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ORIGINAL INVESTIGATION
Endogenous kappa-opioid mediation of stress-induced
potentiation of ethanol-conditioned place preference
and self-administration
Robin E. Sperling &Stacey M. Gomes &
Elizabeth I. Sypek &Amanda N. Carey &
Jay P. McLaughlin
Received: 28 October 2009 /Accepted: 22 March 2010
#Springer-Verlag 2010
Abstract
Rationale Exposure to inescapable stressors increases both
the rewarding properties and self-administration of cocaine
through the signaling of the kappa-opioid receptor (KOR),
but the effect of this signaling on other reinforcing agents
remains unclear.
Objective The objective of this study is to test the
hypothesis that signaling of the KOR mediates the forced
swim stress (FSS)-induced potentiation of ethanol reward
and self-administration.
Methods Male C57Bl/6J mice were tested in a biased
ethanol-conditioned place preference (CPP) procedure, and
both C57Bl/6J and prodynorphin gene-disrupted (Dyn −/−)
mice were used in two-bottle free choice (TBC) assays, with
or without exposure to FSS. To determine the role of the
KOR in the resulting behaviors, the KOR agonist U50,488
(10 mg/kg) and antagonist nor-binaltorphimine (nor-BNI,
10 mg/kg) were administered prior to parallel testing.
Results C57Bl/6J mice exposed to repeated FSS 5 min
prior to daily place conditioning with ethanol (0.8 g/kg)
demonstrated a 4.4-fold potentiation of ethanol-CPP com-
pared to unstressed mice that was prevented by nor-BNI
pretreatment. Likewise, pretreatment with U50,488 90 min
prior to daily ethanol place conditioning resulted in a 2.8-
fold potentiation of ethanol-CPP. In the TBC assay,
exposure to FSS significantly increased the consumption
of 10% (v/v) ethanol by 19.3% in a nor-BNI-sensitive
manner. Notably, Dyn −/−mice consumed a similar volume
of ethanol as wild-type littermates and C57Bl/6J mice, but
did not demonstrate significant stress-induced increases in
consumption.
Conclusions These data demonstrated a stress-induced po-
tentiation of the rewarding effects and self-administration of
ethanol mediated by KOR signaling.
Keywords Dynorphin .Kappa-opioid receptor .Stress .
Ethanol .Conditioned place preference .Self-administration
Introduction
Ethanol is a reinforcing agent, but its consumption is a
complex behavior influenced by multiple factors, including
genetic predisposition, social context, and exposure to
stress (Peele and Brodsky 2000;Koobetal.2004;
Vengeliene et al. 2008). In humans, stressful situations are
thought to increase alcohol intake (Pohorecky 1981), but
the effects of stress on ethanol consumption and reward are
less clear in animal studies. Acute exposure to social defeat
stress has been shown to suppress ethanol self-
administration in rats (van Erp and Miczek 2001; van Erp
et al. 2001; Funk et al. 2005), and acute isolation stress was
recently shown to suppress ethanol self-administration in
squirrel monkeys (McKenzie-Quirk and Miczek 2008). In
contrast, a number of studies have demonstrated increases
in ethanol consumption following chronic exposure to
either physical or psychological stressors in rodents (Mills
Robin E. Sperling and Stacey M. Gomes contributed equally to this
project.
R. E. Sperling :S. M. Gomes :E. I. Sypek :A. N. Carey :
J. P. McLaughlin
Department of Psychology, Northeastern University,
Boston, MA 02115, USA
S. M. Gomes :J. P. McLaughlin (*)
Torrey Pines Institute of Molecular Studies,
11350 SW Village Parkway,
Port St. Lucie, FL 34987, USA
e-mail: jmclaughlin@tpims.org
Psychopharmacology
DOI 10.1007/s00213-010-1844-5
et al. 1977; Wolffgramm 1990; Matsuzawa et al. 1998) and
primates (McKenzie-Quirk and Miczek 2008). Notably,
these latter results are consistent with animal studies
showing that exposure to inescapable stressors increased
[both the rewarding properties and self-administration of a
variety of drugs of abuse (Piazza et al. 1990;Shahamand
Stewart 1994;Willetal.1998). While many of these
effects have been attributed to signaling mediated by
corticosterone (Piazza et al. 1990; Shaham and Stewart
1994), recent studies are extending the neuropharmaco-
logical mechanisms by which stress may modulate drug
reward. For example, repeated exposure to stress was
shown to potentiate the rewarding effects of cocaine
through the prior activation of the endogenous kappa-
opioid system (McLaughlin et al. 2003,2006). However,
while implicated (Matsuzawa et al. 1999), the involvement
of endogenous kappa-opioid system activity in stress-
induced potentiation of ethanol reward has not been
closely examined.
A facilitating role for the kappa-opioid system in drug
reward is paradoxical, given that acute activation of kappa-
opioid receptors (KOR) has been shown to prevent the
rewarding effects induced by reinforcing drugs such as
cocaine and ethanol (Shippenberg and Herz 1986; Herz
1997; Shippenberg et al. 2007). Consistent with established
theory, acute KOR activation with the agonist (trans)-3,
4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]
benzeneacetamide (U50,488) decreased ethanol self-
administration in a two-bottle free choice assay (TBC;
Lindholm et al. 2001) and ethanol-conditioned place
preference (CPP; Logrip et al. 2009), and the KOR
antagonist nor-binaltorphimine (nor-BNI) significantly in-
creased ethanol consumption (Mitchell et al. 2005).
However, pretreatment with the KOR agonist CI-977
increased ethanol consumption over time in ethanol-
experienced but deprived rats (Hölter et al. 2000), and both
female prodynorphin gene-disrupted mice (Dyn −/−) and
male KOR gene-disrupted mice showed lower alcohol
consumption and preference (Blednov et al. 2006; Kovacs
et al. 2005a,b). Consistent with these last findings, U50,488
pretreatment was shown to increase cocaine self-
administration (Negus 2004) and mimic stress-induced
potentiation of cocaine-CPP (McLaughlin et al. 2006) when
the administration of cocaine is timed to coincide with the
observed termination of KOR agonist activity in a matching
antinociceptive assay. Overall, a growing set of evidence
suggests that KOR signaling may bidirectionally modulate
the reinforcing effects of drugs of abuse, including ethanol,
in a time-dependent manner that initially suppresses but
then potentiates ethanol reward.
This study hypothesized that the earlier stress-induced
activation of the endogenous kappa-opioid system would
mediate the later stress-induced potentiation of the reward-
ing effects and self-administration of ethanol. To test this,
C57Bl/6J mice were exposed repeatedly to forced swim
stress (FSS), and the rewarding effects of ethanol were
assessed using the CPP and TBC assays to examine
ethanol-seeking and consumption behaviors, respectively.
To determine the involvement of the endogenous kappa-
opioid system on stress-induced potentiation of ethanol
reward, Dyn −/−and wild-type mice were treated with
vehicle or nor-BNI (10 mg/kg, i.p.) prior to forced
swimming. Together, the results suggest that prior activa-
tion of the KOR may potentiate the rewarding effects of
ethanol.
Material and methods
Animals and housing Two hundred seven (153 for CPP, 54
for TBC) male C57Bl/6J mice (Jackson Labs, Bar Harbor,
Maine, USA), ten prodynorphin gene-disrupted (Dyn −/−)
mice, and nine wild-type littermates (Dyn +/+), 8–12 weeks
old and weighing 19–29 g, were used in these experiments.
The C57Bl/6J mouse strain was selected for this work for
noted responses to stress (Lucki et al. 2001) and drugs of
abuse (Orsini et al. 2005), including a preference for
alcohol (Belknap et al. 1993; Risinger at al. 1998;Liet
al. 2005). They are also established subjects for the study of
the effects of stress on the response to reinforcing drugs
(McLaughlin et al. 2003,2006). Transgenic gene-disrupted
mice lacking the functional gene for prodynorphin were
provided by Dr. Ute Hochgeschwender (Oklahoma Medical
Research Foundation, Oklahoma City, OK, USA; see
Sharifi et al. 2001). Prodynorphin gene-disrupted mice
were backcrossed with C57Bl/6J mice for five generations,
resulting in a mixed strain background of 96.9% C57Bl/6J
and 3.1% 129sv. As such, responses of the prodynorphin
gene-disrupted mice were compared to wild-type littermates
of the same generation. The presence or absence of the
prodynorphin gene was confirmed in genomic DNA
isolated from tail tissue samples taken from each mouse
using polymerase chain reaction analysis as described
previously (Sharifi et al. 2001; McLaughlin et al. 2003).
All mice were housed by fours in self-standing plastic cages
(28 cm long×16 cm wide×13 cm high) within the vivarium
at Northeastern University or TPIMS unless otherwise
noted. Housing rooms were illuminated on a 12-h light/
dark cycle with lights on at 7:00 a.m. Note that testing of
mice was carried out during the light phase of the
photocycle, consistent with previous studies on the effects
of stress on drug-CPP responses (McLaughlin et al. 2003,
2006; Carey et al. 2007). Food pellets and water were
available ad libitum. All mice were housed and cared for in
accordance with the 1996 National Institute of Health
Guide for the Care and Use of Laboratory Animals and all
Psychopharmacology
procedures using mice were approved by the Northeastern
University and TPIMS Institutional Animal Care and Use
Committees.
Drugs Ethanol (95%, density= 0.816) was prepared in
solutions of 5%, 10%, or 20% (v/v) diluted in 0.9% saline
solution for CPP testing or diluted in water for the TBC
paradigm. U50,488 and nor-BNI were provided by the
NIDA Drug Supply. Nor-BNI was dissolved in a vehicle of
0.9% saline and administered once at a dose of 10 mg/kg,
i.p., 24 h prior to initial exposure to stress or testing.
U50,488 was dissolved in 0.9% saline and administered at a
dose of 10 mg/kg, i.p., daily, either 90 min prior to place
conditioning or 24 h prior to fluid measurement in the TBC
assay (see below) as noted.
Conditioned place preference One hundred fifty-three
C57Bl/6J mice were used in place-conditioning studies
using three-compartment boxes as described by Carey et al.
(2007). The Plexiglas compartment consisted of three
sections, two equal-sized outer sections (25×25×25 cm)
joined by a smaller central compartment (8.5×25 × 25 cm)
accessed by a sliding door (25×25 cm). The outer
compartments differed in wall striping (vertical vs horizon-
tal alternating black and white lines, 1.5 cm in width) and
floor texture (smooth vs rough). Mice were tested on day 1
before any treatment to establish initial preference for a
particular compartment. Testing involved placing individual
mice in the central compartment with the sliding doors
raised and allowing them to run freely between the two
outer compartments for 30 min. The apparatus itself is
balanced, with animals on average demonstrating an
equivalent amount of time in each of the three compart-
ments (588±11 s in the left compartment, 602±10 s in the
right compartment, and 609±12 s in the central compart-
ment) that did not statistically differ (one-way analysis of
variance (ANOVA), F
(2,456)
=0.95, p=0.39). For condition-
ing, the order of the ethanol administration was counter-
balanced across subjects (i.e., mice were randomly
separated into two groups, one group receiving saline in
the morning and ethanol in the afternoon and the other
group receiving ethanol in the morning and saline in the
afternoon (Houchi et al. 2005; see also Fig. 1a). Place-
conditioning sessions (saline or ethanol) were separated by
5 h, consistent with the time course of ethanol effects
(Faulkner et al. 1990). Note that order of place conditioning
was not found to significantly affect the final place
preference response at any dose of ethanol utilized
(comparing grouped post-conditioning preferences of the
0.4, 0.8, and 1.6 g/kg mice conditioned with saline first vs
those mice conditioned with ethanol first, F
(1,42)
=1.50,
p=0.23; one-way ANOVA). A biased ethanol place-
conditioning design was then utilized, as it produces
ethanol-CPP in C57Bl/6 mice (Nocjar et al. 1999;
Middaugh and Bandy 2000; Gremel et al. 2006). As
adapted for use here, on days 2–5 mice were administered
10 ml/kg, i.p., of an ethanol solution (5%, 10%, or 20%
(v/v), corresponding to 0.4, 0.8, and 1.6 g/kg ethanol;
Kuzman et al. 2003; Thanos et al. 2005) once daily and
immediately confined in their initially non-preferred side
for 25 min. For alternating place-conditioning sessions
(a.m. or p.m.), mice were administered an equivalent
volume of saline and immediately placed in their initially
preferred side for 25 min. On day 6, testing of place-
conditioned preference was conducted when mice ran freely
through the conditioning apparatus for 30 min. Time spent
in each compartment was measured and compared to the
initial preference.
Alcohol preference test: two-bottle free choice ATBC
paradigm with escalating ethanol concentrations was used
(Thiele et al. 1998; Li et al. 2005; Gabriel and Cunningham
2008) to examine ethanol preference. Mice (54 C57Bl/6J,
Day: 1 6 5 4 3 2
-or- -or- -or- -or-
Preference test, 30 min
Ethanol (0.4, 0.8 or 1.6 g/kg) place conditioning, 25 min
Saline place conditioning, 25 min
b
a
Fig. 1 Ethanol-conditioned place preference (CPP) is dose-
dependent. aUpper schematic: experimental design for ethanol-CPP
testing. After an initial 30-min determination of place preference
(triangle, day 1), mice were place conditioned in the non-preferred
chamber for the next 4 days with ethanol (squares; 0.4, 0.8, or
1.6 g/kg) either 5 h before or after saline place conditioning (0.9%,
circles) in the opposite chamber. Final CPP was determined on day 6.
bAll 44 mice demonstrated similar preconditioning place preferences
(“Pre-CPP,”white bar), which were thus grouped together here. Mice
place conditioned for 4 days with 0.4 g/kg ethanol (gray bar) did not
demonstrate a change from the initial place preference response. In
contrast, place conditioning with 0.8 (black bar) or 1.6 g/kg (gray
thatched bar) ethanol produced a significant preference for the
ethanol-paired environment. Asterisk,p<0.05 from preconditioning
response; analysis of variance+Tukey
Psychopharmacology
ten Dyn −/−, and nine Dyn +/+ littermates) were housed
individually and habituated for 5 days to drink from two
bottles (148 mL) containing sterile water. After habituation,
mice were given 24 h access to two bottles, one containing
tap water and the other containing an ethanol solution. Each
bottle was filled with 100 mL of fluid replaced after every
measurement. The concentration of ethanol (v/v)was
increased every 4 days with mice receiving 3%, 6%, and
finally 10% ethanol over the course of the experiment. To
control for position preference, the position of the bottles
was changed every 24 h (at 1200 hours; Li et al. 2005;
Mitchell et al. 2005). Fluid intake was measured to the
closest milliliter every 2 days (Thiele et al. 1998) or every
day after experimental manipulation. To measure ethanol
preference, ethanol-preference ratios were calculated for
each concentration by dividing the volume of ethanol
solution consumed by the total volume of liquid (ethanol
plus water) consumed for each day (Thiele et al. 1998;
Lindholm et al. 2001). To control for leakage and
evaporation, an empty cage containing one bottle of tap
water and one bottle of ethanol solution was placed
alongside experimental cages. Body weights were mea-
sured at the beginning of each change in ethanol concen-
tration and at the end of the experiment. Unfortunately,
daily weights were not consistently collected for all
animals, precluding a complete set of calculations, but the
C57Bl/6J mice consumed an increasing amount of ethanol
correlated with the solution of ethanol available (3%=4.3 ±
0.31 g/kg/day, 6%=7.7±0.56 g/kg/day, and 10%=11.8±
0.96 g/kg/day at the end of each 4-day training period).
Forced swim stress To measure the effect of FSS on
ethanol reward and consumption, mice were exposed to a
forced swim test, as detailed by McLaughlin et al. (2003)
and as modified below. The FSS paradigm utilized was a
2-day (for TBC) or 4-day (for CPP) procedure in which
mice repeatedly swam without the opportunity to escape.
Mice were treated with vehicle or nor-BNI (10 mg/kg, i.p.)
24 h prior to initial FSS exposure. In all swim trials,
animals were placed in an opaque 5-L cylinder filled with
4 L of 30–32°C water. On day 1 (CPP day 1, or TBC day
17), forced swimming occurred over a single 15-min trial
(Fig. 2a). On day 2 (CPP day 2, or TBC day 18), mice
were exposed to four 6-min trials of forced swimming.
Onday3(CPPday3)animalsagainswaminasingle
15-min trial, but on day 4 (CPP day 4), mice were
exposed to three 6-min trials. Upon completion of each
trial, all mice were removed from the water, towel-
dried, and returned to their home cages for approxi-
mately 6 min. Note that when each day's exposure to all
FSS trials was finished, mice subject to CPP testing were
administered ethanol and place conditioned approximately
5 min later as described above.
To measure the effect of FSS on ethanol self-
administration, mice were exposed to a modified FSS as
stated above. Mice were conditioned to 10% ethanol for
4 days and then either treated with vehicle (ten C57Bl/6J; all
Dyn +/+ and −/−mice) or nor-BNI (10 mg/kg i.p.; nine
C57Bl/6J mice). Two hours later, mice were exposed to FSS
over the next 2 days. On the first day of stress (day 17 of the
TBC protocol), mice were exposed to a single 15-min FSS
and then returned to their TBC cage. On day 18, mice were
again exposed to FSS over four 6-min trials before immediate
return to their two-bottle cage.
Day: 1 6 5 4 3 2
-or- -or- -or- -or-
Preference test, 30 min
Ethanol (0.8 g/kg) place conditioning, 25 min
Saline place conditioning, 25 min
vehicle or nor-BNI (10 mg/kg, i.p.)
Forced swim stress
b
a
Fig. 2 Exposure to stress results in a kappa-opioid receptor-mediated
potentiation of ethanol-conditioned place preference (CPP). aUpper
schematic: experimental design for ethanol-CPP testing. After an
initial 30-min determination of place preference (triangle, day 1), mice
were pretreated on day 1 (arrow) with either vehicle (0.9% saline) or
nor-binaltorphimine (BNI; 10 mg/kg, i.p.). On days 2–5, mice were
then left in home cages or exposed to forced swim stress (FSS, gray
diamonds) and place conditioned daily with 1.6 g/kg ethanol (squares)
either 5 h before or after saline place conditioning (circles) in the
opposite chamber. Final CPP was determined on day 6 (triangle). b
Unstressed mice demonstrated ethanol-CPP after place conditioning
(black bar). Mice administered vehicle before repeated exposure to
forced swim stress demonstrated significant potentiation of ethanol-
CPP when place conditioned 5 min after exposure to FSS (gray bar).
Pretreatment of mice with nor-BNI (striped gray bar)prevented
stress-induced potentiation of ethanol-CPP responses. Notably, nor-
BNI pretreatment alone did not alter ethanol-CPP (thatched dark
gray bar). Asterisk,p<0.05 from preconditioning response; dagger,
p<0.05 from ethanol-CPP of unstressed mice; double dagger,p<
0.05 from ethanol-CPP response of FSS-exposed mice; analysis of
variance+Tukey
Psychopharmacology
Antinociceptive testing using the 55°C warm-water tail-
withdrawal assay The 55°C warm-water tail-withdrawal
assay was used as described previously (McLaughlin et al.
2003), with the latency of the mouse to withdraw its tail
taken as an endpoint. Tail-withdrawal latencies were collect-
ed before and every 10 min after U50,488 (10 mg/kg i.p.)
exposure until the latency to remove the tail returned to
baseline (pre-drug) responses. A cutoff time of 15 s was used
to prevent tissue damage. Note that from this data (see
“Results”), a 90-min pretreatment time with this dose of
U50,488 was used in the CPP (24 mice) and TBC (eight
mice alone and nine mice with nor-BNI pretreatment) assays,
as the waxing and waning time course of antinociceptive
activity indicates the onset and termination of agonist-
induced KOR signaling.
Statistical analysis Antinociception was analyzed by com-
paring pre- and post-U50,488 tail-withdrawal latencies
using paired-samples ttests to test for significant increases
in latency after drug administration. All other results were
compared and analyzed using one-way and two-way
ANOVA as appropriate using the SPSS 14.0 statistical
package (Chicago, IL, USA). Analyses examined the main
effect of CPP phase (e.g., pre- or post-place conditioning)
by dose (0.4, 0.8, 1.6 g/kg) or ethanol consumption by
training solution (0%, 3%, 6%, or 10%) or day (day 17, and
then post-treatment on days 18 and 19) and the interaction
of environmental condition (unstressed or stress-exposed),
genotype (C57Bl/6J or prodynorphin gene-disrupted or
wild-type mice), and drug pretreatment (U50,488, nor-
BNI or vehicle). Significant effects were further analyzed
with Tukey honestly significant difference (HSD) post hoc
testing. Data are presented as mean difference in time spent
on the drug-paired side (seconds), ethanol consumed
(milliliters), or ethanol-preference ratio ± SEM of the animal
treatment group, with significance set at p<0.05.
Results
Optimization of dose in ethanol-conditioned place
preference testing with C57Bl/6J mice
Optimization of ethanol place conditioning was conducted
with ethanol doses of 0.4, 0.8, or 1.6 g/kg once daily for
4 days (Fig. 1a). Results indicated a significant main effect
of ethanol dose (F
(3,87)
=17.5, p<0.001; one-way ANOVA).
Mice place conditioned with 0.4 g/kg ethanol displayed no
significant change in preference from the initially preferred
chamber to the ethanol-paired chamber (Fig. 1b). However,
mice place conditioned 25 min daily with 0.8 or 1.6 g/kg
doses of ethanol spent significantly more time in the
ethanol-paired chamber in final preference testing (p<
0.01, Tukey HSD test; Fig. 1b). Although not significantly
different from the effect of place conditioning with a
1.6-g/kg dose of ethanol, a peak effect was observed with
0.8 g/kg ethanol (132±65.5 s), prompting use of the
0.8-g/kg dose of ethanol in CPP testing for the remainder
of the study.
Stress-induced potentiation of ethanol-conditioned place
preference is mediated by signaling of the kappa-opioid
receptor
The effect of FSS exposure on ethanol-CPP was evaluated.
After obtaining preconditioning responses in the CPP
apparatus for subsequent comparisons, C57Bl/6J mice were
administered vehicle or the KOR antagonist nor-BNI
(10 mg/kg; Fig. 2a). On each of the following 4 days, mice
were left idle in home cages or exposed to FSS daily prior
to ethanol place conditioning. Conditioning with ethanol
produced significant place preference across all conditions
tested (F
(4,137)
=27.7, p<0.0001; one-way ANOVA). Mice
pretreated with vehicle and exposed to daily FSS before
ethanol place conditioning demonstrated a significant
4.4-fold potentiation of ethanol-CPP over the response of
unstressed mice (p<0.01, Tukey HSD test; Fig. 2b).
Pretreatment with nor-BNI prevented the FSS-induced
potentiation of ethanol-CPP, with a response significantly
less than the vehicle-treated, FSS-exposed mice (p< 0.01,
Tukey HSD test), but not different from the response of
unstressed mice (Fig. 2b). Importantly, nor-BNI pretreat-
ment in the absence of FSS exposure did not significantly
affect the place conditioning response to ethanol (p=0.88,
n.s.; Fig. 2b). Moreover, nor-BNI pretreatment alone did
not induce CPP under these conditions, as ten mice
pretreated separately with nor-BNI (10 mg/kg, i.p.) prior
to vehicle place conditioning (i.e., without ethanol) did not
demonstrate a significant change in place preference from
initial preconditioning responses (−141.5 ± 65.9 s precondi-
tioning preference as compared to −176.4±46.6 s post-
CPP; p=0.61, Student's ttest.)
Prolonged prior U50,488-induced activation of KOR
potentiates ethanol-CPP
While acute activation of the KOR prevents cocaine-
(McLaughlin et al. 2006) and ethanol-CPP (Logrip et al.
2009), previous work has shown that prolonged prior
activation of KOR paradoxically potentiated CPP responses
(McLaughlin et al. 2006). To determine if a similar
prolonged prior KOR activation might mimic the effects
of FSS exposure sufficiently to potentiate ethanol-CPP,
mice were administered the KOR agonist U50,488
(10 mg/kg, i.p.) daily 90 min prior to ethanol place
conditioning (Fig. 3a). This duration of pretreatment was
Psychopharmacology
chosen as U50,488-induced antinociception at this dose
was shown to recover at this time point when measured
with the 55°C warm-water tail-withdrawal assay, suggest-
ing the termination of initial agonist activity. Administra-
tion of U50,488 increased the tail-withdrawal latency from
baseline (1.59 ± 0.16 s) to a peak value 40 min after
administration (12.1±1.76 s; p<0.001), returning to laten-
cies similar to baseline responses after 90 min (2.09±0.3 s;
p=0.32). Much like a previous demonstration of KOR
agonist-induced potentiation of cocaine-CPP (McLaughlin
et al. 2006), prior treatment with U50,488 90 min before
ethanol significantly potentiated the resulting CPP com-
pared with vehicle-pretreated mice (F
(3,124)
=30.7, p<
0.0001; one-way ANOVA, followed by Tukey HSD post
hoc test; Fig. 3b). Moreover, the potentiation produced by
prior U50,488-mediated stimulation was itself prevented by
KOR antagonist pretreatment, as mice administered nor-BNI
before daily U50,488 (Fig. 3a) subsequently demonstrated a
normal ethanol-CPP response that was significantly less than
theresponseofU50,488-pretreatedmice(p<0.01; Fig. 3b),
but not significantly different from the ethanol-CPP response
of vehicle-treated, unstressed mice.
Characterization of ethanol consumption in C57Bl/6J
or prodynorphin (+/+) or (−/−) mice using the two-bottle
free choice (TBC) assay
Average consumption of solutions containing ethanol by
C57Bl/6J, prodynorphin gene-disrupted (Dyn −/−)and
wild-type littermate (Dyn +/+) mice was examined with
the TBC assay. Amount of ethanol consumed and the
percentage of ethanol over total fluid consumption were
analyzed at the end of each training period with water (day
5), 3% (day 9), 6% (day 13), or 10% ethanol (day 17)
solutions (Fig. 4). The volume of ethanol consumed at the
end of each training period did not change significantly
across dose or strain (F
(6,279)
=0.84, p=0.54; two-way
ANOVA; Fig. 4a). When consumption of solutions was
analyzed as a percentage of ethanol consumed out of total
fluid intake, no significant differences were observed
among the mouse strains after 4 days' training with each
solution (F
(6,279)
=1.31, p=0.25; two-way ANOVA;
Fig. 4b).
Prior exposure to FSS or a KOR agonist potentiates ethanol
consumption in the TBC assay
Following the measurements completing ethanol-
preference training, C57Bl/6J mice were pretreated with
vehicle (0.9% saline) or nor-BNI (10 mg/kg, i.p.). Two
hours later, half of each group were exposed to FSS and
returned to their two-bottle cages. After 24 h, the amount
of ethanol consumed was measured. Exposure to FSS
significantly increased the percentage of ethanol consumed
over this period (F
(4,84)
=2.61, p<0.05; one-way ANOVA;
Fig. 5). Consistent with the CPP results, nor-BNI
pretreatment prevented the FSS-induced potentiation (p<
0.05; Fig. 5). Importantly, nor-BNI pretreatment did not
significantly alter the amount or percentage of total
ethanol consumed over this time period by ethanol-
trained, unstressed mice (6.1± 1.2 ml of 10% ethanol
consumed over the matching 24-h period, for a percentage
of 50.5±6.1 ethanol of total fluid intake; p=0.07, n.s.).
An additional set of TBC-trained mice was administered
U50,488 (10 mg/kg, i.p.) and returned to their two-bottle
cages for a measurement of 10% ethanol consumed 24 h
later. Consistent with the CPP testing, mice pretreated with
Day: 1 6 5 4 3 2
-or- -or- -or- -or-
Preference test, 30 min
Ethanol (0.8 g/kg) place conditioning, 25 min
Saline place conditioning, 25 min
vehicle or nor-BNI (10 mg/kg, i.p.)
U50,488 (10 mg/kg, i.p., -90 min prior to EtOH)
b
a
Fig. 3 Prolonged exposure to kappa-opioid receptor (KOR) agonist
results in potentiation of ethanol-conditioned place preference (CPP).
aUpper schematic: experimental design for ethanol-CPP testing.
After an initial 30-min determination of place preference (triangle,
day 1), mice were pretreated (arrow) with either vehicle or nor-
binaltorphimine (BNI; 10 mg/kg, i.p.) and injected with KOR agonist
U50,488 (10 mg/kg, i.p.; gray diamond) prior to place conditioning
with 0.8 g/kg ethanol (squares) either 5 h before or after saline place
conditioning (circles, 0.9%, i.p.) in the opposite chamber for the next
4 days. Final CPP was determined on day 6. bMice demonstrated
ethanol-CPP after place conditioning (black bar). Mice pretreated
daily with U50,488 90 min prior to place conditioning with 0.8 g/kg
ethanol demonstrated potentiation of ethanol-CPP (gray bar). Pre-
treatment with nor-BNI prior to U50,488 treatment (striped gray bar)
prevented the agonist-induced potentiation of ethanol-CPP. Asterisk,p
<0.05 from preconditioning response; dagger,p<0.05 from ethanol-
CPP of unstressed mice; double dagger,p<0.05 from ethanol-CPP
response of forced swim stress-exposed mice; analysis of variance +
Tukey
Psychopharmacology
U50,488 consumed a significantly greater percentage of
ethanol than unstressed, vehicle-treated mice (F
(3,77)
=4.54,
p<0.01; one-way ANOVA; Fig. 5). Notably, the percentage
of ethanol consumed was equivalent to the percentage
consumed after a single exposure to FSS (p=0.64, n.s.),
suggesting that prior agonist-induced stimulation of the
KOR was sufficient to increase the percentage of ethanol
consumed.
Interestingly, the effects of both FSS and KOR agonist
stimulation on the percentage of ethanol consumed were
not found to be long lasting. When mice were exposed for
an additional day to FSS or a second administration of
U50,488, no significant differences in ethanol consumption
were observed among all groups tested (F
(4,80)
=0.87, p<
0.05; one-way ANOVA; Fig. 5, rightmost symbols).
Prodynorphin gene-disrupted mice do not demonstrate FSS-
induced potentiation of ethanol consumption in the two-
bottle choice assay
The possible involvement of the endogenous KOR system in
the stress-induced potentiation of ethanol consumption was
assessed using Dyn −/−mice and their wild-type littermates.
Wild-type littermates exposed to FSS demonstrated a signif-
icant increase (to 65.6± 6.0) in the percentage of ethanol
consumed as compared to vehicle-treated, unstressed C57Bl/
6J mice (F
(4,87)
=5.78, p<0.05; one-way ANOVA; Fig. 6).
This effect was statistically equivalent to the FSS-induced
potentiation demonstrated by C57Bl/6J mice (p=0.24, n.s.),
but significantly different from the percentage of ethanol
consumed by Dyn −/−mice exposed to FSS (44.5±5.5%; p
<0.01, Tukey HSD test; Fig. 6), which was not significantly
different from the percentage of ethanol consumed by the
unstressed C57Bl/6J mice (p=0.34).
Notably, the capacity for FSS-induced potentiation of
ethanol consumption was again demonstrated to be brief.
Exposure to an additional day of FSS did not significantly
change the percentage of ethanol consumed across the
groups tested (F
(4.83)
=0.51, p=0.73; one-way ANOVA;
Fig. 6, rightmost points).
Fig. 5 Prior exposure to forced swim stress (FSS) or kappa-opioid
receptor agonist potentiates ethanol consumption in the two-bottle
choice assay. Unstressed mice (circles) were administered vehicle,
while other mice were exposed to forced swim stress (white
diamonds) or treatment with U50,488 (squares)24hpriorto
collection of ethanol consumed. FSS-induced potentiation of 10%
ethanol consumption was blocked by nor-binaltorphimine pretreat-
ment (gray diamonds). Asterisk,p<0.05 from vehicle-treated, un-
stressed mice; dagger,p<0.05 from FSS-exposed mice; two-way
analysis of variance+Tukey
Fig. 4 Ethanol consumption in the two-bottle free choice assay is
consistent among strains and across concentrations of ethanol tested.
Data is shown for fluid intake on last day of training with each
solution, be it water (day 5), 3% ethanol (day 9), 6% ethanol (day 13),
or 10% v/vethanol (day 17). aVolume consumption of ethanol (in
milliliters per day) is consistent on the last day of training with each
solution throughout 17-day training among C57Bl/6J (circles),
prodynorphin wild-type (Dyn +/+, squares), and prodynorphin gene-
disrupted (Dyn −/−,triangles) mice. bPercentage of ethanol
consumed in total fluid intake over 24 h is also consistent throughout
17-day training across C57Bl/6J (circles), prodynorphin wild-type
(Dyn +/+, squares), and prodynorphin gene-disrupted (Dyn −/−,
triangles) mice
Psychopharmacology
Discussion
The principal finding of this study was that repeated
exposure to FSS potentiated ethanol-CPP observed in a
biased procedure. Stress-induced potentiation of ethanol-
CPP was blocked by pretreatment with the KOR antagonist
nor-BNI and mimicked by prior administration of the KOR
agonist U50,488. Exposure to stress also significantly
increased the amount and percentage (of total fluid intake)
of ethanol consumed in a TBC assay, effects again
mimicked by prior exposure to U50,488. These effects
were blocked by nor-BNI pretreatment or prodynorphin
gene disruption.
The demonstration of ethanol-CPP in male C57Bl/6J
mice is perhaps surprising given the failure of these mice to
demonstrate place preference for ethanol in counterbal-
anced place-conditioning designs (Cunningham et al.
1992). However, the present data is consistent with
demonstrations of the successful use of the biased place-
conditioning design (i.e., place-conditioning subjects with
ethanol in the initially non-preferred chamber) to produce
ethanol-CPP with C57Bl/6 mice (Kelley et al. 1997; Nocjar
et al. 1999; Middaugh and Bandy 2000). In fact, the
ethanol-CPP demonstrated by C57Bl/6J mice after place
conditioning in a biased apparatus and subject assignment
procedure was thought to possibly suggest “a greater ability
to detect a smaller rewarding effect in the biased apparatus”
in a recent study (Gremel et al. 2006).
While certain types and exposures to stress have been
noted to acutely suppress ethanol self-administration (van
Erp and Miczek 2001; van Erp et al. 2001; Funk et al.
2005), stress-induced increases in the rewarding effects of
ethanol are also well established in animal models. Rats
exposed to electric foot shock (Mills et al. 1977),
immobilization stress (Rockman et al. 1987), and isolation
stress/psychological stress (Wolffgramm 1990) all demon-
strate increased consumption of ethanol during periods
corresponding with the recovery from stress. Consistent
with this, the present results suggest the activity of the
endogenous kappa-opioid system mediated the stress-
induced potentiation of the rewarding effects and self-
administration of ethanol. Dynorphin, the endogenous
ligand for the KOR, is known to be released in C57Bl/6J
brain in response to exposure to FSS (Shirayama et al.
2004). Since a 24-h pretreatment with nor-BNI prevented
the potentiation of ethanol reward and consumption, it is
plausible that a stress-induced release of dynorphin peptides
activated KOR signaling to paradoxically potentiate ethanol
reward. While not directly tested in the present study, KOR
gene-disrupted mice previously exposed to stress did not
demonstrate increases in cocaine-CPP (McLaughlin et al.
2006). Additional mice in that report pretreated with
U50,488 and place conditioned immediately after the
cessation of the antinociceptive effects of the KOR agonist
mimicked the effect of stress, demonstrating a potentiation
of cocaine-CPP (McLaughlin et al. 2006). Likewise, prior
exposure to U50,488 here also mimicked the effect of
stress, potentiating the rewarding effects of ethanol as well
as the percentage of ethanol consumed. This result is
consistent with a previous report in which chronic
administration of the KOR agonist CI-977 potentiated
ethanol intake and preference in relapse-like drinking
measured in alcohol-deprived, but long-term ethanol-
experienced, rats (Hölter et al. 2000). Earlier work has also
demonstrated that exposure to conditioned fear stress
potentiated ethanol-CPP (Matsuzawa et al. 1998,1999).
The authors concluded that KOR may modulate the
rewarding effect of ethanol under this psychological stress,
but nor-BNI pretreatment prior to ethanol conditioning (but
after exposure to foot shock) slightly increased the
potentiation of ethanol-CPP. It is possible that procedural
differences may account for the contradiction between
studies. In the present study, nor-BNI was administered
prior to FSS exposure, since previous work has shown
KOR antagonists administered after exposure to stress are
ineffective in preventing stress-induced changes in mea-
sured behavior (Carey et al. 2009; Aldrich et al. 2009).
The varied responses to stress by an organism suggest
alternative mechanisms may underlie the potentiation of
Fig. 6 Prodynorphin gene-disrupted mice do not demonstrate forced
swim stress (FSS)-induced potentiation of ethanol consumption in the
two-bottle free choice (TBC) assay. After completion of training on
day 17 of the TBC protocol, C57Bl/6J (circles), prodynorphin gene-
deleted (Dyn −/−,triangles), and wild-type (Dyn +/+, squares) mice
were administered vehicle. Dyn −/−and Dyn +/+ mice were exposed
to FSS (arrows) on both days 17 and 18 and ethanol consumption
measured 24 h after each exposure. Asterisk,p<0.05 from unstressed
C57Bl/6J; dagger,p<0.05 from stress-exposed Dyn +/+; analysis of
variance+ Tukey
Psychopharmacology
ethanol-CPP and consumption observed here, such as the
possible effect of corticosterone (Lê et al. 2000). However,
previous studies have shown that the FSS-induced release
of corticosterone is not affected by nor-BNI pretreatment or
prodynorphin gene disruption (McLaughlin et al. 2006). As
such, it seems unlikely that the prevented potentiation of
ethanol-CPP observed here is due to a disruption of
hypothalamic–pituitary–adrenal axis function, but instead
that a disruption of the kappa system alone might suffice.
Alternatively, the mediation of ethanol reward and con-
sumption by the endogenous mu- and delta-opioid systems
raises the possibility that the present results were due to
interactions at other opioid receptors than KOR. Activation
of delta- and mu-opioid receptors has been implicated in
reward mechanisms and behavioral reinforcement of etha-
nol (Vengeliene et al. 2008), and blockade of these
receptors (Stromberg et al. 1998) or mu-opioid receptor
gene disruption (Roberts et al. 2000) has been shown to
decrease ethanol self-administration. However, this alterna-
tive seems unlikely in the present study, given the
established selectivity of nor-BNI for the KOR at the dose
and duration of pretreatment used (McLaughlin et al. 2003)
and the use of prodynorphin gene-disrupted animals in the
present study, although additional testing with KOR gene-
disrupted mice or additional KOR-selective antagonists
would be of value.
It is conceivable that the potentiation of ethanol reward
demonstrated by the FSS- or U50,488-exposed mice may
result from a stress- and KOR-induced enhancement of
learning and memory performance. KOR agonists have
been demonstrated to improve performance in learning and
memory tasks (Hiramatsu and Hoshino 2005; Kuzmin et al.
2006). However, this seems unlikely given that the memory
performance enhancements have been attributed to agonist
activity through non-opioid sites (Hiramatsu and Hoshino
2005; Kuzmin et al. 2006), and a recent study suggests that
KOR signaling seems to impair novel object recognition,
another animal model of learning and memory (Carey et al.
2009).
In a more plausible alternative, the stress-induced increase
in ethanol-seeking behavior observed here might be attributed
to attempts to counteract symptoms of anxiety. Presentation of
anxiety-like behaviors is predictive of ethanol self-
administration in rats (Spanagel et al. 1995), squirrel monkeys
(McKenzie-Quirk and Miczek 2008), and humans (Willinger
et al. 2002), and ethanol was shown to produce anxiolytic
effects similar to diazepam in rat models of anxiety (Wilson
et al. 2004). KOR antagonists were recently shown to have
anxiolytic effects (Knoll et al. 2007), suggesting that the nor-
BNI used here may have prevented stress-induced potenti-
ation of ethanol effects by reducing anxiety directly, but
further parallel testing in mouse models of anxiety will be
needed to investigate this alternative.
Of interest, the potentiating effects of stress- or U50,488-
exposure on the percentage of ethanol consumed in the
TBC assay were shown to be of short duration, lasting no
more than 1 day. These effects suggest either a brief impact
by stress on reward circuitry, or a possible habituation to
predictable stress. This relatively short duration of action is
of interest, given reports of lengthy stress-induced increases
in ethanol consumption in BALB/cJ mice (Lowery et al.
2008), and worthy of further examination. Use of a set of
chronic unpredictable stressors might conceivably extend
stress-induced potentiation for a longer duration, although
further testing and characterization of the duration of stress-
induced vulnerability to increased ethanol consumption are
needed.
The potentiation of the rewarding effects of ethanol by
the kappa-opioid system seems paradoxical, as acute
administration of kappa agonists along with ethanol are
known to block ethanol-CPP (Matsuzawa et al. 1999;
Logrip et al. 2009) and voluntary ethanol intake (Lindholm
et al. 2001). In addition, kappa-opioid agonists produce
conditioned place aversion in rodents (Shippenberg and
Herz 1986; Suzuki et al 1992; Matsuzawa et al. 1999).
However, these studies with KOR agonists utilized the
acute administration and activity of KOR agonists, whereas
the present study examined the effect of a prior activation
of the KOR with chronic KOR agonist stimulation, which
reports suggest may potentiate drug reward (Hölter et al.
2000; Negus 2004; McLaughlin et al. 2006). Given a recent
report where nor-BNI was found to attenuate ethanol self-
administration in rats previously made dependent on
ethanol, but had no effect on nondependent animals
(Walker and Koob 2008), it could be hypothesized that
repeated exposure to stress or KOR agonists produces a
dysphoric state which subsequently enhances the rewarding
value of euphoric agents that attenuate this effect
(McLaughlin et al. 2006; Walker and Koob 2008).
Alternatively, a direct neurobiological mechanism may
account for the potentiation of drug reward (Sommer et
al. 2006; Zapata and Shippenberg 2006), although neither
theory was directly tested in the present study.
Importantly, nor-BNI inhibition of KOR or KOR gene
disruption increased the ethanol-induced release of dopa-
mine in the mouse nucleus accumbens (Zapata and
Shippenberg 2006), and a single administration of nor-
BNI was reported to increase ethanol consumption, but not
CPP, in long-term ethanol-experienced rats (Mitchell et al.
2005). This finding contradicts the present results, where
nor-BNI did not significantly alter ethanol reward in
unstressed mice, although this is consistent with other
reports (Hölter et al. 2000; Lindholm et al. 2001).
Interestingly, KOR gene-disrupted mice (Kovacs et al.
2005) and female prodynorphin gene-disrupted mice
(Blednov et al. 2006) have exhibited lower rates of ethanol
Psychopharmacology
self-administration than wild-type littermates. Curiously,
male Dyn −/−mice demonstrate no change in ethanol
consumption (Blednov et al. 2006), a finding confirmed
with the present data. It is notable that the effects of stress
on ethanol reward or consumption were not investigated in
these mouse studies, and may suggest an important
procedural difference in these results. Overall, despite
contradictions, these results suggest the significance of the
endogenous kappa-opioid system in the maintenance of
ethanol consumption and suggest a bidirectional regulation
of ethanol reward by the KOR system that requires further
investigation.
In summary, these data demonstrated a stress-induced
potentiation of the rewarding effects and self-administration
of ethanol mediated by prior activation of KOR signaling
and suggest that KOR antagonists may prove beneficial in
preventing stress-induced increases in ethanol consumption.
Acknowledgements We thank Dr. Ute Hochgeschwender for gen-
erously providing the prodynorphin gene-disrupted mice. This
research is supported by R03 DA16415 from the National Institute
on Drug Abuse to JPM and funds from the State of Florida, Executive
Office of the Governor's Office of Tourism, Trade, and Economic
Development. Mice were housed and cared for in the Northeastern
University or TPIMS animal facilities in accordance with the 1996
National Institutes of Health Guide for the Care and Use of
Laboratory Animals. All studies were performed after prior approval
by the local Institutional Animal Care and Use Committee and are in
full compliance with the current laws of the USA.
Conflicts of interest The authors declare that they have full control
of all primary data. Except for income received from our primary
employers, no financial support or compensation has been received
from any individual or corporate entity over the past 3 years for
research or professional services related to this project, and there are
no personal financial holdings that could be perceived as constituting
a potential conflict of interest.
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