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Effects of CP 154,526, a CRF1 receptor antagonist, on behavioral responses to cocaine in rats
Neuropeptides Neuropeptides 39 (2005) 525–533 www.elsevier.com/locate/npep
Effects of CP 154,526, a CRF1 receptor antagonist, on behavioral responses to cocaine in rats Edmund Przegalin´ski *, Małgorzata Filip, Małgorzata Frankowska, Magdalena Zaniewska, Iwona Papla Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Sme˛tna 12, 31-343 Krako´w, Poland Received 4 April 2005; accepted 10 July 2005 Available online 2 September 2005
Abstract We examined the influence of CP 154,526, a selective antagonist of corticotropin-releasing factor (CRF)1 receptors, in the locomotor, sensitizing, discriminative stimulus and rewarding effects of cocaine, as well as on the cocaine-induced reinstatement of cocaine-seeking behavior in male Wistar rats. CP 154,526 in doses of 5, 10 and 20 mg/kg, which did not affect basal locomotor activity, dose-dependently reduced the hyperactivation evoked by cocaine. To assess the effects of CP 154,526 on the expression of cocaine sensitization, the rats were injected with either saline or cocaine (10 mg/kg) for 5 days, and were then challenged with cocaine (10 mg/kg) after pretreatment with saline or CP 154,526 on day 5 of withdrawal. The cocaine-induced hyperactivity in sensitized rats was reduced by CP 154,526 (10 and 20 mg/kg). In rats trained to discriminate cocaine (10 mg/kg) from saline, pretreatment with CP 154,526 (5–20 mg/kg) did not affect the cocaine (1.25–10 mg/kg)-induced discriminative stimulus effects. In a selfadministration model, the rats were trained to self-administer cocaine (0.5 mg/kg/infusion) in the FR 5 schedule of reinforcement. Administration of CP 154,526 (10–20 mg/kg) did not alter the rewarding effects of cocaine, assessed as the number of active-lever presses and infusions; however, following a 10-day extinction phase, CP 154,526 (5–20 mg/kg) significantly decreased in a dosedependent manner the cocaine (10 mg/kg) priming-induced reinstatement of cocaine-seeking behavior. The present study implies that CRF1 receptors control the expression of cocaine hyperactivation and sensitization as well as the cocaine-induced relapse behavior, but do not play any role in cocaine discrimination and self-administration. These findings may suggest that CRF1 receptor antagonists should be considered as possible medications in the treatment of cocaine addiction. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Cocaine; Corticotropin-releasing factor; CP 154,526; Drug discrimination; Locomotor activity; Sensitization, Self-administration; Rats
1. Introduction Corticotropin-releasing factor (CRF), a 41-amino acid peptide initially identified as a hypothalamic agent involved in the secretion of corticotropin (ACTH) from the anterior pituitary (Vale et al., 1981; Cummings et al., 1983), plays an essential role in the regulation of not only endocrine, but also autonomic and immune *
Corresponding author. Tel.: +48 12 662 3293; fax: +48 12 637 4500. E-mail address: [email protected] (E. Przegalin´ski). 0143-4179/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2005.07.002
responses to stress (De Souza, 1995). At the same time, being widely distributed throughout the brain, CRF (which exerts its biological actions via two receptor subtypes known as CRF1 (Chang et al., 1993; Perrin et al., 1993) and CRF2 (Lovenberg et al., 1995)), has also been implicated in the modulation of several behavioral effects (Shibasaki et al., 1991; Koob et al., 1994), including these produced by the psychostimulant cocaine. In fact, the cocaine-induced locomotor hyperactivity and stereotypy, as well as the reinforcing activity of the psychostimulant (assessed in place preference and self-administration models) have been found to be
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blocked by the immunoneutralization of endogenous CRF and/or by CRF receptor antagonists (Sarnyai et al., 1992; Erb et al., 1998; Shaham et al., 1998; Goeders and Guerin, 2000; Lu et al., 2001; Gurkovskaya and Goeders, 2001; Goeders and Clampitt, 2002). The above data are in line with the observations showing that acute treatment with cocaine releases endogenous CRF from not only the hypothalamic (Sarnyai et al., 2001), but also extrahypothalamic structures (Richter et al., 1995). On the other hand, no data are available on the role of CRF in other effects of cocaine, such as discriminative cue or behavioral sensitization. The former one models the subjective effects of cocaine in humans (Schuster and Johanson, 1988), while sensitization, a phenomenon characterized by an increase in – among others – behavioral responses (e.g. locomotor hyperactivity) to subsequent cocaine challenge after its repeated administration is discontinued (Robinson and Berridge, 1993; Kalivas et al., 1998), plays an essential role in the psychostimulant-induced psychoses or craving for drugs of abuse in humans (Segal et al., 1981; Robinson and Berridge, 1993; Kalivas et al., 1998). In the present study we examined the effect of the non-peptide CRF1 receptor antagonist CP 154,526 (Seymour et al., 2003) on the discriminative stimulus effect of cocaine and on the expression phase of sensitization to the cocaine-induced locomotor hyperactivity in rats. The effects of CP 154,526 on the hyperlocomotion evoked by a single dose of the psychostimulant, as well as on cocaine self-administration and the cocaine-induced reinstatement of extinguished cocaine-seeking behavior in rats were also examined.
2. Methods 2.1. Animals The experiments were performed on male Wistar rats (10–12 weeks of age). The rats were housed either 8 per cage (locomotor activity studies) or individually (drug discrimination and self-administration procedures) in standard plastic rodent cages in a colony room maintained at 20 ± 1 °C and at 40–50% humidity under a 12 h light–dark cycle (lights on at 06:00). The animals had free access to food (Labofeed pellets) and water. The rats were habituated to the above conditions for 7 days before experiments. All experiments were conducted during the light phase of the light-dark cycle (between 0800 and 1500 hours) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approval from the Bioethics Commission as compliant with the Polish Law (21 August 1997).
2.2. Drugs Cocaine hydrochloride (Merck, Darmstadt, Germany) and CP 154,526 (Pfizer, Groton, USA) were used. Cocaine was dissolved in sterile 0.9% NaCl, while CP 154,526 was dissolved in a mixture of 5% cremophor0.9% NaCl. Cocaine was given either i.v. (0.05 ml/infusion) or i.p. (1 ml/kg). CP 154,526 was injected i.p. in a volume of 1 ml/kg. 2.3. Locomotor activity The locomotor activity of rats was recorded for each animal as described previously (Przegalin´ski et al., 2001). Briefly, the locomotor activity was measured in Opto-Varimex cages (Columbus Instruments, USA). Horizontal locomotor activity, defined as a distance traveled, was expressed in cm. Before locomotor activity was recorded, rats were habituated in the test cages for 2 h/day on each of the 2 days before the start of the experiment, and on each test day for 1 h before the start of the test session. Locomotor activity was recorded for 60 min. Each group consisted of 7–8 animals. 2.3.1. Acute treatment Locomotor activity was recorded in animals which received either vehicle, or saline, or cocaine (10 mg/kg, i.p.), or CP 154,526 (5–20 mg/kg) alone, or CP 154,526 (5–20 mg/kg) combined with cocaine (10 mg/ kg, i.p.). CP 154,526 and cocaine were given 45 and 5 min, respectively, before the start of locomotor activity recording. 2.3.2. Cocaine repeated treatment (sensitisation) During the first 5 days of the experiment, the animals received either saline or cocaine. On day 10, to assess effects of CP 154,526 on expression of cocaine sensitization they were challenged with vehicle + cocaine (10 mg/kg, i.p.), or CP 154,526 (5–20 mg/kg) + cocaine (10 mg/kg, i.p.). CP 154,526 and cocaine were given 45 and 5 min, respectively, before the start of locomotor activity recording. 2.4. Cocaine discrimination Rats (n = 16), which had limited water amount given during training sessions in operant chambers, after test sessions (15 min) and at weekends, were trained to discriminate cocaine (10 mg/kg, i.p.) from 0.9% NaCl (i.p.), as described previously (Filip et al., 2001; Filip and Cunningham, 2003). Briefly, the drug or saline was administered 15 min before daily (Monday–Friday) sessions (15 min), in two-lever standard operant chambers (Med-Associates; USA), under a fixed ratio (FR) 20 schedule of continuous water reinforcement. That
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phase of training continued until all the animals met the criterion (an individual mean accuracy of at least 80% of correct responses, before the first reinforcer during 10 consecutive sessions). Later, test sessions were conducted once or twice a week. Cocaine and saline sessions intervened between the test sessions to maintain discrimination accuracy. Only the rats that met an 80% performance criterion during the preceding cocaine and saline sessions were used in the tests. After completion of 20 responses to either lever, or after the session time elapsed, a single reinforcer was delivered and the animals were removed from the chamber. Once in their home cages, all the rats were allowed 15 min of free access to water. In substitution tests, the rats were tested with different doses of cocaine (1.25–10 mg/kg, i.p.) or CP 154,526 (5–20 mg/kg). In combination tests, CP 154,526 (5–20 mg/kg) preceded the injection of cocaine (10 mg/kg, i.p.), or CP 154,526 (20 mg/kg) was given before different doses of cocaine (1.25–5 mg/kg, i.p.). CP 154,526 and cocaine were given 45 and 15 min, respectively, before the tests. 2.5. Cocaine self-administration Rats, which had limited water amount during initial training sessions, were trained to press the lever in standard operant chambers (Med-Associates, USA) under a FR 5 schedule of water reinforcement. Two days following lever-press training and free access to water, the rats were anesthetized with ketamine HCl (75 mg/kg, ip; Biowet, Poland) and xylazine (5 mg/kg, i.p.; Biowet, Poland) and chronically implanted with a silastic catheter in the external right jugular vein, as described previously by Filip (2005). Catheters were flushed each day with 0.1 ml of saline solution containing heparin (70 U/mlBiochemie, Austria) and 0.1 ml of solution of cephazolin (10 mg/ml; Biochemie GmbH, Austria). Catheter potency was tested periodically, or whenever an animal displayed behavior outside baseline parameters, with the ultrashort-acting barbiturate anesthetic methohexital (10 mg/kg, i.v.) for loss of consciousness within 5 s. 2.5.1. Maintenance of cocaine self-administration Rats were allowed 10 days to recover before the start of the experiments. Initially, all animals deprived of water for 18 h were trained in one 2-h session to press the lever on an FR 5 schedule for water reinforcement. Then, subjects (n = 8–9 rats/group) began lever pressing for cocaine reinforcement and from that time they were given water ad libitum throughout the remaining period of the experiment. Rats were given access to cocaine during 2-h daily sessions performed 6 days/week (maintenance). Each completion of an FR 5 schedule (i.e. 5 lever presses) on the ‘‘active’’ lever resulted in an infusion of cocaine (0.5 mg/kg over 5 s). Injection speed was adjusted according to the weight of each rat. A tone
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(2000 Hz; 15 dB above ambient) and illumination with the stimulus light directly above the ‘‘active’’ lever (i.e. the conditioned stimulus) was presented for 5 s, concurrently with a successful response for cocaine delivery. Following each injection, there was a 20-s time-out period during which responding was recorded but had no programmed consequences. Response on the ‘‘inactive’’ lever never resulted in cocaine delivery. Each training trial lasted for 2 h or until the subject had self-administered 25 infusions of cocaine. An arbitrary acquisition criterion required that the number of active lever presses varied by 10% or less over the course of 3 consecutive maintenance days. Once stable rates of responding were established, rats were pretreated with either vehicle or CP 154,526 (10–20 mg/kg) before the test sessions. The order of injections was counterbalanced according to a Latin square design, and test sessions were separated by at least two–three baseline days of cocaine selfadministration. 2.5.2. Reinstatement of cocaine self-administration After the 18 days of self-administration, the rats were subjected to extinction trials for 10 days. During extinction, the animals had 2-h daily training sessions; however, active lever presses now resulted in neither the delivery of cocaine (saline was substituted for cocaine) nor the presentation of the conditioned stimulus. Rats remained in extinction until the responding on the active lever fell below 10% of the level observed during maintenance. After extinction, the rats were tested for response reinstatement induced by cocaine (10 mg/kg, i.p.). During reinstatement test (2 h session), active lever presses on the FR 5 schedule resulted only in an intravenous injection of saline and no cocaine was delivered. Rats were pretreated with either vehicle or CP 154,526 (5–20 mg/kg) before cocaine (10 mg/kg, i.p.). Drug combinations were given in a randomized order and test sessions were separated by at least two–three extinction sessions. Each group consisted of 8 animals. 2.6. Statistical analyses 2.6.1. Locomotor activity The data are expressed as a distance traveled during a 60-min observation period. An one-way ANOVA, followed by post-hoc DunnettÕs test, were applied to evaluate the treatment group effect in locomotor activity studies (acute treatment). To evaluate expression of cocaine sensitization, the one-way ANOVA, followed by post-hoc DunnettÕs test, were applied to evaluate the treatment group effect on day 10. 2.6.2. Cocaine discrimination During training and test sessions, accuracy was defined as a ratio of correct responses to the total number of responses before delivery of the first reinforcer.
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Response rates (responses per second), regarded as a measure of behavioral disruption, were calculated as the total number of responses to either lever before completion of the first FR 20, divided by the number of seconds required to complete the FR. Only the data from animals that completed the FR 20 during the test sessions were used. An one-way ANOVA for repeated measures was used to compare the percentage of druglever responding and response rates during the test sessions with the corresponding values of the preceding drug session (substitution tests) or the test dose given alone (combination tests). An ANOVA for repeated measures was used to compare whether the percentage of cocaine-appropriate responses and response rates observed across a fixed dose of cocaine differed in the presence or absence of a dose of CP 154,526 (combination tests); post-hoc comparisons at a dose of cocaine with or without the test drug were made with the StudentÕs t-test. 2.6.3. Cocaine self-administration During maintenance of cocaine self-administration, the number of responses on the active and inactive lever and the number of infusions were analyzed by an oneway ANOVA for repeated measures. During reinstatement of cocaine seeking behavior induced by cocaine priming, the number of responses on the active and inactive lever was analyzed via an one-way ANOVA for repeated measures. Post-hoc DunnettÕs test was used to analyze differences between group means.
3. Results 3.1. Locomotor activity Cocaine (10 mg/kg) significantly (ca. 4.5-fold) increased the ratsÕ basal locomotor activity compared to saline (Fig. 1).
CP 154,526 (5–20 mg/kg) did not alter the ratsÕ basal locomotor activation expressed as distance traveled [cm] (vehicle = 459 ± 96; CP 154,526, 5 mg/kg = 441 ± 89; CP 154,526, 10 mg/kg = 434 ± 92; CP 154,526, 20 mg/ kg = 431 ± 99; F(3,24) = 1.72, p > 0.05). When given in combination with cocaine (10 mg/kg), CP 154,526 (5–20 mg/kg) decreased dose-dependently the cocaine locomotor effect (F(4,35) = 5.16, p < 0.01; Fig. 1). On day 10, the challenge with cocaine of rats treated repeatedly with the psychostimulant (days 1–5) produced a significant increase (ca. 4-fold) in locomotor hyperactivity compared to the effect of acute cocaine injection to saline-treated (days 1–5) animals (locomotor sensitization; Fig. 2). When the animals were given a challenge dose of cocaine in combination with CP 154,526 (10–20, but not 5, mg/kg), a significant decrease in locomotor activity compared to cocaine-treated and cocaine-challenged animals was found on day 10 (F(4,35) = 7.96, p < 0.001; Fig. 2). 3.2. Cocaine discrimination The acquisition of cocaine (10 mg/kg) vs. saline discrimination was reached in an average of 25 sessions (ranging between: 19 and 29). Administration of cocaine (1.25–10 mg/kg)produced a dose-dependent increase in the cocaine-lever responding (Fig. 3); drug-lever responding after cocaine, 1.25–10 mg/kg, ranged from 26% to 94%. Administration of saline evoked less than 10% of drug-lever responding (data not shown). The response rates after saline and all the test doses of cocaine did not differ from those recorded during the preceding cocaine session (data not shown). Following administration of CP 154,526 (5–20 mg/ kg) no substitution (less than 20% of cocaine-lever responding) nor alteration in the animalsÕ response rates were found (data not shown).
Fig. 1. Effect of CP 154,526 (5–20 mg/kg) on the cocaine-stimulated locomotor activity in rats. *p < 0.01 vs. control; #p < 0.01 vs. cocaine.
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Fig. 2. Effect of CP 154,526 on the expression of cocaine sensitization. The rats were treated repeatedly (days 1–5) with either saline or cocaine (10 mg/kg). On day 10, the animals were given a challenge dose of cocaine (10 mg/kg) following saline or CP 154,526 (5–20 mg/kg). *p < 0.01 vs. saline-treated and cocaine-challenged group; #p < 0.05, ##p < 0.01 vs. cocaine-treated and cocaine-challenged group.
Fig. 3. Effect of CP 154,526 on cocaine discrimination in rats trained to recognize cocaine (10 mg/kg) from saline. Left: Effect of CP 154,526 (5– 20 mg/kg) on the cocaine (10 mg/kg)-induced discriminative stimulus effects. Right: Effect of CP 154,526 (20 mg/kg) on the cocaine (1.25–5 mg/kg)induced discriminative stimulus effects.
Combined administration of CP 154,526 (5–20 mg/ kg) did not alter the discriminative stimulus effects produced by cocaine, 10 mg/kg, which per se induced a 94% cocaine-lever responding (F(3,28) = 0.67, p > 0.05; Fig. 3A). The animal response rates were unaltered after the above treatment combinations (F(3,28) = 0.51, p > 0.05). Pretreatment with CP 154,526, 20 mg/kg, in combination with cocaine (1.25–5 mg/kg) did not change ocaine discrimination (F(2,24) = 0.39, p > 0.05; Fig. 3B). The animal response rates were unaltered after the above treatment combinations (F(2,24) = 0.33, p > 0.05).
3.3. Cocaine self-administration Rats showed stable responding on levers during the last 6 self-administration maintenance sessions with an acquisition criterion requiring that the rate of active lever presses varied by less than 10%. The animals had self-administered 36–42 infusions of cocaine with the daily mean cocaine intake between 18 and 21 mg/kg. Rats responded significantly more frequently on the active lever than on the inactive lever (p < 0.05), independently of self-administration test day (Fig. 4). CP 154,526 (10–20 mg/kg) did not change the number of active (F(2,18) = 0.19, p > 0.05) or inactive
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Fig. 4. Effect of CP 154,526 on cocaine self-administration in rats responding under a FR 5 schedule of reinforcement. Left: Effect of CP 154,526 (10–20 mg/kg) on active (associated with cocaine self-administration) and inactive lever presses. Right: Effect of CP 154,526 (10–20 mg/kg) on number of infusions.
(F(2,18) = 0.33, p > 0.05) lever presses (Fig. 4). The number of cocaine infusion also remained unaltered (F(2,18) = 0.47, p > 0.05) (Fig. 4). After 10 days of extinction trials during which active lever presses resulted in the i.v. delivery of saline without the presentation of the conditioned stimulus (cue), the rats were tested for response reinstatement induced by cocaine (10 mg/kg, i.p.). During cocaine-primed reinstatement test, rats responded more often on the active lever in relation to the inactive lever (p < 0.05) and to the extinction period (p < 0.05). The responses on the inactive lever were not different across the days (Fig. 5).
CP 154,526 (5–20 mg/kg) reduced dose-dependently the response reinstatement on active lever induced by cocaine priming, 10 mg/kg, i.p. (F(4,30) = 12.49, p < 0.001; Fig. 5). There was no significant effect of CP 154,526 treatment observed for total inactive lever responding (F(4,30) = 1.79, p > 0.05; Fig. 5).
4. Discussion The results of the present study enrich our knowledge of the role of CRF1 receptors in the behavioral effects of
Fig. 5. Effect of CP 154,526 on the reinstatement of cocaine seeking behavior induced by cocaine priming. Number of active (i.e. previously associated with cocaine self-administration) and inactive lever presses following CP 154,526 (5–20 mg/kg) in combination with cocaine (10 mg/kg, i.p.) or saline (i.p.). *p < 0.001 vs. vehicle-saline; #p < 0.05, ##p < 0.01 vs. vehicle-cocaine.
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cocaine. In fact, the data obtained in our experiments with the selective antagonist of CRF1 receptors, CP 154,526, administered in doses of 5–20 mg/kg sufficient to block those receptors (Seymour et al., 2003), not only confirm some earlier observations about their role in the cocaine-induced reinstatement of cocaine-seeking behavior (Erb et al., 1998) and locomotor hyperactivity (Sarnyai et al., 1992; Lu et al., 2003), but also show the importance of CRF1 receptors in the expression of sensitization to the cocaine-induced hyperlocomotion, but not in the discriminative stimulus effect of the psychostimulant in rats. In accordance with our earlier observations (Przegalin´ski et al., 2001, 2002), the present findings showed cocaine sensitization to its locomotor hyperactivity effect under the experimental conditions used. Actually, when the rats received cocaine in a dose of 10 mg/kg for 5 days and were then challenged with the same dose of the psychostimulant on day 5 after its withdrawal (day 10 of the experiment), their locomotor hyperactivity was more than four times higher than that after cocaine challenge in animals treated repeatedly (5 days) with saline. CP 154,526, administered in doses of 10 and 20 mg/kg (but not 5 mg/kg) before the challenge dose of cocaine, significantly reduced the expression of psychostimulant sensitization. With regard to these results it should be stressed that the inhibitory effect of the CRF1 receptor antagonist appeared after doses which did not affect basal locomotor activity, but dose-dependently inhibited the hyperlocomotion induced by a single dose of cocaine. The latter observation is supported by some earlier results reported by Sarnyai et al. (1992) and Lu et al. (2003), who found that both a-helical CRF9-41 (a non-selective CRF receptor antagonist) and CP 154,526, respectively, antagonized cocaine hyperlocomotion. In other words, the above data and our results indicate that CRF1 receptors are involved not only in the expression of behavioral sensitization to cocaine, but also in the locomotor hyperactivity response to acute treatment with the psychostimulant. Our results also show that CP 154,526 administered in doses of 5–20 mg/kg before the priming dose of cocaine, dose-dependently decreases the ability of the psychostimulant to reinstate responding following extinction from cocaine self-administration. As the reinstatement tests were initiated with cocaine administered by an experimenter, a question arises whether CP 154,526 alters the motivational or other (hyperlocomotion, anxiety) effects of the psychostimulant. Although the CRF1 receptor antagonist inhibited the cocaineinduced locomotor hyperactivity (Lu et al., 2003; present study), it is unlikely that it could inhibit the cocaine-primed reinstatement by a general disruption of operant behavior, since the effective doses of CP 154,526 reduced neither basal locomotor activity nor lever pressing during maintenance of cocaine self-
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administration (present study). Similarly, the potential anxiolytic activity of CP 154,526 does not seem to be responsible for its inhibitory effect on the cocaineinduced reinstatement, since CRF1 receptor antagonists are effective mainly in stress-induced anxiety, especially after their chronic administration (Takahashi, 2001). The priming effect of cocaine may also serve as a discriminative stimulus, and may indirectly affect motivation by signaling the availability of the psychostimulant (de Wit and Stewart, 1981; Schenk and Partridge, 1999). However, CP 154,526 in doses inhibiting cocaine reinstatement did not affect the discriminative stimulus effects of different doses of the psychostimulant. On the other hand, it should be kept in mind that CRF receptor blockade enhances the conditioned aversive action of cocaine (Heinrichs et al., 1998), an effect which may be responsible for the inhibitory action of CP 154,526 on the cocaine-induced reinstatement. As regards our results obtained with cocaine selfadministration and cocaine reinstatement, they differ from those reported by other authors. In fact, Goeders and Guerin (2000) found that CP 154,526 in doses of 20–40 mg/kg (but not lower) produced dose-related decreases in cocaine self-administration maintained especially at its lower doses (0.125–0.25 mg/kg/infusion). At the same time, Lee et al. (2003) found that CP 154,526 did not affect the cocaine-induced reinstatement in squirrel monkeys, while Erb et al. (1998) reported that the non-selective CRF receptor antagonist D-Phe CRF12-41 administered intracerebroventricularly to rats only minimally attenuated cocaine reinstatement, but it blocked the footshock-induced reinstatement. Interestingly, the blockade of the stress-induced reinstatement by CP 154,526 was also observed by other authors (Shaham et al., 1998; Goeders and Clampitt, 2002). Although the cause of such a discrepancy between our and other authorsÕ (Erb et al., 1998; Goeders and Guerin, 2000; Lee et al., 2003) results on cocaine self-administration and the cocaine-induced reinstatement of drug-seeking behavior is not clear, the following questions should be raised: (1) regarding self-administration model, it needs to be stressed that Goeders and Guerin (2000) used a different procedure than that employed in our experiment (alternating access to food reinstatement and cocaine self-administration vs. access to cocaine self-administration only); (2) when compared to our results, the considerably weaker antireinstatement effect of the CRF receptor antagonist (observed by Erb et al., 1998) may result from differences in the CRF antagonists used (D-Phe CRF12-41 vs. CP 154,526), as well as in the route of their administration (intracerebroventricular vs. i.p.); moreover, it is not clear whether the doses of D-Phe CRF12-41 (0.1–1 lg) injected intracerebroventricularly (Erb et al., 1998) correspond to the doses of CP 154,526 (10–20 mg/kg) administered i.p. (present study) in terms of their
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potency to block CRF receptors; (3) the negative results observed by Lee et al. (2003) in squirrel monkeys seem to indicate that the CRF antagonist-induced inhibition of cocaine reinstatement, observed in rats in the present study, is a species-specific effect. As far as the mechanism of CP 154,526 antagonism towards the behavioral effects of cocaine (locomotor hyperactivity, behavioral sensitization, the priming of seeking behavior) is concerned, the following issues should be considered: (1) the behavioral effects of cocaine depend on its inhibitory effect on the dopamine transporter (Reith et al., 1986) and the consequent increase in extracellular dopamine level (Kuczenski and Segal, 1992; Woolverton and Johnson, 1992); (2) CRF1 receptors are distributed in several brain extrahypothalamic areas (Primus et al., 1997) including the ventral tegmental area and the nucleus accumbens (Steckler and Holsboer, 1999), the structures forming the mesolimbic dopaminergic system involved in the behavioral effects of cocaine (Kalivas and Nakamura, 1999); (3) cocaine releases CRF from extrahypothalamic structures (Richter et al., 1995; Sarnyai et al., 1993), and sensitization to this effect has been observed (Richter et al., 1995); the latter finding seems to be particularly important in terms of the inhibitory effect of CP 154,256 on cocaine behavioral sensitization; (4) the cocaine-induced dopamine release in the ventral tegmental area and the nucleus accumbens is completely blocked by intracerebroventricular administration of CP 154,526 (Lu et al., 2003). In other words, the above findings indicate that the inhibitory effect of CP 154,526 on the locomotor hyperactivity, behavioral sensitization and seeking behavior induced by cocaine, observed in the present study, may result from its inhibitory action on dopamine release evoked by the psychostimulant in the mesolimbic system. It should be stressed, however, that the lack of CP 154,526 effect on the cocaine-induced discriminative stimulus and reward suggests that there are behavioral responses to the psychostimulant in which CRF1 receptors are not engaged. Summing up, the present study demonstrates that CRF1 receptors are involved in some behavioral effects of cocaine, and that antagonists of these receptors may be considered as possible medications in the treatment of cocaine addiction and other psychostimulant-related behavioral disorders.
Acknowledgement The authors wish to thank Ms. E. Nowak and K. Wydra for providing technical assistance. CP 154,526 was kindly donated by Pfizer. This study was supported by the Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland.
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Effects of CP 154,526, a CRF1 receptor antagonist, on behavioral responses to cocaine in rats Edmund Przegalin´ski *, Małgorzata Filip, Małgorzata Frankowska, Magdalena Zaniewska, Iwona Papla Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Sme˛tna 12, 31-343 Krako´w, Poland Received 4 April 2005; accepted 10 July 2005 Available online 2 September 2005
Abstract We examined the influence of CP 154,526, a selective antagonist of corticotropin-releasing factor (CRF)1 receptors, in the locomotor, sensitizing, discriminative stimulus and rewarding effects of cocaine, as well as on the cocaine-induced reinstatement of cocaine-seeking behavior in male Wistar rats. CP 154,526 in doses of 5, 10 and 20 mg/kg, which did not affect basal locomotor activity, dose-dependently reduced the hyperactivation evoked by cocaine. To assess the effects of CP 154,526 on the expression of cocaine sensitization, the rats were injected with either saline or cocaine (10 mg/kg) for 5 days, and were then challenged with cocaine (10 mg/kg) after pretreatment with saline or CP 154,526 on day 5 of withdrawal. The cocaine-induced hyperactivity in sensitized rats was reduced by CP 154,526 (10 and 20 mg/kg). In rats trained to discriminate cocaine (10 mg/kg) from saline, pretreatment with CP 154,526 (5–20 mg/kg) did not affect the cocaine (1.25–10 mg/kg)-induced discriminative stimulus effects. In a selfadministration model, the rats were trained to self-administer cocaine (0.5 mg/kg/infusion) in the FR 5 schedule of reinforcement. Administration of CP 154,526 (10–20 mg/kg) did not alter the rewarding effects of cocaine, assessed as the number of active-lever presses and infusions; however, following a 10-day extinction phase, CP 154,526 (5–20 mg/kg) significantly decreased in a dosedependent manner the cocaine (10 mg/kg) priming-induced reinstatement of cocaine-seeking behavior. The present study implies that CRF1 receptors control the expression of cocaine hyperactivation and sensitization as well as the cocaine-induced relapse behavior, but do not play any role in cocaine discrimination and self-administration. These findings may suggest that CRF1 receptor antagonists should be considered as possible medications in the treatment of cocaine addiction. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Cocaine; Corticotropin-releasing factor; CP 154,526; Drug discrimination; Locomotor activity; Sensitization, Self-administration; Rats
1. Introduction Corticotropin-releasing factor (CRF), a 41-amino acid peptide initially identified as a hypothalamic agent involved in the secretion of corticotropin (ACTH) from the anterior pituitary (Vale et al., 1981; Cummings et al., 1983), plays an essential role in the regulation of not only endocrine, but also autonomic and immune *
Corresponding author. Tel.: +48 12 662 3293; fax: +48 12 637 4500. E-mail address: [email protected] (E. Przegalin´ski). 0143-4179/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2005.07.002
responses to stress (De Souza, 1995). At the same time, being widely distributed throughout the brain, CRF (which exerts its biological actions via two receptor subtypes known as CRF1 (Chang et al., 1993; Perrin et al., 1993) and CRF2 (Lovenberg et al., 1995)), has also been implicated in the modulation of several behavioral effects (Shibasaki et al., 1991; Koob et al., 1994), including these produced by the psychostimulant cocaine. In fact, the cocaine-induced locomotor hyperactivity and stereotypy, as well as the reinforcing activity of the psychostimulant (assessed in place preference and self-administration models) have been found to be
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blocked by the immunoneutralization of endogenous CRF and/or by CRF receptor antagonists (Sarnyai et al., 1992; Erb et al., 1998; Shaham et al., 1998; Goeders and Guerin, 2000; Lu et al., 2001; Gurkovskaya and Goeders, 2001; Goeders and Clampitt, 2002). The above data are in line with the observations showing that acute treatment with cocaine releases endogenous CRF from not only the hypothalamic (Sarnyai et al., 2001), but also extrahypothalamic structures (Richter et al., 1995). On the other hand, no data are available on the role of CRF in other effects of cocaine, such as discriminative cue or behavioral sensitization. The former one models the subjective effects of cocaine in humans (Schuster and Johanson, 1988), while sensitization, a phenomenon characterized by an increase in – among others – behavioral responses (e.g. locomotor hyperactivity) to subsequent cocaine challenge after its repeated administration is discontinued (Robinson and Berridge, 1993; Kalivas et al., 1998), plays an essential role in the psychostimulant-induced psychoses or craving for drugs of abuse in humans (Segal et al., 1981; Robinson and Berridge, 1993; Kalivas et al., 1998). In the present study we examined the effect of the non-peptide CRF1 receptor antagonist CP 154,526 (Seymour et al., 2003) on the discriminative stimulus effect of cocaine and on the expression phase of sensitization to the cocaine-induced locomotor hyperactivity in rats. The effects of CP 154,526 on the hyperlocomotion evoked by a single dose of the psychostimulant, as well as on cocaine self-administration and the cocaine-induced reinstatement of extinguished cocaine-seeking behavior in rats were also examined.
2. Methods 2.1. Animals The experiments were performed on male Wistar rats (10–12 weeks of age). The rats were housed either 8 per cage (locomotor activity studies) or individually (drug discrimination and self-administration procedures) in standard plastic rodent cages in a colony room maintained at 20 ± 1 °C and at 40–50% humidity under a 12 h light–dark cycle (lights on at 06:00). The animals had free access to food (Labofeed pellets) and water. The rats were habituated to the above conditions for 7 days before experiments. All experiments were conducted during the light phase of the light-dark cycle (between 0800 and 1500 hours) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approval from the Bioethics Commission as compliant with the Polish Law (21 August 1997).
2.2. Drugs Cocaine hydrochloride (Merck, Darmstadt, Germany) and CP 154,526 (Pfizer, Groton, USA) were used. Cocaine was dissolved in sterile 0.9% NaCl, while CP 154,526 was dissolved in a mixture of 5% cremophor0.9% NaCl. Cocaine was given either i.v. (0.05 ml/infusion) or i.p. (1 ml/kg). CP 154,526 was injected i.p. in a volume of 1 ml/kg. 2.3. Locomotor activity The locomotor activity of rats was recorded for each animal as described previously (Przegalin´ski et al., 2001). Briefly, the locomotor activity was measured in Opto-Varimex cages (Columbus Instruments, USA). Horizontal locomotor activity, defined as a distance traveled, was expressed in cm. Before locomotor activity was recorded, rats were habituated in the test cages for 2 h/day on each of the 2 days before the start of the experiment, and on each test day for 1 h before the start of the test session. Locomotor activity was recorded for 60 min. Each group consisted of 7–8 animals. 2.3.1. Acute treatment Locomotor activity was recorded in animals which received either vehicle, or saline, or cocaine (10 mg/kg, i.p.), or CP 154,526 (5–20 mg/kg) alone, or CP 154,526 (5–20 mg/kg) combined with cocaine (10 mg/ kg, i.p.). CP 154,526 and cocaine were given 45 and 5 min, respectively, before the start of locomotor activity recording. 2.3.2. Cocaine repeated treatment (sensitisation) During the first 5 days of the experiment, the animals received either saline or cocaine. On day 10, to assess effects of CP 154,526 on expression of cocaine sensitization they were challenged with vehicle + cocaine (10 mg/kg, i.p.), or CP 154,526 (5–20 mg/kg) + cocaine (10 mg/kg, i.p.). CP 154,526 and cocaine were given 45 and 5 min, respectively, before the start of locomotor activity recording. 2.4. Cocaine discrimination Rats (n = 16), which had limited water amount given during training sessions in operant chambers, after test sessions (15 min) and at weekends, were trained to discriminate cocaine (10 mg/kg, i.p.) from 0.9% NaCl (i.p.), as described previously (Filip et al., 2001; Filip and Cunningham, 2003). Briefly, the drug or saline was administered 15 min before daily (Monday–Friday) sessions (15 min), in two-lever standard operant chambers (Med-Associates; USA), under a fixed ratio (FR) 20 schedule of continuous water reinforcement. That
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phase of training continued until all the animals met the criterion (an individual mean accuracy of at least 80% of correct responses, before the first reinforcer during 10 consecutive sessions). Later, test sessions were conducted once or twice a week. Cocaine and saline sessions intervened between the test sessions to maintain discrimination accuracy. Only the rats that met an 80% performance criterion during the preceding cocaine and saline sessions were used in the tests. After completion of 20 responses to either lever, or after the session time elapsed, a single reinforcer was delivered and the animals were removed from the chamber. Once in their home cages, all the rats were allowed 15 min of free access to water. In substitution tests, the rats were tested with different doses of cocaine (1.25–10 mg/kg, i.p.) or CP 154,526 (5–20 mg/kg). In combination tests, CP 154,526 (5–20 mg/kg) preceded the injection of cocaine (10 mg/kg, i.p.), or CP 154,526 (20 mg/kg) was given before different doses of cocaine (1.25–5 mg/kg, i.p.). CP 154,526 and cocaine were given 45 and 15 min, respectively, before the tests. 2.5. Cocaine self-administration Rats, which had limited water amount during initial training sessions, were trained to press the lever in standard operant chambers (Med-Associates, USA) under a FR 5 schedule of water reinforcement. Two days following lever-press training and free access to water, the rats were anesthetized with ketamine HCl (75 mg/kg, ip; Biowet, Poland) and xylazine (5 mg/kg, i.p.; Biowet, Poland) and chronically implanted with a silastic catheter in the external right jugular vein, as described previously by Filip (2005). Catheters were flushed each day with 0.1 ml of saline solution containing heparin (70 U/mlBiochemie, Austria) and 0.1 ml of solution of cephazolin (10 mg/ml; Biochemie GmbH, Austria). Catheter potency was tested periodically, or whenever an animal displayed behavior outside baseline parameters, with the ultrashort-acting barbiturate anesthetic methohexital (10 mg/kg, i.v.) for loss of consciousness within 5 s. 2.5.1. Maintenance of cocaine self-administration Rats were allowed 10 days to recover before the start of the experiments. Initially, all animals deprived of water for 18 h were trained in one 2-h session to press the lever on an FR 5 schedule for water reinforcement. Then, subjects (n = 8–9 rats/group) began lever pressing for cocaine reinforcement and from that time they were given water ad libitum throughout the remaining period of the experiment. Rats were given access to cocaine during 2-h daily sessions performed 6 days/week (maintenance). Each completion of an FR 5 schedule (i.e. 5 lever presses) on the ‘‘active’’ lever resulted in an infusion of cocaine (0.5 mg/kg over 5 s). Injection speed was adjusted according to the weight of each rat. A tone
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(2000 Hz; 15 dB above ambient) and illumination with the stimulus light directly above the ‘‘active’’ lever (i.e. the conditioned stimulus) was presented for 5 s, concurrently with a successful response for cocaine delivery. Following each injection, there was a 20-s time-out period during which responding was recorded but had no programmed consequences. Response on the ‘‘inactive’’ lever never resulted in cocaine delivery. Each training trial lasted for 2 h or until the subject had self-administered 25 infusions of cocaine. An arbitrary acquisition criterion required that the number of active lever presses varied by 10% or less over the course of 3 consecutive maintenance days. Once stable rates of responding were established, rats were pretreated with either vehicle or CP 154,526 (10–20 mg/kg) before the test sessions. The order of injections was counterbalanced according to a Latin square design, and test sessions were separated by at least two–three baseline days of cocaine selfadministration. 2.5.2. Reinstatement of cocaine self-administration After the 18 days of self-administration, the rats were subjected to extinction trials for 10 days. During extinction, the animals had 2-h daily training sessions; however, active lever presses now resulted in neither the delivery of cocaine (saline was substituted for cocaine) nor the presentation of the conditioned stimulus. Rats remained in extinction until the responding on the active lever fell below 10% of the level observed during maintenance. After extinction, the rats were tested for response reinstatement induced by cocaine (10 mg/kg, i.p.). During reinstatement test (2 h session), active lever presses on the FR 5 schedule resulted only in an intravenous injection of saline and no cocaine was delivered. Rats were pretreated with either vehicle or CP 154,526 (5–20 mg/kg) before cocaine (10 mg/kg, i.p.). Drug combinations were given in a randomized order and test sessions were separated by at least two–three extinction sessions. Each group consisted of 8 animals. 2.6. Statistical analyses 2.6.1. Locomotor activity The data are expressed as a distance traveled during a 60-min observation period. An one-way ANOVA, followed by post-hoc DunnettÕs test, were applied to evaluate the treatment group effect in locomotor activity studies (acute treatment). To evaluate expression of cocaine sensitization, the one-way ANOVA, followed by post-hoc DunnettÕs test, were applied to evaluate the treatment group effect on day 10. 2.6.2. Cocaine discrimination During training and test sessions, accuracy was defined as a ratio of correct responses to the total number of responses before delivery of the first reinforcer.
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Response rates (responses per second), regarded as a measure of behavioral disruption, were calculated as the total number of responses to either lever before completion of the first FR 20, divided by the number of seconds required to complete the FR. Only the data from animals that completed the FR 20 during the test sessions were used. An one-way ANOVA for repeated measures was used to compare the percentage of druglever responding and response rates during the test sessions with the corresponding values of the preceding drug session (substitution tests) or the test dose given alone (combination tests). An ANOVA for repeated measures was used to compare whether the percentage of cocaine-appropriate responses and response rates observed across a fixed dose of cocaine differed in the presence or absence of a dose of CP 154,526 (combination tests); post-hoc comparisons at a dose of cocaine with or without the test drug were made with the StudentÕs t-test. 2.6.3. Cocaine self-administration During maintenance of cocaine self-administration, the number of responses on the active and inactive lever and the number of infusions were analyzed by an oneway ANOVA for repeated measures. During reinstatement of cocaine seeking behavior induced by cocaine priming, the number of responses on the active and inactive lever was analyzed via an one-way ANOVA for repeated measures. Post-hoc DunnettÕs test was used to analyze differences between group means.
3. Results 3.1. Locomotor activity Cocaine (10 mg/kg) significantly (ca. 4.5-fold) increased the ratsÕ basal locomotor activity compared to saline (Fig. 1).
CP 154,526 (5–20 mg/kg) did not alter the ratsÕ basal locomotor activation expressed as distance traveled [cm] (vehicle = 459 ± 96; CP 154,526, 5 mg/kg = 441 ± 89; CP 154,526, 10 mg/kg = 434 ± 92; CP 154,526, 20 mg/ kg = 431 ± 99; F(3,24) = 1.72, p > 0.05). When given in combination with cocaine (10 mg/kg), CP 154,526 (5–20 mg/kg) decreased dose-dependently the cocaine locomotor effect (F(4,35) = 5.16, p < 0.01; Fig. 1). On day 10, the challenge with cocaine of rats treated repeatedly with the psychostimulant (days 1–5) produced a significant increase (ca. 4-fold) in locomotor hyperactivity compared to the effect of acute cocaine injection to saline-treated (days 1–5) animals (locomotor sensitization; Fig. 2). When the animals were given a challenge dose of cocaine in combination with CP 154,526 (10–20, but not 5, mg/kg), a significant decrease in locomotor activity compared to cocaine-treated and cocaine-challenged animals was found on day 10 (F(4,35) = 7.96, p < 0.001; Fig. 2). 3.2. Cocaine discrimination The acquisition of cocaine (10 mg/kg) vs. saline discrimination was reached in an average of 25 sessions (ranging between: 19 and 29). Administration of cocaine (1.25–10 mg/kg)produced a dose-dependent increase in the cocaine-lever responding (Fig. 3); drug-lever responding after cocaine, 1.25–10 mg/kg, ranged from 26% to 94%. Administration of saline evoked less than 10% of drug-lever responding (data not shown). The response rates after saline and all the test doses of cocaine did not differ from those recorded during the preceding cocaine session (data not shown). Following administration of CP 154,526 (5–20 mg/ kg) no substitution (less than 20% of cocaine-lever responding) nor alteration in the animalsÕ response rates were found (data not shown).
Fig. 1. Effect of CP 154,526 (5–20 mg/kg) on the cocaine-stimulated locomotor activity in rats. *p < 0.01 vs. control; #p < 0.01 vs. cocaine.
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Fig. 2. Effect of CP 154,526 on the expression of cocaine sensitization. The rats were treated repeatedly (days 1–5) with either saline or cocaine (10 mg/kg). On day 10, the animals were given a challenge dose of cocaine (10 mg/kg) following saline or CP 154,526 (5–20 mg/kg). *p < 0.01 vs. saline-treated and cocaine-challenged group; #p < 0.05, ##p < 0.01 vs. cocaine-treated and cocaine-challenged group.
Fig. 3. Effect of CP 154,526 on cocaine discrimination in rats trained to recognize cocaine (10 mg/kg) from saline. Left: Effect of CP 154,526 (5– 20 mg/kg) on the cocaine (10 mg/kg)-induced discriminative stimulus effects. Right: Effect of CP 154,526 (20 mg/kg) on the cocaine (1.25–5 mg/kg)induced discriminative stimulus effects.
Combined administration of CP 154,526 (5–20 mg/ kg) did not alter the discriminative stimulus effects produced by cocaine, 10 mg/kg, which per se induced a 94% cocaine-lever responding (F(3,28) = 0.67, p > 0.05; Fig. 3A). The animal response rates were unaltered after the above treatment combinations (F(3,28) = 0.51, p > 0.05). Pretreatment with CP 154,526, 20 mg/kg, in combination with cocaine (1.25–5 mg/kg) did not change ocaine discrimination (F(2,24) = 0.39, p > 0.05; Fig. 3B). The animal response rates were unaltered after the above treatment combinations (F(2,24) = 0.33, p > 0.05).
3.3. Cocaine self-administration Rats showed stable responding on levers during the last 6 self-administration maintenance sessions with an acquisition criterion requiring that the rate of active lever presses varied by less than 10%. The animals had self-administered 36–42 infusions of cocaine with the daily mean cocaine intake between 18 and 21 mg/kg. Rats responded significantly more frequently on the active lever than on the inactive lever (p < 0.05), independently of self-administration test day (Fig. 4). CP 154,526 (10–20 mg/kg) did not change the number of active (F(2,18) = 0.19, p > 0.05) or inactive
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Fig. 4. Effect of CP 154,526 on cocaine self-administration in rats responding under a FR 5 schedule of reinforcement. Left: Effect of CP 154,526 (10–20 mg/kg) on active (associated with cocaine self-administration) and inactive lever presses. Right: Effect of CP 154,526 (10–20 mg/kg) on number of infusions.
(F(2,18) = 0.33, p > 0.05) lever presses (Fig. 4). The number of cocaine infusion also remained unaltered (F(2,18) = 0.47, p > 0.05) (Fig. 4). After 10 days of extinction trials during which active lever presses resulted in the i.v. delivery of saline without the presentation of the conditioned stimulus (cue), the rats were tested for response reinstatement induced by cocaine (10 mg/kg, i.p.). During cocaine-primed reinstatement test, rats responded more often on the active lever in relation to the inactive lever (p < 0.05) and to the extinction period (p < 0.05). The responses on the inactive lever were not different across the days (Fig. 5).
CP 154,526 (5–20 mg/kg) reduced dose-dependently the response reinstatement on active lever induced by cocaine priming, 10 mg/kg, i.p. (F(4,30) = 12.49, p < 0.001; Fig. 5). There was no significant effect of CP 154,526 treatment observed for total inactive lever responding (F(4,30) = 1.79, p > 0.05; Fig. 5).
4. Discussion The results of the present study enrich our knowledge of the role of CRF1 receptors in the behavioral effects of
Fig. 5. Effect of CP 154,526 on the reinstatement of cocaine seeking behavior induced by cocaine priming. Number of active (i.e. previously associated with cocaine self-administration) and inactive lever presses following CP 154,526 (5–20 mg/kg) in combination with cocaine (10 mg/kg, i.p.) or saline (i.p.). *p < 0.001 vs. vehicle-saline; #p < 0.05, ##p < 0.01 vs. vehicle-cocaine.
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cocaine. In fact, the data obtained in our experiments with the selective antagonist of CRF1 receptors, CP 154,526, administered in doses of 5–20 mg/kg sufficient to block those receptors (Seymour et al., 2003), not only confirm some earlier observations about their role in the cocaine-induced reinstatement of cocaine-seeking behavior (Erb et al., 1998) and locomotor hyperactivity (Sarnyai et al., 1992; Lu et al., 2003), but also show the importance of CRF1 receptors in the expression of sensitization to the cocaine-induced hyperlocomotion, but not in the discriminative stimulus effect of the psychostimulant in rats. In accordance with our earlier observations (Przegalin´ski et al., 2001, 2002), the present findings showed cocaine sensitization to its locomotor hyperactivity effect under the experimental conditions used. Actually, when the rats received cocaine in a dose of 10 mg/kg for 5 days and were then challenged with the same dose of the psychostimulant on day 5 after its withdrawal (day 10 of the experiment), their locomotor hyperactivity was more than four times higher than that after cocaine challenge in animals treated repeatedly (5 days) with saline. CP 154,526, administered in doses of 10 and 20 mg/kg (but not 5 mg/kg) before the challenge dose of cocaine, significantly reduced the expression of psychostimulant sensitization. With regard to these results it should be stressed that the inhibitory effect of the CRF1 receptor antagonist appeared after doses which did not affect basal locomotor activity, but dose-dependently inhibited the hyperlocomotion induced by a single dose of cocaine. The latter observation is supported by some earlier results reported by Sarnyai et al. (1992) and Lu et al. (2003), who found that both a-helical CRF9-41 (a non-selective CRF receptor antagonist) and CP 154,526, respectively, antagonized cocaine hyperlocomotion. In other words, the above data and our results indicate that CRF1 receptors are involved not only in the expression of behavioral sensitization to cocaine, but also in the locomotor hyperactivity response to acute treatment with the psychostimulant. Our results also show that CP 154,526 administered in doses of 5–20 mg/kg before the priming dose of cocaine, dose-dependently decreases the ability of the psychostimulant to reinstate responding following extinction from cocaine self-administration. As the reinstatement tests were initiated with cocaine administered by an experimenter, a question arises whether CP 154,526 alters the motivational or other (hyperlocomotion, anxiety) effects of the psychostimulant. Although the CRF1 receptor antagonist inhibited the cocaineinduced locomotor hyperactivity (Lu et al., 2003; present study), it is unlikely that it could inhibit the cocaine-primed reinstatement by a general disruption of operant behavior, since the effective doses of CP 154,526 reduced neither basal locomotor activity nor lever pressing during maintenance of cocaine self-
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administration (present study). Similarly, the potential anxiolytic activity of CP 154,526 does not seem to be responsible for its inhibitory effect on the cocaineinduced reinstatement, since CRF1 receptor antagonists are effective mainly in stress-induced anxiety, especially after their chronic administration (Takahashi, 2001). The priming effect of cocaine may also serve as a discriminative stimulus, and may indirectly affect motivation by signaling the availability of the psychostimulant (de Wit and Stewart, 1981; Schenk and Partridge, 1999). However, CP 154,526 in doses inhibiting cocaine reinstatement did not affect the discriminative stimulus effects of different doses of the psychostimulant. On the other hand, it should be kept in mind that CRF receptor blockade enhances the conditioned aversive action of cocaine (Heinrichs et al., 1998), an effect which may be responsible for the inhibitory action of CP 154,526 on the cocaine-induced reinstatement. As regards our results obtained with cocaine selfadministration and cocaine reinstatement, they differ from those reported by other authors. In fact, Goeders and Guerin (2000) found that CP 154,526 in doses of 20–40 mg/kg (but not lower) produced dose-related decreases in cocaine self-administration maintained especially at its lower doses (0.125–0.25 mg/kg/infusion). At the same time, Lee et al. (2003) found that CP 154,526 did not affect the cocaine-induced reinstatement in squirrel monkeys, while Erb et al. (1998) reported that the non-selective CRF receptor antagonist D-Phe CRF12-41 administered intracerebroventricularly to rats only minimally attenuated cocaine reinstatement, but it blocked the footshock-induced reinstatement. Interestingly, the blockade of the stress-induced reinstatement by CP 154,526 was also observed by other authors (Shaham et al., 1998; Goeders and Clampitt, 2002). Although the cause of such a discrepancy between our and other authorsÕ (Erb et al., 1998; Goeders and Guerin, 2000; Lee et al., 2003) results on cocaine self-administration and the cocaine-induced reinstatement of drug-seeking behavior is not clear, the following questions should be raised: (1) regarding self-administration model, it needs to be stressed that Goeders and Guerin (2000) used a different procedure than that employed in our experiment (alternating access to food reinstatement and cocaine self-administration vs. access to cocaine self-administration only); (2) when compared to our results, the considerably weaker antireinstatement effect of the CRF receptor antagonist (observed by Erb et al., 1998) may result from differences in the CRF antagonists used (D-Phe CRF12-41 vs. CP 154,526), as well as in the route of their administration (intracerebroventricular vs. i.p.); moreover, it is not clear whether the doses of D-Phe CRF12-41 (0.1–1 lg) injected intracerebroventricularly (Erb et al., 1998) correspond to the doses of CP 154,526 (10–20 mg/kg) administered i.p. (present study) in terms of their
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potency to block CRF receptors; (3) the negative results observed by Lee et al. (2003) in squirrel monkeys seem to indicate that the CRF antagonist-induced inhibition of cocaine reinstatement, observed in rats in the present study, is a species-specific effect. As far as the mechanism of CP 154,526 antagonism towards the behavioral effects of cocaine (locomotor hyperactivity, behavioral sensitization, the priming of seeking behavior) is concerned, the following issues should be considered: (1) the behavioral effects of cocaine depend on its inhibitory effect on the dopamine transporter (Reith et al., 1986) and the consequent increase in extracellular dopamine level (Kuczenski and Segal, 1992; Woolverton and Johnson, 1992); (2) CRF1 receptors are distributed in several brain extrahypothalamic areas (Primus et al., 1997) including the ventral tegmental area and the nucleus accumbens (Steckler and Holsboer, 1999), the structures forming the mesolimbic dopaminergic system involved in the behavioral effects of cocaine (Kalivas and Nakamura, 1999); (3) cocaine releases CRF from extrahypothalamic structures (Richter et al., 1995; Sarnyai et al., 1993), and sensitization to this effect has been observed (Richter et al., 1995); the latter finding seems to be particularly important in terms of the inhibitory effect of CP 154,256 on cocaine behavioral sensitization; (4) the cocaine-induced dopamine release in the ventral tegmental area and the nucleus accumbens is completely blocked by intracerebroventricular administration of CP 154,526 (Lu et al., 2003). In other words, the above findings indicate that the inhibitory effect of CP 154,526 on the locomotor hyperactivity, behavioral sensitization and seeking behavior induced by cocaine, observed in the present study, may result from its inhibitory action on dopamine release evoked by the psychostimulant in the mesolimbic system. It should be stressed, however, that the lack of CP 154,526 effect on the cocaine-induced discriminative stimulus and reward suggests that there are behavioral responses to the psychostimulant in which CRF1 receptors are not engaged. Summing up, the present study demonstrates that CRF1 receptors are involved in some behavioral effects of cocaine, and that antagonists of these receptors may be considered as possible medications in the treatment of cocaine addiction and other psychostimulant-related behavioral disorders.
Acknowledgement The authors wish to thank Ms. E. Nowak and K. Wydra for providing technical assistance. CP 154,526 was kindly donated by Pfizer. This study was supported by the Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland.
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