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Effects of periadolescent ethanol exposure on alcohol preference in two BALB substrains
Alcohol 34 (2004) 177–185
Effects of periadolescent ethanol exposure on alcohol preference in two BALB substrains David A. Blizarda,*, David J. Vandenbergha,b, Akilah L. Jeffersona, Cynthia D. Chatlosa, George P. Voglera,b, Gerald E. McClearna,b a Center for Developmental and Health Genetics, 201 Research Building D, The Pennsylvania State University, University Park, PA 16802, USA b Department of Biobehavioral Health, The Pennsylvania State University, University Park, PA 16802, USA
Received 4 June 2004; received in revised form 2 August 2004; accepted 5 August 2004
Abstract Ethanol exposure during adolescence is a rite of passage in many societies, but only a subset of individuals exposed to ethanol becomes dependent on alcohol. To explore individual differences in response to ethanol exposure, we compared the effects of periadolescent ethanol exposure on alcohol drinking in an animal model. Male and female mice of two BALB substrains were exposed to ethanol in one of three forms—choice [water vs. 10% (volume/volume) ethanol], forced (10% ethanol in a single bottle), or gradual (single bottle exposure, starting with 0.5% ethanol and increasing at 2-day intervals to 10% ethanol)—from the 6th through the 12th week of age and administered twobottle alcohol preference tests (10% ethanol vs. water) for 15 days immediately thereafter. All three forms of ethanol exposure increased alcohol preference in male and female BALB/cByJ mice, relative to findings for ethanol-naive control animals. Only gradual ethanol exposure produced an increase in alcohol preference in BALB/cJ mice. During extended alcohol preference testing (for a total of 39 days) of mice in the gradual ethanol exposure group, the higher alcohol preference of the gradual ethanol-exposed BALB/cByJ male mice persisted, but alcohol preference of control group female mice in this strain—formerly ethanol naive, but at this point having received 10% ethanol in the two-bottle paradigm for 15 days—rose to the level of alcohol preference of female mice in the gradual ethanol exposure group. This finding demonstrated that both adolescent and adult ethanol exposure stimulated alcohol preference in female mice of this strain. Across days of testing in adulthood, alcohol preference of the gradual ethanol-exposed BALB/cJ mice decreased, resulting in a lack of effect of gradual exposure to ethanol on alcohol preference in both male and female mice of this strain during the period of extended testing. These strain differences support a genetic basis for the effects of ethanol exposure on alcohol preference and fit within a body of literature, showing substantial individual differences in the effects of ethanol exposure among genetically undefined rats and differences in response to ethanol exposure among inbred rat strains. Exploration of the mechanisms underlying this gene by environment interaction in a mouse model may help elucidate individual differences in the effects of ethanol exposure in human beings and contribute to the understanding of the causes of alcoholism. 쑖 2005 Elsevier Inc. All rights reserved. Keywords: Periadolescent ethanol exposure; Genetics; Alcohol preference; BALB substrains
1. Introduction In many cultures, ethanol exposure is a rite of passage during adolescence and early adulthood, yet only a subset of individuals thus exposed becomes dependent on alcohol or are alcohol abusers. An understanding of the variability in alcohol preference after ethanol exposure in animal models might yield useful clues to the causes of alcohol dependence and abuse. Efforts to study ethanol exposure have generally
* Corresponding author. Tel.: ⫹1-814-865-3429; fax: ⫹1-814-863-4768. E-mail address: [email protected] (D.A. Blizard). Editor: T.R. Jerrells 0741-8329/05/$ – see front matter 쑖 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2004.08.007
used genetically undefined rat stocks, comparisons of a pair of rat strains, or individual mouse strains. The exposure protocol may consist of two-bottle choice (Ho et al., 1989; Kakihana, 1965), forced consumption (Boyle et al., 1994; Sarviharju et al., 2001), incorporation of ethanol into a liquid diet (Schulteis et al., 1996), ethanol vapor (Aufre`re et al., 1997; Slawecki & Betancourt, 2002), or administration of ethanol by means of intragastric (Marfaing-Jallat & Le Magnen, 1982) or intraperitoneal (Wahlstrom, 1994) routes. The duration of the exposure has varied widely [from 18 to 24 h (Adams et al., 2003) to 19 months (Sarviharju et al., 2001)], as has the age at which ethanol exposure is initiated [prenatal period (Randall et al., 1983); before weaning (McKinzie et al., 1999); after weaning (Ho et al., 1989); adolescence (Kakihana, 1965; Slawecki & Betancourt, 2002);
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and variable ages up to 10 months (Yoshimoto et al., 2002)]. Variable results have been obtained, with the findings of some studies indicating that ethanol exposure increases ethanol intake in a postexposure test period [see, for example, Ho et al. (1989), Kakihana (1965), and Marfaing-Jallat and Le Magnen (1982)], and the findings of other studies revealing either no effect (McKinzie et al., 1999; Slawecki & Betancourt, 2002) or an increase in preference for alcohol in a small proportion of animals exposed to ethanol (Rodgers & McClearn, 1962). Individual differences in response to a variety of ethanol ingestion paradigms have been widely noted. Wahlstrom (1994) reviewed several experiments, in which Sprague– Dawley rats were placed on 24 h of alcohol preference weekly, followed by injection of a 2-g/kg dose of ethanol, for circa 50 weeks, and reported highly reliable individual differences in the amount of ethanol ingested during twobottle preference testing across a range of ethanol concentrations during a posttreatment evaluation period. In summary, the effect of ethanol exposure was both variable between animals and consistent within individuals. Schulteis et al. (1996) tested genetically heterogeneous rats for ethanol intake during several withdrawal episodes after exposure to ethanol in a liquid diet. Animals with blood alcohol concentrations greater than 100 mg% at the end of the exposure period showed sustained intake of ethanol throughout four withdrawal periods. Those with blood alcohol concentrations less than 100 mg% showed progressive decreases in ethanol intake. Implicating genetic background in the differential response to ethanol exposure, Adams et al. (1991, 2001) found that alcohol preference of Maudsley Reactive (MR) rats was markedly stimulated by ethanol exposure, but that the same treatment had a smaller and short-term effect in Maudsley Nonreactive (MNRA) rats. Aufre`re et al. (1997) also found marked strain differences in alcohol preference after 4 weeks of exposure of 10 rat strains/stocks to ethanol vapor. Finally, Sarviharju et al. (2001) found subtle, but different, patterns governing the way in which exposure to 12% ethanol for 19 months affected the alcohol-preferring AA (Alko Alcohol) and alcohol-nonpreferring ANA (Alko Non-Alcohol) rat strains. Individual differences in the effect of ethanol exposure on the drinking of alcohol may help identify biobehavioral processes that will be useful in the analysis of alcoholism and alcohol abuse. In the current study, we compared the effect of several forms of periadolescent ethanol exposure on alcohol preference in early adult life in two BALB substrains, BALB/cByJ and BALB/cJ, whose ancestors were derived from the main BALB/c line between 1937 and 1939 when it had been inbred for approximately 36 generations (Potter, 1985). BALB/cByJ mice were selected for study because we believed this strain to be closely related to the BALB/cCrgl strain, which had shown a positive alcohol preference response to periadolescent ethanol exposure in the studies of Kakihana (1965). Our initial explorations revealed BALB/cJ mice to be unresponsive to the same treatment, and comparisons of the two strains were therefore
extended. We reasoned that the demonstration of straindependent effects would provide a useful model for the exploration of the biologic basis of individual differences in the effects of ethanol exposure by bringing the power of mouse genetics to bear on this important health-related problem.
2. Materials and methods 2.1. Animals and maintenance conditions Male and female mice of the BALB/cByJ and BALB/cJ strains were obtained from The Jackson Laboratory (Bar Harbor, ME) at 28 ⫾ 2 days of age and acclimated to the laboratory for 7 days before use in experiments. Animal rooms were maintained on a 12-h light/12-h dark cycle, with temperature and humidity controlled at 72ºF ⫾ 2ºF and 45%, respectively. Purina Lab Chow (sterilizable chow equivalent to 5001 after sterilization) was available ad libitum throughout. The protocols, as well as the care and use of animals, used in the study were approved by the Pennsylvania State University Institutional Animal Care and Use Committee. 2.1.1. Ethanol exposure conditions The different exposure conditions are shown in Fig. 1 and are based on protocols developed by Kakihana (1965) in her studies with the BALB/cCrgl subline. Animals were weighed at the beginning of the experiment (age 35 ⫾ 2 days) and assigned randomly to ethanol-naive or ethanol exposure groups. Three different forms of ethanol exposure were used from weeks 6 through 12: choice, forced, and gradual. In the choice procedure, two 25-ml graduated cylinders were placed on the cage: one containing 10% (volume/ volume) ethanol in tap water and the other containing tap water. The positions of the tubes were reversed (i.e., from left to right) at 3-day intervals. The forced procedure presented a single tube containing 10% ethanol as the sole source of
Fig. 1. Timing of protocols used to study the effects of periadolescent ethanol exposure on alcohol preference. The concentration of ethanol for the forced and choice ethanol exposure protocols, at the peak of the gradual ethanol exposure protocol and during the testing phase, was 10% (volume/ volume). The protocols were developed by Kakihana (1965).
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fluid. The gradual procedure followed the protocol of Kakihana (1965). On the 35th day of age (⫾ 2 days), a tube containing 0.5% ethanol was placed on the cage as the sole source of fluid, and every 2 days the concentration was increased by 0.5% until it reached 10%. The 10% concentration was subsequently maintained until the end of the exposure period. Tubes were read every day in the middle of the light cycle and refilled when necessary. Ethanol-naive mice drank tap water throughout the experimental period. 2.1.2. Exposure groups Mice in each strain by sex subgroup were given choice (N ⫽ 20), forced (N ⫽ 10), or gradual (N ⫽ 10) ethanol exposure conditions, or they were maintained on tap water (ethanol-naive exposure condition, N ⫽ 30) (Fig. 1). To accommodate the large number of subjects, animals were tested in three experimental test groups. Because of death of animals and spoiled readings in a few cases, final Ns differed slightly from the above. 2.1.3. Alcohol preference procedure A 15-day observation period immediately after the end of the ethanol exposure period was adopted as the standard period of assessment (McClearn & Rodgers, 1959; Rodriguez et al., 1994) (Fig. 1). Alcohol preference was measured by using the same procedure described in the choice ethanol exposure protocol (see above). After the gradual ethanol exposure procedure, this period was extended to 39 days for gradual ethanol-exposed and ethanol-naive groups, so that we could assess the long-term stability of group differences (Fig. 1). The volume of 10% ethanol ingested was expressed as a percentage of total fluid intake for each day, calculated according to the following formula: 10% ethanol (ml)/[water (ml) ⫹ 10% ethanol (ml)] × 100 2.2. Genotyping DNA from mice from the BALB/cByJ, BALB/cJ, and BALB/cCrgl substrains was tested for polymorphisms at genetic marker sites from the MIT database, as previously described (Vandenbergh et al., 2003), except that oligonucleotide primers were purchased from both Research Genetics (Huntsville, AL) and Applied Biosystems (Foster City, CA). Polymerase chain reactions were carried out in a 7 ⫾ l-µl total volume with genomic DNA template (10 ng), magnesium chloride (1.5 mM), 2′-deoxynucleoside 5′-triphosphate [(dNTP) 0.25 mM], 1X buffer, and Taq polymerase (1 U). Both the polymerase and buffer were obtained from Sigma, Inc. (St. Louis, MO). Primers were purchased as fluorescently labeled from Research Genetics and included at 2 µM final concentration. Amplification conditions were as follows: 1 cycle of 94ºC, 60 s, followed by 38 cycles of 94ºC, 30 s/59ºC, 30 s/72ºC 60 s, then 1 cycle at 72ºC, 7 min. An ABI 3100 Genetic Analyzer was used for allele detection.
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2.3. Statistical analysis Mean alcohol preference scores over 15 days were calculated for each animal and used as the principal dependent variable (McClearn & Rodgers, 1959). Mean alcohol intake, corrected for body weight, was also calculated. Analyses of variance (ANOVAs) were carried out by using the SPSS 11.0 software package. Appropriate partitions of variance were made for strain, sex, and exposure group. Post hoc tests used the Bonferroni protection procedure to compensate for multiple comparisons. 3. Results 3.1. Ethanol-naive control animals There were no significant differences in alcohol preference (mean of 15 days) associated with experimental test group or strain among ethanol-naive control animals (Fs ⬍ 1). Female mice had higher alcohol preference than male mice [F(1, 106) ⫽ 5.9, P ⬍ .02]. There were no interactions among the three group variables. Thus, the mean alcohol preference for the 15-day assessment period among male and female animals of the two strains was stable across batches carried out at different times. On the basis of this analysis, data were combined across the different test groups for subsequent analyses. 3.2. Effects of ethanol exposure on alcohol preference/intake Analyses of variance of mean alcohol preference scores of the different groups (Fig. 2) revealed highly significant effects of strain [F(1, 259) ⫽ 42.7, P ⬍ .001] and exposure group [F(3, 259) ⫽ 15.9, P ⬍ .001], as well as an interaction between strain and exposure group [F(3, 259) ⫽ 6.2, P ⬍ .001]. This interaction was explored accordingly by separate ANOVAs within each strain. There was no interaction with sex, but there was a statistically significant effect of exposure group in both strains: BALB/cByJ [F(3, 121) ⫽ 12.9, P ⬍ .001] and BALB/cJ [F(3, 126) ⫽ 3.6, P ⬍ .02]. Bonferroni analysis of the BALB/cByJ strain revealed that all ethanolexposed groups (choice, forced, and gradual forms of ethanol exposure) had higher alcohol preference than ethanol-naive control animals, with no difference in alcohol preference among the different exposure groups. Within BALB/cJ, only the gradual ethanol-exposed group had higher alcohol preference than ethanol-naive animals (P ⬍ .003) and the other groups (P ⬍ .005 vs. forced ethanol exposure, P ⬍ .04 vs. choice ethanol exposure). There were no statistically significant differences among choice or forced ethanol exposure groups and ethanol-naive groups (Fig. 2). These different patterns of group differences within strains seem to account for the statistically significant exposure group × strain interaction found in analyses of the combined data set (see above). Mean alcohol intake relative to body weight was subjected to the same ANOVAs described for preference. In
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Fig. 2. Mean (⫾ standard error of the mean) preference for 10% alcohol with the use of a two-bottle test after three forms of ethanol exposure from 35 days of age for 7 weeks. (See Fig. 1 for illustration of these protocols.) Choice (N ⫽ 20 male and 20 female mice), forced (N ⫽ 10 male and 10 female mice), and gradual (N ⫽ 10 male and 10 female mice) ethanol exposure protocols significantly increased alcohol preference in BALB/cByJ (ByJ) mice above the level of ethanol-naive control animals (N ⫽ 30 male and 30 female mice). Only the gradual ethanol exposure protocol increased alcohol preference of BALB/cJ (cJ) mice above that of ethanol-naive control animals.
all cases, group differences, interactions between groups, follow-up tests, and so forth, that were found to be statistically significant for alcohol preference were also found to be statistically significant for alcohol intake. For the extended alcohol preference testing of the gradual ethanol-exposed group, mean alcohol preference scores for days 16 through 39 were calculated for animals in the gradual ethanol-exposed and ethanol-naive groups and are shown in Fig. 3. Analysis of variance revealed a statistically significant strain × exposure group × sex interaction [F(1, 108) ⫽ 6.2, P ⬍ .01]. Within-strain analyses revealed a statistically significant group × sex interaction [F(1, 53) ⫽ 6.4, P ⬍ .01] in BALB/cByJ mice. Gradual ethanol exposure continued to be associated with increased alcohol preference in BALB/cByJ male mice. However, ethanol-naive BALB/cByJ female mice exhibited increased alcohol preference and reached the same level as that shown by female mice in the gradual ethanol-exposed group. In contrast, the alcohol preference– increasing effect of gradual ethanol exposure seen during the first 15 days of alcohol preference testing in the BALB/cJ strain disappeared. There was no effect of exposure group, nor a sex × exposure group interaction, within the BALB/cJ strain (Fs ⬍ 1). Similar results (levels of significance, interactions) were obtained for alcohol intake relative to body weight. The only discrepant result was that the group × sex interaction found for the BALB/cByJ strain just failed to reach statistical significance (P ⬍ .052).
The emergence of a sex × exposure group interaction within the BALB/cByJ and the disappearance of an effect of gradual ethanol exposure within the BALB/cJ strains during extended alcohol preference testing seem to be due to strain-specific and sex-specific changes in alcohol preference that occurred across days of testing. This was supported by the finding of a statistically significant strain × group × sex × days interaction when a repeated-measures analysis was applied to the 39-day test period [F(38, 4104) ⫽ 31.9, P ⬍ .001]. Different patterns of group × sex × days interactions were found within the two strains: BALB/cByJ [F(38, 2014) ⫽ 2.39, P ⬍ .001] and BALB/cJ [F(38, 2090) ⫽ 1.78, P ⬍ .002]. As shown in the plots of daily alcohol preference scores in Fig. 4, within BALB/cByJ, ethanol-naive female mice showed increases in alcohol preference from about the 13th day of testing, which reached the levels of female mice in the gradual ethanol-exposed group by the 21st day of testing. No such change was seen in the alcohol preference of ethanol-naive male mice in this strain, which continued to exhibit lower alcohol preference than that exhibited by male mice in the gradual ethanol-exposed group. In contrast, within BALB/cJ, alcohol preference of male mice in the gradual ethanol-exposed group was relatively high during the first few days of testing (especially on days 4–6), but decreased during extended testing, so that by the 22nd day of alcohol preference, there was no difference between mice in the gradual ethanol-exposed group and ethanol-naive animals (Fig. 5). By comparison, the higher alcohol preference of
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Fig. 3. Mean (⫾ standard error of the mean) preference for 10% alcohol with the use of a two-bottle test for days 16 through 39 after gradual ethanol exposure from 35 days of age for 7 weeks (See Fig. 1 for illustration of these protocols.) Gradual (N ⫽ 10 male and 10 female mice) ethanol exposure significantly increased alcohol preference in BALB/cByJ (ByJ) male mice above the level of ethanol-naive control animals (N ⫽ 20 male and 20 female mice). However, there was no effect of gradual ethanol exposure on alcohol preference of ByJ female mice. The disappearance of the ethanol exposure effect previously seen in ByJ female mice during the first 15 days of testing (Fig. 2) was the result of the increase in alcohol preference that occurred in ethanolnaive female mice during the choice ethanol exposure procedure (See Fig. 4.) The effect of gradual ethanol exposure on alcohol preference in BALB/cJ (cJ) mice previously seen in the first 15 days after ethanol exposure (Fig. 2) dissipated owing to a decrease in alcohol preference over days in animals in the gradual ethanol exposure group (Fig. 5).
BALB/cJ female mice in the gradual ethanol-exposed was seen between days 8 and 21, with minor differences observed between gradual ethanol-exposed and ethanol-naive groups before or after that time. In summary, three forms of ethanol exposure (choice, forced, and gradual) increased alcohol preference during the first 15 days of testing in the BALB/cByJ strain, but only gradual ethanol exposure increased alcohol preference in BALB/cJ. During extended testing, the higher alcohol preference of BALB/cJ mice in the gradual ethanol-exposed group dissipated, and differences between mice in the gradual ethanol-exposed group and ethanol-naive mice were no longer seen. During the same period, alcohol preference of ethanol naive BALB/cByJ female mice increased and reached the level of mice of that strain given gradual exposure to ethanol during periadolescence. No such increase was seen in BALB/cByJ ethanol-naive male mice, and the effect of periadolescent gradual ethanol exposure was sustained. Similar findings were obtained for alcohol intake relative to body weight. 3.3. Body weight during the period of ethanol exposure Body weights were recorded during administration of the gradual ethanol exposure protocol. Body weight increased throughout the period of ethanol exposure [F(5, 345) ⫽ 2099.3, P ⬍ .0001]. There were highly significant effects
of strain [F(1, 69) ⫽ 39.6, P ⬍ .001]: BALB/cByJ animals of both sexes were heavier than BALB/cJ throughout the period of measurement. There were also highly significant effects of sex [F(1, 69) ⫽ 179.9, P ⬍ .001]. However, there was no effect of ethanol exposure (P ⬍ .61), nor any interaction of ethanol exposure with strain or sex.
3.4. Genetic comparison among BALB substrains DNA from three BALB substrains (BALB/cByJ, BALB/ cJ, and BALB/cCrgl) was compared on 96-Mit microsatellite markers distributed across all autosomes and the X chromosome. Results of an earlier study (Panoutsakopoulou et al., 1997) revealed that 5.2% of microsatellites were polymorphic between BALB/cByJ and BALB/cJ. We found 11% of the genetic markers were polymorphic between the same two strains (i.e., the two used in the current study). Approximately half the chromosomes showed no polymorphisms. Of those that did, chromosomes 8 and 15 contained the highest frequency (40% and 50% of markers, respectively, were polymorphic). The percentage of polymorphisms between BALB/cByJ and BALB/cCrgl and between BALB/cJ and BALB/cCrgl were 20% and 17%, respectively. The Crgl substrain was used by Kakihana (1965) in her previously cited studies of ethanol exposure.
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Fig. 4. Mean alcohol preference scores of BALB/cByJ mice for 10% alcohol with the use of a two-bottle test for days 1 through 39 after gradual ethanol exposure from 35 days of age for 7 weeks (See Fig. 1 for illustration of this protocol.) The higher alcohol preference of gradual ethanol-exposed (N ⫽ 10) versus ethanol-naive (N ⫽ 20) male mice persisted throughout the experimental period. The initially higher alcohol preference of gradual ethanol-exposed (N ⫽ 10) versus ethanol-naive (N ⫽ 20) female mice disappeared because the latter group exhibited increased alcohol preference starting at approximately day 13 of alcohol preference testing.
4. Discussion Choice, forced, or gradual ethanol exposure during periadolescence increased alcohol preference in the 15-day standard test period in BALB/cByJ mice from the 20% to 25% typical of ethanol-naive male and female animals to approximately 50% (averaged over the three ethanol exposure conditions; Fig. 2). These results resembled the effects of ethanol exposure in the BALB/cCrgl strain cited earlier (Kakihana, 1965). Among BALB/cByJ female mice, animals in the forced ethanol exposure group, which received the highest amount of ethanol during the exposure period, had the lowest mean alcohol preference of the various ethanol exposure groups. Forced and gradual ethanol exposure of BALB/ cByJ male mice did result in higher mean alcohol preference during the 15-day test period than observed with choice ethanol exposure, but these group differences were not statistically significant. Thus, the magnitude of the effect of ethanol exposure on alcohol preference in BALB/cByJ mice does not seem to be related simply to the amount of ethanol ingested during the period of ethanol exposure. The differences in the response of the two BALB substrains were striking. Only gradual ethanol exposure increased alcohol preference during the standard assessment period in BALB/cJ mice. Ethanol exposure (at least among animals undergoing the gradual ethanol exposure protocol) had no detectable effect on body weight increases in male and female mice of either strain. Thus, the different effects
of ethanol exposure in the two strains were not related to strain-dependent alterations in body growth. Extended testing of animals that underwent the gradual ethanol exposure protocol revealed additional important information about the effect of ethanol exposure in both substrains. Because of increases in alcohol preference that began late in the standard 15-day test period and were maintained from days 16 through 39, alcohol preference of ethanolnaive female BALB/cByJ mice (that had, at this point, ingested ethanol during the first 15 days of the two-bottle protocol) increased to the level of female mice in the gradual ethanol-exposed group (Figs. 3 and 4). Among ethanol-naive BALB/cByJ male mice, no such increase in alcohol preference was seen, and the higher alcohol preference of male mice in the gradual ethanol-exposed group persisted for at least 39 days. In contrast, among BALB/cJ male and female mice in the gradual ethanol-exposed group, the increase in alcohol preference that was seen in approximately the first half of alcohol preference testing dissipated with time (Figs. 3 and 5). In addition, in contrast with BALB/cByJ female mice, BALB/cJ ethanol-naive female mice showed no increase in alcohol preference during extended testing (Fig. 5). These findings emphasize that results obtained during a relatively short period of alcohol preference testing may not be representative of group differences in long-term alcohol preference. To this point we have used the term periadolescent to refer to the period of ethanol exposure explored in the current
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Fig. 5. Daily mean alcohol preference scores of BALB/cJ mice for 10% alcohol with the use of a two-bottle test for days 1 through 39 after gradual ethanol exposure from 35 days of age for 7 weeks (See Fig. 1 for illustration of this protocol.) Alcohol preference of gradual ethanol-exposed male mice (N ⫽ 10) and female mice (N ⫽ 10) was sporadically higher than that of ethanol-naive animals (N ⫽ 20 male and 20 female mice) (e.g., gradual ethanol-exposed male mice, days 4–6, 10–12; gradual ethanol-exposed female mice, days 8, 9, and 13–18) during the first half of the period, but this difference disappeared owing to converging trends between gradual ethanol-exposed and ethanol-naive groups of both sexes as alcohol preference testing progressed.
study. However, the finding that alcohol preference of BALB/cByJ female mice, which were ethanol naive during periadolescence, increased during extended preference testing in early adulthood raises the possibility that their increased responsiveness to ethanol exposure is not limited to the periadolescent period. In a more general sense, the effect of age at which ethanol exposure is applied needs to be carefully explored in future experiments, especially in the context of strain and sex differences. The results of the current study offer a useful model for further analysis of the genetic basis of the variation in response to ethanol exposure. The results of studies by Adams et al. (1991, 2001) and Aufre`re et al. (1997) are the closest parallel to those of the current study because these investigators also implicate genetic background in the effects of ethanol exposure on alcohol preference in rats. After relatively short periods of ethanol exposure, MR and other strains of rats exhibited sustained increases in alcohol preference, whereas MNRA rats showed no effect of ethanol exposure or short-term increases in alcohol preference, which, like those seen in BALB/cJ mice in the current study, dissipated over the course of testing. Sex differences in response to ethanol exposure were also noted by Adams et al. (2001), with the strain-dependent effects of ethanol exposure being more marked in male animals, an observation that parallels sex differences in ethanol exposure response in BALB/cByJ mice. Aufre`re et al. (1997) found that a non-inbred Wistar line exhibited the highest ethanol intake after 4 weeks of
exposure to ethanol vapor in comparison with findings for 9 inbred strains (WKY had the lowest ethanol intake during the test period). When the concentration of ethanol in the drinking tubes was changed, ethanol-dependent Wistar rats also adjusted their intake appropriately to maintain a relatively constant intake of ethanol over time. They also tended to drink substantial quantities of ethanol during the light phase of the circadian cycle. Both these behaviors were held to support the idea that dependent rats were attempting to maintain a constant concentration of ethanol in blood. Although the observation of consistent differences in the behavioral or physiologic response of inbred strains is accepted as prima facie evidence of genetic involvement, Adams et al. (1991) emphasized that marked individual differences characterized alcohol preference. Although animals in the MR strain exhibited higher mean alcohol preference than MNRA rats, there were wide individual differences in alcohol preference, with some animals exhibiting high, and others exhibiting low, alcohol preference. Similar differences were seen within the BALB/cByJ strain in the current study. After exposure to ethanol, some BALB/cByJ mice exhibited consistently high alcohol preference (close to 90%) over days of testing, whereas others had low alcohol preference scores. We and others have previously noted these striking individual differences in alcohol preference within inbred strains of rats and mice (Adams et al., 1991; Dole et al., 1988; Rodgers & McClearn, 1962), as well as within individual
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animals across time (McClearn, 1972). We are keenly interested in understanding the basis of these individual differences and plan further analyses to elucidate them. However, the existence of within-strain individual differences should not distract one from the important observation that ethanol exposure effects on alcohol preference have been shown to be strain dependent in two species under a variety of carefully controlled ethanol exposure conditions, and that the most likely source of such strain variation is allelic differences between the relevant strains. In all, the different responses of two closely related BALB mouse strains to a variety of periadolescent ethanol exposure regimens provide a new illustration of the significance of individual differences in the manner in which ethanol exposure influences alcohol preference. BALB/cByJ mice exhibited a response profile similar to that exhibited by BALB/ cCrgl mice in an earlier study (Kakihana, 1965) by showing reliable increases in alcohol preference after three different ethanol exposure regimens. BALB/cJ mice showed no effect of choice and forced ethanol exposures and only an erratic and transient increase in alcohol preference after the gradual ethanol exposure condition. These findings complement the findings of several studies in rats, in which a variety of ethanol exposure regimens have been used (Adams et al., 1991, 2001, 2003; Aufre`re et al., 1997; Schulteis et al., 1996; Wahlstrom, 1994) and in which the investigators have found substantial differences in the alcohol preference response of individual animals or strains. The analysis of microsatellite genotypes from the BALB/cJ and BALB/cByJ substrains by us and others (Panoutsakopoulou et al., 1997) draws attention to regions of the genome (e.g., chromosomes 8 and 15) for examination of variants underlying the differences in response to ethanol exposure. In previous studies, investigators have identified important phenotypic differences between BALB/cJ and other BALB sublines [e.g., aggressive behavior and adrenal catecholamine biosynthetic enzymes (Ciaranello et al., 1974) and serine dehydratase in liver and l-glycerol 3-phosphate dehydrogenase in brown fat (Kozak, 1985); for more examples, see Potter (1985)]. Although the connection between these phenotypes and variation in response to ethanol exposure is unclear, the hypotheses that have been considered in the attempts to understand the basis for subline differentiation in the various phenotypes [discussed by Potter (1985)] will be helpful in conducting similar explorations of the alcohol exposure phenotype. Development of a mouse model to study the effects of ethanol exposure on alcohol preference will permit the full armamentarium of molecular genetic techniques to be focused on this important biomedical topic. Future research will need to explore the trends in the current study, indicating that ethanol exposure effects may depend on the interaction of sex and age. Hippocampal structure and function have been shown to be altered by adolescent alcohol use and abuse in studies with human subjects (De Bellis et al., 2000). Findings of parallel studies in rats point to
adolescence as a period of greater susceptibility to alcoholinduced brain damage, vis-a`-vis adults (Crews et al., 2000) and greater susceptibility to alcohol-induced disruption of functional indices such as hippocampal long-term potentiation (Pyapali et al., 1999). Thus, it will be necessary to consider the potential role of the CNS in individual differences in our mouse model. The near ubiquity of periadolescent ethanol exposure in Western societies and the wide range of alcohol-drinking responses to this exposure emphasize the significance of these differences for human health and well being. Preclinical exploration of ethanol-related gene by environment interactions by means of animal models is a valuable strategy for exploration of the biologic basis of such variation.
Acknowledgments We thank Helen Lake for assistance with alcohol preference testing and Karly Wortmann for genotyping. This work was supported by grant AA08125 from the National Institute on Alcohol Abuse and Alcoholism.
References Adams, N., Campbell, S. D., & Mitchell, P. S. (2003). Minimal exposure to ethanol increases ethanol preference in Maudsley reactive male rats. Alcohol 29, 149–156. Adams, N., Oldham, T. D., Briscoe, J. T., Hannah, J. A., & Blizard, D. A. (2001). Ethanol preference in Maudsley and RXNRA recombinant inbred strains of rats. Alcohol 24, 25–34. Adams, N., Shihabi, Z. K., & Blizard, D. A. (1991). Ethanol preference in the Harrington derivation of the Maudsley Reactive and NonReactive strains. Alcohol Clin Exp Res 15, 170–174. Aufre`re, G., Le Bourhis, B., & Beauge´, F. (1997). Ethanol intake after chronic intoxication by inhalation of ethanol vapour in rats: behavioural dependence. Alcohol 14, 247–253. Boyle, A. E., Smith, B. R., Spivak, K., & Amit, Z. (1994). Voluntary ethanol consumption in rats: the importance of the exposure paradigm in determining final intake outcome. Behav Pharmacol 5(4 and 5), 502–512. Ciaranello, R. D., Lipsky, A., & Axelrod, J. (1974). Association between fighting behavior and catecholamine biosynthetic enzyme activity in two inbred mouse sublines. Proc Natl Acad Sci U S A 71, 3006–3008. Crews, F. T., Braun, C. J., Hoplight, B., Switzer, R. C. III, & Knapp, D. J. (2000). Binge ethanol consumption causes differential brain damage in young adolescent rats compared with adult rats. Alcohol Clin Exp Res 24, 1712–1723. De Bellis, M. D., Clark, D. B., Beers, S. R., Soloff, P. H., Boring, A. M., Hall, J., Kersh, A., & Keshavan, M. S. (2000). Hippocampal volume in adolescent-onset alcohol use disorders. Am J Psychiatry 157, 737–744. Dole, V. P., Ho, A., Gentry, R. T., & Chin, A. (1988). Toward an analogue of alcoholism in mice: analysis of nongenetic variance in consumption of alcohol. Proc Natl Acad Sci U S A 85, 827–830. Ho, A., Chin, A. J., & Dole, V. P. (1989). Early experience and the consumption of alcohol by adult C57BL/6J mice. Alcohol 6, 511–515. Kakihana, R. Y. (1965). Developmental study of preference for and tolerance to ethanol in inbred strains of mice. Unpublished doctoral thesis, University of California, Berkeley. Kozak, L. P. (1985). Genetic variation in catecholamine responsive metabolic pathways—a hypothesis for a common regulatory mechanism in BALB/c sublines. Curr Top Microbiol Immunol 122, 66–70.
D.A. Blizard et al. / Alcohol 34 (2004) 177–185 Marfaing-Jallat, P., & Le Magnen, J. (1982). Induction of high voluntary ethanol intake in dependent rats. Pharmacol Biochem Behav 17, 609–612. McClearn, G. E. (1972). The genetics of alcohol preference. Finnish Foundation for Alcohol Studies 20, 113–119. McClearn, G. E., & Rodgers, D. A. (1959). Differences in alcohol preference among inbred strains of mice. Q J Stud Alcohol 30, 691–695. McKinzie, D. L., Cox, R., Murphy, J. M., Li, T.-K., Lumeng, L., & McBride, W. J. (1999). Voluntary ethanol drinking during the first three postnatal weeks in lines of rats selectively bred for divergent ethanol preference. Alcohol Clin Exp Res 23, 1892–1897. Panoutsakopoulou, V., Spring, P., Cort, L., Sylvester, J. E., Blank, K. J., & Blankenhorn, E. P. (1997). Microsatellite typing of CXB recombinant inbred and parental mouse strains. Mamm Genome 8, 357–361. Potter, M. (1985). History of the BALB/c family. Curr Top Microbiol Immunol 122, 1–5. [Also: Potter, M. (Ed.). (1985). The BALB/c mouse. In Current Topics in Microbiology and Immunology (pp. 1–5). Berlin/ New York/Tokyo: Springer-Verlag.] Pyapali, G. K., Turner, D. A., Wilson, W. A., & Swartzwelder, H. S. (1999). Age and dose-dependent effects of ethanol on the induction of hippocampal long-term potentiation. Alcohol 19, 107–111. Randall, C. L., Hughes, S. S., Williams, C. K., & Anton, R. F. (1983). Effect of prenatal alcohol exposure on consumption of alcohol and alcoholinduced sleep time in mice. Pharmacol Biochem Behav 18 Suppl 1, 325–329.
185
Rodgers, D. A., & McClearn, G. E. (1962). Alcohol preference in mice. In E. L. Bliss (Ed.), Roots of Behavior (ch. 5). New York: Harper & Bros. Rodriguez, L. A., Plomin, R., Blizard, D. A., Jones, B. C., & McClearn, G. E. (1994). Alcohol acceptance, preference, and sensitivity in mice. I. Quantitative genetic analysis using BXD recombinant inbred strains. Alcohol Clin Exp Res 18, 1416–1422. Sarviharju, M., Jaatinen, P., Hyytia¨, P., Hervonen, A., & Kiianmaa, K. (2001). Effects of lifelong ethanol consumption on drinking behavior and motor impairment of alcohol-preferring AA and alcohol-avoiding ANA rats. Alcohol 23, 157–166. Schulteis, G., Hyytia¨, P., Heinrichs, S. C., & Koob, G. F. (1996). Effects of chronic ethanol exposure on oral self-administration of ethanol or saccharin by Wistar rats. Alcohol Clin Exp Res 20, 164–171. Slawecki, C. J., & Betancourt, M. (2002). Effects of adolescent ethanol exposure on ethanol consumption in adult rats. Alcohol 26, 23–30. Vandenbergh, D. J., Heron, K., Peterson, R., Shpargel, K. B., Woodroffe, A., Blizard, D. A., McClearn, G. E., & Vogler, G. P. (2003). Simple tests to detect errors in high-throughput genotype data in the molecular laboratory. J Biomol Tech 14, 9–16. Wahlstrom, G. (1994). An animal model of alcoholism developed in the male rat. In T. Palomo, & T. Archer (Eds.), Strategies for Studying Brain Disorders, Vol. 1, Depressive, Anxiety and Drug Abuse Disorders (ch. 28). London: Farrand Press. Yoshimoto, K., Hori, M., Sorimachi, Y., Watanabe, T., Yano, T., & Yasuhara, M. (2002). Increase of rat alcohol drinking behavior depends on the age of drinking onset. Alcohol Clin Exp Res 26(8 Suppl), 63S–65S.
Effects of periadolescent ethanol exposure on alcohol preference in two BALB substrains David A. Blizarda,*, David J. Vandenbergha,b, Akilah L. Jeffersona, Cynthia D. Chatlosa, George P. Voglera,b, Gerald E. McClearna,b a Center for Developmental and Health Genetics, 201 Research Building D, The Pennsylvania State University, University Park, PA 16802, USA b Department of Biobehavioral Health, The Pennsylvania State University, University Park, PA 16802, USA
Received 4 June 2004; received in revised form 2 August 2004; accepted 5 August 2004
Abstract Ethanol exposure during adolescence is a rite of passage in many societies, but only a subset of individuals exposed to ethanol becomes dependent on alcohol. To explore individual differences in response to ethanol exposure, we compared the effects of periadolescent ethanol exposure on alcohol drinking in an animal model. Male and female mice of two BALB substrains were exposed to ethanol in one of three forms—choice [water vs. 10% (volume/volume) ethanol], forced (10% ethanol in a single bottle), or gradual (single bottle exposure, starting with 0.5% ethanol and increasing at 2-day intervals to 10% ethanol)—from the 6th through the 12th week of age and administered twobottle alcohol preference tests (10% ethanol vs. water) for 15 days immediately thereafter. All three forms of ethanol exposure increased alcohol preference in male and female BALB/cByJ mice, relative to findings for ethanol-naive control animals. Only gradual ethanol exposure produced an increase in alcohol preference in BALB/cJ mice. During extended alcohol preference testing (for a total of 39 days) of mice in the gradual ethanol exposure group, the higher alcohol preference of the gradual ethanol-exposed BALB/cByJ male mice persisted, but alcohol preference of control group female mice in this strain—formerly ethanol naive, but at this point having received 10% ethanol in the two-bottle paradigm for 15 days—rose to the level of alcohol preference of female mice in the gradual ethanol exposure group. This finding demonstrated that both adolescent and adult ethanol exposure stimulated alcohol preference in female mice of this strain. Across days of testing in adulthood, alcohol preference of the gradual ethanol-exposed BALB/cJ mice decreased, resulting in a lack of effect of gradual exposure to ethanol on alcohol preference in both male and female mice of this strain during the period of extended testing. These strain differences support a genetic basis for the effects of ethanol exposure on alcohol preference and fit within a body of literature, showing substantial individual differences in the effects of ethanol exposure among genetically undefined rats and differences in response to ethanol exposure among inbred rat strains. Exploration of the mechanisms underlying this gene by environment interaction in a mouse model may help elucidate individual differences in the effects of ethanol exposure in human beings and contribute to the understanding of the causes of alcoholism. 쑖 2005 Elsevier Inc. All rights reserved. Keywords: Periadolescent ethanol exposure; Genetics; Alcohol preference; BALB substrains
1. Introduction In many cultures, ethanol exposure is a rite of passage during adolescence and early adulthood, yet only a subset of individuals thus exposed becomes dependent on alcohol or are alcohol abusers. An understanding of the variability in alcohol preference after ethanol exposure in animal models might yield useful clues to the causes of alcohol dependence and abuse. Efforts to study ethanol exposure have generally
* Corresponding author. Tel.: ⫹1-814-865-3429; fax: ⫹1-814-863-4768. E-mail address: [email protected] (D.A. Blizard). Editor: T.R. Jerrells 0741-8329/05/$ – see front matter 쑖 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j.alcohol.2004.08.007
used genetically undefined rat stocks, comparisons of a pair of rat strains, or individual mouse strains. The exposure protocol may consist of two-bottle choice (Ho et al., 1989; Kakihana, 1965), forced consumption (Boyle et al., 1994; Sarviharju et al., 2001), incorporation of ethanol into a liquid diet (Schulteis et al., 1996), ethanol vapor (Aufre`re et al., 1997; Slawecki & Betancourt, 2002), or administration of ethanol by means of intragastric (Marfaing-Jallat & Le Magnen, 1982) or intraperitoneal (Wahlstrom, 1994) routes. The duration of the exposure has varied widely [from 18 to 24 h (Adams et al., 2003) to 19 months (Sarviharju et al., 2001)], as has the age at which ethanol exposure is initiated [prenatal period (Randall et al., 1983); before weaning (McKinzie et al., 1999); after weaning (Ho et al., 1989); adolescence (Kakihana, 1965; Slawecki & Betancourt, 2002);
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and variable ages up to 10 months (Yoshimoto et al., 2002)]. Variable results have been obtained, with the findings of some studies indicating that ethanol exposure increases ethanol intake in a postexposure test period [see, for example, Ho et al. (1989), Kakihana (1965), and Marfaing-Jallat and Le Magnen (1982)], and the findings of other studies revealing either no effect (McKinzie et al., 1999; Slawecki & Betancourt, 2002) or an increase in preference for alcohol in a small proportion of animals exposed to ethanol (Rodgers & McClearn, 1962). Individual differences in response to a variety of ethanol ingestion paradigms have been widely noted. Wahlstrom (1994) reviewed several experiments, in which Sprague– Dawley rats were placed on 24 h of alcohol preference weekly, followed by injection of a 2-g/kg dose of ethanol, for circa 50 weeks, and reported highly reliable individual differences in the amount of ethanol ingested during twobottle preference testing across a range of ethanol concentrations during a posttreatment evaluation period. In summary, the effect of ethanol exposure was both variable between animals and consistent within individuals. Schulteis et al. (1996) tested genetically heterogeneous rats for ethanol intake during several withdrawal episodes after exposure to ethanol in a liquid diet. Animals with blood alcohol concentrations greater than 100 mg% at the end of the exposure period showed sustained intake of ethanol throughout four withdrawal periods. Those with blood alcohol concentrations less than 100 mg% showed progressive decreases in ethanol intake. Implicating genetic background in the differential response to ethanol exposure, Adams et al. (1991, 2001) found that alcohol preference of Maudsley Reactive (MR) rats was markedly stimulated by ethanol exposure, but that the same treatment had a smaller and short-term effect in Maudsley Nonreactive (MNRA) rats. Aufre`re et al. (1997) also found marked strain differences in alcohol preference after 4 weeks of exposure of 10 rat strains/stocks to ethanol vapor. Finally, Sarviharju et al. (2001) found subtle, but different, patterns governing the way in which exposure to 12% ethanol for 19 months affected the alcohol-preferring AA (Alko Alcohol) and alcohol-nonpreferring ANA (Alko Non-Alcohol) rat strains. Individual differences in the effect of ethanol exposure on the drinking of alcohol may help identify biobehavioral processes that will be useful in the analysis of alcoholism and alcohol abuse. In the current study, we compared the effect of several forms of periadolescent ethanol exposure on alcohol preference in early adult life in two BALB substrains, BALB/cByJ and BALB/cJ, whose ancestors were derived from the main BALB/c line between 1937 and 1939 when it had been inbred for approximately 36 generations (Potter, 1985). BALB/cByJ mice were selected for study because we believed this strain to be closely related to the BALB/cCrgl strain, which had shown a positive alcohol preference response to periadolescent ethanol exposure in the studies of Kakihana (1965). Our initial explorations revealed BALB/cJ mice to be unresponsive to the same treatment, and comparisons of the two strains were therefore
extended. We reasoned that the demonstration of straindependent effects would provide a useful model for the exploration of the biologic basis of individual differences in the effects of ethanol exposure by bringing the power of mouse genetics to bear on this important health-related problem.
2. Materials and methods 2.1. Animals and maintenance conditions Male and female mice of the BALB/cByJ and BALB/cJ strains were obtained from The Jackson Laboratory (Bar Harbor, ME) at 28 ⫾ 2 days of age and acclimated to the laboratory for 7 days before use in experiments. Animal rooms were maintained on a 12-h light/12-h dark cycle, with temperature and humidity controlled at 72ºF ⫾ 2ºF and 45%, respectively. Purina Lab Chow (sterilizable chow equivalent to 5001 after sterilization) was available ad libitum throughout. The protocols, as well as the care and use of animals, used in the study were approved by the Pennsylvania State University Institutional Animal Care and Use Committee. 2.1.1. Ethanol exposure conditions The different exposure conditions are shown in Fig. 1 and are based on protocols developed by Kakihana (1965) in her studies with the BALB/cCrgl subline. Animals were weighed at the beginning of the experiment (age 35 ⫾ 2 days) and assigned randomly to ethanol-naive or ethanol exposure groups. Three different forms of ethanol exposure were used from weeks 6 through 12: choice, forced, and gradual. In the choice procedure, two 25-ml graduated cylinders were placed on the cage: one containing 10% (volume/ volume) ethanol in tap water and the other containing tap water. The positions of the tubes were reversed (i.e., from left to right) at 3-day intervals. The forced procedure presented a single tube containing 10% ethanol as the sole source of
Fig. 1. Timing of protocols used to study the effects of periadolescent ethanol exposure on alcohol preference. The concentration of ethanol for the forced and choice ethanol exposure protocols, at the peak of the gradual ethanol exposure protocol and during the testing phase, was 10% (volume/ volume). The protocols were developed by Kakihana (1965).
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fluid. The gradual procedure followed the protocol of Kakihana (1965). On the 35th day of age (⫾ 2 days), a tube containing 0.5% ethanol was placed on the cage as the sole source of fluid, and every 2 days the concentration was increased by 0.5% until it reached 10%. The 10% concentration was subsequently maintained until the end of the exposure period. Tubes were read every day in the middle of the light cycle and refilled when necessary. Ethanol-naive mice drank tap water throughout the experimental period. 2.1.2. Exposure groups Mice in each strain by sex subgroup were given choice (N ⫽ 20), forced (N ⫽ 10), or gradual (N ⫽ 10) ethanol exposure conditions, or they were maintained on tap water (ethanol-naive exposure condition, N ⫽ 30) (Fig. 1). To accommodate the large number of subjects, animals were tested in three experimental test groups. Because of death of animals and spoiled readings in a few cases, final Ns differed slightly from the above. 2.1.3. Alcohol preference procedure A 15-day observation period immediately after the end of the ethanol exposure period was adopted as the standard period of assessment (McClearn & Rodgers, 1959; Rodriguez et al., 1994) (Fig. 1). Alcohol preference was measured by using the same procedure described in the choice ethanol exposure protocol (see above). After the gradual ethanol exposure procedure, this period was extended to 39 days for gradual ethanol-exposed and ethanol-naive groups, so that we could assess the long-term stability of group differences (Fig. 1). The volume of 10% ethanol ingested was expressed as a percentage of total fluid intake for each day, calculated according to the following formula: 10% ethanol (ml)/[water (ml) ⫹ 10% ethanol (ml)] × 100 2.2. Genotyping DNA from mice from the BALB/cByJ, BALB/cJ, and BALB/cCrgl substrains was tested for polymorphisms at genetic marker sites from the MIT database, as previously described (Vandenbergh et al., 2003), except that oligonucleotide primers were purchased from both Research Genetics (Huntsville, AL) and Applied Biosystems (Foster City, CA). Polymerase chain reactions were carried out in a 7 ⫾ l-µl total volume with genomic DNA template (10 ng), magnesium chloride (1.5 mM), 2′-deoxynucleoside 5′-triphosphate [(dNTP) 0.25 mM], 1X buffer, and Taq polymerase (1 U). Both the polymerase and buffer were obtained from Sigma, Inc. (St. Louis, MO). Primers were purchased as fluorescently labeled from Research Genetics and included at 2 µM final concentration. Amplification conditions were as follows: 1 cycle of 94ºC, 60 s, followed by 38 cycles of 94ºC, 30 s/59ºC, 30 s/72ºC 60 s, then 1 cycle at 72ºC, 7 min. An ABI 3100 Genetic Analyzer was used for allele detection.
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2.3. Statistical analysis Mean alcohol preference scores over 15 days were calculated for each animal and used as the principal dependent variable (McClearn & Rodgers, 1959). Mean alcohol intake, corrected for body weight, was also calculated. Analyses of variance (ANOVAs) were carried out by using the SPSS 11.0 software package. Appropriate partitions of variance were made for strain, sex, and exposure group. Post hoc tests used the Bonferroni protection procedure to compensate for multiple comparisons. 3. Results 3.1. Ethanol-naive control animals There were no significant differences in alcohol preference (mean of 15 days) associated with experimental test group or strain among ethanol-naive control animals (Fs ⬍ 1). Female mice had higher alcohol preference than male mice [F(1, 106) ⫽ 5.9, P ⬍ .02]. There were no interactions among the three group variables. Thus, the mean alcohol preference for the 15-day assessment period among male and female animals of the two strains was stable across batches carried out at different times. On the basis of this analysis, data were combined across the different test groups for subsequent analyses. 3.2. Effects of ethanol exposure on alcohol preference/intake Analyses of variance of mean alcohol preference scores of the different groups (Fig. 2) revealed highly significant effects of strain [F(1, 259) ⫽ 42.7, P ⬍ .001] and exposure group [F(3, 259) ⫽ 15.9, P ⬍ .001], as well as an interaction between strain and exposure group [F(3, 259) ⫽ 6.2, P ⬍ .001]. This interaction was explored accordingly by separate ANOVAs within each strain. There was no interaction with sex, but there was a statistically significant effect of exposure group in both strains: BALB/cByJ [F(3, 121) ⫽ 12.9, P ⬍ .001] and BALB/cJ [F(3, 126) ⫽ 3.6, P ⬍ .02]. Bonferroni analysis of the BALB/cByJ strain revealed that all ethanolexposed groups (choice, forced, and gradual forms of ethanol exposure) had higher alcohol preference than ethanol-naive control animals, with no difference in alcohol preference among the different exposure groups. Within BALB/cJ, only the gradual ethanol-exposed group had higher alcohol preference than ethanol-naive animals (P ⬍ .003) and the other groups (P ⬍ .005 vs. forced ethanol exposure, P ⬍ .04 vs. choice ethanol exposure). There were no statistically significant differences among choice or forced ethanol exposure groups and ethanol-naive groups (Fig. 2). These different patterns of group differences within strains seem to account for the statistically significant exposure group × strain interaction found in analyses of the combined data set (see above). Mean alcohol intake relative to body weight was subjected to the same ANOVAs described for preference. In
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Fig. 2. Mean (⫾ standard error of the mean) preference for 10% alcohol with the use of a two-bottle test after three forms of ethanol exposure from 35 days of age for 7 weeks. (See Fig. 1 for illustration of these protocols.) Choice (N ⫽ 20 male and 20 female mice), forced (N ⫽ 10 male and 10 female mice), and gradual (N ⫽ 10 male and 10 female mice) ethanol exposure protocols significantly increased alcohol preference in BALB/cByJ (ByJ) mice above the level of ethanol-naive control animals (N ⫽ 30 male and 30 female mice). Only the gradual ethanol exposure protocol increased alcohol preference of BALB/cJ (cJ) mice above that of ethanol-naive control animals.
all cases, group differences, interactions between groups, follow-up tests, and so forth, that were found to be statistically significant for alcohol preference were also found to be statistically significant for alcohol intake. For the extended alcohol preference testing of the gradual ethanol-exposed group, mean alcohol preference scores for days 16 through 39 were calculated for animals in the gradual ethanol-exposed and ethanol-naive groups and are shown in Fig. 3. Analysis of variance revealed a statistically significant strain × exposure group × sex interaction [F(1, 108) ⫽ 6.2, P ⬍ .01]. Within-strain analyses revealed a statistically significant group × sex interaction [F(1, 53) ⫽ 6.4, P ⬍ .01] in BALB/cByJ mice. Gradual ethanol exposure continued to be associated with increased alcohol preference in BALB/cByJ male mice. However, ethanol-naive BALB/cByJ female mice exhibited increased alcohol preference and reached the same level as that shown by female mice in the gradual ethanol-exposed group. In contrast, the alcohol preference– increasing effect of gradual ethanol exposure seen during the first 15 days of alcohol preference testing in the BALB/cJ strain disappeared. There was no effect of exposure group, nor a sex × exposure group interaction, within the BALB/cJ strain (Fs ⬍ 1). Similar results (levels of significance, interactions) were obtained for alcohol intake relative to body weight. The only discrepant result was that the group × sex interaction found for the BALB/cByJ strain just failed to reach statistical significance (P ⬍ .052).
The emergence of a sex × exposure group interaction within the BALB/cByJ and the disappearance of an effect of gradual ethanol exposure within the BALB/cJ strains during extended alcohol preference testing seem to be due to strain-specific and sex-specific changes in alcohol preference that occurred across days of testing. This was supported by the finding of a statistically significant strain × group × sex × days interaction when a repeated-measures analysis was applied to the 39-day test period [F(38, 4104) ⫽ 31.9, P ⬍ .001]. Different patterns of group × sex × days interactions were found within the two strains: BALB/cByJ [F(38, 2014) ⫽ 2.39, P ⬍ .001] and BALB/cJ [F(38, 2090) ⫽ 1.78, P ⬍ .002]. As shown in the plots of daily alcohol preference scores in Fig. 4, within BALB/cByJ, ethanol-naive female mice showed increases in alcohol preference from about the 13th day of testing, which reached the levels of female mice in the gradual ethanol-exposed group by the 21st day of testing. No such change was seen in the alcohol preference of ethanol-naive male mice in this strain, which continued to exhibit lower alcohol preference than that exhibited by male mice in the gradual ethanol-exposed group. In contrast, within BALB/cJ, alcohol preference of male mice in the gradual ethanol-exposed group was relatively high during the first few days of testing (especially on days 4–6), but decreased during extended testing, so that by the 22nd day of alcohol preference, there was no difference between mice in the gradual ethanol-exposed group and ethanol-naive animals (Fig. 5). By comparison, the higher alcohol preference of
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Fig. 3. Mean (⫾ standard error of the mean) preference for 10% alcohol with the use of a two-bottle test for days 16 through 39 after gradual ethanol exposure from 35 days of age for 7 weeks (See Fig. 1 for illustration of these protocols.) Gradual (N ⫽ 10 male and 10 female mice) ethanol exposure significantly increased alcohol preference in BALB/cByJ (ByJ) male mice above the level of ethanol-naive control animals (N ⫽ 20 male and 20 female mice). However, there was no effect of gradual ethanol exposure on alcohol preference of ByJ female mice. The disappearance of the ethanol exposure effect previously seen in ByJ female mice during the first 15 days of testing (Fig. 2) was the result of the increase in alcohol preference that occurred in ethanolnaive female mice during the choice ethanol exposure procedure (See Fig. 4.) The effect of gradual ethanol exposure on alcohol preference in BALB/cJ (cJ) mice previously seen in the first 15 days after ethanol exposure (Fig. 2) dissipated owing to a decrease in alcohol preference over days in animals in the gradual ethanol exposure group (Fig. 5).
BALB/cJ female mice in the gradual ethanol-exposed was seen between days 8 and 21, with minor differences observed between gradual ethanol-exposed and ethanol-naive groups before or after that time. In summary, three forms of ethanol exposure (choice, forced, and gradual) increased alcohol preference during the first 15 days of testing in the BALB/cByJ strain, but only gradual ethanol exposure increased alcohol preference in BALB/cJ. During extended testing, the higher alcohol preference of BALB/cJ mice in the gradual ethanol-exposed group dissipated, and differences between mice in the gradual ethanol-exposed group and ethanol-naive mice were no longer seen. During the same period, alcohol preference of ethanol naive BALB/cByJ female mice increased and reached the level of mice of that strain given gradual exposure to ethanol during periadolescence. No such increase was seen in BALB/cByJ ethanol-naive male mice, and the effect of periadolescent gradual ethanol exposure was sustained. Similar findings were obtained for alcohol intake relative to body weight. 3.3. Body weight during the period of ethanol exposure Body weights were recorded during administration of the gradual ethanol exposure protocol. Body weight increased throughout the period of ethanol exposure [F(5, 345) ⫽ 2099.3, P ⬍ .0001]. There were highly significant effects
of strain [F(1, 69) ⫽ 39.6, P ⬍ .001]: BALB/cByJ animals of both sexes were heavier than BALB/cJ throughout the period of measurement. There were also highly significant effects of sex [F(1, 69) ⫽ 179.9, P ⬍ .001]. However, there was no effect of ethanol exposure (P ⬍ .61), nor any interaction of ethanol exposure with strain or sex.
3.4. Genetic comparison among BALB substrains DNA from three BALB substrains (BALB/cByJ, BALB/ cJ, and BALB/cCrgl) was compared on 96-Mit microsatellite markers distributed across all autosomes and the X chromosome. Results of an earlier study (Panoutsakopoulou et al., 1997) revealed that 5.2% of microsatellites were polymorphic between BALB/cByJ and BALB/cJ. We found 11% of the genetic markers were polymorphic between the same two strains (i.e., the two used in the current study). Approximately half the chromosomes showed no polymorphisms. Of those that did, chromosomes 8 and 15 contained the highest frequency (40% and 50% of markers, respectively, were polymorphic). The percentage of polymorphisms between BALB/cByJ and BALB/cCrgl and between BALB/cJ and BALB/cCrgl were 20% and 17%, respectively. The Crgl substrain was used by Kakihana (1965) in her previously cited studies of ethanol exposure.
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Fig. 4. Mean alcohol preference scores of BALB/cByJ mice for 10% alcohol with the use of a two-bottle test for days 1 through 39 after gradual ethanol exposure from 35 days of age for 7 weeks (See Fig. 1 for illustration of this protocol.) The higher alcohol preference of gradual ethanol-exposed (N ⫽ 10) versus ethanol-naive (N ⫽ 20) male mice persisted throughout the experimental period. The initially higher alcohol preference of gradual ethanol-exposed (N ⫽ 10) versus ethanol-naive (N ⫽ 20) female mice disappeared because the latter group exhibited increased alcohol preference starting at approximately day 13 of alcohol preference testing.
4. Discussion Choice, forced, or gradual ethanol exposure during periadolescence increased alcohol preference in the 15-day standard test period in BALB/cByJ mice from the 20% to 25% typical of ethanol-naive male and female animals to approximately 50% (averaged over the three ethanol exposure conditions; Fig. 2). These results resembled the effects of ethanol exposure in the BALB/cCrgl strain cited earlier (Kakihana, 1965). Among BALB/cByJ female mice, animals in the forced ethanol exposure group, which received the highest amount of ethanol during the exposure period, had the lowest mean alcohol preference of the various ethanol exposure groups. Forced and gradual ethanol exposure of BALB/ cByJ male mice did result in higher mean alcohol preference during the 15-day test period than observed with choice ethanol exposure, but these group differences were not statistically significant. Thus, the magnitude of the effect of ethanol exposure on alcohol preference in BALB/cByJ mice does not seem to be related simply to the amount of ethanol ingested during the period of ethanol exposure. The differences in the response of the two BALB substrains were striking. Only gradual ethanol exposure increased alcohol preference during the standard assessment period in BALB/cJ mice. Ethanol exposure (at least among animals undergoing the gradual ethanol exposure protocol) had no detectable effect on body weight increases in male and female mice of either strain. Thus, the different effects
of ethanol exposure in the two strains were not related to strain-dependent alterations in body growth. Extended testing of animals that underwent the gradual ethanol exposure protocol revealed additional important information about the effect of ethanol exposure in both substrains. Because of increases in alcohol preference that began late in the standard 15-day test period and were maintained from days 16 through 39, alcohol preference of ethanolnaive female BALB/cByJ mice (that had, at this point, ingested ethanol during the first 15 days of the two-bottle protocol) increased to the level of female mice in the gradual ethanol-exposed group (Figs. 3 and 4). Among ethanol-naive BALB/cByJ male mice, no such increase in alcohol preference was seen, and the higher alcohol preference of male mice in the gradual ethanol-exposed group persisted for at least 39 days. In contrast, among BALB/cJ male and female mice in the gradual ethanol-exposed group, the increase in alcohol preference that was seen in approximately the first half of alcohol preference testing dissipated with time (Figs. 3 and 5). In addition, in contrast with BALB/cByJ female mice, BALB/cJ ethanol-naive female mice showed no increase in alcohol preference during extended testing (Fig. 5). These findings emphasize that results obtained during a relatively short period of alcohol preference testing may not be representative of group differences in long-term alcohol preference. To this point we have used the term periadolescent to refer to the period of ethanol exposure explored in the current
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Fig. 5. Daily mean alcohol preference scores of BALB/cJ mice for 10% alcohol with the use of a two-bottle test for days 1 through 39 after gradual ethanol exposure from 35 days of age for 7 weeks (See Fig. 1 for illustration of this protocol.) Alcohol preference of gradual ethanol-exposed male mice (N ⫽ 10) and female mice (N ⫽ 10) was sporadically higher than that of ethanol-naive animals (N ⫽ 20 male and 20 female mice) (e.g., gradual ethanol-exposed male mice, days 4–6, 10–12; gradual ethanol-exposed female mice, days 8, 9, and 13–18) during the first half of the period, but this difference disappeared owing to converging trends between gradual ethanol-exposed and ethanol-naive groups of both sexes as alcohol preference testing progressed.
study. However, the finding that alcohol preference of BALB/cByJ female mice, which were ethanol naive during periadolescence, increased during extended preference testing in early adulthood raises the possibility that their increased responsiveness to ethanol exposure is not limited to the periadolescent period. In a more general sense, the effect of age at which ethanol exposure is applied needs to be carefully explored in future experiments, especially in the context of strain and sex differences. The results of the current study offer a useful model for further analysis of the genetic basis of the variation in response to ethanol exposure. The results of studies by Adams et al. (1991, 2001) and Aufre`re et al. (1997) are the closest parallel to those of the current study because these investigators also implicate genetic background in the effects of ethanol exposure on alcohol preference in rats. After relatively short periods of ethanol exposure, MR and other strains of rats exhibited sustained increases in alcohol preference, whereas MNRA rats showed no effect of ethanol exposure or short-term increases in alcohol preference, which, like those seen in BALB/cJ mice in the current study, dissipated over the course of testing. Sex differences in response to ethanol exposure were also noted by Adams et al. (2001), with the strain-dependent effects of ethanol exposure being more marked in male animals, an observation that parallels sex differences in ethanol exposure response in BALB/cByJ mice. Aufre`re et al. (1997) found that a non-inbred Wistar line exhibited the highest ethanol intake after 4 weeks of
exposure to ethanol vapor in comparison with findings for 9 inbred strains (WKY had the lowest ethanol intake during the test period). When the concentration of ethanol in the drinking tubes was changed, ethanol-dependent Wistar rats also adjusted their intake appropriately to maintain a relatively constant intake of ethanol over time. They also tended to drink substantial quantities of ethanol during the light phase of the circadian cycle. Both these behaviors were held to support the idea that dependent rats were attempting to maintain a constant concentration of ethanol in blood. Although the observation of consistent differences in the behavioral or physiologic response of inbred strains is accepted as prima facie evidence of genetic involvement, Adams et al. (1991) emphasized that marked individual differences characterized alcohol preference. Although animals in the MR strain exhibited higher mean alcohol preference than MNRA rats, there were wide individual differences in alcohol preference, with some animals exhibiting high, and others exhibiting low, alcohol preference. Similar differences were seen within the BALB/cByJ strain in the current study. After exposure to ethanol, some BALB/cByJ mice exhibited consistently high alcohol preference (close to 90%) over days of testing, whereas others had low alcohol preference scores. We and others have previously noted these striking individual differences in alcohol preference within inbred strains of rats and mice (Adams et al., 1991; Dole et al., 1988; Rodgers & McClearn, 1962), as well as within individual
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animals across time (McClearn, 1972). We are keenly interested in understanding the basis of these individual differences and plan further analyses to elucidate them. However, the existence of within-strain individual differences should not distract one from the important observation that ethanol exposure effects on alcohol preference have been shown to be strain dependent in two species under a variety of carefully controlled ethanol exposure conditions, and that the most likely source of such strain variation is allelic differences between the relevant strains. In all, the different responses of two closely related BALB mouse strains to a variety of periadolescent ethanol exposure regimens provide a new illustration of the significance of individual differences in the manner in which ethanol exposure influences alcohol preference. BALB/cByJ mice exhibited a response profile similar to that exhibited by BALB/ cCrgl mice in an earlier study (Kakihana, 1965) by showing reliable increases in alcohol preference after three different ethanol exposure regimens. BALB/cJ mice showed no effect of choice and forced ethanol exposures and only an erratic and transient increase in alcohol preference after the gradual ethanol exposure condition. These findings complement the findings of several studies in rats, in which a variety of ethanol exposure regimens have been used (Adams et al., 1991, 2001, 2003; Aufre`re et al., 1997; Schulteis et al., 1996; Wahlstrom, 1994) and in which the investigators have found substantial differences in the alcohol preference response of individual animals or strains. The analysis of microsatellite genotypes from the BALB/cJ and BALB/cByJ substrains by us and others (Panoutsakopoulou et al., 1997) draws attention to regions of the genome (e.g., chromosomes 8 and 15) for examination of variants underlying the differences in response to ethanol exposure. In previous studies, investigators have identified important phenotypic differences between BALB/cJ and other BALB sublines [e.g., aggressive behavior and adrenal catecholamine biosynthetic enzymes (Ciaranello et al., 1974) and serine dehydratase in liver and l-glycerol 3-phosphate dehydrogenase in brown fat (Kozak, 1985); for more examples, see Potter (1985)]. Although the connection between these phenotypes and variation in response to ethanol exposure is unclear, the hypotheses that have been considered in the attempts to understand the basis for subline differentiation in the various phenotypes [discussed by Potter (1985)] will be helpful in conducting similar explorations of the alcohol exposure phenotype. Development of a mouse model to study the effects of ethanol exposure on alcohol preference will permit the full armamentarium of molecular genetic techniques to be focused on this important biomedical topic. Future research will need to explore the trends in the current study, indicating that ethanol exposure effects may depend on the interaction of sex and age. Hippocampal structure and function have been shown to be altered by adolescent alcohol use and abuse in studies with human subjects (De Bellis et al., 2000). Findings of parallel studies in rats point to
adolescence as a period of greater susceptibility to alcoholinduced brain damage, vis-a`-vis adults (Crews et al., 2000) and greater susceptibility to alcohol-induced disruption of functional indices such as hippocampal long-term potentiation (Pyapali et al., 1999). Thus, it will be necessary to consider the potential role of the CNS in individual differences in our mouse model. The near ubiquity of periadolescent ethanol exposure in Western societies and the wide range of alcohol-drinking responses to this exposure emphasize the significance of these differences for human health and well being. Preclinical exploration of ethanol-related gene by environment interactions by means of animal models is a valuable strategy for exploration of the biologic basis of such variation.
Acknowledgments We thank Helen Lake for assistance with alcohol preference testing and Karly Wortmann for genotyping. This work was supported by grant AA08125 from the National Institute on Alcohol Abuse and Alcoholism.
References Adams, N., Campbell, S. D., & Mitchell, P. S. (2003). Minimal exposure to ethanol increases ethanol preference in Maudsley reactive male rats. Alcohol 29, 149–156. Adams, N., Oldham, T. D., Briscoe, J. T., Hannah, J. A., & Blizard, D. A. (2001). Ethanol preference in Maudsley and RXNRA recombinant inbred strains of rats. Alcohol 24, 25–34. Adams, N., Shihabi, Z. K., & Blizard, D. A. (1991). Ethanol preference in the Harrington derivation of the Maudsley Reactive and NonReactive strains. Alcohol Clin Exp Res 15, 170–174. Aufre`re, G., Le Bourhis, B., & Beauge´, F. (1997). Ethanol intake after chronic intoxication by inhalation of ethanol vapour in rats: behavioural dependence. Alcohol 14, 247–253. Boyle, A. E., Smith, B. R., Spivak, K., & Amit, Z. (1994). Voluntary ethanol consumption in rats: the importance of the exposure paradigm in determining final intake outcome. Behav Pharmacol 5(4 and 5), 502–512. Ciaranello, R. D., Lipsky, A., & Axelrod, J. (1974). Association between fighting behavior and catecholamine biosynthetic enzyme activity in two inbred mouse sublines. Proc Natl Acad Sci U S A 71, 3006–3008. Crews, F. T., Braun, C. J., Hoplight, B., Switzer, R. C. III, & Knapp, D. J. (2000). Binge ethanol consumption causes differential brain damage in young adolescent rats compared with adult rats. Alcohol Clin Exp Res 24, 1712–1723. De Bellis, M. D., Clark, D. B., Beers, S. R., Soloff, P. H., Boring, A. M., Hall, J., Kersh, A., & Keshavan, M. S. (2000). Hippocampal volume in adolescent-onset alcohol use disorders. Am J Psychiatry 157, 737–744. Dole, V. P., Ho, A., Gentry, R. T., & Chin, A. (1988). Toward an analogue of alcoholism in mice: analysis of nongenetic variance in consumption of alcohol. Proc Natl Acad Sci U S A 85, 827–830. Ho, A., Chin, A. J., & Dole, V. P. (1989). Early experience and the consumption of alcohol by adult C57BL/6J mice. Alcohol 6, 511–515. Kakihana, R. Y. (1965). Developmental study of preference for and tolerance to ethanol in inbred strains of mice. Unpublished doctoral thesis, University of California, Berkeley. Kozak, L. P. (1985). Genetic variation in catecholamine responsive metabolic pathways—a hypothesis for a common regulatory mechanism in BALB/c sublines. Curr Top Microbiol Immunol 122, 66–70.
D.A. Blizard et al. / Alcohol 34 (2004) 177–185 Marfaing-Jallat, P., & Le Magnen, J. (1982). Induction of high voluntary ethanol intake in dependent rats. Pharmacol Biochem Behav 17, 609–612. McClearn, G. E. (1972). The genetics of alcohol preference. Finnish Foundation for Alcohol Studies 20, 113–119. McClearn, G. E., & Rodgers, D. A. (1959). Differences in alcohol preference among inbred strains of mice. Q J Stud Alcohol 30, 691–695. McKinzie, D. L., Cox, R., Murphy, J. M., Li, T.-K., Lumeng, L., & McBride, W. J. (1999). Voluntary ethanol drinking during the first three postnatal weeks in lines of rats selectively bred for divergent ethanol preference. Alcohol Clin Exp Res 23, 1892–1897. Panoutsakopoulou, V., Spring, P., Cort, L., Sylvester, J. E., Blank, K. J., & Blankenhorn, E. P. (1997). Microsatellite typing of CXB recombinant inbred and parental mouse strains. Mamm Genome 8, 357–361. Potter, M. (1985). History of the BALB/c family. Curr Top Microbiol Immunol 122, 1–5. [Also: Potter, M. (Ed.). (1985). The BALB/c mouse. In Current Topics in Microbiology and Immunology (pp. 1–5). Berlin/ New York/Tokyo: Springer-Verlag.] Pyapali, G. K., Turner, D. A., Wilson, W. A., & Swartzwelder, H. S. (1999). Age and dose-dependent effects of ethanol on the induction of hippocampal long-term potentiation. Alcohol 19, 107–111. Randall, C. L., Hughes, S. S., Williams, C. K., & Anton, R. F. (1983). Effect of prenatal alcohol exposure on consumption of alcohol and alcoholinduced sleep time in mice. Pharmacol Biochem Behav 18 Suppl 1, 325–329.
185
Rodgers, D. A., & McClearn, G. E. (1962). Alcohol preference in mice. In E. L. Bliss (Ed.), Roots of Behavior (ch. 5). New York: Harper & Bros. Rodriguez, L. A., Plomin, R., Blizard, D. A., Jones, B. C., & McClearn, G. E. (1994). Alcohol acceptance, preference, and sensitivity in mice. I. Quantitative genetic analysis using BXD recombinant inbred strains. Alcohol Clin Exp Res 18, 1416–1422. Sarviharju, M., Jaatinen, P., Hyytia¨, P., Hervonen, A., & Kiianmaa, K. (2001). Effects of lifelong ethanol consumption on drinking behavior and motor impairment of alcohol-preferring AA and alcohol-avoiding ANA rats. Alcohol 23, 157–166. Schulteis, G., Hyytia¨, P., Heinrichs, S. C., & Koob, G. F. (1996). Effects of chronic ethanol exposure on oral self-administration of ethanol or saccharin by Wistar rats. Alcohol Clin Exp Res 20, 164–171. Slawecki, C. J., & Betancourt, M. (2002). Effects of adolescent ethanol exposure on ethanol consumption in adult rats. Alcohol 26, 23–30. Vandenbergh, D. J., Heron, K., Peterson, R., Shpargel, K. B., Woodroffe, A., Blizard, D. A., McClearn, G. E., & Vogler, G. P. (2003). Simple tests to detect errors in high-throughput genotype data in the molecular laboratory. J Biomol Tech 14, 9–16. Wahlstrom, G. (1994). An animal model of alcoholism developed in the male rat. In T. Palomo, & T. Archer (Eds.), Strategies for Studying Brain Disorders, Vol. 1, Depressive, Anxiety and Drug Abuse Disorders (ch. 28). London: Farrand Press. Yoshimoto, K., Hori, M., Sorimachi, Y., Watanabe, T., Yano, T., & Yasuhara, M. (2002). Increase of rat alcohol drinking behavior depends on the age of drinking onset. Alcohol Clin Exp Res 26(8 Suppl), 63S–65S.