Increase in Free Choice Oral Ethanol Self-Administration in Catechol-O-Methyltransferase Gene-Disrupted Male Mice

Authors


Author for correspondence: Anne Tammimäki, Division of Pharmacology and Toxicology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, FIN-00014 Helsinki, Finland (fax +358 9 191 59471, e-mail anne.tammimaki@helsinki.fi).

Abstract

Abstract:  The effect of catechol-O-methyltransferase (Comt) gene disruption on the voluntary oral consumption of water, ethanol (2.5–20%, v/v) and cocaine (0.1–0.8 mg/ml) was studied in the free-choice, two-bottle paradigm in male and female mice. Solutions containing ethanol or cocaine, or tap water were available ad libitum from drinking burettes for 4 weeks. Catechol-O-methyltransferase-deficient male mice consumed significantly more ethanol than their wild-type male littermates. In contrast, female mice did not show genotype differences in the consumption of ethanol solutions. During the cocaine experiment, male mice developed either a side preference or an aversion that obscured cocaine consumption. This pattern of drinking was not dependent on Comt genotype. In female mice, Comt genotype was not associated with cocaine consumption. In conclusion, disruption of Comt gene influenced ethanol consumption in a gender-dependent manner in mice, supporting the hypothesis that low catechol-O-methyltransferase activity is one of the predisposing factors for high alcohol consumption in males.

Catechol-O-methyltransferase catalyses the metabolism of catecholamines and other catechols both in the brain and in the peripheral tissues. In striatum, uptake by the dopamine transporter removes dopamine from the synaptic cleft [1–3]. However, in brain areas with a low dopamine transporter density (e.g. in the prefrontal cortex), the role of catechol-O-methyltransferase in the control of dopaminergic transmission is more crucial [4–6]. Recently, it was shown that in catechol-O-methyltransferase (Comt) gene-disrupted mice, elimination of dopamine in prefrontal cortex was two times slower than in wild-type animals, whereas dopamine clearance in the striatum was normal [7]. Prefrontal cortex is involved in certain aspects of cognition [8,9]. It sends projections and modulates dopaminergic transmission in subcortical areas and even dopamine synthesis in the midbrain [10]. These pathways are critically important not only in reward and emotions but also in the development of addiction [11].

The common functional Val108/158Met polymorphism is known to control human catechol-O-methyltransferase activity. The Met allele results in a heat-labile enzyme with reduced enzyme activity [12,13]. Comt Val108/158Met polymorphism has been proposed to affect cognitive functions [14,15], but its role in alcoholism remains unclear. Some studies have shown an association between the high catechol-O-methyltransferase activity and alcoholism in males [16]. In contrast, there are reports claiming an association between the low enzyme activity and high alcohol consumption in non-alcoholic males [17] as well as in individuals with type 1 and type 2 alcoholism [18,19]. Several reports also indicate that catechol-O-methyltransferase polymorphism is not linked to alcoholism [20].

Previous studies with catechol-O-methyltransferase-deficient mice have shown that the gene disruption has only minor effects on striatal dopamine levels under normal conditions [21,22] even though the levels of 3,4-dihydroxyphenylacetic acid (DOPAC) are increased three to four times in homozygous animals [23]. Instead, the catechol-O-methyltransferase-deficient male mice are less sensitive to the motor activation caused by cocaine and GBR 12909, as well as to the initial motor depression caused by high amphetamine doses [21,24].

The aim of the present study was to evaluate the role of Comt gene disruption or catechol-O-methyltransferase activity as a predisposing factor for high alcohol consumption and cocaine use. Thus, voluntary ethanol and cocaine consumptions were assessed in a free-choice two-bottle system in Comt gene-disrupted mice of both genders.

Materials and Methods

Animals.  Male and female Comt gene-disrupted homozygous [Comt(–/–)] and heterozygous [Comt(+/–)] mice as well as their wild-type littermates, age 2–6 months (ethanol treatment) or 6–8 months (cocaine treatment) at the beginning of the experiments, were used. The Comt gene-disrupted mouse strain was originally generated by Gogos et al. [22] on mixed 129Sv × C57BL/6J background and later backcrossed for more than 20 generations on pure C57BL/6J background. Mice were bred in the National Laboratory Animal Center, Kuopio, Finland, and heterozygous males and females were used as breeding couples. To keep the strain viable, it was enriched regularly by mating C57BL/6J females obtained from Harlan, The Netherlands, with heterozygous males, and their heterozygous offspring were used further for breeding. The mice were housed individually at an ambient temperature of 21–23° under a 12-hr light:dark cycle with free and continuous access to food pellets and drinking fluid. The oestrus phase was not determined. All procedures with animals were performed according to European Community Guidelines for the use of experimental animals (European Communities Council Directive 86/609/EEC) and reviewed and approved by the Animal Ethics Committee at the University of Helsinki in conformance with current legislation.

Genotyping.  Genomic DNA was isolated from tail biopsies as described by Laird et al. [25]. For genotyping, a polymerase chain reaction (PCR) method was developed using 5′-ACCATGGAGATTAACCCTGACTACG-3′ (sense), 5′-GTGTGTCTGGAAGGTAGCGGTC-3′ (antisense) primer set to detect catechol-O-methyl transferase gene (Comt) alleles, and 5′-GTGTTC CGGCTGTCAGCGCA-3′ (sense), 5′-GTCCTGATAGCGGTCCGCCA-3′ (antisense) primer set to detect mutant alleles containing the neomycin gene (neo) cassette that replaces exons 2–4 of the Comt gene. Genomic PCR was completed using the FailSafe PCR system (Epicentre Technologies, Madison, WI, USA) using FailSafe buffer B and the following thermal cycles: an initial denaturing at 98° for 1 min. and 35 cycles consisting of denaturing temperature of 94° for 30 sec., annealing temperature of 65° for 1 min., and extension at 72° for 3 min. with a final extension of 70° for 10 min. The amplified fragments were visualized by ethidium bromide staining under ultraviolet light after electrophoresis in a 1.7% agarose gel (fig. 1).

Figure 1.

Detection of catechol-O-methyltransferase gene (Comt) or neo insertion in genomic DNA. On the left 100 bp molecular weight marker. WT, wild-type; HET, heterozygous; HOM, homozygous; 0, sterile water.

Free choice oral consumption of ethanol and cocaine.

Cage arrangements.  Drinking burettes were constructed of custom-made glass tips (Laborexin, Helsinki, Finland) and graded plastic cylinders connected to each other with plastic tubing and capped with rubber plugs. Similar burettes have been used previously in alcohol drinking studies [26]. The burettes were attached to the cage with a mounting of stainless steel wire. The box was divided into two compartments by a plywood partition: on one side, there was bedding and nesting material available, and on the other side, where the burette tips passed through the partition, the floor was fitted with a metal grid consisting of folded stainless steel wire mesh. This system prevented the burettes from becoming blocked by bedding material.

Experimental setup for the free-choice oral self-administration.  Male and female mice of all genotypes had free choice between tap water and ethanol or cocaine solution. Before the presentation of drug solutions, the mice were given tap water in both burettes for 2 days. Evaporation was assessed by measuring the water loss from burettes kept in empty boxes. Evaporation was found to be less than 0.1 ml/day and, therefore, it was ignored. The fluid consumption was registered daily for 4 weeks and the positions of control and drug burettes were interchanged every 4 days to avoid development of place preference.

Drinking solutions.  Ethanol was given in the following concentrations: 2.5% (v/v; 20 mg/ml; days 1–7), 5% (40 mg/ml; days 8–14), 10% (80 mg/ml; days 15–21) and 20% (160 mg/ml; days 22–28). Dilutions were made from 96% ethanol (Altia, Rajamäki, Finland) without any additives. Cocaine was given in concentrations 0.1 mg/ml (days 1–4), 0.2 mg/ml (days 5–12), 0.4 mg/ml (days 13–20) and 0.8 mg/ml (days 21–28). Cocaine hydrochloride (University Pharmacy, Helsinki, Finland) was dissolved in tap water. In order to improve the stability of the solution, the pH was adjusted to 3.2 with hydrochloric acid. Furthermore, the pH of water was adjusted similarly in order to ensure that the mice would make the decision based on the drug's effects and not on any possible aversive taste or smell of the acid. Fresh cocaine or ethanol solutions were prepared every 4 days and stored in a refrigerator. Burettes were refilled either every day (cocaine) or every 4 days (ethanol), depending on the stability of the solution.

Statistical analysis.  Results are given as means ± S.E.M. Daily drinking data and weekly weight data were analysed with two-way anova for repeated measures (sex and genotype as between-subjects variables and time as within-subjects variable). Tukey's honestly significant difference test was used as the post hoc test where appropriate. All the analyses were conducted with SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Results were considered significant at P < 0.05.

Results

Oral self-administration of ethanol.

Consumption of ethanol and water as well as ethanol preference ratios in male and female mice (n = 10 in each group) are shown in fig. 2. Generally, the mice tolerated ethanol well, and they gained weight normally (table 1). Female mice consumed more ethanol (ml/kg) than males and the association of genotype and ethanol consumption was different between sexes [sex effect: F(1, 51) = 26.811, P < 0.001; genotype effect: F(2, 51) = 1.114, P > 0.05; sex × genotype interaction: F(2, 51) = 4.577, P < 0.05]. Females also received a higher ethanol dose (g/kg) than males [sex effect: F(1, 51) = 49.739, P < 0.001; genotype effect: F(2, 51) = 1.069, P > 0.05; sex × genotype interaction: F(2, 51) = 3.058, P > 0.05]. Male Comt(–/–) and Comt(+/–) mice consumed more ethanol (ml/kg) than wild-type males [male Comt(–/–) versus wild-type male P < 0.01; male Comt(+/–) versus wild-type male P < 0.05] and, consequently, they received a higher ethanol dose (g/kg) [male Comt(–/–) versus wild-type male P < 0.05; male Comt(+/–) versus wild-type male P < 0.01]. Male Comt(–/–) mice also displayed a higher preference for ethanol solutions than wild-type males [male Comt(–/–) versus wild-type male P < 0.05]. At the lowest concentrations given during the first 2 weeks, the ethanol preference of homozygous female mice tended to be lower than that of the other genotypes. However, the association between Comt genotype and ethanol consumption or preference was not significant in females. Female mice also consumed more water than males. Again, the Comt-disrupted animals did not differ from wild-type mice [sex effect: F(1, 51) = 14.038, P < 0.001; genotype effect: F(2, 51) = 0.280, P > 0.05; sex × genotype interaction: F(2, 51) = 2.399, P > 0.05]. The weight of animals remained constant throughout the experiment.

Figure 2.

Ethanol consumption in Comt gene-disrupted mice. (A, B) Ethanol dose (g/kg/day) in male and female mice. (C, D) Consumption of ethanol solutions (2.5–20%, v/v; ml/kg/day) in male and female mice. (E, F) Consumption of water (ml/kg/day) in male and female mice. (G, H) Ethanol preference ratio in male and female mice. Preference was calculated as percentage of ethanol solution of the total volume of liquid consumed. Day 0 = water, days 1–7 = 2.5% ethanol, days 8–14 = 5% ethanol, days 15–21 = 10% ethanol, days 22–28 = 20% ethanol. Arrows indicate the days when the burette positions were reversed; n = 10 in each group. WT, wild-type; HET, Comt(+/–); HOM, Comt(–/–). *P < 0.05, **P < 0.01 Comt(–/–) versus wild-type, P < 0.05, P < 0.01 Comt(+/–) versus wild-type. Results are given as mean ± S.E.M.

Table 1. 
Effect of voluntary cocaine and ethanol drinking on mouse weight.
TreatmentGenderGenotypeBefore experiment (g)Week 1 (g)Week 2 (g)Week 3 (g)Week 4 (g)
  1. Values represent means ± S.E.M. of mouse body weights. Repeated measures anova results for cocaine treatment: time effect: F(4, 184) = 49.607, P < 0.001; time × sex interaction: F(4, 184) = 4.552, P < 0.01; time × genotype interaction: F(8, 184) = 0.423, P > 0.05; time × sex × genotype interaction: F(8, 184) = 0.941, P > 0.05. WT, wild-type; HET, Comt(+/–); HOM, Comt(–/–); n = 7–11 in each group.

CocaineMaleWT31.5 ± 1.231.3 ± 1.031.2 ± 0.930.9 ± 0.729.3 ± 0.6
HET30.1 ± 0.830.1 ± 0.930.3 ± 0.829.8 ± 1.029.1 ± 1.0
HOM28.5 ± 0.928.5 ± 0.928.9 ± 0.927.5 ± 0.926.7 ± 1.0
FemaleWT25.1 ± 0.625.1 ± 0.425.0 ± 0.522.8 ± 0.722.8 ± 0.9
HET26.4 ± 1.326.7 ± 1.326.0 ± 1.223.4 ± 1.124.1 ± 1.0
HOM25.1 ± 0.625.0 ± 0.724.9 ± 0.622.0 ± 0.723.3 ± 0.9
EthanolMaleWT29.7 ± 1.129.7 ± 1.129.7 ± 1.229.5 ± 1.331.2 ± 1.5
HET28.2 ± 0.926.9 ± 0.826.5 ± 0.727.2 ± 0.828.9 ± 0.9
HOM29.6 ± 1.129.1 ± 0.928.3 ± 1.128.9 ± 0.829.6 ± 0.8
FemaleWT21.6 ± 0.721.5 ± 0.622.2 ± 0.622.4 ± 0.522.2 ± 0.6
HET20.9 ± 0.521.6 ± 0.421.8 ± 0.421.8 ± 0.421.7 ± 0.3
HOM22.0 ± 0.522.1 ± 0.422.1 ± 0.421.8 ± 0.421.8 ± 0.5

Oral self-administration of cocaine.

Consumption of cocaine and water as well as cocaine preference ratios in male and female mice are shown in fig. 3. Cocaine treatment resulted in weight loss in both genders indicating that the dose of cocaine was high enough to cause anorexia (table 1). The weight loss was more pronounced in females, but it was not dependent on Comt genotype [time effect: F(4, 184) = 49.607, P < 0.001; time × sex interaction: F(4, 184) = 4.552, P < 0.01; time × genotype interaction: F(8, 184) = 0.423, P > 0.05; time × sex × genotype interaction: F(8, 184) = 0.941, P > 0.05].

Figure 3.

Cocaine consumption in Comt gene-disrupted mice. (A, B) Cocaine dose (mg/kg/day) in male and female mice. (C, D) Consumption of cocaine solutions (0.1–0.8 mg/ml; ml/kg/day) in male and female mice. (E, F) Consumption of water (ml/kg/day) in male and female mice. (G, H) Cocaine preference ratio in male and female mice. Preference was calculated as percentage of cocaine solution of the total volume of liquid consumed. Day 0 = water, days 1–4 = 0.1 mg/ml cocaine, days 5–12 = 0.2 mg/ml cocaine, days 13–20 = 0.4 mg/ml cocaine, days 21–28 = 0.8 mg/ml cocaine. Arrows indicate the days when burette positions were reversed; n = 7–11 in each group. WT, wild-type; HET, Comt(+/–); HOM, Comt(–/–). Results are given as mean ± S.E.M.

The overall consumption of cocaine solution (ml/kg) as well as the cocaine dose (mg/kg) was higher in females because of their more stable drinking pattern. However, the consumption [sex effect: F (1, 44) = 17.241, P < 0.001; genotype effect: F(2, 44) = 2.916, P > 0.05; sex × genotype interaction: F(2, 44) = 1.319, P > 0.05; time × sex interaction: F(27, 1188) = 3.044, P < 0.001], dose [sex effect: F(1, 44) = 8.964, P < 0.001; genotype effect: F(2, 44) = 1.130, P > 0.05; sex × genotype interaction: F(2, 44) = 1.203, P > 0.05; time × sex interaction: F(27, 1188) = 2.657, P < 0.001], or preference for cocaine [sex effect: F(1, 44) = 0.736, P > 0.05; genotype effect: F(2, 44) = 0.285, P > 0.05; sex × genotype interaction: F(2, 44) = 2.811, P > 0.05; time × sex interaction: F(27, 1188) = 0.788, P > 0.05] were not related to Comt genotype. Again, female mice also consumed more water than male mice [sex effect: F (1, 44) = 10.240, P < 0.01; genotype effect: F (2, 44) = 0.378, P > 0.05; sex × genotype interaction: F (2, 44) = 1.244, P > 0.05]. After the first two changes of burette position, either a side preference or a side aversion was observed in the male mice of all genotypes. This phenomenon persisted during the remainder of the experiment and obscured any possible preference for the drug. The effect of burette position on cocaine consumption was much smaller in female than in male mice.

Discussion

The main aim of the present study was to explore the effect of Comt gene disruption on the free-choice, two-bottle voluntary consumption of increasing concentrations of ethanol (2.5–20%, v/v) and cocaine (0.1–0.8 mg/ml) solutions in male and female mice. Increasing ethanol and cocaine concentrations were used in order to study differences between Comt genotypes at various concentration levels, and to habituate the mice gradually to the taste of ethanol. This approach has been used earlier to study the preference of different rat or mouse strains for ethanol or drugs [27–32]. We found that male Comt(–/–) and Comt(+/–) mice consumed significantly more ethanol than their wild-type male littermates at all concentration levels. The increased ethanol consumption in these animals may indicate enhanced reinforcing effects of ethanol. Even a partial loss of catechol-O-methyltransferase activity in Comt(+/–) males was enough to induce a significant increase in ethanol consumption. In female mice, ethanol consumption was not associated to Comt genotype. With respect to cocaine consumption in male mice, the peculiar drinking pattern exacerbated the interpretation of the results. In females, also cocaine intake or preference were not associated with Comt genotype. Although the effects of Comt gene disruption in mice cannot be compared directly to catechol-O-methyltransferase polymorphism in human beings, the present results support the concept that the low catechol-O-methyltransferase activity, which enhances dopaminergic neurotransmission in the prefrontal cortex, may act as one of the predisposing factors for high alcohol consumption or alcoholism in males.

A few earlier studies have explored the effect of different gene knockouts related to the dopaminergic system on ethanol consumption. Dopamine transporter gene (Dat) disruption has been found to affect ethanol consumption sex-dependently and in a surprisingly similar manner as found for the Comt gene disruption in the present study [27,28]. The dopamine transporter accounts for the rapid removal of dopamine after its release in striatum and nucleus accumbens [1–3], and the extracellular dopamine levels in Dat gene knockouts are very high [3]. It is likely that the effect of dopamine transporter knockout on ethanol consumption reflects the increased extracellular dopamine levels in the brain. Comt gene disruption affects dopamine clearance in the prefrontal cortex but only minimally in the striatum [7]. Comt gene disrupted males may therefore experience longer lasting and more effective dopaminergic activation in the prefrontal cortex after ethanol intake, which could in turn increase the duration and magnitude of ethanol reward. Unfortunately, the relative importance of the different dopamine elimination pathways in the prefrontal cortex of female Comt gene knockout mice is not known. In human beings, it has been found that reward anticipation-related activation in the prefrontal cortex and nucleus accumbens is higher in Comt Met (low enzyme activity) than in Val allele carriers (high enzyme activity) [29]. Thus, Comt gene disruption or a reduced catechol-O-methyltransferase activity level, which prolongs the dopamine elimination time in the prefrontal cortex, may modulate both cortically and accumbally mediated cognitive control, reward prediction and motivation processes in such a way to increase ethanol intake.

In our studies, ethanol consumption was clearly dependent both on gender and on Comt genotype. Female mice generally consume more ethanol than male mice in the free-choice oral self-administration setup [30,31]. Female C57BL/6 mice have been shown to have higher hepatic alcohol dehydrogenase and aldehyde dehydrogenase activity than males [32,33], which may explain the larger ethanol intake. In the present study, the variation in the ethanol consumption was also larger in females this probably being attributable to their free 4-day oestrous cycle that has been shown to influence operant responding for ethanol [34] as well as ethanol consumption patterns [35]. When the ethanol concentration was increased to 20%, water consumption increased in both genders indicating a need to dilute the ethanol concentration.

Sexually dimorphic effects have been observed previously in catechol-O-methyltransferase-deficient mice. For example, it has been shown that Comt gene disruption decreased the sensitivity to the locomotor effects of psychostimulants such as amphetamine and cocaine in male, but not in female mice [21,24]. Furthermore, the selective dopamine transporter inhibitor GBR 12909 increased striatal extracellular dopamine levels to a lesser degree in male Comt(–/–) mice than their wild-type littermates [24]. In female mice, no genotype effect was found in striatal dopamine levels [21,24]. Therefore, the genotype-dependent differences in the consumption of ethanol solution might reflect the sexually dimorphic effects of catechol-O-methyltransferase on dopaminergic transmission. Gender differences have been also found in Dat gene knockout mice in ethanol drinking [27,28] as well as in pre-pulse inhibition and patterns of locomotor behaviour [36]. Thus, more general differences may exist between males and females in the function of the dopaminergic system.

C57BL/6J mice have been shown to drink ethanol avidly. Male mice have been reported to consume 9–14 g/kg/day and females 12–18 g/kg/day, when 10% ethanol solution is available under continuous access, free-choice oral self-administration conditions [37–40]. In this study, female mice consumed ethanol about 12 g/kg/day at a concentration of 10%, whereas males consumed only about 3 g/kg/day. We have observed similar, relatively low, ethanol consumption levels in the wild-type littermates of Comt gene-disrupted mice in our earlier studies with slightly different experimental conditions (Forsberg et al., unpublished observations, 2006). The reason for the unexpectedly low consumption could be the subpopulation of C57BL/6J mice that was used to enrich our strain. Harlan C57BL/6J mice have a spontaneous chromosomal deletion in the α-synuclein locus [41]. The α-synuclein deletion has been found to result in accelerated recovery of dopamine release after multiple stimuli [42] and increased release from the readily releasable pool of dopamine in striatum [43]. Furthermore, the intracranial self-stimulation rate has been shown to be elevated in α-synuclein-deficient mice [44]. Based on these observations, the α-synuclein deletion would render the mice more sensitive to the rewarding effect of alcohol. Because all our mice bear the same deletion, it can be considered as background noise that evidently cannot modify the genotype-dependent differences induced by different levels of catechol-O-methyltransferase.

We did not find any difference between the genotypes in cocaine consumption (0.1–0.8 mg/ml), although definite conclusions about the male cocaine consumption patterns cannot be drawn due to their peculiar drinking behaviour. Even though cocaine has been administered orally to rodents at similar concentrations [45–48] and oral cocaine has been shown to be behaviourally active at corresponding doses [49–51], one could speculate that oral administration in the present setup may not have resulted in a pharmacologically relevant cocaine concentration in the brain. However, the cocaine dose was high enough to cause anorexia in the mice. As a dopamine and noradrenaline transporter inhibitor, cocaine has a major effect on brain dopamine levels. The effect of cocaine on dopamine neurotransmission is similar in wild-type and Comt(–/–) male mice even in prefrontal cortex, although under normal conditions, the Comt gene disruption clearly prolongs the dopamine elimination time [7]. Cocaine consumption behaviour was bizarre in the male mice most likely because they found the cocaine solution unpalatable and started to avoid the side where the cocaine burette had been placed for the first time. Another explanation could be that the males actually favoured cocaine but were unable to follow the burette location changes for some reason.

In conclusion, catechol-O-methyltransferase-deficient [Comt(–/–) and Comt(+/–)] male mice consumed more ethanol than their wild-type littermates. In female mice, the genotype had no significant effect on ethanol consumption. Cocaine consumption did not seem to be linked to catechol-O-methyltransferase levels in either sex. These results suggest that Comt gene disruption has a sexually dimorphic effect on ethanol consumption in mice.

Acknowledgements

The authors wish to thank Ms. Ritva Ala-Kulju and Ms. Anna Niemi for excellent technical assistance, and Mr. Markku Uronen for help with the design and building of the cage system. The authors are also grateful to Professor Kalervo Kiianmaa from the National Public Health Institute in Finland for the loan of the burettes, and to Dr. Ewen MacDonald for linguistic advice. These studies were supported by the Academy of Finland to Pekka T. Männistö (No. 210758/2004 and 117881/2006), TEKES (No. 221853), Sigrid Juselius Foundation, and Helsinki University Research Grants.

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