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Potentiation of cue-induced reinstatement of cocaine-seeking in rats by the anxiogenic drug yohimbine
Behavioural Brain Research 174 (2006) 1–8
Research report
Potentiation of cue-induced reinstatement of cocaine-seeking in rats by the anxiogenic drug yohimbine Matthew W. Feltenstein, Ronald E. See ∗ Department of Neurosciences, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA Received 24 April 2006; received in revised form 27 June 2006; accepted 29 June 2006 Available online 21 August 2006
Abstract Stress and drug-associated cues can trigger drug desire and relapse in abstinent cocaine users. Although the role of these two factors in relapse is well documented, it remains unclear as to whether an interaction between stress and drug-associated cues can lead to an enhancement in cocaine-seeking behavior. Here, we assessed the effects of the anxiogenic ␣2 -noradrenergic receptor antagonist, yohimbine, on reinstatement of cocaine-seeking in rats either in the presence or absence of cocaine-associated cues. Yohimbine pretreatment in the absence of cocaine-associated cues or cues by themselves reliably reinstated responding on the previously cocaine-paired lever (3–4 times higher than extinction levels). However, animals showed greatly potentiated responding if yohimbine preceded cue-induced reinstatement (10–13 times higher than extinction levels, or 3–5 times over cues or yohimbine alone). While cocaine self-administration produced a significant increase in plasma corticosterone, plasma corticosterone levels did not show a clear relationship to cocaine-paired lever responding during cue and/or yohimbine-induced reinstatement. These results demonstrate that exposure to drug-paired cues during a stressful state can greatly potentiate cocaine-seeking and suggest that future treatment interventions should target multiple modalities. © 2006 Elsevier B.V. All rights reserved. Keywords: Cocaine; Cues; Reinstatement; Relapse; Self-administration; Stress; Yohimbine
1. Introduction One of the most difficult problems for successful treatment of psychostimulant dependence is the high incidence of relapse to drug-seeking and drug-taking behaviors following prolonged periods of abstinence [1,2]. To date, clinical research has indicated several factors that can trigger drug desire and relapse in abstinent drug users, including negative mood states, stress, and exposure to drug-associated environmental stimuli (e.g. the context where the drug was consumed or drug paraphernalia). For example, abstinent cocaine users report craving following exposure to cocaine-associated stimuli [3], or in response to stressful life events [4]. These trigger factors have been experimentally studied using the reinstatement model of relapse in animals [5,6]. Typically, cocaine-paired stimuli or exposure to an environmental stressor following withdrawal from chronic cocaine self-administration will robustly reinstate extinguished drug-
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0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.06.039
seeking as measured by increased responding on a previously cocaine-paired lever. The reinstatement model has advanced our understanding of the contribution of individual relapse factors and the neural mechanisms that underlie relapse [7–10], and provided a test for compounds that may prove beneficial for preventing relapse in cocaine dependent individuals [11,12]. Despite substantial evidence suggesting the role of drugassociated stimuli and stress in triggering relapse, very little attention has focused on the interaction of these variables in drug desire and drug-seeking. Given that multiple risk factors usually precede relapse [13], it is likely that exposure to drug-associated cues during periods of stress may increase the probability and/or intensity of drug craving and cocaine-seeking behavior. To test this hypothesis, we examined the separate and interactive effects of previously drug-paired stimuli and a stressor (the anxiogenic drug, yohimbine) on cocaine-seeking behavior in an animal model of relapse. Yohimbine is a norepinephrine (NE) ␣2 receptor antagonist that increases NE release in several neural structures implicated in stress, including the bed nucleus of the stria terminalis [14] and the amygdala [15]. Yohimbine treatment has been shown to produce anxiety-like states in both humans
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[16,17] and laboratory animals [18,19]. Furthermore, yohimbine induces drug craving in abstinent drug-dependent subjects [20] and reinstates cocaine [21], methamphetamine [22], and alcohol [23] seeking in animal models of relapse. In order to obtain a physiological measure of stress activation, we also assessed plasma corticosterone levels during cocaine self-administration, extinction, and reinstatement testing. 2. Methods 2.1. Subjects Male, Sprague–Dawley rats (initial weight 275–300 g; Charles River, Wilmington, MA, USA) were individually housed in a temperature- and humiditycontrolled vivarium on a normal 12 h light–dark cycle (lights on 6 a.m.–6 p.m.). All experimental procedures (drug self-administration, extinction, and reinstatement testing) occurred between 11 a.m. and 5 p.m. Animals received water ad libitum and were maintained on 20–25 g of standard rat chow (Harlan, Indianapolis, IN, USA) per day for the duration of each experiment. Rats were acclimated to handling and allowed to adapt for a minimum of 3 days prior to the start of the experiment. Housing and care of the rats were carried out in accordance with the “Guide for the Care and Use of Laboratory Rats” (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 1996).
2.2. Lever response training Rats were trained to lever press in standard operant conditioning chambers (30 cm × 20 cm × 20 cm) linked to a computerized data collection program (MED-PC, Med Associates, Inc., St. Albans, VT, USA). The chambers were equipped with two retractable levers, a stimulus light above each lever, a food pellet dispenser between the levers, a speaker linked to a programmable tone generator (ANL-926, Med Associates), and a house light on the wall opposite the levers. Each chamber was contained within a sound-attenuating cubicle equipped with a ventilation fan. Forty-eight hours prior to surgery, rats were food deprived overnight and trained to lever press on a fixed ratio (FR) 1 schedule of food reinforcement (45 mg pellets; Noyes, Lancaster, NH, USA) during a 15 h overnight training session in the absence of explicit conditioned stimulus (CS) presentation (i.e. active lever presses resulted in the delivery of a food pellet only). Lever presses on an inactive lever were recorded, but had no programmed consequence. Following lever response training, food dispensers were permanently removed from the test chambers.
2.3. Surgery Rats were anesthetized using a mixture of ketamine hydrochloride and xylazine (66 and 1.33 mg/kg, respectively, IP) followed by equithesin (0.5 ml/kg with a solution of 9.72 mg/ml pentobarbital sodium, 42.5 mg/ml chloral hydrate, and 21.3 mg/ml magnesium sulfate heptahydrate dissolved in a 44% propylene glycol, 10% ethanol solution, IP). Surgical procedures were conducted using aseptic techniques. Catheters were constructed using previously described methods [24] and consisted of external guide cannulae with screw-type connectors (Plastics One Inc., Roanoke, VA, USA), Silastic tubing (10 cm; i.d. = 0.64 mm; o.d. = 1.19 mm; Dow Corning, Midland, MI, USA), prolite polypropylene monofilament mesh (2 cm diameter, Atrium Medical Corporation, Hudson, NH, USA), and cranioplastic cement. The end of the catheter was inserted into the right jugular vein and secured to surrounding tissue with suture. The catheter ran subcutaneously and exited on the rat’s back, posterior to the shoulder blades. To maintain patency, catheters were flushed once daily for 4 days after surgery with 0.1 ml each of an antibiotic solution of cefazolin (10.0 mg/ml; Schein Pharmaceuticals, Florham Park, NJ, USA) dissolved in heparinized saline (70 U/ml; Elkins-Sinn, Cherry Hill, NJ, USA) and heparinized saline. For the duration of the experiment, each subject then received 0.1 ml of heparinized saline (10 U/ml) immediately prior to self-administration sessions and the cefazolin and 70 U/ml heparinized saline regimen following each session. To verify catheter patency, rats occasionally received a 0.12 ml infusion of methohexital
sodium (10.0 mg/ml IV; Eli Lilly and Co., Indianapolis, IN, USA), a short-acting barbiturate that produces a rapid loss of muscle tone when administered intravenously.
2.4. Cocaine self-administration Rats (n = 29) were trained to self-administer cocaine (cocaine hydrochloride dissolved in 0.9% sterile saline; cocaine provided by the National Institute on Drug Abuse, Research Triangle Park, NC, USA) during daily 2 h sessions according to an FR 1 schedule of reinforcement. At the start of each session, the catheter was connected to a swivel (Instech, Plymouth Meeting, PA, USA) via polyethylene 20 tubing that was encased in steel spring leashes (Plastics One Inc., Roanoke, VA, USA). The house light signaled the initiation of the session and remained illuminated throughout the entire session. Lever presses on the active lever resulted in a 2 s activation of the infusion pump (0.5 mg/kg cocaine per 0.05 ml infusion) and a 5 s presentation of a stimulus complex, consisting of activation of the white stimulus light above the active lever and the tone generator (78 dB, 2 kHz). After each infusion, responses on the active lever had no consequences during a 20 s time-out period. Responses on the inactive lever were recorded throughout each session of the study, but had no programmed consequences. Daily cocaine self-administration continued until each rat had obtained the self-administration criterion of 10 sessions with at least 10 infusions per session.
2.5. Extinction and reinstatement of cocaine-seeking Following chronic self-administration and before the first reinstatement test, rats underwent daily 2 h extinction sessions. To further insure that any putative interaction with yohimbine pretreatment could be attributed to incentive motivational value (rather than non-specific factors) of these stimuli, half the animals were extinguished to the previously cocaine-paired cues. During each session, responses on the active lever resulted in either a 5 s CS presentation in the absence of cocaine reinforcement, followed by a 20 s time-out period in which responses did not result in further CS presentations (i.e. Cue Extinction group, n = 15), or no programmed consequences (i.e. neither cocaine reinforcement nor conditioned stimuli were presented; No-Cue Extinction group, n = 14). Once extinction criterion was reached (a minimum of seven extinction sessions with ≤25 active lever responses per session for the last two consecutive days), each rat underwent six separate tests to examine the effects of yohimbine treatment and cue presentation on reinstatement of responding. We have previously found no decrement in responding when the number of tests is limited to a total of three for a given reinstatement condition [25–27]. Thirty minutes prior to a reinstatement test, each rat received an IP injection of sterile distilled water (vehicle) or yohimbine hydrochloride (1.25 or 2.5 mg/kg; Sigma–Aldrich, St. Louis, MO, USA). The doses of yohimbine were selected based on previous studies of yohimbine effects in rats [22,23,28] and were given in a counterbalanced order according to cocaine intake. For some animals in the Cue (n = 7) and No-Cue (n = 8) Extinction groups, responses on the active lever for the first three reinstatement tests resulted in 5 s CS presentations in the absence of cocaine reinforcement (i.e. yohimbine + cues), while active lever responding for the last three reinstatement tests had no programmed consequences (i.e. yohimbine only). For the remaining animals in each group (n = 8 and 6, respectively for the Cue and No-Cue Extinction groups), the situation was reversed. The order of yohimbine treatment for each animal was the same for both sets of reinstatement tests and animals underwent extinction sessions between reinstatement tests until they reached criterion (i.e. ≤25 active lever responses per session for two consecutive days).
2.6. Corticosterone assay In order to assess any alterations in plasma corticosterone levels due to experimental procedures, 100 l of whole blood was collected through the catheter using 1000 U heparin at the following time points: the day before (pre-SA) and immediately following the first (SA1) and last (SA10) day of cocaine self-administration, immediately following the first (E1) and last (E7) day of extinction, and immediately following all reinstatement tests. Plasma was isolated by centrifugation (10,000 rpm at 4 ◦ C for 20 min) and stored at −80 ◦ C
M.W. Feltenstein, R.E. See / Behavioural Brain Research 174 (2006) 1–8 until assayed. Plasma corticosterone concentrations (ng/ml) were determined using radioimmunoassay (Diagnostic Systems Laboratories, DSL-81100, Webster, TX, USA) according to the manufacturer’s directions.
2.7. Data analysis t-Tests and mixed factors analysis of variance (ANOVA) were used to analyze active and inactive lever responses, and drug infusions, with session (self-administration day and extinction day) and extinction group as factors, where appropriate. One-way ANOVAs were used to analyze active and inactive lever responses and plasma corticosterone levels during reinstatement testing, with drug and reinstatement test session as factors, where appropriate. Interaction effects were further investigated using simple main effects tests (one-way ANOVA) with post hoc analyses conducted using Student–Newman–Keuls with the alpha set at 0.05. Data are expressed as the mean ± S.E.M.
3. Results 3.1. Cocaine self-administration and extinction Rats in both extinction groups readily acquired cocaine self-administration and displayed stable active lever responding and cocaine intake during the maintenance phase (Fig. 1). Accordingly, 2 × 3 ANOVAs of active and inactive lever responding for the last three self-administration sessions revealed no significant effects for group, session, or interaction between group and session. Cocaine intake (Cue Extinction group = 19.43 ± 0.96 mg/kg/session; No-Cue Extinction group = 19.54 ± 0.92 mg/kg/session) and number of sessions required to achieve the self-administration criterion (Cue Extinction group = 10.60 ± 0.31; No-Cue Extinction group = 10.79 ± 0.32) also did not differ between groups. Thus, animals responded more on the active lever than on the inactive lever, regardless of session, and the two groups did not differ in cocaine self-administration history. Following the removal of cocaine reinforcement, active lever responding decreased steadily over the course of daily extinction sessions for both groups (Fig. 1). Although animals in
Fig. 1. Lever responding (mean ± S.E.M.) during the last 3 days of cocaine self-administration (SA) on the active and inactive levers and for 7 days of extinction (large symbols = active lever; small symbols = inactive lever). All animals received cues throughout cocaine SA, but only half received cues during extinction (Cue extinction; n = 15) and the other half did not (No-Cue extinction; n = 14).
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the Cue Extinction group exhibited more responding on the active lever, there was no difference between the two groups in the number of sessions required to achieve the extinction criterion. A 2 × 7 ANOVA of active lever responding during the first seven extinction sessions revealed a significant main effect for group (F(1,27) = 7.88, p < 0.01) and session (F(6,162) = 20.59, p < 0.001). However, the group by session interaction was not significant. Similar analyses for inactive lever responding only revealed a significant main effect for session (F(6,162) = 5.32, p < 0.001). The group main effect and group by session interaction effect were not significant. Finally, there were no significant differences between the two extinction groups in the number of sessions required to reach the extinction criterion of a minimum of seven sessions with ≤25 active lever responses per session for the last two consecutive days (Cue Extinction group = 8.00 ± 0.66; No-Cue Extinction group = 7.21 ± 0.11). Thus, although Cue Extinction animals exhibited higher cocaine-paired lever responding during early extinction, both groups extinguished to similarly low levels prior to reinstatement testing. 3.2. Reinstatement tests Prior to further analyses, the data for both extinction groups were examined for possible order effects of reinstatement testing or drug treatment. Analysis failed to reveal any significant differences in the number of active lever presses during each reinstatement test based on the order of reinstatement testing (yohimbine alone, yohimbine + cues) or the order of drug treatment (vehicle, 1.25 and 2.5 mg/kg). As such, the data were collapsed within extinction group and across test day order. For animals in the Cue Extinction group, yohimbine increased reinstatement responding 3–4 times higher than vehicle treatment under both the “no cues” and “cues” reinstatement conditions (Fig. 2). Similar increases in responding were seen after yohimbine treatment during the “no cues” reinstatement condition and for the “cues” presentation alone for animals in the No-Cue Extinction group. However, yohimbine treatment in combination with the cues produced a robust potentiative effect, with responding rates 10–13 times higher than extinction levels, or 3–5 times over cues or yohimbine alone (Fig. 2). A 2 × 2 × 3 ANOVA of active lever responding revealed significant main effects for group (F(1,27) = 18.96, p < 0.001), reinstatement test condition (F(1,27) = 25.66, p < 0.001), and drug (F(2,54) = 25.08, p < 0.001), as well as significant group by reinstatement test condition (F(1,27) = 25.30, p < 0.001), reinstatement test condition by drug (F(2,54) = 3.34, p < 0.05) and group by reinstatement test condition by drug (F(2,54) = 3.68, p < 0.05) interactions. The group by drug interaction was not significant. Similar analyses of inactive lever responding only revealed a significant main effect for drug (F(2,54) = 17.79, p < 0.001), but no significant effect for group and reinstatement test condition main effects or any interactions. For animals in the Cue Extinction group, a 2 × 3 ANOVA of active lever responding revealed a significant main effect for drug (F(2,84) = 12.20, p < 0.001). The reinstatement test condition main effect and reinstatement test condition by drug inter-
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Fig. 3. Response patterns over time during reinstatement tests. Active lever responding (mean ± S.E.M.) is shown in 30 min bins for the two groups (Cue or No-Cue Extinction) following injection with vehicle (), 1.25 mg/kg yohimbine (), or 2.5 mg/kg yohimbine ().
Fig. 2. Potentiation of conditioned-cued reinstatement by yohimbine. Active and inactive (bottom) lever responding (mean ± S.E.M.) are shown for the two groups (Cue or No-Cue Extinction) following injection with vehicle or yohimbine. Active lever responses during the cues reinstatement test resulted in the presentation of the light + tone stimulus complex, while active lever responses during the no cues reinstatement test resulted in no programmed consequences. Significant differences are indicated for vehicle vs. yohimbine injection (* p < 0.05) and for cues vs. no cues presentation († p < 0.05).
action were not significant. One-way simple effects ANOVAs of active lever responding during each reinstatement test revealed significant main effects for drug (F(2,40) = 5.20–6.41, p < 0.05), with post hoc analyses revealing significant increases in active lever responding after 1.25 mg/kg yohimbine during the “cues” reinstatement test, and after 2.5 mg/kg yohimbine during both reinstatement tests (p < 0.05). However, analyses within drug treatment groups failed to reveal any significant effects for reinstatement test condition (no cues or cues). For animals in the No-Cue Extinction group, a 2 × 3 ANOVA of active lever responding revealed significant main effects for reinstatement test condition (F(1,78) = 39.77, p < 0.001) and drug (F(2,78) = 9.77, p < 0.001), as well as a significant reinstatement test condition by drug interaction (F(2,78) = 3.15, p < 0.05). One-way simple effect ANOVAs of active lever responding also revealed significant main effects for drug (F(2,38) = 4.63–6.42, p < 0.05) with post hoc analyses revealing significant increases in active lever responding after 1.25 mg/kg yohimbine during the “cues” reinstatement test, and after 2.5 mg/kg yohimbine during both reinstatement tests (p < 0.05). Compared to the “no cues” reinstatement test, t test analyses following vehicle treatment revealed a significant increase in active lever presses during the “cues” reinstatement test (t(26) = 4.67, p < 0.001), an effect indicative of conditioned-cued reinstatement. Moreover, similar analyses for each yohimbine treatment group revealed a similar potentiation in active lever responding during “cues” reinstatement testing (t(24,25) = 3.40–4.33, p < 0.005).
In order to examine within session patterns of responding across the 2 h reinstatement test sessions, active lever responding was separated into 30 min bins and further analyzed (Fig. 3). In general, responding remained elevated across the entire session after yohimbine and for cues alone in the No-Cue Extinction group. A 2 × 2 × 3 × 4 ANOVA revealed significant main effects for group (F(1,27) = 19.01, p < 0.001), reinstatement test condition (F(1,27) = 25.68, p < 0.001), drug (F(2,54) = 25.06, p < 0.001), and time (F(3,81) = 3.27, p < 0.05), as well as a significant group by reinstatement test condition (F(1,27) = 25.32, p < 0.001), reinstatement test condition by drug (F(2,54) = 3.35, p < 0.05), drug by time (F(6,162) = 3.01, p < 0.01), group by reinstatement test condition by drug (F(2,54) = 3.69, p < 0.05) and group by drug by time (F(6,162) = 2.33, p < 0.05) interactions. All other interactions were not significant. 3.3. Corticosterone levels No differences between extinction condition groups were found for plasma corticosterone levels for the day before cocaine
Fig. 4. Plasma corticosterone levels (mean ± S.E.M.) during self-administration, extinction, and reinstatement. Left: plasma corticosterone levels prior to self-administration (Pre), at day 1 (S1) and day 10 (S10) of self-administration, and day 1 (E1) and day 7 (E7) of extinction. Significant differences from Pre are indicated (* p < 0.05). Right: plasma corticosterone levels for the two groups (Cue or No-Cue Extinction) following injection with vehicle or yohimbine.
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self-administration (pre-SA), the first and last day of cocaine self-administration (SA1, SA10), and extinction days (E1, E7); thus, the data were collapsed (Fig. 4). One-way ANOVA of these data revealed a significant main effect for session (F(4,116) = 5.24, p < 0.001), with post hoc analyses revealing a significant increase in corticosterone during SA1 and SA10 as compared to pre-SA baseline levels (p < 0.05). However, these levels decreased over the course of extinction training, returning to pre-SA levels by the last day of extinction (Fig. 4). A 2 × 2 × 3 ANOVA of plasma corticosterone levels during reinstatement testing only revealed a significant main effect for extinction group (F(1,79) = 10.80, p < 0.005). The main effects for reinstatement test condition and drug and all interactions were not significant. 4. Discussion Although drug-associated cues and stress have independently been shown to induce craving in humans [3,4,29] and reinstate drug-seeking in animal models of relapse [30,31], minimal evidence exists on whether an interaction between stress and drug-associated cues can lead to an enhancement in drugseeking behavior. Although no clinical studies to date have empirically tested the interaction between these factors, previous research using rats has demonstrated a potentiation in cueinduced reinstatement of ethanol seeking following footshock exposure [32,33]. Another recent study found that footshock presented in combination with drug-paired cues during extinction training facilitated reinstatement of cocaine-seeking, although neither stimulus alone produced reinstatement [34]. The current results demonstrate that while exposure to cocaine-paired stimuli or pretreatment with the anxiogenic drug yohimbine yielded similar degrees of reinstatement (3–4 times higher than extinction levels), combining these stimuli resulted in a supra-additive enhancement in the reinstatement of cocaine-seeking (10–13 times higher than extinction levels, or 3–5 times over cues or yohimbine alone). While it could be argued that this increase in responding was due to some non-specific effects (e.g. stimulatory properties of the drug), this interaction was only noted for animals in which the previously drug-paired stimuli maintained incentive motivational value (i.e. the No-Cue Extinction group). Overall, these findings indicate a potent interaction between different stimuli known to precipitate relapse to cocaine-seeking behavior. Generally, animal models of relapse have employed footshock as a means to induce drug-seeking [35–37]. We utilized the ␣2 -noradrenergic antagonist yohimbine, since it reliably reinstates drug-seeking behavior in rats and monkeys [21,22] and has certain advantages over footshock. For example, the efficacy of yohimbine to reinstate drug-seeking does not appear to be limited by procedural factors that can affect the degree of reinstatement produced by footshock [31]. Moreover, the prolonged half-life (7–8 h) of yohimbine [38] ensures a significant overlap between stress activation and exposure to conditioned cues during reinstatement. This overlap was observed in the prolonged pattern of responding during reinstatement testing following yohimbine pretreatment. While yielding similar levels
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of reinstatement, footshock produces high rates of responding early in testing, followed by a rapid drop to extinction levels after the first 30 min [35]. It has been suggested that the ability of a pharmacological agent to reinstate drug-seeking may be due to drug-induced interoceptive states and/or reinforcing effects that resemble the previously self-administered drug [39]. This issue is especially important given that both yohimbine and cocaine produce sympathetic arousal, suggesting that yohimbine may elicit a “drugprimed”, rather than a “stress-induced”, reinstatement effect. However, several lines of evidence make this an unlikely explanation. First, studies in rats and pigeons have demonstrated that yohimbine lacks drug discriminative properties with cocaine [40,41] and contrary to cocaine, yohimbine induces conditioned place aversion [42]. Moreover, other stimulant drugs (e.g. caffeine) lacking cocaine discriminative stimulus properties [43] induce robust reinstatement of cocaine-seeking [44], whereas dopamine (DA) D1 and D3 receptor agonists that partially mimic the discriminative stimulus effects of cocaine [45–47] do not necessarily result in reinstatement of extinguished cocaine-seeking [48,49]. Finally, recent evidence suggests that yohimbine may result in neuropharmacological activation that more closely resembles footshock, rather than cocaineprimed, reinstatement. Specifically, pretreatment with the NE ␣2 receptor agonist, clonidine, has been shown to attenuate both footshock-induced [50] and yohimbine-induced [21], but not cocaine-primed [50], reinstatement of cocaine-seeking. Conversely, cocaine-primed reinstatement can be attenuated by DA receptor antagonists [51], but similar treatment has a negligible effect on either footshock-stimulated [52] or yohimbinestimulated [21] reinstatement. Overall, these results suggest that discriminative stimulus properties are not the mechanism by which drugs exert their reinstatement effects and that NE, as opposed to DA, may play a more pivotal role in yohimbineinduced reinstatement of cocaine-seeking. Similar to previous reports [53–55], cocaine selfadministration increased plasma corticosterone levels in the current study. Moreover, corticosterone levels returned to baseline levels during extinction, even in animals that continued to receive presentations of the previously cocaine-paired stimuli in the absence of primary reinforcement. In contrast to enhanced corticosterone during cocaine self-administration, no discernable changes in corticosterone levels were observed when animals experienced the cues during reinstatement sessions. Although an increase in plasma corticosterone relative to extinction has been reported following exposure to conditioned stimuli [54], these levels were comparable to animals in the No-Cue Extinction group (i.e. 100–200 ng/ml or ≈25–40% of plasma levels relative to self-administration) and were significantly lower than levels both immediately prior to, or 15 min into, the reinstatement test session. To the degree that corticosterone levels reflect stress activation, our results imply that exposure to cues alone may not be perceived as stressful and as such, suggest that activation of the hypothalamo-pituitary-adrenal (HPA) axis may play a secondary role in conditioned-cued reinstatement of cocaine-seeking [56].
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The absence of a robust increase in plasma corticosterone levels following yohimbine pretreatment was unanticipated. Compared to pre-session baseline levels, previous research using squirrel monkeys pretreated with yohimbine noted a significant increase in cocaine-seeking behavior and salivary cortisol levels [21]. Consistent with these results, our analyses did show a modest main effect for yohimbine (F(2,79) = 2.79, p < 0.07), with mean plasma corticosterone levels of 329, 445, and 519 ng/ml following vehicle, 1.25 mg/kg yohimbine and 2.5 mg/kg yohimbine pretreatment, respectively. Moreover, these levels were comparable to plasma corticosterone previously reported for rats that received similar doses [57]. While the lack of a robust yohimbine effect was unexpected, the overall higher baseline levels (e.g. pre-self-administration, last day of extinction, vehicle treatment) that could have resulted from samples being taken during the ascending limb of the normal corticosterone circadian rhythm [58] or disruption of the normal corticosterone circadian rhythm and/or stress associated with restricted food-availability [59], along with variability within treatment groups likely contributed to the absence of a significant effect for yohimbine treatment. Interestingly, the No-Cue Extinction group exhibited higher levels of plasma corticosterone following yohimbine injections than animals in the Cue Extinction group. Although the reason for this discrepancy is unknown, it is also noteworthy to point out that there were no differences in corticosterone between reinstatement tests in the No-Cue Extinction group, despite far more robust drug-seeking in the presence of cues. The similar levels of corticosterone in the No-Cue Extinction group across the two test conditions demonstrates a dissociation between lever responding and activation of corticosterone release. In agreement with this dissociation, Shaham et al. [60] reported that corticotropin-releasing factor, but not corticosterone, is critical for stress-induced heroin-seeking in rats. Clinical research has also shown a lack of clear associations between subjective measures of cocaine craving and biological markers of stress (e.g. cortisol) in abstinent individuals exposed to stress or drug-associated imagery [61], although both stress and cues alone resulted in comparable increases of these markers. Our results suggest that corticosterone may not provide a clear biological measure for ascertaining drug desire and/or relapse in a reinstatement model. Alternatively, by producing a more vigilant and/or attentive-like state through elevations in NE release, it is possible that yohimbine-induced stress activation potentiates conditioned-cued reinstatement of cocaine-seeking by enhancing attentional processing of cocaine-paired stimuli, or directly increasing the incentive motivational properties of these stimuli. Although relatively selective for NE ␣2 receptors, the affinity of yohimbine for other receptors (e.g. serotonergic 5-HT1A and dopaminergic D2 receptors [62]) may have also contributed to this effect through other processes. Previous research has examined the neuroanatomical substrates that underlie reinstatement. While distinct neural substrates appear to mediate stress-induced (e.g. bed nucleus of the stria terminalis [31]) and conditioned-cued (e.g. basolateral amygdala [63]) reinstatement, clear overlap of the neural circuitry exists. For example, the prefrontal cortex to nucleus accumbens core pathway has been identified as critical for
cocaine-seeking using various reinstatement modalities [64,65]. Selective DA D1 receptor antagonism in the prefrontal cortex blocked the reinstatement of cocaine-seeking following exposure to cocaine-predictive stimuli [66], whereas infusions of DA D1 /D2 receptor antagonists into the prefrontal cortex blocked footshock-induced reinstatement of cocaine-seeking [67,68]. Moreover, footshock [69], yohimbine [70], or exposure to various other stressors [71,72] has been shown to increase prefrontal DA activity. These results suggest that simultaneous exposure to cocaine-associated cues and stress could result in a synergistic increase in prefrontal cortex DA, resulting in enhanced drug-seeking behavior. However, other pathways, including connections between the central amygdala to the ventral tegmental area [68] and the stria terminalis to the amygdala [73], likely contribute to increased drug-seeking. Given the synergistic effects of cocaine-paired stimuli and yohimbine noted in the current study, along with the fact that multiple risk factors often precede relapse [13], the current results highlight the necessity for examining the interaction between these factors when examining antecedents of relapse and possible treatments for prevention of relapse in cocaine dependent individuals [74]. Future studies will examine the interaction between other stimuli that can facilitate relapse (e.g. stress + drug prime) and employ this polymodal approach for examining the efficacy of putative pharmacotherapeutics for reducing craving and relapse in animal models and cocaine dependent individuals. Acknowledgements The authors would like to thank Jordan M. Case and Ritu H. Mehta for excellent technical assistance. This research was supported by National Institute on Drug Abuse grants DA016511 and DA015369 (RES), DA07288 (MWF), and NIH grant C06 RR015455. References [1] Dackis CA, O’Brien CP. Cocaine dependence: a disease of the brain’s reward centers. J Subst Abuse Treat 2001;21:111–7. [2] Wagner FA, Anthony JC. From first drug use to drug dependence; developmental periods of risk for dependence upon marijuana, cocaine, and alcohol. Neuropsychopharmacology 2002;26:479–88. [3] Childress AR, Hole AV, Ehrman RN, Robbins SJ, McLellan AT, O’Brien CP. Cue reactivity and cue reactivity interventions in drug dependence. NIDA Res Monogr 1993;137:73–95. [4] Sinha R, Catapano D, O’Malley S. Stress-induced craving and stress response in cocaine dependent individuals. Psychopharmacology (Berl) 1999;142:343–51. [5] See RE. Neural substrates of conditioned-cued relapse to drug-seeking behavior. Pharmacol Biochem Behav 2002;71:517–29. [6] Shaham Y, Shalev U, Lu L, De Wit H, Stewart J. The reinstatement model of drug relapse: history, methodology and major findings. Psychopharmacology (Berl) 2003;168:3–20. [7] Meil WM, See RE. Lesions of the basolateral amygdala abolish the ability of drug associated cues to reinstate responding during withdrawal from self-administered cocaine. Behav Brain Res 1997;87: 139–48. [8] Neisewander JL, Baker DA, Fuchs RA, Tran-Nguyen LT, Palmer A, Marshall JF. Fos protein expression and cocaine-seeking behavior in rats
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Research report
Potentiation of cue-induced reinstatement of cocaine-seeking in rats by the anxiogenic drug yohimbine Matthew W. Feltenstein, Ronald E. See ∗ Department of Neurosciences, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425, USA Received 24 April 2006; received in revised form 27 June 2006; accepted 29 June 2006 Available online 21 August 2006
Abstract Stress and drug-associated cues can trigger drug desire and relapse in abstinent cocaine users. Although the role of these two factors in relapse is well documented, it remains unclear as to whether an interaction between stress and drug-associated cues can lead to an enhancement in cocaine-seeking behavior. Here, we assessed the effects of the anxiogenic ␣2 -noradrenergic receptor antagonist, yohimbine, on reinstatement of cocaine-seeking in rats either in the presence or absence of cocaine-associated cues. Yohimbine pretreatment in the absence of cocaine-associated cues or cues by themselves reliably reinstated responding on the previously cocaine-paired lever (3–4 times higher than extinction levels). However, animals showed greatly potentiated responding if yohimbine preceded cue-induced reinstatement (10–13 times higher than extinction levels, or 3–5 times over cues or yohimbine alone). While cocaine self-administration produced a significant increase in plasma corticosterone, plasma corticosterone levels did not show a clear relationship to cocaine-paired lever responding during cue and/or yohimbine-induced reinstatement. These results demonstrate that exposure to drug-paired cues during a stressful state can greatly potentiate cocaine-seeking and suggest that future treatment interventions should target multiple modalities. © 2006 Elsevier B.V. All rights reserved. Keywords: Cocaine; Cues; Reinstatement; Relapse; Self-administration; Stress; Yohimbine
1. Introduction One of the most difficult problems for successful treatment of psychostimulant dependence is the high incidence of relapse to drug-seeking and drug-taking behaviors following prolonged periods of abstinence [1,2]. To date, clinical research has indicated several factors that can trigger drug desire and relapse in abstinent drug users, including negative mood states, stress, and exposure to drug-associated environmental stimuli (e.g. the context where the drug was consumed or drug paraphernalia). For example, abstinent cocaine users report craving following exposure to cocaine-associated stimuli [3], or in response to stressful life events [4]. These trigger factors have been experimentally studied using the reinstatement model of relapse in animals [5,6]. Typically, cocaine-paired stimuli or exposure to an environmental stressor following withdrawal from chronic cocaine self-administration will robustly reinstate extinguished drug-
∗
Corresponding author. Tel.: +1 843 792 2487; fax: +1 843 792 4423. E-mail address: [email protected] (R.E. See).
0166-4328/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2006.06.039
seeking as measured by increased responding on a previously cocaine-paired lever. The reinstatement model has advanced our understanding of the contribution of individual relapse factors and the neural mechanisms that underlie relapse [7–10], and provided a test for compounds that may prove beneficial for preventing relapse in cocaine dependent individuals [11,12]. Despite substantial evidence suggesting the role of drugassociated stimuli and stress in triggering relapse, very little attention has focused on the interaction of these variables in drug desire and drug-seeking. Given that multiple risk factors usually precede relapse [13], it is likely that exposure to drug-associated cues during periods of stress may increase the probability and/or intensity of drug craving and cocaine-seeking behavior. To test this hypothesis, we examined the separate and interactive effects of previously drug-paired stimuli and a stressor (the anxiogenic drug, yohimbine) on cocaine-seeking behavior in an animal model of relapse. Yohimbine is a norepinephrine (NE) ␣2 receptor antagonist that increases NE release in several neural structures implicated in stress, including the bed nucleus of the stria terminalis [14] and the amygdala [15]. Yohimbine treatment has been shown to produce anxiety-like states in both humans
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[16,17] and laboratory animals [18,19]. Furthermore, yohimbine induces drug craving in abstinent drug-dependent subjects [20] and reinstates cocaine [21], methamphetamine [22], and alcohol [23] seeking in animal models of relapse. In order to obtain a physiological measure of stress activation, we also assessed plasma corticosterone levels during cocaine self-administration, extinction, and reinstatement testing. 2. Methods 2.1. Subjects Male, Sprague–Dawley rats (initial weight 275–300 g; Charles River, Wilmington, MA, USA) were individually housed in a temperature- and humiditycontrolled vivarium on a normal 12 h light–dark cycle (lights on 6 a.m.–6 p.m.). All experimental procedures (drug self-administration, extinction, and reinstatement testing) occurred between 11 a.m. and 5 p.m. Animals received water ad libitum and were maintained on 20–25 g of standard rat chow (Harlan, Indianapolis, IN, USA) per day for the duration of each experiment. Rats were acclimated to handling and allowed to adapt for a minimum of 3 days prior to the start of the experiment. Housing and care of the rats were carried out in accordance with the “Guide for the Care and Use of Laboratory Rats” (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 1996).
2.2. Lever response training Rats were trained to lever press in standard operant conditioning chambers (30 cm × 20 cm × 20 cm) linked to a computerized data collection program (MED-PC, Med Associates, Inc., St. Albans, VT, USA). The chambers were equipped with two retractable levers, a stimulus light above each lever, a food pellet dispenser between the levers, a speaker linked to a programmable tone generator (ANL-926, Med Associates), and a house light on the wall opposite the levers. Each chamber was contained within a sound-attenuating cubicle equipped with a ventilation fan. Forty-eight hours prior to surgery, rats were food deprived overnight and trained to lever press on a fixed ratio (FR) 1 schedule of food reinforcement (45 mg pellets; Noyes, Lancaster, NH, USA) during a 15 h overnight training session in the absence of explicit conditioned stimulus (CS) presentation (i.e. active lever presses resulted in the delivery of a food pellet only). Lever presses on an inactive lever were recorded, but had no programmed consequence. Following lever response training, food dispensers were permanently removed from the test chambers.
2.3. Surgery Rats were anesthetized using a mixture of ketamine hydrochloride and xylazine (66 and 1.33 mg/kg, respectively, IP) followed by equithesin (0.5 ml/kg with a solution of 9.72 mg/ml pentobarbital sodium, 42.5 mg/ml chloral hydrate, and 21.3 mg/ml magnesium sulfate heptahydrate dissolved in a 44% propylene glycol, 10% ethanol solution, IP). Surgical procedures were conducted using aseptic techniques. Catheters were constructed using previously described methods [24] and consisted of external guide cannulae with screw-type connectors (Plastics One Inc., Roanoke, VA, USA), Silastic tubing (10 cm; i.d. = 0.64 mm; o.d. = 1.19 mm; Dow Corning, Midland, MI, USA), prolite polypropylene monofilament mesh (2 cm diameter, Atrium Medical Corporation, Hudson, NH, USA), and cranioplastic cement. The end of the catheter was inserted into the right jugular vein and secured to surrounding tissue with suture. The catheter ran subcutaneously and exited on the rat’s back, posterior to the shoulder blades. To maintain patency, catheters were flushed once daily for 4 days after surgery with 0.1 ml each of an antibiotic solution of cefazolin (10.0 mg/ml; Schein Pharmaceuticals, Florham Park, NJ, USA) dissolved in heparinized saline (70 U/ml; Elkins-Sinn, Cherry Hill, NJ, USA) and heparinized saline. For the duration of the experiment, each subject then received 0.1 ml of heparinized saline (10 U/ml) immediately prior to self-administration sessions and the cefazolin and 70 U/ml heparinized saline regimen following each session. To verify catheter patency, rats occasionally received a 0.12 ml infusion of methohexital
sodium (10.0 mg/ml IV; Eli Lilly and Co., Indianapolis, IN, USA), a short-acting barbiturate that produces a rapid loss of muscle tone when administered intravenously.
2.4. Cocaine self-administration Rats (n = 29) were trained to self-administer cocaine (cocaine hydrochloride dissolved in 0.9% sterile saline; cocaine provided by the National Institute on Drug Abuse, Research Triangle Park, NC, USA) during daily 2 h sessions according to an FR 1 schedule of reinforcement. At the start of each session, the catheter was connected to a swivel (Instech, Plymouth Meeting, PA, USA) via polyethylene 20 tubing that was encased in steel spring leashes (Plastics One Inc., Roanoke, VA, USA). The house light signaled the initiation of the session and remained illuminated throughout the entire session. Lever presses on the active lever resulted in a 2 s activation of the infusion pump (0.5 mg/kg cocaine per 0.05 ml infusion) and a 5 s presentation of a stimulus complex, consisting of activation of the white stimulus light above the active lever and the tone generator (78 dB, 2 kHz). After each infusion, responses on the active lever had no consequences during a 20 s time-out period. Responses on the inactive lever were recorded throughout each session of the study, but had no programmed consequences. Daily cocaine self-administration continued until each rat had obtained the self-administration criterion of 10 sessions with at least 10 infusions per session.
2.5. Extinction and reinstatement of cocaine-seeking Following chronic self-administration and before the first reinstatement test, rats underwent daily 2 h extinction sessions. To further insure that any putative interaction with yohimbine pretreatment could be attributed to incentive motivational value (rather than non-specific factors) of these stimuli, half the animals were extinguished to the previously cocaine-paired cues. During each session, responses on the active lever resulted in either a 5 s CS presentation in the absence of cocaine reinforcement, followed by a 20 s time-out period in which responses did not result in further CS presentations (i.e. Cue Extinction group, n = 15), or no programmed consequences (i.e. neither cocaine reinforcement nor conditioned stimuli were presented; No-Cue Extinction group, n = 14). Once extinction criterion was reached (a minimum of seven extinction sessions with ≤25 active lever responses per session for the last two consecutive days), each rat underwent six separate tests to examine the effects of yohimbine treatment and cue presentation on reinstatement of responding. We have previously found no decrement in responding when the number of tests is limited to a total of three for a given reinstatement condition [25–27]. Thirty minutes prior to a reinstatement test, each rat received an IP injection of sterile distilled water (vehicle) or yohimbine hydrochloride (1.25 or 2.5 mg/kg; Sigma–Aldrich, St. Louis, MO, USA). The doses of yohimbine were selected based on previous studies of yohimbine effects in rats [22,23,28] and were given in a counterbalanced order according to cocaine intake. For some animals in the Cue (n = 7) and No-Cue (n = 8) Extinction groups, responses on the active lever for the first three reinstatement tests resulted in 5 s CS presentations in the absence of cocaine reinforcement (i.e. yohimbine + cues), while active lever responding for the last three reinstatement tests had no programmed consequences (i.e. yohimbine only). For the remaining animals in each group (n = 8 and 6, respectively for the Cue and No-Cue Extinction groups), the situation was reversed. The order of yohimbine treatment for each animal was the same for both sets of reinstatement tests and animals underwent extinction sessions between reinstatement tests until they reached criterion (i.e. ≤25 active lever responses per session for two consecutive days).
2.6. Corticosterone assay In order to assess any alterations in plasma corticosterone levels due to experimental procedures, 100 l of whole blood was collected through the catheter using 1000 U heparin at the following time points: the day before (pre-SA) and immediately following the first (SA1) and last (SA10) day of cocaine self-administration, immediately following the first (E1) and last (E7) day of extinction, and immediately following all reinstatement tests. Plasma was isolated by centrifugation (10,000 rpm at 4 ◦ C for 20 min) and stored at −80 ◦ C
M.W. Feltenstein, R.E. See / Behavioural Brain Research 174 (2006) 1–8 until assayed. Plasma corticosterone concentrations (ng/ml) were determined using radioimmunoassay (Diagnostic Systems Laboratories, DSL-81100, Webster, TX, USA) according to the manufacturer’s directions.
2.7. Data analysis t-Tests and mixed factors analysis of variance (ANOVA) were used to analyze active and inactive lever responses, and drug infusions, with session (self-administration day and extinction day) and extinction group as factors, where appropriate. One-way ANOVAs were used to analyze active and inactive lever responses and plasma corticosterone levels during reinstatement testing, with drug and reinstatement test session as factors, where appropriate. Interaction effects were further investigated using simple main effects tests (one-way ANOVA) with post hoc analyses conducted using Student–Newman–Keuls with the alpha set at 0.05. Data are expressed as the mean ± S.E.M.
3. Results 3.1. Cocaine self-administration and extinction Rats in both extinction groups readily acquired cocaine self-administration and displayed stable active lever responding and cocaine intake during the maintenance phase (Fig. 1). Accordingly, 2 × 3 ANOVAs of active and inactive lever responding for the last three self-administration sessions revealed no significant effects for group, session, or interaction between group and session. Cocaine intake (Cue Extinction group = 19.43 ± 0.96 mg/kg/session; No-Cue Extinction group = 19.54 ± 0.92 mg/kg/session) and number of sessions required to achieve the self-administration criterion (Cue Extinction group = 10.60 ± 0.31; No-Cue Extinction group = 10.79 ± 0.32) also did not differ between groups. Thus, animals responded more on the active lever than on the inactive lever, regardless of session, and the two groups did not differ in cocaine self-administration history. Following the removal of cocaine reinforcement, active lever responding decreased steadily over the course of daily extinction sessions for both groups (Fig. 1). Although animals in
Fig. 1. Lever responding (mean ± S.E.M.) during the last 3 days of cocaine self-administration (SA) on the active and inactive levers and for 7 days of extinction (large symbols = active lever; small symbols = inactive lever). All animals received cues throughout cocaine SA, but only half received cues during extinction (Cue extinction; n = 15) and the other half did not (No-Cue extinction; n = 14).
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the Cue Extinction group exhibited more responding on the active lever, there was no difference between the two groups in the number of sessions required to achieve the extinction criterion. A 2 × 7 ANOVA of active lever responding during the first seven extinction sessions revealed a significant main effect for group (F(1,27) = 7.88, p < 0.01) and session (F(6,162) = 20.59, p < 0.001). However, the group by session interaction was not significant. Similar analyses for inactive lever responding only revealed a significant main effect for session (F(6,162) = 5.32, p < 0.001). The group main effect and group by session interaction effect were not significant. Finally, there were no significant differences between the two extinction groups in the number of sessions required to reach the extinction criterion of a minimum of seven sessions with ≤25 active lever responses per session for the last two consecutive days (Cue Extinction group = 8.00 ± 0.66; No-Cue Extinction group = 7.21 ± 0.11). Thus, although Cue Extinction animals exhibited higher cocaine-paired lever responding during early extinction, both groups extinguished to similarly low levels prior to reinstatement testing. 3.2. Reinstatement tests Prior to further analyses, the data for both extinction groups were examined for possible order effects of reinstatement testing or drug treatment. Analysis failed to reveal any significant differences in the number of active lever presses during each reinstatement test based on the order of reinstatement testing (yohimbine alone, yohimbine + cues) or the order of drug treatment (vehicle, 1.25 and 2.5 mg/kg). As such, the data were collapsed within extinction group and across test day order. For animals in the Cue Extinction group, yohimbine increased reinstatement responding 3–4 times higher than vehicle treatment under both the “no cues” and “cues” reinstatement conditions (Fig. 2). Similar increases in responding were seen after yohimbine treatment during the “no cues” reinstatement condition and for the “cues” presentation alone for animals in the No-Cue Extinction group. However, yohimbine treatment in combination with the cues produced a robust potentiative effect, with responding rates 10–13 times higher than extinction levels, or 3–5 times over cues or yohimbine alone (Fig. 2). A 2 × 2 × 3 ANOVA of active lever responding revealed significant main effects for group (F(1,27) = 18.96, p < 0.001), reinstatement test condition (F(1,27) = 25.66, p < 0.001), and drug (F(2,54) = 25.08, p < 0.001), as well as significant group by reinstatement test condition (F(1,27) = 25.30, p < 0.001), reinstatement test condition by drug (F(2,54) = 3.34, p < 0.05) and group by reinstatement test condition by drug (F(2,54) = 3.68, p < 0.05) interactions. The group by drug interaction was not significant. Similar analyses of inactive lever responding only revealed a significant main effect for drug (F(2,54) = 17.79, p < 0.001), but no significant effect for group and reinstatement test condition main effects or any interactions. For animals in the Cue Extinction group, a 2 × 3 ANOVA of active lever responding revealed a significant main effect for drug (F(2,84) = 12.20, p < 0.001). The reinstatement test condition main effect and reinstatement test condition by drug inter-
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Fig. 3. Response patterns over time during reinstatement tests. Active lever responding (mean ± S.E.M.) is shown in 30 min bins for the two groups (Cue or No-Cue Extinction) following injection with vehicle (), 1.25 mg/kg yohimbine (), or 2.5 mg/kg yohimbine ().
Fig. 2. Potentiation of conditioned-cued reinstatement by yohimbine. Active and inactive (bottom) lever responding (mean ± S.E.M.) are shown for the two groups (Cue or No-Cue Extinction) following injection with vehicle or yohimbine. Active lever responses during the cues reinstatement test resulted in the presentation of the light + tone stimulus complex, while active lever responses during the no cues reinstatement test resulted in no programmed consequences. Significant differences are indicated for vehicle vs. yohimbine injection (* p < 0.05) and for cues vs. no cues presentation († p < 0.05).
action were not significant. One-way simple effects ANOVAs of active lever responding during each reinstatement test revealed significant main effects for drug (F(2,40) = 5.20–6.41, p < 0.05), with post hoc analyses revealing significant increases in active lever responding after 1.25 mg/kg yohimbine during the “cues” reinstatement test, and after 2.5 mg/kg yohimbine during both reinstatement tests (p < 0.05). However, analyses within drug treatment groups failed to reveal any significant effects for reinstatement test condition (no cues or cues). For animals in the No-Cue Extinction group, a 2 × 3 ANOVA of active lever responding revealed significant main effects for reinstatement test condition (F(1,78) = 39.77, p < 0.001) and drug (F(2,78) = 9.77, p < 0.001), as well as a significant reinstatement test condition by drug interaction (F(2,78) = 3.15, p < 0.05). One-way simple effect ANOVAs of active lever responding also revealed significant main effects for drug (F(2,38) = 4.63–6.42, p < 0.05) with post hoc analyses revealing significant increases in active lever responding after 1.25 mg/kg yohimbine during the “cues” reinstatement test, and after 2.5 mg/kg yohimbine during both reinstatement tests (p < 0.05). Compared to the “no cues” reinstatement test, t test analyses following vehicle treatment revealed a significant increase in active lever presses during the “cues” reinstatement test (t(26) = 4.67, p < 0.001), an effect indicative of conditioned-cued reinstatement. Moreover, similar analyses for each yohimbine treatment group revealed a similar potentiation in active lever responding during “cues” reinstatement testing (t(24,25) = 3.40–4.33, p < 0.005).
In order to examine within session patterns of responding across the 2 h reinstatement test sessions, active lever responding was separated into 30 min bins and further analyzed (Fig. 3). In general, responding remained elevated across the entire session after yohimbine and for cues alone in the No-Cue Extinction group. A 2 × 2 × 3 × 4 ANOVA revealed significant main effects for group (F(1,27) = 19.01, p < 0.001), reinstatement test condition (F(1,27) = 25.68, p < 0.001), drug (F(2,54) = 25.06, p < 0.001), and time (F(3,81) = 3.27, p < 0.05), as well as a significant group by reinstatement test condition (F(1,27) = 25.32, p < 0.001), reinstatement test condition by drug (F(2,54) = 3.35, p < 0.05), drug by time (F(6,162) = 3.01, p < 0.01), group by reinstatement test condition by drug (F(2,54) = 3.69, p < 0.05) and group by drug by time (F(6,162) = 2.33, p < 0.05) interactions. All other interactions were not significant. 3.3. Corticosterone levels No differences between extinction condition groups were found for plasma corticosterone levels for the day before cocaine
Fig. 4. Plasma corticosterone levels (mean ± S.E.M.) during self-administration, extinction, and reinstatement. Left: plasma corticosterone levels prior to self-administration (Pre), at day 1 (S1) and day 10 (S10) of self-administration, and day 1 (E1) and day 7 (E7) of extinction. Significant differences from Pre are indicated (* p < 0.05). Right: plasma corticosterone levels for the two groups (Cue or No-Cue Extinction) following injection with vehicle or yohimbine.
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self-administration (pre-SA), the first and last day of cocaine self-administration (SA1, SA10), and extinction days (E1, E7); thus, the data were collapsed (Fig. 4). One-way ANOVA of these data revealed a significant main effect for session (F(4,116) = 5.24, p < 0.001), with post hoc analyses revealing a significant increase in corticosterone during SA1 and SA10 as compared to pre-SA baseline levels (p < 0.05). However, these levels decreased over the course of extinction training, returning to pre-SA levels by the last day of extinction (Fig. 4). A 2 × 2 × 3 ANOVA of plasma corticosterone levels during reinstatement testing only revealed a significant main effect for extinction group (F(1,79) = 10.80, p < 0.005). The main effects for reinstatement test condition and drug and all interactions were not significant. 4. Discussion Although drug-associated cues and stress have independently been shown to induce craving in humans [3,4,29] and reinstate drug-seeking in animal models of relapse [30,31], minimal evidence exists on whether an interaction between stress and drug-associated cues can lead to an enhancement in drugseeking behavior. Although no clinical studies to date have empirically tested the interaction between these factors, previous research using rats has demonstrated a potentiation in cueinduced reinstatement of ethanol seeking following footshock exposure [32,33]. Another recent study found that footshock presented in combination with drug-paired cues during extinction training facilitated reinstatement of cocaine-seeking, although neither stimulus alone produced reinstatement [34]. The current results demonstrate that while exposure to cocaine-paired stimuli or pretreatment with the anxiogenic drug yohimbine yielded similar degrees of reinstatement (3–4 times higher than extinction levels), combining these stimuli resulted in a supra-additive enhancement in the reinstatement of cocaine-seeking (10–13 times higher than extinction levels, or 3–5 times over cues or yohimbine alone). While it could be argued that this increase in responding was due to some non-specific effects (e.g. stimulatory properties of the drug), this interaction was only noted for animals in which the previously drug-paired stimuli maintained incentive motivational value (i.e. the No-Cue Extinction group). Overall, these findings indicate a potent interaction between different stimuli known to precipitate relapse to cocaine-seeking behavior. Generally, animal models of relapse have employed footshock as a means to induce drug-seeking [35–37]. We utilized the ␣2 -noradrenergic antagonist yohimbine, since it reliably reinstates drug-seeking behavior in rats and monkeys [21,22] and has certain advantages over footshock. For example, the efficacy of yohimbine to reinstate drug-seeking does not appear to be limited by procedural factors that can affect the degree of reinstatement produced by footshock [31]. Moreover, the prolonged half-life (7–8 h) of yohimbine [38] ensures a significant overlap between stress activation and exposure to conditioned cues during reinstatement. This overlap was observed in the prolonged pattern of responding during reinstatement testing following yohimbine pretreatment. While yielding similar levels
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of reinstatement, footshock produces high rates of responding early in testing, followed by a rapid drop to extinction levels after the first 30 min [35]. It has been suggested that the ability of a pharmacological agent to reinstate drug-seeking may be due to drug-induced interoceptive states and/or reinforcing effects that resemble the previously self-administered drug [39]. This issue is especially important given that both yohimbine and cocaine produce sympathetic arousal, suggesting that yohimbine may elicit a “drugprimed”, rather than a “stress-induced”, reinstatement effect. However, several lines of evidence make this an unlikely explanation. First, studies in rats and pigeons have demonstrated that yohimbine lacks drug discriminative properties with cocaine [40,41] and contrary to cocaine, yohimbine induces conditioned place aversion [42]. Moreover, other stimulant drugs (e.g. caffeine) lacking cocaine discriminative stimulus properties [43] induce robust reinstatement of cocaine-seeking [44], whereas dopamine (DA) D1 and D3 receptor agonists that partially mimic the discriminative stimulus effects of cocaine [45–47] do not necessarily result in reinstatement of extinguished cocaine-seeking [48,49]. Finally, recent evidence suggests that yohimbine may result in neuropharmacological activation that more closely resembles footshock, rather than cocaineprimed, reinstatement. Specifically, pretreatment with the NE ␣2 receptor agonist, clonidine, has been shown to attenuate both footshock-induced [50] and yohimbine-induced [21], but not cocaine-primed [50], reinstatement of cocaine-seeking. Conversely, cocaine-primed reinstatement can be attenuated by DA receptor antagonists [51], but similar treatment has a negligible effect on either footshock-stimulated [52] or yohimbinestimulated [21] reinstatement. Overall, these results suggest that discriminative stimulus properties are not the mechanism by which drugs exert their reinstatement effects and that NE, as opposed to DA, may play a more pivotal role in yohimbineinduced reinstatement of cocaine-seeking. Similar to previous reports [53–55], cocaine selfadministration increased plasma corticosterone levels in the current study. Moreover, corticosterone levels returned to baseline levels during extinction, even in animals that continued to receive presentations of the previously cocaine-paired stimuli in the absence of primary reinforcement. In contrast to enhanced corticosterone during cocaine self-administration, no discernable changes in corticosterone levels were observed when animals experienced the cues during reinstatement sessions. Although an increase in plasma corticosterone relative to extinction has been reported following exposure to conditioned stimuli [54], these levels were comparable to animals in the No-Cue Extinction group (i.e. 100–200 ng/ml or ≈25–40% of plasma levels relative to self-administration) and were significantly lower than levels both immediately prior to, or 15 min into, the reinstatement test session. To the degree that corticosterone levels reflect stress activation, our results imply that exposure to cues alone may not be perceived as stressful and as such, suggest that activation of the hypothalamo-pituitary-adrenal (HPA) axis may play a secondary role in conditioned-cued reinstatement of cocaine-seeking [56].
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The absence of a robust increase in plasma corticosterone levels following yohimbine pretreatment was unanticipated. Compared to pre-session baseline levels, previous research using squirrel monkeys pretreated with yohimbine noted a significant increase in cocaine-seeking behavior and salivary cortisol levels [21]. Consistent with these results, our analyses did show a modest main effect for yohimbine (F(2,79) = 2.79, p < 0.07), with mean plasma corticosterone levels of 329, 445, and 519 ng/ml following vehicle, 1.25 mg/kg yohimbine and 2.5 mg/kg yohimbine pretreatment, respectively. Moreover, these levels were comparable to plasma corticosterone previously reported for rats that received similar doses [57]. While the lack of a robust yohimbine effect was unexpected, the overall higher baseline levels (e.g. pre-self-administration, last day of extinction, vehicle treatment) that could have resulted from samples being taken during the ascending limb of the normal corticosterone circadian rhythm [58] or disruption of the normal corticosterone circadian rhythm and/or stress associated with restricted food-availability [59], along with variability within treatment groups likely contributed to the absence of a significant effect for yohimbine treatment. Interestingly, the No-Cue Extinction group exhibited higher levels of plasma corticosterone following yohimbine injections than animals in the Cue Extinction group. Although the reason for this discrepancy is unknown, it is also noteworthy to point out that there were no differences in corticosterone between reinstatement tests in the No-Cue Extinction group, despite far more robust drug-seeking in the presence of cues. The similar levels of corticosterone in the No-Cue Extinction group across the two test conditions demonstrates a dissociation between lever responding and activation of corticosterone release. In agreement with this dissociation, Shaham et al. [60] reported that corticotropin-releasing factor, but not corticosterone, is critical for stress-induced heroin-seeking in rats. Clinical research has also shown a lack of clear associations between subjective measures of cocaine craving and biological markers of stress (e.g. cortisol) in abstinent individuals exposed to stress or drug-associated imagery [61], although both stress and cues alone resulted in comparable increases of these markers. Our results suggest that corticosterone may not provide a clear biological measure for ascertaining drug desire and/or relapse in a reinstatement model. Alternatively, by producing a more vigilant and/or attentive-like state through elevations in NE release, it is possible that yohimbine-induced stress activation potentiates conditioned-cued reinstatement of cocaine-seeking by enhancing attentional processing of cocaine-paired stimuli, or directly increasing the incentive motivational properties of these stimuli. Although relatively selective for NE ␣2 receptors, the affinity of yohimbine for other receptors (e.g. serotonergic 5-HT1A and dopaminergic D2 receptors [62]) may have also contributed to this effect through other processes. Previous research has examined the neuroanatomical substrates that underlie reinstatement. While distinct neural substrates appear to mediate stress-induced (e.g. bed nucleus of the stria terminalis [31]) and conditioned-cued (e.g. basolateral amygdala [63]) reinstatement, clear overlap of the neural circuitry exists. For example, the prefrontal cortex to nucleus accumbens core pathway has been identified as critical for
cocaine-seeking using various reinstatement modalities [64,65]. Selective DA D1 receptor antagonism in the prefrontal cortex blocked the reinstatement of cocaine-seeking following exposure to cocaine-predictive stimuli [66], whereas infusions of DA D1 /D2 receptor antagonists into the prefrontal cortex blocked footshock-induced reinstatement of cocaine-seeking [67,68]. Moreover, footshock [69], yohimbine [70], or exposure to various other stressors [71,72] has been shown to increase prefrontal DA activity. These results suggest that simultaneous exposure to cocaine-associated cues and stress could result in a synergistic increase in prefrontal cortex DA, resulting in enhanced drug-seeking behavior. However, other pathways, including connections between the central amygdala to the ventral tegmental area [68] and the stria terminalis to the amygdala [73], likely contribute to increased drug-seeking. Given the synergistic effects of cocaine-paired stimuli and yohimbine noted in the current study, along with the fact that multiple risk factors often precede relapse [13], the current results highlight the necessity for examining the interaction between these factors when examining antecedents of relapse and possible treatments for prevention of relapse in cocaine dependent individuals [74]. Future studies will examine the interaction between other stimuli that can facilitate relapse (e.g. stress + drug prime) and employ this polymodal approach for examining the efficacy of putative pharmacotherapeutics for reducing craving and relapse in animal models and cocaine dependent individuals. Acknowledgements The authors would like to thank Jordan M. Case and Ritu H. Mehta for excellent technical assistance. This research was supported by National Institute on Drug Abuse grants DA016511 and DA015369 (RES), DA07288 (MWF), and NIH grant C06 RR015455. References [1] Dackis CA, O’Brien CP. Cocaine dependence: a disease of the brain’s reward centers. J Subst Abuse Treat 2001;21:111–7. [2] Wagner FA, Anthony JC. From first drug use to drug dependence; developmental periods of risk for dependence upon marijuana, cocaine, and alcohol. Neuropsychopharmacology 2002;26:479–88. [3] Childress AR, Hole AV, Ehrman RN, Robbins SJ, McLellan AT, O’Brien CP. Cue reactivity and cue reactivity interventions in drug dependence. NIDA Res Monogr 1993;137:73–95. [4] Sinha R, Catapano D, O’Malley S. Stress-induced craving and stress response in cocaine dependent individuals. Psychopharmacology (Berl) 1999;142:343–51. [5] See RE. Neural substrates of conditioned-cued relapse to drug-seeking behavior. Pharmacol Biochem Behav 2002;71:517–29. [6] Shaham Y, Shalev U, Lu L, De Wit H, Stewart J. The reinstatement model of drug relapse: history, methodology and major findings. Psychopharmacology (Berl) 2003;168:3–20. [7] Meil WM, See RE. Lesions of the basolateral amygdala abolish the ability of drug associated cues to reinstate responding during withdrawal from self-administered cocaine. Behav Brain Res 1997;87: 139–48. [8] Neisewander JL, Baker DA, Fuchs RA, Tran-Nguyen LT, Palmer A, Marshall JF. Fos protein expression and cocaine-seeking behavior in rats
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