Treatment of cocaine withdrawal anxiety with guanfacine: relationships to cocaine intake and reinstatement of cocaine seeking in rats

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1 Psychopharmacology (2012) 223: DOI /s ORIGINAL INVESTIGATION Treatment of cocaine withdrawal anxiety with guanfacine: relationships to cocaine intake and reinstatement of cocaine seeking in rats Deanne M. Buffalari & Chelsey K. Baldwin & Ronald E. See Received: 1 November 2011 / Accepted: 26 March 2012 / Published online: 18 April 2012 # Springer-Verlag 2012 Abstract Rationale Successful treatment of cocaine addiction is severely impeded by the propensity of users to relapse. Withdrawal severity may serve as a key predictor of susceptibility to relapse. Therefore, the identification and treatment of cocaine withdrawal symptoms such as anxiety may improve addiction treatment outcome. Objectives The current study examined the role of anxietylike behavior during cocaine withdrawal and anxiolytic treatment in reinstatement of cocaine seeking in an animal model of relapse. Methods Male rats experienced daily IV cocaine selfadministration. One group of animals received the norepinephrine α-2 agonist, guanfacine, or vehicle prior to anxiety testing 48 h after the last self-administration session. In the second group of rats, relationships between cocaine intake, anxiety-like behavior after withdrawal of cocaine, and reinstatement responding were investigated. The third and fourth groups of animals received guanfacine, yohimbine (norepinephrine α-2 antagonist), or vehicle once per day for 3 days 48 h after cessation of cocaine self-administration, followed by extinction and subsequent reinstatement induced by cocaine injections, cocaine-paired cues, and yohimbine administration. Results Cocaine-withdrawn rats at 48 h demonstrated higher levels of anxiety-like behavior as measured on a D. M. Buffalari Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260, USA C. K. Baldwin : R. E. See (*) Department of Neurosciences, Medical University of South Carolina, BSB416B, 173 Ashley Avenue, Charleston, SC 29425, USA seere@musc.edu defensive burying task when compared to yoked saline controls, an effect reversed by guanfacine treatment. Cocaine intake was positively correlated with measures of anxiety-like behavior during early withdrawal, and this anxiety-like behavior was significantly correlated with subsequent cocaine-primed reinstatement. Yohimbine treatment during early withdrawal increased reinstatement to conditioned cues, while guanfacine treatment reduced reinstatement to yohimbine. Conclusions These studies suggest an important role for noradrenergic mediation of anxiety-like behavior that emerges after withdrawal of cocaine and potential risk of relapse as modeled by reinstatement, and suggest that treatment of anxiety symptoms during early abstinence may reduce the risk of relapse. Keywords Anxiety. Cocaine. Guanfacine. Norepinephrine. Reinstatement. Relapse. Withdrawal Introduction A number of factors contribute to poor treatment outcome in cocaine addiction and the cycle of temporary abstinence followed by relapse that occurs in many cocaine users. One of the variables most often associated with cocaine treatment outcome is the severity of cocaine withdrawal symptoms(ahmadietal.2009). Cocaine withdrawal is frequently characterized by anxiety, as well as other symptoms that also define generalized anxiety disorder, such as sleep disturbances, fatigue, and irritability (American Psychiatric Association 1994). Cocaine withdrawal severity has been associated with poor treatment retention (Poling et al. 2007), and patients with severe withdrawal symptoms are less likely to achieve stable abstinence (Kampman et al.

2 180 Psychopharmacology (2012) 223: ). Anxiety has also been specifically linked to high rates of attrition from abstinence early during treatment programs (Kampman et al. 2001), and self-reports of anxiety are negatively correlated with days in treatment and positively correlated with cocaine use (O'Leary et al. 2000). Despite the noted link between severe withdrawal anxiety and likelihood to relapse to drug use, few attempts have been made to directly treat cocaine withdrawal-induced anxiety. Dysfunction in brain norepinephrine (NE) systems likely contributes to cocaine withdrawal anxiety. Cocainedependent subjects are more responsive to challenge with the anxiogenic NE α-2 antagonist, yohimbine (McDougle et al. 1994). Furthermore, during early withdrawal, patients displayed increased levels of fear and nervousness, as well as increased levels of plasma 3-methyl-4-hydroxyphenylethylene glycol in response to yohimbine (McDougle et al. 1994). Limited studies on NE receptor modulation during withdrawal have yielded promising results, with the implication that NE agents may effectively treat withdrawal anxiety, with subsequent improvement in treatment retention and lower cocaine use (McDougle et al. 1994; Sofuoglu and Kosten 2006). The NE β-antagonist, propranolol, reduced withdrawal symptoms, particularly in patients with high symptom severity (Kampman et al. 2001). Furthermore, in this group, propranolol led to better retention rates in treatment, as well as less cocaine use. The dopamine betahydroxylase inhibitor, disulfiram, has also shown promise for increasing treatment retention rates and abstinence from cocaine use (Carroll et al. 2004; Preti 2007). Preclinical studies have shown that withdrawal from noncontingent cocaine administration can lead to increased anxiety-like behavior in rats. Animals displayed less open arm exploration in the elevated plus maze (Fung and Richard 1994; Rudoy and Van Bockstaele 2007; Sarnyai et al. 1995), as well as decreased latency to shock probe burying and increased overall time spent burying in the defensive burying paradigm (Basso et al. 1999; Harris and Aston-Jones 1993). Increased defensive withdrawal (Yang et al. 1992) and increased ultrasonic vocalizations in the 22- khz range, indicative of negative affect, have also been reported in cocaine-withdrawn rats (Covington and Miczek 2003). Furthermore, anxiety-like behavior that emerges after withdrawal of cocaine has been linked to changes in NE function. Chronic cocaine increased NE transporter (NET) binding in regions associated with stress and anxiety, including the bed nucleus of the stria terminalis, the paraventricular nucleus of the hypothalamus, the A1 NE cell groups, and the amygdala (Beveridge et al. 2005). Increased NET labeling in the hypothalamus was also reported in female rats 4 days after cessation of cocaine exposure (Belej et al. 1996). β-1 NE receptor expression was increased in the amygdala 48 h after withdrawal of repeated cocaine (Rudoy and Van Bockstaele 2007), and depressed levels of baseline NE metabolites have been reported after cocaine self-administration (Antkiewicz-Michaluk et al. 2006). In addition, α-2 NE receptor sensitivity was altered in animals 42 h after cessation of chronic cocaine (Baumann et al. 2004). Attempts to alleviate anxiety after cessation of cocaine exposure with NE agents have shown some success as well, in that propranolol and atenolol, a specific β-1 NE antagonist, decreased heightened anxiety behavior in the shock probe burying paradigm (Harris and Aston-Jones 1993). Betaxolol, another β-1 NE antagonist, also reduced anxiety associated with withdrawal as measured by the elevated plus maze (Rudoy and Van Bockstaele 2007). However, as these studies utilized noncontingent cocaine injections, their direct relevance to human addiction is limited. The current study examined early cocaine withdrawal anxiety using a cocaine self-administration and reinstatement model of relapse and investigated the relationship of withdrawal emergent anxiety-like behavior with subsequent reinstatement of cocaine seeking. We also administered guanfacine, a NE α-2 agonist, or yohimbine, a NE α-2 antagonist, during early withdrawal to reduce or exacerbate anxiety respectively and, potentially, alter subsequent reinstatement caused by cocaine-conditioned cues, cocaine priming injections, and yohimbine administration. Cocaine withdrawal anxiety-related behavior was measured 48 h after the last self-administration session using the elevated plus maze and defensive burying paradigms. We predicted that cocaine self-administration would lead to increased anxiety-like behavior when compared to yoked saline controls, that animals with high levels of anxiety-like behavior would demonstrate increased reinstatement, and that guanfacine treatment would reduce later cocaine seeking. Materials and methods Subjects Male, Sprague Dawley rats (n091, initial weight g; Charles River, Wilmington, MA, USA) were individually housed in a temperature- and humidity-controlled vivarium on a reverse 12-h light dark cycle (lights on 6 p.m. 6 a.m.). Animals received water and standard rat chow (Harlan, Indianapolis, IN, USA) ad libitum, with the exception of 2 3 daysoffoodrestrictionduringinitial cocaine self-administration. Procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Rats (Institute of Laboratory Animal Resources on Life Sciences, National Research Council) and approved by the IACUC of the Medical University of South Carolina.

3 Psychopharmacology (2012) 223: Surgery All rats (except those used in the vehicle control group of experiment 1, n013) underwent surgery for jugular catheter implantation. Rats were anesthetized using a mixture of ketamine hydrochloride and xylazine [66 and 1.33 mg/kg, respectively, intraperitoneally (IP)] followed by equithesin (0.5 ml/kg, IP). Surgical procedures were conducted using aseptic techniques. Catheters were constructed using previously described methods (Fuchs et al. 2004) and consisted of external guide cannulae with screw-type connectors (Plastics One Inc., Roanoke, VA, USA), Silastic tubing (10 cm; i.d mm; o.d 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, secured with suture, 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 then heparinized saline (70 U/ml). For the duration of the experiment, each subject received 0.1 ml of heparinized saline (10 U/ml) immediately prior to self-administration 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. Cocaine self-administration Rats self-administered cocaine (cocaine hydrochloride dissolved in 0.9 % sterile saline; 0.5 mg/kg/infusion, cocaine provided by the National Institute on Drug Abuse, Research Triangle Park, NC, USA) during daily 2-h sessions according to a fixed-ratio 1 schedule of reinforcement. We chose this cocaine dose based on extensive experience and prior studies in our laboratory with cocaine self-administration, as well as a previous experiment involving interactions between reinstatement to cocaine seeking and guanfacine (Buffalari and See 2009). 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). Self-administration occurred in standard operant conditioning chambers ( cm) linked to a computerized data collection program (MED-PC, Med Associates Inc., St. Albans, VT, USA). The chambers were equipped with two levers, a stimulus light above each lever, a tone generator (ENV-223HAM, Med Associates), and a house light on the back wall of the chamber. 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 infusion (0.2 mg/50 μl) 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, 4.5 khz). Following each infusion, responding on the active lever had no consequences during a 20-s time-out period. Inactive lever presses had no consequences, but were recorded. Daily cocaine selfadministration continued until each rat had obtained the self-administration criterion of 14 sessions with at least ten infusions per session. Extinction and reinstatement of cocaine seeking Rats used in experiments 2, 3, and 4 underwent extinction and reinstatement testing following chronic cocaine self-administration. These rats experienced daily 2-h extinction sessions, during which active lever presses had no programmed consequences. Once extinction criterion was reached (a minimum of seven extinction sessions with 15 active lever responses per session for the last two consecutive days before testing), each rat underwent three separate reinstatement tests. Prior studies have successfully utilized similar repeated reinstatement testing designs (Feltenstein and See 2006; Kippinetal. 2006). Counterbalanced reinstatement tests were used to measure cocaine seeking in the presence of conditioned cues (contingent presentations of the light+tone), yohimbine (2.5 mg/kg, IP; 30 min prior to testing), or cocaine (10 mg/kg, IP; immediately prior to testing). Animals were extinguished to criterion between reinstatement tests ( 15 active lever responses per session for two consecutive days). Doses for yohimbine and cocaine were based on previous reinstatement studies in rats (Feltenstein and See 2006; Fuchsetal.2004; Shepard et al. 2004). Anxiety testing Elevated plus maze Animals in experiments 1 and 2 underwent anxiety testing 48 h after withdrawal from cocaine self-administration (or yoked saline administration). The elevated plus maze test (Pellow et al. 1985) was carried out according to previously described methods (Cecchi et al. 2002a; Cecchietal. 2002b). The plus maze apparatus (Scientific Design, Pittsburgh) was constructed of black Plexiglas and contained two open arms ( cm) and two closed arms ( cm) with opaque black walls (40.64 cm tall)

4 182 Psychopharmacology (2012) 223: on both sides and the end of the closed arms, and a platform adjoining all the arms in the middle. Rats were transported to a dimly lit testing room, placed in the center of the maze, and allowed to explore freely for 5 min. Open arm entries, time spent in open arms, and percent time spent in open arms were recorded as measures of anxiety behavior, while total arm entries were used to measure general locomotor behavior. An observer who was unaware of the experimental treatment calculated measurements via videotape after testing sessions. Shock probe burying paradigm The defensive burying test was carried out according to previously described methods (Pinel and Treit 1978). The apparatus consisted of a polycarbonate cage with 5 cm of wood chip bedding material evenly spread across the bottom. On test days, an electrical shock probe was inserted through a small opening 2.5 cm above the bedding. The probe was 6 cm in length and was connected to a shock generator set to deliver 2.0 ma of current upon contact. Rats were habituated to the chamber (in the absence of the probe) during 1-h sessions for 3 days prior to testing. On the fourth day, animals were placed in the chamber which now had the shock probe inserted through the opening. Upon contact with the probe, the time to initiate burying behavior was calculated as latency. After probe contact, duration was calculated as the total time spent burying during the 15- min session. An observer who was unaware of the experimental treatment calculated measurements via videotape after testing sessions. Data analysis Mixed repeated measures ANOVA (day group) was used to measure differences in lever pressing between cocaine and saline (experiment 1), or vehicle- and yohimbine-treated (experiment 3), or vehicle- and guanfacine-treated (experiment 4) animals during selfadministration and extinction. Differences in cocaine intake were also examined with mixed repeated measures ANOVA (day group, experiments 3 and 4), and total intake was compared using paired t tests. One-way ANOVA (experiment 1, group) or paired t tests (experiment 2, treatment) were used to measure differences in open arm entries, open arm time, burying latency, and burying duration (experiment 1). Responding during reinstatement was analyzed with the Kruskal Wallis test (due to equivariance violations, experiment 2) or repeated measures ANOVAs (experiments 3 and 4). Pearson s correlation coefficients were calculated to examine relationships between average daily cocaine intake (last 5 days) and burying duration (experiments 1 and 2), and burying duration levels and reinstatement responding (experiment 2). All main effects were analyzed with planned post hoc comparisons for significance. All data are shown as the mean ± SEM, and effects were noted as significant at p<0.05. Rats that did not complete all behavioral testing were not included in data analysis. Experiment 1: measurement of anxiety-like behavior during cocaine withdrawal and acute treatment with guanfacine Rats (n 010) underwent 14 days of cocaine selfadministration as described above, while yoked saline controls (n010) were exposed to the same chambers and had access to levers, but lever presses had no programmed consequences. Each time the cocaine self-administration rat pressed the active lever, the yoked saline control received a presentation of the same light-tone cues and a saline infusion. All groups underwent 2 days of identical extinction sessions, and 2 h after the last extinction session (approximately 48 h of cocaine withdrawal), rats were tested for anxiety-like behavior on the elevated plus maze and shock probe burying test. Rats received either saline vehicle or guanfacine hydrochloride (0.5 mg/kg, IP) 30 min prior to testing, with the tests separated by 15 min. The dose of guanfacine was chosen based on previous experiments that tested guanfacine effects on reinstatement in our (Buffalari and See 2009) and other (Le et al. 2011) laboratories. Guanfacine displays good selectivity for the α-2a noradrenergic receptor (Intengan and Smyth 1997; Uhlen and Wikberg 1991). A control group of animals (n013) without prior cocaine self-administration also underwent anxiety assessment on the two measures. Experiment 2: measurement of anxiety-related behavior and reinstatement Rats (n08) were treated in a manner identical to that described for experiment 1. However, after the testing of anxiety-like behavior, rats continued for subsequent days with extinction and reinstatement testing as described above, and had no drug treatment during extinction. Experiment 3: repeated yohimbine administration during early cocaine withdrawal and subsequent reinstatement testing Animals (n032) underwent self-administration, extinction, and reinstatement testing as described above, with no anxiety testing at 48 h after withdrawal of cocaine (due to possible effects of the acute stress of exposure to the shock probe on subsequent reinstatement). In addition, animals received a single daily injection of yohimbine (2.5 mg/kg, IP) or saline vehicle on the first 3 days of cocaine withdrawal/extinction.

5 Psychopharmacology (2012) 223: Injectionsweregiven4hafterextinctionsessionssoasnot to interfere with extinction responding performance. a Experiment 4: treatment of anxiety-like behavior during cocaine withdrawal with repeated guanfacine and subsequent reinstatement testing Animals (n028) underwent self-administration, extinction, and reinstatement testing as described above, with no anxiety testing at 48 h after withdrawal of cocaine. In addition, animals received a single daily injection of guanfacine (0.5 mg/kg, IP) or saline vehicle on the first 3 days of cocaine withdrawal/extinction. Injectionsweregiven4hafterextinctionsessionssoasnotto interfere with extinction responding performance. b Results Experiment 1: anxiety-like behavior measured 48 h after withdrawal from cocaine self-administration is reduced by acute guanfacine administration Self-administration Animals in the cocaine group acquired self-administration rapidly and maintained stable levels of responding throughout the maintenance phase of the experiment. Yoked saline controls demonstrated minimal active lever responding, indicative of the specificity of the response. Two-way ANOVA revealed a significant effect of drug (cocaine vs. saline, F (1,246) , p<0.0001) and a significant effect of day on responding (F (13,246) 02.26, p<0.01), and a significant interaction (F (13, 246) 02.33, p<0.01). Active lever responding (Fig. 1a) measured on the last 2 days of self-administration reached an average of 50.9±8.5 (cocaine) and 3.9±1.3 (saline), and cocaineintakeaveraged20.9±1.8mg/kg. Anxiety testing Animals with a history of cocaine self-administration demonstrated significantly higher levels of anxiety-like behavior compared to yoked saline controls when tested on the shock probe burying test at 48 h of withdrawal. One-way ANOVA revealed a significant effect of treatment (F (2,19) 03.75, p<0.05), and post hoc analysis showed that non-treated cocaine-experienced animals displayed higher levels of probe burying behavior compared to saline controls or cocaine-experienced animals pretreated with guanfacine (Fig. 1b). However, there were no significant differences in latency to initiate burying (F (2,19) 00.71, p>0.05), or for any measures on the elevated plus maze (p>0.05, data not shown). Further analysis revealed a significant relationship between cocaine intake c Fig. 1 Anxiety-like behavior measured at 48 h after withdrawal of self-administration is increased in cocaine-experienced animals compared to yoked saline controls and reduced by acute guanfacine administration. a Lever responding during the last 2 days of selfadministration for animals in experiment 1. b Average shock probe burying duration in yoked saline controls (Saline), cocaine selfadministration animals treated with vehicle (Cocaine), and cocaine self-administration animals treated with guanfacine (Cocaine+Guanfacine). *p<0.05 for cocaine vs. saline controls and guanfacine-treated rats. c Significant correlation (*p<0.05) between shock probe burying duration and cocaine intake in vehicle-treated rats (animals from experiments 1 and 2) and burying duration in cocaine-exposed animals as well. Cocaine intake measured on the last 2 days of selfadministration was significantly correlated with burying duration (Fig. 1c, r , p<0.05). Burying duration was not correlated with any measure of cocaine intake in animals treated with guanfacine before anxiety testing (ps>0.05). Guanfacine administration also reduced burying duration in

6 184 Psychopharmacology (2012) 223: control rats not exposed to cocaine. Paired t tests revealed a significant reduction in burying duration on the shock probe burying paradigm, indicative of decreased anxiety-like behavior, compared to animals treated with vehicle (p<0.05, data not shown). Experiment 2: anxiety-like behavior measured 48 h after withdrawal from cocaine self-administration is related to subsequent reinstatement of cocaine seeking Self-administration All animals acquired cocaine self-administration rapidly and maintained stable levels of responding throughout the maintenance phase of the experiment. For the last 2 days of selfadministration, active lever responses reached a mean of 52.7±8.9 (Fig. 2a), and daily cocaine intake was 18.6± 2.2 mg/kg. As in experiment 1, there was a significant correlation of burying duration with cocaine intake (r , p<0.05). Extinction One-way repeated measures ANOVA revealed a significant effect of day on active lever pressing (F (6,55) 05.85, p< ). Post hoc analyses demonstrated that responding on day 1 of extinction differed from all other days of extinction (Fig. 2b, p<0.05). Animals experienced an average of 7.9 days of extinction training before reaching criterion (<15 active lever responses over 2 days) and undergoing their first reinstatement test. Inactive lever responding showed uniformly very low levels across all conditions, and no significant differences were found between treatment conditions during extinction or reinstatement trials. Reinstatement and anxiety testing Anxiety-like behavior measured after 48 h of cocaine withdrawal was significantly related to subsequent reinstatement responding. As this reinstatement data set failed the test for equivariance, a nonparametric Kruskal Wallis test revealed a significant main effect of testing condition on active lever responding (Kruskal Wallis statistic012.62, p<0.005). Post hoc Dunn s tests showed that exposure to cocaine-paired cues and cocaine priming injections significantly reinstated active lever responding above extinction levels (Fig. 2c, p< 0.05). Yohimbine did not significantly increase cocaine seeking above extinction, although this effect was nearly significant (p00.08). Burying duration of the probe after shock exposure, which was measured at 48 h of cocaine withdrawal, was positively correlated with subsequent cocaine-induced reinstatement (Fig. 2d, r , p<0.05). Fig. 2 Anxiety-like behavior measured 48 h after the last cocaine self-administration session is related to cocaine intake and subsequent cocaineinduced reinstatement. Lever responding during a the last 2 days of self-administration, b extinction, and c before (Ext) and during reinstatement testing to conditioned cues (Cue), cocaine priming injections (Coc), and yohimbine injections (Yoh). *p<0.05 for responding above extinction levels. d Significant correlation (*p<0.05) between shock probe burying duration and cocaine-induced reinstatement a c b d

7 Psychopharmacology (2012) 223: Experiment 3: repeated yohimbine administration during early withdrawal from cocaine self-administration increases subsequent conditioned cue-induced reinstatement a Self-administration All animals acquired cocaine self-administration rapidly and maintained stable levels of responding throughout the maintenance phase of the experiment. Active lever responses reached a mean of 55.8±3.2 for vehicle animals and 52.5± 5.4 for yohimbine animals on the last 2 days of selfadministration (Fig. 3a), and intake on days averaged 20.6±3.8 mg/kg for vehicle animals and 19.8±2.7 for yohimbine animals. Two-way repeated measures ANOVA revealed a significant effect of day (F (13,390) 03.89, p< 0.05) on active lever presses during self-administration, but no significant effect of treatment (F (1,30) 00.25, p> 0.05) and no significant interaction between day and treatment (F (13,390) p>0.05). Paired t tests revealed no significant differences in total cocaine intake between yohimbine and saline-treated rats (p>0.05) or for average intake over the last 2 days of self-administration (p>0.05). Inactive lever responding declined after day 1 and remained uniformly low throughout the duration of self-administration, extinction, and reinstatement (data not shown). b c Extinction Two-way repeated measures ANOVA revealed a significant effect of day (F (6,180) , p<0.05) on active lever responding during extinction, but no significant effect of treatment (F (1,30) 02.26, p>0.05) and no significant interaction (F (6,180) 01.96, p>0.05). Responding on extinction day 1 was significantly different from responding on all other days (p<0.05), while responding on day 2 was significantly different from days 5, 6, and 7 (p<0.05) for both groups (Fig. 3b). Paired t tests revealed no significant group differences in active lever presses on the last day of extinction before testing (p>0.05) or total days to achieve criterion (p>0.05). Active lever responding reached extinction criterion after 9.8±2.3 (vehicle) or 10.0±3.9 (yohimbine) sessions. Reinstatement Reinstatement data are shown in Fig. 3c. Two-way ANOVA revealed a significant effect of test (F (3,109) , p<0.001), as well as a significant test treatment interaction (F (3,109) , p<0.001), but no significant main effect of drug treatment (F (1,109) 00.14, p>0.05). Post hoc analyses revealed significant reinstatement for conditioned cue-induced, yohimbine-induced, and cocaine priming-induced responding in both groups (Fig. 3c, Fig. 3 Repeated yohimbine injections given h after the last cocaine self-administration session increase subsequent cue-induced reinstatement. Lever responding during a the last 2 days of selfadministration, b extinction, and c active (upper) and inactive (lower) lever responding before (Ext) and during reinstatement to conditioned cues (Cue), cocaine priming injections (Coc), and yohimbine injections (Yoh) for vehicle and yohimbine-treated groups. *p<0.05 for responding above extinction levels; p<0.05 relative to the vehicle group ps< ). Of particular note, only conditioned cueinduced reinstatement showed a between-group difference, with the yohimbine pretreatment group showing significantly greater reinstatement at more than twice the responding of the vehicle pretreatment group (Fig. 3c, p<0.001). Experiment 4: repeated guanfacine administration during early withdrawal from cocaine self-administration attenuates subsequent yohimbine-induced reinstatement

8 186 Psychopharmacology (2012) 223: Self-administration All animals rapidly acquired cocaine self-administration and maintained stable levels of responding throughout the maintenance phase. Active lever responses reached a mean of 38.0±3.2 for vehicle animals and 40.9±5.8 for guanfacine animals on the last 2 days of self-administration (Fig. 4a), and intake on days averaged 21.8±1.8 mg/kg for vehicle animals and 22.8±3.4 for guanfacine animals. A two-way ANOVA revealed a significant effect of day on active lever pressing during self-administration (F (13,362) 0 6.9, p<0.0001), but no significant effect of group (F (1,362) , p>0.05) and no significant interaction (F (13,362) 00.8, p>0.05). Paired t tests revealed no significant differences in intake on the last 2 days of self-administration (p>0.05) or total intake across all 14 days (p>0.05). a b Extinction Two-way repeated measures ANOVA revealed a significant effect of day (F (6,150) , p<0.0001) on active lever responding during extinction, but no significant effect of group (F (1,25) 01.15, p>0.05), and no interaction (F (6,150) 01.13, p>0.05). Post hoc analyses showed that extinction day 1 significantly differed from all other extinction days (ps<0.05), while days 2 and 3 were significantly different from day 7 (Fig. 4b, p<0.05). Active lever responding reached the established criterion (<15 active lever responses over 2 days) in a mean of 9.8±1.4 (vehicle) or 10.3±1.8 (guanfacine) days before subsequent reinstatement testing (no significant differences, paired t tests, p>0.05). Mean lever presses before the first reinstatement test also did not differ between groups. Inactive lever responding showed uniformly very low levels throughout the study, and no significant differences were found between treatment conditions during reinstatement trials (data not shown). Reinstatement A two-way ANOVA revealed a significant main effect of testing condition on active lever responding (F (3,98) , p 0.001), but no significant effect of drug treatment (F (1,98) 00.28, p>0.05) and a significant interaction (F (3,98) 02.73, p<0.05). Post hoc analyses revealed that exposure to cocaine-paired cues and cocaine significantly reinstated active lever responding above extinction baseline (Fig. 4c, p<0.05). Post hoc tests also revealed that active lever responding in response to yohimbine in guanfacine-treated animals was significantly less than yohimbine-induced reinstatement in control animals (p<0.01). c Fig. 4 Repeated guanfacine injections given h after the last cocaine self-administration session decrease subsequent yohimbineinduced reinstatement. Lever responding during a the last 2 days of self-administration, b extinction, and c active (upper) and inactive (lower) lever responding before (Ext) and during reinstatement to conditioned cues (Cue), cocaine priming injections (Coc), and yohimbine injections (Yoh) for vehicle and guanfacine-treated groups. *p< 0.05 for responding above extinction levels; p<0.05 relative to the vehicle group Discussion The current results confirm the emergence of anxiety-like behavior in rats after withdrawal from cocaine selfadministration. Furthermore, these results suggest that cocaine intake is related to the magnitude of anxiety-like

9 Psychopharmacology (2012) 223: behavior, and that anxiety-like behavior is linked to subsequent reinstatement of cocaine seeking in cocainewithdrawn rats. Finally, noradrenergic agents such as guanfacine may be a potential pharmacotherapy for reduction of anxiety that emerges after cocaine use and risk for relapse to drug use. Noradrenergic signaling has a well-documented role in preclinical models of reinstatement of cocaine seeking. Disulfuram reduced cocaine-induced reinstatement of cocaine seeking via inhibition of dopamine beta-hydroxylase (Schroeder et al. 2010), and α-2 agonists such as lofexidine, clonidine, and guanfacine reduced stress (Highfield et al. 2001), cue-induced (Smith and Aston-Jones 2011), and yohimbine-induced (Buffalari and See 2009) reinstatement of cocaine seeking, respectively. Clonidine also reduced cocaine-primed reinstatement in monkeys (Platt et al. 2007). Prazosin, an α-1 receptor antagonist, blocked cocaine-induced reinstatement (Zhang and Kosten 2005), and when administered in combination with propranolol, attenuated cue-induced reinstatement as well (Smith and Aston-Jones 2011). Further, we demonstrated that anxiety reduction through administration of guanfacine, an α-2 noradrenergic agonist, given during early withdrawal decreased subsequent reinstatement. However, this effect was specific to yohimbine-induced reinstatement as we did not see effects on other forms of reinstatement. The effects of NE drugs on cue-induced reinstatement are unclear as atomoxetine, a NE reuptake inhibitor, prevented cue-induced cocaine seeking (Economidou et al. 2011). The results of the atomoxetine are difficult to compare with the above data as the rats in their study did not undergo extinction training, which may play an important role in the neural modulation of drug-seeking behavior. In the current study, our repeated guanfacine treatment may have been either too limited in duration or too temporally removed from reinstatement testing to affect other forms of reinstatement. Future studies mayassessamoreaggressivetreatmentregimenforits effects on cue-induced reinstatement, based on the potentiated cue-induced reinstatement seen in the yohimbinetreated group. Furthermore, repeated guanfacine exposure may have led to selective downregulation of α-2 receptors and, subsequently, reduced sensitivity to yohimbine. Future studies could examine whether a more extended guanfacine treatment regimen will attenuate other forms of reinstatement. Clinical studies have demonstrated that treatment of anxiety during withdrawal may be beneficial to some users (Kampman et al. 2006), particularly a subpopulation of users with higher levels of cocaine intake. We found that pharmacologically increasing anxiety through yohimbine administration increased subsequent reinstatement of cocaine seeking elicited by conditioned cues. Yohimbine has previously been shown to enhance cueinduced drug seeking when given shortly before reinstatement testing (Banna et al. 2010; Feltenstein and See 2006); however, this is the first study to demonstrate enhanced reinstatement when yohimbine is given several days prior to the reinstatement test. Cold swim stress also has prolonged effects on reinstatement, causing potentiated cocaine-seeking behavior that lasted 3 days beyond the stress (Conrad et al. 2010). The current data suggest that stress exposure or anxiety induced during abstinence may contribute to subsequent relapse. However, the animals exposed to anxiety testing during extinction (experiment 2) did not demonstrate significant yohimbineinduced reinstatement, further complicating interpretation as to whether stress exposure during withdrawal may alter subsequent reinstatement to stressors. The nature of these interactions may be stressor specific. We also saw a great deal of variability between experiments, particularly in yohimbineinduced reinstatement. We have seen this before, and it is not surprising given the variable nature of the response to stress. We confirmed there were no statistical differences in reinstatement response to yohimbine across experiments. Previous studies have demonstrated a reduction in anxiety-like behavior measured after cocaine exposure in rats using anxiolytic drugs (Aujla et al. 2008; Basso et al. 1999), some of which target the noradrenergic system (Ambrose-Lanci et al. 2010; Basso et al. 1999; Rudoy and Van Bockstaele 2007). We found that guanfacine administration during early stages of withdrawal from cocaine abolished group differences in the shock probe burying task. In order to determine the specificity of this anxiolytic effect to the cocaine-exposed animals, we also tested and found that guanfacine effectively reduced anxiety-like behavior in noncocaine-exposed controls. These data indicate that noradrenergic agents that reduce anxiety in normal controls may also be useful in reducing withdrawal-specific anxiety. Guanfacine targets the NE α-2a receptor and likely decreases NE transmission due to agonist effects on autoreceptors; however, postsynaptic actions cannot be ruled out. Our results are consistent with previous data suggesting an important role for NE after cocaine withdrawal (Erb 2010) and reinstatement (Smith and Aston-Jones 2011, 2008; Platt et al. 2007) and indicate that increased NE tone may play a role in anxiety-like behavior that emerges after exposure to cocaine. Noradrenergic transmission within the bed nucleus of the stria terminalis has been cited as critical for negative affect during withdrawal and reinstatement (Aston-Jones and Harris 2004; Smith and Aston-Jones 2008). The bed nucleus of the stria terminalis, which plays a role in stress- (Buffalari and See 2011; Leri et al. 2002) and cue-induced (Buffalari and See 2011) reinstatement, is thus a likely potential site of action for the yohimbine and guanfacine effects in the current study. While our animals showed enhanced anxiety-like behavior when tested on the shock probe burying task, we failed to detect any differences on the elevated plus maze. Previous

10 188 Psychopharmacology (2012) 223: studies have demonstrated effects of cocaine exposure on anxiety measured in the elevated plus maze (Fung and Richard 1994; Rudoy and Van Bockstaele 2007; Sarnyai et al. 1995); however, these studies utilized noncontingent cocaine exposure. Other studies have also reported difficulty detecting baseline anxiety differences using elevated plus maze, even specific to drug-related withdrawal anxiety, at short withdrawal periods (Aujla et al. 2008; Basso et al. 1999; Mantsch et al. 2008). Some evidence suggests that differences in anxiety not seen under baseline testing conditions may be revealed through elevated plus maze testing in response to an acute stressor (Lapiz-Bluhm et al. 2008). Our current approach used the elevated plus maze to obtain a measure of baseline anxiety-like behavior and the shock probe paradigm as an indicator of acute stress-evoked anxiety-like behavior. The results suggest that anxiety-like behavior seen after cocaine self-administration may be more dramatically expressed in response to a stressor than at baseline in paradigms using cocaine self-administration. Animals display heightened stress responsivity after exposure to a long-access cocaine self-administration paradigm (6 h daily exposure) when compared to animals given standard short access (2 h daily exposure) (Aujla et al. 2008; Mantsch et al. 2008). This may seem in conflict with our data as our self-administration sessions were more similar to the short access conditions described in these reports. However, cocaine intake in the current study was up to two times higher than the levels achieved by the rats in their short access condition. We demonstrated that animals with higher levels of intake display higher shock probe burying durations, which is consistent with previous reports of increases in anxiety-like behavior (Aujla et al. 2008; Mantsch et al. 2008). The specificity of the effect to direct stress exposure, without effects on baseline anxiety, is also consistent. These data are particularly relevant to the clinical situation, in that consideration of severity of cocaine use may be relevant to the emergence of withdrawal symptoms in patients, with a potential dissociation between baseline and stress-evoked anxiety. Such items may be informative of appropriate treatment considerations. Indeed, in patient populations, heavier psychostimulant users may be more prone to severe symptoms during withdrawal, and be potentially more susceptible to relapse (Sinha 2001). The majority of previous studies have used noncontingent cocaine exposure to assess the emergence of anxietylike in rats after cessation of cocaine administration (Aujla et al. 2008; Basso et al. 1999; DeVries et al. 1998; Sarnyai et al. 1995). The self-administration paradigm allows for the ability to examine relationships between cocaine intake and subsequent anxiety-like behavior, as well as the examination of relationship between anxiety-like behavior and reinstatement to cocaine seeking. While recent studies have suggested a link between anxiety during withdrawal and subsequent reinstatement of cocaine seeking (Erb 2010), this has heretofore been untested. Our results showed that rats with higher levels of anxiety-like behavior reinstated more after cocaine priming injections. This result is consistent with clinical data demonstrating that chronic cocaine users with severe withdrawal symptoms reported greater cocaine use and enhanced subjective experience to a single dose of cocaine during withdrawal (Sofuoglu et al. 2003). The limited clinical attempts to explore the use of noradrenergic agents for treatment of psychostimulant addiction have been modestly successful. Atomoxetine effectively reduced the high and good drug effects of dextroamphetamine and cocaine in humans (Sofuoglu et al. 2008; Stoops et al. 2008), and reboxetine, which also blocks NE reuptake, reduced the high and sympathetic effects of 3,4- methylenedioxymethamphetamine (Newton 2011). Clonidine also blocked stress-induced drug craving in cocaine users (Jobes et al. 2011). One important consideration is that noradrenergic agents may be more effective in individuals with severe withdrawal symptoms (Kampman et al. 2001). However, studies that do not consider withdrawal symptom severity may underestimate the overall effectiveness of such treatments. In fact, it is those patients with the most severe withdrawal symptomatology that are at the higher risk for relapse, and that experience more severe drug-associated problems such as emergency room visits and emotional and psychosocial problems (Schuckit et al. 1999; Sofuoglu et al. 2005). Therefore, particular patient subgroups may receive greater benefits from targeted withdrawal treatment. The present findings demonstrate relationships between cocaine intake, severity of anxiety-like behavior examined after cocaine self-administration, and subsequent reinstatement of cocaine seeking. These results suggest that subpopulations of cocaine users may be particularly prone to cocaine withdrawal anxiety, and therefore at higher risk for relapse to drug use. Anxiety is related to treatment retention and relapse to cocaine use (Ahmadi et al. 2006; Kampman et al. 2006; Kampman et al. 2001), and treatment of anxiety during acute withdrawal may benefit such users, as has been previously shown (Sofuoglu and Sewell 2009). Selective components of the noradrenergic system may offer potential treatment targets for pharmacotherapy. Consideration of drug history, duration and severity of use, comorbid disorders such as anxiety or depression, and withdrawal symptom severity are all important in adapting therapeutic approaches to users seeking treatment. Acknowledgments This research was supported by National Institute on Drug Abuse grants DA16511 and DA21690 (RES), 1F32 DA (DMB), and NIH grant C06 RR The authors thank Shannon Ghee and Bernard Smalls for technical assistance and data collection.

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