Repeated amphetamine administration alters AMPA receptor subunit expression in rat nucleus accumbens...

Repeated Amphetamine AdministrationAlters AMPA Receptor Subunit Expression

in Rat Nucleus Accumbens and MedialPrefrontal Cortex

WENXIAO LU AND MARINA E. WOLF*Department of Neuroscience, Finch University of Health Sciences/The Chicago Medical School,

North Chicago, Illinois

KEY WORDS glutamate receptors; excitatory amino acids; behavioral sensitization;immunocytochemistry

ABSTRACT Glutamate is critical for the induction and maintenance of behavioralsensitization and associated neuroadaptations in the mesocorticolimbic dopamine (DA)system. We have shown previously [Lu et al. (1997) Synapse 26:269–280] that repeatedamphetamine administration alters AMPA receptor subunit mRNA levels in rat nucleusaccumbens (NAc) and medial prefrontal cortex (PFC). The present study determined ifamphetamine elicits corresponding changes in AMPA receptor subunit immunolabeling.Rats were injected with amphetamine sulphate (5 mg/kg/day) or saline for 5 days andperfused 3 or 14 days after the last injection. AMPA receptor subunit immunolabelingwas quantified using autoradiographic immunocytochemistry. In the NAc, GluR1 andGluR2 immunolabeling were unchanged after 3 days of withdrawal, but both weredecreased significantly after 14 days of withdrawal (GluR1, 85.5 6 2.6% of control group,P , 0.01; GluR2, 79.2 6 3.2%, P , 0.01). Analysis of core and shell subregions at the14-day withdrawal time indicated that GluR1 immunolabeling decreased significantly inshell, while GluR2 immunolabeling decreased significantly in both core and shell. Nochanges in GluR2/3, GluR2/4, or GluR4 immunolabeling in NAc were found at eitherwithdrawal time. In the PFC, GluR1 immunolabeling increased after 3 days ofwithdrawal (115.3 6 7.0%, P , 0.01) but returned to control levels after 14 days. Thepresent results correspond well with our previous findings at the mRNA level. Thesealterations in AMPA receptor expression may account for previously described changes inthe electrophysiological responsiveness of NAc and PFC neurons to glutamate andAMPA. Along with alterations in DA function, they may contribute to drug-induceddysregulation of reward-related neurotransmission. Synapse 32:119–131, 1999.r 1999 Wiley-Liss, Inc.

INTRODUCTION

Behavioral sensitization refers to the persistent en-hancement of behavioral responses to psychomotorstimulants that occurs upon their repeated administra-tion. It is important to elucidate the mechanismsunderlying behavioral sensitization in rats, as it mayprovide a useful animal model for intensification ofdrug craving in humans (Robinson and Berridge, 1993).Both the mesolimbic dopamine (DA) system, whichoriginates in the ventral tegmental area (VTA) andprojects to the nucleus accumbens (NAc), and themesoprefrontal DA system, which originates in the VTAand projects to the medial prefrontal cortex (PFC),exhibit numerous adaptations after repeated stimulant

administration (Kalivas and Stewart, 1991; White andWolf, 1991; Self and Nestler, 1995; White et al., inpress). Adaptations within the VTA are transient, andthus likely to be associated with induction mechanisms,whereas adaptations within the NAc show greaterpersistence and are thought to be associated withmaintenance and expression of sensitization.

Glutamate transmission also plays an important rolein behavioral sensitization. NMDA, AMPA, and metabo-

Contract grant sponsor: US Public Health Service; Contract grant number: DA09621.

*Correspondence to: Dr. Marina E. Wolf, Department of Neuroscience, FUHS/The Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064.

Received 1 May 1998; Accepted 18 September 1998

SYNAPSE 32:119–131 (1999)

r 1999 WILEY-LISS, INC.

tropic glutamate receptor antagonists all prevent theinduction of sensitization; NMDA and AMPA receptorantagonists have also been shown to prevent accompa-nying cellular adaptations in the mesoaccumbens DAsystem (see Wolf, 1998, for review). This is not surpris-ing, given the importance of glutamate in other forms ofneuronal plasticity and the convergence of DA andglutamate transmission both at the level of the VTA(White, 1996; Clark and Overton, 1998) and the NAc(Pennartz et al., 1994; Cepeda and Levine, 1998). Whilethe effect of glutamate receptor antagonists on theexpression of sensitization is more controversial, thereis good evidence that glutamate transmission is alteredin sensitized animals (Wolf, 1998). For example, extra-cellular single unit recording studies have revealedthat repeated amphetamine administration alters theresponsiveness of NAc neurons (White et al., 1995b, inpress), VTA DA neurons (White et al., 1995b; Zhang etal., 1997), and PFC neurons (Peterson et al., 1998) tothe excitatory effects of iontophoretically applied gluta-mate agonists.

These and other findings prompted us to examinewhether amphetamine sensitization is accompanied byaltered glutamate receptor expression in the NAc andthe PFC. We focused on AMPA receptors because, undernormal conditions, they are the primary mediators ofexcitatory transmission in medium spiny neurons ofthe NAc (e.g., Pennartz et al., 1990, 1991; Hu andWhite, 1996) and pyramidal neurons of the PFC (e.g.,Jay et al., 1992; Pirot et al., 1994). Four AMPA recep-tors subunits have been identified (GluR1–4). Subunitcomposition determines the functional properties of theoligomeric receptor, including pharmacology, magni-tude of agonist-induced currents, current-voltage rela-tionships, desensitization kinetics and recovery, andCa21 permeability (Seeburg, 1993; Hollmann and Heine-mann, 1994). Using a quantitative method of in situhybridization histochemistry, we found that GluR1 andGluR2, but not GluR3, mRNA levels in the NAc weredecreased after 14 days of withdrawal from repeatedamphetamine administration, whereas no significantchanges were present after 3 days of withdrawal. In thePFC, GluR1 mRNA levels were increased after 3 butnot 14 days of withdrawal, while no changes in GluR2and GluR3 mRNA levels were found at either with-drawal time (Lu et al., 1997).

These results suggest that sensitization may beassociated with alterations in AMPA receptor functionin the NAc and PFC. However, it is important todetermine whether changes at the mRNA level result incorresponding alterations in protein levels of AMPAreceptor subunits. Thus, in the present study we usedautoradiographic immunocytochemistry to investigatethe effects of the same amphetamine regimen on AMPAreceptor subunit immunolabeling in the NAc and PFC.

MATERIALS AND METHODSAnimals and drug treatment

Thirty-six male Sprague-Dawley rats (Harlan, India-napolis, IN), weighing 200–225 g at the beginning of theexperiments, were used. All procedures were performedin strict accordance with the National Institutes ofHealth Guide for the Care and Use of LaboratoryAnimals and were approved by the Institute AnimalCare and Use Committee of the Chicago Medical School.After 3–4 days of handling, rats received 5 daily i.p.injections of amphetamine sulphate (5 mg/kg/day) orsaline (1 ml/kg/day) in their home cages. This regimenresults in robust behavioral sensitization (Wolf andJeziorski, 1993; Wolf et al., 1994). Rats were perfused 3or 14 days after the last injection. Thus, four pretreat-ment groups were generated: amphetamine / 3-daywithdrawal, vehicle / 3-day withdrawal, amphetamine /14-day withdrawal, and vehicle / 14-day withdrawal.Each group consisted of nine rats.

Tissue preparation

Because of the large number of rats involved in thestudy, perfusions were performed on three consecutivedays. All rats were perfused between 9 AM and 1 PM. Tominimize variability, a pair of rats (one from theamphetamine group and one from the vehicle group)was always perfused simultaneously. Rats were anesthe-tized with pentobarbital and perfused with 200 ml ofice-cold saline, followed by 400 ml of fixative solutioncontaining 4% paraformaldehyde (Sigma, St. Louis,MO), 1.5 % sucrose, and 0.1 M phosphate buffer (pH7.2). After perfusion, the brains were immediatelyremoved and immersed in the above fixative solutionfor another hour. Then brains were immersed sequen-tially in solutions containing 0.1 M phosphate buffer,0.1% sodium azide, and either 10, 20, or 30% sucrose.Sections (40 µm) were cut frozen on a sliding microtomeand sequentially placed into 12 wells of a cell cultureplate. At the completion of sectioning, each sectiongroup (1 well) contained sections that sampled theentire rostral–caudal extent of either the PFC or theNAc at 480 µm intervals (2–3 coronal sections for thePFC and 3–4 for the NAc). One section group was usedfor immunocytochemistry with each antibody. Sectionswere stored free-floating in cryoprotectant solution(30% sucrose, 30% ethylene glycol [Fisher, Pittsburgh,PA], and 0.1 M phosphate buffer, pH 7.2) (deOlmos etal., 1978) at -20°C.

Immunocytochemistry

Sections were transferred from cryoprotectant solu-tion into a net in a dish containing rinsing buffer.Sections were rinsed in 0.1 M phosphate buffer (pH 7.2)(PO4) for 4 3 10 min and in 0.1 M phosphate buffercontaining 0.3% Triton X-100 (PO4/T) for 4 3 10 min.After incubating in 10% horse serum (Life Technolo-

120 W. LU AND M.E. WOLF

gies, Grand Island, NY) in PO4/T for 30 min to blockbackground staining, sections were transferred intowells of cell culture plates containing primary antibod-ies in 10% horse serum and PO4/T, then incubated for1–3 days at 4°C with continuous agitation by a mixer(Thermolyne, Dubuque, IA). The concentration ofantibody was 0.5 µg/ml for anti-GluR1 (Chemicon,Temecula, CA) (Wenthold et al., 1992), anti-GluR2(Chemicon) (Vissavajjhala et al., 1996), anti-GluR2/4(Pharmingen, San Diego, CA) and anti-GluR4 (Chemi-con) (Wenthold et al., 1992) antibodies, and 0.25 µg/mlfor anti-GluR2/3 antibody (Chemicon) (Wenthold et al.,1992). After rinsing in PO4/T for 4 3 10 min and inPO4/T containing 10% horse serum for 10 min, sectionswere incubated with 35S-labeled anti-rabbit IgG anti-body or anti-mouse IgG antibody (1:100–1:200 dilution;Amersham, Arlington Heights, IL) at room tempera-ture for 1–2 h with continuous agitation. Finally, sec-tions were rinsed in PO4/T (4 3 10 min) and in PO4 (4 310 min), then mounted onto gelatin-coated microslides.Sections from amphetamine and vehicle groups wereprocessed simultaneously throughout all steps of theimmunocytochemical procedure. All sections weremounted on the same day in random order, to avoiddifferential loss of signals during storage in PO4 prior tomounting.

Autoradiography and image analysis

Sections were exposed to BioMax-MR films (Kodak,Rochester, NY) with 14C-standard microscale strips(Amersham) for 1–2 days. Sections from amphetamineand vehicle groups with the same withdrawal timewere exposed to the same film, to avoid possible differ-ences between films. Films were developed with GBXdeveloper (Kodak) for 4 min and fixed with rapid fixer(Kodak). Autoradiographs on films were scanned by acomputer-scanner system (Macintosh, Cupertino, CA).For each rat in each pretreatment group, 3–4 sectionsbetween bregma 0.7 and 2.2 mm (Paxinos and Watson,1986) were scanned for the NAc. Within each section,the boundary of the entire NAc was defined according toPaxinos and Watson (1986), including both core andshell subregions of the NAc, but carefully excludingsome surrounding areas with high signals, such as theislands of Calleja and the olfactory tubercle. The entireregion of the NAc, defined in this manner, was scannedon both left and right sides of each section. For analysisof subregions of the NAc, the core and shell werescanned separately, according to boundaries illustratedin Figure 5 of our previous study (Lu et al., 1997). Dueto the lack of a clear boundary between the core and theshell subregions, a transitional zone between the coreand the shell was avoided in scanning autoradiographs.In addition, because it is difficult to divide the rostraland caudal poles of the NAc into core and shell, thesesubregions were scanned only in those sections betweenbregma 1.0–2.0 mm (2–3 coronal sections) (Paxinos and

Watson, 1986). Thus, the total area for the core andshell subregions is smaller than that scanned for theentire NAc. For analysis of the PFC, 2–3 coronalsections between bregma 2.7–3.7 mm (Paxinos andWatson, 1986) were scanned, on right and left sides, foreach rat in each pretreatment group. The regionsscanned were restricted to the medial precentral, thedorsal anterior cingulate, and the prelimbic cortices, asdefined by Sesack et al. (1989). All layers of PFC werescanned. NIH Image computer software was used forquantitative analysis of autoradiographs. Within NAcand PFC, there are areas that should not be included inthe analysis of specific signals, such as white matterareas located within these structures (e.g., the anteriorcommissure), blood vessels, and areas where the sec-tion was damaged. To separate such areas from thosewith specific signals, the threshold function of the NIHImage program was employed with a cutoff value. Thecutoff value was determined by making backgroundmeasurements in surrounding white matter regionsand was defined as the mean of these backgroundmeasurements 1 2 standard deviations (to set thecutoff level at a point that would be greater than 95% ofall background measurements). The areas within NAcor PFC that exhibited values lower than this cutoffwere defined as background. In regions with valuesgreater than the cutoff, the specific signal was definedas the total signal minus the mean background signal.Data were expressed as nano-curies per gram drytissue weight, determined using 14C-standard micro-scales.

Data analysis

Statistical comparison of data from amphetamineand vehicle groups was performed using a two-tailedStudent’s t-test. For figures, data for amphetaminepretreatment groups are expressed as percentage of thecorresponding control group (e.g., amphetamine / 3-daywithdrawal rats are compared to vehicle / 3-day with-drawal rats).

RESULTSMethodological considerations

For each rat in each pretreatment group, we deter-mined average levels of immunolabeling for AMPAreceptor subunits by scanning both right and left sidesof several coronal sections spanning the rostral–caudalextent of the NAc or the PFC. To reduce variability,different groups were processed simultaneously to asgreat an extent as possible, including drug injections,perfusions, sectioning, immunocytochemical staining,section mounting, and so on (for details, see Materialsand Methods). Our sampling and processing methodsenabled accurate and reproducible measurements, asdemonstrated by small standard errors for the experi-mental groups depicted in Figures 1–6.

121AMPA RECEPTORS AND AMPHETAMINE SENSITIZATION

Several control experiments were performed to con-firm the specificity of antibodies (data not shown). Forsome primary antibodies, we verified that preadsorp-tion with the corresponding antigen peptides or theirconjugates reduced signals on control sections to back-ground levels. Specific labeling was also abolishedwhen primary antibodies were incubated in a boilingwater bath for 10 min. Labeling with 35S-labeled second-ary antibodies was abolished by 10% rabbit serum or byboiling the antibodies.

Effect of repeated amphetamine administrationon AMPA receptor subunit expression

in the NAc and its subregions

For all experiments, we analyzed the entire NAc aswell as core and shell subregions (see Materials andMethods). Levels of GluR1 immunolabeling in the NAcand its subregions were not significantly differentbetween amphetamine and vehicle pretreatment groupsat the 3-day withdrawal time. However, after 14 days ofwithdrawal GluR1 immunolabeling in the entire NAcwas reduced (85.5 6 2.6% of vehicle group, P , 0.01) inthe amphetamine group (Fig. 1). The reduction inGluR1 immunolabeling was statistically significant inthe shell (81.2 6 2.9%, P , 0.05) and there was a trendtowards reduced GluR1 immunolabeling in the core(90.6 6 4.6%). There were no significant changes inGluR2 immunolabeling in the NAc and its subregionsafter 3 days of withdrawal from repeated amphetamineadministration. At the 14-day withdrawal time, GluR2immunolabeling was decreased significantly in the NAcas a whole (79.2 6 3.2%, P , 0.01) and in both the core(78.8 6 2.3%, P , 0.001) and the shell (78.7 6 2.5%, P ,0.01) (Fig. 2).

Because antibodies selective for GluR3 were notavailable, we used anti-GluR2/3 antibody to indirectlyexamine GluR3 expression in amphetamine-sensitizedrats (Fig. 3). Whereas GluR2 immunolabeling in theNAc was decreased significantly after 14 days of with-drawal, GluR2/3 immunolabeling was not (93.9–96.3%of control levels). GluR2/3 immunolabeling was alsounchanged at the 3-day withdrawal time. These resultssuggest that amphetamine administration does notalter GluR3 expression, consistent with lack of signifi-cant changes at the mRNA level (Lu et al., 1997).

Before the anti-GluR2 antibody became available,the anti-GluR2/4 antibody was used to indirectly exam-ine the GluR2 subunit (Fig. 4). No changes in GluR2/4immunolabeling were found at the 3-day withdrawaltime. At the 14-day withdrawal time, there was a strongtrend towards decreased GluR2/4 immunolabeling inboth the core (89.1 6 2.1%, P 5 0.052) and the shell(90.5 6 3.2%, P 5 0.068). However, in experimentsusing an antibody that recognizes only GluR4, immuno-labeling in the NAc and its subregions was not signifi-cantly altered at either the 3- or 14-day withdrawaltime (Fig. 5). The latter finding, taken together with the

observation that mRNA levels for GluR4 in the NAc aremuch lower than for GluR1–3 (Lu et al., 1997), suggeststhat the trend toward decreased GluR2/4 immunolabel-ing in NAc subregions at the 14-day withdrawal timereflects decreased GluR2 immunolabeling.

Effect of repeated amphetamine administrationon AMPA receptor subunit expression in the PFC

In the PFC, GluR1 immunolabeling was significantlyincreased in the amphetamine group after 3 days ofwithdrawal (115.3 6 7.0%, P , 0.01), but not after 14days (Fig. 6). Immunolabeling for GluR2 or GluR4 was

Fig. 1. Effect of repeated amphetamine administration on GluR1immunolabeling in NAc and its subregions, core, and shell. Levels ofGluR1 immunolabeling were compared between vehicle and amphet-amine pretreated rats after 3 or 14 days of withdrawal. Specific signalswere defined as the total value minus the background in surroundingwhite matter. For each rat in each pretreatment group, average levelsof GluR1 immunolabeling were determined on both sides of severalcoronal sections spanning the rostral–caudal extent of the NAc (seeMaterials and Methods). The bars represent the mean 6 SEM of suchdeterminations from nine rats in each pretreatment group. Eachamphetamine group was compared with a vehicle group from thecorresponding withdrawal time using a two-tailed Student’s t-test.*P , 0.05, **P , 0.01.


not significantly changed after repeated amphetamineadministration at either withdrawal time. Likewise, nosignificant differences were detected using anti-GluR2/3or anti-GluR2/4 antibodies (Fig. 6).

DISCUSSIONAmphetamine produces parallel changesin AMPA receptor subunit mRNA levels

and immunolabeling

This study revealed increased levels of GluR1 immu-nolabeling in the PFC after 3 days of withdrawal fromrepeated amphetamine administration, and decreasedlevels of GluR1 and GluR2 immunolabeling in the NAcafter 14 days of withdrawal. These results extend ourprevious studies of AMPA receptor subunit expressionat the mRNA level (Lu et al., 1997). Table I, which

compares mRNA and protein levels for AMPA receptorsubunits in the NAc and the PFC at the 3- and 14-daywithdrawal times, demonstrates that amphetamine-induced alterations in AMPA receptor subunit expres-sion at the mRNA level are accompanied by correspond-ing changes at the protein level. Other studies havereported a good correlation between mRNA levels forAMPA receptor subunits and functional properties ofAMPA receptor channels within the same cell (Lam-bolez et al., 1992; Bochet et al., 1994; Jonas et al., 1994;Geiger et al., 1995; Angulo et al., 1997). Thus, regula-tion of mRNA levels for individual subunits representsan important mechanism for determining the subunitcomposition and, thus, the functional properties ofheteromeric AMPA receptors. Alterations in mRNAlevels could reflect changes in the rate of transcriptionor changes in RNA stability.

Fig. 2. Effect of repeated amphetamine administration on GluR2immunolabeling in NAc and its subregions, core, and shell. Levels ofGluR2 immunolabeling were compared between vehicle and amphet-amine pretreated rats after 3 or 14 days of withdrawal (n 5 9rats/group). See Figure 1 for details of the analysis. **P , 0.01, ***P ,0.001.

Fig. 3. Effect of repeated amphetamine administration on GluR2/3immunolabeling in NAc and its subregions, core, and shell. Levels ofGluR2/3 immunolabeling were compared between vehicle and amphet-amine pretreated rats after 3 or 14 days of withdrawal (n 5 9rats/group). See Figure 1 for details of the analysis.


Significance of amphetamine-induced decreasesin GluR1 and GluR2 subunits in the NAc:

Medium spiny neurons

In the striatum, 90–95% of total neurons are mediumspiny projection neurons (Smith and Bolam, 1990;Gerfen, 1992). The ability to detect robust decreases inGluR1 and GluR2 subunits at a regional level (i.e., byscanning films) suggests that the decreases are prob-ably occurring in medium spiny neurons. Immunocyto-chemical studies have demonstrated that the majorityof medium spiny neurons in rat striatum are immunore-active for GluR2/3 (Martin et al., 1993; Tallaksen-Greene and Albin, 1994; Chen et al., 1996; Kwok et al.,1997; Bernard et al., 1997) and that the majority ofGluR2/3 immunoreactivity in the striatum is found inthese medium spiny neurons (Martin et al., 1993; Chenet al., 1996). Until recently, the existence of GluR1 inmedium spiny neurons has been more controversial.

While GluR1 immunoreactivity is abundant in thestriatal neuropil and GluR1 mRNA is expressed bymedium spiny neurons, immunocytochemical studieshave generally failed to find GluR1 immunolabeling inthe perikarya of medium spiny neurons (Tallaksen-Greene and Albin, 1994; Chen et al., 1996; Kwok et al.,1997; Ariano et al., 1997; but see Martin et al., 1993;Bernard et al., 1997). An explanation for these findingscomes from a recent study demonstrating that moststriatal projection neurons express GluR1 mRNA (94%of enkephalin-containing neurons, 75% of substanceP-containing neurons, and 87% of enkephalin- andsubstance P-containing neurons) but selectively targetGluR1 to their spines and dendritic shafts (Chen et al.,1998). Less attention has been focused on AMPA recep-tors in the rat NAc and the expression of particularsubunits in chemically identified neuronal populationsof the NAc has not been examined. However, available

Fig. 4. Effect of repeated amphetamine administration on GluR2/4immunolabeling in NAc and its subregions, core, and shell. Levels ofGluR2/4 immunolabeling were compared between vehicle and amphet-amine pretreated rats after 3 or 14 days of withdrawal (n 5 9rats/group). See Figure 1 for details of the analysis.

Fig. 5. Effect of repeated amphetamine administration on GluR4immunolabeling in NAc and its subregions, core, and shell. Levels ofGluR4 immunolabeling were compared between vehicle and amphet-amine pretreated rats after 3 or 14 days of withdrawal (n 5 9rats/group). See Figure 1 for details of the analysis.


data indicate that the pattern of AMPA receptor sub-unit distribution in the rat NAc is similar to thatobserved in striatum, although immunolabeling may bemore intense in the NAc (Petralia and Wenthold, 1992;Martin et al., 1993; Sato et al., 1993; Jakowec et al.,1998).

These anatomical findings are consistent with thepossibility that decreased GluR1 and GluR2 expressionat the 14-day withdrawal time occurred in mediumspiny neurons. If so, decreased GluR1 and GluR2expression could account for previous electrophysiologi-cal results obtained using extracellular single unitrecording, a technique that predominantly samplesresponses of medium spiny neurons. Initial studies,which examined the responsiveness of NAc neurons toiontophoretic glutamate after the present amphet-amine regimen or after repeated cocaine administra-tion, revealed that NAc neurons recorded from sensi-tized rats after 3 days of withdrawal were subsensitiveto the excitatory effects of glutamate (White et al.,1995b). Subsequent studies found that this subsensitiv-ity persists for at least 14 days and reflects decreasedresponsiveness to AMPA and NMDA, but not themetabotropic agonist 1S,3R-t-ACPD (White et al., inpress). Subsensitivity at the 3-day withdrawal timemay reflect decreased sodium currents in NAc neurons(Zhang et al., 1998). However, at the 14-day withdrawaltime, decreased responsiveness to NMDA may reflect

decreases in the expression of the NMDA receptorsubunit NMDAR1, which we have observed in NAcafter the same amphetamine regimen and withdrawalperiod (Lu et al., in press). Subsensitivity to AMPA atthe 14-day withdrawal time may reflect the decreasesin GluR1 and GluR2 expression observed in the presentstudy.

A critical question is whether decreases in GluR1 andGluR2 reflect a reduction in the total number of oligo-meric AMPA receptors or whether they indicate a shiftin subunit composition with no change in total receptornumber. For example, AMPA receptors lacking theGluR2 subunit allow Ca21 influx (Hollman et al., 1991;Jonas et al., 1994; Geiger et al., 1995). Does this implythat the Ca21 permeability of AMPA receptors on NAcneurons is increased after withdrawal from amphet-amine? This would only be the case if the ratio of GluR2to other subunits is actually decreased in individualNAc neurons, resulting in the formation of oligomericreceptors lacking GluR2. The fact that GluR1 expres-sion was also decreased by amphetamine may suggest areduction in the total number of oligomeric receptorsrather than a shift in subunit stoichiometry. Severallines of evidence are consistent with this possibility.Immunoprecipitation studies with subunit-specific an-tibodies have shown that AMPA receptors on hippocam-pal pyramidal neurons fall into two major classes —those made up of GluR1 and GluR2, and those made up

Fig. 6. Effect of repeated amphetamine administration on AMPAreceptor subunit immunolabeling in the medial PFC. Levels of AMPAreceptor subunit immunolabeling were compared between vehicle andamphetamine pretreated rats after 3 or 14 days of withdrawal (n 5 9

rats/group). For each rat in each pretreatment group, average levels ofimmunolabeling were determined on both sides of several coronalsections spanning the rostral–caudal extent of the PFC (see Materialsand Methods). See Figure 1 for details of the analysis. *P , 0.05.


of GluR1 and GluR3 (Wenthold et al., 1996). In situhybridization studies have confirmed the idea thatAMPA receptor subunit stoichiometries fall into dis-crete categories and demonstrated that both hippocam-pus and striatum belong to the AR-1,2 class, in whichGluR1 and GluR2 mRNAs predominate and are pres-ent in approximately equal proportions (Gold et al.,1997). Moreover, ultrastructural studies support atleast partial colocalization of GluR1 and GluR2/3 immu-noreactivity in striatal spines (Bernard et al., 1997). Allof these findings support the idea that NAc neuronsexpressing GluR1 and GluR2-containing AMPA recep-tors experience an overall loss of such receptors as aresult of repeated amphetamine administration.

A reduction in the number of AMPA receptors pro-vides the simplest explanation for the decreased re-sponse to AMPA observed in electrophysiological stud-ies (White et al., 1995a, in press). However, a shift insubunit composition, resulting in decreased GluR1 andGluR2 incorporation, could also account for the electro-physiological findings, since the largest agonist-acti-vated inward currents in oocyte and cultured cellstudies are observed for AMPA receptors assembledwith GluR1 and GluR2 subunits (Boulter et al., 1990;Keinanen et al., 1990).

Medium spiny NAc neurons receive convergent in-puts from DA and glutamate terminals (Sesack andPickel, 1992) and exhibit a high degree of DA andglutamate receptor colocalization (Ariano et al., 1997).While DA/glutamate interactions in the striatal com-plex are complex and controversial, it is now believedthat DA receptors exert neuromodulatory effects onglutamate-mediated synaptic responses (Cepeda andLevine, 1998). The nature of the modulatory effectdepends on the recording preparation, the subtypes ofDA and glutamate receptors involved, and the concen-tration of DA (e.g., Chiodo and Berger, 1986; Cepeda etal., 1993; Hu and White, 1997). In general, DA appearsto reduce the excitability of NAc neurons (O’Donnelland Grace, 1996; Zhang et al., 1998). However, single-unit recording studies in awake rats suggest that themodest inhibition of NAc neuronal activity produced byDA has the effect of amplifying the phasic activationinduced by glutamate (Kiyatkin and Rebec, 1996).These and other findings (see O’Donnell and Grace,1996) support the idea that DAmodulates the ‘‘signal-to-noise’’ ratio in the NAc. This may be related to thehypothesized role of DA transmission in signalingtarget neurons about deviations from predicted reward(Schultz, 1997). It should be noted that some neuro-

TABLE I. Comparison of protein levels with mRNA levels for AMPA receptor subunits in NAc,its subregions and PFC after repeated amphetamine administration*

mRNA Protein

3 days 14 days 3 days 14 days

GluR1NAc 100.1 6 7.2 65.8 6 5.3*** 104.9 6 6.6 85.5 6 2.6**core 113.5 6 10.3 63.7 6 7.8*** 107.6 6 8.2 90.6 6 4.6shell 106.0 6 9.5 70.1 6 5.0*** 105.1 6 9.7 81.2 6 2.9*PFC 138.5 6 8.6* 97.4 6 5.7 115.3 6 7.0* 99.4 6 5.0

GluR2NAc 105.6 6 7.4 77.6 6 9.5* 104.2 6 11.7 79.2 6 3.2**core 108.0 6 15.4 72.8 6 7.6** 108.5 6 13.4 78.8 6 2.3***shell 104.9 6 8.0 70.5 6 1.6*** 105.5 6 11.9 78.7 6 2.5**PFC 110.0 6 4.0 94.0 6 4.7 105.5 6 10.8 93.1 6 3.9

GluR2/3NAc 97.6 6 6.0 94.0 6 2.8core 96.3 6 6.1 93.9 6 3.1shell 100.0 6 6.5 96.3 6 3.0PFC 100.7 6 4.4 101.9 6 3.1

GluR3NAc 104.2 6 5.6 85.9 6 7.6core 92.0 6 12.1 101.9 6 10.0shell 102.1 6 10.7 75.7 6 9.9PFC 105.3 6 5.1 93.4 6 9.6

GluR2/4NAc 96.6 6 6.5 93.3 6 2.4core 93.8 6 7.2 89.1 6 2.1shell 93.9 6 7.7 90.5 6 3.2PFC 114.1 6 8.0 110.6 6 6.6

GluR4NAc 96.8 6 4.8 100.6 6 5.3core 91.5 6 7.1 101.7 6 4.5shell 98.3 6 5.2 98.8 6 6.0PFC 102.5 6 10.0 107.1 6 9.1

*Only data for amphetamine groups are listed, and are expressed as percentage of the corresponding control groups. Aportion of the data on mRNA levels has been reported previously (Lu et al., 1997).*P , 0.05.**P , 0.01.***P , 0.001.


modulatory actions of DA may be exerted presynapti-cally (Nicola and Malenka, 1997, and referencestherein).

Considerable evidence indicates that DA transmis-sion in the NAc is augmented after long withdrawalsfrom repeated stimulant administration. D1 receptorson medium spiny neurons are supersensitive in electro-physiological studies (Henry and White, 1991, 1995;Wolf et al., 1994), D1 receptor-activated signal transduc-tion pathways are upregulated (Self and Nestler, 1995)and stimulant-induced DA release is augmented (e.g.,Robinson et al., 1988; Kalivas and Duffy, 1990; Pettit etal., 1990). The combination of enhanced DA transmis-sion and reduced glutamate transmission (due to de-creased expression of GluR1 and GluR2) might beexpected to profoundly affect the output of the striatalcomplex. The resulting dysregulation of dopaminergicmodulation of goal-directed behavior (see Salamone etal., 1997) could contribute to craving and relapse.

Decreases in GluR1 and GluR2 expression may alsoinfluence DA and glutamate release in the NAc. How-ever, the direction of the effect is difficult to predict,since evidence exists for both facilitatory and inhibitoryeffects of AMPA receptors on glutamate release (e.g.,Liu and Moghaddam, 1995) and on DA release (e.g.,Youngren et al., 1993).

Whereas our electrophysiological, in situ hybridiza-tion, and immunocytochemical results indicate de-creased AMPA receptor function in amphetamine- andcocaine-sensitized rats, the results of behavioral stud-ies suggest that the responsiveness of NAc AMPAreceptors is increased 3 weeks after discontinuation ofrepeated cocaine treatment (Pierce et al., 1996). Re-cently, the same group reported a significant increase inGluR1 and NR1 subunits, measured by Western blotanalysis, in the NAc at the same withdrawal time(Churchill et al., 1997). Different regimens and with-drawal times may contribute to discrepancies betweenthese results and the present findings, but there mayalso be important differences between cocaine andamphetamine with respect to the circuitry involved insensitization (see Pierce et al., 1998). Another possibil-ity is that some of the behavioral findings obtained incocaine-sensitized rats (Pierce et al., 1996) reflectchanges in AMPA receptors on interneurons as well asprojection neurons (see below), whereas our electro-physiological studies (White et al., 1995b, in press) arepredominantly measuring AMPA responses of mediumspiny neurons.

Significance of amphetamine-induceddecreases in GluR1 and GluR2 subunits

in the NAc: Interneurons

According to morphology and neurochemical mark-ers, the interneurons of the striatum can be divided intothree subtypes: 1) medium aspiny neurons containing

somatostatin, neuropeptide Y, and/or NADPH-diapho-rase/nitric oxide synthase (NOS); 2) large aspiny neu-rons containing acetylcholine; and 3) medium aspinyneurons containing parvalbumin (PARV) and GABA(Vincent and Johansson, 1983; Cowan et al., 1990;Dawson et al., 1991; Hope et al., 1991; Kawaguchi,1993). All studies have reported the absence or very lowlevels of expression of AMPA receptor subunits insomatostatin- or NOS-positive interneurons (Tallaksen-Greene and Albin, 1994; Catania et al., 1995; Chen etal., 1996; Kwok et al., 1997; Bernard et al., 1997). Incholinergic interneurons, no GluR2/3 immunoreactiv-ity was found (Chen et al., 1996; Bernard et al., 1997).For GluR1 and GluR4, some studies reported no GluR1and GluR4 immunolabeling in choline acetyltransfer-ase- and muscarinic m2 receptor-positive interneuronsof the striatum (Chen et al., 1996; Kwok et al., 1997),while others reported modest levels of GluR1 andGluR4 immunoreactivity in 40–50% of choline acetyl-transferase-positive interneurons (Martin et al., 1993;Bernard et al., 1997). Modest to low levels of GluR2/3immunoreactivity were found in 50–70% of PARV-positive interneurons (Tallaksen-Greene and Albin,1994; Chen et al., 1996; Bernard et al., 1997). Highlevels of GluR1 immunoreactivity were found in PARV-positive interneurons, and 90–100% of PARV-positiveinterneurons were double-stained for GluR1 (Tallaksen-Greene and Albin, 1994; Chen et al., 1996; Kwok et al.,1997; Bernard et al., 1997). While 86–100% of PARV-positive interneurons were labeled for GluR4, the levelsof GluR4 in striatum as a whole are modest (Chen et al.,1996; Kwok et al., 1997; Bernard et al., 1997).

These results suggest that amphetamine-induceddecreases in GluR1 and GluR2 immunolabeling mightbe occurring in PARV-positive GABAergic interneu-rons. There are important functional interactions be-tween these interneurons and striatal projection neu-rons. For example, electrophysiological studies haveshown that excitatory glutamatergic inputs to the NAccan elicit feed-forward inhibition of medium spinyneurons, likely mediated by GABAergic interneurons(Pennartz and Kitai, 1991). Such interactions could beinfluenced in very complex ways as a result of alteredglutamate receptor expression in striatal projectionneurons and/or interneurons.Added complexity is intro-duced by the likelihood that other transmitter systemsare also altered in sensitized animals. For example,changes in GABAA receptor function and subunit mRNAexpression have been found after repeated cocaineadministration (Carney and Abel, 1995; Peris, 1996).Drug effects on multiple transmitter systems withinthe striatum may account for results indicating thatsensitization involves a functional reorganization ofbasal ganglia circuitry (Moratalla et al., 1996; Curranet al., 1996).


Significance of the amphetamine-inducedincrease in GluR1 in the medial PFC

Many recent studies have implicated the glutamater-gic projection from PFC to VTA in the development ofbehavioral sensitization (Kalivas and Alesdatter, 1993;Banks and Gratton, 1995; Wolf et al., 1995; Tong et al.,1995; White et al., 1995b; Fitzgerald et al., 1996; Cadoret al., 1997; Zhang et al., 1997; Carlezon et al., 1997;Tzschentke and Schmidt, 1998; Li et al., in press) andsome results suggest that the induction of sensitizationinvolves increased activity in this pathway (Ben-Shahar and Ettenberg, 1994; Schenk and Snow, 1994;Sorg and Kalivas, 1993; White et al., 1995a; Sorg et al.,1997). Since glutamate projections from PFC to VTAprovide a major source of excitatory drive to VTA DAneurons, increased activity in the PFC-VTA pathwaywould explain numerous findings indicating that theactivity of VTA DA neurons is increased after shortwithdrawals (1–3 days) from repeated stimulant admin-istration. This transient increase in DA cell activity isbelieved to represent an obligatory step in the sensitiza-tion cascade (Zhang et al., 1997; Clark and Overton,1998; Henry et al., 1998; Wolf, 1998).

The increase in GluR1 expression observed after 3days of withdrawal from amphetamine provides apossible mechanism for this proposed increase in theactivity of PFC neurons. AMPA receptors on PFCpyramidal neurons are primarily responsible for medi-ating excitatory effects of glutamate-containing inputsoriginating in thalamus, hippocampus, and other corti-cal regions (e.g., Jay et al., 1992; Pirot et al., 1994).Increased GluR1 expression would, therefore, be ex-pected to increase the excitability of PFC pyramidalneurons. Consistent with this prediction, we havefound that PFC neurons recorded after 3 days ofwithdrawal from the same amphetamine regimen usedin the present study exhibit increased responsivenessto the excitatory effects of iontophoretically appliedglutamate (Peterson et al., 1998). In addition, microdi-alysis studies indicate that glutamate transmission inthe PFC may be augmented by repeated stimulantadministration (Stephans and Yamamoto, 1995).

No information is available on the cell types in the ratPFC that express particular AMPA receptor subunits.However, in other regions of rat neocortex, pyramidalneurons express GluR1 (Molnar et al., 1993; Martin etal., 1993). GluR1 is particularly abundant in layer V(Martin et al., 1993), which also receives input from DAterminals (e.g., Van Eden et al., 1987). In both rat andprimate PFC, DA terminals form symmetric synapticcontacts onto dendritic spines that also receive asym-metric synapses from presumed glutamatergic termi-nals, forming a triad similar to that observed in thestriatal complex (Goldman-Rakic et al., 1989; Verney etal., 1990; Carr and Sesack, 1996). Consistent with thisanatomical arrangement, DA exerts an inhibitory effect

on excitatory transmission in the PFC (Ferron et al.,1984), decreasing both AMPA and NMDA componentsof monosynaptic EPSPs in PFC pyramidal neurons(Law-Tho et al., 1994). A number of findings suggestthat repeated stimulant administration decreases DAtone in the PFC (Sorg and Kalivas, 1993; White et al.,1995a; Sorg et al., 1997; Peterson et al., 1998). Areduction in inhibitory DA tone might be expected tofurther augment the excitatory effects of glutamate onPFC pyramidal neurons.

While it is appealing to propose that increased gluta-mate tone and decreased DA tone synergize at the levelof PFC pyramidal cells to increase the activity ofexcitatory PFC projections to regions such as the VTA,several caveats should be noted. First, although DAexerts inhibitory effects in PFC under certain condi-tions (see above), it is more properly viewed as amodulator of neuronal excitability (Yang and Seamans,1996, and references therein). Second, this model doesnot account for other types of DA/glutamate interac-tions in the PFC, such as the ability of AMPA receptorstimulation to increase DA release (e.g., Jedema andMoghaddam, 1996). Finally, although intensity of GluR1immunolabeling depended on the cortical region andlayer examined, nonpyramidal neurons were often morestrongly labeled than pyramidal neurons (Molnar et al.,1993; Martin et al., 1993). Increased GluR1 expressionby nonpyramidal neurons would exert complex effectson PFC output that would depend on which subpopula-tion was affected.

CONCLUSIONS

GluR1 expression in the PFC is increased after 3 daysof withdrawal from repeated amphetamine administra-tion, while expression of GluR1 and GluR2 is decreasedin the NAc after 14 days of withdrawal. IncreasedAMPA transmission in the PFC may contribute to theaugmentation of activity in the PFC-VTA pathway thatappears to play a role in the induction of sensitization.Decreased AMPA transmission in the NAc, along withother persistent drug-induced neuroadaptations identi-fied in this region, may contribute to the maintenanceof the sensitized state. More generally, altered DA/glutamate interactions in the NAc may underlie thedysregulation of reward-related neurotransmission thatis thought to underlie addiction and relapse.

ACKNOWLEDGMENTS

This work was supported by US Public Health Ser-vice grant DA 09621 to M.E.W. We are grateful toChang-Jiang Xue for excellent technical assistance.

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Repeated amphetamine administration alters AMPA receptor subunit expression in rat nucleus accumbens...

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