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.
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