3 Agonist LY379268 Attenuate the Expression of Incubation of Cocaine Craving

Systemic and Central Amygdala Injections of the mGluR2/3 Agonist LY379268 Attenuate the Expression of Incubation of Cocaine Craving Lin Lu, Jamie L. Uejima, Sarah M. Gray, Jennifer M. Bossert, and Yavin Shaham Background: We and others reported time-dependent increases in cue-induced cocaine seeking after withdrawal, suggesting that craving incubates over time. Recently, we found that central amygdala extracellular signal-regulated kinases (ERK) and glutamate are involved in this incubation. Here, we further explored the role of central amygdala glutamate in the incubation of cocaine craving by determining the effect of systemic or central amygdala injections of the mGluR2/3 agonist LY379268 (which decreases glutamate release) on cue-induced cocaine seeking during early and late withdrawal. Methods: Rats were trained to self-administer cocaine for 10 days (6 hours/day); infusions were paired with a tone-light cue. Cocaine seeking and craving after systemic or central amygdala injections of LY379268 were then assessed in extinction tests in the presence of the cocaine-associated cues during early (day 3) or late (day 21) withdrawal. Results: Systemic (1.5 or 3 mg/kg) or central amygdala (.5 or 1.0 ␮g/side) injections of LY379268 attenuated enhanced extinction responding on day 21 but had no effect on lower extinction responding on day 3. Conclusions: Results confirm our previous findings on the role of central amygdala glutamate in the incubation of cocaine craving and together with previous reports suggest that mGluR2/3 agonists should be considered in the treatment of drug relapse. Key Words: Basolateral amygdala, drug seeking, glutamate, reinstatement, relapse

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elapse to cocaine use in humans can occur after prolonged abstinence and is often precipitated by exposure to cocaine-associated cues that provoke drug craving, a subjective state that often precedes and accompanies cocaine seeking (O’Brien et al 1992). In an attempt to account for this persistent relapse, Gawin and Kleber (1986) hypothesized that cue-induced cocaine craving (and relapse) progressively increases over the first several weeks of abstinence and remains high over extended drug-free periods (see Kosten et al 2005 for tentative empirical support for this hypothesis in a clinical trial). We and others (Grimm et al 2001; Lu et al 2004b; Neisewander et al 2000; Sorge and Stewart 2005) identified an analogous phenomenon in laboratory rats: time-dependent increases in cueinduced cocaine seeking after withdrawal from drug self-administration, suggesting that cocaine craving incubates over time. In addition, we and others (Di Ciano and Everitt 2004a; Grimm et al 2002, 2005; Shalev et al 2001; Shepard et al 2004) found time-dependent increases in cue-induced reward seeking in rats that previously self-administered heroin, methamphetamine, or sucrose. In initial studies (Grimm et al 2001, 2003), we assessed time-dependent increases in cue-induced responding on a lever previously associated with cocaine self-administration (the operational measure from which we inferred that cocaine craving incubates over time) with extinction tests in the presence of contextual cues previously associated with cocaine availability

From the Behavioral Neuroscience Branch, Intramural Research Program/ National Institute on Drug Abuse/National Institutes of Health/Department of Health and Human Services, Baltimore, Maryland. Address reprint requests to Prof. Lin Lu, National Institute on Drug Dependence, Peking University, 38 Yixueyuan Road, Beijing 100083, China; E-mail: [email protected]. Received March 13, 2006; revised April 13, 2006; accepted April 17, 2006.

0006-3223/07/$32.00 doi:10.1016/j.biopsych.2006.04.011

and subsequent cue-induced reinstatement tests (Meil and See 1996), during which lever presses led to contingent presentations of a discrete 5-sec tone-light cue previously paired with cocaine injections during self-administration training. We found that lever-responding in the extinction and the cue-induced reinstatement tests followed a similar time course and were highly correlated (Lu et al 2004c). These observations suggest that although different sets of cocaine cues induce drug seeking in these tests, they likely provoke a similar motivational state (craving) that incubates over time. Therefore, in more recent studies (and the present report), we and others have assessed the incubation of craving in extinction tests, during which rats were exposed to the drug contextual cues and lever presses resulted in contingent presentations of a discrete cue; the discrete cue serves as a conditioned reinforcer in the extinction tests (Conrad et al 2005; Lu et al 2004a, 2004b, 2005; Sorge and Stewart 2005). Recently, we explored whether the incubation of cocaine craving involves activation of extracellular signal-regulated kinases (ERK) signaling in the amygdala (Lu et al 2005). Cocaine activates the ERK pathway in mesolimbic dopamine projection areas (Berhow et al 1996; Mattson et al 2005; Valjent et al 2000). In the amygdala and other brain areas, this pathway is involved in learning and memory processes (Adams and Sweatt 2002) that were hypothesized to play a role in drug addiction and relapse (Everitt et al 2001; Jentsch and Taylor 1999; Nestler 2001; White 1996; Wise 1988). We found that exposure to cocaine cues in the extinction tests increases ERK phosphorylation in the central but not basolateral amygdala after 30 days but not 1 day of withdrawal from cocaine self-administration. In the basolateral but not the central amygdala, ERK phosphorylation was increased after 1 day but not 30 days of withdrawal from cocaine; this effect, however, was not associated with exposure to the cocaine cues in the extinction tests. After 30 withdrawal days, inhibition of central amygdala ERK phosphorylation by U0126 or the N-methyl–D aspartate (NMDA) antagonist AP-5 decreased cueinduced cocaine seeking. After 1 day of withdrawal from cocaine, stimulation of central amygdala ERK phosphorylation by BIOL PSYCHIATRY 2007;61:591–598 © 2007 Society of Biological Psychiatry

592 BIOL PSYCHIATRY 2007;61:591–598 NMDA increased cocaine seeking, an effect that was reversed by U0126. We interpreted these data to indicate that glutamatemediated, time-dependent increases in the responsiveness of the central amygdala ERK pathway to cocaine cues are critically involved in the expression of incubation of cocaine craving after prolonged withdrawal from the drug. Here, we further explored the role of glutamate in the expression of incubation of cocaine craving by determining the effect of systemic and central amygdala injections of a group II metabotropic glutamate receptor agonist (LY379268) on cueinduced cocaine seeking, as measured in extinction tests that were performed after 3 or 21 days of withdrawal from cocaine self-administration. Group II metabotropic glutamate receptors consist of the mGluR2 and the mGluR3 subtypes (Schoepp 2001). Subtype 2 metabotropic glutamate receptors are expressed primarily on presynaptic glutamate (and ␥-aminobutyric acid) neurons, and when activated they provide negative feedback to decrease evoked neurotransmitter release (Anwyl 1999; Schoepp 2001). Subtype 3 metabotropic glutamate receptors are expressed on both postsynaptic and presynaptic neurons and on glia, but their functional role is largely unknown (Schoepp 2001; Tamaru et al 2001). We studied the effect of LY379268 on the expression of incubation of cocaine craving, because systemic or intracranial injections of LY379268 were reported to attenuate discriminative, cue-induced relapse to cocaine seeking (Baptista et al 2004) and contextual cue-induced relapse to heroin seeking (Bossert et al 2004, Bossert et al, in press), as measured in a reinstatement model (Self and Nestler 1998; Shaham et al 2003; Stewart and de Wit 1987). On the basis of our previous work (Lu et al 2005), we hypothesized that systemic and central amygdala injections of LY379268 would decrease the expression of incubation of craving, as measured in a late withdrawal (day 21) extinction test. As an anatomical control, we also examined the effect of basolateral amygdala injections of LY379268 on extinction responding after 21 days of withdrawal from cocaine.

L. Lu et al planted bilaterally 1 mm above the central or basolateral amygdala. The coordinates (Paxinos and Watson 2005) for the central or basolateral sub-nuclei were: AP ⫺2.5 mm, ML ⫾ 4.5 mm (2° angle), DV ⫺7.5 mm, and AP ⫺2.5 mm, ML ⫾ 5.3 mm (2° angle), DV ⫺7.8 mm, respectively. After cannulae implantation, silastic catheters were inserted into the jugular vein and were passed subcutaneously to the top of the skull, as previously described (Lu et al 2004b; Shaham et al 1997). The catheters were then attached to a modified 22-gauge cannula and mounted to the rat’s skull with dental cement. Buprenorphine (.1 mg/kg, SC) was administered as an analgesic after surgery, and the rats were allowed to recover for 7–10 days. During the recovery and training phases, catheters were flushed every 24 – 48 hours with sterile saline ⫹ Gentamicin (.08 mg/mL). Systemic and Intracranial Injections The LY379268 (a gift from Eli Lilly, Indianapolis, Indiana) was dissolved in 1.2 mol/L NaOH solution (pH adjusted to 7.4). For systemic injections, the rats were injected with vehicle or LY379268 (1.5 or 3.0 mg/kg, IP) 30 min before testing. The doses of LY379268 were based on previous reports (Baptista et al 2004; Bossert et al 2004; Kim and Vezina 2002). Bilateral intracranial injections of LY379268 (0, .5, or 1.0 ␮g/side, injection volume .5 ␮L) were made with 10-␮L Hamilton syringes (Hamilton Co., Reno, Nevada) that were connected via polyethylene-50 tubing to 30-gauge injectors (Plastics One). The LY379268 or vehicle was infused into each side over 1 min, and the injector was left in place for an additional 1 min to allow for diffusion. The intracranial doses of LY379268 are based on our previous studies (Bossert et al 2004, Bossert et al, in press). At the end of Experiments 2–3 (see subsequent sections), the rats were killed, their brains were removed, and coronal sections (40 ␮m) were sliced on a cryostat and stained with Cresyl Violet (ICN Biomedicals, Aurora, Ohio). Cannulae placements were verified under a microscope, and their anatomical location is depicted in Figure 1C.

Methods and Materials Subjects and Apparatus Male Long-Evans rats (total n ⫽ 183, 300 – 400 g, Charles River, Raleigh, North Carolina) were maintained under a reverse 12-hour light– dark cycle (lights off at 9 AM). Food and water were freely available in the home cage. Experimental procedures followed the guidelines of the “Principles of Laboratory Care” (National Institutes of Health publication No. 86-23, 1996) and were approved by the local Animal Care and Use Committee. Fifty-six rats were excluded because of loss of catheter patency, poor health, or cannulae misplacement. The self-administration chambers were located inside soundattenuating cabinets and were controlled by a Med Associates (Georgia, Vermont) system. Each chamber had two levers located 9 cm above the floor. Presses on one (active, retractable) lever activated the infusion pump; presses on the other (inactive, stationary) lever were also recorded. The rat’s catheter was connected via a modified cannula (Plastics One, Roanoke, Virginia) to a liquid swivel (Instech, Plymouth Meeting, Pennsylvania) via polyethylene-50 tubing that was protected by a metal spring. Intravenous and Intracranial Surgery Rats were anesthetized with a mixture of sodium pentobarbital ⫹ chloral hydrate (60 and 25 mg/kg, respectively, IP), and permanent guide cannulae (23-gauge, Plastics One) were imwww.sobp.org/journal

Procedures The experiments consisted of three phases: self-administration training, withdrawal period, and tests for cocaine seeking under extinction conditions (Figure 1A). Training Phase. The rats were chronically housed and trained in the self-administration chambers. They were trained to selfadminister cocaine-HCl (National Institute on Drug Abuse) during six daily 1-hour sessions, separated by 5 min, over 10 days under a fixed-ratio-1 reinforcement schedule. Cocaine was dissolved in saline and self-administered at a dose of .75 mg/kg/infusion (.10 mL/infusion). Active lever responses activated the infusion pump and led to the delivery of a 5-sec tone-light compound cue (2900 Hz, 20 dB above background; 7.5 W white light located 4 cm above the active lever). Each infusion was followed by a 40-sec timeout period during which presses were recorded but did not result in drug delivery. The self-administration sessions started at the onset of the dark cycle and began with the insertion of the active lever and the illumination of a red house light that remained on for the duration of the session. At the end of each 1-hour session, the house light turned off, and the active lever was retracted. To facilitate acquisition of cocaine self-administration, food was removed from the chambers during the 6-hour sessions of the first 3 training days. A maximum of 20 cocaine infusions/hour was set to minimize overdose. At the end of the training phase, the groups to be tested with the different LY379268 doses were matched for their cocaine intake during training.

BIOL PSYCHIATRY 2007;61:591–598 593

L. Lu et al

A. Experimental timeline Cocaine Selfadministration training (10 d)

Withdrawal period (3 or 21 d)

Extinction test Day 21

Extinction test Day 3

B. Training

Infusions (6 h)

100

75

50 Exp. 1 Exp. 2

25

Exp. 3 0

1

2 3 4 5 6 7 8 9 10 Training day

C. Cannulae placements Central amygdala Bregma -2.16 mm

Bregma -2.52 mm

Bregma -2.92 mm

Basolateral amygdala

Bregma -2.16 mm

Withdrawal Phase. After the training phase, rats were returned to the animal facility, where they remained for 3 or 21 withdrawal days; the rats were handled three times per week. Extinction Test. The extinction test in the presence of the cocaine-associated cues consisted of a single 3-hour extinction session, conducted after 3 or 21 withdrawal days. (The extinction tests were conducted over 3 hours rather than 6 hours [the duration of the training sessions], because we initially assessed in several rats the effect of systemic LY379268 injections on cocaine-priming induced reinstatement. These rats were given saline on hour 4 and different doses of cocaine on hours 5– 6. These data, however, are not presented because of differences between the day-3 and day-21 groups in response to saline injections on hour 4; these differences in “baseline” response rates confound the interpretation of the cocaine priming data.) The experimental conditions were the same as those in the training phase except that presses on the previously active lever were not reinforced with cocaine. Tests started at the onset of the dark cycle and began with the insertion of the active lever and the illumination of the red house light, which remained on for the duration of the session. Active lever responses during testing resulted in contingent presentations of the tone-light cue that was previously paired with cocaine infusions. We assessed early withdrawal extinction responding on day 3 rather than on day 1 (as we did in our recent report; Lu et al 2005). The reason for this procedural change is that on withdrawal day 1 extinction responding is very low, which makes it difficult to detect the effects of experimental manipulations on decreases in response rates. In contrast, extinction responding on day 3, although significantly lower than during late withdrawal (Lu et al 2004a), is similar to that typically observed in studies on the effect of pharmacological and neuroanatomical manipulations on reinstatement of lever responding (Bossert et al 2005b; Shaham et al 2003). This is an important methodological consideration, because our aim was to study mechanisms that selectively mediate the enhanced cue-induced cocaine seeking in extinction tests during late withdrawal (what we term the “expression of incubation cocaine craving” in this report) and not the neuronal mechanisms underlying extinction responding in general. Specific Experiments Experiment 1: Systemic Injections of LY379268. Six groups of rats (n ⫽ 9 –10/group) were used in a 2 (withdrawal period: day 3, day 21) ⫻ 3 (LY379268 dose: 0, 1.5, 3.0 mg/kg) factorial design. The LY379268 or its vehicle was injected 30 min before the extinction tests, which were performed after 3 or 21 withdrawal days. Experiment 2: Central Amygdala Injections of LY379268. Six groups of rats (n ⫽ 8 –10/group) were used in a 2 (withdrawal period: day 3, day 21) ⫻ 3 (LY379268 dose: 0, .5, 1.0 ␮g/side) factorial design. The LY379268 or its vehicle was injected 5–10 min before the extinction tests, which were performed after 3 or 21 withdrawal days.

Bregma -2.52 mm

Bregma -2.92 mm

Figure 1. Experimental procedure, training, and cannulae placements. (A) Timeline of the experimental procedure. (B) Training phase: mean ⫾ SEM number of cocaine infusions over the ten 6-hour daily self-administration training sessions in Experiment 1 (n ⫽ 57), Experiment 2 (n ⫽ 56), and Experiment 3 (n ⫽ 14). (C) Cannulae placements: representative pictures of central and basolateral amygdala injector tips and the approximate placements of the injectors’ tips of the rats (Paxinos and Watson 2005).

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594 BIOL PSYCHIATRY 2007;61:591–598

Results The rats (total n ⫽ 127) demonstrated reliable cocaine self-administration, as indicated by the significant effect of training day [F (9,1107) ⫽ 24.5, p ⬍ .01] (Figure 1B); there were no significant differences between the rats that participated in Experiments 1, 2, and 3 (p ⬎ .05). Experiment 1: Systemic Injections of LY379268 Systemic injections of LY379268 decreased active lever responding in the extinction test on withdrawal day 21 but not day 3 (Figure 2A); this effect was most pronounced during the 1st hour of testing (data not shown). The LY379268 had no effect on inactive lever responding, which was very low during testing (Figure 2A). Active lever responses were analyzed with a mixed-factor ANOVA, using the between-subjects factors of LY379268 Dose and Withdrawal Day and the within-subjects factor of Hour. This analysis revealed significant effects of Withdrawal Day [F (1,51) ⫽ 12.1, p ⬍ .01], LY379268 Dose [F (2,51) ⫽ 3.7, p ⬍ .05], and Withdrawal Day LY379268 Dose ⫻ Hour [F (4,102) ⫽ 3.1, p ⬍ .05] and an approaching significant effect of Withdrawal Day ⫻ LY379268 Dose [F (2,51) ⫽ 3.1, p ⫽ .06]. No significant effects were found for inactive lever responding (p values ⬎ .05), with the exception of a significant effect of Withdrawal Day [F (1,51) ⫽ 5.3, p ⬍ .05], due to a small increase in responding on day 21 (Figure 2A). Post hoc group differences (Fisher probable least-squares difference test) are indicated in Figure 2A. Experiment 2: Central Amygdala Injections of LY379268 Intracranial injections of LY379268 into the central amygdala decreased active lever responding in the extinction test on withdrawal day 21 but not day 3 (Figure 2B); this effect was most pronounced during the 1st hour of the extinction tests (data not shown). The LY379268 had no effect on inactive lever responding, which was very low during testing (Figure 2B). Active lever responses were analyzed with a mixed-factor ANOVA, using the between-subjects factors of LY379268 Dose and Withdrawal Day and the within-subjects factor of Hour. This analysis revealed significant effects of Withdrawal Day [F (1,50) ⫽ 22.4, p ⬍ www.sobp.org/journal

Lever presses (3 h)

200

Day 21 Day 3 Active Active Inactive Inactive

150 100

*

# #

50 0

0 1.5 3.0 LY379268 dose (mg/kg, i.p.)

B. Central amygdala injections 200 Lever presses (3 h)

Statistical Analyses Data from Experiments 1 and 2 (systemic and central amygdala injections of LY379268) were analyzed separately for total (non-reinforced) active and inactive lever responses with a mixed-factor analysis of variance (ANOVA), using the betweensubjects factors of LY379268 Dose and Withdrawal Day and the within-subjects factor of Hour. Data from Experiment 3 (basolateral amygdala injections of LY379268) were analyzed with a mixed-factor ANOVA, using the between-subjects factor of LY379268 Dose and the within-subjects factor of Hour. Significant interaction effects (p ⬍ .05) were followed post hoc Fisher probable least-squares difference tests (two-tailed).

A. Systemic injections

Day 21 Day 3 Active Active Inactive Inactive

150 100

* #

*

50 0

0 0.5 1.0 LY379268 dose (µg/side)

C. Basolateral amygdala injections 200 Lever presses (3 h)

Experiment 3: Basolateral Amygdala Injections of LY379268. We found that central amygdala injections of LY379268 decreased lever responding in the extinction tests after 21 days but not 3 days of withdrawal from cocaine self-administration. Therefore, in Experiment 3 we determined the anatomical specificity of this effect by injecting LY379268 into the nearby basolateral amygdala. Two groups of rats (n ⫽ 7/group) were used. The LY379268 (1.0 ␮g/side) or its vehicle was injected 5–10 min before the extinction tests, which were performed after 21 withdrawal days.

L. Lu et al

150

Day 21 Active Inactive

100 50 0

1.0 0 LY379268 dose (µg/side)

Figure 2. Extinction tests: total lever presses. Data are mean ⫾ SEM presses on the previously active lever and on the inactive lever during the extinction tests after (A) systemic (n ⫽ 9 –10/group, Experiment 1), (B) central amygdala (n ⫽ 8 –10/group, Experiment 2), or (C) basolateral amygdala (n ⫽ 7/group, Experiment 3) injections of vehicle or LY379268. During the extinction tests, the rats were exposed to contextual cues in the self-administration chambers that were associated with cocaine availability and effects during training, and active lever presses led to contingent presentation of a 5-sec tone-light cue previously paired with each cocaine injection. *Different from Vehicle, p ⬍ .05; #different from Day 21, p ⬍ .05.

.01], LY379268 Dose [F (2,50) ⫽ 10.1, p ⬍ .01], Withdrawal Day ⫻ LY379268 Dose [F (2,50) ⫽ 8.6, p ⬍ .01], and Withdrawal Day ⫻ LY379268 Dose ⫻ Hour [F (4,100) ⫽ 4.4, p ⬍ .01]. No significant

L. Lu et al effects were found for inactive lever responding (p values ⬎ .05). Post hoc group differences are indicated in Figure 2B. Experiment 3: Basolateral Amygdala Injections of LY379268 Intracranial injections of LY379268 into the basolateral amygdala had no effect on active or inactive lever presses during the extinction tests that were performed after 21 days of withdrawal (p values ⬎ .05) (Figure 2C).

BIOL PSYCHIATRY 2007;61:591–598 595 amygdala. However, LY379268 injections into the nearby basolateral amygdala had no effect on enhanced extinction responding on withdrawal day 21, suggesting that the central amygdala is likely the main target of our intracranial injections. From an anatomical perspective, the lack of effect of basolateral injections of LY379268 on extinction responding is also of significance, because both mGluR2 and mGluR3 are expressed at higher levels in the basolateral amygdala than in the central amygdala (Ohishi et al 1998; Petralia et al 1996; Tamaru et al 2001).

Discussion The main finding in this report was that systemic and central amygdala injections of LY379268 attenuated cue-induced cocaine seeking after 21 days but not 3 days of withdrawal from cocaine. This pattern of results suggests that these LY379268 injections selectively attenuated the enhanced response to the cocaine cues during the late withdrawal extinction test (or the expression of incubation of cocaine craving) rather than inducing a general (time-independent) decrease in the motivational effects of the cocaine cues. These data provide the first demonstration that a pharmacological agent can selectively affect cue-induced drug seeking after prolonged but not short withdrawal period and confirm our recent finding on the role of central amygdala glutamate in the incubation of cocaine craving (Lu et al 2005). Methodological Considerations The selective effect of systemic and central amygdala LY379268 injections on extinction responding on withdrawal day 21 might be due to LY379268-induced motor deficits that were not manifested on withdrawal day 3 because of the lower response rate on this test day (Figures 2A and 2B). The effects of pharmacological manipulations on lever responding are dependent on baseline response rates, and drugs in general are more likely to decrease higher response rates than lower rates (Sanger and Blackman 1976). However, it is unlikely that motor deficits can account for the present results. Systemic injections of LY379268 (1.0 or 3.0 mg/kg) had no effect on the high rate of lever pressing for oral sucrose (about 100 presses/30 min) or condensed milk (about 80 presses/40 min) (Baptista et al 2004; Bossert et al, in press). As for potential non-specific effects of central amygdala injections of LY379268, we found that accumbens core injections of a higher dose of LY379268 (1.5 ␮g/side) than the ones used here had no effect on high response rates for oral sucrose (Bossert et al, in press). In that study, we also found that dorsal striatum injections of LY379268 (1.5 ␮g/side) had no effect on context-induced reinstatement of heroin seeking (Bossert et al, in press). The dorsal striatum and accumbens are critically involved in motor control (Mogenson et al 1980), and anatomical studies report that they express higher levels of mGluR2 and mGluR3 than the central amygdala (Ohishi et al 1998; Tamaru et al 2001). It is also unlikely that systemic or central amygdala LY379268 injections had no effect on extinction responding on withdrawal day 3 because of a floor effect. Systemic and intracranial injections of LY379268 reliably attenuated reinstatement of lever responding by contextual or discriminative drug cues when response rates were similar (Bossert et al 2005a, Bossert et al, in press) or lower (Baptista et al 2004) than the response rates on withdrawal day 3 (Figure 2). Another methodological concern is that the effect of central amygdala injections of LY379268 is due to diffusion of the drug to other amygdala sub-nuclei (Pitkanen 2000). This possibility cannot be ruled out, because it is unlikely that under our experimental procedure LY379268 was confined to the central

Role of Amygdala Glutamate in Cue-Induced Cocaine Seeking The main finding in the present report was that central but not basolateral amygdala LY379268 injections attenuated the enhanced cue-induced cocaine seeking during the late withdrawal (day 21) extinction tests. We interpreted these data to indicate that glutamate transmission in the central amygdala is critical for the expression of incubation of craving when rats are re-exposed to the cocaineassociated cues after prolonged withdrawal from the drug. The present data with LY379268 are in agreement with those from our previous report in which we found that central but not basolateral amygdala injections of the ERK antagonist U0126 attenuates enhanced extinction responding after prolonged withdrawal from cocaine (30 days) (Lu et al 2005). Our data on the lack of effect of basolateral amygdala LY379268 injections on cue-induced cocaine seeking during the late withdrawal extinction tests are in agreement with two previous studies. See et al (2001) found that basolateral amygdala injections of NMDA or amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists have no effect on cue-induced reinstatement of cocaine seeking. Di Ciano and Everitt (2004b) found that basolateral amygdala injections of an AMPA receptor antagonist have no effect on cue-controlled cocaine seeking, as measured in a second-order reinforcement schedule. The authors of these two studies also reported that blockade of basolateral amygdala dopamine receptors attenuate cue-induced cocaine seeking. A framework that might account for our present and previous findings (Lu et al 2005) as well as those of See et al (2001) and Di Ciano and Everitt (2004b) is that basolateral amygdala dopamine transmission mediates cue-induced cocaine seeking but is not critical for the time-dependent enhancement of cue-induced cocaine seeking after withdrawal from the drug. In contrast, central amygdala glutamate selectively mediates the time-dependent enhancement of cue-induced cocaine seeking after withdrawal from the drug or the incubation of cocaine craving. This is a speculative framework, but it is congruent with other reports on the dissociable roles of the central and basolateral amygdala in mediating appetitive and aversive conditioned responses (Holland and Gallagher 2003; Jentsch and Taylor 1999; Killcross et al 1997). For example, lesions of the basolateral amygdala disrupt the ability of cues paired with food or water to serve as conditioned reinforcers (Everitt et al 1999). However, when these lesions were performed after the conditioning phase, they only modestly affected psychostimulant-induced potentiation of the responses to conditioned reinforcers (Burns et al 1993). In contrast, central amygdala lesions have no effect on the cues’ ability to serve as conditioned reinforcers, but they attenuate psychostimulant-induced potentiation of their conditioned responses (Everitt et al 1999; Robledo et al 1996). To our knowledge, our data on the effect of central amygdala injections of LY379368 and the NMDA receptor antagonist AP5 in our previous report (Lu et al 2005) on cue-induced cocaine seeking after prolonged withdrawal from the drug provide the first evidence for a role of amygdala glutamate in cue-controlled www.sobp.org/journal

596 BIOL PSYCHIATRY 2007;61:591–598 cocaine seeking. These data are potentially in agreement with two other reports on the role of central amygdala in cue-induced cocaine seeking, as measured in the cue-induced reinstatement test (See et al 2003). Kruzich and See (2001) and Alleweireldt et al (2006) reported that central amygdala injections of either the sodium channel blocker tetrodotoxin or the D1-like receptor antagonist SCH 23390 attenuate cue-induced reinstatement of cocaine seeking, respectively. However, unlike the present report in which we found a selective effect of LY379268 in the central amygdala, the authors of these previous reports found that central amygdala tetrodotoxin or SCH 23390 injections were either equally effective (Kruzich and See 2001) or more effective in the basolateral amygdala (Alleweireldt et al 2006). Tetrodotoxin and SCH 23390 are lipophyllic drugs, and at the doses used in these studies (5 ng/side and 1–2 ␮g/side, respectively), these agents likely reach brain areas adjacent to the central amygdala (Caine et al 1995; Zhuravin and Bures 1991). Therefore, the effects on cue-induced reinstatement observed in the above two studies might be due to drug diffusion to the basolateral amygdala (see Alleweireldt et al 2006 for a discussion of issues related to the interpretation of data obtained from studies using intracranial injections of SCH 23390). The glutamatergic projections to the central amygdala that mediate the expression of incubation of cocaine craving after prolonged withdrawal from the drug are unknown. Although not conclusive, our data on the lack of effect of basolateral amygdala LY379268 injections on extinction responding after 21 days of withdrawal from cocaine suggest that the basolateral– central amygdala glutamate projection (Pitkanen 2000) is not involved. There are several excitatory projections from the cortex and the hippocampus to the medial part of the central amygdala (where the output neurons of this brain region are located) (Pitkanen 2000; Swanson and Petrovich 1998). In the context of incubation of cocaine craving, it will be of interest to explore the role of central amygdala glutamate projections from the ventral subiculum (Canteras and Swanson 1992; Cullinan et al 1993) and the agranular insular cortex (Ottersen 1982; Reynolds and Zahm 2005). These brain areas play a role in conditioned responses (Bast et al 2001; Hankins et al 1974; Morgan and LeDoux 1999; Rudy and Matus-Amat 2005). In addition, electrical stimulation of the agranular insular cortex induces neuronal firing of selected central amygdala (medial part) neurons (Pascoe and Kapp 1987; Quirk et al 2003), and stimulation of the ventral subiculum induces long-term potentiation of central amygdala neurons (Henke 1990) and reinstatement of psychostimulant seeking (Taepavarapruk and Phillips 2003; Vorel et al 2001). Implications for Treatment In previous work we found that blockade of central amygdala ERK phosphorylation by U0126 or the NMDA receptor antagonist AP-5 prevents the expression of incubation of cocaine craving after prolonged withdrawal from the drug (30 days). In addition, Valjent et al (2000, 2006) reported that systemic injections of an ERK antagonist (SL327) block cocaine and morphine reward, as measured in the conditioned place preference procedure. These findings are of interest from a neurobiological perspective, but their implications for treatment are not straightforward. ERK signaling is critical for many peripheral and central physiological processes (MacCorkle and Tan 2005; Yoon and Seger 2006), and medications that target fast synaptic transmission mediated by ionotropic glutamate receptors are likely to alter normal processes of learning, memory, and reward (Pennartz et al 2000). Furthermore, systemic injections of an NMDA receptor antagowww.sobp.org/journal

L. Lu et al nist potently reinstate cocaine seeking (De Vries et al 1998). Thus, undesirable effects resulting from systemic administration of ERK or NMDA receptor antagonists will likely preclude their use for the prevention of relapse to abused drugs. In contrast, pharmacological manipulations that target extrasynaptic glutamate transmission such as cysteine prodrugs (Baker et al 2002) or group II metabotropic glutamate receptors are less likely to interfere with normal learning and memory processes that are dependent on fast synaptic glutamate transmission and therefore might provide a new pharmacological approach for the treatment of drug relapse (Kalivas 2004). The present findings on the effect of LY379268 injections in a rat relapse and craving model (Epstein and Preston 2003) extend those from previous studies on the effect of this mGluR2/3 agonist on reinstatement of cocaine seeking by cocaine priming injections (Peters and Kalivas 2006) and discriminative cues (Baptista et al 2004). Systemic injections of LY379268 also attenuate context- and discrete cue-induced reinstatement of heroin seeking (Bossert et al 2004, 2005a) and the enhancement of amphetamine-taking behavior by prior sensitizing exposure to the drug (Kim et al 2005). In addition, Rodd et al (in press) recently found that a related mGluR2/3 agonist (LY404039) reduces cue-induced alcohol seeking and the expression of the alcohol deprivation effect. These results as well as other reports on the attenuation of the behavioral effects of abused drugs by systemic injections of LY379268 and related compounds suggest that pharmacological manipulations that target group II metabotropic glutamate receptors should be considered in the treatment of drug addiction (Heidbreder and Hagan 2005; Kenny and Markou 2004; Weiss 2005). Finally, in the present report and in the previous studies mentioned earlier, the behavioral effects of LY379268 on cueinduced relapse behavior were observed after acute systemic injections. Thus, from a medication development perspective, it is important to confirm these findings in studies in which investigators use chronic dosing regimens. Unfortunately, owing to a limited supply of LY379268 and related mGluR2/3 agonists, to our knowledge, such studies have not yet been performed.

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