontingently exhibited the same DAergic re-sponse to the ethanol challenge as naive animals in theprevious experiment (242). The group of AA rats that hadingested the ethanol voluntarily even showed a signifi-cantly smaller increase in DA after the ethanol challenge

(343). The latter result implies that tolerance develops tothe DA releasing effect of ethanol in voluntarily drinkingAA rats. This suggestion is further supported by yet an-other experiment in which DA release in the NAC wasmeasured before and during alcohol drinking in AA rats.Self-administration of the ethanol solution had only aminor effect on DA levels during the first 10 min after theonset of drinking (344). Giving the rats a cue for ethanol,which was part of their daily, routine drinking regime, didnot raise DA levels before ethanol was presented to therats (i.e., during anticipation) (344). Together, this con-sistent set of findings shows that mesolimbic DA is not thecentral substrate that produces the reinforcement fromethanol in AA rats.

Similar findings were obtained in a further line ofalcohol-preferring rats. In alcohol-naive, high alcohol-drinking (HAD) and low-alcohol-drinking (LAD) lines ofrats, alcohol dose-response curves for DA release exhib-ited no difference in the sensitivity to alcohol between thelines (354, 543). In a further comparative study, alcohol-naive HAD/LAD and AA/ANA rats were examined for theirbasal and ethanol-stimulated release of DA in the NAC bymeans of no-net-flux quantitative microdialysis. Aftercompletion of the neurochemical tests, the rats voluntaryalcohol intake and preference in the home cage weretested for 1 mo (233). Analysis of the data across individ-ual animals and different lines revealed that extracellularDA and the percent of baseline increase in DA due toethanol were significant predictors of ethanol preference(233).

With regard to the apparent lack of congruity amongthe aforementioned studies of DA release, the fact thatmost of these experiments were done with experimenter-administered alcohol must be taken into consideration, asthis may explain why no differences are observed be-tween the preferring and nonpreferring AA/ANA andHAD/LAD lines. Further studies are clearly warranted inrat lines where DA measurements are performed at ahigh-time resolution during operant self-administration.However, since the nonpreferring lines hardly respond toethanol, appropriate experimental controls are lacking.The comparative study by Katner and Weiss (233), how-ever, suggests that elevated extracellular levels of DAwithin the NAC and a greater responsiveness to enhance-ments in DA release by ethanol may be factors that con-tribute to high-alcohol preference. Furthermore, the datasuggest that alcohol may be more reinforcing in animalsthat exhibit an enhanced DAergic response to initial eth-anol exposure and, as such, may subsequently be associ-ated with the acquisition of higher ethanol intake andpreference.

The role of DA in mediating alcohol reinforcementhas also been studied in the human brain. In an initialreport by Ahlenius et al. (4), it was shown that -methyl-p-tyrosine, a compound that blocks DA synthesis, de-

6 Various alcohol-preferring and nonpreferring rat lines have beendeveloped within the last 50 yr. Depending on the line, preferring ratsconsume 59 g kg1 day1 ethanol, whereas the nonpreferring linesconsume less than 1 g kg1 day1. These lines are very powerful animalmodels in the study of the neurochemical substrates of alcohol rein-forcement. A comprehensive overview of the different lines has recentlybeen reported (31, 83, 93, 382, 354, 454).

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creases ethanol-induced psychostimulation in humans.Using positron emission tomography (PET) measure-ments, Boileau et al. (60) demonstrated a significant re-duction in [11C]raclopride binding in the NAC in healthyvolunteers after alcohol ingestion. In this study the mag-nitude of the change in [11C]raclopride binding correlatedwith the psychostimulant effects of alcohol. This indi-cates that enhanced DA release occurs in response toalcohol drinking and that the degree of psychostimulationis mediated, at least in part, by augmented extracellularDA levels.

Given that DA plays a crucial role in the acquisitionof alcohol reinforcement in animals and humans, it maybe postulated that neurochemical points of access di-rectly modulating DAergic activity (e.g., GABA, gluta-mate, serotonin, acetylcholine, glycine) must also play acrucial role in the acquisition of alcohol reinforcement.

GABAA receptors also play an important role in al-cohol reinforcement, being both a primary target for al-cohol and a direct neurochemical access point into themesolimbic DAergic system. For instance, pharmacologi-cal manipulations of GABAA receptors with negative al-losteric modulators were shown to reduce alcohol con-sumption in several alcohol-preferring rat lines (386, 523).In addition, antagonism of GABAA receptors within theVTA (342) or an increase in the activity of GABAA recep-tors in NAC (225) suppressed alcohol consumption inalcohol-preferring P-rats, suggesting the particular impor-tance of GABAA receptors in both nuclei in alcohol rein-forcement. Also, knockout mice lacking various GABAAreceptor subunits were examined in several alcohol-re-lated paradigms, and it was shown that 1, 2, 5, and subunit deletion leads to reduced alcohol consumption(53, 102, 226, 316). Furthermore, Sardinian alcohol non-preferring rats, selected for their low alcohol preferenceand consumption (93), as well as ANA rats, carry a pointmutation (R100Q) in the gene coding for the GABAAreceptor 6 subunit, suggesting that the lack or malfunc-tion of this subunit also contributes to reduced alcoholintake (74, 416).

The results of pharmacological studies using gluta-mate receptor antagonists in alcohol self-administrationparadigms are less conclusive. Different NMDA receptorantagonists applied either systemically or locally into theNAC may reduce or have no effect on alcohol intake (40,385, 443). The application of the AMPA/kainate receptorantagonist GYKI 52468 did not selectively alter operantresponse to alcohol (472). Neither did experiments withknockout mice suggest the involvement of AMPA recep-tors in the maintenance of alcohol drinking, as GluR1 andGluR3 deletions had no effect on either home-cage alco-hol drinking or operant self-administration (101, 423).These more or less negative behavioral results do reflectthe observations made at the neurochemical level. Thus,as previously mentioned, a clear modulatory role of glu-

tamatergic input on DAergic A10 neuronal activity has sofar not been established.

The dorsal raphe nucleus 5-HT system modulates theDAergic activity of the VTA and the NAC (333). This 5-HTeffect is mainly mediated via the 5-HT3 receptor (284).Blockade of 5-HT3 receptors, therefore, selectively pre-vents both ethanol-induced DA release in the NAC (71)and the somatodentritic release of DA in the VTA (69).5-HT3 receptor-mediated effects on DA release may bedue to a mixed primary action of ethanol on this receptorand a secondary effect of ethanol-induced serotonin re-lease.

Knockout mouse models and pharmacological ma-nipulations of various components of the 5-HT systemhave indicated a modulatory role for 5-HT in voluntaryalcohol consumption. Deletion of 5-HT transporters (235)or overexpression of 5-HT3 receptors (132) leads to areduction in alcohol self-administration compared withthat observed in control mice. Pharmacological manipu-lations of 5-HT system activity revealed that administra-tion of a variety of serotonergic compounds were capableof reducing alcohol consumption in common stock aswell as alcohol-preferring animals (263, 354, 545). 5-HT3receptor antagonists were shown to suppress the acqui-sition of voluntary alcohol consumption in alcohol-prefer-ring P-rats. Furthermore, the reinforcing effects of etha-nol within the posterior VTA of rats require activation oflocal 5-HT3 receptors (403); a pattern therefore evolveslinking the action of 5-HT3 receptors on DAergic neuronswithin the VTA with alcohol reinforcement.

It has been shown that alcohol-induced stimulationof DAergic A10 neurons also involves central nACh andstrychnine-sensitive glycine receptors, suggesting a pos-sible involvement of these receptors in alcohol reinforce-ment. Infusion of mecamylamine into the VTA reducesvoluntary alcohol consumption (134); however, it remainsto be established which particular nACh receptor subunitcomposition is most important in this respect. It is knownthat 42 and 7 subtypes of nACh receptors do not playan important role in alcohol consumption (135, 265),whereas antagonism of 32 and 3 subunits of the nAChreceptors has been shown to reduce voluntary alcoholconsumption in both rats and mice (218, 260). Modulationof the activity of the glycinergic system also leads toreduced voluntary alcohol consumption. Molander et al.(235) have recently shown that the glycine reuptake in-hibitor Org 25935, acting specifically on the glycine trans-porter 1, decreases alcohol preference and intake in ratsby increasing extracellular glycine levels, which primarilyactivate inhibitory strychnine-sensitive glycine receptors.The picture that emerges once more highlights the impor-tance of cholinergic and glycinergic input onto DAergicneurons in alcohol reinforcement.

In summary, animal research has demonstrated thatmidbrain DA A10 neurons and several modulatory neuro-

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chemical access points, including GABAA, 5-HT3, nACh,and glycine receptors, play an essential role in the acqui-sition of primary alcohol reinforcement processes. Thusmesolimbic DA activation is a property of ethanol andmay possibly mediate its reinforcing effects. However, itmust be emphasized that primary reinforcement pro-cesses do not necessarily reflect the emotional hedoniccomponents of ethanol reward; it seems more probablethat an enhanced DA signal highlights important stimuliand functions as a neurochemical learning signal for re-inforcing stimuli (433, 467). Whether DA also plays a rolein mediating hedonic aspects of alcohol intake is notknown. However, the endocannabinoid and endogenousopioid systems may well serve as neurochemical sub-strates involved in the mediation of these positive moodstates.

C. Are Endogenous Opioids and EndocannabinoidsInvolved in Mediating the Rewarding andPleasurable Effects Induced by Alcohol?

Accumulating evidence indicates a central role forthe endocannabinoid system in the regulation of the re-warding properties of drugs of abuse including alcohol(291). This system participates in drug reward through therelease of endocannabinoids in the VTA. However, endo-cannabinoids are also involved in the motivation to seekdrugs via DA-independent mechanisms (291), and an en-docannabinoid hypothesis of drug reward has been pos-tulated as an alternative to the DA hypothesis of drugreward. Endocannabinoids mediate retrograde signalingin neuronal tissues by the presynaptic cannabinoid (CB)receptors and are thus involved in the regulation of syn-aptic transmission by suppressing classical transmitteraction. This powerful modulatory action on synaptictransmission has significant functional implications andinteracts with the effects of drugs of abuse includingalcohol. The endocannabinoid system includes CB1, CB2,and the orphan receptor GPR55 as a new CB receptor(261), endocannabinoids, e.g., 2-arachidonyl-glycerol (2-AG) and anandamide, their biosynthetic and inactivatingenzymes and, perhaps, transporters for endocannabinoids(146).

Alcohol reinforcement processes are dependent onCB1 receptor activity. Thus CB1 receptors in alcohol-avoiding DBA/2 mice exhibit a lower efficacy than CB1receptors in alcohol-preferring C57BL/6 mice (210). Ge-netically selected Marchigian Sardinian alcohol-preferring(msP) rats or AA rat lines exhibit specific differences inthe organization of the brain endocannabinoid system in anumber of brain regions when compared with unselectedWistars or alcohol-avoiding ANA rats (86, 171), and CB1receptor antagonism has been reported specifically tosuppress acquisition of alcohol-drinking behavior in ro-

dents (96). In general, pharmacological manipulation ofthe CB1 receptor influences ethanol intake and prefer-ence (15, 94, 158). Similarly, CB1 receptor knockout micedisplay reduced alcohol self-administration (488, 521).The study of Wang et al. (521) further demonstrated thatthere is an age-dependent decline in ethanol preferenceand intake in wild-type but not in CB1 knockout mice,which is consistent with reward-dependent mechanismsbecoming less important with age and that a decrease ofactivity within the endocannabinoid system might corre-late with these events. A direct link between alcoholreinforcement and alterations in brain endocannabinoidformation has recently been established. Alcohol self-administration was shown significantly to increase micro-dialysate 2-AG levels within the NAC, and the relativechange in dialysate 2-AG content was significantly corre-lated with the quantity of alcohol consumed (67).

In summary, the endocannabinoid system is involvedin DA-dependent reinforcement processes, but it also elic-its DA-independent effects on reward. Whether these ef-fects are associated with a pleasurable hedonic state in-duced by alcohol is not as yet known. CB1 receptorstimulation in humans can produce euphoric effects.However, it is of key importance to test whether admin-istration of a selective CB1 receptor antagonist in volun-teers, drinking small but stimulatory amounts of alcohol,will blunt the euphoric stimulatory effects of alcohol.

Such an alcohol challenge experiment has been con-ducted in social drinkers using naltrexone, an opioid re-ceptor antagonist, to test whether the endogenous opioidsystem mediates subjective euphoric effects (120). Usinga double-blind design, subjects received naltrexone orplacebo and 1 h later consumed a beverage containingethanol (0.5 g/kg). Breath alcohol levels were measuredover 3 h after the beverage was consumed, and subjectscompleted standardized subjective effects questionnairesat regular intervals. Ethanol under placebo producedits prototypic effects, including positive subjective re-sponses such as euphoria and increased ratings of overallliking. Surprisingly, pretreatment with naltrexone did notalter the positive subjective or any other effects of ethanol(120). The same experiment was repeated in light drink-ers and moderate drinkers with the same outcome: nal-trexone pretreatment had no dampening effect on thesubjective response to ethanol (121). The situation is,however, quite different in heavy-drinking subjects; it hasbeen repeatedly shown that naltrexone decreases subjec-tive (e.g., liking) and psychomotor responses to alcohol inheavy drinkers (122, 309, 388).

It has long been suspected that endogenous opioidpeptides, such as endorphins and enkephalins, are theneurochemical substrates of reward processes and areimportant for mediating the associated euphoric effects.Early studies showed that both enkephalins and endor-phins possess intrinsic rewarding properties and are self-

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administered by rodents directly into the brain ventricles(33, 505) and the NAC (162). The VTA is a further hot spotfor opioids to induce reward, since opioid receptor ago-nists produce conditioned place preference when admin-istered into this brain site (189) and are also self-admin-istered into the VTA (111). Thus / opioid receptors, thetargets of enkephalins and endorphins, in the VTA andNAC appear to be critically involved in the neurobiologi-cal mechanisms underlying reward (458). It has furtherbeen demonstrated that basal DA levels within the NACare modulated by endogenous opioid systems (459). Formany years, however, it was unclear whether drugs ofabuse do, in fact, trigger reward-related processes viarelease of endorphins and enkephalins. In a key publica-tion by Olive et al. (348), it was finally demonstrated by invivo microdialysis that drugs of abuse, including ethanol,release endorphin into the NAC. Importantly, concomi-tant measurement of DA levels demonstrated that afteradministration of alcohol, the increase in extracellularlevels of DA appeared to occur at an earlier time pointthan in the case of endorphin. This suggests that alcoholstimulates DA and endorphin in the NAC, but probablydoes so via independent mechanisms (299). Given thefindings of studies showing the positive reinforcing prop-erties of / agonists when injected into this brain region(162, 504), it is hypothesized that this increase in extra-cellular endorphin levels may play a role in the rewardingproperties of ethanol and other drugs of abuse. The NACreceives endorphinergic input from pro-opiomelanocortin(POMC)-containing neurons in the arcuate nucleus of thehypothalamus (52, 145). It is unclear, however, whetherthe ethanol-induced increase in extracellular NAC endor-phin levels is a result of direct activation of the arcuate-NAC endorphin pathway, as some studies have demon-strated that acute ethanol administration increases POMCmRNA in the arcuate nucleus (290, 383) while others havebeen unable to find any effect of acute ethanol on arcuatePOMC mRNA content (245).

Importantly, the opioid receptor antagonist naltrex-one reverses alcohol-induced DA release in the NAC inrats, and suppression of operant alcohol-reinforced be-havior by naltrexone is associated with attenuation of thealcohol-induced increase in dialysate DA levels in theNAC (164). These findings not only show that alcoholreinforcement depends on the activity of endogenousopioid systems but also confirm that DA output in theNAC is associated with this reinforcement process(189). Furthermore, alcohol-preferring AA rats showlower opioidergic activity in areas involved in alcoholreinforcement (346), and many other studies have alsoreported innate differences in opioid systems in otheralcohol-preferring and alcohol-avoiding lines of ani-mals (189, 507). In addition, -opioid receptor knock-out mice do not self-administer alcohol under severaldifferent test conditions (399) and, in accordance, se-

lective antagonists acting at -opioid receptors are ableto reduce alcohol consumption (211).

In conclusion, animal research clearly indicates thatendocannabinoids and endogenous opioids play a crucialrole in alcohol reward.7 This further demonstrates inter-actions with the mesolimbic DA system as well as DA-independent processes. Owing to the limitation in animalstudies that subjective states cannot be measured in anadequate way renders the translation of this knowledge tothe human context difficult, and an understanding of howthe subjective euphoric and hedonic aspects of rewardssuch as ethanol evolve in humans remains elusive. It maybe speculated that a state of well-being and euphoriainvolves far more complex processes than merely thecentral activation of CB1 and /-opioid receptors, beinglikely to involve the whole body system, including a bal-ance within the stress system and physiological parame-ters driven by the autonomic nervous system. In thisrespect, the hypothalamus, which interfaces the brain-body axis, may prove to be of importance.

D. Signaling Pathways Involvedin Alcohol Reinforcement

In view of the role of DA in the acquisitition ofalcohol reinforcement, over the past two decades variousresearch groups have investigated signal transductionwithin the NAC and other areas receiving input from A10neurons (114, 408). Following the release of DA, variousDA receptors become activated. The D1-like receptors,which include DA D1 and D5 receptors, enhance theactivity of adenylyl cyclase (AC) via coupling to stimula-tory G proteins (Gs). Alternatively, D2-like receptors(D2-D4) inhibit AC through inhibitory Gi. D1-like recep-tor stimulation results in an increase in the concentrationof cAMP and the activation of cAMP-dependent proteinkinase A (PKA) signaling, which then leads to substratephosphorylation. One of the substrates of PKA is thetranscription factor cAMP response element-binding pro-tein (CREB), which eventually results in increased tran-scription of genes containing cAMP response elements(CRE) in their promoter region (280). The cAMP-PKApathway is a primary signaling cascade induced by expo-sure to alcohol (114, 408), and the expression of numer-ous ethanol-responsive genes is regulated by PKA (seesect. IV) (Fig. 6). Voluntary alcohol intake significantlydecreases the expression of Ca2/calmodulin-dependentprotein kinase IV (CaMKIV) and CREB phosphorylation,specifically in the shell of NAC (322), suggesting thatdecreased CaMKIV-dependent CREB phosphorylation in

7 In addition, a functional cross-talk between the endocannabinoidand opioid systems has been found in the mutual modulation of drug/alcohol reinforcement and reward processes (143, 401).

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the shell region of NAC is involved in alcohol reinforce-ment. While the main role of CaMKIV may be activation ofCREB, it has also been reported to regulate histonedeacetylase (HDAC) trafficking (497). Interestingly, alco-hol decreases HDAC activity and increases acetylation ofhistones (357) (Fig. 6 and sect. IVB).

The importance of cAMP-PKA signaling has beendemonstrated in mice with genetically modified Gs func-tion. Mice lacking one Gs allele exhibit low AC activity inthe NAC and show decreased voluntary alcohol consump-tion compared with their wild-type littermates (520). Sim-ilarly, viral delivery into the NAC of a dominant-negativepeptide that inhibits the subunits of G proteins reducesself-administration of alcohol in rats (539). These dataimply that a reduction in cAMP-PKA signaling leads toreduced alcohol consumption. Surprisingly, however,augmented voluntary alcohol consumption is seen inknockout mice that lack a regulatory subunit of PKA(491). These mice also show a reduction in cAMP-stimu-lated PKA activity in the NAC and the amygdala. In linewith this genetic manipulation of PKA activity, infusion of

a PKA inhibitor into the NAC shell significantly increasesvoluntary alcohol consumption (321). Further PKA inhi-bition was shown to lead to decreased protein levels ofthe -catalytic subunit of PKA (PKA-C) and phospho-CREB, indicating that decreased PKA/CREB function isinvolved in high alcohol preference (321). Indeed, innatehigh alcohol preference and excessive alcohol consump-tion, occurring for example in P-rats (31), is associatedwith lower phospho-CREB levels within the central amyg-dala (CeA) compared with NP rats. Infusion of a PKAactivator into the CeA increased CREB function and de-creased the alcohol intake of P-rats, whereas infusion of aPKA inhibitor into the CeA reversed the phenotype of NPrats with enhanced alcohol consumption and decreasedCREB function (358). These results indicate that de-creased CREB function in the CeA may be involved in thehigh alcohol consumption of P rats. In agreement withthis is the finding that heterozygous CREB knockout micealso show enhanced alcohol consumption (358), althoughit remains questionable whether the latter finding is con-clusive since the loss of CREB is readily compensated by

FIG. 6. Following the release of dopamine (DA) induced by ethanol, the DA D1 receptor is stimulated. Subsequently, the activity of adenylylcyclase (AC), through coupling to stimulatory G proteins (Gs), results in an increase in cAMP concentration and in the activation of cAMP-dependent protein kinase A (PKA) signaling. cAMP induces this activation by promoting the dissociation of the regulatory subunit (R) of PKA fromthe catalytic subunit (PKA-C). PKA-C then leads to phosphorylation of the transcription factor cAMP response element-binding protein (CREB).Exposure to ethanol also influences the expression of Ca2/calmodulin-dependent protein kinase IV (CaMKIV) and thereby CREB phosphorylationin the NAC. These events finally result in altered transcription of genes containing a cAMP response element (CRE) in their promoter region suchas corticotrophin-releasing hormone (CRH), neuropeptide Y (NPY), prodynorphin (PDYN), and brain-derived neurotropic factor (BDNF). Not onlyis CREB phosphorylated upon activitvation of D1 cAMP-PKA signaling but also DARPP-32, which is a 32-kDa protein that is expressed predomi-nantly in striatal medium spiny neurons. In its phosphorylated form, it acts as a potent inhibitor of protein phosphatase 1 (PP1). The function ofPP1 is the dephosphorylation of the NR1 subunit of the NMDA receptor. Therefore, PP1 inhibition by DARPP-32 leads to augmented NMDA receptorphosphorylation, which then increases channel function and counteracts the acute inhibitory action of ethanol on this receptor. Deletion orpharmacological blockade of Gs, , PKA, or DARPP-32 leads to alterations in alcohol (ETOH) self-administration as indicated by the arrows. Notethere are inconsistencies between the different knockout models and their alcohol consumption patterns; thus a reduction in cAMP-PKA signalingcan lead to both reduced and enhanced alcohol consumption. These discrepancies are difficult to interpret and are not discussed in the relevantliterature.

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overexpression of CREM (208, 503), another member ofthe CREB family. In summary, regardless of the inconsis-tencies between the different knockout models and theiralcohol consumption patterns, these data provide com-pelling evidence that PKA signaling modulates alcoholreinforcement processes and that reduced CREB functionis seen after chronic alcohol exposure. In this context, afundamental difference in alcohol-related cAMP-PKA sig-naling compared with other drugs of abuse should beemphasized, which is that an upregulation of CREB func-tion is usually observed following chronic exposure todrugs such as cocaine (72, 408).

In addition to CREB, DARPP-32, a 32-kDa protein ex-pressed predominantly in striatal medium spiny neurons, isalso phosphorylated upon activation of D1 cAMP-PKA sig-naling. In its phosphorylated form, it acts as a potent inhib-itor of protein phosphatase-1 (PP1) and, as such, is an im-portant regulator of DAergic signaling (168). The function ofPP1 is the dephosphorylation of the NR1 subunit of theNMDA receptor. PP1 inhibition by DARPP-32 thereforeleads to augmented NMDA receptor phosphorylation, whichthen increases channel function and counteracts the acuteinhibitory action of ethanol on this receptor (292). It shouldbe emphasized that this enhancement of NMDA receptoractivity in response to ethanol occurs only in dopaminocep-tive neurons that contain D1 receptors along with theDARPP-32/PP1 cascade. This casacade may therefore play acritical role in synaptic plasticity induced by alcohol expo-sure, as DARPP-32-mediated enhancement of NMDA recep-tor function in striatal areas is likely to be an importantfactor in NMDA-dependent long-term potentiation(LTP), as outlined in section V. As a result of thesecellular changes, DARPP-32 should be involved in theregulation of alcohol reinforcement. In fact, DARPP-32knockouts voluntarily drink less alcohol than theirwild-type littermates (397) (Fig. 6).

As well as cAMP-PKA signaling, early cell culturestudies implicated the protein kinase C (PKC) pathwayin the mediation of both acute and chronic responses toethanol exposure (114, 339). PKC is a family of kinasesthat is activated by Ca2. Various PKC isoforms havebeen found in the brain. Following activation, theytranslocate to their substrate sites where they bind toscaffolding proteins, i.e., proteins that enable kinasesefficiently to couple to specific targets such as recep-tors or ion channels. Important examples of scaffoldingproteins involved in the actions and neuroadaptationsof alcohol are Homer (482), RACK1 (502), and -arres-tin 2 (43). The two isoforms PKC- and PKC- interactwith these scaffolding proteins, and they seem to be ofparticular importance in mediating alcohol-induced be-havioral responses. PKC- knockout mice show en-hanced alcohol preference (62) compared with wild-type mice, whereas PKC- knockouts exhibit a mark-edly reduced preference for alcohol (192). The latter

phenotype could be rescued by means of inducibleexpression of PKC- in the NAC, and other forebrainareas restored alcohol preference in adult PKC-knockout mice to the level seen in wild-type mice (81).These findings indicate that PKC- signaling in the adultbrain regulates alcohol reinforcement. Both PKCs seemto physically interact via phosphorylation with GABAAreceptors in an opposing manner (339), resulting inreduced enhancement of GABAA receptor function byethanol in PKC- knockout mice (177) or augmentedfunction in PKC- knockouts (192).

As well as GABAA, another key player in mediatingthe effects of alcohol is the glutamate receptor. Theglutamatergic system is strongly linked to the intra- andextracellular messenger nitric oxide (NO) (63). Thusstimulation of NMDA receptors leads to Ca2 influx,and binding of Ca2 to calmodulin activates, amongothers, neuronal NO synthase which produces NO fromarginine. NO is one of the few known gaseous signalingmolecules and can act as a retrograde messenger. Ac-tivation of guanylyl cyclase and the resulting elevationof cGMP is a major downstream signal of NO in neu-rons. The full details of signaling through cGMP havenot yet been clarified. cGMP affects several ion chan-nels and phosphodiesterases in vivo. In many cells, thetarget of cGMP is the cGMP-dependent protein kinase Ior II, abbreviated as cGKI and cGKII, respectively(200). In brain, NO, cGMP, and cGKII are closely re-lated because both enzymes, neuronal NO synthase(nNOS) and cGKII, are frequently coexpressed, eitherdirectly or indirectly with cGKII-expressing neurons,which receive afferents from nNOS-containing neurons(200).

Evidence from pharmacological and knockout stud-ies has implicated nNOS/NO/cGMP/cGKII signaling in theaction of alcohol (Fig. 7); hence, administrations of com-pounds that inhibit all isoforms of NOS influence alcoholconsumption in alcohol-preferring rats (68, 392). Moreimportantly, nNOS knockout mice consumed six timesmore alcohol from high concentrated alcohol solutionsthan did wild-type mice (466).

In conclusion, NO signaling is critically involved inthe regulation of alcohol reinforcement. Moreover, sincenNOS knockout mice exhibit pronounced aggressive be-havior (337), which was even augmented following alco-hol treatment in an intermale aggression test (Spanagel,unpublished results), the close association of aggressive-ness and alcohol drinking might also be related to alter-ations in the nNOS gene. In this respect, it should berealized that in humans aggressive personality is oftenassociated with alcoholism (215) and, vice versa, alcoholconsumption is associated with a high incidence of manydifferent types of aggressive and violent behavior (376).Finally, the downstream components of NO in neurons,cGMP and its kinase, are also mediating some of the

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behavioral responses to alcohol exposure. Thus cGKIIknockout mice voluntarily consume more alcohol com-pared with wild-type littermates (527). Overall, similari-ties of behavioral responses in nNOS and cGMPII knock-outs suggest that the NO/cGMP/cGKII signaling pathwayis involved in controlling alcohol reinforcement and otherbehavioral effects such as alcohol-induced aggressive-ness.

In summary, cAMP-PKA signaling is involved in me-diating effects of alcohol as well as influencing CREB-mediated processes. This altered CREB function affectsmultiple alcohol-responsive target genes that will be re-viewed in section IV. In addition, cAMP-PKA signaling inmedium spiny neurons affects DARPP-32 function whichis, in turn, an important regulator of NMDA receptorfunction within the reinforcement system and may play animportant role in neuroadaptations in response to alcoholexposure. NMDA receptors are closely linked to NO/cGMP signaling, and this pathway also plays a critical rolein mediating alcohol reinforcement as well as other be-havioral responses induced by alcohol. Finally, PKC sig-naling is also strongly affected by alcohol which, in turn,affects GABAA receptor function. Hence, alcohol affectsthe functioning of receptors (NMDA and GABAA) relevantto synaptic plasticity (see sect. V) via various signalingpathways.

IV. GENE TRANSCRIPTION AND EPIGENETICEFFECTS MEDIATED BY ALCOHOL

A. Gene Transcription Induced by Ethanol

The list of putative CREB target genes with CREsequences now exceeds 100 and includes genes that con-

trol neurotransmission, cell structure, signal transduc-tion, transcription, and metabolism (280). Given that sev-eral acute and chronic effects of ethanol are mediated byCREB, it can be assumed that CREB target genes areinvolved in mediating behavioral responses to ethanol. Infact, this has been demonstrated by pharmacological in-tervention studies and appropriate knockout models for avariety of CREB target genes, the most prominent beingcorticotrophin-releasing hormone (CRH) (181), prodynor-phin (45), brain-derived neurotrophic factor (BDNF)(311), neuropeptide Y (NPY) (490), and numerous othergenes (102). However, there are also many CREB-inde-pendent genes that may respond to alcohol, and the ques-tion is how can novel alcohol-responsive target genes andtheir products be identified in a hypothesis-free ap-proach? Using the new -omics technologies, molecularexpression profiles can be assembled and quantified onthe mRNA, protein, and metabolite levels. In particular,there have been great advances in transcriptomics whereexpression levels of mRNAs in a given brain area or cellpopulation are studied by one of the many gene expres-sion profiling approaches (150). In particular, DNA mi-croarrays are more and more applied as high-throughputtechnologies in alcohol research (151, 237).

Mammalian genomes are extensively transcribed butnot necessarily translated (41), and this excessive RNAproduction may be an important contribution to the flowof information in a cell (475). Particularly, in the CNS, thesite of RNA production can be some distance from theactual translation into proteins. Apart from cell bodies,substantial amounts of mRNA transcripts and other non-coding RNA species are found in different microregions ofthe neurons (e.g., dendritic spines, synaptic boutons),ready for activity-dependent translation, modulation byRNA editing, and degradation (380). Aware of the fact that

FIG. 7. Neuronal nitric oxide synthase (nNOS)/NO/cGMP/cGMP-dependent protein kinase II (cGKII) signalingis involved in mediating alcohol reinforcement. The stimu-lation of NMDA receptors leads to Ca2 influx, and bindingof Ca2 to calmodulin activates nNOS which produces NOfrom arginine. NO acts as a retrograde messenger. Theactivation of the guanylyl cyclase and the resulting eleva-tion of cGMP is a major downstream signal of NO inneurons. In neurons, the target of cGMP is the cGKII.Genetic deletion of nNOS and cGKII, respectively, leads toenhanced alcohol (ETOH) self-administration.

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transcriptional changes do not reflect altered proteinfunction, this section explores evidence for specific eth-anol action on gene expression.

Similar to its neurochemical actions, effects of etha-nol on gene expression can be seen, on a much slowertime scale, as waves of subsequent

alcoholism

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