Motivational state determines the functional role of the mesolimbic dopamine system in the mediation of opiate reward processes

Motivational state determines the functional role of the mesolimbic dopamine system in the mediation of opiate reward processes

Behavioural Brain Research 129 (2002) 17 – 29 www.elsevier.com/locate/bbr Research report Motivational state determines the functional role of the m...

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Behavioural Brain Research 129 (2002) 17 – 29 www.elsevier.com/locate/bbr

Research report

Motivational state determines the functional role of the mesolimbic dopamine system in the mediation of opiate reward processes Steven R. Laviolette *, Karim Nader, Derek van der Kooy Department of Anatomy and Cell Biology, Neurobiology Research Group, Uni6ersity of Toronto, Medical Sciences Building, Room 1105, Toronto Ont., Canada, M5S 1A8 Received 8 February 2001; received in revised form 21 June 2001; accepted 22 June 2001

Abstract We have previously reported that mesolimbic dopamine (DA) substrates are critically involved in the rewarding effects of opiates only during states of opiate-dependence and withdrawal. However, in previously drug-naive animals, opiate reward is mediated through a DA-independent neural system. In the present study, we report that bilateral microinjections of a DA receptor antagonist, a-flupenthixol (0.3–3 mg/0.5 ml) into the nucleus accumbens (NAc), blocks morphine reward (10 mg/kg, i.p.) in opiate-withdrawn animals, but not in opiate-naive animals, suggesting that accumbal dopamine receptors are required for opiate reward signaling in drug-deprived motivational states. Next, the role of dopamine was examined in the development of opiate dependence and somatic withdrawal, and expression of withdrawal aversions. Pretreatment with a-flupenthixol (0.8 mg/kg, i.p.) before morphine injections during the development of opiate dependence did not effect expression of withdrawal aversions or the expression of somatic withdrawal. We have previously reported that pretreatment with a dopamine receptor antagonist, a-flupenthixol, blocks the aversive effects of opiate withdrawal. We now report that pretreatment with a direct dopamine receptor agonist, apomorphine (1.0–5.0 mg/kg, i.p.) before conditioning in a state of withdrawal, also blocks the aversive effects of opiate withdrawal. We propose that the aversive motivational effects of opiate withdrawal may be mediated by a specific dopaminergic neuronal signal. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Dopamine; Nucleus accumbens; Opiate withdrawal; Reward

1. Introduction The mesolimbic dopamine (DA) pathway consists of the dopaminergic cell bodies of the ventral tegmental area (VTA) and their ascending projections to the nucleus accumbens (NAc). Considerable evidence suggests that this DA pathway is critically involved in both the rewarding properties of opiate drugs [30,35] and the aversive effects of opiate withdrawal [15,18]. To this end, various studies have reported that blockade of mesolimbic DA transmission with DA receptor antago* Corresponding author. Tel.: + 1-416-978-4539; fax: +1-416-9783844. E-mail address: [email protected] (S.R. Laviolette).

nists, or neurotoxic lesions of DA pathways are sufficient to attenuate the rewarding properties of opiates [1,6,19,32]. In contrast, other reports have suggested that DA signaling is neither necessary nor sufficient for the mediation of opiate reward [3,11,13,22]. We have previously reported that the role of the mesolimbic DA system in opiate motivational processes is dependent upon whether the animal is in an opiatenaive state, or is opiate-dependent and in withdrawal. Thus, in opiate-naive animals, opiate reward is mediated by a DA-independent neural reward system, that is dependent upon the integrity of the tegmental pedunculopontine nucleus (TPP) [3,22]. Conversely, in animals that are opiate-dependent and in withdrawal, the rewarding effects of opiates are mediated by a DA-depen-

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dent neural reward system, independently of the TPP [3,22]. Similarly, within the VTA itself, opiates produce reward through a DA-independent neural substrate in opiate-naive animals, while in opiate-dependent and withdrawn animals, opiate reward is mediated through a DA-dependent neural reward system [22]. Since the ability of opiates to alleviate the aversive effects of opiate withdrawal is highly sensitive to DA receptor blockade during states of opiate dependence and withdrawal, we hypothesized that DA receptor signaling in the NAc (the terminal projection field of the VTA DA neurons) is essential for the mediation of opiate reward during states of opiate dependency and withdrawal. Conversely, in previous opiate naive animals, DA receptor activation in the NAc will not be essential for opiate reward signaling. To test this hypothesis, we examined the effects of bilateral, intra-NAc microinjections of the broad-spectrum DA receptor antagonist, a-flupenthixol, on the rewarding effects of morphine in both opiate-dependent and withdrawn animals or opiate-naive and non-dependent animals. Previous reports have suggested that molecular alterations in the mesolimbic DA system during states of opiate dependency and withdrawal are involved in both the somatic withdrawal symptoms associated with opiate withdrawal (physical symptoms such as teeth chattering, writhing and wet dog shakes), as well as the aversive motivational effects of withdrawal (as measured by the animal’s aversion to an environment previously paired with opiate withdrawal) [9,15,27,29]. Thus, several reports have suggested that decreases in mesolimbic DA activity during opiate withdrawal are responsible for the aversive withdrawal syndrome [9,15,27]. In contrast, other reports have suggested that dopaminergic activity may actually be increased during withdrawal, as demonstrated by increased fos activation in the NAc correlated with opiate withdrawal [34] and the demonstration that during opiate withdrawal, animals become hypersensitive to the behavioral effects of DA receptor agonists [10,34]. Further evidence for the role of DA systems in the opiate withdrawal syndrome come from the demonstration that intraNAc microinjections of the DA receptor antagonist, a-flupenthixol, induced somatic withdrawal signs similar to those observed following treatment with the opiate receptor antagonist, naloxone, in opiate dependent rats [15]. Conversely, systemic administration of the DA receptor agonist, apomorphine, significantly attenuated somatic withdrawal signs associated with opiate withdrawal [15]. Together, these results suggest that alterations in mesolimbic DA activity during opiate dependency and withdrawal are

involved in the opiate withdrawal syndrome. Furthermore, if somatic withdrawal symptoms are indicative of the aversive motivational effects of opiate withdrawal, these results suggest that alterations in a NAc DA substrate during the addiction process may be involved in both the aversive motivational effects of opiate withdrawal and the somatic withdrawal symptoms associated with opiate withdrawal. We have previously reported that neuroleptic pretreatment blocks the aversive motivational properties of opiate withdrawal, but has no effect upon the expression of somatic withdrawal signs [5], suggesting that somatic withdrawal signs and the aversive properties of opiate withdrawal may be mediated by separate neural substrates. If the aversive motivational properties of opiate withdrawal were due to decreased activity at mesolimbic DA receptors [9,15,27,29], then DA receptor antagonists would be expected to potentiate the aversive properties of opiate withdrawal by further blunting mesolimbic DA transmission, rather than alleviate them. One alternative hypothesis is that the aversive properties of opiate withdrawal are mediated by a specific mesolimbic DA neuronal signal, which is present during the opiate withdrawal syndrome. In this case, the aversive effects of opiate withdrawal would be alleviated by blocking the aversive motivational signal mediated through the activity of the mesolimbic DA pathway, either by neuroleptic blockade of DA receptors or by direct activation of postsynaptic DA receptors with a direct DA receptor agonist, apomorphine. We predicted that direct DA receptor activation with apomorphine would mask this hypothesized aversive, dopaminergic signal during opiate withdrawal, and thus block conditioned place aversions to environments paired with opiate withdrawal. To test this hypothesis, opiate dependent and withdrawn rats were pretreated with the direct DA receptor agonist, apomorphine, before being conditioned in a state of withdrawal. We further tested the possible involvement of DA systems in the development of opiate dependence by pretreating rats with a high dose of a-flupenthixol prior to morphine injections during the acquisition of opiate dependence. If DA systems are necessary for the development of morphine dependence and withdrawal, then we predicted that a-flupenthixol pretreatment during the development of opiate dependence would prevent the neuroadaptive changes that mediate morphine dependence, and in turn, withdrawal. We also examined whether neuroleptic pretreatment during the development of opiate dependence would affect the expression of somatic withdrawal signs observed later in these animals in the absence of neuroleptic.

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2. General method

2.1. Animals All animals used in these experiments were adult (350– 400 g) male Wistar rats (Charles River). Animals were individually housed in clear Plexiglas cages in a temperature-controlled room (9 21 °C) with access to food and water ad libitum throughout the duration of the experiments. The animal care and experimental procedures that were performed were in accordance with both institutional and governmental animal care guidelines.

2.2. Place conditioning procedures and apparatus The place conditioning apparatus was identical to that described previously [3,22]. Briefly, place conditioning took place in one of two distinct environments that differed in color, smell and texture. One environment was white, with a wire mesh floor that was covered in wood chips. The other environment was black, with a smooth Plexiglas floor that was wiped down with a 2% acetic acid solution before each conditioning session. These conditioning environments are motivationally balanced such that animals show no initial preference for either environment before conditioning. To confirm this, a separate, control group of animals (n = 5) were treated with saline before being placed in either the black or white environments, in a fully counterbalanced order, as described below. At testing, a narrow, neutral gray zone (on which the rats were placed at the beginning of each test session) separated the two test compartments. All animals in all experiments were tested 1 week after termination of training and were tested in a drug-free state. Each animal was tested for 10 min and times spent in each of the environments were independently scored. Two place conditioning protocols were used in the present set of experiments. For experiments that examined the effects of intra-NAc a-flupenthixol on opiate reward, or the effects of a-flupenthixol on systemic lithium chloride (LiCl), animals were conditioned with a fully counterbalanced place conditioning procedure. In this protocol, animals are exposed to both the black and white conditioning environments in a fully counterbalanced order. The environments are randomly assigned to either drug or saline treatment. All experimental groups (both previous opiate-naive or opiate-dependent and withdrawn) received alternating morphine-environment and saline-environment pairings. Each conditioning session lasted 40 min, for all of the experiments described below. For experiments that examined the effects of apomorphine on the motivational effects of heroin withdrawal or LiCl, or the effects of systemic a-flupenthixol on the development or expression of conditioned place aversions to environments paired with opiate with-

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drawal, a variant of the above described conditioning paradigm was used. The single-side-withdrawal procedure (previously referred to as the W-procedure) [3,5] involves exposing animals to only one of the two conditioning environments. In this procedure, opiate-dependent and withdrawn animals are exposed for 40 min to one conditioning environment (either the black or white box) 21 h after receiving their last injection of the opiate drug. Thus, animals receive an injection of morphine (20 mg/kg, i.p.) 3.5 h after removal from the conditioning environment. The environments that are paired with the aversive effects of opiate withdrawal are counterbalanced within groups. This conditioning cycle is repeated until each animal has been exposed to the assigned conditioning environment four times over an 8-day period. On alternate days, three 20 mg/kg (i.p.) maintenance doses of morphine are administered at irregular intervals through the day. At the termination of training, a recovery and testing phase is instituted, during which time the animals remain undisturbed in their home cages for 1 week in order to recover from any residual effects of either pharmacological challenges during training, or withdrawal itself. After 1 week, animals are tested for 10 min in a drug-free state for their preference for either a novel, neutral environment, and the environment previously paired with any aversive effects of opiate withdrawal. This protocol is sensitive to the aversive effects of both spontaneous and naloxoneprecipitated withdrawal [5]. Previous studies using the identical procedure have shown that any confounding effects of novelty in this paradigm are insignificant. For example, training drug-naı¨ve rats with saline only in one of the environments or food-sated rats with the absence of food in only one environment does not produce any preferences or aversions for either the familiar or novel environment [5]. Furthermore, groups trained concurrently with morphine according to a standard place conditioning procedure (see above) or a single-side-conditioning procedure, demonstrate place preferences for the morphine-paired environments of the same magnitude. If novelty was a confound, then place preferences of different magnitude should have been observed in animals conditioned to both sides of the apparatus compared with animals conditioned to only one of the two environments.

2.3. Opiate dependency and withdrawal For the induction of opiate dependency and withdrawal, two separate protocols were performed. Animals used in central, intra-accumbens a-flupenthixol, systemic apomorphine or lithium chloride experiments, received daily 0.5 mg/kg subcutaneous injections of heroin commencing 4 days before the start of conditioning. Animals were conditioned 21 h after their last heroin injection. During conditioning, this dose of

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heroin was administered daily 3.5 h after the termination of training. Although this regimen of heroin treatment does not induce classical somatic withdrawal signs, they are sufficient to induce a state of motivational dependence, and 20 h after heroin injection, rats are experiencing withdrawal as measured by the conditioned aversions to environments paired with the absence of drug [3,5]. This protocol was used for animals in the central accumbens experiments because longer opiate dependence-inducing protocols (i.e. 21 days or longer) often are not feasible in animals with chronic, indwelling intra-cranial cannulae. Furthermore, this rapid, highly effective regimen reduces the risk of intracranial infection or cannulae blockade in these animals. The aversive effects of withdrawal (as measured by conditioned place aversions to withdrawal-paired environments) induced by this regimen are quantitatively similar to those observed after a 3 week regimen of morphine administration, which produces aversive motivational effects as well as intense somatic withdrawal signs [5]. In experiments examining the development of opiate dependence and the subsequent expression of somatic withdrawal signs, animals were made dependent over a 21 day period. On the day 1, animals received a 20 mg/kg i.p. injection of morphine. On the following 2 days, two injections of 20 mg/kg morphine were administered each day. For the following 18 days, each rat received three injections of 20 mg/kg morphine at irregular intervals during the day. This regimen of morphine exposure induces classic somatic withdrawal symptoms [3,5] and produces aversive behavioral effects that are quantitatively identical to those produced by the heroin protocol described above [5].

2.4. Statistics All data were analyzed with one or two-way analysis of variance (ANOVA) or by student’s t-tests, where appropriate. Post-hoc analyses were performed with Newman Keuls or Fisher’s LSD tests where appropriate.

2.5. Experiment 1 In experiment 1, we examined the effects of intraNAc microinjections of a-flupenthixol (0.3– 30 mg/0.5 ml) on the motivational effects of morphine (10 mg/kg, i.p.) or LiCl (15 mg/kg, i.p.) in either previous opiatenaive animals, or opiate-dependent and withdrawn animals.

2.5.1. Surgery for intra-accumbal microinjection experiments Rats were anesthetized with sodium pentobarbitol (Somnotol) (60 mg/kg, i.p.) and placed in a stereotaxic apparatus with the incisor bar set at − 3.3. All coordi-

nates were taken from [24]. Twenty-two gauge guide cannulae were angled 10° toward the midline and implanted 1.5–2 mm dorsal to the NAc (n= 58) (from bregma: AP + 1.8, L9 3.1; from the dural surface V, − 6.8). The coordinates used for cannulae aimed for the dorsal NAc (n= 5) were AP+ 1.8, L9 3.1 and from the dural surface V, − 5.3. The guide cannulae were anchored to the skull by dental acrylic and stainless steel screws.

2.5.2. Histology At the end of the behavioral experiments, all rats were deeply anesthetized and intracardially perfused with isotonic saline, followed by 10% formalin. The brains were removed and post-fixed in a 25% sucrose solution. The brains were then cut in 32 mm sections and stained with cresyl violet. Cannulae placements were then verified by microscopic examination of the sections. 2.5.3. Microinfusion procedure For bilateral intracranial microinfusions, obturators were removed from the animal’s skullcap, and a 28gauge injector cannula was slowly lowered into the injection site. The injector protruded 1.5– 2 mm ventral to the guide cannulae. Polyethylene tubing (PE-50, Clay–Adams) connected the injector to a 1 ml Hamilton microsyringe, which was loaded with the appropriate solution before microinfusion. Rats were held by the experimenter while infusions were made over a 1 min period. After termination of the infusion, the injector was left in place for an additional 1 min before being removed. Animals received bilateral microinjections of a-flupenthixol into the NAc of either 0, 0.3, 3 or 30 mg/0.5 ml volume per hemisphere. Microinjections were performed 15 min prior to the animals receiving either drug (morphine, 10 mg/kg, i.p. or LiCl, 15 mg/kg, i.p.) or saline, and were then immediately placed in the assigned conditioning environment. 2.5.4. Neuroleptic pretreatment and the a6ersi6e effects of lithium chloride In order to determine if high doses of intra-NAc (30 mg/0.5 ml) or systemic (0.8 mg/kg, i.p.) a-flupenthixol produced any non-specific learning or sensory impairments, that were not specific to opiate-class drugs, separate groups of opiate-naive animals received either intra-NAc a-flupenthixol (30 mg/0.5 ml) (n = 7) or intraNAc saline pretreatment (as described above) (n= 8) before receiving an injection of LiCl (15 mg/kg, i.p.) or saline (1 ml/kg, i.p.) and placed in one of the conditioning environments, as previously described. We also compared the effects of a high systemic dose of a-flupenthixol (0.8 mg/kg, i.p.) between a group of previous drug-naı¨ve animals (n= 5) and a group of heroin-dependent and withdrawn animals (n= 6) (as described

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above) in the acquisition of conditioned place aversions to LiCl (15 mg/kg, i.p.). This dose and pretreatment time of a-flupenthixol potently blocks post-synaptic D1 and D2 receptors [7] and produces no motivational effects in and of itself [14]. The purpose of this experiment was to determine if the ability of DA receptor blockade to attenuate opiate reward in heroin-dependent and withdrawn animals was due to the disruption of a DA-dependent learning process that may only be apparent in the opiate-dependent and withdrawn state, rather than a specific blockade of the rewarding effects of opiates.

2.6. Experiment 2 In experiment 2, we investigated the possible role of the mesolimbic DA system in the development of opiate dependence and the aversive effects of opiate withdrawal. We further examined the possible role of DA systems in the later expression of somatic withdrawal signs associated with opiate withdrawal.

2.6.1. Neuroleptic pretreatment and the de6elopment of morphine dependence In order to assess the possible role of DA in the development of opiate dependence, two groups of animals were made opiate dependent over 21 days (described above) and conditioned using the single-side withdrawal procedure, as previously described. During the development phase of opiate dependence, rats received an injection of either a-flupenthixol (0.8 mg/kg, i.p.) (a-flupenthixol/morphine group, n =7) or saline (saline/morphine group, n = 7) 2.5 h before morphine injections. During the conditioning phase, each group also received additional injections of saline or a-flupenthixol 2.5 h prior to each maintenance dose of morphine. No saline or a-flupenthixol injections were given to either group prior to the placement of each rat in the conditioning environments. Thus, animals were conditioned 21 h after their last morphine maintenance dose and 23.5 h after their last a-flupenthixol or saline dose. 2.6.2. Obser6ation of somatic opiate withdrawal signs Immediately after the last conditioning trial, each animal was placed in a novel environment for 10 min and the frequencies of teeth chattering, jumping, wet dog shakes and writhing were recorded. This novel observation compartment had a green floor with white stripes and plywood walls that were painted with urethane. In order to control for any motivational or somatic effects of chronic a-flupenthixol administration, a third group of rats (a-flupenthixol/saline group, n= 8) received the same regimen of DA antagonist as above but was given saline injections instead of morphine. Thus, on the test day, this group had a choice between a novel, neutral environment, and one paired

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with 23.5 h abstinence from chronic a-flupenthixol treatment.

2.7. Experiment 3 In experiment 3, we investigated the possible effects of DA receptor activation on the aversive effects of opiate withdrawal. To examine this, animals were pretreated with the direct DA receptor agonist, apomorphine (1 –5 mg/kg, i.p.) prior to being conditioned in a state of opiate withdrawal. In a separate control experiment, two groups of opiate-dependent and withdrawn animals were pretreated with either saline or apomorphine (5 mg/kg, i.p.) prior to receiving LiCl (15 mg/kg, i.p.) and then were conditioned in a state of opiate withdrawal, as described below.

2.7.1. Apomorphine pretreatment and the a6ersi6e effects of opiate withdrawal In order to investigate the nature of the dopaminergic mediation of the aversive properties of opiate withdrawal, two groups of animals were made opiate dependent as described above and conditioned according to the single-side withdrawal procedure. During the training phase, (21 h after the last heroin injection) the groups received either a 1 mg/kg (i.p.) (n = 7) or 5 mg/kg (i.p.) (n=7) injection of the direct DA receptor agonist, apomorphine, 10 min prior to being placed in one of the conditioning environments for 40 min. The control group (n= 7) received a 1 ml/kg saline injection prior to being placed in the conditioning environment. 2.7.2. Apomorphine pretreatment and the a6ersi6e effects of lithium chloride In order to control for possible non-specific behavioral effects of apomorphine, we challenged the aversive effects of a separate drug, LiCl (15 mg/kg, i.p.) in the identical single-sided conditioning procedure, described above. In this experiment, two groups of animals were made opiate-dependent, as described above. Then, 21 h following their last heroin injection, animals received an injection of apomorphine (5 mg/kg, i.p.) 5 min prior to receiving an injection of LiCl (15 mg/kg, i.p.) and were then immediately placed in the assigned conditioning environment, as previously described. A second group of animals (n= 8) received an injection of saline (1 ml/kg) and then received an injection of LiCl (15 mg/kg) 5 min later.

3. Results

3.1. Histological analysis All animals that were included in the behavioral analysis had appropriate injector cannulae placements

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within the NAc or dorsal to the NAc for the dorsal control groups. Representative NAc cannulae placements are shown in Fig. 1. Although the majority of cannulae injector tips were found to be within the shell region of the NAc, no behavioral differences were observed between animals that had tip placements within the shell and animals that had tip placements within the core region of the NAc.

3.1.1. Effects of intra-accumbal h-flupenthixol on opiate reward processes In animals that were conditioned in a non-dependent, drug-naı¨ve state, microinjections of a-flupenthixol at the 0.3 and 3 mg doses did not block conditioned place preferences for the morphine-paired environments. However, in animals conditioned in states of opiate withdrawal these identical doses of intra-NAc a-flupenthixol blocked morphine conditioned place preferences. The highest dose of intra-NAc a-flupenthixol (30 mg) blocked morphine conditioned place preferences in both groups of animals. A two-way ANOVA compar-

Fig. 1. Schematic representation of bilateral cannulae placements in the nucleus accumbens. Numbers to the right of a section indicate millimeters rostral to bregma. Open triangles represent cannulae tip placements within the NAc. Black circles represent cannulae tip placements dorsal to the NAc.

ing groups (previously opiate-naive versus opiate-dependent and withdrawn) with intra-NAc a-flupenthixol dose (0– 30 mg/0.5 ml) on difference scores between times spent in saline and morphine-paired environments revealed a significant interaction between group and a-flupenthixol dose (F 3,79 = 5.02, PB 0.05). Post-hoc analysis revealed that in opiate naive animals, preferences for the morphine-paired environment were not significantly reduced from saline control (n= 6) levels at the 0.3 (n= 9) and 3 mg (n= 10) doses of intra-NAc a-flupenthixol (P’s\ 0.05). However, at the higher dose of 30 mg (n= 8), the conditioned place preference for the morphine-paired environment was significantly reduced relative to saline control levels (PB 0.05) (Fig. 2A). In animals that were conditioned while in a heroindependent and withdrawn state, post-hoc analysis revealed that microinjections of a-flupenthixol into the NAc significantly attenuated morphine place preferences relative to saline control (n= 5) levels at the 0.3 (n=7), 3 (n=7) and 30 (n=6) mg doses of intra-NAc a-flupenthixol (P’sB0.05) (Fig. 2B). Thus, DA receptor blockade in the NAc during states of opiate dependence and withdrawal is sufficient to block the rewarding effects of morphine.

3.1.2. Intra-accumbens h-flupenthixol dorsal controls and place conditioning en6ironment controls To control for the possible diffusion of a-flupenthixol dorsally along the cannulae tracks, a control group (n= 5) received 3 mg of bilateral a-flupenthixol 1.5 mm dorsal to the NAc, a likely site of diffusion from the injection sites (Section 2). Since this dose of a-flupenthixol (3 mg) only blocks opiate reward in heroin-dependent and withdrawn animals (Fig. 2B) animals in this experiment were made heroin-dependent and withdrawn as described above. Microinjections of a-flupenthixol 1.5 mm dorsal to the NAc did not block place preferences for morphine, as animals displayed a significant place preference for the morphine-paired environment (t 4 = 4.6, PB 0.05) (Fig. 2B). Histological analysis revealed that dorsal cannulae tip placements were located in the caudate putamen, outside the anatomical boundaries of the NAc (Fig. 1). Thus, the observed behavioral effects of intra-NAc a-flupenthixol are not likely due to dorsal spread of drug from the injection sites but are localized to regions of the NAc surrounding the cannulae tip placements (Fig. 1). To determine that the place conditioning environments were unbiased, a separate, control group of animals (n= 5) received saline pretreatment before being placed into either the black or white conditioning environments, according to the place conditioning procedure described above. At testing, animals demonstrated no preference for either the black or white conditioning environments (t 4 = 1.6, P\0.05) (Fig. 2A, inset). Thus,

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Fig. 2. The effects of intra-accumbal microinjections of a-flupenthixol on the rewarding effects of morphine (10 mg/kg, i.p.). (A). The effects of intra-accumbal a-flupenthixol microinjections (0 –30 mg/0.5 ml/side) hemisphere) on times spent in environments previously paired with morphine minus times spent in environments previously paired with saline (difference scores) in previously drug naive animals. Animals demonstrated significant preferences for the morphine-paired environment at 0, 0.3 and 3 mg/0.5 ml doses of intra-NAc a-flupenthixol, but not at the 30 mg/0.5 ml dose. Inset, absolute times spent in either the black or white place conditioning environments in saline pretreated animals. Animals displayed no preference for either the white or black conditioning environments. Bars represent mean difference scores (time in seconds spent in the drug-paired minus the salinepaired environment) 9S.E.M. for this and all subsequent graphs. (B). The effects of intra-accumbal a-flupenthixol microinjections (0 – 30 mg/0.5 ml/side) on times spent in environments previously paired with morphine minus times spent in environments previously paired with saline in opiate dependent and withdrawn animals. Animals that received intra-NAc saline (0) demonstrated a significant preference for the morphine-paired environment. However, animals demonstrated a strongly attenuated preference for the morphine-paired environment at 0.3, 3 or 30 mg/0.5 ml doses of intra-NAc a-flupenthixol, relative to saline control animals. The effects of dorsal intra-accumbal a-flupenthixol (3.0 mg/0.5 ml/side) on times spent in environments previously paired with morphine minus times spent in environments previously paired with saline. Dorsal a-flupenthixol control animals demonstrated a significant place preference for the morphine-paired environment.

animals display no intrinsic preference for either of the conditioning environments used in the present study.

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3.1.3. Effects of neuroleptic pretreatment on the moti6ational effects of lithium chloride: beha6ioral control experiments If the ability of the higher dose (30 mg) of intra-NAc a-flupenthixol to block morphine conditioned place preferences was due to a specific block of the rewarding effects of morphine rather than a non-specific behavioral impairment, we predicted that this dose of intraNAc a-flupenthixol should have no effect on the acquisition of conditioned place aversions to environments paired with 15 mg/kg LiCl. This low dose of LiCl has previously been shown to produce robust conditioned place aversions that are independent of DA systems [20]. In a separate group of opiate-naive animals (n=7), intra-NAc microinjections of 30 mg a-flupenthixol completely blocked the acquisition of conditioned place aversions to environments previously paired with LiCl (t 6 = 0.4, P\0.05) (Fig. 3A). In contrast, animals that received intra-NAc saline (n=8) demonstrated a significant conditioned place aversion to the environment previously paired with LiCl (t 7 = 2.7, PB 0.05) (Fig. 3A). We further tested whether a high dose of systemic a-flupenthixol (0.8 mg/kg, i.p.) would block the acquisition of conditioned place aversions to environments paired with LiCl in either opiatedependent and withdrawn animals (n= 5) or in previous opiate-naive animals (n= 6). Opiate dependent and withdrawn animals displayed a significant aversion to environments previously paired with LiCl injections (t 4 = 5.8, PB0.05). Similarly, previously opiate-naive animals displayed a significant aversion to environments previously paired with LiCl (t 5 = 3.1, PB0.05) (0.8 mg/kg, i.p.) (Fig. 3B). 3.2. Effects of neuroleptic pretreatment on the de6elopment of opiate dependence Alpha-flupenthixol pretreatment prior to each morphine injection during the development phase and prior to each maintenance dose during the training phase did not attenuate the later acquisition of conditioned place aversions to environments paired with the absence of drug (withdrawal) (Fig. 4A). ANOVA comparing the effects of groups (saline/morphine, a-flupenthixol/morphine, a-flupenthixol/saline) and the times spent in each compartment (withdrawal-paired side or neutral side) revealed a significant interaction of groups with times spent in each compartment (F 2,19 = 5, PB0.05). Posthoc analysis revealed that the rats in both the saline/ morphine and a-flupenthixol/morphine groups spent a significantly greater amount of time in the neutral, previously unexposed environment (P’s B0.05). However, there was no significant difference in how the animals in the a-flupenthixol/saline group partitioned their time between the neutral and 26.5 h post-a-flupenthixol paired environments (P\ 0.05) (Fig. 4A).

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3.2.1. Effects of neuroleptic pretreatment on the expression of somatic withdrawal signs a-flupenthixol pretreatment during the development phase and prior to each maintenance dose in the conditioning phase had no effect on the frequency of morphine-induced somatic withdrawal signs seen in opiate dependent and withdrawn rats (Fig. 4B). An ANOVA comparing the effects of group (saline/morphine, aflupenthixol/morphine, a-flupenthixol/saline) and somatic withdrawal signs revealed a significant interaction of groups with signs (F 6,51 =3.4, P B0.05). Post-hoc analysis revealed that there was no difference in the incidences of any somatic withdrawal signs between the saline/morphine and a-flupenthixol/morphine groups

Fig. 3. The effects of intra-accumbal microinjections of a-flupenthixol on the motivational properties of lithium chloride (15 mg/kg, i.p.) (A). The effects of 0 or 30 mg/0.5 ml/side of intra-accumbal a-flupenthixol on times spent in environments previously paired with lithium chloride (15 mg/kg, i.p.) minus times spent in environments previously paired with saline. Animals receiving intra-NAc saline pretreatment demonstrated a significant aversion to the LiCl-paired environment. Animals receiving intra-NAc a-flupenthixol did not demonstrate an aversion to the LiCl-paired environment. (B). The effects of systemic a-flupenthixol (0.8 mg/kg, i.p.) on times previously spent in LiCl (15 mg/kg) minus times spent in saline-paired environments in previously opiate-naive animals, or in heroin-dependent and withdrawn animals. Both previous drug-naive and heroin-dependent and withdrawn animals displayed a significant aversion to the LiClpaired environment that was quantitatively similar to LiCl withdrawal aversions displayed by saline treated controls (Panel A).

Fig. 4. The effects of saline or a-flupenthixol (0.8 mg/kg, i.p.) pretreatment on the development of opiate dependence and withdrawal aversions; the effects of saline or a-flupenthixol (0.8 mg/kg, i.p.) pretreatment during the development of opiate dependence on the later expression of somatic withdrawal signs during opiate withdrawal. (A). The effects of saline (saline/morphine) or a-flupenthixol (0.8 mg/kg, i.p.) (a-flu/morphine) pretreatment prior to morphine injections (20 mg/kg, i.p.) during the development and maintenance of opiate dependence on times spent in environments paired with morphine withdrawal minus times spent in neutral environments. The effects of a-flupenthixol (0.8 mg/kg, i.p.) pretreatment prior to saline injections (a-flu/saline) on times spent in environments paired with the absence of a-flupenthixol minus times spent in neutral environments. Neither saline nor a-flupenthixol pretreatments had any effects on the development of conditioned place aversions to environments paired with opiate withdrawal. (B). The effects of saline (saline/morphine) or a-flupenthixol (0.8 mg/kg, i.p.) (a-flu/morphine) pretreatments prior to morphine injections (20 mg/kg, i.p.) during the development and maintenance of morphine dependence on the later expression of somatic withdrawal signs. The effects of a-flupenthixol (0.8 mg/kg, i.p.) pretreatment (a-flu/saline) prior to saline injections on the later expression of somatic withdrawal signs in the absence of a-flupenthixol. Neither saline nor a-flupenthixol pretreatments had any effect upon the later expression of somatic opiate withdrawal signs.

(P’s\ 0.05). The non-morphine dependent (a-flupenthixol/saline) group demonstrated fewer incidences of each somatic withdrawal sign than either the a-flupenthixol/morphine or saline/morphine groups. Wet dog

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shakes, which were the highest frequency somatic withdrawal sign observed in the a-flupenthixol/saline group, were still seen less frequently than in either the a-flupenthixol/morphine or saline/morphine groups (P’s B 0.05). Thus, the groups pretreated with either a-flupenthixol or saline and chronically exposed to morphine during the development phase both became morphine dependent and subsequently acquired conditioned place aversions to environments paired with the absence of morphine, and also demonstrated equivalent frequencies of somatic withdrawal signs. Chronic a-flupenthixol administration on its own, however, neither produced conditioned place aversions to environments paired with the absence of a-flupenthixol nor induced significant somatic withdrawal signs.

3.2.2. Effects of apomorphine pretreatment on the a6ersi6e effects of opiate withdrawal Pretreatment with the direct DA receptor agonist, apomorphine (5 mg/kg, i.p.) blocked the acquisition of place aversions to environments paired with opiate withdrawal. A one-way ANOVA comparing the effects of treatment (saline or apomorphine dose) on the difference scores for times spent in each compartment (withdrawal or neutral) revealed a significant main effect of apomorphine dose on difference scores for times spent in each environment (F 2,21 =5.97, PB 0.05). Post-hoc analysis revealed that while conditioned place aversions to opiate-withdrawal paired environments were considerably attenuated at the lower apomorphine dose (1 mg/kg) (n =7), this effect did not reach statistical significance (P \0.05). In contrast, the higher apomorphine dose (5 mg/kg) (n= 7) significantly attenuated aversions to the withdrawalpaired environments (P B 0.05) (Fig. 5A). Thus, apomorphine pretreatment blocked the aversive motivational properties of opiate withdrawal in opiatedependent animals. In a separate control experiment, we tested whether apomorphine pretreatment would block the aversive effects of a separate drug class, LiCl (15 mg/kg) in opiate dependent and withdrawn animals (see above). Comparing difference scores between times spent in environments paired with LiCl and times spent in the neutral environments in saline pretreated animals (n= 8) and apomorphine pretreated (5 mg/kg) animals (n = 8) revealed no significant difference in the place aversions to environments previously paired with LiCl (15 mg/kg) (t 7 =0.16, P \ 0.05) between the two groups. Thus, while apomorphine pretreatment blocked the aversive effects of spontaneous opiate withdrawal (Fig. 5A), this identical dose of apomorphine (5 mg/kg) did not block the aversive effects of LiCl (15 mg/kg) in opiate dependent and withdrawn animals (Fig. 5B).

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4. Discussion The mesolimbic DA system is involved importantly in both the positive reinforcing effects of opiates [1,6,19,30,32,35] as well as the aversive motivational properties associated with opiate dependency and withdrawal [3,5,15]. Previous reports have suggested that increases in DA transmission are responsible for the positive reinforcing effects of opiates [1,6,19,30,32,35] while decreases in DA transmission are responsible for the aversive motivational effects of opiate withdrawal [9,15,18,27]. The present results suggest that the motivational state of the animal (either opiate-naive or

Fig. 5. The effects of apomorphine pretreatment on the development of conditioned place aversions to environments paired with opiate withdrawal, or with LiCl (15 mg/kg). (A). The effects of saline or apomorphine (1.0 mg/kg or 5.0 mg/kg, i.p.) injections on times spent in environments previously paired with morphine withdrawal minus times spent in neutral environments. The aversive effects of opiate withdrawal as measured by aversions to the withdrawal-paired environment were attenuated by a lower dose of apomorphine (1 mg/kg) and were completely blocked by a higher dose of apomorphine (5.0 mg/kg, i.p.). (B). The effects of saline or apomorphine (5 mg/kg, i.p.) on times spent in environments previously paired with LiCl (15 mg/kg) in opiate-dependent and withdrawn animals minus times spent in neutral environments. Both saline and apomorphine pretreated animals demonstrated significant conditioned place aversions to environments paired with LiCl.

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opiate-dependent and in withdrawal) is a critical determining factor for the role of the mesolimbic DA system in the mediation of the rewarding effects of opiates.

4.1. Moti6ational state determines the role of accumbal DA receptors in opiate reward processes The behavioral dissociation we observed between previously opiate-naive versus opiate-dependent and withdrawn animals demonstrated that direct blockade of striatal DA receptors with doses of a-flupenthixol over an order of magnitude dose range (0.3– 3.0 mg) strongly attenuates the rewarding effects of opiates in opiate-withdrawn animals, yet had no effects on opiate reward in previously drug-naive animals. Interestingly, the highest dose of intra-NAc a-flupenthixol (30 mg) blocked the rewarding effects of opiates in both drugnaive and drug-dependent and withdrawn animals. This result would tentatively suggest that the mesolimbic DA system is involved in opiate reward processes in both previous opiate-naive as well as the opiate-dependent and withdrawn states. In this case, while the rewarding effects of opiates in the opiate-dependent and withdrawn states would be dramatically more sensitive to DA receptor blockade (as the present results suggest), the involvement of DA systems in opiate reward processes in opiate-naive animals can also be revealed at the highest dose of intra-NAc a-flupenthixol. However, there are several reasons why this explanation is unlikely. First, we found that the highest dose of intraNAc a-flupenthixol not only blocked the positive rewarding effects of opiates in both drug-naive and drug-dependent and withdrawn animals, but was also sufficient to completely block the acquisition of conditioned place aversions to the aversive motivational effects of LiCl, suggesting that rather than producing specific effects on opiate reward processes, the high dose of intra-NAc a-flupenthixol was sufficient to block the animal’s ability to associate the interoceptive effects of the drug (either rewarding or aversive) with the exteroceptive cues present in the conditioning environments. Second, when we challenged the aversive effects of LiCl with a high, systemic dose of a-flupenthixol (0.8 mg/kg), both previously opiate-naive and opiate-dependent and withdrawn animals demonstrated robust conditioned place aversions to the environments paired with LiCl. This latter finding strongly argues that the apparent blockade of opiate reward observed in both opiate-naive and opiate-dependent and withdrawn animals following the highest dose of intra-NAc a-flupenthixol was likely due to a non-specific effect of the drug at this high concentration, directly in the NAc, as a much higher systemic dose of a-flupenthixol (catalepsy-inducing) had no effects on LiCl conditioned place aversions (present results) and does not block opiate reward in previously opiate-naive animals

[3,5,22]. Furthermore, the fact that opiate-dependent and withdrawn animals were capable of learning a conditioned place aversion to LiCl even when challenged with a high systemic dose of a-flupenthixol, suggests that the blockade of the rewarding effects of opiates in opiate withdrawn animals with behaviorally specific doses of intra-NAc a-flupenthixol (0.3– 3.0 mg) was not likely due to an associative learning deficit in the opiate-dependent and withdrawn state. Nevertheless, the possibility that a sufficiently high dose of intra-NAc a-flupenthixol is capable of specifically blocking the rewarding effects of opiates regardless of motivational state, cannot be entirely ruled out in the present study. However, for the reasons outlined above, we suggest that the present results are better explained by the suggestion that exceptionally high concentrations of neuroleptic directly in the NAc may be sufficient to non-specifically block all motivational signaling, regardless of whether the conditioning stimulus is rewarding, or aversive. An alternative explanation for the present results is that opiate experienced animals have developed sensitization to the motivational effects of opiates. Thus, in the opiate withdrawn state, if opiate reward processes are now in a supersensitive state, the rewarding effects of opiates would be more sensitive to DA receptor blockade due to some neurophysiological alteration in the neural substrates mediating opiate reward processes. We believe there are two reasons why this explanation is unlikely. First, blockade of DA transmission that is sufficient to attenuate the sensitized opiate reward in opiate dependent and withdrawn animals (0.3– 3 mg) (Fig. 2B), should have been sufficient to block the weaker, unsensitized opiate reward in the previously drug-naive animals. Second, opiate reward sensitization involves a long-lasting neurophysiological adaptation [17,26]. Indeed, some studies have reported that the mesolimbic DA system remains sensitized to opiates a month or longer after the initial morphine exposure [31]. These findings would suggest that once the DA system has been sensitized to opiates, then the rewarding effects of opiates should continue to be mediated through a DA-dependent neural reward pathway regardless of whether the animal is in a state of drug withdrawal. However, we have reported previously that the neural substrates mediating opiate reward switch back to a DA-independent neural motivational system (mediated by the TPP) once they have recovered from opiate dependence and withdrawal [22], or if they are taken out of opiate withdrawal with morphine pretreatment prior to conditioning [4]. These results suggest that chronic opiate exposure does not produce any DA-mediated opiate reward sensitization and that the behavioral dissociation observed in the present study is not likely the result of a sensitized mesolimbic DA pathway.

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How might the mesolimbic DA system function to signal both the rewarding effects of opiates in states of opiate dependency and withdrawal as well as the aversive motivational effects of opiate withdrawal? We have reported previously that the activity of the mesolimbic DA system is fundamentally involved in the mediation of various aversive motivational states. Dopamine receptor blockade attenuates the aversive effects of naloxone-induced opiate withdrawal and spontaneous opiate withdrawal [5], as well as the aversive effects of food deprivation [14]. Thus, neuroleptics can block both the aversiveness of a deprivation state and the rewarding properties of stimuli alleviating that state. Finally, direct activation of DA receptors with a DA receptor agonist, apomorphine, blocks the aversive effects of opiate withdrawal (present results). Essentially, in states of opiate withdrawal, the rewarding effects of opiates are dependent upon their ability to remove the aversive motivational effects mediated by a specific dopaminergic signal. An alternative explanation is that during the opiate withdrawal state, accumbal DA receptors become supersensitive to neuroleptic blockade. Since there is a substantial projection from the accumbens GABA neurons to the DA neurons of the VTA [16], one prediction would be that intra-NAc blockade of inhibitory, postsynaptic DA receptors located on these GABAergic projections to the VTA would increase the activity of this inhibitory projection, and effectively shut down the mesolimbic DA system, preventing the transmission of a DA-dependent opiate reward signal during states of opiate dependency and withdrawal. Although some studies have reported that chronic opiate treatment can alter the sensitivity of mesolimbic DA receptors [23,34], other reports have suggested that opiate dependency and withdrawal is not associated with changes in mesolimbic DA receptor levels [25]. Finally, as previously noted, once animals are brought out of an opiate withdrawal state with opiate pretreatment (after a history of chronic opiate exposure), the motivational effects of opiates are no longer sensitive to DA receptor blockade, an effect that occurs within 3.5 h after opiate treatment [4]. These results would not suggest that long-term alterations in mesolimbic DA receptors are responsible for the qualitative switch from a DA-independent to a DA-dependent neural reward system observed in opiate-dependent and withdrawn animals.

4.2. Acti6ation of dopamine receptors blocks the a6ersi6e effects of opiate withdrawal In the present study, systemic administration of apomorphine, at doses that potently activate both pre and post-synaptic DA receptors [12,28], significantly attenuated the aversive effects of opiate withdrawal. Apomorphine is known to produce potent behavioral effects in

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and of itself, and has strong reinforcing motivational properties [2,33]. Thus, one alternative explanation for the present results is that this dose of apomorphine (5 mg/kg) produces rewarding effects that simply overshadow the aversive effects of spontaneous opiate withdrawal. Another possibility is that this dose of apomorphine interferes with associative learning processes, and thus blocks the acquisition of conditioned place aversions to environments paired with opiate withdrawal. However, these explanations are unlikely given that this same dose of apomorphine was not able to block the aversive effects of a separate drug class, LiCl, in animals with an identical history of opiate exposure and withdrawal. This further suggests that apomorphine was not inducing a non-specific blockade of associative learning or any sensory impairments, as animals were fully capable of associating the aversive interoceptive cues of LiCl with the exteroceptive place conditioning cues. The fact that activation of DA receptors during opiate withdrawal can block the aversive effects of withdrawal is particularly interesting given that high doses of DA receptor antagonists have also been demonstrated to block the aversive motivational effects of both spontaneous and naloxone-precipitated opiate withdrawal [5]. As previously noted, one possible explanation for this effect is that the aversive effects of opiate withdrawal are mediated by specific DA neuronal signal. If this were the case, than blockade of this signal by either direct activation or antagonism of post-synaptic DA receptors would be sufficient to attenuate the aversive effects of withdrawal, as the present results suggest.

4.3. Dopamine systems and the de6elopment of opiate dependence and somatic withdrawal signs Although DA systems are critically involved in both the withdrawal-alleviating rewarding properties of opiates as well as the aversive motivational effects of opiate withdrawal, little is known about the possible involvement of DA systems in the development of opiate dependence. The results of the present study suggest that the neuroadaptive changes underlying the development of opiate dependence are separate from those processes that are critical for mediating the aversive motivational properties of opiate withdrawal. In the present study, DA antagonist pretreatment prior to each morphine injection during the development and maintenance of opiate dependence had no effects on the development of dependence as measured by the later display of the aversive motivational effects or somatic signs of opiate withdrawal in opiate dependent animals. These results suggest that the neurobiological substrate(s) mediating the development of opiate dependence is upstream of the DA substrate mediating the

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aversive effects of opiate withdrawal, after opiate dependence has been acquired. However, once opiate dependence has been established, DA antagonists are capable of eliciting pronounced somatic withdrawal signs [15], and also block the aversive motivational properties of opiate withdrawal [5] and food deprivation [14]. Our proposal that the aversive motivational properties of opiate withdrawal are mediated by a specific dopaminergic neuronal signal is in contrast to models of somatic opiate withdrawal which would suggest that the somatic signs of opiate withdrawal are mediated by decreases in DA neurotransmission [9,27,29]. We propose that the somatic withdrawal signs and the aversive properties of opiate withdrawal are mediated by separate neural mechanisms, as neuroleptics block the aversive motivational effects of opiate withdrawal but have no effects upon the development and later expression of somatic withdrawal signs associated with spontaneous withdrawal (present results) or naloxone-induced somatic withdrawal signs [5]. Indeed, Delfs et al. [8] have recently reported that activation of a2 adrenergic receptors directly in the bed nucleus of the stria terminalis attenuates the aversive effects of opiate withdrawal, but does not affect the expression of somatic opiate withdrawal signs. Similarly, systemic administration of the adrenergic receptor agonist, clonidine, blocks opiatewithdrawal aversions and the withdrawal-alleviating effects of opiates in opiate-dependent and withdrawn animals [21]. Together with the present findings, this suggests that both noradrenergic and dopaminergic neural systems, acting either in parallel or in series, are involved in the aversive motivational effects associated with opiate withdrawal states. In conclusion, the present results suggest an interesting role for mesolimbic DA systems in mediating the motivational effects of opiates in states of dependency and withdrawal. Our findings suggest that in the drugnaive state, the rewarding effects of opiates can be mediated through a DA-independent neural reward system, as previous studies have suggested [3,5,11,22]. However, once the animal is opiate-dependent and withdrawn, the rewarding properties of opiates are critically dependent upon specific mesolimbic DA signaling, suggesting that the motivational state of the animal (drug-naive or drug-dependent and withdrawn) is an important determinant of the functional role of the mesolimbic DA system in opiate reward processes.

Acknowledgements Steven R. Laviolette and Karim Nader contributed equally to this work. This work was supported by the Canadian Institutes of Health Research.

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