Nuclear calcium signaling controls CREB-mediated gene expression triggered by synaptic activity

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1 rticles Nucler clcium signling controls CREB-medited gene expression triggered y synptic ctivity Giles E. Hrdinghm, Fion J. L. Arnold nd Hilmr Bding MRC Lortory of Moleculr Biology, Hills Rod, Cmridge CB2 2QH, Englnd Correspondence should e ddressed to H.B. (h1@mrc-lm.cm.c.uk) Informtion storge in the nervous system requires trnscription triggered y synpticlly evoked clcium signls. It hs een suggested tht trnsloction of clmodulin into the nucleus, initited y sumemrnous clcium trnsients, relys synptic signls to CREB. Here we show tht in hippocmpl neurons, signling to CREB cn e ctivted y nucler clcium lone nd does not require import of cytoplsmic proteins into the nucleus. The nucleus is prticulrly suited to integrte neuronl firing ptterns, nd specifies the trnscriptionl outputs through urst frequency-tonucler clcium mplitude conversion. Clcium relese from intrcellulr stores promotes clcium wve propgtion into the nucleus, which is criticl for CREB-medited trnscription y synptic NMDA receptors. Phrmcologicl or genetic modultion of nucler clcium my directly ffect trnscription-dependent memory nd cognitive functions. The regultion of gene trnscription y neuronl ctivity is n integrl prt of the process tht underlies lerning nd memory 1. Electricl impulse ptterns re coded using intrcellulr clcium signls nd linked to specific genomic responses 2,3. Activity-dependent ctivtion of the trnscription fctor CREB is hevily implicted in synptic plsticity, lerning nd survivl 4 8. CREB is trget for two clcium-regulted signling pthwys: the MAP kinse/extrcellulr signl-regulted kinse (ERK1/2) cscde 9 13 nd the clcium/clmodulin (CM) dependent protein kinses, in prticulr, the nucler CM kinse IV Either pthwy cn cuse phosphoryltion of CREB on its ctivtor site serine 133 (refs. 13,19), which is often regrded s mrker for CREB-medited trnscription. However, CREB phosphoryltion on serine 133 does not necessrily indicte tht CREB-dependent gene expression is induced. Activtion of the ERK1/2 pthwy lone, for exmple, y electricl ctivity in the presence of inhiitors of CM kinses or y selective stimultion of ERK1/2 with growth fctors or y genetic mens, leds to CREB phosphoryltion on serine 133 without pprecile induction of CREB-dependent trnscription 13, These results were explined y the discovery of second regultory event tht is criticl for CREB-medited trnscription. This second event involves stimultion of the ctivity of the CREB inding protein (CBP) 13,19,22, trnscriptionl co-ctivtor tht intercts with CREB phosphorylted on serine 133 (ref. 23). The CBP-ctivting signl is provided y nucler clcium nd CM kinse IV, ut not y the ERK1/2 cscde tht consequently fils to induce CREB-dependent trnscription 13,22. Thus, CM kinse IV (or closely relted nucler CM kinse) hs emerged s key regultor of neuronl gene expression. It is necessry nd sufficient to ctivte CREB-dependent gene expression in response to clcium signls, nd is likely to control the ctivity of mny other trnscription fctors tht cn recruit CBP to promoter 19,24. However, it remins uncertin how synptic signls re conveyed to the nucleus nd to nucler CM kinses. Studies hve suggested tht signl-induced trnsloction of clmodulin from the cytoplsm to the nucleus relys electricl ctivity to CREB 25. Here we show tht in hippocmpl neurons, synpse-to-creb signling cn function in the sence of nucler import of clmodulin. Insted, independent clcium signl processing in neuronl nuclei couples synptic ctivity to CREB-medited gene trnscription. RESULTS The sucellulr locliztion of clmodulin ws nlyzed in hippocmpl slice preprtion nd in cultured hippocmpl neurons. Slices from the hippocmpus of dult rts showed strong nucler clmodulin immunorectivity in most neurons in the dentte gyrus nd in the res CA3 nd CA1 (Fig. 1). In unstimulted hippocmpl cultures, virtully ll neurons expressed clmodulin in the nucleus (Fig. 1). Similrly, FITC-leled clmodulin microinjected into the cytoplsm of cultured rt hippocmpl neurons lrgely ccumulted in the nucleus (Fig. 1c). Microinjection per se did not chnge the distriution of the endogenous clmodulin (Fig. 1). The nucler locliztion of the endogenous clmodulin nd nucler ccumultion of FITCleled clmodulin were unffected y tretments of the neurons tht either increse or decrese their electricl ctivity nd thus seem to e intrinsic properties of hippocmpl neurons (Fig. 1d nd dt not shown). These results indicte tht most hippocmpl neurons re unlikely to use signl-induced nucler trnsloction of clmodulin for the rely of synptic signls to the trnscription-ctivting mchinery. We next determined whether nucler import of ny protein is importnt for clcium signling to CREB. Hippocmpl neurons were microinjected into the cytoplsm with whet germ gglutinin (WGA), plnt lectin tht inds to the nucler pore complex nd locks nucler import 26. The inhiition y WGA nture neuroscience volume 4 no 3 mrch

2 rticles d f c e Fig. 1. Dynmics of clmodulin locliztion in hippocmpl neurons: uncoupling of synptic ctivity-induced CREB phosphoryltion from import of proteins into the nucleus. () Immunocytochemicl nlysis of clmodulin expression in hippocmpl neurons. Representtive confocl imges re shown. The percentge of neurons showing strong nucler clmodulin immunorectivity ws 85 ± 5% (dentte gyrus), 76 ± 9% (CA3), nd 64 ± 4% (CA1) (n = 3), > 98% (cultured hippocmpl neurons; n = 5). Scle r, 40 µm. () Nucler clmodulin immunorectivity in cultured hippocmpl neurons microinjected with Texs Red-leled 70 kd dextrn into the cytoplsm or the nucleus. Arrowheds, microinjected neurons. Left, Texs Redleled injection mrker; right, clmodulin immunorectivity. Scle r, 40µm. (c) Whet germ gglutinin (WGA) locks nucler ccumultion of FITC-clmodulin microinjected into the cytoplsm of hippocmpl neurons. Representtive confocl sections re shown. Left, Texs Red-leled injection mrker; right, FITC-clmodulin. Scle r, 20 µm. (d) Quntittive nlysis of nucler trnsloction of FITC-clmodulin. Hippocmpl neurons were microinjected into the cytoplsm with either FITC-clmodulin, FITC-clmodulin/WGA or FITC-clmodulin/WGA/N-cetyl glucosmine (GlcNAc). The ctivity of WGA is quenched y preincution with GlcNAc. The percentge of cytoplsmic FITC-clmodulin trnslocted to nucleus ws quntified in untreted hippocmpl neurons nd in hippocmpl neurons treted either for 20 to 30 min with 1 mm kynurente/12 mm MgCl 2 (KyMg). Stimultion ws with KCl (50 mm) plus APV (100 µm) for 5 min. (e) Clcium-induced CREB phosphoryltion on serine 133 occurs independently of nucler trnsloction of FITC-clmodulin. The Texs Red fluorescence imge (left column) identifies injected cells; rrowheds indicte cells microinjected into the cytoplsm. Locliztion of FITC-CM (middle column) nd CREB phosphoryltion on serine 133 (right column) ws nlyzed in hippocmpl neurons, microinjected into the cytoplsm with either FITC-clmodulin or FITC-clmodulin/WGA. Neurons were stimulted with KCl (50 mm) plus APV (100 µm), or were left unstimulted. Pre-tretment with KN-62 (10 µm) ws done for min. CREB phosphoryltion ws exmined 5 to 10 min or 30 min fter stimultion, giving identicl results. Scle r, 40 µm. (f, g, h) Quntittive nlysis of clcium-induced CREB phosphoryltion s function of nucler trnsloction of FITC-clmodulin in hippocmpl neurons microinjected into the cytoplsm with FITCclmodulin (CON, lck squres) or FITC-clmodulin/WGA (WGA, open tringles). Unstimulted neurons (f); neurons stimulted with KCl (50 mm) plus APV (100 µm) for 5 minutes without (g) or with pre-tretment with 10 µm KN-62 (h). Simple regression nlysis showed reltionship etween inhiition of ctive protein import (mesured y monitoring FITC-clmodulin trnsloction) nd inhiition of CREB phosphoryltion when KN-62 ws present (tht is, only the ERK1/2 pthwy ws in opertion); p < In ddition, comprison of the two dt sets (WGA-injected versus non-injected) showed significnt difference only when KN-62 ws present; p < 0.02, unpired Student s t-test; p < 0.05, Mnn Whitney U test. g h of ctive nucler trnsport of FITC-clmodulin microinjected into the cytoplsm (Fig. 1c nd d) ws used to monitor the efficcy of WGA. Clcium flux through L-type clcium chnnels, potent ctivtor of CRE/CREB-dependent trnscription 13,14,18 20,27 30, ws induced y memrne depolriztion, nd CREB ctivtion ws ssyed using n ntiody tht recognizes CREB in its ctivted (serine 133-phosphorylted) stte 31. Microinjection of FITC-clmodulin or WGA/FITC-clmodulin per se did not induce CREB phosphoryltion (Fig. 1e, top two rows nd f). Upon stimultion, CREB phosphoryltion incresed in uninjected nd FITC-clmodulin-microinjected neurons ut, crucilly, lso incresed in neurons in which import of proteins into the nucleus ws locked y WGA (Fig. 1e, middle two rows nd g). WGA did inhiit clcium-induced CREB phosphoryl- 262 nture neuroscience volume 4 no 3 mrch 2001

3 rticles Fig. 2. The nucler clcium pool specifies the trnscriptionl response. () Exmples of MEA recordings of icuculline-induced ursts of ction potentils in control nd APV-treted hippocmpl neurons. () Bursts of ction potentils induce NMDA receptor-dependent nucler clcium trnsients tht outlst electricl ctivity. MEA recordings nd simultneous clcium imging using CCD cmer. Action potentil firing ws induced y icu- c d culline; hippocmpl neurons were susequently exposed to icuculline nd APV (500 µm). The width nd the height of the rs indicte the durtion of the electricl ctivity nd the numer of spikes in the urst, respectively. (c) Immunolot nlysis of CREB phosphoryltion e in unstimulted hippocmpl neurons nd in hippocmpl neurons treted for 10 min with icuculline (50 µm) in the presence nd sence of APV (250 µm). (d) Exmples of nucler nd corresponding g dendritic clcium peks illustrting the temporl dely of nucler clcium nd differences in the decy of the clcium trnsients. (e) MEA recordings illustrting the f increse in urst frequency of icuculline-stimulted hippocmpl neurons following tretment with the indicted concentrtion of 4-AP. The h totl numer of spikes, mesured over equl periods of time, did not increse in 4- AP-treted neurons (dt not shown). (f) Exmple of nucler clcium trnsients illustrting the effects of 4-AP on the frequency of the trnsients, the pek mplitude nd inter-pek clcium levels. (g, h) Immunocytochemicl detection of GAL4-CREB-medited trnscription (g) nd endogenous c-fos expression (h) in hippocmpl neurons 2 h fter inducing ursts of ction potentils for 10 min with icuculline in the sence or presence of the indicted mount of 4-AP. Hippocmpl neurons were microinjected with GAL4-CREB expression vector nd the c-fos-sed, myc-tgged GAL4 reporter gene pf222 CREmyc 13,29. Numer of cells nlyzed, 306, GAL4-CREB; 451, endogenous c-fos. The sl level of GAL4-CREB medited gene expression ws 34 ± 7% of tht otined fter icuculline tretment. tion when CM kinses were locked using KN-62 (Fig. 1e, ottom two rows nd h). This ws ecuse the remining pthwy to CREB, the Rs-ERK1/2-RSK2 cscde (known to cuse CREB phosphoryltion ut not CREB-medited trnscription 13,20,21 ) ws ctivted in the cytoplsm nd thus depended upon trnsloction to the nucleus. These results indicte tht signling to CREB following trnsmemrne clcium flux into hippocmpl neurons does not require nucler import of proteins. Insted, nucler clcium pool, ctivting n intrnucler CM kinse, seems to control CREB function. Anlysis of nucler clcium homeostsis in electriclly ctivted hippocmpl neurons reveled function of the nucler nture neuroscience volume 4 no 3 mrch

4 rticles c d e Fig. 3. NMDA receptor-induced clcium relese from intrcellulr stores promotes clcium wve propgtion to the nucleus, which is criticl for CREB-medited trnscription y synptic ctivity. () Exmples of icuculline-induced nucler clcium trnsients in hippocmpl neurons with or without CPA-induced depletion of intrcellulr clcium stores efore stimultion. (, c) Depletion of intrcellulr clcium stores locked urst ctivityinduced CREB-medited () ut not TCF/ SRF-medited (c) trnscription. Activtion of synptic NMDA receptors following icuculline tretment ws found to e very potent inducer of CREB-medited trnscription. This is in contrst to the regultion of CREB function y glutmte th ppliction tht only poorly ctivtes CREB-dependent gene expression 19,28 due to ctivtion of extrsynptic NMDA receptors (unpulished oservtion). Hippocmpl neurons were microinjected either with GAL4-CREB expression vector nd the c-fos-sed, myc-tgged GAL4 reporter gene pf222 CREmyc or the TCF/SRF-dependent, myc-tgged reporter gene pfos CREmyc 13,29. Trnscription ctivtion ws ssessed immunocytochemiclly 2 h fter inducing ursts of ction potentils for 10 min with icuculline in control hippocmpl neurons (, 106 cells nlyzed; c, 50 cells nlyzed), or hippocmpl neurons pretreted for 20 min with 10 µm CPA (, 57 cells nlyzed; c, 56 cells nlyzed) or 15 µm thpsigrgin (, 53 cells nlyzed). Induction y icuculline fo GAL4-CREB- nd TCF/SRF-medited trnscription ws 3- to 4-fold. (d, e) Immunolot nlysis of ERK1/2 ctivtion (d) nd immunocytochemicl nlysis of c-fos protein expression (e) in hippocmpl neurons with or without CPA-induced depletion of intrcellulr clcium stores efore stimultion with icuculline. Neurons were pre-treted with the indicted inhiitors. clcium pool s n integrtor of neuronl firing ptterns. Bursts of ction potentil firing were induced using the GABA A receptor locker icuculline nd monitored using microelectrode rry recordings (MEA; Fig. 2). Action potentil ursts occurred with frequencies of 0.05 to 0.15 Hertz, ck-propgted into the dendritic tree (unpulished oservtion), nd elicited NMDA receptor-dependent clcium trnsients tht lsted considerly longer thn the detectle electricl ctivity (Fig. 2). Bicuculline induced rpid phosphoryltion of CREB on serine 133 (within 30 s; dt not shown; Fig. 2c). This phosphoryltion ws triggered y ctivtion of NMDA receptors (Fig. 2c) nd ws medited y oth CM kinses nd the ERK1/2-RSK2 cscde ecuse lockde of CREB phosphoryltion required the inhiition of oth pthwys using KN-62 nd the MEK 1 inhiitor PD98059, respectively (dt not shown). CREB phosphoryltion remined elevted for the durtion of synptic ctivity, nd following its cesstion, dephosphoryltion occurred with t 1/2 of out five minutes (dt not shown). These decy kinetics were lrgely unffected y inhiition of either the CM kinses or the ERK1/2- RSK2 pthwy (dt not shown). This suggests tht dephosphoryltion is limited y the ction of the relevnt phosphtse (likely protein phosphtse 1; refs. 18,32) s opposed to ny differentil shut-down of the two pthwys. Dendritic clcium increses evoked y ction potentil ursts preceded increses in the nucleus consistent with clcium wve propgting from the site of clcium entry into the nucleus 264 nture neuroscience volume 4 no 3 mrch 2001

5 DISCUSSION This study indictes tht clcium itself, rther thn clcium/clmodulin complex, is the messenger tht couples synptic ctivity to the nucler mchinery tht regultes trnscription. Although we fil to oserve ny ctivity-dependent clmodulin trnsloction to the nucleus, we cnnot rule out tht it does occur in certin cell types nd/or experimentl condirticles (Fig. 2d). The unique property of the nucleus to enle clcium trnsients to fr outlst synpticlly evoked dendritic clcium trnsients (Fig. 2d) 33,34 cused conversion of urst frequency-coded electricl signl into n nucler clcium mplitude-coded signl: y incresing the frequency of ction potentil ursts following tretment of hippocmpl neurons with low doses of the K + chnnel locker 4-mino pyridine (4-AP; Fig. 2e), nucler clcium trnsients filed to return to sl levels etween ursts nd remin t n elevted plteu (Fig. 2f). This urst frequency-tonucler clcium mplitude conversion hs implictions for gene regultion. The ffinity of clmodulin for clcium cn increse drmticlly following clcium/clmodulin inding to trget enzymes 35. Therefore, sustined elevted nucler clcium levels my prolong the ctive stte of nucler clcium/clmodulin-dependent enzymes nd fcilitte induction of trnscription. Indeed, we found tht the mplitude of the sustined nucler clcium signls ( function of the urst frequency) correltes with the trnscriptionl response: synpticctivity-induced CREB-medited trnscription nd induction of the endogenous c-fos gene (triggered y NMDA receptor ctivtion; dt not shown) is enhnced upon incresing the urst frequency with 4-AP (Fig. 2g nd h). Thus, the nucler clcium pool cn decode neuronl impulse ptterns nd my specify the trnscriptionl response. The source of nucler clcium is to lrge extent provided y intrcellulr clcium stores. Depletion of intrcellulr clcium stores with cyclopizonic cid (CPA) or thpsigrgin, lthough hving no effect on ction potentil firing (dt not shown), compromised synpticlly evoked nucler clcium trnsients (Fig. 3) nd cused virtully complete lock of CREB-medited gene expression (Fig. 3). In contrst, trnscription medited y the serum response element (SRE)-intercting complex TCF/SRF 36,37, which is ctivted y sumemrnous clcium pool stimulting the ERK1/2 signling cscde (unpulished oservtions), ws unffected y clcium store depletion (Fig. 3c). Thus, clcium relese from intrcellulr stores is criticl for clcium wve propgtion from synptic NMDA receptors to the nucleus nd, consequently, for CREB-medited gene expression. In contrst, ctivtion of the ERK1/2 cscde, which is not compromised y CPA-induced store depletion (Fig. 3d), is evidently not sufficient to induce CREB-medited trnscription, ut increses trnscription medited y TCF/SRF. These findings suggest tht ctivity-induced expression of gene such s c-fos tht is regulted y oth CREB nd TCF/SRF 20,28,38 will e compromised, yet not completely locked, y the depletion of intrcellulr clcium stores. The remining induction of CREB/TCF/SRF-regulted genes is predicted to e medited y the ERK1/2 cscde. This is indeed the cse: depletion of intrcellulr clcium stores with Fig. 4. The nucler clcium pool is sufficient to signl to CREB. () Phse contrst photomicrogrph of hippocmpl neurons efore (top) nd 30 s fter lysis (ottom) in solution contining 0.5% NP-40. Scle r, 60 µm. () Immunolot nlysis of CREB expression, CREB phosphoryltion nd ctivtion of ERK1/2 in exposed nuclei from detergent-soluilized hippocmpl neurons nd in intct unstimulted hippocmpl neurons (uns) or hippocmpl neurons stimulted with KCl (50 mm) plus APV (100 µm) for 5 min. For nlysis of intct cells nd nuclei, 10 µm KN-62 ws dded to the cultures min efore stimultion or lysis, respectively. Inhiition of clcium-induced CREB phosphoryltion y KN-62 in isolted nuclei ws 89 ± 7% (n = 4). CPA reduced the endogenous c-fos response ut left intct residul induction tht ws sensitive to inhiition of the ERK1/2 cscde with PD98059 (Fig. 3e). In contrst, PD98059 did not reduce c-fos induction in control neurons (Fig. 3e), indicting tht ctivtion of CREB y nucler clcium is sufficient to fully ctivte c-fos trnscription. To finlly determine whether nucler clcium pool is sufficient to signl to CREB, we treted hippocmpl neurons with detergent solution contining clcium/egta uffer with free clcium concentrtion of 65 nm. This tretment rpidly soluilized the plsm memrne nd the cytosol leving ehind isolted nuclei (Fig. 4). Compred to nuclei tht remined t 65 nm free clcium, incresing the free clcium concentrtion to 1 µm rpidly induced CREB phosphoryltion (Fig. 4; compre lnes 7 nd 9). As expected, due to detergent soluiliztion of cytoplsmic nd plsm memrne-ssocited proteins, ERK1/2 were not ctivted y incresing the free clcium concentrtion (Fig. 4; compre lnes 7 nd 9). An increse in CREB phosphoryltion nd lck of ERK1/2 ctivtion ws lso oserved in nuclei lysed with detergent solution contining 1 µm free clcium (Fig. 4; compre lnes 4 nd 5). Clcium-induced CREB phosphoryltion in hippocmpl nuclei ws locked y KN-62 (Fig. 4; compre lnes 5 nd 6, nd lnes 9 nd 10) indicting tht the rection is ctlyzed y nucler CM kinses. These results demonstrte tht nuclei of hippocmpl neurons re independent clcium-signl-processing units tht contin everything needed to ctivte CREB. nture neuroscience volume 4 no 3 mrch

6 rticles tions. In these cses it would e likely to ugment or support nucler clcium-dependent signling to CM kinse IV. We hve shown tht clcium influx into hippocmpl neurons through synptic NMDA receptors triggers clcium relese from intrcellulr clcium stores. This mplifies synptic clcium signls nd relys them, perhps in the form of regenertive clcium wve, to the nucleus. Synpticlly induced nucler clcium trnsients my e dependent upon postsynptic firing nd ckpropgting dendritic ction potentils. Dendritic ction potentils (daps) enhnce synptic clcium trnsients y depolriztion of the dendrites, cusing more efficient relief of the mgnesium lock of the NMDA receptors. A mechnism involving metotropic glutmte receptors is lso conceivle 42. In ddition, daps my cuse glol clcium trnsients y fcilitting clcium flux through dendritic or somtic voltge-gted clcium chnnels 43,44. However, in our experimentl system, L-type clcium chnnels do not seem to contriute to the increses in intrcellulr clcium; in most hippocmpl neurons, inhiition of L-type clcium chnnels with nifedipine cused only smll decrese in the pek mplitude of icuculline-induced clcium trnsients nd n unexpected increse in the frequency of the ction potentil ursts (dt not shown). Our experimentl conditions generte vigorous synptic ctivity with ursting of neurons nd daps (unpulished oservtion). But even wek synptic inputs cn, when coinciding with daps, generte lrge glol clcium trnsients tht likely trigger nucler clcium-induced trnscription events. Synptic stimuli coinciding with daps cn cuse roust increses in synptic efficcy 39,40. The genomic responses ssocited with it my help consolidte nd mintin chnges in potentited synpses tht re perhps tgged y the locl synptic clcium trnsients 45. As informtion is likely encoded in the firing rtes of neurons eliciting distinct genomic responses, impulse ptterns must specify ctivity-dependent gene induction. Our study reveled tht the trnsformtion y the nucleus of trnsient dendritic clcium increses into longer-lsting nucler clcium trnsients converts urst frequency-coded signl into nucler clcium mplitudecoded signl. This llows firing ptterns to e integrted to generte n elevted nucler clcium plteu, the mplitude of which is function of the frequency of the electricl signl. Becuse the mplitude of clcium signls is determinnt of the type nd mgnitude of trnscription ctivtion 29,46, this frequency-tomplitude conversion provides mechnism through which neuronl impulse ptterns shpe genomic responses. The importnce of CREB in synptic plsticity nd lerning 4 8 suggests tht its principl regultor, nucler clcium, my e key signl for trnscription-dependent chnges in neuronl function 8. Enhncement or ttenution of nucler clcium signls could therefore represent new strtegy for modulting informtion storge nd memory. One possiility involves drugs, such s the dihydropyridines nimodipine or nifedipine, which, similrly to 4-AP (Fig. 2), increse the urst frequency of neuronl firing nd enhnce gene expression (unpulished oservtions). Such increses my tke plce in the intct rin, nd thus could explin the oserved improvement of cognitive functions with nimodipine in mmmls, non-humn primtes nd humns METHODS Hippocmpl cultures, preprtion of nuclei nd stimultions. Hippocmpl neurons were cultured s descried 9 except tht growth medi ws supplemented with B27 (Life Technologies, Rockville, Mrylnd). Bursts of ction potentil firing were induced y tretment of cultured hippocmpl neurons with 50 µm icuculline. The memrne of hippocmpl neurons ws depolrized y incresing the extrcellulr KCl concentrtion y 50 mm in the presence of 100 µm D( )-APV 19,28. Stimultions were terminted y dding 10 mm MgCl 2 nd 1 mm sodium kynurente to the cultures 19,28. Expression of c-fos protein ws nlyzed 2 hours fter stimultion, nd CREB phosphoryltion on serine 133 nd ctivtion of ERK1/2 ws nlyzed 5 to 10 min fter stimultion unless otherwise indicted 19. PD98059 (50 µm; New Englnd Biols, Beverly, Msschusetts) nd KN-62 (10 µm; CN Biosciences, Drmstdt, Germny) were dded to the cultures 60 min efore stimultion nd 15 to 20 min efore stimultion, respectively. At these concentrtions nd exposure times, KN-62 or PD98059 did not interfere with icuculline-induced clcium trnsients (dt not shown). Nuclei were prepred y lysing hippocmpl neurons t room temperture in PBS (ph 7.2) contining 0.5% (v/v) NP-40, 7.5 mm K + 2EGTA, 2.5 mm C 2+ EGTA nd 2.5 (free clcium concentrtion is 65 nm). The free clcium concentrtion ws incresed to 1 µm y dding 6.2 volumes of 10 mm C 2+ EGTA. After 60 to 90 s, 1.8 volumes of 4 smple uffer ws dded, nuclei were scrped off the dish, oiled for 7 min, nd sujected to SDS-PAGE nd immunolot nlyses. Anlysis of clmodulin in hippocmpl slices nd cultured neurons. Hippocmpi were dissected from the rin of dult Long-Evns rts in ice-cold PBS contining 30 mm glucose, 2 µm TTX, 1 mm sodium kynurente nd 10 mm MgCl 2. After immedite fixtion for 1 to 2 h in PBS contining 4% prformldehyde/4 mm EGTA nd equilirtion for 12 to 36 h t 4 C in PBS contining 30% (w/v) sucrose/0.001% (w/v) sodium zide, slices were cut on cryostt followed y immunohistochemicl stining using n ntiody to clmodulin (Upstte Biotechnology, Lke Plcid, New York). Cultured hippocmpl neurons were fixed for 15 to 20 min in PBS contining 4% prformldehyde/3% (w/v) sucrose/4 mm EGTA followed y immunostining using clmodulin ntiodies. Fixtion t room temperture nd on ice gve identicl results. Microinjection nd imging. Microinjection, Fluo-3 clcium imging nd confocl lser scnning microscopy ws done s descried 13,19,29. Clcium concentrtions re expressed s function of the Fluo-3 fluorescence ((F F min )/(F mx F)). Clmodulin (CN Biosciences) ws leled with FITC using kit (Sigm). Locliztion of FITC-clmodulin ws ssessed 20 to 30 min fter microinjection into the cytoplsm of hippocmpl neurons. The concentrtion of WGA (Sigm) in the injection solution ws 5.5 µg/µl. MEA recordings. Hippocmpl neurons were plted onto polylysine/lminin-coted chmers contining n rry of 60 microelectrodes. MEA recordings (Multichnnel Systems, Reutlingen, Germny) nd Fluo-3 clcium imging using CCD cmer (Hmmtsu Photonics UK, UK; Kinetic Imging Softwre, Kinetc Imging, Bromorough, UK) were done t room temperture using 10 to 11 dys old hippocmpl cultures. Signling nd gene expression. CREB phosphoryltion on serine 133, ctivtion of ERK1/2, nd expression of the endogenous c-fos protein nd c-fos-sed reporter genes ws nlyzed immunocytochemiclly or y immunolotting s descried 13,19,29. ACKNOWLEDGEMENTS We thnk N.R. Hrdinghm nd J. Jck for discussion nd ckground electrophysiologicl dt, E. Gruenstein for dvice on the 4-AP experiments, nd B. Wisden for discussion. This work ws supported y the MRC, Dunhill Medicl Trust, The Royl College of Surgeons of Englnd, the Sckler Medicl Reserch Centre nd Clre College, Cmridge. RECEIVED 18 SEPTEMBER; ACCEPTED 6 DECEMBER Milner, B., Squire, L. R. & Kndel, E. R. Cognitive neuroscience nd the study of memory. Neuron 20, (1998). 2. Ghosh, A. & Greenerg, M. E. Clcium signling in neurons: moleculr mechnisms nd cellulr consequences. Science 268, (1995). 266 nture neuroscience volume 4 no 3 mrch 2001

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