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Involvement of dopamine-dependent and -independent mechanisms in the rewarding effects mediated by δ opioid receptor subtypes in mice
Brain Research 744 Ž1997. 327–334
Research report
Involvement of dopamine-dependent and -independent mechanisms in the rewarding effects mediated by d opioid receptor subtypes in mice Tsutomu Suzuki a
a, )
, Minoru Tsuji a , Tomohisa Mori a , Hiroko Ikeda a , Miwa Misawa a , Hiroshi Nagase b
Department of Pharmacology, School of Pharmacy, Hoshi UniÕersity, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142, Japan b Basic Research Laboratories, Toray Industries, Inc., Kamakura 248, Japan Accepted 10 September 1996
Abstract The rewarding effects of the d 1 opioid receptor agonist wD-Pen2 , Pen5 xenkephalin ŽDPDPE. and the d 2 opioid receptor agonist wD-Ala2 xdeltorphin II ŽDELT. on the activity of mesolimbic and nigrostriatal dopamine ŽDA. neurons were examined in mice. Both DPDPE Ž15 nmol, i.c.v.. and DELT Ž5 nmol, i.c.v.. produced a significant place preference in mice. The DPDPE Ž15 nmol, i.c.v..-induced place preference was abolished by 7-benzylidenenaltrexone ŽBNTX; 0.5 mgrkg, s.c.., a d 1 opioid receptor antagonist, but not by naltriben ŽNTB; 0.5 mgrkg, s.c.., a d 2 opioid receptor antagonist. In contrast, the DELT Ž5 nmol, i.c.v..-induced place preference was antagonized by NTB, but not BNTX. I.c.v. injection of DPDPE, but not DELT, at a dose that produced a significant place preference produced a significant elevation of DA turnover in the mouse limbic forebrain, and this effect of DPDPE was antagonized by BNTX but not by NTB. In addition, i.c.v. injection of DPDPE or DELT did not affect DA turnover in the mouse striatum. These results suggest that the rewarding effects produced by the activation of central d 1, but not d 2 , opioid receptors may be caused through the enhancement of the mesolimbic DA neurotransmission, and confirm our previous hypothesis that the DA-dependent and -independent mechanisms may exist in the rewarding effects produced by the activation of central d opioid receptor subtypes. Keywords: d Opioid receptor; Conditioned place preference; Reward; Dopamine turnover; Mesolimbic dopamine system
1. Introduction The existence of m , k and d opioid receptors in the central nervous system is well documented w5,15,19x. In our previous studies, we showed that wD-Ala2 , N-MePhe 4 , Gly-ol 5 xenkephalin ŽDAGO., a selective m opioid receptor agonist, produced a dose-related place preference w33x. In contrast, U-50,488H and E-2078, selective k opioid receptor agonists, produced a dose-dependent conditioned place aversion w8x. Moreover, there have also been studies on rewarding properties of d opioid receptor agonists. For example, wD-Pen2 , Pen5 xenkephalin ŽDPDPE., a selective d opioid receptor agonist, produces a dose-dependent place preference w2,25,33x, and this rewarding effect of DPDPE is abolished by pretreatment with ICI174,864, a selective d
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opioid receptor antagonist w2,25x. Thus, d opioid receptors, as well as m and k opioid receptors, may be involved in the motivational effects of opioids. A great deal of evidence suggests that the central dopamine ŽDA. system is involved in the motivational effects produced by opioids. For example, the conditioned place preference induced by morphine or heroin is attenuated by either pretreatment with DA receptor antagonist w3,29x or 6-hydroxydopamine lesion of the nucleus accumbens ŽN.Acc.., the terminal projection site of the mesolimbic DA system w29x. Moreover, chronic administration of the dopamine D 1 receptor antagonist SCH23390 during conditioning sessions also attenuates both the morphine-induced place preference w26,27,34x and k opioid receptor agonist U-69593-induced place aversion w26,27x. These results suggest that the central DA system, especially D 1 receptors, may play an important role in the expression of the motivational effects of opioids. It is well known that d opioid receptor agonists, like m opioid receptor agonists, also produce a place preference. However, the role of the
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central DA system in the rewarding effects of d opioid receptor agonists is not yet clear. Previous reports have shown that the involvement of opioid receptors in behavioral or rewarding effects depends on the central DA system, although contradictory reports also exist. Pert and Sivit w21x first reported that administration of morphine or enkephalin analogue, DAla2-Met 5-enkephalinamide ŽDALA., into the N.Acc. produced an increase in locomotor activity; interestingly, this effect was not antagonized by administration of DA receptor antagonists. Kalivas et al. w11x reported a similar result, in that administration of DALA into the N.Acc. did not alter the metabolism of DA. A study using self-administration paradigms also demonstrated that cocaine-reinforced responding was completely eliminated by pretreatment with DA receptor antagonist, while heroin self-administration was maintained at constant rates throughout the course of the test session w7x. Surprisingly, it has also been demonstrated that numerous behavioral responses, including locomotor activity, self-administration and place preference, produced by intra-accumbens DALA and systemic administration of heroin are potentiated either by lesion of DA neurons or by chronic blockade of DA receptors w11,12,30,31x. These reports indicate that the behavioral or rewarding effects produced by the activation of central opioid receptors may be partially mediated via DA-independent mechanisms. Recent evidence regarding the direct antinociceptive effects of d opioid receptor agonists has suggested the existence of d opioid receptor subtypes. This conclusion is based on the differential antagonism of DPDPE- and wDAla2 xdeltorphin II ŽDELT.-induced antinociceptions by wDAla2 , Leu5, Cys 6 xenkephalin ŽDALCE. and naltrindole 5X-isothiocyanate Ž5X-NTII., selective d 1 and d 2 opioid receptor antagonists, respectivelyw10x, and on the lack of cross-tolerance between DPDPE and DELT w18x. These findings suggest that DPDPE and DELT mediate their antinociceptions through different d opioid receptor subtypes. DPDPE- and DALCE-sensitive sites are called d 1 opioid receptors, and DELT- and 5X-NTII-sensitive sites are called d 2 opioid receptors. Although several investigators have shown that central d opioid receptors are involved in the rewarding effects of opioids w2,25,33,35x, the role of d opioid receptor subtypes, i.e. d 1 and d 2 opioid receptors, in these rewarding effects is not yet clear. In a previous study, we found that i.c.v. injection of either DPDPE or DELT produced a dose-dependent place preference, and that the DPDPE- but not the DELT-induced place preference was abolished by pretreatment with D 1 receptor antagonist w36x, which suggests that both DA-dependent and -independent mechanisms are involved in the rewarding effects produced by the activation of central d 1 and d 2 opioid receptor subtypes. The aim of the present study was to clarify this suggestion. The present study was designed to determine the effects of DPDPE and DELT at doses that produce
place preference on the activity of mesolimbic and nigrostriatal DA neurons, and we measured the change in DA turnover in regions of the brain which contain terminals of these neurons Žlimbic forebrain and striatum. after i.c.v. injection of DPDPE and DELT at doses that produce place preference using high-performance liquid chromatography with electrochemical detection ŽHPLC-ECD..
2. Materials and methods The present studies were carried out in accordance with the Guide for Care and Use of Laboratory Animals adopted by the Committee on Care and Use of Laboratory Animals of Hoshi University, which is accredited by the Ministry of Education, Science, Sports and Culture, Japan. 2.1. Animals Male ddY mice ŽTokyo Experimental Animals, Tokyo, Japan. weighing 25 to 35 g were housed in a temperaturecontrolled room. They were maintained on a 12:12 h lightrdark cycle Žlights on 08.00–20.00 h. with laboratory mouse chow and water available ad libitum. 2.2. Place conditioning Place conditioning was conducted as previously described using a minor modification of an unbiased procedure w32x. The apparatus consisted of a shuttle box Ž15 = 30 = 15 cm, width = length = height. which was made acrylic resin board and divided into two equal-sized compartments. One compartment was white with a textured floor, and the other was black with a smooth floor. For conditioning, mice were confined to one compartment after drug injection and to the other compartment after saline injection. The order of injection Ždrug or saline. and compartment Žwhite or black. was counterbalanced across subjects. Conditioning sessions Žthree for drug: three for vehicle. were conducted for a 60-min period once daily. 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. After the conditioning sessions, on day 7, mice were not treated with either drugs or saline, and then were placed on the platform. The time spent in each compartment during a 900-s session was then recorded automatically using an infrared beam sensor ŽKN-80, Natsume Seisakusyo Co., Tokyo, Japan. in a blinded fashion. The position of the mouse was defined by the position of its body Že.g., forelimbs and head.. All sessions were conducted under conditions of dim illumination Ž40 lux lamp. and masking white noise ŽPixie, PXJ43B1, Japan Servo Co., Japan.. DPDPE Ž1–15 nmolrmouse. and DELT Ž0.5–5 nmolrmouse. were injected into the lateral cerebral ventri-
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cle of unanesthetized mice. One day before the beginning the drug or saline injection, the mice were anesthetized with ether and a 2-mm double-needle Žtip: 27 gauge = 2 mm; base: 22 gauge = 10 mm; Natsume Seisakusyo, Tokyo, Japan. attached to a 25-m l Hamilton microsyringe was inserted into the unilateral injection site; as a result, single hole for the injection was made in the skull. The unilateral injection site was approximately 2 mm from either side of the midline between the anterior roots of the ears w9x. The head of the mouse was held against a V-shaped holder, and the drugs were injected into the hole under unanesthetized conditions. Solution was injected in a volume of 2.5 m l per mouse over a period of 10 s. 7-Benzylidenenaltrexone ŽBNTX; 0.5 mgrkg, s.c.. and naltriben ŽNTB; 0.5 mgrkg, s.c.. were given to mice 20 min and 30 min, respectively, prior to treatment with DPDPE or DELT. None of the mice that received i.c.v. saline during the conditioning session exhibited a significant preference for either compartment of the test box.
with electrochemical detection ŽECD.. The HPLC system consisted of a delivery pump Ž880-PU, Jasco, Japan., an analytical column ŽEicompak, MA-50DS, Eicom, Japan. and a guard column ŽEicom.. The electrochemical detector ŽEC-100, Eicom. had a graphite electrode ŽWE-3G, Eicom. and was used at a voltage setting of q0.70 V versus an AgrAgCl reference electrode. The mobile phase consisted of 0.1 M sodium acetater0.1 M citric acid buffer, pH 3.5, containing 13–15% methanol, sodium 1-octanesulfonate and EDTA Ž2Na.. The flow rate was set to 1.0 mlrmin with a column temperature of 258C. DPDPE Ž1–15 nmolrmouse. and DELT Ž0.5–5 nmolrmouse. were injected into the lateral cerebral ventricle of unanesthetized mice. Solution was injected in a volume of 2.5 m l per mouse over a period of 10 s. BNTX Ž0.5 mgrkg, s.c.. and NTB Ž0.5 mgrkg, s.c.. were given to mice 20 min and 30 min respectively, prior to treatment with DPDPE.
2.3. Neurochemical analysis
wD-Pen2 , Pen5 xenkephalin ŽDPDPE. and wD-Ala2 xdeltorphin II ŽDELT. were obtained from Peninsula Laboratories ŽCA, USA.. 7-Benzylidenenaltrexone methanesulfonate hydrate ŽBNTX. and naltriben methanesulfonate hydrate ŽNTB. were synthesized by us. All drugs were dissolved in 0.9% NaCl. All doses refer to the salt forms of the drugs.
The concentrations of dopamine ŽDA., 3,4-dihydroxyphenylacetic acid ŽDOPAC. and homovanillic acid ŽHVA. were determined as described previously w8,20x. Mice were sacrificed by dislocation of cervical vertebrae 60 min after saline Ž2.5 m lrmouse, i.c.v.., DPDPE Ž1–15 nmolrmouse, i.c.v.. or DELT Ž0.5–5 nmolrmouse, i.c.v.. injection, and then immersed into a dry icerethanol solution to minimize the postmortem increase of DA-related substances. The brain was quickly removed and the limbic forebrain, including the N.Acc. and olfactory tubercle, and striatum were dissected on an ice-cold glass plate. Briefly, the brain was turned to expose the dorsal surface and a vertical cut was made through the anterior commissure. The resulting frontal part was turned to expose the ventral surface. A vertical cut was passed through the rhinal fissure and the small part, including the accessory olfactory bulb and olfactory nucleus, was removed. The resulting block of tissue was turned to expose the section, and the area bordered by the caudate putamen and the N.Acc. was cut vertically. The block of tissue that included the N.Acc. and olfactory tubercle was considered to be the main portion of the limbic forebrain. The medial borders of the striatum were cut free, and the striatum was removed without the underlying cortex. The block of tissue was removed and was considered to be the main portion of the striatum. The tissues were frozen at y808C and stored until analysis. The frozen tissues were homogenized in 500 m l 0.2 M perchloric acid containing 100 m M EDTA Ž2Na. and 100 ng isoproterenol, as an internal standard. To remove the proteins completely, the homogenates were placed in cold water for 60 min, and then centrifuged at 20 000 = g for 20 min at 08C. The supernatants were maintained at pH 3.0 using 1 M sodium acetate. Samples were applied to a high-performance liquid chromatography ŽHPLC. system
2.4. Drugs
2.5. Data analysis Conditioning scores represent the time spent in the drug-paired place minus the time spent in the saline-paired place, and are expressed as the mean " S.E.M. A one-way analysis of variance ŽANOVA. followed by a Dunnett’s test was used to determine whether individual doses produced a significant conditioning Ž P - 0.05 and P - 0.01.. DA turnover was determined as the DA ratio: DA ratio s DOPAC Žngrg wet tissue. q HVA Žngrg.4 rDA Žngrg.. Neurochemical data were also evaluated statistically with a one-way ANOVA followed by Dunnett’s test.
3. Results 3.1. Place conditionings produced by the d1 opioid receptor agonist DPDPE and the d 2 opioid receptor agonist DELT Preference for the injection-associated place, which was regarded as a substitute for the drug, was calculated. The place conditionings produced by the d 1 opioid receptor agonist DPDPE and the d 2 opioid receptor agonist DELT are shown in Fig. 1. Both the d 1 and d 2 opioid receptor agonists produced a significant preference for the drug-associated place ŽDPDPE: F Ž3,34. s 5.504; P - 0.01, DELT: F Ž3,35. s 4.711; P - 0.05 and 0.01., compared
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Fig. 3. Change in dopamine turnover in the mouse limbic forebrain after treatment with DELT Ž0.05–5 nmolrmouse, i.c.v... Each column represents the mean with S.E.M. of 8 mice.
3.2. The effects of DPDPE and DELT on the DA turnoÕer in the mouse limbic forebrain
Fig. 1. A: place conditioning produced by DPDPE Ž1–15 nmolrmouse, i.c.v.. and DELT Ž0.5–5 nmolrmouse, i.c.v... Ordinate: mean difference Žs. between the times spent in the drug- and saline-paired sides of the test box. Each point represents the mean with S.E.M. of 8–12 mice. ) ) P 0.01 vs. saline control. B: effects of BNTX Ž0.5 mgrkg, s.c.. and NTB Ž0.5 mgrkg, s.c.. on the DPDPE Ž15 nmolrmouse, i.c.v..- and DELT Ž5 nmolrmouse, i.c.v..-induced place preferences. Each column represents the mean with S.E.M. of 8–12 mice. ) P - 0.05 vs. DPDPE-alone group; a P - 0.05 vs. DELT-alone group.
with the saline control. The DPDPE Ž15 nmol, i.c.v..-induced place preference was abolished by BNTX Ž0.5 mgrkg, s.c.. but not by NTB Ž0.5 mgrkg, s.c... In contrast, the DELT Ž5 nmol, i.c.v..-induced place preference was abolished by pretreatment with NTB but not by BNTX.
Fig. 2. Change in dopamine turnover in the mouse limbic forebrain after treatment with DPDPE Ž1–15 nmolrmouse, i.c.v... Each column represents the mean with S.E.M. of 8 mice. ) ) P - 0.01 vs. saline control.
The effects of DPDPE and DELT on the DA turnover in the mouse limbic forebrain are shown in Fig. 2 and Fig. 3. I.c.v. injection of DPDPE Ž15 nmol. produced a significant increase in DA turnover in the mouse limbic forebrain Ž F Ž3,28. s 10.523; P - 0.01.. In contrast, i.c.v. injection of DELT Ž0.5–5 nmol. did not produce a significant change in the DA turnover in the mouse limbic forebrain Ž F Ž3,28. s 0.567; not significant Žn.s.... 3.3. Effects of d opioid receptor antagonists on the DPDPE-induced increase in DA turnoÕer in the mouse limbic forebrain The effects of d opioid receptor antagonists on the DPDPE-induced increase in DA turnover in the mouse limbic forebrain are shown in Fig. 4. Neither BNTX Ž0.5 mgrkg, s.c.. nor NTB Ž0.5 mgrkg, s.c.. alone affected DA turnover in the mouse limbic forebrain wthe means of DA ratio were 0.168 " 0.004, 0.163 " 0.005 and 0.164 " 0.006 in saline, BNTX and NTB treated groups, respectively; F Ž2,21. s 0.182; n.s.x. The DPDPE Ž15 nmol,
Fig. 4. Effects of BNTX Ž0.5 mgrkg, s.c.. and NTB Ž0.5 mgrkg, s.c.. on the enhancement of dopamine turnover in the mouse limbic forebrain in mice produced by DPDPE Ž15 nmolrmouse, i.c.v... BNTX and NTB were administered 20 and 30 min prior to treatment with DPDPE, respectively. Each column represents the mean with S.E.M. of 8 mice. )) P - 0.01 vs. saline control; aa P - 0.01 vs. DPDPE-alone group.
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Fig. 5. Change in dopamine turnover in the mouse striatum after treatment with DPDPE Ž5–15 nmolrmouse, i.c.v.. and DELT Ž0.5–5 nmolrmouse, i.c.v... Each column represents the mean with S.E.M. of 8 mice.
i.c.v..-induced increase in the DA ratio in the mouse limbic forebrain was abolished by pretreatment with BNTX Ž0.5 mgrkg, s.c.., a d 1 opioid receptor antagonist, but not by NTB Ž0.5 mgrkg, s.c.., a d 2 opioid receptor antagonist. 3.4. The effects of DPDPE and DELT on the DA turnoÕer in the mouse striatum The effects of DPDPE and DELT on DA turnover in the mouse striatum are shown in Fig. 5. I.c.v. injection of neither DPDPE Ž1–15 nmol. nor DELT Ž0.5–5 nmol. affected the DA ratio in the mouse striatum Ž F Ž3,28. s 0.410; n.s. and F Ž3,28. s 0.171; n.s...
4. Discussion The present study demonstrated that the i.c.v. administration of DPDPE Ž15 nmol., a selective d 1 opioid receptor agonist, or DELT Ž5 nmol., a selective d 2 opioid receptor agonist, produced a significant conditioned place preference. Moreover, the place preference induced by DPDPE was abolished by BNTX, a selective d 1 opioid receptor antagonist, but not by NTB, a selective d 2 opioid receptor antagonist. In contrast, the DELT-induced place preference was abolished by NTB, but not by BNTX. We previously reported that both BNTX and NTB alone induced neither significant place preference nor place aversion w35x. Therefore, these results suggest that the DPDPE- and DELT-induced place preferences may be produced via the activation of central d 1 and d 2 opioid receptors, respectively, and that the activation of both central d 1 and d 2 opioid receptors may play a significant role in the expression of the reward. In the present study, i.c.v. injection of DPDPE at a dose that produces a significant place preference produced a
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significant increase in DA turnover in the mouse limbic forebrain, and this effect of DPDPE was abolished by pretreatment with BNTX but not with NTB. We previously reported that NTB alone did not affect the level of DA turnover in the mouse limbic forebrain w20x. Moreover, we confirmed in the present study that both BNTX and NTB alone did not modify it Žsee Section 3.. These results suggest that activation of central d 1 opioid receptors by DPDPE may increase mesolimbic DA neurotransmission. Several previous reports have suggested that the rewarding effects of opioids may be caused by a change in DA transmission in the central DA system. For example, the conditioned place preference produced by morphine and heroin was abolished by pretreatment with DA receptor antagonists in rats and mice w3,29,32x. Similarly, the heroin-induced place preference was blocked by 6-hydroxydopamine lesion of the N.Acc., the terminal projection site of the mesolimbic dopaminergic system w3x. Moreover, chronic administration of SCH23390, a selective dopamine D 1 receptor antagonist, during conditioning sessions attenuates the morphine-induced place preference w13,26,27,34x. Thus, activation of the central DA system, and especially D 1 receptors, may be implicated in the rewarding effects of opioids. In our previous study, the DPDPE-induced place preference was abolished by pretreatment with SCH23390, but not with sulpiride, a selective D 2 receptor antagonist w36x. It has been shown that i.c.v. treatment with DPDPE enhances DA release w28x and the concentration of the DA metabolite DOPAC w16x in the N.Acc. Therefore, the results of the present study provide the further evidence to clarify the our previous hypothesis that the enhancement of DA release and the activation of central DA receptors, especially D 1 receptors, may play an important role in the rewarding effect produced by the activation of central d 1 opioid receptors. In contrast to the results with DPDPE, the i.c.v. injection of DELT at a dose that produces a significant place preference did not modify DA turnover in the mouse limbic forebrain. We previously found that the DELT-induced place preference was not abolished by pretreatment with the DA receptor antagonists SCH23390 and sulpiride w36x, and concluded that the rewarding effect of DELT, which is produced by the activation of central d 2 opioid receptors, does not occur through the enhancement of central DA release, and that some other dopamine-independent mechanism may play an important role in this case. Previous reports have also demonstrated the existence of a DA-independent mechanism in opioid-mediated hyperlocomotion and rewarding effects. Pert and Sivit w21x reported that microinjection of the peptidase-resistant enkephalin analog DALA into the N.Acc. produced behavioral hyperactivity, and that this effect of DALA was not antagonized by DA receptor antagonist. Similarly, Kalivas et al. w11x demonstrated that although DALA injection into the N.Acc. produced behavioral hyperactivity Žlocomotion and rearing., this effect was not blocked by intra-accu-
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mbens injection of fluphenazine, a DA receptor antagonist, or by destruction of the mesolimbic dopaminergic system with 6-hydroxydopamine. Furthermore, they also demonstrated that the injection of DALA into the N.Acc. did not affect the levels of DA or its metabolites in the N.Acc. These data demonstrate that DALA causes behavioral hyperactivity by acting on the N.Acc. via a DA-independent mechanism. However, Longoni et al. w14x have reported that while haloperidol and raclopride fail to affect deltorphin-induced hyperactivity, specific D 1 receptor antagonist SCH23390 effectively blocks it. Although the effect of SCH23390 on the DALA-induced hyperactivity is not investigated, in consideration of this report, it is possible that a specific D 1 receptor-mechanism might be involved on the DALA-induced hyperactivity as in the case of deltorphin. However, Amalric and Koob w1x also reported that heroin produces the behavioral hyperactivity by activating to some extent the DA mesolimbic pathway, but more importantly, by stimulating a postsynaptic pathway in the N.Acc. which is independent of a DA transmission. This report may support the DA-independent hypothesis in opioid-mediated hyperlocomotion. Based on these reports, it is suggested that the expression of opioid-induced behavioral hyperactivity may partially involve a DA-independent mechanism. Moreover, it is previously demonstrated that cocaine-reinforced responding was significantly attenuated by pretreatment with a DA receptor antagonist or the destruction of the DA terminals in the N.Acc. with 6-hydroxydopamine, while heroin self-administration was unaffected in a self-administration paradigm w7,22x, which suggests that the rewarding effects of opiates can be DA-independent under some circumstances. A similar DA-independent effect for both heroin-self-administration and heroininduced place preference was observed using chronic dopamine receptor blockade w31x. Thus, it is also possible that the rewarding effects of d opioid receptor agonists involve a DA-independent mechanism. Based on these previous reports and our present findings, we conclude that the rewarding effect induced by the activation of central d 2 opioid receptors does not occur through the modulation of central DA release, and that some other DA-independent mechanismŽs. may play an important role in the expression of the rewarding effects of d 2 opioid receptor agonists. Contrast to our previous results, Longoni et al. w14x reported that local application by reverse dialysis of DELT to N.Acc. resulted in a motor stimulation and an increase in extracellular DA concentration, and concluded that DELT-induced motor stimulation was mediated by DA-independent function. Although the detailed evidence to clarify the discrepancy between the previous and present findings cannot be provided, one possibility is differences in degrees to activate the d 2 opioid receptors, and therefore, it is possible that treatment with more high doses of DELT might produce the change in DA turnover in the limbic forebrain. Moreover, it is important to note that there are methodological differences to estimate the d 2-
mediated function produced by DELT between these studies, i.e. rewarding properties and motor stimulant behavior. Therefore, although a more detailed investigation might be required to clarify the interaction between d 2 opioid receptors and DA systems in the N.Acc., we concluded that, at least on the dose ranges which produce the rewarding properties, DELT did not affect the DA neuronal activity. In the present study, i.c.v. injection of DPDPE and DELT did not modify the DA turnover in the mouse striatum, the terminal projection site of the nigrostriatal DA system. There is strong evidence that the mesolimbic, rather than the nigrostriatal, DA system plays an important role in mediating the rewarding effects of opioids. Microinjection of morphine into the ventral tegmental area ŽVTA. produces a place preference w4,23,24x, and this effect can be prevented by neurochemical destruction of DA neurons w23,24x. Moreover, morphine at a dose which produces a significant conditioned place preference significantly elevates the level of DA metabolites without changing DA steady-state levels in the limbic forebrain; however, these changes are not observed in the striatum w8x. In vivo microdialysis studies have demonstrated that DA neurotransmission in the N.Acc. is enhanced by treatment with m and d opioid receptor agonists at the same doses that produce rewarding effects w6,28x. These findings suggest that mesolimbic DA neurons rather than nigrostriatal DA neurons may play an important role in the development of the rewarding effect of morphine. In addition, evidence of regional differences in the sensitivity of DA neurons to opioids has also been reported. Electrophysiological studies have shown that morphine elicits a greater increase in the firing rate of DA cells in the VTA than in the substantia nigra w17x. Similarly, the morphine-induced increase in extracellular DA levels is greater in the N.Acc. than in the striatum w6x. I.c.v. injection of DPDPE at the same dose range used in the present study also produced an increase in the concentration of DOPAC and accumulation of DOPA in the N.Acc., but had no effect in the striatum w16x. Along with these previous reports, the present data suggest that activation of mesolimbic, rather than nigrostriatal, DA neurons may be especially important in the development of the rewarding effects of d opioid receptor agonists. In conclusion, the present data demonstrate that i.c.v. injection of DPDPE but not DELT at a dose that produces a place preference also produces a significant elevation of DA turnover in the mouse limbic forebrain. These findings suggest that activation of the mesolimbic DA system may play an important role in the rewarding effects of DPDPE but not of DELT, and confirm our previous hypothesis that the rewarding effects induced by the activation of central d 1 opioid receptors may be produced through the enhancement of DA release and the activation of DA receptors, especially D 1 receptors. In contrast, the rewarding effect induced by the activation of central d 2 opioid receptors may occur through some DA-independent mechanismŽs..
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Acknowledgements This work was supported by a scientific research grant to T.S. from the Ministry of Health and Welfare and by a Research Grant Ž5A-7. for Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan to T.S. We would like to thank Mr. Hiroyuki Goto for his excellent technical assistance.
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w15x Lord, J.A.H., Waterfield, A.A., Hughes, J. and Kosterlitz, H.W., Endogenous opioid peptides: multiple agonists and receptors, Nature, 267 Ž1977. 495–499. w16x Manzanares, J., Durham, R.A., Lookingland, K.J. and Moore, K.E., d-Opioid receptor-mediated regulation of central dopaminergic neurons in the rat, Eur. J. Pharmacol., 249 Ž1993. 107–112. w17x Matthews, W.R. and German, D.C., Electrophysiological evidence for excitation of rat ventral tegmental area dopamine neuron by morphine, Neuroscience, 11 Ž1984. 617–625. w18x Mattia, A., Vanderah, T., Mosberg, H.I. and Porreca, F., Lack of antinociceptive cross-tolerance between wD-Pen2 , D-Pen5 xenkephalin and wD-Ala2 xdeltorphin II in mice: Evidence for delta receptor subtypes, J. Pharmacol. Exp. Ther., 258 Ž1991. 583–587. w19x Mognan, J., Paterson, S.J., Tavani, A. and Kosterlitz, H.W., The bind spectrum of narcotic analgesic drugs with different agonist and antagonist properties, Naunyn-Schmiedeberg’s Arch. Pharmacol., 319 Ž1982. 197–205. w20x Narita, M., Suzuki, T., Funada, M., Misawa, M. and Nagase, H., Involvement of d-opioid receptors in the effects of morphine in locomotor activity and the mesolimbic dopaminergic system in mice, Psychopharmacology, 111 Ž1993. 423–426. w21x Pert, A. and Sivit, C., Neuroanatomical focus for morphine and enkephalin-induced hypermotility, Nature, 265 Ž1977. 645–647. w22x Pettit, H.O., Ettenberg, A., Bloom, F.E. and Koob, G.F., Destruction of dopamine in the nucleus accumbens selectively attenuates cocaine but not heroin self-administration in rats, Psychopharmacology, 84 Ž1984. 167–173. w23x Phillips, A.G. and LePiane, F.G., Reinforcing effects of morphine microinjection into the ventral tegmental area, Pharmacol. Biochem. BehaÕ., 12 Ž1980. 965–968. w24x Phillips, A.G. and LePiane, F.G., Reward produced by microinjection of ŽD-ala- 2 ., met 5-enkephalinamide into the ventral tegmental area, BehaÕ. Brain. Res., 5 Ž1982. 225–229. w25x Shippenberg, T.S., Bals-Kubik, R. and Herz, A., Motivational properties of opioids: evidence that an activation of d-receptors mediates reinforcement proccesses, Brain Res., 436 Ž1987. 234–239. w26x Shippenberg, T.S. and Herz, A., Place preference conditioning reveals the involvement of D1-dopamine receptors in the motivational properties of m- and k-opioid agonists, Brain Res., 436 Ž1987. 169–172. w27x Shippenberg, T.S. and Herz, A., Motivational effects of opioids: influence of D-1 versus D-2 receptor antagonists, Eur. J. Pharmacol., 151 Ž1988. 233–242. w28x Spanagel, R., Herz, A. and Shippenberg, T.S., The effects of opioid peptides on dopamine release in the nucleus accumbens: An in vivo microdialysis study, J. Neurochem., 55 Ž1990. 1734–1740. w29x Spyraki, C., Fibiger, N.C. and Phillips, A.G., Attenuation of heroin reward in rats by disruption of mesolimbic dopamine system, Psychopharmacology, 79 Ž1983. 278–283. w30x Stinus, L., Nadaud, D., Jauregui, J. and Kelley, A.E., Chronic treatment with five different neuroleptics elicits behavioral supersensitivity to opiate infusion into the nucleus accumbens, Biol. Psychiatry, 21 Ž1986. 34–48. w31x Stinus, L., Nadaud, D., Deminiere, J.M., Jauregui, J., Hand, T.T. and Le Moal, M., Chronic flupentixol treatment potentiates the reinforcing properties of systemic heroin administration, Biol. Psychiatry, 26 Ž1989. 363–371. w32x Suzuki, T., Masukawa, Y. and Misawa, M., Drug interactions in the reinforcing effects of over-the-counter cough syrups, Psychopharmacology, 102 Ž1990. 438–442. w33x Suzuki, T., Funada, M., Narita, M., Misawa, M. and Nagase, H., Pertussis toxin abolishes m- and d-opioid agonist-induced place preference, Eur. J. Pharmacol., 205 Ž1990. 85–88. w34x Suzuki, T., Funada, M., Narita, M., Misawa, M. and Nagase, H.,
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Morphine-induced place preference in the CXBK mouse: characterisistics of m opioid receptor subtypes, Brain Res., 602 Ž1993. 45–52. w35x Suzuki, T., Yoshiike, M., Mizoguchi, H., Kamei, J., Misawa, M. and Nagase, H., Blockade of d-opioid receptors prevents morphine-induced place preference in mice, Jpn. J. Pharmacol., 66 Ž1994. 131–137.
w36x Suzuki, T., Tsuji, M., Mori, T., Misawa, M. and Nagase, H., The effects of dopamine D1 and D 2 receptor antagonists on the rewarding effects of d 1 and d 2 opioid receptor agonists in mice, Psychopharmacology, 124 Ž1996. 211–218.
Research report
Involvement of dopamine-dependent and -independent mechanisms in the rewarding effects mediated by d opioid receptor subtypes in mice Tsutomu Suzuki a
a, )
, Minoru Tsuji a , Tomohisa Mori a , Hiroko Ikeda a , Miwa Misawa a , Hiroshi Nagase b
Department of Pharmacology, School of Pharmacy, Hoshi UniÕersity, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142, Japan b Basic Research Laboratories, Toray Industries, Inc., Kamakura 248, Japan Accepted 10 September 1996
Abstract The rewarding effects of the d 1 opioid receptor agonist wD-Pen2 , Pen5 xenkephalin ŽDPDPE. and the d 2 opioid receptor agonist wD-Ala2 xdeltorphin II ŽDELT. on the activity of mesolimbic and nigrostriatal dopamine ŽDA. neurons were examined in mice. Both DPDPE Ž15 nmol, i.c.v.. and DELT Ž5 nmol, i.c.v.. produced a significant place preference in mice. The DPDPE Ž15 nmol, i.c.v..-induced place preference was abolished by 7-benzylidenenaltrexone ŽBNTX; 0.5 mgrkg, s.c.., a d 1 opioid receptor antagonist, but not by naltriben ŽNTB; 0.5 mgrkg, s.c.., a d 2 opioid receptor antagonist. In contrast, the DELT Ž5 nmol, i.c.v..-induced place preference was antagonized by NTB, but not BNTX. I.c.v. injection of DPDPE, but not DELT, at a dose that produced a significant place preference produced a significant elevation of DA turnover in the mouse limbic forebrain, and this effect of DPDPE was antagonized by BNTX but not by NTB. In addition, i.c.v. injection of DPDPE or DELT did not affect DA turnover in the mouse striatum. These results suggest that the rewarding effects produced by the activation of central d 1, but not d 2 , opioid receptors may be caused through the enhancement of the mesolimbic DA neurotransmission, and confirm our previous hypothesis that the DA-dependent and -independent mechanisms may exist in the rewarding effects produced by the activation of central d opioid receptor subtypes. Keywords: d Opioid receptor; Conditioned place preference; Reward; Dopamine turnover; Mesolimbic dopamine system
1. Introduction The existence of m , k and d opioid receptors in the central nervous system is well documented w5,15,19x. In our previous studies, we showed that wD-Ala2 , N-MePhe 4 , Gly-ol 5 xenkephalin ŽDAGO., a selective m opioid receptor agonist, produced a dose-related place preference w33x. In contrast, U-50,488H and E-2078, selective k opioid receptor agonists, produced a dose-dependent conditioned place aversion w8x. Moreover, there have also been studies on rewarding properties of d opioid receptor agonists. For example, wD-Pen2 , Pen5 xenkephalin ŽDPDPE., a selective d opioid receptor agonist, produces a dose-dependent place preference w2,25,33x, and this rewarding effect of DPDPE is abolished by pretreatment with ICI174,864, a selective d
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Corresponding author. Fax: Ž81 . Ž3 . 5498-5787; e-mail: [email protected]
opioid receptor antagonist w2,25x. Thus, d opioid receptors, as well as m and k opioid receptors, may be involved in the motivational effects of opioids. A great deal of evidence suggests that the central dopamine ŽDA. system is involved in the motivational effects produced by opioids. For example, the conditioned place preference induced by morphine or heroin is attenuated by either pretreatment with DA receptor antagonist w3,29x or 6-hydroxydopamine lesion of the nucleus accumbens ŽN.Acc.., the terminal projection site of the mesolimbic DA system w29x. Moreover, chronic administration of the dopamine D 1 receptor antagonist SCH23390 during conditioning sessions also attenuates both the morphine-induced place preference w26,27,34x and k opioid receptor agonist U-69593-induced place aversion w26,27x. These results suggest that the central DA system, especially D 1 receptors, may play an important role in the expression of the motivational effects of opioids. It is well known that d opioid receptor agonists, like m opioid receptor agonists, also produce a place preference. However, the role of the
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central DA system in the rewarding effects of d opioid receptor agonists is not yet clear. Previous reports have shown that the involvement of opioid receptors in behavioral or rewarding effects depends on the central DA system, although contradictory reports also exist. Pert and Sivit w21x first reported that administration of morphine or enkephalin analogue, DAla2-Met 5-enkephalinamide ŽDALA., into the N.Acc. produced an increase in locomotor activity; interestingly, this effect was not antagonized by administration of DA receptor antagonists. Kalivas et al. w11x reported a similar result, in that administration of DALA into the N.Acc. did not alter the metabolism of DA. A study using self-administration paradigms also demonstrated that cocaine-reinforced responding was completely eliminated by pretreatment with DA receptor antagonist, while heroin self-administration was maintained at constant rates throughout the course of the test session w7x. Surprisingly, it has also been demonstrated that numerous behavioral responses, including locomotor activity, self-administration and place preference, produced by intra-accumbens DALA and systemic administration of heroin are potentiated either by lesion of DA neurons or by chronic blockade of DA receptors w11,12,30,31x. These reports indicate that the behavioral or rewarding effects produced by the activation of central opioid receptors may be partially mediated via DA-independent mechanisms. Recent evidence regarding the direct antinociceptive effects of d opioid receptor agonists has suggested the existence of d opioid receptor subtypes. This conclusion is based on the differential antagonism of DPDPE- and wDAla2 xdeltorphin II ŽDELT.-induced antinociceptions by wDAla2 , Leu5, Cys 6 xenkephalin ŽDALCE. and naltrindole 5X-isothiocyanate Ž5X-NTII., selective d 1 and d 2 opioid receptor antagonists, respectivelyw10x, and on the lack of cross-tolerance between DPDPE and DELT w18x. These findings suggest that DPDPE and DELT mediate their antinociceptions through different d opioid receptor subtypes. DPDPE- and DALCE-sensitive sites are called d 1 opioid receptors, and DELT- and 5X-NTII-sensitive sites are called d 2 opioid receptors. Although several investigators have shown that central d opioid receptors are involved in the rewarding effects of opioids w2,25,33,35x, the role of d opioid receptor subtypes, i.e. d 1 and d 2 opioid receptors, in these rewarding effects is not yet clear. In a previous study, we found that i.c.v. injection of either DPDPE or DELT produced a dose-dependent place preference, and that the DPDPE- but not the DELT-induced place preference was abolished by pretreatment with D 1 receptor antagonist w36x, which suggests that both DA-dependent and -independent mechanisms are involved in the rewarding effects produced by the activation of central d 1 and d 2 opioid receptor subtypes. The aim of the present study was to clarify this suggestion. The present study was designed to determine the effects of DPDPE and DELT at doses that produce
place preference on the activity of mesolimbic and nigrostriatal DA neurons, and we measured the change in DA turnover in regions of the brain which contain terminals of these neurons Žlimbic forebrain and striatum. after i.c.v. injection of DPDPE and DELT at doses that produce place preference using high-performance liquid chromatography with electrochemical detection ŽHPLC-ECD..
2. Materials and methods The present studies were carried out in accordance with the Guide for Care and Use of Laboratory Animals adopted by the Committee on Care and Use of Laboratory Animals of Hoshi University, which is accredited by the Ministry of Education, Science, Sports and Culture, Japan. 2.1. Animals Male ddY mice ŽTokyo Experimental Animals, Tokyo, Japan. weighing 25 to 35 g were housed in a temperaturecontrolled room. They were maintained on a 12:12 h lightrdark cycle Žlights on 08.00–20.00 h. with laboratory mouse chow and water available ad libitum. 2.2. Place conditioning Place conditioning was conducted as previously described using a minor modification of an unbiased procedure w32x. The apparatus consisted of a shuttle box Ž15 = 30 = 15 cm, width = length = height. which was made acrylic resin board and divided into two equal-sized compartments. One compartment was white with a textured floor, and the other was black with a smooth floor. For conditioning, mice were confined to one compartment after drug injection and to the other compartment after saline injection. The order of injection Ždrug or saline. and compartment Žwhite or black. was counterbalanced across subjects. Conditioning sessions Žthree for drug: three for vehicle. were conducted for a 60-min period once daily. 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. After the conditioning sessions, on day 7, mice were not treated with either drugs or saline, and then were placed on the platform. The time spent in each compartment during a 900-s session was then recorded automatically using an infrared beam sensor ŽKN-80, Natsume Seisakusyo Co., Tokyo, Japan. in a blinded fashion. The position of the mouse was defined by the position of its body Že.g., forelimbs and head.. All sessions were conducted under conditions of dim illumination Ž40 lux lamp. and masking white noise ŽPixie, PXJ43B1, Japan Servo Co., Japan.. DPDPE Ž1–15 nmolrmouse. and DELT Ž0.5–5 nmolrmouse. were injected into the lateral cerebral ventri-
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cle of unanesthetized mice. One day before the beginning the drug or saline injection, the mice were anesthetized with ether and a 2-mm double-needle Žtip: 27 gauge = 2 mm; base: 22 gauge = 10 mm; Natsume Seisakusyo, Tokyo, Japan. attached to a 25-m l Hamilton microsyringe was inserted into the unilateral injection site; as a result, single hole for the injection was made in the skull. The unilateral injection site was approximately 2 mm from either side of the midline between the anterior roots of the ears w9x. The head of the mouse was held against a V-shaped holder, and the drugs were injected into the hole under unanesthetized conditions. Solution was injected in a volume of 2.5 m l per mouse over a period of 10 s. 7-Benzylidenenaltrexone ŽBNTX; 0.5 mgrkg, s.c.. and naltriben ŽNTB; 0.5 mgrkg, s.c.. were given to mice 20 min and 30 min, respectively, prior to treatment with DPDPE or DELT. None of the mice that received i.c.v. saline during the conditioning session exhibited a significant preference for either compartment of the test box.
with electrochemical detection ŽECD.. The HPLC system consisted of a delivery pump Ž880-PU, Jasco, Japan., an analytical column ŽEicompak, MA-50DS, Eicom, Japan. and a guard column ŽEicom.. The electrochemical detector ŽEC-100, Eicom. had a graphite electrode ŽWE-3G, Eicom. and was used at a voltage setting of q0.70 V versus an AgrAgCl reference electrode. The mobile phase consisted of 0.1 M sodium acetater0.1 M citric acid buffer, pH 3.5, containing 13–15% methanol, sodium 1-octanesulfonate and EDTA Ž2Na.. The flow rate was set to 1.0 mlrmin with a column temperature of 258C. DPDPE Ž1–15 nmolrmouse. and DELT Ž0.5–5 nmolrmouse. were injected into the lateral cerebral ventricle of unanesthetized mice. Solution was injected in a volume of 2.5 m l per mouse over a period of 10 s. BNTX Ž0.5 mgrkg, s.c.. and NTB Ž0.5 mgrkg, s.c.. were given to mice 20 min and 30 min respectively, prior to treatment with DPDPE.
2.3. Neurochemical analysis
wD-Pen2 , Pen5 xenkephalin ŽDPDPE. and wD-Ala2 xdeltorphin II ŽDELT. were obtained from Peninsula Laboratories ŽCA, USA.. 7-Benzylidenenaltrexone methanesulfonate hydrate ŽBNTX. and naltriben methanesulfonate hydrate ŽNTB. were synthesized by us. All drugs were dissolved in 0.9% NaCl. All doses refer to the salt forms of the drugs.
The concentrations of dopamine ŽDA., 3,4-dihydroxyphenylacetic acid ŽDOPAC. and homovanillic acid ŽHVA. were determined as described previously w8,20x. Mice were sacrificed by dislocation of cervical vertebrae 60 min after saline Ž2.5 m lrmouse, i.c.v.., DPDPE Ž1–15 nmolrmouse, i.c.v.. or DELT Ž0.5–5 nmolrmouse, i.c.v.. injection, and then immersed into a dry icerethanol solution to minimize the postmortem increase of DA-related substances. The brain was quickly removed and the limbic forebrain, including the N.Acc. and olfactory tubercle, and striatum were dissected on an ice-cold glass plate. Briefly, the brain was turned to expose the dorsal surface and a vertical cut was made through the anterior commissure. The resulting frontal part was turned to expose the ventral surface. A vertical cut was passed through the rhinal fissure and the small part, including the accessory olfactory bulb and olfactory nucleus, was removed. The resulting block of tissue was turned to expose the section, and the area bordered by the caudate putamen and the N.Acc. was cut vertically. The block of tissue that included the N.Acc. and olfactory tubercle was considered to be the main portion of the limbic forebrain. The medial borders of the striatum were cut free, and the striatum was removed without the underlying cortex. The block of tissue was removed and was considered to be the main portion of the striatum. The tissues were frozen at y808C and stored until analysis. The frozen tissues were homogenized in 500 m l 0.2 M perchloric acid containing 100 m M EDTA Ž2Na. and 100 ng isoproterenol, as an internal standard. To remove the proteins completely, the homogenates were placed in cold water for 60 min, and then centrifuged at 20 000 = g for 20 min at 08C. The supernatants were maintained at pH 3.0 using 1 M sodium acetate. Samples were applied to a high-performance liquid chromatography ŽHPLC. system
2.4. Drugs
2.5. Data analysis Conditioning scores represent the time spent in the drug-paired place minus the time spent in the saline-paired place, and are expressed as the mean " S.E.M. A one-way analysis of variance ŽANOVA. followed by a Dunnett’s test was used to determine whether individual doses produced a significant conditioning Ž P - 0.05 and P - 0.01.. DA turnover was determined as the DA ratio: DA ratio s DOPAC Žngrg wet tissue. q HVA Žngrg.4 rDA Žngrg.. Neurochemical data were also evaluated statistically with a one-way ANOVA followed by Dunnett’s test.
3. Results 3.1. Place conditionings produced by the d1 opioid receptor agonist DPDPE and the d 2 opioid receptor agonist DELT Preference for the injection-associated place, which was regarded as a substitute for the drug, was calculated. The place conditionings produced by the d 1 opioid receptor agonist DPDPE and the d 2 opioid receptor agonist DELT are shown in Fig. 1. Both the d 1 and d 2 opioid receptor agonists produced a significant preference for the drug-associated place ŽDPDPE: F Ž3,34. s 5.504; P - 0.01, DELT: F Ž3,35. s 4.711; P - 0.05 and 0.01., compared
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Fig. 3. Change in dopamine turnover in the mouse limbic forebrain after treatment with DELT Ž0.05–5 nmolrmouse, i.c.v... Each column represents the mean with S.E.M. of 8 mice.
3.2. The effects of DPDPE and DELT on the DA turnoÕer in the mouse limbic forebrain
Fig. 1. A: place conditioning produced by DPDPE Ž1–15 nmolrmouse, i.c.v.. and DELT Ž0.5–5 nmolrmouse, i.c.v... Ordinate: mean difference Žs. between the times spent in the drug- and saline-paired sides of the test box. Each point represents the mean with S.E.M. of 8–12 mice. ) ) P 0.01 vs. saline control. B: effects of BNTX Ž0.5 mgrkg, s.c.. and NTB Ž0.5 mgrkg, s.c.. on the DPDPE Ž15 nmolrmouse, i.c.v..- and DELT Ž5 nmolrmouse, i.c.v..-induced place preferences. Each column represents the mean with S.E.M. of 8–12 mice. ) P - 0.05 vs. DPDPE-alone group; a P - 0.05 vs. DELT-alone group.
with the saline control. The DPDPE Ž15 nmol, i.c.v..-induced place preference was abolished by BNTX Ž0.5 mgrkg, s.c.. but not by NTB Ž0.5 mgrkg, s.c... In contrast, the DELT Ž5 nmol, i.c.v..-induced place preference was abolished by pretreatment with NTB but not by BNTX.
Fig. 2. Change in dopamine turnover in the mouse limbic forebrain after treatment with DPDPE Ž1–15 nmolrmouse, i.c.v... Each column represents the mean with S.E.M. of 8 mice. ) ) P - 0.01 vs. saline control.
The effects of DPDPE and DELT on the DA turnover in the mouse limbic forebrain are shown in Fig. 2 and Fig. 3. I.c.v. injection of DPDPE Ž15 nmol. produced a significant increase in DA turnover in the mouse limbic forebrain Ž F Ž3,28. s 10.523; P - 0.01.. In contrast, i.c.v. injection of DELT Ž0.5–5 nmol. did not produce a significant change in the DA turnover in the mouse limbic forebrain Ž F Ž3,28. s 0.567; not significant Žn.s.... 3.3. Effects of d opioid receptor antagonists on the DPDPE-induced increase in DA turnoÕer in the mouse limbic forebrain The effects of d opioid receptor antagonists on the DPDPE-induced increase in DA turnover in the mouse limbic forebrain are shown in Fig. 4. Neither BNTX Ž0.5 mgrkg, s.c.. nor NTB Ž0.5 mgrkg, s.c.. alone affected DA turnover in the mouse limbic forebrain wthe means of DA ratio were 0.168 " 0.004, 0.163 " 0.005 and 0.164 " 0.006 in saline, BNTX and NTB treated groups, respectively; F Ž2,21. s 0.182; n.s.x. The DPDPE Ž15 nmol,
Fig. 4. Effects of BNTX Ž0.5 mgrkg, s.c.. and NTB Ž0.5 mgrkg, s.c.. on the enhancement of dopamine turnover in the mouse limbic forebrain in mice produced by DPDPE Ž15 nmolrmouse, i.c.v... BNTX and NTB were administered 20 and 30 min prior to treatment with DPDPE, respectively. Each column represents the mean with S.E.M. of 8 mice. )) P - 0.01 vs. saline control; aa P - 0.01 vs. DPDPE-alone group.
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Fig. 5. Change in dopamine turnover in the mouse striatum after treatment with DPDPE Ž5–15 nmolrmouse, i.c.v.. and DELT Ž0.5–5 nmolrmouse, i.c.v... Each column represents the mean with S.E.M. of 8 mice.
i.c.v..-induced increase in the DA ratio in the mouse limbic forebrain was abolished by pretreatment with BNTX Ž0.5 mgrkg, s.c.., a d 1 opioid receptor antagonist, but not by NTB Ž0.5 mgrkg, s.c.., a d 2 opioid receptor antagonist. 3.4. The effects of DPDPE and DELT on the DA turnoÕer in the mouse striatum The effects of DPDPE and DELT on DA turnover in the mouse striatum are shown in Fig. 5. I.c.v. injection of neither DPDPE Ž1–15 nmol. nor DELT Ž0.5–5 nmol. affected the DA ratio in the mouse striatum Ž F Ž3,28. s 0.410; n.s. and F Ž3,28. s 0.171; n.s...
4. Discussion The present study demonstrated that the i.c.v. administration of DPDPE Ž15 nmol., a selective d 1 opioid receptor agonist, or DELT Ž5 nmol., a selective d 2 opioid receptor agonist, produced a significant conditioned place preference. Moreover, the place preference induced by DPDPE was abolished by BNTX, a selective d 1 opioid receptor antagonist, but not by NTB, a selective d 2 opioid receptor antagonist. In contrast, the DELT-induced place preference was abolished by NTB, but not by BNTX. We previously reported that both BNTX and NTB alone induced neither significant place preference nor place aversion w35x. Therefore, these results suggest that the DPDPE- and DELT-induced place preferences may be produced via the activation of central d 1 and d 2 opioid receptors, respectively, and that the activation of both central d 1 and d 2 opioid receptors may play a significant role in the expression of the reward. In the present study, i.c.v. injection of DPDPE at a dose that produces a significant place preference produced a
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significant increase in DA turnover in the mouse limbic forebrain, and this effect of DPDPE was abolished by pretreatment with BNTX but not with NTB. We previously reported that NTB alone did not affect the level of DA turnover in the mouse limbic forebrain w20x. Moreover, we confirmed in the present study that both BNTX and NTB alone did not modify it Žsee Section 3.. These results suggest that activation of central d 1 opioid receptors by DPDPE may increase mesolimbic DA neurotransmission. Several previous reports have suggested that the rewarding effects of opioids may be caused by a change in DA transmission in the central DA system. For example, the conditioned place preference produced by morphine and heroin was abolished by pretreatment with DA receptor antagonists in rats and mice w3,29,32x. Similarly, the heroin-induced place preference was blocked by 6-hydroxydopamine lesion of the N.Acc., the terminal projection site of the mesolimbic dopaminergic system w3x. Moreover, chronic administration of SCH23390, a selective dopamine D 1 receptor antagonist, during conditioning sessions attenuates the morphine-induced place preference w13,26,27,34x. Thus, activation of the central DA system, and especially D 1 receptors, may be implicated in the rewarding effects of opioids. In our previous study, the DPDPE-induced place preference was abolished by pretreatment with SCH23390, but not with sulpiride, a selective D 2 receptor antagonist w36x. It has been shown that i.c.v. treatment with DPDPE enhances DA release w28x and the concentration of the DA metabolite DOPAC w16x in the N.Acc. Therefore, the results of the present study provide the further evidence to clarify the our previous hypothesis that the enhancement of DA release and the activation of central DA receptors, especially D 1 receptors, may play an important role in the rewarding effect produced by the activation of central d 1 opioid receptors. In contrast to the results with DPDPE, the i.c.v. injection of DELT at a dose that produces a significant place preference did not modify DA turnover in the mouse limbic forebrain. We previously found that the DELT-induced place preference was not abolished by pretreatment with the DA receptor antagonists SCH23390 and sulpiride w36x, and concluded that the rewarding effect of DELT, which is produced by the activation of central d 2 opioid receptors, does not occur through the enhancement of central DA release, and that some other dopamine-independent mechanism may play an important role in this case. Previous reports have also demonstrated the existence of a DA-independent mechanism in opioid-mediated hyperlocomotion and rewarding effects. Pert and Sivit w21x reported that microinjection of the peptidase-resistant enkephalin analog DALA into the N.Acc. produced behavioral hyperactivity, and that this effect of DALA was not antagonized by DA receptor antagonist. Similarly, Kalivas et al. w11x demonstrated that although DALA injection into the N.Acc. produced behavioral hyperactivity Žlocomotion and rearing., this effect was not blocked by intra-accu-
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mbens injection of fluphenazine, a DA receptor antagonist, or by destruction of the mesolimbic dopaminergic system with 6-hydroxydopamine. Furthermore, they also demonstrated that the injection of DALA into the N.Acc. did not affect the levels of DA or its metabolites in the N.Acc. These data demonstrate that DALA causes behavioral hyperactivity by acting on the N.Acc. via a DA-independent mechanism. However, Longoni et al. w14x have reported that while haloperidol and raclopride fail to affect deltorphin-induced hyperactivity, specific D 1 receptor antagonist SCH23390 effectively blocks it. Although the effect of SCH23390 on the DALA-induced hyperactivity is not investigated, in consideration of this report, it is possible that a specific D 1 receptor-mechanism might be involved on the DALA-induced hyperactivity as in the case of deltorphin. However, Amalric and Koob w1x also reported that heroin produces the behavioral hyperactivity by activating to some extent the DA mesolimbic pathway, but more importantly, by stimulating a postsynaptic pathway in the N.Acc. which is independent of a DA transmission. This report may support the DA-independent hypothesis in opioid-mediated hyperlocomotion. Based on these reports, it is suggested that the expression of opioid-induced behavioral hyperactivity may partially involve a DA-independent mechanism. Moreover, it is previously demonstrated that cocaine-reinforced responding was significantly attenuated by pretreatment with a DA receptor antagonist or the destruction of the DA terminals in the N.Acc. with 6-hydroxydopamine, while heroin self-administration was unaffected in a self-administration paradigm w7,22x, which suggests that the rewarding effects of opiates can be DA-independent under some circumstances. A similar DA-independent effect for both heroin-self-administration and heroininduced place preference was observed using chronic dopamine receptor blockade w31x. Thus, it is also possible that the rewarding effects of d opioid receptor agonists involve a DA-independent mechanism. Based on these previous reports and our present findings, we conclude that the rewarding effect induced by the activation of central d 2 opioid receptors does not occur through the modulation of central DA release, and that some other DA-independent mechanismŽs. may play an important role in the expression of the rewarding effects of d 2 opioid receptor agonists. Contrast to our previous results, Longoni et al. w14x reported that local application by reverse dialysis of DELT to N.Acc. resulted in a motor stimulation and an increase in extracellular DA concentration, and concluded that DELT-induced motor stimulation was mediated by DA-independent function. Although the detailed evidence to clarify the discrepancy between the previous and present findings cannot be provided, one possibility is differences in degrees to activate the d 2 opioid receptors, and therefore, it is possible that treatment with more high doses of DELT might produce the change in DA turnover in the limbic forebrain. Moreover, it is important to note that there are methodological differences to estimate the d 2-
mediated function produced by DELT between these studies, i.e. rewarding properties and motor stimulant behavior. Therefore, although a more detailed investigation might be required to clarify the interaction between d 2 opioid receptors and DA systems in the N.Acc., we concluded that, at least on the dose ranges which produce the rewarding properties, DELT did not affect the DA neuronal activity. In the present study, i.c.v. injection of DPDPE and DELT did not modify the DA turnover in the mouse striatum, the terminal projection site of the nigrostriatal DA system. There is strong evidence that the mesolimbic, rather than the nigrostriatal, DA system plays an important role in mediating the rewarding effects of opioids. Microinjection of morphine into the ventral tegmental area ŽVTA. produces a place preference w4,23,24x, and this effect can be prevented by neurochemical destruction of DA neurons w23,24x. Moreover, morphine at a dose which produces a significant conditioned place preference significantly elevates the level of DA metabolites without changing DA steady-state levels in the limbic forebrain; however, these changes are not observed in the striatum w8x. In vivo microdialysis studies have demonstrated that DA neurotransmission in the N.Acc. is enhanced by treatment with m and d opioid receptor agonists at the same doses that produce rewarding effects w6,28x. These findings suggest that mesolimbic DA neurons rather than nigrostriatal DA neurons may play an important role in the development of the rewarding effect of morphine. In addition, evidence of regional differences in the sensitivity of DA neurons to opioids has also been reported. Electrophysiological studies have shown that morphine elicits a greater increase in the firing rate of DA cells in the VTA than in the substantia nigra w17x. Similarly, the morphine-induced increase in extracellular DA levels is greater in the N.Acc. than in the striatum w6x. I.c.v. injection of DPDPE at the same dose range used in the present study also produced an increase in the concentration of DOPAC and accumulation of DOPA in the N.Acc., but had no effect in the striatum w16x. Along with these previous reports, the present data suggest that activation of mesolimbic, rather than nigrostriatal, DA neurons may be especially important in the development of the rewarding effects of d opioid receptor agonists. In conclusion, the present data demonstrate that i.c.v. injection of DPDPE but not DELT at a dose that produces a place preference also produces a significant elevation of DA turnover in the mouse limbic forebrain. These findings suggest that activation of the mesolimbic DA system may play an important role in the rewarding effects of DPDPE but not of DELT, and confirm our previous hypothesis that the rewarding effects induced by the activation of central d 1 opioid receptors may be produced through the enhancement of DA release and the activation of DA receptors, especially D 1 receptors. In contrast, the rewarding effect induced by the activation of central d 2 opioid receptors may occur through some DA-independent mechanismŽs..
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Acknowledgements This work was supported by a scientific research grant to T.S. from the Ministry of Health and Welfare and by a Research Grant Ž5A-7. for Nervous and Mental Disorders from the Ministry of Health and Welfare, Japan to T.S. We would like to thank Mr. Hiroyuki Goto for his excellent technical assistance.
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