Enrichment induces structural changes and recovery from nonspatial memory deficits in CA1 NMDAR1-knockout mice

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1 rtiles Enrihment indues struturl hnges nd reovery from nonsptil memory defiits in CA1 NMDAR1-knokout mie Clire Rmpon, Y-Ping Tng, Joe Goodhouse, Eiji Shimizu, Mureen Kyin nd Joe Z. Tsien Deprtment of Moleulr Biology, Prineton University, Wshington Rod, Prineton, New Jersey , USA The first two uthors ontriuted eqully to this work. Correspondene should e ddressed to J.Z.T. (jtsien@molio.prineton.edu) We produed CA1-speifi NMDA reeptor 1 suunit-knokout () mie to determine the NMDA reeptor dependene of nonsptil memory formtion nd of experiene-indued struturl plstiity in the CA1 region. mie were profoundly impired in ojet reognition, olftory disrimintion nd ontextul fer memories. Surprisingly, these defiits ould e resued y enrihing experiene. Using stereologil eletron mirosopy, we found tht enrihment indued n inrese of the synpse density in the CA1 region in knokouts s well s ontrol littermtes. Therefore, our dt indite tht CA1 NMDA reeptor tivity is ritil in hippompus-dependent nonsptil memory, ut is not essentil for experiene-indued synpti struturl hnges. Comprtive ntomil studies of hippompl ytorhiteture revel seletive expnsion of the CA1 region in the hippompus in primtes with respet to rodents nd in humns with respet to monkeys, suggesting tht this suregion is importnt in humn hippompl funtion 1. Dmge to the hippompus or the CA1 suregion in humns leds to defiits in memories of people, ojets, ples nd events ( delrtive memory ) 2 4. Similr sptil nd nonsptil memory defiits re lso oserved in vriety of lortory nimls with hippompl lesions 4,5. Reordings of neuronl tivity in the CA1 suregion of rodents during ehviorl tests show the importne of this suregion in the enoding of nonsptil informtion 6. However, the moleulr mehnisms underlying these proesses remin unknown. N-methyl-D-sprtte (NMDA) reeptors re widely distriuted in the rin 7 nd re essentil for the indution of mjor forms of long-term potentition (LTP) nd long-term depression (LTD) 8,9. Enhned NMDA reeptor funtion in the forerin improves lerning nd memory 10, inditing its ruil role in these proesses. Using the Cre/loxP-medited reomintion system, we developed region-speifi gene-knokout tehnique to generte onditionl-knokout mie in whih NMDAR1, key suunit of NMDA reeptor, ws seletively deleted in the CA1 suregion 11. These CA1-speifi NMDAR1 knokout mie lked NMDA-medited urrents nd plstiity in the CA1 region nd were profoundly impired in sptil memory tsks 12,13. In this study, our first gol ws to exmine the role of CA1 NMDA reeptor tivity in the formtion of nonsptil memory nd the effets of enrihed experiene on these memory funtions. Our seond gol ws to exmine whether enrihed experiene during dulthood ould led to struturl hnges in the CA1 region, nd whether CA1 NMDA reeptor-medited responses re required for suh struturl modifitions. Synpti struturl hnges re ssumed to e the ntomil sustrte of long-term storge of lerned experiene 14,15. For exmple, numers of dendriti spines in the hippompus inrese fter sptil lerning in dult rts 16. Bsed on findings of NMDA reeptor funtion in the developing rin 17,18, it is postulted tht NMDA reeptors might e required for ehviorl experiene-indued struturl plstiity in dult rin. However, new dendriti spines n form on mture hippompl neurons in vitro in the sene of synpti tivity 19, nd LTP hs no effet on synpse numer in the CA1 region 20. These findings imply tht the role of the NMDA reeptor in struturl hnges in dult rin might differ from tht in the developing rin. By using unised stereologil eletron mirosopy, we exmined the struturl hnges in our mie efore nd fter enrihed experiene. RESULTS NMDAR1 knokout in the CA1 region We onfirmed the omplete deletion of the NMDAR1 gene in mie using in-situ hyridiztion histohemistry. No NMDAR1 mrna ws deteted in the CA1 suregion (Fig. 1), inditing tht the geneti deletion of NMDAR1 in young dult mie ws heritle fter three yers of reeding nd ws omplete. Nonsptil lerning nd memory defiits nd ontrol mie used in ll ehviorl tests were 2 4 month-old littermtes. We used three hippompus-dependent ehviorl tsks to ssess ssoitive, nonsptil memory funtions. The hippompus is importnt in the formtion of reognition memory in oth humn ptients nd nimls 3 5. However, little is known out its ntomil sis nd its moleulr nd ellulr mehnisms 21. We evluted reognition memory with novelojet reognition-memory tsk 10. During the trining session, the totl mount of time spent exploring two ojets ws ± 3.94 seonds in ontrol mie (n = 17) nd ± 3.56 seonds in CA1- KO mie (n = 12), nd no signifint explortory preferene ws found etween ontrol nd mie (dt not shown). These oservtions indite tht these two types of mie hve the sme levels of motivtion, uriosity, nd interest in exploring novel ojets. 238 nture neurosiene volume 3 no 3 mrh 2000

2 rtiles Fig. 1. Cre/loxP-medited deletion of NR1 gene in mie (2.5 months old) is omplete. An ntisense 42-mer oligonuleotide reognizing the NR1 gene ws used for in situ hyridiztion. CTX, ortex; ST, stritum; DG, dentte gyrus. For retention tests, t 30 minutes, 2 hours nd 24 hours fter the trining (Fig. 2), one ojet ws repled y novel ojet. As expeted, ontrol mie showed signifint preferene for exploring the novel ojet during eh retention test. In ontrst, mie showed only mrginl preferene for the novel ojet (Fig. 2). A self-anova etween trining nd retention tests reveled signifint differene in ontrol mie (F 3,64 = 7.744, p < 0.001) ut not in mie. Repeted mesures of ANOVA on retention tests showed highly signifint differene etween ontrol nd mie (F 2,27 = , p < 0.001). A post-ho nlysis using Dunnett s test demonstrted signifine of this differene in retention t 30 minutes, 2 hours nd 24 hours (p < 0.05, p < 0.01 nd p < 0.01, respetively; Fig. 2). These results indite profound defiits in novel ojet reognition memory in. We then exmined olftory disrimintion memory using soil trnsmission of food preferene 22. Rodents develop preferene for foods they hve reently smelled on the reth of other individuls 23. Lesions restrited to the hippompus impir this kind of memory 24 hours fter soil intertion (trining) 24. However, the moleulr mehnisms underlying this kind of memory remin unknown 22,24. The tsk used here onsisted of three stges. First, nimls eme ustomed to eting from food up pled on the ge floor. Seond, in trining sessions, oserver mie were llowed to intert with demonstrtor mie fed with either innmon-sented (1% per weight) or oo-sented (2% per weight) food. Third, oserver mie were then tested y presenttion of oth sented foods, nd onsumption of eh food ws reorded. mie showed signifintly higher preferene for food smelled during the trining session over unsmelled food thn did mie (F 1,26 = 5.291; Fig. 3), inditing tht olftory-disrimintion memory ws impired in mie. To ensure tht this oservtion ws not due to differene in feeding ehviors, we mesured the totl mount of food tken during the retention session; this quntity did not signifintly differ Fig. 2. Impired novel-ojet reognition memory in mie. Reognition memory is expressed in terms of explortory preferene in the retention tests. The memory for ontrol (n = 17) or (n = 12) mie ws mesured t three different time intervls fter trining. The dt re expressed s men ± s.e.; p < 0.05, p < 0.01, determined y post-ho nlysis. We oserved similr results in seprte groups of nd ontrol mie. etween ontrol nd mie (1.723 ± g versus ± g, respetively). Beuse these two types of sented food were ounterlned throughout the experiments, these results indited tht the defiits oserved in mie were due neither to norml food intke nor to sent is per se. We then exmined nonsptil memory using ontextul feronditioning tsk 10. In this hippompus-dependent tsk 25, nimls lern to fer n environment y ssoiting it with n versive stimulus (foot shok). nd mie signifintly differed in ontextul freezing, ut not in freezing responses immeditely fter the shoks, in retention tests t 2 hours nd 24 hours (p < 0.05, p < 0.01, respetively; Fig. 4), inditing tht mie were impired in ontextul fer memory. However, in ued-onditioning test, whih is hippompus independent 25, freezing responses of mie to tone were similr to those of ontrol mie in retention tests t 2 hours (dt not shown) nd 24 hours (Fig. 4). In ddition, no norml noieptive responses were found in mie: urrent required to eliit flinhing/running, jumping or voliztion in the mie ws the sme s in ontrol mie (dt not shown). These dt lerly showed tht hippompus-dependent, ut not hippompusindependent, fer memory ws impired in mie. Explortory preferene (%) Explortory preferene (%) Trining session 30 min 2 h 24 h Retention intervl nture neurosiene volume 3 no 3 mrh

3 rtiles Fig. 3. Soil trnsmission of food preferene. Olftion-disrimintion memory is expressed in terms of food preferene. () The memory of either ontrol (n = 15) or (n = 13) mie ws mesured 24 h fter trining. () Food onsumption dt re expressed s men ± s.e. p < 0.05 determined y one-wy ANOVA. Enrihment-indued reovery of memory defiits These results indited profound defiit in three forms of nonsptil memory in mie. Enrihed experiene n signifintly improve performne in sptil mze tests in norml nimls 26 nd ttenute memory defiits in nimls with hippompl lesions 27. However, it is not known whether enrihed experiene n enhne performne of the nimls in the three nonsptil memory tsks we used, nor if it is le to resue the memory defiits oserved in mie. To ddress these issues, we evluted nonsptil memory using the sme tsks with dditionl groups of dult nimls fter they were trined dily in n enrihed environment for two months. In the novel-ojet reognition tsk, we oserved n inrese in preferene for exploring novel ojets in oth ontrol nd mie (n = 14; n = 12, respetively; Fig. 5). A self-anova etween trining nd retention tests reveled signifint differenes for ontrol (F 3,52 = , p < 0.001) nd mie (F 3,44 = 3.797, p < 0.05), inditing tht trining in the enrihed-environment not only enhned the performne of norml mie ut lso ttenuted the defiits in mie. An integrted sttistil nlysis of nimls with the sme genotype onfirmed the effets of trining on memory in ontrol mie (F 1,29 = 7.674, p < 0.05) nd in mie (F 1,22 = 6.674, p < 0.05). However, repeted mesures ANOVA on retention tests still reveled highly signifint group differene etween Contextul freezing (%) Cued freezing (%) Immedite freezing Pre-CS CS Totl food onsumption (grms) Food preferene (%) enrihed ontrol nd enrihed mie (F 2,24 = , p < 0.01), inditing tht enrihing experiene ould only prtilly resue the defiits oserved in the mie. In the test of soil trnsmission of food preferene, we found tht enrihed ontrol nimls (n = 15) did not inrese food preferene more thn nive nimls. This possily refleted either eiling effet or delite lne etween hoosing the preferred food nd eting ny food fter 24 hours of food deprivtion. However, food preferene of enrihed mie (n = 12) ws drmtilly inresed over tht of nive nimls (F 1,22 = 4.746, p < 0.05, Fig. 5), inditing tht the enrihed experiene ompletely resued the memory defiits in the mie. We lso oserved tht the enrihment trining signifintly enhned ontextul freezing in ontrol mie (F 1,25 = 7.366, p < 0.05) nd ompletely resued the defiits oserved in the mie (F 1,23 = , p < 0.001). No signifint differene ws found etween enrihed ontrol nd enrihed mie (Fig. 5). In ddition, ued freezing lso drmtilly inresed following enrihed experiene in oth ontrol (F 1,25 = 6.213, p < 0.05) nd mie (F 1,23 = 4.372, p < 0.001; Fig. 5d). These results lerly indite tht the enrihed experiene ould enhne these two kinds of fer memory in ontrol nimls nd ould resue the defiits in mie. Fig. 4. Fer-onditioning tsks. () Contextul fer onditioning. Memory is expressed s perent of freezing responses. Immedite lerning indites the freezing response during the 30 s immeditely fter shok in the trining session. Contextul fer memory in ontrols (n = 14) nd (n = 12) ws mesured 24 h fter trining. () Cued fer onditioning. Memory is expressed s perentge of freezing responses. There ws no signifint differene in proportion of freezing responses either t pre-cs or retention test etween ontrol mie (n = 14) nd the mie (n = 12). All dt re expressed s men ± s.e.; p < 0.01, determined y one-wy ANOVA. Similr results were otined from nother set of experiments. 240 nture neurosiene volume 3 no 3 mrh 2000

4 rtiles Explortory preferene (%) Food preferene (%) Nive Enrihed Nive Enrihed Enrihment-indued ntomil hnges Wht re the moleulr nd struturl mehnisms underlying the enrihment-indued effets? Enrihed experiene promotes vrious iohemil nd morphologil hnges in severl rin regions 26, Synpti struturl hnges re elieved to e the ntomil sustrte of long-term storge of lerned experiene, nd it is ssumed tht NMDA reeptors re required for experiene-dependent ntomil hnges in the dult rin. As the first step towrd the geneti dissetion of the moleulr mehnisms of experiene-indued struturl plstiity in the dult hippompl CA1 region, we used light nd eletron mirosopy to exmine the effets of enrihment on ntomil hnges in the CA1 region nd the role of the NMDA reeptor in this effet. Nissl-stined tissue of mie shows no gross ntomil normlity 12. We used the Golgi-impregntion tehnique 32 to initilly ssess dendriti strutures. Golgi-impregnted CA1 pyrmidl neurons of ontrol nd nive nimls presented similr dendriti morphology (Fig. 6 nd ). Quntittive nlysis of dendriti-spine density on CA1 pyrmidl ells reveled no signifint differene etween nive ontrol (8.1 ± 0.8 spines per 10 µm of pil dendrite, men ± s.e.) nd nive mie (8.6 ± 0.6 spines; Fig. 6). This suggests tht onditionl knokout of the NMDAR1 gene in the CA1 region, whih ours in the postntl third nd fourth weeks, did not result in norml spine density. To hrterize the effets of enrihment on dendriti spine density, we then ounted the numer of spines on dendrites of Fig. 6. Enrihment-indued inrese in CA1 spine density in oth norml ontrol nd mie. Photomirogrphs of Golgi-impregnted pil dendriti segments of CA1 pyrmidl neurons of nive ontrol () nd nive () mie. () Brs represent stter plot of individul verge numers of dendriti spines per 10 µm length of CA1 pyrmidl dendriti segments. Dimonds represent group mens ± s.e. (n = 3 for eh group). The sexes (M or F) nd ges in months of nimls in eh olumn (from top to ottom) re nive norml ontrol (M, 3.5; M, 4; M, 3.5), enrihed norml ontrol (M, 3.5; M, 5; M, 4.5), nive CA1- KO (M, 4.5; M, 5; M, 4) nd enrihed (M, 5; M, 5; M, 4.5). p < 0.05, Mnn-Whitney U-test. d Cued freezing (%) Contextul freezing (%) Nive Enrihed Nive Enrihed Fig. 5. Effets of enrihed experiene on nonsptil memory in oth ontrol nd mie. () Enrihed experiene inresed explortory preferene in oth ontrol (n = 14) nd (n = 12) mie in the novelojet reognition tsk. () Enrihed experiene resued memory defiits of (n = 12) mie in the soil trnsmission of food preferene. For ontrol mie, n = 15. () Enrihed experiene enhned ontextul freezing response in oth ontrol (n = 13) nd (n = 11) mie in the fer-onditioning tsk. There ws no signifint differene in immedite freezing etween the two groups (dt not shown). (d) Enrihed experiene inresed ued freezing in the fer-onditioning tsk. There ws no signifint differene in pre-cs freezing or ued freezing etween ontrol mie (n = 13) nd (n = 11) mie. All dt re expressed s men ± s.e. p < 0.05, p < 0.01, p < in one-wy ANOVA. CA1 pyrmidl ells of enrihed nimls. We oserved signifintly higher density of dendriti spines in enrihed ontol mie ompred with nive nimls (10.8 ± 1.1 versus 8.1 ± 0.8; p < 0.05; Fig. 6). This is onsistent with previous report of inresed spine density on CA1 neurons in rts fter enrihment 16. Surprisingly, enrihment similrly inresed spine density in mie (10.0 ± 0.3 versus 8.6 ± 0.6; p < 0.05). Sttistil nlysis reveled no signifint differene etween enrihed ontol nd enrihed nimls (Fig. 6). Dendriti spines per 10 µm Nive Enrihed Nive Enrihed nture neurosiene volume 3 no 3 mrh

5 rtiles Fig. 7. Enrihment-indued inrese in CA1 synpse density in oth ontrol nd mie. Representtive eletron photomirogrphs illustrting synpses (indited y rrows) in the strtum rditum of the CA1 region of nive ontrols (), nive mie (), enrihed ontrols () nd enrihed mie (d). Sle rs, 1 µm. (e) Dimonds represent group mens ± s.e. of estimted synpti densities in the strtum rditum of the CA1 region efore nd fter enrihment, lulted with the stereologil disetor. Brs represent stter plot of the CA1 synpti density in individul nimls in eh group. The sexes (M or F) nd ges in months of nimls in eh olumn re (from top to ottom) nive ontrol (M, 4.5; M, 5; M, 4; M, 4; M, 3; M, 3.5; M, 4), enrihed ontrol (M, 4.5; M, 5; M, 5.5; M, 4; M, 5), nive (M, 4; M, 4.5; F, 3.5; M, 4) nd enrihed (M, 3.5; F, 4; M, 4.5; M, 5). p < 0.05, determined y Mnn-Whitney U-test. d These dt indite tht enrihed experiene resulted in hnges in dendriti-spine density nd suggest tht this inrese in spine density n our in the sene of NMDA reeptor tivity. These results re onsistent with the finding tht mture CA1 pyrmidl neurons in vitro n grow spines in sene of NMDA-reeptor tivtion 19. However, Golgi stining should e interpreted utiously. First, this method does not revel spines hidden eneth or sitting ove the dendriti segment, thus, spine density mesured this wy proly underestimtes the tul numer of spines. Seond, some rnhed spine heds reeive no innervtion 33 ; therefore, spine density should not e simply extrpolted to synpse density. To ddress these onerns, we used eletron mirosopy to quntittively nlyze synpse density in the strtum rditum of the CA1 hippompl region (Fig. 7 d). We ensured unised smpling y using the stereologil disetor tehnique 34,35. No signifint differene in synpti density ws oserved etween nive ontrol mie (76.9 ± 1.9 synpses per 100 µm 3, men ± s.e.) nd nive mie (70.0 ± 4.4 synpses, Fig. 7e). However, fter the enrihment trining, ontrol nimls showed signifint inrese of CA1 synpti density (93.8 ± 5.3) over the nive ontrol group (p < 0.05). Synpti density ws lso strikingly higher in trined mie (91.2 ± 2.9) ompred to the nive group (p < 0.01, Fig. 7e). Furthermore, no signifint differene in synpti density ws found etween enrihed ontrol nd enrihed mie. These dt indite tht enrihed experiene promotes synpti struturl hnges in the CA1 hippompl region. Furthermore, these results suggest tht the NMDA reeptor is not required for the inrese of CA1 synpti density. e Synpses per 100 µm 3 Nive Enrihed Nive Enrihed (n = 7) (n = 5) (n = 4) (n = 4) mie To investigte the possile differentil effet of enrihment nd NMDA reeptor tivity on hnges in the density of synpti sutypes, we exmined the distriution of CA1 synpses in three mjor sulsses (non-perforted, perforted nd shft synpses) tegorized ording to the profile of the postsynpti density (PSD) in pirs of seril setions. The PSD ws lssified s non-perforted if the thikening ws ontinuous or s perforted if the PSD ws interrupted y eletron-luent regions (Fig. 8). Synpses with symmetri PSDs on dendriti shfts were identified using Gry s riteri 36 Fig. 8. Mjor sulsses of CA1 synpses in the CA1 region. (, ) Eletron mirogrphs illustrting the ultrstruture of different types of synpses in the strtum rditum of CA1. Sle rs, 0.5 µm. () Non-perforted xospinous synpse (np) nd perforted xospinous synpse (p). () Axodendriti synpses involving dendriti shfts (s). (, d) Estimted distriution of CA1 synpses in the tegories desried ove for ontrol () nd (d) nimls, either nive or rered in n enrihed environment. Synpti densities were lulted per surfe re nd then expressed s the numer of synpses per 100 µm 3 y using the stereologil oeffiient otined with the disetor method. The dt re expressed s men ± s.e.; p < 0.05, Mnn-Whitney U-test. Synpses per 100 µm 3 Synpses per 100 µm 3 Perforted Non-perforted Shft d Nive ontrol Enrihed ontrol Nive Enrihed 242 nture neurosiene volume 3 no 3 mrh 2000

6 rtiles nd were ounted seprtely from those on dendriti spines (Fig. 8). We found signifintly higher density of non-perforted synpses in ontrol mie fter enrihment (77.1 ± 5.2, men ± s.e.) thn in nive ontrol nimls (62.6 ± 2.3, p < 0.05; Fig. 8). Remrkly, enrihment lso signifintly inresed the density of non-perforted synpses in mie (78.3 ± 5.4 versus 60.7 ± 5.1; p < 0.05; Fig. 8d), wheres the densities of perforted nd shft synpses remined unhnged fter enrihment in oth ontrol nd mie. For etter oservtion of the synpti struturl hnges, sophistited pproh using three-dimensionl reonstrution of synpses viewed through seril eletron mirogrphs would e vlule 33. Possile tissue shrinkge during the emedding proess n e ruled out s explntion for these findings. First, the effet is synpse speifi. If there were generlized shrinkge in the enrihed nimls with no other hnge, then ll types of synpses should e ffeted uniformly. Seond, the mitohondril rosssetionl dimeters were uniform ross the nive nd enrihed onditions, showing tht no generlized shrinkge of individul proess ourred (dt not shown). DISCUSSION Here we exmined three kinds of nonsptil memory in mie lking NMDAR1 in the CA1 region. Our nlysis provides evidene supporting the importnt role of NMDA reeptor tivity in the CA1 region for the formtion of hippompus-dependent nonsptil memory. Tking into ount the defiit in sptil lerning in the mie 12, we onlude tht NMDA reeptor tivity in CA1 is essentil for the formtion of oth sptil nd nonsptil memory. More interestingly, we found tht the memory defiits ssoited with the disruption of the hippompl NMDA reeptor ould e resued y dily trining in n enrihed environment. We show tht ehviorl experiene n enhne lerning nd memory in the mutnt nimls. Thus, the memory defiits oserved in genti mutnt nimls re not neessrily irreversile, nd enrihed experiene ould promote reovery of some of the defiits. To investigte further the possile ellulr mehnisms underlying these enrihment effets, we systemtilly evluted the ntomil hnges using light nd eletron mirosopy. We showed tht enrihed experiene promotes signifint inrese in synpse density in the CA1 hippompl field. Beuse there is no neurogenesis in the CA1 region 26, the inresed synpse density is likely to reflet higher numer of synpses per neuron rther thn n inresed numer of pyrmidl ells in CA1. At the ultrstruturl level, we showed tht mie lking the NMDA reeptor in the CA1 region lso inrese synpti density following exposure to n enrihed environment. This suggests tht NMDA-reeptor tivity is not required for struturl plstiity in the CA1 region of dult nimls indued y ehviorl experiene, nd thus the moleulr mehnism underlying dult tivitydependent struturl plstiity is very different from mehnisms tive in the developing rin 17,18. As synpti plstiity n lso e indued in the CA1 region y n NMDA reeptor-independent proess 37,38 suh s LTP dependent on voltge-gted lium hnnels, other mehnisms might serve s puttive ndidtes for experiene-indued struturl plstiity in dult rin. It remins to e determined whether the enrihment-indued inrese in CA1 synpti density hs ny funtionl role in the onomitnt ehviorl effets. It is likely tht other rin regions my undergo similr iohemil nd struturl hnges fter enrihment, nd lso might prtiipte in the enrihment-indued ehviorl improvement. Enhned synpti oinidene detetion in the forerin of trnsgeni mie vi upregultion of suunit 2B of the NMDA reeptor lso leds to overll enhnement of lerning nd memory 10. Thus, hnges in the omposition (for instne, rtio of NR2A to NR2B suunits) of the NMDA reeptor omplex my e possile mehnism for the enrihmentindued effets. Tken together, these findings indite tht lerning nd memory might e enhned in mmmls y geneti ftors s well s ehviorl experiene. METHODS Animls. The nd ontrol mie were produed s desried 11. Throughout the ehviorl nd histologil experiments, oservers were lind to the genotype of n individul niml. In situ hyridiztion. Desried proedures 10 were used. Briefly, n ntisense 42-mer oligonuleotide proe (5 -ACCACTCTTTCTATC CTG CAG GTT CTT CCT CCA CAC GTT-3 ), whih reognizes NR1 exon 20, ws end leled with [ 35 S]-dATP. After hyridizing with the oligonuleotide proe ( pm per slide) t 47 C for 24 h, rin setions (20 µm) were wshed in 2 SSC t room temperture (RT) followed y two wshes in 0.2 SSC t 60 C nd one wsh in 0.1 SSC t RT. Interestingly, preliminry oservtions indited tht the deletion of the NR1 gene in six-month-old knokouts seemed to spred to other forerin regions suh s the ortex, proly refleting the reomintion threshold effet s the CMKII promoter-driven Cre umulted. Enrihed environment trining. Adult littermtes (1.5 2 months old) were rndomly distriuted into two experimentl groups. One group ws kept in stndrd ges (nive group) nd the other group trined in n enrihed environment for three hours dily for two months (enrihed group). The enrihment trining ws rried out in speilly designed oxes (1.5 m 0.8 m 0.8 m), in whih vrious toys, running wheels nd smll houses were hnged every other dy to enourge explortion. Food nd wter were lso ville in the oxes. Beuse eletrophysiologil, ehviorl nd ntomil experiments showed similr results for wild-type nd Cre trnsgeni mie, these two genotypes of mie were pooled together s ontrols. Novel-ojet reognition tsk nd fer-onditioning tsks. The experimentl protool ws the sme s desried previously 10, exept tht the durtion of the trining period in the ojet-reognition tsk ws 15 min. Soil trnsmission of food preferene tsk. In this experiment, oserver mie were tested for memory nd demonstrtor wild-type mie were used to intert with oservers ording to desried proedure 22. After intertion, the oservers were deprived of food for 24 h nd then tested for memory. During testing, oservers were offered oth innmon- nd oo-sented food for 2 h, nd onsumption of eh food ws susequently mesured. The preferred food hd the sme sent s food eten y the demonstrtor with whom the oserver interted. Preferene ws lulted s perentge rtio of the mount of preferred food onsumed over 50% of the totl mount of food onsumed. Histology. For eletron mirosopy, ontol groups were omposed of mles, months old. The nive mie were 3 mles (4.5, 4 nd 4 months) nd 1 femle (3.5 months), nd the enrihed mie were 3 mles (5, 4.5 nd 3.5 months) nd 1 femle (4 months). Beuse CA1 dendriti spine density in rts flututes over the estrous yle 39, femles were killed during proestrus, the stge t whih the spine numers of femle rts pproh those of mle rts (lthough it is not known whether the sme phenomenon exists in mie). Animls were nesthetized nd perfused with 2% prformldehyde, 2% glutrldehyde nd 1.5% sturted piri id in 0.1 M phosphte uffer (ph 7.4) nd postfixed overnight in the sme solution. Following modified version of the single-setion Golgi impregntion tehnique 32, 100 µm-thik oronl setions were ut with virtome into th of 3% potssium dihromte in distilled wter nd inuted overnight in this solution. The slide ssemlies were inuted in the drk in solution of 1.5% silver nitrte for 2 dys. For eletron mirosopy, we used virtome to ut 250 µm-thik oronl setions into th of 0.1 M sodium odylte uffer. Hippompl nture neurosiene volume 3 no 3 mrh

7 rtiles setions orresponding to figures of the mouse rin tls 40 were disseted, rinsed in distilled wter nd postfixed in 1% osmium tetroxide followed with 1% ferroynide-redued osmium tetroxide. Tissues were rinsed, stined en lo overnight in 1% queous urnyl ette, rinsed nd dehydrted nd susequently infiltrted nd flt emedded with Emed 812 resin. Setions (0.5 µm) were ut nd stined with 1% toluidine lue. Setions were exmined to determine how the lok should e trimmed for ultrthin setioning to inlude the pex of the CA1 pyrmidl ell lyer, from the strtum rditum to the strtum lunosum moleulre. Ultrthin setions were ut with dimond knife, pled on ron Formvroted slot grids nd stined with 1% urnyl ette nd led itrte. Setion thikness ws mesured y snning eletron mirosope mesurements to rnge from to µm, depending on the series. Dt nlysis: spine density. Golgi-impregnted dendriti segments seleted for nlysis were loted µm from the pyrmidl ell odies in the strtum rditum, elonged to thoroughly impregnted pyrmidl neuron, remined pproximtely in the plne of fous nd were >10 µm in length nd µm in width. Clultions of spine density were otined y mesuring dendriti segments mthing the ove riteri (ontrol enrihed n = 37, nive n = 38, enrihed n = 41 nd ontrol nive n = 43) from ge- nd sex-lned sets of 3 nimls of eh group. These segments represented totl dendriti length of µm per group of mie. Dt nlysis: synpse density. Synpse density ws estimted y the disetor method 34,35. From eh rin, 15 eletron mirogrphs of seprte regions within the strtum rditum, µm from the pyrmidl ell lyer, were tken from different setions t 10,000 on JEOL 100C eletron mirosope to rete 15 referene plnes. The mirogrphs overed n re of pproximtely 2100 µm 2 per niml. Mirogrphs of extly the sme regions were tken on n djent setion to rete look-up plnes. The numer of synpses ontined in referene plne ut sent in the orresponding look-up plne (Q i ) ws ounted to determine the numer of synpses present within the volume defined y the referene plne, the look-up plne nd the distne etween them (h i ). Two sides of the retngulr piture were ssigned rndomly s inlusion or exlusion edges to rete ounting frme tht minimized potentil edge effets ross smples 41. To inrese the smpling reliility, synpses were ounted over totl re of 8,400 14,700 µm 2 from 600 eletron mirogrphs for eh experimentl group of mie. Setion thikness (h i ) ws determined y snning eletron mirosope mesurements (see Histology). Estimted synpse density (est N v ) ws lulted s est N v = ΣQ i / Σ (A i h i ) Note: Additionl methods detils n e found on the Nture Neurosiene we site ( ACKNOWLEDGEMENTS We thnk E. Gould for help with stereology nd sttistis nd reding the mnusript, nd P. Tnpt for tehnil dvie onerning the Golgi method. This work ws supported in prt y postdotorl fellowship from Fondtion pour l Reherhe Medile to C.R. nd y grnts from Prineton University, Bekmn Foundtion nd NIH to J.Z.T. RECEIVED 12 OCTOBER; ACCEPTED 28 DECEMBER Rosene, D. L. & Vn Hoesen, G. W. in Cererl Cortex Vol. 6: Further Aspets of Cortil Funtion, Inluding Hippompus (eds. Jones, E. G. & Petes, A.) (Plenum, New York, 1987). 2. Soville, W. B. & Milner, B. Loss of reent memory fter ilterl hippompl lesion. J. Neurol. Neurosurg. Psyhitry 20, (1957). 3. Zol-Morgn, S., Squire, L. R. & Amrl, D. G. Humn mnesi nd the medil temporl region: enduring memory impirment following ilterl lesion limited to field CA1 of the hippompus. J. Neurosi. 6, (1986). 4. Squire L. R. Memory nd the hippompus: synthesis from findings with rts, monkeys, nd humns. Psyhol. Rev. 99, (1992). 5. Mumy, D. G. et l. Ishemi-indued ojet-reognition defiits in rts re ttenuted y hippompl ltion efore or soon fter ishemi. Behv. Neurosi. 110, (1996). 6. Wood, E. R., Dudhenko, P. A. & Eihenum, H. The glol reord of memory in hippompl neuronl tivity. Nture 397, (1999). 7. Moriyoshi, K. et l. Moleulr loning nd hrteriztion of the rt NMDA reeptor. Nture 354, (1991). 8. Nioll, R. A. & Mlenk, R. C. Expression mehnisms underlying NMDA reeptor-dependent long-term potentition. Ann. NY Ad. Si. 868, (1999). 9. Ber, M. F. & Mlenk, R. C. Synpti plstiity: LTP nd LTD. Curr. Opin. Neuroiol. 4, (1994). 10. Tng, Y.-P. et l. Geneti enhnement of lerning nd memory in mie. Nture 401, (1999). 11. Tsien, J. Z. et l. Suregion- nd ell type-restrited gene knokout in mouse rin. Cell 87, (1996). 12. Tsien, J. Z., Huert, P. T. & Tonegw, S. The essentil role of hippompl CA1 NMDA reeptor-dependent synpti plstiity in sptil memory. Cell 87, (1996). 13. MHugh, T. J., Blum, K. I., Tsien, J. Z., Tonegw, S. & Wilson, M. A. Impired hippompl representtion of spe in CA1-speifi NMDAR1 knokout mie. Cell 87, (1996). 14. Woolf, N. J. A struturl sis for memory storge in mmmls. Prog. Neuroiol. 55, (1998). 15. Biley, C. H. & Kndel, E. R. Struturl hnges ompnying memory storge. Annu. Rev. Physiol. 55, (1993). 16. Moser, M. B., Trommld, M. & Andersen, P. An inrese in dendriti spine density on hippompl CA1 pyrmidl ells following sptil lerning in dult rts suggests the formtion of new synpses. Pro. Ntl. Ad. Si. USA 91, (1994). 17. Goodmn, C. S. & Shtz, C. J. Developmentl mehnisms tht generte preise ptterns of neuronl onnetivity. Cell 72, Suppl (1993). 18. Li, Y., Erzurumlu, R. S., Chen, C., Jhveri, S. & Tonegw, S. Whisker-relted neuronl ptterns fil to develop in the trigeminl rinstem nulei of NMDAR1 knokout mie. Cell 76, (1994). 19. Kirov, S. A. & Hrris, K. M. Dendrites re more spiny on mture hippompl neurons when synpses re intivted. Nt. Neurosi. 10, (1999). 20. Sorr, K. E. & Hrris, K. M. Stility in synpse numer nd size t 2 hr fter long-term potentition in hippompl re CA1. J. Neurosi. 18, (1998). 21. Mnsuy, I. M., Myford, M., Jo, B., Kndel, E. R. & Bh, M. E. Restrited nd regulted overexpression revels lineurin s key omponent in the trnsition from short-term to long-term memory. Cell 92, (1998). 22. Kogn, J. H. et l. Sped trining indues norml long-term memory in CREB mutnt mie. Curr. Biol. 7, 1 11 (1996). 23. Strupp, B. J. & Levitsky, D. A. Soil trnsmission of food preferenes in dult hooded rts (Rttus norvegius). J. Comp. Psyhol. 98, (1984). 24. Bunsey, M. & Eihenum, H. Seletive dmge to the hippompl region loks long-term retention of nturl nd nonsptil stimulus-stimulus ssoition. Hippompus 5, (1995). 25. Phillips, R. G. & LeDoux, J. E. Differentil ontriution of mygdl nd hippompus to ued nd ontextul fer onditioning. Behv. Neurosi. 106, (1992). 26. Kempermnn, G., Kuhn, G. H. & Gge, F. H. More hippompl neurons in dult mie living in n enrihed environment. Nture 386, (1997). 27. Dlrymple-Alford, J. C. & Benton, D. Preopertive differentil housing nd dorsl hippompl lesions in rts. Behv. Neurosi. 98, (1984). 28. Rosenzweig, M. R. Environmentl omplexity, ererl hnge, nd ehvior. Am. Psyhol. 21, (1966). 29. Dimond, M. C. Enrihing Heredity: The Impt of the Environment on the Antomy of the Brin (Free Press, New York, 1988). 30. Fil, B. A., Joye, J. N. & Greenough, W. T. Environmentl omplexity modultes growth of grnule ell dendrites in developing ut not dult hippompus of rts. Exp. Neurol. 59, (1978). 31. Greenough, W. T., Withers, G. S. & Wlle, C. S. in The Biology of Memory (eds. Squire, L. R. & Lindenum, E.) (Shttuer, Stuttgrt, 1990). 32. Gott, P. L & Somogyi, J. The single Golgi impregntion proedure: methodologil desription. J. Neurosi. Methods 11, (1984). 33. Sorr, K. E., Fil, J. C. & Hrris, K. M. Critil ssessment of the involvement of perfortions, spinules, nd spine rnhing in hippompl synpse formtion. J. Comp. Neurol. 398, (1998). 34. Sterio, D. C. The unised estimtion of numer nd sizes of prtiles using the disetor. J. Miros. 134, (1984). 35. DeGroot, D. M. G. & Biermn, E. P. B. A ritil evlution of methods for estimting the numeril density of synpses. J. Neurosi. Methods 18, (1986). 36. Gry, E. G. Axosomti nd xodendriti synpses of the ererl ortex: An eletron mirosopi study. J. Ant. 83, (1959). 37. Nioll, R. A. & Mlenk, R. C. Contrsting properties of two forms of longterm potentition in the hippompus. Nture 377, (1995). 38. Stevens, C. F. & Sullivn, J. Synpti plstiity. Curr. Biol. 8, R (1998). 39. Woolley, C. S., Gould, E., Frnkfurt, M. & MEwen, B. S. Nturlly ourring flutution in dendriti spine density on dult hippompl pyrmidl neurons. J. Neurosi. 10, (1990). 40. Frnklin, K. B. J. & Pxinos, G. The mouse rin in stereotxi oordintes. (Ademi, Sn Diego, 1997). 41. Gundersen, H. J. G. Notes on the estimtion of the numeril density of ritrry profiles: the edge effet. J. Miros. 111, (1977). 244 nture neurosiene volume 3 no 3 mrh 2000