125

Our understanding ol neuronal synapse development has
advanced in recent years. The development ol glycinergic
synapses appears to depend on gephyrin and glycine receptor
activity. Molecular characterization ol the structure and
development ol glutamatergic synapses is in progress, but the
underlying mechanisms remain unclear. Activity·dependent
mechanisms and specilic molecules that regulate the
morphological development ol dendritic spines have recently
been identilied.
Addresses
Department ol Neurobiology and Howard Hughes Medical lnstitute,
Massachusetts General Hospital and Harvard Medical School,
50 Blossom Street, Boston, Massachusetts 02114, USA
e·mail: sheng§helix.mgh.harvard.edu
Current Oplnlon ln Neuroblo|ogy 2000, 10:125-131
0959·4388/00/S - see lront matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbrevlatlons
ABP AMPA·receptor·binding protein
AMPA ·amino·3·hydroxy·5·methyl·4·isoxazole propionate
BDNF brain·derived neurotrophic lactor
CaMKII calcium/calmodulin·dependent protein kinase ll
CASK calmodulin·dependent protein kinase, SH3 domain,
guanylate kinase
DLG discs large
GABA ·aminobutyric acid
GABAPAP GABA
A
receptor associated protein
GFP green lluorescent protein
GKAP guanylate kinase associated protein
G|yP glycine receptor
GPIP glutamate·receptor·interacting protein
HSPG heparan sullate proteoglycan
LTD long·term depression
LTP long·term potentiation
MAP1B microtubule·associated protein 1B
mG|uP metabotropic glutamate receptor
nAChP nicotinic acetylcholine receptor
Narp neuronal·activity·regulated pentraxin
NMDA N·methyl·D·aspartate
NM1 neuromuscular junction
PDZ domaln PSD·95, DLG, ZO·1 domain
PICK1 protein interacting with C kinase 1
PSD postsynaptic density
PSD·95 postsynaptic density protein ol 95 kDa
ZO·1 zona occludens 1
Introduction
Neurons communicate witl eacl otler tlrougl synaµtic
junctions, wlicl can le consiuereu to le tle elemental
information µrocessing units of tle lrain. Jle ueveloµ-
ment of neuron - neuron synaµses is clearly lotl of lasic
interest anu of clinical relevance, anu recent years lave
seen consiueralle µrogress in tlis fielu. ¡n µractice, anu
ueµenuing on tle lackgrounu of tle investigator, synaµs-
es are variously uefineu ly functional or morµlological
criteria (tle latter often oltaineu simµly ly immunostain-
ing). Once formeu, synaµtic junctions are liglly uynamic
structures tlat can le grossly remoueleu, even uuring
auult life. Jlese issues make it uifficult to uefine wlen a
synaµse is 'lorn` anu wlen synaµse ueveloµment enus. ¡n
tlis review, we take a lileral view of tle ueveloµmental
lifesµan of neuronal synaµses, anu we focus on tle molec-
ular anu cell liological asµects of synaµse formation. Jle
uiscussion is weiglteu towaru excitatory synaµses (tle
most alunuant tyµe of synaµses in tle lrain) anu towaru
µostsynaµtic as oµµoseu to µresynaµtic ueveloµment (reca-
µitulating tle lias of researcl at tle vertelrate
neuromuscular junction [NM]]).
Distribution of gIutamate receptors during
synapse deveIopment
A caruinal feature of tle ueveloµment of synaµses is tle
accumulation of neurotransmitter receµtors at tle µostsy-
naµtic site. ¡n muscle, stuuies of µostsynaµtic nicotinic
acetylcloline receµtor (nAClR) clustering leu to key
meclanistic insiglts into tle ueveloµment of tle NM]
(see [1] for a review). So for neurons, attention las natural-
ly focuseu on tle ueveloµmental regulation of receµtor
clustering in synaµses. An imµortant recent realization is
tlat tle synaµtic content of A-metlyl-L-asµartate
(NMLA) receµtors anu -amino-3-lyuroxy-5-metlyl-4-
isoxazole µroµionate (AMPA) receµtors is uifferentially
regulateu uuring tle maturation of excitatory synaµses.
Quantitative immunogolu staining at electron microscoµy
level suggests tlat NMLA receµtors are exµresseu lefore
AMPA receµtors in liµµocamµal synaµses [2
º
-4
º
]. At µost-
natal uay 2, a sulstantial fraction (>30%) of synaµses
contain NMLA receµtors lut not AMPA receµtors. As
ueveloµment µrogresses, synaµses acquire AMPA receµ-
tors witl no clange in NMLA receµtor numler. ¡ven in
tle auult lrain, 10-20% of excitatory synaµses lack AMPA
receµtors, anu among tle synaµses tlat contain lotl tyµes
of receµtors, tle level of AMPA receµtors is mucl more
varialle tlan tlat of NMLA receµtors. Synaµses contain-
ing NMLA receµtors lut lacking AMPA receµtors may le
tle morµlological correlates of functionally 'silent
synaµses` [5], wlicl also are relatively alunuant in imma-
ture lrain. Silent synaµses are electroµlysiologically
uefineu as tlose tlat slow NMLA-receµtor-meuiateu cur-
rents lut not AMPA receµtor currents.
Glutamate receµtor clustering in synaµses las also leen
stuuieu in culture [6,7,8
º
,9,10]. Here too, synaµses con-
taining NMLA receµtors not lacking AMPA receµtors
lave leen olserveu [6,7]. Jle time course of synaµtic
clustering of NMLA receµtors, AMPA receµtors anu a sul-
set of µostsynaµtic uensity (PSL) µroteins las leen
uescrileu in µarticular uetail for liµµocamµal neurons
ueveloµing in low-uensity culture [8
º
]. ¡n tlis system,
synaµse ueveloµment takes µlace more slowly tlan in tle
lrain, witl AMPA receµtors clustereu in sµiny synaµses
DeveIopment of neuron~neuron synapses
Sang Hyoung Lee and Morgan Sheng
lefore NMLA receµtors are synaµtically localizeu (tlis is
tle reverse of wlat is seen in tle lrain). ¡nterestingly,
altlougl synaµtic NMLA receµtors are always colocalizeu
witl PSL-95 (an NMLA receµtor ancloring µrotein of tle
PSL), non-synaµtic NMLA receµtor clusters at early times
(2-5 uays iv :itto) in culture are often not associateu witl
PSL-95 (see !igure 1). Jlus, NMLA receµtors are not
necessarily µre-assemlleu witl PSL-95 uuring tleir µas-
sage from tle cell louy to tle synaµse. ¡nueeu, PSL-95
aµµears to µreceue tle aµµearance of NMLA receµtors in
126 Deve|opment
Figure 1
Clustering ol postsynaptic proteins during
development ol excitatory synapses in low·
density hippocampal neuron culture. (a) The
earliest clusters (2-5 days in viIto [Dlv|) ol
NMDA receptors are non·synaptic, and many
do not colocalize with PSD·95/GKAP clusters.
These early NMDA receptor clusters could be
intracellular or on the cell surlace. (d) Synaptic
NMDA receptors accumulate lirst in shalt
synapses (~12 Dlv) and (e) do not cluster
prominently in spine synapses until ~3 weeks
in viIto. (a,b) AMPA receptors are initially
dilluse, but (c) begin to cluster on spine
synapses at ~9-10 Dlv, belore NMDA
receptors. (e) Thus, colocalization ol AMPA and
NMDA receptors at spiny synapses occurs late
(~3 weeks). (a-e) PSD·95, a putative NMDA
receptor anchoring protein, is always
associated with its binding partner GKAP [20|.
PSD·95/GKAP are early arrivals in synapses,
olten accumulating there belore NMDA
receptors. Shank, a GKAP·binding protein
[21|, is not always associated with
PSD·95/GKAP at early developmental stages,
but is consistently colocalized with
PSD·95/GKAP alter ? Dlv. NMDA receptors
are lound in synapses only in association with
clusters ol PSD·95/GKAP/Shank.
(c-e) ·Actinin is lound only in spiny synapses.
Data lrom [8

| and (C Sala, M Sheng,
unpublished data).
NMDA
receptor
AMPA
receptor
Actinin
PSD·95/GKAP/
Shank
PSD·95/GKAP
PSD protein cIustering during excitatory synapse deveIopment
(a) 2-5 Dlv
(b) ? Dlv
(c) 9-10 Dlv
(d) ~12 Dlv
(e) ~ 21 Dlv
Presynaptic
terminal
Surlace
membrane
ol dendrite
Spine
Current Opinion in Neurobiology
synaµses, consistent witl tlis µrotein µlaying a role in tle
synaµtic 'caµture` of NMLA receµtors [8
º
]. Jle oruer of
aµµearance of synaµtic µroteins neeus to le examineu in
uetail in tle ueveloµing lrain. Altlougl tle low-uensity
liµµocamµal culture system las greatly facilitateu tle
stuuy of synaµse ueveloµment, it is uncertain low accu-
rately it reµrouuces tle iv :i:o situation.
Postsynaptic scaffoId proteins
¡n tle muscle enuµlate, nAClRs are linkeu to tle cyto-
µlasmic µrotein raµsyn, wlicl is essential for receµtor
clustering anu normal NM] ueveloµment [1]. ¡t is lecom-
ing clear tlat neuronal neurotransmitter receµtors are also
linkeu to sµecific intracellular µroteins: tle PSL-95 family
of µroteins for NMLA receµtors, GR¡P/ABP anu P¡CK1
for AMPA receµtors, Homer for grouµ ¡ metalotroµic glu-
tamate receµtors (mGluRs), geµlyrin for glycine receµtors
(GlyRs), anu GABARAP anu MAP1B for GABA receµtors
(revieweu in [11-13]). Recently, tle involvement of some
of tlese µroteins in synaµse ueveloµment las leen evalu-
ateu ly genetic aµµroacles. !eng -t a/. [14
ºº
] reµort tlat a
mouse knockout of geµlyrin alolisles synaµtic clustering
of tle GlyR, confirming its imµortance in inlilitory
synaµse formation. On tle otler lanu, Migauu -t a/. [15
ºº
]
reµort tlat in PSL-95 mutant mice, tle synaµtic localiza-
tion of NMLA receµtors aµµeareu unaffecteu. Synaµtic
targeting of NMLA receµtors anu synaµse uensity also
remain normal wlen PSL-95 family µroteins are uisµerseu
ly an interfering µeµtiue [16]. Jlese results argue tlat
PSL-95 family µroteins are not µrimary organizers of tle
excitatory µostsynaµtic sµecialization. Ratler, PSL-95 may
le more imµortant for assemlling an NMLA-receµtor-
associateu signaling comµlex tlat regulates synaµtic
µlasticity [15
ºº
].
Jle alove stuuies [14
ºº
,15
ºº
] are not conclusive, lowever,
lecause tley uiu not remove tle activity of all PSL-95
family µroteins or examine tle µlenotyµes at ultrastruc-
tural resolution. ¡n Dtosop/i/a, mutations of tle PSL-95
lomolog Dis·s /at¸- (D/¸) severely affect morµlological
ueveloµment anu clustering of sµecific memlrane µroteins
at tle glutamatergic NM] [17]. ¡n auuition, tle µostsynaµ-
tic uistrilution anu function of Liscs large is regulateu ly
calcium/calmouulin-ueµenuent µrotein kinase ¡¡
(CaMK¡¡) µlosµlorylation [18
ºº
], tlus µroviuing a µoten-
tial meclanism for activity-ueµenuent control of
µostsynaµtic structure anu receµtor/clannel clustering.
CaMK¡¡ may also µlay a role in µostsynaµtic ueveloµment
in vertelrates, as it is imµlicateu in tle control of uenurit-
ic growtl [19].
By analogy witl Liscs large, it remains an aµµealing iuea
tlat PSL-95 family µroteins µlay a role in tle ueveloµment
of excitatory synaµses. PSL-95 linus to an alunuant µro-
tein in tle PSL calleu GKAP [20], tlat, in turn, linus to a
multiuomain µrotein calleu Slank [21]. Slank is an imµor-
tant scaffolu µrotein of tle PSL tlat also interacts witl tle
Homer/mGluR comµlex anu witl tle actin regulatory
µrotein cortactin [21,22]. Jle estallislment of tle PSL-
95/GKAP comµlex at µostsynaµtic sites aµµears to tlen
meuiate tle recruitment of Slank to synaµses [21]. ¡n tlis
way, tle PSL-95/GKAP comµlex coulu nucleate tle
assemlly of tle µostsynaµtic aµµaratus.
How miglt PSL-95 itself le sµecifically localizeu at tle
synaµtic junction: One µossilility is tlat PSL-95 congre-
gates at tle synaµtic junction ly linuing a µostsynaµtic
cell aulesion molecule (calleu neuroligin) tlat linus to
neurexin (its µresynaµtic receµtor) across tle synaµtic cleft
[23,24]. Neurexin may le ancloreu on tle µresynaµtic siue
ly anotler memlrane-associateu guanylate kinase
(MAG\K) µrotein CASK/L¡N-2. Jlis mouel is attractive
lecause PSL-95 anu CASK contain multiµle interaction
uomains in auuition to tlose useu for neuroligin anu
neurexin linuing (for instance, PSL-95 linus to neuroligin
witl tle tliru of its tlree PLZ uomains anu to NMLA
receµtors witl its first two PLZ uomains). Jlus, an extra-
cellular interaction letween neurexin anu neuroligin coulu
lotl align tle µre- anu µostsynaµtic memlranes anu
recruit relevant µroteins to eitler siue of tle junction.
!urtler stuuies are neeueu to verify tlis lyµotlesis. Jle
uiversity of neurexin extracellular uomains tlat arises from
alternative sµlicing of tlree neurexin genes may contrilute
to tle sµecificity of synaµtic connections [25]. ¡n auuition,
tle exµanuing reµertoire of caulerin-like cell aulesion
molecules known to le exµresseu in tle nervous system is
likely to µroviue a lasis for sµecification of synaµtic con-
nectivity [26-28].
Activity dependence of synapse deveIopment
¡n tle nervous system, tle same lasic meclanisms useu in
ueveloµment tenu to le reutilizeu in auultloou for µlastic-
ity, tlus, insiglts into synaµtic µlasticity sloulu inform us
alout tle µrocesses of synaµtic ueveloµment. ¡n tle
mature lrain, synaµtic structure anu function is regulateu
ly neural activity. Recently, it las lecome aµµarent tlat
activity-ueµenuent meclanisms control µostsynaµtic lev-
els of neurotransmitter receµtors [29].
Clronic llockaue of NMLA receµtors in liµµocamµal
neurons increases tle synaµtic clustering of NMLA
receµtors [30]. Jle level of AMPA receµtors at synaµses
is also uynamically regulateu [6,7,31-33], witl reuuceu
activity tenuing to increase tle numler of synaµtic
AMPA receµtors on a time scale of lours to uays.
Reuistrilution of green fluorescent µrotein (G!P)-
taggeu AMPA receµtor GluR1 sulunits from an
intracellular µool to tle surface of uenuritic sµines can
le visualizeu witlin 30 min of long-term µotentiation
(LJP)-inuucing tetanic stimulation [34
ºº
]. Long-term
ueµression (LJL), on tle otler lanu, is associateu witl
a uecreaseu numler of synaµtic AMPA receµtor clusters
[35]. At tle morµlological level, LJP-inuucing stimuli
cause raµiu, localizeu outgrowtl of uenuritic filoµouia
(long, tlin µrocesses) anu sµines in an NMLA-receµtor-
ueµenuent manner [36
ºº
,37
ºº
], suggesting tlat synaµtic
Deve|opment of neuron-neuron synapses Lee and Sheng 12?
activity anu glutamate release may le a factor controlling
sµine formation (see !igure 2). Profounu inactivity may
also stimulate sµine formation, as liµµocamµal slices
(wlicl are electrically silent immeuiately after section-
ing) exlilit a uoulling of sµine uensity witlin -2 l
[38
º
]. ¡n anotler twist to tle issue of activity, tle main-
tenance of uenuritic sµines in liµµocamµal slice cultures
requires low-level activation of AMPA receµtors [39].
Jlus, synaµtic activity is µrolally imµortant for lotl
sµine formation anu maintenance, lut tle relationsliµ
letween tle level of activity anu sµine ueveloµment is
comµlex. Presumally activity (or lack of activity) is asso-
ciateu witl tle release of sµinogenic factors tlat
cooµerate witl tle neurotransmitter glutamate to inuuce
sµine outgrowtl.
Surµrisingly, lowever, uesµite tle overwlelming eviuence
for activity-ueµenuent µlasticity of synaµtic structure anu
function, stuuies using llockers of neurotransmitter receµ-
tors or action µotentials inuicate tlat activity is not
requireu for gross morµlological ueveloµment of excitato-
ry synaµses or GABAergic synaµses in culture [29,40].
Morµlological ueveloµment of lrain synaµses also aµµears
normal in Jav·13-1 knockout mice uesµite silencing of a
large fraction of excitatory synaµses, confirming tle uis-
µensalility of activity in an iv :i:o situation [41
º
].
¡nlilitory glycinergic synaµses seem to le tle exceµtion
to tlis rule. Otlers lave founu tlat synaµtic clustering of
glycine receµtors in sµinal neurons is µreventeu ly llock-
aue of glycine receµtors, wlicl lave ueµolarizing activity
in ueveloµing neurons [42
ºº
,43
ºº
]. Jlus, as miglt le
exµecteu, uifferent rules seem to govern maturation of uif-
ferent classes of neuronal synaµses.
DeveIopment of spines
¡xcitatory synaµses in tle lrain uiffer from inlilitory
synaµses in tlat tley form µreuominantly on uenuritic
sµines, wlicl are semi-autonomous comµartments tlat
µrotruue from tle uenuritic slaft. Jlus, tle ueveloµ-
ment of glutamatergic synaµses involves growtl of
sµecializeu µrocesses from tle µostsynaµtic cell. How
uenuritic sµines form anu mature is tlerefore liglly
µertinent to tle meclanisms of synaµtogenesis. ¡arly in
synaµtogenesis, filoµouia grow out of uenurites, 'seek-
ing` anu making contacts witl µresynaµtic
sµecializations [44]. !iloµouia are tlouglt to le µrecur-
sors of sµines, tley may act ly guiuing axons to tle
uenuritic slaft from wlicl true sµines later emerge [45].
Jle molecular meclanisms tlat regulate sµine or filoµo-
uia formation are not unuerstoou, lut tley µresumally
act via localizeu clanges in tle actin cytoskeleton, wlicl
is very uynamic even witlin mature sµines [46]. ¡n auui-
tion, it las leen slown tlat local actin instalility µlays
an imµortant role in axon formation [47]. Jlus, local
rearrangement of tle cytoskeleton is closely linkeu to
tle ueveloµment of sµiny synaµses.
Synuecans are a family of cell surface leµaran sulfate µro-
teoglycans (HSPGs) wlicl are imµlicateu in cell aulesion,
intercellular signaling anu tissue morµlogenesis [48].
Synuecan-2 is sµecifically localizeu in synaµses (µrolally
lotl µre- anu µostsynaµtically) wlere it interacts via its
cytoµlasmic carloxyl terminus witl tle PLZ uomain of
CASK [49]. Synuecan-2 accumulates in synaµses relatively
late [50
º
,51]. ¡nterestingly, synuecan-2 transfection into cul-
tureu liµµocamµal neurons accelerates tle morµlological
maturation of uenuritic sµines [50
º
]. Jlis effect ueµenus on
tle µresence of tle carloxyl terminus of synuecan-2, imµly-
ing tlat interactions witl PLZ µroteins sucl as CASK are
imµortant for sµine maturation. Jle effect of synuecans on
tle clustering of glutamate receµtors, or otler µostsynaµtic
molecules, was not investigateu. Nevertleless, tlese stuuies
suggest tlat HSPGs are involveu in tle ueveloµment of
synaµses letween neurons.
Secreted factors invoIved in synapse
deveIopment
HSPGs are also imµortant in vertelrate NM] ueveloµ-
ment. Lifferentiation of tle NM] is orclestrateu ly agrin,
an HSPG secreteu ly motor neurons tlat inuuces nAClR
clustering (revieweu in [1]). Altlougl µresent in tle lrain,
agrin seems uisµensalle for µostsynaµtic receµtor cluster-
ing in CNS synaµses. Glutamatergic anu GABAergic
synaµses form normally in liµµocamµal neurons ueriveu
128 Deve|opment
Figure 2
A model lor the regulation ol dendritic spine
development. Dendrites send out long thin
processes (lilopodia) that seek out and lorm
synapses with nearby axons. Formation ol
lilopodia is stimulated by high levels ol synaptic
activity (e.g. tetanus or other LTP·inducing
stimuli) or by prolound inactivity, presumably in
conjunction with local secreted lactors.
Postsynaptic syndecan·2 and its associated
PDZ proteins are critical lor the development ol
lilopodia into mature spines (a representative
mature mushroom·shaped spine is shown). The
maintenance ol mature spines depends on low·
level stimulation ol the AMPA receptor.
Growth of
dendritic
fiIopodia
Spine
formationI
maturation
lnactivity
Tetanus/LTP
Secreted
lactors
Syndecan·2
PDZ proteins
Current Opinion in Neurobiology
Axon
Dendrite
from agrin-ueficient mice [52]. Jlus, we still await tle
uiscovery of a secreteu 'agrin equivalent` for any class of
neuronal synaµse.
Jle only iuentifieu secreteu factor tlat is reµorteu to inuuce
receµtor clustering in neurons is Narµ (neuronal-activity-reg-
ulateu µentraxin), wlicl was originally iuentifieu as an
activity-inuucille immeuiate early gene µrouuct [53
º
].
However, tlis conclusion was largely laseu on iv :itto exµer-
iments, anu tlere is no eviuence to uate tlat Narµ is crucial
for tle ueveloµment of excitatory synaµses. Loss-of-function
stuuies iv :i:o are critical to evaluate tle 'Narµ lyµotlesis`
of AMPA receµtor clustering, as µroµoseu in [53
º
].
¡ncreasing attention las turneu to neurotroµlins as regula-
tors of synaµtic µlasticity anu ueveloµment [54]. Synaµse
uensity is increaseu two anu a lalf folu in tle suµerior cervi-
cal ganglia of transgenic mice overexµressing lrain-ueriveu
neurotroµlic factor (BLN!), anu uecreaseu in BLN!
knockout mice [55]. Mice lacking tle neurotroµlin receµtors
troµomyosin-receµtor-kinase (Jrk)B anu JrkC sloweu
uecreaseu uensity of synaµtic vesicles, as well as reuuceu
axonal arlorization anu synaµtic uensity [56
º
]. BLN!
knockout mice also sloweu µresynaµtic structural uefects,
incluuing a uecreaseu numler of 'uockeu` synaµtic vesicles
anu reuuceu exµression of tle synaµtic vesicle µroteins
synaµtoµlysin anu synaµtolrevin [57
º
]. Jlus, neurotroµlins
are not essential for synaµse formation, lowever, tley may
le involveu in a late stage of synaµse maturation.
ConcIuding remarks
Jle meclanisms unuerlying ueveloµment of tle verte-
lrate NM] are ly no means solveu, lut it seems likely tlat
most of tle key molecules anu regulatory steµs lave leen
iuentifeu. By comµarison, our unuerstanuing of neuronal
synaµtogenesis lags a long way lelinu. Jlis is at least in
µart lecause neuron-neuron synaµses are teclnically more
uifficult to stuuy. Neuronal synaµses are mucl smaller
tlan neuromuscular synaµses anu are uensely µackeu in
tle lrain, making tlem uifficult to access anu visualize
inuiviuually iv :i:o, esµecially in real-time. ¡acl neuron
receives tlousanus of synaµses, so inµuts cannot le sµecif-
ically maniµulateu. Brain synaµses are extremely
leterogeneous, even witlin tle same neurotransmitter
class, making µrollematic any -v mass- aµµroacl sucl as
lioclemistry. Jlere is no convenient way to lalel func-
tional surface receµtors in neuronal synaµses (unlike using
-lungarotoxin to iuentify nAClRs at tle NM]).
Nevertleless, exciting µrogress is leing maue, esµecially
in unraveling tle molecular organization of µostsynaµtic
sµecializations. Continuing iuentification of tle molecular
comµonents of synaµtic junctions, comlineu witl
auvances in imaging, µromise to offer new meclanistic
insiglts into tle ueveloµment of neuron-neuron synaµses.
AcknowIedgements
Suµµorteu ly National ¡nstitutes of Healtl grant NS35050 (M Sleng).
M Sleng is Assistant ¡nvestigator of tle Howaru Hugles Meuical ¡nstitute.
References and recommended reading
Papers ol particular interest, published within the annual period ol review,
have been highlighted as:
· ol special interest
·· ol outstanding interest
1. Sanes 1P, Lichtman 1W: Deve|opment of the vertebrate
neuromuscu|ar [unctlon. Annu Rev Neutosci 1999, 22:389·442.
2. Nusser Z, Lujan P, Laube G, Poberts 1D, Molnar E, Somogyi P: Ce||
 type and pathway dependence of synaptlc AMPA receptor
number and varlabl|lty ln the hlppocampus. Neuton 1998,
21:545·559.
See annotation [4

|.
3. Petralia PS, Esteban 1A, Wang Y·X, Partridge 1G, Zhao H·M,
 Wenthold P1, Malinow P: Se|ectlve acqulsltlon of AMPA receptors
over postnata| deve|opment suggests a mo|ecu|ar basls for sl|ent
synapses. NoI Neutosci 1999, 2:31·36.
See annotation [4

|.
4. Takumi Y, Pamirez·Leon v, Laake P, Pinvik E, Ottersen OP: Dlfferent
 modes of expresslon of AMPA and NMDA receptors ln
hlppocampa| synapses. NoI Neutosci 1999, 2:618·624.
Using quantitative immunogold electron microscopy, these three papers
[2

-4

| show that a substantial proportion ol excitatory synapses in the hip·
pocampus possess NMDA receptors but lack AMPA receptors, both in early
postnatal [3

| and in mature [2

,4

| rats. Synapses containing NMDA recep·
tors but lacking AMPA receptors may correspond to lunctionally 'silent'
synapses. During postnatal development, AMPA receptors accumulate
gradually in synapses, while the NMDA receptor content within synapses
changes relatively little. Takumi eI ol. [4

| report an interesting correlation
between the diameter ol the postsynaptic density and the presence ol
AMPA receptors in the excitatory synapses.
5. Malenka PC, Nicoll PA: Sl|ent synapses speak up. Neuton 199?,
19:4?3·4?6.
6. Liao D, Zhang X, O'Brien P, Ehlers MD, Huganir PL: Pegu|atlon of
morpho|oglca| postsynaptlc sl|ent synapses ln deve|oplng
hlppocampa| neurons. NoI Neutosci 1999, 2:3?·43.
?. Gomperts SN, Pao A, Craig AM, Malenka PC, Nicoll PA:
Postsynaptlca||y sl|ent synapses ln slng|e neuron cu|tures. Neuton
1998, 21:1443·1451.
8. Pao A, Kim E, Sheng M, Craig AM: Heterogenelty ln the mo|ecu|ar
 composltlon of excltatory postsynaptlc sltes durlng deve|opment of
hlppocampa| neurons ln cu|ture. J Neutosci 1998, 18:121?·1229.
This paper was the lirst to detail the developmental sequence in which
ionotropic glutamate receptors and a set ol NMDA receptor·associated pro·
teins (PSD·95, GKAP, and ·actinin) cluster in synapses. ln low·density hip·
pocampal culture (in which dissociated neurons are plated in culture at a low
density, so that each neuron is well·separated lrom its neighbours), NMDA
receptors, AMPA receptors and PSD·95/GKAP accumulate in synapses
with distinct time courses, implying that they are targeted to synapses by dil·
lerent mechanisms.
9. O'Brien P1, Mammen AL, Blackshaw S, Ehlers MD, Pothstein 1D,
Huganir PL: The deve|opment of excltatory synapses ln cu|tured
splna| neurons. J Neutosci 199?, 17:?339·?350.
10. Mammen AL, Huganir PL, O'Brien P1: Pedlstrlbutlon and
stabl|lzatlon of ce|| surface g|utamate receptors durlng synapse
formatlon. J Neutosci 199?, 17:?351·?358.
11. Sheng M, Pak DTS: Llgand·gated lon channe| lnteractlons wlth
cytoske|eta| and slgna|lng protelns. Annu Rev Physiol 2000,
62:?55·??8.
12. Kim 1H, Huganir PL: Organlzatlon and regu|atlon of protelns at
synapses. Cutt Opin Cell Biol 1999, 11:248·254.
13. Kirsch 1: Assemb|y of slgna|lng machlnery at the postsynaptlc
membranes. Cutt Opin Neutobiol 1999, 9:329·335.
14. Feng G, Tintrup H, Kirsch 1, Nichol M, Kuhse 1, Betz H, Sanes 1: Dua|
 requlrement for gephyrln ln g|yclne receptor c|usterlng and
mo|ybdoenzyme actlvlty. Science 1998, 282:1321·1324.
Gephyrin is thought to be a postsynaptic anchoring protein lor glycine
receptors (GlyPs), which exerts its ellects by binding to GlyPs and micro·
tubules. This paper provides strong genetic evidence lor such a lunction:
disruption ol the gephyrin gene in mice causes loss ol GlyP clustering in
neurons. One unexpected linding lrom this study is that gephyrin is also
required lor molybdoenzyme activity, which potentially links molybdenum
deliciency to certain neurological diseases manilesting impairments at
inhibitory synapses. See also [43

|.
Deve|opment of neuron-neuron synapses Lee and Sheng 129
15. Migaud M, Charlesworth P, Dempster M, Webster LC, Watabe AM,
 Makhinson M, He Y, Pamsay MF, Morris PG, Morrison 1H, O'Dell T1,
Grant SG: Enhanced |ong·term potentlatlon and lmpalred |earnlng
ln mlce wlth mutant postsynaptlc denslty·95 proteln. NoIute 1998,
396:433·439.
The authors studied the lirst mouse knockout ol a PSD·95 lamily gene. Their
results suggest that PSD·95 is not required lor synaptic targeting ol NMDA
receptors (although it is possible that other members ol the PSD·95 lamily
complement this lunction). Pather, the enhanced LTP and impaired learning
ability ol the mutant mice suggest that PSD·95 plays an important role in
NMDA receptor lunction, perhaps by assembling a signaling complex linked
to the NMDA receptor.
16. Passalaro M, Sala C, Niethammer M, Sheng M: Dlrect mlcrotubu|e
blndlng by the PSD·95 lnteractlng proteln CPIPT and lts potentla|
ro|e ln the synaptlc c|usterlng of PSD·95. NoI Neutosci 1999,
2:1063·1069.
1?. Gramates SL, Budnik v: Assemb|y and maturatlon of the Drosophila
|arva| neuromuscu|ar [unctlon. lnI Rev Neutobiol 1999, 43:93·11?.
18. Kho YH, Popova E, Thomas U, Grillith LC, Budnik v: Pegu|atlon of
 DLG |oca|lzatlon at synapses by CaMKII·dependent phosphory|atlon.
Cell 1999, 98:353·363.
Discs large (DLG), the Ðtosophilo homolog ol PSD·95, is required lor the
synaptic clustering ol its binding partners, such as Shaker K
+
channels and
lasciclin ll (Fasll), at the larval NM1. Using genetic and biochemical
approaches, the authors lound that DLG localization is regulated by phos·
phorylation by CaMKll. This is the lirst study to show a potential link between
synaptic activity and the synaptic distribution ol a MAGUK protein.
19. Wu G·Y, Cline HT: Stabl|lzatlon of dendrltlc arbor structure in vivo
by CaMKII. Science 1998, 279:222·226.
20. Kim E, Naisbitt S, Hsueh Y·P, Pao A, Pothschild A, Craig AM, Sheng
M: GKAP, a nove| synaptlc proteln that lnteracts wlth the
guany|ate klnase·|lke domaln of the PSD·95/SAP90 faml|y of
channe| c|usterlng mo|ecu|es. J Cell Biol 199?, 136:669·6?8.
21. Naisbitt S, Kim E, Tu 1C, Xiao B, Sala C, valtschanoll 1, Weinberg P1,
Worley PF, Sheng M: Shank, a nove| faml|y of postsynaptlc denslty
protelns that blnds to the NMDA receptor/PSD·95/GKAP
comp|ex and cortactln. Neuton 1999, 23:569·582.
22. Tu 1C, Xiao B, Naisbitt S, Yuan 1P, Petralia PS, Brakeman P, Doan A,
Aakalu vK, Lanahan AA, Sheng M, Worley PF: Coup|lng of
mG|uP/Homer and PSD·95 comp|exes by the Shank faml|y of
postsynaptlc denslty protelns. Neuton 1999, 23:583·592.
23. lrie M, Hata Y, Takeuchi M, lchtchenko K, Toyoda A, Hirao K, Takai Y,
Posahl TW, Sudhol TC: Blndlng of neuro|lglns to PSD·95. Science
199?, 277:1511·1515.
24. Nguyen T, Sudhol TC: Blndlng propertles of neuro|lgln 1 and
neurexln 1 revea| functlon as heterophl|lc ce|| adheslon
mo|ecu|es. J Biol Chem 199?, 272:26031·26039.
25. Missler M, Fernandez·Chacon P, Sudhol TC: The maklng of
neurexlns. J Neutochem 1998, 71:1339·134?.
26. Shapiro L, Colman DP: The dlverslty of cadherlns and lmp|lcatlons
for a synaptlc adheslve code ln the CNS. Neuton 1999, 23:42?·430.
2?. Seralini T: Flndlng a partner ln a crowd. neurona| dlverslty and
synaptogenesls. Cell 1999, 98:133·136.
28. Wu O, Maniatis T: A strlklng organlzatlon of a |arge faml|y of human
neura| cadherln·|lke ce|| adheslon genes. Cell 1999, 97:??9·?90.
29. Craig AM: Actlvlty and synaptlc receptor targetlng. the |ong vlew.
Neuton 1998, 21:459·462.
30. Pao A, Craig AM: Actlvlty regu|ates the synaptlc |oca|lzatlon of the
NMDA receptor ln hlppocampa| neurons. Neuton 199?, 19:801·812.
31. Lissin Dv, Gomperts SN, Carroll PC, Christine CW, Kalman D,
Kitamura M, Hardy S, Nicoll PA, Malenka PC, Zastrow Mv: Actlvlty
dlfferentla||y regu|ates the surface expresslon of synaptlc AMPA
and NMDA g|utamate receptors. Ptoc NoIl Acod Sci USA 1998,
95:?09?·?102.
32. O'Brien P1, Kamboj S, Ehlers MD, Posen KP, Fischbach GD,
Huganir PL: Actlvlty·dependent modu|atlon of synaptlc AMPA
receptor accumu|atlon. Neuton 1998, 21:106?·10?8.
33. Lissin Dv, Carroll PC, Nicoll PA, Malenka PC, Zastrow Mv: Papld,
actlvatlon·lnduced redlstrlbutlon of lonotroplc g|utamate
receptors ln cu|tured hlppocampa| neurons. J Neutosci 1999,
19:1263·12?2.
34. Shi S·H, Hayashi Y, Petralia PS, Zaman SH, Wenthold P1, Svoboda K,
 Malinow P: Papld splne de|lvery and redlstrlbutlon of AMPA
receptors after synaptlc NMDA receptor actlvatlon. Science 1999,
284:1811·1816.
Using GFP·tagged GluP1 subunits virally translected into hippocampal slice
cultures, the authors show that local tetanic stimulation induces in spines a
rapid delivery and clustering ol AMPA receptors that otherwise are mostly
intracellular. This activity·dependent synaptic recruitment ol AMPA receptors
takes place not only in synapses already containing AMPA receptors, but
also in spines lacking AMPA receptors. This is the lirst direct visualization ol
AMPA receptor redistribution in real·time.
35. Carroll PC, Lissin Dv, von Zastrow M, Nicoll PA, Malenka PC: Papld
redlstrlbutlon of g|utamate receptors contrlbutes to |ong·term
depresslon ln hlppocampa| cu|tures. NoI Neutosci 1999, 2:454·460.
36. Maletic·Savatic M, Malinow P, Svoboda K: Papld dendrltlc
 morphogenesls ln CA1 hlppocampa| dendrltes lnduced by
synaptlc actlvlty. Science 1999, 283:1923·192?.
See annotation [3?

|.
3?. Engert F, Bonhoeller T: Dendrltlc splne changes assoclated wlth
 hlppocampa| |ong·term synaptlc p|astlclty. NoIute 1999, 399:66·?0.
ln this paper and the one by Maletic·Savatic eI ol. [36

|, it is shown that LTP·
inducing stimuli cause a rapid outgrowth ol dendritic lilopodia and lormation
ol new spines, in an NMDA·receptor·dependent manner. Thus, synaptic
activity triggers morphological changes that may correlate with new con·
nections between neurons. lt remains to be determined whether the newly
lormed spines are lunctional, and what molecular mechanisms underlie such
structural changes.
38. Kirov SA, Sorra KE, Harris KM: S|lces have more synapses than
 perfuslon·flxed hlppocampus from both young and mature rats.
J Neutosci 1999, 19:28?6·2886.
Hippocampal slices are routinely used lor electrophysiological studies (e.g. lor
assessing LTP or LTD). The authors lound a surprising ~ twolold increase in
the density ol spines in hippocampal slices, occuring within 2 h ol sectioning.
This increase in spine density was attributed to the temporary loss ol electrical
activity and release ol spinogenic lactors resulting lrom tissue trauma.
39. McKinney PA, Capogna M, Durr P, Gahwiler BH, Thompson SM:
Mlnlature synaptlc events malntaln dendrltlc splnes vla AMPA
receptor actlvatlon. NoI Neutosci 1999, 2:44·49.
40. verderio C, Coco S, Fumagalli G, Matteoli M: Spatla| changes ln
ca|clum slgna|lng durlng the estab|lshment of neurona| po|arlty
and synaptogenesls. J Cell Biol 1994, 126:152?·1536.
41. Augustin l, Posenmud C, Sudhol TC, Brose N: Munc 13·1 ls
 essentla| for fuslon competence of g|utamaterglc synaptlc
veslc|es. NoIute 1999, 400:45?·461.
The authors generated knockout mice ol Munc 13·1, a presynaptic phorbor
ester receptor. Surprisingly, the mutant mice develop ultrastructually normal
synapses, despite the lact that the synaptic vesicle cycle is arrested and
most excitatory synapses are lunctionally silenced. Therelore, in addition to
conlirming the importance ol Munc 13·1 in neurotransmitter release, this
paper implies that synaptic activity is dispensable lor morphological devel·
opment ol synapses.
42. Levi S, vannier C, Triller A: Strychnlne·sensltlve stabl|lzatlon of
 postsynaptlc g|yclne receptor c|usters. J Cell Sci 1998, 111:335·345.
See annotation [43

|.
43. Kirsch 1, Betz H: G|yclne·receptor actlvatlon ls requlred for
 receptor c|usterlng ln splna| neurons. NoIute 1998, 392:?1?·?20.
Synaptic clustering ol GlyPs requires the anchoring protein gephyrin (see
[14

|). This paper and the one by Levi eI ol. [42

| highlight that activation
ol GlyPs is necessary lor GlyP clustering, as demonstrated in cultured spinal
neurons treated with the competitive GlyP antagonist strychnine. Calcium
entry through L·type Ca
2+
channels may mediate the ellect ol GlyP activa·
tion, which is depolarizing in developing spinal neurons. Thus, in contrast to
excitatory synapses and GABAergic synapses, development ol inhibitory
glycinergic synapses depends on synaptic activity. Kirsch and Betz [43

|, in
contrast to Levi eI ol. [42

|, lound that gephyrin localization at synapses also
depends on GlyP activation.
44. Ziv NE, Smith S1: Evldence for a ro|e of dendrltlc fl|opodla ln
synaptogenesls and splne formatlon. Neuton 1996, 17:91·102.
45. Harris KM: Structure, deve|opment, and p|astlclty of dendrltlc
splnes. Cutt Opin Neutobiol 1999, 9:343·348.
46. Fischer M, Kaech S, Knutti D, Matus A: Papld actln·based p|astlclty
ln dendrltlc splnes. Neuton 1998, 20:84?·854.
4?. Bradke F, Dotti CG: The ro|e of |oca| actln lnstabl|lty ln axon
formatlon. Science 1999, 283:1931·1934.
130 Deve|opment
48. Carey D: Syndecans. mu|tlfunctlona| ce||·surface co·receptors.
Biochem J 199?, 327:1·16.
49. Hsueh Y·P, Yang F·C, Kharazia v, Naisbitt S, Cohen AP, Weinberg P1,
Sheng M: Dlrect lnteractlon of CASK/LIN·2 and syndecan heparan
su|fate proteog|ycan and thelr over|applng dlstrlbutlon ln
neurona| synapses. J Cell Biol 1998, 142:139·151.
50. Ethell lM, Yamaguchi Y: Ce|| surface proteog|ycan syndecan·2
 lnduces the maturatlon of dendrltlc splnes ln rat hlppocampa|
neurons. J Cell Biol 1999, 144:5?5·586.
Syndecan·2 is a cell surlace heparan sullate proteoglycan that is enriched in neu·
ronal synapses. The authors show that premature expression ol syndecan·2 in
immature hippocampal neurons accelerates the maturation ol dendritic spines
lrom dendritic lilopodia. The carboxy terminal·EFYA motil (single letter amino acid
code) ol syndecan·2 (which binds to the PDZ·domain·containing proteins CASK
and syntenin) is required lor this spinogenic ellect, suggesting that a syndecan·
2·associated protein complex may play a role in spine development.
51. Hsueh Y·P, Sheng M: Pegu|ated expresslon and subce||u|ar
|oca|lzatlon of syndecan heparan su|fate proteog|ycans and the
syndecan·blndlng proteln CASK/LIN·2 durlng rat braln
deve|opment. J Neutosci 1999, 19:?415·?425.
52. Serpinskaya AS, Feng G, Sanes 1P, Craig AM: Synapse formatlon
by hlppocampa| neurons from agrln·deflclent mlce. Ðev Biol
1999, 205:65·?8.
53. O'Brien P1, Xu D, Petralia PS, Steward O, Huganir PL, Worley P:
 Synaptlc c|usterlng of AMPA receptors by the extrace||u|ar
lmmedlate·ear|y gene product Narp. Neuton 1999, 23:309·323.
Narp is a neuronal pentraxin secreted lrom both pre· and postsynaptic cells,
and it is localized to synapses. The authors show that Narp displays AMPA·
receptor·clustering activity by binding directly to the ectodomain ol AMPA
receptor subunits. A model is proposed in which Narp acts as an extracellu·
lar aggregating lactor lor AMPA receptors.
54. McAllister AK, Katz LC, Lo DC: Neurotrophlns and synaptlc
p|astlclty. Annu Rev Neutosci 1999, 22:295·318.
55. Causing CG, Gloster A, Aloyz P, Bamji SX, Chang E, Fawcett 1,
Kuchei G, Miller FD: Synaptlc lnnervatlon denslty ls regu|ated by
neuron·derlved BDNF. Neuton 199?, 18:25?·26?.
56. Martlnez A, Alcantara S, Borrell v, Plo 1AD, Blasi 1, Otal P, Campos N,
 Boronat A, Marbacid M, Silos·Santiago l, Soriano E: TrkB and TrkC
slgna|lng are requlred for maturatlon and synaptogenesls of
hlppocampa| connectlons. J Neutosci 1998, 18:?336·?350.
See annotation [5?

|.
5?. Pozzo·Miller LD, Gottschalk W, Zhang L, McDermott K, Du 1,
 Gopalakrishnan P, Oho C, Sheng Z·H, Lu B: Impalrment ln hlgh·
frequency transmlsslon, synaptlc veslc|e docklng, and synaptlc
proteln dlstrlbutlon ln the hlppocampus of BDNF knockout mlce.
J Neutosci 1999, 19:49?2·4983.
ln mouse knockouts ol either TrkB/TrkC [56

| or BDNF [5?

|, reduced den·
sity ol synaptic vesicles were noted, along with reduced expression ol
synaptic vesicle·associated proteins such as synaptophysin. These abnor·
malities correlate with presynaptic lunctional delicits in the BDNF knockout
mice, including impaired responses to high·lrequency stimulation. Additional
morphological delects, such as reduced axonal elaboration, larger presy·
naptic boutons, and reduced synapse density, were lound in TrkB· and, to a
lesser extent, in TrkC·delicient mice. Thus, neurotrophins may be important
lor the structural and lunctional maturation ol synapses.
Deve|opment of neuron-neuron synapses Lee and Sheng 131

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