I. Introduction

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High rates of relapse to drug use following prolonged withdrawal periods characterize the behavior of experienced heroin and cocaine users (Mendelson and Mello, 1996; O'Brien, 1997). In heroin- or cocaine-free individuals, drug craving and relapse to drug use can be triggered by exposure to the self-administered drug (Meyer and Mirin, 1979; Jaffe et al., 1989; de Wit, 1996), by stimuli previously associated with drug taking (Childress et al., 1992; Carter and Tiffany, 1999), or by exposure to stressors (Kosten et al., 1986; Kreek and Koob, 1998; Sinha et al., 1999).

Over the last several decades, some laboratories have been using an animal model, termed the reinstatement model, to study factors that underlie relapse to heroin and cocaine seeking induced by exposure to the self-administered drug, drug cues, and stressors. The use of this procedure has become increasingly popular as indicated in Fig. 1, which depicts the number of studies that used the reinstatement model from 1971 to 2001 in laboratory animals. In the learning literature, reinstatement refers to the resumption of a previously extinguished conditioned response after acute noncontingent exposure to the unconditioned stimulus (Bouton and Swartzentruber, 1991; Catania, 1992). In the studies reviewed below, reinstatement typically refers to the resumption of extinguished lever-pressing behavior after noncontingent exposure to drug or nondrug stimuli (Stewart and de Wit, 1987). In studies on cue-induced reinstatement (Section III.), reinstatement also refers to the resumption of drug seeking after exposure to the drug cues following extinction of the lever-pressing behavior in the absence of these cues. Common terms used in reinstatement studies are defined in Table 1.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Number of publications of studies which used reinstatement models in laboratory animals (mice, rats, and monkeys). Studies of reinstatement of heroin, cocaine, nicotine, and alcohol seeking are included.

In the reinstatement model, laboratory animals are initially trained to self-administer drugs by pressing a lever for intravenous drug infusions in operant conditioning chambers. Subsequently, the drug-reinforced behavior is extinguished by substituting the drug solutions with saline or by disconnecting the infusion pumps. After extinction of the drug-reinforced behavior, the ability of acute exposure to drugs (i.e., drug priming) or nondrug stimuli to reinstate drug seeking is determined under extinction conditions (Stretch et al., 1971; Stewart and de Wit, 1987). There are two main dependent variables during tests for reinstatement: nonreinforced responses on a lever that previously delivered the drug, the active lever; and responses on a lever not associated with drug infusions, the inactive lever. Responses on the active lever are interpreted to reflect reinstatement of drug seeking. Inactive lever responses are typically interpreted to reflect nonspecific activity, but they may also reflect response generalization (Catania, 1992).

It has been argued that the reinstatement model does not mimic most situations in humans that lead to drug abstinence and thus may not be suitable to model relapse (Marlatt, 1996; Bergman and Katz, 1998). In addition, based on data demonstrating different neurochemical and behavioral effects of contingent versus noncontingent drug injections (Dworkin et al., 1995; Hemby et al., 1997; Markou et al., 1999), it has been argued that the effect of priming drug injections in the reinstatement model may not be relevant to drug addiction (Everitt and Robbins, 2000). Furthermore, it has not been established that opioid withdrawal, which is associated with relapse in humans (Himmelsbach, 1943; O'Brien et al., 1986; Wikler, 1973), can reinstate heroin seeking in the reinstatement model. Naloxone-precipitated withdrawal does not reinstate heroin seeking following extinction (Stewart and Wise, 1992; Shaham and Stewart, 1995b; Shaham et al., 1996). In contrast, spontaneous 24-h heroin withdrawal was found to reinstate heroin seeking, but it cannot be concluded from this study whether this effect was due to the motivational effects of opioid withdrawal or from state-dependent mechanisms (see Shaham et al., 1996).

Despite these limitations, the reinstatement model has good predictive validity because conditions that reliably reinstate heroin and cocaine seeking in laboratory animals such as drug re-exposure, drug cues, and stress (Self and Nestler, 1998; Shaham et al., 2000a; Stewart, 2000) also were reported to provoke drug relapse and craving in humans (see above). Thus, the model can be used to study neuronal mechanisms underlying relapse to drugs despite the fact that the conditions that lead to drug abstinence in laboratory animals are different from those in humans.

In this review we will summarize data from studies that used the reinstatement model in laboratory rats on the role of specific neurotransmitter systems in relapse to heroin and cocaine seeking. Although most studies used heroin or cocaine as the self-administered drug, several studies in which rats were trained to self-administer amphetamine or morphine also are reviewed. Studies using the reinstatement model with monkeys or those using this model with alcohol-trained rats are not reviewed here Lê and Shaham, 2002); for recent reviews, see (Spealman et al., 1999 . The section below provides an overview of several procedures used in reinstatement studies.

Using monkeys, initial studies on reinstatement of amphetamine or cocaine seeking by drug priming were conducted by Stretch and Gerber in the early 1970s (Stretch et al., 1971; Stretch and Gerber, 1973; Gerber and Stretch, 1975). Goldberg, Schuster, and colleagues also have shown that stimuli paired with morphine or cocaine injections in monkeys reinstate drug-taking behavior after the lever-pressing behavior is extinguished in their absence (Schuster and Woods, 1968; Kelleher and Goldberg, 1977; Goldberg et al., 1981). Davis and Smith (1976) and de Wit and Stewart (1981, 1983) were the first to study reinstatement of drug seeking in rats. Over the years, several types of reinstatement procedures were used (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   A depiction of the timeline of within-session, between-session, and between-within-session reinstatement procedures. See Table 1 and text for a full description of these procedures.

Stretch et al. (1971) and Davis and Smith (1976) used a "between-session" reinstatement method, in which training for drug self-administration, extinction of the drug-reinforced behavior, and tests for reinstatement were conducted during different daily sessions. The advantage of the between-session model is that it mimics somewhat the human situation of relapse to drugs at times that are beyond the acute withdrawal phase. A limitation of this method, however, is that repeated testing under extinction conditions results in the attenuation of responding to the reinstating stimulus. Thus, subjects that are trained for prolonged periods cannot be tested after extinction more than two to three times, leading to the use of many subjects.

de Wit and Stewart (1981) introduced a "within-session" reinstatement method. In this method, rats are initially trained to self-administer cocaine or heroin. Subsequently, tests for reinstatement are carried out several times a week in a daily session of 1 to 2 h of drug self-administration, 3 to 4 h of extinction of the drug-reinforced behavior, and a subsequent test for reinstatement. Rats are given regular drug self-administration training between the test days. The advantage of this method is that rats can be repeatedly tested for reinstatement (de Wit and Stewart demonstrated that neither tolerance nor sensitization is evident after repeated testing with priming drug injections). The limitations of the within-session method are that it does not simulate long-term relapse in humans, and the rats are not "truly" drug-free at the time of testing (i.e., they are tested several hours after last exposure to drug).

A more recent variation of the reinstatement procedure is the "between-within" method (Tran-Nguyen et al., 1998). In this method, rats are initially trained for drug self-administration. Subsequently, extinction training and tests for reinstatement are conducted on the same day after different days of drug withdrawal. The advantage of this method is that it can be used to study the relationship between the duration of the drug withdrawal period and reinstatement of drug seeking (see Section V. Discussion). At present, however, it is not clear whether this method is suitable for repeated testing. Thus, different groups of rats, tested at each withdrawal period, are needed for a clear interpretation of the data (Tran-Nguyen et al., 1998).

Two other reinstatement models were developed in recent years. Ettenberg and colleagues introduced a "runway " reinstatement model to study the role of discriminative drug cues in relapse (Ettenberg, 1990; Ettenberg et al., 1996; McFarland and Ettenberg, 1997). In this model, the dependent measure is the "run time " from a "start box " to a "goal box " where a drug infusion is given. During the initial discrimination training (Catania, 1992), rats are given a drug injection when they reach the goal box in the presence of one discriminative cue (e.g., specific odor) or saline injections in the presence of a different cue. The discriminative cues are presented at the start box. Over time, rats decrease their run time in the presence of the drug predictive cue, but not the saline cue. Rats are then given sessions in the absence of the discriminative cues and the drug during which run time progressively increases (extinction). During testing, a single presentation of the drug-associated cue leads to a decrease in the run time to reach the goal box (cue reinstatement). In addition, a single drug infusion in the goal box during extinction decreases the run time on the subsequent drug-free day (drug reinstatement).

The advantage of the runway method is that the impact of drug priming on behavior is studied on a subsequent, drug-free day. Thus, alternative interpretations for the effects of pharmacological manipulations given prior to drug priming (e.g., locomotor activation/sedation) on behavior during testing can be ruled out. The limitation of the runway model is that rats are not exposed to more than several drug infusions/day, and consequently drug intake is much lower than in self-administration studies. Thus, the runway model can only mimic some aspects of recreational drug use but not compulsive use of high amounts of drugs. This model also reveals a complex behavioral pattern in cocaine-trained animals, which do not decrease their run time during training, presumably due to the "anxiogenic" effects of cocaine (Ettenberg and Geist, 1991, 1993). Thus, this model is not suitable for studying relapse to cocaine because it is difficult to establish that cocaine serves as a reinforcer in this model. Finally, rats are given the drug priming contingently when they reached the goal box during an extinction session. Thus, the priming manipulation in the runway method is different than that of the traditional reinstatement method.

Most recently, several laboratories have developed a conditioned place preference (CPP²) reinstatement model (Mueller and Stewart, 2000; Parker and McDonald, 2000). In these studies, rats are initially trained to associate one distinctive environment with a drug injection and a different environment with a saline (vehicle) injection. Following training, rats spend more time in the drug-paired environment, when given a choice between the two environments, on a drug-free test day. This acquired preference for the drug-paired environment can be extinguished by daily injections of saline in the previously drug-paired environment (extinction). It was found that after extinction, injections of cocaine (Mueller and Stewart, 2000) or morphine (Parker and McDonald, 2000) reinstate the extinguished CPP for the drug. Finally, it was recently reported that both morphine injections and footshock stress "reactivate" CPP for morphine (that is no longer observed) after 9 (Wang et al., 2000) or 36 (Lu et al., 2000) drug-free days, during which the rats are not exposed to extinction conditions. The advantage of the CPP reinstatement model is that nonspecific motor effects of pharmacological manipulations are less likely to influence behavior because the dependent measure is not lever-pressing behavior. This model also does not require the expertise of intravenous surgery and the need to maintain catheter patency. However, as in the case of the runway model, total drug exposure is low and thus the relevance of this model to compulsive and chronic drug use is limited.

In conclusion, several experimental procedures can be used to study reinstatement of drug seeking in rats, each with its advantages and disadvantages. In the sections below, we will review studies that used these different procedures.

	II. Drug Priming-Induced Reinstatement

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Many studies have reported reliable heroin- or cocaine-induced reinstatement using the different reinstatement methods described above (Self and Nestler, 1998; Stewart, 2000). The drug priming effect also was reported in alcohol- and nicotine-trained rats (Chiamulera et al., 1996; Shaham et al., 1997a; Lê et al., 1998). As described below, this effect is demonstrated following systemic and intracranial administration. Agents from the same pharmacological class of the self-administered drug reliably reinstate heroin and cocaine seeking (Carroll and Comer, 1996; de Wit, 1996). Several studies, however, also demonstrated "cross-reinstatement" with drugs that are from different classes than the self-administered drug (Davis and Smith, 1976; De Vries et al., 1999). The magnitude of drug-induced reinstatement is positively correlated with the priming dose. In addition, doses that are higher than the unit dose of the self-administered drug are needed to reliably reinstate the behavior (de Wit, 1996). Also, at higher doses, peak responding occurred later and continued for longer periods than with low doses (de Wit and Stewart, 1981). Finally, Lynch and Carroll (2000) reported that female rats are more responsive to cocaine-induced reinstatement than male rats. However, the priming effect is reliably observed with male rats, which were used in most of reinstatement studies. In the section below, we describe studies in which pharmacological and neurochemical methods were used to elucidate the role of specific neurotransmitter systems underlying reinstatement by heroin and cocaine priming. Table 2 summarizes data from substitution (cross-reinstatement) studies in which the effect of pharmacological agents on reinstatement of heroin and cocaine seeking was determined. Table 3 summarizes data on the effect of pharmacological agents on reinstatement induced by heroin or cocaine priming.

A large body of evidence indicates that the mesocorticolimbic dopamine (DA) system (Fallon and Moore, 1978) contributes to the acute reinforcing effects of heroin and cocaine (Koob and Bloom, 1988; Wise, 1996b). Cocaine, an indirect DA agonist, increases DA release by blocking the DA transporter (Heikkila et al., 1975). Heroin and other µ-opioid receptor agonists increase DA release in terminal regions by inhibiting GABAergic neurons in the VTA, which provide tonic inhibition of DA neurons, resulting in increased DA release in terminal regions (Di Chiara and North, 1992). The data reviewed indicate that the mesocorticolimbic DA system also is involved in reinstatement by cocaine or heroin priming.

1. Cocaine Priming. The effect of cocaine priming on reinstatement is mimicked by systemic injections of amphetamine (a DA reuptake blocker and a DA releaser), DA reuptake blockers (GBR-12909, methylphenidate) and D2-like receptor agonists (7-hydroxy-2-dipropylaminotetralin, quinpirole, bromocriptine) (de Wit and Stewart, 1981; Wise et al., 1990; Self et al., 1996; De Vries et al., 1999; Schenk and Partridge, 1999). On the other hand, mixed DA agonists (apomorphine) or direct D1-like agonists (SKF 82958, SKF 81297, ABT 431) do not mimic the effect of cocaine priming on reinstatement (Self et al., 1996, 2000; De Vries et al., 1999) (Figs. 3 and 4). Surprisingly, these direct D1-like agonists, some of which are self-administered by rats and monkeys (Self and Stein, 1992; Weed et al., 1993; but see Caine et al., 1999b), also block cocaine-induced reinstatement in rats (Self et al., 1996, 2000) and monkeys (Khroyan et al., 2000). Norman et al. (1999) reported that the D1-like antagonist, SCH 23390, attenuates cocaine-induced reinstatement. In addition, using the runway model in amphetamine-trained rats, Ettenberg (1990) reported that the D2-like receptor antagonist, haloperidol, attenuates drug-induced reinstatement. These pharmacological data indicate that DA plays a crucial role in cocaine-induced reinstatement. In addition, although activation of D2-like receptors provokes cocaine seeking, activation of D1-like receptors inhibits it. The reasons for this pharmacological dissociation are not clear in light of the literature on the similar behavioral effects of the D1- and D2-like agonists on locomotor activity (Self et al., 1996) and cocaine reinforcement (Self and Nestler, 1995).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Reinstatement following long-term extinction (3 weeks) of heroin and cocaine self-administration: mean (± S.E.M.) number of nose poke responses in the previously active (drug-paired) and inactive hole during the 2-h test for reinstatement. A, priming effects in rats previously trained to self-administer heroin; B, priming effects in rats previously trained to self-administer cocaine. Saline, cocaine (10 mg/kg, i.p.), amphetamine (1.0 mg/kg, i.p.), or heroin (0.25 mg/kg, s.c.) were injected 10 min before the start of the reinstatement session. *, significantly different from preceding saline injection (p < 0.05). Data are from De Vries et al., 1998, reprinted ©1998 with permission from Blackwell Science Ltd.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of intraperitoneal (i.p.) injections with vehicle (saline), the D2-like dopamine agonist 7-hydroxy-2-dipropylaminotetralin, or the D1-like agonist SKF 82958 on reinstatement of nonreinforced lever-press responding. Priming injections were given after extinction from 2 h of intravenous cocaine self-administration when only intravenous saline injections were available. A, lever-press responding in a representative animal. Hatch marks denotes the times of each self-infusion of cocaine in the cocaine phase and of saline in the saline phase. B and C, mean number (± S.E.M.) of nonreinforced lever-press responses during the final hour of the saline phase in the drug-reinstatement paradigm. Asterisks indicate significant differences from saline treatment (*, p < 0.05; **, p < 0.01). Data are from Self et al., 1996. Copyright 1996 with permission from Science (Wash DC).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Responses (mean ± S.E.M.) on the previously active (cocaine- or heroin-paired) lever following priming injections of morphine (Mor) into the ventral tegmental area (cell body region of the mesolimbic dopaminergic neurons), in rats previously trained to self-administer cocaine (left panel) or heroin (right panel). Rats were tested with (Nal-Mor) or without (Mor) pretreatment with the opioid receptor antagonist naltrexone (Nal), administered i.p. Data are from Stewart, 1984, reprinted ©1984 with permission from Elsevier Science.

2. Heroin Priming. Indirect DA agonists (cocaine, amphetamine) were found to reinstate heroin seeking after prolonged withdrawal periods (3 weeks) (De Vries et al., 1998a, 1999) (Fig. 3). Intra-NAc infusions of amphetamine also reinstate heroin seeking in the within-session reinstatement procedure (Stewart and Vezina, 1988) (Fig. 5). In contrast, using the within-session procedure, it was reported that cocaine does not reliably reinstate heroin seeking (de Wit and Stewart, 1983). The discrepant results between the effect of cocaine in the between- and within-reinstatement procedures may be related to the development of sensitization to the DA-dependent behavioral activating effects of cocaine following prolonged, but not short, withdrawal periods (De Vries et al., 1999; Vanderschuren and Kalivas, 2000).

Activation of µ-opioid receptors is critically involved in heroin reinforcement (Mello and Negus, 1996). The rewarding effects of heroin are mediated via dopamine-dependent mechanisms within the VTA (Wise, 1996b) and dopamine-independent mechanisms within the NAc (Koob, 1992). On the other hand, despite the fact that manipulations of opioid receptors were found to alter cocaine self-administration and CPP in several studies (Shippenberg and Elmer, 1998; Van Ree et al., 1999), it has not been established that activation of opioid receptors is critical for cocaine reward and in many studies opioid receptor antagonists failed to alter cocaine reward (Mello and Negus, 1996). Several studies have examined the effect of opioid receptor agonists and antagonists on reinstatement of heroin and cocaine seeking.

1. Cocaine Priming. The role of opioid receptors in cocaine priming has not been clearly established and the data reviewed below mirror the conflicting literature on the role of opioid receptors in cocaine reinforcement. The preferentially µ-opioid antagonist, naltrexone, has no effect on cocaine-induced reinstatement (Comer et al., 1993). In addition, systemic injections of µ-opioid receptor agonists (heroin, morphine, etonitazene), a mixed µ-agonist/-antagonist (buprenorphine) or a mixed µ/-agonist (butorphanol) do not reliably reinstate cocaine seeking (de Wit and Stewart, 1981; Comer et al., 1993; De Vries et al., 1998a; Lynch et al., 1998). Furthermore, butorphanol, etonitazene, and buprenorphine (Comer et al., 1993; Lynch et al., 1998), but not morphine (Lynch et al., 1998), attenuate cocaine-induced reinstatement. However, the interpretation of these data is complicated because in opioid-naive rats, systemic injections of opioid agonists have a biphasic effect on behavior: an initial sedative effect that is followed by behavioral activation (Babbini et al., 1975). These initial sedative effects may have masked the expression of the motivational effects of the opioid receptor agonists on reinstatement. Thus, when morphine is infused acutely into the VTA, where it induces behavioral activation (Joyce and Iversen, 1979), it reinstates cocaine seeking (Stewart, 1984). More recently, Schenk et al. (1999, 2000) reported that the -opioid receptor agonist, U69593, decreases cocaine-induced reinstatement. This effect may be related to the inhibitory effect of -opioid receptor activation on DA release (Spanagel et al., 1990; Shippenberg and Elmer, 1998).

2. Heroin Priming. The effect of heroin priming on reinstatement is dependent on activation of µ-opioid receptors. Priming injections of µ-opioid receptor agonists such as morphine mimic the effect of heroin on reinstatement when given systemically (de Wit and Stewart, 1983; Stewart and Wise, 1992) or intra-VTA but not intra-NAc (Stewart, 1984) (Fig. 5). Naltrexone, a preferentially µ-opioid receptor agonist, blocks reinstatement induced by either systemic injections of heroin (Shaham and Stewart, 1996) or intra-VTA morphine (Stewart, 1984). Finally, chronic occupation of the opioid receptors with heroin given via Alzet osmotic minipumps attenuates heroin-induced reinstatement (Shaham et al., 1996). These data suggest that activation of DA neurons in the VTA mediates heroin-induced reinstatement. However, there are recent reports that infusions of opioid and GABAergic agents into the VTA also have DA-independent rewarding effects (Nader and Van der Kooy, 1997; McBride et al., 1999). Thus, it cannot be ruled out that DA-independent mechanisms within the VTA are involved in heroin-induced reinstatement.

Glutamate neurotransmission is involved in the development and expression of behavioral and neurochemical sensitization to opioid and psychostimulant drugs (Pierce and Kalivas, 1997; White and Kalivas, 1998). Based on these reports, several recent studies investigated the effect of systemic or intracranial injections of agonists and antagonists of ionotropic glutamate receptors (NMDA and AMPA/kainate) on reinstatement of cocaine seeking.

De Vries et al. (1998b) reported that systemic injections of the noncompetitive NMDA antagonist, MK-801, reinstate cocaine seeking. In contrast, Bespalov et al. (2000) reported that the competitive NMDA receptor antagonist, D-CPPene, and the low-affinity NMDA receptor channel blocker, memantine, do not reinstate cocaine seeking. These discrepant results are one of many examples of the different effects of noncompetitive and competitive NMDA receptor antagonists on behavior (Willetts et al., 1990). Another finding in the study of Bespalov et al. was that pretreatment with the NMDA receptor antagonists led to increased responding on the inactive lever. The reasons for this nonspecific effect are not clear.

Evidence for the role of glutamate neurotransmission in cocaine reinstatement comes from two studies by Cornish and Kalivas (1999, 2000). They found that intra-VTA infusions of AMPA selectively reinstate cocaine but not sucrose seeking. The NMDA receptor agonist (cis-ACDA) increased responding on the active lever during testing, but also somewhat increased responding on the inactive lever. In addition, the AMPA receptor antagonist, CNQX, blocked reinstatement of cocaine seeking induced by cocaine priming (given systemically) and intra-NAc infusions of DA. In contrast, the NMDA receptor antagonist, CPP, had no effect on cocaine priming (Fig. 6). Thus, although activation of AMPA and NMDA receptors in the NAc can induce reinstatement, only the former is directly involved in cocaine-induced reinstatement.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of intra-accumbens treatment with the mixed dopamine receptor antagonist fluphenazine (FLU; 10 nmol/side), the NMDA receptor antagonist CPP (0.1 nmol/side), and the AMPA receptor antagonist CNQX (1.0 nmol/side) on cocaine- (10.0 mg/kg, i.p.) induced reinstatement of nonreinforced lever-press responding. Data are expressed as mean ± S.E.M. *, significantly different from the vehicle-treated group (p < 0.05). Data are from Cornish and Kalivas, 2000, reprinted ©2000 with permission from the Society for Neuroscience.

A study by Vorel et al. (2001) provides additional evidence for the role of glutamate in cocaine reinstatement. Using stimulation parameters previously shown to evoke glutamate receptor-mediated changes in dopamine efflux in the NAc (Blaha et al., 1997), these investigators found that stimulation of the hippocampal-containing glutamatergic neurons in the ventral subiculum reinstates cocaine seeking. They also found that hippocampal stimulation-induced reinstatement is blocked by an intra-VTA infusion of the nonselective ionotropic glutamate antagonist, kynurenic acid, and is mimicked by intra-VTA infusions of NMDA, a manipulation that increases DA release in the NAc (Westerink et al., 1996). The relevance of these provocative data to reinstatement induced by cocaine priming, however, is not known.

Recent data indicate that glutamate action on AMPA receptors within the NAc plays a critical role in cocaine-induced reinstatement. In addition, activation of NMDA receptors within both the NAc and the VTA can reinstate cocaine seeking. The relationship between the glutamatergic mechanisms within the VTA and NAc in cocaine reinstatement is an interesting question for future research. Finally, acamprosate (calcium-acetyl homotaurinate), a compound that alters glutamatergic neurotransmission (Spanagel and Zieglgänsberger, 1997), had no effect on heroin-induced reinstatement (Spanagel et al., 1998). Studies on the effect of specific NMDA or AMPA receptor ligands on heroin-induced reinstatement have not been published.

1. 5-Hydroxytryptamine. Manipulations of brain 5-HT systems can alter the behavioral effects of cocaine, including drug self-administration and discrimination (Walsh and Cunningham, 1997). Several studies examined the effect of 5-HT agents on cocaine priming-induced reinstatement. The selective serotonin reuptake inhibitor (SSRI), fluoxetine, which increases 5-HT levels in terminal regions (Perry and Fuller, 1992), has no effect on cocaine-induced reinstatement (Baker et al., 2001). The 5-HT_1a antagonist, WAY 100635, which increases 5-HT cell firing and release (Mongeau et al., 1997), attenuates cocaine-induced reinstatement. In addition, the 5-HT_2C agonist Ro 60-0175 (Grottick et al., 2000), but not the 5-HT₂ antagonist ritanserin (Schenk, 2000), attenuates cocaine-induced reinstatement. Ro 60-0175 may attenuate cocaine-induced reinstatement by decreasing DA levels in the NAc and frontal cortex (Millan et al., 1998; Di Matteo et al., 1999). Finally, Tran-Nguyen et al. (2001) reported that 5-HT lesions by 5,7-dihydroxytryptamine shift the dose-response curve of cocaine priming to the left. These data suggest that 5-HT acts on 5-HT_2C receptors to attenuate cocaine-induced reinstatement. However, the observation that fluoxetine has no effect on cocaine priming is not in agreement with this idea. Thus, the role of 5-HT in cocaine-induced reinstatement remains to be determined.

2. Corticosterone. The stress hormone, corticosterone, which is released following activation of the hypothalamic-pituitary adrenal (HPA) axis (Selye, 1956), plays an important role in cocaine reinforcement (Piazza and Le Moal, 1998). Cocaine activates the HPA axis (Sarnyai et al., 2001), and inhibition of circulating corticosterone decreases intravenous cocaine self-administration in rats (Goeders, 1997; Piazza and Le Moal, 1997). The data reviewed below, however, suggest that corticosterone secretion does not play a major role in cocaine- (or heroin-) induced reinstatement. The removal of corticosterone by adrenalectomy (ADX) or the administration of CRF receptor antagonists had no effect on cocaine- or heroin-induced reinstatement (Shaham et al., 1997b; Erb et al., 1998). In addition, synthesis inhibitors of corticosterone (ketoconazole or metyrapone) had no effect on cocaine- or heroin-induced reinstatement (Shaham et al., 1997b; Mantsch and Goeders, 1999b). Recently, however, the nonselective CRF receptor antagonist, -helical CRF, and the selective CRF₁ receptor antagonist, CP-154,526 (Schulz et al., 1996) but not the CRF₂ receptor antagonist antisauvagine-30, were reported to attenuate reactivation of morphine CPP by drug priming after 28 days of withdrawal (Lu et al., 2000). Lu et al. (2001a) also reported that -helical CRF, but not CP-154,526 or antisauvagine-30, attenuates reactivation of cocaine CPP by cocaine priming. The reasons for the different effects of the CRF receptor antagonists in the CPP model versus the self-administration model are not clear.

3. -Aminobutyric Acid. Roberts and Brebner (2000) found that the GABA_B receptor agonist, baclofen, attenuates cocaine reward. Campbell et al. (1999) reported that baclofen also attenuates cocaine-induced reinstatement (Campbell et al., 1999). This effect may be due to the inhibitory action of baclofen on DA neurons in the VTA (Westerink et al., 1996).

4. Noradrenaline. Several studies found that manipulations of central NA have an effect on opioid and psychostimulant self-administration behavior (Davis et al., 1975; Harris et al., 1996). However, based on other studies, it is generally believed that the involvement of central NA neurons in drug reinforcement is minimal (Wise, 1978, 1996b). Erb et al. (2000) found that the -2 adrenoceptor agonists, clonidine and lofexidine, have no effect on cocaine-induced reinstatement at doses that decrease NA release in the amygdala and prefrontal cortex. These data suggest that the action of cocaine on the NA transporter (Blakely et al., 1994) is not involved in its effect on reinstatement. The role of NA in heroin-induced reinstatement has not been determined.

5. Acetylcholine. Cholinergic neurons modulate mesocorticolimbic DA neurotransmission in the NAc and the VTA (Di Chiara et al., 1994; Sarter et al., 1999). Nicotine increases DA release in the NAc (Damsma et al., 1989), an effect mediated via the activation of nicotine receptors in the VTA (Nisell et al., 1994). The available data, however, do not implicate nicotinic acetylcholine receptors in cocaine-induced reinstatement. Schenk et al. (1999) found some effect of nicotine on reinstatement of cocaine seeking, but Wise et al. (1990) did not.

6. Endocannabinoids. The endocannabinoid system has been implicated in several neuropsychiatric conditions, including drug addiction (Gardner and Vorel, 1998; Piomelli et al., 2000). The active ingredient of marijuana, 9-THC, activates the mesocorticolimbic DA system (Chen et al., 1991; Tanda et al., 1997). In addition, DA through activation of D2-like receptors, releases endogenous cannabinoids in the striatal complex (Giuffrida et al., 1999). Based on these findings, two studies determined the effect of activation or blockade of cannabinoid receptors on cocaine seeking. Using the within-session method, Schenk and Partridge (1999) found that 9-THC has no effect on cocaine seeking. In contrast, using the between-session method, De Vries et al. (2001) found that the nonselective cannabinoid agonist, HU210, potently reinstates cocaine seeking following 2 to 3 weeks of withdrawal, whereas the CB1 receptor antagonist SR141716A attenuates cocaine-induced reinstatement. These discrepant results may be due to the time of testing (several hours versus several weeks of withdrawal) and the longer duration of action of HU210 compared with 9-THC.

	III. Cue-Induced Reinstatement

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In an initial study Davis and Smith (1976) trained rats to press a lever for intravenous injections of morphine; each injection was accompanied by a buzzer presentation (a discrete conditioned stimulus, CS). Lever pressing for morphine was then extinguished by replacing morphine infusions with saline in the absence of the CS. During testing, lever presses resulted in response-contingent presentations of the CS (a conditioned reinforcer), and rats increased their lever-pressing behavior. In contrast, de Wit and Stewart (1981) found that noncontingent exposure to a tone cue following extinction of the lever-pressing behavior for cocaine in the absence of the CS has a weak effect on reinstatement. Similarly, two recent studies found that noncontingent presentations of discrete CSs have a minimal effect on cocaine seeking following extinction of lever pressing in their absence (Fuchs et al., 1998; Tran-Nguyen et al., 1998). It appears that two features are important for obtaining a reliable effect of discrete drug CS on reinstatement (See et al., 1999; Grimm et al., 2000). First, a compound (i.e., tone + light) cue is more effective in inducing reinstatement than a simple tone or light cue (See et al., 1999). Second, as predicted from the studies above, the drug cues should be presented contingently during tests for reinstatement (Grimm et al., 2000).

More recently, Ettenberg et al. (1996) and Weiss et al. (2000) have developed discrimination procedures (Catania, 1992) to characterize the role of discriminative cues, which predict drug availability, in relapse. In these studies, discrete environmental cues (e.g., sound, smell) predict whether drug or no drug (saline) is available during drug self-administration training. These investigators showed that discriminative cues that predict drug availability provoke relapse when they are introduced after the drug-reinforced behavior is extinguished in their absence (McFarland and Ettenberg, 1997; Gracy et al., 2000).

Using a renewal procedure (Bouton and Bolles, 1979), we examined the role of contextual stimuli (e.g., physical characteristics of the test environment), which predict drug availability, in relapse to drug seeking (Crombag and Shaham, 2002). In the renewal procedure, conditioned responses to discrete CSs are recovered when they are reintroduced in the original conditioning context (where they were paired with the primary reinforcer) after extinction in a different context. We found that in rats with a history of speedball (a heroin-cocaine combination) self-administration, drug seeking is reinstated when rats are exposed to the drug self-administration context following extinction of the lever-pressing behavior in the presence of drug-contingent CSs (stimulus light, sound of the pump) in a different context (Crombag and Shaham, 2002).

Finally, learning theorists view responding in the absence of a primary reinforcer during extinction as a behavior that is controlled by the CSs previously paired with the reinforcer (Pavlov, 1927; Skinner, 1953). From this perspective, the effect of pharmacological/lesion manipulations on rate of extinction (or resistance to extinction) can be used to study neuronal substrates involved in discrete CS-induced drug seeking (Fuchs et al., 1998). Studies of this type are reviewed below. However, data from studies in which in vivo electrophysiology, microdialysis, and electrochemistry were used during drug self-administration, and thus samples were occasionally taken in response to the drug cues prior to drug infusions, are not reviewed here. Table 4 describes the data from the pharmacological studies on cue-induced reinstatement.

Bespalov et al. (2000) reported a decrease in cocaine cues-induced reinstatement following pretreatment with the NMDA receptor antagonist, D-CPPene, but not the low affinity NMDA channel blocker, memantine. See et al. (2001) found that basolateral amygdala (BLA) intra-infusions of an NMDA receptor antagonist (AP-5) or kainate/AMPA receptor antagonist (CNQX) have no effect on cocaine cues-induced reinstatement. These data suggest that NMDA receptors in regions other than the BLA may be involved in cocaine cues-induced reinstatement. Most recently, De Vries et al. (2001) found that the CB1 receptor antagonist SR141716A attenuates cocaine cues-induced reinstatement. These data suggest that the brain endocannabinoid system is involved in neuronal processes underlying cue-induced relapse to cocaine seeking.

Alleweireldt et al. (2002) reported that the D1-like receptor antagonist, SCH 23390, attenuates cocaine cues-induced reinstatement. See et al. (2001) also found that SCH 23390 but not raclopride (a D2-like antagonist) injected into the BLA attenuates cue-induced reinstatement of cocaine seeking. These data extend previous reports by See and colleagues on the effect of permanent (excitotoxic) or reversible (tetrodotoxin, TTX; 3 ng/µl) lesions of the BLA on cue-induced reinstatement of cocaine seeking (Meil and See, 1997; Grimm and See, 2000). Interestingly, Grimm and See (2000) found that intra-NAc of TTX (3 ng/µl), which blocks cocaine self-administration, has no effect on cue-induced reinstatement, whereas the BLA reversible lesions had no effect on cocaine self-administration (Fig. 7). These data are of theoretical importance as they demonstrate a double neuroanatomical dissociation between responding controlled by the primary (cocaine) versus the secondary conditioned reinforcer. Most recently, Kruzich and See (2001) demonstrated that TTX infusions (5 ng/µl) into both the BLA and the CeA decreased cocaine cues-induced reinstatement. However, these data cannot be clearly interpreted because a high dose of TTX was used, anatomical controls for spread of the toxin were not used (see Wise and Hoffman, 1992), and the authors did not assess whether the lesions led to motor deficits.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Responses (mean ± S.E.M.) on the previously active (cocaine-paired lever) during the last week of self-administration, extinction and the two test days. Animals were microinjected with vehicle or TTX immediately prior to test days indicated as "tone + light" and "cocaine". A, lever responses for rats implanted with bilateral cannulae in the basolateral amygdala and microinjected with vehicle or TTX. Rats microinjected with TTX failed to reinstate responding for the tone + light (p < 0.05). B, lever responses for rats implanted with bilateral cannulae in the nucleus accumbens and microinjected with vehicle or TTX. Responding of TTX-treated rats for the primary (cocaine) reward was selectively attenuated when compared with vehicle infusion (p < 0.05). *, significant group difference for the test session (p < 0.05). Data are from Grimm and See, 2000, reprinted ©2000 with permission from Elsevier Science.

It appears that the BLA and, in particular, D1-like receptors in this area are involved in cue-induced reinstatement of cocaine seeking. These data are in agreement with those from studies on the role of conditioned drug cues in cocaine seeking as measured by the second-order schedule procedure and with those from studies on the effect of BLA lesions on the ability of stimuli paired with natural rewards to control behavior (Robbins et al., 1989; Everitt et al., 1999). Finally, studies on the neuronal mechanisms underlying cue-induced reinstatement of heroin seeking have not been reported. Thus, an interesting question, in light of recent data on the lack of effect of BLA lesions on responding for heroin-associated cues under the second-order schedule (Alderson et al., 2000), is whether the findings from studies on cocaine cues-induced reinstatement generalize to reinstatement induced by heroin cues.

Fuchs et al. (1998) found that chronic administration of the NA reuptake blocker, desmethylimipramine, decreases lever pressing during extinction. In two other studies it was found that the tryptophan hydroxylase inhibitor, para-chlorophenylalanine, 5-HT lesions with 5,7-dihydroxytryptamine and chronic exposure to fluoxetine also decrease extinction behavior (Tran-Nguyen et al., 1999, 2001; Baker et al., 2001). It is not clear, however, how to interpret these data as both decreases and increases in 5-HT neurotransmission decrease resistance to extinction.

Using in vivo microdialysis, several studies have examined the effect of exposure to discrete cocaine cues during extinction on DA release in the NAc. Meil et al. (1995) described a precipitous drop in DA levels in the NAc when cocaine was removed from the syringe pumps, even when rats continued to press for a discrete CS light during extinction. Ranaldi et al. (1999) reported a decrease in DA levels in the NAc during extinction when amphetamine was removed, despite an increase in lever pressing for the drug CSs. These data are somewhat difficult to interpret because any effect of the cues on DA release may be masked by the decrease in DA levels due to the clearance of cocaine from the DA transporter. Neisewander et al. (1996) reported that DA levels in the NAc are not altered when the cocaine-associated cues are reintroduced during extinction testing following 7 days of withdrawal. Together, these data suggest that alterations in DA levels in the NAc are not associated with the lever-pressing behavior controlled by the cocaine cues during extinction. This conclusion is in agreement with those from two recent studies in rats and monkeys, using the second-order procedure, on the lack of effect of contingent cocaine cues on DA release in the NAc (Bradberry et al., 2000; Ito et al., 2000). In contrast, Tran-Nguyen et al. (1998) reported a significant elevation in DA levels in the amygdala when rats returned to a self-administration chamber and were allowed to press the lever for the discrete cocaine cues following one month of withdrawal.

In vivo electrochemistry methods (e.g., chronoamperometry) could potentially clarify the importance of DA in the NAc in cue-induced reinstatement due to the enhanced temporal resolution of the technique. It is possible that the larger sample intervals and sampling of extrasynaptic versus synaptic space used in microdialysis mask the brief alterations in neurotransmitter release. One study used in vivo chronoamperometry to study changes in DA signal in the NAc in response to discrete amphetamine cues (Di Ciano et al., 2001). The authors reported that whereas the amphetamine priming increased the DA signal, the CS had no effect. The limitation of this study, however, is that the DA response to the CS was determined after the behavioral response to the cue was extinguished. Thus, the rats were not actively involved in drug seeking during testing.

Recent studies examined neurochemical and genomic correlates of extinction behavior and exposure to discrete cocaine cues during withdrawal periods. Neisewander et al. (2000) studied Fos protein activation following 21 days of withdrawal after one session of extinction. Exposure to the self-administration environment enhanced Fos expression in several brain areas, including the anterior cingulate, BLA, hippocampal CA1 region, dentate gyrus, and NAc. Thomas and Everitt (2001) used in situ hybridization to image  PKC (an intracellular signal correlated with neuronal activity) expression in several brain areas during exposure to a cocaine-paired cue. They found selective activation by the discrete CSs previously paired with cocaine infusions in regions of the amygdala and cortex but not the NAc. The relevance of these data to extinction behavior and cue-induced relapse, however, is not clear because the discrete CS were given noncontingently and lever pressing-behavior was not measured following cue exposure.

Schmidt et al. (2001) reported that 12 days of cocaine self-administration reduced tyrosine hydroxylase (TH) immunoreactivity in the NAc shell but not core after 7 days of withdrawal. However, TH immunoreactivity in the NAc was restored in rats that experienced extinction training during this period. Extinction training also increased TH levels in the VTA, whereas TH was not altered in the VTA by cocaine withdrawal alone. The authors concluded that extinction-induced normalization of NAc TH levels could involve increased TH synthesis, stability, and/or transport from the VTA to the NAc. It should be pointed out, however, that because rats in the no extinction group were not exposed to the self-administration environment during the 1-week withdrawal period, it is not known whether repeated exposure to the cocaine self-administration context or the active experience of extinction training (or both) are involved in the effects described above.

Finally, Crespo et al. (2001) studied the expression of proenkephalin mRNA (PENK mRNA) in several brain areas following 0, 1, 5, or 10 days of extinction. One group of rats had previously self-administered cocaine, whereas the other two groups of rats had received either cocaine or saline injections yoked to the rats self-administering cocaine. The main finding in this study was a decrease in PENK mRNA in the CeA of the rats of the contingent group following 5 and 10 days of extinction and withdrawal and a similar decrease in the ventromedial hypothalamus following 5 days but not 10 days. However, because in the paired group both the duration of cocaine withdrawal and the experience of extinction were manipulated (i.e., the rats in the late withdrawal periods also had more extinction training), the relative contribution of these factors to the changes in PENK mRNA is not known.

Although several neurochemical and genomic correlates of extinction behavior were reported, because of the correlational nature of these studies, the neuronal mechanisms underlying drug seeking during extinction remained unknown. In addition, recent studies have shown that manipulations that alter 5-HT utilization can alter extinction behavior. However, as both increases and decreases in 5-HT levels are associated with decreased lever pressing during extinction, the role of 5-HT in extinction behavior remained unclear. Finally, recent data suggest that extinction training can alter the neuroadaptive changes associated with chronic cocaine use.

C. Discriminative and Contextual Drug Cues

1. Discriminative Drug Cues. Using a runway model with heroin-trained rats, McFarland and Ettenberg (1995, 1997, 1998) found that the opioid antagonist naloxone or the preferentially D2-like receptor antagonist haloperidol have no effect on heroin seeking provoked by discriminative heroin cues. Naloxone and haloperidol, however, blocked drug seeking on a test day conducted 24 h after last exposure to heroin (Fig. 8). These data suggest that the motivational processes underlying relapse induced by the discriminative cues and the drug itself are dissociable (McFarland and Ettenberg, 1997). Ciccocioppo et al. (2001) and Weiss et al. (2001) reported that SCH 23390 and SCH 39166 (a newer selective D1-like receptor antagonist that