Blockade of o-opioid Receptors Prevents Morphine-Induced Place Preference in Mice

Blockade of o-opioid Receptors Prevents Morphine-Induced Place Preference in Mice Tsutomu Suzuki', Michiharu Yoshiike', Hirokazu Mizoguchi', Junzo Kamei', Miwa Misawa' and Hiroshi Nagase2 'Department of Pharmacology, School of Pharmacy, Hoshi University, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142, Japan 2Basic Research Laboratories, Toray Industries, Inc., 111 Tebiro, Kamakura 248, Japan Received May 12, 1994 Accepted June 18, 1994 ABSTRACT-Effects of highly selective 5-opioid receptor antagonists on the morphine-induced place preference in ddy and p,-opioid receptor deficient CXBK mice were investigated. Pretreatment with naltrin dole (NTI: a non-selective 5-opioid receptor antagonist), 7-benzylidenenaltrexone (BNTX: a selective 5, opioid receptor antagonist) or naltriben (NTB: a selective 52-opioid receptor antagonist) abolished the mor phine-induced place preference in ddy mice in a dose-dependent manner. These findings suggest that the morphine-induced place preference may be mediated by both d, and 52-opioid receptors. On the other hand, in p,-opioid receptor deficient CXBK mice, pretreatment with these selective 5-opioid receptor an tagonists did not affect the morphine-induced place preference, although pretreatment with ;3-funaltrex amine (13-FNA: a selective p-opioid receptor antagonist) significantly inhibited the morphine-induced place preference. [D-Pen 2,D-Pen5]enkephalin (DPDPE: a 0,-opioid receptor agonist) and [D-Ala2, Glu4]deltorphin (deltorphin II: a 52-opioid receptor agonist) induced a significant place preference in ddy mice, but not in CXBK mice. These results suggest that d, and 52-opioid receptors in the nucleus accumbens that are related to the DPDPE and deltorphin II-induced place preference may be dysfunctional and/or poor in CXBK mice. These findings also indicate that 5, and 52-opioid receptors may be involved in the modulation of the reinforcing effect of morphine. Keywords: Conditioned place preference, Morphine, 5-Opioid receptor agonist, 5-Opioid receptor antagonist, CXBK mouse Work over several years has suggested interactions be tween p and 5-opioids. Recently, Porreca and colleagues have confirmed that at sub-antinociceptive doses 5-opioid receptor agonists modulate antinociceptive responses to p-opioid receptor agonists (1). Thus administration of the selective 5-agonist [D-Pen 2,D-Pen']enkephalin (DPDPE) at a dose that by itself produces no anti nociception potentiates the potency of i.c.v. morphine. Numerous attempts have been made to study the role of various opioid-receptor types in the development of morphine tolerance and physical dependence., -Opioid receptors, especially, are known to play a major role in the development of morphine tolerance and dependence (2-4). Morphine is a,u-preferring agonist, but it also in teracts with 5 and K-opioid receptors both in vitro and in vivo (5). Recently, the involvement of 5 and K-opioid receptors in the development of morphine tolerance and dependence has been reported (6 8). More recently, evi dence for the presence of 5-opioid receptor subtypes, i.e., 5, and 52-receptors, has been reported (9-11). More recently, Miyamoto et al. (12, 13) have reported that 52 opioid receptors are involved in the development of acute and chronic dependence on morphine and that continu ous blockade rather than intermittent blockade of 52 receptors is necessary to inhibit morphine dependence. The p and 5-agonists may have similar effects on the mesolimbic dopaminergic (DAergic) system, which may account for their similar effects on motivation, motor be havior and biochemical analyses (14-16). Activation of the mesolimbic DAergic system by,o-agonists and 5 agonists can elicit a reinforcing effect in addition to the motor-activating effect (17, 18). Furthermore, Koob (19) proposed that the mesolimbic DAergic, projection is a key component of the opioid reward mechanism. DPDPE acts as a S,-opioid receptor agonist (9), while [D Ala2,Glu4]deltorphin (deltorphin II) acts as a 52-opioid receptor agonist (10). While DPDPE has been shown to produce a significant place preference (14), the motiva

tional effect of deltorphin II is not yet clear. It has been shown that the morphine-induced place preference is not antagonized by the peptide o-opioid receptor antagonist ICI 174,864 (3 tag, i.c.v.) (14). However, the effects of o1 and o2-opioid receptor antagonists on the reinforcing effect of morphine are not yet clear. On the other hand, it is well known that the CXBK strain of mouse, which is derived from a cross between the C57BL/6 and BALB/c strains, is deficient in central pl-opioid receptors (20). It is possible that the role of the interaction between p and 5-opioid receptors in mor phine-induced place preference can be clarified by using p,-opioid receptor deficient CXBK mice. In the present study, the effects of the non-selective o opioid receptor antagonist naltrindole (NTI), the selec tive o1-opioid receptor antagonist 7-benzylidenenal trexone (BNTX) and the selective d2-opioid receptor antagonist naltriben (NTB) on the morphine-induced place preference in ddy mice and i1-opioid receptor deficient CXBK mice were investigated. Moreover, we also examined the motivational effects of o-opioid recep tor agonists in ddy and CXBK mice. MATERIALS AND METHODS Animals Male ddy mice (Tokyo Experiment Animal Co., Ltd., Tokyo) weighing 20 to 30 g were housed in groups of 10 15 in a temperature-controlled room at 22 ± 1 C with a 12 hr light-dark cycle (light on 8:00 a.m. to 8:00 p.m.). Male CXBK mice weighing 18 to 31 g at the beginning of the experiments were obtained from Jackson Labora tories (Bar Harbor, ME, USA). Laboratory chow and water were available ad libitum. Place conditioning Conditioning was conducted by an unbiased procedure according to Suzuki et al. (21, 22). The apparatus used was a shuttle box (15 x 30 x 15 cm: w x 1 x h) that was divided into two compartments of equal size. One com partment was white with a textured floor and the other was black with a smooth floor. For conditioning, mice were immediately confined to one compartment after drug injections and to another compartment after saline injections. The pairings of injec tion (drug or saline) and compartment (white or black) were counterbalanced across all of the subjects. Con ditioning sessions (3 for drug: 3 for saline) were conduct ed for a 60-min period once a day. On day 7, conditioning was tested as follows: the partition separating the two compartments was raised 7 cm above the floor, and a neu tral platform was inserted along the seam separating the compartments. The time spent in each compartment dur ing a 900-sec session was then measured automatically by an infrared beam sensor (KN-80; Natsume Seisakusyo, Tokyo) in a blinded fashion. The position of the mouse was defined by the position of its forelimbs and head. All sessions were conducted under conditions of dim illumina tion (40 lux) and white masking noise. Mice exhibited no preference for either of the place states under these condi tions. Effects of NTI, BNTX and NTB on the morphine-in duced place preference The control mice were injected s.c. with saline (10 ml/kg) instead of drugs in each of the conditioning ses sions. After their daily injection of saline, these mice were confined to one compartment on one day and to the other compartment on the next day. This process was repeated 3 times. Before the start of the experiments, either saline control (black or white-floored compartment place) was randomly chosen to be the substitute for the drug-associat ed place. Morphine (1, 5 and 10 mg/kg) and saline were injected s.c. into ddy mice on alternate days. The ddy mice were immediately confined to each compartment after the injec tion. NTI (0.5 and 1 mg/kg), BNTX (0.35 and 0.7 mg/kg) and NTB (0.1 and 0.5 mg/kg) were injected s.c. at 30, 20 and 30 min before the conditioning or the morphine injec tion, respectively. Drugs and saline were injected on alter nate days. For CXBK mice, morphine (5 mg/kg) and saline were injected s.c. on alternate days. NTI (0.5 mg/kg), BNTX (0.7 mg/kg) and NTB (0.5 mg/kg) were injected s.c. at 30, 20 and 30 min before the morphine injection, respec tively. Furthermore, the selective p-opioid receptor an tagonist l-funaltrexamine ((3-FNA; 10 mg/kg) was inject ed s.c. 24 hr before the morphine injection. Drugs and sa line were injected on alternate days. Place conditioning produced by DPDPE and deltorphin II DPDPE (15 nmol) or deltorphin II (4 nmol) and saline were injected i.c.v. to ddy and CXBK mice on alternate days. Several opioids were given into the lateral cerebral ventricle of mice (2.5 tal/mouse) according to the method of Haley and McCormick (23). The control mice were in jected i.c.v. with saline (2.5 tat/mouse) instead of drugs in each of the conditioning sessions. Drugs The drugs used in the present study were morphine hydrochloride (Sankyo Co., Tokyo), [D-Pen 2,D-Pen 5] enkephalin (DPDPE; Peninsula Laboratories, CA, USA), [D-Ala2, Glu4]deltorphin (deltorphin II, Peninsula Laboratories), naltrindole methanesulfonate hydrate

(NTI), 7-benzylidenenaltrexone tartrate (BNTX), naltriben methanesulfonate (NTB) and 13-funaltrexamine hydrochloride (j3-fna). NTI, BNTX, NTB and (3-FNA were synthesized by us. All of the drugs were dissolved in saline. Data analysis Conditioning scores represent the time spent in the drug-paired place minus the time spent in the vehicle paired place and are expressed as the mean±s.e.m. Dose-response curves were analyzed by one-way random factorial analysis of variance. The Wilcoxon test was used to determine whether individual doses produced a sig nificant conditioning. RESULTS Effect of NTI, BNTX and NTB on the morphine-induced place preference As shown in Figs. 1 and 2, the s.c.-saline control mice exhibited no preference for either compartment. The mean conditioning scores in MY and CXBK mice were 20.5 ± 40.1 sec and 37.5 ± 45.5 sec, respectively. Fur thermore, the i.c.v.-saline control mice also exhibited no preference (Fig. 3). The mean conditioning scores in ddy and CXBK mice were 8.3 ± 48.6 sec and 8.9 ± 56.0 sec, respectively. NTI (0.5 and 1 mg/kg), BNTX (0.35 and 0.7 mg/kg) and NTB (0.1 and 0.5 mg/kg) induced neither significant place preference nor place aversion in ddy mice (Fig. 1); the mean conditioning scores associated Fig. 1. Effects of NTI (d-opioid receptor antagonist), BNTX (o1-opioid receptor antagonist) and NTB (d2-opioid receptor an tagonist) on morphine-induced place preference in ddy mice. NTI, BNTX or NTB was given s.c. at 30, 20 or 30 min, respective ly, prior to injection of morphine. Each column represents the mean with S.E.M. of 8-21 mice. The asterisk denotes significant preference conditioning (Wilcoxon test: P<0.05 vs. saline). The sharp denotes a significant difference from morphine alone (Wilcoxon test: P<0.05).

Fig. 2. Effect of NTI (0.5 mg/kg), BNTX (0.7 mg/kg), NTB (0.5 mg/kg) and rl-fna (FNA; 10 mg/kg) on the morphine (5 mg/kg) induced place preference in CXBK mice. Each column represents the mean with S.E.M. of 8-21 mice. The asterisk denotes significant preference conditioning (Wilcoxon test: P<0.05 vs. saline). The sharp denotes a significant difference from morphine alone (Wilcoxon test: P < 0.05). with 0.5 and 1 mg/kg NTI were -1.5 ± 50.3 sec and -6.0± 75.2 sec, respectively; the mean conditioning scores associated with 0.35 and 0.7 mg/kg BNTX were 21.7:±_ 34.2 sec and 26.2 ± 54.7 sec, respectively; and the mean conditioning scores associated with 0.1 and 0.5 mg/kg NTB were 36.1 ± 41.6 sec and 26.5 ± 63.9 sec, respectively. Morphine induced a dose-dependent place preference (F(4, 77)=3.01; P<0.05), and significant conditioning was observed at doses of 5 mg/kg (123.5 ± 38.6 sec, Fig. 1) and 10 mg/kg (122.3±26.7 sec). The morphine-in duced place preference was antagonized by pretreatment with NTI, BNTX or NTB in a dose-dependent manner. The morphine (5 mg/kg)-induced place preference was sig nificantly suppressed by pretreatment with NTI (0.5 and 1 mg/kg). The mean conditioning scores of morphine in ddy mice treated with 0.5 and 1 mg/kg NTI were -48.3±37.3 sec and 96.6 ± 12.6 sec, respectively (P<0.05, Fig. 1). BNTX at 0.7 mg/kg significantly sup pressed the morphine (5 mg/kg)-induced place prefer ence, with a mean conditioning score of 53.9 ± 51.1 sec (P < 0. 05, Fig. 1). NTB at 0.5 mg/kg also significantly sup pressed the morphine (5 mg/kg)-induced place prefer ence, with a mean conditioning score of -104.2 ± 42.6 sec (P < 0.05, Fig. 1). In CXBK mice, morphine (5 mg/kg) induced a sig nificant place preference, with a mean conditioning score of 241.0±53.6 sec (Fig. 2). Pretreatment with NTI (0.5 Fig. 3. Place conditioning produced by [D-Pen 2,D-Pen']enkephalin (DPDPE: 15 nmol) and [D-Ala2, Glu4]deltorphin (DELT II: 4 nmol) in two strains of mice. Each column represents the mean with S.E.M. of 10 mice. The asterisk denotes a significant difference from saline (Wilcoxon test: P <0.05). mg/kg), BNTX (0.7 mg/kg) or NTB (0.5 mg/kg) did not affect the morphine (5 mg/kg)-induced place preference, with mean conditioning scores of 320.0---L91.3 sec, 225.5 ± 64.6 sec and 200.5 ± 84.9 sec, respectively. However, pretreatment with 13-FNA (10 mg/kg) sig nificantly blocked the morphine-induced place prefer ence, with a mean conditioning score of 102.5 --'-112.7 sec (P < 0.05). Place conditioning produced by DPDPE and deltorphin II As shown in Fig. 3, i.c.v. DPDPE (15 nmol) and deltor phin II (4 nmol) induced a significant place preference in ddy mice. The mean conditioning scores produced by DPDPE and deltorphin II were 297.0± 118.8 sec and 158.3 -L 40.3 sec, respectively. However, under the same conditions as with ddy mice, neither DPDPE (15 nmol) nor deltorphin II (4 nmol) produced a place preference in CXBK mice, with mean conditioning scores of 41.6±101.5 sec and 38.0±81.2 sec (Fig. 3). DISCUSSION In the present study, the morphine (5 mg/kg)-induced place preference was completely prevented in ddy mice by all of the highly selective non-peptide 3-opioid recep tor antagonists tested, including NTI (24, 25) for d-, BNTX (26) for 61 and NTB (27) for o2-opioid receptors. By themselves, these o-opioid receptor antagonists, NTI (0.5 and 1 mg/kg), BNTX (0.35 and 0.7 mg/kg) and NTB (0.1 and 0.5 mg/kg), induced neither place preference nor

place aversion. However, these doses of d-opioid receptor antagonists selectively antagonize the antinociceptive effects of the respective d-opioid receptor-subtype agonists but not p-opioid receptor agonists (11, 26, 28). These findings suggest that both d, and d2-opioid recep tors may be partially involved in the morphine-induced place preference in ddy mice. Morphine is a p-opioid receptor-preferring agonist, but it also interacts with 5 and K-opioid receptors both in vitro and in vivo (5). Shippenberg et al. (14) and our re cent study (16) demonstrated that the reinforcing effects of d-opioid receptor agonists may result from the activa tion of central d-opioid receptors. Furthermore, we previ ously reported that the morphine-induced place prefer ence, locomotor-enhancing effect and enhancement of do pamine-turnover were antagonized by pretreatment with j3-funaltrexamine (13-FNA)(21, 28, 29). These results sug gest that 13-FNA-sensitive sites may play an important role in the expression of dopamine-dependent actions, such as place preference and locomotor-enhancing effect, by mor phine in mice. Although r3-fna selectively antagonizes p-opioid receptors (30), i.c.v. 13-FNA interferes with the binding of both p and d-ligands in mouse brain mem branes (31). Rothman et al. (32) proposed that /3-FNA selectively antagonizes p-opioid receptors that exist within a functional complex with 6-receptors. Thus, 13-FNA may have a influence on d-opioid receptors. Recently, evi dence for the presence of d-opioid receptor subtypes, d, and d2-opioid receptors, has been reported (9-11). More recently, Miyamoto et al. (12, 13) have found that d2 opioid receptors are involved in the development of physi cal dependence on morphine. In the present study, we found that pretreatment with the non-selective d-opioid receptor antagonist NTI, the selective d1-opioid receptor antagonist BNTX or the selective d2-opioid receptor an tagonist NTB abolishes the morphine-induced place preference in ddy mice in a dose-dependent manner. These results suggest that suppression of the morphine-in duced place preference by pretreatment with NTI, BNTX and NTB may result from antagonism of d-opioid recep tor subtypes, and that d, and 52-receptors are involved in the reinforcing effect of morphine. However, it has been shown that the morphine-induced place preference is not antagonized by the peptide d-opioid receptor antagonist ICI 174,864 (14). More recently, Comer et al. (33) have reported that the discriminative stimulus effects of the nonpeptide 5-opioid receptor agonist BW373U86 are an tagonized by NTI but not ICI 174,864. Therefore, there may be some differences between NTI and ICI 174,864. Several investigations have indicated that the ventral tegmental area (VTA), from which mesolimbic DA neu rons originate, is an anatomical substrate of the reward ing effect of opioids. Intra-VTA morphine has been shown to induce a conditioned place preference (34) and a reinforcing effect in a self-administration procedure (35). In a microdialysis study, [D-Ala2,N-MePhe4,Gly-ol5] enkephalin (DAGO), a selective p-opioid receptor agonist, produced a significant dose-related increase in DA release in the nucleus accumbens (N. Acc) following injection into the VTA, while injection into the N. Acc had no effect (36). Thus, activation of p-opioid receptors on the VTA may play an important role in the activation of the mesolimbic DAergic system by morphine. Further more, an autoradiography study indicated that d-opioid receptors are located in the N. Acc, but not in the VTA (37). Longoni et al. (38) recently reported that deltorphin II, a selective 52-opioid receptor agonist, enhances loco motor activity and DA release in the N. Acc, but not in the striatum. Furthermore, the d-opioid receptor an tagonist NTI inhibits the discriminative stimulus effects of morphine (33). These reports indicate that the regions of the N. Acc where d-opioid receptors appear in high con centrations are important for mediating the DA-related behaviors that are induced by p and d-opioid receptor agonists. When d-opioid receptors in the N. Acc are masked by d-opioid receptor antagonists, activation of mesolimbic DA neurons that are related to the morphine induced place preference may be reduced. Therefore, the morphine-induced place preference may be mediated primarily by p-opioid receptors and partially by d-opioid receptors, including d, and d2-opioid receptor subtypes. On the other hand, we have previously reported that f1,-opioid receptor deficient CXBK mice show a sig nificant morphine-induced place preference, which sug gests that the morphine-induced place preference may be mediated by fit-opioid receptors (21). In the present study, pretreatment with the non-selective d-opioid recep tor antagonist NTI, the selective d1-opioid receptor an tagonist BNTX or the selective d2-opioid receptor an tagonist NTB did not affect the morphine-induced place preference in CXBK mice. Furthermore, the morphine-in duced place preference in CXBK mice was antagonized by the selective p-opioid receptor antagonist r3-fna. These results suggest that inhibition of the morphine-induced place preference by d-opioid receptor antagonists may be related to the interaction between p,-opioid receptors and d-opioid receptors, and that p2-opioid receptors that medi ate the morphine-induced place preference may be regulat ed by this interaction. To demonstrate this hypothesis, we examined the reinforcing effect of the d1-opioid receptor agonist DPDPE and the d2-opioid receptor agonist deltor phin II in CXBK mice. In the present study, we found that DPDPE and deltorphin II both produce a significant place preference in ddy mice, but not in CXBK mice. The dose of DPDPE and deltorphin II used in the present study is known to produce antinociception in mice (9, 10,

39), and DPDPE or deltorphin-ii-induced antinocicep tion was antagonized by pretreatment with the respective o-opioid receptor subtype antagonists but not p-opioid receptor antagonists (40). Furthermore, Raffa et al. (41) recently reported that DPDPE, but not deltorphin II, produces antinociception in CXBK mice. It is believed that the density of o2-opioid receptors in CXBK mice may be low. These results suggest that 61 and o2-opioid recep tors in the N. Acc that are related to DPDPE and deltor phin II-induced place preference may be dysfunctional and/or poor in CXBK mice. In conclusion, we found that 61 and o2-opioid receptor antagonists antagonize the morphine-induced place preference, suggesting that 61 and o2-opioid receptors may be involved in the modulation of the reinforcing effect of morphine. We also found that DPDPE and del torphin II produce a significant place preference in ddy mice, but not in CXBK mice, suggesting that 51 and o2 opioid receptors in the N. 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