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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 290:535–542, 1999
Vol. 290, No. 2
Printed in U.S.A.
Caffeine, Acting on Adenosine A1 Receptors, Prevents the
Extinction of Cocaine-Seeking Behavior in Mice1
A. KUZMIN, B. JOHANSSON, E. E. ZVARTAU, and B. B. FREDHOLM
Department of Pharmacology, Pavlov Medical University, St. Petersburg, Russia (A.K., E.Z.); and Section of Molecular Neuropharmacology,
Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden (A.K., B.J., B.B.F.)
Accepted for publication April 22, 1999
This paper is available online at http://www.jpet.org
Cocaine-dependent individuals tend to relapse into cocaine
use even after long periods of abstinence. It is important to
find factors that contribute to such a relapse. Animal studies
have shown that drug-seeking behavior can be elicited not
only by a low dose of the original self-administered drug
(Gerber and Stretch, 1975; de Wit and Stewart, 1981; Stewart and Wise, 1992; Self et al., 1996), but also by drugs from
pharmacological groups other than that of the original selfadministered drug (Slikker et al., 1984).
Previously, it was shown that caffeine reinstated responding during an extinction phase in rats trained to self-administer cocaine (Worley et al., 1994; Schenk et al., 1996) or
Received for publication October 7, 1998.
1
This work was supported by Physiological Effects of Coffee Committee
(PEC), Swedish Medical Research Council (Projects 2553 and 12587), Socialstyrelsens Fonder, the Åke Wiberg Foundation, Magn. Bergvall Foundation,
O. E. and Edla Johansson Scientific Foundation, Syskonen Svenssons Fund for
Medical Research, and Fredrik and Ingrid Thuring Foundation. A. K. received
visiting scientist support from the Karolinska Institute and from the Royal
Swedish Academy of Sciences. B. J. was supported by a grant from the Brain
Fund.
sponding in the active group than in the yoked control group
during a single extinction trial. The adenosine A1-receptor antagonists DPCPX and 8-CPT and the nonselective antagonist
CGS 15943 partially reproduced the effect of a low dose of
caffeine on the cocaine-associated behavior in a dose-dependent manner and did not alter the nose-poke activity of yoked
control mice in the extinction experiment. In contrast, the adenosine A2A antagonist SCH 58261, in doses above 1 mg/kg,
reduced nose-poke activity equally in active and yoked control
animals. This confirms that a drug from a different pharmacological class (adenosine-receptor antagonist) can induce behavior changes similar to the effects of the original self-administered drug (indirect dopamine-receptor agonist). The data
also suggest that the effects of caffeine on cocaine-seeking
behavior might be related to interaction with adenosine A1
receptors, but not A2A receptors.
potentiated cocaine effects on the reinstatement of self-administration behavior (Self et al., 1996). The nature of this
cue is not understood presently. Caffeine does not generalize
well to cocaine in a drug-discrimination paradigm, and it is
poorly self-administered (Griffiths and Woodson, 1988; Gauvin et al., 1990). However, cocaine substituted for the caffeine-discriminative stimulus in rats trained to discriminate
caffeine from saline (Holtzman, 1986). Thus, how caffeine
and cocaine can substitute for each other, and to what extent,
remains to be determined.
Of all of the known primary biochemical mechanisms of
action of caffeine, only antagonism of adenosine effects on
adenosine A1 and A2A receptors is known to be important at
concentrations of caffeine that produce behavioral stimulation (see Fredholm, 1995). Adenosine A1 receptors are widely
distributed in the brain (Johansson et al., 1993) and regulate
the release of several neurotransmitters (Fredholm and Dunwiddie, 1988). In contrast, adenosine A2A receptors are
largely restricted to neurons that release g-aminobutyric
ABBREVIATIONS: DPCPX, 1,3-dipropyl-8-cyclopentyl xanthine; 8-CPT, 8-cyclopentyl theophylline; SCH 58261, 5-amino-7-(2-phenylethyl)-2-(2furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; CGS 15943, 9-chloro-2(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine; DMSO, dimethyl sulfoxide.
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ABSTRACT
Drug-naive DBA/2 mice were trained to self-administer cocaine
(40 mg/kg/infusion) i.v. by nose poking. The number of nosepoke responses was higher in mice receiving response-contingent injections of cocaine (active group) than in yoked controls
or in animals receiving response-contingent saline injections.
Twenty-four hours after the training session (cocaine or saline
self-administration), mice were injected i.p. with saline, cocaine, caffeine, 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX),
8-cyclopentyl theophylline (8-CPT), 5-amino-7-(2-phenylethyl)2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine
(SCH 58261), or 9-chloro-2(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS 15943) and placed again in exactly the
same operant boxes as during the training session but without
response-contingent i.v. infusions. Saline injection elicited similar responding in animals from the active group and from the
yoked control group. A low dose of cocaine (5 mg/kg) or caffeine (3 mg/kg), but not higher doses, produced greater re-
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Kuzmin et al.
Materials and Methods
Animals. All experiments were carried out on male DBA/2 mice
(18 –22 g). Animals were obtained from the breeding farms in Rappolovo, Russia and Bomholtgård, Denmark and kept under standard
laboratory conditions with unlimited access to food and water. They
were housed 12 mice per cage in a light-controlled room (12 h light/
dark cycle; light on at 10:00 AM) at 21°C and 60% humidity. The
experiments were approved by the respective regional animal ethics
boards.
Drugs. Caffeine (as free base; Sigma Chemical Co., St. Louis, MO)
and cocaine (as hydrochloride; Sigma Chemical Co.) were dissolved
in warm saline and injected i.p. just before the experiment. SCH
58261 (a gift from Dr. Ennio Ongini, Schering-Plough, Milan, Italy),
CGS 15943 (Research Biochemicals International, Natick, MA) and
DPCPX (Research Biochemicals International) were dissolved in dimethyl sulfoxide (DMSO), and stock solutions were diluted in saline
so that the final concentration of DMSO was 20% (v/v). 8-CPT (Research Biochemicals International) was dissolved in 0.01 N NaOH
and the pH of the solution was adjusted to 7.2 with 0.01 N HCl.
Cocaine HCl for i.v. self-administration was dissolved in saline and
the pH of the solution was adjusted to 7.2 with 0.01 N NaOH. Doses
of cocaine refer to the salt.
Intravenous Self-Administration Method. The model of onesession initiation of i.v. self-administration of cocaine was used (40
mg/kg/infusion; 1.6 ml per infusion; fixed ratio 5 1; naive mice;
Kuzmin et al., 1997). This unit dose was chosen on the basis of
preliminary testing in the range from 10 to 80 mg/kg/infusion. Mice
were tested in pairs in cages (8 3 8 3 8 cm) that had a frontal hole
(1.5 cm) for nose poking fitted with infrared sensors (3 mm into the
hole from the inner surface of the cage) interfaced to an operating
computer that controlled the automatic syringe pump. Mice were
partially immobilized by fixing their tails with adhesive tape to the
horizontal surface. The tails protruded through a vertical slot in the
back wall of the box.
First, the number of nose-poke reactions during a 10-min period
was recorded for all experimental animals. On the basis of this
10-min test, pairs of mice were chosen so that animals in pairs
exhibited an approximately equal level of nose-poke activity.
Thereafter, the matched pairs were placed into the experimental
boxes, and cannulas (external diameter, 0.4 mm) were introduced
into the lateral tail veins of both of the animals in the pair. To verify
the proper placement of the cannulas, test infusions (3 3 1.6 ml) were
made. Improper positioning of the needle resulted in a local paling of
the tail. In this case, the needle was removed and reintroduced.
When all of the cannulas were properly placed, the response-contingent injections were activated. Each nose poke of one mouse (called
the active mouse) resulted in a response-contingent infusion of 1.6 ml
of cocaine solution (40 mg/kg/infusion) or saline to both the active
mouse and the yoked control. The injection had a constant duration
of 1 s. Nose pokes of the yoked control animal were counted but had
no programmed consequences. The self-administration session
lasted 30 min. After this period, the animals were labeled and returned to their home cages.
The number of nose pokes by both active and yoked control animals was counted and analyzed. Because of the differences in the
initial (i.e., before drug treatment) nose-poke activity between pairs,
a ratio (r) criterion was used as a quantitative measure of the
reinforcing effect of the drug solution (Kuzmin et al., 1997). This was
calculated as the log10 of the ratio between the cumulative number of
nose-pokes by the active and the yoked control mouse in the matched
pair during a 30-min self-administration session. Logarithms were
used to normalize the distribution of the data. The rationale of this
criterion is that if the infused solution has a reinforcing effect, the
number of operant reactions (nose pokes) in active mice will exceed
that in the yoked control animal, independent of the stimulatory
effects of the drug, and the log10 of the ratio will become a positive
value.
Not all of the pairs of mice acquired cocaine self-administration
according to the r criterion (about 15–25% of pairs in groups failed to
reach self-administration criterion). In the present study, only those
pairs of mice with the r criterion greater than the upper 95% confidence limit of the mean r criterion obtained in the saline self-administration experiments were selected for the extinction experiments.
Extinction of Cocaine Reward-Associated Responding. On
day 2, extinction sessions were carried out. Just before placement
into boxes, both active and yoked control mice were injected i.p. (1
ml/kg) with saline or one of the compounds investigated: cocaine HCl
(2.5–20 mg/kg); caffeine (3–30 mg/kg); CGS 15943 (1.25–5 mg/kg);
SCH 58261 (0.312–2.5 mg/kg); DPCPX (1.25–5.0 mg/kg); or 8-CPT
(5–20 mg/kg). There were six pairs in the treatment groups except in
the groups given cocaine and caffeine treatment (n 5 8). After the
injection, pairs of mice were placed into exactly the same experimental boxes and partially immobilized as in the initiation experiments
(day 1), but the needles were not introduced and cocaine infusions
were not activated. As in the initiation session, the number of nose
pokes of each mouse in the pair was counted and analyzed.
Data Analysis and Statistics. The number of nose pokes for
active and yoked control mice in each treatment group was analyzed
with two-way ANOVA; mode (active mice versus yoked control mice)
and dose (saline/vehicle and drug treatments) were the independent
factors. Next, paired comparison (two-tailed Student’s t test) was
performed to compare active and yoked control activity within each
group of drug-injected animals and nose-poke activity in active and
yoked control animals between the drug-treated group and a saline/
vehicle-treated group. The whole study was designed as a betweensubjects (independent groups) experiment (i.e., each treatment we
describe was done on a single set of animals).
Next, the r criterion was analyzed in a similar way: one-way
ANOVA for the r criteria (with drug treatment as an independent
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acid that also express dopamine D2 receptors (Fink et al.,
1992; Svenningsson et al., 1997a), and there is a functionally
important negative interaction between adenosine A2A and
dopamine D2 receptors (see Ferré et al. 1997). Perhaps secondarily to its primary effects, caffeine also influences and
interacts with a host of transmitter systems in the brain (see
Daly, 1993).
Recently, it was shown that an agonist at dopamine D2
receptors, but not one at D1 receptors, could reinstate cocaine-seeking behavior (Self et al., 1996). Given that D2 and
A2A receptors are colocalized, that they are mutually antagonistic (Ongini and Fredholm, 1996; Ferré et al., 1997), and
that an A2A-receptor antagonist can mimic the stimulation of
motor behavior produced by a D2-receptor agonist (Svenningsson et al., 1995), it seemed possible that the ability of
caffeine to reinstate the extinguished cocaine-seeking behavior is mainly associated with its ability to block A2A receptors.
To test this possibility, we used a technique of the extinction
of cocaine-associated behavior based on a model of one-session
initiation of i.v. cocaine self-administration in naive mice
(Kuzmin et al., 1997). With this model, we tested the ability of
cocaine and caffeine to prevent the extinction of cocaine rewardassociated responding in mice withdrawn from contingent reinforcing infusions of cocaine. Also, we examined the ability of
selective adenosine A1-receptor antagonists, 1,3-dipropyl-8cyclopentyl xanthine (DPCPX) and 8-cyclopentyl theophylline
(8-CPT), a selective A2A-receptor antagonist, 5-amino-7-(2phenylethyl)-2-(2-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo[1,5c]pyrimidine (SCH 58261; see Ongini and Fredholm, 1996), and
a nonselective, nonxanthine, adenosine-receptor antagonist,
9-chloro-2(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine
(CGS 15943) to mimic the effect of caffeine.
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1999
Adenosine and Cocaine-Seeking Behavior
537
factor), followed by paired comparison of the r criteria (two-tailed
Student’s t test) in an extinction experiment with corresponding
ratios in the acquisition experiment and the extinction ratio in the
control group (treated with saline/vehicle).
Results
Fig. 1. Influence of cocaine and caffeine on the extinction of cocaineseeking behavior in mice. Top, nose-poke responses (mean 6 S.E.M.; n 5
8). Dark bar and filled circle, active mice. Light bar and open circles,
yoked control mice. Doses of cocaine (left) and caffeine (right) are given in
mg/kg. *p , .05; Student’s t test, comparison between active and yoked
control mice within group of animals injected with drug during day 2 of
the experiment. Bottom, r criteria in animals injected with cocaine or
caffeine on day 2; mean 6 S.E.M.; n 5 8. The r criterion is defined as the
logarithm (Lg) of the ratio in nose-poke activity of active (A) animals over
yoked control (YC) animals. The mean 6 S.E.M. of the r criterion in the
acquisition experiment (day 1) is presented as a crossed field. Zp , .05;
Student’s t test, comparison between the ratio on day 2 in the drugversus saline-treated groups of pairs of mice. #p , .05; Student’s t test,
comparison between the r criterion obtained in the extinction experiment
(day 2) and the r criterion obtained in the acquisition experiment (day 1).
pattern in mice trained to self-administer cocaine (Fig. 1).
Administration of cocaine at the dose of 5.0 mg/kg increased
specifically the nose-poking response of the active animals
that had learned to self-administer cocaine on day 1 (p , .05
as compared with the saline-treated group). ANOVA revealed a significant effect of cocaine on the number of nose
pokes in the active mice (F(4,29) 5 4.31; p 5 .007; n 5 8
animals per group). However, this effect of cocaine (5.0 mg/
kg) was not seen in the active animals trained for saline
self-administration on day 1 (data not shown) or in yoked
control animals that were trained with either cocaine or
saline infusion on day 1. With the higher doses of cocaine (10
and 20 mg/kg), there were no significant differences in nosepoking activity between the animals in the pair.
ANOVA also showed a significant influence of cocaine dose
on r criteria (F(4,29) 5 8.79; p 5 .00008). Paired comparison
revealed a significant difference between r criteria in the
initiation experiment versus the extinction experiment in the
groups injected with saline (p 5 .002) or the two highest
doses of cocaine (10 mg/kg, p 5 .008; 20 mg/kg, p 5 .00001).
In groups injected with 2.5 or 5.0 mg/kg cocaine, the r criterion in the extinction experiment did not differ significantly
from that in the initiation experiment. However, it was only
in the group injected with 5.0 mg/kg cocaine that the r criterion during extinction differed significantly from that in the
group injected with saline (p , .05). Of special importance is
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Initiation of Cocaine/Saline Self-Administration. The
initial nose-poke activities (in a 10-min trial without infusions) did not differ significantly between the different
groups of animals. The mean number of nose-poke responses
in the various groups ranged from 23.88 6 14.13 to 31 6
12.87 during 10 min of testing (lowest and highest levels for
different groups; mean 6 S.E.M.). However, it is important to
note that the difference in nose-poke activity between individual pairs was significant (from 16 to 88 in 10 min; lowest
and highest activity in pairs). Two-way ANOVA showed no
difference between treatment groups (p 5 .49) or between
active and yoked control mouse subgroups (p 5 .86), and
there was no interaction between group and subgroup factors. From this, it can be concluded that the matching between and within experimental groups was successful. Thus,
any reinforcing action of the contingent infusions of the drug
solution was expected to result in a larger number of nose
pokes by the active mouse than by the yoked control.
Cocaine (40 mg/kg/infusion), when made contingent to the
nose pokes of one animal in the pair, selectively and significantly increased the operant responses of active animals (t 5
13.55; p , .001; 276 degrees of freedom), whereas the responses of the yoked control animals remained unchanged
and did not differ from the activity of the control animals in
groups with saline self-administration. This translates into a
highly significant (p , .01) increase of the r criterion (i.e., the
logarithm of the ratio of nose-poke responses in treatment
versus control group; see Materials and Methods) in mice
given cocaine on day 1 (0.27 6 0.01, 148 selected pairs) as
compared with that in mice offered saline self-administration
(20.02 6 0.01; 32 selected pairs). This was interpreted as a
strong reinforcing effect of this particular unit dose of cocaine. In addition, the results show that a 30-min experimental session with the nose-poke operant procedure is sufficient
for mice to acquire cocaine self-administration behavior. For
the extinction experiments, only pairs of mice that reached
the criterion for the acquisition of cocaine self-administration
were used (see Materials and Methods).
Extinction. On day 2, mice were given i.p. injections of
different drugs or saline/vehicle and placed into exactly the
same experimental boxes as in the initiation experiment. In
the groups injected with saline/vehicle, the number of nose
pokes was not significantly different between active and
yoked control mice (see Figs. 1-3). The ratio between the
response rate in the two paired mice was close to 1 and,
hence, the r criterion did not differ from 0. This was interpreted as the extinction of the behavior acquired on day 1 in
the absence of response-contingent cocaine infusions. Injection of the vehicle for adenosine antagonists (20% DMSO)
produced a parallel decrease in nose pokes in both active and
yoked control mice as compared with saline-treated animals.
However, the r criterion remained on the same level after
vehicle treatment as after saline treatment.
Effects of Cocaine in the Extinction Phase. Injection
of cocaine had a dose-dependent influence on the operant
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Kuzmin et al.
Fig. 2. Influence of CGS 15943 and SCH 58261 on the extinction of
cocaine-seeking behavior in mice. Top, nose-poke responses (mean 6
S.E.M.; n 5 6). Dark bar and filled circles, active mice. Light bar and open
circles, yoked control mice. Doses of CGS 15943 (left) and SCH 58261
(right) are given in mg/kg. *p , .05; Student’s t test, comparison between
active and yoked control mice within the group of animals injected with
a drug during day 2 of the experiment. 1p , .05; Student’s t test,
comparison between the drug-treated group and the vehicle-treated
group. Bottom, r criteria in animals injected with CGS 15943 (CGS) or
SCH 58261 (SCH) on day 2 (mean 6 S.E.M.; n 5 6). The mean 6 S.E.M.
of the r criterion in the acquisition experiment is presented as a crossed
field. #p , .05; Student’s t test, comparison between the r criterion
obtained in the extinction experiment (day 2) and the r criterion obtained
in the acquisition experiment (day 1).
receptor antagonist, SCH 58261, which is structurally related to CGS 15943 (see Ongini and Fredholm, 1996). This
compound is at least as potent as CGS 15943 as an antagonist at A2A receptors, but at least 100 times less potent at A1
receptors. Therefore, it was given in doses similar to those of
CGS 15943. As seen in Fig. 2, lower doses of this compound
had little effect on nose-poke responding, whereas higher
doses produced a marked decrease. ANOVA revealed a significant effect of SCH 58261 injection on nose-poke responses
in both active and yoked control mice (F(4,25) 5 22.5; p ,
.0005 and F(2,25) 5 20.5; p , .0005, respectively; 6 animals
per group). There was a significant reduction (compared with
vehicle-injected group) of the operant activity in the groups
injected with SCH 58261 at doses of 1.25 or 2.5 mg/kg (p ,
.01 for both doses). Treatment with 0.312 and 0.625 mg/kg
had no effect on operant responses in either active or yoked
control mice (comparison with vehicle-treated group).
ANOVA also failed to reveal any significant effect of SCH
58261 on the r criteria in the extinction experiment. Paired
comparison of the levels of r criteria in the initiation versus
the extinction experiments revealed that in all of the groups
injected with SCH 58261, the r criteria in the extinction
experiments differed significantly from those in the initiation
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the fact that cocaine (5.0 mg/kg) did not increase the value of
the r criterion in mice that had been trained to self-administer saline during the initiation experiment.
Effects of Caffeine in the Extinction Phase. ANOVA
failed to reveal a significant effect of caffeine treatment on
the number of nose pokes in groups. However, there was a
significant influence of caffeine dose on the level of the r
criterion in the extinction experiment (F(3,26) 5 7.04; p 5
.001; n 5 8 animals per group) with a significant increase of
the r criterion (p , .01, compared with the saline-injected
group) in the group injected with caffeine (3 mg/kg). Paired
comparison revealed a significant difference between r criteria in the initiation versus the extinction experiment in the
groups injected with saline (p 5 .002) or with caffeine at the
doses of 10 mg/kg (p 5 .0002) and 30 mg/kg (p 5 .002; Fig. 1,
bottom). There was no difference between the r criteria in
initiation and extinction sessions in the group treated with
caffeine at the dose of 3 mg/kg. Thus, it was concluded that
i.p. administration of caffeine at a dose of 3 mg/kg maintained the cocaine-oriented pattern of behavior in mice in the
absence of contingent, reinforcing cocaine infusions, and prevented the extinction of cocaine-oriented behavior, whereas
higher doses of caffeine lacked this effect. To find the reason
for these effects, paired comparison of the nose pokes in
particular groups was performed. Among the active mice
trained to self-administer cocaine, those injected with caffeine (3 mg/kg) exhibited more nose-poke responses than did
those injected with saline (p 5 .05; Fig. 1). No such difference
was seen in active mice trained for saline self-administration
or in either of the yoked control groups. The highest dose of
caffeine (30 mg/kg) increased responding in both active mice
and yoked controls, but the effect did not reach statistical
significance (p 5 .08). Thus, caffeine at the low dose (3 mg/kg)
tended to increase specifically nose-poke responses of the
active animals that had learned to self-administer cocaine on
day 1.
Effects of the Nonselective Adenosine-Receptor
Antagonist CGS 15943 in the Extinction Phase. First,
we examined whether the effect of caffeine could be reproduced by CGS 15943, a structurally unrelated antagonist, at
adenosine A1 and A2A receptors. CGS 15943 is approximately
1000-fold more potent than caffeine at both adenosine A1 and
A2A receptors (see Ongini and Fredholm, 1996). However, it
is much less water-soluble and, hence, its apparent volume of
distribution is much higher. For this reason, it must be given
in vivo in doses close to those of caffeine. As seen in Fig. 2, in
none of the CGS 15943-treated groups (6 animals per group)
was the r criterion different from that obtained in the initiation experiments, whereas such a difference was observed
in the vehicle-treated group. Although ANOVA failed to show
a significant influence of the drug on the nose-poke activity in
the extinction experiment, we tested whether the increase in
the r criterion with the highest dose of CGS 15943 was
because of an increase in nose-poke responding in active mice
or because of a decrease in activity in yoked control mice. The
former was the case (p , .05; active and yoked control groups
were compared with Student’s t test). Thus, we concluded
that CGS 15943 tends to prevent the extinction of the cocaine-oriented behavior.
Effects of the Adenosine A2A-Receptor Selective
Antagonist SCH 58261 in the Extinction Phase. Next,
we examined the effect of a highly selective adenosine A2A-
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1999
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crease of the level of the r criterion at the dose of 5.0 mg/kg
(p , .05, comparison with vehicle level). Paired comparison
revealed a significant difference between the r criteria in the
initiation experiment versus the extinction experiment in the
groups injected with vehicle (p 5 .03) or with DPCPX at the
doses of 1.25 mg/kg (p 5 .005) and 2.5 mg/kg (p 5 .02). There
was no difference between r criteria in initiation and extinction sessions in the group treated with DPCPX at a dose of
5.0 mg/kg. Thus, it could be concluded that i.p. administration of DPCPX at the dose of 5.0 mg/kg prevented the extinction of the cocaine-oriented behavior.
Next, we examined the effects of 8-CPT (Fig. 3). 8-CPT is
some 10-fold less potent than DPCPX on both A1 and A2A
receptors (see Daly, 1993). It also is more water soluble, and
therefore was given in 4-times higher doses than DPCPX.
There was a significant dose-dependent influence of 8-CPT
on nose-poke responding in mice (F(3.44) 5 3.37; p , .05).
Among active mice trained to self-administer cocaine, those
injected with 8-CPT at 5 and 10 mg/kg showed a tendency
(p , .1) to increased nose-poke responding compared with
those injected with vehicle. Paired comparison of the levels of
r criteria in initiation versus the extinction experiments revealed that in groups treated with 8-CPT at 5 and 20 mg/kg,
r criteria in the extinction experiments differed significantly
from those in the initiation experiments (p 5 .008 and p 5
.03, respectively), whereas in the group injected with 10
mg/kg 8-CPT, there was no difference between r criteria in
initiation and in the extinction experiments. Thus, 8-CPT
tends to prevent the extinction of the cocaine-oriented behavior.
Discussion
Fig. 3. Influence of DPCPX and 8-CPT on the extinction of cocaineseeking behavior in mice. Top, nose-poke responses (mean 6 S.E.M.; n 5
6). Dark bar and filled circles, active mice. Light bar and open circles,
yoked control mice. Doses of DPCPX (left) and 8-CPT (right) are given in
mg/kg. *p , .05; Student’s t test, comparison between active and yoked
control mice within the group of animals injected with drug during day 2
of the experiment. Bottom, r criteria in animals injected with DPCPX or
8-CPT on day 2 (mean 6 S.E.M.; n 5 6). The mean 6 S.E.M. of the r
criterion in the acquisition experiment is presented as a crossed field.
Zp , .05; Student’s t test, comparison between the ratio on day 2 in the
drug- versus saline-treated groups of pairs of mice. #p , .05; Student’s t
test, comparison between the r criterion obtained in the extinction experiment (day 2) and r criterion obtained in the acquisition experiment
(day 1).
This study shows that the initiation and the extinction of
cocaine-related behavior can be studied not only in rats, but
also in mice. A single, 30-min session in mice established
self-administration of cocaine by means of directed nose
pokes. This behavior was rapidly extinguished in mice that
received only saline/vehicle injections during testing on a
subsequent day. However, an i.p. injection of cocaine at 5.0
mg/kg, a dose known to exert a reinforcing effect in a conditioned place-preference paradigm in mice (Kuzmin et al.,
1997), was able to stabilize the higher level of nose-poke
activity in active animals compared with the yoked control
animals even when withdrawn from the contingent cocaine
infusions. The ability of cocaine to effectively maintain cocaine-oriented behavior in the absence of response-contingent injections of cocaine was not surprising because others
have shown that noncontingent administration of the selfadministered drug produces potent reinstatement of extinguished drug-taking behavior (Gerber and Stretch, 1975; de
Wit and Stewart, 1981; Slikker et al., 1984; Self et al., 1996).
Conversely, the highest dose of cocaine used in the present
study (20 mg/kg), a dose that does not induce conditioned
place preference in mice (Kuzmin et al., 1997), lacked a
priming effect during the extinction session. Thus, it can be
speculated that only reinforcing doses of the drugs are able to
prevent an extinction of drug-related behavior.
In our experiments, caffeine at the dose of 3 mg/kg i.p. also
effectively prevented the extinction of a cocaine-induced operant pattern. This finding confirms and extends previous
data showing the ability of caffeine to reinstate cocaine-
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experiments. Thus, at none of the doses used did the selective
adenosine A2A-receptor antagonist SCH 58261 prevent the
extinction of cocaine-associated behavior.
Effects of the Adenosine A1 Receptor Selective
Antagonists DPCPX and 8-CPT in the Extinction
Phase. DPCPX is slightly (about 3-fold) more potent than
CGS 15943 on adenosine A1 receptors, but almost 1000-fold
less potent than CGS 15943 at A2A receptors (see Ongini and
Fredholm, 1996). Because the physicochemical properties of
the two compounds are similar, the doses used were also
similar. The results of experiments with DPCPX are presented in Fig. 3. ANOVA revealed significant differences
between nose-poke responses in active and yoked control
animals after treatment with DPCPX (F(1,40) 5 5.68; p 5 .02;
6 animals per group), although the effect was not dependent
on the dose of DPCPX administered. Subsequent paired comparison showed that at 5.0 mg/kg DPCPX, there was a significant increase in the active group of mice (p 5 .04, comparison with the vehicle-treated group; p 5 .005, comparison
with the corresponding yoked control group).
ANOVA also revealed a significant influence of DPCPX
treatment on the level of the r criteria in the extinction
experiments (F(3,20) 5 4.09; p 5 .02) with a significant in-
Adenosine and Cocaine-Seeking Behavior
540
Kuzmin et al.
bens nucleus (Parkinson and Fredholm, 1990; Johansson et
al., 1997), where they can modulate striatal dopaminergic
neurotransmission. Furthermore, there are antagonistic
adenosine A2A-dopamine D2 receptor interactions: activation
of adenosine A2A receptors decreases both the affinity of
dopamine D2 receptors and the signal transduction from
dopamine D2 receptors to the guanine nucleotide-binding
protein (see Ferré et al., 1997). The finding that a dopamine
D2 agonist, but not a dopamine D1 agonist, reinstated cocaine-seeking behavior in rats (Self et al., 1996) raised the
possibility that A2A-receptor antagonism in this terminal
region of the mesolimbic dopamine system underlies the reinstatement effects produced by caffeine. Therefore, it was
surprising that in our study, the selective adenosine A2A
antagonist could not exhibit the same behavioral effect as a
low dose of caffeine in the extinction experiment despite the
fact that this dose of the antagonist can mimic the stimulatory effect of caffeine on motor behavior in rats (Bertolli et al.,
1996; Pinna et al., 1996). In fact, the A2A-receptor antagonist
caused a dose-dependent decrease in nose-poke responses in
both active and yoked control mice and actually decreased
the difference between them. In contrast, the drugs that
inhibit adenosine A1 receptors (i.e., DPCPX, CGS 15943, and,
to a lesser extent, certain doses of 8-CPT) did prevent, or
tended to prevent, the extinction of cocaine-induced behavior
and maintained cocaine-associated responses in active mice
on a higher level than the responses of the yoked control
mice.
A critically important question is whether the present
results are confounded by behavioral activation unrelated
to cocaine seeking. It can be argued that a nonspecific
motor stimulation might enhance any drug-related activity. This can only be partially controlled for by yoked
controls for the initiation of such behaviors, and it would
be particularly important if drug-induced activity persists
at the time of the experiment. Unpublished experiments
with the present method show that drug-related behavior
is lost 10 to 20 min after the beginning of the extinction
session and that the level of activity remaining is not
different from that observed during a second or third extinction session (48 or 72 h after initiation; A.K., E.E.Z.,
unpublished data). Thus, the present method is, in this
respect, not significantly different from other methods
used to study reinstatement. Of course, it also could be
argued that a motor stimulant would be able to induce an
activity that the animal has learned to perform previously,
even if that activity is not overt under basal conditions.
This is true, and we have not been able to control for this.
However, the same problem occurs in every method for the
study of reinstatement. There is, finally, one important
finding from a previous study from our laboratory that
provides strong evidence against nonspecific motor stimulation being the explanation for the major finding in our
study. Svenningsson and coworkers (Svenningson et al.,
1997b) showed that DPCPX, the adenosine A1 antagonist
that enhanced cocaine-seeking behavior in the present
study, is not a motor stimulant, but actually tended to
reduce locomotor activity. Conversely, SCH 58261, which
did not enhance cocaine-seeking behavior, acted as a motor
stimulant in that study. For all of these reasons, we feel
that nonspecific motor activation cannot explain the ef-
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 12, 2017
seeking behavior in rats (Worley et al., 1994; Schenk et al.,
1996). However, Self and coworkers (Self et al., 1996) found
that caffeine at the dose we used for this study (3 mg/kg)
lacked appreciable priming ability, whereas caffeine at 10
mg/kg tended to enhance cocaine priming. A probable explanation is that in our mouse strain, the caffeine dose-response
curve is slightly shifted to the left compared with the rat
strain used by Self and coworkers (Self et al., 1996), but there
also are differences in the experimental procedure. Whereas
in our study there was a 24-h delay after the cocaine selfadministration session, in the cited study, the extinction and
the effects of priming caffeine injections were studied 2 to 4 h
after the cocaine self-administration session.
Whereas the lower dose (3 mg/kg) of caffeine had a clearcut priming effect, a high dose (30 mg/kg) of caffeine induced
an increase in nose poking in both active and yoked control
mice, but this activation was not selective and lacks priming
effect, according to the r criteria. Because caffeine has a
biphasic reinforcing/aversive activity depending on the dose
used (Brockwell et al., 1991), the dose of 30 mg/kg in mice
might have some aversive properties. In fact, our preliminary
experiments with the aid of the conditioned place-preference
technique also showed that whereas caffeine at the dose of 3
mg/kg produced conditioned place-preference in mice, the
dose of 30 mg/kg produced a strong place-aversion reaction
(A.K., unpublished observation).
The ability of a low dose of caffeine to prevent the extinction of a cocaine-related behavior cannot be explained simply
by its psychomotor stimulant properties (Schenk et al., 1989)
because an increased responding was observed only in active
animals and not in the yoked control mice. Thus, the effect of
caffeine is related to an association of the operant responses
with cocaine infusions on day 1 of the experiment. Moreover,
it has been shown previously that the motor-activating effect
of caffeine is not correlated with its ability to reinstate cocaine-associated responding (Schenk et al., 1989), and that
with repeated administration, the effectiveness of a dose of
caffeine in increasing motor activity remains unchanged,
whereas its ability to reinstate cocaine-associated responding
decreases (Schenk et al., 1996).
It is important to note that the nose-poke activity does
not mirror the general motor activity in mice. In fact,
administration of high doses of cocaine (Kuzmin et al.,
1997) that produce definite stimulatory effect usually resulted in a decrease in the number of nose pokes in the
technique used. This gives a unique opportunity to study
selectively the reinforcing effects of drugs because the
increase in nose-poke activity was found only in mice that
actively self-administer cocaine and not in animals from
the yoked control group.
It is possible that some component of the effects of caffeine
can modify cocaine-sensitive systems, possibly by the activation of the central dopaminergic systems (Ferré et al., 1991;
Garrett and Holtzman, 1994). As mentioned, it is generally
believed that a major primary neurochemical effect of caffeine is the blockade of adenosine receptors (Daly, 1993;
Fredholm, 1995), and this effect has been demonstrated as
critical for the acute stimulant effects of caffeine (Kaplan et
al., 1989; Holtzman, 1991). It also has been shown that
activation of adenosine receptors can antagonize the effects
of dopamine at both D1 and D2 receptors (see Ferré et al.,
1997). There is a high density of A2A receptors in the accum-
Vol. 290
1999
Acknowledgment
We thank Dr. Ennio Ongini at the Schering-Plough pharmaceutical company in Milan for the gift of SCH 58261.
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Adenosine A1 receptors have a widespread distribution in
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present in the nucleus accumbens, where they selectively
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(Ferré et al., 1996). In addition, adenosine A1 receptors are
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might increase the release of endogenous dopamine (Stoner
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findings have shown that reinstatement of cocaine-associated behavior by cocaine priming does not correlate with the
dopaminergic neurotransmission in the accumbens nucleus
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al., 1996), although D1 agonists were shown to maintain
self-administration behavior in rats (Weed et al., 1997).
Thus, the schemes for explaining the actions of caffeine as
well as selective adenosine-receptor antagonists in terms of
interactions with known mechanisms involving dopamine
receptors, schemes that work very well in explaining the
actions of these compounds on motor behavior (see Ferré et
al., 1997), break down when trying to explain the effects of
these drugs in the extinction model of cocaine-seeking behavior. Consequently, the mechanism of the maintenance of
drug-seeking behavior also must incorporate other neurotransmitter systems and probably other neuronal circuits
(Koob and Le Moal, 1997). It also might be speculated that
the receptors involved in reinforcement (D1 for cocaine and,
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involved in the processes of extinction and reinstatement (D2
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can be successfully used to study the mechanisms of the
extinction of drug-seeking behavior during withdrawal from
the response-contingent reinforcement. Given the increasing
availability of genetically modified mouse strains, this type of
experimental model might prove valuable in future studies of
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effect on the extinction of cocaine-seeking behavior when
using both the self-administered drug (cocaine) and a drug
from a completely different drug class (caffeine). Finally, low,
behaviorally stimulant doses of caffeine can prevent the extinction of cocaine-seeking behavior, possibly via the blockade of A1 adenosine receptors.
Adenosine and Cocaine-Seeking Behavior
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Vol. 290
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Send reprint requests to: Dr. Bertil B. Fredholm, Section of Molecular
Neuropharmacology, Department of Physiology and Pharmacology, Karolinska Institute, S-171 77 Stockholm, Sweden. E-mail: Bertil.Fredholm@
fyfa.ki.se
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