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BEHAVIORAL PHARMACOLOGY
Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland (Z.J., S.F., P.N.S., M.S., L.A.P., J.L.H., P.M., S.R.G.); Department of Pharmacology, Medical School, University of Athens, Goudi Athens, Greece (K.A.); and Pharmaceutical Chemistry, Pharmaceutical Institute, University of Bonn, Bonn, Germany (J.H.)
Received July 8, 2003; accepted August 28, 2003.
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Abstract |
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). Increased dopaminergic neurotransmission appears to be a main mechanism mediating the discriminative-stimulus effects of both methamphetamine (Sasaki et al., 1995; Tidey and Bergman, 1998; Munzar and Goldberg, 2000) and cocaine (Baker et al., 1993; Caine et al., 2000; Howell et al., 2000), although there is evidence for the involvement of nondopaminergic systems (e.g., Kuczenski et al., 1995; Munzar and Goldberg, 1999; Mori et al., 2002; Munzar et al., 2002b). Although methamphetamine and cocaine have similar behavioral effects and similar effects on dopaminergic neurotransmission, their neurochemical mechanisms of action are different. Methamphetamine facilitates the release of dopamine, norepinephrine, and serotonin from nerve terminals (e.g., Kuczenski et al., 1995), whereas cocaine binds to monoamine transporters and inhibits synaptic dopamine, norepinephrine, and serotonin reuptake (for a review, see Uhl et al., 2002). Furthermore, there is evidence suggesting that cocaine, but not d-amphetamine, increases cerebral extracellular levels of adenosine (Herrera-Marschitz et al., 1994; Fiorillo and Williams, 2000).
Antagonistic and reciprocal interactions are known to exist between different subtypes of adenosine and dopamine receptors in the brain (Ferré et al., 1997). Adenosine A1 receptors are colocalized with dopamine D1 receptors, and adenosine A2A receptors are colocalized with dopamine D2 receptors and form functional heteromeric complexes (Gines et al., 2000; Hillion et al., 2002). In general, stimulation of adenosine receptors counteracts the behavioral effects of dopamine receptor stimulation (Ferré et al., 1997). Accordingly, adenosine receptor agonists counteract, whereas adenosine receptor antagonists potentiate, the pharmacological effects of psychostimulants like cocaine and amphetamines (Heffner et al., 1989; Popoli et al., 1994; Rimondini et al., 1997; Poleszak and Malec, 2000, 2002; Shimazoe et al., 2000; Knapp et al., 2001). Therefore, the discriminative-stimulus effects of cocaine and the analog of d-amphetamine methamphetamine should be altered by agents acting at adenosine receptors. In a recent report, we suggested that A2A receptors play a more important role than A1 receptors in adenosine-mediated modulation of the discriminative-stimulus effects of methamphetamine (Munzar et al., 2002a). However, this interpretation was based on results obtained with the A1 receptor antagonist DPCPX and the two nonselective A2A antagonists, caffeine and 3,7-dimethyl-1-propargylxanthine. Moreover, the role of A1 and A2A adenosine receptors in the discriminative-stimulus effects of cocaine is still unknown.
The aim of the present study was to compare involvement of adenosinergic receptors in the discriminative-stimulus effects of cocaine and methamphetamine. We characterized the role of A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of psychostimulants by studying the ability of selective adenosine A1 and A2A receptor agonists [N6-cyclopentyladenosine (CPA) and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680), respectively] and adenosine A1 and A2A receptor antagonists [8-cyclopentyl-1,3-dimethylxanthine (CPT) and 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthin phosphate disodium salt (MSX-3), respectively] to mimic or modulate the discriminative-stimulus effects of both methamphetamine and cocaine in rats.
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Materials and Methods |
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Apparatus. Twelve standard operant-conditioning chambers (Coulbourn Instruments, Allentown, PA) were used. Each chamber contained a white house light and two levers, separated by a recessed tray into which a pellet dispenser could deliver 45-mg food pellets (F0021; Bioserv, Frenchtown, NJ). Each press of a lever with force of 0.4 N through 1 mm was recorded as a response and was accompanied by an audible click. The operant-conditioning chambers were controlled by microcomputers using the MED Associates MED-PC software package (MED Associates, St. Albans, VT).
Drug-Discrimination Procedure. Rats were trained as described previously (Yasar et al., 1994; Munzar and Goldberg, 1999) under a discrete-trial schedule of food-pellet delivery to respond on one lever after an injection of a training dose of 1 mg/kg methamphetamine (n = 18) or 10 mg/kg cocaine (n = 12) and on the other lever after an injection of 1 ml/kg saline vehicle. Injections of methamphetamine, cocaine, or saline were given intraperitoneally 15 min before the start of the session. At the start of the session, a white house light was turned on, and in its presence, the rats were required to make 10 consecutive responses (fixed-ratio 10 schedule of food delivery) on the lever appropriate to the presession treatment. The completion of 10 consecutive responses on the correct lever produced delivery of a 45-mg food pellet and initiated a 45-s timeout during which lever-press responses had no programmed consequences and the chamber was dark. Responses on the incorrect lever had no programmed consequences other than to reset the fixed-ratio requirement on the correct lever. After each timeout, the white house light was again turned on, and the next trial began. Each session ended after completion of 20 fixed-ratio trials or after 30 min elapsed, whichever occurred first. Discrimination-training sessions were conducted 5 days per week under a double alternation schedule (i.e., DDSSDDSS etc., where D = drug and S = saline). Training continued until there were eight consecutive sessions during which rats completed at least 90% of their responses during the session on the correct lever and no more than four responses occurred on the incorrect lever during the first trial. Test sessions with other doses and other drugs were then initiated.
During test sessions, a range of doses of different adenosinergic compounds were substituted for the training dose of methamphetamine or cocaine. Adenosine receptor agonists were also administered together with training doses of these psychostimulants to assess possible alteration of their discriminative-stimulus effects. Subsequently, the effects of selected doses of adenosinergic compounds on the methamphetamine and cocaine dose-response curves were established. Methamphetamine and cocaine dose-response curves were first determined after the discrimination was acquired before testing other drugs and were redetermined three times in each subject. The last determination was performed after completion of testing of all pretreatments on both dose-response curves. Test sessions were identical to training sessions, with the exception that 10 consecutive responses on either one of the two levers ended the trial. Switching responding from one lever to the other lever reset the ratio requirement. In a test phase, a single alternation schedule was introduced, and test sessions were usually conducted on Tuesdays and Fridays. Thus, a 2-week sequence starting on Monday was: DTSDTSTDST (T = test). In this way, test sessions occurred with equal probability after saline and drug sessions. Test sessions were conducted only if the criterion of 90% accuracy and not more than four incorrect responses during the first trial was maintained in the two preceding training sessions.
Drugs. Methamphetamine, adenosine A1 receptor agonist CPA, adenosine A1 receptor antagonist CPT, and adenosine A2A receptor agonist CGS 21680 were purchased from Sigma-Aldrich (St. Louis, MO). Adenosine A2A receptor antagonist MSX-3 was synthesized at the Pharmaceutical Institute, University of Bonn, Germany. (–)-Cocaine HCl was obtained from the National Institute on Drug Abuse, National Institutes of Health (Rockville, MD).
Doses of methamphetamine, cocaine, MSX-3, and CGS 21680 refer to the weight of the salt, whereas doses of CPT and CPA refer to the weight of the drug. One milligram of the salt form of MSX-3 is equivalent to 0.74 mg of base, and 1 mg of CGS 21680 is equivalent to 0.93 mg of base. All drugs were dissolved in saline (0.9% NaCl) with a minimal amount of 1 N NaOH for MSX-3 and CPT (final pH 7.4) and sonicated or slightly heated (CGS 21680) if needed. Most drugs were injected in a volume of 1.0 ml/kg. The highest tested doses of CPT (13.3 and 23.7 mg/kg) and MSX-3 (30 and 56 mg/kg) were injected in a volume of 2 or 3 ml/kg due to solubility constraints. All drugs were administered intraperitoneally.
A range of doses of each drug was tested in the drug-discrimination study, and dose was increased until there was either complete substitution for the methamphetamine- or cocaine-training stimulus or until the test drug produced a significant decrease in response rates. Effects of adenosine antagonists alone were tested first followed by testing of the effects of selected doses of each antagonist (and their vehicle) on the methamphetamine and cocaine dose-response curves. After completing studies with adenosinergic antagonists, the adenosinergic agonists were tested alone and then in combination with the training dose of methamphetamine or cocaine. Finally, selected doses of each agonist were then tested in combination with different doses of methamphetamine or cocaine to assess possible alterations in dose-response curves (combination tests). Not all of the compounds were tested in all subjects. Generally, CPA and CPT or CGS 21680 and MSX-3 were tested in the same subjects.
Injections of methamphetamine, cocaine, or saline were given 15 min before the start of the session. Due to their quick initial effect, a short time interval (from 0–15 min) is usually used to study the ability of systemically administered CPA, CGS 21680, CPT, or MSX-3 to modulate the effects of psychostimulants or other central-acting substances (Rimondini et al., 1997; Poleszak and Malec, 2000, 2002; Karcz-Kubicha et al., 2003). In generalization tests, the adenosine agonists and antagonists were administered 10 min before the session. In combination tests, all tested compounds were administered 10 min before methamphetamine or cocaine (i.e., 25 min before the session). Immediately preceding all combination tests, the selected dose of each of the adenosinergic compounds and also its vehicle was administered 10 min before saline (i.e., 25 min before the session) to obtain proper controls for assessment of the interactions during combination tests (additive versus superadditive effects; see Figs. 2, 3, 5, and 6).
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Data Analysis. Discriminative-stimulus data were expressed as the percentage of the total responses on both levers that were made on the methamphetamine- or cocaine-appropriate lever. Complete substitution for the methamphetamine training dose was defined as 90% or more of responses on the methamphetamine-appropriate lever, whereas no generalization was defined as less than 20% of responses on the methamphetamine- or cocaine-appropriate lever. Response-rate data were expressed as responses per second averaged over the session, with responding during timeout periods not included in calculations. The data from sessions during which rats did not complete at least one fixed ratio were excluded from analysis of drug-lever selection. All results are presented as group means ± S.E. values.
Statistical analysis of generalization and pretreatment tests was done by using one-way ANOVA for repeated measures. Significant main effects were analyzed further by subsequent paired comparisons with vehicle control (responding after vehicle injections in substitution tests or after vehicle pretreatment in tests with training stimuli) using post hoc Dunnett's test. ED50 values and 95% confidence intervals (CIs) for methamphetamine and cocaine dose-response curves after different pretreatments were calculated by linear regression using three or four points on the ascending portions of the dose-response curves. The A50 values and 95% confidence intervals were calculated for each antagonist by fitting the data to a sigmoidal dose-response equation using Prism3 software (GraphPad Software Inc., San Diego, CA).
As dose-response curves were not always parallel, dose-response curves were further evaluated by using two-way ANOVA for repeated measures. Shifts in dose-response curves were considered significant only if 95% CIs did not overlap and if two-way ANOVA for repeated measures revealed significant difference (p < 0.05). To evaluate whether the drug combinations produced simple additive or superadditive effects, theoretically additive values were individually calculated for each rat (as described in our previous reports, Munzar et al., 2002a,b) and compared with experimental values actually obtained. Theoretically additive values were calculated by adding the effect of each pretreatment drug when administered alone to the effects of each dose of methamphetamine or cocaine when administered together with the vehicle. Since 100% was the maximal achievable value, all sums greater than 100% were adjusted to this value. Changes were considered significant when p < 0.05. The SigmaStat program (SPSS Inc., Chicago, IL) was used.
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, 2000) or 10.0 mg/kg training dose of cocaine (Munzar et al., 2002b). When the dose of methamphetamine was varied, there was a dose-dependent increase in drug-lever selection with maximal selection (99.91%) at the 1.0 mg/kg training dose of methamphetamine (one-way ANOVA for repeated measures; F5,75 = 101.70, p < 0.001; Fig. 1, left upper panel). Similarly, when the dose of cocaine was varied, there was a dose-dependent increase in drug-lever selection with maximal selection (99.79%) at the 10.0 mg/kg training dose of cocaine (F5,55 = 64.43, p < 0.001; Fig. 1, right upper panel). The methamphetamine and cocaine dose-response curves remained stable throughout the study. The ED50 values for dose-response curves calculated after stable discrimination performance was acquired and before the testing of other drugs (first test) were similar to the ED50 values recalculated after testing of all pretreatments (last test), as revealed by overlapping 95% CIs (Table 1).
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Generalization Tests with Adenosine Receptor Antagonists. Figure 1 shows the percentage of responses made on the drug lever and overall rates of responding obtained during sessions when different doses of two adenosine antagonists were tested for their ability to substitute for the training dose 1 mg/kg of methamphetamine or 10 mg/kg of cocaine. The selective A1 receptor antagonist CPT produced a partial, but statistically significant, substitution for both the methamphetamine-(F6,66 = 28.26, p < 0.001) and cocaine-(F6,49 = 13.68, p < 0.001) training stimuli at doses of 7.5, 13.3, and 23.7 mg/kg. A50 values and 95% CIs for substitution for the methamphetamine- and cocaine-training stimuli were 7.38 (6.11–8.908 CI) mg/kg and 6.25 (3.11–12.58 CI) mg/kg, respectively. The level of drug-lever selection at the 13.3 mg/kg dose of CPT was about 65% in methamphetamine-trained rats and 75% in cocaine-trained rats. Increasing the dose of CPT to 23.7 mg/kg did not produce further increases in drug-lever selection in cocaine-trained animals and increased drug-lever selection by only about 10% in methamphetamine-trained rats, but it significantly decreased rates of responding to very low levels (one-way ANOVA for repeated measures; methamphetamine-trained rats: F6,72 = 16.07, p < 0.001; cocaine-trained rats: F6,54 = 20.77, p < 0.001), and 50% of the rats in each group failed to complete at least one fixed ratio (Fig. 1, bottom panels).
The selective A2A receptor antagonist MSX-3 also produced partial substitution for both the methamphetamine- and cocaine-training stimuli, although with lower potency than CPT. A50 values and 95% CIs for substitution for the methamphetamine- and cocaine-training stimuli were 14.50 (5.47–38.45) mg/kg and 9.98 (4.98–24.93) mg/kg, respectively. In methamphetamine-trained rats, MSX-3 produced significant substitution (F5,37 = 15.27, p < 0.001) for the methamphetamine-training stimulus only after high doses (30.0 and 56.0 mg/kg; Fig. 1, left panels). The 56.0 mg/kg dose of MSX-3 significantly decreased rates of responding (F5,37 = 6.38, p < 0.001). In cocaine-trained rats, MSX-3 produced partial substitution for the cocaine-training stimulus at doses of 10.0 and 30.0 mg/kg (F4,28 = 10.27, p < 0.001), with 30.0 mg/kg of MSX-3 significantly decreasing rates of responding (F4,28 = 7.77, p < 0.001; Fig. 1, right panels). We were not able to test a higher dose of 56.0 mg/kg MSX-3 in this group due to limited availability of the substance. There is a possibility that MSX-3 would substitute completely for the cocaine-training stimulus at a dose of 56 mg/kg, since the level of cocaine-appropriate responding at the 30 mg/kg dose was 70% (compared with 60% in the methamphetamine group). However, increasing the dose of MSX-3 in the methamphetamine group of rats only increased the level of the substitution for the methamphetamine stimulus by 5%, so we would not expect a significant increase in generalization in the cocaine group either.
Effects of Pretreatments with Antagonists on Methamphetamine and Cocaine Dose-Response Curves. Figures 2 and 3 show effects of the A1 receptor antagonist CPT and the A2A receptor antagonist MSX-3 on methamphetamine (left panels) and cocaine (right panels) dose-response curves. ED50 values for drug-lever selection with 95% CIs are presented in Table 1. A 4.2-mg/kg dose of CPT, which did not produce significant substitution for the methamphetamine- or cocaine-training stimuli and did not significantly change response rates when given alone, produced shifts to the left of both the methamphetamine and cocaine dose-response curves (Fig. 2). These leftward shifts were significant as indicated by nonoverlapping 95% CIs of ED50 values for vehicle and CPT pretreatments (Table 1) as well as by two-way ANOVA for repeated measures (methamphetamine: F1,40 = 23.86, p < 0.001; cocaine: F1,24 = 0.34, p < 0.001). The effect of CPT-methamphetamine and CPT-cocaine combinations were superadditive, since there was a statistically significant difference between the curves actually obtained and the calculated additive curves (methamphetamine: F1,40 = 17.68, p = 0.002; cocaine: F1,32 = 44.63, p < 0.001).
Pretreatment with 10.0 mg/kg MSX-3 (dose chosen under the same criterion as dose of CPT) shifted the methamphetamine dose-response curve markedly to the left (Fig. 3), as revealed by both nonoverlapping 95% CIs of ED50 values (Table 1) and two-way ANOVA for repeated measures (F1,24 = 17.80, p < 0.001). The cocaine dose-response curve was also significantly shifted to the left by doses of 3.0 and 10.0 mg/kg of MSX-3 (Fig. 3), as revealed by nonoverlapping 95% CIs of ED50 values (Table 1) and two-way ANOVA for repeated measures (3.0 mg/kg: F1,14 = 51.86, p < 0.001; 10 mg/kg: F1,14 = 145.85, p < 0.001). The effect of MSX-3 (10 mg/kg)-methamphetamine and MSX-3 (10 mg/kg)-cocaine combinations were clearly superadditive, since there was a statistically significant difference between the values actually obtained and the calculated additive values (methamphetamine: F1,24 = 15.56, p = 0.004; cocaine: F1,14 = 44.55, p < 0.001). Response rates after administration of a range of doses of both psychostimulants were markedly increased in combinations with 4.2 mg/kg of CPT (methamphetamine: F1,40 = 17.12, p = 0.002; cocaine: F1,32 = 7.44, p = 0.026) or with 10 mg/kg of MSX-3, although the effect in this case was statistically significant only in the cocaine-trained group (F1,14 = 7.01, p = 0.033).
Generalization and Pretreatment Tests with Adenosine Receptor Agonists. Figure 4 shows the percentage of responses made on the drug lever and overall rates of responding when a range of doses of two selective adenosine receptor agonists were administered alone or together with the 1.0 mg/kg training dose of methamphetamine (left panels) or the 10.0 mg/kg training dose of cocaine (right panels). When the selective A1 receptor agonist CPA was administered alone, no methamphetamine-lever selection was observed, even at doses of 0.03 and 0.1 mg/kg CPA, which significantly decreased response rates (3 of 7 rats did not complete a single fixed ratio; F3,18 = 15.82, p < 0.001). The high 0.1 mg/kg dose of CPA also did not produce significant substitution for the cocaine-training stimulus but markedly and significantly decreased response rates (2 of 7 rats did not complete a single fixed ratio; F3,18 = 25.56, p < 0.001).
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The selective A2A receptor agonist CGS 21680 did not produce substitution for the methamphetamine-training stimulus, even at the highest dose tested, 0.177 mg/kg, which markedly decreased response rates (2 of 6 rats did not complete a single fixed ratio; F3,15 = 6.12, p = 0.006). However, CGS 21680 produced partial, but statistically significant (F3,14 = 4.31, p = 0.024), substitution for the cocaine-training stimulus at doses of 0.1 and 0.177 mg/kg, which, however, produced significant decrease of response rates (F3,15 = 10.91, p < 0.001).
Neither CPA (0.01–0.1 mg/kg) nor CGS 21680 (0.03–0.177 mg/kg) significantly attenuated the discriminative-stimulus effects of training doses of either methamphetamine or cocaine (Fig. 4, upper panels). However, doses of CPA 0.018 to 0.056 mg/kg produced small, but not significant, reductions in drug-lever selection (in 2 of the 7 cocaine-trained rats) when administered with the training dose of cocaine.
Effects of Pretreatments with Agonists on Methamphetamine and Cocaine Dose-Response Curves. Figures 5 and 6 show effects of the A1 receptor agonist CPA and the A2A receptor agonist CGS 21680 on methamphetamine (left panels) and cocaine (right panels) dose-response curves. ED50 values for drug-lever selection with 95% CIs are presented in Table 1.
The A1 receptor agonist CPA produced different effects in the methamphetamine-versus the cocaine-trained rats. A 0.03-mg/kg dose of CPA, which did not produce substitution for the methamphetamine- or cocaine-training stimuli but produced a slight reduction in drug-lever selection when administered with the training doses of both methamphetamine and cocaine, did not produce any shift of the methamphetamine dose-response curve in the methamphetamine-trained rats (Fig. 5; overlapping 95% CIs of the ED50 values are shown in Table 1). In cocaine-trained rats, however, pretreatment with 0.03 mg/kg CPA shifted the cocaine dose-response curve markedly to the left (Fig. 5), and the shift was significant according to nonoverlapping 95% CIs of the ED50 values (Table 1) and two-way ANOVA for repeated measures (F1,18 = 15.045, p = 0.008). The effect of the CPA-cocaine combination was clearly superadditive, since there was a statistically significant difference between values actually obtained and the calculated additive values (F1,18 = 17.11, p = 0.006).
The A2A agonist CGS 21680 also produced different effects in the methamphetamine- and cocaine-trained rats. A 0.056-mg/kg dose of CGS 21680, which did not produce significant substitution for the methamphetamine- or cocaine-training stimuli and did not significantly change response rates when given alone, did not produce any shift of the methamphetamine dose-response curve in methamphetamine-trained rats (Fig. 6; overlapping 95% CIs of the ED50 values are shown in Table 1). However, pretreatment with 0.056 mg/kg CGS 21680 shifted the cocaine dose-response curve markedly and significantly to the left (Fig. 6), as revealed by nonoverlapping 95% CIs of the ED50 values (Table 1) and two-way ANOVA for repeated measures (F1,15 = 11.86, p = 0.018). The effect of the CGS 21680-cocaine combination was superadditive, since there was a statistically significant difference between values actually obtained and calculated additive values (F1,15 = 15.82, p = 0.011).
Response rates after administration of a range of doses of both methamphetamine and cocaine were consistently decreased when the psychostimulants were administered in combination with 0.03 mg/kg of CPA or 0.056 mg/kg of CGS 21680. However, the effects reached statistical significance only in the methamphetamine-trained group (CPA-methamphetamine: F1,20 = 33.14, p = 0.002; CGS 21680-methamphetamine: F1,20 = 8.41, p = 0.034).
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). MSX-3 was developed as a phosphoric acid ester prodrug of MSX-2, with an in vitro A2A:A1 receptor selectivity of about 100, also demonstrated in rat striatal membranes (Sauer et al., 2000). Both adenosine antagonists easily penetrate into the brain after systemic administration (Baumgold et al., 1992; Muller et al., 2000), and we have recently reported that the motor-activating effects of CPT and MSX-3 in rats are related to their selective blockade of A1 and A2A receptors, respectively (Karcz-Kubicha et al., 2003). Thus, both CPT and MSX-3 produce motor activation at the same doses that selectively counteract motor depression induced by A1 and A2A receptor agonists, respectively (Karcz-Kubicha et al., 2003). At motor-activating doses, MSX-3 produced partial substitution for both the methamphetamine- and cocaine-training stimuli, and there were no significant differences between methamphetamine- and cocaine-trained rats. These results confirm our previous suggestion that A2A receptors play an important role in adenosine-mediated modulation of the discriminative stimulus effects of methamphetamine (Munzar et al., 2002a) and extend this finding to cocaine. Motor-activating doses of CPT also produced a partial substitution for both the methamphetamine- and cocaine-training stimuli, and there were no significant differences between methamphetamine- and cocaine-trained rats. The high level of partial substitution for both the methamphetamine- and cocaine-training stimuli produced by CPT implies that A1 receptors are also directly involved in the discriminative-stimulus effects of these psychostimulants.
The present results are in disagreement with our previous conclusion that A1 receptors play a less important role than A2A receptors in adenosine-mediated modulation of the discriminative-stimulus effects of methamphetamine (Munzar et al., 2002a), which was based on our previous findings that the A1 receptor antagonist DPCPX did not produce any substitution for a methamphetamine-training stimulus but shifted the methamphetamine dose-response curve to the left. Both CPT and DPCPX are widely used selective A1 receptor antagonists, with good penetration into the brain, and DPCPX has higher in vitro A1:A2A receptor selectivity than CPT (about 4-fold higher; Maemoto et al., 1997). However, a repeatedly reported differential behavioral effect of both antagonists is the lack of motor activation induced by DPCPX (reviewed in Karcz-Kubicha et al., 2003). Our present and previous studies (Munzar et al., 2002a) show the existence of significant differences in the discriminative-stimulus properties of CPT and DPCPX. Since the selective A1 receptor antagonism of both compounds seems to be clearly established, these differences could be explained by the existence of an additional nonshared mechanism of action. In fact, DPCPX has been reported to bind with very high affinity to binding sites other than A1 receptors, such as "nonstriatal atypical A2A receptors" (atypical A2A receptors; Cunha et al., 1996) and the cystic fibrosis transmembrane conductor regulator (Jacobson et al., 1995; Cohen et al., 1997). Since both atypical A2A receptors and cystic fibrosis transmembrane conductor regulator are expressed in the rat brain (Cunha et al., 1996; Johannesson et al., 1997), they could therefore be involved in the unique profile responsible for the motor and discriminative-stimulus effects of DPCPX.
The involvement of A1 and A2A receptors in the adenosine-mediated modulation of discriminative-stimulus effects of both methamphetamine and cocaine is in agreement with previous drug-discrimination studies showing involvement of both D1 and D2 receptors in the subjective effects of psychostimulants (see Introduction). The partial substitution produced by both adenosine antagonists is very likely mediated through enhancement of dopaminergic neurotransmission. It is known that at a presynaptic level, adenosine, mostly by acting on adenosine A1 receptors localized in nerve terminals, inhibits dopamine and glutamate release (Okada et al., 1996; Flagmeyer et al., 1997; Golembiowska and Zylewska, 1997). A recent study (Solinas et al., 2002) demonstrated that CPT, but not the selective A2A receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1, 5-c]pyrimidine (SCH 58261), significantly increased extracellular levels of dopamine and glutamate (approximately 100% increases in both cases) in the shell of the nucleus accumbens. At a postsynaptic level, adenosine decreases dopaminergic neurotransmission via specific antagonistic interactions between adenosine and dopamine receptors (Ferré et al., 1991, 1997), suggesting that CPT (in addition to enhancing dopamine release) and MSX-3 can counteract an endogenous adenosine tone on central adenosine receptors, thus removing negative adenosinergic tone from D1 and D2 receptors, respectively, and mimicking the effects of psychostimulants. In the present experiments, we found CPT to be more potent than MSX-3 in producing substitution for both the methamphetamine-(2x) and cocaine-training stimuli (1.5x). This is in contrast with their relative potencies as motor stimulants in the same rat strain, where MSX-3 is about twice as potent as CPT (Karcz-Kubicha et al., 2003). The greater potency of the A1 receptor antagonist at mimicking the discriminative-stimuli of methamphetamine and cocaine could be related to the ability of A1, but not A2A, receptor antagonists to increase the extracellular concentration of dopamine (e.g., Solinas et al., 2002).
In view of the clear effects of adenosine antagonists described above, it was expected that adenosine agonists would not produce stimulant-like discriminative-stimulus effects, and that they, very likely, would attenuate the discriminative-stimulus effects of psychostimulants. In agreement with this hypothesis, neither the A1 receptor agonist CPA nor the A2A receptor agonist CGS 21680 produced significant substitution for the methamphetamine-training stimulus, but they also did not produce any shifts in the methamphetamine dose-response curve. Unexpectedly, CGS 21680 produced significant partial substitution (50% of drug-lever selection) for the cocaine-training stimulus, although this occurred at doses that decreased rates of responding, making it difficult to evaluate the effects. Both CPA and CGS 21680 potentiated the discriminative-stimulus actions of cocaine, as shown by significant leftward shifts of the cocaine dose-response curve. Although substitution for the cocaine-training stimulus was only significant for the A2A receptor agonist, the common potentiating effects of both CPA and CGS 21680 on cocaine discrimination suggest that these effects could be explained by the ability of cocaine, but not methamphetamine, to increase extracellular concentrations of adenosine. In fact, repeated cocaine treatment (which is also a condition present in drug-discrimination experiments) produces an increase in extracellular adenosine tone in the ventral tegmental area (Fiorillo and Williams, 2000). On the other hand, the acute systemic administration of d-amphetamine does not significantly modify the striatal extracellular concentration of adenosine (Herrera-Marschitz et al., 1994). Nevertheless, more experiments need to be carried out to establish differential effects of methamphetamine and cocaine on the extracellular levels of adenosine in the brain.
In conclusion, the present findings obtained with selective drugs at A1 and A2A adenosine receptors provide evidence that adenosinergic mediation or modulation of the discriminative-stimulus effects of methamphetamine and cocaine involves both receptor subtypes. A1 and A2A adenosine receptors appear to play important, although differential, roles in the discriminative-stimulus effects of the studied psychostimulants. Interestingly, adenosine receptor agonists produced different discriminative-stimulus effects in cocaineversus methamphetamine-trained rats and modulated only the discriminative-stimulus effects of cocaine. Neurobiological mechanisms responsible for these effects remain to be determined.
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ABBREVIATIONS: METH, S(+)-methylamphetamine hydrochloride; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; ANOVA, analysis of variance; CI, confidence interval; CPA, N6-cyclopentyladenosine; CPT, 8-cyclopentyl-1,3-dimethylxanthine; CGS 21680, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride; MSX-3, 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthin phosphate disodium salt.
1 Current address: ALEXZA Molecular Delivery Corporation, 1001 East Meadow Circle, Palo Alto, CA 94303. 
Address correspondence to: Dr. Steven R. Goldberg, Preclinical Pharmacology Section, NIDA, NIH, 5500 Nathan Shock Drive, Baltimore, MD 21224. E-mail: sgoldber{at}intra.nida.nih.gov
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, p < 0.05, 