Parallel Loss of Hippocampal LTD and Cognitive Flexibility in a Genetic Model of Hyperdopaminergia

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1 (27) 32, & 27 Nture Pulishing Group All rights reserved X/7 $3. Prllel Loss of Hippocmpl LTD nd Cognitive Flexiility in Genetic Model of Hyperdopminergi Elise Morice 1, Jen-Mrie Billrd 2,Cécile Denis 1, Flvie Mthieu 1, Ctlin Betncur 1, Jcques Epelum 2, Bruno Giros 1 nd Mrik Nosten-Bertrnd*,1 1 INSERM U513, Neuroiologie et Psychitrie, Créteil Cedex, Frnce; 2 INSERM U549, Neuroiologie de l Croissnce et de l Sénescence, IFR Broc Sinte-Anne, Pris, Frnce Dopmine-medited neurotrnsmission hs een implicted in the modultion of synptic plsticity nd in the mechnisms underlying lerning nd memory. In the present study, we tested different forms of ctivity-dependent neuronl nd ehviorl plsticity in knockout mice for the dopmine trnsporter (DAT-KO), which constitute unique genetic model of constitutive hyperdopminergi. We report tht DAT-KO mice exhiit slightly incresed long-term potentition nd severely decresed long-term depression t hippocmpl CA3 CA1 excittory synpses. Mutnt mice lso show impired dpttion to environmentl chnges in the Morris wtermze. Both the electrophysiologicl nd ehviorl phenotypes re reversed y the dopmine ntgonist hloperidol, suggesting tht hyperdopminergi is involved in these deficits. These findings support the modultion y dopmine of synptic plsticity nd cognitive flexiility. The ehviorl deficits seen in DAT-KO mice re reminiscent of the deficits in executive functions oserved in dopmine-relted neuropsychitric disorders, suggesting tht the study of DAT-KO mice cn contriute to the understnding of the moleculr sis of these disorders. (27) 32, ; doi:1.138/sj.npp ; pulished online 7 Mrch 27 Keywords: dopmine trnsporter; synptic plsticity; Morris wtermze; hloperidol; methylphenidte INTRODUCTION In ddition to its key role in motor nd rewrd systems, rin dopmine (DA) hs een een implicted in integrtive functions contriuting to dptive ehviors such s ttention, lerning, nd memory (Nieoullon, 22; Cools, 26). Wheres DA or DA gonists improve memory, selective depletion, dysfunction, or lesion of forerin DA systems strongly impir cquisition nd retention, s well s the ility to focus nd dpt to environmentl chnges. In support of its role in cognitive processes, DA hs een shown to exert mjor modultory input in synptic plsticity in different rin res, including the stritum (Clresi et l, 1992; Aruthnott et l, 2), hippocmpus (Lismn nd Otmkhov, 21), nd prefrontl cortex (Gurden et l, 1999; Jy, 23). However, clinicl nd experimentl models of oth excessive nd deficient DA trnsmission hve reveled n inverted U-shped reltionship etween DA levels nd cognitive performnce, nd suggested tht the eneficil or detrimentl effects of DA *Correspondence: Dr M Nosten-Bertrnd, INSERM U513, Neuroiologie et Psychitrie, 8 rue du Générl Srril, 941 Créteil Cedex, Frnce, Tel: , Fx: , E-mil: nosten@creteil.inserm.fr Received 25 July 26; revised nd ccepted 1 Jnury 27 depend on sl DA levels, tsk demnds, nd regionl differences (Cools, 26). Thus, the specific contriution of DA to the cognitive impirments oserved in DA-relted neuropsychitric disorders such s schizophreni, Prkinson s disese, nd ttention-deficit hyperctivity disorder (ADHD) remins to e elucidted. In the present study, we exmined the role of DA in synptic plsticity, lerning, nd memory in mice lcking the dopmine trnsporter (DAT), which constitute unique genetic model of persistent functionl hyperdopminergi (Giros et l, 1996; Ginetdinov nd Cron, 23). Mutnt mice (DAT-KO) exhiit extremely high levels of DA synthesis nd turnover (Jones et l, 1998), with gretly prolonged (3-fold) extrcellulr lifetime of DA (Giros et l, 1996), resulting in five- to ten-fold increse in sl extrcellulr DA levels in the stritum nd nucleus ccumens (Jones et l, 1998; Spielewoy et l, 2; Shen et l, 24). Besides their ovious hyperctivity, DAT-KO mice exhiit heightened rtes of stereotypies nd persevertive ptterns of nongol-directed ehvior, distorting their motor, explortory, nd socil ehviors (Ginetdinov et l, 1999; Spielewoy et l, 2; Rlph et l, 21; Rodriguiz et l, 24). Bsed on their constitutive hyperdopminergi, DAT-KO mice were used s genetic model of long-term exposure to psychostimulnts nd were shown to exhiit n enhnced long-term potentition (LTP) of the synptic strength in the

2 nucleus ccumens, mjor component of the rin rewrd system (Yo et l, 24). Here, we studied whether constitutive hyperdopminergi impired ctivity-dependent synptic plsticity in CA1 hippocmpl slice preprtions nd lerning nd memory processes in the Morris wtermze. MATERIALS AND METHODS Animls DAT knockout (KO) mice were otined y homologous recomintion (Giros et l, 1996) nd mintined on two inred genetic ckgrounds: C57BL/6JOrl (B6) nd DBA/ 2JOrl (D2), s previously descried (Morice et l, 24). In the present study, homozygous null mutnts nd their wildtype (WT) nd heterozygous (HT) littermtes were otined from the mting of B6-DAT-HT femles with D2-DAT-HT mles to test the effect of the muttion on the hyrid B6 D2F 1 genetic ckground. The genotype of the mice ws determined y polymerse chin rection (PCR) nlysis s descried previously (Croni et l, 21). Experiments were conducted during the light phse of 12-h light/drk schedule, with lights on t 73 h. All ehviorl nd electrophysiologicl studies were performed on independent groups of nive nimls. The studies were performed in ccordnce with the Europen Communities Council Directive (86/89/EEC) regrding the cre nd use of nimls for experimentl procedures nd pproved y the locl ethicl committee. Electrophysiologicl Assessments Mice were decpitted under deep hlothne nesthesi, nd the hippocmpus ws quickly removed nd plced in cold rtificil cererospinl fluid (CSF) continuously uled with 95% O 2 /5% CO 2 gs mixture (ph 7.4). The composition of the CSF ws s follows (in mm): NCl, 124; KCl, 3.5; MgSO 4, 1.5; CCl 2, 2.3; NHCO 3, 26.2; NHPO 4, 1.2; nd glucose, 11. Slices (4 mm thick) were cut nd plced in holding chmer to recover. Then, single slice ws trnsferred on net to the test chmer nd continuously perfused with CSF (2 ml/min). Extrcellulr recordings were otined t 25 31C from the picl dendritic lyer of CA1 re using micropipettes filled with 2 M NCl (resistnce of 2 6 MO). Field excittory postsynptic potentils (fepsps) were evoked every 1 s y electricl stimultion (1 ms durtion) of fferent fiers locted in the strtum rditum. To investigte the potentition of synptic trnsmission, test stimuli were pplied every 15 s nd djusted to otin n fepsp slope t 3% of the mximum response. The initil slope of three verged fepsps ws mesured for 15 min efore the delivery of thet-urst stimultion consisting of five trins of four pulses t 1 Hz seprted y 2 ms. This sequence ws repeted three times with n interurst intervl of 1 s. In the cse of high-frequency stimultion, the conditioning stimultion consisted of two trins of 1 pulses t 1 Hz seprted y 2 s. Testing with single pulse ws then resumed for 6 min to determine the level of LTP. Synptic depression ws induced y low-frequency stimultion consisting of 12 pulses t 2 Hz. This conditioning stimultion ws induced fter 15 min of seline recording with current intensity djusted to otin n fepsp slope t 6% of the mximum response. Testing with single pulse ws then resumed for 4 min fter low-frequency stimultion to determine the level of longterm depression (LTD). The effects of hloperidol (Hldol, Jnssen-Cilg, Issy-Les-Moulineux, Frnce, 1 mm) perfused for 2 min nd methylphenidte (kindly supplied y Novrtis Phrm AG, Rueil Mlmison, Frnce, 2 mm) preincuted for 2 h efore the seline were investigted on LTD in WT nd KO mice. In these experiments, perfusion with the drugs ws mintined throughout the recording. Locomotor Activity Nive nimls were individully ssessed for their horizontl ctivities in trnsprent cges ( cm), with utomtic monitoring of photocell em reks (Imetronic, Frnce). The nimls were first hituted to the ctivity oxes for 3 min, then given n injection of sline (WT nd KO mice) or hloperidol (KO mice), nd immeditely plced ck into the pprtus for 2 h. For the dose response to methylphenidte, fter n initil ssessment of spontneous ctivity for 3 min, mice were injected with sline or methylphenidte (3, 6, or 8 mg/kg) nd locomotor ctivity ws recorded for 2 h. Morris Wtermze The wtermze consists of circulr stinless-steel pool (15 cm dimeter, 29 cm height) filled to depth of 16 cm with wter mintined t 2 221C nd mde opque using white queous emulsion (Acusol OP 31 opcifier, Rohm Ihs, Frnce). The escpe pltform, mde of rough stinless steel, ws sumerged 1 cm elow the wter surfce. A video trcking system (View Point, Frnce) ws used to monitor ctivity. Hidden Pltform Version During the trining phse of the stndrd plce lerning version of the Morris wtermze, mice lerned the fixed position of smll hidden pltform (6 cm dimeter), using prominent distl extr-mze cues rrnged in the room round the pool. Ech tril strted with the mice fcing the interior wll of the pool nd ended when they climed onto the pltform or fter mximum serching time of 9 s. The strting position ws chnged pseudorndomly etween trils. Animls tht did not find the pltform were gently guided nd plced on it for 2 s. The nimls received one hitution tril on the first dy nd then two trils per dy for 5 consecutive dys. The mice were left undistured in their home cge for the 9-min intertril intervl. On the seventh dy, mice were given the lst trining tril efore the 6 s proe test in which the pltform hd een removed. The distnce trveled in ech qudrnt nd the numer of times the niml crossed ech of the four possile pltform sites (nnulus) ws utomticlly clculted from the video trcking system. After the first proe tril, ll mice were given reversl test in which the hidden pltform ws moved to new position. The mice were trined for 7 dys 219

3 211 following the sme trining procedure, nd then tested for the second proe tril. Cued Pltform Version During the cued version of the wtermze, n independent group of nive mice were trined to find nd escpe onto wider pltform (9 cm dimeter), mde visile to the mice y smll lck ll (4.5 cm dimeter) fixed 11 cm ove the pltform. The trining procedure ws identicl to tht of the sptil version except tht: (i) nimls were trined for only 5 consecutive dys nd (ii) oth the pltform s position nd the niml s strting position were pseudorndomized for ech tril. Cued Pltform Version under Hloperidol or Methylphenidte Tretment Nive mice were given cued trining trils in the wtermze s descried previously, except tht KO mice were injected (i.p. in volume of 1 ml/kg ody weight) with hloperidol (Hldol, diluted in NCl.9%,.75 mg/kg) or methylphenidte (dissolved in NCl.9%, 8 mg/kg), nd WT mice were injected with sline, 3 nd 15 min, respectively, efore the test. Sttisticl Anlysis Repeted-mesures nlysis of vrince ws performed to ssess the interction etween genotypes (etween fctor) nd time (within fctor). Significnt min effects were further nlyzed y post hoc comprisons of mens using Newmn Keuls tests. The one-tiled two-smple Kolmogorov Smirnov test ws used to determine if the distriution of the proportion of nimls succeeding in tril differed significntly depending on genotype. Comprison of mice ccording to their genotypes t the end of the trining phse ws crried out using the w 2 test (or the Mntel Henzel w 2 test when necessry). Only significnt sttisticl tests re reported in the text, with the significnce estlished t P-vlue o.5. RESULTS Selective nd Reversile Impirment in Hippocmpl LTD Electrophysiology experiments were conducted using extrcellulr recordings in ex vivo hippocmpl slices from nive WT, HT, nd DAT-KO mice. Our results showed tht sl glutmtergic trnsmission, including N-methyl-D-sprtte (NMDA) nd non-nmda receptor-medited synptic potentils, did not differ etween genotypes (dt not shown). The induction of LTP y thet-urst stimultion ws significntly fcilitted in DAT-KO reltive to their WT littermtes (lst 15 min: F 1,27 ¼ 4.96, Po.5; Figure 1), suggesting reduced threshold in mutnt mice. The mgnitude of high-frequency stimultion-induced LTP during the lst 15 min of recording ws lso enhnced in DAT-KO, ut the verge LTP during the sme period ws not sttisticlly significnt etween oth genotypes (Figure 1). In contrst, LTD induced y lowfrequency stimultion ws severely impired in DAT-KO fepsp slope (% of seline) fepsp slope (% of seline) c fepsp slope (% of seline) LONG-TERM POTENTIATION THETA 14 LONG-TERM POTENTIATION 2x1Hz WT (n=11) KO (n=1) WT (n=12) HT (n=7) KO (n=17) LONG-TERM DEPRESSION 6 WT (n=11) 4 HT (n=7) KO (n=14) WT HT KO mice (Figure 1c). Low-frequency stimultion induced roust nd stle NMDA receptor-dependent LTD in WT, wheres LTD ws drmticlly reduced in DAT-KO (lst 15 min: F 1,23 ¼ 12.9, Po.1). HT mice showed n intermedite phenotype compred to WT nd KO mice, indicting tht the LTP nd LTD deficit re fine-tuned y the dopminergic tone. To ssess whether enhnced DA levels t the synptic cleft were directly responsile for the decresed LTD, we exmined the effect of the DA ntgonist hloperidol. In hippocmpl slices of oth WT nd KO mice, hloperidol did not ffect sl glutmtergic neurotrnsmission (dt not shown). Interestingly, hloperidol ws le to fully prevent the deficit of LTD in mutnt mice. After dministrtion of the dopminergic ntgonist, the LTD in DAT-KO mice ws significntly fcilitted nd did not differ ny longer from WT mice (Figure 2). The role of NMDA receptor ctivtion on synptic plsticity ws investigted fter locking NMDA receptors Men of the lst 15 min (%) Men of the lst 15 min (%) Men of the lst 15 min (%) * WT HT KO ** * Figure 1 DAT-KO mice exhiit enhnced LTP ut impired LTD. (, left) Time course of men thet-urst stimultion-induced LTP (rrow) in hippocmpl slices of WT (n ¼ 7), HT (n ¼ 4), nd KO (n ¼ 13) mice. (, right) Summry histogrms of the percent in fepsp slope verged from the lst 15 min of recordings. (, left) Time course of men high frequency stimultion-induced LTP (rrow) in WT (n ¼ 7) nd KO (n ¼ 6) mice. (, right) Summry histogrms of the percent in fepsp slope verged from the lst 15 min of recordings. (c, left) Time course of men low frequency stimultion-induced LTD (rrow) in WT (n ¼ 8), HT (n ¼ 5), nd KO (n ¼ 9) mice. (c, right) Summry histogrms of the percentge in fepsp slope verged from the lst 15 min of recordings. The dotted line represents percentge of seline. Vlues represent mens7sem (n ¼ numers of slices per group). *Po.5 nd **Po.1.

4 with the competitive ntgonist DL-2-mino-5-phosphonovlerte cid (D-APV, 5 mm). D-APV completely locked the induction of oth LTP nd LTD (with or without fepsp slope (% of seline) fepsp slope (% of seline) LTD AFTER HALOPERIDOL WT (n=8) KO (n=7) WT (n=9) KO (n=1) Men of the lst 15 min (%) LTD AFTER METHYLPHENIDATE Men of the lst 15 min (%) ** Figure 2 Hloperidol, ut not methylphenidte, fully restores LTD in DAT-KO mice. (, left) Time course of men low-frequency stimultioninduced LTD (rrow) clculted from slices of WT (n ¼ 8) nd KO (n ¼ 5) mice in the presence of hloperidol. (, right) Summry histogrms of the percent in fepsp slope verged from the lst 15 min of recordings performed in the presence of hloperidol. (, left) Time course of men low-frequency stimultion-induced LTD (rrow) in WT (n ¼ 6) nd KO (n ¼ 6) mice in the presence of methylphenidte. (, right) Summry histogrms of the percent in fepsp slope verged from the lst 15 min of recordings performed in the presence of methylphenidte. The dotted line represents percentge of seline. Vlues represent mens7sem (n ¼ numers of slices per group). **Po.1. hloperidol) in DAT-KO nd WT mice, indicting tht these processes were NMDA receptor dependent. DAT-KO mice hve een proposed s n useful model of ADHD, sed on the oservtion tht the psychostimulntlike methylphenidte, one of the most frequently prescried tretments for ADHD, remrkly ttenutes their hyperctivity (Ginetdinov et l, 1999). We thus tested whether methylphenidte could lso improve the electrophysiologicl impirment in synptic plsticity in DAT-KO mice. However, we found tht methylphenidte did not improve LTD in KO mice (Figure 2), which remined significntly reduced compred to WT mice (lst 15 min: F 1,34 ¼ 8.9, Po.1). Delyed Acquisition of Plce Nvigtion ut Intct Sptil Memory in the Morris Wtermze DAT-KO mice were tested for sptil lerning nd memory performnce in the Morris wtermze. As DAT-KO mice swm fster thn their WT littermtes (speed: F 1,98 ¼ 18.1, Po1 4 ), distnce rther thn ltency to find the pltform ws chosen s lerning vrile. Compred with their WT nd HT littermtes, KO mice swm longer distnce to find the hidden escpe pltform (F 2,147 ¼ 15.24, Po1 4 ; Figure 3). However, the distnce decresed during trining in the three genotypes, indicting tht lerning hd occurred (F 6,147 ¼ 18.39, Po.1), nd y the lst 2 dys, ll mice exhiited the sme level of performnce. Furthermore, during the proe tril, when the pltform ws removed, the three genotypes showed strong preference for the trget qudrnt (F 3,84 ¼ 4.12, Po.1; Figure 3) nd for the plce where the pltform ws locted (nnulus crossings, F 3,84 ¼ 16.64, Po.1). These results demonstrte tht DAT-KO mice re le to utilize sptil strtegy to lern the position of the pltform Men distnce trveled (cm) PLACE LEARNING ACQUISITION TRIALS Successful trils (proportion) WT c Men distnce trveled (cm) HT KO 6 7 REVERSAL LEARNING ACQUISITION TRIALS Successful trils (proportion) Distnce trveled (%) NW NE SW SE NW SE NE SW Qudrnts PROBE TRIAL Numer of nnulus crossings NW NE 3 SW SE 2 1 NW SE NE SW Pltforms WT d Distnce trveled (%) HT KO SE NW NE SW SE NW SW Qudrnts NE PROBE TRIAL Numer of nnulus crossings SE NW NE SW SE NW SW Pltforms NE Figure 3 DAT-KO mice exhiit norml sptil reference memory, ut show disrupted reversl lerning in the Morris wtermze. Performnce of WT, HT, nd KO mice during cquisition trils of the plce () nd reversl (c) lerning. The results re expressed s distnce trveled (cm) nd proportion of successful trils. Proe tril of the plce () nd reversl lerning (d). Mice were scored for the percentge of distnce trveled in the four qudrnts: northest (NE), south-est (SE), south-west (SW), nd north-west (NW), nd for the numer of nnulus crossings. The trgeted qudrnt nd pltform re indicted in lck. The horizontl line indictes the distnce trveled using rndom serch strtegy. Vlues represent mens7sem (n ¼ 8 mice per group).

5 2112 Distured Reversl Lerning in the Morris Wtermze We then exmined lerning flexiility on the reversl test, when the hidden pltform ws moved to new loction. Although ll groups improved with trining (F 6,147 ¼ 8.72, Po.1; Figure 3c), DAT-KO mice swm greter distnce to find the pltform (F 2,147 ¼ 12.5, Po.1), nd this deficit persisted until the end of the trining phse (for trining dys 6 nd 7: genotype, F 2,42 ¼ 7.65, Po.1, nd successful trils, KO vs WT, w 2 2df ¼ 14.93, Po.1). Moreover, during the proe tril, WT nd HT mice exhiited sptil strtegy, fvoring the trget qudrnt nd the plce where the pltform ws locted, wheres DAT-KO mice swm n equl distnce in ll qudrnts (qudrnt: F 3,84 ¼ Po.1, nd genotype qudrnt interction; F 6,84 ¼ 3.48, Po.1; Figure 3d) nd crossed the four virtul pltforms s often (nnulus crossings, F 3,84 ¼ 6.2, Po.1, nd genotype, F 2,84 ¼ 2.2, Po.3). Therefore, DAT-KO mice filed to dpt their ehvior following chnge of the pltform position, indicting lck of ehviorl flexiility. The deficit of KO mice ws not due to inility to shift wy from the previously reinforced qudrnt, ecuse during the first tril following the qudrnt test they did not visit the previous reinforced qudrnt more often thn the other qudrnts. Insted, when they did not find the pltform during the reversl trining, their ehvior ecme chotic nd not focused to serching the pltform, s illustrted y the vriility in the distnce swm to rech the pltform nd in the numer of successful trils (Figure 3c). These findings demonstrte tht the distured reversl lerning of DAT-KO mice is not the expression of persevertive ehvior in the wtermze. Severe Impirment in the Cued Version of the Morris Wtermze In the cued version of the wtermze, mice were trined to swim to pltform mounted with visile cue nd plced in different sptil loction on ech tril. The distnce trveled to escpe decresed significntly with time in oth WT nd HT mice, wheres DAT-KO mice showed hrdly ny improvement over the course of trining (genotype, F 2,75 ¼ 37.74, Po.1; trining, F 4,75 ¼ 1.32, Po.1; nd interction, F 8,75 ¼ 2.77, Po.1; Figure 4). DAT-KO mice lso succeeded in escping on fewer trils thn WT nd HT mice (w 2 2df ¼ 29., Po.1 nd w 2 2df ¼ 14.8, Po.1, respectively). To test whether this deficit ws due to n inility of DAT-KO mice to ssocite the cue ll to the escpe pltform, new group of mice ws trined in the cued version following the sme trining procedure except tht the pltform ws kept in fixed position. In this condition, we found tht DAT-KO mice escped successfully on twice s mny trils s efore, nd their performnce improved with trining (KO, F 4,3 ¼ 3.8, Po.5; Figure 4), demonstrting tht deficits oserved in the previous test were due to the lck of dpttion to rpid chnges in the environment rther thn to deficit of ssocitive lerning. To test whether the excess of DA neurotrnsmission contriuted to the loss of ehviorl flexiility exhiited y DAT-KO mice, n independent group of nimls ws Men distnce trveled (cm) Successful trils (proportion) treted with hloperidol nd tested on cued nvigtion. The dose of hloperidol ws sed on previous experiments (Spielewoy et l, 2). The dose of.75 mg/kg ws found to restore norml locomotor ctivity in DAT-KO, not only in the ctimeter (Figure 5) ut lso in the wtermze, s illustrted y the swimming speed tht did not differ ny longer mong the KO nd WT genotypes nd incresed in similr wy with trining (F 4,7 ¼ 8., Po.1; Figure 5). In the wtermze, following hloperidol dministrtion, DAT-KO ecme s successful s WT mice in finding the pltform, nd their performnce mrkedly improved with trining (KO, F 1,35 ¼ 4.35, Po.5; Figure 5). We then exmined whether, in ddition to its clming effect (decrese in hyperlocomotion) (Ginetdinov et l, 1999), methylphenidte could improve the cognitive performnce of DAT-KO mice in the cued version of the Morris wtermze. Methylphenidte exerts its psychostimulnt-like effects in WT mice vi the DAT, wheres its mechnism of ction in DAT-KO mice is not yet understood. Thus, the purpose of this experiment ws not to compre DAT-KO mice to psychostimulted nimls ut to test whether methylphenidte restored cognition in DAT-KO mice to the level of control nimls. A dose response experiment confirmed its inhiitory effect on horizontl ctivity of mutnt mice nd reveled tht the dose of 8 mg/kg reduced the locomotion of DAT-KO mice to the level of WT nimls (Figure 6). However, in the Morris wtermze (Figure 6), DAT-KO showed hrdly ny improvement with trining fter methylphenidte tretment (genotype, F 1,84 ¼ 17.5, Po.1; trining, F 5,84 ¼ 4.8, Po.1; nd interction, F 5,84 ¼ 11.5, Po.1) nd escped successfully on twice s fewer trils s their WT littermtes (w 2 1df ¼ 21.8, Po.1). DISCUSSION SPONTANEOUS CUED VERSION WT CUED VERSION The present study illustrtes profound modifiction of vrious forms of ctivity-dependent plsticity in DAT-KO mice, highlighted y enhnced LTP, mrkedly impired LTD, nd distured cognitive dpttion to environmentl 5 HT FIXED PLATFORM KO Figure 4 DAT-KO mice re severely impired in the cued version of the Morris wtermze. () Performnce of WT, HT, nd KO mice on the spontneous cued version. () Performnce of WT nd KO mice in the cued version with the pltform kept t fixed position. Results re expressed s men distnce trveled (top) nd proportion of successful trils (ottom). Vlues represent mens7sem (n ¼ 5 7 mice per group).

6 Men swim speed (cm/min) Men distnce trveled (cm) Photocell em reks/12 min Successful trils (proportion) LOCOMOTOR RESPONSE TO HALOPERIDOL Sline KO s Hloperidol.75 mg/kg CUED VERSION AFTER HALOPERIDOL Figure 5 Hloperidol improves the performnces of DAT-KO mice in the cued version of the Morris wtermze. () Totl locomotor horizontl ctivity in WT nd KO mice utomticlly recorded over 2-h period fter cute injection of sline or hloperidol (n ¼ 5 8 mice per group). () Performnce of WT nd KO mice in the cued version of the wtermze. Thirty minutes efore ech dily session, mutnt mice were injected with hloperidol (.75 mg/kg, i.p.) nd WT mice received sline injection. Results re expressed s men distnce trveled (top) nd proportion of successful trils (ottom). Vlues represent mens7sem (n ¼ 8 mice per group). Photocell em reks /5 min Men distnce trveled (cm) Successful trils (proportion) DOSE-RESPONSE TO METHYLPHENIDATE WT, sline KO, sline KO, 3 mg/kg KO, 6 mg/kg KO, 8 mg/kg CUED VERSION AFTER METHYLPHENIDATE Figure 6 Methylphenidte does not improve the performnce of DAT- KO mice in the cued version of the Morris wtermze. () Locomotor dose response to dministrtion of methylphenidte in DAT-KO mice over 2-h period (n ¼ 5 15 mice per group). () Performnce of the WT nd KO in the cued version following methylphenidte tretment. Fifteen minutes efore ech dily session, mutnt mice were injected with methylphenidte (8 mg/kg, i.p.), nd WT mice received sline injection. All results re expressed s men distnce trveled (top) nd proportion of successful trils (ottom). Vlues represent mens7sem (n ¼ 8 mice per group). chnges in the wtermze. Notly, oth impired LTD nd cued lerning re reversed y hloperidol. This result strongly suggests tht the hyperdopminergic tone during the test, rther thn compenstory mechnisms occurring during development, is directly involved in these deficits, lthough other mechnisms in DAT-KO mice, such s direct effect of hloperidol cnnot e excluded (Arvnov et l, 1997; Gemperle et l, 23). In ddition, this prllel loss/rescue strongly supports the hypothesis of common mechnisms underlying oth forms of cellulr nd ehviorl plsticity (Mlenk nd Ber, 24). In vitro electrophysiologicl studies hve provided cler evidence tht DA cts s mjor modultor of fst glutmtergic neurotrnsmission (Jy, 23). The modultory role of DA in synptic plsticity is further supported y in vivo studies showing tht repeted systemic exposure to psychostimulnts fcilittes LTP induction in the midrin 2113

7 2114 (Liu et l, 25), reduces LTD in the nucleus ccumens (Thoms et l, 21), nd converts LTD into LTP in the stritum (Nishioku et l, 1999). Interestingly, enhnced LTP of the cortico-ccuml synpses ws recently reported in DAT-KO mice, used s genetic model of long-term exposure to psychostimulnts (Yo et l, 24). Altogether, these dt nd ours, demonstrting tht oth LTP nd LTD re ltered in hippocmpl CA1 synpses, suggest tht hyperdopminergi medites loss of idirectionl synptic plsticity in high-order pthwys nd strengthen the emerging consensus tht drug ddiction nd memory processes my rely on similr cellulr mechnisms (Nestler et l, 1993; Berke nd Hymn, 2; Hymn nd Mlenk, 21; Mlenk nd Ber, 24). Our results indicte tht the hyperdopminergi of DAT- KO mice produces cognitive deficits similr to those oserved following lesion or phrmcologiclly induced hypodopminergi of the mesocorticolimic DA system (Cools, 26), nd re thus in greement with the oservtion tht oth excessive nd insufficient DA levels impir cognitive performnces (Arnsten, 1998). The hyperdopminergi of DAT-KO mice does not suppress the ility to perform ehvior, they re not deilitted nd they interct socilly with their littermtes (Spielewoy et l, 2). Moreover, they do not exhiit generl defect in lerning. In self-dministrtion prdigm, where food is used s reinforcer, DAT-KO mice re le to lern n opernt ehvior within the sme trining sessions s controls (Roch et l, 1998). Similrly, KO mice re le to discriminte olfctory stimuli, nd show no deficits in ssocitive lerning s tested in the plce preference test (Sor et l, 1998; Rodriguiz et l, 24). Here, we show tht their sptil nvigtion nd motivtion re spred: DAT-KO mice ctively serch for the pltform, clim on it s soon s they find it, nd do not exhiit ny floting ehvior. Rther, they pper drmticlly impired in their cpcity to dpt their ehvior to environmentl chnges, s illustrted y their distured reversl lerning, impired lerning in the cued version, nd improved performnce only when the cued pltform ws kept t fixed position. Together with previous studies showing their persevertive ptterns of socil ehviors nd their greter distrctiility (Rlph et l, 21; Rodriguiz et l, 24), these dt support permissive rther thn mnemonic role of centrl DA (Whishw nd Dunnett, 1985). Complex cognitive functions, referred to s executive functions, which contriute to the continuous orchestrtion of well-dpted nd gol-directed ehviors, rely on oth prefrontl cortex nd stritl DA trnsmission (Cools, 26). Severl lines of evidence suggest tht the cognitive deficits oserved in DAT-KO mice re more likely to depend on stritl DA rther thn on prefrontl cortex DA. First, wheres the DAT is mjor determinnt of the intensity nd durtion of DA neurotrnsmission in the stritum, microdilysis experiments demonstrte less prominent role of the DAT in DA clernce in the prefrontl cortex (Css nd Gerhrdt, 1995), which correltes with the reltive sprse distriution of the DAT in the prefrontl cortex (Sesck et l, 1998), s well s with severl studies showing heterologous DA uptke y the norepinephrine trnsporter in this region (Croni et l, 199; Tnd et l, 1997). Second, in DAT-KO mice, dilyste DA is pproximtely 1-fold higher in oth the stritum nd nucleus ccumens, wheres the sl level of extrcellulr DA is not ffected in the prefrontl cortex (Shen et l, 24). Furthermore, the rte of DA uptke in the prefrontl cortex hs lso een shown to e norml in DAT-KO mice (Mundorf et l, 21). Not surprisingly, the drmtic moleculr ltertions of oth pre- nd postsynptic homeostsis following inctivtion of the DAT hve een extensively descried in the stritum (Giros et l, 1996; Roch et l, 1998; Jones et l, 1999; Shen et l, 24), wheres no dt re ville in these mutnt mice concerning the mesencephlo-hippocmpl DA projection. We show here tht synptic plsticity is clerly impired t the Schffer collterl CA1 synpses. Even though we demonstrted tht hippocmpldependent sptil nvigtion is clerly preserved in DAT- KO mice, impired DA trnsmission in the hippocmpus could e involved in their initil delyed cquisition. The potentil impliction of the hippocmpus in the oserved deficits is further supported y growing ody of evidence demonstrting tht oth stritl nd hippocmpl systems interct intimtely in vrious cognitive functions (Poldrck nd Pckrd, 23; Voermns et l, 24), nd tht the hippocmpus nd the midrin form DA-dependent loop tht contriutes to the role of the hippocmpus in novelty detection (Lismn nd Grce, 25). However, further moleculr chrcteriztion of DA homeostsis within the hippocmpl region of DAT-KO mice remins to e crried out. It ws previously reported tht ADHD ptients suffer primrily from n inility to inhiit their ehviorl response, suggesting tht their impired cognitive function could e secondry to the hyperkinesi (Brkley, 1997). The oservtion tht psychostimulnts exerted clming effect in DAT-KO mice suggested tht similr mechnisms my exist in DAT-KO mice nd they were thus proposed s model of ADHD (Ginetdinov et l, 1999). The present finding demonstrting tht methylphenidte, despite its efficient ttenution of the hyperctivity in DAT-KO mice, neither improved cued lerning nor restored LTD chllenges this hypothesis. Our results suggest tht the serotoninergic trnsmission, shown to medite the clming effect of methylphenidte on locomotor ctivity of DAT-KO mice (Ginetdinov et l, 1999), is not likely to e involved in their cognitive deficit. Mentl rigidity, persevertion, inility to shift, to dpt, nd to djust ehviors to the context re common fetures of DA-relted disorders such s schizophreni, Prkinson s disese, nd ADHD. We show here tht similr ehviorl phenotypes re present in DAT-KO mice, s the result of geneticlly-induced hyperdopminergi. Thus, DAT-KO mice offer n unprecedented niml model to study the neurl nd moleculr mechnisms underlying these trnsnosogrphic symptoms for which no stisfctory tretment is yet ville. ACKNOWLEDGEMENTS We thnk S Dvis nd S Cooke for helpful comments on the mnuscript. We lso thnk L Hillrd for niml cre. This work ws supported y INSERM nd Fondtion pour l

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