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Blockade of morphine-induced place preference through the activation of κ-opioid receptors in mice
$201 BLOCKADE OF MORPHINE-INDUCED PLACE PREFERENCE THROUGH THE ACTIVATION OF ~-OPIOID RECEPTORS IN MICE Funada M, Suzuki T, Narita M, Misawa M and Nagase H.*, Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo 142 and *Basic Research Laboratories, Toray Industries, Inc., Kamakura 248, Japan
Summary. The effects of pretreatment with the ~-agonists U-50,488H (U-50) and E-2078 on the morphine (MRP)-induced conditioned place preference (CPP) were examined in mice. Both U-50 (1 mg/kg, s.c.) and E-2078 (0.1 mg/kg, s.c.) induced a slight, non-significant place aversion. Pretreatmerit with U-50 (1 mg/kg) or E-2078 (0.1 mg/kg) abolished the MRP (3 mg/kg, s.c.)-induced CPP. In addition, MRP (3 mg/kg, s.c.) significantly increased the levels of the DA metabolites, DOPAC and HVA in the limbic forebrain (nucleus accumbens and olfactory tubercle). These effects were reduced by pretreatment with U-50. These results suggest that K-agonists suppress the MRPinduced CPP, and that activation of K-receptors could suppress the reinforcing effects of MRP which may be induced by enhanced DA transmission in the mesolimbic DA system. INTRODUCTION The rewarding properties of opioid agonists have been examined using the self-administration and conditioned place preference paradigms. Preferencially Ix-opioid receptor agonists exert the reinforcing effects in both paradigms (1). In contrast, ~-opioid receptor agonists such as U-50,488H and the stable dynorphin analog E-2078, produce aversive effects (1,2). These opposing effects of opioids appear to be caused by changes in dopamine (DA) transmission in the central DA system (1). Based on these findings, we hypothesized that ~-opioid agonists might be useful for the chronic pain states, especially when ~x-agonists are employed. In the present study, the effects of pretreatment with •-opioid agonists, U-50,488H and E-2078, on the morphine-induced place preference were investigated in mice. In addition, the effects of U-50,488H on the morphine-induced elevation of DA metabolites in the mesolimbic DA systems were also examined in mice. MATERIALS AND METHODS Animals: Male ddY mice (Tokyo Experimental Animals Ltd., Tokyo) weighing 20 to 30 g were housed in groups of 10 in a temperature-controlled room. The mice maintained on a 12 h light/dark cycle with laboratory mouse chow and water available ad libitum. Place conditioning: Place conditioning was conducted as previously described (4). The apparatus used was a shuttlebox (15x30x15 cm: wxlxh) which was divided into two compartments of equal size. One compartment was white with a textured floor and the other was black with a smooth floor. Conditioning sessions (3 for drug: 3 for vehicle, 60 min in duration) were conducted once a day. In a combination study, the influence of pretreatment with the •-agonists U-50,488H and E-2078 on the morphine-induced place preference was investigated. Mice were injected with U-50,488H and E-2078 10 min before treatment with morphine. However, with apomorphine (1 mg/kg, s.c.), conditioning sessions (15 min in duration) were conducted once a day and mice were injected with U-50,488H or E-2078 45 min before treatment with apomorphine. Saline was administered twice via the corresponding route to these drugs. Drugs and saline were injected on alternate days. On day 7, tests of conditioning were performed as follows: The partition separating the two compartments was raised to 7 cm above the floor, and a neutral platform was inserted along the seam separating the compartments. The time spent in each compartment during a 900 s session was then measured automatically using a sensor (KN-80, Natsume Seisakusyo, Tokyo, Japan). Neurochemical analysis: Using HPLC-ECD system, the concentrations of DA and DA metabolites in the limbic forebrain (containing nucleus accumbens (N.Acc) and olfactory tubercles) were determined. U-50,488H (1 mg/kg, s.c.) was given to mice 10 min prior to the s.c. morphine injection. Mice were sacrificed 60 min after s.c. morphine (3 mg/kg). The time of sacrifice corresponded to the duration of the conditioning session. Data analysis: Conditioning scores represent the time spent in the drug-paired place minus the time spent in the vehiclepaired place and are expressed as the mean + S.E.M. The Wilcoxon test was used to determine whether individual doses produced a significant conditioning. Neurochemical data were statistically
$202 evaluated with a one-way ANOVA followed by a Dunnett's test. RESULTS
Morphine caused a dose-related preference for the drag-associated place; the effects at doses of 3 and 5 mg/kg were significant. On the other hand, U-50,488H or E-2078 produced a dose-related conditioned place aversion. Both U-50,488H (1 mg/kg, s.c.) and E-2078 (0.1 mg/kg, s.c.) induced a slight, non-significant place aversion. Pretreatment with U-50,488H or E-2078 abolished the morphine (3 and 5 mg/kg)-induced place preference. The inhibitory effects of the K-agonists were antagonized by a K-opioid antagonist nor-binaltorphimine (nor-BNI; 3 mg/kg, s.c.). In contrast, pretreatment with U-50,488H did not affect the place preference induced by the DA receptor agonist apomorphine (1 mg/kg, s.c.). In addition, morphine (3 mg/kg, s.c.) increased the levels of the DA metabolites (DOPAC by 28.4%; and HVA by 20.8%). Pretreatment with U-50,488H significantly reduced the morphine-induced elevation of DA metabolites in the limbic forebrain. DISCUSSION
The results of the present study demonstrated that morphine produces place preference, and it did significantly elevate the DA metabolites (DOPAC and HVA) without changing the DA steadystate levels in the limbic forebrain. These findings suggest that the activation of mesolimbic DA system may be critically linked to the expression of morphine-induced place preference. Pretreatment with K-agonists abolished either the place preference conditioning or DA metabolites elevations produced by morphine. The blocking effect of K-agonists on morphine-induced place preference suggests that the activation of central ~ opioid system may suppress the reinforcing effects of morphine. From the in vitro studies, it has been demonstrated that the electrically evoked release of the [3H]DA from N.Acc slice was inhibited by U-50,488H, but not either a selective ~t-agonist [D-Ala 2, MePhe 4, Gly-olS]enkephalin nor a 6-agonist [D-Pen 2, D-PenS]enkephalin (5). This report indicates that the inhibitory effect of opioids on the DA release from dopaminergic nerve terminals projected from the ventral tegmental area may be mediated only by presynaptic ~-opioid receptors. In the present study, we demonstrated that the DA agonist apomorphine-induced place preference was not reduced by pretreatment with U-50,488H. This result suggests that the motivational effects of apomorphine may be mediated through the activation of postsynaptical DA receptors, while morphine-induced motivational effects occurred through modulation of the activity of DA neurons. Our findings suggest that the K-opioid receptors which are located on dopaminergic terminals may play an important role in the modulation of DA transmission in the limbic forebrain. Finally, in our previous study, it was indicated that the inactivation of •-opioid system by nor-BNI may potentiate the development of tolerance to morphine analgesia, and may aggravate the naloxone-precipitated withdrawal signs (6). The present findings demonstrated that morphine-induced place preference was blocked by K-agonist treatment. In view of the present findings and previous studies, it seems that the activation of endogenous ~-opioid systems may reduce some of morphine's side effects such as analgesic tolerance, physical dependence and psychic dependence. REFERENCES
(1) (2) (3) (4) (5) (6)
Shippenberg T.S. and Herz A. (1987) Brain Res. 436: 169-172. Bals-Kubik R., Herz A. and Shippenberg T.S. (1989) Psychopharmacology 98: 203-206. Suzuki T., Shiozaki Y., Masukawa Y., Misawa M. and Nagase H. (1992) Jpn. J. Pharmacol. 58: 435-442. Suzuki T., Funada M., Narita M., Misawa M. and Nagase H. (1991) Eur. J. Pharmacol. 205: 85-88. Heijna M.H., Padt M., Hogenboom F., Portoghese P.S., Mulder A.H. and Schoffelmeer A.N.M. (1990) Eur. J. Pharmacol. 18: 267-278. Suzuki T., Narita M., Takahashi Y., Misawa M. and Nagase H. (1992) Eur. J. Pharmacol. 213: 91-97.
Summary. The effects of pretreatment with the ~-agonists U-50,488H (U-50) and E-2078 on the morphine (MRP)-induced conditioned place preference (CPP) were examined in mice. Both U-50 (1 mg/kg, s.c.) and E-2078 (0.1 mg/kg, s.c.) induced a slight, non-significant place aversion. Pretreatmerit with U-50 (1 mg/kg) or E-2078 (0.1 mg/kg) abolished the MRP (3 mg/kg, s.c.)-induced CPP. In addition, MRP (3 mg/kg, s.c.) significantly increased the levels of the DA metabolites, DOPAC and HVA in the limbic forebrain (nucleus accumbens and olfactory tubercle). These effects were reduced by pretreatment with U-50. These results suggest that K-agonists suppress the MRPinduced CPP, and that activation of K-receptors could suppress the reinforcing effects of MRP which may be induced by enhanced DA transmission in the mesolimbic DA system. INTRODUCTION The rewarding properties of opioid agonists have been examined using the self-administration and conditioned place preference paradigms. Preferencially Ix-opioid receptor agonists exert the reinforcing effects in both paradigms (1). In contrast, ~-opioid receptor agonists such as U-50,488H and the stable dynorphin analog E-2078, produce aversive effects (1,2). These opposing effects of opioids appear to be caused by changes in dopamine (DA) transmission in the central DA system (1). Based on these findings, we hypothesized that ~-opioid agonists might be useful for the chronic pain states, especially when ~x-agonists are employed. In the present study, the effects of pretreatment with •-opioid agonists, U-50,488H and E-2078, on the morphine-induced place preference were investigated in mice. In addition, the effects of U-50,488H on the morphine-induced elevation of DA metabolites in the mesolimbic DA systems were also examined in mice. MATERIALS AND METHODS Animals: Male ddY mice (Tokyo Experimental Animals Ltd., Tokyo) weighing 20 to 30 g were housed in groups of 10 in a temperature-controlled room. The mice maintained on a 12 h light/dark cycle with laboratory mouse chow and water available ad libitum. Place conditioning: Place conditioning was conducted as previously described (4). The apparatus used was a shuttlebox (15x30x15 cm: wxlxh) which was divided into two compartments of equal size. One compartment was white with a textured floor and the other was black with a smooth floor. Conditioning sessions (3 for drug: 3 for vehicle, 60 min in duration) were conducted once a day. In a combination study, the influence of pretreatment with the •-agonists U-50,488H and E-2078 on the morphine-induced place preference was investigated. Mice were injected with U-50,488H and E-2078 10 min before treatment with morphine. However, with apomorphine (1 mg/kg, s.c.), conditioning sessions (15 min in duration) were conducted once a day and mice were injected with U-50,488H or E-2078 45 min before treatment with apomorphine. Saline was administered twice via the corresponding route to these drugs. Drugs and saline were injected on alternate days. On day 7, tests of conditioning were performed as follows: The partition separating the two compartments was raised to 7 cm above the floor, and a neutral platform was inserted along the seam separating the compartments. The time spent in each compartment during a 900 s session was then measured automatically using a sensor (KN-80, Natsume Seisakusyo, Tokyo, Japan). Neurochemical analysis: Using HPLC-ECD system, the concentrations of DA and DA metabolites in the limbic forebrain (containing nucleus accumbens (N.Acc) and olfactory tubercles) were determined. U-50,488H (1 mg/kg, s.c.) was given to mice 10 min prior to the s.c. morphine injection. Mice were sacrificed 60 min after s.c. morphine (3 mg/kg). The time of sacrifice corresponded to the duration of the conditioning session. Data analysis: Conditioning scores represent the time spent in the drug-paired place minus the time spent in the vehiclepaired place and are expressed as the mean + S.E.M. The Wilcoxon test was used to determine whether individual doses produced a significant conditioning. Neurochemical data were statistically
$202 evaluated with a one-way ANOVA followed by a Dunnett's test. RESULTS
Morphine caused a dose-related preference for the drag-associated place; the effects at doses of 3 and 5 mg/kg were significant. On the other hand, U-50,488H or E-2078 produced a dose-related conditioned place aversion. Both U-50,488H (1 mg/kg, s.c.) and E-2078 (0.1 mg/kg, s.c.) induced a slight, non-significant place aversion. Pretreatment with U-50,488H or E-2078 abolished the morphine (3 and 5 mg/kg)-induced place preference. The inhibitory effects of the K-agonists were antagonized by a K-opioid antagonist nor-binaltorphimine (nor-BNI; 3 mg/kg, s.c.). In contrast, pretreatment with U-50,488H did not affect the place preference induced by the DA receptor agonist apomorphine (1 mg/kg, s.c.). In addition, morphine (3 mg/kg, s.c.) increased the levels of the DA metabolites (DOPAC by 28.4%; and HVA by 20.8%). Pretreatment with U-50,488H significantly reduced the morphine-induced elevation of DA metabolites in the limbic forebrain. DISCUSSION
The results of the present study demonstrated that morphine produces place preference, and it did significantly elevate the DA metabolites (DOPAC and HVA) without changing the DA steadystate levels in the limbic forebrain. These findings suggest that the activation of mesolimbic DA system may be critically linked to the expression of morphine-induced place preference. Pretreatment with K-agonists abolished either the place preference conditioning or DA metabolites elevations produced by morphine. The blocking effect of K-agonists on morphine-induced place preference suggests that the activation of central ~ opioid system may suppress the reinforcing effects of morphine. From the in vitro studies, it has been demonstrated that the electrically evoked release of the [3H]DA from N.Acc slice was inhibited by U-50,488H, but not either a selective ~t-agonist [D-Ala 2, MePhe 4, Gly-olS]enkephalin nor a 6-agonist [D-Pen 2, D-PenS]enkephalin (5). This report indicates that the inhibitory effect of opioids on the DA release from dopaminergic nerve terminals projected from the ventral tegmental area may be mediated only by presynaptic ~-opioid receptors. In the present study, we demonstrated that the DA agonist apomorphine-induced place preference was not reduced by pretreatment with U-50,488H. This result suggests that the motivational effects of apomorphine may be mediated through the activation of postsynaptical DA receptors, while morphine-induced motivational effects occurred through modulation of the activity of DA neurons. Our findings suggest that the K-opioid receptors which are located on dopaminergic terminals may play an important role in the modulation of DA transmission in the limbic forebrain. Finally, in our previous study, it was indicated that the inactivation of •-opioid system by nor-BNI may potentiate the development of tolerance to morphine analgesia, and may aggravate the naloxone-precipitated withdrawal signs (6). The present findings demonstrated that morphine-induced place preference was blocked by K-agonist treatment. In view of the present findings and previous studies, it seems that the activation of endogenous ~-opioid systems may reduce some of morphine's side effects such as analgesic tolerance, physical dependence and psychic dependence. REFERENCES
(1) (2) (3) (4) (5) (6)
Shippenberg T.S. and Herz A. (1987) Brain Res. 436: 169-172. Bals-Kubik R., Herz A. and Shippenberg T.S. (1989) Psychopharmacology 98: 203-206. Suzuki T., Shiozaki Y., Masukawa Y., Misawa M. and Nagase H. (1992) Jpn. J. Pharmacol. 58: 435-442. Suzuki T., Funada M., Narita M., Misawa M. and Nagase H. (1991) Eur. J. Pharmacol. 205: 85-88. Heijna M.H., Padt M., Hogenboom F., Portoghese P.S., Mulder A.H. and Schoffelmeer A.N.M. (1990) Eur. J. Pharmacol. 18: 267-278. Suzuki T., Narita M., Takahashi Y., Misawa M. and Nagase H. (1992) Eur. J. Pharmacol. 213: 91-97.