Lutske Tampier

The UChA and UChB rat lines : metabolic and genetic differences influencing ethanol 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 × 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+) (Aldh22), while drinker rats present two Aldh2 alleles (Aldh21 and Aldh23) with four‐ to fivefold higher affinities for NAD+. Further, the ALDH2 encoded by Aldh21 also shows a 33% higher Vmax than those encoded by Aldh22 and Aldh23. Maximal voluntary ethanol intakes are the following: UChA Aldh22/Aldh22 = 0.3–0.6 g/kg/day; UChB Aldh23/Aldh23 = 4.5–5.0 g/kg/day; UChB Aldh21/Aldh21 = 7.0–7.5 g/kg/day. In F2 offspring of UChA × UChB, the Aldh22/Aldh22 genotype predicts a 40–60% of the alcohol consumption. Studies also show that the low alcohol consumption phenotype of Aldh22/Aldh22 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.

Ethanol as a Prodrug: Brain Metabolism of Ethanol Mediates its Reinforcing effects

BACKGROUND  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 (ADH), 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 ADH greatly stimulated (2 to 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.

Effects of Aminotriazole on Ethanol, Water, and Food Intake and on Brain Catalase in UChA and UChB Rats

Aminotriazole (AT), a catalase inhibitor, was administered to UChA (low ethanol consumer) and UChB (high ethanol consumer) rats. Ethanol, water, and solid food intake were measured during basic, treatment, and posttreatment periods. The effects of AT on brain catalase activity and acetaldehyde recovered during incubation of brain homogenates with ethanol were also studied in rats of both strains. Results showed that AT decreased voluntary ethanol intake in UChB rats, and also diminished the consumption of food by rats of both strains. No strain difference in brain catalase activity and acetaldehyde recovered during ethanol incubation was observed. The results suggest that AT effect on ethanol consumption is secondary to a reduction in the appetite for calories and not related to its catalase blocking effect.

Baclofen reduces ethanol intake in high-alcohol-drinking University of Chile bibulous rats.

Treatment with γ‐aminobutiric acid (GABAB) receptor agonist, ±baclofen, has been shown to reduce ethanol intake in selectively bred Sardinian alcohol‐preferring rats. The general goal of the present study was to characterize the high ethanol consumption high‐alcohol‐drinking University of Chile bibulous (UChB) rats with regard to the anti‐alcohol effect of GABAB receptor stimulation. UChB rats were treated with the more active enantiomer of baclofen [R(+)‐baclofen; at a dose of 1.0, 2.0 or 3.0 mg/kg] administered intraperitoneally once daily for four consecutive days or a single dose. When comparing ethanol and saccharin consumption in a free‐choice regimen with unlimited access 24 hours/day, the dose of baclofen required to attenuate ethanol consumption significantly was 1.0 mg/kg administered once a day for three consecutive days while the dose that was sufficient to affect saccharin consumption significantly was 2.0 mg/kg, indicating that baclofen was more potent in reducing ethanol intake by UChB rats than reducing saccharin consumption. The reduction of ethanol or saccharin intake can not be attributed to baclofen‐induced motor impairment, since baclofen (1.0, 2.0 or 3.0 mg/kg) did not alter spontaneous locomotor activity in UChB rats. Baclofen dose‐dependently suppressed the motor activity stimulated by ethanol administration, a phenomenon mediated by activation of the mesolimbic dopamine system. In conclusion, these results showed that the activation of GABAB receptor by R(+)‐baclofen reduced ethanol and saccharin consumption, as well as ethanol‐induced motor stimulation, implicating the GABAB receptor in the neural substrates mediating effects that sustain voluntary ethanol in take in UChB rats.

Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.

Humans who carry a point mutation in the gene coding for alcohol dehydrogenase‐lB (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 Vmax 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.—Rivera‐Meza, M., Quin‐tanilla, M. E., Tampier, L., Mura, C. V., Sapag, A., Israel, Y. Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model. FASEB J. 24, 266–274 (2010). www.fasebj.org

Ethanol induces stronger dopamine release in nucleus accumbens (shell) of alcohol-preferring (bibulous) than in alcohol-avoiding (abstainer) rats

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 (1 g/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.

Effect of 3-amino-1,2,4-triazole on narcosis time and lethality of ethanol in UChA rats

The capacity of rat brain homogenates to oxidize ethanol by catalase peroxidative system, previously reported, was reevaluated in experiments using lower ethanol concentration, showing that the effect of this system can be observed even with a concentration of 50 mM, equivalent to non lethal blood level. The involvement of catalase was confirmed by its blocking by aminotriazole (AT) or methanol but not by pyrazole or butanol. Evidence for a functional role of ethanol oxidation by brain catalase in the action of this substance was given by the fact that rats pretreated with AT (1 g/kg IP) exhibited a significant shorter narcosis than untreated controls, strongly suggesting the mediation of acetaldehyde in this effect. Previous results with doses of 60 mmole/kg IP were confirmed with 70 mmole/kg IP, but not with 90 mmole/kg IP. A significant prolonging of narcosis time was observed when AT was administered after any of these doses by an unknown mechanism. Furthermore it was observed that AT pretreatment reduced significantly the lethal effect of 110 mmole/kg IP ethanol; but when AT was given after ethanol (90 mmole/kg IP) it enhanced the lethality. These results suggest that catalase peroxidative pathway might play a role not only in narcosis time but also in ethanol toxicity.

Aversion to acetaldehyde: differences in low-alcohol-drinking (UChA) and high-alcohol-drinking (UChB) rats.

We have previously found the existence of a relation between activity of the brain mitochondrial aldehyde dehydrogenase (ALDH2) and consumption of ethanol in rats of the low-alcohol-drinking (UChA) and the high-alcohol-drinking (UChB) strains. The aim of the present study was to determine whether UChA and UChB rats also differed in sensitivity to the aversive effects of acetaldehyde (AcH). Aversion to AcH was studied by using a conditioned taste aversion (CTA) paradigm. Ethanol naive UChA and UChB rats were administered AcH intraperitoneally (50, 100, or 150 mg/kg) or saline and exposed to a banana-flavored solution during five conditioning trials. A strong dose-dependent CTA to AcH was found in UChA rats, whereas UChB rats did not show a CTA to any dose of AcH. At equal doses of AcH, cerebral venous blood AcH levels in UChA rats were consistently higher than in UChB rats, a finding that may reflect the previously observed differences in the activity of ALDH2 between these strains. However, this observation is unlikely to explain fully the differences observed because aversion to AcH was developed in the UChA strain at blood levels of AcH that did not produce any aversion in the UChB strain. These results support the suggestion that, for the first time, differences in central or systemic effects of AcH per se may play a major role in determining the aversion to AcH in drinker and nondrinker animals.

Reward and Relapse: Complete Gene-Induced Dissociation in an Animal Model of Alcohol Dependence

BACKGROUND In animal models of continuous alcohol self-administration, in which physical dependence does not constitute the major factor of ethanol intake, 2 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 1-hour basis, after the animals had reached high levels of ethanol intake for 60 to 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 of an anticatalase vector into the VTA of naïve animals blocked reward and alcohol self-administration, 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 of the anticatalase vector on ethanol intake 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 long-lasting pharmacological treatment to reduce ethanol reward, may have protracted effects in reducing alcohol self-administration.

Polymorphisms in the mitochondrial aldehyde dehydrogenase gene (Aldh2) determine peak blood acetaldehyde levels and voluntary ethanol consumption in rats

Dependence on alcohol, a most widely used drug, has a heritability of 50–60%. Wistar-derived rats selectively bred as low-alcohol consumers for many generations present an allele (Aldh22) of mitochondrial aldehyde dehydrogenase that does not exist in high-alcohol consumers, which mostly carry the Aldh21 allele. The enzyme coded by Aldh22 has a four- to five-fold lower affinity for NAD+ than that coded by Aldh21. The present study was designed to determine whether these polymorphisms account for differences in voluntary ethanol intake and to investigate the biological mechanisms involved. Low-drinker F0Aldh22/Aldh22 rats were crossed with high-drinker F0Aldh21/Aldh21 rats to obtain an F1 generation, which was intercrossed to obtain an F2 generation that segregates the Aldh2 alleles from other genes that may have been coselected in the breeding for each phenotype. Data show that, with a mixed genetic background, F2Aldh21/Aldh21 rats voluntarily consume 65% more alcohol (P<0.01) than F2Aldh22/Aldh22 rats. A major phenotypic difference was a five-fold higher (P<0.0025) peak blood acetaldehyde level following ethanol administration in the lower drinker F2Aldh22/Aldh22 compared to the higher drinker F2Aldh21/Aldh21 animals, despite the existence of identical steady-state levels of blood acetaldehyde in animals of both genotypes. Polymorphisms in Aldh2 play an important role in: (i) determining peak blood acetaldehyde levels and (ii) modulating voluntary ethanol consumption. We postulate that the markedly higher peak of blood acetaldehyde generated in Aldh22/Aldh22 animals is aversive, leading to a reduced alcohol intake in Aldh22/Aldh22 versus that in Aldh21/Aldh21 animals.