Brain Research Bulletin,Vol. 29, pp. 675- 680, 1992 Printed in the USA. All rights reserved. Copyright RAPID 0361- 9230/92 $5.00 + .OO 0 1992 Pergamon Press Ltd. COM M UNICATION Evidence for Opiate-Dopamine CrossSensitization in Nucleus Accumbens: Studies of Conditioned Reward S. TIFFANY CUNNINGHAM* AND ANN E. KELLEY’ *Department of Psychology, Harvard University, Cambridge, MA 02138 fDepartment of Psychology, Northeastern University, Boston, MA 02115 Received 20 March 1992; Accepted 12 April 1992 CUNNINGHAM, S. T. AND A. E. KELLEY. Evidence for opiate-dopamine cross- sensitization in nucleus accumbens: Studies ofcondifioned reward. BRAIN RES BULL. 29(5), 675-680, 1992.-We investigated opiate-amphetamine interactions within the nucleus accumbens in responding for conditioned reward. Separate groups of animals received 4-day intra-accumbens treatment with either saline, morphine (0.5 &0.5 r.d), [D-Ala2 NMe-Phe4 Gly-olS]-Enkephalin (DAMGO; I.0 &0.5 PI), or [D-Pen2,5]Enkephalin (DPEN; 2.0 pg/O.5 ~1). On two subsequent test days, these rats were given a challenge of d-amphetamine (2.0 and 10.0 pg/O.5 ~1) and responding for conditioned reward was measured. In the conditioned reinforcement (CR) procedure, fooddeprived animals were trained in an initial phase to associate a food reward (primary reinforcement) with a compound stimulus (light/click). In the next phase, a lever was introduced and responding on the lever produced the compound stimulus alone (secondary reinforcement). Previous evidence shows that psychostimulants but not opiates markedly potentiate responding for conditioned reward. In the present design, animals previously treated with either morphine or DAMGO (preferential mu agonists) showed potentiated lever responding following amphetamine challenges, relative to either DPEN- or saline-treated animals. These findings show that prior exposure of nucleus accumbens neurons to /l-selective opiates induces sensitization to the effects of amphetamine. The results are discussed in terms of opioid effects on dopamine transmission and second messenger systems. Opioids Receptor subtypes Dopamine Cross-sensitization OPIATE and psychostimulant drugs share several characteristics regarding their behavioral and rewarding effects in animals. Peripheral or central administration of these substances can result in hypermotility (7,9,14,2 1,24,25,32). In addition, these drugs are self-administered by animals (IO, 13,15,43) and are capable of inducing behavioral sensitization ( 18,19). Behavioral sensitization is defined as a potentiated behavioral response to a drug following prior chronic treatment with the drug. For example, multiple exposure to opiates produces a progressively enhanced locomotor response to the drug (16,19,41). One region which has been implicated in both stimulant- and opiate-induced sensitization is the ventral tegmental area (VTA), the cell body region for A10 dopamine neurons (4 1). For example, repeated microinjection of morphine or the opioid peptide, [D-Ala2-NMe-Phe4-Gly-o15]Enkephalin (DAMGO) (41) or enkephalin (16,19) into the VTA results in a progressively augmented locomotor response. Conditioned reinforcement Similar behavioral effects have been reported with multiple injections of amphetamine, such that chronic injection of amphetamine into the VTA but not the nucleus accumbens sensitizes rats to systemic amphetamine and cocaine (20). More recent data suggest the nucleus accumbens, a major terminal field for A 10 dopamine neurons, may also contribute to stimulant-induced sensitization (29). Paulson and Robinson (29) found that repeated systemic administration of amphetamine results in an enhanced locomotor response to amphetamine injected into the nucleus accumbens. Interestingly, opiates and psychostimulants also cross-sensitize to one other. For example, multiple intra-VTA treatments of d-amphetamine will result in an augmented response to a subthreshold dose of systemic morphine, as measured by locomotor activity (36,42). The reverse situation has also been demonstrated, such that multiple systemic morphine injections sensitizes animals to systemic amphetamine (40). In addition, intra- ’ Requests for reprints should be addressed to Ann E. Kelley, Department of Psychology, Northeastern University, 125 Nightingale Hall, Boston, MA 02115. 675 zyxwvu 676 CUNNINGHAM VTA enkephalin infusions sensitize animals to systemic amphetamine or cocaine ( 12,I6). Most of the investigations that have examined the effects of cross-sensitization between opiates and psychostimulants have focused on motor activity (12,16,36,40). However, little is known about the effects of opiate-psychostimulant cross-sensitization on reinforcement processes. In the following experiments, the effects of prior treatment with opiates on sensitivity to amphetamine were examined using a conditioned reinforcement procedure. In the conditioned reinforcement (CR) paradigm, an animal’s reactivity to reward-related stimuli is determined (34). Hungry rats are trained using classical conditioning to associate a compound stimulus with a food reward. The animals are then tested for operant responding for presentation of the conditioned stimulus. Acquisition of this new behavior, lever pressing, is a measure of the potency of the compound stimulus as the conditioned reinforcer (26). The nucleus accumbens is an important neural site for psychostimulant-potentiated responding for CR (5,23,38). Intra-accumbens morphine and opioid peptides, however, fail to elevate responding for conditioned reward (6), suggesting that psychostimulants and opiates affect reward-related responding differentially. However, in a previous dose-response study with intra-accumbens morphine. we noted that following several days treatment with intra-accumbens morphine, animals showed elevated responding to intra-accumbens amphetamine (6). Therefore, in the present study. sensitivity to amphetamine was compared in animals previously treated with morphine, selective opioid peptides, or saline injections in the nucleus accumbens. ME-I'HOD Thirty-eight male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were used for these experiments. Animals were handled by the experimenter on arrival and housed in pairs in Plexiglas cages with wire grid floors. Eight to 15 g of food were given to the rats daily to maintain them at 85% of their free-feeding body weight and water was freely available. The ambient photoperiod was 12 h, with lights on from 07:OO to 19:OO h. On the day of surgery (which took place after the classical conditioning phase) animals were anesthetized with nembutal (50 mg/kg, IP) and given atropine (0.1 ml. s.c.). A Kopf stereotaxis was used to implant stainless steel cannula guides (23-gauge) 2.5 mm above nucleus accumbens. Based on the atlas of Pellegrino and Cushman (30) with incisor bar 5 mm above interaural zero, the coordinates were, in millimeters: antero-posterior $3.5 from bregma; lateral-medio 21.7 from midline; dorsoventral -5.7 from skull surface. Liquid acrylic and a light-curable dental resin were used to affix the cannulae to skull screws. Following surgery, wire stylets were placed in cannulae to prevent occlusion and animals were allowed a minimum of 2 days recovery. The following drugs were used for these experiments: Morphine sulfate (Penick Corp.. Lyndhurst. NJ), d-amphetamine (Sigma Chemical Co., St. Louis, MO), [D-Ala2 NMe-Phe4 Glyol5]-Enkephalin (DAMGO) (Bachem Inc.. Torrance, CA), [DPen2.5]-Enkephalin (DPEN) (Bachem), and bovine serum albumin (BSA. 10% solution) (Sigma). BSA was used to coat the microinjection tubing (PE-10, Clay Adams) to prevent the pep- AND KELLEY tides from adhering to the walls of the tubing. On each test day, wire stylets were removed and a precut dental square broach was used to clear the cannulae. Stainless steel injector needles (30-gauge), 12.5 mm in length, were used to deliver the drugs. A microdrive pump (Harvard Apparatus) connected to the injectors via tubing delivered the drugs over 1 min 33 s with I min diffusion. Prior to the onset of testing, animals were given a preliminary saline infusion to familiarize them with the procedure. A detailed description of the conditioned reinforcement paradigm has been published elsewhere (22). To summarize, fooddeprived rats were initially trained in a classical conditioning phase to associate a compound stimulus (light/click) with a food reward. In the next phase. a lever was placed in the apparatus and operant responding delivered the conditioned stimulus alone (no food). All drugs were administered during this second phase and total lever presses for the conditioned stimulus was recorded over a 45-min test session. On four separate test days, separate groups of animals received either intra-accumbens saline, morphine (0.5 pg/O.5 ccl), [D-Ala2 NMe-Phe4 Gly-olS]-Enkephalin (DAMGO, a p agonist: I .O pg/O.5 PI), or [D-Pen2,5]-Enkephalin (DPEN. a 6 agonist: 2.0 Kg/O.5 ~1). On two subsequent test days, challenges with intra-accumbens d-amphetamine (2.0 and 10.0 pg/O.5 hl) were administered. Animals in the morphine and DAMGO pretreatment groups were given a 30-min delay (based on previous observations of maximum enhancement of motor activity) (6) before introduction to the testing apparatus. All other groups were placed in chambers immediately following drug infusion. Data were analyzed using an IBM-compatible CRunch Interactive Statistical Package (CRISP). A between-subjects analysis of variance (ANOVA) was performed to test for overall differences between pretreatment groups. When appropriate, a twofactor ANOVA (group X treatment day) was used to determine treatment X day interactions. At the termination of the experiment, subjects were given an overdose of nembutal and perfused transcardially first with saline followed by 10% formalin. Following in situ fixation, the brains were removed and stored in formalin. Coronal sections (60 pm) were made and stained with cresyl violet to verify cannula track and injector tip location. Fig. 2 (discussed in detail later) is a photomicrograph of the histology of a representative subject. RESULTS Animals given the 4-day treatments of morphine showed a sensitized response to amphetamine challenges. This pretreatment group had potentiated amphetamine-induced responding relative to the other treatment groups. The first analysis was a pretreatment group X dose ANOVA on the scores following amphetamine challenge. This analysis indicated a significant difference between the saline and morphine groups, F( 1, 18) = 13.96, p < 0.002 (Fig. 1A and B). There was no group X dose interaction indicating that both groups responded similarly to the two doses of amphetamine. In addition, a between-groups ANOVA on the scores of the 4-day pretreatments was carried out for the morphine and saline groups. No significant difference OPIATE-DOPAMINE 617 CROSS-SENSITIZATION A B 250 (N=ll) 250 1 (N=S) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1 200 fn 1 3 g 150- E E loo- ? 0 0 DOSE 2 0 SALINE AMPH (pg10.5 250 * l T zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0.5 10 0.5 0.5 ~1) DOSE 2 10 AMP” AMPH 0.5 MORPHINE AMPH C * l (pg10.5 pl) D zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA (N=lO) (Nd) 250 1 I 200 - 200 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML z 3 150- : 1 2 AMPH DAM GO DOSE (pg10.5 AMPH 2 2 OPEN DOSE ~1) (pg10.5 2 10 AMPH AMP” pl) FIG. 1. (A-D) Effects of 4day intra-accumbens (A) saline, (B) morphine, (C) DAMGO (p agonist), or (D) DPEN (6 agonist) pre-exposure on amphetamine-induced responding for conditioned reward. Bars represent mean lever presses y SEM. zyxwvutsrqponmlkjihgfedcbaZYXWVUTS * p < 0.05, **p < 0.01, relative to salinepretreated amphetamine response. was found suggesting that morphine itself does not potentiate CR responding. Figure 1 (C and D) shows the response to amphetamine in animals pretreated with either DAMGO (p agonist) or DPEN (6 agonist). Amphetamine responding for conditioned reward was potentiated in the DAMGO pretreatment animals. A twofactor ANOVA on the amphetamine scores of the DAMGO and saline pretreatment groups showed a significant group effect, F( I, 19) = 5.48, p < 0.03 and no group X dose interaction. Analysis of the DAMGO and saline 4-day pretreatment scores revealed no significant differences between groups. An ANOVA performed on amphetamine scores in the DPEN pretreatment group did not reveal any significant effect relative to amphetamine scores in the saline pretreatment group. A between-groups ANOVA on the 4-day treatment for DPEN and saline, however, revealed a significant difference, F( 1, 17) = 4.93, p < 0.04. DISCUSSION In the present experiments, either morphine or DAMGO repeated prior administration into the nucleus accumbens of po- tentiated amphetamine-induced responding for conditioned reward. There are several points to address in consideration of this finding. First, the effects of the opioids themselves; second, the enhanced response to amphetamine; third, the possible mechanisms underlying cross-sensitization. In the current paradigm, neither morphine nor DAMGO (both ~1agonists) elevated responding for conditioned reward; response levels on all test days were similar to those following saline infusion. This profile is in agreement with a recent dose-response study of opiate infusion into nucleus accumbens (6). That study demonstrated that nucleus accumbens infusion ofopiates increased locomotor activity but did not affect CR responding. DPEN (a 15agonist) did induce a significant elevation in responding (compared with the saline group). This increase is somewhat surprising because in the previous dose-response study (6) no increase in CR responding was found following DPEN infusion. This finding may be related to evidence that &agonists are more potent in eliciting dopamine release than ~1agonists (3 1). Also, a-agonists induce more robust motor activation relative to pm-agonists (67). Note, however, that the level of DPEN- 678 CUNNINGHAM AND KELLEY FIG. 2. Photomicrograph of coronal section depicting cannula tracks and injection tips within nucleus accumbens. group. The cresyl violet-stained section is from a representative induced responding is well below that observed for amphetamine. Thus, based on knowledge of previous work, we do not consider this increase to be a true potentiation effect (6). We did not observe progressive sensitization to opioid infusion into the accumbens; responding was similar on all 4 test days. This finding is in agreement with previous studies using locomotor activity as a measure for sensitization to intra-accumbens opiates and DAMGO following repeated exposure to these drugs (4 1). For example, Vezina and colleagues (41) reported that multiple exposure to opiates in the ventral tegmental area but not in the nucleus accumbens resulted in a progressively greater locomotor response with repeated injections. The most significant finding in the present experiments was that preexposure of the nucleus accumbens to opiates enhances the animal’s response to amphetamine injections in that site. As noted earlier, there have been reports of opiate-amphetamine cross-sensitization in measurements of motor activity and employing systemic injections. Here we have demonstrated crosssensitization utilizing a reward-related paradigm and have shown that this effect may be induced at the level of the nucleus accumbens. In support of this general idea, Paulson and Robinson (29) found potentiated behavioral response to intra-accumbens amphetamine 21 days following the termination of multiple systemic injections of amphetamine. These authors concluded that the nucleus accumbens is important for the expression of sensitization to amphetamine while the A10 cell body region may mediate its induction (29). In consideration of the present data, we suggest that the nucleus accumbens may also mediate long-term changes associated with the induction of sensitization. The observed cross-sensitization was manifested differentially via the opiate receptor subtypes because previous exposure to subject in the DPEN pretreatment the delta agonist, DPEN, did not enhance amphetamine-induced responding. Morphine binds both mu and delta receptor subtypes whereas DAMGO binds selectively at the mu receptor; pretreatment with either drug potentiated lever pressing elicited by amphetamine. Thus, it is likely that the mu receptor subtype mediates opiate-amphetamine cross-sensitization in nucleus accumbens. Another distinction between the different receptorspecific peptides and their role in behavior has been cited in locomotor activity studies. Preferential mu agonists induce hypomotility followed by hypermotility wheras delta agonists induce an immediate onset of hyperactivity (6,7,14). Moreover, b-agonist-induced activity may involve dopamine release, whereas p-agonist-induced activity is independent of dopamine release in nucleus accumbens (2 I ,32,37). Psychostimulants, particularly those which potently release dopamine, have been shown to potentiate responding for conditioned reward (4,23,33,35). The nucleus accumbens has been shown to be a site sensitive to amphetamine-induced responding for conditioned reward (22,38). In the aforementioned experiments, CR responding following intra-accumbens amphetamine challenges in the saline pretreatment group was lower than that previously observed in this laboratory (23). Partial extinction may underlie this diminished responding because animals did not receive amphetamine challenges until test days 5 and 6. The responding did not completely extinguish but repeated testing without amphetamine may have resulted in decreased magnitude of the impact of the CR. In the present experimental design, animals received morphine pretreatment and subsequent amphetamine challenges in the same test environment. There is evidence in the literature that environmental conditioning contributes to opiate-amphetamine cross-sensitization in locomotor activity (36,40). Further research is currently being done to determine whether condi- OPIATE-DOPAMINE 679 CROSS-SENSITIZATION tioning factors contribute to the observed cross-sensitization in the CR paradigm. Although the precise neural mechanisms underlying crosssensitization are not yet known, one may speculate about several possibilities. The first possibility to consider is whether multiple injections of opiates into the nucleus accumbens induce an alteration in dopamine transmission. There is abundant evidence that the dopamine system regulates activity of the opioid peptides in the striatum (1,2,17,27,37,44). It may follow that repeated stimulation of the opiate system elicits changes in dopaminergic activity. Chronic morphine administration has inhibitory effects on the phosphorylation of tyrosine hydroxylase in the nucleus accumbens, implicating a decrease in dopamine production at the terminal region (3). A decrease in dopamine production may result in increased sensitivity of dopamine receptors. Opiate-induced changes in second messenger systems may also contribute to cross-sensitization. Recent biochemical data suggest that chronic morphine treatment results in a variety of changes in signal transduction mechanisms. For example, chronic prenatal exposure to morphine elevated striatal in vitro D 1 receptor-mediated adenylate cyclase activity in offspring (8). Terwilliger and colleagues (39) demonstrated that daily subcutaneous implantations of morphine pellets increased both ade- nylate cyclase activity as well as cyclic AMP-dependent protein kinase activity in the nucleus accumbens and other brain regions. In another study, long-term exposure to morphine elevated pertussis toxin-induced adenosine diphosphate (ADP)-ribosylation of G-proteins (28) which exert inhibitory actions on secondmessenger systems. 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