NUCLEUS ACCUMBENS AND EFFORT-RELATED FUNCTIONS:BEHAVIORAL AND NEURAL MARKERS OF THE INTERACTIONSBETWEEN ADENOSINE A2A AND DOPAMINE D2 RECEPTORS

A. M. FARRAR,a1 K. N. SEGOVIA,a P. A. RANDALL,a

E. J. NUNES,a L. E. COLLINS,a C. M. STOPPER,a

R. G. PORT,a J. HOCKEMEYER,b C. E. MÜLLER,b

M. CORREAa2 AND J. D. SALAMONEa*aDepartment of Psychology, University of Connecticut, Storrs, CT06269-1020, USAbPharma-Zentrum Bonn, Universität Bonn, Pharmazeutisches Institut,Pharmazeutische Chemie I, Bonn, Germany

Abstract—Nucleus accumbens dopamine (DA) is a criticalcomponent of the brain circuitry regulating work output inreinforcement-seeking behavior and effort-related choice be-havior. Moreover, there is evidence of an interaction betweenDA D2 and adenosine A2A receptor function. Systemic admin-istration of adenosine A2A antagonists reverses the effects ofD2 antagonists on tasks that assess effort related choice. Thepresent experiments were conducted to determine if nucleusaccumbens is a brain locus at which adenosine A2A and DAD2 antagonists interact to regulate effort-related choice be-havior. A concurrent fixed ratio 5 (FR5)/chow feeding proce-dure was used; with this procedure, rats can choose betweencompleting an FR5 lever-pressing requirement for a preferredfood (i.e., high carbohydrate operant pellets) or approachingand consuming a freely available food (i.e., standard rodentchow). Rats trained with this procedure spend most of theirtime pressing the lever for the preferred food, and eat verylittle of the concurrently available chow. Intracranial injec-tions of the selective DA D2 receptor antagonist eticlopride(1.0, 2.0, 4.0 !g) into nucleus accumbens core, but not adorsal control site, suppressed FR5 lever-pressing and in-creased consumption of freely available chow. Either sys-temic or intra-accumbens injections of the adenosine A2A

receptor antagonist MSX-3 reversed these effects of eticlo-pride on effort-related choice. Intra-accumbens injections ofeticlopride also increased local expression of c-Fos immuno-reactivity, and this effect was attenuated by co-administra-tion of MSX-3. Adenosine and DA systems interact to regulateinstrumental behavior and effort-related processes, and nu-cleus accumbens is an important locus for this interaction.These findings may have implications for the treatment ofpsychiatric symptoms such as psychomotor slowing, aner-gia and fatigue. © 2010 IBRO. Published by Elsevier Ltd. Allrights reserved.

Key words: anergia, reward, motivation, decision making,fatigue, depression.

An important aspect of motivation is that organisms areable to overcome work-related response costs that sepa-rate them from motivationally relevant stimuli (Salamone,1992; Salamone et al., 1991, 1997, 2003, 2007; Salamoneand Correa, 2002; van den Bos et al., 2006; Niv et al.,2007). The work requirements necessary for obtainingaccess to reinforcing stimuli can vary in several ways (e.g.ratio, force or distance requirements; Collier and Jennings,1969; Aberman and Salamone, 1999; Ishiwari et al., 2004;van den Bos et al., 2006). In addition, organisms oftenmake effort-related choices based upon cost/benefit anal-yses, allocating responses in relation to several factors,including assessments of motivational value and work re-quirements (Salamone et al., 1991, 1997, 2003, 2005;Salamone and Correa, 2002, 2009; Rushworth et al.,2004; Ernst and Paulus, 2005; Phillips et al., 2007; Crox-son et al., 2009). Considerable evidence indicates thatnucleus accumbens dopamine (DA) is involved in behav-ioral activation and effort-related processes (Salamone etal., 1991, 2003, 2005, 2007; Vezina et al., 2002; Zhang etal., 2003; Wakabayashi et al., 2004; Barbano and Cador,2006, 2007; Cagniard et al., 2006; Denk et al., 2005;Phillips et al., 2007; Floresco et al., 2008; Salamone,2010). The effect of nucleus accumbens DA depletionsdepends greatly upon the ratio requirement of the operantschedule (McCullough et al., 1993; Aberman and Salam-one, 1999; Salamone et al., 2001; Ishiwari et al., 2004).Furthermore, nucleus accumbens DA also plays an impor-tant role in effort-related choice behavior. Several studiesin this area have employed maze tasks to assess effort-related choice (Salamone et al., 1994; Cousins et al.,1996; Floresco and Ghods-Sharifi, 2007; Mott et al., 2009;Bardgett et al., 2009), while others have used a concurrentfixed ratio 5 (FR5)/chow feeding procedure (Salamone etal., 1991, 2002, 2003, 2007; Cagniard et al., 2006; Koch etal., 2000). With the FR5/chow feeding task, rats canchoose between responding on an FR5 schedule of rein-forcement for a highly preferred food (i.e., high carbohy-drate operant pellets) or approaching and consumingfreely available food (i.e., less preferred laboratory rodentchow). Typically, untreated rats that are trained with thisprocedure spend most of their time pressing the lever forthe preferred food, and consume very little of the concur-rently available free chow. Relatively low doses of eitherD1 or D2 DA receptor antagonists, including haloperidol,cis-flupenthixol, SCH 23390, SCH 39166, raclopride, andeticlopride, all suppress lever pressing for food, but actu-ally increase chow intake (Salamone et al., 1991; Cousinset al., 1994; Salamone et al., 2002; Sink et al., 2008). A

1 Present address: Center for Molecular and Behavioral Neuroscience,Rutgers University, Newark, NJ 07102, USA.2 Present address: Àrea de Psicobiologia, Campus de Riu Sec, Uni-versitat Jaume I, 12071 Castelló, Spain.*Corresponding author. Tel: !1-860-486-4302; fax: !1-860-486-2760.E-mail address: john.salamone@uconn.edu (J. D. Salamone).Abbreviations: DA, dopamine; FR5, fixed ratio 5.

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0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2009.12.056

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brain region that is closely associated with these effects isnucleus accumbens. DA depletion and injection of D1 orD2-family antagonists into dorsomedial shell and core sub-regions of accumbens have been shown to decrease le-ver-pressing and increase chow consumption on this task(Salamone et al., 1991; Cousins et al., 1993; Sokolowskiand Salamone, 1998; Koch et al., 2000; Nowend et al.,2001).

In addition to nucleus accumbens DA, other brain areasand transmitters are involved in effort-related processes,including prefrontal/anterior cingulate cortex, amygdala,and ventral pallidal GABA (Walton et al., 2002, 2003, 2006;Denk et al., 2005; Schweimer et al., 2005; Schweimer andHauber, 2006; Floresco and Ghods-Sharifi, 2007; Farrar etal., 2008; Desmurget and Turner, 2008; Hauber and Som-mer, 2009; Croxson et al., 2009). Recent research hasbegun to describe the role of the purine nucleoside aden-osine in this type of function (Farrar et al., 2007; Font et al.,2008; Mingote et al., 2008; Salamone and Correa, 2009).Adenosine A2A receptors are highly concentrated in striatalareas, including both neostriatum and nucleus accumbens(Jarvis and Williams, 1989; Schiffmann et al., 1991; DeMetand Chicz-DeMet, 2002; Ferre et al., 2004), where they arelargely co-localized with DA D2 receptors on enkephalinpositive neurons (Fink et al., 1992; Ferré, 1997; Hillion etal., 2002; Fuxe et al., 2003). Interactions between adeno-sine A2A and DA D2 receptors have typically been inves-tigated in connection with neostriatal motor functions thatare potentially related to Parkinsonism (Ferre et al., 1997,2001; Hauber and Munkle, 1997; Svenningsson et al.,1999; Hauber et al., 2001; Wardas et al., 2001; Morelli andPinna, 2001; Correa et al., 2004; Pinna et al., 2005; Salam-one et al., 2008a,b). Evidence indicates that DA-adenosineinteractions also are involved in behavioral functions re-lated to nucleus accumbens. Stimulation of accumbensadenosine A2A receptors by local injections of the agonistCGS 21680 decreased locomotor activity (Barraco et al.,1993, 1994), and the suppression of locomotion inducedby the DA antagonist haloperidol was reversed by injec-tions of the adenosine A2A receptor antagonist MSX-3 intoaccumbens core, but not into the shell or the ventrolateralneostriatum (Ishiwari et al., 2007). Intra-accumbens injec-tions of CGS 21680 also affected lever pressing and effort-related choice behavior in a manner that closely resembledthe effects of accumbens DA depletion or antagonism(Mingote et al., 2008; Font et al., 2008). Furthermore,systemic administration of adenosine A2A antagonists hasbeen shown to reverse the effort-related effects of system-ically administered DA D2 antagonists (Farrar et al., 2007;Worden et al., 2009; Mott et al., 2009; Salamone et al.,2009b). Nevertheless, it remains uncertain if nucleus ac-cumbens is a brain locus at which adenosine A2A and DAD2 antagonists interact to regulate effort-related choicebehavior.

The present experiments were conducted to study theability of systemic or intra-accumbens co-administration ofthe selective adenosine A2A antagonist, MSX-3, to reversethe effects of intra-accumbens core injections of the DA D2

antagonist eticlopride on the concurrent FR5/chow feeding

procedure. For these experiments, eticlopride was usedbecause of recent data indicating that adenosine A2A an-tagonism is capable of fully reversing the effects of a D2

antagonist on effort-related choice behavior, but had onlyminimal impact on the actions of a D1 antagonist (Wordenet al., 2009). In addition, the core subregion of nucleusaccumbens was targeted because of data indicating thatthe core is involved in mediating some of the effort-relatedeffects of adenosine A2A agonists (Font et al., 2008; Min-gote et al., 2008), and because it sends direct projectionsto the ventral pallidal area that was the target in a relatedseries of experiments (Farrar et al., 2008; Mingote et al.,2008). Experiments 1 and 2 assessed the effects of eticlo-pride alone on the concurrent choice procedure when in-jected into accumbens core and a control site dorsal toaccumbens core, respectively. Experiments 3 and 4 exam-ined the ability of intra-accumbens and systemic injectionsof MSX-3 to reverse the effects of eticlopride on the con-current choice procedure. In order to provide a neuralmarker of the interactions between eticlopride and MSX-3,experiment 5 assessed the effect of intra-accumbens eti-clopride on expression of c-Fos, and the ability of MSX-3 toreverse this effect. These experiments were conducted inorder to determine if nucleus accumbens is an importantlocus for the effort-related behavioral functions regulatedby DA-adenosine interactions.

EXPERIMENTAL PROCEDURES

Animals

For all experiments, 132 male Sprague–Dawley rats (Harlan–Sprague–Dawley, Indianapolis, IN, USA) were used. The animalswere housed in a colony maintained at 22–24 °C with a 12 hlight/12 h dark cycle (lights on at 0700), and housed in pairs beforesurgery and placed in single cages afterwards. Water was avail-able ad lib in the home cages at all times. The rats that were testedin operant boxes were food restricted to 85% of their free-feedingweight for initial operant training and allowed modest weight gainduring the studies. Animal protocols have been approved by theUniversity of Connecticut Institutional Animal Care and Use Com-mittee, and the studies were conducted according to NIH guide-lines for animal care and use.

Operant choice procedure

Behavioral sessions were conducted in operant conditioningchambers (28"23"23 cm3; Med Associates, Georgia, VT, USA)during the light period. Animals were trained in 30 min sessions, 5days per week. In the first week of training, all rats were trained tolever press for 45 mg pellets (Research Diets, Inc., New Bruns-wick, NJ, USA) on an FR1 schedule. In the second week, animalswere shifted to an FR5 schedule, which was maintained for 3–4weeks to ensure stable performance. At this point, rats weretrained on the concurrent FR5/chow feeding choice procedure.Weighed amounts of laboratory chow (Prolab, Lab Diet, Brent-wood, MO, USA; typically 15–20 g, three large pieces) wereconcurrently available on the floor of the chamber during the 30min FR5 sessions. At the end of the session, rats were immedi-ately removed from the chambers. Food intake was determined byweighing the remaining food, including spillage, while lever press-ing was recorded by a computer program.

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Pharmacological agents

The dopamine D2 antagonist eticlopride (S(#)-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamidehydrochloride) was obtained from Sigma Chemical (St. Louis, MO,USA) and was dissolved in a 0.9% saline solution. Doses ofeticlopride were determined on the basis of pilot studies. Theadenosine A2A antagonist used was MSX-3 ((E)-phosphoric acidmono-[3-[8-[2-(3-methoxyphenyl)vinyl]-7-methyl-2,6-dioxo-1-prop-2-ynyl-1,2,6,7-tetrahydropurin-3-yl] propyl] ester disodium salt),which was synthesized at the laboratory of Dr. Christa Müller atthe Pharmazeutisches Institut, Universität Bonn, in Bonn, Ger-many. Preparation of the drug solution consisted of dissolvingMSX-3 (free acid) in 0.9% saline and adjusting the pH by titratingwith microliter quantities of 1.0 N NaOH until the drug was insolution. The final pH was typically 7.5$0.2, but never exceeded7.8. MSX-3 is a pro-drug that is cleaved in vivo to the pharmaco-logically active compound MSX-2 (Hockemeyer et al., 2004).Doses of MSX-3 were selected based upon pilot studies andprevious research (Ishiwari et al., 2007; Farrar et al., 2007; Wor-den et al., 2009).

Surgical and intracranial injection procedures

Rats were anesthetized with a solution (1.0 ml/kg, IP) that con-tained ketamine (Bioniche Pharma, Lake Forest, IL, USA; 100mg/ml) and xylazine (Phoenix Pharmaceuticals, St. Joseph, MO,USA; 0.75 ml of a 20 mg/ml solution added to 10.0 ml of ketaminesolution), and placed in a stereotaxic apparatus. The incisor baron the stereotaxic apparatus was set to 5.0 mm above the inter-aural line. All animals received bilateral implantations of stainlesssteel guide cannulae (25 ga, extra-thin wall). For the nucleusaccumbens site, guide cannulae were implanted 1.0 mm dorsal totarget at the following coordinates: AP!2.8 mm (from bregma),ML$1.6 mm (from midline), and DV–6.8 mm (from the skullsurface). For the dorsal control site, guide cannulae were im-planted at the following coordinates: AP!2.8 mm (from bregma),ML$1.6 mm (from midline), and DV–4.8 mm (from the skullsurface). The guide cannulae were secured to the skull withstainless steel screws and cranioplastic cement. To maintain pa-tency of the cannulae prior to injection, stainless steel stylets wereinserted. All animals were housed in separate cages after surgery,and were allowed 7–10 days to recover. After recovering fromcannulae implantation surgery, rats in experiments 1–4 resumedtraining on the concurrent FR5/chow feeding procedure for twoadditional weeks. For bilateral intracranial (IC) injections throughthe cannulae, 30 ga stainless steel injectors were used. Theinjectors were set to extend 1.0 mm beyond the tip of the guidecannulae. The injectors were connected to 10.0 !l Hamilton sy-ringes with PE-10 tubing, and the injections were driven by asyringe pump (Harvard Apparatus). For experiments 1, 2, 4 and 5the infusion rate was 0.125 !l/min., and each side received 0.5 !ltotal volume. In experiment 3 the infusion rate was 0.25 !l/min.,because each side received 1.0 !l total volume for experiment inwhich eticlopride and MSX-3 were dissolved in the same intracra-nial solution. Following all intracranial infusions, the injectors wereleft in place for an additional 1 min to allow diffusion of the drug.Each animal received an injection of only one drug treatmentcondition.

Experimental procedures

Experiment 1: Effect of intra-accumbens microinjection of theD2 receptor antagonist eticlopride on the concurrent choiceprocedure. Rats (n%33) were trained on the concurrent FR5/chow feeding procedure as described above. After training, ratswere implanted with cannulae in nucleus accumbens, and trainingwas resumed after the post-surgical recovery period. Drug testingwas conducted using a between-groups design, with each rat

receiving only one drug treatment. Rats were randomly assignedto receive intracranial injections of saline vehicle, 1.0, 2.0 !g, or4.0 !g eticlopride per side, in 0.5 !l total volume per side. Directlyfollowing the injection procedure, the animals were placed in theoperant chambers for a 30-min FR5/chow feeding session. Be-havioral measures included the total number of lever presses andthe total amount of chow consumed.

Experiment 2: Effect of intracranial injections of eticloprideinto a dorsal control site on the concurrent choice procedure.Rats (n%15) were trained on the concurrent FR5/chow feedingprocedure. After training, rats were implanted with cannulae in acontrol site dorsal to nucleus accumbens and training was re-sumed after post-surgical recovery. Rats were randomly assignedto receive saline vehicle, 2.0 !g eticlopride or 4.0 !g eticloprideper side. The drug injection procedure, behavioral procedures andmeasures for this experiment were identical to those used inexperiment 1.

Experiment 3: Effect of intra-accumbens MSX-3 and eticlo-pride on the concurrent choice procedure. Rats (n%29) weretrained on the concurrent FR5/chow feeding procedure as de-scribed above. After training, rats were implanted with cannulae innucleus accumbens, and training was resumed after the post-surgical recovery period. Drug testing was conducted using abetween-groups design, with each rat receiving only one drugtreatment. Rats were randomly assigned to receive intracranialinjections of saline vehicle, 4.0 !g eticlopride, 4.0 !g eticlo-pride!1.25 !g MSX-3, 4.0 !g eticlopride !2.5 !g MSX-3, or 4.0!g eticlopride!5.0 !g MSX-3, in 1.0 !l total volume. Directlyfollowing the injection procedure, the animals were placed in theoperant chambers for a 30-min FR5/chow feeding session. Be-havioral measures included the total number of lever presses andthe total amount of chow consumed.

Experiment 4: Effect of systemic MSX-3 and intra-accumbenseticlopride on the concurrent choice procedure. Rats (n%32)were trained on the concurrent FR5/chow feeding procedure asdescribed above. After training, rats were implanted with cannulaein nucleus accumbens, and training was resumed after the post-surgical recovery period. Drug testing was conducted using abetween-groups design, with each rat receiving only one drugtreatment. Rats were randomly assigned to the following treat-ment conditions: IP saline vehicle (20 min before testing)!intracranial saline vehicle (immediately before testing), IP salinevehicle!intracranial eticlopride (2.0 !g in 0.5 !l per side), or IPMSX-3 (0.5, 1.0, or 2.0 mg/kg)!intracranial eticlopride. Directlyfollowing the intracranial injection procedure, the animals wereplaced in the operant chambers for a 30-min FR5/chow feedingsession. Behavioral measures included the total number of leverpresses and the total amount of chow consumed.

Experiment 5: Effect of intra-accumbens microinjection ofeticlopride alone and eticlopride!systemic MSX-3 on immediateearly gene expression (c-Fos immunoreactivity) in nucleus ac-cumbens neurons. Experimentally naïve rats (n%23) were im-planted with cannulae in nucleus accumbens. Following the post-surgical recovery period, rats were randomly assigned to thefollowing treatment conditions: IP saline vehicle (140 min beforeperfusion)!intracranial saline vehicle (120 min before perfusion),IP saline vehicle!intracranial eticlopride (2.0 !g in 0.5 !l perside), or IP MSX-3 (2.0 mg/kg)!intracranial eticlopride. All ani-mals were anesthetized and perfused with physiological salinefollowed by 3.7% formaldehyde 120 min after the IC injections andstored at 4 °C in formaldehyde until processing.

c-Fos visualization and quantification

The processing for c-Fos visualization was performed using ausing a standard immunohistochemistry protocol modified for

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the detection of c-Fos in free-floating sections. Free floatingcoronal sections (50 !m) were serially cut using a microtomecryostat (Weymouth, MA, USA), rinsed in 0.01 M PBS (pH 7.4)and incubated in 0.3% hydrogen peroxide (H2O2) for 30 min toblock endogenous staining. Sections were then rinsed in PBS(3" for 5 min) and transferred into the primary antibody, anti-c-Fos (Calbiochem, Germany) for a 48 h incubation. Followingthe primary antibody treatment, the sections were rinsed in PBSand incubated in the secondary antibody, anti-rabbit HRP con-jugate, envision plus (DAKO, Denmark) for 2 h. The immuno-histochemical reaction was developed using diaminobenzidine(DAB) as the chromagen. Processed sections were thenmounted to gelatin-coated slides, air dried, and cover-slippedusing Cytoseal 60 (Thermo Scientific) as a mounting medium.The sections were examined and photographed using a NikonEclipse E600 (Melville, NY, USA) upright microscope equippedwith an Insight Spot digital camera (Diagnostic Instruments, Inc).Images of the region of interest were magnified at 20X and cap-tured digitally using SPOT software; the target area was the0.5 mm2 region immediately ventral to the site of the injection.Brains with injection sites outside nucleus accumbens, or thosewith extensive damage or uneven staining across sections wereexcluded from statistical analysis. Cells that were positively la-beled for c-Fos were quantified with the aid of ImageJ software (v.1.42, National Institutes of Health sponsored image analysis pro-gram). The analysis was carried out on two sections per animaland the average value for both sections was used for statisticalanalysis.

Nissl staining procedure

All animals from experiments 1–4 were anesthetized with CO2

and perfused with physiological saline followed by 3.7% formal-dehyde solution. The brains were extracted and stored in formal-dehyde solution for 48 h. Prior to sectioning, the brains wereplaced in cryoprotectant solution consisting of 30% sucrose, andwere subsequently sectioned with a cryostat in 50 !m slices andmounted on glass microscope slides. Following mounting, tissuewas stained with Cresyl Violet and cover-slipped, allowing verifi-cation of cannula placements using a light microscope. Only an-imals with verified cannula placements were used for statisticalanalysis (24% of animals were rejected). Cannula placements for

all animals receiving the 4.0 !g dose of eticlopride in experiments1 and 2 are shown in Fig. 1.

Data analyses

Data for experiments 1–4, including total lever presses and chowintake quantities, were analyzed using a one-factor (drug treat-ment) between subjects analysis of variance (ANOVA). Non-or-thogonal planned comparisons using the overall error term wereused, and data from each treatment condition were compared witheither the data from the vehicle condition (experiments 1–2) or theeticlopride plus vehicle treatment condition (experiments 3–4).For the planned comparisons, the number of comparisons wasrestricted to the number of treatments minus one (Keppel, 1991).For experiments 1 and 2, effect size calculations (R2 values;Keppel, 1991; p. 66) were performed to assess the magnitude ofthe effect (i.e., the size of the treatment effect sum of squaresexpressed as the proportion of total sum of squares, which is amarker of the total variance accounted for by treatment variance;for example R2%0.3 reflects 30% of the variance explained).Additionally, correlational analyses were used to measure therelation between lever pressing and chow consumption in exper-iments 1 and 2. Data for experiment 5 (number of c-Fos positivecells) were analyzed using a one-factor (drug treatment) betweensubjects ANOVA. Non-orthogonal planned comparisons wereused to compare the eticlopride condition to both the vehicle andMSX-3 plus eticlopride conditions.

RESULTS

Experiments 1 and 2

The results of experiment 1 are shown in Fig. 2A, B. Intra-accumbens injections of eticlopride dose-dependently de-creased lever-pressing [F(3, 29)%14.06, P&0.001] andincreased consumption of the concurrently available chow[F(3, 29)%10.75, P&0.001]. Planned comparisons re-vealed that both the 2.0 and 4.0 !g doses of eticlopridesignificantly decreased lever-pressing and increased chowintake. Furthermore, there was a significant inverse corre-lation between lever presses and chow intake (r%#0.84,

Fig. 1. (A) Composite photomicrograph of Nissl-stained section indicating representative accumbens core cannula placement. (B) Drawings indicatingcannula placements for animals that received 4.0 !g eticlopride in accumbens core in experiment 4.1 (open circles). (C) Cannula placements foranimals that received 4.0 !g eticlopride in the dorsal control site in experiment 4.2 (black circles). Coronal section diagrams adapted from Pellegrinoet al. (1979).

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df%29; P&0.001). The results of experiment 2 are shownin Fig. 2C, D. Injections of eticlopride into the dorsal controlsite failed to produce a significant shift from lever pressingto chow intake; there was no significant decrease in lever-pressing [F(2, 12)%2.08, n.s.] or increase in chow con-sumption [F(2, 12)%0.59, n.s.]. Furthermore, there was nosignificant inverse correlation between lever pressingand chow intake (r%#0.45, df%12; n.s.). Table 1 showsthe effect size calculations (R2 values) for experiments 1and 2.

Experiment 3

The results of experiment 3 are shown in Fig. 3. There wasa significant overall effect of drug treatment on lever-press-ing [F(4, 24)%6.53, P&0.01] and chow consumption [F(4,24)%3.83, P&0.05]. Planned comparisons revealed thateticlopride significantly reduced lever pressing (P&0.01)and increased chow consumption (P&0.05) relative to ve-hicle alone. In addition, co-administration of MSX-3 with

eticlopride significantly increased lever pressing comparedto eticlopride alone (P&0.05), and decreased chow intakeat the 1.25 !g (P&0.05) and 5.0 !g (P&0.01) doses.

Experiment 4

The results of experiment 4 are shown in Fig. 4. There wasa significant overall effect of drug treatment on lever-press-ing [F(4, 27)%4.74, P&0.01] and chow consumption [F(4,27)%3.86, P&0.05]. Planned comparisons revealed thateticlopride significantly reduced lever pressing (P&0.01)and increased chow intake (P&0.05) when compared tothe vehicle-only condition. Furthermore, systemic co-ad-ministration of MSX-3 significantly increased lever press-ing at the 0.5 (P&0.05), 1.0 (P&0.01), and 2.0 (P&0.01)mg/kg doses, and decreased chow intake at the 1.0(P&0.05) and 2.0 (P&0.01) mg/kg doses relative to theeticlopride alone condition.

Experiment 5

The results of experiment 5 are shown in Fig. 5. There wasa significant overall effect of drug treatment on the numberof c-Fos positive cell counts [F(2, 20)%12.8, P&0.01]. Inaddition, planned comparisons revealed that eticlopridealone significantly increased c-Fos positive cell countsrelative to vehicle alone (P&0.001), whereas co-adminis-

Fig. 2. (A, B) The effects of accumbens core injections of eticlopride on performance of the concurrent lever pressing/chow feeding choice procedure.Rats received treatment with either saline vehicle, 1.0, 2.0 or 4.0 !g eticlopride. (A) Mean ($SEM) number of lever presses (FR5 schedule) duringthe 30 min session. (B) Mean ($SEM) gram quantity of chow intake. (* P&0.05, different from vehicle, planned comparisons). (C, D) The effects ofinjections of eticlopride into the dorsal control site on performance of the concurrent lever pressing/chow feeding choice procedure. Rats receivedtreatment with either saline vehicle, 2.0 or 4.0 !g eticlopride. (C) Mean ($SEM) number of lever presses (FR5 schedule) during the 30 min session.(D) Mean ($SEM) gram quantity of chow intake.

Table 1. Effect size calculations (R2 values) for eticlopride dose re-sponse studies

Site Lever pressing Chow intake

Accumbens core 0.61 0.40Dorsal control 0.17 0.02

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tration of MSX-3 significantly reduced c-Fos positive cellcounts relative to eticlopride alone (P&0.05).

DISCUSSION

In the present studies, a concurrent lever pressing/chowfeeding task was used to characterize the effects of intra-cranial administration of the D2 DA receptor antagonisteticlopride, and to examine the interaction between eticlo-pride and the selective adenosine A2A receptor antagonistMSX-3. These studies were undertaken to determinewhether nucleus accumbens is an important locus for theD2/A2A receptor interactions that are involved in effort-related choice behavior. Experiment 1 demonstrated thatlocal injections of eticlopride into the nucleus accumbenscore produced a dose-related decrease in lever pressing,and a concomitant increase in chow intake. In addition,there was a high inverse correlation between lever press-ing and chow intake. These results are consistent with

previous studies demonstrating that the shift from leverpressing to chow intake on the concurrent lever pressing/chow feeding choice procedure can be produced by injec-tions of low systemic doses of either D1 or D2-type DAreceptor antagonists (Salamone et al., 1991, 2002; Sink etal., 2008), intra-accumbens injection of the D2 DA receptorantagonists sulpiride (Koch et al., 2000) and raclopride(Nowend et al., 2001), and by accumbens DA depletions(Cousins et al., 1993, 1994; Cousins and Salamone, 1994;Sokolowski and Salamone, 1998). Considerable evidenceindicates that the shift from lever pressing to chow intakeinduced by interference with DA transmission is not due toa suppression of appetite for food or a change in foodpreference. Systemic administration of the D2 antagonisthaloperidol at the same doses that cause the shift fromlever pressing to chow intake did not affect intake of either

Fig. 4. The effects of systemic MSX-3 on intra-accumbens eticlopride-induced changes in performance on the concurrent lever pressing/chow feeding procedure. Rats received of I.P. injections of vehicle plusintra-accumbens injections of vehicle (Veh!Veh), 2.0 !g eticloprideplus I.P. vehicle (Etic!Veh), and 2.0 !g eticlopride plus 0.5, 1.0 and2.0 mg/kg doses of systemic MSX-3 (Etic!0.5, 1.0 and 2.0 M).(A) Mean ($SEM) number of lever presses (FR5 schedule) during the30 min session. (B) Mean ($SEM) gram quantity of chow intake.Eticlopride significantly decreased lever pressing and increased chowintake relative to vehicle (# P&0.05; ## P&0.01). MSX-3 systemicallyadministered to eticlopride-treated rats significantly increased leverpressing and decreased chow intake relative to treatment with eticlo-pride alone (* P&0.05; ** P&0.01).

Fig. 3. The effects of intra-accumbens MSX-3 on eticlopride-inducedchanges in performance on the concurrent lever pressing/chow feed-ing procedure. Rats received intra-accumbens injections of vehicleplus vehicle (Veh!Veh), 4.0 !g eticlopride plus vehicle (Etic!Veh),and 4.0 !g eticlopride plus 1.25, 2.5 and 5.0 !g doses of MSX-3(Etic!1.25, 2.5 and 5.0 M). (A) Mean ($SEM) number of lever presses(FR5 schedule) during the 30 min session. (B) Mean ($SEM) gramquantity of chow intake. Eticlopride significantly decreased leverpressing and increased chow intake relative to vehicle (# P&0.05).MSX-3 administered to eticlopride-treated rats significantly increasedlever pressing and decreased chow intake relative to treatment witheticlopride alone (* P&0.05; ** P&0.01).

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operant pellets or chow, nor did it alter preference for thetwo types of food (Salamone et al., 1990, 1991). Intra-accumbens injections of sulpiride at doses that affectedperformance on the concurrent choice task also failed toaffect either intake of the preferred operant pellets or thepreference for the operant pellets over chow (Koch et al.,2000). In addition, chow intake was not affected by accum-bens DA depletions (Koob et al., 1978; Salamone et al.,1993), or by intra-accumbens injections of D1 or D2 familyantagonists into core or shell subregions (Baldo et al.,2002). Finally, considerable evidence has demonstrated

that the actions of DA antagonists on the concurrent choiceprocedure do not resemble the effects of appetite suppres-sant drugs or pre-feeding to reduce appetite (Salamone etal., 1991, 2002; Cousins et al., 1994; Sink et al., 2008).This pattern of findings, together with other evidence, hasbeen interpreted to mean that low-to-moderate doses ofDA antagonists are not acting as appetite suppressantsthat generally blunt primary food motivation or alter foodpreference, but instead are acting on other processes(e.g., behavioral activation, instrumental response output,response allocation, effort-related processes; Salamone et

Fig. 5. The effects of systemic MSX-3 on c-Fos expression induced by intra-accumbens eticlopride. (A) Left: Photomicrograph showing representativecannula placement in nucleus accumbens, and box indicating location of the area of the photos that were used for counting of c-Fos positive cells.Right: Diagram of coronal section from Pellegrino et al. (1979), showing location of the area of the photomicrograph containing the cannula placement.(B) Photomicrograps of c-Fos staining from representative animals in each group (I.P. injections of vehicle plus intra-accumbens injections of vehicle(Veh-Veh), I.P. vehicle plus 2.0 !g eticlopride (Veh-Etic), and 2.0 mg/kg MSX-3 plus 2.0 !g eticlopride (MSX-Etic)). (C) Mean ($SEM) number of c-Fospositive cells; Eticlopride alone significantly increased c-Fos expression relative to vehicle (## P&0.01). MSX-3 significantly reduced the c-Fosexpression induced by intra-accumbens eticlopride (* P&0.05).

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al., 1991, 1997, 2003, 2005, 2007, 2009a; Salamone andCorrea, 2002, 2009; Kelley et al., 2005; Baldo and Kelley,2007; Barbano and Cador, 2007; Niv et al., 2007; Phillipset al., 2007; Floresco et al., 2008; Sink et al., 2008; Salam-one, 2010). Dopaminergic modulation of effort-related pro-cesses appears to be bi-directional, as studies have shownthat DA transporter knockdown and injection of amphet-amine can increase selection of higher effort alternatives(Cagniard et al., 2006; Bardgett et al., 2009). Although fastphasic release of accumbens DA does not seem to beincreased in response to cues associated with higher re-sponse costs (Gan et al., 2009), accumbens DA doesappear to be involved in setting the threshold for work-related cost expenditures (Salamone et al., 2007; Phillipset al., 2007; Gan et al., 2009). Moreover, recent imagingstudies have linked nucleus accumbens to effort-relatedprocesses in humans (Croxson et al., 2009; Botvinick etal., 2009).

Experiment 2 assessed the effects of injections of eti-clopride into a control site dorsal to accumbens core. Pre-vious studies of effort-related choice behavior that involvedintra-accumbens injections of DA antagonists (e.g. Koch etal., 2000; Nowend et al., 2001) did not provide this impor-tant anatomical control. In the present experiments, therewere no significant effects on either lever pressing or chowintake following injections of either the 2.0 or 4.0 !g dosesof eticlopride into the control site. This is in marked con-trast to the '60%–75% reduction in lever pressing, andthe substantial increase in chow intake, seen when thesedoses were injected into the accumbens core. The fact thateticlopride produced substantial and significant decreasesin lever pressing and increases in chow intake when in-jected into accumbens core, while these effects were en-tirely absent when the same dose was injected into adorsal control site, provides evidence for site-specificity ofthe effects observed in experiment 1. Thus, it does notappear that the behavioral effects of intra-accumbens in-jections of eticlopride are occurring because of general ornonspecific effects that could occur with drug injectionsinto any forebrain region. Furthermore, despite the fact thatdorsomedial neostriatal areas do have functions related toaspects of instrumental behavior (e.g. action-outcomelearning; Yin et al., 2008), control site injections of eticlo-pride that were placed into the dorsal aspect of this regiondid not affect performance on the concurrent FR5 choiceprocedure. These results are consistent with Cousins et al.(1993), who reported that DA depletion in nucleus accum-bens produced the shift from lever pressing to chow con-sumption, while DA depletion in overlying medial neostri-atum had no effect. Nevertheless, it should be stressedthat site specificity for studies involving intracranial druginjections should always be viewed as relative, rather thanabsolute, possibly because of spread of the drug to neigh-boring brain areas. As observed previously (Trevitt et al.,2001), DA antagonists can produce effects when highenough doses are injected into control sites such as over-lying cortex or dorsal brainstem. For this reason, a rela-tively lower dose of eticlopride (2.0 !g) was used in thereversal experiments.

Experiments 3 and 4 examined the behavioral effectsof the adenosine A2A antagonist MSX-3 when co-adminis-tered with intra-accumbens eticlopride. Both intra-accum-bens and systemic co-administration of MSX-3 reversedthe behavioral effects of local interference with DA neuro-transmission in nucleus accumbens; that is, MSX-3 atten-uated the decrease in lever pressing and increase in chowintake induced by eticlopride. The present data are con-sistent with recent studies from our laboratory indicatingthat systemic administration of adenosine A2A antagonistssuch as MSX-3 and KW-6002 was able to reverse theeffects of systemic administration of the DA antagonistshaloperidol and eticlopride in rats responding on the con-current lever pressing/chow intake task (Farrar et al., 2007;Worden et al., 2009; Salamone et al., 2009b). Although thepresent paper did not include an assessment of the effectsof MSX-3 alone, previous work has examined the effects ofsystemic administration of MSX-3 (Farrar et al., 2007) andKW-6002 (Salamone et al., 2009b) on performance of theconcurrent choice task; neither drug administered in theabsence of a DA antagonist had any effect on either leverpressing or chow intake. In addition, MSX-3 had a muchgreater reversal effect when combined with a D2 antago-nist than a D1 antagonist (Worden et al., 2009). Takentogether, these data indicate that adenosine A2A antago-nists are actually reversing the effects of D2 antagonism ina relatively specific manner, and not merely producingnon-specific stimulant effects.

In the present studies, eticlopride was injected directlyinto accumbens core in order to specifically address thehypothesis that nucleus accumbens is an important locusfor D2/A2A interactions involved in effort-related functions.Experiment 3 involved simultaneous co-administration ofboth eticlopride and MSX-3 into accumbens core. Intra-accumbens co-administration of MSX-3 produced a com-plete reversal of the suppression of lever pressing andincrease in chow intake induced by eticlopride at all dosestested. Experiment 4 was conducted to determine whethersystemic administration of MSX-3 could reverse the effectsof intra-accumbens eticlopride on the concurrent FR5/chow feeding procedure. Fig. 4 clearly shows that systemicMSX-3 attenuated the eticlopride-induced suppression oflever pressing (Fig. 4A) and increase in chow consumption(Fig. 4B). Considered together, the findings from experi-ments 3 and 4, which used two different routes of admin-istration of MSX-3, indicate that nucleus accumbens coreis an important site of action for D2-A2A receptor interac-tions involved in regulating performance on the concurrentchoice procedure. This conclusion is consistent with arecent study showing that injections of the adenosine A2A

receptor agonist CGS 21680 into nucleus accumbenscore, but not a dorsal control site, decreased lever press-ing and increased chow intake on the FR5/chow feedingprocedure in a manner that closely resembles the effectsof interference with accumbens DA neurotransmission(Font et al., 2008).

Experiment 5 examined the effects of eticlopride andMSX-3 on c-Fos expression in accumbens core. Intra-accumbens injection of eticlopride, at the same dose used

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in experiment 4 (2.0 !g), induced a robust and significantincrease in c-Fos immunoreactivity in nucleus accumbenscore neurons relative to vehicle-treated animals. This find-ing is consistent with several earlier reports indicating thatsystemic administration of D2 DA antagonists can increasec-Fos expression in striatal areas, including nucleus ac-cumbens (Dragunow et al., 1990; Miller, 1990; Robertsonand Fibiger, 1992; Fibiger, 1994; MacGibbon et al., 1994;Wan et al., 1995; Pinna et al., 1999). Adenosine A2A

receptors are located on enkephalin-positive neurons thatalso express DA D2 receptors (Schiffmann et al., 1991;Fink et al., 1992; Ferré, 1997; Svenningsson et al., 1999;Wang et al., 2000; Hettinger et al., 2001; Chen et al.,2001). DA D2 and adenosine A2A receptors converge ontothe same signal transduction mechanisms and show thecapacity for forming heteromers (Fink et al., 1992; Ferré,1997; Ferre et al., 1997, 2004; Ferré et al., 2008; Sven-ningsson et al., 1999; Hillion et al., 2002; Fuxe et al.,2003). Because D2 and adenosine A2A receptor stimula-tion has opposite effects on stimulation of cAMP-relatedsignal transduction pathways, it was thought that adeno-sine A2A antagonism should blunt the ability of D2 antag-onists to affect transcription of immediate early genes andinduce formation of Fos-related proteins. The A2A recep-tor agonist, CGS 21680, induced c-Fos expression instriatal areas in a manner similar to D2 antagonism (Pinnaet al., 1997). Furthermore, it has been shown that systemicadministration of A2A receptor antagonists (DMPX, CSC,SCH 58261) can attenuate the increase in c-Fos expres-sion induced by systemic administration of the D2 antago-nist haloperidol (Boegman and Vincent, 1996; Pinna et al.,1999). Betz et al. (2009) recently demonstrated that theadenosine A2A antagonist KW 6002 could reduce theneostriatal c-Fos expression induced by the D2 antagonistpimozide. On the basis of these findings, it was hypothe-sized that c-Fos expression could serve as a reliable cel-lular marker of D2-A2A interactions in the accumbens, andthus MSX-3 would reverse the expression of c-Fos in-duced by eticlopride. In support of this, the present resultsdemonstrated that intra-accumbens injection of eticlopriderobustly induced c-Fos expression in accumbens neuronssurrounding the injection site, and that this effect wasattenuated with systemic co-administration of MSX-3. Theeffects of this drug treatment combination on c-Fos immu-noreactivity are particularly relevant for behavior, becausethey mirrored the conditions used in the parallel behavioralstudy (experiment-4), that is, the dose of eticlopride thatwas used to induce the shift from lever pressing to chowintake was also shown to robustly increase c-Fos expres-sion, and the dose of MSX-3 that reversed the behavioraleffects of eticlopride was shown to attenuate the c-Fosimmunoreactivity induced by eticlopride. These results arein agreement with previous findings demonstrating an an-tagonistic relationship between D2 and A2A receptor func-tion, and suggest a cellular basis in accumbens core forthe involvement of D2-A2A interactions in effort-relatedprocesses.

CONCLUSION

The present experiments indicate that nucleus accumbenscore is an important locus for the involvement of D2-A2A

receptor interactions in effort-related choice. Nucleus ac-cumbens core injections of eticlopride, but not those giveninto a dorsal control site, induced the shift from leverpressing to chow intake in rats performing on the concur-rent choice procedure. Both systemic and local intra-ac-cumbens core administration of MSX-3 reversed the de-crease in lever pressing and increase in chow intake in-duced by intra-accumbens eticlopride. These findings areconsistent with a recent report that intra-accumbens injec-tions of the A2A receptor agonist CGS 21680 can inducethe shift from lever pressing to chow intake on the concur-rent choice procedure (Font et al., 2008). Furthermore, theinduction of c-Fos expression by intra-accumbens eticlo-pride was attenuated by co-administration of MSX-3, whichdemonstrated a cellular marker for D2-A2A interactionswithin nucleus accumbens core. Taken together, thesefindings provide substantial evidence for the hypothesisthat nucleus accumbens is an important locus for the ef-fort-related behavioral manifestations of DA D2/adenosineA2A receptor interactions. This research may have impli-cations for clinical studies of energy-related disorders suchas psychomotor slowing, anergia and fatigue in depressionand other disorders (Salamone et al., 2006, 2007, 2009a;Botvinick et al., 2009).

Acknowledgments—This research was supported by a grant toJDS from the USA NIH/NIMH (R01MH78023). Merce Correa wassupported by a grant of Fundació Caixa Castelló-Bancaixa. Manythanks to Adam Penarolla for his technical assistance.

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(Accepted 23 December 2009)(Available online 20 January 2010)

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