The Role of Acetaldehyde in the Central Effects of Ethanol

The Role of Acetaldehyde in the Central Effectsof Ethanol

Etienne Quertemont, Kathleen A. Grant, Mercè Correa, Maria N. Arizzi, John D. Salamone, Sophie Tambour,Carlos M.G . Aragon, William J. McBride, Zachary A. Rodd, Avram Goldstein, Alejandro Zaffaroni, Ting-Kai Li,

Milena Pisano, and Marco Diana

This article represents the proceedings of a symposium at the 2004 annual meeting of the ResearchSociety on Alcoholism in Vancouver, Canada. The symposium was organized by Etienne Quertemont andchaired by Kathleen A. Grant. The presentations were (1) Behavioral stimulant effects of intracranialinjections of ethanol and acetaldehyde in rats, by Mercè Correa, Maria N. Arizzi and John D. Salamone; (2)Behavioral characterization of acetaldehyde in mice, by Etienne Quertemont and Sophie Tambour; (3)Role of brain catalase and central formed acetaldehyde in ethanol’s behavioral effects, by Carlos M.G.Aragon; (4) Contrasting the reinforcing actions of acetaldehyde and ethanol within the ventral tegmentalarea (VTA) of alcohol-preferring (P) rats, by William J. McBride, Zachary A. Rodd, Avram Goldstein,Alejandro Zaffaroni and Ting-Kai Li; and (5) Acetaldehyde increases dopaminergic transmission in thelimbic system, by Milena Pisano and Marco Diana.

Key Words: Acetaldehyde, Ethanol, Brain, Catalase, Self-administration, Dopamine.

ACETALDEHYDE, THE FIRST product of ethanolmetabolism, is a biologically active compound. As

such, it is speculated to play a significant role in alcoholabuse and alcoholism (Quertemont, 2004). However, thebehavioral properties of acetaldehyde itself are largely un-defined. In particular, its motivational and hedonic prop-erties have been highly debated for the last two decades.Blood acetaldehyde accumulation generally deters alcoholconsumption in humans, as, for example, in subjects carry-ing the ALDH2*2 allele that reduces the activity of theenzyme aldehyde dehydrogenase (Quertemont, 2004).Based on this latter observation, acetaldehyde is usuallyconsidered a highly aversive substance. However, a number

of animal studies have challenged this common view andsuggested instead that acetaldehyde might be reinforcing.Indeed, laboratory rats self-administer acetaldehyde di-rectly into the brain (Rodd-Henricks et al., 2002) as well asthe periphery (Myers et al., 1984). Other experimentalevidence in support of a significant role for acetaldehyde inethanol’s effects come from studies that manipulate theactivity of the enzyme catalase. The H2O2-catalase enzy-matic pathway metabolizes ethanol into acetaldehydewithin the brain (Zimatkin et al., 1998), and changes inbrain catalase activity alter the behavioral effects of ethanoladministration (Quertemont and Tambour, 2004; Smith etal., 1997). Although it is very likely that acetaldehyde me-diates these effects, this later assumption still awaits an invivo experimental demonstration. All these results suggestadditional studies are needed to clarify the behavioral ef-fects of acetaldehyde and, especially, to unravel the neuro-chemical basis of such effects. If acetaldehyde plays a sig-nificant role in ethanol’s effects, it is expected to induceethanol-like behavioral effects and to share similar neuro-chemical mechanisms with ethanol. The present sympo-sium summarizes the latest results on the behavioral andneurochemical effects of acetaldehyde in mice and rats andtheir role in comparison to ethanol’s central effects.

BEHAVIORAL STIMULANT EFFECTS OF INTRACRANIALINJECTIONS OF ETHANOL AND ACETALDEHYDE IN RATS

Mercè Correa, Maria N. Arizzi and John D. Salamone

Ethanol typically is classified as a sedative/hypnotic. Nev-ertheless, as is the case with many sedative or depressant

From Neuroscience Comportementale et Psychopharmacologie, Universityof Liège, Liège, Belgium (EQ, ST); Department of Physiology and Pharma-cology, Wake Forest University School of Medicine, Winston-Salem, NorthCarolina (KAG); Department of Psychology, University of Connecticut,Storrs, Connecticut (MC, MNA, JDS); Area de Psicobiologia, UniversitatJaume I, Castelló, Spain (MC, CMGA); Institute of Psychiatric Research,Department of Psychiatry, Indiana University School of Medicine, Indianap-olis, Indiana (WJM, ZAR); Stanford University, Stanford, California (AG);Technofyn Associates, Palo Alto, California (AZ); NIAAA, Bethesda, MD(T-KL); Laboratory of Cognitive Neuroscience, Dept. of Drug Sciences,University of Sassari, Italy (MP, MD).

Received September 15, 2004; accepted December 6, 2004.Supported by the Belgian National Funds for Scientific Research (EQ,

ST); NIAAA grants AA07611, AA12262 and AA14437 (WJM, ZAR, AG,AZ, T-KL).

Reprint requests: Etienne Quertemont, Neuroscience Comportementale etPsychopharmacologie, Univesrity of Liege, Boulevard du Rectorat 5/B32, B-4000,Liege, Belgium; Fax: (32)04-366-28-59; E-mail: [email protected]

Copyright © 2005 by the Research Society on Alcoholism.

DOI: 10.1097/01.ALC.0000156185.39073.D2

0145-6008/05/2902-0221$03.00/0ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 29, No. 2February 2005

Alcohol Clin Exp Res, Vol 29, No 2, 2005: pp 221–234 221

drugs, ethanol can have complex biphasic effects acrosstime and dose (Pohorecky, 1977). Ethanol generally pro-duces sedative effects at moderate to high doses, but isreported to have psychomotor stimulant properties at rel-atively low doses, and at relatively short times after injec-tion (Pohorecky, 1977). Although locomotor activation of-ten is reported to occur after systemic administration of lowdoses of ethanol in mice, peripheral injections of ethanol inrats generally produce a monophasic suppression of loco-motor activity (Correa et al., 2003a). The basis of thisphenomena is not known but several factors may becontributing.

It has been postulated that a number of the centraleffects of ethanol are mediated through the ethanol me-tabolites acetaldehyde and acetate. The oxidative metabo-lism of ethanol into its first metabolite, acetaldehyde, caninvolve several organs (e.g., brain, liver) and multiple en-zymes, including alcohol dehydrogenase (ADH), cyto-chrome P-450 2E1, and catalase (Smith et al., 1997). Acet-aldehyde is then metabolized into acetic acid (acetate),primarily by aldehyde dehydrogenase (ALDH). Althoughethanol and peripherally-produced acetate reach the brainin significant amounts, the presence of acetaldehyde in thebrain after moderate levels of ethanol intake is a topic ofsome controversy (Smith et al., 1997; Quertemont andTambour, 2004). In the liver ADLH rapidly converts acet-aldehyde into acetate, and therefore very low levels ofacetaldehyde are detected in blood after the administrationof moderate to low doses of ethanol (Quertemont andTambour, 2004). Acetaldehyde derived from the peripheralmetabolism of ethanol penetrates from blood to brain withdifficulty because of the metabolic barrier presented byALDH. Nevertheless, it has been suggested that high quan-tities of blood acetaldehyde can saturate this enzymaticbarrier between blood and brain, and therefore cross intothe brain (Quertemont and Tambour, 2004). Another plau-sible source of brain acetaldehyde is the direct synthesis ofacetaldehyde from the ethanol that escapes peripheral me-tabolism. It has been suggested that acetaldehyde is formeddirectly in the brain in part via the enzyme catalase (Smithet al., 1997). Considerable evidence indicates that braincatalase is involved in ethanol-induced changes in locomo-tor activity (Correa et al., 2001). The putative central oxi-dation of ethanol may have important biological implica-tions, and more strategies are needed to demonstrate thecontribution of acetaldehyde to the effects of ethanol. Inthis regard, behavioral studies of the direct effects of acet-aldehyde are of critical importance.

Considerable evidence indicates that acetaldehyde is anactive metabolite that contributes to the motor effects ofethanol. In mice and rats it has been shown that peripheralinjections of acetaldehyde decrease locomotion and rearingin an open field (Holtzman and Schneider, 1974), producea loss of righting reflex (Tampier and Quintanilla, 2002),and reduce lever pressing for food in a discriminationparadigm (Quertemont and Grant, 2002). In agreement

with these previous data, recent studies from our laboratoryhave shown that acetaldehyde peripherally administered ismore potent than ethanol for suppressing locomotion insmall stabilimeter cages, reducing lever pressing, and in-creasing the interresponse time on a fixed ratio 5 operantschedule (FR5) (Correa et al., 2003b). The FR5 scheduletypically generates a relatively high rate of responding andtherefore is very sensitive to the rate suppressing effects ofdrugs. It has been demonstrated that i.p. ethanol adminis-tration decreases performance on different FR schedulesand increases the latency to respond (Hiltunen and Jarbe,1988). From our experiments, it appears that ethanol, ac-etaldehyde, and acetate have response-suppressing effectswhen injected i.p. Furthermore, ethanol metabolites aremore potent than ethanol in producing the rate-decreasingeffects. Acetaldehyde appears to be more potent than eth-anol or acetate for suppressing locomotion and lever press-ing. The lowest effective dose of ethanol was 1.0 g/kg, whilethe lowest effective dose of acetaldehyde was 0.05 g/kg.These results, in agreement with previous data (Correa etal., 2003a; Hiltunen and Jarbe, 1988), show that the rate-decreasing effect of ethanol is present across a variety ofdistinct behavioral paradigms when ethanol is peripherallyadministered, but in addition, they show how peripheralacetaldehyde or acetate have the same pattern of effects asethanol.

Central administration of these substances avoids theissue of brain penetrability, and therefore more directlyassesses the intracerebral actions of ethanol and ethanolmetabolites on different aspects of motor activity. In con-trast to the peripheral effects of ethanol administration, inrecent studies we have demonstrated that intraventricular(ICV) infusions of low doses of ethanol in rats can inducesigns of behavioral activation in small activity cages, such asincreases in acute locomotion and locomotor sensitizationafter repeated administration (Correa et al., 2003a). ICVadministration of ethanol over an 8-fold dose range pro-duced an inverted U-shape dose response curve for loco-motion in an open field (Correa et al., 2003c). Ethanol-induced increases in total motor activity observed after ICVinjections were statistically significant in a dose range of0.35 to 2.8 �mol, while 5.6 �mol ICV ethanol failed tostimulate motor activity. The reliability of this effect issupported by parallel studies demonstrating that on a DRLschedule, which produces a low rate of responding and issensitive to rate increasing effects of drugs, ICV ethanolinjections produced an inverted “U” shaped dose responsecurve (Arizzi et al., 2003), with 2.8 �mol being the dose ofethanol that significantly increased responding, whilehigher doses (up to 17.6 �mol) had no significant effects.These studies show that the biphasic effects of ethanol inrats are present when the route of administration avoidsthe periphery. It is not clear why peripheral administra-tion of ethanol in rats is less likely to lead to increases inactivity than infusions directly into the brain. It is possi-ble that peripheral ethanol administration leads to sed-

222 QUERTEMONT ET AL.

ative or depressant effects in rats that mask the centralactivity-inducing effects, and that ICV infusions do notinstigate these peripheral actions.

More interesting is the fact that, in addition to ethanol,acetaldehyde administered into the lateral ventricles alsoincreased locomotor activity both in the open field and insmall stabilimeter cages (Correa et al., 2003a, 2003c). ICVacetaldehyde infusions can produce a pattern of motoractivation at low to moderate doses that is similar to theeffect produced by ICV ethanol. Rats that received 0.7, 1.4and 2.8 �moles of acetaldehyde showed an induction oflocomotion that was significantly different from vehicle,while rats that received the highest dose (5.6 �mol) did not.In addition, acetaldehyde at the same doses induced in-creased exploration of the unprotected interior part of thearena in the open field (Correa et al., 2003c), an effect thatoften is interpreted to reflect a disinhibitory or anxiolytictype of drug effect. These effects of acetaldehyde appear tobe more robust than the anxiolytic effects of ethanol, be-cause only the 0.7 �mole dose of ethanol increased activityin the interior part of the open field. Acetaldehyde ICValso increased lever pressing rate on a DRL30 sec schedule(Arizzi et al., 2003) over a broader dose range than ethanolin this paradigm, since ICV acetaldehyde infusions in-creased DRL responding at the 0.7, 2.8, and 5.6 �molesdoses. As with ethanol, higher doses of acetaldehyde failedto have either rate-increasing or rate-decreasing effects onDRL responding. With the FR5 schedule, neither ethanolnor acetaldehyde had any suppressant effects on leverpressing in the dose range of 0.7–17.6 �moles (Arizzi et al.,2003), contrary to what was found in the same operantschedule when the drugs were injected peripherally (Cor-rea et al., 2003b). In contrast, acetate injected in the ven-tricles had suppressive effects on locomotion, rearing andlever pressing for food in the FR5 schedule (Arizzi et al.,2003; Correa et al., 2003c).

These studies show that the biphasic motor effects ofethanol and acetaldehyde in rats are present when theroute of administration is central, and not peripheral. How-ever, independently of the route of administration, acetateconsistently produces motor suppressant effects. The neu-rochemistry and neuroanatomy of this effect is still un-known, and although several brain sites have been sug-gested to have an effect on ethanol induced locomotion(Dar, 2002; Sanchis-Segura et al., 2000), until recently nostudies have been done with direct administration of etha-nol or acetaldehyde.

We have started by investigating the possible involve-ment of Substantia Nigra pars reticulata (SNr) in the motoreffects of ethanol and acetaldehyde. This nucleus is a partof the basal ganglia circuitry that has been implicated inseveral types of motor activity, including muscle rigidity,lever pressing, tremor, catalepsy, circling, and locomotion(Correa et al., 2003d; Trevitt et al., 2001, 2002). Althoughthe SNr was once viewed as a region that simply providedfeedback regulation of dopamine neurons in the substantia

nigra pars compacta, it has become evident in the last fewyears that the SNr is one of the two major output nuclei forthe basal ganglia (Scheel-Kruger et al., 1981). SNr appearsto be a brain site at which several neurotransmitter systemsinteract to regulate motor activity. GABAergic manipula-tion of the SNr produced profound effects on various as-pects of locomotion (Scheel-Kruger et al., 1981). It also hasbeen shown that direct infusion of the D1 agonist SKF82958 into the SNr increased locomotor behavior in a smallactivity chamber, and also increased GABA release in theSNr as measured by microdialysis (Trevitt et al., 2002).Moreover, SNr is the brain area at which GABAA agonistsand antagonists exert their most potent effects on locomo-tion (Trevitt et al., 2002). Ethanol also has been demon-strated to affect the physiology and neurochemistry of SNrneurons. Ethanol enhances GABA function within SNr byaffecting a GABAA receptor with specific structural com-ponents (Criswell et al., 1993). In view of the findingsindicating that SNr is a basal ganglia site regulating motoroutput, and that ethanol exerts modulatory effects over thephysiology of SNr neurons, we have started to investigatethe possibility that local injections of ethanol into SNr willaffect motor activity. Moreover, previous research indicat-ing that substantia nigra is one of the brain areas with thehighest concentration of catalase (Brannan et al., 1981)also suggests that SNr may be an important brain locus atwhich acetaldehyde would modulate locomotor activity.We have found that infusions of ethanol directly into SNrresulted in a significant induction of locomotor activity.This is the first study reporting that locomotor activity canbe induced by local infusions of ethanol into a discretebrain locus. In the SNr the overall dose response curve forthe effect of ethanol had an inverted-U shape, and SNrinjections up to 1.4 �moles of ethanol resulted in a signif-icant increase in locomotion relative to vehicle. Additionalexperiments demonstrated that infusions of ethanol intocontrol sites dorsal and posterior to SNr did not affectlocomotion, which demonstrates some degree of site spec-ificity for this effect. However, SNr is not the only placewere ethanol injections induced locomotion. Posterior ven-tral tegmental area (pVTA) infusions of 1.4 �moles etha-nol also significantly increased locomotor activity in smallstabilimeter cages compared to vehicle injections (vehicle� 62.5 � 3.9, ethanol � 93.4 � 10.3 counts in 10 min,F(1,21) � 6.23, p � 0.05).

As suggested previously (Correa et al., 2001; Smith et al.,1997), the acute motor effect of ethanol on locomotion maybe mediated by centrally formed acetaldehyde. In our re-sults, the induction of locomotion produced by intranigraladministration of ethanol was blocked by peripheral admin-istration of sodium azide, a catalase inhibitor that acts toreduce brain ethanol metabolism and to block ethanolinduced locomotion in mice (Correa et al., 2004; Sanchis-Segura et al., 1999a). This suppressive effect of catalaseinhibition on intranigral ethanol-induced locomotor stimu-lation agrees with pilot studies in rats showing that the

ACETALDEHYDE AND CENTRAL ETHANOL EFFECTS 223

induction of open field locomotion produced by ICV infu-sions of ethanol was blocked by sodium azide injectedperipherally (Correa, unpublished observations). This sug-gests that the transformation of ethanol by catalase in thesubstantia nigra, and hence the presence of acetaldehyde, isa plausible mechanism for some of the behavioral effectsobserved after central ethanol administration. This state-ment is supported by our observation of a potent androbust increase in locomotion after infusions of acetalde-hyde into the SNr. Acetaldehyde was both more potent andmore efficacious than ethanol in terms of the stimulation oflocomotion induced by SNr injections. These results indi-cate that SNr is one of the sites at which ethanol may beacting to induce locomotor activity. Together with the datashowing that acetaldehyde is a biologically active com-pound that stimulates locomotor activity after injections inthe SNr, it is hypothesized that the conversion of ethanolinto acetaldehyde in the brain is an important biochemicalstep involved in the locomotor stimulation induced by lowdoses of ethanol.

These studies have implications for understanding thebrain mechanisms involved in mediating the ascending limbof the biphasic dose response curve that characterizes themotor effects of ethanol administration. The specific neuralmechanisms through which low doses of ethanol can inducelocomotor activity are unknown. Based upon our resultsand on the data reviewed above, it is possible that ethanolfacilitates GABAA receptor function on SNr output neu-rons (Criswell et al., 1993). These neurons in turn project tovarious brainstem motor areas, including reticular forma-tion and the pedunculopontine nucleus (Scheel-Kruger etal., 1981). Thus, it is possible that local infusions of ethanolor acetaldehyde into SNr are stimulating locomotionthrough a mechanism that is similar to the one that isthought to be involved in muscimol-induced locomotion(Trevitt et al., 2002). Further research is necessary to testthis hypothesis, and to determine if other brain systemssuch as ascending dopamine projections also play a role.

BEHAVIORAL CHARACTERIZATION OF ACETALDEHYDEIN MICE

Etienne Quertemont and Sophie Tambour

In recent years, a number of studies have investigated therole of acetaldehyde in the neurochemical and behavioraleffects of alcohol (Quertemont and Tambour, 2004). How-ever, most of these studies investigated the behavioral ef-fects of acetaldehyde by pharmacological alterations ofethanol metabolism, although the specificity of several ofthe pharmacological substances that were used has beenquestioned. In contrast, the behavioral properties of thisfirst ethanol metabolite have been scarcely investigatedwith direct administrations of acetaldehyde. In the presentstudies, the locomotor, anxiolytic and memory-impairingeffects of intraperitoneal (i.p.) acetaldehyde injections wereinvestigated in mice. The effects of various ethanol doses

were also tested to compare the behavioral effects of eth-anol with those of its first metabolite, acetaldehyde. Finally,the behavioral effects of ethanol were tested in cyanamide-pretreated mice. Cyanamide inhibits the enzyme aldehydedehydrogenase, thereby delaying acetaldehyde eliminationafter ethanol intake. Therefore, this compound stronglyincreases blood and brain acetaldehyde concentrations af-ter ethanol administration (Hillbom et al., 1983; Jamal etal., 2003). If the behavioral effects of ethanol under inves-tigation are mediated in part by acetaldehyde, cyanamidepretreatments should potentiate these behavioral effects.In the present studies, the effects of ethanol and acetalde-hyde were also compared in two strains of mice, inbredC57BL/6J (C57) and outbred CD1 mice. Previous studiesclearly showed that C57 and CD1 mice respond differen-tially to ethanol. For example, ethanol induces biphasiclocomotor effects in CD1 mice with stimulant effects atdoses between 1 and 2 g/kg and depressant effects at higherdoses, whereas C57 mice generally fail to show ethanol-induced stimulant effects and only display locomotor de-pression after ethanol administration (Frye and Breese,1981). Relative to CD1 mice, the C57 strain is character-ized by lower levels of catalase (Correa et al., 2004). Sincecatalase is strongly involved in the metabolism of ethanolwithin the brain (Zimatkin et al., 1998), C57 mice arebelieved to produce lower brain acetaldehyde concentra-tions after ethanol administration, although this hypothesishas never been tested in vivo. According to this hypothesis,the magnitude of ethanol-induced behaviors should belower in these mice whenever ethanol’s effects are medi-ated by acetaldehyde. Such an explanation has been givento explain the lack of ethanol-induced locomotor-stimulanteffects in C57 mice (Correa et al., 2004).

Locomotor Activity. Mice locomotor activity was recordedin 10 chambers (19.5 � 19.5 cm surface x 20.5 cm height)encased in a sound-attenuating shell. The locomotor activ-ity was measured by a pair of infrared light-beam sensorslocated on each side of the enclosure. A locomotor activitycount was recorded each time a mouse crossed the fulldistance (at least 6.5 cm) between two beams. Immediatelybefore testing, mice from different groups were injectedwith various doses of either ethanol (0, 1, 2, 3 or 4 g/kg, i.p.)or acetaldehyde (0, 30, 56, 100, 170 or 300 mg/kg, i.p.). Inagreement with previous studies (Frye and Breese, 1981),ethanol induced a biphasic locomotor effect in CD1 mice(F4,45 � 12.33, p � 0.001) with a significant increase inlocomotor activity at 2 g/kg ethanol and significant depres-sions after the administration of 3 and 4 g/kg ethanol. Incontrast, the locomotor activity of C57 mice was signifi-cantly depressed at all ethanol doses (F4,45 � 24.67, p �0.001). Such results are consistent with the idea that acet-aldehyde contributes to the locomotor stimulant effects ofethanol. Indeed, ethanol’s stimulant effects are difficult toshow in C57 mice, which have lower levels of catalaseactivity and presumably lower brain acetaldehyde concen-trations after ethanol administration. However, the present


studies found no evidence of the locomotor stimulant ef-fects of acetaldehyde when injected peripherally. Acetalde-hyde injections induced a very significant dose-dependentlocomotor depression in both CD1 (F4,45 � 62.88, p �0.001) and C57 (F4,45 � 27.89, p � 0.001) mice at doseshigher than 100 mg/kg. In addition, the aldehyde dehydro-genase inhibitor cyanamide potentiated the locomotor de-pressant effects of ethanol. Different groups of mice werepretreated with either saline or cyanamide (25 mg/kg, i.p.)and injected 90 min later with either 0, 2 or 3 g/kg ethanol.The two-way ANOVA computed on mice locomotor activ-ity showed a significant main effect of both cyanamidepretreatment (F1,40 � 18.44, p � 0.001) and ethanol dose(F2,40 � 8.99, p � 0.001) and a significant interactionbetween both factors (F2,40 � 5.60, p � 0.001). In cyana-mide pretreated mice, significantly lower levels of activitywere observed after the administration of both 2 and 3 g/kgethanol relative to their respective control groups pre-treated with saline and injected with ethanol. These resultsclearly show that increases in blood and brain acetaldehydeconcentrations resulting from the inhibition of the enzymealdehyde dehydrogenase potentiate the locomotor depres-sant effects of ethanol. Together the results of the presentstudy indicate that acetaldehyde is mainly a depressantdrug and might therefore contribute to the sedative anddepressant effects of ethanol. However, a recent studydemonstrated that acetaldehyde induces locomotor stimu-lant effects only when infused directly into the brain (Cor-rea et al., 2003c). Therefore, it remains possible that thelocomotor stimulant effects of brain acetaldehyde weremasked in the present studies by stronger peripheralacetaldehyde-induced depressant effects. Future studiesshould help to clarify this question.

Anxiolytic Effects

Drug anxiolytic effects were tested in an elevated plus-maze, consisting of two open (39 � 5 x 0.25 cm) and twoclosed (39 � 5 � 15 cm) arms emanating from a centralplatform (5 � 5 cm) to form a plus shape (Quertemont etal., 2004). The entire apparatus was elevated at a height of80 cm above the floor. 5 min after the injection of eitherethanol (0, 1, 1.5 or 2 g/kg, i.p.) or acetaldehyde (0, 30, 56,100 or 170 mg/kg, i.p.), the mice were placed into thecentral platform and their exploratory behavior was re-corded by a videocamera. Videotapes were later scored bya trained observer blind to the drug treatment. As micetend to avoid open spaces, the percentage of entries intothe open arms was used as a conventional parameter forthe assessment of the anxiolytic-like effects of ethanol andacetaldehyde, whereas the total number of arm entries(open arm � closed arm entries) was used as a measure ofgeneral activity. Ethanol at doses between 1 and 2 g/kginduced similar increases in the percentage of open armentries in both CD1 (F3,28 � 5.8, p � 0.01) and C57 (F3,28� 7.47, p � 0.001) mice. These results clearly indicate that

ethanol has similar anxiolytic effects in both strains of mice.In contrast, acetaldehyde at doses up to the toxic limitfailed to alter the percentage of open arm entries in eitherCD1 (F4,35 � 2.41, p � 0.05) or C57 (F4,35 � 0.63, p � 0.05)mice, although it depressed mice general activity at thehighest doses in both strains of mice. Therefore, the resultsof the plus-maze experiments demonstrate that acetalde-hyde has no anxiolytic properties, but confirm its locomotordepressant effects. This lack of acetaldehyde anxiolytic ef-fects cannot be explained by acetaldehyde difficulties tocross the blood brain barrier, since we had previouslyshown very significant brain acetaldehyde concentrationsafter i.p. acetaldehyde injections at doses between 100 and300 mg/kg in mice (Quertemont et al., 2004). The lack ofacetaldehyde anxiolytic effects is further confirmed in thecyanamide experiments. Indeed, a cyanamide pretreatmentfailed to alter the anxiolytic effects of 1.5 g/kg ethanol,although it potentiated the locomotor depressant effects ofethanol. The two-way ANOVA computed on the percent-age of open arm entries indicated a significant effect ofethanol (F1,27 � 31.65, p � 0.001), whereas the effect ofcyanamide pretreatment (F1,27 � 2.02, p � 0.05) and theinteraction between both factors (F1,27 � 0.13, p � 0.05)failed to reach statistical significance. In contrast, the two-way ANOVA computed on the number of total arm entriesshowed a significant main effect of cyanamide pretreatment(F1,27 � 15.11, p � 0.001) and a significant interactionbetween both factors (F1,27 � 19.43, p � 0.001). In conclu-sion, these results indicate that acetaldehyde is devoid ofanxiolytic properties and is not involved in the anxiolyticeffects of its parent drug ethanol. The dissociation betweenthe locomotor depressant and the (lack of) anxiolytic ef-fects of acetaldehyde strongly supports this conclusion.

Amnesic Effects. In a preliminary experiment, we testedthe amnesic effects of various acetaldehyde doses with theone-trial passive avoidance test (Quertemont et al., 2004).C57 mice were trained to avoid a compartment in whichthey previously experienced an electrical foot shock (0.3mA). After a single acquisition trial, the subsequent in-crease in step-through latency is used as an index of mem-ory retention. On the acquisition trial, different groups ofmice were injected with various acetaldehyde doses (0, 56,100, 170 or 300 mg/kg, i.p.) immediately after the footshock, i.e., during the memory consolidation phase. Mem-ory retention was tested 24 hr later. The one-way ANOVAcomputed on the step-through latencies on the retentiontest indicated a highly significant effect of acetaldehydedoses (F4,44 � 66.85, p � 0.001). In the saline controlgroup, the step-through latency was greatly enhanced, in-dicating a vivid memory of the foot shock experiencedduring the acquisition trial. Acetaldehyde dose-dependently impaired this retention performance. A signif-icant impairment was already noted after the administra-tion of 100 mg/kg acetaldehyde, a dose without significantsedative effects. In mice injected with 300 mg/kg acetalde-hyde, no changes in the step-through latency were observed


relative to the acquisition trial, indicating a very strongamnesia. These results indicate that acetaldehyde impairsmemory consolidation at doses below the sedative/hypnoticthreshold. As similar results were obtained with ethanol inprevious studies (Aversano et al., 2002), such results sug-gest that acetaldehyde might mediate or be involved in theamnesic effects observed after acute ethanol intoxications.

ROLE OF BRAIN CATALASE AND CENTRAL FORMEDACETALDEHYDE IN ETHANOL’S BEHAVIORAL EFFECTS

Carlos M.G. Aragon

Evidence from several studies suggest that some of thepsychopharmacological effects classically attributed to eth-anol administration are mediated by its primary metabolite,acetaldehyde (Hunt, 1996; Smith et al., 1997). Acetalde-hyde when administered directly into the brain (re)pro-duces the same activating and reinforcing effects observedafter ethanol administration, including an increase of loco-motion and sensitization (Correa et al., 2003a, 2003b,2003c), place preference (Smith et al., 1984) and self-administration (Brown et al., 1979; Myers et al., 1984;Rodd-Henricks et al., 2002). However, it is accepted thatacetaldehyde derived from liver ethanol metabolism pene-trates from the blood to the brain with difficulty because ofthe presence of the enzyme aldehyde dehydrogenase in theblood-brain barrier (Hunt, 1996). However, a source forcentral acetaldehyde formation through ethanol metabo-lism in the brain itself has been demonstrated in severalstudies. Experimental evidence point to the central oxida-tion of ethanol by the catalase-H2O2 system, as an impor-tant factor explaining the behavioral effects observed afterethanol administration. It has been shown that ethanol ismetabolized to acetaldehyde in rodent brain homogenates(Aragon and Amit, 1993; Aragon et al., 1992a; Gill et al.,1992; Zimatkin et al., 1998) as well as in neural tissuecultures (Eysseric et al., 1997; Hamby-Mason et al., 1997;Reddy et al., 1995) via the peroxidative activity of catalase.In addition to these in vitro data, it has been reported thatin vivo ethanol administration effectively protects braincatalase from several inhibitors (Aragon et al., 1991). Pro-tection of catalase by ethanol constitutes indirect evidencefor the oxidation of ethanol by the peroxidatic activity ofcatalase in the brain in vivo (Aragon et al., 1991).

Interestingly, brain catalase activity has also been impli-cated in several behavioral effects of ethanol (for review,see Smith et al., 1997). Thus, it has been demonstrated thatthe catalase inhibitor 3-amino-1,2,4-triazole reduces volun-tary ethanol consumption in mice (Koechling and Amit,1994) and rats (Aragon and Amit, 1992; Rotzinger et al.,1994; Tampier et al., 1994). Moreover, correlations be-tween brain catalase activity and voluntary ethanol con-sumption have been reported (Amit and Aragon, 1988;Aragon et al., 1985; Correa et al., 2004). In addition, it hasbeen demonstrated that changes in brain catalase activityresult in an alteration in several ethanol’s behavioral ef-

fects, such as conditioned taste aversion, the loss of rightingreflex, hypothermia, lethality and locomotor changes. Overthe last years, we have focused our work on the locomotoreffects of ethanol showing that, after the administration ofseveral catalase inhibitors and/or potentiators, brain cata-lase activity and the stimulating effects of ethanol in micelocomotion closely correlate. Indeed, rodents treated withcatalase inhibitors, such as 3-amino-1,2,4-triazole, sodiumazide, cyanamide, and chronic lead acetate, have lowerethanol-induced locomotion than control animals (Aragonand Amit, 1993; Correa et al., 1999a; Escarabajal et al.,2000; Sanchis-Segura et al., 1999b). Conversely, acute leadand chronic cyanamide administrations enhance brain cata-lase activity and increase ethanol-induced locomotor activ-ity (Correa et al., 1999b; Sanchis-Segura et al., 1999c). Thechanges observed in the behavioral effects of ethanol aftermodifications of brain catalase activity are bi-directionaland the locomotor output is directly related to the amountof brain catalase activity. Moreover, the results of thesestudies are also consistent with previous reports obtainedfrom genetically acatalasemic mice, which showed lowerethanol-induced locomotor activity in acatalasemic com-pared to normal mice (Aragon et al., 1992b; Aragon andAmit, 1993; Correa et al., 2004). Together, all these resultshave been understood as experimental support for thenotion that acetaldehyde plays a role in the mediation ofthe psychopharmacological effects of ethanol. Thus, it hasbeen suggested that the higher the activity of catalase, thehigher the rate of acetaldehyde formation in the brain and,consequently, the higher ethanol stimulates mice locomo-tor activity.

However, the catalase-mediated oxidation of ethanol toacetaldehyde is H2O2 dependent. Therefore, any variationin H2O2 levels should produce a change in the final statusof this enzymatic system and should accordingly alter brainacetaldehyde formation as well as the behavioral conse-quences of ethanol administration. We have recently shown(Pastor et al., 2002) how increases in brain H2O2 levels (butwhich do not change the enzymatic properties of catalase)result in an increase in ethanol-, but not saline-, cocaine- orcaffeine-induced locomotion in mice. These latter resultsshow that the behavioral consequences of ethanol are notonly related to the levels of brain catalase, but also tochanges in brain H2O2 levels, probably because both com-pounds equally determine the rate of ethanol oxidation inthe brain and, consequently, the formation of acetaldehydein the central nervous system.

Given these findings, it seemed logical to extend thesestudies on the role of the enzyme catalase and centralformed acetaldehyde in the mediation of the central effectsof ethanol. The following studies are an attempt to examinethis possibility by extending the range of behavioral andpharmacological variables studied.

Effects of Brain Catalase on Ethanol-Induced AnxiolyticEffects. One of the typical responses to the acute adminis-tration of low doses of ethanol in humans and rodents is an


anxiolytic effect. Previous results show that ethanol andacetaldehyde (the first metabolite of ethanol) administeredin the lateral ventricles produced anxiolytic effects in ratsexposed to an open arena (Correa et al., 2003c). We inves-tigated the role of catalase on ethanol anxiolytic effectsmeasured in an elevated x-maze. CD1 mice were pretreatedwith catalase inhibitors (Aminotriazole, 0.0 or 0.5 g/kg orSodium Azide, 0 or 10 mg/kg, i.p.) or with the catalasepotentiator lead acetate (0 or 100 mg/kg) and then injectedwith different doses of ethanol (0.0, 0.5, 1.0 or 1.5 g/kg, i.p.).After the ethanol injection, the animals returned to theirhome cage for 10 min and then were placed into the centerof the x-maze where several parameters were recorded for5 min. Control mice injected with ethanol at different dosesshowed an increase in the time spent and entries in theopen arms. However, the inhibition of catalase and, there-fore, the reduction in acetaldehyde production in the brain,reduced these ethanol anxiolytic effects. The opposite ef-fect was found when catalase activity was potentiated bylead acetate; animals showed an increase in time spent inthe open arms compared to control vehicle mice. Theseeffects were found in absence of motor effects. These re-sults show that the anxiolytic properties of ethanol can bemodulated when ethanol conversion into acetaldehyde isinhibited by catalase inhibitors or potentiated by catalaseenhancers.

Effects of Brain Catalase on Ethanol-Induced Improve-ment on Short-Term Memory. In the last few years a verynaturalistic test has been successfully employed to study theeffects of ethanol on social memory in rats (Thor andHolloway, 1982). This test has been widely used to measureshort-term memory, and is based on rodents’ innate pref-erence to explore unknown conspecifics more intenselythan familiar ones. Thus, when a juvenile rodent is exposedto an adult at two different moments, the amount of timethe adult spends exploring the juvenile in the second pre-sentation reflects the memory for that particular animal,the longer the exploration the weaker the memory of thatjuvenile. Using this paradigm, the capacity of ethanol toenhance memory when administered immediately aftertraining has been successfully proved in rats. However,studies addressing the same phenomenon in mice are lack-ing. In a recent study, we used the social recognition test toinvestigate the capacity of ethanol to enhance the socialmemory of mice and to evaluate the implication of catalasein this ethanol’s effect. Exploration ratios (ER) were cal-culated to evaluate the recognition capacity of mice. Eth-anol (0.0, 0.5, 1.0 or 1.5 g/kg, i.p.) was administered imme-diately after the first juvenile presentation and two hourslater the juvenile was re-exposed to the adult. Additionally,adult mice received aminotriazole (AT) or sodium azidepretreatments. Ethanol (1.0 and 1.5 g/kg) was able to re-duce ER, indicating an improving effect over memory. Thisimprovement was prevented by either AT or sodium azidepretreatments. However, neither AT nor sodium azide at-tenuated the memory-enhancing capacity of NMDA or

nicotine, suggesting a specific interaction between catalaseinhibitors and ethanol in their effects over memory. Thepresent results suggest that brain catalase activity couldmediate the memory-enhancing capacity of ethanol.

Effects of D-Penicillamine, an Acetaldehyde SequesteringAgent, on Ethanol Behaviors

D-penicillamine, a sulfhydryl amino acid derived frompenicillin, is a highly selective agent for sequestering acet-aldehyde in vivo. The knowledge about the precise mech-anism by which D-penicillamine attenuates the acetalde-hyde and ethanol-induced behavioral effects is elucidatedby earlier evidence indicating that D-penicillamine can con-dense, in vivo, with ethanol-derived acetaldehyde and formadducts. The acetaldehyde condensation product is thecyclic amino acid, 2,5,5-trimethylthiazolidine-4-carboxylicacid that shows stability enough to be excreted in the urine(Nagasawa et al., 1975, 1987). These results suggest thatonce this condensation product is formed, the reactivity ofacetaldehyde is lost. Thus, it is expected that D-penicillamineprevents the behavioral effects of acetaldehyde. Our resultsrevealed that behavioral depression caused by acetalde-hyde in mice (200 and 300 mg/kg) can be attenuated bya D-penicillamine treatment. Thus, we established thatD-penicillamine is an effective tool for preventing thebehavioral effects of exogenous injected acetaldehyde. Inaddition, D-penicillamine was also effective in loweringbehavioral locomotion induced by ethanol (1.8 and2.4 g/kg), without altering spontaneous locomotor activ-ity. Our data yield clear evidence for the behavioralre levance of seques ter ing ace ta ldehyde wi thD-penicillamine and, more importantly, they provide, forthe first time, information about the effectiveness ofD-penicillamine in preventing stimulant effects inducedby ethanol. The aim of a second study was to investigatethe possible role of acetaldehyde in voluntary ethanolconsumption in rats by means of its selective inactivationby D-penicillamine. Rats were offered ethanol and waterunder a two-bottle free choice procedure. Daily avail-ability of the ethanol solution was limited to a 15 minperiod per day. Chronic treatment with D-penicillamine(25, 50 or 75 mg/kg) resulted in the extinction of ethanolconsumption. Drinking of ethanol significantly decreased. Onthe other hand, following cessation of the D-penicillaminetreatment, animals returned to baseline levels of ethanol con-sumption. These findings indicate that the selective inactivatorof acetaldehyde blocks ethanol consumption.

In conclusion, the results obtained in the present seriesof experiments tend to support the assumption that the roleof the enzyme catalase in ethanol’s effects is mediated by itsability to oxidize ethanol in the brain and that this capacityexerts at least some influence on ethanol-induced behav-iors. We have shown in in vivo and in vitro experiments thecapacity of catalase to oxidize ethanol in brain tissues. Wefurther demonstrated in vivo that rodents differing in their


behavioral responses to ethanol also differed in their brainlevels of catalase activity and, finally we reported findingsproviding evidence for the behavioral relevance of seques-tering acetaldehyde in the brain. These results show thatwhen acetaldehyde levels are diminished, by catalase inhi-bition or acetaldehyde sequestration, ethanol’s behavioralconsequences are reduced.

CONTRASTING THE REINFORCING ACTIONS OFACETALDEHYDE AND ETHANOL WITHIN THE VENTRAL

TEGMENTAL AREA (VTA) OF ALCOHOL-PREFERRING(P) RATS

William J. McBride, Zachary A. Rodd, Avram Goldstein,Alejandro Zaffaroni, and Ting-Kai Li

Acetaldehyde, the first metabolite of ethanol, is a highlyreactive biological compound. Many behavioral and phar-macological effects of ethanol have been hypothesized tobe mediated by acetaldehyde formed in the CNS (Aragonand Amit, 1992; Aragon et al., 1986; Smith et al., 1984;1997). Because of the very low alcohol dehydrogenase ac-tivity in the brain (Aragon et al., 1992a; Gill et al., 1992), itis unlikely that acetaldehyde is formed by this pathway inbrain tissue. Moreover, under physiological conditions, theacetaldehyde formed peripherally from ethanol is not likelyto enter the brain because of the high aldehyde dehydro-genase activity associated with the blood brain barrier (Sip-pel 1974; Smith et al., 1997; Zimatkin 1991). However,there is evidence that acetaldehyde can be formed in braintissue via a catalase reaction (Aragon et al., 1992a; Hamby-Mason et al., 1997). Several pharmacological studies sug-gested that the effects attributed to ethanol might resultfrom the formation of acetaldehyde through the catalasepathway (Aragon and Amit, 1992; Aragon et al., 1986;Brown et al., 1979; Smith et al., 1984; 1997). For example,inhibition of catalase activity decreases ethanol intake inLong-Evans rats (Aragon and Amit, 1992). Brown et al.,(1979) reported that rats self-administered 1 – 5% acetal-dehyde into the cerebral ventricles, and Smith et al. (1984)found that repeated intracerebroventricular injections ofacetaldehyde produced conditioned place preference.

Acetaldehyde could contribute to the reinforcing actionsof ethanol in several ways. Within the CNS, ethanol couldbe converted to acetaldehyde, and the acetaldehyde is pro-ducing the effects attributed to ethanol, or acetaldehyde couldbe producing its own effects in addition to the reinforcingactions of ethanol. This could occur extracellularly at certainligand gated ion-channels (e.g., nicotinic and/or 5-HT3 recep-tors), or intracellularly via condensation with free aminegroups on membrane proteins, which then alters membranefunction within the ventral tegmental area (VTA). The for-mation of acetaldehyde could also enhance the actions ofethanol at certain receptors by reacting with free aminogroups on receptors and membrane proteins. In addition,acetaldehyde could react with biogenic amines to form activecompounds (e.g., salsolinol), which could enhance or contrib-

ute to the actions of ethanol. The objective of this presenta-tion is to compare the reinforcing actions of acetaldehyde andethanol within the VTA of alcohol-preferring (P) rats, usingthe intracranial self-administration technique.

Alcohol- and experimentally-naive female P rats werestereotaxically implanted with guide cannulae aimed ateither the anterior or posterior VTA. One week later, ratswere placed in 2-lever (active and inactive) operant cham-bers and connected to the electrolytic microinjection trans-ducer system as previously described (Rodd-Henricks et al.,2000). Depression of the ‘active lever’ (FR1 schedule ofreinforcement) caused the delivery of a 100-nl bolus ofinfusate over 5 sec followed by a 5-sec time-out period.During both the 5-sec infusion period and 5-sec time-outperiod, responses on the active lever were recorded but didnot produce further infusions. Responses on the ‘inactivelever’ did not result in infusions. The number of infusionsand responses on the active and inactive lever were re-corded. Sessions were 4 hr in duration and were conductedevery other day. There were usually a total of 7 sessions: 4acquisition sessions, followed by 2 treatment sessions (ses-sions 5 and 6), and 1 recovery session (session 7).

A previous study (Rodd-Henricks et al., 2000) demon-strated that Wistar rats self-infused ethanol into the poste-rior but not anterior VTA. Ethanol concentrations between0 and 300 mg% were tested to determine the response-contingent behaviors of female P rats with injections sites inthe posterior or anterior VTA. Reducing the analysis to theaverage number of infusions received during the 4 sessionsrevealed that there was a significant effect of placement(F1,70 � 220.6; p � 0.0001), concentration (F6,70 � 22.2; p� 0.0001), and a placement x concentration interaction(F6,70 � 20.2; p � 0.0001). In rats with injection tips locatedwithin the posterior VTA, there was a significant effect ofethanol concentration (F6,35 � 26.6; p � 0.0001), withposthoc comparisons (Tukey’s b; p � 0.05) indicating thatP rats given 75, 100, 150, 200 or 300 mg% (17 – 66 mM)ethanol received significantly more infusions than ratsgiven 0 or 50 mg % ethanol. In contrast to the posteriorVTA data, 50 – 300 mg% concentrations of ethanol werenot significantly self-infused into the anterior VTA.

A previous study indicated that acetaldehyde was self-infused into the posterior VTA by P rats. However, theanterior VTA was not tested. In the present study, acetal-dehyde concentrations between 3 and 360 �M were testedto determine the response-contingent behaviors of femaleP rats with injection sites in the posterior or anterior VTA.Reducing the analysis to the average number of infusionsreceived during the 4 sessions revealed that there was asignificant effect of placement (F1,111 � 88.9; p � 0.0001),concentration (F8,111 � 17.6; p � 0.0001), and a placementx concentration interaction (F8,111 � 8.7; p � 0.0001). Inrats with injection tips located within the posterior VTA,there was a significant effect of acetaldehyde concentration(F8,55 � 39.4; p � 0.0001), with posthoc comparisons(Tukey’s b; p � 0.05) indicating that P rats given 6, 11, 23,


45 or 90 �M acetaldehyde received significantly more in-fusions than rats given 0, 3, 180 or 360 �M acetaldehyde. Incontrast to the posterior VTA data, none of the concentra-tions of acetaldehyde was significantly self-infused into theanterior VTA. Moreover, the effective reinforcing concen-trations of acetaldehyde were approximately 1000-foldlower than ethanol and were within a range that could begenerated with brain alcohol levels of 45 – 225 mg% (Zi-matkin et al., 1998).

P rats readily self-administered 150 mg% ethanol intothe posterior VTA and learned to discriminate the activefrom the inactive lever by the second session; respondingon the active lever was maintained from session 2 throughsession 8. To determine if the formation of acetaldehydemight be contributing to the reinforcing effects of ethanol,coinfusion experiments were conducted with the catalaseinhibitor, 3-amino-1,2,4-triazole (triazole). Coinfusion of 8to 64 mM triazole had no significant effect on respondingon the active lever for the self-infusion of 150 mg% ethanolby P rats. These concentrations have been shown to effec-tively inhibit catalase activity in brain tissue preparations(Zimatkin et al., 1998), suggesting that formation of acet-aldehyde may not be contributing to the local self-infusionof ethanol in the posterior VTA.

A previous study with Wistar rats indicated that dopa-mine neuronal activity is needed for the self-infusion ofethanol into the posterior VTA (Rodd et al., 2004b). How-ever, a similar study has not been carried out with P rats,and, in addition, no studies have been conducted to deter-mine the involvement of VTA dopamine neuronal activityin the local reinforcing effects of acetaldehyde. Coadmin-istration of 100 �M quinpirole (a D2,3 agonist) duringsessions 5 and 6 reduced both ethanol and acetaldehydeself-infusions, and reduced responses on the active lever tolevels observed on the inactive lever (for active lever re-sponses in session 4 versus sessions 5 and 6, all F values2,10� 12.3; all p values �0.002). During session 7, when etha-nol and acetaldehyde alone were given, P rats reinstatedresponding on the active lever (all F values2,12 � 14.6; all pvalues �0.001 for session 7 versus sessions 5 and 6) to levelsobserved in session 4.

Previously, we had determined that the self-infusion ofethanol into the posterior VTA by Wistar rats was depen-dent upon activation of local 5-HT3 receptors (Rodd-Henricks et al., 2003). However, similar studies have notbeen conducted with P rats, and, in addition, it is not knownif the self-infusion of acetaldehyde into the posterior VTAby P rats is mediated by a similar mechanism. Therefore, weexamined the effects of coadministration of ICS205–930(ICS) on the self-infusion of ethanol into the posteriorVTA by P rats. Coadministration of 100 �M ICS did notalter the acquisition or maintenance of 150 mg% ethanolself-infusion into the posterior VTA by P rats. However,coadministration of 200 �M ICS prevented the acquisitionand maintenance of 150 mg % ethanol self-infusion. Thiseffective concentration of the ICS compound was 4-fold

higher in the P than Wistar rat, suggesting differences inthe 5-HT3 receptor system within the posterior VTA be-tween the two rat strains. The effect of ICS on maintenanceof ethanol self-infusion was reversible, because P rats rein-stated responding on the active lever when 150 mg% eth-anol alone was given in session 7.

In contrast, coadministration of 100 or 200 �M ICS didnot inhibit the acquisition of 23 �M acetaldehyde self-infusion into the posterior VTA. P rats self-administering23 �M acetaldehyde with either 100 or 200 �M ICS dis-criminated between the active and inactive levers for all 7sessions (p values �0.026). For the maintenance experi-ment, during sessions 2 - 4, rats readily self-infused 23-�Macetaldehyde and discriminated the active from the inactivelever (all p values �0.05). However, coadministration of200 or 400 �M ICS did not alter responding on the activelever for the self-infusion of 23 �M acetaldehyde. Theseresults suggest that different mechanisms are involved inregulating the reinforcing effects of ethanol and acetalde-hyde within the posterior VTA of P rats.

In conclusion, the major findings of this study suggestthat ethanol and acetaldehyde can produce independentreinforcing effects within the posterior VTA of P rats.However, acetaldehyde is a 1000-fold more potent rein-forcer than ethanol in this subregion. Furthermore, thereinforcing effects of ethanol and acetaldehyde within theposterior VTA require activation of DA neurons. Finally,the reinforcing effects of ethanol within the posterior VTArequire activation of local 5-HT3 receptors, whereas thereinforcing effects of acetaldehyde do not, suggesting dif-ferent mechanisms are involved in mediating their effects.

ACETALDEHYDE INCREASES DOPAMINERGICTRANSMISSION IN THE LIMBIC SYSTEM

Milena Pisano and Marco Diana

Alcoholism is a major addictive disorder with profoundreflections on the individual and society. Among the vari-ous pharmacological treatments available for this disorder,disulfiram (Antabuse@) is the oldest (Chick et al., 1992;Fuller et al., 1986; Litten et al., 1996) and, perhaps, themost widely utilized. Its mechanism of action is thought toreside on the property to inhibit aldehyde dehydrogenasethrough which it should raise acetaldehyde blood levels,produced by ethanol ingested and metabolized by the al-cohol dehydrogenase normally found in gastric and hepatictissue of human beings (Baraona et al., 1991). In turn, theaugmented blood acetaldehyde concentrations are consid-ered to be aversive (Eriksson, 2001; Litten et al., 1996) andto form the basis for the well known “flushing syndrome,”commonly observed in many Orientals, an ethnic groupwith low incidence of alcoholism after ethanol ingestion.

On the other hand, at least some of the motivationalproperties of ethanol are thought to be mediated by themesolimbic dopamine (DA) system whose cell bodies arelocated in the ventro tegmental area (VTA) in the mid-


brain. Accordingly, acute ethanol administration increaseselectrophysiological activity of these neurons (Brodie et al.,1990; Gessa et al., 1986) and augments DA extracellularconcentrations in terminal areas (Imperato et al., 1986).Conversely, ethanol withdrawal decreases dopaminergicneuronal activity (Diana et al., 1993) and reduces DAconcentrations in the nucleus accumbens (Diana et al.,1993; Rossetti et al., 1992; Weiss et al., 1996). All thesestudies have suggested that both positive (reinforcing) andnegative (dysphoriant) properties induced by acute ethanoland by its withdrawal, respectively, are mediated, at leastpartially, by increments and decrements of DA neuronsprojecting to the nucleus accumbens (Pulvirenti and Diana,2001).

In contrast, studies suggest that acetaldehyde may par-ticipate in the motivational properties of ethanol (Aragonet al., 1986; Eriksson, 2001; Smith et al., 1997). Indeed,acetaldehyde is self-administered directly into the VTA ofalcohol-preferring rats (Rodd-Henricks et al., 2002) andinto the cerebral ventricles (Brown et al., 1979) of un-selected rodents. Further, when administered intracere-broventricularly, acetaldehyde is able to induce place-preference in rats (Smith et al., 1984) and to produce aconditioned stimulus preference even when administeredperipherally (Quertemont and De Witte, 2001). All thesestudies lend support to the hypothesis that central actionsof ethanol might be mediated by its metabolite acetalde-hyde instead.

In the present study, we sought to determine directly ifacetaldehyde administration alters DA neuronal activity inthe VTA and if this action bears any relationship withexogenously administered ethanol. To this aim, we blockedethanol metabolism with the alcohol dehydrogenase inhib-itor 4-methyl-pyrazole (4-MP) and studied the effect ofethanol and acetaldehyde on the electrophysiological prop-erties of DA-containing VTA neurons.

Male Sprague-Dawley albino rats (200/300 g) were usedin all experiments. Rats were divided into subgroups asfollows: 1) Acetaldehyde (n � 19) which received exponen-tially increasing doses (5–40 mg/kg/i.v.) of acetaldehyde. 2)Ethanol (n � 10) which received exponentially increasingdoses of ethanol (250–1000 mg/kg/i.v.). 3) Pretreated eth-anol (n � 5) which received a single dose of the alcoholdehydrogenase inhibitor 4-MP (90 mg/kg/i.p.) dissolved insaline and ethanol (250–1000 mg/kg/i.v.) 48 hr later. 4)Pretreated acetaldehyde (n � 5) which received a singledose of the alcohol dehydrogenase inhibitor 4-MP (90 mg/kg/i.p.) dissolved in saline and acetaldehyde (5–40 mg/kg/i.v.) 48 hr later. 5) Controls (n � 9) which received an equalvolume (0.1 ml/hg of body weight) of vehicle (saline i.p.)and 48 hr later ethanol (n � 4) or acetaldehyde (n � 5). Allgroups underwent identical surgical procedure. Subjectswere anesthetized with urethane (1.3 g/kg) i.p., the femoralvein was exposed and a catheter inserted into the lumento allow intravenous administration of pharmacologicalagents. Rats were then mounted on a stereotaxic appa-

ratus (Kopf, Tujunga CA) for the placement of a record-ing electrode filled with 0.5 M NaCl, above the VTA (AP1.8/2.0 from lambda; L 0.2/0.5 from midline). Presump-tive dopaminergic neurons were identified according towell established electrophysiological characteristics, i.e.,-Action potentials with biphasic or triphasic waveformsgreater than 2.5 msec. in duration.-A typically slow spon-taneous firing rate (2–5 Hz).-Occurrence of single andburst spontaneous firing pattern. The extracellular neu-ronal signal from single neurons was amplified (Neu-rolog System) and displayed on a digital oscilloscope(Tektronix TDS 3012) before storage on magnetic tapefor off-line analysis of the data. Data were logged on astandard PC computer through CED 1401 interface andfiring rate and pattern analysis were performed by CEDSpike2 system utilizing firing rate histograms generatedby CED Spike2 software. A burst was defined accordingto Grace and Bunney (1984) as a train of at least twospikes with the first interspike interval of 80 msec or lessand a termination interval greater than or equal to 160msec. Burst activity was analyzed according to the totalpercent of firing occurring in bursts called percentage ofbursts, and by the mean number of spikes within a burst(Diana et al., 1989). The analysis of these parameters(spikes/sec, spikes/burst and percentage of burst firing) isan important index for the activity of DA cells and allowsone to evaluate the influences that a putative drug exertsin the cell pattern. After five min of stable neuronalrecording (basal activity), exponentially increasing dosesof ethanol (0.25/0.25/0.5 g/kg) or acetaldehyde (5/5/10/20mg/kg) were injected i.v. every 2 min., so that last ad-ministered dose was equal to the sum of the drug alreadyreceived, and cell activity was recorded. Only one cellwas recorded per rat. Drug-induced modifications of thebasal activity were calculated in percentage for the 2 minperiod following each administration and compared withthe predrug baseline. Statistical significance of the datawas evaluated by one-way analysis of variance for re-peated measures (ANOVA). At the end of each record-ing section, DC current (5 �A for 15 min) was passedtrough the recording electrode to eject Pontamine skyblue which allowed the identification of the recordedcells. Brains were removed and fixed in 8% formalinsolution. The position of the electrodes was microscop-ically verified on sections (60 �m) stained with Cresylviolet.

The effect of acetaldehyde on VTA dopaminergic neuronalactivity was studied in a total of 19 VTA neurons. In 13 casesacetaldehyde was administered up to the cumulative dose of20 mg/kg i.v. and in the remaining 6 neurons a cumulativedose 40 mg/kg was reached. Since no statistical difference wasfound, basal activity values were pooled and analyzed fordifferences between pre- and post acetaldehyde.

Baseline firing rate was 3.08 � 0.25 (mean � SEM) andit was increased dose-dependently by intravenous acetalde-hyde administration. Acetaldehyde administration pro-


duced also an increment in the number of spikes containedin each burst (spikes/burst) and in the percentage of spikesdelivered in bursts (burst/firing). Intravenous ethanol ad-ministration (0.25–1 g/kg) produced increments in all threeparameters studied of similar magnitude.

To gain some further insight on the relative contributionof the two drugs (i.e., acetaldehyde and ethanol) to theactivation of VTA neurons, an additional group of rats (n� 10) was pretreated with the alcohol dehydrogenase in-hibitor 4-MP (Waller et al., 1982). Ethanol was then ad-ministered in 4-MP pretreated rats and relative controls(pretreated with saline) at the same doses administered innormal (untreated) animals. Ethanol-stimulating capacityupon VTA neuronal activity was completely abolished in4-MP rats. In contrast acetaldehyde administration in-creased neuronal activity in 4-MP pretreated rats to adegree similar to that observed in untreated rats.

The results presented here strongly suggest that the en-hancement of dopaminergic transmission after ethanol ad-ministration is, in fact, produced by acetaldehyde. Accord-ingly, acetaldehyde administration readily and dose-dependently increased firing rate, spikes/burst and burstfiring of DA-containing neurons of the VTA, brain regionwhich is known to be involved in the positive motivationalproperties of drugs of abuse in general, including ethanol.In addition, acetaldehyde stimulated electrophysiologicalparameters of DA neurons in animals in which ethanolmetabolism was pharmacologically blocked by the alcoholdehydrogenase inhibitor 4-MP, whereas ethanol was totallyineffective under this condition. This experiment indicatesthat conversion of ethanol into acetaldehyde is essential toobserve an enhancement of DA transmission after ethanoladministration. Further, acetaldehyde (5 �M) produces aninward current in DA neurons recorded in vitro in thewhole-cell configuration of the patch clamp technique (Me-lis and Bonci, unpublished results) suggesting a direct ef-fect on the membrane of DA neurons.

These results add significantly to a growing body ofevidence which lend support to the hypothesis that acetal-dehyde might be an active metabolite of ethanol in theeuphoriant properties of alcoholic beverages. Indeed, acet-aldehyde is self-administered directly into the VTA (Rodd-Henricks et al., 2002), and into the cerebral ventricles(Brown et al., 1979), produces place preference when ad-ministered intracerebroventricularly (Smith et al., 1984)and produces a conditioned stimulus preference even whenadministered peripherally (Quertemont and De Witte, 2001).

These results may also bear important consequences onthe therapeutic side of alcoholism and drug addiction, ingeneral. Indeed, according to the present results, blockadeof ethanol metabolism should deprive ethanol of its re-warding properties and, possibly, discourage individualsfrom intake. Accordingly, 4-MP has been found to be ef-fective in reducing spontaneous alcohol intake in rodentlines selected for high alcohol preference (Waller et al.,1982) and similar results were recently observed in human

nicotine addicts with lower metabolic capacity for nicotine(Pianezza et al., 1998). This would suggest that a reducedmetabolism of drugs of abuse, either pharmacologicallyobtained or genetically determined, may reduce the risk ofaddiction.

In conclusion the present results suggest that ethanolstimulates dopaminergic transmission in the limbic systemthrough its by-product acetaldehyde, previously thoughtonly as an aversive compound useful in the pharmacologi-cal treatment of alcoholics.

CONCLUSIONS

The results presented here clearly confirm that acetalde-hyde is a biologically active substance that induces a rangeof behavioral and neurochemical effects. Acetaldehydegreatly affects the locomotor activity of both rats and mice.While peripheral acetaldehyde injections are mainly de-pressant, Correa et al. showed that acetaldehyde also exertsstimulant effects when infused directly into the brain and inparticular into the substantia nigra pars reticula. Acetalde-hyde was also shown to induce strong amnesic effects atdoses below its sedative threshold. Additionally, this etha-nol’s metabolite was self-administered by rats into the pos-terior VTA, thereby indicating its reinforcing properties.McBride et al. also showed that acetaldehyde self-infusionrequires the activation of dopamine neurons. This conclu-sion was further supported by the results of Pisano andDiana, who showed that acetaldehyde injections increasethe firing rate of the VTA dopamine neurons. Altogetherthe results presented here indicated that acetaldehyde ex-hibits many of the properties that are usually expressed byaddictive drugs, i.e., locomotor stimulation, self-administration and activation of dopamine neurons, andtherefore suggested that acetaldehyde may play a signifi-cant role in alcohol addiction.

The present symposium also emphasized several ques-tions that remain unresolved or controversial.

1. The physiological significance of the behavioral effectsof acetaldehyde remains to be demonstrated in vivo. Inparticular, it remains to be shown that ethanol adminis-tration leads to brain acetaldehyde levels in sufficientconcentrations to produce significant pharmacologicaland behavioral effects.

2. It remains unclear whether acetaldehyde induces anxio-lytic effects. Correa et al. reported that brain acetalde-hyde infusions increased the exploration of the unpro-tected interior part of an open field, an effect generallyinduced by anxiolytic drugs. Additionally, Aragon et al.showed that the inhibition of catalase activity reducedethanol’s anxiolytic effects in an elevated x-maze, sug-gesting that brain acetaldehyde mediates the anxiolyticproperties of ethanol. However, Quertemont and Tam-bour found no evidence of the anxiolytic effects of ac-etaldehyde in the elevated plus-maze and no effect ofaldehyde dehydrogenase inhibition on ethanol’s anxio-


lytic properties. The reason for such discrepancies re-mains unknown and requires further studies.

3. It is unclear why peripheral acetaldehyde administrationinduced opposite locomotor effects from brain acetalde-hyde infusion. These discrepancies do not seem to berelated to the blood brain barrier permeability of acet-aldehyde, as it was recently shown that i.p. acetaldehydeinjections lead to very significant brain acetaldehydeconcentrations (Quertemont et al., 2004). It is possiblethat peripheral acetaldehyde induces depressant effectsthat mask its central activating effects, although suchperipheral effects remain to be defined.

4. The nature of the contribution of acetaldehyde to eth-anol’s effects also remain controversial. Some resultspresented here suggest that acetaldehyde mediatessome, but not all, of ethanol’s behavioral effects. Forexample, the inhibition of brain catalase was shown toreduce or even prevent several effects of ethanol, sug-gesting that brain acetaldehyde mediates these effects.In contrast, other results attribute a less ambitious rolefor acetaldehyde in ethanol’s effects. Although bothacetaldehyde and ethanol are self-infused into the pos-terior VTA, McBride et al. showed that different neu-rochemical mechanisms underlie their respective rein-forcing actions. Ethanol, but not acetaldehyde, self-administration involves the activation of local 5-HT3receptors. Therefore, acetaldehyde effects may potenti-ate rather than mediate ethanol’s reinforcing effects.[Correa et al., 2003d, Quertemont and De Witte, 2001]

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The Role of Acetaldehyde in the Central Effects of Ethanol

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