Both Glutamate Receptor Antagonists andPrefrontal Cortex Lesions Prevent

Induction of Cocaine Sensitization andAssociated Neuroadaptations

YONG LI,1 XIU-TI HU,2 TIMOTHY G. BERNEY,1 A. JOHN VARTANIAN,1 CHRISTY D. STINE,1MARINA E. WOLF,1 AND FRANCIS J. WHITE2*

1Department of Neuroscience, Finch University of Health Sciences/The Chicago Medical School,North Chicago, Illinois

2Department of Cellular and Molecular Pharmacology, Finch University of Health Sciences/The Chicago Medical School, North Chicago, Illinois

KEY WORDS dopamine; excitatory amino acids; ventral tegmental area; nucleusaccumbens; dopamine D1 receptors; dopamine D2 receptors

ABSTRACT Behavioral sensitization to psychomotor stimulants is accompanied by anumber of alterations in the mesoaccumbens dopamine (DA) system, including DAautoreceptor subsensitivity in the ventral tegmental area (VTA) and DA D1 receptorsupersensitivity in the nucleus accumbens (NAc). We investigated the role of excitatoryamino acid (EAA) transmission in the induction of cocaine sensitization and theseaccompanying DA receptor alterations. To do so, we used three glutamate receptorantagonists, the noncompetitive NMDA receptor antagonist MK-801 (0.1 mg/kg), thecompetitive NMDA receptor antagonist CGS 19755 (10.0 mg/kg), and the AMPA receptorantagonist NBQX (12.5 mg/kg). Rats received daily double injections of either one ofthese antagonists or saline with either cocaine (15.0 mg/kg) or saline for 5 days. Cocainesensitization was defined as an increase in horizontal locomotor activity in response tococaine challenge (7.5 mg/kg) on the third day of withdrawal. All three antagonistsprevented the induction of cocaine sensitization. Extracellular single cell recordingsrevealed that these antagonists also prevented the induction of DA autoreceptorsubsensitivity in the VTA and DA D1 receptor supersensitivity in the NAc. To determinewhether the relevant glutamate receptors were under regulation by medial prefrontalcortex (mPFC) EAA efferents, we next lesioned the mPFC bilaterally with ibotenic acid atleast 7 days before repeated cocaine treatment began. These lesions also prevented theinduction of cocaine sensitization and the associated neuroadaptations. Our findingsindicate that glutamate transmission from mPFC to the mesoaccumbens DA system iscritical for the induction of cocaine sensitization and its cellular correlates. Synapse34:169–180, 1999. r 1999 Wiley-Liss, Inc.

INTRODUCTION

Repeated administration of psychomotor stimu-lants (e.g., amphetamine or cocaine) to rodents leadsto an enhanced ability of such drugs to produce lo-comotor stimulation. This phenomenon, termed be-havioral sensitization, may be related to processesinvolved in both psychosis and drug addiction inhumans (Post et al., 1992; Robinson and Berridge,1993). The mesoaccumbens dopamine (DA) path-way projecting from the ventral tegmental area (VTA)to the nucleus accumbens (NAc) is critically involvedin the induction and expression of behavioral sensiti-zation.

The VTA is implicated in the induction of behavioralsensitization because repeated administration of am-phetamine directly into the VTA, but not the NAc ormedial prefrontal cortex (mPFC), induces sensitizationto subsequent systemic (or intra-NAc) injections of apsychomotor stimulant (Vezina et al., 1987; Kalivas

Contract grant sponsor: USPHS; Contract grant number: DA04093; Contractgrant sponsor: the National Institute on Drug Abuse (NIDA); Contract grantnumbers: DA09621, DA00207; Contract grant sponsor: the National Institute ofMental Health; Contract grant number: MH11388.

*Correspondence to: Francis J. White, Department of Cellular and MolecularPharmacology, Finch University of Health Sciences/The Chicago Medical School,3333 Green Bay Rd., North Chicago, IL 60064. E-mail: whitef@mis.finchcms.edu

Received April 17, 1998; Accepted July 31, 1998

SYNAPSE 34:169–180 (1999)

r 1999 WILEY-LISS, INC.

and Weber, 1988; Vezina and Stewart, 1990; Hooks etal., 1992; Cador et al., 1995; Bjijou et al., 1996). BothDA D1 receptors and NMDA receptors within the VTAhave been implicated in induction processes (Vezinaand Stewart, 1989; Kalivas and Alesdatter, 1993; Ve-zina, 1996; Bjijou et al., 1996; Pierce et al., 1996b). Inaddition, some of the earliest cellular alterations associ-ated with sensitization occur within the VTA. Theseinclude somatodendritic DA autoreceptor subsensitiv-ity and increased DA neuronal activity, both of whichare relatively transient (White and Wang, 1984; Henryet al., 1989; Ackerman and White, 1990).

The expression of behavioral sensitization appears tobe mediated in the NAc. Acute injection of amphet-amine or cocaine into the NAc induces locomotor activ-ity (Pijnenburg et al., 1976; Delfs et al., 1990). Rats thatreceive systemic or intra-VTA injections of amphet-amine exhibit sensitization to intra-NAc administra-tion of amphetamine (Kolta et al., 1989; Paulson andRobinson, 1991; Perugini and Vezina, 1994; Cador etal., 1995). Correspondingly, sensitization-related cellu-lar changes in the NAc, especially functional supersen-sitivity of postsynaptic DA D1 receptors, parallel thepersistence of behavioral sensitization (Henry andWhite, 1991, 1995).

Excitatory amino acid (EAA) transmission also playsa critical role in sensitization inasmuch as both NMDAand AMPA receptor antagonists prevent the inductionof behavioral sensitization (Karler et al., 1989; Wolf andKhansa, 1991; Wolf and Jeziorski, 1993; Stewart andDruhan, 1993; Karler et al., 1994; Wolf et al., 1995; Li etal., 1997). Cellular studies have shown that NMDAreceptor antagonists also prevent the development ofVTA DA autoreceptor subsensitivity and NAc D1 recep-tor supersensitivity which accompany amphetaminesensitization (Wolf et al., 1994). In addition, behavioralsensitization is accompanied by altered sensitivity ofVTA and NAc neurons to glutamate (White et al.,1995a; Zhang et al., 1997) and altered glutamatereceptor subunit expression in the VTA (Fitzgerald etal., 1996) and mPFC (Lu et al., 1997).

Major EAA projections to both the VTA and NAcoriginate in the mPFC (Sesack and Pickel, 1992).Involvement of the mPFC in behavioral sensitizationhas been suggested because: 1) the excitatory responsesof VTA DA neurons to mPFC stimulation are increasedin chronic amphetamine treated rats (Tong et al., 1995);2) chronic electrical kindling of the mPFC sensitizescocaine’s locomotor stimulating properties (Schenk andSnow, 1994); 3) bilateral lesions of mPFC prevent thedevelopment of amphetamine sensitization (Wolf et al.,1995); and 4) manipulation of DA, GABA, or EAAsystems in the frontal cortex can modulate inductionand expression of amphetamine sensitization (Karler etal., 1997). These results suggest that the mPFC isnecessary for sensitization and accompanying neuroad-aptations.

We designed a series of experiments to determinewhether both the induction of cocaine-induced behav-ioral sensitization and its accompanying cellular corre-lates (DA autoreceptor subsensitivity in VTA and D1receptor supersensitivity in the NAc) are prevented bycoadministration of NMDA and AMPA receptor antago-nists during the induction phase or by ibotenic acidlesions of mPFC prior to the induction phase. Ourpresent results indicate an essential role of the mPFCEAA system in the induction of cocaine sensitizationand its cellular correlates.

MATERIALS AND METHODSAnimals

Subjects were male Sprague-Dawley rats (Harlan,Indianapolis, IN) weighing 200 to 224 g at the begin-ning of the drug treatment or at the time of mPFClesions. They were housed 2–3/cage in a colony roommaintained at constant temperature and humiditywith a 12-h light/dark cycle. Food and water wereavailable ad libitum. The experiments were carried outin accordance with the Guide for the Care and Use ofLaboratory Animals (NIH publication No. 85–23) andwere approved by our Institutional Animal Care andUse Committee.

Repeated drug treatment protocols andbehavioral testing

Rats were handled for 2–3 days and then assigned toone of the following groups: saline/saline, saline/cocaine, MK-801/saline, MK-801/cocaine, CGS 19755/saline, CGS 19755/cocaine, NBQX/saline, NBQX/co-caine, mPFC lesions/saline, mPFC lesions/cocaine, shamlesions/cocaine. Double injections (separated by 30 min)were given i.p. once a day for 5 days. Drug doses were:cocaine (15.0 mg/kg), MK-801 (0.1 mg/kg), CGS 19755(10.0 mg/kg), and NBQX (12.5mg/kg).

Bilateral excitotoxic lesions of the mPFC

Pentobarbital- (55 mg/kg) anesthetized rats werefixed in a stereotaxic frame. Ibotenic acid (5 µg / 0.5 µl /2.5 min) or an equal volume of vehicle (0.1 M phosphatebuffered saline, pH 7.4) was infused into each side ofthe mPFC using a 1.0 µl Hamilton syringe at thefollowing coordinates (relative to Bregma and to thebony surface): A 13.2, L 60.7, V 23.4. The syringeremained in place for 5 min after each infusion. Ratswere allowed at least 7 days to recover from surgery.

Behavioral tests

Twelve photobeam frames (30 3 50 cm; PAS monitor-ing system, San Diego Instruments, San Diego, CA)were mounted around standard rectangular transpar-ent rodent cages. Each frame was equipped with threephotoelectric beams that divided the long axis of thecage into four quadrants. Interruption of photobeams

170 Y. LI ET AL.

generated digital pulses that lead to a 286-based PCwhich determined the total number of photobeam inter-ruptions as well as interruptions of consecutive photo-beams (ambulations). All behavioral tests occurredbetween 12:00 and 14:00 h in a separate room in theanimal colony, which was environmentally distinctfrom the room used for animal housing. Each testconsisted of a 30-min habituation period, followed bydrug or vehicle injection and a 60-min test period.Three days after the last of the repeated injections (day8), all rats were transferred to testing cages andchallenged with cocaine (7.5 mg/kg, i.p.) to determinewhether sensitization had developed. This low chal-lenge dose was used because it is at the threshold forambulatory activation and thus provides excellent dis-crimination of sensitized rats. In addition, this dosefails to induce signs of cocaine-induced stereotyped‘‘head-bobbing,’’ even in sensitized rats (Henry andWhite, 1995).

In vivo extracellular single-unit recording

Rats were anesthetized with chloral hydrate (400mg/kg, i.p.) and mounted in a stereotaxic frame. Bodytemperature was maintained at 36–37°C by a thermo-statically controlled heating pad. A lateral tail vein wascatheterized for administration of additional anes-thetic, as needed. A burr hole was drilled in the skulland the dura was retracted from the area overlying theVTA or NAc so that the electrode could be lowered to therecording area. The stereotaxic coordinates for the VTAwere (relative to lambda and cortical surface): A 3.0–3.5, L 0.4–1.0, V 6.0–8.0; and for the NAc were (relativeto bregma and cortical surface): A 0.7–2.7, L 0.8–2.0, V6.0–8.0. Electrode potentials were passed to an ampli-fier, displayed on an oscilloscope, and monitored with awindow discriminator. Spike activity was recorded on apolygraph. Digital counts were obtained for off-lineanalysis.

Recording from VTA DA neurons

Recordings were made with single-barrel glass elec-trodes filled with 2M NaCl saturated with 1% FastGreen dye. DA neurons were identified by well-characterized electrophysiological criteria (see White,1996, for review). Each DA neuron was recorded for 3–5min to establish a stable baseline firing rate. To deter-mine the sensitivity of DA autoreceptors in the VTA,the nonselective DA receptor agonist apomorphine wasapplied through the catheterized tail vein and cumula-tive dose–response curves were generated. To confirmreceptor-mediated suppression of activity, the apomor-phine-induced inhibition was reversed with the selec-tive DA D2-class receptor antagonist eticlopride (i.v.).

Recording from NAc neurons

Five-barrel glass microelectrodes were used in theseexperiments. The center barrel was filled with the same

solution as in the VTA single-barrel electrodes. Oneside barrel was filled with 2 M NaCl solution forautomatic current balancing. A second side barrel wasfilled with L-glutamate monosodium salt (100 mM in 50mM NaCl, pH 8) and the other two side barrels werefilled with the D1 receptor-selective agonist SKF 38393HCl (10 mM in 150 mM NaCl, pH 4). Glutamate wasejected as an anion, whereas SKF 38393 was ejected asa cation. Retaining currents of 8–10 nA were applied tothe drug barrels during nonejection periods to mini-mize passive diffusion. Under anesthetized conditions,most NAc neurons are either quiescent or fire at veryslow and irregular rates. Therefore, during each elec-trode penetration in the NAc glutamate was appliedcontinuously. Once a neuron was identified, glutamatewas ejected in a pulse pattern of 30 sec on / 30 sec off.Glutamate current was adjusted so that the firing rateof NAc neurons was 3–6 spikes/sec. Less than 10% ofrecordings in these studies were performed from spon-taneously firing neurons. Previous studies in our labora-tory have found no differences between glutamatedriven neurons and spontaneously active neurons intheir response to SKF 38393 (Henry and White, 1991).After 3–5 min of baseline recording, SKF 38393 wasadministered continuously. The ejecting current (1–128nA) was doubled every 2 min so that a current–response curve could be generated for each neuronrecorded. All the neurons in this study recovered frominhibition by SKF 38393. A total of 1–4 neurons wasrecorded from each rat.

Histology

At the end of each recording, the position of the tip ofthe electrode was marked by depositing a spot of FastGreen dye. Rats were then deeply anesthetized andtranscardially perfused with saline followed by 10%phosphate-buffered formalin. Sections were cut andstained with cresyl violet. Recording sites were verifiedby routine light microscopic examination. For NAcexperiments, the coordinates of each neuron were re-corded and the location of the neuron was estimated bycomparing its coordinates with those of the Fast Greenspot. For mPFC lesion studies, the extent of the lesionswas determined using the same histological proce-dures. Lesion boundaries were defined as the area ofneuronal loss.

Statistics

The behavioral data for antagonist tests were ana-lyzed using one-way ANOVA or, when assumptions ofnormality failed, using Kruskal-WallisANOVAon ranks.The behavioral data from the lesion experiment wereanalyzed with Student’s t-test. The electrophysiologicaldata were analyzed using a two-way ANOVA withrepeated measures on one variable (drug dose or ionto-phoretic current). Planned comparisons between treat-

171GLUTAMATE AND COCAINE SENSITIZATION

ment and control means were conducted using eitherDunn’s or Dunnett’s test with a 5 0.05.

RESULTSGlutamate receptor antagonists prevent the

induction of cocaine sensitization

Previous studies have demonstrated that the cocaine-induced increase in horizontal locomotor activity ismonophasic, with peak activity at about 20 min aftercocaine challenge and a return to basal levels within 50min (Henry and White, 1995), Accordingly, we mea-sured locomotor activity, expressed as ambulations, for1 h after cocaine administration. Figure 1 compares thelocomotor activity of different treatment groups tococaine challenge injection. Rats treated repeatedlywith cocaine (sal/coc group) showed a highly sensitizedlocomotor response as compared to controls (sal/salgroup). The noncompetitive NMDA receptor antagonistMK-801, the competitive NMDA receptor antagonistCGS 19755, and the AMPA receptor antagonist NBQXeach prevented the induction of cocaine sensitizationwhen administered 30 min before cocaine during re-

peated treatment. Repeated administration of MK-801with saline failed to alter the response to cocaine (notshown). Although CGS 19755 1 saline and NBQX 1saline groups were not tested in these experiments,previous studies have shown that repeated administra-tion of the antagonists does not alter the response ofrats to psychomotor stimulants (Jeziorski et al., 1994;Li et al., 1997). These findings indicate that bothNMDA and AMPA receptor activation are required forthe induction of cocaine sensitization.

Glutamate receptor antagonists prevent theinduction of DA autoreceptor subsensitivity

Our recordings were conducted 2–3 days after thecessation of drug treatment because autoreceptor sub-sensitivity is maximal at this time point (Ackermanand White, 1990). As shown in Figure 2, repeatedcocaine treatment significantly reduced the sensitivityof VTA DA neurons to the inhibitory effects of apomor-phine. As with the behavioral results, this effect wasprevented by coadministration of MK-801 (Fig. 2A),CGS 19755 (Fig. 2B) or NBQX (Fig. 2C). Thus, iono-tropic glutamate receptor antagonists, when coadminis-tered with cocaine, prevented repeated cocaine-inducedDA autoreceptor subsensitivity in VTA DA neurons.When coadministered with saline, these glutamatereceptor antagonists exerted no effect on DA autorecep-tor sensitivity.

Glutamate receptor antagonists preventfunctional supersensitivity of DA D1

receptors in the NAc

Previous studies from our laboratory have demon-strated that after repeated cocaine treatment, NAcneurons show increased sensitivity to cocaine and theDA D1 receptor agonist SKF 38393 (Henry and White,1991, 1995). In addition, the presence of this effectparallels the persistence of behavioral sensitization tococaine, i.e., both phenomena are apparent followingwithdrawal periods of 1 day to 1 month, but not 2months. The present electrophysiological studies wereconducted 5–9 days after drug treatment ceased. Asshown in Figure 3, iontophoretic administration of SKF38393 inhibited the firing of NAc neurons in a current-dependent manner. The inhibitory effect of SKF 38393was markedly enhanced in the cocaine-pretreated group,but not when glutamate receptor antagonists (MK-801,CGS 19755, and NBQX) were coadministered withcocaine during the induction phase. Neither CGS 19755nor NBQX, when administered with saline, altered theresponse of NAc neurons to SKF 38393. However,coadministration of MK-801 and saline produced aslight but statistically significant reduction in theresponse of NAc neurons to SKF 38393. This result isopposite to that which we previously observed with ahigher dose of MK-801 (0.25 mg/kg) and a 7-day with-drawal period (Wolf et al., 1993).

Fig. 1. Glutamate receptor antagonists prevent the development ofcocaine sensitization. All results are presented as mean 6 SEM (n 512/group). Separate sal/sal and sal/coc groups were used in the NBQXexperiment, which was conducted several months after the experi-ment with the NMDA receptor antagonists. The left set of bars showthat both NMDA receptor antagonists MK-801 and CGS-19755 pre-vented the development of cocaine sensitization. The right set of barsshow a similar result using the AMPA receptor antagonist NBQX.Within each experiment, comparisons were made between the sal/salgroup and each treatment group using a one-tailed Student’s t-test. Inthe NMDA receptor antagonist experiment, the sal/coc group showedsensitized locomotor responses to cocaine as compared to the sal/salgroup (H3 5 14.4, P 5 0.002, Kruskal-Wallis ANOVA on ranks, *P ,0.05 with Dunn’s multiple comparisons test). Similar sensitizationwas observed in the NBQX experiment (F2,33 5 36.3, P , 0.001, *P ,0.05 with Dunnett’s test) No significant increases in locomotor activitywere observed in the rest of the groups. Thus, repeated cocainetreatment induced locomotor sensitization to cocaine, which wasprevented by coadministration of EAA receptor antagonists (MK-801,CGS-19755, or NBQX).

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Fig. 2. Glutamate receptor antagonists prevent the induction ofDA autoreceptor subsensitivity by repeated cocaine administration.Cumulative dose–response curves showing the inhibitory effects of theDA receptor agonist apomorphine (given i.v.) on the spontaneousactivity of VTA DA neurons in different drug treatment groups. Eachdata point represents the mean 6 SEM. DA neurons recorded fromsal/coc treated rats were markedly less sensitive to apomorphineinhibition as compared to the sal/sal group in each of the threeexperiments. ANOVA revealed significant group effects (A: F3,42 522.4, P , 0.00001; B: F3,40 5 15.7, P , 0.00001; C: F3,47 5 12.1, P ,0.00001). Planned comparison analyses showed that the group effectin each case was due to the sal/coc group (*P , 0.05, Dunnett’s test).Thus, repeated cocaine treatment reduced the sensitivity of DAautoreceptors, an effect that was completely prevented by coadminis-tration of the noncompetitive NMDA receptor antagonist MK-801 (A),the competitive NMDA receptor antagonist CGS 19755 (B), or theAMPA receptor antagonist NBQX (C). When coadministered withsaline, these drugs exerted no effect on VTA DA autoreceptor sen-sitivity.

Fig. 3. Glutamate receptor antagonists prevent the ability ofrepeated cocaine administration to enhance the inhibitory effects ofDA D1 receptor stimulation on NAc neurons. Current–response curvesshowing the inhibitory effects of the DA D1 receptor agonist SKF-38393 (administered iontophoretically) on the firing rate of NAcneurons in different treatment groups. Data points represent themeans 6 SEM. In each of three experiments, NAc neurons recordedfrom sal/coc rats were significantly more sensitive to the inhibitoryeffects of SKF-38393 as compared to sal/sal rats. (A: F3,40 5 16.7, P ,0.00001; B: F3,42 5 9.1, P , 0.001; C: F3,41 5 6.6, P , 0.0025). Plannedcomparison analyses revealed that the significant groups effects wereprimarily due to the saline/cocaine groups in each experiment (*P ,0.05). Thus, the ability of cocaine to induce apparent DA D1 receptorsupersensitivity in NAc neurons was blocked by coadministration ofMK-801 (A) CGS-19755 (B) or NBQX (C). When coadministered withsaline, CGS-19755 and NBQX exerted no effect on the sensitivity ofNAc neurons to SKF 38393. However, there was a slight but statisti-cally significant decrease in the sensitivity to SKF 38393 followingrepeated administration of MK-801 alone (*P , 0.05).

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Lesions of the mPFC prevent cocainesensitization and its cellular correlates

Bilateral ibotenic acid-induced lesions of the mPFCwere performed 7–8 days prior to beginning repeateddrug treatments. As shown in Figure 4A, repeatedcocaine treatment produced significant behavioral sen-

sitization in sham-lesioned rats. The failure of mPFC-lesioned rats to exhibit sensitization cannot be attrib-uted to these lesions preventing expression of thesensitized behavior, because our previous studies havedirectly demonstrated that mPFC lesions do not pre-vent expression of cocaine sensitization once it has beenestablished (Li, Wolf, and White, submitted). Figure4B,C shows that lesions of the mPFC also prevented theinduction of VTA DA autoreceptor subsensitivity andthe enhanced response of NAc neurons to D1 receptorstimulation.

Representative examples of the bilateral mPFC le-sions are shown in Figure 5. The average lesionsextended from Bregma 14.7 to 12.2 (Paxinos andWatson, 1986). In all rats, an area of neuronal loss andgliosis surrounded an area of central cavitation. Withinthe PFC, destruction of cingulate cortex area 3 andinfralimbic cortex was observed in all rats. Portions ofthe medial and ventral orbital cortices, cingulate cortexareas 1 and 2, and small portions of frontal cortex area2 were also affected by most lesions. Based on aprevious study of the topographical organization of theefferent projections of the rat PFC (Sesack et al., 1989),the present lesions should have eliminated the majorityof projections to the NAc and midbrain DA areas.

DISCUSSION

We found that either NMDAorAMPAreceptor antago-nists, when coadministered with cocaine during re-peated treatment, prevent the induction of behavioralsensitization, as well as DA D2 autoreceptor subsensi-tivity in the VTA and DA D1 receptor supersensitivityin the NAc. We also observed that bilateral ibotenicacid-induced lesions of the mPFC produce the sameeffects as those observed with the EAA antagonists.These results clearly establish a causal link betweenEAA transmission originating in the mPFC and the

Fig. 4. Ibotenic acid-induced lesions of the mPFC prevent cocainesensitization and its cellular correlates. Lesions were made 7–8 daysbefore beginning repeated cocaine (or saline) administration. A: Thesham-lesioned, cocaine-treated group (n 5 11) showed a sensitizedlocomotor response to cocaine as compared to the mPFC-lesioned,saline-treated group (*t21 5 2.07, P , 0.05), or as compared to any ofthe previous saline/saline pretreated groups (see Fig. 1). Lesions of themPFC completely prevented the induction of cocaine sensitization. B:Cumulative dose–response curves showing that bilateral ibotenic acidlesions of mPFC prevent the development of VTA DA autoreceptorsubsensitivity induced by repeated cocaine treatment, and measuredas inhibition of firing by apomorphine (i.v.). Each data point representsthe mean 6 SEM. ANOVA revealed a significant group effect (F2,29 513.5, P , 0.001), which was completely due to the sham lesion/cocainegroup (*P , 0.05, Dunnett’s test). C: Current–response curves show-ing that bilateral excitotoxic lesions of mPFC prevent the ability ofrepeated cocaine treatment to enhance the sensitivity of NAc neuronsto the inhibitory effects of the DA D1 receptor agonist SKF 38393(administered iontophoretically). Each data point represents themean 6 SEM. ANOVA revealed a significant group effect (F2,35 5 21.7,P , 0.00001), which was totally due to the sham lesion/cocaine group(*P , 0.05, Dunnett’s test).

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induction of cocaine sensitization and its cellular corre-lates in the mesoaccumbens DA system.

EAA receptor antagonists and mPFC lesions:Induction of cocaine sensitization

The present results, showing that CGS 19755 andMK-801 prevent the induction of cocaine sensitization,replicate many previous studies that used differentclasses of NMDAreceptor antagonists, including NMDAreceptor channel blockers (Karler et al., 1989; Wolf andJeziorski, 1993; Kalivas and Alesdatter, 1993; Haraczet al., 1995; Ida et al., 1995), glycine site antagonists(Morrow et al., 1995; Shoaib et al., 1995), and competi-tive NMDA receptor antagonists (Kalivas and Alesdat-ter, 1993; Karler et al., 1994; Haracz et al., 1995). Theability of ibotenic acid lesions of mPFC to preventinduction of cocaine sensitization has not been previ-ously demonstrated, although we have reported thatsimilar lesions prevent the induction of amphetamine

sensitization (Wolf et al., 1995) but not its expression(Li and Wolf, 1997). In the only other study examiningthe role of AMPA receptors in the induction of cocainesensitization, partial prevention was observed in micewith the AMPA receptor antagonist DNQX (Karler etal., 1994). Our findings with the AMPA receptor antago-nist NBQX differ somewhat in that we observed com-plete prevention of cocaine sensitization in rats.Whether this is related to the use of different antago-nists, different species, or different behavioral mea-sures (ambulation vs. stereotypy) is unclear. Moreimportant is our new observation that all treatmentsthat prevented induction (NMDA antagonists, AMPAantagonists, mPFC lesions) also prevented the develop-ment of two established cellular correlates of cocainesensitization (see White et al., 1995b; White, 1996, forreviews), i.e., VTA DA autoreceptor subsensitivity andNAc D1 receptor supersensitivity. Along with otherresults (Wolf and Jeziorski, 1993), these findings argue

Fig. 5. Schematic representation of a typical ibotenic acid-induced lesions of mPFC. Lesion boundariesare defined as the area of neuronal loss (stippled area) as determined by routine light microscopy.Coordinates are from Paxinos and Watson (1986).

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that ionotropic glutamate receptor antagonists do notmerely mask the presence of sensitization, but interferewith fundamental cellular changes associated with itsinduction.

EAA receptor antagonists and mPFC lesions:Cellular correlates of sensitization

Although NMDA receptor antagonists were shownpreviously to prevent the development of cellular corre-lates of amphetamine sensitization (Wolf et al., 1995)the effects of mPFC lesions and AMPA receptor antago-nists on these cellular correlates were not determinedin that study. Moreover, until the present study noinformation was available about the effect of any ofthese manipulations on cellular correlates of cocainesensitization, a significant point because of the impor-tant differences that exist with respect to mechanismsresponsible for the induction of cocaine and amphet-amine sensitization. For example, DA D1 receptorantagonists prevent induction of amphetamine sensiti-zation but not of cocaine sensitization (Kuribara andUchihashi, 1993; Mattingly et al., 1994, 1996; Kurib-ara, 1995) whereas inhibitors of nitric oxide synthasecompletely prevent sensitization to cocaine (Pudiakand Bozarth, 1993; Kim and Park, 1995; Haracz et al.,1997; Itzhak, 1997) but either fail to prevent or onlypartially prevent sensitization to amphetamines (Stew-art et al., 1994; Abekawa et al., 1995; Inoue et al., 1996;Itzhak, 1997). In contrast, the present findings indicatethat amphetamine and cocaine sensitization have incommon a requirement for EAA transmission originat-ing in mPFC and involving NMDA and AMPA receptorstimulation.

The role of VTA EAAs in cocaine sensitization

Many studies have suggested that the VTA is theprimary brain site involved in processes responsible forthe induction of behavioral sensitization (see Introduc-tion). Our previous studies indicate that a criticaladaptation for the induction of sensitization is anincrease in the basal activity of VTA DA neurons. Thiscan be brought about by DA autoreceptor subsensitivityor by other mechanisms such as increased responsive-ness to glutamate (see White, 1996, for review). Wehave argued that, despite the transient nature ofincreased basal DA neuronal activity and associatedchanges within the VTA, these effects may be necessaryto produce subsequent adaptations that maintain sensi-tization (Ackerman and White, 1990; Henry and White,1991; Wolf et al., 1994). For example, decreased inhibi-tory tone due to DA autoreceptor subsensitivity couldpermit increased NMDA receptor activation, perhapseliciting ‘‘LTP-like’’ changes in DA neurons that contrib-ute to induction of sensitization. Although some investi-gators have argued against a role for DA D2 autorecep-tor subsensitivity in the initiation of sensitization,

based on the inability of D2 receptor antagonists toprevent its induction (Vezina and Stewart, 1989; Bjijouet al., 1996), they overlook the fact that D2 receptorantagonists also disinhibit the activity of VTA DAneurons and induce behavioral supersensitivity withrepeated administration (see White, 1996, for reviewand White et al., 1998 for additional relevant discus-sion).

The ability of EAA antagonists and mPFC lesions toprevent both cocaine sensitization and DA autoreceptorsubsensitivity argue further that the latter is criticallyinvolved in the induction processes responsible forsensitization. But how do EAA antagonists and mPFClesions prevent the development of DA autoreceptorsubsensitivity? The most likely possibility is that bothinterfere with cocaine-induced increases in EAA trans-mission between mPFC and VTA that are required toelicit DA autoreceptor subsensitivity and other compen-satory changes in the VTA. It is likely that interferenceoccurs at the level of the VTA. This would be consistentwith the ability of intra-VTA injections of NMDA recep-tor antagonists to prevent sensitization resulting fromrepeated intra-VTA amphetamine administration (Ca-dor et al., 1997) as well as enhanced behavioral sensitiv-ity to cocaine in a single administration (‘‘one-shot’’)context-dependent treatment paradigm (Kalivas andAlesdatter, 1993).

What would be the effect of mPFC lesions or EAAantagonists on EAA transmission within the VTA? Atleast three possibilities can be envisioned (see Wolf,1998, for detailed discussion). First, both manipula-tions would interrupt direct EAA projections to VTA DAcells (Sesack and Pickel, 1992) that may be required forhypothesized increases in excitatory drive (above) and‘‘LTP-like’’ changes in DA cell responsiveness. Thiswould be consistent with findings that coadministra-tion of EAAantagonists with stimulants prevents upreg-ulation of tyrosine hydroxylase activity and expression(Berhow et al., 1996; Masserano et al., 1996) becausesuch upregulation is generally associated with in-creased synaptic activation of catecholamine-contain-ing neurons (Guitart et al., 1990). Second, given thatnon-DA (GABAergic) cells in VTA are the primarytargets of excitatory afferents from mPFC (Sesack andPickel, 1992) and are known to regulate DA neuronactivity (see White, 1996, for review), depriving theVTA of excitatory drive may lead to disinhibition of DAcell activity, which would oppose DA autoreceptor-mediated inhibition in response to each injection ofcocaine, and thereby prevent the development of DAautoreceptor subsensitivity and ensuing neuroadapta-tions. Finally, glutamate receptors have been shown tomodulate DA D2 receptor gene expression in the mid-brain (Healy and Meador-Woodruff, 1996), raising thepossibility of a direct mechanism for glutamatergiccontrol of DA autoreceptor function.

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If EAA antagonists and mPFC lesions prevent sensi-tization by interrupting cocaine-induced increases inexcitatory transmission between mPFC and VTA, thenit follows that mPFC-VTA transmission might be en-hanced in sensitized rats. Indeed, there is evidence thatcocaine sensitization is accompanied by disinhibition ofmPFC neurons and that this may involve adaptationsat the level of mPFC. Repeated cocaine administrationreduces both the cocaine-evoked increases in extracellu-lar DA in the mPFC (Sorg et al., 1997) and thesensitivity of mPFC neurons to inhibition by DA (Whiteet al., 1995b). Consistent with a role for disinhibition ofmPFC neurons in sensitization is the finding thatelectrical kindling of mPFC sensitizes rats to cocaine(Schenk and Snow, 1994). Other studies indicate thatcocaine-induced neuroadaptations within the VTA it-self contribute to increased activity of DA neurons,including decreased levels of inhibitory G-protein a-sub-units (Nestler et al., 1990; Striplin and Kalivas, 1993)increased levels of glutamate receptor (GluR1 andNR1) subunits (Fitzgerald et al., 1996), and disinhibi-tion due to alterations in adenosine-DA interactionscontrolling GABA release (Bonci and Williams, 1996).

Role of NAc EAAs in cocaine sensitization

The NAc is intricately involved in the production oflocomotion by psychomotor stimulants as well as theexpression of sensitization to such behaviors (see Intro-duction). We have argued that sensitization to cocaineis related to the functional sensitivity of DAD1 receptor-mediated processes because enhanced sensitivity ofNAc neurons to DA D1 receptor stimulation parallelsthe persistence of cocaine sensitization (Henry andWhite, 1995). This idea is supported by findings indicat-ing that when cholera toxin is injected into the NAc toactivate the Gs-cAMP transduction pathway used by D1receptors, rats exhibit heightened locomotor responsesto cocaine (Cunningham and Kelley, 1993). Althoughchanges in NAc D1 receptor density have not beenroutinely observed after repeated cocaine administra-tion, upregulation of the DA D1 receptor signalingsystem has been reported, including increased levels ofadenylyl cyclase and cAMP-dependent protein kinase(see Fitzgerald and Nestler, 1995, for review). This, inturn, results in reduced sodium current density of NAcneurons, rendering them less excitable and biasingtheir response to hyperpolarization (White et al., 1997;Zhang et al., 1998). In addition, direct alterations inglutamate receptors within the NAc may also contrib-ute to the diminished activity of NAc neurons insensitized rats (White et al., 1995a; Lu et al., 1997).

The present findings indicate that intact EAA sys-tems originating in the mPFC are critical for theinduction of enhanced DA D1 receptor function in theNAc. How might mPFC lesions or systemic administra-tion of EAA antagonists prevent the induction of D1receptor supersensitivity? Based on the accepted role of

VTA as the site of the initiation of sensitization, a likelymechanism is that mPFC lesions or systemic adminis-tration of EAA antagonists decreases glutamatergictone in the VTA. This prevents cocaine-induced adapta-tions in VTA DA cell activity, and thereby preventsresultant downstream adaptations such as DA D1receptor supersensitivity in the VTA. However, themPFC also sends a substantial EAA projection directlyto the NAc (Sesack and Pickel, 1992) and it is wellestablished that corticostriatal projections are criticalfor synaptic plasticity exhibited by dorsal striatal andNAc neurons (Calabresi et al., 1992; Kombian andMalenka, 1994; Garcia-Munoz et al., 1996). Thus, it isconceivable that EAA projections to the NAc also play acritical role in sensitization-related changes in theexcitability of NAc neurons, including DA D1 receptorsupersensitivity. The idea that EAA antagonists (ormPFC lesions) prevent sensitization and associatedneuroadaptations in part through actions in the NAc issupported by a report that microinjection of (1)-HA966,an antagonist of the glycine site on the NMDA receptor,into the NAc prior to each of three daily cocaineinjections prevented the induction of cocaine sensitiza-tion (Khan and Shoaib, 1996). The fact that a similarresult was not obtained with intra-NAc injection ofMK-801 using the one-shot cocaine sensitization para-digm (Kalivas and Alesdatter, 1993) raises furtherquestions about the generality of this paradigm to moretraditional repeated cocaine treatments (see White etal., 1998, for further discussion). The idea that the NAcplays at least a partial role in the ability of EAAsystems to modulate induction of cocaine sensitizationis not necessarily inconsistent with the prevailing viewthat sensitization can be elicited by drug actions in theVTA. Rather, it simply fortifies the conceptualization ofsensitization as a complex cascade of processes ulti-mately requiring adaptations with the NAc and otherforebrain areas such as mPFC, and involving gluta-mate as a ubiquitous regulator at all levels of therelevant neuronal circuitry.

Comparisons between amphetamineand cocaine sensitization with respect

to the involvement of EAAs

As noted above, previous studies have revealed differ-ences in dopaminergic involvement in the induction ofcocaine and amphetamine sensitization, so it was impor-tant to determine if glutamate similarly played differ-ent roles in the induction of sensitization to eachstimulant. The present results, combined with earlierfindings (Wolf et al., 1994, 1995), demonstrate thatglutamatergic tone, originating at least in part frommPFC, is essential for the induction of cocaine andamphetamine sensitization and associated neuroadap-tations. Because sensitization is a form of neuronalplasticity, it is not surprising that glutamatergic trans-mission is required for its induction in both instances.

177GLUTAMATE AND COCAINE SENSITIZATION

However, the present results do not rule out moresubtle differences between the exact mechanisms bywhich glutamate contributes to induction of amphet-amine and cocaine sensitization. Indeed, such differ-ences appear to exist with respect to psychomotorstimulant-induced effects on glutamate efflux withinthe VTA (Kalivas and Duffy, 1995; Xue et al., 1996; Wolfand Xue, 1998) and the nature of AMPA receptorinvolvement in the induction of sensitization (Li et al.,1997). More marked differences appear to exist withrespect to glutamatergic involvement in the mainte-nance and expression of amphetamine and cocainesensitization. For example, mPFC lesions performedafter repeated amphetamine administration fail to pre-vent the expression of amphetamine sensitization (Liand Wolf, 1997), whereas lesions targeting dorsal, butnot ventral, mPFC disrupt the expression of cocainesensitization (Pierce et al., 1998). It is likely that thisreflects a more important role for disinhibition of mPFCprojections in the expression of cocaine sensitization ascompared to amphetamine sensitization (Sorg and Kali-vas, 1993; White et al., 1995b; Xue et al., 1996; Pierce etal., 1996a, 1998; Sorg et al., 1997). Moreover, repeatedadministration of cocaine and amphetamine lead todifferent patterns of alteration in glutamate receptorexpression in the NAc when examined after relativelylong withdrawal periods (Churchill et al., 1997;Ghasemzadeh et al., 1997; Lu et al., 1997; Lu and Wolf,1997).

Conclusions

Our findings add to a growing body of evidenceimplicating the mPFC as an important site involved inthe induction of cocaine (and amphetamine) sensitiza-tion. In recent years, the VTA has been the major focusof sensitization research based primarily on the factthat intra-VTA injections of various drugs either inducesensitization or modulate its induction (see Kalivas,1995, for review). However, direct injections of cocaineinto the VTA do not induce sensitization (Steketee,1998) and it is known that the mPFC is a critical site forthe induction of cocaine self-administration behavior(Goeders and Smith, 1983; Goeders et al., 1986). Evenwith amphetamine and other drugs, for which it isknown that actions in the VTA are sufficient for theinduction of sensitization, it is likely that additionalneuroadaptations within the mPFC (and elsewhere)contribute to essential features of sensitization such aspersistence. Indeed, amphetamine sensitization is ac-companied by time-dependent alterations in EAA recep-tor subunit expression in the mPFC (Lu et al., 1997).Additional studies regarding neuroadaptations in themPFC during induction of cocaine sensitization areclearly warranted and are likely to provide new in-sights into cortical involvement in cocaine addictionand withdrawal.

ACKNOWLEDGMENTS

We thank Lorinda Baker for technical assistance.This work was supported by USPHS grants DA04093(FJW) and DA09621 (MEW) from the National Insti-tute on Drug Abuse (NIDA). FJW is also the recipient ofan Independent Scientist Award from NIDA (DA00207).CDS is supported by a predoctoral National ResearchService Award from the National Institute of MentalHealth (MH11388).

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