rtiles A new form of long-term epression in the perirhinl ortex K. Cho 1, N. Kemp 1, J. Noel 1,2, J. P. Aggleton 3, M. W. Brown 1 n Z. I. Bshir 1 1 MRC Centre for Synpti Plstiity, Dept. of Antomy, University of Bristol, Bristol BS8 1TD, UK 2 Present ress: Biologie ellulire es omprtiments liques, Universite e Nie-Sophi Antipolis, Fulte es sienes, 06108 Nie, Frne 3 Shool of Psyhology, Criff University, Criff CF10 3YJ, UK Corresponene shoul e resse to Z.I.B. (z.i.shir@ris..uk) We emonstrte form of long-term epression (LTD) in the perirhinl ortex tht relies on intertion etween ifferent glutmte reeptors. Group II metotropi glutmte (mglu) reeptors filitte group I mglu reeptor-meite inreses in intrellulr lium. This filittion plus NMDA reeptor tivtion my e neessry for inution of LTD t resting memrne potentils. However, epolriztion enhne NMDA reeptor funtion n remove the requirement of synergy etween group I n group II mglu reeptors: uner these onitions, tivtion of only NMDA n group I mglu reeptors ws require for LTD. Suh glutmte reeptor intertions potentilly provie new rules for synpti plstiity. These forms of LTD our in the perirhinl ortex, where long-term ereses in neuronl responsiveness my meite reognition memory. There re goo resons for elieving tht ereses in synpti effiy in neurons of the perirhinl ortex, the region of temporl ortex jent to the rhinl sulus, re relte to reognition memory 1. Lesions of perirhinl ortex in rts n primtes impir performne of reognition-memory tsks 2 4. Importntly, perirhinl neuronl responses to novel visul stimuli erese mrkely one suh stimuli eome fmilir 1,5,6. The erement in responsiveness n lst mny hours, n this hnge my provie the informtion require to solve suh tsks. Ativity-epenent long-term epression of synpti trnsmission in perirhinl ortex provies potentil mehnism for explining ereses in neuronl responsiveness. Inution of homosynpti LTD epens on postsynpti inreses in lium 7 9 rought out y ifferent mehnisms tht inlue tivtion of NMDA reeptors 10 12 or mglu reeptors 13 17. However, there is no eviene for role of mglu reeptor tivtion uner onitions in whih NMDA reeptor tivtion unerlies the inution of tivity-epenent LTD. Furthermore, there seems to e no role for NMDA reeptor tivtion when mglu reeptors re involve in the inution of tivity-epenent LTD. Group I mglu reeptors n NMDA reeptors use istint forms of LTD y oupling to the tivtion of protein kinse C n protein phosphtses, respetively 14 16,18. An lterntive potentil mens of LTD inution y group I mglu reeptor tivtion is through phosphoinositieinue relese of lium from intrellulr stores 19 22. A presynpti role for group II mglu reeptors in LTD t mossy fier synpses 23,24 seems to epen on AMP turnover 25, ut the mehnisms unerlying the role of group II mglu reeptors in other regions re not well unerstoo 26 28. Perirhinl ortex is ritilly involve in numer of ifferent types of memory. NMDA reeptor-epenent LTP 29,30 n e inue in perirhinl ortex in vitro; in ition, investigting mehnisms of LTD my provie insights into funmentl mehnisms of lerning n memory in this n other regions of ortex. Here we esrie onitions for the inution of tivity-epenent LTD in ult perirhinl ortex. We emonstrte new form of LTD in this ortex tht relies on the tivtion of NMDA n group I n group II mglu reeptors. RESULTS In the perirhinl ortex, stimultion elivere to either sie of the rhinl sulus in lyers II/III resulte in synpti trnsmission tht ws epenent on AMPA/kinte n NMDA reeptors 30 (Fig. 1). Low-frequeny stimultion (LFS; 200 stimuli, 1 Hz) elivere to either the entorhinl or the temporl sie of the rhinl sulus omine with epolriztion of the postsynpti neuron to 40 mv inue roust homosynpti LTD mesure 30 35 minutes fter stimultion. No ifferenes were foun etween the mgnitue of LTD with stimultion to the entorhinl or the temporl ortex sie (p > 0.05); therefore, t from the two inputs were poole (men epression of 47 ± 8%, n = 10; Fig. 2) in this n susequent experiments. Voltge lmp of neurons t 40 mv for 200 s without LFS i not result in LTD (+1 ± 8%; n = 8, p > 0.05; t not shown). LTD ws lso inue y LFS elivere while the postsynpti ell ws voltge lmpe t 70 mv (epression of 37 ± 8%, n = 8; Fig. 2). LTD inue y either of the ove protools ws mintine for s long s the reoring ws ontinue (up to 180 min; t not shown). The NMDA reeptor ntgonist AP5 (50 µm) loke the inution of LTD mesure 30 35 min fter LFS elivere t 40 mv (2 ± 5%, n = 7, p > 0.05; Fig. 2). In three experiments, AP5 ws wshe out, n susequent LFS resulte in LTD (52 ± 11%, n = 3, p < 0.05; Fig. 2). In seprte series of experiments, AP5 loke LTD t 70 mv (1 ± 5%, n = 7; p > 0.05; Fig. 2) in reversile mnner (48 ± 9%, p < 0.05, n = 3; Fig. 2). In keeping with the voltge epenene of NMDA reeptor-meite synpti trnsmission, LTD ws prevente y hyperpolrizing neurons to 90 mv (7 ± 6%, n = 3, not shown) or 110 mv (3 ± 11%, n = 3, not shown). To etermine if postsynpti rise 150 nture neurosiene volume 3 no 2 ferury 2000
rtiles Fig. 1. The perirhinl ortex. () Lterl view of the rt rin (from ref. 44) initing the position of the perirhinl ortex (res 35 n 36) to either sie of the rhinl sulus. Approximte ngles of in vitro slies re illustrte y igonl lines. () Representtion of the perirhinl ortex slie initing eletroe positions. Whole-ell reorings (Re) were otine from single neurons in lyer II/III. Stimulting eletroes (S1 n S2) were positione in lyer II/III either sie (temporl n entorhinl) of the reoring eletroe. () Synpti EPSCs in perirhinl ortex neurons voltge lmpe t 70 mv rely on tivtion of AMPA/kinte n NMDA reeptors, s emonstrte y the effets of NBQX n AP5 (ref. 30). in lium ws neessry for LTD, we use whole-ell filling solution ontining 10 mm EGTA. Uner these reoring onitions, inution of LTD ws prevente when LFS ws elivere t either 40 or 70 mv (Fig. 2e n f). To etermine if metotropi glutmte (mglu) reeptors hve role in LTD, we use the ro-spetrum (group I/II) mglu reeptor ntgonist MCPG 31,32. MCPG (500 µm) prevente the inution of LTD when LFS ws elivere t 40 mv (Fig. 3; 1 ± 6%, n = 7, p > 0.05). Following MCPG wshout in 3 experiments, LFS resulte in LTD (47 ± 7%, n = 3, p < 0.05; Fig. 3). MCPG lso loke inution of LTD t 70 mv (Fig. 3; 1 ± 9%, n = 7, p > 0.05) in reversile mnner (46 ± 6%, n = 3, p < 0.05; Fig. 3). The group I mglu reeptor ntgonist AIDA 33,34 loke LTD t 40 mv (Fig. 3; +1 ± 7%, n = 7, p > 0.05) in reversile mnner (35 ± 7%, n = 3, p < 0.05). At 70 mv, AIDA lso proue lok of LTD inution (Fig. 3; 1 ± 11%, n = 6, p > 0.05) tht ws reversile (35 ± 10%, n = 2). MAP4, group III mglu reeptor ntgonist 35, file to prevent LTD t either 40 or 70 mv (43 ± 11%, n 41 ± 10%, respetively, n = 4, p < 0.05 for eh; Fig. 3e n f). These results suggest tht oth group I mglu reeptors n NMDA reeptors re require for the inution of LTD. This result ws unexpete euse in previously reporte ses of tivity-epenent LTD, inution relies on either NMDA or mglu reeptors ut not on tivtion of oth. The tivtion of group II metotropi glutmte (mglu) reeptors y pplition of DCG-IV n result in long-lsting epression of fiel EPSPs in perirhinl ortex 36. We investigte whether synpti tivtion of group II mglu reeptors hs role in LTD. The group II mglu reeptor ntgonist EGLU 37 i not lok the inution of LTD when LFS ws elivere t 40 mv (epression of 40 ± 9%, n = 4; p < 0.01, Fig. 4). Surprisingly, however, EGLU loke LTD when LFS ws elivere t 70 mv (epression of 3 ± 7%, n =7; p > 0.05; Fig. 4). In 3 experiments, EGLU ws wshe out, n susequent LFS inue LTD (46 ± 12%, n = 3, p < 0.05; Fig. 4). LTD ws lso loke y the group II/III mglu reeptor ntgonist CPPG (200 µm) when LFS ws elivere t 70 mv (2 ± 10% in CPPG n 47 ± 14% following wshout; n = 3; t not shown). Beuse the role of group II mglu reeptors in LTD epene on memrne potentil, we investigte whether group II mglu reeptors were involve in LTD uner protools in whih the memrne potentil ws not voltge lmpe, thus more losely pproximting onitions in vivo. LFS ws elivere while the postsynpti neuron ws hel in urrent lmp t 70 mv, vlue lose to norml resting potentil. With the memrne potentil free to hnge uring LFS, LTD ws still relily inue (epression of 39 ± 7%, n = 3; p < 0.05, Fig. 4). Interestingly, uner these onitions LTD still epene on the tivtion of group II mglu reeptors, s LTD ws loke y EGLU (3 ± 14%, n = 3; p > 0.05, Fig. 4). The voltge epenene of involvement of group II mglu reeptors in LTD ws unexpete. We resone tht group II mglu reeptors might e importnt uring erese NMDA reeptor tivtion. Therefore, their involvement might relte to voltge epenene of NMDA reeptor tivtion, n not to voltge epenene of group II mglu reeptors per se. If this were the se, inresing NMDA reeptor tivtion n lium influx t 70 mv shoul remove the requirement for group II mglu reeptors in LTD inution. In keeping with this suggestion, when extrellulr lium ws inrese (from 2 to 4 mm), LTD inue t 70 mv ws not loke y EGLU (63 ± 7%, n = 3; p < 0.01, Fig. 5). When extrellulr mgnesium ws reue (from 1 to 0.01 mm) to enhne NMDA reeptor funtion, LTD inue t 70 mv ws not loke y EGLU (41 ± 9% epression, n = 3; p < 0.01, Fig. 5). Thus enhning NMDA reeptor tivtion removes group II mglu reeptor involvement in LTD. Conversely, eresing NMDA reeptor tivtion t 40 mv might proue requirement for group II mglu reeptor tivtion in LTD t epolrize memrne potentils. In low onentrtion of AP5 (2 µm), LTD ws inue y piring LFS with epolriztion of the postsynpti neuron to 40 mv. LTD inue t 40 mv uner these onitions ws loke y EGLU (+6 ± 10%, mesure 10 min fter LFS; n = 3; p > 0.05, Fig. 5 n ). These results suggest tht the levels of NMDA reeptor tivtion etermine the requirement of group II mglu reeptors in LTD. One explntion for these results is tht oopertivity etween group I n II mglu reeptors n NMDA reeptors my e require to inrese lium levels for inution of LTD t resting memrne potentils. Beuse LTD is loke uring ntgonism of either NMDA or group I mglu reeptors n euse group II mglu reeptors o not iretly ouple to lium signling, it is possile tht group II mglu reeptors intert with nture neurosiene volume 3 no 2 ferury 2000 151
rtiles e f Fig. 2. LTD relies on tivtion of NMDA reeptors n postsynpti inrese in lium. () LFS pire with epolriztion to 40 mv results in LTD in the input reeiving LFS ut not in the ontrol input (not shown). () Delivering LFS with the neuron voltge lmpe t 70 mv lso results in homosynpti LTD, with no effet on the ontrol input (not shown). () LTD is loke y the NMDA reeptor ntgonist AP5 when LFS is pire with epolriztion to 40 mv. LFS following AP5 wshout in three of these experiments inue LTD. () LTD ws loke y AP5 when LFS ws elivere t 70 mv. In three experiments, LFS inue LTD fter AP5 ws wshe out. (e, f) The lium heltor EGTA (10 mm) loks the inution of LTD 20 25 min fter LFS t oth 40 mv (e) n 70 mv (f). The ontrol onentrtion of EGTA (0.5 mm) h no effet on LTD in interleve ontrol experiments (not shown). Illustrte synpti responses re tken from pproprite time points, s inite (1, 2). The ontrol n epresse responses (n voltge steps; 5 mv) re lso shown superimpose (1, 2). The perio of LFS (200 stimuli, 1 Hz) is inite y the horizontl r joine y the two rrows. In this n susequent figures, fille irles inite experiments in whih the neuron ws epolrize to 40 mv only for the urtion of LFS. Open irles inite experiments in whih LFS ws elivere while the neuron ws voltge lmpe t 70 mv. Breks in the x xes (, ) inite the time t whih AP5 ws wshe out. NMDA or group I mglu reeptors. We emonstrte synergisti intertion etween group II n group I mglu reeptors (Fig. 6). At 70 mv, the inwr urrent proue y the group I mglu reeptor gonist DHPG 37 ws signifintly (p < 0.05) inrese y the group II mglu reeptor gonist DCG-IV (Fig. 6; 15 ± 6 without DCG-IV; 59 ± 17 pa with DCG-IV, n = 4). Beuse DCG-IV ts s n gonist t NMDA reeptors 38 (leit t higher onentrtions thn 0.5 µm), these experiments were rrie out in the presene of 50 µm AP5. In ontrol experiments without DCG-IV, seon pplition of DHPG proue inwr urrents of similr mgnitues to tht proue y first pplition of DHPG (t not shown). To investigte how intertion etween group I n group II mglu reeptors might filitte LTD, we monitore intrellulr lium levels. In ulture perirhinl ortex neurons, DHPG (20 µm) inrese fluoresene of Fluo-3-AM fille neurons (21 ± 6% inrese; n = 16 neurons). This ws signifintly (p < 0.05) enhne (45 ± 10%; n = 16, Fig. 6 n ) y o-pplition of DHPG with the group II mglu reeptor gonist DCG-IV (1 µm). DCG-IV itself h no effet on fluoresene levels (Fig. 6). In seprte experiments (Fig. 6 n e), the enhnement y DCG-IV of DHPG-inue lium moiliztion (44 ± 7%; n = 23 neurons) ws reversily loke y the group II mglu reeptor ntgonist EGLU (15 ± 4%; n = 23 neurons). Therefore, group II n group I mglu reeptors n intert to inrese intrellulr lium levels in neurons of the perirhinl ortex. We postulte tht this synergisti inrese in lium my unerlie the oserve inution of LTD t resting memrne potentils. DISCUSSION This stuy esries n tivity-epenent LTD in the ult perirhinl ortex. This fining is signifint euse this LTD my reflet mehnisms unerlying reognition memory-relte ereses in neuronl responses in perirhinl ortex in vivo 1,5,6. The mehnisms of homosynpti tivity-epenent LTD in the perirhinl ortex re notle euse these epen on synpti tivtion of oth NMDA reeptors n mglu reeptors, in mrke ontrst to previously esrie forms of tivity-epenent LTD tht epen either on the tivtion of NMDA reep- 152 nture neurosiene volume 3 no 2 ferury 2000
ever, t resting memrne potentils, when lium influx through NMDA reeptor hnnels is restrite, the itionl tivtion of group I mglu reeptors is insuffiient for LTD. Uner these onitions, inution of LTD requires tivtion of group II mglu reeptors. It is not known if this mehnism onfers unique properties on perirhinl ortil plstiity or if it lso ours in wiespre ortil regions. Thus, lthough group II mglu reeptors hve role in LTD in other rin regions 26 28, it is not known if this lso ours t resting memrne potentils, s reporte here. Although tivtion of postsynpti group II mglu reeptors proly explins our results, the possiility tht presynpti group II mglu reeptors lso hve some role in LTD nnot e rule out 23,24. It is interesting to note tht the mglu reeptor ntgonists MCPG n EGLU reue the epression of trnsmission uring LFS, n effet more evient when LFS ws elivere t 70 mv. This suggests tht synpti tivtion of mglu reeptors my trnsiently erese synpti trnsmission, possily vi presynpti mehnism, s hs een emonstrte t mossy fier synpses 39. At 40 mv, the erese riving fore lso results in EPSP epression, mking it iffiult to etermine the effets of mglu reeptor ntgonists on epression uring LFS. Group II mglu reeptors re lote oth pre- n postsynptilly n re known to erese forskolin-stimulte AMP turnover 23,24. However, we provie eviene tht the tivtion of group II mglu reeptors n inrese group I mglu reeptormeite lium moiliztion. This result extens previous work 40,41 showing tht group II mglu reeptors inrese group I mglu reeptor-epenent phosphoinositie turnover. The preise mehnisms unerlying the synergy etween these groups of mglu reeptors remin to e ientifie. However, this interrtiles e f Fig. 3. LTD epens on group I ut not group III mglu reeptor tivtion. () LTD is loke y the mglu reeptor ntgonist MCPG when LFS is elivere t 40 mv. In three experiments, MCPG ws wshe out, n LTD ws susequently inue. () LTD ws loke y MCPG t 70 mv; in 3 experiments, LFS resulte in LTD following wshout. () The group I mglu reeptor ntgonist AIDA prevente the inution of LTD t 40 mv; in three experiments, LFS fter AIDA wshout resulte in LTD. () LTD ws loke y AIDA t 70 mv. In two experiments, AIDA ws wshe out n LTD susequently inue y LFS. (e, f) LTD ws not loke y MAP4 either t 40 mv (e) or t 70 mv (f). tors 10 12 or on the tivtion of mglu reeptors 13,14,16,17, ut not on the onjoint tivtion of oth. Inee, istint NMDA n mglu reeptor-epenent forms of LTD oexist in the CA1 region of the hippompus 15. Although we hve not investigte iohemil mehnisms of perirhinl LTD, NMDA reeptor-epenent LTD relies on protein phosphtse tivtion 18, wheres mglu reeptor-epenent LTD epens on protein kinse C tivtion 14,16 in the hippompus. The onjoint tivtion of NMDA n mglu reeptors provies n itionl rule for the inution of synpti plstiity. The ererl ortex n potentilly exploit the vriety of suh rules, either to effet ifferent types of memory or to inrese the onitions for memory storge. The most intriguing fining is tht Group II mglu reeptors re involve in LTD t resting memrne potentils ut not t epolrize potentils. Our results inite tht the role for group II mglu reeptors epens on the level of NMDA reeptor tivtion, rther thn memrne potentil per se. We propose tht t epolrize potentils, when NMDA reeptor tivtion is inrese, o-tivtion of NMDA n group I mglu reeptors is suffiient n neessry to trigger the inution of LTD. How- nture neurosiene volume 3 no 2 ferury 2000 153
rtiles Fig. 4. The role of group II mglu reeptors in LTD is voltge epenent. () The group II mglu reeptor ntgonist EGLU i not lok the inution of LTD when LFS ws elivere t 40 mv. () However, when LFS ws elivere t 70 mv, LTD ws loke y EGLU ut oul e inue in three experiments following wshout. () Delivering LFS to the neuron in urrent lmp (t 70 mv) results in LTD inution tht is lso loke y the group II mglu reeptor ntgonist EGLU (). tion my our iretly etween Gβγ suunits (tivte y group II mglu reeptors) n G q/11 α (tivte y group I mglu reeptors) 40 rther thn involve group II mglu reeptor-meite hnges in AMP turnover 41. Whtever the unerlying mehnism, our results emonstrte tht this intertion inreses intrellulr lium. Given the essentil role of lium in the inution of synpti plstiity, this synergy my e require for LTD in the perirhinl ortex t resting memrne potentils. urrent lmp urrent lmp Therefore, these results suggest mehnism for the requirement of synpti tivtion of group II mglu reeptors in LTD. Our results emonstrte tht LTD in the perirhinl ortex n epen on severl ifferent types of glutmte reeptor, thus rising the question s to the physiologil relevne of vrious meh- Fig. 5. The role of group II mglu reeptors in LTD is influene y the level of NMDA reeptor tivtion. (, ) EGLU oes not lok LTD t 70 mv in 4 mm extrellulr C 2+ () or in 0.01 mm extrellulr Mg 2+ (). (, ) Single exmple () n poole t () show tht EGLU prevents the inution of LTD t 40 mv when NMDA reeptor tivtion is erese y 2 µm AP5. Inution of LTD ws not loke y this onentrtion of AP5 lone (). 154 nture neurosiene volume 3 no 2 ferury 2000
rtiles Fig. 6. Group II mglu reeptor tivtion y DCG-IV enhnes group I mglu reeptor (DHPG)-inue inwr urrent n lium moiliztion. () DHPG inue n inwr urrent tht ws signifintly enhne y DCG-IV. To prevent ny gonist effet of DCG-IV t NMDA reeptors, we use low onentrtion of DCG-IV (0.5 µm) in the presene of 50 µm AP5. () Single exmple of fluoresene inrese in ulture perirhinl ortex neuron in response to pplition of 20 µm DHPG. The lium rise in response to DHPG is synergistilly inrese in reversile mnner y the opplition of DCG-IV. () Single exmple illustrtes reversile lok y EGLU of DCG-IV s enhnement of DHPG-inue fluoresene inrese. () Poole t illustrte tht DHPGinue lium moiliztion ws signifintly enhne y DCG-IV. (e) Poole t show tht DCG-IV s enhnement of the DHPG response is loke y EGLU. Neither EGLU nor DCG-IV lone h ny effet on fluoresene. nisms. One possiility is tht the erese neuronl tivity with stimulus repetition in perirhinl ortex 1 rises from vriety of fferent-tivtion ptterns ppropritely enoe y the vrious synpti n moleulr mehnisms we ientifie. It is lso possile tht the ifferent mehnisms unerlying synpti plstiity re neessry for the vriety of forms of lerning n memory involving neurons in perirhinl ortex 1 4. In onlusion, the results of this stuy ientify new mehnisms of tivity-epenent synpti plstiity tht rely on funtionl intertion etween ifferent lsses of synptilly tivte glutmte reeptors. METHODS Eletrophysiology. Slies of perirhinl ortex were prepre from ult mle DA rts (150 270 g, 7 12 weeks, Bntin n Kingmn, UK). All efforts were me to minimize numers of nimls use. Animls were nesthetize with hlothne, epitte in orne with the UK Animls (Sientifi Proeures) At 1986, the rin rpily remove n ple in ie-ol rtifiil ererospinl flui (CSF; ule with 95% 0 2 /5% CO 2 ), whih omprise 124 mm NCl, 3 mm KCl, 26 mm NHCO 3, 1.25 mm NH 2 PO 4, 2 mm CCl 2, 1 mm MgSO 4 n 10 mm D-gluose. A misgittl setion of the rin ws me, the rostrl n ul prts were remove y single slpel uts pproximtely 45 to the orsoventrl xis, n eh hlf ws glue y its ul en to viroslie stge (Cmpen Instruments, Siley, UK). Slies (400 µm) tht inlue perirhinl, entorhinl n temporl orties were store sumerge in CSF (20 25 C). A single slie ws ple in sumerge reoring hmer (28 30 C; flow rte, 2 ml per min) when require. Pirotoxin (5 µm) ws present throughout the experiment. Blin whole-ell reorings were otine from neurons in lyer II/III. Pipet (4 7 MΩ) solutions (280 mosm, ph 7.2) omprise 130 mm esium methyl sulfte, 8 mm NCl, 4 mm Mg-ATP, 0.3 mm N-GTP, 0.5 mm EGTA, 10 mm HEPES n 6 mm QX-314. In some experiments, potssium methyl sulfte ws sustitute for esium methyl sulfte, n QX-314 ws omitte. High-EGTA filling solution ontine 10 mm EGTA, sustitute for equimolr esium methyl sulfte to mintin osmolrity. One stimulting eletroe ws ple orsorostrlly on the temporl ortex sie (re 36) n one ventroully on the entorhinl ortex sie (re 35/entorhinl ortex) of the rhinl sulus. Stimuli (onstnt voltge) were elivere lterntely to the two eletroes (eh eletroe 0.033 Hz). Neurons were voltge lmpe t 70 mv unless otherwise inite. To inue LTD in eh slie, lowfrequeny stimultion (LFS; 200 stimuli, 1 Hz) ws elivere to only one input. LFS ws elivere t 70 mv, pire with epolriztion to 40 mv or elivere uner urrent lmp t 70 mv, s pproprite for ifferent experiments. Altertions of memrne potentil were me only uring the LFS. Amplitues of the evoke EPSCs were mesure n Fluoresene ( % of seline) Fluoresene Chnge in holing urrent (pa) Time (s) Fluoresene e Fluoresene Time (s) expresse reltive to the normlize pre-onitioning seline. Effets of LFS were mesure t pproprite time points (verge over five min) fter elivering LFS. Dt were nlyze from only one slie per rt unless otherwise inite. Dt poole ross slies re expresse s mens ± s.e. n signifine (p < 0.05) teste using either pire or unpire t-tests s pproprite. Only experiments in whih there ws little seline rift (<10%) were inlue in the poole t. Dt were reore using n Axopth 200 mplifier (Axon Instruments, Foster City, Cliforni), monitore n nlyze on line n re-nlyze off line (W.W. Anerson, G. L. Collingrige, So. Neurosi. Astr. 23, 665, 1997). Agonists n ntgonists were pplie y ition to the perfuste. Cell ulture. Perirhinl ultures were prepre oring to esrie methos 42 for preprtion of hippompl ultures. Briefly, the perirhinl ortex ws remove from rts (three to five ys ol) n neurons reovere y enzymti igestion n mehnil issoition. Cells were plte onto overslips in 35-mm petri ishes. Cultures were mintine t 37 C in 95% O 2 /5% CO 2 -humiifie inutor. The ulture mei ws ompose of miniml essentil mei (Gio; 30 mm gluose, 2 mm glutmine; 15 mm HEPES, 100 µg per ml ovine trnsferrin n 30 µg per ml insulin, omplemente with 5 10% fetl lf serum). From the seon y in ulture, the mei were supplemente with ytosine-β-drinofurnosie (2.5 µm). Neurons were use for lium-imging stuies 14 30 ys fter plting. Clium imging. Imging tehniques were use s esrie previously 32,43.Briefly, ells were wshe three times in HBS uffer (119 mm NCl, 5 mm KCl, 25 mm HEPES, 33 mm gluose, 2 mm CCl 2, 2 mm MgCl 2, 500 nm TTX, 1 µm glyine, 100 µm pirotoxin, ph 7.4; osmolrity, nture neurosiene volume 3 no 2 ferury 2000 155
rtiles 300 310 mosm) n loe with 5 µm of the memrne-permele C 2+ initor fluo-3-am me up in 1 mg per ml ovine serum lumin/hbs t 37 C for 20 min. Cells were then wshe 3 times in HBS n inute for 30 min in 5% CO 2 tmosphere t 22 C to llow for the e-esterifition of the flurophore. Cells were then viewe on BioR (Hemel Hempste, UK) MRC600 onfol mirosope equippe with n rgon ion lser using stnr green filter sets n perfuse ontinuously t 2 ml per min with HBS uffer to whih AP5 (50 µm) ws e. Agonists were th pplie. Integrtions of five iniviul imges were otine every 10 s efore, uring n fter gonist pplition. In six of seven over slips use in this stuy, DHPG-inue fluoresene ws signifintly enhne y DCG- IV. All neurons from these six over slips tht initilly respone to DHPG with n inrese in fluoresene were use in susequent nlysis. The fluoresene of iniviul ells in eh preprtion ws mesure using puli-omin NIH progrm (http://rs.info.nih.gov/nihimge/) n expresse reltive to seline. The men pek fluoresene ws then lulte. Drugs (ll from Toris, Bristol, UK) use were the N-methyl-D-sprtte (NMDA) reeptor ntgonist D-2-mino-5-phosphonopentnote (AP5), the metotropi glutmte (mglu) reeptor ntgonists (S)-αmethyl-4-roxyphenylglyine (MCPG) (R,S)-1-minoinn-1,5-iroxyli i (AIDA) (S)-2-mino-2-methyl-4-phosphonoutnoi i (MAP4) n (2S)-α-ethylglutmi i (EGLU) n the mglu reeptor gonists (R,S)-3,5-ihyroxyphenylglyine (DHPG) n (2S,2 R,3 R)- 2-(2,3 -iroxyylopropyl)glyine (DCG-IV). ACKNOWLEDGEMENTS We thnk A. Doherty, L. Pikr n V. Collett for help with ell ulture n lium imging n G.L. Collingrige for isussions. This work ws supporte y the BBSRC, MRC n Wellome Trust. RECEIVED 29 OCTOBER, ACCEPTED 23 NOVEMBER 1999 1. Brown, M. W. & Xing, J.-Z. Reognition memory: neuronl sustrtes of the jugement of prior ourrene. Prog. Neuroiol. 55, 149 189 (1998). 2. Meunier, M., Bhevlier, J., Mishkin, M. & Murry, B. 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