Since the observation that inbred mouse strains exhibit marked differences in voluntary EtOH consumption (e.g., Belknap et al. 1993; McClearn and Rodgers 1959), inbred mice have been utilized as a tool to study the genetics of multiple alcohol-related phenotypes, including sensitivity to intoxication (e.g., Bachmanov et al. 2002; Crabbe 1983; Crabbe et al. 2005; Kakihana et al. 1966; Milner and Buck 2010; Tabakoff et al. 2008). However, the underlying genetics of these traits is still not well understood despite the increasing availability of techniques for studying gene–phenotype relationships.

In this context, we previously reported that two inbred mouse strains, C57BL/6J (hereafter abbreviated B6) and 129S1/SvImJ (hereafter abbreviated S1), differ markedly in sensitivity to acute EtOH intoxication (Chen and Holmes 2009; Palachick et al. 2008). In these studies, this difference in sensitivity was evidenced by increased loss of righting reflex (LORR) responses in S1, relative to B6, in response to a moderate–high dose (3 g/kg) of EtOH. By contrast, B6 and S1 did not vary in hypothermic responses to the same (3 g/kg) dose or in ataxia responses to a 1.75 g/kg dose, consistent with a specific pharmacodynamic, rather than general pharmacokinetic, difference between the two strains. These data are generally consistent with the findings of Crabbe and colleagues obtained from a larger inbred strain comparison in which the authors also observed relatively greater responses in S1 than in B6 on various EtOH behaviors (Crabbe et al. 2003a, b, 2005; Metten et al. 2004; Rustay et al. 2003).

The current study was based upon previous observations that the S1 parental strain was significantly more sensitive to the sedative/hypnotic, but not the ataxic or hypothermic, effects of a 3-g/kg dose of EtOH than the B6 parental strain (Chen and Holmes 2009; Palachick et al. 2008; L. DeBrouse et al. (unpublished)). Here, we replicated this difference and further found that a higher EtOH dose (3.5 g/kg) produced not only a greater sedative/hypnotic response but also a greater hypothermic response in S1 compared to B6 mice. We also found that while the strains showed an equivalent ataxic response to a 1.75-g/kg EtOH dose, S1 mice showed modestly greater ataxia to a 2.0-g/kg EtOH dose than B6 that was statistically significant in only one of two experiments. Previous studies using variations on these methods have generally found similar results, including some inconsistency in ataxia measures, in the context of larger inbred strain panels (Crabbe et al. 2003a, b, 2005; Metten et al. 2004; Rustay et al. 2003). Thus, these data confirm our earlier observations that S1 mice are more sensitive to acute EtOH challenge than B6 mice, and extend them by demonstrating that the strains differ across a broader range of measures at increasing EtOH doses.

It is important to note that the current study was conducted in mice that had previously been assessed for Pavlovian fear conditioning and extinction (results to be presented in a future article). To minimize potential carry-over effects, an interval of at least 1 week was interposed between the completion of fear testing and the start of EtOH testing. However, the possibility remains that by virtue of its stressful nature, prior fear testing impacted measures of sensitivity. Discounting, but not fully excluding this possibility, we have previously shown that while sensitivity to EtOH-induced hypothermia and LORR duration was increased in B6 mice by exposure to chronic (14 days) swim stress that ended the day prior to testing, neither acute (1 day) nor subchronic (3 days) stress was sufficient to alter these measures (Boyce-Rustay et al. 2007, 2008a).

The measures of EtOH sensitivity used in the current study cannot dissociate between the initial response to EtOH challenge and acute functional tolerance (AFT) to that response. AFT has a strong genetic component (for review, see Tabakoff et al. 2008). Prior work has shown that S1 mice have a similar AFT as that of B6 to LORR duration (Ponomarev and Crabbe 2004), but have a greater AFT to EtOH-induced ataxia in the rotarod (Rustay and Crabbe 2004) and dowel test (Hu et al. 2008). Greater AFT would be expected to be associated with a decreased, not increased, sensitivity which is opposite to that shown by S1; these data suggest that AFT is unlikely to explain the strain differences. However, more direct examination of AFT in our assays would be necessary to fully exclude a contribution of this process.

QTL analysis was conducted on all three measures of behavioral intoxication as well as pre-EtOH baseline rotarod and motor learning (summarized in Fig. 7). We have the greatest confidence in the QTLs for hypothermia and LORR, which were apparent in a simple main-effect scan. These were found on Chr 16 for hypothermia and on Chrs 4 and 6 for LORR. These QTLs were of generally small effect. While this is typical for QTLs for behavioral traits, it does suggest a significant nongenetic source of variance and/or a degree of insensitivity of our mapping analysis. Nonetheless, the hypothermia and LORR QTLs may be amenable to refinement (e.g., with introgressed-congenic strategies) and experimental validation (e.g., via building convergent evidence across studies), although this will be complicated by our finding that these phenotypes also showed main effects (hypothermia) or interactions (LORR) with sex.

Our analysis revealed a number of other QTLs that overlap with those previously linked to EtOH-related phenotypes in various mouse populations. For example, a query of the MGD revealed that the hypothermia QTL we found on Chr 7 overlaps Ethm3 (Crawshaw et al. 2001) and our Chr 2 locus for LORR overlaps with Alcrsp2 (Erwin et al. 1997). In addition, the Chr 16 hypothermia QTL we found overlaps with that previously linked to similar phenotypes in other mouse populations. This QTL is in the same region as a LORR QTL (Browman and Crabbe 2000) and EtOH drinking phenotype QTL (Gehle and Erwin 1998) previously found in B6 × DBA/2J recombinant inbreds (BXD RI). Interestingly, this same locus has been recently linked to a measure of EtOH AFT in long-sleep/short-sleep mice (Bennett et al. 2007) and EtOH drinking in a B6 × C3H/HeJ F2 population (Drews et al. 2010).

Of the other QTLs we found, loci for BEC at LORR recovery on Chrs 9 and 11 overlap with a number of preference-related loci (Bachmanov et al. 2002; Bice et al. 2006; Erwin et al. 1997; Malmanger et al. 2006; Melo et al. 1996; Phillips et al. 1994; Tarantino et al. 1998), and the Chr 9 locus also overlaps with loci for acute alcohol locomotor activation (Erwin et al. 1997; Malmanger et al. 2006) and conditioned taste aversion (Risinger et al. 1998) and our locus for ataxia. The other locus we found for ataxia on Chr 8 does not overlap any previously discovered alcohol-related loci, but interestingly, it does overlap Cbm2, a QTL for cerebellum weight (Airey et al. 2001). Likewise, our QTLs for LORR overlap with alcohol-drinking loci on Chrs 2, 4, 8, and 12 (Bachmanov et al. 2002; Belknap et al. 1997; Bice et al. 2006; Fernandez et al. 1999; Gill and Boyle 2005; Melo et al. 1996; Phillips et al. 1994; Tarantino et al. 1998). Also of particular note is the large number of traits that we found to map to Chr 2, given previous reports that a locus in this region has been linked to various EtOH-related traits in various mouse lines (Crabbe et al. 1994; Gill and Boyle 2005; Malmanger et al. 2006). Candidate gene studies implicate Stxbp1 as a candidate for consumption-related traits at this locus (Fehr et al. 2005). More broadly, the finding that our LORR QTL overlapped with regions consistently linked to EtOH drinking suggests a common genetic influence on these behaviors. This provides important, albeit indirect, evidence supporting the hypothesis that variation in sensitivity to high-dose (aversive) EtOH intoxication is a factor driving the propensity to drink and, by extension, relative risk for alcohol abuse (Krystal et al. 2003).

The convergent loci across studies could facilitate the reduction of positional candidates using a multiple-cross mapping strategy or other comparison of strain haplotypes, or through the integration of other functional studies. For this reason, we have deposited all QTL positional candidates into the GeneWeaver database (Baker et al. 2011). Using this system, we have identified priority candidates for the Chr 3 EtOH-induced hypothermia locus that may influence multiple EtOH-related responses. The most highly connected candidate is Hs2st1, a heparin sulfate sulfotransferase. A search of the Allen Brain Atlas reveals that this gene is highly expressed in the hippocampus. A GeneNetwork query reveals that it is coexpressed with alcohol preference in BXD RI strains. Another compelling candidate is Prkacb, interesting because of the already known role of the protein kinase A pathway in both LORR and hypothermia (Yang et al. 2003).

There are several instances where our data do not correspond to prior studies. For example, with the exception of QTLs on Chr 6 previously found for ethanol consumption in a B6.BALB/cJ-introgressed line (Vadasz et al. 2007) and for ethanol-induced locomotor activity (Downing et al. 2003), the LORR QTLs we report are largely distinct from those reported for LORR duration in BXD RI (Browman and Crabbe 2000), LXS (Haughey et al. 2005), and long-sleep/short-sleep mice (Bennett et al. 2002, 2008) populations, EtOH drinking in BXDs (Phillips et al. 1998), as well as EtOH drinking (Belknap et al. 1997; McClearn et al. 1997) and EtOH-induced locomotor stimulation in a B6 × D2 intercross (Hitzemann et al. 1998).

QTL mapping studies rarely have sufficient power to reveal all causative loci underlying complex phenotypes, and in our study we also failed to reproduce the QTL on a Chr 1 “hotspot” previously linked to multiple EtOH phenotypes in other mouse crosses (Ehlers et al. 2010; Mozhui et al. 2008). These situations could be the result of differences in segregating alleles in each of these populations and heterozygosity in our B6 × S1 F2 population, methodological differences in the measurement of EtOH-related phenotypes between studies, or simply genuine false negatives. Although we performed genome-wide searches, it is computationally prohibitive to search the entire multiple-locus model space. Future studies using alternative statistical QTL models may have better fidelity. It will also be important to take some of our provisional findings further by testing for convergent evidence from other crosses.

A consistent finding in the QTL–EtOH literature that was also a major pattern in our data is the largely nonoverlapping QTLs across phenotypes (e.g., see Browman and Crabbe 2000; Drews et al. 2010; Gehle and Erwin 1998; Phillips et al. 1998). This was echoed by our phenotypic correlational analysis, which found few significant correlations between phenotypes. One exception was a significant negative correlation between ataxia and LORR, indicating that high sensitivity to EtOH’s ataxic effects predicted high sensitivity to LORR. Longer LORR time was also associated with lower BECs on awakening. This is a general relationship and is not unexpected if LORR duration is a function of EtOH clearance as opposed to being modulated independently of LORR time by, for example, pharmacokinetic factors. Nonetheless, both measures can provide useful measures of EtOH sensitivity and are best considered together. The more general conclusion from these correlational analyses across the various end-point measures we made is that the pattern of largely noncorrelations is consistent with largely independent genetic influences.