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Differential effects of morphine and cocaine on locomotor activity and sensitization in μ-opioid receptor knockout mice
Neuroscience Letters 344 (2003) 37–40 www.elsevier.com/locate/neulet
Differential effects of morphine and cocaine on locomotor activity and sensitization in m-opioid receptor knockout mice Ji-Hoon Yooa, Eun-Mi Yanga, Seok-Yong Leea, Horace H. Lohb, Ing K. Hoc, Choon-Gon Janga,* a
Department of Pharmacology, College of Pharmacy, Sungkyunkwan University, Cheoncheon-dong, Jangan-ku, Suwon, 440-746, South Korea b Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA c Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA Received 4 December 2002; received in revised form 20 March 2003; accepted 27 March 2003
Abstract The present study was undertaken to investigate the hypothesis that the m-opioid receptors play a crucial role in locomotor activity and sensitization to cocaine and morphine in wild-type and m-opioid receptor knockout mice. Our results show that morphine and cocaine increased locomotor activity in wild-type mice, but failed to increase locomotor activity in m-opioid receptor knockout mice, suggesting a contribution of m-opioid receptor. Repeated morphine treatment induced sensitization in wild-type mice, but this was not observed in mopioid receptor knockout mice. In contrast repeated cocaine treatment produced sensitization in m-opioid receptor knockout mice, but not in wild-type mice on day 6. However, the sensitization to cocaine was observed in m-opioid receptor knockout and wild-type mice on day 12. These results suggest that the expression of m-opioid receptor may contribute to locomotor sensitization induced by morphine, but that mopioid receptor does not play an important role in mediating sensitization to cocaine. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: m-Opioid receptors; Knockout mice; Cocaine; Morphine; Locomotor activity; Behavioral sensitization
Drugs of abuse, such as opiates and psychostimulants, induce enhanced locomotor activity after repeated treatment [8]. This phenomenon is termed behavioral sensitization and is thought to underlie certain aspects of addiction. Behavioral sensitization is thought to be due to the repeated use of drugs of abuse produced by the incremental neuroadaptation of the neural system, rendering it increasingly, and perhaps permanently, hypersensitive to drugs [15]. Recent successes in developing knockout mice with the m-opioid receptor gene deletions [11] have demonstrated that m-opioid receptor plays a role in the locomotor activity induced by morphine [2], whereas acute administration of cocaine did not change locomotor activity enhancement in m-opioid receptor knockout mice [3]. However, previous investigations have demonstrated that the blockade of opioid receptor with naloxone wholly antagonizes the locomotor activating effect of cocaine [7]. In the present study, we examined whether the m-opioid receptor plays a crucial role in locomotor responses to the
* Corresponding author. Tel.: þ 82-31-290-7780; fax: þ82-31-292-8800. E-mail address: [email protected] (C.G. Jang).
acute and repeated administration of cocaine and morphine using m-opioid receptor knockout and wild-type mice. The generation of m-opioid receptor knockout mice has been described previously [11]. The nature of the genomic DNA in the animals used in the present study was confirmed by Southern blot hybridization (data not shown). All mice used were between 8 and 10 weeks of age. They were maintained in an animal room on a 12 h light/dark cycle and at constant temperature (22 ^ 2 8C). All procedures for animal care and breeding were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by our Institutional Animal Care and Use Committee. m-Opioid receptor knockout and wild-type mice were used between 8 and 10 weeks of age. The doses of drugs used in the experiments were based on previously published work [9,10]. The mice were introduced to the testing room 1 h before being tested. Each mouse was habituated for 10 min in an activity cage before treatment. All mice were injected with 10 mg/kg of morphine hydrochloride (Keukdong Pharm. Co. Ltd., Inchon, South Korea) subcutaneously or 15 mg/kg of cocaine hydrochloride (Macfarlan Smith Ltd., Edinburgh, UK) intraperitoneally, or saline. After
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00410-5
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treatment, each mouse was placed back in the activity cage, locomotor activity was recorded for 25 min using a video camera placed above the activity cage (opaque plastic, 25 £ 32 £ 32 cm), and video-recordings were made under red illumination (25 lux). Horizontal activity was analyzed by using a computer-based video-tracking system (NeuroVision, Pusan National University, Pusan, South Korea). For repeated treatment, morphine (10 mg/kg, s.c.) or cocaine (15 mg/kg, i.p.) was injected once daily at 09:00 and 10:00 h for 6 consecutive days. Generally, because the expression of sensitization is more robust following a drugfree interval [1], the animals did not receive any treatment for 5 days following the last injection (Fig. 1). On days 6 and 12, animals received challenge of morphine (10 mg/kg, s.c.), cocaine (15 mg/kg, i.p.) or saline, and locomotor activity was measured. All drugs were dissolved in physiological saline (0.9%), and injections were performed during the light phase of the light/dark cycle. Data are expressed as means ^ SEM for 9 –12 mice. Statistical analyses were performed using two-way analysis of variance (ANOVA) after Student – Newman –Keuls post hoc testing using Prism 3.0 (Graphpad Software, Inc.) On day 1, treatment with morphine and cocaine significantly increased locomotor activity in wild-type mice. Morphine treatment significantly altered the amplitude of the locomotor response by the genotype (treatment, Fð1;40Þ ¼ 16:13, P , 0:001; genotype, Fð1;40Þ ¼ 11:28, P , 0:01; treatment – genotype interaction, Fð1;40Þ ¼ 5:74, P , 0:05; two-way ANOVA) (Fig. 2A). Cocaine treatment also significantly altered the amplitude of the locomotor response by the genotype (treatment, Fð1;41Þ ¼ 55:77, P , 0:001; genotype, Fð1;41Þ ¼ 24:18, P , 0:001; treatment – genotype interaction, Fð1;41Þ ¼ 17:61, P , 0:001; two-way ANOVA) (Fig. 2B). The post hoc analysis showed that locomotor responses to morphine or cocaine were significantly lower in the m-opioid receptor knockout mice than in the wild-type mice (morphine: P , 0:001; cocaine: P , 0:001). The behavioral sensitization induced by the repeated administration of morphine and cocaine was evaluated on days 6 and 12. After repeated treatment with saline, locomotor responses were similar on day 1 in m-opioid receptor knockout and wild-type mice (data not shown). However, repeated treatment with morphine showed locomotor sensitization in wild-type mice on day 6 (P , 0:05,
Fig. 1. The procedure used to evaluate the sensitization to the locomotor effects of morphine and cocaine in the m-opioid receptor knockout mice.
Fig. 2. Effects of morphine (10 mg/kg, s.c.) and cocaine (15 mg/kg, i.p.) on locomotor activity in the wild-type and m-opioid receptor knockout mice. Data are shown as means ^ SEM for 9–12 mice. (A) Morphine-induced locomotor activity in the wild-type and m-opioid receptor knockout mice on day 1. ***P , 0:001, compared to the saline treatment group (wild-type mice); ###P , 0:001, compared to the morphine treatment group (wild-type mice). (B) Cocaine-induced locomotor activity in the wild-type and mopioid receptor knockout mice on day 1. ***P , 0:001, compared to the saline treatment group (wild-type mice); ###P , 0:001, compared to the cocaine treatment group (wild-type mice).
Fig. 3A), which was not observed in m-opioid receptor knockout mice. Post hoc analysis showed that locomotor sensitization was significantly lower in m-opioid receptor knockout mice than in the wild-type mice (P , 0:001). Repeated treatment with cocaine showed locomotor sensitization in m-opioid receptor knockout mice (P , 0:05), but not in the wild-type mice on day 6 (Fig. 3B). The expressions of behavioral sensitization induced by morphine or cocaine challenge were evaluated on day 12. The locomotor response to morphine challenge was maintained in the wild-type mice (Fig. 3A), but this expression of sensitization did not reach statistical significance. Locomotor response after morphine challenge was significantly lower in m-opioid receptor knockout mice than in the wild-type mice (P , 0:001). Locomotor response to cocaine challenge was significantly increased in wild-type mice on day 12 versus day 6 (Fig. 3B). Locomotor response to cocaine challenge was maintained in m-opioid receptor knockout mice, but this was significantly lower than that shown by wild-type mice (P , 0:05). In the present study we investigated the role of the m-
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Fig. 3. Effects of morphine (10 mg/kg, s.c.) and cocaine (15 mg/kg, i.p.) on the behavioral sensitization in wild-type and m-opioid receptor knockout mice. Data are shown as means ^ SEM for 6–12 mice. (A) Locomotor activity of repeated morphine injection. *P , 0:05, **P , 0:01, compared to wild-type mice on day 1; ###P , 0:001, compared to wild-type mice on day 6; þ þ þ P , 0:001, compared to wild-type mice on day 12. (B) Locomotor activity of repeated cocaine injection. *P , 0:05, compared to wild-type mice on day 1; %P , 0:05, compared to m-opioid receptor knockout mice on day 1; þP , 0:05, compared to wild-type mice on day 6; # P , 0:05, compared to wild-type mice on day 12.
opioid receptor in the locomotor response induced by two drugs of abuse, morphine and cocaine, using m-opioid receptor knockout mice. As shown in Fig. 2A,B, basal locomotor responses to saline injection were similar in wildtype and m-opioid receptor knockout mice. Locomotor response to repeated saline treatment was similar on day 1 in both m-opioid receptor knockout and wild-type mice (data not shown). These results are in line with those of previous studies showing that wild-type and m-opioid receptor knockout mice show similar patterns of locomotor activity [2,4,19] and that no genotype differences are detectable at the level of horizontal and vertical locomotion during the habituation period using activity cages [4]. As expected, acute and repeated morphine injection failed to increase the locomotor activity of m-opioid receptor knockout mice (Figs. 2A and 3A). These results are in agreement with those of previous studies [2,19] and confirm
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that the m-opioid receptor plays an important role in modulating morphine-induced locomotor activity. In our study, acute cocaine treatment significantly decreased locomotor activity in m-opioid receptor knockout mice as compared to wild-type mice. This result differs from that of a previous study, which found no changes in locomotor activity after cocaine treatment in m-opioid receptor knockout mice or wild-type mice [3]. However, another study showed that the blockade of the opioid receptor with naloxone abrogates the locomotor activating effect of cocaine in rats [7]. Furthermore, available evidence suggests that the endogenous opioid system is involved in mediating or modulating some of the effects of cocaine [3]. It is well known that the locomotor effect of cocaine depends on a number of factors, such as the treatment paradigm, the dose and the genetic background [14,22]. Therefore, differences between results could be due to different experimental conditions, i.e. differences between test doses (20 mg/kg, 40 mg/kg) and measurement times (45 min) [3], and our test dose (15 mg/kg) and measurement time (25 min). In our behavioral sensitization study, the repeated administration of morphine failed to induce behavioral sensitization in m-opioid receptor knockout mice on days 6 and 12 (Fig. 3A). In line with this result, other studies have shown the absence of morphine-induced motor stimulation in m-opioid receptor knockout mice [19,21]. These results suggest that locomotor activity and sensitization to morphine are mediated directly through the m-opioid receptor. In our present study, repeated administration of cocaine induced behavioral sensitization in wild-type mice on day 12, whereas m-opioid receptor knockout mice displayed robust behavioral sensitization on day 6 and maintained this sensitization until day 12. These results raise two important issues. First, the presence of cocaineinduced sensitization in m-opioid receptor knockout mice indicates that the m-opioid receptor has a minor role in cocaine sensitization. In line with this conclusion, other studies found no changes in cocaine-induced place preference between wild-type and m-opioid receptor knockout mice [4]. Furthermore, the m-opioid receptor selective antagonist b-funaltrexamine did not influence cocaine selfadministration in rats [12]. Second, a display of robust behavioral sensitization to cocaine in m-opioid receptor knockout mice indicates that the lack of m-opioid receptor may augment cocaine-induced sensitization. The lack of mopioid receptor can also generally induce a compensatory modulation in other neurotransmitter systems. For example, the lack of m-opioid receptor can induce the up- or downregulation of other opioid receptor subtypes, which leads to altered cocaine sensitization. Recently it was shown that knocking out the genes of special opioid receptor subtypes changes the activity of the others genes. However, the results obtained have not been uniform [13]. Moreover, various studies have demonstrated that manipulations of dopamine neurotransmission can regulate the expressions of opioid peptides and opioid receptors. More specifically,
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dopamine depletion or neuroleptic treatment enhances enkephalin mRNA and peptide levels in striatopallidal neurons [5]. And recently it was reported that the loss of mopioid receptor diminished D2 receptor binding [3], suggesting compensatory modulation. The down-regulation of the dopaminergic system may enhance the d- or k-opioid receptor systems. Additionally, cocaine treatment was also found to elevate striatonigral dynorphin content [17,18] and striatal prodynorphin mRNA levels [20]. It has also been reported that the d- [6] and k-opioid receptors [16] are involved in cocaine sensitization. m-Opioid receptor knockout mice that we used here have a tone of d- and k-opioid receptor system [11]. Therefore, we speculate that subchronic cocaine treatment may enhance the endogenous opioid activity, and thus enhanced levels of d- and k-opioid activity may be responsible for increased cocaine sensitization in m-opioid receptor knockout mice. We noted with interest that the cocaine-induced expression of sensitization was lower in m-opioid receptor knockout mice than in the wild-type mice on day 12 (P , 0:05) (Fig. 3B), which may be due to the reduced initial locomotor activity to cocaine in m-opioid receptor knockout mice. Taken together our results suggest that the m-opioid receptor plays an important role in modulating the acute locomotor activity induced by morphine or cocaine. Our behavioral sensitization study suggests that the m-opioid receptor plays a role in modulating the adaptive responses induced by morphine, and that the m-opioid receptor systems do not play a key role in mediating the development of cocaine sensitization.
Acknowledgements This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (02-PJ1-PG3-21301-0017). The authors thank Dr E.Y. Cha and Mr S.-G. Lee for providing a automated analysis system (NeuroVison).
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Differential effects of morphine and cocaine on locomotor activity and sensitization in m-opioid receptor knockout mice Ji-Hoon Yooa, Eun-Mi Yanga, Seok-Yong Leea, Horace H. Lohb, Ing K. Hoc, Choon-Gon Janga,* a
Department of Pharmacology, College of Pharmacy, Sungkyunkwan University, Cheoncheon-dong, Jangan-ku, Suwon, 440-746, South Korea b Department of Pharmacology, University of Minnesota Medical School, Minneapolis, MN 55455, USA c Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA Received 4 December 2002; received in revised form 20 March 2003; accepted 27 March 2003
Abstract The present study was undertaken to investigate the hypothesis that the m-opioid receptors play a crucial role in locomotor activity and sensitization to cocaine and morphine in wild-type and m-opioid receptor knockout mice. Our results show that morphine and cocaine increased locomotor activity in wild-type mice, but failed to increase locomotor activity in m-opioid receptor knockout mice, suggesting a contribution of m-opioid receptor. Repeated morphine treatment induced sensitization in wild-type mice, but this was not observed in mopioid receptor knockout mice. In contrast repeated cocaine treatment produced sensitization in m-opioid receptor knockout mice, but not in wild-type mice on day 6. However, the sensitization to cocaine was observed in m-opioid receptor knockout and wild-type mice on day 12. These results suggest that the expression of m-opioid receptor may contribute to locomotor sensitization induced by morphine, but that mopioid receptor does not play an important role in mediating sensitization to cocaine. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: m-Opioid receptors; Knockout mice; Cocaine; Morphine; Locomotor activity; Behavioral sensitization
Drugs of abuse, such as opiates and psychostimulants, induce enhanced locomotor activity after repeated treatment [8]. This phenomenon is termed behavioral sensitization and is thought to underlie certain aspects of addiction. Behavioral sensitization is thought to be due to the repeated use of drugs of abuse produced by the incremental neuroadaptation of the neural system, rendering it increasingly, and perhaps permanently, hypersensitive to drugs [15]. Recent successes in developing knockout mice with the m-opioid receptor gene deletions [11] have demonstrated that m-opioid receptor plays a role in the locomotor activity induced by morphine [2], whereas acute administration of cocaine did not change locomotor activity enhancement in m-opioid receptor knockout mice [3]. However, previous investigations have demonstrated that the blockade of opioid receptor with naloxone wholly antagonizes the locomotor activating effect of cocaine [7]. In the present study, we examined whether the m-opioid receptor plays a crucial role in locomotor responses to the
* Corresponding author. Tel.: þ 82-31-290-7780; fax: þ82-31-292-8800. E-mail address: [email protected] (C.G. Jang).
acute and repeated administration of cocaine and morphine using m-opioid receptor knockout and wild-type mice. The generation of m-opioid receptor knockout mice has been described previously [11]. The nature of the genomic DNA in the animals used in the present study was confirmed by Southern blot hybridization (data not shown). All mice used were between 8 and 10 weeks of age. They were maintained in an animal room on a 12 h light/dark cycle and at constant temperature (22 ^ 2 8C). All procedures for animal care and breeding were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by our Institutional Animal Care and Use Committee. m-Opioid receptor knockout and wild-type mice were used between 8 and 10 weeks of age. The doses of drugs used in the experiments were based on previously published work [9,10]. The mice were introduced to the testing room 1 h before being tested. Each mouse was habituated for 10 min in an activity cage before treatment. All mice were injected with 10 mg/kg of morphine hydrochloride (Keukdong Pharm. Co. Ltd., Inchon, South Korea) subcutaneously or 15 mg/kg of cocaine hydrochloride (Macfarlan Smith Ltd., Edinburgh, UK) intraperitoneally, or saline. After
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00410-5
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treatment, each mouse was placed back in the activity cage, locomotor activity was recorded for 25 min using a video camera placed above the activity cage (opaque plastic, 25 £ 32 £ 32 cm), and video-recordings were made under red illumination (25 lux). Horizontal activity was analyzed by using a computer-based video-tracking system (NeuroVision, Pusan National University, Pusan, South Korea). For repeated treatment, morphine (10 mg/kg, s.c.) or cocaine (15 mg/kg, i.p.) was injected once daily at 09:00 and 10:00 h for 6 consecutive days. Generally, because the expression of sensitization is more robust following a drugfree interval [1], the animals did not receive any treatment for 5 days following the last injection (Fig. 1). On days 6 and 12, animals received challenge of morphine (10 mg/kg, s.c.), cocaine (15 mg/kg, i.p.) or saline, and locomotor activity was measured. All drugs were dissolved in physiological saline (0.9%), and injections were performed during the light phase of the light/dark cycle. Data are expressed as means ^ SEM for 9 –12 mice. Statistical analyses were performed using two-way analysis of variance (ANOVA) after Student – Newman –Keuls post hoc testing using Prism 3.0 (Graphpad Software, Inc.) On day 1, treatment with morphine and cocaine significantly increased locomotor activity in wild-type mice. Morphine treatment significantly altered the amplitude of the locomotor response by the genotype (treatment, Fð1;40Þ ¼ 16:13, P , 0:001; genotype, Fð1;40Þ ¼ 11:28, P , 0:01; treatment – genotype interaction, Fð1;40Þ ¼ 5:74, P , 0:05; two-way ANOVA) (Fig. 2A). Cocaine treatment also significantly altered the amplitude of the locomotor response by the genotype (treatment, Fð1;41Þ ¼ 55:77, P , 0:001; genotype, Fð1;41Þ ¼ 24:18, P , 0:001; treatment – genotype interaction, Fð1;41Þ ¼ 17:61, P , 0:001; two-way ANOVA) (Fig. 2B). The post hoc analysis showed that locomotor responses to morphine or cocaine were significantly lower in the m-opioid receptor knockout mice than in the wild-type mice (morphine: P , 0:001; cocaine: P , 0:001). The behavioral sensitization induced by the repeated administration of morphine and cocaine was evaluated on days 6 and 12. After repeated treatment with saline, locomotor responses were similar on day 1 in m-opioid receptor knockout and wild-type mice (data not shown). However, repeated treatment with morphine showed locomotor sensitization in wild-type mice on day 6 (P , 0:05,
Fig. 1. The procedure used to evaluate the sensitization to the locomotor effects of morphine and cocaine in the m-opioid receptor knockout mice.
Fig. 2. Effects of morphine (10 mg/kg, s.c.) and cocaine (15 mg/kg, i.p.) on locomotor activity in the wild-type and m-opioid receptor knockout mice. Data are shown as means ^ SEM for 9–12 mice. (A) Morphine-induced locomotor activity in the wild-type and m-opioid receptor knockout mice on day 1. ***P , 0:001, compared to the saline treatment group (wild-type mice); ###P , 0:001, compared to the morphine treatment group (wild-type mice). (B) Cocaine-induced locomotor activity in the wild-type and mopioid receptor knockout mice on day 1. ***P , 0:001, compared to the saline treatment group (wild-type mice); ###P , 0:001, compared to the cocaine treatment group (wild-type mice).
Fig. 3A), which was not observed in m-opioid receptor knockout mice. Post hoc analysis showed that locomotor sensitization was significantly lower in m-opioid receptor knockout mice than in the wild-type mice (P , 0:001). Repeated treatment with cocaine showed locomotor sensitization in m-opioid receptor knockout mice (P , 0:05), but not in the wild-type mice on day 6 (Fig. 3B). The expressions of behavioral sensitization induced by morphine or cocaine challenge were evaluated on day 12. The locomotor response to morphine challenge was maintained in the wild-type mice (Fig. 3A), but this expression of sensitization did not reach statistical significance. Locomotor response after morphine challenge was significantly lower in m-opioid receptor knockout mice than in the wild-type mice (P , 0:001). Locomotor response to cocaine challenge was significantly increased in wild-type mice on day 12 versus day 6 (Fig. 3B). Locomotor response to cocaine challenge was maintained in m-opioid receptor knockout mice, but this was significantly lower than that shown by wild-type mice (P , 0:05). In the present study we investigated the role of the m-
J.-H. Yoo et al. / Neuroscience Letters 344 (2003) 37–40
Fig. 3. Effects of morphine (10 mg/kg, s.c.) and cocaine (15 mg/kg, i.p.) on the behavioral sensitization in wild-type and m-opioid receptor knockout mice. Data are shown as means ^ SEM for 6–12 mice. (A) Locomotor activity of repeated morphine injection. *P , 0:05, **P , 0:01, compared to wild-type mice on day 1; ###P , 0:001, compared to wild-type mice on day 6; þ þ þ P , 0:001, compared to wild-type mice on day 12. (B) Locomotor activity of repeated cocaine injection. *P , 0:05, compared to wild-type mice on day 1; %P , 0:05, compared to m-opioid receptor knockout mice on day 1; þP , 0:05, compared to wild-type mice on day 6; # P , 0:05, compared to wild-type mice on day 12.
opioid receptor in the locomotor response induced by two drugs of abuse, morphine and cocaine, using m-opioid receptor knockout mice. As shown in Fig. 2A,B, basal locomotor responses to saline injection were similar in wildtype and m-opioid receptor knockout mice. Locomotor response to repeated saline treatment was similar on day 1 in both m-opioid receptor knockout and wild-type mice (data not shown). These results are in line with those of previous studies showing that wild-type and m-opioid receptor knockout mice show similar patterns of locomotor activity [2,4,19] and that no genotype differences are detectable at the level of horizontal and vertical locomotion during the habituation period using activity cages [4]. As expected, acute and repeated morphine injection failed to increase the locomotor activity of m-opioid receptor knockout mice (Figs. 2A and 3A). These results are in agreement with those of previous studies [2,19] and confirm
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that the m-opioid receptor plays an important role in modulating morphine-induced locomotor activity. In our study, acute cocaine treatment significantly decreased locomotor activity in m-opioid receptor knockout mice as compared to wild-type mice. This result differs from that of a previous study, which found no changes in locomotor activity after cocaine treatment in m-opioid receptor knockout mice or wild-type mice [3]. However, another study showed that the blockade of the opioid receptor with naloxone abrogates the locomotor activating effect of cocaine in rats [7]. Furthermore, available evidence suggests that the endogenous opioid system is involved in mediating or modulating some of the effects of cocaine [3]. It is well known that the locomotor effect of cocaine depends on a number of factors, such as the treatment paradigm, the dose and the genetic background [14,22]. Therefore, differences between results could be due to different experimental conditions, i.e. differences between test doses (20 mg/kg, 40 mg/kg) and measurement times (45 min) [3], and our test dose (15 mg/kg) and measurement time (25 min). In our behavioral sensitization study, the repeated administration of morphine failed to induce behavioral sensitization in m-opioid receptor knockout mice on days 6 and 12 (Fig. 3A). In line with this result, other studies have shown the absence of morphine-induced motor stimulation in m-opioid receptor knockout mice [19,21]. These results suggest that locomotor activity and sensitization to morphine are mediated directly through the m-opioid receptor. In our present study, repeated administration of cocaine induced behavioral sensitization in wild-type mice on day 12, whereas m-opioid receptor knockout mice displayed robust behavioral sensitization on day 6 and maintained this sensitization until day 12. These results raise two important issues. First, the presence of cocaineinduced sensitization in m-opioid receptor knockout mice indicates that the m-opioid receptor has a minor role in cocaine sensitization. In line with this conclusion, other studies found no changes in cocaine-induced place preference between wild-type and m-opioid receptor knockout mice [4]. Furthermore, the m-opioid receptor selective antagonist b-funaltrexamine did not influence cocaine selfadministration in rats [12]. Second, a display of robust behavioral sensitization to cocaine in m-opioid receptor knockout mice indicates that the lack of m-opioid receptor may augment cocaine-induced sensitization. The lack of mopioid receptor can also generally induce a compensatory modulation in other neurotransmitter systems. For example, the lack of m-opioid receptor can induce the up- or downregulation of other opioid receptor subtypes, which leads to altered cocaine sensitization. Recently it was shown that knocking out the genes of special opioid receptor subtypes changes the activity of the others genes. However, the results obtained have not been uniform [13]. Moreover, various studies have demonstrated that manipulations of dopamine neurotransmission can regulate the expressions of opioid peptides and opioid receptors. More specifically,
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dopamine depletion or neuroleptic treatment enhances enkephalin mRNA and peptide levels in striatopallidal neurons [5]. And recently it was reported that the loss of mopioid receptor diminished D2 receptor binding [3], suggesting compensatory modulation. The down-regulation of the dopaminergic system may enhance the d- or k-opioid receptor systems. Additionally, cocaine treatment was also found to elevate striatonigral dynorphin content [17,18] and striatal prodynorphin mRNA levels [20]. It has also been reported that the d- [6] and k-opioid receptors [16] are involved in cocaine sensitization. m-Opioid receptor knockout mice that we used here have a tone of d- and k-opioid receptor system [11]. Therefore, we speculate that subchronic cocaine treatment may enhance the endogenous opioid activity, and thus enhanced levels of d- and k-opioid activity may be responsible for increased cocaine sensitization in m-opioid receptor knockout mice. We noted with interest that the cocaine-induced expression of sensitization was lower in m-opioid receptor knockout mice than in the wild-type mice on day 12 (P , 0:05) (Fig. 3B), which may be due to the reduced initial locomotor activity to cocaine in m-opioid receptor knockout mice. Taken together our results suggest that the m-opioid receptor plays an important role in modulating the acute locomotor activity induced by morphine or cocaine. Our behavioral sensitization study suggests that the m-opioid receptor plays a role in modulating the adaptive responses induced by morphine, and that the m-opioid receptor systems do not play a key role in mediating the development of cocaine sensitization.
Acknowledgements This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (02-PJ1-PG3-21301-0017). The authors thank Dr E.Y. Cha and Mr S.-G. Lee for providing a automated analysis system (NeuroVison).
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