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BEHAVIORAL PHARMACOLOGY

Noradrenergic Mechanisms in Cocaine-Induced Reinstatement of Drug Seeking in Squirrel Monkeys

New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts

Received for publication February 21, 2007
Accepted May 14, 2007.

	Abstract

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Norepinephrine (NE) uptake and NE receptor mechanisms play important modulating roles in the discriminative stimulus and stimulant effects of cocaine. The present study investigated the role of NE mechanisms in cocaine priming-induced reinstatement of extinguished drug seeking. Squirrel monkeys (Saimiri sciureus) were trained to stability under a second-order fixed interval, fixed ratio schedule of drug self-administration in which operant responding was maintained jointly by i.v. cocaine injections and presentations of a cocaine-paired stimulus. Drug seeking was then extinguished by replacing cocaine with vehicle and eliminating the cocaine-paired stimulus. In test sessions during which the cocaine-paired stimulus was reintroduced but only vehicle was available for self-administration, priming with cocaine, the dopamine transport inhibitor 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine (GBR 12909), and the NE transport inhibitors nisoxetine and talsupram induced dose-dependent reinstatement of drug seeking. The maximum effect of the NE transport inhibitors was less than half that of cocaine. Both nisoxetine and talsupram augmented the priming effects of a low but not a high dose of cocaine. The priming effects of nisoxetine were blocked by the 1-adrenoceptor antagonist prazosin, the 2-adrenoceptor agonist clonidine, and the -adrenoceptor antagonist propranolol, but not by the dopamine receptor antagonist flupenthixol. The priming effects of cocaine were antagonized by clonidine and flupenthixol. Neither nisoxetine nor cocaine increased physiological (salivary cortisol) or behavioral (self-directed behaviors) markers of stress. These findings suggest that NE transporter inhibition and 2-adrenoceptor mechanisms play a significant role in cocaine-induced reinstatement of drug seeking that is not secondary to activation of brain stress pathways.

Although inhibition of DA uptake and subsequent stimulation of DA receptors are established mechanisms mediating the abuse-related effects of cocaine, there is evidence that NE uptake and NE receptor mechanisms can serve important modulating roles in cocaine's behavioral effects in laboratory animals. In pigeons, rats, and monkeys trained to discriminate relatively low doses of cocaine from vehicle, NE transport inhibitors partially mimic the discriminative stimulus effects of cocaine (Cunningham and Callahan, 1991; Baker et al., 1993; Johanson and Barrett, 1993; Terry et al., 1994; Spealman, 1995), and when combined with cocaine or the selective DA transport inhibitor GBR 12909, NE transport inhibitors have been shown to enhance their discriminative stimulus effects (Cunningham and Callahan, 1991; Spealman, 1995; Kleven and Koek, 1998). Furthermore, the discriminative stimulus effects of cocaine and the cocaine-like effects of the NE transport inhibitors talsupram and tomoxetine can be attenuated by the 1-adrenoceptor antagonist prazosin (Johanson and Barrett, 1993; Spealman, 1995).

Clinical evidence points to incidental re-exposure to cocaine (priming), environmental stimuli associated with previous cocaine use, and stress as triggers of relapse in people. These same triggers have been found to induce reinstatement of cocaine-seeking behavior in laboratory animals, providing potentially useful models for investigating the biological basis of relapse (Spealman et al., 2004; Bossert et al., 2005). The strongest evidence for a role for the noradrenergic system in cocaine relapse comes from studies of stress-induced reinstatement of drug seeking. For example, the 2-adrenoceptor antagonists yohimbine and RS-79948 can reinstate cocaine-seeking behavior in monkeys at doses that induce physiological and behavioral indicators of stress (Lee et al., 2004). Moreover, the priming effects of yohimbine can be reversed with the 2-adrenoceptor agonist clonidine (Lee et al., 2004). Clonidine and other 2-agonists also have been shown to inhibit footshock-induced reinstatement of cocaine seeking in rats (Erb et al., 2000). -Adrenergic mechanisms also may play a role in stress-induced reinstatement, because central administration of 1- and 2-adrenoceptor antagonists block footshock-induced reinstatement of cocaine seeking (Leri et al., 2002).

The purpose of the present study was to investigate the role of NE mechanisms in cocaine priming-induced reinstatement in monkeys by evaluating the ability of the selective NE transport inhibitors talsupram and nisoxetine to mimic or modulate the relapse-inducing effects of cocaine and the cocaine-like priming effects of GBR 12909, a selective DA transport inhibitor. Additional studies were conducted to assess the role of specific subtypes of adrenergic receptors in cocaine priming-induced reinstatement by investigating the degree to which selective 1- and -adrenoceptor antagonists (prazosin and propranolol, respectively) and a selective 2-adrenoceptor agonist (clonidine) attenuated reinstatement of drug seeking induced by nisoxetine, GBR 12909, and cocaine priming. Finally, the ability of nisoxetine and cocaine to engender physiological and behavioral indices of stress was evaluated in an additional group of monkeys. Understanding the neurobiological mechanisms mediating reinstatement of cocaine seeking in animals may aid development of pharmacotherapies to combat relapse to cocaine use in people.

	Materials and Methods

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Subjects and Surgical Procedure. Twelve adult squirrel monkeys (Saimiri sciureus), weighing 650 to 1000 g, were studied in daily experimental sessions (Monday–Friday). Eight monkeys served as subjects in the reinstatement studies, and four monkeys served as subjects in the observation/salivary cortisol studies. Between sessions, monkeys lived in individual home cages where they had unrestricted access to food [Teklad Monkey Diet (Harlan Teklad, Madison, WI) supplemented with fresh fruit] and water. All animals were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School and the Guide for Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources (National Research Council, Department of Health, Education and Welfare Publication No. 85-23, revised 1996). Research protocols were approved by the Harvard Medical School Institutional Animal Care and Use Committee.

Subjects in the reinstatement studies were prepared with a chronic indwelling venous catheter (polyvinyl chloride; i.d., 0.38 mm; o.d., 0.76 mm) using the general surgical procedures described in Platt et al. (2005). Under isoflurane anesthesia and aseptic conditions, one end of a catheter was passed to the level of the right atrium by way of a femoral or jugular vein. The distal end of the catheter was passed subcutaneously and exited in the mid-scapular region. Catheters were flushed daily with saline and were sealed with stainless steel obturators when not in use. Monkeys wore custom-made nylon-mesh jackets (Lomir Biomedical, Toronto, ON, Canada) at all times to protect the catheter.

Apparatus. Self-administration and reinstatement sessions were conducted in ventilated and sound-attenuating chambers that were equipped with white noise to mask external sounds. Within the chamber, monkeys were seated in primate chairs with a response lever mounted on the panel in front of the monkey (MED Associates, Georgia, VT). Each press of a lever with a minimum downward force of approximately 0.25 N was recorded as a response. Colored lights mounted above the levers could be illuminated to serve as visual stimuli. Catheters were connected to a syringe pump (MED Associates) located outside of the chamber. Each operation of the pump lasted 1 s and delivered 0.18 ml of vehicle or drug solution into the catheter.

Observational studies followed by saliva collection were conducted in a ventilated, transparent Plexiglas arena (114 cm in length x 122 cm in width x 213 cm in height) located in a lighted room isolated from other animals. The arena was equipped with perches, suspended plastic chains, and a wood-chip foraging substrate to allow for the expression of a range of species-typical behaviors. A videocamera and a videocassette recorder operated continuously during the observation session.

Cocaine Self-Administration and Extinction. Monkeys were trained to respond under a second-order fixed interval (FI), fixed ratio (FR) schedule of reinforcement. In the presence of a white light, completion of every nth response (FR 10 or 30; depending on the monkey) during a 10-min interval of time (FI 10) resulted in a 2-s change in illumination from white to red light. Completion of the first FR after the 10-min interval elapsed produced both the 2-s light change and an i.v. injection of cocaine. A 60-s time-out period, during which all lights were off and responses had no scheduled consequences, followed each injection. If the FR requirement was not completed within 8 min following the expiration of the FI, the component ended automatically without an injection. Daily sessions ended after five cycles of the second-order schedule. Initially, the dose of cocaine was varied over a 10-fold or greater range to determine the dose that maintained maximum rates of responding for each monkey. The selected cocaine doses were 0.18 or 0.3 mg/kg/injection depending on the particular subject, and these doses of cocaine were kept constant for the remainder of the study.

After a 6- to 8-month period of stable cocaine self-administration under the second-order schedule, responding was extinguished by substituting saline for cocaine and omitting all presentations of the 2-s stimulus. Extinction sessions were otherwise identical to those described above for cocaine self-administration. Extinction sessions were conducted daily until response rate declined and stabilized at 10% of the rate maintained during active cocaine self-administration (3–10 sessions depending on the subject).

Reinstatement of Drug Seeking. Tests for priming-induced reinstatement of extinguished cocaine seeking were begun once the criteria for extinction were satisfied. Reinstatement test sessions used procedures identical to those during cocaine self-administration except that only saline was available for self-administration. Response-contingent presentations of the cocaine-paired stimulus were restored during reinstatement test sessions because earlier studies showed that reinstatement of drug seeking induced by cocaine priming was greatest when priming injections were accompanied by restoration of the cocaine-paired stimulus (Spealman et al., 2004). Between reinstatement test sessions with different doses of a particular drug, extinction sessions were conducted to ensure that monkeys maintained low rates of responding. Between experiments with different drugs, cocaine self-administration was re-established using the procedures described previously until responding stabilized (defined as no upward or downward trend in response rate across at least three daily self-administration sessions). Depending upon the subject, it took between three and nine sessions to achieve stable self-administration behavior. Previous studies have shown that by periodically re-establishing and then extinguishing drug-seeking behavior, reinstatement of drug seeking can be reliably induced by cocaine priming over several years of testing.

To evaluate their capacity to reinstate cocaine-seeking behavior, tests were conducted with saline and a range of doses of cocaine (0.1–1 mg/kg), the selective DA transport inhibitor GBR 12909 (0.3–1.8 mg/kg), and the selective NE transport inhibitors nisoxetine (0.3–3 mg/kg) and talsupram (1–5.6 mg/kg). Drugs were administered i.v. immediately before the session and followed by a saline flush to clear the catheter of residual drug solution. Different doses of a particular drug were tested on different days, and each test session was separated by two or more extinction sessions. The order of doses tested within each drug was varied across monkeys.

To assess the possibility of interactions between the transport inhibitors and cocaine, a subgroup of four monkeys received i.v. injections of either 1 mg/kg GBR 12909, 3 mg/kg nisoxetine, or 5.6 mg/kg talsupram immediately before priming injections of either a low (0.1 mg/kg) or a high (1.0 mg/kg) dose of cocaine. Additionally, these animals received an i.v. injection of either 3 mg/kg nisoxetine or 5.6 mg/kg talsupram immediately before priming injections of either a low (0.1 mg/kg) or high (1.0 mg/kg) dose of GBR 12909. Different drug combinations were studied on different days, and each test session was separated by two or more extinction sessions. The order of testing was varied across monkeys.

Antagonism experiments evaluated the degree to which DA and/or NE receptor mechanisms contributed to the cocaine-like priming effects of GBR 12909 and nisoxetine. A subgroup of four to five monkeys received i.m. injections of the 1-adrenoceptor antagonist prazosin (1 or 1.8 mg/kg, 30-min pretreatment; see Spealman, 1995), the 2-adrenoceptor agonist clonidine (0.01 and 0.03 mg/kg, 10-min pretreatment; cf. Lee et al., 2004), the -adrenoceptor antagonist propranolol (1 or 3 mg/kg, 30-min pretreatment; see Spealman, 1995), the nonselective DA antagonist flupenthixol (0.01 or 0.03 mg/kg, 60-min pretreatment; cf. Lee et al., 2004), or their vehicles before receiving an i.v. priming injection of either 1 mg/kg GBR 12909 or 3 mg/kg nisoxetine. Based on results from these experiments, selected doses of the pretreatment drugs also were administered before a maximally effective priming dose of 1 mg/kg i.v. cocaine. Two or more extinction sessions separated testing with each dose of a pretreatment drug. Doses of the pretreatment drugs were administered to individual subjects in irregular order.

Observation Study with Nisoxetine and Cocaine. After habituation to the observation arena, handling, and injection procedures, 30-min observational sessions were conducted daily during which the behavior of an animal was videotaped continuously. Drug test sessions were conducted once or twice per week, with saline control sessions on intervening days. Nisoxetine (0.3–3 mg/kg i.m.) or cocaine (0.1 to 1 mg/kg i.m.) was administered 5 min before placing the subject in the observation arena.

Scoring of videotapes was conducted by two observers who were trained in the use of the behavioral scoring system described in Lee et al. (2004), but they were not informed about the drugs under investigation. Before beginning the study, each observer underwent at least 20 h of training until he or she reached an interobserver reliability criterion of 90% based on percent of agreement scores. The behavioral scoring system included 10 categories (Table 1), which were scored by recording the presence or absence of each behavior in 15-s intervals during three 5-min observation periods across the session (0–5, 7–12, and 24–29 min). Frequency scores were calculated from these data as the number of 15-s intervals in which a particular behavior was observed.

In addition, during the 6th, 18th, and 30th min of each session, the monkey was removed from the observational arena by a trained handler, and it was evaluated for ataxia [defined as the inability to balance on and/or grasp a stainless steel pole (56 cm in length; 1 cm in diameter) held in a horizontal plane] and muscle resistance (defined as resistance to hind limb extension). For ataxia, a score of 0 indicated that the monkey was able to balance normally on the pole, a score of 1 indicated inability to balance effectively, and a score of 2 indicated that the monkey could neither balance on nor grasp the pole. For muscle resistance, a score of 0 indicated no change in resistance to hind limb extension, a score of +1 indicated increased resistance to extension and/or clinging to the grid floor, and a score of –1 indicated decreased resistance to extension and/or flaccidity.

Salivary Cortisol Study with Nisoxetine. The effects of 0.3 to 3 mg/kg i.m. nisoxetine and saline vehicle on circulating levels of cortisol, a physiological stress marker, were measured in saliva pre- and 90 min postinjection. A comparable study with cocaine was conducted in the context of an earlier experiment (Lee et al., 2003). Saliva was collected noninvasively in the observation arena between 8:30 AM and noon using the techniques described in Lee et al. (2004). In brief, a device consisting of a polyvinyl chloride pole with a dried cotton dental rope (3/8 in. in diameter; 5 cm in length, Richmond Dental, Charlotte, NC) flavored with concentrated Kool-Aid solution was given to a monkey. After the monkey chewed and dampened the dental rope with saliva, the device was retrieved, and the dental rope was placed in a salivette tube (Sarstedt AG & Co., Nümbrecht, Germany). The saliva was extracted by centrifugation at 1000 rpm for 12 min, and it was frozen at –80°C for later cortisol assay. Cortisol levels in saliva were determined in duplicate using a commercially available radioimmunoassay kit (Diagnostic Products, Los Angeles, CA). The intra-assay coefficient of variation for cortisol was 3.9%.

Data Analysis. For reinstatement studies, the rate of responding in individual monkeys was computed for each session by dividing the total number of responses by the total elapsed time (excluding responses and time during time-out periods). For each experimental condition, the mean response rate ± S.E.M. was calculated for groups of four to eight monkeys. To compare the effects of drug pretreatments on GBR 12909- and nisoxetine-induced reinstatement, response rates following pretreatment with different doses of prazosin, clonidine, propranolol, flupenthixol, or their vehicles were converted to percentage of response rates obtained with GBR 12909 and nisoxetine in the absence of any pretreatment. Data from reinstatement tests were analyzed with separate one- or two-way repeated measures ANOVAs followed by Bonferroni t tests.

In the observation study, for each drug, individual scores for each behavior were averaged over the three 5-min observation periods, because no systematic trends were observed across the three observation periods of the test session (as determined by separate repeated measures ANOVAs). Scores were then averaged across subjects to provide group means. Group means for each behavior were analyzed with separate one- or two-way repeated measures ANOVAs followed by Bonferroni t tests as appropriate. The effect of nisoxetine on salivary cortisol levels was assessed with a one-way repeated measures ANOVA. The level for all statistical tests was p 0.05.

Drugs. (–)-Cocaine HCl, GBR 12909 dihydrochloride, nisoxetine HCl, cis-z-flupenthixol dihydrochloride, prazosin HCl, and clonidine HCl (Sigma-Aldrich, St. Louis, MO), as well as talsupram HCl (H. Lundbeck A/S, Valby, Denmark) and propranolol HCl (Ayerst, New York, NY) were dissolved in small amounts of 95% ethanol or 0.1 N HCl as required and then diluted to the desired concentrations with sterile water or 0.9% saline solution. Injection volumes were 0.1 to 0.3 ml/kg body weight.

	Results

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Cocaine Self-Administration and Priming-Induced Reinstatement. Self-administered cocaine maintained consistently high rates of responding in individual monkeys under the second-order schedule of i.v. drug injection, averaging 0.5 to 2.2 responses/s in individual subjects, with a group mean of 1.0 ± 0.2 (S.E.M.) (n = 8). During extinction sessions, in which saline was substituted for cocaine and the cocaine-paired stimulus was omitted, responding declined and stabilized at low rates, averaging 0.01 to 0.2 responses/s depending upon the animal (mean ± S.E.M.; 0.06 ± 0.01; n = 8). Priming injections of cocaine (0.1–1 mg/kg) before test sessions in which the cocaine-paired stimulus was restored but only saline was available for self-administration produced reliable, dose-dependent reinstatement of extinguished cocaine-seeking behavior [Fig. 1a; F(3,21) = 18.3, p < 0.001; Bonferroni t tests]. Average response rates engendered by priming with 0.3 and 1 mg/kg cocaine approached response rates maintained by active cocaine self-administration (see above). Priming injections of vehicle did not reinstate extinguished drug seeking above levels observed during extinction (Fig. 1a, point above VEH).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Dose-dependent reinstatement of extinguished drug-seeking behavior induced by cocaine (a), GBR 12909 (b), nisoxetine (c), or talsupram (d) combined with restoration of the cocaine-paired stimulus. Points above VEH show response rates following a vehicle prime. Data are means ± S.E.M. from five (talsupram) or eight (cocaine, GBR 12909, and nisoxetine) monkeys. *, p < 0.05 compared with VEH.

The NE transport inhibitors nisoxetine and talsupram also reinstated extinguished cocaine seeking (Fig. 1, c and d, respectively). Priming injections of the highest doses of nisoxetine (3 mg/kg) and talsupram (5.6 mg/kg) engendered significantly higher response rates than rates observed following a vehicle prime [nisoxetine: F(3,20) = 4.9, p < 0.01; talsupram: F(3,12) = 3.5, p < 0.05; Bonferroni t tests]. However, the maximal rates of responding engendered by the two NE transport inhibitors averaged 36 to 55% of the maximal rate engendered by cocaine or GBR 12909. Higher doses of the NE transport inhibitors were not evaluated due to the emergence of untoward side effects (e.g., emesis) in at least one subject.

Interactions between Cocaine and Selective DA and NE Transport Inhibitors. Pretreatment with a maximally effective dose of 1 mg/kg GBR 12909 before cocaine priming resulted in an augmentation of drug-seeking behavior (Fig. 2a). Specifically, combinations of GBR 12909 with a low, ineffective priming dose of cocaine (0.1 mg/kg) engendered significantly higher response rates compared with cocaine alone (p < 0.05; Bonferroni t tests). GBR 12909 did not significantly increase the rate of reinstated cocaine seeking induced by a maximally effective priming dose of cocaine (1 mg/kg).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Enhancement of the priming effects of a low, but not high, dose of cocaine by GBR 12909 (a), nisoxetine (b), or talsupram (c). Data are means + S.E.M. from four monkeys. *, p < 0.05 compared with cocaine alone.

Interactions between GBR 12909 and Selective NE Transport Inhibitors. In contrast to the effects observed when either nisoxetine or talsupram was combined with cocaine, pretreatment with maximally effective doses of nisoxetine (3 mg/kg) and talsupram (5.6 mg/kg) did not reliably alter reinstatement induced by either a low or high dose of GBR 12909 (Fig. 3, a and b, respectively). In no case was the effect of the combination of the NE uptake inhibitors and GBR 12909 greater than the sum of the individual drug effects.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Lack of enhancement of the priming effects of GBR 12909 by nisoxetine (a) or talsupram (b). Data are means + S.E.M. from four monkeys. *, p < 0.05 compared with GBR 12909 alone.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Attenuation of the cocaine priming effects of nisoxetine or GBR 12909 by flupenthixol (FLU) (a), prazosin (PRZ) (b), clonidine (CLN) (c), or propranolol (PRP) (d). Nisoxetine dose, 3.0 mg/kg; GBR 12909 dose, 1.0 mg/kg. Data are means + S.E.M. from four (PRZ and PRP) or five (CLN and FLU) monkeys. *, p < 0.05 compared with VEH.

The opposite pattern of effects was observed when a range of doses of the 1-adrenoceptor antagonist prazosin was administered as a pretreatment before priming with GBR 12909 or nisoxetine (Fig. 4b). That is, the cocaine-like priming effects of nisoxetine, but not GBR 12909, were significantly decreased by prazosin [F(3,7) = 128.9, p < 0.001; Bonferroni t test]. Maximum decreases in the priming effects of nisoxetine (76% compared with vehicle) were observed after pretreatment with 1.8 mg/kg prazosin. This dose of prazosin produced slight, nonsignificant decreases in the effect of GBR 12909 compared with vehicle. When tested alone, no dose of prazosin reliably reinstated cocaine seeking (maximal response rate engendered by prazosin, 0.17 ± 0.12 responses/s).

Pretreatment with the 2-adrenoceptor agonist clonidine also produced dose-dependent reductions in the priming effects of nisoxetine but not GBR 12909 (Fig. 4c). A dose of 0.03 mg/kg clonidine significantly inhibited the effects of nisoxetine [F(3,10) = 26.6, p < 0.001; Bonferroni t test], reducing response rate by 74% compared with vehicle. When evaluated alone, clonidine did not reliably reinstate extinguished cocaine seeking (maximal response rate engendered by clonidine, 0.17 ± 0.09 responses/s).

Finally, pretreatment with the -adrenoceptor antagonist propranolol produced effects similar to those of the -adrenoceptor ligands. Propranolol reliably reduced the cocaine-like priming effects of nisoxetine but not GBR 12909 (Fig. 4d). A 3 mg/kg dose of propranolol significantly inhibited the effects of nisoxetine to 62% of vehicle [F(3,7) = 16.3, p < 0.003; Bonferroni t test], whereas this dose of propranolol did not reliably alter the priming effects of GBR 12909. Alone, propranolol did not reinstate cocaine-seeking behavior (maximal response rate engendered by propranolol, 0.12 ± 0.06 responses/s).

Interactions of Subtype-Selective Adrenergic Receptor Ligands and Cocaine. The priming effects of 1 mg/kg cocaine were redetermined after pretreatment with vehicle and selected doses of flupenthixol (0.03 mg/kg), prazosin (1.8 mg/kg), clonidine (0.03 mg/kg), and propranolol (3 mg/kg). Doses of pretreatment drugs were chosen to be those that exhibited selectivity for effects against either GBR 12909 or nisoxetine in results described above. As shown in Fig. 5, cocaine-induced reinstatement was significantly decreased following pretreatment with flupenthixol and clonidine but not prazosin or propranolol [F(4,12) = 4.3, p < 0.02; Bonferroni t tests]. Both flupenthixol and clonidine reduced response rates engendered by cocaine priming by about 75% compared with vehicle.

View larger version (14K): [in this window] [in a new window]   Fig. 5. Effects of cocaine priming following treatment with saline (VEH), FLU, PRZ, CLN, or PRP. Cocaine dose, 1.0 mg/kg. Data are means + S.E.M. from four monkeys. *, p < 0.05 compared with VEH.

Likewise, cocaine had few significant effects on observed behavior. Cocaine (0.1–1 mg/kg) did not alter measures of locomotion, object exploration, foraging, visual scanning, vocalization, rest posture, static posture, balance, or muscle resistance. However, cocaine did reliably decrease measures of self-directed behavior (Table 2). That is, all doses of cocaine reduced scratching levels [F(3,9) = 8.5, p < 0.005; Bonferroni t test], and the two highest cocaine doses eliminated self-grooming [F(3,9) = 4.6, p < 0.032; Bonferroni t test]. These effects are opposite of what has been shown for yohimbine, a pharmacological stressor (cf. Lee et al., 2004). Salivary cortisol data for cocaine were obtained from Lee et al. (2003), and they show that, like nisoxetine, 0.3 and 1 mg/kg cocaine had no reliable effect on salivary cortisol levels.

	Discussion

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DA mechanisms undoubtedly play a critical role in the effects of cocaine related to its abuse. However, less is known regarding the role of NE mechanisms in the behavioral effects of cocaine. In this study, priming injections of the NE transport inhibitors nisoxetine and talsupram engendered reliable reinstatement of cocaine-seeking behavior, although not to the same degree as the DA transport inhibitor GBR 12909 or cocaine itself. Maximally effective doses of cocaine and GBR 12909 engendered response rates comparable with those observed during active cocaine self-administration, whereas the NE transport inhibitors engendered rates approximately half of those engendered by cocaine. In drug combination studies, nisoxetine and talsupram augmented the priming effects of cocaine. These findings support the idea that inhibition of DA uptake plays a key role in the priming effects of cocaine and also are consistent with the hypothesis that NE uptake inhibition can contribute to these effects of cocaine.

Our results supporting a role for NE mechanisms in the priming effects of cocaine differ from the majority of data reported in rats, which show a minimal role for these mechanisms in the behavioral effects of cocaine, including its reinstatement-inducing effects (Schmidt and Pierce, 2006). These dissimilar findings may reflect underlying species differences in the NE system of primates compared with rodents. For example, Paczkowski et al. (1999) showed quantitative differences in the pharmacological profiles for human and rat NE transporters, including an increased affinity for cocaine for the human transporter compared with the rat transporter. In addition, the distribution of NE transporters in nonhuman primate brain has been shown to differ in some significant respects from the distribution in rats (Smith et al., 2006). Along with studies that show significant up-regulation of NE transporters as a consequence of long-term cocaine exposure in monkeys (Macey et al., 2003; Beveridge et al., 2005) but not rats (Belej et al., 1996; Arroyo et al., 2000), these findings provide a basis for the speculation that NE uptake inhibition plays a more important role in the priming effects of cocaine in primates compared with rodents.

It has been suggested that shared discriminative stimulus effects may contribute to the degree to which a drug can reinstate cocaine-seeking behavior (for reviews, see Katz and Higgins, 2003; Shaham et al., 2003). Indeed, in addition to reinstating cocaine seeking, NE transport inhibitors share discriminative stimulus effects with cocaine (Cunningham and Callahan, 1991; Baker et al., 1993; Johanson and Barrett, 1993; Terry et al., 1994; Spealman, 1995). Cocaine also induces cardiovascular effects (e.g., increases in heart rate and diastolic and systolic blood pressure; Tella et al., 1993) that mimic those of some NE transport inhibitors, including nisoxetine, raising the possibility that common peripheral effects might contribute to a discriminable cue. However, the discriminative stimulus, as well as cardiovascular effects, of cocaine can be attenuated by antagonists with selectivity for 1-adrenoceptors only or in addition to -adrenoceptors (Johanson and Barrett, 1993; Spealman, 1995; Sofuoglu et al., 2000a,b). Although the cocaine-like priming effects of nisoxetine were attenuated by both prazosin (selective for 1 adrenoceptors) and propranolol (selective for adrenoceptors) in the present study, the priming effects of cocaine itself were not, suggesting that common discriminable cues (whether peripheral or central) do not underlie the ability of NE transport inhibitors to reinstate cocaine seeking.

Both nisoxetine and talsupram are selective for NE transporters compared with DA transporters (50- to >1000-fold selective, respectively; e.g., Czoty et al., 2004; McConathy et al., 2004). However, for nisoxetine at least, its affinity for the DA transporter is within the range of that of GBR 12909 and other drugs that share behavioral effects with methamphetamine, a stimulant whose effects are prominently mediated by dopaminergic mechanisms (e.g., Czoty et al., 2004). The latter finding raises the possibility that the ability of NE transport inhibitors to partially mimic the priming effects of cocaine is due to their ability to increase DA directly via interactions with the DA transporter. However, the priming effects of nisoxetine were not attenuated by dopamine receptor-selective doses of the antagonist flupenthixol, suggesting that mechanisms other than direct actions at the DA transporter underlie the priming effects of NE transport inhibitors.

Alternatively, the cocaine-like priming effects of NE transport inhibitors may reflect their capacity to indirectly regulate midbrain DA levels via prefrontal cortical adrenergic receptors. Stimulant-induced increases in NE transmission in this brain region have been linked to increased DA cell bursting in the ventral tegmental area as well as increased DA release in the nucleus accumbens, critical brain regions mediating drug priming-induced reinstatement (Darracq et al., 1998; Ventura et al., 2003, 2005). A similar effect on DA cells was observed following intravenous administration of nisoxetine (Shi et al., 2000). Additional evidence suggests that the increase in DA release observed in the nucleus accumbens is mediated by 1-adrenoceptors in the prefrontal cortex and not DA receptors, because the changes in DA neuron firing and DA release could be attenuated by the 1-adrenoceptor antagonist prazosin but not the DA receptor antagonists SCH 23390 or raclopride (Darracq et al., 1998; Shi et al., 2000). Our data are consistent with these neurochemical findings in that the cocaine-like priming effects of nisoxetine could be attenuated by prazosin, but not the DA antagonist flupenthixol. A DA augmenting mechanism also may underlie the ability of nisoxetine and talsupram to increase drug seeking induced by low priming doses of cocaine. However, the NE transport inhibitors did not enhance the reinstatement of drug seeking induced by GBR 12909, suggesting the possibility that the increase in extracellular DA induced by DA uptake inhibition was sufficient to mask the presumably smaller effects of nisoxetine and talsupram on DA neuron firing.

In antagonism studies, nisoxetinebut not GBR 12909-induced priming also was attenuated by the 2-adrenoceptor agonist clonidine and the -adrenoceptor antagonist propranolol. Both 2-and -adrenoceptor mechanisms have been implicated as factors underlying stress-induced reinstatement (Erb et al., 2000; Leri et al., 2002; Lee et al., 2004). Thus, it is possible that nisoxetine and talsupram induced reinstatement as a consequence of their ability to increase synaptic levels of NE and thereby activate stress pathways in the brain. The bed nucleus of the stria terminalis is a brain region that is enriched by NE neurons as well as sensitive to neuroadaptations induced by long-term cocaine exposure (up-regulation of NE transporters; Macey et al., 2003; Beveridge et al., 2005). This region also is critical for autonomic responses to stressors. Heightened noradrenergic tone in this region can stimulate release of corticotropin-releasing factor from the hypothalamus, an important initial response to stress (Koob, 1999; Beveridge et al., 2005). However, unlike the anxiogenic 2-adrenoceptor antagonist yohimbine, nisoxetine failed to increase either physiological or behavioral indices of stress (Table 2) (see Lee et al., 2004). The present results provide no evidence to suggest that activation of stress pathways played a major role in the ability of the NE transport inhibitors to reinstate cocaine-seeking behavior.

In antagonism studies with cocaine, flupenthixol and clonidine effectively attenuated priming-induced reinstatement. Our finding that flupenthixol antagonized the priming effects of cocaine at a dose that was effective against GBR 12909-induced priming is in agreement with the large body of evidence indicating a primary role for DA receptor mechanisms in cocaine-induced reinstatement (for review, see Bossert et al., 2005). However, the ability of clonidine to attenuate cocaine priming was unexpected in light of the finding that clonidine and other 2-adrenoceptor agonists did not alter cocaine priming-induced reinstatement in rats (Erb et al., 2000). The effect of clonidine in the present study does not seem to be related to potential cocaine-induced activation of brain stress pathways, because cocaine neither engendered behavioral signs of stress in observation studies nor increased salivary cortisol levels (Table 2) (Lee et al., 2003). It remains a possibility that species differences in the regional distribution, receptor heterogeneity, and pharmacological properties of 2-adrenoceptors underlie the differential effects of clonidine in primates versus rats (Zilles et al., 1993; Molderings et al., 2000).

In contrast to the results with flupenthixol and clonidine, neither prazosin nor propranolol altered cocaine priming-induced reinstatement. These findings were unexpected given the effectiveness of these adrenergic compounds against cocaine in other species and/or procedures. For example, prazosin has been shown to reduce cocaine-induced reinstatement in rats (Zhang and Kosten, 2005) and to attenuate the discriminative stimulus effects of cocaine in monkeys (Spealman, 1995). Likewise, propranolol has been shown to decrease cocaine self-administration in both rats and monkeys (Goldberg and Gonzalez, 1976; Harris et al., 1996). The reasons underlying the divergent effects of prazosin and propranolol in the present study are not clear. Nevertheless, they highlight the fact that diverse receptor mechanisms probably contribute to the different behavioral effects of cocaine. For example, whereas pharmacological inhibition of NE uptake seems to play a role in cocaine priming-induced reinstatement, this mechanism seems to have a less significant impact in the reinforcing effects of cocaine. Along these lines, in rhesus monkeys previously trained to self-administer cocaine, the selective NE transport inhibitors nisoxetine, desipramine, and atomoxetine do not maintain self-administration above vehicle levels (Woolverton, 1987; Wee and Woolverton, 2004; Wee et al., 2006). Moreover, when combined with cocaine, neither nisoxetine nor desipramine systematically alters cocaine self-administration, even at doses that produce virtually complete inhibition of NE uptake (Tella, 1995; Wee et al., 2006).

In summary, this study presents evidence of a significant role for NE inhibition and 2-adrenoceptor mechanisms in cocaine-induced reinstatement of drug-seeking. These mechanisms seem not to contribute universally to the behavioral effects of cocaine, but rather are specific to cocaine-induced reinstatement in monkeys. The specificity may reflect underlying neurobiological differences between monkeys and rodents and/or unique mechanisms associated with drug-induced reinstatement versus other behavioral effects including the discriminative stimulus and reinforcing effects of cocaine.

	Acknowledgements

 
We thank Kristen Bano, Annemarie Duggan, Sharon Malinak, Julie Silva, and Noah Rosenberg for expert technical assistance.

	Footnotes

 
This research was supported by U.S. Public Health Service Grants DA11054 and RR00168.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.121806.

ABBREVIATIONS: DA, dopamine; NE, norepinephrine; VEH, vehicle; GBR 12909, 1-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine; FI, fixed interval; FR, fixed ratio; ANOVA, analysis of variance; FLU, flupenthixol; PRZ, prazosin; CLN, clonidine; PRP, propranolol; RS-79948, (8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-dechydro-3-mexthoxy-12-(ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine.

Address correspondence to: Dr. Donna M. Platt, New England Primate Research Center, Harvard Medical School, One Pine Hill Dr., Box 9102, Southborough, MA 01772-9102. E-mail: donna_platt{at}hms.harvard.edu

	References

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