Selective acquisition of AMPA receptors over postnatal development suggests a molecular basis for silent synapses

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1 rticles Selective cquisition of AMPA receptors over postntl development suggests moleculr sis for silent synpses R. S. Petrli 1, J. A. Esten 2, Y.-X. Wng 1, J. G. Prtridge 3, H.-M. Zho 1, R. J. Wenthold 1 nd R. Mlinow 2 1 L. of Neurochemistry, NIDCD, NIH, Bethesd, Mrylnd 20892, USA 2 Cold Spring Hror Lortory, Cold Spring Hror, New York 11724, USA 3 Dept. Phrmcol., Vnderilt Univ., Nshville, Tennessee 37232, USA The first two uthors contriuted eqully to this project. Correspondence should e ddressed to R.M. (mlinow@cshl.org) Erly in postntl development, glutmtergic synpses trnsmit primrily through NMDA receptors. As development progresses, synpses cquire AMPA receptor function. The moleculr sis of these physiologicl oservtions is not known. Here we exmined single excittory synpses with immunogold electron-microscopic nlysis of AMPA nd NMDA receptors long with electrophysiologicl mesurements. Erly in postntl development, significnt frction of excittory synpses hd NMDA receptors nd lcked AMPA receptors. As development progressed, synpses cquired AMPA receptors with little chnge in NMDA receptor numer. Thus, synpses with NMDA receptors ut no AMPA receptors cn ccount for the electrophysiologiclly oserved silent synpse. Excittory trnsmission in the verterte centrl nervous system is medited y ctions of glutmte on AMPA (α-mino-3- hydroxy-5-methyl-4-isoxzolepropionte) nd NMDA (Nmethyl-D-sprtte) receptor sutypes. These receptors hve different roles in trnsmission, plsticity nd toxicity 1. In the hippocmpus 2 4 nd other rin regions 5,6, the rtio of AMPAreceptor- to NMDA-receptor-medited trnsmission is initilly low nd increses over development. Furthermore, mny synptic events, prticulrly erly in development, re medited y the ctivtion of only NMDA receptors 2,3,5 8. Such responses re proposed to occur t structures termed postsynpticlly silent synpses, ecuse trnsmission cn only e detected if the postsynptic memrne potentil is rised ove the resting level. At resting memrne potentils, the NMDA receptor pore is lrgely locked y extrcellulr mgnesium 9, nd hence trnsmitter reching only NMDA receptors will produce little response. The prevlence of pure NMDA responses mesured electrophysiologiclly decreses during development 2,3,5,6. Long-term potentition (LTP), form of ctivity-dependent synptic plsticity, cn dd n AMPA-receptor-medited component of trnsmission to pure NMDA-receptor-medited responses 7,8. These electrophysiologicl oservtions suggest tht NMDA receptors re present in synpses efore AMPA receptors nd tht during development AMPA receptors re progressively dded, possily through n LTP-like process. However, lterntive theories re possile. One scenrio proposes tht oth AMPA nd NMDA receptors coincide in their ppernce t synpses, ut erly in development only low concentrtions of glutmte rech synpses, concentrtions sufficient to ctivte primrily NMDA receptors. As these receptors hve higher ffinity for glutmte thn AMPA receptors, this view is iophysiclly plusile 10. A low concentrtion of trnsmitter could rech receptors either ecuse of low concentrtion relesed y vesicles or ecuse of geometricl reltions etween relese site nd receptor loction (for exmple, spillover from nery synpses 11 or incresed synptic cleft seprtion). Another possiility is tht oth receptors re on synpses, ut erly in development AMPA receptors re functionlly silent, either ecuse they lck n ssocited protein (such s glutmte receptor intercting protein 12 ) or ecuse they lck necessry posttrnsltionl modifiction (such s phosphoryltion 13 ). We hve directly tested the hypothesis tht NMDA receptors (NMDA-Rs) re present t synpses efore AMPA receptors (AMPA-Rs) nd tht the ltter re progressively dded during development. Results We hve used post-emedding immunogold electron microscopy (Methods) to exmine glutmte receptors t CA1 hippocmpl symmetric synpses t different times in postntl development (postntl dy (P)2, P10 nd 5 weeks). Most synpses showed immunorectivity for NMDA-Rs (Fig. 1, Tle 1). Furthermore, there were no significnt chnges over the ges exmined. We found similr numer of gold prticles per synpse, numer of gold prticles per µm of postsynptic memrne nd percentge of synpses leled t ll ges studied (Fig. 1, Tle 1, see lso Fig. 4). This ws true for ntiodies directed ginst NR1 nd NR2A/B suunits. These results rgue tht the numer of NMDA- Rs per synpse remins constnt etween P2 nd 5 weeks. In contrst to NMDA-Rs, AMPA-R immunorectivity ws low t P2 nd P10 nd high t 5 weeks (Fig. 2, Tle 1). For nture neuroscience volume 2 no 1 jnury

2 rticles NR1 NR2A/B c d e Fig. 1. Immunogold leling of NMDA receptors in the CA1 strtum rditum of the hippocmpus. Postemedding immungold leling ws done using ntiodies to NR1 (, d, f) nd NR2A/B (,c,e,g)with 10 nm gold, t postntl dy 2 (P2; c), postntl dy 10 (P10; d, e) nd 5 weeks (f, g). p, presynptic terminl. Line scle is 0.2 µm. Microgrphs were chosen to illustrte the moderte level of NMDA-R leling mintined from P2 to 5 weeks. instnce, for AMPA-R suunit GluR1 immunorectivity, the verge numer of gold grins per synpse incresed 1.7-fold from P2 to P10 nd incresed 2-fold further from P10 to 5 weeks. In ddition, the frction of synpses contining AMPA-R immunorectivity incresed with development (from 16% t P2 to 50% t 5 weeks, in the cse of GluR1, nd from 44% to 75% in the cse of the AMPA-R suunits GluR2/3, see Tle 1). Similr results were otined with two dditionl ntiodies, one recognizing GluR1 nd the other GluR2 (not shown). This indictes tht the chnges in immunorectivity t different ges re not likely to e due to developmentl chnges in epitopes of AMPA-Rs. This developmentl difference in leling etween AMPA-Rs nd NMDA-Rs ws verified with doule leling. Erly in development, synpses contining NMDA-R immunorectivity nd little or no AMPA-R immunorectivity were common (Fig. 3f nd g), wheres t 5 weeks, immunoleling for AMPA- Rs nd NMDA-Rs ws comprle (Fig. 3h). These dt strongly support the view tht the developmentl increse in AMPA-R-medited trnsmission 2 6 is due to n increse in the numer of AMPA-Rs t synpses. Wht do these dt sy regrding the nture of silent synpses? Cn one conclude directly from these ntomicl dt tht mny synpses contin NMDA-Rs nd lck AMPA-Rs? Immunogold electron microscopy only detects frction of receptors on ny synpse. Thus, it is possile tht ll synpses hve AMPA-Rs, ut ecuse of low detection, some postsynptic densities fil to show immunorectivity. This difficulty in determining unmiguously the existence of synpses tht lck AMPA-Rs hs een recognized in other studies on AMPA-R nd NMDA-R expression t synpses 14,15. We hve tken severl pproches to determine if there exist synpses lcking AMPA-Rs nd whether their prevlence chnges during postntl development. First, we nlyzed the frequency distriution of immunoprticles (Fig. 4). The generl fetures indicted in Tle 1 re shown in greter detil in these distriutions. For instnce, tissue nlyzed for GluR1 or GluR2/3 generlly hd mny more synpses lcking immunorectivity compred to tissue nlyzed for NR1. The oserved frction of synpses lcking immunoprticles (in f g zero) decreses with ge for GluR1 nd GluR2/3 while remining constnt for NR1 (Fig. 4). From these oserved distriutions, we hve estimted the frction of synpses truly lcking receptors. We hve fitted the oserved frequency distriutions to simple model (Methods), ssuming tht the oserved prticle distriution is rndom low-detection smpling of the true receptor distriution. From these fits, we cn estimte the frction of synpses truly lcking receptors, FLR (Fig. 4). For GluR1 immunoleling, we found tht FLR is high t P2 (71%) nd decreses with ge (P10, 50%; 5 weeks, 29%). Similr ge-relted chnges were oserved for GluR2/3 immunoleling (P2, 44%; P10, 34%; 5 weeks, 12%). The lower FLR estimted using GluR2/3 immunoleling is consistent with findings tht significnt frction of AMPA receptors lck GluR1 suunit, wheres most of them contin GluR2 suunit 16. Thus, some synpses my hve AMPA receptors only contining GluR2/3. For NR1, FLR ws low t ll ges (<10%). We cn exmine this model s roustness y clculting the estimted FLR fter simulted lowdetection smpling from the oserved distriution (which itself is lredy low-detection smpling from the true distriution). For GluR1, the clculted FLR ws lrgely independent of detection level (Fig. 4), supporting the ccurcy of the estimted vlue. For GluR2/3 (P10 nd 5 weeks) nd for NR1 (ll ges), the FLR incresed somewht with lower detection levels, suggesting tht the true FLR vlue my e slightly lower thn the estimted one (Methods). These nlyses of the oserved immunogrin distriution therefore support the view tht significnt frction of synpses hve NMDA-Rs nd lck AMPA-Rs, prticulrly erly in postntl development. To complement the estimtes reched from the low-detection nlysis, we mximized AMPA-R detection y comining five AMPA-R ntiodies nd y modifying severl steps in the immunoleling procedure (see Fig. 3 legend). With these modifictions, the leling ws considerly incresed (Fig. 3). However, t P10, 49% of synpses still lcked immunoleling. At 5 weeks, only 9% lcked immunoleling (Fig. 5). These vlues coincide well with the FLR estimtes reched from individul Tle 1. Summry of immunogold leling for NMDA-Rs nd AMPA-Rs. NR1 Prticles/synpse Percent synpses leled Prticles/leled synpse Numer of synpses NR2A/B Prticles/synpse Percent synpses leled Prticles/leled synpse Numer of synpses GluR1 Prticles/synpse Percent synpses leled Prticles/leled synpse Numer of synpses GluR2/3 Prticles/synpse Percent synpses leled Prticles/leled synpse Numer of synpses The verge numer of prticles per synpse nd the percentge of leled synpses were significntly different (t-test nd chi-squre, respectively) etween 5 weeks nd the other two ges in the cse of GluR1 nd GluR2/3 ntiodies. For GluR1 immunoleling, the percentge of leled synpses ws lso significntly different etween P2 nd P10. No significnt difference mong P2, P10 or 5 weeks ws found for NMDA-R leling. 32 nture neuroscience volume 2 no 1 jnury 1999

3 rticles Glu R1 GluR2/3 c d e f Fig. 2. Immunogold leling of AMPA receptors in the CA1 strtum rditum of the hippocmpus. Postemedding immungold leling ws done using ntiodies to GluR1 C-terminus (, d, g) nd GluR2/3 (,c,e,f,h,i)with 10 nm gold, t postntl dy 2 (P2; c), postntl dy 10 (P10; d f), nd 5 weeks (g i). p, presynptic terminl. Line scle is 0.2 µm. Microgrphs were chosen to illustrte the mjor trend, tht is, lrge increse in leling for AMPA-Rs t 5 weeks compred to P2/P10 ( f versus g i). g h i AMPA-R ntiodies. Simulted low-detection smpling from these dt (Fig. 5, inset) produced similr estimtes for frction of synpses lcking AMPA-Rs, regrdless of detection level. Thus, these results further support the presence of synpses lcking AMPA-Rs erly in development. In the trnsition from P10 to 5 weeks, there ws n increse in the verge numer of immunoprticles in synpses contining leling (Fig. 5). We wished to relte these ntomicl oservtions to physiologicl responses from single synpses. Wholecell recordings were otined from CA1 neurons from rt hippocmpl slices t P2, P10 nd 5 weeks. AMPA-R-medited responses from single synpses were evoked y loclly pplying hypertonic sucrose solution under visul guidnce nd holding the postsynptic cell t hyperpolrized potentils (Methods). The sucrose ws directed t dendritic regions in strtum rditum ~100 µm from the cell ody with ptch pipette (regions comprle to those from which ntomicl mesurements were otined). The resulting miniture excittory postsynptic currents (mepscs) vried in mplitude (Fig. 6), s reported 17. The frequency of responses decresed more thn fourfold when the sucrose pipette ws moved 50 µm wy from the recorded neuron, indicting tht the ctivted synpses were close to the pipette (dt not shown). There ws little difference in men mepsc mplitude cross the different ges exmined (Fig. 6). This result grees with recent oservtions of spontneous mepscs recorded from the whole cell 4. The sme men mepsc mplitude t younger ges, despite the smller numer (nd density) of AMPA-Rs t leled synpses, indictes tht the impct of individul AMPA-Rs on single-synpse response is lrger erlier in development. Thus, these oservtions rgue ginst the view tht the lower AMPA-R-medited component of trnsmission in young tissue results from AMPA-Rs of diminished function, either from receptor modultion or low trnsmitter delivery. Rther, our dt indicte tht receptor function is greter in young nimls nd tht the lower level of AMPA-R-medited trnsmission is due to the smll frction of synpses contining AMPA-Rs. This interprettion is lso in greement with n increse in mepsc frequency over development 4 (lso see Fig 6). Discussion In this study, we hve exmined the moleculr sis for the developmentl profile of AMPA nd NMDA responses t glutmtergic synpses. In the hippocmpus nd other rin regions, trnsmission is initilly medited lrgely y NMDA-Rs, nd over development the AMPA-R-medited component of the synptic response increses. We hve used the post-emedding immunogold technique to test quntittively the hypothesis tht this is due to n initil synptic presence of NMDA-Rs nd tht during development the presence of AMPA-Rs t synpses increses. The most direct line of evidence supporting this view is tht the mount of immunoleling per synpse (verged c d e f g h Fig. 3. AMPA-R immunoleling in high-sensitivity conditions. Immunogold leling for AMPA-R ws optimized y using five ntiodies recognizing the N-terminl epitope of GluR1 suunit, GluR2, GluR2/3, GluR4 suunits nd AMPA-extrcellulr loop (Methods). The incution temperture for primry nd secondry ntiodies ws incresed to 37 C. Immunogold leling of the CA1 strtum rditum of the hippocmpus using ntiodies to n AMPA-R mix ( e, 5-nm gold), nd doule-leling (f h) with AMPA-R mix (15- nm gold) plus NR2A/B (5 nm; rrowheds), t P10 ( c, f, g; no gold seen in ) nd 5 weeks (Ad; d, e, h). p, presynptic terminl. Line scles re 0.2 µm (, ) nd 0.1 µm (c e, f h). Microgrph (c) is high mgnifiction of the synpse (rrow) in microgrph (). Microgrphs were chosen to illustrte the mjor trends, including lrge increse in leling for AMPA receptors t 5 weeks compred to P10 nd moderte level of NMDA receptors mintined t P10 nd t 5 weeks. Pnel (g) contins synpse showing NMDA-R nd no AMPA-R leling. nture neuroscience volume 2 no 1 jnury

4 Methods ELECTRON MICROSCOPY. The postemedding immunogold method hs een descried 22,23 nd is modified from previous method 24. Briefly, mle Sprgue-Dwley rts were perfused with 4% prformldehyde plus 0.5% glutrldehyde in 0.1 M phosphte uffer. Two nimls were used per ge; dditionl nimls were used in preliminry studies (not shown). Prsgittl sections (250 µm) of the hippocmpus were cryoprotected in 30% glycerol nd frozen in liquid propne in Leic EM CPC. Frozen sections were immersed in 1.5% urnyl cette in methnol t 90 C in Leic AFS freeze-sustitution instrument, infiltrted with Lowicryl HM 20 resin t 45 C, nd polymerized with ultrviolet light. Thin sections of the hippocmpus were incuted in 0.1% sodium orohydride plus 50 mm glycine in Tris-uffered sline/0.1% Triton X-100 (TBST), followed y 10% norml got serum (NGS) in TBST, primry ntiody in 1% NGS/TBST, immunogold (10 nm; Amershm) in 1% NGS/TBST plus 0.5% polyethylene glycol, nd finlly stining in urnyl cette nd led citrte. For doule leling, the first primry ntiody nd correspondrticles Fig. 4. Frequency distriution of AMPA nd NMDA-R immunogold leling, oserved nd fit y the low-detection model. GluR1 GluR1 () Open rs show frequency of numer of immunogold prticles per synpse detected t P2, P10 nd 5 weeks for GluR1 C-terminus (top), GluR2/3 (middle) nd NR1 (ottom). Frequency distriutions hve een GluR2/3 fitted y lest-squre regression nlysis to GluR2/3 model with two components: Poisson frction (gry rs) nd frction contining only zero (lck rs; FLR; Methods). N is numer of synpses counted. () The roustness of NR1 the model ws ssessed y Monte Crlo simultions of low-detection smpling from the NR1 experimentl immunogold distriutions (Methods). Averge vlues of FLR otined from the simultions re plotted s function of the detection level pplied to the experimentl distriution for GluR1 (top), GluR2/3 (middle) nd NR1 (ottom) distriutions. Immunogold prticles per synpse Reltive detection Error rs represent stndrd devition. Note tht the vriility in FLR estimtion ecomes lrger s detection ecomes lower. This results from the reduced numer of ins in the low-detection distriutions. For instnce, simulted distriutions otined t reltive detection of 0.1 were typiclly composed of 90% zeroes. over ll excittory synpses) is initilly low nd increses during development for AMPA-Rs, while remining high nd constnt for NMDA-Rs. These findings were lrgely independent of the prticulr AMPA or NMDA-R ntiody, indicting tht these re properties of the receptors nd not chnges in epitopes or suunits during development. We hve lso tested the hypothesis tht synpses exist with NMDA-Rs ut no AMPA-Rs nd tht over postntl development the frequency of such pure NMDA-R synpses decreses y the synptic incorportion of AMPA-Rs. This view is supported y the finding tht t P2 most synpses lck AMPA-R immunorectivity, wheres most hve NMDA-R leling. This disprity decreses with development, so tht t five weeks similr frction lcks AMPA-R nd NMDA-R immunorectivity. We lso modeled the effect of the low detection efficiency of the immunogold technique. Appliction of this model to the oserved distriutions of immunoprticles indicted tht the frction of synpses lcking AMPA-Rs is initilly high nd progressively drops with development. This nlysis lso indicted tht most synpses hve NMDA-Rs throughout development. Lstly, we hve incresed the sensitivity of detection y modifying the immunoleling procedure. Agin we sw significnt frction of synpses lcking AMPA immunoleling in young nimls, wheres t five weeks most synpses hve AMPA immunoleling. This study provides moleculr explntion for previous physiologicl oservtions tht glutmtergic trnsmission is initilly lrgely medited y NMDA-Rs, nd over development AMPA-Rs increse in their prticiption. Our dt suggest tht these properties of trnsmission cn e lrgely ttriutle to n initil synptic incorportion of NMDA-Rs, with progressive incorportion of AMPA-Rs during postntl development. A similr moleculr mechnism, nmely the delivery of AMPA-Rs to synpses tht hve only NMDA-Rs or hve oth AMPA nd NMDA-Rs, cn explin much of long-term potentition in this region of hippocmpus, cellulr model of lerning nd memory 2,3,7,8. This provides further support for the notion tht LTP nd erly postntl development re mechnisticlly relted Frequency Frequency Fig. 5. Frequency distriutions of AMPA-R immunogold prticles with high-sensitivity immunoleling conditions. Filled rs represent P10; open rs represent 5 weeks. Inset, simultions of rndom low-detection smpling done s descried for Fig. 4. Averge vlues nd stndrd devitions for FLR re plotted versus detection reltive to the experimentl distriution. Frction lcking receptors Immunogold prticles per synpse 34 nture neuroscience volume 2 no 1 jnury 1999

5 rticles Fig. 6. Amplitude of foclly evoked miniture excittory postsynptic currents does not chnge with development. () Representtive smples of miniture synptic responses in CA1 hippocmpl neurons to focl ppliction of sucrose from P2 (left), P10 (middle) nd 5 weeks (right). Six trces of 500 ms ech re shown superimposed. The increse in mepsc frequency with development ws generl finding 4 nd consistent with n increse in the numer of synpses contining AMPA-Rs. () Amplitude distriutions of ll responses from P2 (5 cells, 312 events), P10 (5 cells, 656 events) nd 5 weeks (3 cells, 360 events). Neither men mplitude nor cumultive distriutions (not shown) were significntly different. Frequency men = 9.6 pa N = 312 men = 9.9 pa N = 656 Miniture mplitude, pa men = 10.1 pa N = 360 ing immunogold-conjugted ntiody (5-nm gold) were pplied, sections were exposed to prformldehyde vpor t 80 C for one hour, nd the second primry nd secondry (15-nm gold) ntiodies were pplied the following dy For the single-leled AMPA-R ntiody mix study, primry nd secondry ntiody incutions were done t 37 C, nd 5 nm gold ws used. Immunogold prticles were counted in the postsynptic density nd cleft, s descried 22,23. In ech section, ll synpses in selected portion of the CA1 strtum rditum were photogrphed nd included in the study. At ll ges, synpses (even immture ones) used in this study showed the chrcteristic fetures of symmetric synpses: postsynptic density, cleft nd presynptic vesicles. Nevertheless, synpses were uncommon t P2. Mny ppered immture, with smll, poorly developed postsynptic densities. Spines typiclly were short, nd mny ppered to e only protrusions from dendrites. In spite of the immture ppernce of some spects of presynptic nd postsynptic structures t P2, synpses were identified redily y the sic chrcteristics typicl of ll symmetric synpses, s noted ove. At P10, synpse development ws intermedite etween P2 nd 5 weeks. Controls included sence of primry ntiody for single leling nd sence of the second primry ntiody for doule leling; controls lwys showed little or no gold leling. Most primry ntiodies were chrcterized previously Primry ntiodies used were GluR1-C terminus (4.1 µg/ml), GluR1-N terminus (3.2 µg/ml), GluR2 (4 µg/ml), GluR2/3 (2 µg/ml), NR1 (4 µg/ml) nd NR2A/B (4 µg/ml). The AMPA-R mix is comintion of ntiodies to GluR1-N terminus (1.6 µg/ml), GluR2 (2 µg/ml), GluR2/3 (2 µg/ml), GluR4 (1.9 µg/ml) nd AMPA-R-extrcellulr loop. To develop pn-ampa-r ntiody, the extrcellulr loop of GluR2 ws mplified y PCR nd inserted into pet vector (Novgen, Mdison, WI) to crete C-terminus polyhistidine-tgged fusion protein. The fusion protein ws purified nd injected into rits. Antiserum ws ffinity purified nd shown to recognize flip nd flop vrints of GluR1-4 y western lot nlysis of memrnes from cells trnsfected with GluR1 4 cdnas. All microgrphs used in the figures were processed with Adoe Photoshop 4.0 to optimize rightness nd contrst. LOW-DETECTION ANALYSIS. Becuse the frction of receptors detected y immunogold electron microscopy depends on rndom fctors like thin sectioning of postsynptic densities or incomplete epitope exposure, we hve modeled the oserved immunogrin distriutions s rndom lowdetection smpling of the true receptor distriutions. The true distriution of receptors t synpses (efore low-detection smpling) cn e divided into two sets: those synpses truly hving receptors, R +, nd those synpses truly without receptors, R. (This ltter popultion my hve only few receptors, in which cse these synpses would likely e electrophysiologiclly silent.) After simulted low-detection smpling (see elow), we find tht the resulting distriution of receptors rising from R + pproches Poisson distriution if the coefficient of vrition (c.v.) of R + is less thn ~0.6 or gmm distriution otherwise (see prt of supplementry figure t The synpses without receptors, R, remin without receptors fter low-detection smpling. We fitted (nonliner lest-squres nlysis 31 ) the oserved distriutions to model distriution mde of Poisson plus extr zeroes (model hs two degrees of freedom) or gmm plus extr zeroes (model hs three degrees of freedom). In most cses, oserved distriutions were ccurtely descried y Poisson plus zeroes (p > 0.99, Kolmogorov-Smirnov test). In the reminder of cses, relile fit (p > 0.99) ws otined y using gmm plus zeroes. Nevertheless, the vlue for extr zeroes ws similr (within 5%) using either distriution. We use the vlue for extr zeroes s the estimted frction of synpses lcking receptors (FLR). Confidence limits for the estimted prmeters were determined y Monte Crlo simultions 32 (see elow). The ccurcy of the method, tht is, its ility to find the correct vlue for FLR, ws evluted y modeling ritrry receptor distriutions with different frction of synpses lcking receptors. Low-detection simultions (see elow) from these distriutions showed tht the estimted vlue for FLR coincides with the correct frction of synpses lcking receptors when the originl distriution is not very rod (low c.v.). In this cse, the estimtion for FLR ws firly independent from the detection vlue. On the contrry, the estimted FLR vlue tends to e lrger thn the correct one when the originl distriution is highly dispersed (high c.v.), nd this overestimtion increses s detection ecomes smller (supplementry figure, prt ). An overestimtion of FLR cn e detected y otining experimentl dt t different detection levels or y rndomly smpling the experimentl distriution with different detection vlues. If the clculted FLR vlue increses with decresing detection levels, we cn conclude tht the frction of synpses lcking receptors is eing overestimted. Conversely, the estimted FLR is ccurte when it is independent from the detection level. This test ws shown to e vlid for receptor distriutions with different verge numer of receptors per synpse or different frction lcking receptors (supplementry figure, prt ). LOW-DETECTION SIMULATIONS AND ESTIMATES OF CONFIDENCE LIMITS. Rndom low-detection smpling ws simulted s follows. A rndom numer etween 0 nd 1 ws ssigned to ech receptor in prticulr synpse. When the rndom numer ws higher thn the chosen detection vlue, the corresponding receptor ws rejected, tht is, not detected. Thus, the lower the detection vlue, the more likely it is tht receptor will e rejected. By repeting this process for every synpse in the originl distriution, low-detection distriution is generted with the non-rejected, tht is, detected receptors. In the cse of detection rte of one, simulted distriutions were generted y rndomly choosing synpses from the originl dt without ltering their numer of receptors. As mny synpses were chosen s were present in the originl distriution, ut the rndom selection llowed some synpses to e chosen severl times, wheres others my not hve een selected in one prticulr simultion. One thousnd independent simultions were crried out for nture neuroscience volume 2 no 1 jnury

6 rticles ech detection level. Then, the resulting distriutions were fitted individully to the model to estimte the corresponding vlue for the frction of synpses lcking receptors. With these independent vlues, proility distriutions nd confidence limits were generted for the estimted prmeter 32. ELECTROPHYSIOLOGY. Rt hippocmpl slices were prepred, nd whole-cell recordings were otined from CA1 neurons under visul guidnce with stndrd thing nd internl solutions s descried 7. Bthing medium contined 1 µm tetrodotoxin. Neuronl somt were held t 60 mv in voltge-clmp mode. Series resistnce ws not significntly correlted with men mepsc mplitude, nor ws it significntly different for the different ge groups. A ptch pipette contining 500 mm sucrose ws positioned ~100 µm from the cell odies in strtum rditum, nd pulses of pressure were pplied (10 psi, 100 ms, 0.3 Hz). Note: Further detils of methods nd supplementry figure my e found on the Nture Neuroscience we site t Acknowledgements We thnk A. Allen nd N. Dwkins-Pisni for technicl support, nd J. Lismn nd memers of the Mlinow nd Wenthold lortories for comments. RECEIVED 1 SEPTEMBER; ACCEPTED 20 NOVEMBER Hollmnn, M. & Heinemnn, S. Cloned glutmte receptors. Annu. Rev. Neurosci. 17, (1994). 2. Lio, D. & Mlinow, R. Deficiency in induction ut not expression of LTP in hippocmpl slices from young rts. Lern. Mem. 3, (1996). 3. Durnd, G., Kovlchuk, Y. & Konnerth, A. Long-term potentition nd functionl synpse induction in developing hippocmpus. Nture 381, (1996). 4. Hsi, A. Y., Mlenk, R. C. & Nicoll, R. A. Development of excittory circuitry in the hippocmpus. J. Neurophysiol. 79, (1998). 5. Wu, G.-Y., Mlinow, R. & Cline, H. T. Mturtion of centrl glutmtergic synpse. Science 274, (1996). 6. Isc, J. T., Crir, M. C., Nicoll, R. A. & Mlenk, R. C. Silent synpses during development of thlmocorticl inputs. Neuron 18, (1997). 7. Lio, D., Hessler, N. A. & Mlinow, R. 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Science 276, (1997). 14. Khrzi, V. N., Phend, K. D., Rustioni, A. & Weinerg, R. J. EM colocliztion of AMPA nd NMDA receptor suunits t synpses in rt cererl cortex. Neurosci. Lett. 210, (1996). 15. Desmond, N. L. & Weinerg, R. J. Enhnced expression of AMPA receptor protein t perforted xospinous synpses. Neuroreport 9, (1998). 16. Wenthold, R. J., Petrli, R. S., Blhos II, J. & Niedzielski, A. S. Evidence for multiple AMPA receptor complexes in hippocmpl CA1/CA2 neurons. J. Neurosci. 16, (1996). 17. Bekkers, J. M., Richerson, G. B. & Stevens, C. F. Origin of vriility in quntl size in cultured hippocmpl neurons nd hippocmpl slices. Proc. Ntl. Acd. Sci. USA 87, (1990). 18. Shtz, C. J. Impulse ctivity nd the ptterning of connections during CNS development. Neuron 5, (1990). 19. Fox, K. The criticl period for long-term potentition in primry sensory cortex. Neuron 15, (1995). 20. Ktz, L. C. & Shtz, C. J. Synptic ctivity nd the construction of corticl circuits. Science 274, (1996). 21. 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