A Line of Mice Selected for High Blood Ethanol
Concentrations Shows Drinking in the Dark to
John C. Crabbe, Pamela Metten, Justin S. Rhodes, Chia-Hua Yu, Lauren Lyon Brown, Tamara J. Phillips,
and Deborah A. Finn
models aimed at increasing alcohol self-administration have used genetic or environmental manipulations, or their combination. Strictly
that will regularly and voluntarily drink alcohol to the point of intoxication. Although some behavioral manipulations (e.g., scheduling or
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.
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.
Key Words: DID, ethanol consumption, genetic animal models,
HDID, intoxication, mouse, pharmacogenetics, selective breeding
neurobiological research targeting alcoholism has employed
rodents, and the usual measure of self-administration is the
two-bottle preference test in which animals are offered a bottle of
tap water versus a bottle containing an alcohol solution. That
genotype strongly influences preference drinking has been
known for many years (2,3), and differences in preference for
drinking alcohol among inbred mouse strains are stable across
laboratories and over decades (4). However, inbred strains differ
for many phenotypes, and not all alleles leading to high drinking
are overrepresented in the genotypes of even the highest drink-
ing strains. Thus, alcohol research has benefited from frequent
use of the technique of selective breeding. By mating highly
preferring individuals repeatedly over generations, several lines
of rats and mice have been produced that preferentially drink
nearly all their daily fluid from a bottle of unflavored 10% ethanol
despite the availability of unadulterated water. Because all of
these lines have been selected for nearly identical drinking
phenotypes, the comparison of results across such lines as
Preferring (P) versus Non-Preferring (NP) rats, High and Low
lcoholism is a complex psychiatric disorder with strong
genetic as well as environmental risk factors, the interac-
tions of which affect individual risk (1). Most laboratory
Alcohol Drinking (HAD/LAD) rats, Alko Alcohol (AA) and Non-
Alcohol (ANA) rats, and High and Low Alcohol Preferring
(HAP/LAP) mice has been very informative (see several recent
reviews of these lines in Addiction Biology 2006, Vol 11). We
have learned, for example, that low levels of brain serotonin are
associated with high-preferring genotypes (5).
One of the limits of alcohol preference drinking studies has
been that rodents, unlike humans, rarely self-administer enough
alcohol to become intoxicated. Rodents drink in bouts rather
than continuously, and most intake occurs during the circadian
dark. It appears that as their rate of intake approaches the
maximal rate at which they can metabolize ethanol and eliminate
it, they slow their drinking (6,7). Many humans would like to
reach this level of self-regulation! Even those lines genetically
selected for preference generally stop drinking when their blood
ethanol concentration (BEC) reaches about 50 to 70 mg% (7).
These levels correspond roughly to the legal driving limit in most
However, some of the genetically predisposed rodent lines
will self-administer significant amounts of alcohol under certain
conditions (8,9). Thus, selection can produce animals that will
voluntarily exceed BECs of 100 mg% and that become physically
dependent. However, the existing protocols for achieving this
behavior involve selectively examining those animals at the
extremely high end of the drinking distribution, as well as either
many weeks of testing or technically challenging procedures
such as gastric cannulation. Many other procedures for achieving
high BECs in rats or mice are also available, but they either
involve food or water restriction, relatively complicated operant
schedules, or long periods of study. For discussion of these
requirements, see Supplement 1.
Therefore, there remains the need for rodent models of one of
the key behavioral aspects of abusive human drinking, the
tendency to drink excessively. In a recent study, C57BL/6J mice
were exposed to gradually increasing concentrations of ethanol
in water solutions for a 30-min period restricted to their circadian
From the Portland Alcohol Research Center (JCC, PM, C-HY, LLB, TJP, DAF),
Department of Behavioral Neuroscience, Oregon Health & Science Uni-
versity, and VA Medical Center (JCC, PM, C-HY, LLB, TJP, DAF), Portland,
Oregon; Department of Psychology (JSR), Beckman Institute, University
of Illinois at Urbana–Champaign, Urbana, Illinois.
Address reprint requests to John C. Crabbe, Ph.D., Portland VA Medical
Center, R&D-12, 3710 SW Veterans Hospital Road, Portland, OR 97239;
BIOL PSYCHIATRY 2009;65:662–670
© 2009 Society of Biological Psychiatry
dark phase, when they are normally engaged in their highest
levels of eating, drinking, and activity. After about a week, these
mice were drinking enough alcohol to show signs of intoxication
(10,11). We adapted these procedures substantially, and devel-
oped a procedure in which mice would drink ethanol to
intoxication by the second day of exposure (12,13). Inbred
strains differed in their propensity for drinking in the dark (DID),
and the behavior appeared to be heritable (13). Here, we report
a new genetic animal model, High Drinking in the Dark
(HDID-1) mice, developed by selectively breeding for high BEC
after a short period of access to an alcohol solution during the
Methods and Materials
Animals and General Husbandry
See Supplement 1. All procedures were approved by our
Institutional Animal Care and Use Committee.
Drinking in the Dark
The mouse colony was illuminated during the “dark” phase
with a red bulb (2 lumen/square foot, about 21.5 lux). Mice were
individually housed in the same type of caging for 5–9 days
before testing. During this period, mice were trained to drink
from a water spout using a polycarbonate bottle with a stainless
steel drinking spout (Ancare Corp., Bellmore, New York). For
DID testing, the same type of drinking spout was inserted into 9
mL, calibrated polystyrene tubes for measurement of intake (for
details, see Supplement 1). Testing was conducted starting
between 50 and 119 days of age with the exception of one family
of selected generation 9 (S9) tested at 47 days of age. Details
regarding the drinking in the dark phenotype and how it is
ascertained have been published (12,13). In addition, a detailed
procedural protocol is available at http://www.scripps.edu/
pdf or from the authors on request. Previous studies showed that
individual intake values for C57BL/6J mice for DID on the first
day of exposure were not highly correlated with DID intakes on
Days 2–4 but that intakes on the second day were well correlated
with Days 3 and 4 (12) and for up to 12 further days of drinking
(data not shown). Therefore, we elected to use a 2-day DID test.
Each mouse was weighed on Day 1 before the lights went out.
On the same day, starting at 3 hours after lights off, the single
water bottle was removed from the cage of each mouse and
replaced with a single tube containing 20% ethanol in tap water
(v/v). Tubes were read again at 2 hours, the volume change
recorded, and each tube was replaced by the standard water
bottle. On Day 2, the procedure was repeated exactly, but tubes
were left in place at the 2 hours reading for an additional 2 hours.
At 4 hours, a 20-?L blood sample was drawn from the periorbital
sinus with a capillary tube. Care was taken to be as quiet as
possible and to disturb the mice minimally.
The Selection Phenotype
We wanted to select mice drinking to intoxication without
behaviorally testing them for intoxication. Because intake was
imperfectly predictive of BEC and because the many lines
selected for high intake rather than for intoxication do not readily
drink to intoxicating BECs, we elected to breed selectively on
BEC attained rather than intake in g/kg.
We initiated this selection with a founding population (selec-
tion generation zero, S0), of 82 female and 76 male HS/Npt mice,
all the offspring of 25 HS/Npt pregnant dams (see Supplement 1).
All mice were tested for DID for 2 days and the BEC determined.
Generations S0 through S2 were maintained and tested within
the Oregon Health & Science University (OHSU) facility. When
S3 offspring were about 12–17 days old, the colony was moved
to the Portland VA Medical Center Veterinary Medical Unit. VA
and OHSU husbandry conditions were very similar. For the first
five generations, we employed within-family selection, using a
method we have employed for prior selections for alcohol
phenotypes (14,15). Because response to selection was slower
than anticipated, when selecting the S5 breeders to produce S6,
we shifted to the use of individual selection, which we have since
employed. Details for numbers of mice tested each generation
are given in Table 1. For methods, see the Supplement 1.
Mice are deemed “HDID-1” because we are in the process of
breeding a replicate line, using generally the same procedures.
Data from HDID-2 mice, which are being selected from the
outset using individual selection, will be reported after the
selection has proceeded for more than the current five genera-
Naive HDID-1 mice from the S11 generation were compared
with HS/Npt mice after an injection of 2 g/kg ethanol (20% v/v in
saline, intraperitoneally). Mice were 132–143 days old and
comprised half male and half female per genotype (n ? 6–7/
line). Blood samples (20-?L) were obtained from the periorbital
sinus from alternating eyes at four time points after injection: 15,
30, 60, and 120 min.
Naive male mice from second litters of the S9 generation,
aged 55–68 days, were given a two-bottle preference version of
the DID test (13). Thirty-eight mice were tested for 3 days with 2
hours access starting 3 hours after lights off. On the fourth day,
access was extended for 4 hours. Half the mice were offered a
single tube of 20% ethanol (v/v) each day (standard DID group).
The other half of the mice were offered two identical tubes, one
containing water and one containing ethanol, each day (two-
bottle choice DID group). Mice within the latter groups were
assigned to either left or right position of the ethanol tube, which
remained the same each day, to mimic the procedure previously
followed (13). A blood sample was taken from all mice at the end
of the test on Day 4.
Naive mice from second litters of the S9 generation were
pretested on the balance beam, tested for DID as described
earlier, and then tested for performance on the balance beam
and accelerating rotarod immediately following the second day
of DID testing. For testing procedures, see Supplement 1.
Response to Selection for DID
Selection on BEC at the end of the 4 hours drinking session on
Day 2 of the DID test resulted in a 3.6-fold increase in the average
BEC across 11 generations. Figure 1A shows the average BEC for
each generation. BEC in the foundation population of HS/Npt
mice (S0) averaged .30 mg/mL. Inverted open triangles indicate
the average BEC of individuals selected as parents for each
subsequent generation. For example, by S4, BEC in HDID-1 mice
averaged .43 mg/mL. From this population, BEC in the 13 mating
pairs that were chosen to produce the S5 generation averaged .83
J.C. Crabbe et al.
BIOL PSYCHIATRY 2009;65:662–670 663
mg/mL. Their S5 offspring had average BECs of .61 mg/mL. By
S11, the average BEC was 1.07 mg/mL for the offspring popula-
tion, an increase over the foundation population of 262%.
Table 1 gives the number of offspring that were tested each
generation and their average BEC, 4 hours consumption in g/kg,
age, and body weight (at the time of DID testing). The mean BEC
and consumption values for the selected parents of each gener-
ation also are provided. The selection differentials (S) were
calculated from the mean BECs of the mice selected to breed the
next generation minus the mean of the population from which
they were selected. The number of breeder pairs (families) varied
slightly across generations because some pairs were infertile or
offspring were too few or died before testing. Figure 2 shows the
realized response to selection R, the change in BEC from the S0
foundation mean value (e.g., S2–S1), plotted against the cumu-
lative S. The regression of R on cumulative S gave an estimate of
Table 1. Generational Data on Selection for BEC after Drinking in the Dark
Generations of SelectionNBEC (mg/mL)4-Hour Consumption (g/kg)Age (days) Body Weight (g)No. FamiliesNe
.35 ? .05
.24 ? .04
.97 ? .07
.31 ? .04
.34 ? .06
.78 ? .08
(S ? .460)
.47 ? .05
.25 ? .04
.77 ? .07
(S ? .398)
.61 ? .08
.58 ? .08
1.09 ? .10
(S ? .496)
.40 ? .06
.45 ? .05
.83 ? .08
(S ? .397)
.62 ? .05
.60 ? .06
1.22 ? .08
(S ? .608)
.56 ? .05
.50 ? .05
1.23 ? .05
(S ? .697)
.66 ? .06
.63 ? .06
1.38 ? .05
(S ? .733)
.70 ? .05
.63 ? .04
1.54 ? .06
(S ? .877)
.90 ? .07
.84 ? .08
1.54 ? .05
(S ? .677)
.83 ? .07
.66 ? .06
1.53 ? .05
(S ? .800)
1.12 ? .05
1.02 ? .07
1.78 ? .04
(S ? .703)
4.7 ? .26
3.2 ? .20
5.0 ? .36
87.8 ? .91
87.0 ? .88
20.9 ? .26
25.8 ? .32
4.6 ? .23
4.0 ? .31
6.5 ? .60
66.3 ? .47
67.2 ? .45
20.1 ? .23
25.7 ? .46
5.1 ? .27
4.0 ? .37
5.7 ? .43
65.7 ? .53
66.3 ? .52
21.2 ? .19
26.7 ? .34
5.6 ? .27
4.7 ? .26
6.5 ? .30
56.1 ? .14
56.1 ? .13
19.3 ? .19
24.4 ? .33
5.5 ? .29
4.9 ? .27
6.5 ? .39
82.4 ? .37
82.9 ? .24
22.0 ? .27
26.5 ? .27
6.5 ? .31
5.2 ? .30
7.4 ? .48
65.5 ? .48
62.6 ? .71
20.3 ? .21
24.6 ? .32
5.9 ? .24
4.5 ? .23
6.8 ? .44
70.7 ? .26
70.7 ? .21
21.0 ? .26
25.0 ? .28
6.3 ? .37
5.6 ? .36
7.4 ? .53
70.3 ? .48
70.7 ? .40
19.0 ? .24
24.3 ? .32
15 32 .076
6.0 ? .21
4.4 ? .17
6.3 ? .43
103.3 ? .90
101.9 ? .96
22.2 ? .16
27.6 ? .25
6.1 ? .26
5.6 ? .29
7.2 ? .27
60.7 ? .87
60.3 ? .86
18.9 ? .20
23.5 ? .33
6.6 ? .28
5.4 ? .28
7.9 ? .38
73.4 ? .63
73.6 ? .53
20.3 ? .21
25.3 ? .33
7.1 ? .26
5.7 ? .26
7.5 ? .33
71.2 ? .60
70.4 ? .65
20.2 ? .27
25.0 ? .32
BEC, blood ethanol concentration.
aNe, the “effective” breeding population size, changes as a function of number of breeders and breeding scheme (see text).
b?F is the inbreeding coefficient; Cum ?F is the cumulative inbreeding over generations.
cS is selection differential (see text).
dIn this generation, first and second litters were tested.
664 BIOL PSYCHIATRY 2009;65:662–670
J.C. Crabbe et al.
heritability from the slope of the regression line (h2? .09).
Response to selection has not slowed, suggesting that additive
genetic variability remains in the population (see Supplement 1).
Correlated Response to Selection for DID
Selection strictly on BEC at the end of the 4 hours drinking
session on Day 2 of the DID test also produced concomitant
increases in g/kg consumption across generations (Figure 1B),
which are considered a correlated response to selection.
Whereas BEC had more than tripled over generations, intake
increased from 4.00 g/kg in S0 to 6.43 g/kg in S11, an increase of
60.8%. Figure 1C shows the percentage of animals in each
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
sented 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
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
(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 SNparents (in-
verted open triangle) minus the SNpopulation mean (black dot), or
[(.97–.30) ? .67] for S0. This value is added to [(.78–.32) ? .46], [(.77–.37) ?
in Table 1. Units for both axes are in mg EtOH/ml blood, but axes of R on S
ment 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 re-
sponse. 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.
J.C. Crabbe et al.
BIOL PSYCHIATRY 2009;65:662–670 665
generation for which BEC exceeded 100 mg%. This value is
consistent with behavioral intoxication in mice. By S11, 53% of
males and 58% of females exceeded this threshold. There has
been no apparent change in average body weight across gener-
ations, nor has the general health of the colonies appeared to
change (data not shown).
Sex Differences and Estimate of Inbreeding Coefficient
From Table 1, it appeared that females achieved slightly
higher BECs than males and drank more. Although both mea-
sures increased over generations, the small sex differential
remained stable (see Supplement 1). By S11, the estimate of
inbreeding (see Supplement 1) was 12.8% through S11. These
values are also given in Table 1. The rate of inbreeding during
S6–S11 (approximately 1.4% per generation) is what would be
expected for at least the next several generations.
Intake Across the 4-hour Session
Mice achieving higher BECs displayed modestly greater in-
takes, so we asked whether ethanol intake during the first versus
the second 2-hour portions of the DID test had changed over
generations. Figure 3 shows the (2 hours) intake on the first day
of DID testing, and the intake on Day 2 during the first and last
2 hours of testing, after which the BEC was taken for selection.
Three S11 mice were excluded from this analysis because their
total consumption values on Day 2 were excessive as discussed
in the Supplement 1. Generation 11 mice drank more on the first
day of testing than S0 mice [F(1,319) ? 41.5, p ? .0001]. When
we analyzed the two 2-hour periods of drinking on Day 2 across
generations, there was a significant overall increase in S11 versus
S0 mice when data were collapsed on time [F(1,319) ? 96.8, p ?
.0001]. Mice of both generations drank more during the second
2-hour period on Day 2 [F(1,319) ? 114.9, p ? .0001], and the
interaction was significant [F(1,319) ? 7.2, p ? .01]. Mice of S11
increased their drinking during the second period relative to the
first by about .5 g/kg as compared to S0 mice (increases of 1.37 ?
.16 and .82 ? .13 g/kg, respectively).
Figure 4 shows the modest predictive value of intake in deter-
mining BEC in S11 mice. The left panel shows that intake from the
final 2 hours was modestly associated with BEC, predicting 17% of
the variance. Total intake (right panel) predicted 20% of the
C57BL/6J mice with the DID procedure (12).
Figure 5 shows the results of this study. BECs were somewhat
higher (about 10%) in the HDID-1 than in HS/Npt mice, but lines
did not differ in rate of metabolism (see Supplement 1).
In this study, mice from S9 had 4 days of ethanol access (2
hours on Days 1–3, 4 hours on Day 4), with separate groups of
animals having a single ethanol bottle (standard DID) versus two
bottles (ethanol vs. water). An initial analysis compared place-
ment of ethanol tube (left vs. right) on intake on Day 4 for mice
whom the ethanol tube was placed in the position of the water
bottle versus mice for whom ethanol was placed on the other
side of the cage top. Placement of the ethanol tube did not
significantly alter ethanol intake in either the standard DID group
[F(1,16) ? .03, p ? .86] or the two-bottle choice group [F(1,18) ?
.45, p ? .51]. Thus, data for the counterbalanced groups were
combined for analyses of intake (see Figure 6). Mean BEC in the
single-bottle group was .47 mg/mL and average intake on the last
day of testing was 4.91 g/kg ethanol. Mean BEC in the two-bottle
group was .12 mg/mL, and intake averaged 2.98 g/kg. BEC and
ethanol intake differed significantly in the single-bottle versus
two-bottle groups [F(1,36) ? 5.6 and 9.9, respectively, p ? .05].
Only 3 of the 20 mice in the two-bottle choice group had
detectable BEC (?.05 mg/mL), whereas 15 of the 18 mice in the
single-bottle group had detectable BEC values. Total fluid con-
sumption also differed significantly between groups. The two-
bottle group drank 1.15 ? .11 mL fluid, whereas the single-bottle
group drank .71 ? .06 mL [F(1,36) ? 11.6, p ? .01].
Results are given in Figure 7. A preliminary analysis showed
that mice tested first on the balance beam and subsequently on
the rotarod had BECs at the end of testing equivalent to mice
tested in the opposite order. Mean BECs were .64 ? .15 and
.84 ? .13 mg/mL, respectively [F(1,40) ? 1.03, p ? .10]. Analysis
of foot slips showed that mice from S9 of the HDID-1 selection
were clearly intoxicated, averaging more than three foot slips on
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.
666 BIOL PSYCHIATRY 2009;65:662–670
J.C. Crabbe et al.
the balance beam. Comparable mice given access only to water
averaged fewer than one foot slip, and these group differences
were significant [F(1,55) ? 12.3, p ? .001]. Neither the effect of
test order on foot slips (F ? 1.19) nor the group ? test order
interaction (F ? 1) were significant.
For the rotarod, even though BECs were equivalent for those
mice tested first and second, the results were more complex. A
preliminary analysis of mean trial latencies showed a trend
toward significant effects of test order [F(1,53) ? 3.6, p ? .07].
Those mice tested first on the rotarod performed more poorly
than those tested after balance beam testing, and there also was
a trend toward a significant interaction of group and test order
[F(1,53) ? 2.9, p ? .10]. We therefore performed separate
analyses of the mice tested first and those tested second on the
rotarod. In all analyses, performance improved significantly over
trials, assessed by calculating the difference between Trial 1 and
Trial 3 latencies. This improvement index was slightly skewed to
the left, so we performed a reflection of the data (largest score
?1) and then took the square root of the reflected difference
score to obtain a normal distribution (16). Analyses of these
transformed data showed that ethanol drinking in the dark led to
nearly significant impairment for those mice tested second on the
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.
ethanol (intraperitoneal) in High Drinking in the Dark (HDID-1) mice from
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.
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.
J.C. Crabbe et al.
BIOL PSYCHIATRY 2009;65:662–670 667
rotarod [F(1,25) ? 3.96, p ? .058] but not in those tested first
[F(1,28) ? .5, p ? .47; see Figure 8].
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 popula-
tions (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, Figure 3 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
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
beam are shown in the left panel, and those tested first on the rotarod on the right. For statistical comparisons, see Results.
Figure 7. Intoxication on the balance beam in High Drinking in the Dark
shown for mice offered ethanol versus those offered water (Control). For
statistical comparisons, see Results.
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J.C. Crabbe et al.
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% (Figure 1C), which is a value that has been
shown to produce behavioral intoxication in mice (18,20).
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 intoxi-
cated 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 substan-
tially lower performance than the water-drinking control mice
tested second (Figure 8). 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 improve-
ments in the HDID-1 mice. In a study that employed a different
ethanol drinking schedule and C57BL/6J mice, mice were im-
paired 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 previ-
ous 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-ran-
domly mated) control line is maintained. The rationale, advan-
tages, and disadvantages of the various mating schemes have
been discussed in detail elsewhere (23). Because most geneti-
cally 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 communica-
tion). 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. Unfortu-
nately, the HS/Npt colony experienced an outbreak of mouse
parvo virus (MPV) during 2007. The pathogen has been elimi-
nated, 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 drink-
ing (for reviews, see refs. 26,27). 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 (28–30).
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 Integrated Neuroscience
J.C. Crabbe et al.
BIOL PSYCHIATRY 2009;65:662–670 669
Initiative on Alcoholism (INIA-West), a consortium effort sup-
ported by the National Institute on Alcohol Abuse and Alcohol-
ism. 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 laborato-
ries. For example, the phenotype (DID) has been used in
INIA-West and other laboratories to analyze the pharmacology of
high DID (33–36). We anticipate the use of HDID-1 mice in
studies exploring the neurocircuitry, neurophysiology, and neu-
rochemistry 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 inves-
tigators are invited to contact us with ideas for the use of these
mice and/or requests for their provision.
These studies were conducted as part of the Integrative
Neuroscience Initiative on Alcoholism of the National Institute on
Alcohol Abuse and Alcoholism and were supported by Grant Nos.
AA010760, AA013478, and AA013519 from the National Insti-
tutes of Health and by the Department of Veterans Affairs. We
thank Andy Jade Cameron, Alex Henry, Katie Mordarski, Jason
Schlumbohm, Michelle Sorensen, and Stephanie Spence for ex-
pert technical assistance.
All of the authors report no biomedical financial interests or
potential conflicts of interest.
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670 BIOL PSYCHIATRY 2009;65:662–670
J.C. Crabbe et al.
A Line of Mice Selected for High Blood Ethanol Concentrations Shows Drinking in the Dark
John C. Crabbe et al. MS BPS-D-08-0548
Supplementary Online Material
Ethanol drinking in preference selected lines.
Nearly all the existing selected lines of rodents have been bred for relative total
intake of a 10% alcohol solution vs water over a 24 hr period. All animals are first exposed
to four days where the only available fluid is 10% ethanol in water. Thereafter, they are
tested for two-bottle preference (10% ethanol vs water) for three weeks and those animals
with the highest g/kg ethanol intake and preference ratios (alcohol solution/total fluid
intake) are chosen for mating to produce the next generation of (for example) Preferring
Further steps are required to maximize intake of the preference selected lines. For
example, Murphy et al. (1) studied P rats after the 3-week long selection protocol. They
selected from the population 28 of the highest drinking P rats (those whose intake during
the previous week had exceeded 5.0 g ethanol/kg body weight and with a preference ratio
greater than 67%). After an additional week of free-choice drinking, they sampled blood
levels at 1, 3, 6, 12, 16, or 19 hr after the onset of the circadian dark phase. Peak average
blood ethanol concentration (BEC) of just under 90 mg% was seen 3 hr into the dark
phase. Two of the 6 animals tested at 3 hr showed BECs between 100 and 120 mg%. A
few individual rats showed BECs between 120 and 150 mg% at later time points, though
group averages were less than 60 mg%. During the next week, access to ethanol and
water was restricted to a 4 hr period, and average BECs increased somewhat, to as high
as 120 mg% with 3 rats exceeding 150 mg%. The authors reported virtually no intake of
water during periods of scheduled access (1). Another line of rats (the high alcohol
consumption line from the Alcohol Research Foundation, or HARF) has been selectively
bred to drink substantial amounts of ethanol in a 20 minute long period of (2) (Le, Israel,
Juzytsch, Quan, and Harding 2001). A pilot study of HARF rats from the fourth selected
generation showed that they averaged BECs of 63 mg% (range 16 to 166) immediately
after drinking. However, these animals do not drink a great deal under conditions of
continuous access, and there are few reported studies (2-4). Finally, High Alcohol
Preferring (HAP) mice have been selected to drink 10% ethanol vs water under conditions
mimicking the selection of P rats (5). During continuous 24 hr access conditions, these
mice drink more than 12 g ethanol/kg body weight per day. When tested under conditions
limiting ethanol access to 30 min during the dark, HAP mice drank about 1.4 g/kg ethanol
in 30 min and these animals reached an average BEC of nearly 60 mg/ml, with a range of
After long-term exposure to ethanol preference drinking, if alcohol is withheld for a
period of time, rodents will generally show an increase in drinking when it is reintroduced, a
phenomenon termed the alcohol deprivation effect (ADE) (7). P rats were given four-bottle
free choice access to 10%, 20%, and 30% ethanol versus water for 6 weeks, and then
alcohol was withheld for 2 or 8 weeks before being reintroduced. Rats were then subjected
to three additional ADE cycles (2 weeks on, 2 weeks off alcohol). Intake more than
doubled after the final deprivation, and the ADE increase was maintained for several days.
BECs in similarly-treated animals averaged 120 mg% after the fourth round of ADE, and
150 mg% after a fifth round (8). A similar study used four rounds of ADE (but only offered
10% ethanol versus water) to High Alcohol Drinking (HAD) rat lines of both replicates. HAD
rats showed significant escalations in drinking (approximately 50% increases), but the
escalation lasted 2 days at most. BECs were not reported in this study (9).
There is some evidence that genetically-predisposed P rats will self-administer
enough alcohol to become physically dependent, as evidenced by withdrawal signs. P rats
from the 12th-18thselected generation were tested for preference using the 3-week
selection protocol described above. After selecting for further study those highest drinking
P rats that exceeded a 5 g/kg intake and 67% preference ratio criterion, rats were allowed
to continue to drink 10% ethanol versus water for 20 weeks. Ethanol was then withdrawn
and the animals were observed for signs of withdrawal such as wet-dog shakes and
muscle fasciculation. P rats showed significant signs of withdrawal (10). P rats will also
self-administer ethanol by the intragastric route, and four rats so studied attained BECs
ranging from 116 to 303 mg% (average = 199 mg%) (11). Finally, P rats offered 10%
ethanol for 6 weeks and withdrawn showed 30% reductions in threshold seizure
susceptibility to bicuculline infusions, consistent with withdrawal (12).
Supplementary Methods and Materials:
Animals and general husbandry
All procedures were approved by the Portland VA Medical Center or Oregon Health
& Science University (OHSU) Institutional Animal Care and Use Committee and were
performed according to NIH Guidelines for the Care and Use of Laboratory Animals. Mice
from the foundation population were from a genetically heterogeneous stock (HS/Npt)
created by Dr. Robert Hitzemann at the State University of New York - Stony Brook and
moved to the OHSU Department of Comparative Medicine animal facility in 2000. The
HS/Npt stock was created by the systematic intercrossing of 8 inbred mouse strains (A/J,
AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, CBA/J, DBA/2J, and LP/J) derived from 4 of the 8
inbred mouse strain lineages (13). It is maintained by rotational mating of approximately 48
breeding pairs. Pregnant females from 25 families of the 50th filial generation were
obtained from Dr. Hitzemann and moved into a colony room maintained on a reversed
light:dark cycle as described below. The 25 females produced 158 offspring who were
weaned and separated into same sex cages with siblings at 21 – 28 days and tested at
adulthood (see below) to form the foundation (S0) generation.
All mice were maintained in standard polycarbonate or polysulfone (the two plastic
types were used interchangably) cages (19 X 31 X 13 cm) with stainless steel wire bar tops
with a recess for chow in groups of 2-5 females or 2-4 males on Bed-o-cob bedding
(Andersons, Maumee, OH, USA). Cages were changed once weekly, but were not
changed during the drinking experiments.
The colony was maintained on a 12 hr:12 hr light:dark schedule with lights on at
21:30 and lights off at 09:30. The breeding colony room was kept on this schedule
throughout the lifetimes of all generations: this strategy was adopted simply for our
convenience. The colony room was maintained at a temperature of 21±1°C, and Purina
5001 chow (PMI Nutrition International, Brentwood, MO, USA) was available at all times.
The colony room served as the testing room. Most generations were housed during
mating and offspring production on Thoren racks with automatic lixit water spouts always
available. Some generations were housed on flat racks with square, stoppered
polycarbonate water bottles placed on the cage tops. Water was obtained from these
bottles by licking a pinhole in the bottom side, through the wire cage top.
Drinking tubes and drinking in the dark procedure
Disposable 10 mL Falcon clear polystyrene serological pipets (Fisher Scientific)
were altered to form the drinking tubes. These are cut off at both ends, leaving a cylinder
with the original graduated markings from about 2 mL to 9 mL into which a sipper tube with
a 2.5" stainless steel drinking spout was fitted with heat-shrink tubing. The sipper tubes
have a 5/16" aperture and a ball bearing (Ancare Corp.). The top of the cylinder was
plugged with a silicone stopper (Fisher Scientific). Tubes were clipped to the cage top with
a medium binder clip (ACCO) to prevent spillage when animals were eating, and volume
was read from the meniscus immediately after all tubes were in place (to approximately 0.1
ml accuracy). For reading tube volumes under red light, a standard AA mini-maglite with a
3 LED aftermarket conversion was used (http://www.thinkgeek.com/gadgets/lights/852c).
One individual can read about 80 tubes in 5 minutes while a second records softly-spoken
volume readings. At 4 hr, tubes were read again and each mouse was rapidly removed
from its cage and carried to the other side of the room for placement under a hood, also
illuminated with red light.
Selection of mice for breeding
We tested the total S1 offspring population of 81 female and 57 male mice. From S1
through S5, we chose for breeding the male and female mouse with the highest BEC from
each family. The female from Family #1 was mated to the male from Family #2, and so on
using a rotational scheme in common practice for other selections, until the female from
Family #15 was mated to the male from Family #1. Their offspring (deemed the first
selected, or “S1" generation), were weaned into same-sex groups at 21 - 25 days and
raised until testing as described above. The rotational scheme described above, when
repeated in successive generations, reduces inbreeding by minimizing the variance in the
contribution of each family’s genes to the next generation, at the cost of slowing the
response to selection (14).
This is a somewhat idealized description of the mating schemes. Occasionally,
families failed to produce offspring, and other mice had to be substituted as breeders.
When choosing mice, we also examined their g/kg intake and their body weight, and we
rejected an occasional mouse with very low body weight and other mice with high BECs
when we already had a large number of representatives of that family selected as
breeders. The latter decision was made to slow trait-irrelevant inbreeding in the population.
Consumption in g/kg over the 4 hr test is generally fairly well-correlated with the
BEC ascertained. These correlations typically explain about 25% of the variance in BEC,
whether in individual differences within a genotype, across genetically segregating
populations, or in correlations derived from inbred strain means [(15,16), and unpublished
observations]. However, according to the readings, some mice will ‘drink’ more than 5 mL
of 20% alcohol in 4 hr. Five mL amounts to a g/kg alcohol intake of 40 or more g/kg, which
we believe to be impossible (as these mice are not grossly intoxicated, either by behavioral
observation or by BEC). This may represent leakage, or the animal’s ‘playing’ with the ball
bearing, or a combination of the two. While this is not a major problem, it is seen in one or
two animals in almost every generation or experiment.
Because of the potential problem with apparently aberrant measures of extreme
“consumption,” we paid close attention to ensure that g/kg values corresponded to BEC in
each generation. For example, in the S7 generation, we tested 66 female mice. Although
their average intake was calculated as 7.1 ± 0.7 g/kg, intake data for 3 animals were
viewed as problematic since the ethanol dose consumed was considered non-physiological
(e.g., 21.7 or 47 g/kg) and/or the BEC was not representative of the ethanol dose
consumed (e.g., 16.6 g/kg dose with 0.53 mg/ml BEC). To address this problem, we took a
conservative approach. First, because the selection index was BEC, we analyzed BEC
data using a generation X sex two-factor ANOVA. After determining that there was no
significant interaction of sex with generation and that the main effect of generation was
stronger than that of sex (Fs reported below under Sex differences), we calculated for each
generation the mean total g/kg consumed over the 4 hr period on day 2 and flagged mice
with consumption values in excess of 3 standard deviations above the mean. The number
of mice flagged in each generation ranged from 0 to 5 and included the three S7 female
mice described above. We then calculated for each generation regression coefficients
using least squares regression of BEC on g/kg consumed with these animals excluded
from the regression analyses. Next, we calculated the expected consumption of the
flagged mice using the generation-appropriate regression line equation using their BECs.
There were 32 animals total (of 1852, or less than 2%, across all generations) that
exceeded the consumption threshold defined above, and their consumption values were
replaced with their predicted consumption as described. All other data for these animals
were retained without alteration. These values are used in reporting the 4-hr total
consumption data in Table 1 and in Figure 1B in the main text; however, these animals’
data for consumption were excluded from analysis of the two 2-hr intake periods and the
predictive value of intake in determination of BEC that are described in the main text in the
section on “Intake across the 4-hr session” and from Figures 3 & 4.
For individual selection, family is not considered when selecting breeders (i.e., more
than two members of a family may be chosen). We first rank-ordered the BEC values for
female and male mice separately. We then established 15-20 families by selecting the
mice with the 15-20 largest BEC values from each sex and selecting female and male
partners for breeding randomly. Throughout the selection we have excluded sibling
matings (as this greatly increases inbreeding) and tried to minimize the number of common
grandparents when setting up mating pairs.
Procedures for intoxication assays
Sixty female mice (aged 49-74 days) were individually housed with standard
drinking sipper tubes. Forty-four mice were assigned to the Ethanol group and 16 to the
Control group. Behavioral testing was conducted under a second red light on the opposite
side of the room from the mice still drinking. On Day 1, each mouse was pre-trained on a
3/4" wide balance beam (Flair Plastics, Portland, OR), using previously described
equipment and procedures (17). Each mouse traversed the beam once, was weighed, and
returned to its cage. On Days 2 and 3, the two-day DID test was administered. Ethanol
group mice were exposed to 20% ethanol for 2 hr (Day 2) and 4 hr (Day 3), whereas
Control group mice were exposed to water. Mice were then immediately given two
intoxication tests. The order of testing for balance beam and rotarod was counterbalanced,
with half the mice in each group tested in each order (one order is described). Each mouse
was given a single trial on the balance beam and the number of hind foot slips was
recorded. Mice were then immediately tested on an accelerating rotarod (18). The
AccuRotor Rota Rod (Accuscan Instruments, Columbus, OH) was modified to a 63 cm fall
height and the diameter was 6.3 cm. Groups of 4 mice were placed on the stationary rod,
which was then immediately accelerated at 30 RPM/minute until all mice fell (within 2 min).
After a 30 sec inter-trial interval, the group was given two additional trials. Latencies to fall
were recorded. Immediately following the second intoxication test, each mouse had a 20 l
blood sample drawn from its periorbital sinus. Blood samples were therefore drawn within
15-20 min after the end of DID testing.
Limits of selection and a replicate (HDID-2) selected line
That R on S fits a linear function well (see Figure 2) implies that additive genetic
variance (that genetic variability which can be inherited) has not yet been exhausted by
selection pressure. Estimates of heritability from the linear regression of R on S are
generally most accurate early during selection (14). This is because as allelic forms of
genes leading to high drinking are enriched in the selected parents, the allele frequency at
each such gene moves closer to a value of 1. Ultimately, the responsible genes are fixed in
the homozygous state for the favorable allele, and additive genetic variability in the trait is
decreased. As this begins to happen during later selected generations, the slope of the R
on S function begins to decline and will eventually plateau. When no further response to
selection is seen despite repeated generations of selective breeding, one may conclude
that the limits of selection have been reached, and that the selected line is now inbred for
all genes relevant for the trait. The continued linear response to selection we see here (and
the relatively low heritability) implies that many genes influence this trait, and that further
response will be seen to continued selection pressure (14).
The ultimate limits of our selection (in this case, the plateau average BEC) cannot
be predicted from current information. Long Sleep and Short Sleep mice were differentially
selected for long- and short-duration loss of the righting reflex after an intraperitoneal