Backround While the molecular entity responsible for the rewarding effects of virtually all drugs of abuse is known; that for ethanol remains uncertain. Some lines of evidence suggest that the rewarding effects of alcohol are mediated not by ethanol per se but by acetaldehyde generated by catalase in the brain. However, the lack of specific inhibitors of catalase has not allowed strong conclusions to be drawn about its role on the rewarding properties of ethanol. The present studies determined the effect on voluntary alcohol consumption of two gene vectors; one designed to inhibit catalase synthesis and one designed to synthesize alcohol dehydrogenase, to respectively inhibit or increase brain acetaldehyde synthesis. Methods The lentiviral vectors, which incorporate the genes they carry into the cell genome, were: (i) one encoding a shRNA anticatalase synthesis and (ii) one encoding alcohol dehydrogenase (rADH1). These were stereotaxically microinjected into the brain ventral tegmental area (VTA) of Wistar-derived rats bred for generations for their high alcohol preference (UChB), which were allowed access to an ethanol solution and water. Results Microinjection into the VTA of the lentiviral vector encoding the anticatalase shRNA virtually abolished (-94% p<0.001) the voluntary consumption of alcohol by the rats. Conversely, injection into the VTA of the lentiviral vector coding for alcohol dehydrogenase greatly stimulated (2-3 fold p<0.001) their voluntary ethanol consumption. Conclusions The study strongly suggests that to generate reward and reinforcement, ethanol must be metabolized into acetaldehyde in the brain. Data suggest novel targets for interventions aimed at reducing chronic alcohol intake.

Ethanol non-drinker (UChA) and drinker (UChB) rat lines derived from an original Wistar colony have been selectively bred at the University of Chile for over 70 generations. Two main differences between these lines are clear. (1) Drinker rats display a markedly faster acute tolerance than non-drinker rats. In F2 UChA x UChB rats (in which all genes are 'shuffled'), a high acute tolerance of the offspring predicts higher drinking than a low acute tolerance. It is further shown that high-drinker animals 'learn' to drink, starting from consumption levels that are one half of the maximum consumptions reached after 1 month of unrestricted access to 10% ethanol and water. It is likely that acquired tolerance is at the basis of the increases in ethanol consumption over time. (2) Non-drinker rats carry a previously unreported allele of aldehyde dehydrogenase-2 (Aldh2) that encodes an enzyme with a low affinity for Nicotinamide-adenine-dinuclectide (NAD+) (Aldh2(2)), while drinker rats present two Aldh2 alleles (Aldh2(1) and Aldh2(3)) with four- to fivefold higher affinities for NAD+. Further, the ALDH2 encoded by Aldh2(1) also shows a 33% higher Vmax than those encoded by Aldh2(2) and Aldh2(3). Maximal voluntary ethanol intakes are the following: UChA Aldh2(2)/Aldh2(2) = 0.3-0.6 g/kg/day; UChB Aldh2(3)/Aldh2(3) = 4.5-5.0 g/kg/day; UChB Aldh2(1)/Aldh2(1) = 7.0-7.5 g/kg/day. In F2 offspring of UChA x UChB, the Aldh2(2)/Aldh2(2) genotype predicts a 40-60% of the alcohol consumption. Studies also show that the low alcohol consumption phenotype of Aldh2(2)/Aldh2(2) animals depends on the existence of a maternally derived low-activity mitochondrial reduced form of nicotinamide-adenine-dinucleotide (NADH)-ubiquinone complex I. The latter does not influence ethanol consumption of animals exhibiting an ALDH2 with a higher affinity for NAD+. An illuminating finding is the existence of an 'acetaldehyde burst' in animals with a low capacity to oxidize acetaldehyde, being fivefold higher in UChA than in UChB animals. We propose that such a burst results from a great generation of acetaldehyde by alcohol dehydrogenase in pre-steady-state conditions that is not met by the high rate of acetaldehyde oxidation in mitochondria. The acetaldehyde burst is seen despite the lack of differences between UChA and UChB rats in acetaldehyde levels or rates of alcohol metabolism in steady state. Inferences are drawn as to how these studies might explain the protection against alcoholism seen in humans that carry the high-activity alcohol dehydrogenase but metabolize ethanol at about normal rates.

A quantitative genetic approach, which involves correlation of transcriptional networks with the phenotype in a recombinant inbred (RI) population and in selectively bred lines of rats, and determination of coinciding QTLs for gene expression and the trait of interest, has been applied in the current study. In this analysis, a novel approach was used that combined DNA-Seq data, data from brain exon array analysis of HXB/BXH RI rat strains and six pairs of rat lines selectively bred for high and low alcohol preference, and RNA-Seq data (including rat brain transcriptome reconstruction) to quantify transcript expression levels, generate co-expression modules, and identify biological functions that contribute to the predisposition to consume varying amounts of alcohol. A gene co-expression module was identified in the RI rat strains that contained both annotated and unannotated transcripts expressed in brain, and was associated with alcohol consumption in the RI panel. This module was found to be enriched with differentially expressed genes from the selected lines of rats. The candidate genes within the module and differentially expressed genes between high and low drinking selected lines were associated with glia (microglia and astrocytes), and could be categorized as being related to immune function, energy metabolism and calcium homeostasis, and glial-neuronal communication. Our results illustrate that there are multiple combinations of genetic factors that can produce the same phenotypic outcome. While no single gene accounts for predisposition to a particular level of alcohol consumption in every animal model, coordinated differential expression of subsets of genes in the identified pathways produce similar phenotypic outcomes.

Individuals who carry the most active alcohol dehydrogenase (ADH) isoforms are protected against alcoholism. This work addresses the mechanism by which a high ADH activity leads to low ethanol intake in animals. Male and female ethanol drinker rats (UChB) were allowed access to 10% ethanol for 1 h. Females showed 70% higher hepatic ADH activity and displayed 60% lower voluntary ethanol intake than males. Following ethanol administration (1 g/kg ip), females generated a transient blood acetaldehyde increase ("burst") with levels that were 2.5-fold greater than in males (P Ͻ 0.02). Castration of males led to 1) an increased ADH activity (ϩ50%, P Ͻ 0.001), 2) the appearance of an acetaldehyde burst (3-to 4-fold vs. sham), and 3) a reduction of voluntary ethanol intake comparable with that of naïve females. The ADH inhibitor 4-methylpyrazole blocked the appearance of arterial acetaldehyde and increased ethanol intake. Since the release of NADH from the ADH ⅐ NADH complex constitutes the rate-limiting step of ADH (but not of ALDH2) activity, endogenous NADH oxidizing substrates present at the time of ethanol intake may contribute to the acetaldehyde burst. Sodium pyruvate given at the time of ethanol administration led to an abrupt acetaldehyde burst and a greatly reduced voluntary ethanol intake. Overall, a transient surge of arterial acetaldehyde occurs upon ethanol administration due to 1) high ADH levels and 2) available metabolites that can oxidize hepatic NADH. The acetaldehyde burst is strongly associated with a marked reduction in ethanol intake. male; female; orchidectomy; arterial; pyruvate; limited access ETHANOL IS METABOLIZED IN THE LIVER by alcohol dehydrogenase (ADH) into acetaldehyde, a metabolite that is subsequently oxidized into acetate by aldehyde dehydrogenase (ALDH2). Humans who carry a dominant negative mutation of the gene that codes for mitochondrial aldehyde dehydrogenase (33) display a marked elevation in blood acetaldehyde levels following ethanol intake, which curtails further alcohol use (15). Individuals carrying this allele (ALDH2*2) are protected by 66 to 99% against alcohol abuse and alcoholism (7,8,30,31). Although it has been suggested that acetaldehyde generated in the brain may be a rewarding metabolite (20), high levels of blood acetaldehyde are aversive, as shown by the marked inhibition of ethanol intake in animals in which peripheral ALDH2 activity is reduced and blood acetaldehyde levels are increased by antisense drugs that do not penetrate the bloodbrain barrier (6).Individuals carrying an ADH allele (ADH1B*2) that codes for an isoform that is one order of magnitude more active than that coded by the usual ADH1B*1 (9) are also protected against heavy alcohol use and alcoholism (26; see also meta-analysis, Ref. 34). The highly active ADH is found in some East Asians and Ashkenazi Jews. The steady-state rate of ethanol metabolism in individuals carrying the ADH1B*2 allele is only 8 -13% greater than that of subjects carrying ADH1B*1 (18), indicating that in humans the maxima...

Background In animal models of continuous alcohol self-administration, in which physical dependence does not constitute the major factor of ethanol intake, two factors likely contribute to the perpetuation of alcohol self-administration: (i) the rewarding effects of ethanol and (ii) the contextual conditioning cues that exist along with the process of self-administration. Present studies are aimed at understanding the relative contribution of these factors on the perpetuation of heavy alcohol self-administration, as an indication of relapse. Methods Wistar derived UChB high ethanol drinker rats were allowed access to 10% ethanol and water on a 24-hour basis. In initial studies, an anticatalase shRNA gene coding lentiviral vector aimed at inhibiting acetaldehyde generation was administered into the ventral tegmental area (VTA) of the animals prior to ethanol access. In subsequent studies the lentiviral vector was administered to animals, which had consumed ethanol on a 24-hour basis, or a one-hour basis, after the animals had reached high levels of ethanol intake for 60–80 days. In final studies, quinine (0.01%) was added to the ethanol solution to alter the conditioning taste/smell cues of alcohol that animals had chronically ingested. Results Data indicate that the administration into the VTA of an anti-catalase vector to naïve animals blocked reward and alcohol self-administration in naive animals while it was, nevertheless, inactive in inhibiting alcohol self-administration in rats that had been conditioned to ingest ethanol for over 2 months. The lack of inhibitory effect on ethanol intake of the anti-catalase vector in animals that had chronically self-administered ethanol was fully reversed when the contextual conditioning cues of the alcohol solution were changed. Conclusions Data highlight the importance of conditioning factors in relapse and suggest that only abolishing or blunting it, along with appropriate pharmacological treatments to reduce ethanol reward, may have protracted effects in reducing alcohol self-administration.

Previous studies suggest that acetaldehyde generated from ethanol in the brain is reinforcing. The present studies tested the feasibility of achieving a long-term reduction of chronic and post-deprivation binge ethanol drinking by a single administration into the brain ventral tegmental area (VTA) of a lentiviral vector that codes for aldehyde dehydrogenase-2 (ALDH2), which degrades acetaldehyde. The ALDH2 gene coding vector or a control lentiviral vector were microinjected into the VTA of rats bred for their alcohol preference. In the chronic alcohol administration model, naïve animals administered the control vector and subsequently offered 10% ethanol and water ingested 8-9 g ethanol/kg body weight/day. The single administration of the ALDH2-coding vector prior to allowing ethanol availability reduced ethanol drinking by 85-90% (P < 0.001) for the 45 days tested. In the post-deprivation binge-drinking model, animals that had previously consumed ethanol chronically for 81 days were administered the lentiviral vector and were thereafter deprived of ethanol for three 7-day periods, each interrupted by a single 60-minute ethanol re-access after the last day of each deprivation period. Upon ethanol re-access, control vector-treated animals consumed intoxicating 'binge' amounts of ethanol, reaching intakes of 2.7 g ethanol/kg body weight in 60 minutes. The administration of the ALDH2-coding vector reduced re-access binge drinking by 75-80% (P < 0.001). This study shows that endowing the ventral tegmental with an increased ability to degrade acetaldehyde greatly reduces chronic alcohol consumption and post-deprivation binge drinking for prolonged periods and supports the hypothesis that brain-generated acetaldehyde promotes alcohol drinking.

Several studies on the differences between ethanol-preferring versus non-preferring rat lines suggest an innate deficit in the mesolimbic dopaminergic system as an underlying factor for ethanol volition. Rats would try to overcome such deficit by engaging in a drug-seeking behaviour, when available, to drink an ethanol solution over water. Thus, in the present study we compared the effect of a single dose of ethanol (1g /kg, i.p.) on the extracellular levels of monoamines measured by microdialysis in the shell of nucleus accumbens of University of Chile bibulous (UChB) and University of Chile Abstainer (UChA) rats, bred for 79 and 88 generations to prefer or reject ethanol, respectively. It is reported that under basal conditions extracellular dopamine levels are lower in the bibulous than in the abstainer rats, while ethanol induced a 2-fold greater increase of dopamine release in bibulous than in abstainer rats. The greater effect of ethanol in bibulous rats was not associated to differences in blood ethanol levels, since the concentration and elimination of ethanol were virtually identical in both rat lines, indicating that bibulous rats are more sensitive to the stimulation of dopamine release by ethanol than abstainer rats. No differences were observed in 5-hydroxytryptamine or metabolites measured simultaneously under basal or ethanol-stimulating conditions in bibulous and abstainer rats. Overall, the present results suggest that a low dopaminergic tone and a strong mesolimbic dopamine response to ethanol are concerted neurochemical features associated to an ethanol-seeking behaviour in rats.

Humans who carry a point mutation in the gene coding for alcohol dehydrogenase-1B (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100-fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild-type rat cDNA encoding rADH-47Arg was mutated to encode rADH-47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol-preferring rats. The V(max) of rADH-47His was 6-fold higher (P<0.001) than that of the wild-type rADH-47Arg. Animals transduced with rAdh-47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P<0.001) in voluntary ethanol intake. In animals transduced with rAdh-47His, administration of ethanol (1g/kg) produced a short-lived increase of arterial blood acetaldehyde concentration to levels that were 3.5- to 5-fold greater than those in animals transduced with the wild-type rAdh-47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.