Impulse: the Premier Journal for Undergraduate Publications in the Neurosciences
2007
Oral Self-Administration Of Ethanol In Transgenic Mice
Lacking β-Endorphin
Sidney B. Williams, Ashley Holloway, Kevin Karwan, Stephani Allen, Judith E.
Grisel
Department of Psychology, Furman University, Greenville, SC 29613
Ethanol (EtOH) modifies the production and/or release of endogenous opioid peptides, including
β-endorphin (Gianoulakis, 2004; Przewlocka et al., 1994; Schulz et al., 1980). Opioids
subsequently influence the reinforcing properties of EtOH and the development of alcoholism
(Terenius, 1996; Van Ree, 1996). In this study, β-endorphin deficient mutant mice were used to
examine the effects of a specific opioid peptide on EtOH consumption. A two-bottle free choice
EtOH oral self-administration paradigm was administered to homozygous mutant mice (void of
all β-endorphin), heterozygous mice (50% β-endorphin expression), and sibling wildtype mice
(C57BL/6J; B6). Subjects received increasing concentrations of EtOH (0%, 3%, 6%, 12%, and
15%) each given over an eight day span, and were evaluated for preference and consumption each
day. Overall, females drank more than males. Homozygous mutant mice (KO) showed
decreased preference for EtOH at all concentrations, and self-administered significantly less than
heterozygous mice (HT) and wildtype mice (B6). The HTs had a tendency to drink the most
followed by the B6s, and the KOs drank the least. These data support the hypothesis that βendorphin influences the reinforcing effects of EtOH.
Keywords: Ethanol, β-endorphin, self-administration, transgenic mice
Introduction
Biological contributions to
excessive ethanol (EtOH) drinking that can
lead to alcoholism have been investigated
for decades (Nestler and Aghajanian, 1997;
Cowen et al., 2004). Animal models
evaluating the chemical and genetic
substrates for self-administration of alcohol
(EtOH) have been used in an attempt to
identify mechanisms that promote the
development of alcoholism in humans.
The relationship between EtOH and
endogenous opioid peptides is of particular
interest. For instance, EtOH modifies the
synthesis and release of β-endorphin
(Scanlon et al., 1992; Froehlich, 1995), and
these changes in turn affect dopamine
release in the mesolimbic pathway, a critical
neural substrate for reward (Widdowson and
Holman, 1992). Numerous studies in
animals (Altshuler et al., 1980; Froehlich et
al., 1990) and in humans (O’Malley et al.,
1992; Volpicelli et al., 1992) have shown
that administration of an opiate antagonist
can decrease EtOH consumption. The
EtOH-mediated release of dopamine in the
nucleus accumbens can also be blocked by
opiate antagonists (Gonzalez and Weiss,
1998). Thus, β-endorphin is thought to play
an important role in the reinforcing effects
of EtOH, mediating consumption (Grisel et
al., 1999; Roberts et al., 2000), craving (Van
Ree, 1996; Marinelli et al., 2000), and
relapse (Terenius, 1996).
Some genotypes of rodents that
prefer EtOH have lower endogenous βendorphin when compared to non-preferring
lines (Aguirre et al., 1995; del Arbol et al.,
1995). Moreover, humans with a positive
family history for alcoholism also tend to
have lower basal levels of β-endorphin, as
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Oral Self-Administration of EtOH In Transgenic Mice Lacking β-Endorphin
2007
well as an exaggerated EtOH-mediated
release of this peptide, than those without a
genetic liability for alcoholism (Gianoulakis
et al., 1989). From data such as these it has
been argued that those prone to alcoholism
are “self-medicating” an opioid deficiency
and/or especially benefiting from an opioid
surge, following administration of EtOH
(Oswald and Wand, 2004; Reid et al., 1991).
In an earlier study, Grisel et al.
(1999) found evidence to support the
hypothesis that EtOH-mediated reward
depends upon β-endorphin. We investigated
EtOH consumption in a two-bottle, freechoice paradigm in transgenic mice with
varying levels of endogenous β-endorphin.
Although strain differences were small,
there was a tendency for mice with low
levels of β-endorphin to consume the most
EtOH, supporting the idea that consumption
would be especially reinforcing for subjects
deficient in this peptide. Unfortunately, in
this study, we only evaluated two
concentrations of EtOH: 7 and 10%, and so
were unable to fully elucidate the doseresponse relationship as it varied with
respect to β-endorphin levels. In the present
experiment we expanded the range of
concentrations tested and also included
sufficient numbers of male and female
subjects to test for sex-dependent effects.
Furthermore, we evaluated sucrose
preference to test for differences in sweet
responsivity. We predicted that mice
lacking all β-endorphin (knockout; KO)
would consume the least, because they
would not be able to benefit from any βendorphin release. Mice with half the
normal amounts of β-endorphin
(heterozygotes; HT) were predicted to drink
the most EtOH and wildtype mice (B6) were
expected to drink intermediate levels.
Our data support our hypothesis that
β-endorphin does influence the reinforcing
properties of EtOH. HT mice with 50% βendorphin expression prefer and consume
the most EtOH, KO the least, and B6 in
between. With this data we can further
examine the neurological substrates that
modulate EtOH consumption and addiction.
Methods
Progenitor mice used to obtain
subjects for this study were derived from
those made by Rubinstein et al. (1996) and
obtained from The Jackson Laboratories,
Bar Harbor, ME. The original lines were
constructed by inserting a point mutation in
exon 3 of the POMC gene, causing a
shortened proopiomelanocortin (POMC)
prohormone. The gene has since been fully
backcrossed onto the C57BL/6J inbred line.
All experimental animals were born
and reared at Furman University in the
animal care facilities. Mating pairs were
arranged and offspring produced by either
homozygous (B6, KO) or heterozygous
mating pairs. Mice were group housed by
sex and genotype following weaning at 2021 days, and maintained on a reverse 12:12
light: dark cycle at 21° C ± 2° with ad
libetum access to food and water. All
experimental procedures were in accordance
with the Furman University Institutional
Animal Care and Use Committee and the
principles of laboratory animal care from the
National Institutes of Health guidelines.
On Day 1, adult (50-103 day old,
experimentally naïve) subjects were taken
from the colony room to a procedural room
where they were weighed and single housed
in corn-cob bedding lined Plexiglas cages
with wire lids. Two 25 mL graduated
cylinders containing tap water were placed
on each cage, food hoppers were filled with
rodent block chow, and the tube volumes
were recorded. Cage locations were
counterbalanced so that genotype and sex
were equally distributed on the rack. A
sentinel cage was added to each side of the
rack in order to obtain control volumes of
leakage/evaporation. Approximately 24 h
later, tube volumes were recorded again, and
refilled as necessary. For the following 48
days, tube readings occurred every day, and
after every 48 h tube positions were
switched to control for development of side
preferences. Following 8 days of water
drinking, one of the tubes (counterbalanced
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Impulse: the Premier Journal for Undergraduate Publications in the Neurosciences
2007
for side across cages) was filled with 3%
EtOH for 8 days. Remaining concentrations
of EtOH and a test for sweet preference
(6%, 12%, 15% EtOH, and 8% sucrose)
were also tested for 8 days each. EtOH
consumption was expressed as g/kg/day, and
preference ratios were calculated. The
experiment was conducted in three separate
runs, which were procedurally identical, but
separated by 12 days. A total of 77 test
subjects were used in the study. Twentyfour of these subjects were wildtype (B6),
28 heterozygous (HT), and 25 knockout
(KO). There were 37 females and 40 males,
approximately equally divided across
genotype and test run.
Data were analyzed by two-way
(genotype and sex) repeated measure
ANOVA on average g/kg of EtOH
administered and preference at each dose.
Because of significant effects of sex, males
and females were subsequently evaluated
separately by single factor ANOVAs at each
concentration. Post-hoc analysis of
significant differences was evaluated using
the Scheffe test. Criterion for significance
was set at p ≤ .05.
Results
There was no difference in any
measure of EtOH consumption across
runs, so data from all three runs were
combined following two-way analysis
with run and genotype. No differences
in water or sucrose intake were observed
among the runs or between strains (data
not shown), nor were there any
significant interactions with sucrose
consumption.
Preference
β-endorphin levels influenced the
preference for EtOH as evidenced by a
significant effect of genotype in the two way
repeated measures ANOVA on preference
across the dose range: (F(2,69) = 8.361, p <
.01). As is evident in Figure 1, the KOs
tended to drink the least, the HTs drank the
most, and the B6s were intermediate
drinkers, with this pattern becoming more
evident at higher concentrations of available
EtOH. Although there was no main effect
of sex in this analysis (F(1,69) = .103, p > .05),
there was a significant sex x genotype
interaction (F(2,69) = 3.496, p < .05). In
addition, although there was no overall
effect of EtOH concentration (F(3,207) =
1.898, p > .05), there was a significant
interaction between EtOH concentration and
strain (F(6,207) = 6.306, p < .01), but no
significant interactions with sex (F(3,207) =
.693, p > .05).
Post hoc analysis of EtOH
preference was evaluated in males and
females separately at each concentration.
KO males drank significantly less than
either B6 or HT males at 3 or 6 % EtOH,
and less than B6 males at 12 or 15 % EtOH,
and there was a strong tendency for them to
drink less than HTs at these concentrations
(p = .056 and .091, respectively). In
addition, there was a tendency for HT males
to prefer 6% EtOH more than B6 males (p =
.056).
There were only genotypic
differences in females at 6% EtOH, as KO’s
consumed relatively less EtOH than either
other line.
Dosage
Figure 2 shows the dosage
consumed by different groups across
experimental days. There was no main effect
of genotype on dose (F(2,64) = 1.477, p > .05).
As expected, there was a significant effect of
sex on the amount of EtOH administered
(F(1,64) = 20.357, p < .01), as females are
well known to drink more than males in
rodent studies (Jones and Whitfield, 1995).
There was also no genotype by sex
interaction (F(2,64) = 1.568, p = .216).
However, animals self-administered
different amounts of EtOH depending upon
the concentration available, as there was a
significant effect of dose (F3,192) = 107.904,
p < .01). Of particular interest to us was the
significant interaction between the dose
(g/kg) administered across concentrations
and genotype, indicating that the different
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Oral Self-Administration of EtOH In Transgenic Mice Lacking β-Endorphin
2007
lines of mice were differentially affected by
the change in concentration (F(6,192) = 2.321,
p < .05). This interaction reflects the fact
that at higher EtOH concentrations, KO's
tended to drink less than B6, and especially
HT mice. There was also a significant
interaction between concentration and sex
(F(6,192) = 5.467, p < .01). There was not a
significant 3-way interaction.
A.
Average Preference (%)
B6
HT
KO
EtOH Preference (%)
95
85
75
65
55
45
35
3
6
12
15
EtOH Concentration (%)
B.
C.
Male Average Preference (%)
B6
HT
KO
100
90
80
EtOH Preference (%)
EtOH Preference (%)
Female Average Preference (%)
70
60
50
40
30
3
6
12
15
B6
HT
KO
100
90
80
70
60
50
40
30
20
3
EtOH Concentration (%)
6
15
EtOH Concentration (%)
D.
E.
Female Daily Preference (%)
Male Daily Preference (%)
B6
HT
KO
90
80
100
70
60
50
40
30
20
B6
HT
KO
90
EtOHPreference (%)
100
EtOHPreference (%)
12
80
70
60
50
40
30
9
11 13
3%
15
17
19 21
6%
23
25
Day
27 29
12%
31
33
35 37
15%
39
20
9
11 13
3%
15
17
19 21
6%
23
25
27 29
12%
31
33
35 37
15%
39
Day
Fig.1. EtOH preference ratio for two-bottle choice of 3%, 6%, 12% and 15% EtOH in wildtype (B6), heterozygous
(HT) and β-endorphin knockout mice (KO). Data represent mean ± SEM. Panel A shows the average preference ratio
at all four concentrations of EtOH for each genotype. Panel B shows the female average preference ratio at all four
concentrations of EtOH for each genotype. Panel C shows the male average preference ratio at all four concentrations
of EtOH for each genotype. Panel D shows the female preference ratios across the 40 day testing period. Panel E
shows the male preference ratios across the 40 day testing period.
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Impulse: the Premier Journal for Undergraduate Publications in the Neurosciences
2007
A.
Average Dose
15
B6
HT
KO
Dose (g/kg)
12
9
6
3
0
3
6
12
15
EtOH Concentration (%)
B.
C.
Female Average Dose
Male Average Dose
B6
HT
KO
3
6
12
14
12
Dose (g/kg)
Dose (g/kg)
20
18
16
14
12
10
8
6
4
2
B6
HT
KO
10
8
6
4
2
0
15
3
EtOH Concentration (%)
D.
12
15
E.
Female Daily Dose
B6
HT
KO
22
20
Male Daily Dose
B6
HT
KO
15.0
18
12.5
16
14
Dose (g/kg)
Dose (g/kg)
6
EtOH Concentration (%)
12
10
8
6
4
10.0
7.5
5.0
2.5
2
0.0
0
9
11
3%
13
15
17
19
21
23
25
6%
27
12%
Day
29
31
33
35
15%
37
39
9
11 13
3%
15
17
19 21
6%
23
25
27 29
12%
31
33
35 37
15%
39
Day
Fig. 2. EtOH consumption (g/kg) in two-bottle choice of 3%, 6%, 12%, and 15%EtOH in wildtype (B6),
heterozygous (HT), and Knockout (KO) mice. Data represent mean ± SEM. Panel A shows average amount of EtOH
consumption by each genotype at each concentration in a 48 hour period. Panel B shows average amount of EtOH
consumption by females of each genotype. Panel C shows average amount of EtOH consumption by males of each
genotype. Panel D shows consumption by females across the 40 day testing period as measured every day. Panel E
shows consumption by males across the 40 day testing period.
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Oral Self-Administration of EtOH In Transgenic Mice Lacking β-Endorphin
2007
Discussion
Current theories of addiction
emphasize the rewarding effects
produced by the drug (Wise, 1988). One
mediator of these rewarding effects is βendorphin (Herz, 1997). Many studies
show that EtOH drinking in mice is
correlated with the animal’s genetic
ability to release β-endorphin
(Gianoulakis et al., 1989). It has also
been shown that people with a family
history of alcoholism have an increase in
β-endorphin release after EtOH
administration compared to controls with
no family history of alcoholism
(Gianoulakis et al., 1989).
The purpose of this study was to
see how β-endorphin influenced EtOH
drinking in an animal model. The results
support our hypothesis that β-endorphin
modulates the rewarding properties of
EtOH. Mice
completely lacking β-endorphin (KO)
self-administered less EtOH in a freechoice paradigm.
Overall, females drank more
EtOH than the males, which is a wellknown result in rodents (Jones and
Whitfield, 1995). Notably, the effect of
β-endorphin on drinking was also sexspecific. This peptide did not influence
EtOH self-administration in females, but
did effect drinking in males. The male
KOs, entirely lacking β-endorphin,
appeared to
be insensitive to the rewarding effects of
EtOH as they self administered at chance
levels (50% of the fluid they consumed
was from the EtOH-spiked tube). On the
other hand, male HTs showed a pattern
of drinking consistent with enhanced
EtOH reward, and tended to drink more
than control mice. Both of these findings
are consistent with a theory implicating
β-endorphin release in EtOH
reinforcement, though why this effect
should be sex-specific is unclear at
present. One possibility is that sex
specific hormones, such as progesterone
metabolites, make up for the
lack of β-endorphin in females (Morrow,
2007; Morrow et al., 2006) though
further research is needed to clarify the
mechanisms of this sex-dependent
effect.
There is a main concern that
should be acknowledged when
interpreting data collected from induced
mutant mice. It is possible that the
observable phenotypic behaviors could
be the cause of a “hitchhiking” gene
polymorphism and not purely a
consequence of the intentional genetic
mutation (Gerlai, 1996; Low et al.,
1998). A more likely possibility is that
some compensatory reaction to the
genetic mutation could cause the
different phenotypic behaviors and not
the mutation alone (Mogil and Grisel,
1998). The use of inducible mutant mice
in future studies will lead to a better
understanding of the mechanism by
which β-endorphin induces behavior,
including behaviors related to
alcoholism.
Further investigation should be
done to evaluate the role of β-endorphin
in EtOH sensitivity more generally.
Currently, we are performing place
conditioning tests with the β-endorphin
KO and HT mice. This is also a test for
EtOH reinforcement, but β-endorphin
may affect other consequences of EtOH
too.
The main purpose of this study
was to evaluate the neurobiological
substrates that modulate EtOH
consumption in an animal model where
β-endorphin levels vary. We
demonstrated that β-endorphin,
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Impulse: the Premier Journal for Undergraduate Publications in the Neurosciences
2007
especially in males, influences the
reinforcing properties of EtOH. A more
complete assessment of a possible
vulnerability to alcoholism, giving us a
broader understanding of the
predispositions to addiction and aiding
in the development of successful
interventions to that would ameliorate
the negative consequences of excessive
alcohol use.
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