Rewarding effects of ethanol and cocaine in μ opioid receptor-deficient mice

Abstract

To investigate the role of mu opioid receptors in the reinforcing effects of psychotropic drugs, the voluntary ethanol intake and ethanol- and cocaine-induced conditioned place preference in mu opioid receptor-deficient mice and their wild-type counterpartners was tested. Moreover, dopamine D1 and D2 receptor binding was measured. It was found that ethanol intake was significantly lower in deficient mice. Conditioned place preference in wild-type animals was induced with 5.0 mg/kg cocaine and this dose was ineffective in the knockouts. In this group conditioned place preference occurred after injection of 10.0 mg/kg cocaine. Cocaine induced a similar increase in locomotor activity in both groups of mice. There was no difference in dopamine D1 receptor binding, whereas dopamine D2 receptor binding was significantly lower in the hippocampus of deficient animals. This suggests that interaction between opioid systems and dopaminergic systems may account for the differences in responding to the drugs.

Full text

Abstract To investigate the role of µopioid receptors in
the reinforcing effects of psychotropic drugs, the volun-
tary ethanol intake and ethanol- and cocaine-induced con-
ditioned place preference in µopioid receptor-deficient
mice and their wild-type counterpartners was tested.
Moreover, dopamine D1 and D2 receptor binding was mea-
sured. It was found that ethanol intake was significantly
lower in deficient mice. Conditioned place preference in
wild-type animals was induced with 5.0 mg/kg cocaine
and this dose was ineffective in the knockouts. In this
group conditioned place preference occurred after injec-
tion of 10.0 mg/kg cocaine. Cocaine induced a similar in-
crease in locomotor activity in both groups of mice. There
was no difference in dopamine D1 receptor binding,
whereas dopamine D2 receptor binding was significantly
lower in the hippocampus of deficient animals. This sug-
gests that interaction between opioid systems and dopamin-
ergic systems may account for the differences in respond-
ing to the drugs.
Keywords µopioid receptor · Transgenic mice ·
Ethanol · Cocaine · Self-administration · Conditioned
place preference · Dopamine binding
Introduction
A body of evidence suggests that µopioid receptors are in-
volved in the reinforcing effects of ethanol (Herz 1997,
1998; Hyytia 1993; Krishnan-Sarin et al. 1998; Matsuzawa
et al. 1998, 1999). Studies with opioid receptor antago-
nists have shown that ethanol drinking was reduced by
µ1- but not by δreceptor blockade (Honkanen et al. 1996;
Hyytia 1993; Krishnan-Sarin et al. 1998). µand dopamine
D2 receptor antisense nucleotides injected in the nucleus
accumbens (Nacc) suppressed high ethanol intake in ge-
netic drinking HEP rats (Myers and Robinson 1999). This
is in line with results by others (Hall et al. 2001; Roberts
et al. 2000) demonstrating that µopioid receptor knockout
mice did not self-administer ethanol either in operant pro-
cedures or a two bottle-choice paradigm.
Addictive drugs have rewarding properties and also
produce locomotor stimulating effects. It is commonly ac-
cepted that both locomotor stimulation and rewarding ef-
fects of addictive drugs are associated with the activation
of the mesolimbic dopaminergic system, originating from
the ventral tegmental area (VTA) to the Nacc. There is ev-
idence that the Nacc is a heterogeneous structure and can
be divided into subregions termed the shell and core. It
was proposed that the reinforcing and sensitising proper-
ties of addictive drugs seem to be differentially regulated
in these subregions (Pierce and Kalivas 1995; Pontieri et
al. 1995; David et al. 1998).
There is little doubt that midbrain dopaminergic sys-
tems play an essential role in the acquisition of reward
and drug-seeking behaviour (Spanagel and Weiss 1999)
but other neurotransmitters were also suggested to be im-
plicated in the acquisition of reward and drug-seeking be-
haviour. This is mediated by either increase in extracellu-
lar dopamine by inhibiting the re-uptake of released dopa-
mine by the dopamine transporter (Amara and Kuhar
1993; Giros and Caron 1993; Horn 1990) or, alternatively,
by release of dopamine from presynaptic nerve terminals
in addition to inhibiting re-uptake (Le Pen et al. 1998;
Seiden et al. 1993; Sora et al. 1998, 2001). Thus, ethanol
avoidance in dopamine D2 receptor-deficient mice might
be easily explainable (Phillips et al. 1998). Interestingly,
dopamine D3 receptor blockade was reported to enhance
ethanol reward (Boyce and Risinger 2000).
Given the role attributed to complex µopioid receptor
effects and dopamine effects it was of interest to investi-
Axel Becker · Gisela Grecksch · Jürgen Kraus ·
Horace H. Loh · Helmut Schroeder · Volker Höllt
Rewarding effects of ethanol and cocaine
in µopioid receptor-deficient mice
Naunyn-Schmiedeberg’s Arch Pharmacol (2002) 365:296–302
DOI 10.1007/s00210-002-0533-2
Received: 25 July 2001 / Accepted: 14 January 2002 / Published online: 21 February 2002
ORIGINAL ARTICLE
A. Becker (✉) · G. Grecksch · J. Kraus · H. Schroeder · V. Höllt
Institute of Pharmacology and Toxicology,
Otto-von-Guericke University, Leipziger Strasse 44,
39120 Magdeburg, Germany
e-mail: axel.becker@medizin.uni-magdeburg.de,
Tel.: +49-391-6715351, Fax: +49-391-67190149
H.H. Loh
Department of Pharmacology,
University of Minnesota Medical School, 3-249 Millard Hall,
435 Delaware Street S.E., Minneapolis, MN 55455, USA
© Springer-Verlag 2002

gate further the role of this interaction. For that purpose,
ethanol and cocaine were selected as pharmacological
agents. As referred above, µopioid receptor effects and
dopamine effects mediate rewarding action of ethanol.
Rewarding effects of cocaine were thought to rely on do-
pamine effects and interaction resulting from interactions
with opioid systems (Gerrits et al. 1995; Houdi et al.
1989; Izenwasser et al. 1996; Kuzmin et al. 1997; Rowlett
and Spealman 1998). Therefore, in the present study we
tested rewarding effects of ethanol and cocaine in the
model conditioned place preference in µopioid receptor-
deficient mice and their wild-type counterpartners. In ad-
dition, voluntary ethanol drinking in a two bottle-choice
test was investigated in these animals.
Materials and methods
Animals. For the experiments, µopioid receptor knockout mice
were used (Loh et al. 1998). Breeding of the animals, genotyping,
[
3
H]DAMGO receptor autoradiography, and results of binding
studies have been published earlier (Becker et al. 2000). After
genotyping, homozygote animals were bred according to a standard
breeding protocol.
The animals were kept under controlled laboratory conditions
with lighting regime LD=12:12 (light on at 06.00 a.m.), tempera-
ture 20±2°C, and relative air humidity 50%–60%. The animals had
free access to standard rat pellets (Altromin 1326) and tap water.
After weaning on day 21 post partum, the animals were separated
according to sex and sheltered litter-wise in Macrolon III cages.
Behavioural testing. Male mice aged 7–8 weeks at the beginning
of the experiments.
Voluntary ethanol intake. In this experiment six µopioid receptor-
deficient mice (-/-) and ten wild-type mice (+/+) were used. The
animals were matched according to body weight. Mean body weight
in the -/- group was 27.6±5.3 g and in the +/+ group 27.7±1.7 g. To
measure individual fluid intake, the animals were singly housed in
Macrolon III cages. Each cage was equipped with two bottles con-
taining either water or 10% v/v ethanol solution. Fluid intake was
calculated based on bottle weight measured twice a week. Total
fluid intake was defined as water intake + ethanol solution intake
and it was expressed as g/kg body weight. The position of the bot-
tles was changed at weighing. This experiment ran over a period of
4 weeks.
Conditioned place preference. The apparatus (TSE, Bad Homburg,
Germany) was T-shaped. The central alley (40×11 cm) consisted
of grey polyvinyl chloride (PVC). The two end-compartments
(50×11 cm) were also made from PVC. One end-compartment had
white walls and the floor was covered with a wire mesh whereas
the other end-compartment had black walls and the floor was cov-
ered with a wire grid. The arms were equipped with guillotine
doors. Number of arm crossings and time spent in each arm were
measured via infrared photocells mounted next to the arm entry. A
personal computer controlled the experimental sessions and col-
lected data.
On day 1, the animals were habituated for 30 min with the ap-
paratus. The day after habituation, a session was performed to de-
termine the animals’ arm preference. For that purpose, a mouse
was placed in the apparatus for 30 min. The end-compartment
where the animals spent less than 50% of the test time was consid-
ered as the non-preferred compartment. On the following 4 days
the animals were alternatively intraperitoneally (i.p.) injected with
either ethanol (2.0 g/kg or 4.0 g/kg) or saline. After ethanol mice
were placed for 30 min in the non-preferred end-compartment and
after saline they were placed in the preferred arm. Control animals
received saline prior to placing in the apparatus. During condition-
ing sessions the guillotine doors were closed. Pilot studies have
shown a strong preference for the black arm of the apparatus.
Therefore we decided to use the biased protocol. Data obtained us-
ing biased compartments can be a valid reflection of drug reward
(Carr et al. 1989). On day 7 place preference was measured. The
animals were allowed to explore all arms of the apparatus over a
period of 15 min. The difference in % time spent in the non-pre-
ferred arm on day 2 and day 7 was used as an index of place pref-
erence. For control, two groups of mice injected with saline alone
were used. Similarly, different doses of cocaine (5.0 mg/kg and
10.0 mg/kg) were tested in this paradigm in another group of mice
according to the protocol as described above.
Locomotor activity. Locomotor activity was measured using a fully
computerised activity meter (Moti-Test; TSE, Bad Homburg, Ger-
many). First, for basal locomotor activity during a habituation pe-
riod locomotor activity was measured for 15 min. After habitua-
tion, the animals were injected with either saline, 20 mg/kg or
40 mg/kg cocaine i.p., and the locomotion was measured for fur-
ther 45 min.
Substances. Cocaine (free base) was obtained from Sigma. It was
prepared as a 5.0 mg/kg and 10.0 mg/kg (conditioned place prefer-
ence) or 20.0 mg/kg and 40.0 mg/kg (locomotor activity) solution
in isotonic saline (sal) adding one drop of 1 N HCl. The drug was
i.p. injected at a volume of 1 ml/kg body weight. For control, the
solvent alone or the solvent added with a drop of 1 N HCl was
given.
In the conditioned place preference (CPP) and locomotor activ-
ity experiment physiological saline served as solvent.
Ethanol dilution (10%, v/v) was made up with 95% ethyl alco-
hol and tap water.
Dopamine D1 and D2 receptor binding assay. The dissection of
the brain regions was done according to Popov et al. (1973).
The [
3
H]L-SCH 23 390 and [
3
H]spiroperidol binding was mea-
sured using a method described by Köhler et al. (1994). A crude
synaptic membrane suspension was incubated with 30 mM Tris-
HCl buffer (pH 8.0, containing 120 mM NaCl, 5 mM KCl, 2 mM
CaCl
2
, 1 mM MgCl
2
) and [
3
H]SCH 23 390 or [
3
H]spiroperidol
(+ 50 nmol unlabelled cinanserin to block 5-HT
2
receptors) for
40 min and 30 min at 37°C, respectively. Specific binding was cal-
culated by subtracting non-specific binding – defined as that seen
i
n the presence of 0.5 nM [
3
H]SCH 23 390 (specific activity:
1.43 TBq/mmol; NEN-Dupont, USA) or 1 nM [
3
H]spiroperidol
(specific activity: 800 GBq/mmol; NEN-Dupont, USA) plus 1 µM
unlabelled cis-flupenthixol or 2 µM d-butaclamol (Serva, Heidel-
berg, Germany) – from total binding obtained with [
3
H]SCH 23 390
or [
3
H]spiroperidol alone.
The reaction was terminated by rapid filtration under reduced
pressure through 0.1% polyethyleneimine-treated GF 10 glass-fiber
filters using an Inotech harvester (Berthold, Wildbad, Germany).
Filters were washed with buffer and taken for liquid scintillation
counting in a toluene containing solvent. The data were determined
as fmol bound radioligand per mg protein.
Statistics. To evaluate total liquid intake and ethanol consumption,
the repeated measure model was applied. Locomotor activity and
results obtained in the CPP experiment were first analysed using a
two-way ANOVA, with genetic background (i.e. +/+ and -/-) and
treatment (i.e. saline, 5.0 mg/kg and 10.0 mg/kg cocaine) as de-
pendent variables. One-way ANOVA with Tukey post-hoc test was
performed in both groups of mice. Data obtained in the binding as-
say were analysed by Mann-Whitney U-test. SPSS+ software was
used and the significance level was fixed at 0.05.
297

Results
Voluntary ethanol intake
As shown in Fig.1 (upper panel) there was no difference
in total liquid intake in both groups of mice (F
1,14
=0.744,
P=0.43). Interestingly, ethanol intake was significantly
lower in -/- mice (F
1,14
=4.955, P=0.042; Fig.1, lower panel).
Conditioned place preference (ethanol)
In Fig.2 it is demonstrated that in the conditioned place
preference test both groups of mice showed preference to
that arm which was associated with ethanol. There was no
effect of the genetic background (F
1,59
=0.196, P=0.66) and
no genetic background ×treatment interaction (F2,59=0.167,
P=0.846) but there was a significant effect of ethanol
treatment (F
2,59
=3.5, P=0.037). This suggests that the ani-
mals reacted in a comparable manner to the treatment re-
gardless of the genetic background.
Conditioned place preference (cocaine)
In the CPP (Fig.3) experiment using cocaine, there was
no effect of treatment (F
2,82
=2.1, P=0.129), but a signifi-
cant effect of the genetic background (F
1,82
=1.89, P=7.43)
and a significant genetic background ×treatment interac-
tion (F
2,82
=5.187, P=0.008).
In response to cocaine, +/+ mice differed (Fig.3, left
panel) significantly (F
2,48
=4.5, P=0.016). Tukey test re-
vealed that the difference between sal-injected control
mice (P=0.024) and mice injected with 10.0 mg/kg (P=0.02)
was lower than in animals injected with 5.0 mg/kg co-
caine. There was no difference between sal-injected con-
trol animals and those mice which received 10.0 mg/kg
cocaine (P=0.99). This U-shaped dose-response curve
clearly reflects appetitive and aversive properties of the
substance.
Significant differences (Fig.3, right panel) were found
in the -/- group (F
2,34
=3.94, P=0.029). It is the more so in-
teresting that in -/- mice the difference between sal-in-
jected animals (P=0.042) and 5.0 mg/kg cocaine-injected
mice (P=0.49) is significantly lower compared with ani-
298
Fig.1 Total liquid intake (upper panel) and voluntary ethanol in-
take (lower panel) in wild-type (+/+) mice and their µopioid re-
ceptor-deficient counterpartners (-/-). The test ran over a period
of 4 weeks and liquid intake was measured twice a week. The dif-
ference in ethanol intake is significant (F
1,14
=5.39, P=0.036).
Means ± SEM
Fig.2 Conditioned place preference in wild-type (+/+) and µopi-
oid receptor-deficient mice (-/-) in response to ethanol. Means ±
SEM; n= number of animals used
Fig.3 Conditioned place preference in wild-type (+/+) and µopi-
oid receptor-deficient mice (-/-) in response to cocaine. Means ±
SEM; n= number of animals used. *P<0.05

mals which received 10.0 mg/kg cocaine. The difference
between sal-injected controls and animals which received
5.0 mg/kg cocaine is insignificant (P=0.99). This indi-
cates a shift in the dose-response curve to the right.
Locomotor activity
In the habituation period wild-type animals showed higher
activity scores (F
1,69
=7.622, P=0.007) compared with de-
ficient mice (Fig.4). There were no differences between
the three groups (i.e. saline, cocaine 20 mg/kg, cocaine
40 mg/kg) from each genetic background (wild type:
F
2,31
=0.036, P=0.96; deficient animals: F
2,34
=0.066,
P=0.93). In reaction to cocaine, there was effect of dose
(F
2,65
=24.78, P<0.001), but there was no effect of the ge-
netic background (F
1,65
=3.17, P=0.08) and no genetic
background ×treatment interaction (F
2,65
=0.09, P=0.91).
Animals from both groups reacted in a similar manner to
cocaine injection (saline vs. 20 mg/kg, 20 mg/kg vs. 40 mg/
kg; P<0.001, Tukey test).
Binding study
As shown in Fig.5 (upper panel) there were no differ-
ences in dopamine D1 binding either in the frontal cortex,
the striatum or the hippocampus of drug-naive +/+ and
-/- animals. Similarly, we did not find differences in D2
binding in the frontal cortex and the striatum (Fig.5,
lower panel). However, D2 receptor binding in the hip
po-
campus
is significantly lower in -/- mice compared with
their wild-
type counterpartners.
Discussion
The present results confirm that voluntary ethanol intake
in µopioid receptor-deficient mice is lower than in wild-
type mice (Fig.1). The animals showed a conditioned
place preference to ethanol regardless of the genetic back-
ground (Fig.2). Cocaine induces CPP in +/+ animals at a
dose of 5.0 mg/kg whereas the same dose had no effect in
the knockouts (Fig.3). Deficient mice showed CPP when
cocaine was administered in a higher dose of 10.0 mg/kg.
From literature it is well known that effects of ethanol
in the conditioning place task depend on a number of fac-
tors such as dose, application schedule, and genetic back-
299
Fig.4 Locomotor activity in wild-type (+/+) and µopioid recep-
tor-deficient (-/-) mice in response to cocaine. Means ± SEM; n=
number of animals used
Fig.5 [
3
H]SCH 23 390 (upper panel) and [
3
H]spiroperidol (lower
panel) binding to synaptic membranes from the frontal cortex and
the hippocampus of wild-type (black bars) and µopioid receptor-
deficient (open bars) mice [CRT cortex (n=6), HIP hippocampus
(n=7), STR striatum (n=6)]. *P<0.05

ground (Cunningham and Henderson 2000; Risinger and
Oakes 1996). Thus, it is difficult to compare experimental
data obtained in different laboratories.
Our findings are in line with others (Hall et al. 2001;
Roberts et al. 2000) who found that in mice lacking the
µopioid receptor ethanol consumption is decreased, either
in operant or bottle-choice paradigms. Similar results
were obtained in experiments using dopamine D2 recep-
tor-deficient mice (Phillips et al. 1998). This suggests that
complex interaction between both types of receptors con-
tributes to the rewarding effect of ethanol.
It is evident from the ethanol drinking experiment that
the acutely rewarding effect is the same in +/+ and
-/- mice up to the fourth measurement. After this point the
wild-type animals gradually increase their ethanol intake
and preference since total liquid intake is not changed
(Fig.1). In contrast, -/- remain on the same ethanol drink-
ing level. Thus, the two groups display a difference in the
acquisition of ethanol preference (Fig.1) but not in the re-
sponse to the acute rewarding effects of ethanol (Fig.2).
Dopamine receptors have been implicated in the be-
havioural effects of drugs of abuse (Harris and Aston-
Jones 1994; Koob 1996; Spanagel and Weiss 1999; Uhl et
al. 1998). These effects are thought to be mediated by the
mesolimbic dopaminergic pathway arising from the ven-
tral tegmental area and projecting to the nucleus accum-
bens and prefrontal cortex. Ethanol was shown to increase
the dopamine level after intake (Bunney et al. 2000; Kat-
ner and Weiss 2001; Nurmi et al. 1998; Yavich and Tiiho-
nen 2000; Yim and Gonzales 2000; Yoshimoto et al. 2000)
and excites dopaminergic ventral tegmental area reward
neurones directly (Brodie et al. 1999). Cocaine increases
dopamine levels by blocking the dopamine transporter
(Ferraro et al. 2000; Rocha et al. 1998). To shed light on
opioid-dopamine interaction, we measured D1 and D2
binding to synaptic membranes from neurones of the fron-
tal cortex and the hippocampus. As shown in Fig.5, there
was no difference in D1 binding in the frontal cortex,
striatum and hippocampus in both groups of mice. Simi-
larly, D2 binding was comparable in the frontal cortex and
the striatum of +/+ and -/- mice. Interestingly, in the hip-
pocampus as part of the limbic system D2 receptor bind-
ing is significantly diminished in deficient mice. It was
speculated that the reduction in D2 binding sites occurs in
response to an enhanced dopamine release measured ex
vivo by K
+
-stimulated release of [
3
H]dopamine from hip-
pocampal slices of drug-naive wild-type and µopioid-de-
ficient mice (Schroeder et al. 1999). The reduced D2 re-
ceptor binding in the hippocampus might result in a re-
duction of the rewarding effect of ethanol resulting in
sig
nificantly lower voluntary ethanol consumption in
-/- mice. This is favourably in line with other results (Phillips
et al. 1998; Risinger and Oakes 1996). It was found that
D2 receptor knockout mice have reduced ethanol con-
sumption and lacked of operant ethanol self-administra-
tion, respectively. The authors concluded that reduced re-
sponding in the knockout animals for several reinforcers
including ethanol indicates a more general role for dopa-
mine D2 receptors in motivated responding rather than a
specific role in ethanol reinforcement. Reduced D2 bind-
ing would also explain the higher dose of cocaine needed
to induce CPP in these animals. On the other hand, the
hippocampus was reported to be an important structure
for the mediation and modulation of rewarding effects of
substances. It was speculated that the hippocampal CA3
region may be an important target site for opioid reward
and opioid dependence (Stevens et al. 1991; Wise 1989).
This hypothesis was underlined by lesion experiments.
Morphine rewarding properties were modulated by frontal
cortical and hippocampal lesions (Glick and Cox 1978;
Kelley and Mittleman 1999).
Subchronic cocaine treatment resulted in a significant
µreceptor upregulation in different brain regions of Fisher
rats (Unterwald et al. 1994). Beside an involvement of the
µopioid receptor in cocaine-induced sensitisation to toxic
effects (Braida et al. 1997) there is also evidence that lo-
comotor activity, conditioned motivational, discrimina-
tory, and reinforcing effects of the substance are mediated
by opioid systems (Gerrits et al. 1995; Houdi et al. 1989;
Kuzmin et al. 1997; Mitchem et al. 1999; Rowlett and
Spealman 1998; Suzuki et al. 1992). However, some stud-
ies failed to demonstrate these interactions (Broadbent et
al. 1995; Schad et al. 1995). As shown in Fig.3, wild-type
mice showed a conditioned place preference in reaction to
5.0 mg/kg cocaine, whereas in deficient animals the effec-
tive dose was 10.0 mg/kg. At this dose, cocaine is neutral
to wild-type animals. This shift in the dose-response
curve to the right well matches with findings as referred
above demonstrating complex opioid-dopamine interac-
tions underlying the reinforcing effect of cocaine.
The conflict between CPP data (ethanol vs. cocaine)
and decreased voluntary ethanol intake in the -/- mice is
apparent (Figs.2, 3). Both procedures are being advocated
as possible measures of drug reward (Carr et al. 1989).
Thus, one would expect that the data generated by these
models are consistent. However, there are several reports
on dissociative effects (e.g. DeWitt and Wise 1977; Roberts
et al. 1980). Carr et al. (1989) explained these discrepan-
cies in terms of procedural differences. Operant responses
are necessary for self-administration but not in the CPP
paradigm during conditioning. Moreover, the motiva-
tional background (i.e. voluntary vs. forced drug intake)
might affect the results (Heyne 1996; Ufer et al. 1999).
Acknowledgements The skilful technical assistance of Mrs. D.
Apel,
P. Dehmel, B. Reuter, G. Schulze, I. Schwarz and I. Gräbe-
dünkel is gratefully acknowledged. We are grateful to Mr. A. Toms
(UK) for linguistic improvement.
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