Opiate withdrawal induces dynamic expressions of AMPA receptors and its

Opiate withdrawal induces dynamic expressions of AMPA receptors and its

regulatory molecule CaMKII αin hippocampal synapses

Weixia Zhong a,1,Zhifang Dong b,1,Meng Tian b ,Jun Cao b ,Tianle Xu c ,

Lin Xu b,c,d,?,Jianhong Luo a,?

a

Department of Neurobiology,Zhejiang University School of Medicine,Hangzhou 310006,PR China

b Laboratory of Learning and Memory,Kunming Institute of Zoology,the Chinese Academy of Sciences,Kunming 650223,PR China c

Institute of Neuroscience,Shanghai Institutes for Biological Sciences,the Chinese Academy of Sciences,Shanghai 200031,PR China d

Mental Health Institute,the 2nd Hospital of Xiangya Medical College,Central South University,Changsha 410011,PR China

Received 30December 2005;accepted 28February 2006

Abstract

Adaptive changes in brain areas following drug withdrawal are believed to contribute to drug seeking and relapse.Cocaine withdrawal alters the expression of GluR1and GluR2/3subunits of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)receptors in nucleus accumbens or amygdala,but the influence of drug withdrawal on hippocampus is little known.Here,we have examined the expression of GluR1and GluR2/3in hippocampal membrane and synaptic fractions following repeated morphine exposure and subsequent withdrawal.Repeated morphine exposure for 12d increased GluR1and GluR2/3in synaptosome but not in membrane fraction.Interestingly,CaMKII α,known to be able to regulate the function of AMPA receptors,was decreased in synaptosome but not in membrane fraction;pCaMKII α,the phosphorylated form of CaMKII α,was increased in both fractions.However,during opiate withdrawal,GluR1was generally reduced while GluR2/3was prominently increased in both fractions;pCaMKII αwas strongly decreased immediately after withdrawal,but detectably increased in late phase of morphine withdrawal in both fractions.Importantly,the opiate withdrawal-induced increase in GluR2/3was dependent on the activation of glucocorticoid receptors and NMDA receptors,as it was prevented by the glucocorticoid receptor antagonist RU38486,or intrahippocampal injection of the NMDA receptor antagonist AP-5or the antagonist to NR2B-containing NMDA receptors,Ro25-6981.These findings indicate that opiate withdrawal induces dynamic expression of GluR1and GluR2/3subunits of AMPA receptors in hippocampal synapses,possibly revealing an adaptive process of the hippocampal functions following opiate withdrawal.?2006Elsevier Inc.All rights reserved.

Keywords:AMPA receptor;Phosphorylation;CaMKII α;Opiate withdrawal;Synaptosome

Introduction

Drugs of abuse are known to cause maladaptive synaptic plastic changes in brain areas such as nucleus accumbens

(NAc),the ventral tegmental area (VTA),prefrontal cortex (PFC),and amygdala as well as hippocampus (Hyman and Malenka,2001;Nestler,2001a;Pu et al.,2002;Glass et al.,2005;Lu et al.,2005).Recent evidence suggests that drug withdrawal enhances the incentive value of the drug,leading to compulsive drug seeking behavior (Lu et al.,2003;Bowers et al.,2004;Frenois et al.,2005).The α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPA)type of glutamate receptors are composed of various combinations of four homologous subunits GluR1to GluR4(Nuriya et al.,2005)to form distinct receptor subtypes (Song and Huganir,2002).The majority of AMPA receptors in adult hippocam-pal pyramidal cells are GluR1/GluR2and

GluR2/GluR3

Life Sciences 79(2006)861–

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?Corresponding authors.Luo is to be contacted at Department of Neurobiology,Zhejiang University School of Medicine,Hangzhou 310006,PR China.Tel.:+8657187217010;fax:+8657187217044.Xu,Laboratory of Learning and Memory,Kunming Institute of Zoology,the Chinese Academy of Sciences,Kunming 650223,PR China.Tel.:+868715139165;fax:+868715191823.

E-mail addresses:lxu@https://www.360docs.net/doc/484819660.html, (L.Xu),luojianhong@https://www.360docs.net/doc/484819660.html, (J.Luo).1

These authors contributed equally to this work.0024-3205/$-see front matter ?2006Elsevier Inc.All rights reserved.doi:10.1016/j.lfs.2006.02.040

heteromeric complexes(Wenthold et al.,1996;Kakegawa et al., 2004).The function of the AMPA receptor(AMPAR)is regulated in multiple ways,including dynamic changes in the density of receptors(Banke et al.,2000;Nuriya et al.,2005), which is believed to be one of the mechanisms playing key roles in manifesting synaptic plasticity(Carroll et al.,2001;Shi et al., 2001;Nuriya et al.,2005).In addition to receptor trafficking, the function of AMPA receptors is regulated by Ca2+/ calmodulin-dependent protein kinase II(CaMKII),a ubiquitous protein kinase that acts as a key factor in synaptic plasticity (Bayer et al.,2001).Ca2+-calmodulin complex binding to thr286in the regulatory domain ofαisoform of CaMKII triggers autophosphorylation of this kinase,leading to its activation in the phosphorylated form of CaMKII(pCaMKII) (Mahadev et al.,2001).Following cocaine withdrawal,the expression of GluR1and GluR2/3in NAc is strongly increased (Boudreau and Wolf,2005).Moreover,it has been demonstrat-ed that CaMKII plays important roles in the maladaptive processes of drug addiction(Suenaga et al.,2004).Repeated morphine exposure is associated with changes in several signaling pathways,including CaMKII,that affect the function and targeting of GluR1(Sharma et al.,2003).

However,it is little known how opiate withdrawal will influence the synaptic expression of GluR1and GluR2/3 subunits of AMPA receptors and the regulatory molecule CaMKIIαin the hippocampus.Thus,it is possible that the expression or phosphorylation state of CaMKIIαin hippocam-pus synapses will be changed,which may then affect the synaptic expression of GluR1-and GluR2/3-containing AMPA receptors.In the present study,we examined the effects of repeated morphine exposure and subsequent withdrawal on the level of GluR1,GluR2/3,CaMKIIαand pCaMKIIαin hippocampal membrane and synaptosomal fractions.We then examined whether these changes could be affected by behavioral stress or administration of NMDA receptors antagonists.

Materials and methods

Animal treatment

Experiments were carried out on male Sprague–Dawley rats(inbred strain,Animal House Center,Kunming Medical College,Kunming),weighing200–250g.Animals were group-housed with free access to water and food,in a thermo-regulated environment with a12-h light/dark cycle.Animal care and experimental protocols were approved by the Chinese Academy of Sciences and Zhejiang University School of Medicine,PR China.Animals were chronically treated with morphine(Mor,10mg/kg,s.c.)twice per day for 12consecutive days,a procedure known to produce significant tolerance and dependence to the drug(Pu et al., 2002;Yang et al.,2004)and their body weight and food consumption was monitored.Control animals were injected with saline(Sal,0.6ml/kg,s.c.)according to the same procedure.Then,the animals were withdrawn from morphine for2and18h,and4,7and20d,respectively.(1)Behavioral stress was evoked by elevated platform stress as described previously(Xu et al.,1997,1998;Xiong et al.,2003;Rocher et al.,2004;Yang et al.,2004).Animals withdrawn from morphine for15.5h were placed on an elevated platform for 30min,and returned to their home cage for2h,after which the expression levels of GluR1,GluR2/3,CaMKIIαand pCaMKIIαat18h of withdrawal were analyzed.(2) RU38486(20mg/kg,s.c.)was given twice per day for the first three days of morphine withdrawal,and then the protein expression levels on4d of withdrawal were analyzed.(3) The NMDA receptor antagonist AP-5(10mM per cannula, 1μl,6min)or Ro25-6981,an antagonist at NR2B-containing NMDA receptors(1mM per cannula,1μl,6min)was infused to the surface of the dorsal hippocampus through bilateral guide cannulae that had been implanted2weeks (recovery period)before the procedure of repeated morphine treatment,using techniques similar as those described previously(Padovan and Guimaraes,2004).The infusions were given twice per day for the first three days of morphine withdrawal and the protein level on4d of withdrawal was analyzed.The guide cannula placement was histologically verified post hoc in all rats by infusion of methylene blue dye,and only data obtained from rats with correctly inserted cannulae were included in statistical analysis.After treatment described as above,animals were sacrificed immediately and hippocampal tissues were dissected on ice.

Membrane protein extraction

Membrane protein extraction was carried out using techni-ques described previously(Luo et al.,1997;Zhu et al.,2004;Xu et al.,2005).Briefly,the hippocampal tissue was homogenized twice in100vol.(100×wet weight)of ice-cold10mM Tris–HCl(pH7.4)containing0.32M sucrose at speed5for10s with a20s interval between bursts.The tissue homogenate was centrifuged at700×g for10min at4°C.The supernatant was collected and centrifuged at37,000×g for40min at4°C.The pellets were resuspended in10mM Tris–HCl(pH7.4). Synaptosome preparation

Standard homogenization and ultracentrifugation procedures were used to isolate synaptosomes(Hensley et al.,1994; Witzmann et al.,2005),a subcellular fraction enriched in synaptic proteins(Nayak et al.,1996).Homogenization was performed using a glass homogenizer and a teflon pestle in homogenization buffer containing(in mM):10HEPES, 1EGTA,1EDTA,10Na4P2O7,150NaCl,50NaF,with the protease inhibitors,2μg/mL aprotinin,10μg/mL leupeptin, 4μg/ml pepstatin,and10μg/ml trypsin inhibitor.Homogenates were centrifuged at1000×g for10min to obtain the crude nuclear pellets(P1)and the supernatant(S1).The S1fraction was centrifuged at17,000g for15min to obtain the crude plasma membrane fraction(P2),which was used for the preparation of synaptosomal fractions.The P2fraction was resuspended in the homogenizing buffer,and layered on top of a discontinuous sucrose density gradient consisting of1.2M

862W.Zhong et al./Life Sciences79(2006)861–869

sucrose and0.8M sucrose.The gradient was centrifuged at 54,000×g for90min at4°C in an SW28rotor in a Beckman L-XP refrigerated ultracentrifuge,and the synaptosomal fraction was removed from the interface between0.8and1.2M sucrose. This fraction was slowly diluted with10vol.of ice-cold0.32M sucrose,centrifuged at20,000×g for15min,and the resulting pellets were used for synaptosome protein extraction.Synap-tosome pellets were solubilized in the homogenizing buffer.The protein concentration of synaptosome protein extracts and membrane protein extracts was determined using Folin phenol reagent with bovine serum albumin as a standard(Lowry method)and adjusted to10mg/ml.The samples were frozen at ?80°C until used.

Semi-quantitative Western blotting analysis

Samples containing equal protein amounts(typically10μg/ lane)were loaded onto10%sodium dodecyl sulfate(SDS) polyacrylamide gels(4%stacking gel)and resolved by standard electrophoresis(Bio-Rad,Hercules,CA,USA).According to the previous methods(Luo et al.,1997;Zhu et al.,2004;Xu et al.,2005),we transferred proteins to nitrocellulose membranes in transfer buffer.After blocking,the membranes were then incubated with anti-GluR1(1:200),anti-GluR2/3(1:500),anti-CaMKIIα(1:500)and anti-pT286-CaMKII(1:8500)antibodies (all obtained from Chemicon,Temecula,CA,USA).Immuno-reactive membranes were incubated with horseradish peroxi-dase-conjugated secondary antibody(1:5000).The light emission was detected with enhanced chemiluminescence (Amersham Life Science).Autoradiographs were scanned using a Bio-Rad imaging densitometer and quantified using Quantity One-4.4.0Analysis software.

Data analysis and statistics

In order to make different gel samples comparable,every gel was run with three lanes of the same cortex proteins as a standard(Luo et al.,1997;Zhu et al.,2004;Xu et al.,2005).The values from saline control were used as a basal level for normalization of the experimental data.So after standardized with the average cortex on the same gel,the values for the experimental animals are presented as the percentage of the mean values of saline control that were not included in the behavioral experiments and expressed as the mean±S.E.M%. Statistical comparisons in Western blot studies were made by using unpaired t-test or least significant difference test of one-way ANOV A followed by post hoc analysis.Significance level was set at p<0.05.

Results

Dynamic expression of AMP A receptor subunits GluR1 and GluR2/3in hippocampal synapses during morphine withdrawal

We used specific antibodies to GluR1and GluR2/3to study their expression following repeated morphine exposure and subsequent morphine withdrawal by Western blot technique.

The total membrane fraction and the synaptic fractions

(synaptosomes)were extracted from the hippocampal tissues

taken from animals that were repeatedly treated with saline

(Sal)or morphine(Mor),and withdrawn from morphine for

2h,18h,4d,7d or20d.Characterization revealed an

enrichment of postsynaptic density protein95(PSD95)in

synaptosomes;the cytoskeletal protein(tubulin)was also

weakly detected(not shown).Compared with saline control,

repeated morphine exposure significantly increased the

expression of GluR1and GluR2/3subunits of AMPA

receptors in hippocampal synaptic fraction,but did not affect

their expression in membrane fraction(Fig.1,?p<0.05Mor vs.Sal).This implies that repeated morphine exposure may

increase the insertion of GluR1and GluR2/3into synapses,

consistent with a previous report that acute morphine elicited

synaptic potentiation in the CA1area of the hippocampus in

vivo(Yang et al.,2004).Remarkably,following morphine

withdrawal,GluR1level was generally reduced,but GluR2/3

expression was robustly increased in both fractions,compared

with either saline control or chronic morphine treated

group Sal Mor 2 h18 h 4 d7 d20 d

R

e

l

a

t

i

v

e

l

e

v

e

l

(

%

o

f

s

a

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i

n

e

)

* #

GluR1

GluR2/3

Sal

m s

Mor 2 h18 h 4 d7 d20 d

m s m s m s m s m s m s

Fig.1.Dynamic expression of GluR1and GluR2/3in hippocampal synapses during morphine withdrawal.Repeated morphine exposure and subsequent withdrawal altered expression levels of AMPA receptor subunits GluR1and GluR2/3in hippocampus.Upper panel:representative immunoblots of GluR1 and GluR2/3in membrane(m)and synaptosome(s)fractions from hippocampus of saline control(Sal)and chronic morphine treated rats(Mor)and rats withdrawn from2h to20d(2h,18h,4d,7d,20d).Lower panel:expression status of GluR1and GluR2/3subunits in synaptosome(s)and membrane(m) from hippocampus of saline control(Sal)and chronic morphine treated rats (Mor)and rats withdrawn from2h to20d.(?p<0.05Mor,2h,18h,4d,7d, and20d vs.Sal,n=6;#p<0.052h,18h,4d,7d,and20d vs.Mor,n=6;

a p<0.054d vs.2h,18h,7d,and20d,n=6).One-way ANOV A followed by post hoc analysis;n,number of animals.

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(Fig.1,?p <0.052h to 20d vs.Sal;#p <0.052h to 20d vs.Mor).Among morphine withdrawal groups (2h to 20d),GluR2/3expression of 4d group was at the highest level in membrane but at the lowest level in synaptosomal fraction (Fig.1,a p <0.054d vs.2h,18h,7d,20d).These findings suggest that GluR2/3might be withdrawn from synapses during the early phase of morphine withdrawal (2h to 4d).Conversely,GluR2/3might be inserted into synapses during the late phase of morphine withdrawal (7to 20d).Dynamic expression of CaMKII αand pCaMKII αin hippo-campal synapses during morphine withdrawal

The findings that repeated morphine exposure and subse-quent morphine withdrawal caused dynamic expression of GluR1and GluR2/3of AMPA receptors in hippocampal synapses led us to examine the levels of the general CaMKII and pCaMKII in hippocampal membrane and synaptosomal fractions by Western blot technique.Immunoblots of pCaM-KII αshow a major band at 50kDa and a nonspecific minor band,which might be produced by cross-reactivity between its polyclonal antibody and other CaMKII https://www.360docs.net/doc/484819660.html,pared to saline control,repeated morphine exposure did not influence CaMKII αexpression in the membrane fraction (p >0.05)but moderately decreased the expression in the synaptic fraction

(Fig.2,?p <0.05Mor vs.Sal).Remarkably,repeated morphine exposure slightly increased the expression of pCaMKII αin the membrane fraction and strongly enhanced the expression in the synaptic fraction (Fig.2,?p <0.05Mor vs.Sal).Following morphine withdrawal,compared with saline control,the level of CaMKII αwas reduced in membrane and synaptosomal fractions at 4d of withdrawal (Fig.2,?p <0.054d vs.Sal),and remained unchanged in both fractions at other time points of withdrawal (Fig.2,p >0.052h,18h,7d and 20d vs.Sal).However,the expression of pCaMKII αin the membrane fraction was strongly decreased on the first day after withdrawal (2and 18h)and then gradually recovered to a level similar to that in saline control (Fig.2,?p <0.052and 18h vs.Sal;p >0.054to 20d vs.Sal).Moreover,if compared with repeated morphine group,CaMKII αwas decreased only on 4d after withdrawal (Fig.2,#p <0.054d vs.Mor;p >0.052h,18h,7d,20d vs.Mor).The level of pCaMKII αin the synaptosomal fraction was strongly decreased during the early phase of withdrawal (2h to 4d)but did not show significant difference through the late phase of withdrawal (7to 20d)(Fig.2,#

p <0.052h,18h,4d vs.Mor;p >0.057d,20d vs.Mor).These results suggest that repeated morphine exposure or prolonged morphine withdrawal might induce the transforma-tion of CaMKII αinto pCaMKII αin hippocampal synapses,converting transient signals into long-term changes in the hippocampal

function.

CamKII α-m R e l a t i v e l e v e l (% o f s a l i n e )

CaMKII α pCaMKII α

Sal m s

Mor 2 h 18 h m s

4 d 7 d 20 d m s

m s

m s

m s

m s

Fig.2.Dynamic expression of CaMKII αand pCaMKII αin hippocampal synapses during morphine withdrawal.Repeated morphine exposure and subsequent withdrawal altered expression levels of the general CaMKII αand pCaMKII αin hippocampus.Upper panel:representative immunoblots of the general CaMKII αand pCaMKII αin membrane (m)and synaptosome (s)fractions from hippocampus of saline control (Sal)and chronic morphine treated rats (Mor)and rats withdrawn from 2h to 20d (2h,18h,4d,7d,20d).Lower panel:summary of the general CaMKII αand pCaMKII αexpression in synaptosome (s)and membrane (m)from hippocampus of experimental animals.(?p <0.05Mor,2h,18h,4d,7d,and 20d vs.Sal,n =6;#p <0.052h,18h,4d,7d,and 20d vs.Mor,n =6;a p

<0.054d vs.2h,18h,7d,and 20d,n =6).One-way ANOV A followed by post hoc analysis;n ,number of animals.

R e l a t i v e l e v e l (% o f s a l i n e )

GluR1GluR2/3

18 h m s

Stress 4 d RU38486m s

m

s m s

Fig. 3.GluR1and GluR2/3expression in hippocampal synapses during withdrawal affected by behavioral stress.Behavioral stress and glucocorticoid receptors antagonist changed the dynamic expression levels of AMPA receptor subunits GluR1and GluR2/3in hippocampus.Upper panel:representative immunoblots of membrane fraction (m)and synaptosome (s)prepared from hippocampus of the withdrawal for 18h group (18h),the stressed group (Stress)and the withdrawal for 4days group (4d),the RU38486administration group (RU38486)characterized with specific antibodies to GluR1and GluR2/3respectively.Lower panel:summary graph showing data expressed as average percent of Sal (mean±S.E.M.)in GluR1and GluR2/3.(#p <0.05stress vs.18h,n =6;?p <0.05RU38486vs.4d,n =6).Unpaired t -test;n ,number of animals.

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Behavioral stress affected the synaptic expressions of GluR1and GluR2/3and the regulatory molecule CaMKII αin hippocampus during withdrawal

Then we examined whether additional behavioral stress during withdrawal could influence the synaptic expression of GluR1,GluR2/3,CaMKII αand pCaMKII αin hippocampus.The animals undergoing behavioral stress are denoted as Stress.We found that GluR1in the membrane but not the synaptosomal fraction from these animals was moderately reduced and GluR2/3in both membrane and synaptosomal fractions was markedly decreased,compared with those obtained 18h after withdrawal in animals not exposed to elevated platform stress (Fig.3,#p <0.05Stress vs.18h;unpaired t -test).Conversely,CaMKII αwas slightly increased but pCaMKII αwas strongly increased in both fractions (Fig.4,#p <0.05Stress vs.18h;unpaired t -test).These findings suggested that the synaptic expression of GluR2/3and pCaMKII α,but not GluR1and CaMKII αin hippocampus,was regulated by additional behavioral stress during withdrawal.The hippocampus is enriched with glucocorticoid receptors,which are crucially involved in regulating stress effects (McEwen and Sapolsky,1995;Kim et al.,2001).Therefore,we further examined the effect of the glucocorticoid receptor antagonist RU38486on the expression of GluR1,GluR2/3,CaMKII αand pCaMKII αin both fractions.Remarkably,GluR1at membrane and synaptic fractions did not change,but GluR2/3at membrane and

synaptosomal fractions was strongly decreased,compared to levels on 4d after withdrawal without the administration of RU38486(Fig.3,?p <0.05RU38486vs.4d;unpaired t -test).On the other hand,CaMKII αin the synaptosomal fraction was slightly decreased and pCaMKII αin the membrane fraction was mildly increased (Fig.4,?p <0.05RU38486vs.4d;unpaired t -test).These findings implied that the expression of GluR2/3in hippocampal synapses following morphine withdrawal,but not that of GluR1,CaMKII αand pCaMKII α,was strongly regulated by the activation of glucocorticoid receptors.Taken together,these findings suggest that stress during morphine withdrawal might contribute,at least partially,to the long-term withdrawal-related changes of hippocampal synaptic plasticity.Blockade of NR2B-containing NMDA receptors regulated the synaptic expression of GluR1and GluR2/3and the regulatory molecule CaMKII αin hippocampus during withdrawal We further examined whether the blockade of NMDA receptors (with AP-5)or NR2B-containing NMDA receptors (with Ro25-6981)affected the dynamic expression of GluR1,GluR2/3,CaMKII αand pCaMKII αduring withdrawal.We found that the expression of GluR1in both membrane and synaptosomal fractions remained unchanged (p >0.05),com-pared with the expression seen on 4d of withdrawal without

any

100

200

300

400

500

R e l a t i v e l e v e l (% o f s a l i n e )

CamKII αpCamKII α

18 h m s

Stress 4 d Ru38486m s

m s

m s

Fig.4.The general CaMKII α,and pCaMKII αexpression in hippocampal

synapses during withdrawal affected by behavioral stress.Behavioral stress and glucocorticoid receptors antagonist changed the expression levels of the general CaMKII α,and pCaMKII αin hippocampus during withdrawal.Upper panel:representative immunoblots of membrane fraction (m)and synaptosome (s)prepared from hippocampal of the withdrawal for 18h group (18h),the stressed group (Stress)and the withdrawal for 4days group (4d),the RU38486administration group (RU38486)characterized with specific antibodies to CaMKII

α,and pCaMKII αrespectively.Lower panel:summary graph showing data expressed as average percent of Sal (mean±S.E.M.)in general CaMKII α,and pCaMKII α.(#p <0.05stress vs.18h,n =6;?p <0.05RU38486vs.4d,n =6).Unpaired t -test;n ,number of

animals.

4 d Ap-5Ro25-6981

50

100

150

200

250

R e l a t i v e l e v e l (% o f s a l i n e )

GluR2/3

GluR1

4 d m s

AP-5m s

Ro25-6981m s

Fig. 5.Blockade of NR2B-containing NMDA receptors regulates the hippocampal synaptic expression of GluR1and GluR2/3during withdrawal.Administration of the NMDA receptor antagonist AP-5,and NR2B-specific antagonist Ro25-6981partially altered amounts of AMPA receptor subunits GluR1and GluR2/3in hippocampus.Upper panel:representative immunoblots of hippocampal membrane fraction (m)and synaptosome (s)from the withdrawal for 4days group (4d),the AP-5infusion group (AP-5),the Ro25-6981infusion group (Ro25-6981)characterized with specific antibodies to GluR1and GluR2/3respectively.Lower panel:summary graph showing amount of GluR1and GluR2/3.(#p <0.05AP-5and Ro25-6981vs.4d,n =6).Unpaired t -test;n ,number of animals.

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treatment.Similar to the effects of RU38486,the expression of GluR2/3in membrane and synaptosomal fractions was strongly decreased by both AP-5and Ro25-6981(Fig.5,#p <0.05AP-5or Ro25-6981vs.4d;unpaired t -test).Unlike the effects of RU38486,the expression of CaMKII αwas moderately enhanced and pCaMKII αwas strongly increased in membrane and synaptosomal fraction by both AP-5and Ro25-6981(Fig.6,#p <0.05AP-5or Ro25-6981vs.4d;unpaired t -test).Thus,these findings suggest that the blockade of hippocampal NMDA receptors or hippocampal NR2B-containing NMDA receptors also influenced the dynamic expression of GluR2/3,CaMKII αand pCaMKII αin hippocampal synapses during morphine withdrawal.Discussion

Adaptive process of the hippocampal functions following opiate withdrawal

Hippocampal synaptic plasticity plays important roles in learning and memory (Malenka and Nicoll,1999;Martin et al.,2000;Yang et al.,2004).However,recent evidence from behavioral (Fan et al.,1999;Lu et al.,2000;Mitchell et al.,2000)and electrophysiological studies (Mansouri et al.,1999;

Pu et al.,2002)has suggested that the hippocampus is also involved in drug addiction (Berke and Hyman,2000;Hyman and Malenka,2001;Nestler,2001b ).In the present study,by comparing the protein level between the membrane and synaptosomal fractions prepared from the hippocampus,we demonstrated protein insertion into synapses or withdrawal from synapses,and we have found that there also may be a substantial pool of proteins located at extrasynaptic sites.Here,we found that repeated morphine exposure increased prominently the hippocampal synaptic expression of GluR1,GluR2/3and pCaMKII α,consistent with a previous report that AMPAR subunit levels were increased after cocaine self-administration (Boudreau and Wolf,2005;Lu et al.,2003;Tang et al.,2004).Here,our evidence suggested that opiate withdrawal could produce long-term changes in GluR1and GluR2/3of AMPARs and CaMKII αas well as pCaMKII αat hippocampal synapses,i.e.changes in signaling molecules known to play crucial roles in synaptic plasticity and learning and memory (Bevilaqua et al.,2005),indicating alteration of the hippocampal function after chronic morphine treatment and subsequent withdrawal.However,different subtypes of AMPARs play distinct roles in regulating AMPAR trafficking and synaptic plasticity (Meng et al.,2003).In this study,following opiate withdrawal,GluR1was generally reduced but GluR2/3was further increased in hippocampus (Fig.1),suggesting that some sorts of synaptic plasticity were induced by opiate withdrawal.CaMKII αwas slightly decreased throughout the withdrawal period,but pCaMKII αat hippo-campal synapses,after an initial immediate reduction,gradually recovered to a level similar to that seen in after repeated morphine treatment,a level which was distinctly higher than that seen in the saline group (Fig.2).Since CaMKII may drive extrasynaptic AMPARs into synapses (Oh et al.,2006),and this activity-dependent GluR1-containing AMPARs trafficking is crucial for long-lasting changes in synaptic strength (Kakegawa and Yuzaki,2005),the changed level of the general CaMKII αand pCaMKII αat hippocampal synapses during withdrawal may regulate the synaptic targeting of AMPA receptors.As has previously been shown,increased abundance of the phosphokinase suggests that increased activity is likely as well (Liang et al.,2004).Our results indicate that the upregulated synaptic expression of GluR1and GluR2/3in hippocampus induced by repeated morphine exposure was correlated to the enhanced expression of pCaMKII α,the active form of CaMKII,in hippocampal synapses.The finding of altered total protein levels of glutamate-receptor subunits has not been replicated by all groups to date and might be highly dependent on the treatment regimen (Fitzgerald et al.,1996;Jang et al.,2002;Glass et al.,2005).It is likely that the total protein expression will be changed under our experimental conditions;therefore protein expression change in membrane and synaptosome at least partly owe to the alteration of transcription and expression.Although the underlying mechanisms for the dynamic expression of AMPA receptors and the regulatory molecule CaMKII αat hippocampal synapses are unclear here,our study provides evidence that morphine withdrawal has

a

4 d AP-5Ro25-6981

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CamKII αpCamKII α

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s

AP-5m s

Ro25-6981

m s

Fig. 6.Blockade of NR2B-containing NMDA receptors regulates the hippocampal synaptic expression of the general CaMKII αand pCaMKII αduring withdrawal.AP-5and Ro25-6981partially altered amounts of the general CaMKII αand pCaMKII αin hippocampus.Upper panel:representative immunoblots of hippocampal membrane fraction (m)and synaptosome (s)prepared from the withdrawal for 4days group (4d),the AP-5infusion group (AP-5),the Ro25-6981infusion group (Ro25-6981)characterized with specific antibodies to the general CaMKII α,and pCaMKII αrespectively.Lower panel:summary graph showing data expressed as average percent of na?ve (mean±S.E.M.)in the general CaMKII α,and pCaMKII α.(#p <0.05AP-5and Ro25-6981vs.4d,n =6).Unpaired t -test;n ,number of animals.

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profound impact on the molecular aspects of AMPARs and their regulatory molecule CaMKIIαin the hippocampus, which may influence opiate withdrawal-induced adaptation of hippocampal functions.

Withdrawal-regulated protein expression in hippocampal synapses regulated by other factors

The hippocampus may be a target of behavioral stress (McEwen and Sapolsky,1995;Kim and Diamond,2002),the consequences of which may contribute to triggering or increasing drug seeking and relapse following drug withdrawal. Glucocorticoid receptors are the major target of glucocorticoids released under stressful conditions(Deroche-Gamonet et al., 2003;Yang et al.,2004).Conflicting results have suggested either an increased or decreased AMPA expression after single stressful experiences.Furthermore,glucocorticoid treatment has been shown to induce changes in glutamate receptor expression in hippocampus(Rosa et al.,2002).In the present study, behavioral stress strongly decreased GluR2/3but slightly downregulated GluR1expression in the hippocampal synapses and the withdrawal-induced increase in GluR2/3was also prevented by the glucocorticoid receptor antagonist RU38486 (Fig.3).It has been reported that different stress paradigms produce different changes in phosphorylation of CaMKII,but no significant effects on CaMKII levels in the hippocampus of normal rats(Suenaga et al.,2004).Here we found that acute behavioral stress promoted conversion of CaMKIIαinto pCaMKIIαin hippocampal synapses and that the low level of pCaMKIIαin both fractions was not significantly affected by RU38486(Fig.4).Thus,both behavioral stress and the glucocorticoid receptor antagonist partially changed the hippo-campal synaptic expression of AMPARs and CaMKIIαfollowing morphine withdrawal.The effects of RU38486on GluR2/3were similar but not in contrast to behavioral stress, indicating that more complicated mechanisms might regulate the involvement of glucocorticoid receptors in withdrawal. Although it remains unclear how behavioral stress following drug withdrawal could trigger drug seeking and relapse,these findings suggest that behavioral stress may have an impact on the adaptive process of the hippocampal functions following drug withdrawal,possibly to trigger or increase drug seeking and relapse.

It should also be noted that N-methyl-D-aspartate(NMDA) receptor activation played an important role in opiate tolerance and dependence;Dizocilpine,a non-competitive NMDA receptor antagonist,had been shown to prevent the morphine dependence in rodents(Hamdy et al.,2004).Thus,we used antagonists to examine whether the changes of GluR1and GluR2/3or CaMKIIαand pCaMKIIαin hippocampal mem-brane and synaptic fractions could be prevented.We found that the NMDAR antagonist AP-5had an effect similar to that of the NR2B-containing NMDAR antagonist Ro25-6981,both leading to a significant downregulation of GluR2/3(Fig.5)and a prominent upregulation of pCaMKIIα(Fig.6)in both fractions. This result suggested that the activation of NR2B-containing NMDARs might be one of the reasons the GluR2/3expression increases following morphine withdrawal.It is possible that both alteration in protein expression of NMDA receptors and decrease in the regulation of NMDA produced by AP-5or Ro25-6981occur during withdrawal,and could contribute to the regulation of the expression of AMPA receptor subunits during withdrawal.The speculation is reasonable since it has been reported that NMDAR activation can have differential effects on AMPAR trafficking depending on the subunit composition of NMDARs,and that NR2B-NMDARs have a subtype-specific function in removal of synaptic AMPARs and weakening of synaptic transmission(Kim et al.,2005).AP-5or Ro25-6981 induced a great increase of pCaMKIIαbut a subtle increase of total CaMKIIαin both fractions(Fig.6),demonstrating that an alteration in protein phosphatase and/or CaMKII kinase activity might be induced by blockade of NR2B-containing NMDA receptors and be responsible for the phosphorylation level of thr286.

In summary,our major finding was that,following opiate withdrawal,GluR1was generally decreased but GluR2/3was strongly increased in hippocampal membrane and synapto-somal fractions.The increased expression of GluR2/3could be prevented by the glucocorticoid receptor antagonist RU38486,the NMDA receptor antagonist AP-5or the antagonist to NR2B-containing NMDA receptors,Ro25-6981.However,although AP-5or Ro25-6981increased the expression of CaMKIIαand pCaMKIIα,RU38486had little influence.Thus,blockade of the activation of glucocorticoid and NMDA receptors might regulate the synaptic expression of GluR1,GluR2/3,CaMKIIαand pCaMKIIαin hippocam-pus.These findings suggest that the adaptive changes in the synaptic expression of calcium sensitive AMPA receptors and their regulatory molecule CaMKII within hippocampus, produced by opiate withdrawal,may be an important neural substrate for alterations that occur in opiate addiction, indicating a maladaptive process of the hippocampal func-tions.However further studies are required to clarify the underlying mechanisms.

Acknowledgements

This work was supported by grants from the National Science Foundation of China(30530250)and the National Basic Research Program(2006CB500800)to L.X.and J.H.L. References

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