Ethanol Tolerance and Withdrawal Severity in High
Drinking in the Dark Selectively Bred Mice
John C. Crabbe, Alexandre M. Colville, Lauren C. Kruse, Andy J. Cameron, Stephanie E.
Spence, Jason P. Schlumbohm, Lawrence C. Huang, and Pamela Metten
Background: Mouse lines are being selectively bred in replicate for high blood ethanol concentra-
tions (BECs) achieved after limited access of ethanol (EtOH) drinking early in the circadian dark phase.
High Drinking in the Dark-1 (HDID-1) mice are in selected generation S21, and the replicate HDID-2
line in generation S14. Tolerance and withdrawal symptoms are 2 of the 7 diagnostic criteria for alcohol
dependence. Withdrawal severity has been found in mouse studies to be negatively genetically corre-
lated with EtOH preference drinking.
Methods: To determine other traits genetically correlated with high DID, we compared naı¨ve ani-
mals from both lines with the unselected, segregating progenitor stock, HS/Npt. Differences between
HDID-1 and HS would imply commonality of genetic influences on DID and these traits.
Results: Female HDID-1 and HDID-2 mice tended to develop less tolerance than HS to EtOH
hypothermia after their third daily injection. A trend toward greater tolerance was seen in the HDID
males. HDID-1, HDID-2, and control HS lines did not differ in the severity of acute or chronic with-
drawal from EtOH as indexed by the handling-induced convulsion (HIC). Both HDID-1 and HDID-2
mice tended to have greater HIC scores than HS regardless of drug treatment.
Conclusions: These results show that tolerance to EtOH’s hypothermic effects may share some com-
mon genetic control with reaching high BECs after DID, a finding consistent with other data regarding
genetic contributions to EtOH responses. Withdrawal severity was not negatively genetically correlated
with DID, unlike its correlation with preference drinking, underscoring the genetic differences between
preference drinking and DID. HDID lines showed greater basal HIC scores than HS, suggestive of
greater central nervous system excitability.
Key Words: Selected Mouse Lines, High Drinking in the Dark (HDID) Mice, Hypothermia,
Tolerance, Dependence, Genetic Correlation, Withdrawal.
mal model of binge-like ethanol (EtOH) drinking. The assay
is called Drinking in the Dark (DID). When offered a 4-hour
period of access to a 20% EtOH solution shortly after lights
out, some mice will drink enough to become intoxicated
(Rhodes et al., 2005, 2007). Studies of the DID phenomenon
established that there were significant genetic influences on
DID and that they were partially (but not completely) in
common with genetic influences on 2-bottle EtOH preference
drinking (Rhodes et al., 2007). We have been breeding High
Drinking in the Dark-1 (HDID-1) mice selectively for the
blood EtOH concentrations (BECs) attained after a 2-day
HE COMPANION PAPER (Crabbe et al., 2012)
describes our rationale for developing a new genetic ani-
DID test. The initial selection response was reported else-
where (Crabbe et al., 2009), and we are also breeding a repli-
cate line, HDID-2, for the same trait (Crabbe et al., 2010b).
The animals drink to the point of behavioral intoxication
(Crabbe et al., 2009), but show relatively normal preference
for alcohol solutions in a preference test (Crabbe et al.,
2011b). We review findings from both the selection and the
preference drinking studies in the companion paper (Crabbe
et al., 2012).
We are characterizing the selected lines for other responses
to EtOH to determine domains of EtOH response that might
share common genetic determinants with DID BEC. Studies
of the relative sensitivity to EtOH using several behavioral
assays across a range of EtOH doses from 1.4 to 4.0 g/kg,
reported in a companion manuscript (Crabbe et al., 2012),
revealed that some, but not all, indices of behavioral sensitiv-
ity to EtOH were correlates of DID BEC. We restricted
those analyses to the behavioral domains of motor stimula-
tion, various movement-based measures of intoxication (bal-
ance beam, rotarod, parallel rod floor), and more general
sedation (hypothermia, loss of righting reflex). All tests were
conducted following single injections of EtOH in naı¨ve ani-
mals. Our rationale for selecting these traits was grounded in
the finding of Marc Schuckit’s group that a “low level of
response” to alcohol predicted risk for eventual alcohol
From the Portland Alcohol Research Center, Department of
Behavioral Neuroscience, Oregon Health & Science University, and the
VA Medical Center (JCC, AMC, LCK, AJC, SES, JPS, LCH, PM),
Received for publication September 28, 2011; accepted November 8,
Reprint requests: John C. Crabbe, PhD, VA Medical Center (R&D
12), Portland, OR 97239; Tel.: 503-273-5298; Fax: 503-721-1029;
Copyright © 2012 by the Research Society on Alcoholism.
1152Alcohol Clin Exp Res, Vol 36, No 7, 2012: pp 1152–1161
ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH
Vol. 36, No. 7
dependence diagnosis (Schuckit, 2009; Schuckit and Smith,
2001). We discuss in the companion paper and elsewhere
why it is difficult to know which particular measures of acute
intoxication in rodents might best “model” human low level
of response (Crabbe et al., 2010a).
When EtOH is administered chronically, tolerance gener-
ally develops. This is revealed as either a reduction in
response to the same, repeated dose or the need to raise the
dose to maintain the original level of response. Either mani-
festation is the result of a shift to the right of the dose–effect
curve (Kalant et al., 1971). Tolerance may arise for pharma-
cokinetic reasons (e.g., an induction of metabolizing enzyme
activity); when this occurs, the proximate cause is a reduction
in the EtOH concentration at the target organ site. Because
the effector organ for most or all behavioral responses to
EtOH is the brain, pharmacokinetic (metabolic) tolerance is
not particularly informative as to neural mechanisms. Most
studies of EtOH tolerance have, therefore, focused on phar-
macodynamic (functional) tolerance, where the same BEC is
now less effective because of neuroadaptations to repeated
challenges with EtOH (Kalant et al., 1971). EtOH tolerance
has frequently been studied in rodents (for reviews, see
Kalant, 1996, 1998).
Several investigators have suggested that a propensity to
develop tolerance to EtOH may predispose one to the devel-
opment of dependence. One way this could occur is that indi-
viduals become tolerant to the stimulant, euphorigenic
effects of EtOH and so escalate their dose in an attempt to
recover the pleasurable “high.” Alternatively, or in addition,
development of tolerance to the more sedative and unpleas-
ant effects of EtOH may facilitate the escalation of drinking.
Dependence is thought to ensue in either case as the total,
repeated dose gradually results in neurotoxic effects (Koob
et al., 1998; Le ˆ and Mayer, 1996; Tabakoff and Hoffman,
Dependence is inferred when revealed by signs of with-
drawal, which are remarkably similar across mammalian
species (Friedman, 1980). Manifestations of tolerance and
withdrawal are 2 of the 7 possible contributing symptoms
leading to a diagnosis of alcohol dependence according to
the DSM-IV-R. In mice, the most frequently studied with-
drawal sign is the handling-induced convulsion (HIC), a sign
of central nervous system excitability that waxes and wanes
for several hours after cessation of EtOH administration.
This very sensitive behavioral index is exacerbated for several
hours after even a single anesthetic EtOH dose (Crabbe
et al., 1991a; Goldstein, 1972).
In rodents, sensitivity, tolerance, and dependence are all
influenced by genetic background. They share some overlap
in genetic influences as well, based on studies that estimate
genetic correlations from panels of inbred strains or by
examining correlated responses in selectively bred lines.
However, the areas of commonality do not generally fall
along the lines of the behavioral construct. Thus, animals
sensitive to one effect of EtOH are not necessarily sensitive
to others (Crabbe et al., 2005). There are different forms of
tolerance to EtOH, usually distinguished by how quickly
they develop: they are called acute (within-session, e.g., Mel-
lanby, 1919), rapid (reduced response to a second adminis-
tration, e.g., Crabbe et al., 1979), and chronic (following
multiple doses or long-term chronic exposure, e.g., Chen,
1979). Genetic contributions to rapid and chronic EtOH tol-
erance are reasonably consistent, suggesting that they might
be early and more fully developed aspects of the same under-
lying neuroadaptive processes (Hanchar et al., 2005; Kalant,
1998; Le ˆ and Mayer, 1996; Rustay and Crabbe, 2004). Acute
tolerance seems to be a form more genetically distinct from
rapid and chronic tolerance (Kalant, 1998; Ponomarev,
2002). However, there is also evidence that acute and rapid
tolerance may share some genetic determinants (Radcliffe
et al., 2006). Sensitivity and tolerance are sometimes posi-
tively genetically correlated in that strains and selected lines
more sensitive to an EtOH effect tend to develop more
tolerance (Crabbe et al., 1982; Deitrich, 1993; Phillips and
Crabbe, 1991) but not all findings are consistent (e.g., Aufrere
et al., 1994; Bennett et al., 2007; Deitrich et al., 2000; Drews
et al., 2010; Gehle and Erwin, 2000; Radcliffe et al., 2005).
The literature on this topic is large and has not been reviewed
systematically for many years (Phillips and Crabbe, 1991).
Sensitivity and withdrawal severity are not generally corre-
lated (Crabbe et al., 1983, 1994; Drews et al., 2010). Chronic
tolerance and dependence (i.e., withdrawal severity) may be
negatively genetically coupled, but the evidence for this is
also mixed (Crabbe, 1996; Crabbe et al., 1983, 1988; Drews
et al., 2010; for review, see Crabbe et al., 2011, in press).
Overall, it is likely that the genetic relationships among sensi-
tivity, tolerance, and dependence vary somewhat not only
with the behaviors and the specific forms of tolerance studied
but also with the specific genetic animal models employed
(Deitrich et al., 2000; Radcliffe et al., 2004).
In contrast to the mixed results regarding genetic correla-
tions of sensitivity, tolerance, and dependence, studies com-
paring withdrawal severity and preference drinking have
consistently reported negative genetic correlations between
these phenotypes in both rats and mice (Chester et al., 2002,
2003; for reviews, see Belknap et al., 2008; Metten et al.,
1998). Thus, for example, standard inbred mouse strains that
prefer to drink EtOH solutions tend to display low with-
drawal HICs after being rendered physically dependent. And
lines of mice selectively bred for severe withdrawal HICs tend
to drink less than those selected for mild withdrawal (Metten
et al., 1998). In all these studies, animals are tested for 1 trait
only; thus, the negative genetic relationship does not address
postdependence drinking, of which there are virtually no
We elected to compare HDID-1 mice with their controls, a
heterogeneous stock (HS), for their development of EtOH
tolerance. On the basis of not entirely consistent evidence
reviewed above, and the fact that DID and preference drink-
ing are imperfectly genetically correlated, we predicted that
HDID-1 mice would develop greater tolerance than HS. We
also compared the genotypes for both acute and chronic
GENETIC CORRELATES OF ETHANOL DRINKING IN THE DARK
EtOH withdrawal severity. Again, with the caveat regarding
DID and preference, we predicted that HDID-1 would dis-
play less severe withdrawal HICs than HS. We elected to
employ both HDID-1 and HDID-2 lines, as we were inter-
ested in collecting the archival data for HDID-2. The ratio-
nale is discussed in the Introduction to the companion
manuscript and is not reiterated here (Crabbe et al., 2012).
MATERIALS AND METHODS
Animals and Husbandry
All mice were bred and maintained 2 to 5 to a polycarbonate or
polysulfone cage (used interchangeably) with Bedocob bedding in
ventilated (Thoren Caging Systems, Inc., Hazleton, PA) racks with
ad libitum access to tap water and food (Purina 5001; PMI Nutri-
tion International, St. Louis, MO) in our colonies at the Portland
VA Veterinary Medical Unit, an AAALAC-approved facility.
Lights were on from 07:00 to 19:00 hours, and all behavioral testing
was conducted between 09:00 and 14:00 hours except as noted.
Temperature was maintained at 21 ± 1°C.
We tested approximately equal numbers of males and females in
each genotype and treatment group in Experiments 1 and 2; only
males were available for Experiment 3. Mice were HDID-1 and
HDID-2 selected lines and the heterogeneous stock (HS/Npt; HS)
from which the selections were initiated (Crabbe et al., 2009,
2010b). HDID-1 mice were from the 18th and 19th selected genera-
tions, HDID-2 mice from the 11th and 12th selected generations,
and HS were from the 70th and 71st filial generations. The HDID
lines were selected for the high BEC they displayed at the end of a 4-
hour session of drinking 20% (v/v) EtOH starting 3 hours after the
beginning of their circadian dark period. All mice were naı¨ve at the
beginning of the behavioral test (or pair of tests) and ranged in age
from 8 to 15 weeks.
Drugs and Injections
Ethanol was mixed 20% (v/v) in physiological saline on the
morning of the test. Injections were given intraperitoneally (ip) at a
volume adjusted for dose of EtOH (g EtOH/kg body weight). BECs
were determined using a standard gas chromatographic procedure
(Rustay and Crabbe, 2004). Pyrazole HCl (Sigma-Aldrich, St.
Louis, MO) was given to all mice in Experiment 3.
Asdiscussed in the Introduction tothe companion paper (Crabbe
et al., 2012), we were not interested in assessing potential differences
between HDID-1 and HDID-2 lines. Because the HDID-2 line had
been selected for many fewer generations, expectations about the
relative response of the HDID-2 line versus control HS are less clear
than for HDID-1 versus HS. Thus, we conducted 2 sets of statistical
comparisons for each experiment and independently evaluated
HDID-1 versus HS and HDID-2 versus HS. We reasoned that the
benefit of capturing archival data on the HDID-2 mice was worth
enduring the potential statistical confound of using the control HS
data twice. Analyses of variance (ANOVAs) or t-tests were used to
evaluate all dependent variables. Significant interactions were
pursued with Tukey’s HSD tests. Initial ANOVAs included sex as a
factor. Differences at p < 0.05 were considered significant.
Experiment 1. Tolerance to EtOH Hypothermia
Tolerance to EtOH hypothermia was tested using a variant of
our standard procedures (Crabbe et al., 1982). These were as
described for the acute hypothermia test conducted in Experiment 2
of the companion manuscript (Crabbe et al., 2012). Two groups of
mice were tested for each genotype. On Days 1 and 2, one group
was given a saline injection (Group saline saline ethanol [SSE]) and
the other group was given EtOH (3.0 g/kg; Group ethanol ethanol
ethanol [EEE]). On Day 3, both groups were given 3.0 g/kg EtOH.
Testing for hypothermia before and 30, 60, 90, and 120 minutes
after injection was conducted on Days 1 and 3: only injections were
given on Day 2 and mice were returned to their home cage. On Day
3, a 20-µl blood sample was taken from the tip of the tail to assess
BEC after the 120-minute body temperature.
Experiment 2. Acute Withdrawal Severity
These were the same animals tested simultaneously for loss of
righting reflex in Experiment 3 in the companion manuscript (Crab-
be et al., 2012). Mice were first scored twice for basal HIC severity,
30 minutes apart. These scores were averaged to provide a baseline
HIC score. To test for HICs, mice were gently picked up by the tip
of the tail. If this failed to elicit a convulsion, mice were gently spun
in a 360° arc and scored. The HIC scale ranges from 0 to 7 where 7
is lethal (Crabbe et al., 1991a). Scores after acute withdrawal typi-
cally range from 0 to 5. Because most mice had regained righting
reflex within 2 hours (see Experiment 3 Results from companion
manuscript), mice were tested for EtOH withdrawal HICs starting
at hour 2 after injection of 4.0 g/kg, unless they were still on their
backs. HICs were scored hourly from hours 2 to 12, and again the
next day at hours 24 and 25. After each HIC test, mice were placed
back in their home cages.
Experiment 3. Chronic Withdrawal Severity
Dependence Induction. This study examined withdrawal severity
following continuous administration of EtOH vapor by inhalation
for 72 hours (Metten and Crabbe, 2005). Male mice of each geno-
type were divided into 2 groups: one group was exposed only to air
in the chambers, while the other group was exposed to volatilized
EtOH. Within each genotype, mice within a cage were pseudo-ran-
domly assigned to either the air or EtOH condition. Mice in the
EtOH groups were initially injected with EtOH (1.75 g/kg, 20% v/v
in saline) to establish elevated BECs. Pyrazole HCl (68.1 mg/kg, in
saline, ip), an alcohol dehydrogenase inhibitor, was given combined
with the EtOH injection to inhibit EtOH metabolism and stabilize
BECs. Mice in the Control groups were injected with saline with
pyrazole on Day 1 and placed in identical chambers where they
inhaled only air. Twenty-four (Day 2) and 48 (Day 3) hours later,
all mice were removed from the chambers, injected with pyrazole
again, and replaced in the chambers. After 72 hours of inhalation,
all mice were removed from the chambers and a 20-µl blood sample
was drawn from the tip of their nicked tail. Control mice had their
tails nicked but no blood was withdrawn.
Each morning during inhalation, a few animals had blood
samples taken to assess their BEC before injection of all mice with
pyrazole. Adjustments to the EtOH vapor concentration were made
in an attempt to maintain average BEC at 2.00 mg/ml.
Withdrawal Testing. Immediately after removal from vapor at
72 hours and before blood sampling, all mice were scored for HIC
severity and weighed. HIC scoring was repeated each hour for
12 hours and at hours 24 and 25, using the same procedure
described above. Because scores at hour 25 had not returned to
baseline (see Results), we scored mice again at hours 30 and 31. To
index HIC withdrawal severity, the area under the curve (AUC) was
computed. A peak value was also computed, defined as the highest
running average of 3 consecutive scores that contained the maxi-
mum HIC score for that animal. If there was a tie, the earliest occur-
rence was considered “peak.” Latency to peak was defined as the
withdrawal hour of the first maximum score within the peak.
CRABBE ET AL.
Experiment 1. Hypothermic Tolerance
Day 1 temperature response time curves for the EEE
group receiving their first EtOH injection were very similar
to those seen in Experiment 2 in the companion manuscript,
while animals given saline showed little temperature change
from baseline. Day 3 response time courses were attenuated
in the EEE groups, consistent with tolerance development
(data and analyses not shown). To assess differences in toler-
ance between groups, we calculated and analyzed total hypo-
thermic response as the AUC on Day 3, first including sex as
a factor. For both HDID-1 versus HS and HDID-2 versus
HS comparisons, EEE groups showed less hypothermia than
SSE groups, Fs(1, 47 to 48) ? 10.3, ps < 0.01, and there
were significant 3-way interactions of genotype, treatment,
and sex, Fs(1, 47 to 48) ? 4.5, ps < 0.05. We therefore ana-
lyzed the data from the sexes separately (Fig. 1). Main effects
of treatment within sex were always significant, all Fs(1, 21
to 27) ? 5.0, ps < 0.05. For the first replicate, interactions
of genotype and treatment tended to reach significance only
for the HDID-1 versus HS females, F(1, 27) = 3.1, p = 0.09,
where HS (p = 0.04) but not HDID-1 female mice showed
significant tolerance (Fig. 1A: AUC day 3). The apparently
similar results in female HDID-2 versus HS mice were not
supported by a significant interaction. The apparent differ-
ence between HDID-1 and HS males (Fig. 1B: AUC day 3),
with HDID-1 tending to show greater tolerance than HS,
also was not supported by a significant interaction
(p = 0.17), but the second replicate versus HS males tended
to show an interaction of genotype 9 treatment, F(1, 22)
= 3.2, p = 0.09. HDID-2 (p = 0.02) but not HS males
BECs taken after the 120-minute temperature on Day 3
were analyzed to see whether metabolic tolerance could have
developed. Across the 77 animals, average BEC was
2.16 ± 0.05 mg/ml. Mean BECs ranged between 1.77 and
2.61 mg/ml across the 12 genotype 9 treatment 9 sex con-
ditions (total range = 1.08 to 3.36 mg/ml). ANOVAs includ-
ing sex showed significant main effects of treatment and
sex in both replications, all but one Fs(1, 43 to 44) ?
3.8, ps < 0.05; however, the main effect of sex only tended
toward significance for the HDID-2 versus HS comparison,
F(1, 44) = 3.4, p = 0.07. Neither interaction was significant,
Fig. 1. Mean ± total hypothermic response on Day 3 in SSE groups receiving their first ethanol (EtOH) injection (dark bars) and EEE groups receiving
their third (light bars). Panel A shows data for females, Panel B for males. Left half of each panel shows uncorrected scores [Total hypothermic response
(AUC day 3)], and right half shows residuals from regression of uncorrected scores on blood EtOH concentrations [Total hypothermic response (residual
day 3)]. N = 5-8 per sex/genotype/treatment group. Note that the bars depicting HS scores for residuals [total hypothermic response (residual day 3)] are
a composite, based on averaging the 2 values derived from the separate regressions of each replicate High Drinking in the Dark (HDID) line and HS.
These 2 values did not differ by more than 11% for any averageshown. SSE, saline saline ethanol; EEE, ethanol ethanol ethanol.
GENETIC CORRELATES OF ETHANOL DRINKING IN THE DARK
both Fs(1, 43 to 44) ? 2.0, ps > 0.10. Females had higher
BECs than males within each genotype 9 treatment group
comparison. Consistent with the development of metabolic
tolerance, EEE groups had lower average BECs than SSE
groups within each genotype 9 sex comparison.
To estimate the contribution of metabolic tolerance, we
regressed Day 3 total hypothermic response on BEC sepa-
rately for each replicate, including data from both SSE and
EEE groups. For HDID-1 versus HS, BEC explained 18%
of the variance (R2) in total hypothermic response
(p = 0.002), and the relationship was even stronger in
HDID-2 versus HS (R2= 0.31, p < 0.0001). To parallel the
analyses of uncorrected temperature responses, we then
recalculated the regressions for each sex and replicate combi-
nation separately. These relationships showed that BEC
explained between 10% (HDID-1 vs. HS males) and 41%
(HDID-2 vs. HS females) of the variance in total hypother-
To estimate the importance of functional tolerance after
metabolic tolerance had been accounted for, we therefore
expressed each animal’s total hypothermic response as a
residual from the regression on BEC and analyzed these val-
ues on the assumption that comparisons between SSE and
EEE groups after the contribution of BEC had been statisti-
cally removed provided a reasonable estimate of functional
tolerance. For HDID-1 versus HS females, a significant
interaction of genotype 9 treatment was found, F(1, 24)
= 6.0, p < 0.05; Tukey’s post hoc tests showed that HS
(p = 0.04) but not HDID-1 females showed lower total
hypothermic responses in their EEE group versus SSE
group, that is, functional tolerance (Fig. 1A: residual day 3).
For the comparison of male HDID-1 versus HS mice, no
main effects or interactions reached significance, but a geno-
type 9 treatment trend, F(1, 19) = 3.2, p = 0.09, suggested
that some of the tolerance seen in uncorrected scores in
HDID-1 males may have been functional in nature (Fig. 1B:
residual day 3). Analyses of residual scores for the second
replicate yielded results qualitatively similar to those seen in
the HDID-1 versus HS comparisons; that is, HDID-2
females appeared to show less, and HDID-2 males greater,
of genotype and treatment approached significance for the
HDID-2 males, F(1, 21) = 3.3, p = 0.08; a post hoc test
showedthatHDID-2(p = 0.03)butnotHSshowedtolerance.
Across the 2 analyses, these data suggest that significant
tolerance developed to EtOH’s hypothermic effects. For the
most part, that tolerance appeared to be functional, although
a small degree of metabolic tolerance also developed. The
genotypic differences in tolerance appeared to be sex depen-
dent (see Discussion).
Mean body weights at the beginning of tolerance testing
were analyzed by genotype and treatment within each sex
and replicate comparison to parallel the analyses of hypo-
thermic tolerance. Weights at the beginning were well
matched for the main effects of treatment groups, all
Fs ? 1.2, ps > 0.10. Female HDID-1s weighed significantly
less than HS [19.8 ± 0.4 g vs. 23.0 ± 0.6 g, respectively;
F(1, 27) = 17.7, p < 0.001], and the interaction of genotype
and treatment was not significant (F = 1.1, NS). For male
HDID-1 versus HS mice, neither main genotypic nor treat-
ment group effects nor their interaction were statistically sig-
nificant, all Fs(1, 20) < 1.8, NS. For HDID-2 versus HS,
there were no significant weight differences across treatment
groups as either main effects or interactions with genotype
for either females or males before injections began, all
Fs(1, 22 to 25) ? 1.2, ps > 0.10. There were trends toward
genotypic differences for both sexes (females: F = 2.7, p =
0.12; males, F = 3.6, p < 0.10). Female HDID-2 mice tended
to weigh less than HS, while males tended to weigh more.
Weight loss during the experiment was approximately
?3.3% across all 82 mice. The main effect of treatment with
EtOH for 3 days versus 1 day was to yield significantly
greater weight loss in all genotypes and sexes, all Fs(1, 20 to
27) ? 5.3, ps < 0.05. Neither genotype nor the interaction
with treatment was significant in either replicate (all Fs < 1).
The exception was HDID-2 versus HS males, where geno-
type (F = 5.2, p < 0.05) was significant: male HDID-2 mice
did not lose weight overall (+0.5 ± 1.7%) while HS males did
(?3.3 ± 0.4%). We conclude that weight loss did not con-
tribute to the EtOH tolerance differences seen.
Experiment 2. Acute Withdrawal Severity
The HIC time course before EtOH injection (Time 0) and
during EtOH withdrawal is depicted in Fig. 2A. We first
analyzed baseline HIC scores. Analyses including sex as a
(males > females) for HDID-1 versus HS, F(1, 59) = 6.8,
p < 0.01, and an interaction of sex and genotype for HDID-
2 versus HS, F(1, 60) = 5.0, p < 0.05. However, all data are
reported collapsed on sex because sex-specific analyses did
not differ in outcome (data not shown). Figure 2A shows
that both HDID-1 and HDID-2 mice had significantly
higher baseline HIC scores than HS, ts ? 3.7, df = 61 to 62,
ps < 0.001.
HIC scores were reduced to near zero when first assessed
at 2 hours after EtOH injection, and animals reached their
peak withdrawal between 6 and 9 hours after injection.
Scores had declined to near baseline by 24 hours later.
Figure 2B depicts the AUC of the 25-hour HIC curve over
time, corrected for baseline scores as described in the
Methods. All 3 genotypes showed significant withdrawal (vs.
an area score of zero, which would represent scores equal to
baseline). Neither selected line differed significantly from HS
in withdrawal severity (both ts < 1). Neither corrected peak
scores nor latency to peak differed between genotypes for
any comparison (data not shown).
Experiment 3. Chronic Withdrawal Severity
The HIC time course during EtOH withdrawal is depicted
in Fig. 3A. Peak withdrawal was reached between hours 4
CRABBE ET AL.
and 7 after removal from the chambers, had subsided some-
what by hours 24 to 25, and even more by hours 30 to 31. To
facilitate comparison with data collected in many other geno-
types (Crabbe, 1994; Metten and Crabbe, 2005; Metten
et al., 2010), we indexed withdrawal severity using the AUC
of the first 25 hours of the withdrawal curve (Fig. 3B).
HDID-1 and HS mice displayed equivalent withdrawal
severity. Analyses revealed significant main effects of treat-
ment, F(1, 43) = 197.2, p < 0.0001, but not genotype,
F(1, 43) = 2.8, p = 0.10; nor was the interaction significant
(F < 1). For HDID-2 versus HS, a similar pattern was seen.
Main effects of treatment and genotype were significant,
Fs(1, 45) = 226.6 and 8.2, respectively, ps < 0.01, but the
interaction was not (F < 1). Given the significantly higher
baseline HIC scores seen in both HDID-1 and HDID-2 mice
in Experiment 2, we also compared the AUC for the air-trea-
ted mice in each replicate. As suggested from Fig. 3B, HIC
scores for air-treated control mice appeared to be higher in
both selected lines than in the HS. The difference in control
AUCs was significant for the HDID-2 versus HS compari-
son, t(22) = 2.38, p = 0.03, but not for HDID-1 versus HS,
t(22) = 1.28, p > 0.10. Neither corrected peak scores nor
latency to peak differed between genotypes for any compari-
son (data not shown).
The range of genotypic mean BECs from mice sampled
during inhalation was 1.27 to 2.20 mg/ml (Day 1) and 1.69
to 2.23 mg/ml (Day 2). HDID-1 mice tended to reach
slightly higher, and HDID-2 mice slightly lower, BECs than
HS during the first 2 days. BECs at the time of withdrawal
for the EtOH-treated groups were 1.99 ± 0.08, 1.91 ± 0.08,
and 1.84 ± 0.07 mg/ml for HDID-1, HDID-2, and HS,
respectively; neither selected line differed significantly from
HS in BEC at withdrawal, ts < 1.46, ps > 0.10.
Fig. 2. Panel A: Mean ± SE handling-induced convulsion (HIC) scores
before (Time 0) and after injection (arrow) with 4.0 g/kg EtOH. N = 32, 31,
and 32 for HS, High Drinking in the Dark-1 (HDID-1) and HDID-2, respec-
tively. Mice were scored for LORR between hours 0 and 2 (see companion
manuscript, Crabbe et al., 2012). Panel B: Corrected AUC for data
depicted in Panel A. EtOH, ethanol; LORR, loss of righting reflex.
Fig. 3. Panel A: Mean ± SE handling-induced convulsion (HIC) scores
from the time of removal from the inhalation chambers (Time 0) and
31 hours later. N = 11, 12, and 14 for HS, HDID-1, and HDID-2 EtOH
groups, respectively. N = 11, 10, and 11 for the 3 respective air control
groups. Panel B: Area under the HIC curve for hours 0 to 25 for the data
depicted in Panel A. EtOH-treated mice are shown in dark bars and air-
treated controls in light bars. EtOH, ethanol; HDID, High Drinking in the
GENETIC CORRELATES OF ETHANOL DRINKING IN THE DARK
Mean body weights at the beginning of inhalation testing
were analyzed by genotype and treatment within each repli-
cate comparison to parallel the analyses of withdrawal sever-
ity. Weights at the beginning were well matched across
treatment groups, F(1, 43) = 1.3, but were significantly lower
in male HDID-1 than HS mice 29.1 ± 0.7 g versus
31.8 ± 1.0 g, respectively, F(1, 43) = 4.5, p < 0.05, and the
interaction of genotype and treatment was not significant
(F < 1). For HDID-2 versus HS, there were no apparent
(or significant) weight differences before inhalation, all
Fs(1, 45) ? 2.7, ps > 0.10. Mice lost approximately ?13%
body weight during inhalation exposure. For the HDID-1
versus HS comparison, the main effects of genotype and
treatment, and their interactions, were all nonsignificant, Fs
(1, 42) ? 2.5, ps > 0.10. HDID-2 mice lost less weight than
HS [?10.0 ± 0.9% vs. ?14.0 ± 0.8%, respectively, F(1, 45)
= 7.8, p < 0.01], but percent weight loss did not differ as a
function of treatment or the treatment 9 genotype interac-
tion (Fs < 1).
We conclude that neither any effects of the expected mod-
est weight loss nor differences in delivered EtOH dose could
have masked a genotype difference in withdrawal severity.
Neither could have the differences in air-treated HIC scores
led us to spurious inferences regarding relative sensitivity to
In the hypothermia tolerance study, a complex pattern of
results was seen, evidenced by significant 3-way interactions
of treatment, genotype, and sex for both replicates of the
DID selection. Consideration of only uncorrected tempera-
ture scores showed clear tolerance development when look-
ing at data within sex for each replicate. Thus, the
experiment was successful at engendering EtOH tolerance.
Interpretation becomes less clear when the data are consid-
ered within each sex separately, because what appear to be
substantial genotypic effects interacted with sex. BEC pre-
dicted a significant proportion of total hypothermic response
in most cases, so we tried to estimate the possible contribu-
tions of metabolic versus functional tolerance by comparing
uncorrected hypothermia scores with those expressed as a
residual from regression on BEC. We acknowledge that sta-
tistical rigor for these comparisons was limited by relatively
low N for assessing the interactions of genotype and treat-
ment within sex and replicate. We believe that most of the
tolerance shown here was functional, and to the extent that
metabolic tolerance contributed, it did not affect interpreta-
tion of treatment, sex, or genotypic differences. To convince
the reader of this interpretation, we suggest visual compari-
sons of the left with the right half of Fig. 1A (for females)
and 1B (for males). The pattern of differences for each sex is
strikingly similar. When the contribution of BEC was statis-
tically removed (i.e., the residual scores), the geno-
type 9 treatment interactions for females versus HS in both
replicates were significant; in contrast, analyses of uncor-
rected scores resulted only in a statistical trend toward an
interaction for HDID-2 versus HS females. The analogous
interactions for males yielded only statistical trends; we
believe this was likely due to the relatively greater within-
group variability of the male versus the female groups. Power
to detect interactions in factorial ANOVAs is less than that
needed to detect main effects (Wahlsten, 2007), and we were
not expecting to encounter 3-way interactions.
A formal and powerful assessment of metabolic versus
functional tolerance would require additional experiments
and could best be addressed by administering multiple injec-
tions to 1 set of groups (like our EEE groups) and then com-
paring their response on the last day with several groups of
“SSE” mice, with each pair of SSE versus EEE groups given
different doses of EtOH. This would allow construction of
the dose–effect curves and allow comparison of their slopes
(see Crabbe et al., 1979). It should be noted that the overall
degree of tolerance seen in the current studies was not large.
The 3 daily injections we employed resemble both rapid and
chronic tolerance, but most studies of chronic tolerance use
several more daily (or twice daily) injections or a period of
chronic exposure through drinking or vapor inhalation.
Thus, extending EtOH injections for more days to elicit max-
imal chronic rather than rapid tolerance might clarify the
picture. Tolerance is seen in nearly all genotypes we have
studied when 5 to 8 daily EtOH injections are used (Crabbe,
1994; Crabbe et al., 1982, 2006).
Whether EtOH hypothermic tolerance is a correlated
response to selection on DID BEC is a more difficult ques-
tion to answer. Here, interpretation is first made difficult by
the obvious interactions of sex with genotype. Considering
only the control HS mice, females clearly developed toler-
ance, while males did not. HDID-1 and HDID-2 female mice
failed to develop significant tolerance, with or without
accounting for BEC (Fig. 1A). This pattern of results sup-
ports the hypothesis that resistance to the development of
hypothermic tolerance is a correlated response. Because the
degree of hypothermic tolerance development in mice is
genetically correlated with initial hypothermic sensitivity
(Browman et al., 2000; Crabbe et al., 1982, 2006; but see
Radcliffe et al., 2005), this agrees with our findings in the
companion paper (Crabbe et al., 2012) that selection for
DID BEC resulted in reduced sensitivity to EtOH hypother-
mia in females. Thus, the lack of hypothermic tolerance in
HDID-1 and HDID-2 females may result from their lesser
initial response to EtOH than HS. In contrast, both male
HDID-1 and HDID-2 mice appeared to develop tolerance
while HS did not (Fig. 1B), although these differences did
not reach statistical significance. It is possible that the males
from the selected lines developed tolerance more rapidly than
HS males, as they did not differ in initial response in this
experiment. We do not know why our earlier study showed
less initial hypothermic sensitivity in both female and male
HDID mice than in HS (Crabbe et al., 2012). We tentatively
conclude that hypothermic tolerance is a correlated response,
but that it is sex dependent.
CRABBE ET AL.
We found only 4 papers reporting tolerance to EtOH
hypothermia in rodents of both sexes given multiple injec-
tions of EtOH. One paper with rats showed greater tolerance
in females (Light et al., 1990) while 2 others favored males
(Khanna et al., 1985; Webb et al., 2002). The Webb study
also found greater tolerance in males for the loss of righting
reflex, and their data suggest that males developed more
acute functional tolerance; this could be taken to support the
hypothesis of faster tolerance development in male HDID-1
and HDID-2 mice. A study with mice lacking functional
adenosine A2A receptors showed rapid tolerance develop-
ment to EtOH hypothermia only in female wild types, but
not in either knockout females or males of either genotype
(Naassila et al., 2002).
We found no studies reporting a crossover interaction of
sex with genotype for EtOH tolerance. Gehle and Erwin
(2000) found that female mice of some LSxSS RI strains
showed greater acute functional tolerance on the dowel test
than males, while for other strains no sex differences were
seen. Gill and Deitrich (1998) also examined acute functional
tolerance on a fixed speed rotarod task in LS and SS mice of
both sexes. They found a genotype 9 sex interaction favor-
ing a greater acute functional tolerance difference (SS > LS)
between female mice than the difference seen in males.
The “differentiator hypothesis” (Newlin and Thomson,
1990) suggests that human studies of acute alcohol challenge
show differences between subjects at low or high risk for
developing dependence later in life for 2 reasons. First, at-
risk subjects are more sensitive to effects of EtOH that occur
soon after drinking: these include stimulation, elevated
mood, and subjective “high.” Some time later during the
acute session, at-risk subjects report experiencing less seda-
tion and intoxication and display other signs of blunted
response to EtOH. It is hypothesized that they are less sensi-
tive later owing to the development of more acute functional
tolerance (Newlin and Thomson, 1990). Our companion
report (Crabbe et al., 2012) found that HDID mice were less
sensitive than HS to acute EtOH hypothermia. However,
there was sparse evidence for differential sensitivity of HDID
versus HS mice across a range of other tasks probing stimu-
lant and sedative responses to acute EtOH challenge. In the
current studies, HDID mice tended to develop less, not more,
tolerance than HS to EtOH hypothermia. However, the form
of tolerance probed here resembles rapid or chronic tolerance
more than acute functional tolerance. It would be of interest
to test HDID and HS mice for acute functional tolerance to
EtOH (Erwin and Deitrich, 1996; Ponomarev and Crabbe,
2004) as this is the closest equivalent to tolerance that could
affect apparent sensitivity differences shortly after injections.
The extant data from this and the companion report (Crabbe
et al., 2012) do not align well with the postulate derived from
studies of at-risk humans (Schuckit, 2009; Schuckit and
Smith, 2001) that low level of response to EtOH predicted
alcoholism risk. However, the animal tests we have employed
are not clearly related to the human traits tested, as noted
elsewhere (Crabbe et al., 2010a, in press).
There were clear EtOH withdrawal effects in all 3 geno-
types, after either a single, acute high-dose injection or fol-
lowing chronic vapor inhalation. However, there were no
genetic differences in withdrawal severity. It would not have
been a surprise to find that HDID mice displayed less severe
acute and chronic EtOH withdrawal HIC scores than HS. A
survey of several genetic animal models has revealed a sub-
stantial negative genetic correlation between 2-bottle prefer-
ence drinking and withdrawal severity (Metten et al., 1998),
and subsequent studies in both rats and mice have strength-
ened the hypothesis of similar genetic control of these traits
(e.g., Chester et al., 2002, 2003). However, DID and 2-bottle
preference drinking are only partially influenced by common
genetic factors (Crabbe et al., 2009, 2011b). The current data
suggest that the genetic influence shared between withdrawal
and preference drinking is largely different from that shared
between the 2 drinking phenotypes.
It is interesting that both selected lines seem to have
developed greater basal HIC scores than HS, a sign of a
correlated response. The HIC was first reported under that
name by Goldstein (Goldstein, 1972) as a sensitive sign of
EtOH withdrawal severity. The HIC resembles signs from
earlier reports by Chance (Chance, 1953a,b; Chance and
Yaxley, 1950), who elicited a convulsion by holding a
mouse by the tail and jerking his hand down rapidly. That
the HIC reflects central nervous system excitability may be
inferred from several reports that it is exacerbated by a wide
range of doses of convulsant drugs that are too low to elicit
the full tonic and/or clonic convulsions characteristic of the
particular drug (Crabbe et al., 1981, 1991b). HICs are exac-
erbated during withdrawal from many agents that de‘press
central nervous system activity, including barbiturates,
benzodiazepines, alcohols, and other sedative-hypnotics
(Belknap et al., 1987; Crabbe, 1992; Crabbe et al., 1991a;
Goldstein, 1972; Reilly et al., 2000), and withdrawal-related
HICs are suppressed by those same agents and other drugs
(Beadles-Bohling and Wiren, 2006; Littleton et al., 1990;
Olive and Becker, 2008).
Despite the wide use of the HIC to index drug withdrawal,
little is known about its physiology, and its pharmacology is
obviously promiscuous. Elevated baseline HICs emerged as
a correlated response to selection for chronic EtOH with-
drawal HIC severity in both Withdrawal Seizure-Prone
corresponding Withdrawal Seizure-Resistant (WSR) lines
(Crabbe et al., 1985). This likely occurred because selection
was not based on a difference score between EtOH-with-
drawing mice and saline-treated mice. Withdrawal Seizure-
Prone (WSP) mice are generally about 10% more sensitive
than WSR mice to the effects of convulsants, but this is not
true for all drugs and the pharmacological differences sug-
gested from seizure susceptibility are complex (for review,
see Crabbe, 1996). It would be of interest to compare the
HDID selected lines and HS for susceptibility to a range of
convulsants and other seizure-inducing treatments to explore
the basis for the elevated basal HIC further.
GENETIC CORRELATES OF ETHANOL DRINKING IN THE DARK
We found that differences in EtOH metabolism could not
explain the observed differences between HDID lines and
HS in acute sensitivity to EtOH (Crabbe et al., 2012). For
the chronic withdrawal data in Experiment 3, there was no
serious possibility that differences in metabolism could affect
results, as such differences were explicitly disallowed by
matching BECs across genotypes during each day of inhala-
tion. Neither could BEC at the time of the test explain differ-
ences in hypothermic tolerance in Experiment 1. Much prior
data suggest that genetic differences in behavioral response
to EtOH are unrelated to genetic differences in BEC. The
modest reductions in body weight seen during the chronic
hypothermia and withdrawal tests also could not explain the
patterns of genotypic differences.
As we explained in the companion manuscript (Crabbe
et al., 2012), it is somewhat difficult to interpret the results
from the studies with HDID-2 at this relatively early point in
selection. Thus, it is possible that some of the responses we
tested here could come to differ between HDID-2 and HS in
future generations. In conclusion, selection for high BEC
after DID has not yielded genotypes that differ in withdrawal
severity from unselected HS mice. However, female mice
from these selected lines appear to develop less EtOH hypo-
thermic tolerance than HS. Whether male HDID mice
develop more tolerance or develop it faster than HS will
require further experiments. Finally, many other behavioral
domains remain unexplored in HDID mice. Anxiety- or
depression-like behaviors, impulsive behaviors, sensitivity to
reward or punishment, learning ability, and the sensitivity
to effects of EtOH in any of these domains could be related
to the elevated DID-BEC phenotype and will be the subject
of future experiments.
These studies were supported by Grants AA010760 and
AA013519 from the NIAAA and by a grant from the U.S.
Department of Veterans Affairs. LCK and AMC were sup-
ported by grant AA07468 from the NIAAA. We thank Mark
Rutledge-Gorman for assistance with the manuscript.
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