enhanced extinction of cocaine seeking in brain-derived neurotrophic factor val66met knock-in mice

Enhanced extinction of cocaine seeking in brain-derivedneurotrophic factor Val66Met knock-in mice

Lisa. A. Briand,1,2 Francis S. Lee,3 Julie A. Blendy1 and R. Christopher Pierce2

1Department of Pharmacology, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA2Department of Psychiatry, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA3Department of Psychiatry, Pharmacology, Weill Medical College of Cornell University, New York, NY, USA

Keywords: brain-derived neurotropic factor, cocaine, extinction, neurotrophin, self-administration, single-nucleotide polymorphism

Abstract

The Val66Met polymorphism in the brain-derived neurotropic factor (BDNF) gene results in alterations in fear extinction behavior inboth human populations and mouse models. However, it is not clear whether this polymorphism plays a similar role in extinction ofappetitive behaviors. Therefore, we examined operant learning and extinction of both food and cocaine self-administration behavior inan inbred genetic knock-in mouse strain expressing the variant Bdnf. These mice provide a unique opportunity to relate alterationsin aversive and appetitive extinction learning as well as provide insight into how human genetic variation can lead to differences inbehavior. BDNFMet ⁄ Met mice exhibited a severe deficit in operant learning as demonstrated by an inability to learn the food self-administration task. Therefore, extinction experiments were performed comparing wildtype (BDNFVal ⁄ Val) animals to miceheterozygous for the Met allele (BDNFVal ⁄ Met), which did not differ in food or cocaine self-administration behavior. In contrast tothe deficit in fear extinction previously demonstrated in these mice, we found that BDNFVal ⁄ Met mice exhibited more rapid extinction ofcocaine responding compared to wildtype mice. No differences were found between the genotypes in the extinction of food self-administration behavior or the reinstatement of cocaine seeking, indicating that the effect is specific to extinction of cocaineresponding. These results suggest that the molecular mechanisms underlying aversive and appetitive extinction are distinct from oneanother and BDNF may play opposing roles in the two phenomena.

Introduction

Addiction is a characterized by compulsive drug use that persists inthe face of negative consequences and desire to stop use (Kalivas &O’Brien, 2008; O’Brien, 2011). Conditioned drug craving elicited bydrug-paired cues is among the many factors that contribute to thiscompulsive use. Addicts report that these conditioned cues are one ofthe most potent factors contributing to relapse (Heather et al., 1991).Understanding how to extinguish this cue-elicited craving is a majorgoal in addiction research. Genetically modified mice provide a usefulmodel system in which to examine molecular mechanisms underlyingcocaine extinction behavior and how this might lead to alterations invulnerability in human populations.

Brain-derived neurotrophic factor (BDNF), a member of theneurotrophin family of polypeptide growth factors, is widelyexpressed throughout the brain and is regulated in an activity-dependent manner (Goodman et al., 1996; Egan et al., 2003). BDNFhas been implicated in synaptic plasticity, learning and addiction.Manipulations of BDNF levels in the brain have been shown tomodulate addictive behaviors, such as cocaine-induced locomotoractivity (Pierce & Bari, 2001), cocaine-conditioned place preference

(Horger et al., 1999; Hall et al., 2003) and cocaine self-administration(Lu et al., 2004). Furthermore, upregulation of BDNF expression isobserved in various limbic nuclei during abstinence from cocaine self-administration (Sadri-Vakili et al., 2010), and levels of BDNFexpression correlate with measures of cue-elicited craving (Grimmet al., 2003).Along with this evidence from the preclinical literature, there is also

a relationship between substance abuse and BDNF in humanpopulations. At a genetic level, single-nucleotide polymorphisms(SNPs) conferring vulnerability for polysubstance abuse have beenindentified flanking the BDNF gene (Uhl et al., 2001). More recently,a common SNP in the BDNF gene that leads to a valine (Val)-to-methionine (Met) substitution at codon 66 (Val66Met) was linked tosubstance abuse vulnerability. A higher frequency of the Val allele isfound among methamphetamine addicts compared to non-addictedindividuals, suggesting the Met allele may confer resistance toaddiction (Cheng et al., 2005). However, there is an increase in 66Metallele frequency in smokers, and healthy Met carriers consume morealcohol per week than 66Val homozygotes (Lang et al., 2007; Colzatoet al., 2011). Parsing the role of this SNP in addictive behavior usingan animal model could provide unique insight into the pathophysi-ology of addiction.The current study utilized an inbred genetic knock-in mouse strain

that expresses the variant BDNF allele to recapitulate the specific

Correspondence: L. A. Briand, as above.E-mail: [email protected]

Received 27 October 2011, revised 29 December 2011, accepted 4 January 2012

European Journal of Neuroscience, pp. 1–8, 2012 doi:10.1111/j.1460-9568.2012.08021.x

ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd

European Journal of Neuroscience

phenotypic properties of the human polymorphism in vivo. Previouswork has demonstrated that the BDNF Val66Met genotype isassociated with a resistance to the extinction of fear memories(Soliman et al., 2010). While there is considerable overlap betweenthe neurocircuitry underlying the extinction of aversive and appetitivememories (Ressler et al., 2004; Davis et al., 2006; Peters et al., 2009;Myers & Carlezon, 2010b), it is clear that the molecular mechanismsare not identical (Chhatwal et al., 2009; Ward et al., 2009; Bernardi &Lattal, 2010). The objective of this study was to assess the acquisition,stability and extinction of an appetitive behavior (cocaine self-administration) in Vall66Met knock-in mice.

Materials and methods

Subjects

Mice bred and maintained on a C57Bl ⁄ 6 background as describedpreviously (Chen et al., 2006) were single-housed with food and wateravailable ad libitum. Adult wildtype (BDNFVal ⁄ Val), littermateBDNFVal ⁄ Met and littermate BDNFMet ⁄ Met male and female micederived from BDNFVal ⁄ Met · BDNFVal ⁄ Met crossed parents were usedfor all experiments. All animals (2–4 months of age) were housed in atemperature- and humidity-controlled animal care facility with a 12-hlight–dark cycle (lights on a 07.00 h). All procedures were approvedby the University of Pennsylvania Animal Care and Use Committee.

Drugs

Cocaine was obtained from the National Institutes of Drug AbuseDrug Supply Program (Bethesda, MD, USA) and dissolved in sterile0.9% saline.

Operant food training

Prior to catheterization, mice were trained to perform an operantresponse for sucrose pellets. Although prior food training can lead tocross-sensitization, mice will not self-administer reliably without thistraining (Thomsen & Caine, 2007). The mice were placed in operantchambers (Med-Associates) and trained to spin a wheel manipulan-dum to receive a sucrose pellet. A compound cue stimulus consistingof a cue light above the active lever, a 2.9 kHz tone and the houselight being switched off was concurrent with each pellet administra-tion; this was followed by an additional 8-s time-out during whichresponding had no programmed consequences and the house lightremained off. Mice were allowed to self-administer a maximum of 50pellets per 60 min operant session. During the food self-administrationphase, mice were food restricted to �90% of their free-feeding weight.Mice received 10 training sessions and animals that did not distinguishbetween the active and inactive wheel (> 60% active responding) wereexcluded from further study.

Jugular catheterization surgery

Prior to surgery, mice were anesthetized with 80 mg ⁄ kg ketamine and12 mg ⁄ kg xylazine. An indwelling silastic catheter was placed intothe right jugular vein and sutured in place. The catheter was thenthreaded subcutaneously over the shoulder blade and was routed to amesh backmount platform (Strategic Applications, Inc.) that securedthe placement. Catheters were flushed daily with 0.2 mL of anantibiotic (Timentin; 0.93 mg ⁄ mL) dissolved in heparinized saline.The catheters were sealed with plastic obturators when not in use.

Cocaine self-administration

After surgery, mice were allowed 3–4 days to recover beforebeginning behavioral testing. Mice were tested for cocaine self-administration behavior in 2-h sessions (6 days per week) in the samechamber used for sucrose pellet self-administration. During testing, aquarter turn of a wheel (FR1) on the same wheel used for sucrosetraining now delivered an intravenous cocaine injection (0.5 mg ⁄ kgper infusion). Each session began with a single infusion of cocaineaccompanied by the presentation of the tone and light cue. Acompound cue stimulus consisting of a cue light above the activelever, a 2.9 kHz tone and house-light off was concurrent with eachinjection; this was followed by an additional 8-s time-out whenresponding had no programmed consequences and the house lightremained off. Following 10 days of cocaine self-administration,cocaine-seeking behavior was extinguished by replacing the cocainewith 0.9% saline. Daily 2-h extinction sessions continued for 10 days.Following this 10-day extinction phase, animals that had not reached acriterion of < 25% of their self-administration responding continuedextinction for an additional 5 days. Animals underwent cue-inducedreinstatement after either meeting the extinction criterion or 15 days ofextinction, whichever was shorter. During the cue-induced reinstate-ment session, the light and tone cues were presented non-contingentlyfor 20 s every 2 min during the first 10 min of the session. After thistime period, the cues were presented contingent with operantresponding, just as was done during the cocaine self-administrationphase. During the reinstatement session, animals received salineinfusions following responses on the active wheel.

Sucrose self-administration

Separate groups of mice were trained to lever press for sucrose asdescribed above, and then received and additional 10 days of 2-hsucrose self-administration sessions (6 days per week) in the samechamber used for sucrose training. A compound cue stimulusconsisting of a cue light above the active lever, a 2.9 kHz tone andhouse-light off was concurrent with each pellet administration; thiswas followed by an additional 8-s time-out when responding had noprogrammed consequences and the house light remained off. Eachsession began with a pellet delivery accompanied by the presentationof a tone and light cue. Following 10 days of food self-administration,food-seeking behavior was extinguished by eliminating the fooddelivery following the operant response. Daily 2-h extinction sessionscontinued for 10 days.

Data analysis

Total active operant responses over the course of the 10 days of foodtraining were analyzed using a repeated-measures two-way anova

with genotype (Val ⁄ Val, Val ⁄ Met, Met ⁄ Met) and session as theindependent variables and active responses as the dependent variable.Bonferonni’s post hoc comparisons were conducted when main effectsor interactions were present. Similar analyses were performed for thecocaine self-administration, extinction and reinstatement phases of theexperiment.

Results

Acquisition of sucrose pellet self-administration

The number of pellets earned and active responses during eachsucrose self-administration session was measured. BDNFVal ⁄ Val and

2 L. A. Briand et al.

ª 2012 The Authors. European Journal of Neuroscience ª 2012 Federation of European Neuroscience Societies and Blackwell Publishing LtdEuropean Journal of Neuroscience, 1–8

BDNFVal ⁄ Met mice acquired stable responding for food, readilyresponding for 20 or more pellets after 5 days of training. However,BDNFMet ⁄ Met mice failed to acquire the operant response for fooddespite continued training. While no differences were seen betweenBDNFVal ⁄ Val and BDNFVal ⁄ Met mice with either variable,BDNFMet ⁄ Met mice performed significantly fewer active responses(Fig. 1; two-way repeated-measures anova, effect of genotype:F2,441 = 4.41, P = 0.017; Bonferroni post hoc BDNFMet ⁄ Met vs.BDNFVal ⁄ Val and BDNFVal ⁄ Met, P < 0.05) and received significantlyfewer pellets (Fig. 1; two-way repeated-measures anova, effect ofgenotype: F2,441 = 11.57, P = 0.0001; interaction: F18,441 = 1.73,P = 0.032; Bonferroni post hoc BDNFMet ⁄ Met v. BDNFVal ⁄ Val andBDNFVal ⁄ Met, P < 0.05). The BDNFMet ⁄ Met mice were not includedin the cocaine or food extinction experiments because they wereunable to acquire the operant response for food during training.

Cocaine self-administration behavior

The number of infusions earned, active responses and per cent activeresponding during each cocaine self-administration session in shown

in Fig. 2. Both BDNFVal ⁄ Val and BDNFVal ⁄ Met mice acquired stablecocaine self-administration behavior after 10 sessions. AlthoughBDNFVal ⁄ Met mice showed a trend toward slower acquisition on days1–4, statistical analyses indicated no differences between the groups inthe number of infusions earned or active responses performed over the10 sessions. Additionally, no differences were seen in the per centactive responding, with both groups stabilizing at �70% correct(Fig. 2).

Extinction and reinstatement of cocaine-seeking

The number of active responses as well as the percentage ofresponding compared to the average responding on the last 2 daysof cocaine self-administration was measured. BDNFVal ⁄ Met miceexhibited significantly faster extinction of cocaine responding com-pared to BDNFVal ⁄ Val mice, as measured by both a decrease in activeresponding during extinction (Fig. 3; two-way repeated-measuresanova, effect of genotype: F1,198 = 4.46, P = 0.04; effect of session:F9,198 = 8.81, P = 0.0001) and a decrease in the percentage respond-ing compared to the cocaine self-administration phase (Fig. 3; two-

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Fig. 1. BDNFMet ⁄ Met mice exhibited impaired instrumental learning. While wildtype (BDNFVal ⁄ Val) and BDNFVal ⁄ Met mice gradually learned to spin the wheel toacquire sucrose pellets, the BDNFMet ⁄ Met mice did not. (A) BDNFMet ⁄ Met mice performed significantly fewer active responses (anova; genotype: F2,441 = 4.41,*P = 0.017; post hoc BDNFMet ⁄ Met vs. BDNFVal ⁄ Val and BDNFVal ⁄ Met P < 0.05) compared to the wildtype and heterozygous mice. (B) BDNFMet ⁄ Met micereceived significantly fewer pellets than the wildtype or heterozygous mice (anova, genotype: F2,441 = 11.57, ***P = 0.0001; interaction: F18,441 = 1.73,P = 0.032; post hoc BDNFMet ⁄ Met vs. BDNFVal ⁄ Val and BDNFVal ⁄ Met, P < 0.05). No differences were seen between BDNFVal ⁄ Val and BDNFVal ⁄ Met mice the ineither variable.

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Fig. 2. BDNFVal ⁄ Val and BDNFVal ⁄ Met mice exhibited equivalent levels of cocaine self-administration. Both wildtype and heterozygous mice acquired cocaineself-administration behavior and no differences were seen between the two genotypes in (A) their active responses, (B) infusions earned or (C) per cent activeresponding.

Enhanced extinction of cocaine seeking 3


way repeated-measures anova, effect of genotype: F1,198 = 24.56,P = 0.0001; effect of session: F9,198 = 11.73, P = 0.0001). BothBDNFVal ⁄ Val and BDNFVal ⁄ Met mice exhibited significant reinstate-ment of responding compared to the prior extinction day; however, nodifferences were seen between the genotypes (Fig. 4; two-wayrepeated-measures anova, effect of test: F1,17 = 31.42, P = 0.0001).

Extinction of food seeking

The number of active responses as well as the percentage ofresponding compared to the food self-administration phase wasmeasured. BDNFVal ⁄ Val and BDNFVal ⁄ Met mice exhibited similarrates of extinction of food responding as measured by equivalentactive responding during extinction and equivalent percentageresponding compared to the food self-administration phase (Fig. 5;

two-way repeated-measures anova, effect of session: F9,99 = 15.04,P = 0.0006).

Discussion

BDNF has been implicated in many facets of addiction in preclinicalmodels, including cocaine conditioned reward (Horger et al., 1999;Lobo et al., 2010), cocaine craving (Grimm et al., 2003), cocainereinforcement (Sadri-Vakili et al., 2010) and cocaine reinstatement(Berglind et al., 2007). Variants in the BDNF gene have also beenimplicated in vulnerability to addiction in human addicts (Krebs et al.,2000; Uhl et al., 2001; Tsai, 2007). Using a mouse model system thatcontains the human SNP in the BDNF gene that leads to a Val-to-Metsubstitution at codon 66 (Val66Met), the present study providesseveral new insights into phenotypes associated with the variantBDNFMet. First, we provide evidence for learning impairment in thatBDNFMet ⁄ Met mice failed to acquire food self-administration. Thislearning impairment was specific to the Met homozygotes, as therewas no detectable learning deficit in the BDNFVal ⁄ Met mice. Second,when examining acquisition and stability of cocaine self-administra-tion behavior, we found no differences between BDNFVal ⁄ Val andBDNFVal ⁄ Met mice. However, BDNFVal ⁄ Met mice exhibited morerapid extinction of cocaine responding compared to BDNFVal ⁄ Val

controls. This is in direct contrast to the impaired fear extinctionbehavior seen in both human and mouse BDNF Met carriers (Solimanet al., 2010). Furthermore, there was no difference between thegenotypes in extinction of food seeking. As all mice were food-trainedprior to cocaine self-administration, it seems unlikely that the cocaineeffects resulted from alterations in extinction of food respondingduring the initial cocaine self-administration sessions. Additionally,this suggests that the molecular mechanisms underlying appetitive andaversive fear extinction are at least somewhat distinct as BDNFappears to play different roles in these two forms of learning.

Role of BDNF Val66Met polymorphism in operant learning

The growth factor BDNF is the most abundant neurotrophin in themammalian central nervous system and is involved in mediatingsynaptic plasticity associated with various forms of learning (Patterson

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Fig. 3. BDNFVal ⁄ Met mice exhibited a decrease in cocaine-seeking under extinction conditions. (A) Although both wildtype (BDNFVal ⁄ Val) and BDNFVal ⁄ Met

mice gradually decreased their active responding during the extinction phase (session: F9,198 = 4.03, P < 0.001), the BDNFVal ⁄ Met mice decreased their respondingmore rapidly than BDNFVal ⁄ Val mice (anova, genotype: F1,198 = 4.46, *P = 0.04). (B) This was particularly evident when examining the per cent respondingduring extinction compared to the average active responses during the last 2 days of cocaine self-administration. After ten extinction sessions the BDNFVal ⁄ Met micewere only responding at �20% of their cocaine self-administration levels whereas the BDNFVal ⁄ Val mice were still responding at �100% (anova, genotype:F1,198 = 24.56, ***P = 0.0001).

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Fig. 4. BDNFVal ⁄ Val and BDNFVal ⁄ Met mice exhibited similar reinstatementof cocaine seeking. Both wildtype and heterozygous mice exhibited significantreinstatement of responding during a cue-induced reinstatement session andwere responding at > 125% of their cocaine self-administration levels (anova,test: F1,17 = 31.42, ***P = 0.0001). No differences were seen between the twogenotypes in their per cent responding during reinstatement.



et al., 1996; Mu et al., 1999; Mizuno et al., 2000; Barco et al., 2005).BDNF plays a critical role in hippocampus-dependent spatial learning(Tyler et al., 2002) as well as amygdala-dependent Pavlovian fearconditioning (Rattiner et al., 2004; Ou & Gean, 2006); however, itsrole in operant learning is less clear. While learning an operant task,BDNF mRNA levels increase in the medial prefrontal cortex(Rapanelli et al., 2010). Despite this, infusions of BDNF into thisbrain region do not alter established food-seeking behavior (Berglindet al., 2007). BDNF function in the nucleus accumbens, while clearlyinvolved in addiction, does not seem to be related to operant learningin that the increases in accumbens BDNF seen following cocaine self-administration are not seen following food self-administration (Grimmet al., 2003). Furthermore, infusions into the nucleus accumbens ofeither BDNF or an antibody to BDNF had no effect on establishedoperant responding for food (Graham et al., 2007). Additionally,knockdown of TrkB, the primary receptor for BDNF, in the nucleusaccumbens had no effect on the acquisition of operant behavior foreither sucrose or cocaine (Graham et al., 2009). In contrast, infusionsof BDNF into the dorsomedial striatum led to a decrease in establishedsucrose self-administration (Jeanblanc et al., 2009). However, indi-rectly decreasing BDNF levels within the striatum, through knock-down of the transcription factor MeCP2 or directly manipulatingBDNF through infusions of a BDNF antibody, leads to a decrease incocaine intake during extended access cocaine self-administration (Imet al., 2010). Furthermore, viral overexpression of BDNF in thestriatum increases cocaine intake during extended access self-admin-istration but not during limited access paradigms (Im et al., 2010).This suggests that the role of BDNF within operant behavior in thestriatum is quite complex and may depend not only on the reinforcerbut also on the training regimen.

Although no studies have specifically examined instrumentallearning, the BDNF Val66Met polymorphism has been studiedclinically in other forms of learning. BDNF Met carriers exhibitdeficits in episodic memory (Egan et al., 2003; Dempster et al., 2005),recognition memory (Hariri et al., 2003; Goldberg et al., 2008) andspatial navigation (Banner et al., 2011). Furthermore, these deficits inmemory performance are accompanied by a decrease in hippocampalactivity in BDNF Met carriers during the performance of memorytasks (Hariri et al., 2003; Hashimoto et al., 2008; Banner et al.,2011). The results of the current study clearly indicate that the deficits

in learning seen in BDNF Met carriers may extend beyond tasksdependent upon the hippocampus, and demonstrate a need forexamining the role of this BDNF gene variant in striatal-dependentlearning.

BDNF Val66Met polymorphism plays distinct roles in differentforms of extinction learning

Recent work has demonstrated a role for the BDNF Val66Metpolymorphism in aversive extinction learning. Using the same mousestrain utilized in the current studies, the Met allele was associated withboth increased anxiety-related behaviors (Chen et al., 2006) andimpairment of conditioned fear and conditioned taste aversionextinction (Yu et al., 2009; Soliman et al., 2010). This deficit inextinction learning has also been documented in human BDNF Metcarriers (Soliman et al., 2010). In contrast, the current study demon-strated facilitation of drug-related extinction learning in BDNFVal ⁄ Met

mice compared to wildtype controls. This suggests that the role ofBDNF in these two forms of extinction is distinct.Understanding the role that BDNF may play in these two

phenomena may require a better understanding of how the BDNFVal66Met polymorphism affects the levels of BDNF in different brainregions and how BDNF in different brain regions may play diverseroles in appetitive and aversive extinction learning. Knockdown ofBDNF within the hippocampus or the amygdala leads to impairedaversive extinction behavior, suggesting that BDNF is necessary inthese regions for normal extinction (Chhatwal et al., 2006; Heldtet al., 2007). Furthermore, infusions of BDNF into the infralimbiccortex, following fear conditioning, mimic the effects of extinctiontraining (Peters et al., 2010). These findings are all consistent with theidea that it is a decrease in regulated BDNF that is mediating theimpaired aversive extinction behavior seen in the BDNF Met carriers(Soliman et al., 2010).The effects of BDNF on the extinction of appetitive learning are less

clear. Similarly to what is seen with aversive extinction, infusions ofBDNF into the prefrontal cortex lead to facilitation of extinctionof cocaine seeking (Berglind et al., 2007). However, an infusion ofBDNF into the ventral tegmental area blocks extinction of cocaineseeking (Lu et al., 2004). These results suggest that a decrease inBDNF may lead to a decrease in cocaine craving, which is consistent

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Fig. 5. BDNFVal ⁄ Val and BDNFVal ⁄ Met mice exhibited similar extinction of food-seeking. Both wildtype and heterozygous mice gradually decreased their activeresponding to the same extent during the extinction phase and were responding at < 20% of their food self-administration levels, and no differences were seenbetween the two genotypes in (A) their active responses during extinction or (B) their per cent responding during extinction, compared to the average activeresponses during the last 2 days of food self-administration.



with other preclinical data. During withdrawal from cocaine self-administration, BDNF levels within the ventral tegmental areaincrease and this increase has been correlated with levels of cocainecraving (Grimm et al., 2003). Furthermore, knockdown of BDNFwithin either the VTA or the nucleus accumbens leads a decrease incocaine-conditioned reward (Graham et al., 2009). Taken together,these data indicate that the decrease in regulated BDNF secretionwithin the VTA of BDNF Met mice may predominate over any otheralterations in BDNF and thus lead to facilitation of the extinction ofcocaine seeking behavior.Although there is some overlap in the brain regions thought to

mediate aversive and appetitive extinction (Peters et al., 2009), it isapparent that the exact mechanisms underlying these forms of learningare not identical. Just as BDNF seems to play opposing roles inaversive and appetitive extinction, other signaling systems also lead todiscriminate effects in these two phenomena. For example, while thecannabinoid receptor antagonist rimonabant blocks fear extinction(Chhatwal et al., 2009) it facilitates the extinction of operantresponding for food or cocaine (Ward et al., 2009). Similarly, thealpha-adrenergic receptor antagonist prazosin inhibits fear extinctionwhile having no effect on appetitive extinction (Bernardi & Lattal,2010). However, the administration of a partial NMDA agonist, a drugthat arguably has more widespread effects than either rimonabant orprazosin, facilitates both aversive and appetitive extinction (Ressleret al., 2004; Parnas et al., 2005; Botreau et al., 2006; Davis et al.,2006; Shaw et al., 2009; Myers & Carlezon, 2010a; Thanos et al.,2011a,b). These studies, along with inactivation studies (Morganet al., 1993; Morgan & LeDoux, 1995; Peters et al., 2008), suggestthat there is some overlap between the circuits mediating these twotypes of extinction learning but the underlying molecular mechanismsdiffer.Furthermore, the mechanisms underlying different types of appe-

titive extinction may also be distinct. The facilitation of drug-relatedextinction behavior seen in the BDNFVal ⁄ Met mice in the current studydid not extend to all forms of appetitive learning, as no effect of theBDNF SNP was seen in the extinction of food-seeking behavior. Thisis consistent with the literature demonstrating a clear role for BDNF incocaine seeking that is distinct from food seeking. For example,increases in BDNF within the ventral tegmental area, nucleusaccumbens and amygdala are seen following withdrawal from cocaineseeking but not from sucrose self-administration (Grimm et al., 2003).Furthermore, manipulations of BDNF that alter cocaine seeking do nothave any effect on food-seeking behavior (Berglind et al., 2007).

Implications for clinical populations

In recent years, BDNF gene variants, most commonly the Val66MetSNP examined in the current study, have been implicated in a widerange of psychiatric disorders. BDNF Met carriers are at a higher riskfor depression (Schumacher et al., 2005; Hwang et al., 2006;Verhagen et al., 2010), aggressive forms of schizophrenia (Spallettaet al., 2010), severe depressive episodes in bipolar depression(Hosang et al., 2010) and anxiety-related traits (Montag et al.,2010). Furthermore, BDNF Met carriers have a higher susceptibilityto stress, which may contribute to the effect of this genotype ondepression and anxiety (Shalev et al., 2009; Alexander et al., 2010).In contrast, the current study found that BDNFVal ⁄ Met mice exhibit anincreased rate of extinction of cocaine responding. Despite thisincreased rate of extinction, BDNFVal ⁄ Met mice exhibited equivalentlevels of cue-induced reinstatement. Extinction is believed to be a newform of learning that is dependent upon the ventral prefrontal cortex,whereas cue-induced reinstatement depends on projections from the

amygdala to the dorsal prefrontal cortex. Therefore, our data suggestthat BDNF Met carriers may have an increase in the ability to formnew associations. In support of this hypothesis, human studies ofBDNF Met carriers have demonstrated enhanced behavioral flexibilityin memory-based switching tasks as well as enhanced responseinhibition (Beste et al., 2010; Gajewski et al., 2011). Although BDNFMet carriers clearly exhibit some learning deficits, our study, as wellas these human studies, suggest that this SNP can also confer anadvantage in some cognitive domains.

Conclusion

Through the use of a mouse model system, we determined that thevariant BDNFMet ⁄ Met leads to impaired instrumental learning. Thisabnormality is not seen in BDNFMet ⁄ Val heterozygotes, suggestingthat there may be a threshold level of activity-dependent BDNFrelease that is needed for learning in this task and only theBDNFMet ⁄ Met mice are below this threshold. Furthermore, wedemonstrate that BDNFMet ⁄ Val heterozygotes exhibit a facilitation ofextinction of cocaine seeking. This is in contrast to previous findingsdemonstrating impairment in extinction of aversive learning (Chenet al., 2006; Soliman et al., 2010). To our knowledge, this is the firstevidence that a functionally relevant polymorphism can lead toopposing behavioral phenotypes in these two types of extinctionlearning. These findings provide the basis for further studies todetermine whether human BDNF Met carriers exhibit a similardisparity in behavioral extinction phenotypes.

Acknowledgements

We thank Ted Huang, Blake Kimmey and Jennifer Xue for technical assistance.Research supported by NIDA F32 DA026660 (L.B.), R01DA15214 (R.C.P.),K02 DA18678 (R.C.P.), R01 DA-011649 (J.A.B.) and NINDS R01 NS052819(F.S.L.). The authors report no biomedical financial interests or potentialconflicts of interest.

Abbreviations

BDNF, brain-derived neurotrophic factor; Met, methionine; SNP, single-nucleotide polymorphism; Val, valine; Val66Met, Val-to-Met substitution atcodon 66.

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enhanced extinction of cocaine seeking in brain-derived neurotrophic factor val66met knock-in mice

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