These results confirm and extend previous studies (Blednov et al. 2005
) showing that hybrid mice from the cross of B6 and FVB strains drink substantially more ethanol than either progenitor strain when given a choice of ethanol solution or water. We found that hybrid mice from a cross of SJL (genealogically and genetically close to FVB strain, Beck et al. 2000
; Festing 1994
; Morse 1978
; Petkov et al. 2004
) with B6 also showed higher ethanol consumption than either progenitor strain. It should be noted that increased consumption was observed mainly for high concentrations of ethanol (above 9%). This suggests that the common genetics of FVB and SJL inbred mouse strains may be important in determining the increased ethanol consumption for both hybrids over the already high level of ethanol drinking in B6 mice. It is of interest to note that in this study and many others, mice ‘titrate’ their intake by reducing preference for more concentrated alcohol solutions. This sets a ‘ceiling’ for alcohol intake and suggests that continuous two bottle choice drinking may model social drinking rather than binge or abuse patterns of intake for most strains. However, the hybrids of B6 with either FVB or SJL show altered ‘titration’ such that presentation of more concentrated alcohol solutions results in higher alcohol intake. Titration of alcohol intake to different levels was also seen in longitudinal studies of human alcoholics (Young 1994
). Thus, these hybrids provide a new approach to understand the genetics and neurobiology of regulation of alcohol intake by titration.
Hybrid lines were traditionally evaluated in terms of heterosis or hybrid “vigour”, which describes the deviation of the hybrid line from the two parental or progenitor strains. This phenomenon was extensively studied in plants (Shull 1948
), and in animals, where “behavioral heterosis” was documented (Bruell 1964a
). The genetic basis of heterosis remains murky but ‘dominance’ and ‘overdominance’ are usually invoked as mechanisms. In addition, epistatic interactions between non-allelic genes at two or more loci may also contribute to the phenotypic expression of a trait in hybrids (see Hochholdinger and Hoecker 2007
, for review). However, it is important to note that overdominance reported here refers to the aggregate effect of one to many loci, and cannot be ascribed to any one locus based on the data presented. Nonetheless, because overdominance at known single loci or QTL is relatively rare (Valdar et al. 2006
), this suggests that the observed overdominance is due to relatively few loci. The present findings also demonstrate that alleles do not always affect alcohol drinking behavior in a simple additive or dominant fashion in all crosses. Indeed, hybrids from the intercross of B6 and NZB inbred strains demonstrated either additivity or partial dominance, whereas hybrids from the intercross of B6 and BUB inbred strains showed full or complete dominance, i.e., d
Data obtained in this study clearly show that the range of ethanol consumption in a standard two bottle preference test is not restricted to that seen in standard inbred strains but is substantially broader when hybrids are included. Previous studies of ethanol consumption in BXD recombinant inbred strains (Tarantino et al. 1998
; Phillips et al. 1998
; Gill et al. 1996
) found that the distribution of ethanol consumption is skewed towards low consumption and falls within the range of ethanol consumption of the two parental strains. Similarly, the F1 hybrid cross of 129P3/JxC57BL/6ByJ (Bachmanov et al. 1996
) showed lower ethanol preference than C57BL/6ByJ. Other F1 crosses reported to date include C57BL/Crgl by DBA/NCrgl, A/Crgl/2, C3H/Crgl/2, and BALB/cCrgl (McClearn and Rodgers 1961) and DBA/2JxA/J, DBA/2JxC3HeB/FeJ, C57BL/6JxDBA/2J, C57BL/6JxC3HeB/FeJ, and C57BL/6JxA/J (Fuller 1964
). In these studies, preference for ethanol instead of consumption was reported, but the hybrids in all of these crosses showed lower preference than B6. This conclusion, in conjunction with the present results, is that alcohol preference drinking does not show overdominance as a rule, but rather is restricted to specific progenitor strain crosses, specifically B6 crossed with FVB or SJL in the present study. These new hybrid models should prove useful for exploring the underlying genetic basis of overdominance and its’ contribution to individual differences in alcohol drinking in mice.
Our data also show a maternal effect which increases ethanol consumption. Indeed, both pairs of reciprocal hybrid mice with B6 mothers (B6xFVB and B6xSJL) consumed significantly more ethanol than hybrids with B6 fathers (FVBxB6 and SJLxB6) (). It should be noted that for hybrids obtained from B6 and SJL inbred strains, the effect of overdominance was significant only for the B6xSJL mice. This suggests the possible importance of cytoplasmic heredity, the particular role of some genes located on the X chromosome or epigenetic effects of maternal environment. For example, hybrids obtained from B6 and DBA/2J inbred strains reared by B6 dams consumed more ethanol during forced exposure than did hybrids reared by DBA dams (Gabriel and Cunningham 2008
Summary of consumption data for all inbred strains and hybrids
It is well documented that taste perception is a critical factor in determining ethanol consumption in the two-bottle choice test. A positive relationship between ethanol and sweet intake had been known for more than 40 years (Rodgers et al. 1963
; Rodgers and McClearn 1964
). These findings have been confirmed in many studies in inbred strains of mice (Bachmanov et al. 1996
; Belknap et al. 1993
; Yoneyama et al. 2008
), congenic mouse strains (Blizard and McClearn 2000
), outbred rats (Gosnell and Krahn 1992
), genetically selected alcohol preferring rats (Kampov-Polevoy et al. 1995
; Sinclair et al. 1992
; Stewart et al. 1994
) and monkeys (Higley and Bennett 1999
). Furthermore, rats selected for high or low saccharin consumption consumed more or less ethanol, respectively (Dess et al. 1998
). Recently, we directly showed that the deletion of any one of three different genes expressed in taste buds and involved in detection of sweet taste leads to a substantial reduction of alcohol intake without any changes in the pharmacological actions of ethanol (Blednov et al. 2008
). Despite the limited number of genotypes used in our study (five inbred strains and eight F1 hybrids), we were able to detect the well established positive correlation between preference for saccharin and preference for ethanol. However, despite this correlation, sensitivity to sweet taste cannot explain the increased ethanol consumption observed in hybrids from B6 and FVB strains or B6 and SJL strains because both pairs of parents and reciprocal hybrids show similar, high, preference for saccharin solutions. Moreover, overdominance was seen only for ethanol and not for saccharin preference drinking.
Differences between FVB and B6 strains in preference for some other tastants were noted previously (Bachmanov et al. 2002
). Thus, B6 mice display greater preference for solutions of potassium chloride and ammonium chloride, while FVB mice display greater preference for solutions of sodium chloride and sodium lactate. Preference for sodium chloride in the SJL inbred strain was similar to FVB and significantly higher than in B6 (Tordoff et al. 2007
). Little information about a possible connection between sensitivity to salt and ethanol consumption is available. Two human studies reported that individuals with a paternal history of alcoholism showed significantly enhanced unpleasant response to concentrated sodium chloride and citric acid compared to subjects with no family history of alcoholism (Scinska et al. 2001
; Sandstrom et al. 2003
). Hellekant et al. (1997)
showed that high concentrations of ethanol specifically stimulated individual taste fibers with selective response to sodium chloride in rhesus monkey. Consistent with this possibility, we found a correlation between consumption of alcohol and sodium chloride, particularly for the higher concentrations of alcohol and the higher concentrations of sodium chloride. This relationship is illustrated by the B6xSJL mice which showed higher preference for sodium chloride solutions than B6 mice, whereas no differences were found between SJLxB6 mice and B6 mice. Furthermore, B6xSJL, but not SJLxB6, hybrids consumed more ethanol than B6 (). Consistent with earlier published results (Bachmanov et al. 2002
; Tordoff et al. 2007
), BUB mice, like FVB and SJL mice, showed higher preference for sodium chloride than B6 mice. However, BUBxB6 and B6xBUB mice did not differ from B6 mice in ethanol preference and consumption. Although similar in preference for sodium chloride, the BUB strain is genealogically different from the FVB and SJL strains (Beck et al. 2000
; Petkov et al. 2004
). Therefore, probably both genealogical origin and sensitivity to the salty taste are factors which regulate to some degree ethanol consumption in these hybrids.
It is generally thought that the avoidance of more concentrated ethanol solutions can be related to bitterness. For example, the alcohol consumption in rats was positively correlated with intake of quinine, suggesting that sensitivity to bitter taste influences alcohol acceptance (Kampov-Polevoy et al. 1990
; Goodwin et al. 2000
). Using conditioned taste aversion, Blizard (2007)
showed that B6 mice generalized taste aversions from sucrose and quinine solutions to 10% ethanol and, reciprocally, aversions to 10% ethanol generalized to each of these solutions presented separately. Thus, considering these two gustatory qualities, 10% ethanol should taste both sweet and bitter to B6 mice. However, under conditions of free choice drinking, quinine intake (Phillips et al. 1991
) and ethanol consumption (Fernandez et al. 1999
) were not correlated for the BXD recombinant inbred mouse strains (WebQTL, The Gene Network; http://www.genenetwork.org/
). In agreement with results from this analysis, we did not find any clear correlations between quinine and ethanol consumption for our inbred strains and hybrid mice. Specifically, the high ethanol consuming hybrids (FVB and B6 crosses; SJL and B6 crosses) did not differ from the B6 progenitor strain in avoidance of quinine solutions. Also, hybrids (BUB and B6 crosses) showed significantly lower avoidance of bitter solutions of quinine than B6 mice but were not different from B6 mice in ethanol preference and consumption.
One would expect to find a large number of polymorphisms between two pairs of inbred strains—FVB vs. B6 and SJL vs. B6, as their genealogies are quite different (Beck et al. 2000
; Petkov et al. 2004
). We searched several public databases for genetic polymorphisms between these strains. Indeed, the Mouse Genome Database (searched March 21, 2009) found 158 polymorphisms identified by polymerase chain reaction between B6 and FVB inbred strains (http://www.informatics.jax.org/searches/polymorphism_form.shtml
). The search for genetic polymorphisms between B6 and SJL strains found 189 identified polymorphisms. Some of these polymorphic minisatellites are located within quantitative trait loci (QTL) for ethanol preference on chromosomes 1, 2 and 9 (for B6 and FVB comparison) and on chromosomes 1, 2 (for B6 and SJL comparison) (Tarantino et al. 1998
; Melo et al. 1996
). However, it should be noted that the QTL for ethanol preference mentioned above were obtained for crosses between B6 and DBA inbred strains and we do not know if crosses between B6 and FVB inbred strains will have similar QTL. Consistent with their common genealogy, only three polymorphisms were found between FVB and SJL inbred strains. The Center for Inherited Disease Research Mouse Microsatellite Studies website was searched March 18 (2009) (http://www.cidr.jhmi.edu/mouse/mouse_strp.html
). One hundred and ninety-one polymorphic markers between B6 and FVB were identified with a mean distance of 8.0 cM between markers, 186 polymorphic markers between B6 and SJL were identified with a mean distance of 8.2 cM between markers, and 116 polymorphic markers between FVB and SJL were identified with a mean distance of 12.5 cM between markers.
It is of potential interest to evaluate the emerging SNP databases for differences between the B6 and FVB strains. For chromosome 2, which is strongly implicated in genetic differences in alcohol consumption, the Mouse Phenome Database Mouse SNP site (http://aretha.jax.org/pub-cgi/phenome/mpdcgi?rtn=snps/door
) shows 20008 SNPs between FVB and B6 strains. However, it is important to note that the QTL on chromosome 2 (as well as other QTLs) for alcohol consumption are from B6 and DBA recombinant inbred mice consuming 10% ethanol (Tarantino et al. 1998
; Melo et al. 1996
) and our data suggest different genetic determinants for intake of low (6–10%) and high (30%) concentrations of ethanol. To explore the genetic differences important for the high intake of 30% ethanol in the B6xFVB hybrids with SNP data will require mapping of QTLs in these mice using a range of alcohol consumption.
It should be noted that ethanol consumption in the two-bottle choice test is not always stable over time. In our study, repeated presentation of ethanol after two 1-week periods of abstinence (ethanol deprivation) dramatically reduced consumption, especially of previously highly preferred concentrations of ethanol. However, the genetic dependence of this behavior in ethanol-experienced mice is very different from genetic influences on consumption in ethanol-naïve mice. Thus, genetically similar FVB and SJL inbred strains show opposite changes in ethanol preference and intake after repeated presentation of ethanol. Also, reduction of ethanol preference and intake after ethanol deprivation was found in two other genetically unrelated strains: B6 and NZB. The very low ethanol intake and preference for ethanol observed in BUB mice makes it impossible to evaluate changes in alcohol consumption in this strain in contrast to the other inbred strains. For the hybrids, six of the eight showed stable ethanol preference and intake after repeated ethanol deprivation (). The slight reduction of ethanol intake (but not preference) found in both B6 and SJL reciprocal hybrids after ethanol deprivation can be explained by reduced total fluid intake in these mice. Only the B6xNZB and NZBxB6 reciprocal hybrids showed strong reduction of ethanol preference and intake after ethanol deprivation. This could represent the additive effects of ethanol deprivation observed in both progenitor strains, B6 and NZB although SJL showed a reduction in ethanol consumption after periods of ethanol deprivation, but the hybrids did not show this reduction.
Presentation of high ethanol concentrations and repeated ethanol presentation/deprivation pairings are key challenges known to produce experience dependent changes in ethanol consumption in mice (Melendez et al. 2006
; Y. A. Blednov, unpublished data). Without challenges such as these, some strains will stably drink ethanol for long periods of time; this behavior is thought to model controlled drinking (Melendez et al. 2006
; Y. A. Blednov, unpublished data). After forced deprivations, subsequent increased ethanol consumption is referred to as a positive alcohol deprivation effect (ADE) and is thought to model uncontrolled drinking, whereas decreased ethanol consumption has been referred to as a negative ADE and could represent a change in the threshold for the aversive properties of ethanol (Sinclair and Senter 1968
; Sinclair and Sheaff 1973
; DiBattista 1991
; Melendez et al. 2006
). The contribution of taste learning should also be considered, as it is critical for survival to develop associations between taste and safe/unsafe outcomes. Gutiérrez et al. (2003)
showed that the taste memory trace is simultaneously processed by two mechanisms in the insular cortex, and that their interaction determines the degree of preference or aversion learned to a novel taste. The possible importance of aversive memory in regulating alcohol consumption is supported by data in our companion paper showing differences in development of conditioned taste aversion to ethanol between B6xFVB and B6xNZB mice (A. R. Ozburn et al., companion paper). Future studies will further characterize behaviors of these hybrids to define differences in innate and ethanol-related responses which can cause these differences in ethanol preference.
In conclusion, mice derived from the hybrid crosses of B6 and FVB and B6 and SJL drank higher levels of ethanol than their progenitor strains in the two bottle choice test. The B6 and FVB hybrid is noteworthy for two reasons. First, it demonstrates the occurrence of overdominance in two-bottle choice drinking in mice (i.e., whereby alleles interact to cause the hybrids to score outside the range of the inbred progenitors, where the interaction could occur at alleles within a locus (dominance) or between loci (epistasis), or (more likely) a combination across all loci which influence alcohol preference drinking). Second, it identifies a mouse genotype that shows sustained alcohol preference and consumption in response to the challenges of repeated high ethanol concentrations and periods of abstinence. The hybrid of B6 and NZB demonstrates genetic additivity in two-bottle choice drinking in mice, but shows markedly reduced alcohol preference in response to the challenges of repeated high ethanol concentrations and periods of abstinence. The differences in these phenotypes are explored in the accompanying manuscript (A. R. Ozburn et al., companion paper). It is interesting to note that the inbred mouse strains reduced their ethanol consumption after repeated presentation of ethanol whereas most of the hybrids showed stable drinking. Although inbred mouse strains are a pillar of alcohol genetics research, humans are heterozygous at many loci and we speculate that hybrid mice will provide a wider range of alcohol responses and perhaps a better model of some human responses to alcohol than inbred strains.