A line of mice selected for high blood ethanol concentrations shows drinking in the dark to intoxication

Abstract

Background: Many animal models of alcoholism have targeted aspects of excessive alcohol intake (abuse) and dependence. In the rodent, models aimed at increasing alcohol self-administration have used genetic or environmental manipulations, or their combination. Strictly genetic manipulations (e.g., comparison of inbred strains or targeted mutants, selective breeding) have not yielded rat or mouse genotypes that will regularly and voluntarily drink alcohol to the point of intoxication. Although some behavioral manipulations (e.g., scheduling or limiting access to alcohol, adding a sweetener) will induce mice or rats to drink enough alcohol to become intoxicated, these typically require significant food or water restriction or a long time to develop. We report progress toward the development of a new genetic animal model for high levels of alcohol drinking.

Methods: High Drinking in the Dark (HDID-1) mice have been selectively bred for high blood ethanol concentrations (BEC, ideally exceeding 100 mg%) resulting from the ingestion of a 20% alcohol solution.

Results: After 11 generations of selection, more than 56% of the population now exceeds this BEC after a 4-hour drinking session in which a single bottle containing 20% ethanol is available. The dose of ethanol consumed also produced quantifiable signs of intoxication.

Conclusions: These mice will be useful for mechanistic studies of the biological and genetic contributions to excessive drinking.

Figure 1

Response to selection for high blood ethanol concentration (BEC) across 11 selected generations in High Drinking in the Dark (HDID-1) mice. (A) Mean ± SEM BEC is shown. Solid circles represent the total population tested each generation. Open inverted triangles give values of the animals chosen as parents from the preceding generation: their offspring are represented in solid circles directly below. For numbers of mice, see Table 1. (B) Corresponding ethanol intake (g/kg) for the mice depicted in panel A is shown. (C) Increase in the frequency of HDID-1 subjects with BEC > 1.0 mg/mL across generations is shown. Solid circles depict females, inverted triangles depict males.

Figure 2

Realized response to selection in High Drinking in the Dark (HDID-1) mice. Total realized response to selection in each generation (R) is plotted versus the cumulative selection differential (S) at that generation. R_N is the difference between population mean BEC at the Nth generation and mean BEC in generation S0. S is the difference between BEC of individuals selected as parents and the population from which they were selected (see Figure 1A, Table 1). Thus, for example, as described in the text, the mean BEC of generation S4 was .43, whereas that of the foundation population was .30 (see Table 1). The fourth dot from the left depicts R₄, the total realized response to selection for S0–S4, as .13 mg/mL. The values for S can be estimated from Figure 1A as the difference between the S_N parents (inverted open triangle) minus the S_N population mean (black dot), or [(.97–.30) = .67] for S0. This value is added to [(.78 –.32) = .46], [(.77–.37) = .40], [(1.09 –.60) = .49] to obtain cumulated S₄ = 2.02. This value appears on the × axis for generation S4. The linear regression of R on cumulative S values is shown. From the slope of this line, heritability is estimated to be h² = .096. Data from males and females were combined for this estimate and are given in Table 1. Units for both axes are in mg EtOH/ml blood, but axes of R on S plots are usually not labeled as such by convention. As explained in Supplement 1, the goodness of fit to a linear regression (r = .91, p < .0001) is an indication that additive genetic variability has not yet been exhausted by selective pressure and that the line will continue to show increased response. Once additive variability begins to diminish significantly, the R/S plot will begin to flatten as it reaches an asymptote, and this method of estimating heritability will no longer be valid.

Figure 3

Increase in intake from S0 to S11 in High Drinking in the Dark mice. Each bar represents the mean ± SEM ethanol intake (g/kg) during the 2-hour drinking in the dark (DID; Day 1) and the first and last 2 hours of the 4-hour DID test on Day 2. Inset gives key. Data for all generations for total intake on Day 2 are provided in Table 1. For statistical analyses, see Results.

Figure 4

Blood ethanol concentration (BEC) at the end of drinking in the dark testing displayed versus ethanol intake in 163 High Drinking in the Dark mice from generation S11. Individual BECs are plotted versus intake during hours 2–4 (left panel) or hours 0–4 (right panel). Data from males and females are combined, and the linear regression lines are depicted.

Figure 5

Blood ethanol concentration (BEC) at four time points after 2g/kg ethanol (intraperitoneal) in High Drinking in the Dark (HDID-1) mice from S11 and HS/Npt mice. Closed circles depict HS/Npt mice. Open circles depict HDID-1 mice. Symbols and y-error bars represent the mean ± SEM BEC for each group at each time point. For statistical comparisons, see Results.

Figure 6

Comparison of intake and blood ethanol concentration (BEC) in High Drinking in the Dark mice from S9 with a single-bottle or two-bottle tests (see inset key). Closed circles depict mice tested with a single ethanol tube (standard drinking in the dark [DID] test) and the linear regression of their BEC on their intake (solid line). Open circles depict mice also offered a tube containing water, with a dashed line reflecting the linear regression of their BEC and intake. Symbols with x- and y-error bars represent the mean ± SEM intake and BEC for each group. For statistical comparisons, see Results.

Figure 7

Intoxication on the balance beam in High Drinking in the Dark mice following drinking in the dark testing. Mean ± SEM foot slip errors are shown for mice offered ethanol versus those offered water (Control). For statistical comparisons, see Results.

Figure 8

Intoxication on the accelerating rotarod in High Drinking in the Dark mice following drinking in the dark testing in groups offered ethanol versus water. The improvement in rotarod performance is given as the increase in latency to fall (sec) between the third and first trial. Mice tested first on the balance beam are shown in the left panel, and those tested first on the rotarod on the right. For statistical comparisons, see Results.