HDID-1 mice drink substantial amounts of a relatively high concentration of ethanol (20%) in limited access tests during the circadian dark. The realized heritability of the BEC developed from this behavior across 11 generations of selection is low (approximately 9%), which has undoubtedly contributed to the slow increase in BEC and ethanol intake over generations. The approximate realized heritability of h2
= .096 was lower than that estimated from inbred strains, in which it ranged from .46 to .74 (13
). Differences between such estimates assessed in inbred strains and selected lines are not unexpected. They can arise from many features that distinguish the experimental populations (e.g., no heterozygotes in inbreds, many in selected lines; for discussion see ref. 17
This selection represents the first to our knowledge in which the blood level of a drug served as the selection index. Selection has commonly been employed by the drug abuse (particularly alcohol) research community, but the target of selection has always been either a behavioral response to or the amount of a drug consumed. We elected to target BEC rather than amount consumed because we were interested in developing an animal model of self-intoxication. We reasoned that there were many ways an animal might pattern its intake over a 4-hour test period, and not all of those would be consistent with behavioral intoxication at the end. Indeed, it might have been expected that by targeting high BEC, we would have been choosing those animals that drank more overall, and especially later during the session. However, shows that mice in the foundation population also tended to drink more in the second half of the 4-hour DID test and that the allocation of consumption changed only mildly over selected generations. HDID-1 mice are clearly drinking more overall. A more fine-grained temporal analysis of intake such as the lickometer-derived data we reported for C57BL/6J mice (13
) will be required to determine the role of pattern of intake on BEC. HDID-1 mice reached BEC levels greater than those seen in C57BL/6J mice tested under similar conditions (see Supplement 1
HDID-1 mice were clearly intoxicated when tested on the balance beam. The sensitivity of this task to detect intoxication is high (18
), and the effective dose range across multiple inbred strains was between 1.0 and 1.4 g/kg ethanol (19
). The lower end of this dose range would be expected to yield BECs in the range of many of the tested HDID-1 mice. Consistent with this notion, 39%–46% of mice in the S9 generation exhibited BECs that exceeded 100 mg% (), which is a value that has been shown to produce behavioral intoxication in mice (18
). However, results with the accelerating rotarod (ARR) were more equivocal, because only mice tested second on the ARR showed signs of intoxication. We speculate that the very limited testing (three trials) may have contributed and that those mice for which the ARR experience was their first behavioral test while intoxicated may have been adapting nonspecifically to handling and novelty. One reason we suspect this may be true is that the control group tested first on the rotarod also showed substantially lower performance than the water-drinking control mice tested second (). Also, we have observed that BECs in the range we obtained here cause improvements
in performance on the ARR in some genotypes (21
), so it is possible that we were assessing a mixture of performance decrements and improvements in the HDID-1 mice. In a study that employed a different ethanol drinking schedule and C57BL/6J mice, mice were impaired in a different variant of the rotarod task (fixed speed) after extensive pretraining on the accelerating rotarod. In that study, average BECs were 1.3–1.4 g/kg (22
). We also note that levels of intake and BECs were lower in the second litters of HDID-1 mice that were tested for intoxication than those seen across the first litters of S9 HDID-1 mice tested for selection. We usually do not see such a difference between first and second litters; one potential explanation for the lower intakes (and BECs) is that concurrent testing for intoxication and intake in the same room may have disrupted ethanol consumption.
The test of two-bottle DID intake was consistent with previous studies with inbred mice. Multiple inbred strains of mice were given three DID tests, each lasting 4 days. The first two, a week apart, were with a single ethanol bottle. The final test, 2 weeks later, was with two bottles (ethanol vs. water). In that study, mean strain intake of ethanol was quite stable across all three tests. However, mice also drank some water in the two-bottle test, and mean BECs were considerably lower than in the single-bottle DID test (13
). Given that only about 43% of animals in the HDID-1 line were drinking to intoxication by S9, it is perhaps understandable that the phenotype did not generalize to a two-bottle choice situation in S9. However, we plan to test future generations on a regular basis as their DID response becomes more extreme.
Most selection programs perform bidirectional selection, with two lines selected from the same starting population for the opposite responses. Occasionally, a nonselected (quasi-randomly mated) control line is maintained. The rationale, advantages, and disadvantages of the various mating schemes have been discussed in detail elsewhere (23
). Because most genetically heterogeneous mice drink very little (and therefore reach very low BECs; in S0, only 8.9% had BECs of 1 mg/mL or more) in the DID procedure, we did not deem it useful to try to breed a line selectively for low DID. Rather, we have elected to use the foundation population of HS/Npt mice as a control group for comparisons with HDID-1 mice. This colony is maintained with 48 mating pairs and, in generation 44, was genotyped using a panel of 1532 single nucleotide polymorphism markers. Allele loss was estimated at 5.1% (R. Hitzemann, personal communication). This index suggests that inbreeding by G50 is likely to be low. HS/Npt mice are currently maintained at the original animal facility (Department of Comparative Medicine, OHSU, Portland, Oregon). Both facilities are Association for Assessment and Accreditation of Laboratory Animal Care–approved. Unfortunately, the HS/Npt colony experienced an outbreak of mouse parvo virus (MPV) during 2007. The pathogen has been eliminated, but we were only able to move HS/Npt mice into the VA facility in April, 2008. Our plan is to maintain this subset of HS/Npt breeders by quasi-random mating (excluding common grandparents) so that matched sets of HDID-1 and control mice can be made available for experiments.
Our data suggest that the HDID phenotype is polygenic, and that the limits of selection have not been reached. Greater expansion of the phenotype, as well as the existence of a replicate selected line (HDID-2 mice) will be useful for detecting other correlated responses and understanding the biological basis of the excessive drinking (see Supplement 1
We have discussed elsewhere many other procedures that have been effectively used to increase drinking in rodents (12
) (see also Supplement 1
). These have their uses, and some can lead to very high BECs, but nearly all require a significantly greater degree of training over a longer period. Alternatively, they may require either food or water deprivation (or both). The animals in the DID procedure are never food or fluid deprived. A nonpreferring mouse must withhold drinking for the 4-hour period of the test, but this is easily tolerated without adverse physiological consequences (24
). We do not know why some mice elect to drink a great deal during the DID procedure and others do not. Taste is a complex phenotype, and genetic influences are an important contributor to taste preferences for various tastants (e.g., salt, sweet) (25
). An extensive literature supports a role for taste in two-bottle ethanol preference drinking (for reviews, see refs. 26
). Thus, it will be important to explore taste sensitivity and preferences in the HDID-1 mice. We would predict that a genotype that voluntarily drinks 20% ethanol solutions also will ingest sucrose solutions avidly, on the basis of the substantial common genetic influences on alcohol and sucrose preference drinking (28
We reiterate that the HDID-1 mouse is not intended to serve as a genetic model for alcoholism. Like McClearn (31
), we do not believe that a plausible rodent model that fully resembles clinical alcoholism is a feasible goal (32
). This is primarily because many of the diagnostic criteria for alcohol dependence are behavioral and are defined in ways that undermine the face validity of rodent models (e.g., difficulty with relationships or work). Rather, we are attempting to model one salient feature, a single binge episode. The DID model is rapid and simple, and this is its greatest strength. It is clearly different from human alcoholic drinking in several obvious ways. Whatever its pattern, alcoholic drinking is developed after years, and we would not expect the neurobiological changes seen after DID in mice to reflect the same changes achieved by a chronic alcoholic. In the current generation of HDID-1 mice, DID intakes are reduced when there is water available. However, if intakes continue to increase with further selection, we may well see significant intoxication in HDID-1 mice even when water is available. Maximal intakes are seen during the circadian dark, when feeding and drinking are normally highest. We do not know whether prandial drinking differs in significant ways from drinking at other times during the day, although one might suspect that some prandial drinking is motivated by feeding-associated thirst. Despite these limitations, the genes predisposing to high DID may well influence other alcohol-related traits; this remains to be demonstrated in future studies.
One of the daunting features of undertaking a selective breeding project is the need to convince the relevant research community that the resultant selected lines will be useful. These studies were conducted as a part of the Integrative Neuroscience Initiative on Alcoholism (INIA-West), a consortium effort supported by the National Institute on Alcohol Abuse and Alcoholism. Because one goal is to provide tools to the research community (http://www.scripps.edu/cnad/inia/
), and because the idea of creating these lines emerged consensually, there is broad interest in studying HDID-1 mice among other laboratories. For example, the phenotype (DID) has been used in INIA-West and other laboratories to analyze the pharmacology of high DID (33
). We anticipate the use of HDID-1 mice in studies exploring the neurocircuitry, neurophysiology, and neurochemistry underlying the drinking response, as well as in further behavioral analyses (e.g., will future generations of HDID-1 mice drink sufficient ethanol to display withdrawal signs on cessation? Are there other responses genetically correlated with their propensity to drink to intoxication?). Interested investigators are invited to contact us with ideas for the use of these mice and/or requests for their provision.