Evidence against AMPA Receptor-Lacking Glutamatergic Synapses in the Superficial Dorsal Horn of the Rat Spinal Cord

Beha io al/Sys ems/Cogni i e E idence agains AMPA Recep o -Lacking Glu ama e gic Synapses in he Supe icial Do sal Ho n o he Ra Spinal Co d Toshiha u Yasaka, 1 Da id I. Hughes, 1 E ika Polga´ , 1 Ge gely G. Nagy, 1 Masahiko Wa anabe, 2 John S. Riddell, 1 and And ew J. Todd 1 1 Neu oscience and Molecula Pha macology, Facul y o Biomedical and Li e Sciences, Uni e si y o Glasgow, Glasgow G12 8QQ, Uni ed Kingdom, and 2 Depa men o Ana omy, Hokkaido Uni e si y School o Medicine, Sappo o 060-8638, Japan Pu e NMDA ecep o (NMDA )-media ed EPSCs, hough o co espond o “silen ” glu ama e gic synapses ha lack AMPA ecep o s (AMPA s), ha e been obse ed in supe icial spinal do sal ho n o neona al bu no adul a s. Recen ana omical s udies sugges ha AMPA s a e p esen a i ually all glu ama e gic synapses in his egion in adul s. We used an igen e ie al o examine colocaliza ion o AMPA s and PSD-95 (a ma ke o glu ama e gic synapses) in laminae I–II o neona al and adul a s. We ound a high deg ee o colocaliza ion in all cases, which sugges s ha AMPA s a e p esen in he g ea majo i y o glu ama e gic synapses e en in neona al animals. We he e o e eexamined e idence o silen synapses by pe o ming blind whole-cell eco dings om supe icial do sal ho n neu ons in slices om neona al o adul a s, wi h ocal s imula ion o ac i a e glu ama e gic synapses. On some occasions in bo h neona al (10 o 109, 9%) and adul (9 o 77, 12%) slices, NMDA -media ed EPSCs we e obse ed when he holding po en ial was aised o ⫹50 mV a a s imulus s eng h ha had ailed o e oke AMPA -media ed EPSCs. Howe e , in all cases es ed, AMPA -media ed EPSCs we e hen obse ed when he cell was e u ned o ⫺70 mV; his and o he p ope ies o he EPSCs sugges ha hey do no ep esen genuine silen synapses. When compa ed wi h p e ious indings, ou esul s indica e ha he appea ance o silen synapses depends on expe imen al p o ocol. This sugges s ha pu e NMDA -media ed EPSCs seen in p e ious s udies do no co espond o AMPA -lacking synapses bu esul om ano he mechanism, o example, loss o labile AMPA s om ecen ly o med synapses. In oduc ion Glu ama e is he main exci a o y ansmi e in he spinal do sal ho n and is eleased by p ima y a e en s, axons o exci a o y in e neu ons, and ce ain descending axons (B oman, 1994; Todd e al., 2003; Todd and Koe be , 2005). Glu ama e ac s on iono opic and me abo opic ecep o s, which a e widely dis ib- u ed h oughou he do sal ho n. Iono opic ecep o s o he AMPA ype (AMPA s) media e as exci a o y ansmission (Yoshimu a and Jessell, 1990; Yoshimu a and Nishi, 1992) and con ey in o ma ion pe cei ed as pain (Dickenson e al., 1997; Ga y and Flee wood-Walke , 2004). In con as , NMDA ecep- o s (NMDA s) a e blocked by Mg 2⫹ a es ing po en ial bu ac i a ed when he memb ane is depola ized, allowing en y o Ca 2⫹ , which leads o cen al sensi iza ion o do sal ho n neu ons. NMDA ac i a ion unde lies ce ain o ms o hype algesia and allodynia (Dickenson e al., 1997). Elec ophysiological s udies ha e demons a ed EPSCs medi- a ed by NMDA bu no AMPA ecep o s in b ain (Kullmann, 1994; Isaac e al., 1995; Liao e al., 1995; Du and e al., 1996; Mon gome y e al., 2001) and spinal co d (Ba doni e al., 1998; Li and Zhuo, 1998; Baba e al., 2000; Jung e al., 2005). The mos widely accep ed explana ion o his phenomenon is ha he e a e silen synapses ha possess NMDA s bu lack unc ional AMPA s. I is hough ha “unsilencing” o hese by inse ion o AMPA s con ibu es o long- e m po en ia ion and ma u a ion o glu a- ma e gic synapses. S udies o silen synapses in spinal co d ha e used “minimal s imula ion” p o ocols, in which elec ical s imuli a e applied nea he eco ded cell o in he do sal oo (Ba doni e al., 1998; Li and Zhuo, 1998; Baba e al., 2000) and s imulus ampli ude is educed o a le el a which AMPA -media ed EPSCs a e no longe de ec ed. Changing he holding po en ial o a posi i e alue while con inuing s imula ion a his in ensi y some imes esul ed in NMDA -media ed EPSCs. Li and Zhuo (1998) and Ba doni e al. (1998) obse ed pu e NMDA EPSCs in neu ons in spinal co d slices om a s aged ⬍15 d. Baba e al. (2000) e- po ed ha , al hough such EPSCs could be de ec ed in slices om neona al a s, hey we e no seen in hose om adul s and con- cluded ha silen synapses we e de elopmen ally egula ed. We ecen ly used an an igen e ie al me hod ha e eals syn- ap ic p o eins and demons a ed a e y high deg ee o colocaliza- ion o AMPA s wi h PSD-95 in laminae I–III o adul a spinal co d (Polga´ e al., 2008). PSD-95, a membe o he memb ane- associa ed guanyla e kinase (MAGUK) amily, is hough o be an Recei ed June 5, 2009; e ised Sep . 9, 2009; accep ed Sep . 15, 2009. This wo k was suppo ed by he Wellcome T us . We hank R. Ke , C. Wa , and M. McGill o expe echnical assis ance and D s. R. Ba doni, I. Vida, M. Bea o, and Z. Puska´ o help ul discussion and ad ice. Co espondenceshouldbeadd essed oP o .And ewJ.Todd,SpinalCo dG oup,Wes MedicalBuilding,Uni e - si y o Glasgow, Uni e si y A enue, Glasgow G12 8QQ, UK. E-mail: [email p o ec ed]. DOI:10.1523/JNEUROSCI.2628-09.2009 Copy igh © 2009 Socie y o Neu oscience 0270-6474/09/2913401-09$15.00/0 The Jou nal o Neu oscience, Oc obe 21, 2009 •29(42):13401–13409 • 13401 in eg al pa o he pos synap ic densi y a glu ama e gic syn- apses, and ou inding he e o e sugges s ha AMPA s a e p esen a i ually all glu ama e gic synapses in his egion in he adul . In his s udy, we ha e analyzed he ela ionship be ween AMPA s and PSD-95 in adul and neona al a s o look o e i- dence o AMPA -lacking synapses in young animals. Because ou esul s did no suppo he sugges ion ha signi ican numbe s o glu ama e gic synapses lack AMPA s a any o he ages examined, we pe o med elec ophysiological s udies on slices om neona- al and adul a s, o eexamine he e idence o silen synapses. Ma e ials and Me hods All expe imen s we e app o ed by he E hical Re iew P ocess Applica- ions Panel o he Uni e si y o Glasgow and we e pe o med in acco - dance wi h he Eu opean Communi y di ec i e 86/609/EC and he Uni ed Kingdom Animals (Scien i ic P ocedu es) Ac 1986. Immunocy ochemical de ec ion o AMPA s. Wis a a s o ei he sex we e used in his pa o he s udy. These included adul s (230–280 g; Ha lan), as well as animals aged 2 weeks and 1 week (Glasgow Uni e si y Biological Se ices). In all cases, he animals we e deeply anes he ized wi h pen oba bi one and pe used h ough he le en icle wi h ixa i e ha con ained 4% eshly depolyme ized o maldehyde (200 ml o 1- o 2-week-old a s, 1000 ml o adul s). Midlumba spinal co d blocks we e dissec ed ou , s o ed in he same ixa i e o 2–8 h a 4°C, and cu in o 60 ␮ m ans e se sec ions wi h a Vib a ome. The sec ions we e ea ed in 50% e hanol o 30 min o enhance an ibody pene a ion and p ocessed o an igen e ie al by incuba ing hem o 10 min a 37°C in 0.2 MHCl con aining 1 mg/ml pepsin (Dako) (Wa anabe e al., 1998; Nagy e al., 2004). The sec ions we e hen incuba ed o 3dinamix u e o abbi an ibody agains PSD-95 (1:200) (Fukaya and Wa anabe, 2000) and guinea pig an ibody ha ecognizes all ou AMPA subuni s (pan- AMPA an ibody; 1:100) (Fukaya e al., 2006; Polga´ e al., 2008), ol- lowed by1din heapp op ia e species-speci ic seconda y an ibodies, which we e aised in donkey and conjuga ed o Alexa 488 (1:500; In i o- gen) o Rhodamine Red (1:100; Jackson ImmunoResea ch). They we e moun ed in an i ade medium (Vec ashield; Vec o Labo a o ies) and s o ed a ⫺20°C. All an ibodies we e dilu ed in PBS ha con ained 0. 3 M NaCl and 0.3% T i on X-100, and incuba ions we e a 4°C. Sec ions om six adul , i e 2-week-old, and i e 1-week-old a s we e used o analyze immuno eac i e punc a in laminae I and II. These sec- ions we e scanned wi h a Bio-Rad Radiance 2100 con ocal mic oscope, and all o he analysis was pe o med wi h Me aMo ph so wa e (Uni- e sal Imaging Co po a ion) on s acks o con ocal images scanned sequen ially ( o a oid luo escen bleed- h ough) wi h a 60⫻oil- imme sion lens and a z-sepa a ion o 0.3 ␮ m. Wi h he pan-AMPA an ibody, we ound ha he e was a low le el o s aining o e he cy o- plasm o ce ain neu ons, and, o each con ocal image s ack, we mea- su ed he mean pixel in ensi y o e he cy oplasm o he i e cells ha showed he s onges s aining and mul iplied his by h ee o ob ain a h eshold alue. Any punc um wi h a leas one pixel ha exceeded his h eshold alue was de ined as pan-AMPA immuno eac i e. F om each o he a s, we opened con ocal image s acks ep esen ing PSD-95 and selec ed 100 immuno eac i e punc a in lamina I and 200 such punc a in lamina II. To a oid bias, a g id was applied o each s ack, and he punc- um nea es he bo om igh -hand co ne o each g id squa e on a p e- de e mined op ical sec ion was selec ed (Nagy e al., 2004). We hen me ged image s acks o PSD-95 and pan-AMPA and de e mined whe he each o he selec ed PSD-95-immuno eac i e punc a was pan- AMPA posi i e (i.e., had a leas one pixel ha exceeded he h eshold o pan-AMPA s aining). Fo hose PSD-95-immuno eac i e punc a in which pan-AMPA immunos aining was below h eshold, we also no ed whe he weak pan-AMPA immuno eac i i y was p esen . The p opo - ion o pan-AMPA -posi i e punc a ha we e PSD-95 immuno eac i e was de e mined by selec ing 50 and 100 such punc a om laminae I and II o each animal, espec i ely, using a simila me hod. The pan-AMPA an ibody was aised agains esidues 727-745 o he mouse GluR1 (a egion ha is iden ical in all ou AMPA subuni s) and de ec ed each o he ou AMPA subuni s in ans ec ed cells, wi h a ace o c oss- eac i i y o he kaina e ecep o subuni GluR6 (Fukaya e al., 2006). We ha e shown ha , a e an igen e ie al, all punc a ha a e immunos ained wi h an ibodies agains GluR1, GluR2, GluR3, o GluR4 a e also immuno eac i e wi h his an ibody (Polga´ e al., 2008). The PSD-95 an ibody was aised agains he N- e minal egion ( esidues 1-64) o mouse PSD-95 and ecognizes a band o 87–97 kDa on Wes e n blo s o a b ain homogena es (Fukaya and Wa anabe, 2000). This an- ibody does no show synap ic labeling in sec ions om a PSD-95 mu an mouse in which a unca ed o m o he p o ein is no a ge ed o syn- apses (Migaud e al., 1998; Polga´ e al., 2008). P epa a ion o slices o whole-cell eco ding. Neona al (aged 4–14 d; Glasgow Uni e si y Biological Se ices) and young adul (aged 6–9 weeks; 140–290 g; Ha lan) Wis a a s o ei he sex we e used in his pa o he s udy. The me hods used o p epa ing spinal co d slices we e simila o hose desc ibed p e iously (Yasaka e al., 2007), wi h mino modi ica ions. B ie ly, he a s we e deeply anes he ized wi h halo hane, and, a e ho acolumba laminec omy, he lumbosac al spinal co d was excised and placed in a p eoxygena ed cold (1–3°C) low-sodium K ebs’ solu ion con aining he ollowing (in mM): 250 suc ose, 2.5 KCl, 0.5 CaCl 2 , 6 MgCl 2 , 1.25 NaH 2 PO 4 , 26 NaHCO 3 , and 25 glucose. The a s we e hen immedia ely killed by decapi a ion. All o he en al and do sal oo s we e cu , and he pia-a achnoid memb ane was emo ed. T ans e se slices (500 ␮ m) we e cu on a ib a ing-blade mic o ome (VT1000S; Leica Mic osys ems) and s o ed in he same solu ion. A e a eco e y pe iod o a leas 1 h, he slice was ans e ed o he eco ding chambe and pe used con inuously a a low a e o 5 ml/min wi h no mal K ebs’ solu ion equilib a ed wi h 95% O 2 and 5% CO 2 a oom empe a u e. The K ebs’ solu ion con ained he ollowing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 1.25 NaH 2 PO 4 , 26 NaHCO 3 , and 25 glucose. Reco ding and s imula ion. Blind whole-cell eco dings we e made om neu ons in he supe icial do sal ho n (laminae I–II) using pa ch pipe es illed wi h a solu ion con aining he ollowing (in mM): 110 Cs 2 SO 4 , 5 e a- e hylammonium (TEA)-Cl, 0.5 CaCl 2 , 2 MgCl 2 , 5 EGTA, 5 HEPES, 5 Mg-ATP, and 5 QX-314 [N-(2,6-dime hylphenylca bamoylme hyl) ie h- ylammonium chlo ide], as desc ibed p e iously (Yasaka e al., 2007). The ip esis ance o he pa ch pipe es was 8–12 M⍀. Signals we e acqui ed wi h an Axopa ch 200B ampli ie (Molecula De ices). Da a we e digi- ized wi h an analog- o-digi al con e e (Digida a 1321A; Molecula De ices), s o ed on a pe sonal compu e using a da a acquisi ion p o- g am (Clampex e sion 9; Molecula De ices), and analyzed wi h Clamp i so wa e ( e sion 9; Molecula De ices). In one se ies o expe - imen s, 1,2-bis(2-aminophenoxy)e hane-N,N,N⬘,N⬘- e aace ic acid (BAPTA) was used in he pipe e solu ion, which con ained he ollowing (in mM): 50 Cs 2 SO 4 , 40 Cs-BAPTA, 5 TEA-Cl, 4 CaCl 2 , 2 MgCl 2 , 5 EGTA, 5 HEPES, 5 Mg-ATP, and 5 QX-314. A minimal s imula ion p o ocol was used o de ec silen synapses (see Fig. 3A). Glass pipe es o he same ype as he eco ding elec odes we e used o ocal s imula ion. In ol age-clamp mode, cells we e ini ially held a ⫺70 mV. A s imula ing elec ode was hen placed as close as possible o he eco ded cell, and ocal s imula ion was applied a low equency (0.2 Hz, 100 ␮ s du a ion). I EPSCs we e no e oked in he eco ded cell, he s imula ing elec ode was mo ed o ano he loca ion nea he cell. The dis ance be ween s imula ing and eco ding elec odes was gene ally ⬍100 ␮ m. Once e oked EPSCs we e obse ed, he s imulus in ensi y was educed o jus below h eshold, so ha he cell ailed o espond o he ocal s imula ion. A e a ce ain numbe o ailu es, he memb ane po en ial o he eco ded cell was aised o ⫹50 mV o es whe he NMDA -media ed EPSCs we e p esen . In mos cases, he mem- b ane po en ial was hen e u ned o ⫺70 mV o check whe he AMPA - media ed EPSCs we e p esen , and, in many cases, he h eshold s imulus in ensi y o e oking AMPA -media ed EPSCs was measu ed again. IPSCs we e blocked by adminis e ing bicuculline (10–20 ␮ M) and s ychnine (5 ␮ M) con inuously h oughou he pe iod o eco ding. The absence o IPSCs, which migh ha e been mis aken o NMDA - media ed EPSCs a ⫹50 mV, was checked o each cell o he i s si e om which ocal s imula ion e oked EPSCs, by aising he holding po- en ial o ⬃0 mV and applying be ween 1 and 20 s imuli. This es was pe o med jus be o e inc easing he holding po en ial o ⫹50 mV. A e 13402 •J. Neu osci., Oc obe 21, 2009 •29(42):13401–13409 Yasaka e al. •Silen Synapses in Do sal Ho n each change in he holding po en ial, es s imuli we e esumed as soon as memb ane cu en had s abilized. Iden i ica ion o supe icial do sal ho n neu ons. As epo ed p e iously (Yoshimu a and Jessell, 1989), he supe icial do sal ho n was easily iden- i ied wi h a binocula mic oscope as a anslucen band. Neu ons we e eco ded wi hin his egion a a dep h o 100–300 ␮ m om he su ace o he slices, and, in mos cases, hei iden i y was u he con i med by labeling wi h Neu obio in (0.2% in he elec ode solu ion; Vec o Labo- a o ies) om he pa ch pipe e. A e comple ion o he elec ophysio- logical eco dings, he spinal co d slices we e imme sed o e nigh in 4% eshly depolyme ized o maldehyde in 0.1 Mphospha e bu e , pH 7.4, a 4°C, insed in phospha e bu e , and hen sec ioned ans e sely a 60 ␮ m hickness wi h a Vib a ome. F ee- loa ing sec ions we e incuba ed o e nigh a 4°C in PBS ha con ained 0.3% T i on X-100 and s ep a- idin conjuga ed o Rhodamine Red (1:1000; Jackson ImmunoResea ch) and hen washed se e al imes in PBS. The slices we e moun ed in Vec ash- ield and scanned wi h a con ocal mic oscope (Bio-Rad MRC1024) o de e - mine he loca ion o he cell body. D ug applica ion. D ugs dissol ed in K ebs’ solu ion we e applied by exchanging solu ions ia a h ee-way s opcock wi hou al e ing he pe - usion a e o empe a u e. The d ugs used we e s ychnine (5 ␮ M; Sigma), bicuculline (20–40 ␮ M; Sigma), 2-amino-5-phosphono ale ic acid (APV) (50–100 ␮ M; Toc is Bioscience), 2,3-dioxo-6-ni o-1,2,3,4- e ahyd obenzo[ ]quinoxaline-7-sul onamide (NBQX) (20 ␮ M; Toc is Bioscience), and BAPTA e acesium sal (40 mMin pipe e solu ion; In i ogen). S ock solu ions o s ychnine and APV we e p epa ed in wa e . The s ock solu ions o bicuculline and NBQX we e p epa ed in dime hylsul- phoxide (DMSO). The inal concen a ion o DMSO used in he expe imen did no exceed 0.1%. Resul s Immunocy ochemis y A e an igen e ie al wi h pepsin, bo h PSD-95 and pan- AMPA immuno eac i i y we e p esen h oughou he g ay ma e o he spinal co d. As epo ed p e iously (Polga´ e al., 2008), he densi y o s aining wi h bo h an ibodies was highes in lamina II (Fig. 1). A e y simila lamina dis ibu ion o s aining o bo h PSD-95 and pan-AMPA was al eady p esen in he do sal ho n by 1 week o age, and, a low magni ica ion, he main di e ence in he appea ance a his s age was he absence o my- elin bundles ha pass h ough he medial pa o he do sal ho n in he adul (Fig. 1). A high magni ica ion, he labeling wi h bo h an ibodies was seen o be punc a e, and he wo ypes o immuno eac i i y showed ex ensi e colocaliza ion a each de elopmen al s age (Fig. 2). In he adul , immunos ained punc a in lamina II o en appea ed in clus e s ha we e a anged a ound an unlabeled cen- al space (Fig. 2, a ows). These clus e s a e likely o ep esen synap ic glome uli, because we ha e shown ha he cen al a ea may be occupied by an axonal bou on ha can be immuno- s ained wi h an ibody agains VGLUT1 o labeled wi h he lec in Bandei aea simplici olia isolec in B4, which a e ma ke s o cen- al e minals o low- h eshold myelina ed and nonpep ide gic unmyelina ed p ima y a e en e minals, espec i ely (Nagy e al., 2004). These clus e s appea ed o be less common in he spinal co ds o 2-week-old animals and we e seldom seen in hose om 1-week-old a s. Quan i a i e analysis o immuno eac i e punc a in he supe - icial do sal ho n con i med a high deg ee o colocaliza ion o he wo ypes o immuno eac i i y (Table 1, Fig. 2). Be ween 93 and 94% o PSD-95-posi i e punc a in laminae I and II we e classi ied as pan-AMPA immuno eac i e, because s aining wi h he pan- AMPA an ibody exceeded he h eshold alue (see Ma e ials and Me hods). Howe e , his is p obably an unde es ima e because weak pan-AMPA immunos aining was obse ed a many o he punc a ha we e below h eshold. When hese we e included, he p opo ions o PSD-95 punc a wi h pan-AMPA s aining we e be ween 97 and 99% ( o laminae I and II a each age g oup). Be ween 94 and 99% o pan-AMPA punc a we e PSD-95 immu- no eac i e (Table 1). The e was no signi ican di e ence in he p opo ion o PSD-95 punc a ha we e pan-AMPA immu- no eac i e a any age in lamina I o II, ei he when using he h eshold o de e mine he p esence o pan-AMPA s aining o judging his by eye ( wo-way ANOVA, p⬎0.05). The e was also no di e ence in he p opo ion o pan-AMPA punc a in lamina I o II ha we e PSD-95 immuno eac i e a any age (one-way ANOVA, p⬎0.05). Elec ophysiology The sample o cells om which eco dings we e made Blind whole-cell eco dings we e made om supe icial do sal ho n neu ons in ans e se slices p epa ed om 30 adul and 40 neona al a s. Fo 126 o he eco ded neu ons (59 in slices om adul a s, 67 in slices om neona al a s), EPSCs we e e oked by ocal s imula ion (Fig. 3A), and hese we e subsequen ly es ed wi h he minimal s imula ion p o ocol o he p esence o “silen synapses” (see below). Six y- ou o hese cells we e labeled wi h Neu obio in o con i m hei loca ion. These cells we e si ua ed h oughou he supe icial do sal ho n and we e simila ly dis ib- u ed in slices om adul and neona al animals (Fig. 3C). Figu e 1. The dis ibu ion o PSD-95 and pan-AMPA immuno eac i i y in he do sal ho n o 1-week-old and adul a s, seen a e an igen e ie al wi h pepsin. In each ow, PSD-95 ( ed) is on hele ,pan-AMPA (pAMPA ,g een)isin hemiddle,andame gedimageisshownon he igh .No e ha hedis ibu iono each ypeo s ainingissimila a he woages,wi h hehighes densi y ina egiono hesupe icialdo salho n ha co esponds olaminaII.Themyelinbundles(whichappea asuns aineda easin hemedialpa o hedo salho n)a eonlyp esen in headul .Each image was ob ained om a single op ical sec ion scanned wi h a con ocal mic oscope. Scale ba , 100 ␮ m. Yasaka e al. •Silen Synapses in Do sal Ho n J. Neu osci., Oc obe 21, 2009 •29(42):13401–13409 • 13403 EPSCs e oked by ocal s imula ion The p esence o AMPA - and NMDA - media ed componen s o he EPSCs e oked by ocal s imula ion was con i med pha - macologically in an addi ional sample o 11 neu ons ( i e om adul and six om neo- na al animals) (Fig. 3B). APV subs an ially educed he ampli ude o EPSCs e oked a a holding po en ial o ⫹50 mV, whe eas sub- sequen applica ion o NBQX abolished he esidual as componen o hese EPSCs. APV had no a ec on he ampli ude o EPSCs eco ded a a holding po en ial o ⫺70 mV, bu hese we e comple ely abol- ished by subsequen applica ion o NBQX. All expe imen s we e conduc ed in he p es- ence o bicuculline and s ychnine, and IPSCs we e ne e seen in esponse o ocal s imula ion. Minimal s imula ion es o silen synapses Responses o ocal s imula ion we e es ed using he minimal s imula ion p o ocol. Tes s we e pe o med a be ween 1 and 5 si es pe neu on (a e age o 1.5), and he esul s ob ained a e summa ized in Table 2. In o al, es s we e made a e s imula- ion a 186 si es: 77 si es in slices om adul animals and 109 si es in slices om neona al animals. In some o hese expe - imen s, BAPTA was included in he eco ding pipe e, bu , be- cause his was ound o ha e no signi ican e ec on he appea ance o EPSCs when he holding po en ial was aised o ⫹50 mV (see below), da a om eco dings wi h and wi hou BAPTA we e pooled o his pa o he analysis. An example o he minimal s imula ion p o ocol is illus a ed in Figu e 4A. Focal s imuli e oking EPSCs we e ini ially applied while eco ding wi h a holding po en ial o ⫺70 mV. The s imu- lus in ensi y was hen educed un il he cell esponded o ewe han 5% o a se ies o consecu i e s imuli (5–172 s imuli, a e age o 39). The holding po en ial was hen inc eased o ⫹50 mV, and he esponse o he cell o ocal s imula ion a he same in ensi y was es ed (6–98 s imuli, a e age o 34). Fo he majo i y o syn- ap ic connec ions es ed, no esponse occu ed when s imuli we e applied a he posi i e holding po en ial. Howe e , in a mino i y o es s (19 o 186, 10%), EPSCs we e e oked by s imuli applied while eco ding a a posi i e holding po en ial (Fig. 4). The p opo ion o s imula ion si es a which EPSCs we e e oked while he neu on was held a a posi i e po en ial did no di e signi ican ly be ween slices om adul (11.7%) and neona al (9.2%) animals (Table 2) ( p⫽0.8, Fishe ’s exac p obabili y es ). Fu he mo e, wi hin he neona al g oup, he e was no co - ela ion be ween he p opo ion o es ed connec ions ha showed his phenomenon and age o he animal om which he slice was ob ained ( ⫽⫺0.62, p⫽0.1). All o he 19 connec ions o which EPSCs we e seen when he neu on was held a a posi i e po en ial we e ob ained in di e en slices, and all bu wo o hese we e om di e en animals. Thi een o hese connec ions we e a he i s ocal s imula ion si e used o he neu on unde s udy, whe eas i e we e ob ained a he second and one a he hi d s imula ion si e. In some cases, he e was a ia ion in he la ency o EPSCs e oked by s imuli close o h eshold in ensi y while he cell was held a ⫺70 and ⫹50 mV. Some o his may be a ibu ed o a ia ion in he iming o ansmi e elease as has been e- po ed o occu a ce ain synapses (Feldmeye and Sakmann, 2000; He and Jonas, 2005). Howe e , a some connec ions, i may ha e esul ed om he ac ha ocal s imula ion a h esh- old in ensi y could ac i a e mo e han one p esynap ic elemen wi h simila h esholds, and any combina ion o hese may be ac i a ed by successi e s imuli applied a his in ensi y. The e was also some a ia ion in la ency ea lie in he s imula ion p o ocol, when he cell was held a ⫺70 mV and s imula ion was abo e h eshold. EPSC esponses o en occu ed wi hin he i s ew s imuli a e aising he holding po en ial. In 15 o he 19 cases in which EPSCs we e seen a ⫹50 mV, he holding po en ial had been s epped o ⬃0 mV (no co ec ion made o liquid junc ion po- en ial) be o e being aised o ⫹50 mV o con i m he absence o IPSCs (see abo e). In six o hese cases, EPSCs we e seen wi hin he i s six s imuli while he cell was held a 0 mV, and, in 14 o he 19 cases, he i s EPSCs we e seen wi hin ou s imuli a e he holding po en ial was aised o ⫹50 mV. In hose cells ha showed EPSCs when held a a posi i e po- en ial, he EPSCs we e no usually e oked by e e y s imulus. The esponse a e o he 19 connec ions anged om 15 o 100% Figu e 2. Colocaliza ion o punc a e s aining wi h he PSD-95 and pan-AMPA (pAMPA ) an ibodies in lamina II a di e en de elopmen als ages. In each ow, PSD-95( ed)is on he le ,pan-AMPA (pAMPA ,g een) is in hemiddle,anda me ged image is shown on he igh . No e ha mos punc a a e s ained wi h bo h an ibodies. A ows indica e clus e s o punc a ha p obably co espond osynap icglome uli.Eachimageisap ojec iono wo con ocalop icalsec ionsa 0.3 ␮ mz-spacing.Scaleba ,5 ␮ m. Table 1. Colocaliza ion o PSD-95 and pan-AMPA punc a e s aining in lamina II a di e en ages Lamina % PSD-95 also pan-AMPA % pan-AMPA also PSD-95 IIIIII 1 week (n⫽5) 92.6 (88–96) 93.9 (92–98) 95.2 (92–98) 97.4 (97–99) 2 week (n⫽5) 93.4 (89–98) 92.8 (88–95) 93.6 (90–98) 98.2 (96–99) Adul (n⫽6) 94.3 (89–97) 94.0 (91–97) 96.7 (94–98) 98.8 (96–100) Numbe s in pa en heses a e he anges wi hin each g oup. 13404 •J. Neu osci., Oc obe 21, 2009 •29(42):13401–13409 Yasaka e al. •Silen Synapses in Do sal Ho n (a e age 35%) (Fig. 4B), and esponses gene ally occu ed h oughou he pe iod o s imula ion (Figs. 4A,5C). Howe e , hey we e some imes seen mainly in he ea ly (Fig. 5A) o la e (Fig. 5B) pa o he pe iod du ing which he cell was held a ⫹50 mV. To es ablish whe he EPSCs occu ed mo e o en du ing he ea ly o la e pa o his pe iod, we de e mined he p opo ion o hese ha occu ed in esponse o he i s 50% o s imuli ha we e applied while he cell was held a ⫹50 mV. The mean alue was 0.54 ( ange o 0.05–1), and his did no di e signi ican ly om he alue o 0.5 ha would be expec ed i esponses oc- cu ed in a andom manne h oughou he pe iod ( p⫽0.52, one-sample es ). The ampli udes o he a e aged EPSC esponses seen a posi- i e holding po en ials anged om 7 o 80 pA and we e simila in neu ons om adul and neona al animals. In one case eco ded in an adul slice, in which he pha macology o hese EPSCs was in es iga ed, hey we e subs an ially educed by applica ion o he NMDA blocke APV (Fig. 6). E ec o e u ning o a nega i e holding po en ial Fo 109 o he 186 connec ions in es i- ga ed, he holding po en ial was e u ned o ⫺70 mV immedia ely a e es s a ⫹50 mV, and ocal s imula ion was con inued a he same s imulus in ensi y. Ten o he connec ions es ed in his way ( i e om adul animals and i e om neona al ani- mals) belonged o he mino i y ha e- sponded wi h EPSCs on swi ching o he posi i e holding po en ial. In all o hese cases, ocal s imula ion con inued o e oke EPSCs on e u ning o he nega i e holding po en ial (Figs. 4, 5). In mos cases (8 o 10), he esponse equency a ⫺70 mV was g ea e han ha a ⫹50 mV (a e age inc ease om 27 o 45%) (Fig. 4B). Fo one o hese cases, an EPSC was seen when he holding po en ial was e- u ned o ⫺70 mV du ing applica ion o APV, which had supp essed he NMDA - media ed componen o he EPSC seen a ⫹50 mV (Fig. 6). To con i m ha he EPSCs seen when e u ning o ⫺70 mV we e AMPA media ed, we compa ed he hal -wid hs o he EPSCs eco ded a ⫹50 mV and hose eco ded when he holding po en ial was e u ned o ⫺70 mV in all 10 cases in which his phenomenon oc- cu ed. The a ios o hese hal -wid hs (⫹50/⫺70 mV) a ied om 3 o 17.4 (median o 9.5), e lec ing he much longe du a ion o NMDA EPSCs. This did no di e signi i- can ly om he a io o hal -wid hs a ⫹50 and ⫺70 mV ob ained om he 11 connec ions in which APV and NBQX we e used o con i m he iden i y o he wo ypes o EPSC ( ange o 3–25.5, median o 13.5; Mann–Whi ney U es , p⫽0.42). This s ongly suppo s he sugges ion ha he EPSCs eco ded a ⫺70 mV a e he cells had been held a ⫹50 mV we e media ed by AMPA s. We compa ed ampli udes o EPSCs e oked du ing he i s hal o he es pe iod when he cell was e u ned o ⫺70 mV (ea ly) wi h hose in he second hal o his pe iod (la e). The mean ⫾SD a io o la e/ea ly ampli udes was 0.94 ⫾0.18, and his did no di e signi ican ly om 1 (one-sample es , p⫽0.33), indica ing ha he ampli udes o EPSCs did no change du ing he cou se o he s imula ion a ⫺70 mV. The emaining 99 connec ions ha we e es ed using his pa - adigm showed no EPSCs a a posi i e holding po en ial. How- e e , 10 o hese showed EPSCs on e u ning o a nega i e holding po en ial. Fo 86 o he 89 connec ions ha did no espond o ocal s imula ion a a posi i e holding po en ial o when he memb ane po en ial was e u ned o ⫺70 mV, he h eshold s imulus in ensi y was measu ed again a e e u ning o a hold- ing po en ial o ⫺70 mV in he same manne as a he s a o he p o ocol. Fo 12 o he 86 connec ions, he e had been no change in he h eshold, whe eas o he o he 64, he h eshold s imulus in ensi y was ound o ha e inc eased. When he p ecise h esh- Figu e 3. Reco ding om supe icial do sal ho n neu ons and applica ion o ocal s imula ion. A, Schema ic diag am o expe - imen ala angemen . Whole-cell eco dings we e ob ained omlamina I/II neu ons, andEPSCse okedby ocal s imula ion we e eco ded while IPSCs we e blocked by bicuculline and s ychnine. B, Pha macological demons a ions o EPSC componen s. A a holdingpo en ialo ⫺70mV(bo om eco ds),inwa dlydi ec edEPSCswi h as kine icswe eabolishedbyNBQXbu no APV.A aholding po en ial o ⫹50mV( op 2 owso eco ds)ou wa dly di ec ed EPSCswi hslow kine ics we e blockedbyAPV, whe eas EPSCs wi h as kine ics we e sensi i e o NBQX. No e di e en imescales o op and middle ows o eco ds. C, Loca ions o he in acellula ly labeled neu ons. The plo s show he loca ions o 32 neu ons eco ded in slices om neona al animals ( op) and 32 neu ons eco ded in slices om adul animals (bo om). In each case, cells ha showed EPSCs a posi i e holding po en ials a e shown wi h illed ci cles, and he o he cells a e shown wi h open ci cles. Table 2. Summa y o incidence o “silen synapses” in elec ophysiological expe imen s Neona e Adul To al Numbe o cells 67 59 126 Numbe o connec ions 109 77 186 Numbe o silen synapses 10 (9%) 9 (12%) 19 (10%) This shows he incidence o connec ions a which EPSCs we e obse ed a a holding po en ial o ⫹50 mV while s imula inga ale el ha wasini iallysub h esholdwhile hecellwashelda ⫺70mV(“silen synapses”).Resul s ob ained in slices om neona es (4–14 d) o adul s (6–9 weeks) a e shown sepa a ely. Yasaka e al. •Silen Synapses in Do sal Ho n J. Neu osci., Oc obe 21, 2009 •29(42):13401–13409 • 13405 old s imulus in ensi y was de e mined (n⫽63), i was ound o ha e inc eased on a e age o 130% o he h eshold alue de e mined du ing he i s s imula ion pe iod a a nega i e holding po en ial. E ec o BAPTA To es he possibili y ha he appea ance o EPSCs when he holding po en ial was e u ned o ⫺70 mV in ol ed a Ca 2⫹ - dependen mechanism, BAPTA was in- cluded in he eco ding pipe e solu ion in la e expe imen s in he se ies. The esul s ob ained wi h and wi hou BAPTA in he eco ding elec ode a e summa ized in Table 3. A o al o 70 o he 186 connec- ions in es iga ed we e eco ded wi h BAPTA-con aining elec odes (42 om young animals and 28 om adul s). The g ea majo i y o connec ions es ed wi h BAPTA (66 o 70) showed no EPSC e- sponses a he posi i e holding po en ial. Howe e , as wi h eco dings ha we e made wi hou BAPTA in he elec ode, EPSCs we e e oked in a small numbe o connec ions. O hese, 2 o 42 (4.8%) we e eco ded in young animals and 2 o 28 (7.1%) in adul animals. The o al p opo - ion o connec ions ha showed his phe- nomenon when eco dings we e made wi h (4 o 70, 5.7%) o wi hou (15 o 115, 13%) BAPTA did no di e signi ican ly (Fishe ’s exac p obabili y es , p⫽0.21). In each o hese ou cases, EPSCs we e seen on e u ning o he nega i e holding po en ial (Figs. 4, 5). Al hough we canno be ce ain ha BAPTA eached an adequa e concen a ion o p e en ises in Ca 2⫹ con- cen a ion h oughou he dend i ic ees o he cells, he e a e wo easons why we belie e ha his is e y likely o ha e been he case. Fi s , all o he cells had been in whole-cell con igu a ion wi h BAPTA-con aining pipe es o ⬎20 min be o e he mem- b ane po en ial was aised o ⫹50 mV du ing ocal s imula ion, and we ha e ound ha 20 min is adequa e o allow di usion o Neu obio in (which has a molecula weigh o 323 compa ed wi h ha o 478 o BAPTA), h oughou he en i e dend i ic ees o lamina II neu ons (T. Yasaka, unpublished obse a ions). Second, he concen a ion o BAPTA in ou pipe es (40 mM) was conside ably highe han ha gene ally used in whole-cell pa ch- clamp eco ding s udies (5–20 mM). Discussion The main indings o his s udy we e as ollows: (1) he e was a high deg ee o colocaliza ion o punc a e s aining o AMPA s and PSD-95 in laminae I and II o adul and neona al a s, wi h ew PSD-95 punc a lacking pan-AMPA immuno eac i i y, and (2) genuinely silen (pu e NMDA -media ed) synapses we e no obse ed in neu ons eco ded in his egion a e ocal s imula- ion in slices om ei he adul o neona al animals. Ana omical de ec ion o synap ic AMPA s Con en ional immunocy ochemis y does no e eal synap ic e- cep o s because o masking o epi opes a e ixa ion (F i schy e al., 1998). Wa anabe e al. (1998) showed ha an igen e ie al wi h pepsin ga e punc a e AMPA immuno eac i i y in hip- pocampus and concluded ha his ep esen ed synap ic ecep- o s. We epo ed ha , a e pepsin ea men o spinal co d sec ions, punc a e s aining can be seen wi h an ibodies agains each AMPA subuni (Nagy e al., 2004) and wi h a pan-AMPA an ibody ha de ec s all ou subuni s (Polga´ e al., 2008). We con i med ha hese punc a co espond o synap ic ecep o s by demons a ing hei absence in knock-ou animals, hei associ- a ion wi h glu ama e gic bou ons, and hei ul as uc u al loca- ion in pos synap ic densi ies (Nagy e al., 2004; Polga´ e al., 2008). PSD-95, which is p esen in he pos synap ic densi y o glu ama e gic synapses (Hun e al., 1996; Val schano e al., 1999; Sans e al., 2000), can also be e ealed a e pepsin ea - men (Fukaya and Wa anabe, 2000). We epo ed p e iously ha ⬃98% o PSD-95 punc a in laminae I–III o adul a do sal ho n we e pan-AMPA immuno eac i e, whe eas ⬎99% o pan- AMPA -posi i e punc a in his egion we e PSD-95 immuno e- ac i e (Polga´ e al., 2008). An al e al. (2008) used he highly sensi i e eeze- ac u e eplica labeling me hod and demon- s a ed ha GluR2 subuni s we e p esen a 99% o glu ama e gic synapses in supe icial laminae o adul a s. These esul s sugges ha i ually all glu ama e gic synapses in his egion con ain PSD-95 and AMPA s in he adul . In he p esen s udy, we se a h eshold o pan-AMPA s aining o allow eliable quan i ica ion o synap ic exp ession a di e en ages. Al hough his esul ed in a somewha lowe p opo ion o PSD-95 punc a (93–94%) being de ined as pan-AMPA posi i e, he p opo ion did no di e be ween neona al and adul ani- Figu e 4. Minimal s imula ion p o ocol and ailu e a e o esponses o ocal s imula ion. A, Example o a minimal s imula ion es .AMPA -media edEPSCswe eini ially eco ded,and hes imulusin ensi yca e ully educed oapoin jus below ha e oking EPSCs. While s imula ing a his in ensi y, he memb ane po en ial was inc eased o ⫹50 mV o es o he p esence o slow NMDA -media ed EPSCs. Fo mos cells, he holding po en ial was hen e u ned o ⫺70 mV o check o any change in he AMPA -media ed esponses. The op panel shows 20 indi idual eco ds ( op ow) and a e ages o eco ds made h oughou he eco dingpe ioda each holding po en ial. Two se s o eco ds a e shown o heini ialpe iodo eco ding a ⫺70 mV: one while s imuli eliably e oked EPSCs and one made a e he s imulus in ensi y had been educed below he h eshold le el. The middle wo owsshow hes imulus in ensi yand heholdingpo en ial,and he bo omplo shows heampli udeo EPSCs e okedby es ocal s imuli. This example was eco ded in an adul slice wi h BAPTA in he elec ode. B, Failu e a e o EPSCs in esponse o ocal s imula ion. In he op g aph, each line ep esen s da a o an indi idual connec ion. The ailu e a e was de e mined ini ially a ⫺70 mV (le ), hen a ⫹50 mV (middle), and hen on e u ning o ⫺70 mV ( igh ). Connec ions es ed in slices om adul and neona alanimalsandwi ho wi hou BAPTAin he eco ding elec odea eshown.Thebo omba g aphshowsa e agedda a o he same connec ions. No e he simila i y o esul s ob ained om adul and neona al animals and wi h o wi hou BAPTA. 13406 •J. Neu osci., Oc obe 21, 2009 •29(42):13401–13409 Yasaka e al. •Silen Synapses in Do sal Ho n mals, sugges ing ha , e en in neona al a s, he g ea majo i y o PSD-95-con aining synapses in laminae I–II possess AMPA s. I has been epo ed ha he e is a de elopmen al change in exp es- sion o MAGUK p o eins in a hippocampus, wi h SAP-102 be- ing he p edominan o m and low exp ession o PSD-95 un il pos na al day 10 (Sans e al., 2000). Howe e , he e is e idence ha PSD-95 is impo an o synap ic clus e ing o NMDA s du - ing synap ogenesis in cul u es om emb yonic o neona al hip- pocampus (Rao e al., 1998; F iedman e al., 2000). Toge he wi h ou inding ha he e is al eady a high le el o PSD-95 in 7-d-old a s, his makes i unlikely ha many glu ama e gic synapses in supe icial do sal ho n o neona al animals lack PSD-95. Absence o pu e NMDA -media ed EPSCs Se e al s udies epo ed pu e NMDA -media ed EPSCs in supe - icial do sal ho n neu ons in esponse o do sal ho n o do sal oo s imula ion (Ba doni e al., 1998; Li and Zhuo, 1998; Baba e al., 2000; Jung e al., 2005), and hese indings ha e been in e - p e ed as ep esen ing ac i a ion o synapses ha possess NMDA s bu lack AMPA s. Al hough a i s sigh he EPSCs e oked by p e iously sub h eshold ocal s imula ion ha we obse ed in some cells when he holding po en ial was aised o ⫹50 mV esembled hose epo ed p e iously, he e a e impo an di e - ences. Fi s , we obse ed hese in ela i ely ew cases and wi h Figu e 5. Examples o connec ions p oducing EPSCs a posi i e holding po en ials. The op ow o each sec ion shows indi idual eco dings om a neu on a di e en holding po en ials: ⫺70mV, ⫹50mV, andon e u ning o⫺70 mV.The second owo eachsec ion indica es he holding po en ial, and he bo om plo shows he ampli udes o EPSCs e oked by ocal s imu- la ion. A, Reco ding om an adul slice using a pipe e wi hou BAPTA. B, Reco ding om a neona alsliceusing apipe ewi hou BAPTA.C,Reco ding oma neona alsliceusingapipe e wi h BAPTA. Figu e 6. The e ec o APV on EPSCs e oked by ocal s imula ion while he cell was held a ⫹50 and ⫺70 mV, a e educ ion o he s imulus s eng h o below he le el ha p oduced EPSCswhen he cellwasini ially es ed a ⫺70mV. The op acesshow ha APVsubs an ially educed he EPSC eco ded a ⫹50 mV, lea ing a small esidual cu en ha was p esumably media edbyAMPA s.Thebo om aceshowsaclea EPSC eco dedin hep esenceo APVa e he cell was e u ned o a holding po en ial o ⫺70 mV. This eco ding was made in a slice ob ained om an adul a . Table 3. Compa ison o esul s ob ained wi h and wi hou BAPTA in he eco ding elec ode in expe imen s in which he holding po en ial was e u ned o ⴚ70 mV Wi hou BAPTA Wi h BAPTA To al Neona e Adul To al Neona e Adul Numbe o cells 39 21 18 49 28 21 Numbe o connec ions 53 30 23 70 42 28 Numbe “silen ” 6 (11%) 3 3 4 (6%) 2 2 Numbe o pe sis en EPSCs on e u n o ⫺70 mV 6 (100%) 3 3 4 (100%) 2 2 A summa y o esul s ob ained in slices om neona es (aged 4–14 d) o adul s (aged 6 –9 weeks), wi h o wi hou BAPTA in he eco ding elec ode. Numbe “silen ” shows he numbe (and pe cen age) o connec ions a which EPSCs we e obse ed a a holding po en ial o ⫹50 mV while s imula ing a a le el ha was ini ially sub h eshold when hecellwasheld a ⫺70 mV(“silen synapses”).Thebo om owgi es henumbe (andpe cen age) o hese “silen synapses” o whichAMPA -media edEPSCswe eseenwhen heholdingpo en ialwas e u ned o⫺70mV. Yasaka e al. •Silen Synapses in Do sal Ho n J. Neu osci., Oc obe 21, 2009 •29(42):13401–13409 • 13407 equal equency in slices om neona al and adul animals. Sec- ond, we ound ha , in all cases in which he holding po en ial was subsequen ly e u ned o ⫺70 mV, EPSCs we e s ill de ec ed, sugges ing ha his phenomenon did no esul om ac i a ion o AMPA -lacking synapses. Because some o he EPSCs seen a ⫹50 mV migh ha e esul ed om ac i a ion o mo e han one p esynap ic axon, we canno ule ou he possibili y ha one o hese o med a genuinely silen synapse ha was no de ec ed because o he eappea ance o EPSCs a he o he synapse(s) when he holding po en ial was e u ned o ⫺70 mV. Howe e , i his is he case, hen such synapses a e p esumably e y a e. I is e y unlikely ha he eappea ance o EPSCs a ⫺70 mV was a ibu able o apid inse ion o AMPA s, because in many cases, eco dings had been made om he cells o a leas 30 min, whe eas hippocampal long- e m po en ia ion (which is hough o esul om AMPA inse ion) is no seen in neu ons ha ha e been held in he whole-cell con igu a ion o his long (Malinow and Tsien, 1990). In addi ion, ac i i y-dependen inse ion o AMPA s is Ca 2⫹ media ed, and his phenomenon occu ed wi h BAPTA in he pipe e. One possible explana ion o ou obse a ions is ha he e was a p og essi e al e a ion in he e ec i eness o he ocal s im- ula ion. Howe e , we ound ha , in cases in which EPSCs ap- pea ed when he holding po en ial was aised, his o en occu ed e y apidly (Figs. 4A,5A), sugges ing ha a change in e ec i e- ness may ha e esul ed om depola iza ion. Vo onin e al. (2004) used a minimal s imula ion p o ocol in hippocampal slices and also obse ed EPSCs ha appea ed when he holding po en ial was changed o a posi i e alue and hen emained when i was e u ned o he o iginal nega i e le el. Howe e , his was no seen when he pa ch pipe e con ained 20 mMBAPTA, and he au ho s concluded ha i was Ca 2⫹ dependen . Ou inding ha nei he he appea ance o EPSCs a ⫹50 mV no hei pe sis ence when he holding po en ial was e u ned o ⫺70 mV was p e en ed by 40 mMBAPTA in he pipe e sugges s ha nei he phenomenon was Ca 2⫹ media ed. A po en ial Ca 2⫹ - independen mechanism ha could explain changes in exci abil- i y o p esynap ic neu ons in ol es leakage o Cs ⫹ ou o he eco ded cell when he holding po en ial was aised (Liao e al., 1995; Vo onin e al., 2004). This leakage could occu h ough NMDA ecep o s, as well as h ough Cs ⫹ -pe meable K ⫹ chan- nels ac i a ed by depola iza ion, which ha e been iden i ied in neu ons (Ru¨sch and Ea ock, 1996; Sanchez e al., 1998; Wigmo e and Lacey, 2000). Leakage o Cs ⫹ would ha e he e ec o block- ing K ⫹ channels in nea by neu onal memb anes, he e o e in- c easing he exci abili y o he p esynap ic glu ama e gic axons ha media e he EPSCs seen a e ocal s imula ion. Silen synapses in he supe icial do sal ho n Ou indings canno accoun o he pu e NMDA -media ed EPSCs seen in all p e ious s udies in do sal ho n, because Li and Zhuo (1998) and Ba doni e al. (1998) (R. Ba doni, pe sonal communica ion) ound ha , in hose cases in which EPSCs ap- pea ed when a posi i e holding po en ial was applied, hey we e no obse ed when he holding po en ial was e u ned o ⫺70 mV. Howe e , al hough bo h s udies epo pu e NMDA - media ed EPSCs in neona al animals, he e was a conside able di e ence in hei p e alence: Li and Zhuo ound hem in ⬎60% o cells om animals ⬍10 d old, whe eas Ba doni e al. obse ed hem in ⬃20% o synapses es ed. Al hough some o his disc ep- ancy may esul om di e ences in s imula ion si e (do sal oo s o ocal s imula ion), oge he wi h he p esen indings, i sug- ges s ha he phenomenon shows conside able dependence on expe imen al p o ocol. Assuming ha ou in e p e a ion o he ana omical da a is co ec and ha he e a e ew i any AMPA -lacking glu ama- e gic synapses in supe icial do sal ho n a ages olde han 7 d, i is necessa y o accoun o he obse a ion o pu e NMDA - media ed EPSCs in p e ious s udies. One explana ion is ha i esul s om he p esence o low le els o glu ama e ha a e able o ac i a e NMDA bu no AMPA ecep o s (because he NMDA s ha e a much highe glu ama e a ini y), and i has been sugges ed ha his can esul om glu ama e spillo e om nea by synapses (Kullmann e al., 1996). Glu ama e up- ake mechanisms may be comp omised in slices, o example a ibu able o auma in ol ed in sec ioning (Asz ely e al., 1997), and a ia ion in slice p epa a ion me hods could he e- o e lead o di e ences in he amoun o glu ama e spillo e . A ecen p oposal o explain disc epancies be ween he ind- ing o pu e NMDA -media ed EPSCs and ana omical s udies ha ailed o de ec signi ican numbe s o AMPA -lacking glu ama- e gic synapses is ha AMPA s may be p esen in newly o med synapses bu in a labile s a e (Xiao e al., 2004; G oc e al., 2006). I has been sugges ed ha hese labile AMPA s may be d i en ou o he synapses by a mechanism simila o long- e m dep ession. Xiao e al. (2004) we e able o e oke his “silencing” by s imula - ing a low equency and demons a ed ha i was Ca 2⫹ depen- den bu no NMDA media ed. One di e ence be ween he p esen s udy and hose o Li and Zhuo (1998) and Ba doni e al. (1998) is ha we eco ded om cells ha we e a leas 100 ␮ m below he cu su ace o he slice, whe eas in hese o he s udies eco dings we e appa en ly om mo e supe icially loca ed neu- ons. Because mos supe icial do sal ho n neu ons ha e os o- caudally elonga ed dend i es, many neu ons close o he su ace will ha e unde gone ansec ion o p oximal dend i es in he ans e se slices ha we e used in hese s udies. I is possible ha signi ican Ca 2⫹ en y h ough hese dend i es led o synap ic “silencing” a ibu able o emo al o labile synap ic AMPA s. Re e ences An al M, Fukazawa Y, Eo¨ do¨gh M, Muszil D, Molna´ E, I aku a M, Takahashi M, Shigemo o R (2008) Numbe s, densi ies, and colocaliza ion o AMPA- and NMDA- ype glu ama e ecep o s a indi idual synapses in he supe icial do sal ho n o a s. 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