Chronic morphine exposure and spontaneous withdrawal are associated with modifications of dopamine...

European Journal of Neuroscience, Vol. 11, pp. 481–490, 1999 © European Neuroscience Association

Chronic morphine exposure and spontaneous withdrawalare associated with modifications of dopamine receptorand neuropeptide gene expression in the rat striatum

Francois Georges, Luis Stinus,1 Bertrand Bloch and Catherine Le MoineLaboratoire d’Histologie-Embryologie and 1Laboratoire de Neuropsychobiologie de Desadaptations, Centre National de laRecherche Scientifique, Unite Mixte de Recherche 5541, Universite Victor Segalen Bordeaux 2, 146 rue Leo Saignat, 33076Bordeaux Cedex, France

Keywords: dependence, dopamine–opioid interactions, in situ hybridization, mRNA, opioid peptides

Abstract

The influence of chronic morphine and spontaneous withdrawal on the expression of dopamine receptors and neuropeptide genesin the rat striatum was investigated. Morphine dependence was induced by subcutaneous implantation of two morphine pellets for6 days. Rats were made abstinent by removal of the pellets 1, 2 or 3 days before they were killed. The mRNA levels coding forD1- and D2-dopamine receptors, dynorphin, preproenkephalin A and substance P were determined by quantitative in situhybridization. The caudate putamen and the nucleus accumbens showed equivalent modifications in dopamine receptor andneuropeptide gene expression. After 6 days of morphine, a decrease in D2-dopamine receptor and neuropeptide mRNA levelswas observed (– 30%), but there was no change in D1-dopamine receptor mRNA. In abstinent rats, both D1- and D2-dopaminereceptor mRNA levels were decreased 1 day after withdrawal (– 30% compared with chronic morphine). In contrast, neuropeptidemRNA levels were unaffected when compared with those observed after 6 days of morphine. During the second and third day ofwithdrawal, there was a gradual return to the levels seen in the placebo-treated group, for both dopamine receptor and neuropeptidemRNAs. Phenotypical characterization of striatal neurons expressing µ and κ opioid receptor mRNAs showed that, in striatonigralneurons, both mRNAs were colocalized with D1-receptor and Dyn mRNAs. Our results suggest that during morphine dependence,dopamine and morphine exert opposite effects on striatonigral neurons, and that effects occurring on striatopallidal neurons areunder dopaminergic control. We also show that withdrawal is associated with a down regulation of the postsynaptic D1 and D2receptors.

Introduction

Prolonged exposure to morphine leads to tolerance and dependence.Tolerance is manifested by a reduced effect after repeated exposureto a drug and the need for an increased amount of drug to achievethe same effect. In the dependent state, continuous exposure to adrug is required to avoid withdrawal symptoms (Koob & Bloom,1988; Koob, 1992; Nestler, 1992). Opiates exert their biologicaleffects by interacting with three classes of opioid receptors:δ, µand κ (for review, see Reisine & Bell, 1993). The neurochemicalmechanisms involved in the development of opiate dependence andin the expression of withdrawal include homologous regulationaffecting the endogenous opioid system along with heterologousregulation involving other neurotransmitter systems (Koob & LeMoal, 1997). Numerous nonopioid systems have been proposed toparticipate to this regulation. Among those, dopamine receptors havebeen implicated in the behavioural responses to drugs of abuse (Harris& Aston-Jones, 1994; Koob, 1996; Uhlet al., 1998), which arethought to be mediated by the mesolimbic dopaminergic pathwayarising from the ventral tegmental area and projecting to the limbicsystem, especially the nucleus accumbens. Dopamine differentially

Correspondence: Francois Georges, as above.E-mail: [email protected]

Received 26 June 1998, revised 7 September 1998, accepted 10 September 1998

regulates the two major striatal output pathways, the striatonigralneurons via D1-dopamine (D1) receptors and the striatopallidalneurons via D2-dopamine (D2) receptors (Gerfenet al., 1990; LeMoine & Bloch, 1995). These pathways express different opioidpeptides, dynorphin and enkephalin, respectively (Gerfen & Young,1988). The rewarding properties of opiates (Wise & Bozarth, 1987)and the somatic expression of abstinence (Harris & Aston-Jones,1994) have been related to changes in mesolimbic dopaminergicactivity that could constitute, at least in part, the neural substrate foropioid addiction (Koob, 1992; Harris & Aston-Jones, 1994). Forinstance, injection of opiates into the ventral tegmental area elicitsself-administration (Devine & Wise, 1994; Selfet al., 1995; Kiyatkin& Rebec, 1997), potentiates rewarding brain stimulation and producesa conditioned place preference (Mamoonet al., 1995; Tsuji et al.,1996). Adaptative changes in the regulation of gene expression inneurons of the striatal complex have been suggested to be involvedin the long-term behavioural effects of drugs (Nestler, 1992). Ourstudy focuses on the changes observed in the dopaminergic andopioidergic systems in order to determine which regulation processesoccurred following chronic exposure to morphine and spontaneouswithdrawal. To gain more understanding of the action of morphine(which exhibits affinity predominantly forµ andκ opioid receptors,as compared withδ opioid receptors), we also carried out a phenotypecharacterization of the striatal neurons expressing the different opioid

482 F. Georgeset al.

FIG. 1. Experimental schedule for Experiments 1 and 2. P indicates the placebotreatment. M6, M7, M8 and M9 are the 6-, 7-, 8- and 9-day treatments withmorphine, respectively, W1, W2 and W3 indicate morphine withdrawal statesfollowing 1, 2 or 3 days of abstinence, respectively. The results for the M6group were duplicated as they were needed in both sets of experiments.

receptor mRNAs. We employed quantitativein situ hybridization toexamine the influence of chronic exposure to morphine (M) and ofspontaneous withdrawal (W) on the gene expression of D1 and D2receptors, and on the striatal peptides preproenkephalin A (PPA),dynorphin (Dyn) and substance P (SP).

Materials and methods

Animals

Eighty nine male Sprague–Dawley rats (CERJ, Le Genest Saint-Isle,France) (150–175 g, at the beginning of the experiment) were usedthroughout this study. Rats were adapted to housing in individualcages under controlled conditions (22–23 °C, 40% relative humidity,12 h light/12 h dark illumination cycle (lights from 06.00 to 18.00 h)for at least 1 week before use. Rats were allowed free access tocommercial chow and tap water. After the adaptation period, 24 ratswere individually housed in order to record locomotor activitycontinuously, and 65 rats were housed in groups of five and randomlyassigned into eight groups (five to eight per group) for two sets ofin situ hybridization experiments (Fig. 1).

Induction of morphine dependence

Rats were made morphine dependent by subcutaneous implantationof two morphine (M) pellets, each containing 75 mg of morphinefree base (National Institute of Drug Abuse, NIH, Rockville, MD,USA). The control rats received placebo (P) pellets containing theexcipient without morphine. All surgical procedures (implantation orremoval of pellets) were performed under deep halothane anaesthesiafor 30 s. In Experiment 1, rats were killed 6 (M6), 7 (M7), 8 (M8)or 9 (M9) days following the implantations of morphine pellets. InExperiment 2, withdrawal (W) was induced by removal of the pelletson day 6, and the rats were killed 1 (W1), 2 (W2) or 3 (W3) daysafter the removal (Fig. 1). This procedure has been shown to producea high degree of physical dependence on morphine (Goldet al.,1994). All surgical and experimental procedures were performed inaccordance with the European Communities Council Directive of 24November 1986 (86/609/EEC).

© 1999 European Neuroscience Association,European Journal of Neuroscience, 11, 481–490

Recording spontaneous locomotor activity

Three groups of rats (placebo-treated, morphine-treated and with-drawal: eight per group) were specifically used for the behaviouralmeasurements (Fig. 2). Locomotor activity was measured as describedby Stinuset al. (Stinuset al., 1998) in activity cages ressembling thehome cages. Food and water were continuously available in theseactivity cages. Two beams of infrared lights were aligned across thelong axis of the cage; they were 14 cm apart and 3 cm above thefloor and connected to photoelectric cells so that each passage of theanimal interrupted a beam. Each photocell count was recorded on acomputer. A computer program analysed the total number of activitycounts (total beam interruptions on both cells) representing the totalmotor activity of the animal. Data were analysed by a three-way ANOVA with one between-factor comparison (pharmacologicaltreatment) and two within-factor comparisons (the time in days afterwithdrawal and time of day).

Tissue preparation

Animals of each group (P, M6, M7, M8, M9, W1, W2 and W3) wereanaesthetized with urethane (1.15 g/kg i.p.) and perfused through theheart with 1% paraformaldehyde (PFA). All the animals were killedbetween 9.00 and 12.00 h. The brains were dissected out, immersedfor 1 h in 1% PFA and then in (sucrose 15%) phosphate buffer (0.1M,pH 7.4) for 16 h. Brains were frozen over liquid nitrogen, then cutinto 10µm frontal sections that were collected on gelatin-coated slidesand stored at – 80 °C until use.

In situ hybridization procedures

Quantitativein situ hybridization.

The quantitativein situ hybridization procedure was performed asdescribed by Le Moineet al. (Le Moine et al., 1994a) with somemodifications. The probes used were oligonucleotides labelled bytailing with [35S] dATP (Life Sciences Products, Paris, France) to aspecific activity of 53 108 c.p.m./µg. They were designed to recognizeeither PPA (Tanget al., 1983), SP (Nawaet al., 1984), Dyn, D1-(Dearryet al., 1990) or D2- (Dal Tosoet al., 1989) receptor mRNAs.After labelling, the probes were precipitated in absolute ethanol and3 M sodium chloride, dried and resuspended at the appropriateconcentration (5 pg/µL) in the hybridization buffer (50% deionizedformamide, 10% dextran sulphate, 500µg/mL denatured salmonsperm DNA, 1% Denhardt, 5% sarcosyl, 250µg/mL yeast tRNA,200 mM dithiothreitol, 20 mM NaH2PO4 in 23 SSC (sodium chloride–sodium citrate buffer)). The sections were fixed with 4% PFA (inphosphate buffer 0.1M, pH 7.4) for 5 min at room temperature, rinsedtwice for 30 min in 43 SSC/1% Denhardt, acetylated into 0.25%acetic anhydride in 0.1M triethanolamine/43 SSC (pH 8) for 10 minat room temperature and then dehydrated in graded alcohol. All theslides were then incubated vertically overnight at 40 °C in a bathcontaining the hybridization solution with the appropriate labelledprobe (final volume, 40 mL) and under gentle agitation. At the endof the incubation, slides were washed in decreasing concentrationsof SSC (31 at room temperature for 45 min,31 at 40 °C for 45 min,30.1 at 40 °C for 45 min), then dehydrated in ethanol. Sections wereexposed at room temperature to Biomax film (Kodak, Polylabo,Strasbourg, France) over 5–6 days for PPA, Dyn and SP mRNAdetection, and 7 days for D1- and D2-receptor mRNA detection.Probe concentration and exposure times were chosen in order to staywithin the linear range of the film to allow appropriate quantification.

Dopamine–opioid interactions in the striatum 483

FIG. 2. Time course of total motor activity (total photocell activity counts per 6-h period) recorded in rectangular activity cages during the 9 days followingimplantation of the pellets (black arrowhead). Placebo-treated rats (n 5 8): white squares and dotted lines. Chronic morphine-treated rats (n 5 8): black circlesand thin solid lines. Abstinent rats (n 5 8) white arrowhead indicate removal of the pellets (black circle and thick solid lines). The spontaneous locomotoractivity of morphine-dependent rats was similar to that of placebo-treated rats, but it was disrupted during the first and second days of withdrawal.

Simultaneous detection ofµ, δ and κ opioid-receptor mRNAs witheither D1, Dyn, D2 or PPA mRNAs in normal rats

Multiple combination of probes were used for the simultaneousdetection of two mRNAs on a single striatal section:35S-labelledcRNA probes forµ, δ, κ opioid receptors (Mansouret al., 1994) incombination with digoxigenin-labelled cRNA probes for D1 receptors,Dyn, D2 receptors, PPA and choline acetyltransferase. The doubleinsituhybridization procedure was performed as described by Le Moineet al. (Le Moine et al., 1997). After being dipped into Ilford K5emulsion (Ilford, Armitec, Saint Priest, France; diluted 1 : 3 in 13SSC), the sections were exposed for 3 months in the dark, developedand mounted without counterstaining.

mRNA quantification and statistical analysis

Quantitative measurements of the mRNA levels were obtained byautoradiographic densitometry with a BIOCOM 200 image analyser(BIOCOM, Les Ulis, France) using the RAG program. Radioactivestandards were generated using brain paste incorporated with dilutionsof 35S-labelled nucleotide. A calibration curve representing opticaldensities (OD) as a function of the radioactivity concentration wascalculated from these radioactive standards. The OD were measuredin the caudate putamen and the nucleus accumbens, at the sameanteroposterior level. These values were representative of the quantit-ies of probe hybridized, and therefore correspond to the relativemRNA levels. The OD were measured in a blind way and convertedinto concentrations of radioactivity using the calibration curve. Foreach set of experiments, these values were subjected to a one-wayANOVA followed bypost-hocNewman’s Keuls test. All the experimentswere carried out at least twice, and the two groups P and M6 wereduplicated in our two distinctin situ hybrization experiments (Fig. 1).

Results

Spontaneous locomotor activity

Alterations in spontaneous locomotor activity has been shown toserve as a behavioural measure of the symptoms of opiate withdrawal(Stinuset al., 1998). Opiate dependence and opiate withdrawal weremonitored using continuous recording of locomotor activity for 9 days.Figure 2 presents the time course of total photocell counts per 6-hperiod, for control (placebo-treated) and morphine-dependent rats.


Control rats as well as morphine-dependent rats developed a biphasicpattern of locomotor activity characterized by nocturnal hyperactivitywhich was markedly reduced during the light phase. TheANOVA forthe first 6 days of opiate dependence (M,n 5 16; P,n 5 8) indicatedno overall morphine effect. TheANOVA for the groups W1, W2 andW3 indicates a withdrawal3 day interaction (F3,395 5.5,P , 0.01)and a withdrawal3 time-of-day interaction (F6,785 2.49,P , 0.05).During the first and second days, and part of the third following thebeginning of abstinence, the overall motor activity remained at a verylow level, close to the average activity of control rats during the lightphase [group3 time interaction,F3,395 8.0 (first day),F3,395 3.6(second day) andF3,395 1.9 (third day);P , 0.05 in each case].During the third day after the removal of morphine pellets, themorphine-abstinent rats slowly resumed a normal circadian cycle(Fig. 2).

Morphine dependence

Dopamine receptor mRNA levels in the caudate putamen andnucleus accumbens of morphine-dependent rats

Chronic morphine treatment had no effect on the D1-receptor mRNAlevel in the caudate putamen or nucleus accumbens of the M6, M7,M8 and M9 groups (Table 1; Figs 3 and 4A and B). In contrast, adecrease in the D2-receptor mRNA level was observed in Experiment1 (Fig. 1) for the M6, M7, M8 and M9 groups in comparison withthe control group (P) (Table 1,P , 0.001 for each comparison). Asshown in Fig. 4 (Experiment 2), the M6 group also presented a 27%decrease in the caudate putamen (P , 0.001) and a 26% reductionin the nucleus accumbens (P , 0.001) (Figs 3 and 4C and D).

Neuropeptide mRNA levels in the caudate putamen and nucleusaccumbens of morphine-dependent rats

Chronic morphine impregnation led to a reduction in the expressionof Dyn, SP and PPA mRNAs in the caudate putamen and nucleusaccumbens (Table 1). The effects were similar for groups M6–M9(P , 0.001). As shown in Fig. 5 for the M6 group (Experiment 2),there was a 32% decrease in Dyn mRNA in the caudate putamen(P , 0.001) and a 28% decrease in the nucleus accumbens(P , 0.001). Likewise, there was a 28% fall in SP mRNA in thecaudate putamen (P , 0.001) and a 37% fall in the nucleus accumbens(P , 0.001), along with a 34% decrease in PPA mRNA in the caudate


TABLE 1. Quantitativein situ hybridization analysis of the relative level of D1- and D2-receptor, dynorphin (DYN), substance P (SP) and preproenkephalin A(PPA) mRNAs in the caudate putamen (CPu) and in the nucleus accumbens (NAc) after chronic morphine treatment. Chronic morphine treatment during 6, 7,8 and 9 days caused a decrease of D2 dopamine receptor and neuropeptide mRNA levels. No change of the D1-receptor mRNA level was observed. Data wereexpressed in arbitrary units of radioactivity and as the mean6 SEM of five rats.†P , 0.001 vs. control rats (P). Statistical analysis performed were one-wayANOVA followed by posthocNewman’s test

D1 D2 DYN SP PPA

CPu NAc CPu NAc CPu NAc CPu NAc CPu NAc

Placebo (n 5 5) 100.06 1.5 100.06 2.1 100.06 1.9 100.06 3.4 100.06 2.5 100.06 4.4 100.06 3.6 100.06 3.4 100.06 2.3 100.06 2.9Morphine: 6 days 109.66 4.1 102.26 4.4 78.96 2.3† 71.46 3.3† 69.66 4.7† 68.86 4.9† 76.26 2.7† 73.46 2.4† 68.26 4.3† 67.06 3.4†

(n 5 5)Morphine: 7 days 109.46 4.4 113.46 4.1 79.16 4.1† 70.16 3.4† 75.76 2.3† 70.66 3.4† 89.36 3.8† 86.16 2.5† 95.46 7.5† 83.96 5.7†

(n 5 5)Morphine: 8 days 111.16 6.0 111.86 7.4 84.86 3.1† 79.36 2.7† 75.76 2.1† 69.66 3.2† 97.06 5.9† 87.06 3.5† 98.66 4.6 91.16 4.6(n 5 5)Morphine: 9 days 102.96 2.7 102.46 1.6 75.96 3.1† 68.66 3.7† 69.26 4.2† 64.96 3.4† 69.26 2.5† 60.86 2.5† 72.26 3.0† 63.26 2.5†

(n 5 5)

NAc 5 nucleus accumbens; CPu5 caudate putamen.

putamen (P , 0.001) and a 35% decrease in the nucleus accumbens(P , 0.01), relative to the P group (Figs 3 and 5).

Morphine abstinenceDopamine receptor mRNA levels in the caudate putamen andnucleus accumbens following opiate withdrawal

A specific temporal pattern of D1- and D2-receptor mRNA levelswas observed after withdrawal with two characteristic features: adecrease on the first day of withdrawal, and a progressive return tocontrol levels during the second and third days. Indeed, at W1, D1-and D2-receptor mRNA levels were significantly decreased comparedwith the M6 group. Figure 4 shows a decrease of 32% for D1-(P , 0.001) and of 31% for D2-receptor mRNA (P , 0.001) in thecaudate putamen, and a decrease of 28% for D1- (P , 0.001) and of33% for D2-receptor mRNA (P , 0.01) in the nucleus accumbens(Figs 3 and 4). The W2 and W3 groups showed an increase inthe D1-receptor mRNA level in the caudate putamen and nucleusaccumbens, compared with the W1 group (P , 0.001), returning tothe control group level at W3 (Fig. 4A and B). Similarly, the D2-receptor mRNA levels of the W3 group were above those of the W1group (43% increase (P , 0.001) in the caudate putamen and 42%(P , 0.001) in the nucleus accumbens, Fig. 4C and D).

Neuropeptide mRNA levels in the caudate putamen and nucleusaccumbens following opiate withdrawal

The effect of withdrawal on peptide mRNA levels also exhibited aspecific temporal pattern with a characteristic increase during thesecond and third day, followed by a return to control levels. Indeedthe W1 group showed no change in Dyn, SP and PPA mRNA levelswith respect to the M6 group (Figs 3 and 5). There were a gradualincrease of Dyn, SP and PPA mRNA levels in the caudate putamenand the nucleus accumbens in the W2 and W3 groups relative to theW1 group (P , 0.001), which subsequently returned to levels of theP group (Fig. 5). Moreover, for the W3 group, these mRNA levelswere significantly elevated relative to those at M6. In the caudateputamen these increases were:1 25% for Dyn (P , 0.01), 1 14%for SP (not significant) and1 23% for PPA (P , 0.05); in the nucleusaccumbens they were:1 19% (P , 0.01) for Dyn,1 20% (P , 0.05)for SP and1 29% (P , 0.01) for PPA, Fig. 4).

Phenotypical characterization of striatal neurons expressingthe µ, δ and κ opioid receptor mRNAsPhenotypical characterization of the striatal neurons expressingµ, δand κ receptor mRNAs was performed by combining radioactive


(for µ, δ and κ receptor mRNA detection) with nonradioactive (forD1 receptors, Dyn, D2 receptors, PPA and choline acetyltransferasemRNA detection) cRNA probes. Microscopic analysis showed aspecific expression ofµ andκ opioid-receptor genes in the Dyn/SP/D1 striatonigral neurons. Indeed, we observed a colocalization ofµand κ receptor mRNAs with both Dyn and D1-receptor mRNAs(Fig. 6A, C and E). These results were corroborated by the absenceof colocalization betweenµ or κ receptor mRNAs and D2-receptoror PPA mRNAs (Fig. 6C, D, G and H). In addition,δ receptor mRNAwas mainly detected in choline acetyltransferase-mRNA-expressingneurons as described by Le Moineet al. in mice (Le Moineet al.,1994b) (data not shown).

Discussion

We have investigated the effects of chronic morphine treatment andwithdrawal on dopamine receptor and neuropeptide mRNA levels inthe caudate putamen and in the nucleus accumbens. The behaviouraldata showed no apparent effect of chronic morphine treatment onlocomotor activity in our model, in contrast to the other model ofmorphine dependence, induced by injections (Stinuset al., 1998).This is probably related to the constant and low rate of drug releaseinduced by subcutaneous morphine implantation (Yoburnet al., 1985)and supports a homeostatic hypothesis of morphine action. In contrast,during the first 24 h following the removal of the pellets, the overallmotor activity during the dark and light period was similar to themotor activity of control rats during the light phase. Behaviouralinstability could be detected by measurement of the total photocellcounts (per 6-h period) which were recorded over the first two daysfollowing removal of the pellets. This was assumed to indicate thestate of withdrawal in our model.

Dependence

We demonstrated here, for the first time, that chronic exposure tomorphine is associated with a marked concomitant decrease of geneexpression for dopamine receptors and neuropeptides in the twostriatal output pathways, i.e. the striatopallidal and striatonigralneurons. In particular, we observed a decrease in D2-receptor andPPA mRNA levels, no change in D1-receptor mRNA level, and adecrease in Dyn and SP mRNA levels. The inhibitory effect ofmorphine on D2-receptor mRNA is consistent with the increase ofdopamine release during the development of morphine dependence(Acquaset al., 1991; Pothoset al., 1991; Crippens & Robinson, 1994;


FIG. 3. Representative autoradiograms ofin situ hybridization showing the distribution of D1- and D2-receptor, and neuropeptide mRNAs in the caudate putamenand nucleus accumbens. P corresponded to placebo. M6 indicates treatment for 6 days with morphine. W1 indicates morphine withdrawal on the 1 day ofabstinence. After 6 days of morphine, decreases in D2-receptor and neuropeptide mRNA levels were observed. The levels of both D1- and D2-receptor mRNAsdecreased after 1 day of withdrawal when compared with M6. By contrast the neuropeptide mRNA levels were unaffected. Quantitative data are shown inFigs 4 and 5. Bar5 0.25 cm.



FIG. 4. Quantitativein situ hybridizationanalysis of D1- (A and B) and D2-receptormRNAs (C and D) in the caudate putamen (Aand C) and the nucleus accumbens (B and D)after chronic morphine treatment andwithdrawal. The P and M6 group data arepresented as black bars, and the those from themorphine withdrawal groups (W1, W2 andW30 with hatched bars. The data are expressedin arbitrary units of radioactivity, and as themean6 SEM of eight rats. After 6 days ofmorphine, a decrease of D2- receptor mRNAlevels and no change of D1-receptor mRNAlevels were observed. During the withdrawal,both D1- and D2-receptor mRNA levelsshowed a decrease on the first day, and aprogressive increase during the second andthird days towards the control levels.n 5P , 0.001 vs. placebo-treated rats.s 5P , 0.01 vs. M6 group.m 5 P , 0.001 vs.W1 group.d 5 P , 0.001 vs. W2 group.Statistical analyses performed were a one-wayANOVA followed by aposthocNewman’s test.

Spanagel & Shoaib, 1994; Pontieriet al., 1995). Indeed, morphineindirectly stimulates dopamine neurons in the ventral tegmental areavia µ opioid receptors (Di Chiara & North, 1992; Johnson & North,1992; Klitenicket al., 1992; Spanagelet al., 1992). A similar decreasein D2-receptor mRNA is observed in other situations of hyperdopami-nergy, such as that following the disruption of the dopamine transportergene (Giroset al., 1996). It has also been shown, after chronicmorphine, that striatal D2 receptors are reduced in number, and thatthere is a corresponding increase in cAMP concentration (Navarroet al., 1992). This is in line with the D2-receptor-mediated inhibitoryeffect of dopamine on adenylate cyclase activity (Kebabian & Calne,1979) and on D2-receptor gene expression (Le Moineet al., 1990;Bernard et al., 1991). As we show here that the opioid receptormRNAs are not expressed in striatopallidal neurons, we suggest thatthe decrease observed in the level of D2-receptor mRNA may be dueto the morphine-induced hyperdopaminergy leading to the saturationof the D2 receptors. The decrease of PPA mRNA levels followingmorphine treatment is also in agreement with previous results ofNorthern blot analysis (Basheer & Tempel, 1993; Uhlet al., 1988;Gudehithlu & Bhargava, 1995). Thus, our results provide additionalevidence for an inhibitory influence of chronic morphine treatmenton the gene expression of both D2 receptors and enkephalins in thestriatopallidal neurons.

Our results also showed that the chronic morphine treatment didnot alter D1-receptor mRNA levels, but did reduce the mRNA levelsof Dyn and SP (coexpressed in the same neurons), both in the caudateputamen and the nucleus accumbens. These results for the D1receptor correlate well with similar results obtained in binding studies(Bhargavaet al., 1989). Regarding the possible molecular mechanismsunderlying opiate effects on striatal dopaminergic activity, one possibleexplaination for the lack of effect on D1-receptor mRNA is thatstimulation of opioid receptors by morphine alters the efficiency ofthe coupling between the D1 receptor and adenylate cyclase, viainhibition of G proteins. Indeed, studies on striatal primary culturesand on striatal slices (Erikssonet al., 1991; Schoffelmeeret al., 1993)have indicated that opioid ligands inhibit adenylate cyclase activitylinked to D1 receptors. These results are also consistent within vivo


studies indicating thatκ receptors on striatal neurons contribute tothe inhibition of some D1-receptor-mediated responses (Steiner &Gerfen, 1996). The distribution of opioid receptor mRNAs andbinding sites have been described in the central nervous system(Mansouret al., 1987; Mansouret al., 1994, 1995). Moreover, as themorphine effects are linked to the nature of the neurons expressingµ,δ, or κ opioid receptors, we have determined here thatµ andκ opioid-receptor mRNAs were colocalized in striatonigral neurons containingD1-receptor and Dyn mRNAs. This is consistent with the fact thatactivation of opioid receptors during chronic morphine treatment,reduces the postsynaptic D1-receptor-mediated effect of dopamine onadenylate cyclase (Erikssonet al., 1991; Heijnaet al., 1992). Anotherpossibility would be that the magnitude of the dopamine increaseduring the morphine dependence is not sufficient to induce changesin the D1-receptor mRNA level.

It has been shown that D1 receptors mediate increases in Fos andJunB expression (Steiner & Gerfen, 1993; Liuet al., 1994; Wanget al., 1996; Le Moineet al., 1997) and that a possible target genefor the Fos/Jun B complex is the preprodynorphin gene (Naranjoet al., 1991). Accordingly, an increase in the level of Dyn mRNAwould be expected following chronic morphine treatment, as shownin the chronic hyperdopaminergic model of mice lacking the dopaminetransporter (Giroset al., 1996). Our results, like those of otherNorthern blot studies (Romualdiet al., 1991; Przewlockaet al., 1996)showed the opposite. These data raises the question of whether ornot the down-regulation of the Dyn mRNA synthesis is indicative ofopiate dependence. High doses of morphine bind to bothµ and κopioid receptors (Woodet al., 1987), which may be relevant to theinterpretation of the present results. Accordingly, the synthesis andrelease of dynorphin are both down-regulated (Romualdiet al., 1991;Przewlockaet al., 1996). The decrease in Dyn mRNA levels isconsistent with the expression of opioid receptor mRNAs in striatonig-ral neurons, and may correspond to a mechanism of down-regulationfollowing the saturation ofκ opioid receptors by morphine.

WithdrawalMicrodialysis studies have shown that morphine-dependent animalsexhibit depressed dopamine release in the nucleus accumbens during


FIG. 5. Quantitativein situ hybridizationanalysis of Dyn (A and B), SP (C and D) andPPA mRNAs (E and F), respectively, in thecaudate putamen (A, C and E) and in thenucleus accumbens (B, D and F) after chronicmorphine treatment and withdrawal. P and M6groups: black bars; morphine withdrawal (W1,W2 and W3): hatched bars. After 6 days ofmorphine, a decrease of striatal neuropeptidemRNA levels was observed. During thewithdrawal, neuropeptide mRNA levels wereunaffected on the first day, and progressivelyincreased during the second and third daystowards the control levels. The data areexpressed as arbitrary units of radioactivity,and are the mean6 SEM of eight rats.n 5P , 0.01 vs. placebo-treated rats.s 5P , 0.05 vs. M6 group.m 5 P , 0.001 vs.W1 group.d 5 P , 0.001 vs. W2 group.Statistical analyses performed were a one-wayANOVA followed by aposthocNewman’s test.

withdrawal episodes as compared with basal level (Acquaset al.,1991; Pothoset al., 1991; Acquas & Di Chiara, 1992; Rossettiet al.,1992; Spanagelet al., 1994). Our results show that changes in theexpression of D1- and D2-receptors, and Dyn, SP and PPA mRNAsoccur within 3 days of withdrawal. Indeed, 1 day after the removalof the pellets, D1- and D2-receptor mRNA levels were significantlyreduced in comparison with levels observed after 6 days of chronicmorphine. However, during the second and third day of withdrawal,the level of dopamine receptor mRNAs returned to the control level.As mentioned previously, it is likely that during the dependent state,dopamine and morphine exert a balanced control on D1-receptor-mediated effects on adenylate cyclase (Erikssonet al., 1991;Schoffelmeeret al., 1993). The deactivation of opioid receptors duringthe withdrawal could allow a more effective action of dopamine onD1 receptors despite the decrease of dopamine release. It is possiblethat the decrease in striatal dopamine levels described during with-drawal is induced by the activation of the striatonigral neurons which,in turn, inhibits nigrostriatal dopamine neurons (Suaud-Chagnyet al.,1992). Thus, we suggest that the decrease of D1-receptor mRNArepresents a compensatory mechanism following saturation of the D1receptors. The activation of D2 receptors within the nucleusaccumbens has been reported to reduce the severity of the naloxone-precipated withdrawal syndrome, whereas its blockade in morphine-dependent animals precipitates abstinence (Harris & Aston-Jones,1994). Our results are consistent with these findings, as 1 day ofabstinence induces a decrease in the D2-receptor mRNA level.

Our present study shows that, between 1 and 3 days of withdrawal,


the level of peptide mRNAs in the caudate putamen and the nucleusaccumbens increases and returns to the control level. These resultsare consistent with previous studies showing similar increases of Dynand PPA mRNAs in morphine-abstinent rats (Gudehithlu & Bhargava,1995; Przewlockaet al., 1996). It has been postulated that thestimulation of the opioid receptors exert tonic inhibition on striatalopioid-peptide biosynthesis (Morriset al., 1988). Therefore attenu-ation of this inhibition may be expected during morphine withdrawal.We have shown above that chronic administration of morphine isassociated with a decrease in the levels of endogenous opioid-peptidemRNAs in the nucleus accumbens and caudate putamen. A possibleexplanation for the changes in the peptide mRNA levels associatedwith morphine withdrawal could be a response of the neurons to adeactivation ofκ andµ opioid receptors. This could represent acompensatory mechanism necessary for the increase in biosynthesisof endogenous opioid peptides to return to a normal level.

Conclusion

The present results provide evidence that chronic morphine treatmentand spontaneous withdrawal evoke changes of gene expression in thetwo striatal output pathways. Interestingly, these changes occur tothe same extent in the caudate putamen and in the nucleus accumbens,the structure particularly involved in the rewarding properties ofopiates. Our data strongly suggest that striatal dopamine and morphineexert opposite effects on striatonigral neurons. Thus, D1 receptorsmay contribute to the development of withdrawal symptoms in


FIG. 6. Phenotype characterization of the striatal neurons expressingµ and κ receptor mRNAs. Doublein situ hybridization detected either D1-receptor, Dyn,D2-receptor or PPA mRNA with digoxigenin-labelled riboprobe (stained cells), together withµ or κ receptor mRNA, with a35S-labelled riboprobe (silvergrains). A, C and E show thatµ and κ receptor mRNAs were present in Dyn and D1-receptor mRNA-containing neurons (double arrows). B, D and F showthatµ andκ receptor mRNAs (arrowheads) were not present in PPA- and D2-receptor-mRNA-containing neurons. Bars5 10µm.

animals treated with chronic morphine. The blockade of this receptorwill perhaps allow new perspectives for the therapy of opiatewithdrawal syndrome. Interestingly, morphine withdrawal has beenfound to be unaffected in mice lacking D2 receptors (Maldonadoet al., 1997). Thus, the decrease in D2-receptor mRNA observed inour study does not appear to be crucial to the morphine withdrawalsyndrome, but instead may represent an adaptative mechanism tooppose its aversive symptoms. Further study of opioid-receptor-deficient or dopamine-depleted animals will open new perspectivesto understand the functional roles of opioid receptors and theirinteraction with the dopamine system.


AcknowledgementsThe authors thank Drs S. J. Watson and A. Mansour for the gift of the opioid-receptor cDNA clones. We also thank C. Vidauporte for expert photographicartwork, M. Manse for excellent and skilful technical assistance and M. Jaberfor helpful comments on the manuscript. This work was supported by fundsfrom the INSERM (MILDT N°96C02).

AbbreviationsD1, D1 dopamine (receptor); D2, D2 dopamine (receptor); Dyn, dynorphin;OD, optical densities; P, placebo; PPA, preproenkephalin A; PFA, paraformal-dehyde; M, morphine; M6–9, groups with morphine implants for 6–9 days;


SP, substance P; SSC, sodium chloride–sodium citrate buffer; W, withdrawal;W1–3, groups experiencimg 1–3 days of withdrawal.

ReferencesAcquas, E., Carboni, E. & Di Chiara, G. (1991) Profound depression of

mesolimbic dopamine release after morphine withdrawal in dependent rats.Eur. J. Pharmacol., 193, 133–134.

Acquas, E. & Di Chiara, G. (1992) Depression of mesolimbic dopaminetransmission and sensitization to morphine during opiate abstinence.J. Neurochem., 58, 1620–1625.

Basheer, R. & Tempel, A. (1993) Morphine-induced reciprocal alterations inG alpha s and opioid peptide mRNA levels in discrete brain regions.J. Neurosci. Res., 36, 551–557.

Bernard, V., Le Moine, C. & Bloch, B. (1991) Striatal neurons expressincreased level of dopamine D2 receptor mRNA in response to haloperidoltreatment: a quantitative in situ hybridization study.Neuroscience, 45,117–126.

Bhargava, H.N., Ramarao, P., Gulati, A., Gudehithlu, K.P. & Tejwani, G.A.(1989) Methionine-enkephalin and beta-endorphin levels in spleen andthymus gland of morphine tolerant-dependent and abstinent rats.Life Sci.,45, 2529–2537.

Crippens, D. & Robinson, T.E. (1994) Withdrawal from morphine oramphetamine: different effects on dopamine in the ventral-medial striatumstudied with microdialysis.Brain Res., 650, 56–62.

Dal Toso, R., Sommer, B., Ewert, M., Herb, A., Pritchett, D.B., Bach, A.,Shivers, B.D. & Seeburg, P.H. (1989) The dopamine D2 receptor: twomolecular forms generated by alternative splicing.EMBO. J, 8, 4025–4034.

Dearry, A., Gingrich, J.A., Falardeau, P., Fremeau, R.T., Bates,M.D., Jr &Caron, M.G. (1990) Molecular cloning and expression of the gene for ahuman D1 dopamine receptor.Nature, 347, 72–76.

Devine, D.P. & Wise, R.A. (1994) Self-administration of morphine. DAMGO.and DPDPE into the ventral tegmental area of rats.J. Neurosci., 14,1978–1984.

Di Chiara, G. & North, R.A. (1992) Neurobiology of opiate abuse.Trends.Pharmacol. Sci., 13, 185–193.

Eriksson, P.S., Hansson, E. & Ronnback, L. (1991) Mu and delta opiatereceptors in neuronal and astroglial primary cultures from various regionsof the brain – coupling with adenylate cyclase. localisation on the sameneurones and association with dopamine (D1) receptor adenylate cyclase.Neuropharmacology, 30, 1233–1239.

Gerfen, C.R. & d. Young, W.S. (1988) Distribution of striatonigral andstriatopallidal peptidergic neurons in both patch and matrix compartments:an in situ hybridization histochemistry and fluorescent retrograde tracingstudy.Brain Res., 460, 161–167.

Gerfen, C.R., Engber, T.M., Mahan, L.C., Susel, Z., Chase, T.N. & Monsma,F.J., Jr & Sibley, D.R. (1990) D1: and D2 dopamine receptor-regulatedgene expression of striatonigral and striatopallidal neurons [see comments].Science, 250, 1429–1432.

Giros, B., Jaber, M., Jones, S.R., Wightman, R.M. & Caron, M.G. (1996)Hyperlocomotion and indifference to cocaine and amphetamine in micelacking the dopamine transporter.Nature, 379, 606–612.

Gold, L.H., Stinus, L., Inturrisi, C.E. & Koob, G.F. (1994) Prolonged tolerance.dependence and abstinence following subcutaneous morphine pelletimplantation in the rat.Eur. J. Pharmacol., 253, 45–51.

Gudehithlu, K.P. & Bhargava, H.N. (1995) Modulation of preproenkephalinmRNA levels in brain regions and spinal cord of rats treated chronicallywith morphine.Peptides, 16, 415–419.

Harris, G.C. & Aston-Jones, G. (1994) Involvement of D2 dopamine receptorsin the nucleus accumbens in the opiate withdrawal syndrome.Nature, 371,155–157.

Heijna, M.H., Bakker, J.M., Hogenboom, F., Mulder, A.H. & Schoffelmeer,A.N. (1992) Opioid receptors and inhibition of dopamine-sensitive adenylatecyclase in slices of rat brain regions receiving a dense dopaminergic input.Eur. J. Pharmacol., 229, 197–202.

Johnson, S.W. & North, R.A. (1992) Opioids excite dopamine neurons byhyperpolarization of local interneurons.J. Neurosci., 12, 483–488.

Kebabian, J. & Calne, D.B. (1979) Multiple receptors for dopamine.Nature,277, 93–96.

Kiyatkin, E.A. & Rebec, G.V. (1997) Activity of presumed dopamine neuronsin the ventral tegmental area during heroin self-administration.Neuroreport,8, 2581–2585.

Klitenick, M.A., DeWitte, P. & Kalivas, P.W. (1992) Regulation ofsomatodendritic dopamine release in the ventral tegmental area by opioidsand GABA: an in vivo microdialysis study.J. Neurosci., 12, 2623–2632.


Koob, G.F. (1992) Drugs of abuse: anatomy. pharmacology and function ofreward pathways.Trends Pharmacol. Sci., 13, 177–184.

Koob, G.F. (1996) Drug addiction: the yin and yang of hedonic homeostasis.Neuron, 16, 893–896.

Koob, G.F. & Bloom, F.E. (1988) Cellular and molecular mechanisms of drugdependence.Science, 242, 715–723.

Koob, G.F. & Le Moal, M. (1997) Drug abuse: hedonic homeostaticdysregulation.Science, 278, 52–58.

Le Moine, C., Bernard, V. & Bloch, B. (1994a) Quantitative in situhybridization using radioactive probes in the study of gene expression inheterocellular systems.Methods Mol. Biol., 33, 301–311.

Le Moine, C. & Bloch, B. (1995) D1: and D2 dopamine receptor geneexpression in the rat striatum: sensitive cRNA probes demonstrate prominentsegregation of D1 and D2 mRNAs in distinct neuronal populations of thedorsal and ventral striatum.J. Comp. Neurol., 355, 418–426.

Le Moine, C., Kieffer, B., Gaveriaux-Ruff, C., Befort, K. & Bloch, B. (1994b)Delta-opioid receptor gene expression in the mouse forebrain: localizationin cholinergic neurons of the striatum.Neuroscience, 62, 635–640.

Le Moine, C., Normand, E., Guitteny, A.F., Fouque, B., Teoule, R. & Bloch, B.(1990) Dopamine receptor gene expression by enkephalin neurons in ratforebrain.Proc. Natl Acad. Sci. USA, 87, 230–234.

Le Moine, C., Svenningsson, P., Fredholm, B.B. & Bloch, B. (1997) Dopamine–adenosine interactions in the striatum and the globus pallidus: inhibition ofstriatopallidal neurons through either D2 or A2A receptors enhances D1receptor-mediated effects on c-fos expression.J. Neurosci., 17, 8038–8048.

Liu, J., Nickolenko, J. & Sharp, F.R. (1994) Morphine induces c-fos and junBin striatum and nucleus accumbens via D1 and N-methyl-D-aspartatereceptors. Proc. Natl. Acad. Sci. USA, 91, 8537–8541.

Maldonado, R., Saiardi, A., Valverde, O., Samad, T.A., Roques, B.P. &Borrelli, E. (1997) Absence of opiate rewarding effects in mice lackingdopamine D2 receptors.Nature, 388, 586–589.

Mamoon, A.M., Barnes, A.M., Ho, I.K. & Hoskins, B. (1995) Comparativerewarding properties of morphine and butorphanol.Brain Res. Bull., 38,507–511.

Mansour, A., Fox, C.A., Akil, H. & Watson, S.J. (1995) Opioid-receptormRNA expression in the rat CNS: anatomical and functional implications.Trends Neurosci, 18, 22–29.

Mansour, A., Fox, C.A., Burke, S., Meng, F., Thompson, R.C., Akil, H. &Watson, S.J. (1994) Mu. delta. and kappa opioid receptor mRNA expressionin the rat CNS: an in situ hybridization study.J. Comp. Neurol., 350,412–438.

Mansour, A., Khachaturian, H., Lewis, M.E., Akil, H. & Watson, S.J. (1987)Autoradiographic differentiation of mu. delta. and kappa opioid receptorsin the rat forebrain and midbrain.J. Neurosci., 7, 2445–2464.

Morris, B.J., Hollt, V. & Herz, A. (1988) Opioid gene expression in ratstriatum is modulated via opioid receptors: evidence from localized receptorinactivation.Neurosci. Lett.,89, 80–84.

Naranjo, J.R., Mellstrom, B., Achaval, M. & Sassone-Corsi, P. (1991)Molecular pathways of pain: Fos/Jun-mediated activation of a noncanonicalAP-1 site in the prodynorphin gene.Neuron, 6, 607–617.

Navarro, M., Fernandez-Ruiz, J.J., Rodriguez de Fonseca, F., Hernandez,M.L., Cebeira, M. & Ramos, J.A. (1992) Modifications of striatal D2dopaminergic postsynaptic sensitivity during development of morphinetolerance-dependence in mice.Pharmacol Biochem. Behav., 43, 603–608.

Nawa, H., Kotani, H. & Nakanishi, S. (1984) Tissue-specific generation oftwo preprotachykinin mRNAs from one gene by alternative RNA splicing.Nature, 312, 729–734.

Nestler, E.J. (1992) Molecular mechanisms of drug addiction.J. Neurosci.,12, 2439–2450.

Pontieri, F.E., Tanda, G. & Di Chiara, G. (1995) Intravenous cocaine.morphine. and amphetamine preferentially increase extracellular dopaminein the ‘shell’ as compared with the ‘core’ of the rat nucleus accumbens.Proc. Natl Acad. Sci. USA, 92, 12304–12308.

Pothos, E., Rada, P., Mark, G.P. & Hoebel, B.G. (1991) Dopamine microdialysisin the nucleus accumbens during acute and chronic morphine. naloxone-precipitated withdrawal and clonidine treatment.Brain Res., 566, 348–350.

Przewlocka, B., Turchan, J., Lason, W. & Przewlocki, R. (1996) The effectof single and repeated morphine administration on the prodynorphin systemactivity in the nucleus accumbens and striatum of the rat.Neuroscience,70, 749–754.

Reisine, T. & Bell, G.I. (1993) Molecular biology of opioid receptors.TrendsNeurosci,16, 506–510.

Romualdi, P., Lesa, G. & Ferri, S. (1991) Chronic opiate agonists down-regulate prodynorphin gene expression in rat brain.Brain Res., 563, 132–136.


Rossetti, Z.L., Hmaidan, Y. & Gessa, G.L. (1992) Marked inhibition ofmesolimbic dopamine release: a common feature of ethanol. morphine.cocaine and amphetamine abstinence in rats.Eur. J. Pharmacol., 221,227–234.

Schoffelmeer, A.N., De Vries, T.J., Hogenboom, F. & Mulder, A.H. (1993)Mu- and delta-opioid receptors inhibitorily linked to dopamine-sensitiveadenylate cyclase in rat striatum display a selectivity profile towardendogenous opioid peptides different from that of presynaptic mu. deltaand kappa receptors.J. Pharmacol. Exp. Ther., 267, 205–210.

Self, D.W., McClenahan, A.W., Beitner-Johnson, D., Terwilliger, R.Z. &Nestler, E.J. (1995) Biochemical adaptations in the mesolimbic dopaminesystem in response to heroin self-administration.Synapse, 21, 312–318.

Spanagel, R., Almeida, O.F., Bartl, C. & Shippenberg, T.S. (1994) Endogenouskappa-opioid systems in opiate withdrawal: role in aversion andaccompanying changes in mesolimbic dopamine release.Psychopharmacology (Berl),115, 121–127.

Spanagel, R., Herz, A. & Shippenberg, T.S. (1992) Opposing tonically activeendogenous opioid systems modulate the mesolimbic dopaminergic pathway.Proc. Natl Acad. Sci. USA, 89, 2046–2050.

Spanagel, R. & Shoaib, M. (1994) Involvement of mesolimbic kappa-opioidsystems in the discriminative stimulus effects of morphine.Neuroscience,63, 797–804.

Steiner, H. & Gerfen, C.R. (1993) Cocaine-induced c-fos messenger RNA isinversely related to dynorphin expression in striatum.J Neurosci, 13,5066–5081.

Steiner, H. & Gerfen, C.R. (1996) Dynorphin regulates D1 dopamine receptor-mediated responses in the striatum: relative contributions of pre- andpostsynaptic mechanisms in dorsal and ventral striatum demonstrated byaltered immediate-early gene induction.J. Comp. Neurol., 376, 530–541.

Stinus, L., Robert, C., Karasinski, P. & Limoge, A. (1998) Continuousquantitative monitoring of spontaneous opiate withdrawal: locomotoractivity and sleep disorders.Pharmacol. Biochem. Behav., 59, 83–89.


Suaud-Chagny, M.F., Chergui, K., Chouvet, G. & Gonon, F. (1992)Relationship between dopamine release in the rat nucleus accumbens andthe discharge activity of dopaminergic neurons during local in vivoapplication of amino acids in the ventral tegmental area.Neuroscience, 49,63–72.

Tang, F., Costa, E. & Schwartz, J.P. (1983) Increase of proenkephalin mRNAand enkephalin content of rat striatum after daily injection of haloperidolfor 2–3 weeks.Proc. Natl Acad. Sci. USA, 80, 3841–3844.

Tsuji, M., Nakagawa, Y., Ishibashi, Y., Yoshii, T., Takashima, T., Shimada, M.& Suzuki, T. (1996) Activation of ventral tegmental GABA-B receptorsinhibits morphine-induced place preference in rats.Eur. J. Pharmacol., 313,169–173.

Uhl, G.R., Ryan, J.P. & Schwartz, J.P. (1988) Morphine alters preproenkephalingene expression.Brain Res., 459, 391–397.

Uhl, G.R., Vandenbergh, D.J., Rodriguez, L.A., Miner, L. & Takahashi, N.(1998) Dopaminergic genes and substance abuse.Adv. Pharmacol., 42,1024–1032.

Wang, X.Q., Imaki, T., Shibasaki, T., Yamauchi, N. & Demura, H. (1996)Intracerebroventricular administration of beta-endorphin increases theexpression of c-fos and of corticotropin-releasing factor messengerribonucleic acid in the paraventricular nucleus of the rat.Brain Res., 707,189–195.

Wise, R.A. & Bozarth, M.A. (1987) A psychomotor stimulant theory ofaddiction.Psychol. Rev., 94, 469–492.

Wood, P.L., Kim, H.S., Cosi, C. & Iyengar, S. (1987) The endogenous kappaagonist. dynorphin (1–13). does not alter basal or morphine-stimulateddopamine metabolism in the nigrostriatal pathway of the rat.Neuropharmacology, 26, 1585–1588.

Yoburn, B.C., Chen, J., Huang, T. & Inturrisi, C.E. (1985) Pharmacokineticsand pharmacodynamics of subcutaneous morphine pellets in the rat.J. Pharmacol. Exp. Ther., 235, 282–286.

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