glycogen synthase kinase 3β in the basolateral amygdala is critical for the reconsolidation of...

Exposure to drug-associated cues can provoke craving andrelapse to drug seeking even after prolonged abstinence(Wikler 1973; Stewart et al. 1984; O’Brien et al. 1992;Everitt and Robbins 2000; See 2002; Ciccocioppo et al.2004; Lu et al. 2006). The intense and pathologic memoriesthat develop between drug-paired contextual cues and therewarding stimuli or drug withdrawal-associated aversivefeelings may contribute to the high rate of relapse amongaddicts (Nestler 2001, 2004; Hyman et al. 2006; Robbinset al. 2008). Similar to other types of memories (Nader et al.2000a; Sara 2000; Milekic and Alberini 2002; Tronson andTaylor 2007), the reactivation of a consolidated drug rewardmemory returns this memory to a labile state, rendering itonce again vulnerable to amnestic treatment (Miller andMarshall 2005; Lee et al. 2006; Wang et al. 2008; Lee 2009;Zhao et al. 2009; Li et al. 2010) and the stabilization of thelabile state needs reconsolidation.

Glycogen synthase kinase-3b (GSK-3b) is an evolutiona-rily conserved, ubiquitous serine/threonine protein kinase,which was first isolated and purified as a protein kinase

capable of phosphorylating and inhibiting glycogen synthase(Embi et al. 1980). GSK-3b is widely expressed in the brain,including the amygdala, nucleus accumbens (NAc), andhippocampus (Woodgett 1990; Leroy and Brion 1999), whichare considered key dopamine projection areas (Robbins et al.2008). Previous studies have elucidated the involvement ofthe b-arrestin-2–Akt–GSK-3 pathway in the regulation ofdopamine-associated behaviors (Beaulieu et al. 2007b,2008). Our previous study reported that GSK-3b is required

Received March 13, 2011; revised manuscript received April 7, 2011;accepted April 11, 2011.Address correspondence and reprint requests to Prof. Lin Lu, National

Institute on Drug Dependence, Peking University, 38, Xue Yuan Road,Beijing 100191, China. E-mail: [email protected] used: BLA, basolateral amygdala; CeA, central amyg-

dala; CPP, conditioned place preference; GSK-3b, glycogen synthasekinase 3b; LiCl, lithium chloride; LTD, long-term depression; LTP, long-term potentiation; NAc, nucleus accumbens; pGSK-3b, phosphorylatedGSK-3b; Post-C, post-conditioning test; Post-T, post-treatment; Pre-C,pre-conditioning test.

National Institute on Drug Dependence, Peking University, Beijing, China

Abstract

Exposure to cocaine-associated conditioned stimuli elicits

craving and increases the probability of cocaine relapse in

cocaine users even after extended periods of abstinence.

Recent evidence indicates that cocaine seeking can be in-

hibited by disrupting the reconsolidation of the cocaine cue

memories and that basolateral amygdala (BLA) neuronal ac-

tivity plays a role in this effect. Previous studies demonstrated

that glycogen synthase kinase 3b (GSK-3b) plays a role in the

reconsolidation of fear memory. Here, we used a conditioned

place preference procedure to examine the role of GSK-3b in

the BLA in the reconsolidation of cocaine cue memories.

GSK-3b activity in the BLA, but not central amygdala (CeA), in

rats that acquired cocaine (10 mg/kg)-induced conditioned

place preference increased after re-exposure to a previously

cocaine-paired chamber (i.e., a memory reactivation proce-

dure). Systemic injections of the GSK-3b inhibitor lithium

chloride after memory reactivation impaired the reconsolida-

tion of cocaine cue memories and inhibited subsequent cue-

induced GSK-3b activity in the BLA. Basolateral amygdala,

but not central amygdala, injections of SB216763, a selective

inhibitor of GSK-3b, immediately after the reactivation of

cocaine cue memories also disrupted cocaine cue memory

reconsolidation and prevented cue-induced increases in GSK-

3b activity in the BLA. The effect of SB216763 on the

reconsolidation of cocaine cue memories lasted at least

2 weeks and was not recovered by a cocaine priming injec-

tion. These results indicate that GSK-3b activity in the BLA

mediates the reconsolidation of cocaine cue memories.

Keywords: addiction, amygdala, cocaine, GSK-3b, memory,

reconsolidation.

J. Neurochem. (2011) 118, 113–125.

JOURNAL OF NEUROCHEMISTRY | 2011 | 118 | 113–125 doi: 10.1111/j.1471-4159.2011.07277.x

� 2011 The AuthorsJournal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2011) 118, 113–125 113

for the initiation and expression of cocaine-induced locomo-tor sensitization (Xu et al. 2009), indicating that GSK-3b wasinvolved in neuroadaptive changes in response to repeatedcocaine exposure. However, the role of GSK-3b activity incue-induced drug-seeking behavior is not yet known.

Glycogen synthase kinase-3b is involved in the regulationof synaptic plasticity, including the regulation of NMDAreceptor-dependent long-term potentiation (LTP) (Hooperet al. 2007; Peineau et al. 2007) and long-term depression(LTD) (Peineau et al. 2007, 2009), which are considered theunderlying mechanisms of learning and memory (Malenka1994; Stevens 1998; Maren 1999; Braunewell and Manahan-Vaughan 2001; Kemp and Manahan-Vaughan 2007; Masseyand Bashir 2007). Kimura et al. (2008) recently found thatheterozygote GSK-3b knockout mice had impaired memoryreconsolidation, and intraperitoneal injection of a GSK-3binhibitor before memory reactivation impaired memoryreconsolidation in wildtype mice. In the present study, weexamined the role of GSK-3b in the basolateral amygdala(BLA) in the reconsolidation of cocaine cue memories. Weused a Pavlovian conditioned place preference (CPP)procedure, in which the preference of rats for the drug contextcue serves as a measure of drug reward (Mucha et al. 1982)and the incentive motivational effects of drug cues (Muellerand Stewart 2000). The CPP paradigm has been widely usedto assess drug cue memory reconsolidation (Miller andMarshall 2005; Wang et al. 2008; Li et al. 2010). We focusedon the amygdala because it plays a critical role in thereconsolidation of aversive and appetitive memories (Naderet al. 2000b; McGaugh 2004; Lee et al. 2005, 2006; Tronsonand Taylor 2007; Fuchs et al. 2009; Sanchez et al. 2010).

Materials and methods

SubjectsThe subjects were male Sprague–Dawley rats (Department of

Laboratory Animal Science, Peking University Health Science

Center). The rats weighed 220–240 g upon arrival and were housed

five rats per cage before surgery. A constant temperature (21 ± 2�C)and humidity (approximately 60%) was maintained throughout the

experiment. The rats were allowed free access to food and water

under a 12 h/12 h light/dark cycle (lights on at 8:00 AM) throughout

the experiment. The rats were allowed to habituate for approxi-

mately 7 days prior to the experiments. A total of 218 rats were used

in the present study, of which 12 were excluded because of strong

unconditioned preference, four died during surgery, and four were

excluded because of cannula implantation failure. All experimental

procedures were performed in compliance with the National

Institutes of Health Guide for the Care and Use of Laboratory

Animals and the Local Committee of Animal Use and Protection.

DrugsCocaine hydrochloride was purchased from Qinghai Pharmaceu-

ticals (Xining, China) and dissolved in 0.9% saline. Lithium

chloride (LiCl) was purchased from Beijing Chemical Plant

(Beijing, China) and dissolved in distilled water with a working

concentration of 9 mg/mL. SB216763 was purchased from Sigma

(St. Louis, MO, USA) and dissolved in dimethyl sulfoxide with

concentrations of 0.2 or 2 ng/lL. All drugs were prepared

immediately before use. The drug doses used were according to

our previous report (Xu et al. 2009).

SurgerySodium pentobarbital (60 mg/kg, i.p.) was used to anesthetize rats

(weighing 280–320 g on surgery) before they were bilaterally

implanted permanent guide cannulae (23 gauge; Plastics One,

Roanoke, VA, USA) which were located 1 mm above the BLA or

central amygdala (CeA). The coordinates (Paxinos and Watson

2005; Wang et al. 2008; Li et al. 2010) for the BLA were the

following: anterior/posterior: )2.9 mm and medial/lateral: ±5.0 mm

from bregma, dorsal/ventral: )8.5 mm from the surface of the skull.

The coordinates for the CeA were the following: anterior/posterior:

)2.9 mm and medial/lateral: ±4.2 mm from bregma, dorsal/ventral:

)7.8 mm from the surface of the skull. Stainless steel screws and

dental cement were used to anchor the cannulae. The cannulae were

kept patent by inserting a stainless steel stylet blocker into each

cannula. The rats were used for subsequent procedures after 5–

7 days’ recovery from surgery.

Intracranial injectionsThe injection of SB216763 into the BLA or CeA (0.5 lL/side) wasperformed as described in our previous report (Xu et al. 2009).Specifically, Hamilton syringes (Hamilton, Reno, NV, USA)

connecting to 30-gauge injectors (Plastics One) was used

intracranial injections. The injection was performed in 1 min with

a rate of 0.5 lL/min. The injection needle was not removed until

1 min following completion of injection, which make the drug

completely diffuse from the tips. Cannula placements were verified

with Nissl staining with a section thickness of 40 lm under light

microscopy. Rats with misplaced cannulae were excluded from the

statistical analysis. Schematic representations of the injection sites in

the BLA and CeA are shown in Fig. 1.

Conditioned place preferenceThe CPP procedure was performed using an unbiased, counter-

balanced protocol. The apparatus used for CPP training and

testing were ten identical three-chamber polyvinyl chloride boxes

as described in our previous reports (Wang et al. 2008; Zhai

et al. 2008; Li et al. 2010). During the pre-conditioning test (Pre-

C), the rats were initially placed in the middle chamber for

15 min free shuttle between the three chambers. The time spent

in every chamber was measured by a computer during the test by

recording infrared beam interruptions. Twelve rats were excluded

because of a strong unconditioned preference to one chamber

(> 540 s).

During the conditioning training for 8 days, each rat received

alternate injections of cocaine (10 mg/kg, i.p.) and saline (1 mL/kg,

i.p.) which was followed by 45 min confinement to the correspond-

ing conditioning chamber. The rats in the saline groups received

saline every day. On the day following completion of conditioning,

cocaine induced CPP was tested [post-conditioning (Post-C) test]

under conditions identical to those described in the Pre-C test. We

Journal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2011) 118, 113–125� 2011 The Authors

114 | P. Wu et al.

defined the CPP score by subtracting the time spent in the cocaine-

unpaired (saline-paired) chamber from the time spent in the cocaine-

paired chamber (Wang et al. 2008).

Cocaine reward memory reactivationCocaine reward memory was selectively reactivated by confining

the rats to the cocaine-paired chamber without cocaine administra-

tion for 10 min (Milekic et al. 2006; Wang et al. 2008; Zhai et al.2008; Li et al. 2010). The rats of no memory reactivation in control

groups remained in the home cages at the same time.

Retesting of cocaine-induced CPPCocaine induced CPP was retested 1 day [post-treatment (Post-T) 1]

or 2 weeks (Post-T 14) after memory reactivation. The rats showed

no cocaine CPP in the Post-T 14 were tested again (priming test) on

the following day immediately after administration of a priming

injection of cocaine (5 mg/kg, i.p.).

Tissue sample preparationThe tissue sample preparation procedure used was based on our

previous reports (Li et al. 2008; Xu et al. 2009). After decapitationwithout anesthesia 10 min after CPP test, the brains were quickly

extracted and frozen in )60�C N-hexane and then transferred to a

)80�C freezer. A freezing cryostat ()20�C; Reichert-Jung 2800

Frigocut E; Leica, Heerbrugg, Switzerland) was used to make 1 mm

thick coronal sections located approximately )2.5–3 mm from

bregma. We took bilateral tissue punches (16 gauge) of the BLA and

CeA and then homogenized them (10–15 s · 3, 5-s interval) on ice

with an electrical disperser (Wiggenhauser, Sdn Bhd, Berlin,

Germany) after 30 min in ristocetin-induced platelet agglutination

lysis buffer [Beyotime Biotechnology, Haimen, China; 20 mM Tris

(pH 7.5), 150 mM NaCl, 1% Triton X-100, 2.5 mM sodium

pyrophosphate, 1 mM EDTA, 1% Na3VO4, 0.5 lg/mL leupeptin,

1 mM phenylmethanesulfonyl fluoride]. After that, the Tissue

homogenates were centrifuged by 10 000 g at 4�C for 5 min. The

protein concentrations of all samples were determined using a

bicinchoninic acid assay (Beyotime Biotechnology) and equalized

by diluting in ristocetin-induced platelet agglutination lysis buffer.

Western blot assaysThe samples were treated and assayed according to our previous

study (Xu et al. 2009) except that the antibody used were anti-

phospho-GSK-3b (1 : 1000; Cell Signaling Technology, Danvers,

MA, USA) and anti-GSK-3b (1 : 2000; Cell Signaling Technology).

The Quantity One software v. 4.4.0 (Bio-Rad Corporation, Hercules,

CA, USA) was used to determine the band intensities for b-actin,GSK-3b and phosphorylated GSK-3b (pGSK-3b) by two observers.

Values of GSK-3b and pGSK-3b of rats in experimental groups

were normalized to b-actin and those of naı̈ve group.

Experimental design

Experiment 1: Effect of cocaine reward memory reactivation onGSK-3b expression in the amygdalaWe assessed the effect of exposure to the cocaine-paired context on

the expression of GSK-3b in the BLA and CeA in rats that

underwent CPP training with cocaine or saline (Fig. 2a). Because

previous studies have shown that GSK-3b activity can be reduced

by phosphorylation at the N-terminal serine-9 residue (Plyte et al.1992; Wang et al. 1994) through several kinases (Bhat et al. 2004;Peineau et al. 2008), the present study assessed GSK-3b phosphor-

ylation at the serine-9 residue to reflect GSK-3b activity (Xu et al.2009). We used four groups of rats (n = 10 per group) in an

experimental design that included the between-subjects factors

cocaine dose (0 and 10 mg/kg) and exposure (exposure and no

exposure) (Fig. 2). All rats underwent a baseline preference test

(Pre-C). The test for the expression of cocaine-induced CPP was

performed 1 day after the last training/injection day for all rats

(Post-C). One day later, half of the rats were exposed to the cocaine-

paired context and were decapitated 10 min later. Their brains were

extracted for subsequent determination of pGSK-3b in the BLA and

CeA by western blotting.

Experiment 2: Effect of LiCl-induced GSK-3b inhibition on thereconsolidation of cocaine reward memoryTo determine the functional role of GSK-3b in the reconsolidation

of CPP, we inhibited GSK-3b activity using LiCl (Frame and

Cohen 2001; Bhat et al. 2004; Wada 2009; Xu et al. 2009)

immediately after cocaine memory reactivation and tested the rats

for the expression of cocaine-induced CPP and amygdala GSK-3bexpression 1 day later (Fig. 3a). We used three groups of rats

(n = 8–11 per group) in an experimental design that included the

between-subjects factor LiCl dose (0, 30, and 100 mg/kg, i.p.).

The dependent measures were CPP score and pGSK-3b level in

the BLA and CeA. All rats were trained for cocaine-induced CPP

for 8 days and tested for the expression of CPP on day 9 without

any injections (Post-C). On day 10, four groups of rats were

confined to the cocaine-paired context for 10 min to reactivate

cocaine reward memory (Wang et al. 2008; Li et al. 2010) and

then injected with either vehicle or LiCl (30 and 100 mg/kg, i.p.)

immediately after cocaine-context exposure. On day 11, these rats

were tested again for cocaine-induced CPP without any injections

(Post-T). Ten minutes after the 15 min Post-T test, all rats were

decapitated, and their brains were extracted for subsequent pGSK-

3b determination.

To exclude the possibility that post-reactivation administration of

LiCl produced an aversive memory between the cocaine-paired

context and LiCl injection that counteracted the cocaine reward

Fig. 1 Schematic representation of the regions of the basolateral

amygdala (BLA: )2.8 mm from bregma) and central amygdala (CeA:

)2.8 mm from bregma) into which the cannula was placed and from

which brain tissue was dissected for the western blot assays. Only the

rostral faces of each coronal section are shown.

� 2011 The AuthorsJournal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2011) 118, 113–125

Role of GSK-3b in reconsolidation of cocaine memory | 115

memory, three additional groups of rats (n = 8–9 per group) were

trained and tested for CPP as described above, with the exception

that the rats always received saline injections before being placed in

both side chambers during the 8-day CPP training (Fig. 3). The day

after Post-C (day 10), the rats received LiCl (0, 30, and 100 mg/kg,

i.p.) immediately after exposure to one designated chamber. Twenty-

four hours after LiCl administration (day 11), the rats were re-tested

for the expression of CPP.

Experiment 3: Further confirmation of the effect of LiCl on thereconsolidation of cocaine reward memoryTo further confirm the effect of LiCl on the reconsolidation of

cocaine reward memory, four additional groups were used to

examine the effect of LiCl on the subsequent expression of cocaine-

induced CPP when injected without exposure to the cocaine-paired

context or injected 6 h after exposure to the cocaine-paired context

(Fig. 4). All rats were trained for cocaine-induced CPP for 8 days

and tested for the expression of CPP on day 9 without any injections

(Post-C). On day 10, two groups of rats (n = 6 per group) were

injected with either saline or LiCl (100 mg/kg, i.p.) without cocaine-

context exposure (Fig. 4a). Another two groups of rats (n = 6 per

group) were confined to the cocaine-paired context for 10 min to

reactivate cocaine reward memory and then injected with either

saline or LiCl (100 mg/kg, i.p.) 6 h after cocaine-context exposure

(Fig. 4c). On day 11, these rats were tested again for cocaine-

induced CPP without any injections (Post-T).

Experiment 4: Effect of SB216763-induced GSK-3b inhibition in theamygdala on the reconsolidation of cocaine reward memoryTo confirm the potential role of GSK-3b in the amygdala in the

reconsolidation of the memories for cocaine cues in the CPP

procedure, we examined whether GSK-3b inhibition in the BLA or

CeA induced by the selective GSK-3b antagonist SB216763

(Coghlan et al. 2000) would impair the subsequent expression of

cocaine-induced CPP (Fig. 5a). Six groups of rats (n = 8–11 per

group) were used in an experimental design that included the

between-subjects factors SB216763 dose (0, 0.1, and 1 ng/side) and

amygdala nucleus (central, basolateral). The dependent measures

were CPP score and pGSK-3b level in the CeA and BLA. All rats

were trained for cocaine-induced CPP for 8 days and tested for the

expression of CPP on day 9 without any injections (Post-C). On day

10, four groups of rats were confined to the cocaine-paired context

for 10 min to reactivate cocaine reward memory and received intra-

BLA or CeA injections of either vehicle or SB216763 (0.1 and 1 ng/

side) immediately after cocaine-context exposure. On day 11, these

rats were tested again for cocaine-induced CPP without any

injections (Post-T). Ten minutes after the 15 min Post-T test, all

rats were decapitated, and their brains were extracted for subsequent

determination of pGSK-3b.

Experiment 5: Further confirmation of the effect of intra-BLASB216763 microinfusion on the reconsolidation of cocaine rewardmemoryTo determine whether the inhibitory effect of SB216763 on cocaine

reward memory was reactivation dependent, four additional groups

of rats (n = 8–10 per group) received the same treatment as

described in Experiment 4 with the exception that they were given

intra-BLA injections of either vehicle or SB216763 (1 ng/side)

without cocaine-context exposure (Fig. 6a) or 6 h after cocaine-

context exposure (Fig. 6c).

Experiment 6: Long-term effect of GSK-3b inhibition in the BLA onthe reconsolidation of cocaine reward memoryWe used two groups of rats (n = 10 per group) to determine the

persistence of the effect of SB216763 injections in the BLA on the

reconsolidation of learned cocaine-induced CPP (Fig. 7a). The rats

were trained for cocaine-induced CPP for 8 days and tested for the

expression of CPP on day 9 without any injections (Post-C). On day

10, the rats were confined to the cocaine-paired context for 10 min

to reactivate the cocaine reward memory and then received either

vehicle or SB216763 (1 ng/side) in the BLA immediately after

cocaine-context exposure. On days 11 and 25, these rats were tested

again (Post-T 1 and Post-T 14) for cocaine-induced CPP without

any injections. On day 26, all rats were given a priming injection of

cocaine (10 mg/kg, i.p.) and tested again for cocaine-induced CPP.

Cocaine was injected immediately before the test session.

Data analysisThe data are expressed as mean ± SEM and were analyzed with

ANOVA using appropriate between- and within-subjects factors for

the different experiments (see Results). Because our multifactorial

ANOVAs yielded multiple main effects and interaction effects, we

only report significant effects that are critical for the interpretation of

the data shown in the Results. All post hoc comparisons were made

using the Least Significant Difference test. Values of p < 0.05 were

considered statistically significant. For clarity, post hoc analyses areindicated by asterisks in the figures but are not described in the

Results.

Results

Effect of cocaine memory reactivation on GSK-3bexpression in the amygdalaIn Experiment 1, rats exposed to a previously cocaine-pairedchamber following cocaine-induced CPP training showedsignificantly higher GSK-3b activity in the BLA but not CeA(i.e., significantly decreased pGSK-3b; Fig. 2c and d). Theexposure had no effect on total GSK-3b in the CeA or BLA(Fig. 2c and d; statistical analyses not presented).

Cocaine-induced CPPFigure 2(b) showed the CPP scores in the cocaine and salinegroups. A two-way ANOVA, with test condition (Pre-C andPost-C) as the within-subjects factor and cocaine dose (0 and10 mg/kg) as the between-subjects factor, revealed asignificant cocaine dose · test condition interaction (F1,18 =8.075, p < 0.05), indicating that rats in the cocaine (10 mg/kg) group but not saline group acquired CPP after 8 days ofconditioning training.

pGSK-3bAnalyses were performed separately for the CeA and BLAand included the between-subjects factors cocaine dose


116 | P. Wu et al.

(0 and 10 mg/kg) and exposure (exposure and no exposure).The statistical analysis for pGSK-3b in the BLA revealed asignificant effect of cocaine dose (F1,19 = 42.684, p < 0.01)

and exposure (F1,19 = 38.703, p < 0.01) and a cocainedose · exposure interaction (F1,19 = 32.115, p < 0.01). Theinteraction was attributable to the fact that pGSK-3b levels inthe BLA in cocaine-exposed rats that underwent cocaine-paired chamber exposure were lower than those of the otherthree groups (p < 0.01; Fig. 2c). The analysis for pGSK-3bin the CeA, however, revealed no significant effects ofcocaine dose and exposure and no cocaine dose · exposureinteraction (p > 0.05; Fig. 2d).

Effect of LiCl-induced GSK-3b inhibition on thereconsolidation of cocaine reward memoryIn Experiment 2, LiCl administered at a dose of 100 mg/kg immediately following exposure to the previouslycocaine-paired chamber impaired the reconsolidationof cocaine reward memory (Fig. 3b), and this effectaccompanied restored GSK-3b activity in the BLA(Fig. 3c and d). LiCl had no effect on total GSK-3b inthe CeA or BLA (Fig. 3c and d; statistical analyses notpresented).

Cocaine-induced CPPThe ANOVA for CPP scores included the between-subjectsfactor LiCl dose (0, 30, and 100 mg/kg) and the within-subjects factor test condition (Pre-C, Post-C, and Post-T).This analysis of CPP scores in the cocaine groups revealeda significant LiCl dose · test condition interaction (F4,60 =3.989, p < 0.01). The post hoc analysis showed that aftercocaine-induced CPP training, all cocaine groups acquiredCPP (p < 0.01), with no significant differences in CPPscores between any two groups during Post-C (p > 0.05).Compared with the Post-C test, CPP scores significantlydecreased only in the group of rats that received 100 mg/kg LiCl after exposure to the previously cocaine-pairedchamber (p < 0.001) in the Post-T test (Fig. 3b). LiCl hadno effect on CPP scores in the saline groups (statisticalanalyses not presented), suggesting that LiCl had noaversive effect itself, and the inhibition of cocaine-inducedCPP expression was attributable to an impairment ofreconsolidation of cocaine reward memory.

pGSK-3bAnalyses were performed separately for the CeA and BLAand included the between-subjects factors cocaine dose (0and 10 mg/kg) and LiCl dose (0, 30, and 100 mg/kg). TheANOVA for pGSK-3b in the BLA revealed a significantLiCl dose · cocaine dose interaction (F2,47 = 8.349,p < 0.01). This interaction was attributable to the fact thatpGSK-3b levels in the BLA in rats that received 100 mg/kg LiCl were higher than those of the 0 and 30 mg/kgLiCl groups (p < 0.01; Fig. 3c). The analysis for pGSK-3bin the CeA, however, revealed no significant effect ofcocaine dose or LiCl dose and no cocaine dose · LiCldose interaction (p > 0.05; Fig. 3d).

Timeline(a) (b)

(c) (d)Basolateral amygdala Central amygdala

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cent

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Fig. 2 Effect of reactivation of cocaine-induced CPP on GSK-3b

activity in the amygdala. Data are expressed as mean ± SEM. (a) Be-

havioral procedure. (b) CPP scores for rats during the Pre-C and Post-C

phases. Rats in the cocaine group acquired significant place pre-

ference. Data are shown as preference scores (in seconds) during the

CPP preference tests (n = 10 per group). *p < 0.05, compared with

Pre-C in cocaine group and compared with saline group in the Post-C

phase. (c–d) Phosphorylated and total GSK-3b protein levels and re-

presentative western blots in the basolateral and central amygdale

following exposure to cocaine-paired context. Data are expressed as a

percentage of phosphorylated and total GSK-3b in naive control rats

(n = 5). In the basolateral amygdala, exposure to the cocaine-paired

context decreased phosphorylated GSK-3b in the cocaine group.

*p < 0.05, compared with no-exposure group in the cocaine-condi-

tioned group. Pre-C, pre-conditioning; Post-C, post-conditioning.



Further confirmation of the effect of LiCl on thereconsolidation of cocaine reward memoryAs shown in Fig. 4(b), systemic administration of LiClwithout cocaine-paired context exposure on day 10 had noeffect on the subsequent expression of cocaine-induced CPPon day 11. The ANOVA for CPP scores included the between-subjects factor LiCl dose (0 and 100 mg/kg) and the within-subjects factor test condition (Pre-C, Post-C, and Post-T).This analysis of CPP scores revealed a significant main effectof test condition (F2,18 = 16.92, p < 0.01) but no main effectof LiCl dose and no test condition · LiCl dose interaction(p > 0.05). The post hoc analysis showed that after cocaine-induced CPP training, all cocaine groups acquired CPP(p < 0.01), with no differences in CPP scores between thetwo groups during the Post-C or Post-T phases (p > 0.05).Compared with the Post-C test, CPP scores did not decreasein either group in the Post-T test on day 11 (p > 0.05).

As shown in Fig. 4(d), systemic administration of LiCl 6 hafter exposure to the cocaine-paired context on day 10 had noeffect on the subsequent expression of cocaine-induced CPPon day 11. The ANOVA for CPP scores included the between-subjects factor LiCl dose (0 and 100 mg/kg) and the within-subjects factor test condition (Pre-C, Post-C, and Post-T).This analysis of CPP scores revealed a significant main effectof test condition (F2,18 = 12.52, p < 0.01) but no main effectof LiCl dose and no test condition · LiCl dose interaction(p > 0.05). The post hoc analysis showed that after cocaineCPP training, all cocaine groups acquired CPP (p < 0.01)with no differences in CPP scores between the two groupsduring the Post-C or Post-T phase (p > 0.05). Comparedwith the Post-C test, CPP scores did not decrease in eithergroup in the Post-T test on day 11 (p > 0.05).

Effect of SB216763-induced GSK-3b inhibition in theamygdala on the reconsolidation of cocaine rewardmemorySB216763-induced GSK-3b inhibition in the BLA but notCeA following exposure to the cocaine-paired chamberimpaired the reconsolidation of cocaine reward memory(Fig. 5). Neither BLA nor CeA microinjection of SB216763had an effect on total GSK-3b in the CeA or BLA (Fig. 5band c; statistical analyses not presented).

Cocaine-induced CPPThe ANOVA for CPP scores included the within-subjects factortest condition (Pre-C, Post-C, and Post-T) and the between-subjects factors SB216763 dose (0, 0.1, and 1 ng/side) andamygdala nucleus (CeA and BLA). This analysis revealed asignificant test condition · SB216763 dose · amygdalanucleus interaction (F4,98 = 3.012, p < 0.05). The post hocanalysis showed that after cocaine CPP training, all six groupsacquired CPP (p < 0.01), with no differences in CPP scoresbetween any two groups during Post-C (p > 0.05). Compared

Timeline(a)

(b)

(c)

(d)

CPP scores

Basolateral amygdala

Central amygdala

CPP training

(8 sessions)0 1 8 9 10 11 days

Pre-C

Pre-C



Total GSK-3β

Total GSK-3β

Pre-C

Saline Cocaine

Li 0 mg/kg

Li 30 mg/kg

Li 100 mg/kg

Saline Cocaine CocaineSaline

Saline Cocaine CocaineSaline

Per

cent

age

of n

aive

rat

s

150

*

100

50

0

Per

cent

age

of n

aive

rat

s

150

100

50

0

CP

P s

core

(s)

200

150

100

50

0

–50

Post-C

Post-C Post-C

Post-T

Post-T Post-T

Exposure

Lithiumor saline

Fig. 3 LiCl impaired the reconsolidation of cocaine reward memory

and this effect accompanied restored GSK-3b activity in the baso-

lateral amygdala. Data are expressed as mean ± SEM. (a) Beha-

vioral procedure. (b) Systemic administration of LiCl immediately

after exposure to the cocaine-paired context impaired the

reconsolidation of cocaine reward memory (n = 8–11 per group).

*p < 0.05, compared with Post-C in the 100 mg/kg LiCl group and

compared with the 0 mg/kg LiCl group in the Post-T test phase. (c–d)

Phosphorylated and total GSK-3b protein levels and representative

western blots in the basolateral and central amygdala following post-

treatment test. Data are expressed as a percentage of phosphory-

lated and total GSK-3b in naive control rats (n = 8). *p < 0.05,

compared with 0 mg/kg LiCl group in cocaine-conditioned rats. Pre-

C, pre-conditioning; Post-C, post-conditioning; Post-T, post-treat-

ment.


118 | P. Wu et al.

with the Post-C test, CPP scores significantly decreased only inthe group of rats that received a BLA microinjection of 1 ng/side SB216763 after cocaine reward memory reactivation(p < 0.001) in the Post-T test (Fig. 5b).

pGSK-3bAnalyses were performed separately for the data from thewestern blot assays after CeA and BLA microinjection ofSB216763 and included the between-subjects factorsSB216763 dose (0, 0.1, and 1 ng/side) and amygdalanucleus (CeA and BLA). The analysis of pGSK-3b in theamygdala after BLA infusions of SB216763 revealed asignificant SB216763 dose · amygdala nucleus interaction(F2,47 = 15.485, p < 0.01), which was attributable to the factthat SB216763 dose-dependently increased pGSK-3b only inthe BLA but not CeA (Fig. 5b). The analysis of pGSK-3b inthe amygdala after CeA infusions of SB216763 revealed nosignificant SB216763 dose · amygdala nucleus interaction(F2,47 = 0.678, p > 0.05; Fig. 5c).

Further confirmation of the effect of SB216763 on thereconsolidation of cocaine reward memoryAs shown in Fig. 6(b), microinjection of SB216763 into theBLA without reactivation of cocaine memory had noinfluence on cocaine-induced CPP. The ANOVA for CPPscores included the within-subjects factor test condition(Pre-C, Post-C, and Post-T) and between-subjects factorSB216763 dose (0 and 1 ng/side). This analysis revealed nosignificant test condition · SB216763 dose interaction(p > 0.05). Thus, the inhibitory effect of SB216763 on thereconsolidation of cocaine reward memory was dependent onthe reactivation of cocaine-induced CPP.

As shown in Fig. 6(d), intra-BLA SB216763 microinfu-sion 6 h after exposure to the cocaine-paired context on day10 had no effect on the subsequent expression of cocaine-induced CPP on day 11. The ANOVA for CPP scores includedthe between-subjects factor LiCl dose (0 and 1 ng/side) andthe within-subjects factor test condition (Pre-C, Post-C, andPost-T). This analysis of CPP scores revealed no main effectof test condition · SB216763 dose interaction (p > 0.05).The post hoc analysis showed that after cocaine CPPtraining, all cocaine groups acquired CPP (p < 0.01) withoutdifferences in CPP scores between the two groups during thePost-C or Post-T phase (p > 0.05). Compared with the Post-C test, CPP scores did not decrease in either group in thePost-T test on day 11 (p > 0.05).

Long-term effect of GSK-3b inhibition in the BLA on thereconsolidation of cocaine reward memoryAs shown in Fig. 7(b), SB216763-induced GSK-3b inhibi-tion in the BLA following cocaine memory reactivation hadlong-term effects on the reconsolidation of cocaine rewardmemory. In the group of rats that received SB216763,

Timeline (no exposure)(a)

(b)

(c)

(d)

(8 sessions)

CPP training

0 1 8 9 10 11 days

Lithiumor saline

Pre-C Post-C Post-T

CPP scores

Li 0 mg/kg Li 100 mg/kg

CP

P s

core

(s)

300

250

200

150

100

50

0

–50 Post-C Post-TPre-C

Pre-C

Timeline (6 h after exposure)Lithium

or saline

CPP training

(8 sessions)

Post-C

Post-C

Post-T

Post-T

Pre-C

0 1 8 9 106 h 11 days

Exposure

CPP scores

Li 0 mg/kg Li 100 mg/kg

CP

P s

core

(s)

300

250

200

150

100

50

0

–50

Fig. 4 LiCl had no effect on subsequent cocaine-induced CPP ex-

pression when injected without or 6 h after cocaine-paired context

exposure. Data are expressed as mean ± SEM. (a) Behavioral pro-

cedure of no-exposure experiment. (b) Systemic administration of LiCl

(100 mg/kg) without exposure to the cocaine-paired context had no

effect on the expression of cocaine-induced CPP on day 11 (n = 6 per

group). (c) Behavioral procedure of 6 h post-exposure experiment. (d)

Systemic administration of LiCl (100 mg/kg) 6 h after exposure to the

cocaine-paired context had no effect on the expression of cocaine-

induced CPP on day 11 (n = 6 per group). Pre-C, pre-conditioning;

Post-C, post-conditioning; Post-T, post-treatment.



cocaine-induced CPP did not recover when tested 14 dayspost-reactivation (Post-T 14). The effect of post-reactivationSB216763 administration was not attributable to theextinction of CPP because no significant differences inCPP scores were found between Post-T 14 and Post-C orbetween Post-T 14 and Post-T 1 (p > 0.05) in the vehicle-exposure groups and all non-exposure groups.

To examine whether the original cocaine reward memorywas persistently disrupted by post-reactivation administrationof SB216763, the rats received a priming dose of cocaine(5 mg/kg, i.p.), and CPP was tested again (i.e., priming test).A one-way ANOVA showed no significant differences in CPPscores between the priming test and Post-T 14 or Pre-C(p > 0.05), indicating that a priming injection of cocaine didnot reinstate cocaine-induced CPP. Altogether, these resultsindicate that the inhibitory effect of post-reactivationadministration of SB216763 on the reconsolidation ofcocaine reward memory lasted at least 2 weeks.

Discussion

The main findings of the present study were the following.Reactivation of cocaine reward memory increased GSK-3bactivity in the BLA, but not CeA. Systemic administration of

the GSK-3b inhibitor LiCl impaired the reconsolidation ofcocaine reward memory and restored the increased GSK-3bactivity in the BLA, and this effect was not because of theaversive effects of LiCl. Intra-BLA, but not intra-CeA,infusion of the selective GSK-3b inhibitor SB216763 had thesame effect as LiCl and impaired the reconsolidation ofcocaine reward memory. The disruption of cocaine rewardmemory lasted at least 2 weeks and was not reinstated by acocaine priming injection. Altogether, these findings suggestthat GSK-3b activity in the BLA is critical for thereconsolidation of cocaine reward memory. The effects inthe BLAwere anatomically and temporally specific. First, theactivation of cocaine reward memory was associated withincreased GSK-3b activity in the BLA but not CeA. Second,LiCl-induced GSK-3b inhibition immediately, but not 6 h,after cocaine reward memory reactivation disrupted thereconsolidation of cocaine reward memory, and the impair-ment effect was associated with changes in GSK-3b activityin the BLA, but not CeA. Third, GSK-3b inhibition in theBLA, but not CeA, disrupted the expression of cocaine-induced CPP in the Post-T test. Fourth, inhibition of GSK-3bactivity by LiCl or intra-BLA SB216763 injection withoutreactivation of cocaine reward memory had no effect oncocaine-induced CPP.

Timeline(a)

(b)

(c)

SB216763or vehicle

CPP training(8 sessions)

0 1 8 9 10 11 days

Post-TPost-CPre-C

Exposure

DMSOCPP test

BLA SB216763 microinjection

CeA SB216763 microinjection


CPP test Phosphorylated GSK-3β Total GSK-3β

Total GSK-3β

SB 0.1 ng

SB 1 ng

*

*

DMSO

SB 0.1 ng

SB 1 ng

200

150

100

50

–50

CP

P s

core

(s)

0

200

150

100

50

–50

CP

P s

core

(s)

0

150

100

50

Per

cent

of n

aive

rat

s

0

150

100

50

Per

cent

of n

aive

rat

s

0

BLA CeA BLA CeA

BLA CeA BLA CeA

Post-C Post-TPre-C

Post-C Post-TPre-C

Fig. 5 SB216763-induced GSK-3b inhibi-

tion in the basolateral but not central

amygdala impaired the reconsolidation of

cocaine reward memory. Data are ex-

pressed as mean ± SEM. (a) Behavioral

procedure. (b) Basolateral amygdala mi-

croinjection of SB216763 (1 ng/side) im-

mediately after exposure to the cocaine-

paired context impaired the reconsolidation

of cocaine reward memory with decreasing

GSK-3b activity in the basolateral but not

central amygdala (n = 8–11 per group).

*p < 0.05, compared with Post-C in SB 1

group and compared with dimethyl sulf-

oxide (DMSO) and SB 0.1 group in the

Post-T test phase. (c) Central amygdala

microinjection of SB216763 immediately

after exposure to the cocaine-paired con-

text had no effect on the reconsolidation of

cocaine reward memory and GSK-3 activity

in the basolateral and central amygdala

(n = 8–10 per group). Data from the

western blot assays are expressed as a

percentage of phosphorylated and total

GSK-3b in naive control rats. The western

blots of phosphorylated and total GSK-3b

shown are representative individual

samples. Pre-C, pre-conditioning; Post-C,

post-conditioning; Post-T, post-treatment.


120 | P. Wu et al.

Role of GSK-3b in addiction-associated behaviorsGlycogen synthase kinase-3b is widely expressed indopaminergic projection areas (Winder et al. 2002; Morganeet al. 2005; Robbins et al. 2008), such as the NAc, prefrontalcortex, and amygdala (Leroy and Brion 1999) and has beenshown to be involved in the regulation of dopamine-associated behaviors (Beaulieu et al. 2007a). Thus, somestudies investigated the role of GSK-3b in addiction-associated behaviors. For example, mice injected withcocaine showed a reduction in the phosphorylation ofGSK-3b in the caudate putamen (Miller et al. 2009), andvalproate- or SB216763-induced GSK-3b inhibition reducedlocomotor hyperactivity induced by acute exposure toamphetamine or cocaine (Beaulieu et al. 2004; Gould andManji 2005; Miller et al. 2009). In addition, Perrine et al.(2008) reported that 14 days, but not 1 day, of chroniccocaine administration increased GSK-3b activity in theamygdala. Our recent study also found that GSK-3b activityin the NAc core was elevated by chronic cocaine exposure,

Timeline(a)

(b)

SB216763or vehicle


0 1 8 9 10 11 25 26 days

Priming

Priming

Post-T1

Post-T14

Post-T14Post-C

CPP score

Post-C

Post-T1

* * *

Pre-C

Pre-C

Exposure

DMSO

SB 1 ng

300

250

200

150

100

50

–50

CP

P s

core

(s)

0

Fig. 7 SB216763-induced GSK-3b inhibition in the basolateral

amygdala immediately following cocaine memory reactivation caused

long-lasting impairment of the expression of cocaine reward memory,

which was not restored by acute priming cocaine injections. (a) Be-

havioral procedure. (b) The inhibitory effect of SB216763 on the ex-

pression of cocaine-induced CPP lasted at least 14 days (n = 10).

When given 5 mg/kg cocaine, rats that received a microinjection of

SB216763 (1 ng/side) into the BLA immediately after exposure did not

exhibit cocaine-induced CPP. Pre-C, pre-conditioning; Post-C, post-

conditioning; Post-T 1, post-treatment 1; Post-T 14, post-treatment 14;

Priming, priming by 5 mg/kg cocaine.

Timeline (no exposure)(a)

(b)

(c)

(d)

Timeline (6 h after exposure)

Exposure

SB216763or vehicle


0 1 8 9 10 11 days

Post-TPost-CPre-C

CPP score

DMSO

SB 1 ng

DMSO

SB 1 ng

200

150

100

50

0

–50

CP

P s

core

(s)

Post-C Post-TPre-C

Post-C Post-TPre-C

Post-C Post-TPre-C

SB216763or vehicle


0 1 8 9 10 6 h 11 days

CPP score

250

200

150

100

50

–50

CP

P s

core

(s)

0

Fig. 6 Microinjection of SB216763 into the basolateral amygdala

without exposure or 6 h after exposure to the cocaine-paired context

had no effect on the reconsolidation of cocaine reward memory. (a)

Behavioral procedure of no-exposure experiment. SB212763 was in-

jected once, 24 h after the Post-C test. (b) Microinjection of SB216763

into the basolateral amygdala without exposure to the cocaine-paired

context did not impair the reconsolidation of cocaine reward memory

(n = 8–10 per group). (c) Behavioral procedure of 6 h post-exposure

experiment. SB212763 was injected once, 6 h after the reactivation of

cocaine reward memory. (d) Microinjection of SB216763 into the ba-

solateral amygdala 6 h after exposure to the cocaine-paired context

did not impair the reconsolidation of cocaine reward memory (n = 8–9

per group). Pre-C, pre-conditioning; Post-C, post-conditioning; Post-T,

post-treatment.



and inhibition of GSK-3b activity by systemic LiCladministration or intra-NAc core injections of SB216763suppressed the initiation and expression of cocaine-inducedbehavioral sensitization (Xu et al. 2009). But all thesestudies focus on the alterations of GSK-3b after acute orchronic drug exposure. Our present study extends thesefindings and showed that exposure to cocaine-pairedcontextual cues increased GSK-3b activity in the BLA, andinhibition of GSK-3b activity by systemic LiCl administra-tion or intra-BLA injection of SB216763 immediately afterthe reactivation of cocaine cue memory impaired thesubsequent preference of rats for the drug context cue. Toour knowledge, this is the first study to reveal the critical roleof GSK-3b in the persistence of the incentive value ofcocaine cue. Our study also supports the notion that the BLAis a site involved in the processing of emotional eventsrelated to environmental stimuli that guide motivatedbehavior (Cardinal et al. 2002; Everitt et al. 2003).

Lithium chloride has been used for its anti-manic andantidepressant effects (Sachs 1996; Kleindienst and Greil2003). Increasing evidence suggests that LiCl can be used inthe treatment of acute brain injury (e.g., ischemia) (Chuang2005; Wada et al. 2005) and chronic neurodegenerativediseases (e.g., Alzheimer’s disease) (Barcikowska 2004; Giese2009). Recently,multiple lines of evidence have shown that theeffects of LiCl on mood stabilization and neurogenesis areattributable to LiCl-induced GSK-3b inhibition (Wada 2009).A previous study (Xu et al. 2009) and our present resultsindicate that GSK-3b is involved in neuroadaptation andbehavioral adaptation after repeated cocaine exposure andmediates the conditioned response to cocaine contextual cues.These results are consistent with and extend previous studies,in which LiCl significantly reduced self-stimulation facilitatedby morphine (Liebman and Segal 1976), reduced voluntarymorphine ingestion in addicted rats (Tomkiewicz and Stein-berg 1974), decreased amphetamine- and cocaine-inducedhyperactivity (Beaulieu et al. 2004; Xu et al. 2009), attenu-ated the morphine withdrawal syndrome (Dehpour et al.1995), and had anti-euphoric effects in drug-free opiate addicts(Jasinski et al. 1977). Our study also demonstrated that GSK-3b may be a possible molecular mechanism of the effect ofLiCl on addiction-associated behavior, and LiCl, a GSK-3binhibitor, may be useful in the treatment of drug addiction(Flemenbaum et al. 1979; Abrahamson 1983).

Role of GSK-3b in the BLA in learning and memoryGlycogen synthase kinase-3b is abundant in the brain, bothin neurons and glia (Ferrer et al. 2002). GSK-3b has beenshown to regulate many transcription factors, includingactivator protein-1, cyclic adenosine monophosphate re-sponse element binding protein, b-catenin, CCAAT/enhancerbinding protein, and nuclear factor j-light-chain-enhancer ofactivated B cells (NF-jB) (Frame and Cohen 2001), whichhave been implicated in fundamental brain functions, such

as neurogenesis, the development of neuronal tissue, theregulation of synaptogenesis, axonal growth cone collapse(Lucas et al. 1998; Eickholt et al. 2002; Packard et al.2003), neurotransmitter signaling (Beaulieu et al. 2004,2007b; Li et al. 2004), circadian rhythms (Yin et al. 2006),synaptic plasticity, and memory (Hooper et al. 2007;Peineau et al. 2007, 2008, 2009). Specifically, GSK-3bactivity is involved in the regulation of both NMDAreceptor-dependent LTP and LTD, as well as the cross-talkbetween them (Peineau et al. 2007, 2008, 2009). GSK-3bactivity increased following LTD induction, and GSK-3binhibitors blocked LTD induction (Peineau et al. 2007,2009). Conversely, GSK-3b activity decreased followingLTP (Hooper et al. 2007; Peineau et al. 2007), and theinduction of LTP disrupted the ability of synapses toundergo LTD for up to 1 h in rat hippocampal slices(Peineau et al. 2007). In addition, increased GSK-3b activityinduced by wortmannin or transient over-expression ofwildtype GSK-3b suppressed the induction of LTP in thehippocampus in rats, whereas simultaneous inhibition ofGSK-3b by lithium or SB216763 or transient expression of adominant-negative GSK-3b mutant preserved LTP (Zhuet al. 2007). Furthermore, a recent study using heterozygoteGSK-3b knockout (GSK-3b+/)) mice and a GSK-3binhibitor showed that GSK-3b is required for memoryreconsolidation in adult brain (Kimura et al. 2008). Hetero-zygote GSK-3b knockout mice had impaired memoryreconsolidation and intraperitoneal injection of a GSK-3binhibitor before memory reactivation impaired fear memoryreconsolidation in wildtype mice. Although these resultsindicate that GSK-3b activation might be required during thereconsolidation process, but the brain sites involved in thisprocess have not yet known. Our present study found thatsystemic administration of the GSK-3b inhibitor LiClimpaired the reconsolidation of cocaine reward memoryand inhibited the BLA GSK-3b activation that wouldotherwise be induced by exposure to cocaine paired context.Intra-BLA infusion of SB216763, a selective GSK-3binhibitor, had the same effect as LiCl and impaired thereconsolidation of cocaine reward memory. These effectswere BLA-specific because intra-CeA infusion of SB216763had no effect on memory. Our present study also supportsour previous study and others’ evidence that the BLA is acritical substrate for the reconsolidation of emotionalmemories, including fear memories (Nader et al. 2000a;Duvarci et al. 2005), drug-conditioned withdrawal memories(Hellemans et al. 2006), and appetitive drug memories(Thomas et al. 2003; Tronson et al. 2006; Milton et al.2008; Wang et al. 2008; Fuchs et al. 2009; Li et al. 2010;Sanchez et al. 2010).

Concluding remarksIn summary, we demonstrated that established cocaine-related memories are disrupted by GSK-3b inhibition in


122 | P. Wu et al.

the BLA after memory reactivation via reconsolidationimpairment. Furthermore, our results indicate that the BLAis a critical brain region involved in the integration ofthe influence of GSK-3b activity on cocaine-relatedmemory. Our results suggest the potential therapeuticvalue of GSK-3b inhibitors in the treatment of cocaineaddiction.

Acknowledgements

This work was supported in part by the National Basic Research

Program of China (No. 2009CB522004), and Natural Science

Foundation of China (No. 31070958 and 30725016). The funders

had no role in study design, data collection and analysis, decision to

publish, or preparation of the manuscript. The authors would like to

thank Dr. Yavin Shaham for his comments on the manuscript. The

authors declare that they do not have any conflicts of interest

(financial or otherwise) related to the data presented in this

manuscript.

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