ORIGINAL INVESTIGATION

New operant model of reinstatement of food-seekingbehavior in mice

Elena Martín-García & Aurelijus Burokas & Elzbieta Kostrzewa & Agnieszka Gieryk &

Michal Korostynski & Barbara Ziolkowska & Barbara Przewlocka &

Ryszard Przewlocki & Rafael Maldonado

Received: 28 May 2010 /Accepted: 23 November 2010 /Published online: 14 December 2010# Springer-Verlag 2010

AbstractRationale A major problem in treating obesity is the highrate of relapse to abnormal food-taking behavior whenmaintaining diet.Objectives The present study evaluates the reinstatement ofextinguished palatable food-seeking behavior induced by cuespreviously associated with the palatable food, re-exposure tothis food, or stress. The participation of the opioid anddopamine mechanisms in the acquisition, extinction, and cue-induced reinstatement was also investigated.Materials and methods C57BL/6 mice were first trained ona fixed-ratio-1 schedule of reinforcement to obtainchocolate-flavored pellets during 20 days, which wasassociated to a stimulus light. Operant behavior was thenextinguished during 20 daily sessions. mRNA levels ofopioid peptide precursors and dopamine receptors wereevaluated in the brain by in situ hybridization and RT-PCRtechniques.Results A reinstatement of food-seeking behavior was onlyobtained after exposure to the food-associated cue. Adown-regulation of prodynorphin mRNA was found in thedorsal striatum and nucleus accumbens after the acquisi-tion, extinction, and reinstatement of the operant behavior.Extinction and reinstatement of this operant response

enhanced proenkephalin mRNA in the dorsal striatumand/or the nucleus accumbens core. Down-regulation ofD2 receptor expression was observed in the dorsal striatumand nucleus accumbens after reinstatement. An up-regulation of PDYN mRNA expression was found in thehypothalamus after extinction and reinstatement.Conclusions This study provides a new operant model inmice for the evaluation of food-taking behavior and revealsspecific changes in the dopamine and opioid systemassociated to the behavioral responses directed to obtain anatural reward.

Keywords Operant behavior . Priming . Cue . Stress .

Dopamine receptors . Prodynorphin . Proenkephalin

AbbreviationsArc Activity-regulated cytoskeleton-associated proteinBDNF Brain-derived neurotrophic factorCartpt Prepropeptide cartCrh Corticotropin-releasing hormoneGhrl GhrelinHcrt HypocretinPENK ProenkephalinPDYN Prodynorphin and POMC, proopiomelanocortin

Introduction

Abnormal food-taking habits leading to overweight andobesity are a worldwide health problem with major socio-economic consequences for the healthcare system ofdeveloped countries (Peterson and Mitchell 1999). A majorconcern in treating these disorders is the high rate of relapseto abnormal intake habits when maintaining diet. Recently,

E. Martín-García :A. Burokas : R. Maldonado (*)Departament de Ciencies Experimentals i de la Salut,Universitat Pompeu Fabra, PRBB,C/Dr. Aiguader 88,08003 Barcelona, Spaine-mail: rafael.maldonado@upf.edu

E. Kostrzewa :A. Gieryk :M. Korostynski : B. Ziolkowska :B. Przewlocka :R. PrzewlockiInstitute of Pharmacology, Polish Academy of Sciences,ul. Smetna 12,31-343 Krakow, Poland

Psychopharmacology (2011) 215:49–70DOI 10.1007/s00213-010-2110-6

the concept of addictive behavior associated to relapse toanomalous intake habits is gaining attention, although it isstill a very controversial issue (Volkow and O’Brien 2007;Lutter and Nestler 2009). This kind of addictive-likebehavior has been widely investigated and shares severalsimilarities with drug addiction (Corwin and Grigson2009). Both disorders have in parallel the compulsion toseek and take the reinforcer, the loss of control over intakedespite consciousness of its negative consequences, and therelapse to seek for the reinforcer (Volkow et al. 2008b). Inaddition, the learned habits and preferences related tofeeding and drug use are strengthened by the repetitiveexposure to these powerful rewarding stimuli (Volkow et al.2008a). Not only genetic influence but also environmentalfactors and the interaction between them are involved in thecomplex etiology of these disorders. The development of areliable model in mice of reinstatement to palatable food-seeking behavior would be useful to study in depth themechanisms underlying eating disorders.

Both food and drug reward share in common theinvolvement of similar neuroanatomical and neurochemistrymechanisms and neuroadaptations of learning processes.Several studies have demonstrated that the motivated behav-iors induced by drugs of abuse and highly palatable foods aremediated by the mesolimbic dopaminergic system (Lutter andNestler 2009). Hence, animal studies have demonstrated thathighly palatable food induces the release of dopamine intothe nucleus accumbens, which has been directly related tothe reinforcing effects of this natural stimulus (Mitchell andGratton 1992). The endogenous opioid system also plays animportant role in the mechanisms underlying the behavioralresponses directed to obtain food or drugs of abuse (Kelley2004; Shippenberg et al. 2007). However, the main brainstructure that controls the regulatory signals for foodconsumption is the hypothalamus. In this brain area, multipleneurotransmitters, including endogenous opioids and dopa-mine, participate in the integration of the peripheral signals,which leads to appetite and satiety responses that will controlfeeding behavior (Kelley and Berridge 2002).

In this study, we have developed a new behavioral modelof reinstatement of an operant responding maintained bypalatable food in mice. For this purpose, we have adaptedmodels recently validated to induce drug-seeking behaviorrelapse in mice (Soria et al. 2008; Martin-Garcia et al.2009) and reinstatement of food-seeking behavior devel-oped in rats (Ghitza et al. 2006) in order to establish a newoperant model of reinstatement to food-seeking behaviorinduced by food-associated cues, re-exposure to palatablefood, or stress. Additionally, we have examined changes ingene expression in the mesolimbic system and thehypothalamus in the different phases of this operantresponding by using in situ hybridization and RT-PCRtechniques.

Materials and methods

Animals

One hundred eight male C57BL/6 mice (Charles River,France), weighing 24–26 g at the beginning of theexperiment, were used in this study. The mice were housedindividually in controlled laboratory conditions with thetemperature maintained at 21±1°C and the humidity at 55±10%. The mice were tested during the first hours of thedark phase of a light reversal circadian cycle (lights off at8.00 h and on at 20.00 h). Food and water were available adlibitum except during 5 days before starting the sessionsand the first 10 days of the operant behavior training.During this first period, the animals were food-deprived tomaintain their weight at 90% of initial values. Animalprocedures were conducted in strict accordance with theguidelines of the European Communities Directive 86/609/EEC regulating animal research and were approved by thelocal ethical committee (CEEA-PRBB).

Operant self-administration apparatus

Operant responding maintained by food was investigated inmouse operant chambers (Model ENV-307A-CT, MedAssociates, Georgia, VT, USA) equipped with two retract-able levers, one randomly selected as the active and theother as the inactive. Pressing on the active lever resulted ina pellet delivery together with a stimulus light (associatedcue), while pressing on the inactive had no consequences.The chambers were made of aluminum and acrylic, hadgrid floors connected to an electrical shocker (EVV-414,Med. Associates Inc., St Albans, VT, USA), and werehoused in sound- and light-attenuated boxes equipped withfans to provide ventilation and white noise. A stimuluslight, located above the active lever, was paired contin-gently with the delivery of the reinforcer (chocolate-flavored pellet). A food dispenser equidistant between thetwo levers permitted the delivery of food pellets whenrequired.

Acquisition of operant responding maintained by food

Training sessions of operant responding maintained by foodwere performed in accordance to protocols previouslydescribed (Soria et al. 2008; Martin-Garcia et al. 2009;Barbano et al. 2009). Briefly, responding was maintainedby chocolate-flavored pellets of 20 mg (2% pure unsweet-ened cocoa), containing 21% protein, 13% fat, and 67%carbohydrate with a caloric value of 3.48 kcal/g. One-hourdaily operant responding sessions were conducted 7 daysper week during 20 days. The animals were food-deprived5 days before starting the sessions and during the first ten

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sessions of the operant behavior training to maintain theirweight at 90% of their ad libitum initial weight adjusted forgrowth. The animals were fed ad libitum during theremaining operant behavior training. The houselight wason at the beginning of the session for 3 s and off during theremaining duration of the session. The side of active andinactive lever was counterbalanced between animals. Eachdaily session started with a priming delivery of onechocolate-flavored pellet to facilitate associative learningand to maintain the same experimental conditions duringthe whole acquisition period. An FR1 has been used in thisstudy in order to facilitate the comparison with similarprevious experiments of validity of reinstatement of cocaine(Soria et al. 2008) or nicotine (Martin-Garcia et al. 2009).The stimulus light (associated cue) signaled the delivery ofthe chocolate-flavored pellet. A time-out period of 10 s wasestablished after each pellet delivery. During this period, thecue light was off and no reinforcer was provided afterresponding on the active lever. Responses on the inactivelever and all the responses elicited during the time-outperiod were also recorded. The session was terminated after100 reinforcers were delivered or after 1 h, whicheveroccurred first. As previously described (Soria et al. 2005;Barbano et al. 2009), the criteria for the achievement of theoperant responding were acquired when all of the followingconditions were met: (1) mice maintained a stable respond-ing with less than 20% deviation from the mean of the totalnumber of reinforcers earned in three consecutive sessions(80% of stability), (2) at least 75% responding on the activehole, and (3) a minimum of ten reinforcers per session.After each session, the mice were returned to their homecages. The chambers were cleaned at the end of eachsession to prevent the presence of odor of the previousmouse. After 20 days of operant responding sessions, themice were moved to the extinction phase.

Extinction phase

During the extinction phase, the experimental conditionswere similar to the acquisition sessions except that thechocolate-flavored pellets were not available and thestimulus light was not presented after responding in theactive lever. The mice were fed ad libitum and were givendaily 1-h sessions 7 days per week during 20 days. Thecriterion for extinction was achieved when the mice made amean number of lever presses, during three consecutivesessions, in the active lever of less than 30% of theresponses obtained during the mean of the 3 days ofachievement of the acquisition criteria. After 20 days ofextinction sessions, the mice were tested under reinstate-ment conditions. The animals were divided in sevendifferent groups to evaluate reinstatement induced by thedifferent stimuli.

Reinstatement phase

Different experimental conditions were applied to reinstatefood-seeking behavior: the presentation of conditionedenvironmental cue (stimulus light), a non-contingentlypriming delivery of chocolate-flavored pellets, and theexposure to stressful situations. The reinstatement criterionfor the experiments was achieved when responding in theactive lever was double than during the three consecutivedays that the mice acquired extinction criteria.

Cue-induced reinstatement (one group) Test for cue-induced reinstatement was conducted under the sameconditions used in the training phase except that food wasnot available. Each pressing in the active lever led to thepresentation of the stimulus light (cue) for 2 s.

Chocolate pellet priming-induced reinstatement (twogroups) Test for pellet priming-induced reinstatement wasconducted under the same conditions used in the extinctionphase (chocolate-flavored pellets and cue were not avail-able after responses in the active lever). The chocolatepellets were delivered non-contingently at the beginning ofthe session. In a first experiment, ten pellets were used asstimulus priming. In a second group of mice, two pelletswere used as priming.

Stress-induced reinstatement (four groups) Tests for stress-induced reinstatement were conducted under the sameconditions used in the extinction phase (pellets and cuewere not available after responses in the active lever). Twokinds of stress (electric footshock and pharmacologicalstressor with three different doses) were applied in differentgroups of mice.

Electric footshock Immediately before the reinstatementsession, intermittent electric footshock stimuli were appliedduring 5 min. The mice received five electric footshocksseparated by a 1-min period without shock (0.22 mA during2 s). The intensity of 0.22 mA was selected based onprevious studies conducted in our laboratory on stress-induced reinstatement of cocaine- (Soria et al. 2008) andnicotine-seeking behavior (Martin-Garcia et al. 2009) inmice.

Pharmacological stressor We tested in our experimentalconditions the effects induced by yohimbine, an α-2adrenoceptor antagonist that provokes anxiety-likeresponses (Bremner et al. 1996) and reinstates drug- andfood-seeking in rats (Lê et al. 1998; Ghitza et al. 2006,2007; Nair et al. 2006). At the dose tested in the presentstudy (2 mg/kg, i.p.), yohimbine has been reported toproduce anxiogenic-like effects in the mouse light/dark

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model (Shimada et al. 1995) and the mouse defense testbattery (Blanchard et al. 2001). This reinstatement test wasconducted in four consecutive sessions under the sameexperimental conditions used in the extinction sessions.Two first consecutive sessions of reinstatement wereperformed, followed by one extinction day and by twoadditional consecutive sessions of reinstatement (Ghitzaet al. 2006). Yohimbine was injected 30 min before eachreinstatement session. A day before the first yohimbine(2 mg/kg, i.p.) reinstatement session, the mice received aninjection of vehicle and the operant behavior was evaluated30 min later to verify that the reinstatement was not due tounspecific factors such as the injection procedure. Twoadditional doses of yohimbine (1 mg/kg, i.p., and 4 mg/kg,i.p) were tested using a similar experimental design.

Priming and stress-induced reinstatement (one group) Thisreinstatement test was also conducted in four consecutivetest sessions under the same experimental conditions usedin the extinction sessions. The conditions of priming (twopellets) and pharmacological stressor (yohimbine 2 mg/kg)were both applied together following the same experimentalschedule as reported in the previous paragraph.

Contingent versus non-contingent administrationof chocolate pellets

A different group of mice was trained to obtain chocolateunder the same experimental conditions explained for thephase of acquisition of operant responding maintained byfood under FR1. To determine the consequences of non-contingent chocolate administration, an additional group ofmice was yoked to the previous group during the wholeacquisition training period. These mice received chocolatepellets non-contingently, without receiving the conditionedstimulus at the same time that each master mouse obtaineda contingent administration of chocolate. A control groupof mice that was exposed to the same experimentalconditions than the yoked group, but receiving normalpellets non-contingently, was included.

Quantitative RT-PCR and in situ hybridizationin cue-induced reinstatement and contingent versusnon-contingent chocolate self-administration

At the end of the behavioral experiments, the mice weresacrificed by decapitation 3 h after the last operantresponding session; the brains were removed and quicklyfrozen on dry ice and stored at −70°C. For the cue-inducedreinstatement experiment, the in situ hybridization andquantitative RT-PCR analysis was conducted in four groupsof mice: (1) operant control group, mice that were placed in

the operant boxes with all the stimuli except the delivery offood reinforcement and conditioned stimulus during thewhole training acquisition period, (2) acquisition group,mice that completed the experimental sequence after theacquisition of the operant behavior to obtain the palatablefood, (3) extinction group, mice that completed theexperimental sequence after the extinction of the operantbehavior, (4) cue-induced reinstatement group, mice thatcompleted the experimental sequence after the reinstate-ment of the operant behavior.

For the contingent versus yoked chocolate administra-tion experiment, the in situ hybridization and quantitativeRT-PCR analysis were conducted in three groups of mice:(1) operant control mice that were placed in the operantboxes with all the stimuli except the delivery of conditionedstimulus and receiving of normal pellets non-contingently,(2) contingent chocolate mice that completed the experi-mental sequence after the acquisition of the operantbehavior to obtain the palatable food (same characteristicsas the previous acquisition group), and (3) yoked mice tothe previous group that received chocolate pellets non-contingently, without receiving the conditioned stimulus,and completed the experimental sequence during the wholeacquisition training of the master animal.

Tissue collection After decapitation, the brains were re-moved from the skulls and processed rapidly on ice. First,the hypothalamus was isolated and the hypothalamussamples were placed in individual tubes containing thetissue storage reagent RNAlater (Qiagen Inc., Valencia, CA,USA), frozen on dry ice, and stored at −70°C until RNAisolation for the RT-PCR analysis. The remaining brainparts from the same animals were frozen on dry ice andstored at −70°C for in situ hybridization analysis.

RNA isolation Samples containing the hypothalamus werethawed at room temperature and homogenized in 1 mlTrizol reagent (Invitrogen, Carlsbad, CA, USA). RNAisolation was performed in accordance with the manufac-turer’s protocol. The quality of the total RNA was assessedby the intensity of 28S and 18S bands during denaturatingagarose electrophoresis and by the spectrophotometric ratioA260/A280 (1.9 to 2.1). The total RNA concentration wasmeasured using the NanoDrop ND-1000 spectrometer(NanoDrop Technologies Inc.).

RT-PCR Reverse transcription was performed using Omni-script reverse transcriptase (Qiagen Inc.) at 37°C for 60 min.The reaction was carried out in the presence of the RNaseinhibitor rRNAsin (Promega, Madison, WI, USA), and oligo(dT16) primer (Qiagen Inc.) was used to selectively amplifymRNA. cDNAs were diluted 1:10 with H2O and for eachPCR reaction we used about 50 ng of cDNA obtained from a

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single animal. Quantitative PCR reactions were performedusing Assay-On-Demand Taqman probes, in accordance withthe manufacturer’s protocol (Applied Biosystems), and wererun on the iCycler device (BioRad) with the 3.0a softwareversion. The analysis was conducted on the hypothalamicsamples for the following genes: brain-derived neurotrophicfactor (Bdnf Taqman assay - Mm01334042_m1), activity-regulated cytoskeleton associated protein (Arc -Mm00479619_g1); endogenous opioid peptides precursors:proopiomelanocortin (Pomc - Mm00435874_m1), proenke-phalin (Penk - Mm01212875_m1) and prodynorphin (Pdyn -Mm00457572_m1); genes involved in food intake:corticotropin-releasing hormone (Crh - Mm01293920_s1),CART prepropeptide (Cartpt - Mm00489086_m1), hypocre-t in (Hcrt - Mm01964030_s1), ghrelin (Ghrl -Mm00445450_m1). The threshold cycle values were calcu-lated automatically by iCycler IQ 3.0a software with defaultparameters. The abundance of RNA was calculated as 2-(threshold cycle). The expression of hypoxanthine guaninephosphoribosyl transferase 1 (Hprt1 - Mm03024075_m1),which had a stable mRNA level, was quantified to control forvariations in cDNA amounts. The usefulness of Hprt1 as areference transcript was determined in our previous studies(Korostynski et al. 2007; Piechota et al. 2010).

In situ hybridization Brain tissues for in situ hybridizationwere cut into 12-μm-thick coronal sections on a cryostat-microtome (CM 3050S Leica, Nucloch, Germany). Thesections were thaw-mounted on gelatin-chrom-alum-coatedslides and processed for in situ hybridization according tothe method of Young et al. (1986). Briefly, the sectionswere fixed with 4% paraformaldehyde, washed in PBS, andacetylated by incubation with 0.25% acetic anhydrite (in0.1 M triethanolamine and 0.9% sodium chloride). Thesections were then dehydrated using increasing concen-trations of ethanol (70–100%), treated with chloroform for5 min, and rehydrated with decreasing concentrations ofethanol.

The sections were hybridized for approximately 15 hat 37°C with oligonucleotide probes for PENK, PDYN,D1, and D2 dopamine receptors. The probes for opioidpropeptides were complementary to residues 388–435 ofthe rat PENK mRNA (Yoshikawa et al. 1984) and toresidues 862–909 of the rat PDYN mRNA (Civelli et al.1985). Each probe contained one mismatch with respect tothe corresponding mouse sequence (Zurawski et al. 1986;GenBank, NM_018863.2). The probes for dopaminereceptors were complementary to residues 1224–1271 ofthe mouse D1 receptor mRNA (Drago et al. 1994) and toresidues 553–600 of the mouse D2 receptor mRNA (Shortet al. 2006). All probes were labeled with 35S-dATP bythe 3′-tailing reaction using terminal transferase (MBIFermentas, Vilnius, Lithuania).

The specificity of the PENK and PDYN probes had beenextensively documented elsewhere and was confirmed by aNorthern blot analysis and competition experiments (Younget al. 1986). The patterns of hybridization signal found in thisstudy in the different brain sections (Fig. 5) fully agreed withthe well-known distribution of the PENK and PDYNmRNA. The distribution of the hybridization signal for theD1 and D2 receptors and their relative intensities in discretebrain regions (Fig. 5) were also similar to those reported byother authors (Mengod et al. 1989; Le Moine et al. 1990;Drago et al. 1994), and our results obtained using at leastone additional probe complementary to a different portion ofthe same mRNA molecule (data not shown). In addition,control hybridizations were performed with sense probes toPENK, PDYN, D1, and D2 receptor mRNAs. No specificsignals were obtained with these different sense probes inany of the brain regions of interest evaluated in the presentstudy. This result confirms the specificity of the probes usedunder the present experimental conditions.

After hybridization, the slices were washed three timesfor 20 min with 1×SSC/50% formamide at 40°C and twicefor 50 min with 1×SSC at room temperature. Then, theslices were dried and exposed to Fujifilm (Tokyo, Japan)phosphorimager imaging plates for 3 days for PENK or5 days for PDYN, D1, and D2 receptors. The hybridizationsignal was digitized using the Fujifilm BAS-5000 phos-phorimager and the Image Reader software.

Image analysis In situ hybridization signal was analyzedusing the MCID Elite system (Imaging Research, St.Catharines, Ontario, Canada). Mean signal density,expressed in photostimulated luminescence units per squaremillimeter, was measured in selected brain regions in theFujifilm BAS-5000 images. The regions, which are out-lined in Fig. 5, included dorsal striatum, nucleus accumbenscore and shell (for PENK, PDYN, D1 and D2 receptors),the central nucleus of amygdala (for PENK and PDYN),and substantia nigra and ventral tegmental area (for D2receptor). For each brain structure, data were collected fromthree to five sections, bilaterally, per animal. Backgroundsignal was measured over the white matter (corpuscallosum) and was subtracted from the hybridization signalin the regions of interest.

Statistical analysis

Data analysis was carried out independently for eachexperiment: (1) evaluation of different types of reinstate-ment, (2) quantitative RT-PCR and in situ hybridization inthe condition of cue-induced reinstatement, and (3) contin-gent versus yoked chocolate administration experiment. Toevaluate the different types of reinstatement, six groups of

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mice were used: associated cue (n=12), ten pellets priming(n=7), two pellets priming (n=6), footshock (n=7),yohimbine (n=6), and yohimbine and two pellets priming(n=9). For the quantitative RT-PCR and in situ hybridiza-tion in the condition of cue-induced reinstatement, fourgroups of mice were used: operant control group (n=8),acquisition group (n=8), extinction group (n=8), and cue-induced reinstatement group (n=8). For the contingentversus yoked chocolate administration experiment, the insitu hybridization and quantitative RT-PCR analysis wasconducted in three groups of mice: control (n=8), acquisi-tion contingent chocolate (n=8), and yoked non-contingentchocolate (n=8).

An analysis of the data obtained during the acquisitionand extinction phases was conducted using two-wayANOVA with repeated measures in the factors day (dailysessions) and lever (active/inactive). Post-hoc analyseswere performed when required (Newman–Keuls).

To evaluate the reinstatement of food-seeking behavior,the animals were divided in different groups correspondingto each experimental condition for reinstatement (associatedcue, ten pellets priming, two pellets priming, footshock,yohimbine, and yohimbine two pellets priming). One-wayANOVA was first used to compare the responses of the sixdifferent groups of mice trained to acquire an operantresponding maintained by chocolate-flavored pellets inorder to verify the consistency of responses in all thegroups. Two-way ANOVA with repeated measures in thefactors experimental phase and lever was then performedfollowed by post-hoc analyses (Newman–Keuls) whenrequired. ANOVA with repeated measures and polynomialpost-hoc analyses were also realized when required.Correlations between the number of active lever pressingresponses in the first day of extinction and (1) the meannumber of active lever presses during the 10 days oftraining ad libitum and (2) the mean number of active leverpresses during the 3 days required to achieve the self-administration acquisition criteria were performed using aPearson correlation analysis.

Data derived from quantitative RT-PCR and in situhybridization were analyzed using one-way ANOVA fol-lowed by Newman–Keuls post-hoc test when required. RT-PCR data are expressed as fold change to the control group.

All results are expressed in mean±SEM. Differenceswere considered significant at P<0.05. The statisticalanalysis was performed using the Statistical Package forSocial Science program SPSS® 15.0 (SPSS Inc, Chicago,IL, USA).

Drugs

Yohimbine HCl (Sigma, Madrid, Spain) was dissolved indistilled water. The injection volume was 10 ml/kg. The

yohimbine doses (1, 2, and 4 mg/kg, i.p.) were chosenbased on previous studies reported in the literature(Shimada et al. 1995; Blanchard et al. 2001; Ghitza et al.2006, 2007).

Results

Acquisition and maintenance of operant respondingmaintained by palatable food

Six different groups of mice were first trained to acquire anoperant responding maintained by chocolate-flavored pelletswith the aim of testing the ability of different stimuli to inducereinstatement to food-seeking behavior. One-way ANOVA tocompare the responses of the six different groups did not revealsignificant differences on active or inactive lever pressingduring acquisition training when animals were food-deprived(active [F(5,54)=1.32; n.s.], inactive [F(5,54)=1.01; n.s.]), whenanimals were fed ad libitum (active [F(5,53)=0.97; n.s.],inactive [F(5,53)=0.93; n.s.]), nor during the extinction phase(active [F(5,54)=1.27; n.s.], inactive [F(5,54)=1.98; n.s.]). Theresults were then pooled for all the experimental groupsduring the acquisition and extinction phases and arerepresented in Fig. 1 (see Table 1 for two-way ANOVA).

During the first 10 days of training under food-deprivedconditions, the acquisition criteria were achieved in 4.79±0.22 days by 97% of mice. Therefore, most of the miceacquired the criteria after one (from day 2 to day 4) or2 days (from day 3 to day 5) of non-stable responding, asshown in Fig. 1. The mean number of active lever pressesduring the 3 days when the acquisition criteria wereachieved was 92.63±1.42 and the number of inactive leverpresses was 10.27±0.75. Similar results were obtained inthe last 3 days of training (day 8, 9, and 10) in the active(89.92±1.64) and the inactive (12.18±1.15) lever. Polyno-mial post-hoc analysis revealed an increase in the numberof active lever press responding across sessions during thisfirst training period [polynomial (linear) F(1,75)=35.17; P<0.001]. In contrast, the number of inactive lever pressingresponses remained stable across sessions [polynomial(linear) F(1,75)=3.73; n.s.].

During the 10 days of training under ad libitum conditions,the number of active lever presses significantly decreased whencompared to the previous value obtained on the last day oftraining under food deprivation [polynomial (linear) F(1,68)=39.48; P<0.001]. On the contrary, the number of inactivelever presses on the first day of training under ad libitumconditions significantly increased when compared to the valueobtained on the last day of training under food deprivation[polynomial (linear) F(1,68)=39.48; P<0.001]. Despite thesechanges, 83% of the animals reach all the acquisition criteriaagain after 4.94±0.31 days. The mean number of active lever

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presses during this stable phase of self-administration was67.65±1.98 and 17.44±2.81 in the inactive lever. During thelast 3 days of training on this phase, the mean number of leverpresses was 67.61±1.80 and 16.02±1.87 in the active andinactive, respectively. Polynomial post-hoc analysis revealedthat the number of active lever press responding acrosssessions during this training period remained stable [polyno-mial (linear) F (1,71)=35.17; n.s.]. In contrast, the number ofinactive lever presses responses decreased across sessions[polynomial (linear) F (1,71)=3.73; P<0.05].

In the contingent versus non-contingent chocolateadministration experiments, the number of active leverpresses was lower in the yoked and control mice than in thecontingent group as expected since the lever presses did notresult in reinforcement delivery in these two former groups.In the control group, the number of active lever presses was15.55±1.40 and the number of inactive lever presses was20.84±3.76 during the first 10 days of training under food-deprived conditions. Two-way ANOVA revealed that themice did not discriminate between the active and inactivelevers [F (9,63)=0.56; n.s.]. In the following 10 days oftraining under ad libitum conditions, the number of activelever presses was 13.69±1.87 and the number of inactivelever presses was 20.07±2.97 without significant discrim-ination between levers [F (9,63)=0.50; n.s.]. Similar resultswere obtained in the yoked group during the first 10 days of

training under food-deprived conditions (active, 10.56±1.28; inactive, 16.70±3.11) or under ad libitum conditions(active, 16.71±2.22; inactive, 22.04±4.02). In the sameline, two-way ANOVA revealed that yoked mice did notdiscriminate between levers during food deprivation:[F (9,63)=1.30; n.s.] or under ad libitum [F (9,63)=1.26;n.s.] conditions.

Extinction

The extinction criterion (less than 30% of previous activeresponses during three consecutive sessions of the stable phaseof self-administration training) was achieved by 70% of mice.This exigent criterion was reached after an average of 10.98±6.25 daily sessions of extinction. However, the discriminationbetween levers only occurred in the first day of extinction(Fig. 1). During the first session of extinction, the miceshowed a significant reduction of the number of responses inthe active lever (56.53±2.31) in comparison to the responsesof the last day of food self-administration training (67.01±2.95) [polynomial (linear) F (1,63)=27.24; P<0.001]. A non-significant enhancement in the inactive lever pressingresponses was also observed in the first day of extinction(last day of training: 14.53±1.93, first day of extinction:24.88±4.48) [polynomial (linear) F (1,63)=2.03; n.s.]. Subse-quently, the responses in the active lever went down across

Fig. 1 Acquisition and extinction of operant behavior to obtainchocolate-flavored pellets. Mean number of lever presses in the activeand the inactive lever. a Acquisition of food in deprived and adlibitum animals. Mice were trained daily on 1-h sessions to obtainchocolate-flavored pellets during 20 days under a fixed-ratio-1

schedule of reinforcement in a 1-h session (n=76). b Extinction offood self-administration. Sessions were conducted daily and lasted 1 hduring 20 days (n=68). The animals were no longer reinforced afteractive lever presses. ★★★P<0.001, comparison between levers (New-man–Keuls). Data are expressed as mean±SEM

Psychopharmacology (2011) 215:49–70 55

sessions [polynomial (linear) F (1,67)=147.62; P<0.001]. Thenumber of active lever pressing responses in the first day ofextinction was positively correlated with the mean number ofactive lever presses during the period of training ad libitum(R=0.47; P<0.001) and during the 3 days required toachieve the self-administration acquisition criteria in thistraining phase (R=0.34; P<0.01). A positive correlationbetween the responses during acquisition and extinction wasrevealed, indicating that the mice with a high number of leverpressing responses during acquisition presented a similarhigh number of lever pressing during the first session ofextinction. Thus, correlation reveals that the mice showing ahigh performance during the acquisition phase were resistantto extinction.

Cue-induced reinstatement

After the achievement of the extinction criterion, the cuelight was presented to evaluate cue-induced reinstatement.The exposure to the cue reinstated food-seeking behavior in85% of the animals (Fig. 2a). Post-hoc Newman–Keuls testrevealed that the number of active lever pressing responseswhen reaching the extinction criterion was significantlylower than the responses during the acquisition of the self-administration criteria (P<0.001). During the reinstatementtest, the number of active lever pressing responses wassignificantly higher than that obtained on the days when theextinction criterion was achieved (P<0.01). No significantchanges on the number of inactive lever presses were foundwhen comparing the acquisition, extinction, and reinstate-ment phases (see Table 1 for two-way ANOVA).

Priming-induced reinstatement

After the achievement of extinction criterion, priming-induced reinstatement was evaluated in two different groupsof animals: the first group was exposed to ten pellets (Fig. 2b)and the second to two pellets (Fig. 2c) that were deliverednon-contingently before starting the session. The delivery often pellets of food priming induced a reliable reinstatementof food-seeking behavior only in 14% of mice. When theextinction criterion was reached, the number of active leverpressing responses was significantly lower than that duringthe self-administration acquisition criteria (P<0.001). Duringthe reinstatement test, the number of active lever pressingresponses was not different to that obtained on the dayswhen the extinction criterion was achieved. No significantdifferences on inactive lever presses were found whencomparing the acquisition, extinction, and reinstatementphases (see Table 1 for two-way ANOVA).

None of the animals exposed to two-pellet primingreached the criterion of reinstatement. The number ofactive lever pressing responses, when the extinctionT

able

1Operant

respon

ding

maintainedby

food

during

acqu

isition

,extin

ction,

andrelapse

Two-way

ANOVA

Acquisitio

n

food

deprived

Acquisitio

n

adlib

itum

Extinction

Cue-induced

reinstatem

ent

Priming-induced

reinstatem

ent

(ten

pellets)

Priming-induced

reinstatem

ent

(twopellets)

Electricfootshock-

inducedreinstatem

ent

Yohim

bine-induced

reinstatem

ent

Yohim

bine

andprim

ing

(twopellets)-induced

reinstatem

ent

F-value

P-value

F-value

P-value

F-value

P-value

F-value

P-value

F-value

P-value

F-value

P-value

F-value

P-value

F-value

P-value

F-value

P-value

Lever

F(1,75)=1617.81

P<0.001

F(1,71)=603.77

P<0.001

F(1,67)=0.94

n.s.

F(1,12)=242.41

P<0.001

F(1,6)=24.59

P<0.01

F(1,5)=14.47

P<0.05

F(1,6)=147.94

P<0.001

F(1,8)=4.75

n.s

F(1,12)=418.90

P<0.001

Day

F(5,432)=26.10

P<0.001

F(6,426)=2.42

P<0.05

F(7,498)=18.75

P<0.001

Lever

×day

F(6,459)=39.30

P<0.001

F(5,387)=5.21

P<0.001

F(6,426)=9.65

P<0.001

Experim

ental

phase

F(4,48)=27.40

P<0.001

F(1,6)=14.74

P<0.01

F(4,20)=25.61

P<0.001

F(4,24)=16.56

P<0.001

F(4,32)=7.02

P<0.001

F(4,48)=50.74

P<0.001

Lever

×

experimental

phase

F(4,48)=51.67

P<0.001

F(1,8)=13.54

P<0.01

F(4,20)=24.20

P<0.001

(4,24)=13.26

P<0.001

F(1,10)=4.42

P<0.01

F(2,23)=70.99

P<0.001

“ Materialsandmetho

ds”fordetails

56 Psychopharmacology (2011) 215:49–70

Two-way

ANOVAwith

repeated

measuresin

thefactorsday/experimentalph

aseandlever.See

n.s.no

tsign

ificant

criterion was reached, was significantly lower than thatduring self-administration acquisition (P<0.001). Thenumber of active lever pressing during reinstatement wasnot different to the responses obtained on the days whenthe extinction criterion was achieved. No significanteffects were found when comparing inactive lever pressesduring acquisition, extinction, and reinstatement phases(see Table 1 for two-way ANOVA).

Stress-induced reinstatement

A brief exposure to intermittent electric footshock at theintensity of 0.22 mA or to a pharmacological stressor(yohimbine, 1, 2, and 4 mg/kg, i.p.) was applied to differentgroups of animals to evaluate stress-induced reinstatementafter the achievement of the extinction criterion. Theexposure to the footshock stressor failed to reinstate the

Fig. 2 Cue- and priming-induced reinstatement. Meannumber of lever presses in theactive and the inactive leverduring the different experimen-tal phases: acquisition of foodself-administration behavior(mean of the 3 days of theacquisition criteria under fooddeprivation or ad libitum condi-tion), mean of 3 days of extinc-tion criterion and reinstatementinduced by a cue (n=12), bpriming with ten pellets (n=7),and c priming with two pellets(n=6). ★P<0.05; ★★P<0.01;★★★P<0.001, differences be-tween levers within the sameexperimental phase. ☆☆P<0.01;☆☆☆P<0.001, differences whencompared to the response in thesame lever in different phases(Newman–Keuls). Data areexpressed as mean±SEM

Psychopharmacology (2011) 215:49–70 57

food-seeking behavior (Fig. 3a). Post-hoc Newman–Keulstest revealed that the number of active lever pressing, whenthe extinction criterion was reached, was significantly lowerthan the responses obtained during the acquisition of theself-administration criteria (P<0.001). The number ofactive lever pressing responses, when the extinctioncriterion was reached, was not statistically different to thatobtained during the reinstatement session. No significantdifferences on inactive lever presses were found whencomparing the acquisition, extinction, and reinstatementphases (see Table 1 for two-way ANOVA).

The pharmacological stressor (yohimbine, 2 mg/kg, i.p.)was also not effective in reinstating the food-seekingbehavior. Post-hoc Newman–Keuls test revealed that activelever pressing, when the extinction criterion was reached,was significantly lower than the active responses during theacquisition of self-administration criteria (P<0.001). On theday before the first yohimbine reinstatement session, theanimals received an injection of vehicle and operantbehavior was evaluated 30 min later in a first session. Inthis control session, the number of responses in the activelever (19.13±3.08) was similar to that obtained during

Fig. 3 Stress-induced reinstate-ment. Mean number of leverpresses in the active and theinactive lever during the differ-ent experimental phases: acqui-sition of food self-administrationbehavior (mean of the 3 days ofthe acquisition criteria underfood deprivation or ad libitumcondition), mean of 3 days ofextinction criterion and rein-statement induced by a 0.22-mAfootshock (n=7), b yohimbine(n=6), and c priming withyohimbine (n=9). ★P<0.05;★★P<0.01; ★★★P<0.001, dif-ferences between levers withinthe same experimental phase.☆P<0.05; ☆☆P<0.01;☆☆☆P<0.001, differencesbetween experimental phaseswhen considering the same lever(Newman–Keuls). Data areexpressed as mean±SEM

58 Psychopharmacology (2011) 215:49–70

acquisition of extinction criterion (17.61±2.76). The num-ber of responses in the inactive lever was also similar on theday of vehicle injection (20.75+4.04) to the days ofextinction criterion (27.94±11.69). During the reinstate-ment test with the dose of 2 mg/kg, the number of activelever pressing responses was not significantly higher thanthat obtained on the days when the extinction criterion wasachieved. No significant differences on inactive leverpresses were found when comparing the acquisition,extinction, and yohimbine reinstatement phases [F (1,9)=2.25; n.s.], (see Fig. 3b and Table 1 for two-way ANOVA).

Two additional doses of yohimbine (1 mg/kg, i.p., and4 mg/kg, i.p) were tested in different groups of mice andwere also not effective in reinstating the food-seekingbehavior. Indeed no significant differences were revealedbetween the number of active lever pressing duringreinstatement with these two doses of yohimbine comparedto the days when the extinction criterion was achieved. Nosignificant differences were either observed in the numberof inactive lever pressing during extinction and reinstate-ment in these experimental groups (data not shown).

Priming and stress-induced reinstatement

Both priming (two pellets) and pharmacological stressor(yohimbine, 2 mg/kg, i.p.) were presented together toevaluate the reinstatement of food-seeking behavior afterthe achievement of the extinction criteria (Fig. 3c). Thenumber of active lever pressing responses, when theextinction criterion was reached, was significantly lowerthan the responses during the acquisition of the self-administration criteria (P<0.001). No significant changeson the number of inactive lever presses were found whencomparing the acquisition, extinction, and reinstatementphases (see Table 1 for two-way ANOVA).

Quantitative real-time PCR

No significant changes in the gene expression of Crh,Cartpt, Hcrt, ghrelin, Arc, POMC, PENK, or BDNF werefound in the hypothalamus of the different groups of thecue-induced reinstatement experiment as well as in thecontingent versus non-contingent chocolate administrationexperiment (see Table 2). However, significant differencesamong groups were observed in the PDYN gene expressionin the cue-induced reinstatement experiment [F (3,25)=9.15;P<0.001]. Post-hoc Newman–Keuls test revealed increasedPDYN gene expression in extinction (P<0.001) andreinstatement (P<0.05) groups compared with the controlgroup (Fig. 4a). The extinction (P<0.001) and reinstate-ment (P<0.05) groups also presented a higher PDYN geneexpression than the acquisition group. No significantdifferences were obtained in the PDYN gene expression

[F (2,23)=0.09; n.s.] in the hypothalamus in the differentgroups included in the contingent versus non-contingentchocolate administration experiment (Fig. 4b).

In situ hybridization experiments

Prodynorphin gene expression In the cue-induced reinstate-ment experiment, one-way ANOVA revealed differences inthe PDYN mRNA levels among the experimental groups indorsal striatum [F (3,24)=5.36; P<0.01], central amygdala[F (3,25)=7.53; P<0.001], nucleus accumbens shell [F (3,24)=6.85; P<0.01], and core [F (3,25)=7.19; P<0.01] (Figs. 5 and6). Newman–Keuls post-hoc test revealed decreased PDYNmRNA levels in the acquisition group in dorsal striatum (P<0.05), nucleus accumbens shell (P<0.01), and core (P<0.05)when compared to the control group. In the extinction group,decreased PDYN mRNA levels were found in dorsalstriatum (P<0.01), central amygdala (P<0.05), nucleusaccumbens shell (P<0.01), and core (P<0.001) whencompared to the control group. Similarly, decreased PDYNmRNA levels were obtained in the cue-induced reinstatementgroup in dorsal striatum (P<0.05), central amygdala (P<0.01), nucleus accumbens shell (P<0.01), and core (P<0.05)(Fig. 6) when compared to the control group. In addition,PDYN mRNA levels were significantly lower in the centralnucleus of amygdala in the extinction (P<0.05) andreinstatement group (P<0.01) when compared to theacquisition group.

In contingent versus non-contingent chocolate adminis-tration experiment, one-way ANOVA revealed differencesin PDYN mRNA levels among the experimental groups indorsal striatum [F (2,20)=3.58; P<0.05], nucleus accumbensshell [F (2,19)=9.57; P<0.01], and core [F (2,19)=8.31; P<0.01], whereas no significant effects were obtained incentral amygdala [F (2,20)=0.40; n.s.] (Fig. 4). Newman–Keuls post-hoc test revealed decreased PDYN mRNAlevels in the contingent chocolate group in nucleusaccumbens shell (P<0.01) and core (P<0.01) whencompared to the control group. In the yoked group,decreased PDYN mRNA levels were found in dorsalstriatum (P<0.01), nucleus accumbens shell (P<0.01),and core (P<0.001) when compared to the control group.

Proenkephalin gene expression In the cue-induced reinstate-ment experiment, one-way ANOVA revealed significantdifferences in PENK gene expression among groups in dorsalstriatum [F (3,25)=3.18; P<0.05] and nucleus accumbenscore [F (3,24)=3.59; P<0.05], whereas no significant effectswere obtained in central amygdala [F (3,24)=0.34; n.s.] nornucleus accumbens shell [F (3,26)=2.53; n.s.]. Newman–Keuls post-hoc test revealed an increase of PENK geneexpression in the dorsal striatum in the cue-inducedreinstatement group as compared to the control group (P<

Psychopharmacology (2011) 215:49–70 59

0.05). In the nucleus accumbens core, an up-regulation ofPENK gene was observed in the extinction (P<0.05) andcue-induced reinstatement groups (P<0.05) in comparison tothe control group (Fig. 7).

In the contingent versus non-contingent chocolateadministration experiment, one-way ANOVA revealedsignificant differences in PENK gene expression amonggroups in dorsal striatum [F (2,22)=5.53; P<0.05], centralamygdala [F (2,23)=15.36; P<0.01], and nucleus accum-bens shell [F (2,22)=3.54; P<0.05], whereas no significanteffects were obtained in nucleus accumbens core [F (2,22)=3.14; n.s.]. Newman–Keuls post-hoc test revealed adecrease of PENK gene expression in the dorsal striatum,central amygdala, and nucleus accumbens shell in theyoked group as compared to the control group (P<0.05).

D1 and D2 receptors expression No significant changes inD1 receptor gene expression were detected in the brain regionsevaluated in the different experimental groups included in thecue-induced reinstatement and contingent versus non-contingent chocolate administration experiments (see Table 3).

In the cue-induced reinstatement experiment, one-wayANOVA revealed significant differences in D2 receptormRNA levels among groups in dorsal striatum [F (3,25)=

3.78; P<0.05], nucleus accumbens shell [F (3,23)=4.82; P<0.01], nucleus accumbens core [F (3,24)=3.59; P<0.05],ventral tegmental area [F (3,24)=8.48; P<0.001], andsubstantia nigra [F (3,24)=5.53; P<0.01]. Newman–Keulspost-hoc test revealed a decrease of D2 gene expression inthe dorsal striatum in the cue-induced reinstatement group(P<0.05) when compared to the control group. In thenucleus accumbens shell, a decrease of D2 gene expressionwas observed in the cue-induced reinstatement groupcompared to control (P<0.01), acquisition (P<0.05), andextinction groups (P<0.05). In the nucleus accumbens core,a decrease of D2 gene expression was also observed in thecue-induced reinstatement group (P<0.05) compared to thecontrol group. This down-regulation of D2 gene expressionwas also observed in the ventral tegmental area (P<0.01)when compared to control and acquisition groups as well asin the substantia nigra (P<0.05) when compared to theacquisition group. The extinction group also showeddecreased D2 gene expression in the ventral tegmental areawhen compared to the control (P<0.01) and acquisitiongroups (P<0.01) and in the substantia nigra compared tothe acquisition group (P<0.01) (Fig. 8).

In the contingent versus non-contingent chocolateadministration experiment, one-way ANOVA did not reveal

Table 2 Gene expression levels in the hypothalamus

Mean±SEM One-way ANOVA

Control Acquisition Extinction Reinstatement Acquisitioncontingent chocolate

Yoked non-contingentchocolate

F-value P-value

Reinstatement experiment

Arc 1.00±0.12 1.03±0.12 1.26±0.12 1.25±0.13 F(3,27)=1.38 n.s.

BDNF 1.00±0.07 0.86±0.07 0.98±0.07 0.88±0.07 F(3,27)=0.92 n.s.

Proenkephalin 1.00±0.05 0.91±0.05 1.05±0.05 0.92±0.04 F(3,28)=1.90 n.s.

POMC 1.00±0.08 0.77±0.08 1.00±0.08 0.97±0.08 F(3,28)=1.92 n.s.

Crh 1.00±0.07 1.02±0.07 0.89±0.07 1.01±0.07 F(3,28)=0.72 n.s.

Ghrl 1.00±0.07 0.95±0.07 1.01±0.07 1.13±0.08 F(3,25)=1.28 n.s.

Hcrt 1.00±0.08 0.91±0.08 1.16±0.08 0.01±0.08 F(3,28)=1.93 n.s.

Cartpt 1.00±0.06 0.81±0.06 0.98±0.06 0.90±0.06 F(3,28)=2.25 n.s.

Contingent versus non-contingent administration experiment

Arc 1.00±0.19 1.29±0.29 1.09±0.25 F(2,23)=0.36 n.s.

BDNF 1.00±0.17 0.96±0.22 1.36±0.44 F(2,23)=0.54 n.s.

Proenkephalin 1.00±0.27 0.86±0.25 0.74±0.17 F(2,23)=0.30 n.s.

POMC 1.00±0.17 1.01±0.24 1.14±0.28 F(2,23)=0.12 n.s.

Crh 1.00±0.31 0.24±0.15 0.66±0.27 F(2,21)=2.39 n.s.

Ghrl 1.00±0.39 0.99±0.23 1.10±0.60 F(2,22)=0.018 n.s.

Hcrt 1.00±0.16 0.61±0.16 0.72±0.19 F(2,23)=1.38 n.s.

Cartpt 1.00±0.39 0.85±0.14 1.01±0.39 F(2,23)=0.07 n.s.

One-way ANOVA with group as the between-subjects factor at different time-points of the experimental protocol. Data are expressed as foldchange in comparison to control (mean±SEM). See “Materials and methods” for details

n.s. not significant

60 Psychopharmacology (2011) 215:49–70

significant differences in D2 receptor mRNA levels amonggroups in dorsal striatum [F (2,22)=2.16; n.s.], nucleusaccumbens shell [F (2,22)=2.66; n.s.], nucleus accumbenscore [F (2,22)=1.90; n.s.], ventral tegmental area [F (2,21)=3.37; n.s.], and substantia nigra [F (2,21)=3.13; n.s.].

Discussion

In this study, we have validated a new operant model inmice to evaluate the reinstatement of extinguished behaviorto obtain palatable food. An environmental associated cuewas the most effective stimulus to reinstate the seekingbehavior after extinction in animals previously trained toobtain chocolate-flavored pellets (85% of reinstatement). Incontrast, non-contingent priming of two pellets failed toinduce food-seeking behavior. A non-contingent priming often pellets, exposure to intermittent electric footshock(0.22 mA), or the pharmacological stressor yohimbineproduced minor effects on this reinstatement paradigmand also failed to significantly induce food-seeking behav-ior. The non-contingent exposure of this palatable food

already decreased PDYN and PENK mRNA expressionmainly in the dorsal striatum and nucleus accumbens. Asimilar decrease of PDYN mRNA expression was revealedin these brain structures and also in the amygdala after theacquisition, extinction, and reinstatement of the operantbehavior. Interestingly, the opposite regulation of PENKgene was revealed in the striatum and nucleus accumbenscore after the extinction and/or reinstatement of the operantbehavior. Also, an enhancement of PDYN gene expressionwas revealed in the hypothalamus after the extinction of theoperant behavior. In addition, the reinstatement of theoperant behavior produced a consistent down-regulation ofD2 receptor gene in most of the limbic and motor areasstudied.

Interestingly, the exposure to the conditioned stimuluspaired with palatable food administration was the mosteffective condition to reinstate chocolate-seeking behaviorin mice. This finding supports the critical role played byenvironmental factors in the reinstatement of seekingbehavior to obtain a reward, in agreement with the resultspreviously reported in the reinstatement of nicotine-seekingbehavior in mice (Martin-Garcia et al. 2009). The procedureof cue-induced reinstatement was similar when using foodor drugs of abuse as primary reinforcer, and the conditionedstimuli is acting in both experimental conditions as asecondary reinforcer. Studies in rats have also pointed outthe relevance of conditioned cues in inducing reinstatementto drug- (Caggiula et al. 2001; Fuchs et al. 2004; Bossertet al. 2005, 2007) or food-seeking behavior (Ghitza et al.2006, 2007). Cue-induced reinstatement appears to beparticularly robust in mice compared to the other twoclassical stimuli leading to reinstatement, i.e., priming andstress, which seem to be both less effective in mice than inrats (Yan and Nabeshima 2009).

In this study, priming was not effective in reinstatingpalatable food-seeking behavior in mice. On the contrary,studies in rats have obtained reinstatement to food-seekingbehavior after pellet priming (Ghitza et al. 2006, 2007). Inour experimental conditions, the mice were food-deprivedduring the first training phase to facilitate learning to leverpressing response. However, the mice were fed ad libitumduring the remaining phases of the experiment in order toreturn to the normal physiological situation. The absence offood deprivation during the reinstatement phase could havedecreased the probability of food-seeking behavior inducedby priming with palatable food. Indeed under theseconditions mice could have decreased motivation to seekfood after the re-exposure to this natural reward. Foodrestriction is classically considered to be stressful in rodents(Piazza and Le Moal 1998) and elevates the basal levels oforexigenic messengers, such as preprohypocretin (Sakuraiet al. 1998). In contrast, nutritional deprivation was notnecessary to produce cue-induced food-seeking reinstate-

Fig. 4 Prodynorphin gene expression in the hypothalamus. a Valuesobtained after the completion of each phase of the operant training toobtain chocolate-flavored pellets and b in mice receiving these pelletscontingently or non-contingently. Data are expressed as fold change incomparison to control (mean±SEM, n=8 in each group). ★★P<0.01;★★★P<0.001, significant differences compared to the control group.☆P<0.05; ☆☆☆P<0.001, significant differences compared to theacquisition group (post-hoc Newman–Keuls test)

Psychopharmacology (2011) 215:49–70 61

ment. In our study, the mice were not exposed to fooddeprivation during reinstatement to avoid a possibleinterference of stress and orexigenic stimuli with theintrinsic effects of non-contingent priming with palatablefood. There are few reports related to drug-primedreinstatement of drug-seeking behavior in mice. Thus, thepriming injection of cocaine (Fuchs et al. 2003) or nicotine(Martin-Garcia et al. 2009) fails to induce reinstatement inthe C57BL/6 strain, whereas drug priming was the mosteffective stimulus to reinstate cocaine-seeking behavior inoutbreed mice (Soria et al. 2008).

The stress produced by the exposure to electric foot-shock did not reinstate palatable food-seeking behavior. Inagreement with this result, electric footshock exposure hasbeen reported to be selectively efficient in reinstating drug-seeking, but not food-seeking, behavior in rodents (Ahmedand Koob 1997). Stressors, such as electric footshock,could generate an internal condition partly similar to thestate induced by drugs of abuse, which would reinstatedrug-seeking behavior (Ahmed and Koob 1997). The onlystressor that has been reported to reliably reinstate food-seeking behavior in rodents is yohimbine (Nair et al. 2009).Yohimbine is an α-2 adrenoceptor antagonist that increasesbrain noradrenaline release (Abercrombie et al. 1988) andthe firing of noradrenergic cells (Aghajanian and Vander-Maelen 1982). In contrast, yohimbine did not significantlyreinstate food seeking in our study. This result can beexplained by the absence of food deprivation at the momentof reinstatement testing. Stressors such as electric footshockincrease Crh expression and release in several brain areas(Funk et al. 2006), including the ventral tegmental area(Ward and Dykstra 2005). This Crh release has beenreported to promote the release of glutamate and dendriticdopamine in the ventral tegmental area, but only in ratswith a previous history of cocaine intake. This wouldexplain the selectivity of stress in inducing relapse in

animals exposed to a drug, such as cocaine (Wang et al.2005). Further studies are required with different stressorsand experimental conditions to clarify the mechanismsunderlying food reinstatement induced by stress. In thepresent experiment, the combination of stress and palatablefood priming was not more effective than acute stress alone.Therefore, this result further emphasizes that non-contingent priming with palatable food and stress have nomajor influence on the reinstatement of food-seekingbehavior under the different experimental conditions eval-uated in these mice fed ad libitum.

Chronic consumption of highly palatable foods canproduce adaptive changes in the reward circuits that sharesimilarities to those produced by drugs of abuse (Lutter andNestler 2009). Two particular neurotransmitters, dopamineand endogenous opioids, play a crucial role in the rewardcircuits activated by both food and drugs of abuse (Pelchat2009). Dopamine controls various physiological responsesin the brain, including motivation, emotion, and food intakeamong others (Le Foll et al. 2009). Dopamine mechanismsare thought to play a critical role in the incentivemotivational effects of natural rewards and drugs of abuse(Robinson and Berridge 1993). Food seems to stimulatedopamine affinity through different mechanisms that can beindependent from caloric consumption and taste. Thus,taste receptors are not required to activate dopamine circuitssince mice lacking sweet receptors develop place prefer-ence for sucrose solutions, which also produces an increaseof dopamine release in the nucleus accumbens of thesedeficient mice (de Araujo et al. 2008). Therefore, the role ofdopamine in overeating and obesity is not restricted to theoral hedonic taste (de Araujo et al. 2008). In the presentstudy, a down-regulation of D2 receptor gene was found inthe dorsal striatum and nucleus accumbens in the miceexposed to cue-induced reinstatement, whereas no changesin D1 receptor expression were detected. These changes

Fig. 5 Representative in situhybridization images showingsignals obtained for the prody-norphin (Pdyn), proenkephalin(Penk), D1 (D1R), and D2(D2R) receptor mRNAs senseand antisense probes. Outlinesover the representative imagesindicate the brain regions se-lected for quantitative analysis.DS, dorsal striatum; NAc core,nucleus accumbens core; NACshell, nucleus accumbens shell;CeA, central amygdala

62 Psychopharmacology (2011) 215:49–70

were not due to the exposure to the palatable food sincethey were absent in the mice that received this kind of foodcontingently or non-contingently. Decreased levels of D2dopamine receptors have been previously identified after

repeated morphine (Przewlocka and Lason 1995; Georgeset al. 1999) or sucrose exposure (Bello et al. 2002).

The endogenous opioid peptides enkephalins and dynor-phins, synthesized from the PENK and PDYN gene, are

Fig. 6 Prodynorphin mRNA level. a–d Values obtained after thecompletion of each phase of the operant training to obtain chocolate-flavored pellets and e–h in mice receiving contingently or non-contingently, in a, e dorsal striatum, b, f central amygdala, c, gnucleus accumbens shell, and d, h nucleus accumbens core. Data are

presented as mean±SEM of optical densities of the hybridizationsignal in the indicated brain regions (n=8 in each group) ★P<0.05;★★P<0.01; ★★★P<0.001, significant differences compared tothe control group. ☆P<0.05; ☆☆P<0.01, significant differencescompared to the acquisition group (post-hoc Newman–Keuls test)

Psychopharmacology (2011) 215:49–70 63

abundant in the forebrain regions expressing D1 and D2dopamine receptors (Spangler et al. 2004). Met- and leu-enkephalin present a preferential affinity for delta-opioidreceptor (DOR), although they also bind mu-opioid

receptor (MOR), whereas PDYN derivatives are supposedto be the endogenous ligands for kappa-opioid receptor(KOR) (Roth-Deri et al. 2008). The dynorphin/KOR systemparticipates in the reinforcing properties of drugs of abuse

Fig. 7 Proenkephalin mRNA level. a–d Values obtained after thecompletion of each phase of the operant training to obtain chocolate-flavored pellets and e–h in mice receiving these pellets contingently ornon-contingently, in a, e dorsal striatum, b, f central amygdala, c, gnucleus accumbens shell, and d, h nucleus accumbens core. The data

are presented as mean±SEM of optical densities of the hybridizationsignal in the indicated brain regions (n=8 in each group). ★P<0.05,significant difference compared to the control group (post-hoc New-man–Keuls test)

64 Psychopharmacology (2011) 215:49–70

and other stimuli (Przewlocki 2004; Shippenberg et al.2007) by opposing their rewarding effects. In contrast, anactivation of MOR and sometimes also DOR is required toobtain the rewarding responses produced by drugs of abuseand natural rewarding stimuli (Berrendero et al. 2002,2005; Trigo et al. 2009). In agreement, KOR activation inthe ventral tegmental area produces behavioral actionsopposite to those elicited by MOR activation, providingan additional argument for the opposite role of KOR andMOR/DOR receptors in the control of reward (Margoliset al. 2003). In the present study, the non-contingentexposure to chocolate-flavored palatable food decreasedthe mRNA expression of both PDYN and PENK mainly inthe dorsal striatum and nucleus accumbens. These changesmay be due to the activation of the reward circuit by therepeated exposure to this novel natural stimulus thatpossesses a strong reinforcing value. In agreement, reducedPENK gene expression in several striatal regions includingthe nucleus accumbens was found in rats that had beenrepeatedly exposed to a liquid palatable diet containingchocolate (Kelley et al. 2003). Interestingly, an oppositeregulation of PDYN and PENK gene expression was foundin limbic subregions during the acquisition, extinction, andreinstatement of the operant behavior directed to obtainpalatable food. Thus, the mRNA expression of PDYN wasdecreased in the dorsal striatum and nucleus accumbensafter the acquisition of the operant behavior and this PDYNdown-regulation was expanded to the amygdala after theextinction and reinstatement of this behavior. In contrast tothe results obtained in yoked animals, increased PENKmRNA levels were found in the nucleus accumbens coreand dorsal striatum after the reinstatement of the operantbehavior. Therefore, the endogenous opioid component thatpromotes the rewarding responses, i.e., PENK, was up-regulated mainly after the reinstatement of palatable food-seeking behavior, whereas the opioid component thatcounteracts this reward process, i.e., PDYN, was down-regulated during the different phases of this operantlearning. These results are in agreement with the previouslydescribed functional opposite role of MOR and KOR in thecontrol of rewarding stimuli. In this sense, opioid peptidesderived from PDYN have been reported to participate in theaversive effects of several drugs of abuse (Mendizabal et al.2006; Shippenberg et al. 2007), whereas PENK-derivedpeptides participate in the rewarding effects of these drugs(Berrendero et al. 2005). In the present study, D2 receptorexpression was decreased in dorsal striatum whereas PENKgene expression is increased during cue-induced reinstate-ment. This inverse regulation is in agreement with previouspharmacological studies showing that repeated treatmentwith D2 receptor antagonists up-regulates the PENKtranscript, which is expressed in the dorsal striatum (Tanget al. 1983; Sivam et al. 1986; Angulo 1992). In agreement,T

able

3D1receptor

mRNA

level

Reinstatementexperiment(m

ean±

SEM)

One-w

ayANOVA

Con

tingent

versus

non-contingent

administrationexperiment

(mean±

SEM)

One-w

ayANOVA

Con

trol

Acquisitio

nExtinction

ReinstatementF-value

P-value

Con

trol

Acquisitio

ncontingent

chocolate

Acquisitio

nyo

ked

non-contingent

chocolate

F-value

P-value

Dorsalstriatum

71.35±4.02

69.26±2.39

72.42±3.75

71.30±2.87

F(3,28)=1.78

n.s.

80.06±2.30

78.87±1.89

77.15±2.12

F(2,22)=2.02

n.s.

Nucleus

accumbens

core

61.02±5.19

62.13±3.14

60.59±5.23

57.87±2.64

F(3,28)=0.18

n.s.

70.04±3.07

65.73±3.59

65.14±1.87

F(2,22)=0.80

n.s.

Nucleus

accumbens

shell66

.86±5.92

65.30±4.36

65.05±5.80

58.97±3.72

F(3,28)=0.48

n.s.

73.30±2.25

73.55±6.05

69.05±3.31

F(2,22)=0.35

n.s.

One-w

ayANOVAwith

grou

pas

thebetween-subjectsfactor

indifferenttim

e-po

intsof

theexperimentalprotocol.The

data

arepresentedas

mean±

SEM

ofop

tical

densities

ofthehy

bridization

sign

alin

theindicatedbrainregion

s,expressedin

photostim

ulated

luminescenceun

itspersquare

millim

eter

(n=8).See

“Materialsandmetho

ds”fordetails

n.s.no

tsign

ificant

Psychopharmacology (2011) 215:49–70 65

66 Psychopharmacology (2011) 215:49–70

chronic D2 antagonist treatment enhances the pharmacolog-ical responses produced by the endogenous enkephalins(Maldonado et al. 1990). Therefore, the decreased D2 mRNAduring extinction and relapse could promote an enhancementin the expression of PENK gene, which could be associatedto an increase of the release of enkephalins. In accordance,dopamine has been reported to exert a tonic inhibition of thefiring of enkephalin neurons in the striatum (Tang et al.1983). On the other hand, PDYN gene is expressed instriatonigral neurons that are regulated by dopamine D1receptors (Le Moine et al. 1990; Gerfen et al. 1990; Angulo1992). After cocaine administration, increases in PDYNmRNA levels in the nucleus accumbens have been described(Turchan et al. 1998). This activation of the PDYN synthesisrepresents an adaptive response that may counteract theexcessive dopaminergic stimulation produced by cocaine.D1 receptor and PDYN gene expression, respectively, werenot up-regulated in the different phases of operant training toobtain chocolate-flavored pellets. Therefore, dopaminergicactivation promoted by the palatable food could be of lowermagnitude than that produced by cocaine. PDYN geneexpression was even down-regulated during food operanttraining, mainly during the extinction and reinstatementphases, probably to promote the seeking of chocolate pellets.Whereas the limbic structures are mainly involved in therewarding and hedonic aspects of food intake, the hypothal-amus is the main brain region involved in the primaryregulation of appetite. The expression of several key genesinvolved in the regulation of appetite was not modified in thehypothalamus of the mice exposed to the different phases ofthis operant behavior to obtain palatable food. These animalswere not deprived of food at the moment of the biochemicalmeasurement, which could have influence in the resultobtained here. Orexigenic and anorexigenic hypothalamicpeptides exert a control on food intake based on energyhomeostasis rather than on the motivation to obtain the food,which represents a rewarding stimulus. In the presentexperiments, the amount of food consumed during the daily1-h sessions was rather limited and should not have anyinfluence on metabolic balance. Indeed the mice were notoverfed and body weight was not significantly modified bythis operant training. In agreement, changes on geneexpression were produced by this operant responding in the

brain regions involved in the control of motivation andreward, such as the nucleus accumbens and the VTA. Indeedthe hypothalamus is known to regulate food intake accordingto the energetic needs of the organism, whereas operantresponding maintained by palatable food in non-deprivedanimals may be mainly driven by the rewarding effects offood taste rather than the energetic needs. However, an up-regulation of PDYN mRNA expression was found in thehypothalamus of mice after the extinction and reinstatementof the operant behavior. Therefore, the expression of PDYNin the hypothalamus was previously reported to be dramat-ically reduced after food deprivation and chronic foodrestriction (Johansson et al. 2008). It could be hypothesizedthat the induction of PDYN expression is region-specific anddepends on previous basal levels. PDYN is known to beexpressed in the neurons of the lateral hypothalamus thatalso express hypocretins (Chou et al. 2001), which areinvolved in the reinstatement of drug- and food-seeking(Harris et al. 2005; Dayas et al. 2008; Plaza-Zabala et al.2010), and exert orexigenic actions within the hypothalamus(Olszewski and Levine 2007). This functional role ofdynorphins may suggest that the increased expression ofPDYN in the hypothalamus could contribute to the drive toobtain chocolate pellets during the extinction period whenpalatable food is not available. The differential regulation ofPDYN mRNA gene expression in the limbic region and thehypothalamus could correspond to the different role ofdynorphins in these brain areas in mediating aversiveresponses and appetite, respectively.

Measurement of mRNA for peptide precursors can beused as an excellent tool to interpret neuropeptide accumu-lation in a selected brain region in functional terms. Thus, itis well accepted that an increase in mRNA striatalproenkephalin is associated with an increased utilizationof enkephalins (Tang et al. 1983). However, additionalmeasurements on tissue levels or direct measures of releasewould also provide valuable information for a betterinterpretation of the changes on gene expression reportedhere. Therefore, further studies would be required tocorroborate the involvement of the dopamine and opioidsystem on food-seeking behavior considering the possiblelimitations of interpreting the changes in gene expression.

In conclusion, the present study validates a new operantmodel of reinstatement to palatable food-seeking in miceafter the extinction of this specific behavior. This modelprovides an excellent tool to examine the neurobiologicalmechanisms of food-taking habits by using geneticallymodified mice and selected candidate gene approaches infuture studies. These results also identify the specificchanges within the dopamine and opioid systems occurringin the mesolimbic areas and the hypothalamus during theacquisition, extinction, and reinstatement of this operantbehavior directed to obtain palatable food.

Fig. 8 D2 receptor mRNA level. a–e Values obtained after thecompletion of each phase of the operant training to obtain chocolate-flavored pellets and f–j in mice receiving these pellets contingently ornon-contingently, in a, f dorsal striatum, b, g nucleus accumbens shell,c, h nucleus accumbens core, d, i ventral tegmental area, and e, jsubstantia nigra. The data are presented as mean±SEM of opticaldensities of the hybridization signal in the indicated brain regions (n=8 in each group). ★P<0.05; ★★P<0.01 significant differences comparedto the control group. ☆P<0.05; ☆☆P<0.01, significant differencescompared to the acquisition group. a P<0.05 significant differencecompared to the extinction group (post-hoc Newman–Keuls test)

�

Psychopharmacology (2011) 215:49–70 67

Acknowledgements This work was supported by the USA NationalInstitutes of Health—National Institute of Drug Abuse (NIH-NIDA)(No. 1R01-DA01 6768-0111), the DG Research of the EuropeanCommission (PHECOMP, No. LHSM-CT-2007-037669 and GEN-ADDICT, No. LSHM-CT-2004-05166), the Spanish ‘Instituto deSalud Carlos III’ (No. RD06/001/001), the Spanish ‘Ministerio deEducación y Ciencia’ (No. SAF2007-64062), the Catalan Government(SGR2009-00131), the ICREA Foundation (ICREA Academia-2008),the statutory activity funds of the Institute of Pharmacology, PolishAcademy of Sciences and the Polish Ministry of Science and HigherEducation subsidiary grant 478/6. PR UE/2007/7. E.M.G. wassupported by a post-doctoral fellowship from the Spanish ‘Institutode Salud Carlos III’.

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