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Published in final edited form as: Drug Alcohol Depend. 2011 Oct 10;122(1-2):119–126. doi: 10.1016/j.drugalcdep.2011.09.017

Inhibiting Glycine Transporter-1 Facilitates Cocaine-Cue Extinction and Attenuates Reacquisition of Cocaine-Seeking Behavior*

Bríd Á Nic Dhonnchadha 1, Emmanuel Pinard 3, Daniela Alberati 3, Joseph G Wettstein 3, Roger D Spealman 2, Kathleen M Kantak 1
PMCID: PMC3288199  NIHMSID: NIHMS333172  PMID: 21992874

Abstract

Background

Combining extinction training with cognitive-enhancing pharmacotherapy represents a novel strategy for improving the efficacy of exposure therapy for drug relapse prevention. We investigated if the selective glycine transporter-1 (GlyT-1) inhibitor RO4543338 could facilitate extinction of cocaine-conditioned responses and attenuate reacquisition of cocaine-seeking behavior.

Methods

Rats were trained to self-administer cocaine (0.3 mg/kg), which was associated with a 2-sec light cue under a second-order schedule of i.v. drug injection. Rats received vehicle, 30 or 45 mg/kg of RO4543338 prior to three 1-hr extinction-training sessions spaced at weekly intervals. Responses were extinguished by substituting saline for cocaine while maintaining response-contingent cue presentations. Reacquisition of cocaine-seeking behavior during self-administration sessions began one week after the last extinction session. Control experiments were conducted under conditions that precluded explicit extinction of cocaine-conditioned responses.

Results

Compared to vehicle, 30 and 45 mg/kg RO4543338 significantly decreased responding early in extinction training and during subsequent reacquisition sessions. The latter effect persisted for at least five sessions. In control studies, reacquisition of cocaine-seeking behavior was not altered when RO4543338 was administered either prior to weekly self-administration control sessions or prior to weekly control sessions in which cocaine and cues were omitted and the levers retracted.

Conclusions

As the GlyT-1 inhibitor facilitated cocaine-cue extinction learning and attenuated subsequent reacquisition of cocaine-seeking behavior, this class of compounds may have utility as a pharmacological adjunct to cocaine-cue exposure therapy in addicts.

Keywords: Cocaine-seeking behavior, Cognitive enhancer, Glycine transporter-1 inhibitor, Extinction training, Relapse

1. Introduction

Drug addiction is a brain disease thought to represent pathological usurpation of the mechanisms of reward-related learning and memory (Hyman, 2007). Through the process of associative learning, environmental stimuli (cues) paired with drugs of abuse gain enhanced salience to exert long-lasting influences over behavior of addicts (Volkow et al., 2004). Extinguishing such abnormally strengthened learned responses remains a key problem for treating drug addiction. Exposure-based behavioral therapy makes use of an extinction strategy in which individuals are confronted with cues in a controlled setting and without access to drug in an effort to reduce craving and forestall relapse to drug-seeking behavior. Exposure therapy, however, is not effective consistently as a stand-alone treatment for drug addiction (Conklin and Tiffany, 2002), which may be due in part to drug-induced deficits in memory systems important for extinction learning (c.f., Kantak and Nic Dhonnchadha, 2011). Combining extinction training with cognitive-enhancing pharmacotherapy has been proposed as a novel strategy for improving the efficacy of exposure therapy for drug relapse prevention (c.f., Nic Dhonnchadha and Kantak, 2011).

Activation of N-methyl-D-aspartate (NMDA) receptors induces long-term potentiation and/or depression, which are mechanisms of synaptic plasticity associated with learning and memory formation (Kemp and Manahan-Vaughan, 2007), as well as its extinction (Quirk, 2006; Dalton et al., 2008). D-cycloserine (DCS) is a partial agonist at the glycine site of NMDA receptors that enhances glutamate neurotransmission (Bowery, 1987; Hood et al., 1989). Studies showing facilitative effects of DCS on extinction learning have focused mainly on conditioned fear in animals and anxiety disorders in humans (Davis et al., 2006). However, recent studies have shown a facilitative effect of DCS on extinction of cocaine-conditioned responses in animals (Botreau et al., 2006; Paolone et al., 2009; Torregrossa et al., 2010; Yang et al., 2010). A previous study that used an extinction procedure conceptually similar to exposure therapy in humans showed that prior DCS treatment significantly attenuated reacquisition of cocaine-seeking behavior in both rodents and monkeys by augmenting consolidation of cocaine-cue extinction learning (Nic Dhonnchadha et al., 2010). Recent work has replicated our findings in rats (Thanos et al., 2011). These preclinical findings encourage continued development of strategies and drug targets for facilitating extinction of cocaine-conditioned responses and preventing relapse.

One such drug target is glycine transporter-1 (GlyT-1), which regulates the synaptic levels of glycine (Aragon and Lopez-Corcuera, 2005). GlyT-1 sites are located on glia and on postsynaptic glutamatergic neurons in close proximity to NMDA receptors (Cubelos et al., 2005; Raiteri and Raiteri 2010). Pharmacological blockade or genetic deletion of GlyT-1 has been shown to increase glycine levels in glutamatergic synapses and, consequently, to augment NMDA-receptor transmission (Berger et al., 1998; Bergeron et al., 1998; Kinney et al., 2003; Depoortere et al., 2005; Dubroqua et al., 2010). Rodent studies have shown improvements in cognitive function after treatment with GlyT-1 inhibitors (Hashimoto et al., 2008; Karasawa et al., 2008; Singer et al., 2009), and clinical trials with GlyT-1 inhibitors are currently evaluating their efficacy to ameliorate cognitive dysfunction associated with schizophrenia (Wallace et al., 2011). To assess the effectiveness of GlyT-1 inhibition for facilitating extinction learning and attenuating reacquisition of cocaine-seeking behavior, we tested the selective GlyT-1 inhibitor RO4543338 ([1-(4-fluoro-phenyl)-8-[2-(4-fluoro-phenyl)-2-hydroxy-cyclohexyl]-1,3,8-triaza-spiro[4.5]decan-4-one]; Ceccarelli et al., 2006) using an animal model of cocaine-cue exposure therapy (three weekly sessions) analogous to clinical treatment protocols (Hofmann, 2007). RO4543338 inhibits Gly-T1 [3H]-glycine uptake in cells expressing the human transporter at nanomolar concentrations and shows negligible affinity for the glycine site on the NMDA receptor (Alberati et al., 2010).

2. Material and methods

2.1 Subjects

Male Wistar rats (Crl(WI)BR; 275–300 g) were housed individually in plastic cages (43 × 22 × 20 cm) in a temperature- (21–23 °C) and light- (08:00 h on, 20.00 h off) controlled facility. Rats were maintained in accordance with the 1996 NIH Guide for Care and Use of Laboratory Animals. The Boston University Institutional Animal Care and Use Committee approved research protocols. Details of catheter surgery, daily maintenance and experimental chambers are provided in supplementary material and methods.

2.2 Drugs

Cocaine HCl (NIDA, Bethesda, MD) was dissolved in a sterile 0.9% saline containing 3 IU heparin/ml to a concentration of 1.6 mg/ml. For intravenous (i.v.) self-administration of cocaine, a 0.3 mg/kg unit dose was delivered at a rate of 0.03 ml/sec. This dose produces the peak rate of responding on the active lever under our second-order schedule (Kantak et al., 2009). RO4543338 (Hoffmann-LaRoche, Basel, Switzerland) was suspended in 0.3% Tween 80 (vehicle) and injected via the i.p. route of administration in a volume of 1 ml/kg of body weight.

2.3 Procedure

2.3.1 Cocaine Self-Administration Training

During daily 1-hr cocaine self-administration sessions, rats were trained to press a lever to obtain 0.3 mg/kg i.v. injections of cocaine paired with the simultaneous presentation of a visual stimulus (2-sec light) under a fixed-ratio (FR) 1 schedule. Responses on a second (inactive) lever had no scheduled consequences. Training continued until rats self-administered cocaine reliably under a second-order schedule. Under this schedule, every fifth press (FR5) on the active lever during a 5-min fixed-interval (FI 5-min) resulted in illumination of the 2-sec stimulus light (cocaine-paired stimulus: S). An i.v. injection of cocaine was made contingent on the completion of the first FR5 after expiration of the FI, and was simultaneous with the onset of the 2-sec stimulus light. Previous work has shown that this FI 5-min[FR5:S] schedule maintains high rates of cocaine-seeking behavior and is sensitive for assessing changes in conditioned responding maintained by cocaine-paired cues (Kantak et al., 2002). During 1-hr daily sessions, rats could self-administer a maximum 11 cocaine injections. Rats also received an i.p. saline injection (1 ml/kg) 0.5 hr prior to randomly selected self-administration sessions to prevent development of a specific association between receiving i.p. injections and undergoing weekly extinction or control sessions (see sections 2.3.2 and 2.3.3). Once stable performances were achieved, rats underwent one or two cycles of training and testing, with each cycle consisting of a series of baseline self-administration sessions followed by extinction (or control) and reacquisition test sessions. The first of the weekly extinction or control sessions occurred 24 hr after the last baseline self-administration session. Two rats exhibited unstable levels of baseline responding (i.e., increasing or decreasing trends and ≥ 10% variability day-to-day) and were dropped from the experiment before testing began.

2.3.2 Effects of RO4543338 Pretreatment Prior to Extinction Sessions

This experiment determined if RO4543338 administered prior to weekly extinction sessions could augment extinction learning and attenuate subsequent reacquisition of cocaine-seeking behavior. Twelve rats were randomized to treatment across one or two cycles of training and testing (Supplementary Table 11). Doses of 30 mg/kg (n=7) and 45 mg/kg (n=5) RO4543338 or vehicle (n=7) were each injected 0.5 hr prior to three 1-hr extinction sessions spaced at weekly intervals. The 30 mg/kg dose of RO4543338 was tested first based on the previous findings of Alberati et al. (2010). Once stable baseline self-administration was established, lever pressing was extinguished by substituting saline for cocaine injections while maintaining response-contingent presentations of the 2-sec visual stimulus. Completion of every fifth response produced the visual stimulus, and the first FR 5 completed after the FI 5-min elapsed produced the visual stimulus and an i.v. injection of saline. After each session, rats were handled gently for 3 min before being returned to their home cages because this form of arousal was previously found to promote consolidation of cocaine-cue extinction following DCS treatment (cf. Nic Dhonnchadha et al., 2010). During the six intervening days between successive extinction training sessions, subjects remained in their home environments.

Seven days after the third and last extinction training session, reacquisition of cocaine-seeking behavior (in the absence of RO4543338) was evaluated. Reacquisition tests were conducted using conditions identical to those in the cocaine self-administration phase, and no drug priming or other inducements to initiate lever pressing were given. In addition, rats were given an i.p. injection of saline (1 ml/kg) 0.5 hr prior to the first reacquisition session to mimic the injection procedure used during extinction training. A minimum of 15 consecutive reacquisition sessions was conducted.

2.3.3 Effects of RO4543338 Pretreatment Prior to Control Sessions

This experiment investigated the effects of RO4543338 in two complementary control conditions that precluded explicit extinction of cocaine-conditioned cues. A new group of 12 rats first was trained to self-administer 0.3 mg/kg cocaine under the second-order schedule described in section 2.3.1. The control conditions consisted of either weekly cocaine self-administration sessions or weekly abstinence sessions (placement into the chambers with the levers retracted and without presentation of either the cocaine-paired stimulus or the delivery of cocaine). Because the remaining supply of RO4543338 was sufficient to inject a maximum of six rats, the 45 mg/kg dose was injected into three rats for each of the two control conditions. An additional six rats received vehicle, with three rats tested in each of the two control conditions. Seven days after the third and last control session, reacquisition (resumption) of cocaine-seeking behavior (in the absence of RO4543338) was evaluated for at least 5 sessions. All other conditions, including post-session handling, were identical to those described in section 2.3.2.

2.3.4 Data Analyses

The last five cocaine self-administration sessions of each cycle were used to establish baseline responses on the active and inactive levers. Due to the 3.5-fold differences in baseline responding in individual rats, responses during the 1-hr test sessions were expressed as percentage of the individual subject’s baseline responses. To evaluate within-session changes in responding, the absolute number of responses in sequential 5-min bins was used. The latency to the first response also was analyzed. Mixed-model ANOVAs were performed. For significant main and interaction effects, the Tukey test was used for post-hoc comparisons. Dunnett t-tests also were used to further delineate treatment differences. This procedure controls for type-I error and is appropriate for post-hoc analysis of cell means when the F value of the interaction is not significant (Winer, 1971).

3. Results

3.1 Baseline Responding

Despite differences in response rates for individual subjects, the mean number of active lever responses during baseline cocaine self-administration sessions was not significantly different across groups in each experiment before pretreatments were initiated (Table 1). Inactive lever responses were ≤ 15% of active lever responses and latency to the first response was ≤ 26 sec, with no significant group differences (Table 1). During subsequent experimental phases (sections 3.2 and 3.3), inactive lever responses (≤ 23 % of active lever responses) and latency to the first response (≤ 28 sec) were not significantly different across groups in each experiment as well (Supplementary Table 22).

Table 1.

Latency (sec) to the first response and number of responses on the active and inactive levers during 1-hr baseline cocaine self-administration sessions conducted prior to initiation of vehicle and RO4543338 pretreatments. Values are the mean ± SEM during extinction and control experiments.

Experiment Pretreatment Dose Latency Responses
Active Inactive
Extinction 0 mg/kg (vehicle) 25.5 ± 8.3 244.3 ± 30.0 30.9 ± 14.5
30 mg/kg 20.8 ± 8.3 325.7 ± 64.7 34.5 ± 13.8
45 mg/kg 23.9 ± 4.1 294.6 ± 43.0 18.0 ± 2.9
Control 0 mg/kg (vehicle) 9.5 ± 4.8 295.0 ± 64.0 43.2 ± 18.8
45 mg/kg 10.4 ± 5.2 350.3 ± 19.8 40.3 ± 9.9
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3.2 Effects of RO4543338 Pretreatment Prior to Extinction Sessions

Pretreatment with RO4543338 prior to each of the three weekly extinction sessions facilitated extinction learning (Figure 1a–f). Active lever pressing was significantly altered by RO4543338 [F(2, 16) = 7.0; p ≤ 0.007; treatment main effect], and both 30 and 45 mg/kg RO4543338 decreased responding compared to vehicle (p ≤ 0.025; Tukey test). A significant treatment × session interaction was not observed; however, compared to vehicle, 45 mg/kg RO4543338 decreased responding during extinction sessions 1 and 2 (p ≤ 0.03 and 0.003; Dunnett test), and 30 mg/kg RO4543338 decreased responding during extinction session 2 (p ≤ 0.03; Dunnett test).

Figure 1.

Figure 1

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Effects of RO4543338 or vehicle pretreatment during weekly extinction sessions (n=5–7/dose). Values are the mean ± SEM percent of baseline responses on the active lever for entire 1 hr sessions (panels a–c) and number of responses for sequential 5-min bins (panels d–f). * p ≤ 0.05 and ** p ≤ 0.01 compared to the corresponding 0 mg/kg vehicle control treatment.

Within-session patterns of responding during extinction also were analyzed using data from sequential 5-min bins of each session. Vehicle pretreatment produced a within-session pattern in which responding was greater later than earlier in the session for the first two sessions. Pretreatment with RO4543338 significantly altered this pattern for both session 1 [F(2, 16) = 3.7; p ≤ 0.047; treatment main effect] and session 2 [F(2, 16) = 6.5; p ≤ 0.009; treatment main effect]. Further analysis during session 1 (Figure 1d) revealed that, compared to vehicle, 30 mg/kg RO4543338 decreased responding in bins 4–5 and 8–9 (p ≤ 0.05; Dunnett test), and 45 mg/kg RO4543338 decreased responding in bins 4–6, and 8–12 (p ≤ 0.05; Dunnett test). It is noteworthy that responding during the first 5 min of session 1 (a point in time during extinction training that is indistinguishable from the first 5 min of a self-administration session) did not differ amongst treatments. Analysis of session 2 (Figure 1e) revealed that, compared to vehicle, 30 mg/kg RO4543338 decreased responding in bins 3 and 7–11 (p ≤ 0.05; Dunnett test), and 45 mg/kg RO4543338 decreased responding in bins 5–12 (p ≤ 0.05; Dunnett test). By session 3 of extinction training (Figure 1f), responding was maintained at low levels throughout the session after both RO4543338 and vehicle, and the treatments did not differ significantly.

Pretreatment with RO4543338 in conjunction with extinction training attenuated subsequent reacquisition of cocaine-seeking behavior (Figure 2a–b). The entire series of 15 reacquisition sessions (Supplementary Figure 13) first was analyzed in sequential 5-session blocks. As shown in Figure 2a, there was a slow return to baseline levels of responding after treatment with RO4543338, whereas vehicle-treated rats re-attained baseline levels of drug seeking immediately. Responding differed across blocks [F(2, 32) = 3.6; p ≤ 0.04; block main effect], with block 1 significantly different from block 3 (p ≤ 0.03; Tukey test), but not block 2.

Figure 2.

Figure 2

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Effects of RO4543338 or vehicle pretreatment on subsequent reacquisition of cocaine-seeking behavior (n=5–7/dose). Values are the mean ± SEM percent of baseline responses on the active lever for sequential 5-session blocks (panel a) and for the first five reacquisition sessions (panel b). * p ≤ 0.05 and ** p ≤ 0.02 compared to the corresponding 0 mg/kg vehicle control treatment.

Responding during first five reacquisition sessions was analyzed further using a separate ANOVA to isolate the effects of RO4543338 more on a day-to-day basis. RO4543338 significantly altered reacquisition of cocaine-seeking behavior during the first five sessions [F(2, 16) = 4.9; p ≤ 0.02; treatment main effect], and rats previously treated with either 30 mg/kg or 45 mg/kg RO 4543338 exhibited significantly decreased responding compared to vehicle (p ≤ 0.05 and 0.03, respectively; Tukey test). A significant treatment × session interaction was not observed, but as shown in Figure 2b, prior treatment with 45 mg/kg RO4543338 decreased responding during reacquisition sessions 1, 3 and 4 compared to vehicle (p ≤ 0.05; Dunnett test). Prior treatment with 30 mg/kg RO4543338 decreased responding during reacquisition sessions 1 and 2 compared to vehicle (p ≤ 0.02; Dunnett test), with a trend for decreased responding during reacquisition session 5 (p ≤ 0.06; Dunnett t-test).

3.3 Effects of RO4543338 Pretreatment Prior to Control Sessions

Pretreatment with RO4543338 prior to each of the three weekly self-administration or abstinence control sessions did not alter subsequent resumption of cocaine-seeking behavior compared to vehicle (Figure 3a). Vehicle- and RO4543338-treated rats re-attained baseline levels of self-administration responding during the first session. ANOVA confirmed that RO4543338 did not significantly alter active lever responding across sessions (no treatment or session main effects and no treatment × session interaction).

Figure 3.

Figure 3

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Effects of RO4543338 or vehicle pretreatment on subsequent resumption of cocaine-seeking behavior (panel a, n=6/dose); responding during weekly cocaine self-administration control sessions (panel b, n=3/dose); and responding during sequential 5-min bins of the weekly cocaine self-administration control sessions (panels c–e, n=3/dose). Values are the mean ± SEM percent of baseline active lever responses or the number of responses on the active lever. * p ≤ 0.05 compared to the corresponding 0 mg/kg vehicle control treatment.

In the cohort of rats tested in the self-administration control condition, pretreatment with RO4543338 also did not significantly alter the level of active lever responding during the weekly 1hr sessions (Figure 3b). Analysis of sequential 5-min bins (Figure 3c–e) revealed that vehicle pretreatment produced a relatively stable pattern of responding across bins during each of the three weekly self-administration control sessions. For sessions 1 and 2, RO4543338 did not significantly alter this pattern (no treatment main effects and no treatment × bin interactions), though there was a tendency for a relatively greater number of responses later than earlier in the sessions after RO4543338. During session 3, however, a significant treatment × bin interaction was observed [F(11, 44) = 3.8; p ≤ 0.001]. After RO4543338, responding during bins 8–9 and 12 was significantly greater than during bins 1–3 and 1–5, respectively (p ≤ 0.04; Tukey test), and responding during bins 8–11 was significantly greater than vehicle (p ≤ 0.05; Tukey test).

4. Discussion

4.1 RO4543338 Pretreatment Facilitates Extinction Learning

The extinction protocol employed in this study can be viewed as a laboratory approximation of cue exposure therapy in which drug-associated stimuli (e.g., drug images and paraphernalia) are presented to patients in a controlled drug-free environment during weekly therapy sessions (Hofmann, 2007). Our results show that pretreatment with the selective GlyT-1 inhibitor RO4543338 facilitated cocaine-cue extinction learning and attenuated reacquisition of cocaine-seeking behavior. Both doses of RO4543338 facilitated extinction learning relative to vehicle, as reflected by a more rapid decline in responding across the three weekly extinction sessions. Further analysis of responding during extinction sessions showed that vehicle-treated rats exhibited a normal pattern of between-session extinction learning in that there was less responding during the last session compared to the first session. The within-session pattern of responding in vehicle-treated rats revealed a greater level of responding during the latter portion of sessions 1 and 2 than during the initial 15 to 30 min of these sessions. The lower level of responding following pretreatment with 30 and 45 mg/kg RO4543338 during the latter portion of extinction session 1 is consistent with our interpretation that GlyT-1 inhibition facilitated extinction learning. Furthermore, because the extinction-facilitating effects of both doses of RO4543338 persisted into session 2 of extinction training, RO4543338 likely enhanced both acquisition and consolidation of extinction learning. This view is consistent with previous findings in rats and monkeys demonstrating blunted reacquisition of cocaine-seeking behavior after subjects received DCS either prior to or immediately following extinction training (Nic Dhonnchadha et al., 2010).

After completing extinction training and without further RO4543338 pretreatments, at least five sessions were needed for RO4543338-treated rats to re-attain baseline levels of responding during the reacquisition phase. In our previous work using a single-session extinction protocol in rats, DCS pretreatment also facilitated cocaine-cue extinction learning, but it attenuated reacquisition of cocaine seeking behavior for a maximum of three sessions under an FR5 schedule of i.v. drug injection (Nic Dhonnchadha et al., 2010). Future studies will need to address the issue of whether the apparently longer lasting effect of RO4543338 compared to DCS is related to differences in drug efficacy for facilitating extinction learning per se, differences in the total amount and spacing of cocaine-cue extinction training, differences in the schedule of reinforcement used in training and testing, or other factors. Notably, DCS pretreatment in monkeys prior to a single session of extinction training also deterred reacquisition of cocaine seeking behavior for a maximum of three sessions under a second-order schedule similar to the one used here (Nic Dhonnchadha et al., 2010). Thus, the more extended delay of reacquisition following RO4543338 compared to DCS may be related more to differences in drug efficacy and/or dosing regimen than to other procedural differences.

4.2 Specificity of the Effects of RO4543338 Pretreatment

Although it is possible that the decreased responding exhibited by RO4543338-treated rats during extinction training reflects motor deficits resulting from GlyT-1 inhibition instead of facilitated extinction learning, several findings suggest that this is not the case. First, latency to the first response during weekly extinction or cocaine self-administration control sessions was similar after both RO4543338 and vehicle treatments. Additionally, with regard to the self-administration control condition, responding during the latter portion of the third self-administration control session was significantly greater following pretreatment with RO4543338 than vehicle. These findings provide no evidence that RO4543338 impaired the ability of subjects to press a lever. They may, however, suggest that during self-administration control sessions, RO4543338 facilitated the association between cocaine and the 2-sec light cue to actually augment cocaine seeking. This interpretation should be treated cautiously, however, because of the small number of subjects tested under the self-administration control condition (due to the limited availability of RO4543338). Nonetheless, the possibility that the cognitive-enhancing effect of RO4543338 facilitates associative learning in general is consistent with a previous report with the glycine partial agonist DCS showing that cocaine-seeking behavior is attenuated when the drug is administered in conjunction with extinction training but enhanced when it is administered in conjunction with cue reactivation training (Lee et al., 2009; Nic Dhonnchadha et al., 2010; Torregrossa et al., 2010). Thus, to avoid an enhancement of cocaine seeking, it may be critical that cognitive-enhancing pharmacotherapy be applied only in sufficiently long therapy sessions during which cocaine-conditioned cues are explicitly extinguished.

Treatment with RO4543338 did not attenuate the resumption of responding under conditions that precluded explicit extinction of cocaine-conditioned responses (i.e. the self-administration and abstinence control conditions). The lack of significant effects of RO4543338 on resumption of cocaine-seeking behavior under the control conditions also makes it unlikely that RO4543338 treatment during extinction training reduced the motivational properties of cocaine per se. Collectively, these findings suggest that RO4543338 facilitated cocaine-cue extinction learning during extinction training to produce an enhanced reduction in cue salience and deter relapse to drug seeking when cocaine is once more made available.

4.3 Potential Mechanisms for Facilitated Extinction by RO4543338 Pretreatment

The primary pharmacological effect of GlyT-1 inhibition is an increase in synaptic levels of glycine. However, neuronal mechanisms underlying facilitated cocaine-cue extinction learning after RO4543338 pretreatment remain unknown. Previous studies have shown that acute administration of GlyT-1 inhibitors results in increased dopamine release in prefrontal cortex (Depoortere et al., 2005; Yang and Svensson, 2008), an important locus for consolidation and subsequent retrieval of extinction memory (Peters et al., 2009). Thus, modulation of dopamine neurotransmission within prefrontal cortex may have played a significant role in the effects of RO4543338 in our study. It also is possible that enhancement of NMDA-mediated α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor endocytosis in the amygdala played a role in facilitating cocaine-cue extinction learning by RO4543338. Along these lines, the combination of extinction training and intra-amygdalar infusion of the GlyT-1 inhibitor (R)-(N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)pro-pyl])sarcosine has been shown to facilitate extinction of conditioned fear and to reverse conditioning-induced increases in GluR1- and GluR2-containing AMPA receptors (Mao et al., 2009). Similarly, preliminary evidence suggests that cocaine-cue extinction learning may be associated with internalization of AMPA receptors as shown by significant reductions in the expression of total GluR1 and phosphorylated GluR1 at serine residue 845 in basolateral amygdala of rats (Nic Dhonnchadha et al., 2011).

4.4 Translation to Exposure Therapy in People

While use of animal models consistently indicates a facilitation of cocaine-cue extinction learning following extinction training combined with cognitive-enhancing pharmacotherapy, translation of these preclinical findings to treatment of addiction is uncertain. A case in point is the human laboratory study examining reactivity to cocaine cues following two sessions of exposure therapy combined with DCS treatment (Price et al., 2009). Subjects treated with DCS reported no significant differences in craving compared to placebo-treated subjects either during therapy or at 1-wk follow-up, although the follow-up rating was lower in the DCS group than in the placebo group. One possible reason for these inconclusive findings is that the clinical protocol (i.e., administration of DCS for two consecutive days of exposure therapy) may not have been optimal to facilitate cocaine-cue extinction. For example, fear conditioning studies in rats have shown that repeated daily administration of DCS actually desensitizes the NMDA receptor and prevents facilitation of extinction learning (Parnas et al., 2005; Werner-Seidler and Richardson, 2007). Furthermore, the time between cue exposure sessions has been shown to be relevant in animal models (Bouton, 1993; Rescorla, 2004; Li and Westbrook, 2008), and exposure therapy in people may be optimal when sessions are spaced at weekly intervals (Hofmann, 2007). Similarly, a greater decrease in conditioned craving in response to methamphetamine-paired cues was reported when exposure therapy sessions were spaced >4 days apart than when spaced more closely together (Price et al., 2010). Furthermore, when extinction sessions were spaced two weeks apart, positive effects of DCS administration on reducing reactivity to smoking cues in nicotine-dependent smokers were observed (Santa Ana et al., 2009). These results support the possibility of successful cocaine-cue extinction if optimal exposure therapy protocols are combined with optimal cognitive-enhancing pharmacotherapy.

4.5 Conclusion

Our experimental protocol in which multiple extinction sessions are spaced at weekly intervals may serve as a platform for designing a potentially new clinical strategy in cocaine addicts. RO4543338 works by a mechanism different from DCS and other currently available drugs being examined in clinical or human laboratory studies for augmenting exposure therapy (Hofmann et al., 2011). The positive benefit of R04543338 for forestalling relapse to cocaine-seeking behavior in rats is an original finding and opens the possibility that inhibition of GlyT-1 is a novel pharmacological target for augmenting exposure therapy in addicts.

Supplementary Material

Acknowledgments

Role of Funding Source

This study was funded by grant NIH R01 DA024315 with facilities and services supported in part by NIH grant P51 RR00168. NIH had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.

The authors thank Hoffmann La Roche Ltd for the generous gift of RO4543338 and the National Institute on Drug Abuse for the generous gift of cocaine. We thank Mr. David Tassin for assistance with data collection.

Footnotes

*

Supplementary material can be found by accessing the online version of this paper at http://dx.doi.org and by entering doi:…

1

Supplementary Table 1 can be found by accessing the online version of this paper at http://dx.doi.org and by entering doi: …

2

Supplementary Table 2 can be found by accessing the online version of this paper at http://dx.doi.org and by entering doi:…

3

Supplementary Figure 1 can be found by accessing the online version of this paper at http://dx.doi.org and by entering doi:…

Contributors

Authors Nic Dhonnchadha, Spealman and Kantak designed the study, analyzed and interpreted data and wrote the manuscript. Author Kantak additionally wrote the IACUC protocol. Authors Pinard, Alberati and Wettstein were involved in the synthesis of RO4543338 and contributed to the design of the study and to the writing of the final draft of the manuscript. All authors contributed to and have approved the final manuscript.

Conflict of Interest

All authors declare that, except for income received from their primary employers, have received no financial support or compensation from any individual over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.

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References

  1. Alberati D, Moreau JL, Mory R, Pinard E, Wettstein JG. Pharmacological evaluation of a novel assay for detecting glycine transporter 1 inhibitors and their antipsychotic potential. Pharmacol Biochem Behav. 2010;97:185–191. doi: 10.1016/j.pbb.2010.07.016. [DOI] [PubMed] [Google Scholar]
  2. Aragon C, Lopez-Corcuera B. Glycine transporters: crucial roles of pharmacological interest revealed by gene deletion. Trends Pharmacol Sci. 2005;26:283–286. doi: 10.1016/j.tips.2005.04.007. [DOI] [PubMed] [Google Scholar]
  3. Berger AJ, Dieudonne S, Ascher P. Glycine uptake governs glycine site occupancy at NMDA receptors of excitatory synapses. J Neurophysiol. 1998;80:3336–3340. doi: 10.1152/jn.1998.80.6.3336. [DOI] [PubMed] [Google Scholar]
  4. Bergeron R, Meyer TM, Coyle JT, Greene RW. Modulation of N-methyl-D-aspartate receptor function by glycine transport. Proc Natl Acad Sci U S A. 1998;95:15730–15734. doi: 10.1073/pnas.95.26.15730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Botreau F, Paolone G, Stewart J. D-Cycloserine facilitates extinction of a cocaine-induced conditioned place preference. Behav Brain Res. 2006;172:173–178. doi: 10.1016/j.bbr.2006.05.012. [DOI] [PubMed] [Google Scholar]
  6. Bouton ME. Context, time, and memory retrieval in the interference paradigms of Pavlovian learning. Psychol Bull. 1993;114:80–99. doi: 10.1037/0033-2909.114.1.80. [DOI] [PubMed] [Google Scholar]
  7. Bowery NG. Glycine-binding sites and NMDA receptors in brain. Nature. 1987;326:338. doi: 10.1038/326338a0. [DOI] [PubMed] [Google Scholar]
  8. Ceccarelli SM, Pinard E, Stalder H, Alberati D. Discovery of N-(2-hydroxy-2-aryl-cyclohexyl) substituted spiropiperidines as GlyT1 antagonists with improved pharmacological profile. Bioorg Med Chem Lett. 2006;16:354–357. doi: 10.1016/j.bmcl.2005.09.067. [DOI] [PubMed] [Google Scholar]
  9. Conklin CA, Tiffany ST. Applying extinction research and theory to cue-exposure addiction treatments. Addiction. 2002;97:155–167. doi: 10.1046/j.1360-0443.2002.00014.x. [DOI] [PubMed] [Google Scholar]
  10. Cubelos B, Gimenez C, Zafra F. Localization of the GLYT1 glycine transporter at glutamatergic synapses in the rat brain. Cereb Cortex. 2005;15:448–459. doi: 10.1093/cercor/bhh147. [DOI] [PubMed] [Google Scholar]
  11. Dalton GL, Wang YT, Floresco SB, Phillips AG. Disruption of AMPA receptor endocytosis impairs the extinction, but not acquisition of learned fear. Neuropsychopharmacol. 2008;33:2416–2426. doi: 10.1038/sj.npp.1301642. [DOI] [PubMed] [Google Scholar]
  12. Davis M, Ressler K, Rothbaum BO, Richardson R. Effects of D-cycloserine on extinction: translation from preclinical to clinical work. Biol Psychiatry. 2006;60:369–375. doi: 10.1016/j.biopsych.2006.03.084. [DOI] [PubMed] [Google Scholar]
  13. Depoortere R, Dargazanli G, Estenne-Bouhtou G, Coste A, Lanneau C, Desvignes C, Poncelet M, Heaulme M, Santucci V, Decobert M, Cudennec A, Voltz C, Boulay D, Terranova JP, Stemmelin J, Roger P, Marabout B, Sevrin M, Vige X, Biton B, Steinberg R, Francon D, Alonso R, Avenet P, Oury-Donat F, Perrault G, Griebel G, George P, Soubrie P, Scatton B. Neurochemical, electrophysiological and pharmacological profiles of the selective inhibitor of the glycine transporter-1 SSR504734, a potential new type of antipsychotic. Neuropsychopharmacol. 2005;30:1963–1985. doi: 10.1038/sj.npp.1300772. [DOI] [PubMed] [Google Scholar]
  14. Dubroqua S, Singer P, Boison D, Feldon J, Mohler H, Yee BK. Impacts of forebrain neuronal glycine transporter 1 disruption in the senescent brain: evidence for age-dependent phenotypes in Pavlovian learning. Behav Neurosci. 2010;124:839–850. doi: 10.1037/a0021556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hashimoto K, Fujita Y, Ishima T, Chaki S, Iyo M. Phencyclidine-induced cognitive deficits in mice are improved by subsequent subchronic administration of the glycine transporter-1 inhibitor NFPS and D-serine. Eur Neuropsychopharmacol. 2008;18:414–421. doi: 10.1016/j.euroneuro.2007.07.009. [DOI] [PubMed] [Google Scholar]
  16. Hofmann SG. Enhancing exposure-based therapy from a translational research perspective. Behav Res Ther. 2007;45:1987–2001. doi: 10.1016/j.brat.2007.06.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hood WF, Compton RP, Monahan JB. D-cycloserine: a ligand for the N-methyl-D-aspartate coupled glycine receptor has partial agonist characteristics. Neurosci Lett. 1989;98:91–95. doi: 10.1016/0304-3940(89)90379-0. [DOI] [PubMed] [Google Scholar]
  18. Hyman SE. The neurobiology of addiction: implications for voluntary control of behavior. Am J Bioeth. 2007;7:8–11. doi: 10.1080/15265160601063969. [DOI] [PubMed] [Google Scholar]
  19. Hofmann SG, Smits JA, Asnaani A, Gutner CA, Otto MW. Cognitive enhancers for anxiety disorders. Pharmacol Biochem Behav. 2011;99:275–284. doi: 10.1016/j.pbb.2010.11.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hood WF, Compton RP, Monahan JB. D-cycloserine: a ligand for the N-methyl-D-aspartate coupled glycine receptor has partial agonist characteristics. Neurosci Lett. 1989;98:91–95. doi: 10.1016/0304-3940(89)90379-0. [DOI] [PubMed] [Google Scholar]
  21. Hyman SE. The neurobiology of addiction: implications for voluntary control of behavior. Am J Bioeth. 2007;7:8–11. doi: 10.1080/15265160601063969. [DOI] [PubMed] [Google Scholar]
  22. Kantak KM, Black Y, Valencia E, Green-Jordan K, Eichenbaum HB. Dissociable effects of lidocaine inactivation of the rostral and caudal basolateral amygdala on the maintenance and reinstatement of cocaine-seeking behavior in rats. J Neurosci. 2002;22:1126–1136. doi: 10.1523/JNEUROSCI.22-03-01126.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kantak KM, Mashhoon Y, Silverman DN, Janes AC, Goodrich CM. Role of the orbitofrontal cortex and dorsal striatum in regulating the dose-related effects of self-administered cocaine. Behav Brain Res. 2009;201:128–136. doi: 10.1016/j.bbr.2009.02.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kantak KM, Nic Dhonnchadha BÁ. Pharmacological enhancement of drug cue extinction learning: translational challenges. Ann N Y Acad Sci. 2011;1216:122–137. doi: 10.1111/j.1749-6632.2010.05899.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Karasawa J, Hashimoto K, Chaki S. D-Serine and a glycine transporter inhibitor improve MK-801-induced cognitive deficits in a novel object recognition test in rats. Behav Brain Res. 2008;186:78–83. doi: 10.1016/j.bbr.2007.07.033. [DOI] [PubMed] [Google Scholar]
  26. Kemp A, Manahan-Vaughan D. Hippocampal long-term depression: master or minion in declarative memory processes? Trends Neurosci. 2007;30:111–118. doi: 10.1016/j.tins.2007.01.002. [DOI] [PubMed] [Google Scholar]
  27. Kinney GG, Sur C, Burno M, Mallorga PJ, Williams JB, Figueroa DJ, Wittmann M, Lemaire W, Conn PJ. The glycine transporter type 1 inhibitor N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine potentiates NMDA receptor-mediated responses in vivo and produces an antipsychotic profile in rodent behavior. J Neurosci. 2003;23:7586–7591. doi: 10.1523/JNEUROSCI.23-20-07586.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee JL, Gardner RJ, Butler VJ, Everitt BJ. D-cycloserine potentiates the reconsolidation of cocaine-associated memories. Learn Mem. 2009;16:82–85. doi: 10.1101/lm.1186609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Li SH, Westbrook RF. Massed extinction trials produce better short-term but worse long-term loss of context conditioned fear responses than spaced trials. J Exp Psychol Anim Behav Process. 2008;34:336–351. doi: 10.1037/0097-7403.34.3.336. [DOI] [PubMed] [Google Scholar]
  30. Mao SC, Lin HC, Gean PW. Augmentation of fear extinction by infusion of glycine transporter blockers into the amygdala. Mol Pharmacol. 2009;76:369–378. doi: 10.1124/mol.108.053728. [DOI] [PubMed] [Google Scholar]
  31. Nic Dhonnchadha BÁ, Kantak KM. Cognitive enhancers for facilitating drug cue extinction: insights from animal models. Pharmacol Biochem Behav. 2011;99:229–244. doi: 10.1016/j.pbb.2011.01.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nic Dhonnchadha BÁ, Lovascio B, Shrestha N, Kirkman C, Lin A, Leite-Morris KA, Man HY, Kaplan GB, Kantak KM. c-Fos and AMPA Receptor Expression Following Cocaine Cue Extinction Learning. College on Problems of Drug Dependence Annual Meeting; 2011. p. Abstract # 520. [Google Scholar]
  33. Nic Dhonnchadha BÁ, Szalay JJ, chat-Mendes C, Platt DM, Otto MW, Spealman RD, Kantak KM. D-cycloserine deters reacquisition of cocaine self-administration by augmenting extinction learning. Neuropsychopharmacol. 2010;35:357–367. doi: 10.1038/npp.2009.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Paolone G, Botreau F, Stewart J. The facilitative effects of D: -cycloserine on extinction of a cocaine-induced conditioned place preference can be long lasting and resistant to reinstatement. Psychopharmacology (Berl) 2009;202:403–409. doi: 10.1007/s00213-008-1280-y. [DOI] [PubMed] [Google Scholar]
  35. Parnas AS, Weber M, Richardson R. Effects of multiple exposures to D-cycloserine on extinction of conditioned fear in rats. Neurobiol Learn Mem. 2005;83:224–231. doi: 10.1016/j.nlm.2005.01.001. [DOI] [PubMed] [Google Scholar]
  36. Peters J, Kalivas PW, Quirk GJ. Extinction circuits for fear and addiction overlap in prefrontal cortex. Learn Mem. 2009;16:279–288. doi: 10.1101/lm.1041309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Price KL, Rae-Clark AL, Saladin ME, Maria MM, DeSantis SM, Back SE, Brady KT. D-cycloserine and cocaine cue reactivity: preliminary findings. Am J Drug Alcohol Abuse. 2009;35:434–438. doi: 10.3109/00952990903384332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Price KL, Saladin ME, Baker NL, Tolliver BK, DeSantis SM, Rae-Clark AL, Brady KT. Extinction of drug cue reactivity in methamphetamine-dependent individuals. Behav Res Ther. 2010;48:860–865. doi: 10.1016/j.brat.2010.05.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Quirk GJ. Extinction: new excitement for an old phenomenon. Biol Psychiatry. 2006;60:317–318. doi: 10.1016/j.biopsych.2006.05.023. [DOI] [PubMed] [Google Scholar]
  40. Raiteri L, Raiteri M. Functional ‘glial’ GLYT1 glycine transporters expressed in neurons. J Neurochem. 2010;114:647–653. doi: 10.1111/j.1471-4159.2010.06802.x. [DOI] [PubMed] [Google Scholar]
  41. Rescorla RA. Spontaneous recovery. Learn Mem. 2004;11:501–509. doi: 10.1101/lm.77504. [DOI] [PubMed] [Google Scholar]
  42. Santa Ana EJ, Rounsaville BJ, Frankforter TL, Nich C, Babuscio T, Poling J, Gonsai K, Hill KP, Carroll KM. D-Cycloserine attenuates reactivity to smoking cues in nicotine dependent smokers: a pilot investigation. Drug Alcohol Depend. 2009;104:220–227. doi: 10.1016/j.drugalcdep.2009.04.023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Singer P, Feldon J, Yee BK. The glycine transporter 1 inhibitor SSR504734 enhances working memory performance in a continuous delayed alternation task in C57BL/6 mice. Psychopharmacology (Berl) 2009;202:371–384. doi: 10.1007/s00213-008-1286-5. [DOI] [PubMed] [Google Scholar]
  44. Thanos PK, Bermeo C, Wang GJ, Volkow ND. D-cycloserine facilitates extinction of cocaine self-administration in rats. Synapse. 2011;65:938–944. doi: 10.1002/syn.20922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Torregrossa MM, Sanchez H, Taylor JR. D-cycloserine reduces the context specificity of pavlovian extinction of cocaine cues through actions in the nucleus accumbens. J Neurosci. 2010;30:10526–10533. doi: 10.1523/JNEUROSCI.2523-10.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Volkow ND, Fowler JS, Wang GJ. The addicted human brain viewed in the light of imaging studies: brain circuits and treatment strategies. Neuropharmacol. 2004;47(Suppl 1):3–13. doi: 10.1016/j.neuropharm.2004.07.019. [DOI] [PubMed] [Google Scholar]
  47. Wallace TL, Ballard TM, Pouzet B, Riedel WJ, Wettstein JG. Drug targets for cognitive enhancement in neuropsychiatric disorders. Pharmacol Biochem Behav. 2011;99:130–145. doi: 10.1016/j.pbb.2011.03.022. [DOI] [PubMed] [Google Scholar]
  48. Werner-Seidler A, Richardson R. Effects of D-cycloserine on extinction: consequences of prior exposure to imipramine. Biol Psychiatry. 2007;62:1195–1197. doi: 10.1016/j.biopsych.2007.04.010. [DOI] [PubMed] [Google Scholar]
  49. Winer B. Statistical Principles in Experimental Design. McGraw-Hill Inc; New York: 1971. [Google Scholar]
  50. Yang FY, Lee YS, Cherng CG, Cheng LY, Chang WT, Chuang JY, Kao GS, Yu L. D-cycloserine, sarcosine and D-serine diminish the expression of cocaine-induced conditioned place preference. J Psychopharmacol. 2010 doi: 10.1177/0269881110388333. (originally published online Nov. 24, 2010) [DOI] [PubMed] [Google Scholar]
  51. Yang CR, Svensson KA. Allosteric modulation of NMDA receptor via elevation of brain glycine and D-serine: the therapeutic potentials for schizophrenia. Pharmacol Ther. 2008;120:317–332. doi: 10.1016/j.pharmthera.2008.08.004. [DOI] [PubMed] [Google Scholar]

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