Download Molecular heterogeneity of central synapses: afferent and target

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© 2001 Nature Publishing Group http://neurosci.nature.com
review
Molecular heterogeneity of central
synapses: afferent and target
regulation
Ann Marie Craig and Hélène Boudin
Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid, Campus Box 8108,
958 McDonnell Sciences Building, St. Louis, Missouri 63110, USA
Correspondence should be addressed to A.M.C. ([email protected])
Electrophysiological recordings show a functional spectrum even within a single class of synapse, with
individual synapses ranging widely in fundamental properties, including release probability, unitary
response and effects of previous stimulation on subsequent response. Molecular and cellular biological
approaches have shown a corresponding diversity in the complement of ion channels, receptors,
scaffolds and signal transducing proteins that make up individual synapses. Indeed, we believe that each
individual synapse is unique, a function of presynaptic cell type, postsynaptic cell type, environment,
developmental stage and history of activity. We review here the molecular diversity of glutamatergic
and GABAergic synapses in the mammalian brain in the context of potential cell biological mechanisms
that may explain how individual cells develop and maintain such a mosaic of synaptic connections.
The mammalian brain possesses a wonderful diversity and richness of synaptic connections. Machinery for regulated neurotransmitter release and response must be present at all synapses.
However, from the earliest morphological studies classifying brain
synapses into Gray’s type I and type II (ref. 1), it has been clear that
all synapses are not equivalent. Synaptic composition is initially
determined and continuously modified by ongoing bidirectional
communication between presynaptic and postsynaptic partners.
Nevertheless, individual features of synapses can sometimes result
from a dominating influence, and it seems useful to consider major
classes of cellular influences on synaptic composition (Fig. 1).
One pattern of synaptic protein localization is ubiquitous
expression and distribution (Fig. 1a). A few molecules, particularly some that are involved in synaptic vesicle fusion, are
expressed by all neurons and distributed to all synapses. An
alternative is cell-specific expression (Fig. 1b), in which molecular components of synapses are selectively transcribed and/or
translated only in certain subsets of neurons. In the simplest
form of heterogeneity, these components are targeted to all
synapses made by the expressing neurons. Another pattern
reflects the influence of presynaptic terminals on postsynaptic
development. For example, glutamatergic versus GABAergic
neurotransmitter phenotype is a feature of presynaptic neuron
development that occurs independent of target contact. Thus,
the precise localization of glutamate receptors in dendrites
opposite glutamatergic terminals and GABA receptors in dendrites opposite GABAergic terminals must require some form
of anterograde communication (Fig. 1c). Influences from the
presynaptic cell determine not only the chemical nature of the
receptive field but often also the complement of specific receptor subtypes and associated signaling proteins (Fig. 1d).
Postsynaptic events can also influence presynaptic development (Fig. 1e). The target cell can strongly influence the molecular composition and release properties of individual
nature neuroscience • volume 4 no 6 • june 2001
presynaptic sites of a given axon, a phenomenon that clearly
requires retrograde signaling. Distribution of some proteins is
regulated by cellular domain (Fig. 1f). Some aspects of synaptic composition are determined by synapse location on the
cell, and this in turn can be determined intrinsic features of the
neuron. Thus certain domains of axons or dendrites are permissive or instructive for specific classes or features of
synapses. Finally, heterogeneity also occurs within one synapse
type (Fig. 1g). Activity dynamically regulates the density of
receptors and other signaling components at postsynaptic
sites. Such regulation of AMPA-type receptors has been a focus
of study as a mechanism underlying long-term changes in
synaptic efficacy. Surprisingly, the molecular composition and
release and response properties made by an individual axon
onto an individual dendrite are still quite heterogeneous
(Fig. 1h). Thus, some element of randomness, perhaps in
addition to geometrical influences, is required to explain such
heterogeneity even among autapses of an isolated neuron.
Ubiquitous expression and distribution
Surprisingly few proteins are common to all synapses. Indeed,
we can think of no postsynaptic or cleft components that fit this
category, although much of the machinery of synaptic vesicle
fusion is conserved among synapse types. The v-SNARE
VAMP-2/synaptobrevin II, t-SNARES syntaxin 1A and 1B and
SNAP-25, calcium sensor synaptotagmin I, and regulatory proteins rab3A, SV2A,B, Munc 13-1 and Munc18-1 are widely
expressed and function in all forebrain terminals2 (Fig. 2). However, even these core regulators of vesicle fusion are not ubiquitous. First, different isoforms of many of these proteins are
expressed in other brain regions. VAMP-1 and synatotagmin II
are expressed instead of VAMP-2 and synaptotagmin I in many
parts of the brainstem and spinal cord3,4, and ribbon synapses
in the retina contain syntaxin 3 instead of syntaxin 1 (ref. 5). Sec569
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cell
a Pyramidal
(glutamatergic)
Interneuron
(GABAergic)
Dendrite
Axon
Presynaptic protein
Postsynaptic protein
© 2001 Nature Publishing Group http://neurosci.nature.com
Glu-R: Glutamate receptor
gr.III-mGluR: group III metabotropic
glutamate receptor
GABA-R: GABA receptor
Ubiquitous expression and distribution
(presynaptic protein)
b
Cell specific expression:
presynaptic protein
c
Glu-R
GABA-R
Cell specific expression:
postsynaptic protein
d
Presynaptic influences postsynaptic:
transmitter specificity
(postsynaptic protein)
e
gr.III-mGluR
Glu-R
Presynaptic influences postsynaptic:
glutamatergic synapses
(postsynaptic protein)
f
GABA-R
Regulation by cellular domain
(postsynaptic protein)
Postsynaptic influences presynaptic
(presynaptic protein)
g
Glu-R
h
Heterogeneity within one synapse type:
activity regulation (postsynaptic protein)
Autaptic heterogeneity
(post- and presynaptic proteins)
ond, related isoforms of several of these proteins are also
expressed in forebrain, although they may be functionally nonredundant. Hippocampal pyramidal neurons express several
synaptotagmin isoforms including high levels of synaptotagmin
III (ref. 3), but none can rescue the fast component of calciumdependent release that is abolished by deletion of synaptotagmin
I (ref. 6). Presynaptic cytomatrix organizing proteins have not
been characterized as fully; current evidence suggests they are
also widely expressed, but not ubiquitous. For example, bassoon
and piccolo are present at all forebrain glutamatergic and
GABAergic synapses but not at the neuromuscular junction7.
Cell-specific expression
The simplest mechanism underlying synaptic heterogeneity is
cell-type specific expression of a protein, and targeting of that
protein to all presynaptic or postsynaptic sites made by a cell
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Fig. 1. Cellular mechanisms for the generation of synaptic heterogeneity. Model cell pairs are drawn to show how the molecular composition
of synapses might be influenced by the presynaptic and postsynaptic
partners. The presence of a protein at a synapse is indicated by a red or
blue circle (presynaptic proteins), or a red or blue square (postsynaptic
proteins). Expression of a protein is indicated by color fill of the soma.
(a) Ubiquitous expression and distribution. (b) Cell-specific expression.
(c) Presynaptic terminal influences postsynaptic: transmitter specificity.
(d) Presynaptic terminal influences postsynaptic: glutamatergic synapses.
(e) Postsynaptic influences presynaptic. (f) Regulation by cellular
domain. (g) Heterogeneity within one synapse type: activity regulation.
(h) Autaptic heterogeneity.
(Fig. 1b). On the presynaptic side, neurotransmitter synthetic
enzymes and vesicular and plasma membrane transporters that
determine the chemical nature of a synapse generally fall into this
category. The GABA synthetic enzyme glutamic acid decarboxylase (GAD65 and GAD67), the vesicular inhibitory amino acid
transporter responsible for loading GABA into vesicles
(VIAAT/VGAT), and the major plasma membrane transporter
responsible for reuptake of GABA (GAT-1) are found at symmetric but not asymmetric synapses8–11. In situ hybridization
studies indicate this is due primarily to expression by GABAergic but not glutamatergic neurons9,12,13. Direct evidence for targeting of GAD to all presynaptic terminals of expressing cells has
been shown by colocalization of GAD and synaptophysin
immunofluorescence puncta in isolated neuron cultures14. Glutamatergic phenotype seems to be mainly conferred by expression of the vesicular glutamate transporter BNPI (refs. 15, 16).
Exogenous expression of BNPI in GABAergic neurons resulted
in release of both glutamate and GABA. Thus, expression of a
small number of genes or even a single gene can determine presynaptic transmitter phenotype.
There are many instances of cell-type-specific expression of
postsynaptic components, resulting in differential signaling from
a single axon to individual targets expressing a different complement of receptors or synaptic signaling molecules. Selective gene
expression in pyramidal neurons versus interneurons of the hippocampus is thought to underlie major differences in properties
of synaptic transmission and plasticity at glutamatergic
synapses onto these two types of target cell. Whereas CA1 pyramidal neurons undergo homosynaptic long-term potentiation
(LTP) in response to high-frequency stimulation of CA3 afferents, interneurons exhibit long-term depression (LTD) of the
activated and non-activated synapses with the same stimulation
method17. A major contributing factor to the absence of LTP in
the interneurons may be the absence of the calcium-activated
kinase CaMKIIα. CaMKIIα is highly expressed by pyramidal
neurons, concentrated at spiny synapses, and required for LTP
in pyramidal neurons18, but it is not expressed by interneurons19.
Activated CaMKII enhances synaptic delivery of GluR1 (ref. 20)
and potentiates AMPA receptor function by direct phosphorylation of GluR1 (refs. 21, 22). Other differences that likely contribute to differential signaling include higher levels of AMPA
receptor, the postsynaptic scaffolding protein GKAP, and the activated Rho target citron, and lower levels or an absence of the calcium-activated phosphatase calcineurin, the synaptic Ras GTPase
activating protein SynGAP, and the actin- and NMDA- receptorbinding protein α-actinin-2 in hippocampal interneurons compared with pyramidal neurons 19,23–25 . The mechanisms of
localization of common components may also differ between cell
types, as revealed by the greater detergent extractability and
dependence on actin of synaptic AMPA receptor clusters on pyramidal neurons versus on interneurons26.
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Fig. 2. Molecular components of glutamate
synapses and GABA synapses. Components are
color coded according to function. Red, neurotransmitter receptors; pink, enzymes; blue,
scaffolds; turquoise, cell adhesion proteins;
green, vesicle fusion machinery. Gray lines, protein–protein interactions. Synaptic components
not discussed in this review are not shown.
Synaptobrevin II
Syntaxin 1A,B
SNAP-25
Synaptotagmin I
Rab3A
SV2A,B
Munc13-1
Munc18-1
Glutamate
BNPI
Piccolo
Bassoon
Glutamate
PRESYNAPTIC
N-cadherin
PICK1
Narp
AMPAR
Piccolo
GABA
VIAAT/VGAT
Bassoon
GABA
mGluR7
N-cadherin
Glutamate
GAD
Neurexin
GABA
NMDAR Neuroligin mGluR1
Cadherin
GAT-1
NSF
Cadherin
GABA AR
Thus, selective gene expression is a way
SynGAP
Stargazin
PSD-95
GRIP
that intrinsic features of a differentiated
GRIP
ABP
Citron
POSTSYNAPTIC
neuron influence the molecular composiActinin GKAP
Gephyrin
Homer
CaMKII
tion of synaptic connections. Such intringephyrin
Shank
Calcineurin F-actin
sic influences extend also to controlling
GABA SYNAPSE
synaptic morphology. Excitatory synapses
GLUTAMATE SYNAPSE
on hippocampal pyramidal neurons occur
primarily on dendritic spines, whereas
excitatory synapses on the interneuron dendrites are on shafts.
tional pairing of presynaptic and postsynaptic elements: GABAA
This morphological difference may be determined at least parreceptors are concentrated along with glutamate receptors at glutially by differential expression of actin regulatory proteins, and
tamatergic mossy fiber synapses onto cerebellar granule cells and
has major consequences for postsynaptic calcium dynamics27.
are thought to be activated by GABA spillover from nearby
synapses39. Group I metabotropic glutamate receptors are conCell-type regulation of postsynaptic architecture may be a widespread phenomenon; for example, inhibitory Renshaw cells show
centrated at GABAergic postsynaptic specializations in monkey
larger and more complex glycinergic postsynaptic specializations
pallidus40; glycine receptors are concentrated adjacent to acetylthan neighboring motoneurons28.
choline receptors at cholinergic synapses on chick ciliary ganglion neurons and can be activated by non-vesicular glycine
release41. Presynaptic glutamic acid decarboxylase, which conPresynaptic influences: transmitter specificity
verts glutamate to GABA and is generally specific for GABAerAn obvious and ubiquitous type of afferent-specific differentiagic terminals, is surprisingly required for development of the
tion is the chemical matching of postsynaptic receptor with transpostsynaptic glutamate receptor field at the Drosophila glutamitter released (Fig. 1c). Immunoelectron microscopy shows that
matergic neuromuscular junction42. These observations of misreceptors cluster only at a subset of postsynaptic sites, GABAA and
glycine receptors generally at symmetric synapses29,30 and AMPA
matched and unconventional appositions raise the idea that
synapse development may occur by initial formation of non-speand NMDA glutamate receptors at asymmetric synapses31,32. Doucific appositions followed by selective maintenance of physioble-label immunofluorescence of hippocampal neurons in lowlogically appropriate appositions and destabilization of
density culture shows that GABA receptors cluster selectively
inappropriate appositions36. This model would imply an active
opposite GABAergic terminals, whereas AMPA receptors cluster
33
opposite glutamatergic terminals . Thus, mechanisms must exist
role of both presynaptic and postsynaptic partners.
An attractive idea is that activity, by means of release of transfor targeting GABA versus glutamate receptors to separate postmitter and activation of the appropriate receptor, may be a key
synaptic sites, often a few microns apart on a single dendrite.
signal for synaptogenesis or synapse stabilization. Indeed, for
Because neurons develop glutamatergic versus GABAergic
glycinergic synapses, receptor activation is required for receptor
phenotypes in the absence of target contact, anterograde signals
stabilization at synapses 43,44. However, although one study
must be involved in matching receptor to transmitter type. The
role of presynaptic innervation was directly demonstrated by culreported that activation of AMPA receptors is required for mainture of purified motoneurons alone or with GABAergic innertenance of dendritic spines45, all studies of receptor distributions
vation: isolated motoneurons lacked synaptic GABAA receptor
by immunocytochemistry, calcium imaging or functional mapping report that postsynaptic clusters of GABAA and NMDAclusters, but contact with GABAergic axons locally induced
synaptic GABAA receptor clustering34. However, experiments in
and AMPA-type glutamate receptors form normally opposite
appropriate terminals under conditions of chronic receptor
other systems implicate retrograde signals: the targets of sympablockade33,36,46–48. This does not rule out a role for competitive
thetic neurons direct cholinergic versus noradrenergic neuro35
transmitter selection , and even in hippocampal neurons a
activity in selective synapse elimination, but the evidence indicates that activity per se is not a basic requirement for synaptosimple anterograde signaling model does not account for some
genesis. The formation of some morphological synapses in vivo
recent observations36. Hippocampal neurons grown in isolation
in the absence of Munc18-1, a protein essential for synaptic vesion a small dot of permissive substrate form functional autapscle fusion, also supports this idea49.
es37,38. However, in addition to the appropriately matched connections, these isolated neurons also form mismatched
Potential molecular families that may serve as signals for
appositions of receptor and transmitter 36 . By triple-label
synaptogenesis include neurexins and neuroligins50,51, cadimmunocytochemistry, isolated glutamatergic pyramidal neuherins52,53, cadherin-related neuronal receptors54, and ephrins
rons were shown to form clusters of NMDA glutamate receptor
and Eph receptors55,56. One can imagine how binding of presyopposite some terminals, as expected, but also to form clusters
naptic and postsynaptic partners could lead to costabilization of
of GABAA receptor opposite different terminals of the same
protein scaffolds or activation of signaling pathways thus triggering common aspects of synapse development, or triggering
axon36. The specific aggregation of GABAA receptors opposite
development of specialized synaptic properties if the participatthese glutamatergic terminals could not be accounted for by raning molecules exhibit selective expression patterns. All of these
dom apposition. In vivo, some synapse types also use unconven-
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Fig. 3. Mechanisms of postsynaptic protein targeting and potential sites
of afferent-specific influence (*). Potential trafficking routes are diagrammed for different glutamate receptor subunits (white and black triangles) and different GABA receptor subunits (white and black squares).
This figure is modified from ref. 130.
required for postsynaptic clustering of both glycine and GABAA
receptors68,69. Motoneurons participate postsynaptically in pure
glycinergic, pure GABAergic, and mixed glycinergic and GABAergic synapses, raising the interesting question of how receptor and
gephyrin distribution is regulated34.
protein families have been localized to subsets of synapses and/or
bind to synaptic components and have multiple family members
that could mediate selective interactions. Neuroligins presented
on the surface of a fibroblast can induce presynaptic specializations in glutamatergic axons51; it is not yet known whether this
ability extends to other classes of axons. N-cadherin shows an
interesting localization to both GABAergic and glutamatergic
synapses early in development and a restriction to glutamatergic synapses later in development in hippocampal cultures57.
Studies are no doubt underway to determine the roles of these
protein families in synapse matching. Another protein that may
be involved in such a mechanism at a specific type of synapse
from hippocampal pyramidal neurons to interneurons is Narp
(neuronal activity-regulated pentraxin). Narp self-associates,
clusters AMPA receptors when expressed on the same or an
opposing cell, and is selectively localized to pyramidal-interneuron synapses and to pyramidal axons but not GABAergic axons in
culture58. Based on these properties, it is tempting to speculate
that Narp secreted by pyramidal cell axons and by interneuron
dendrites may be stabilized at these points of contact and induce
the high levels of AMPA receptor at such synapses.
Within the postsynaptic cell, most scaffolding proteins and
some signal transducing enzymes are also concentrated at specific chemical synapse types. The PSD-95 family, stargazin,
PICK1, GKAP and Shank scaffolding proteins and the enzymes
CaMKIIα and SynGAP are concentrated opposite glutamatergic
but not GABAergic terminals24,59–63. One exception to this idea
is the AMPA receptor binding protein GRIP, which has been
found at glutamatergic and at GABAergic synapses64,65. The
transmitter-specific localization of most of these postsynaptic
density proteins is presumably controlled by the same transsynaptic signals as for the glutamate receptors. Furthermore, local
aggregation and regulation of these scaffolding proteins is thought
to contribute to the selective localization of the receptors. Synaptic targeting of AMPA receptors requires stargazin59, and stabilization of AMPA receptors at synapses requires GRIP or ABP66.
The major inhibitory scaffolding protein gephyrin binds glycine
receptors and colocalizes with both glycine and GABAA receptors but not glutamate receptors67. Furthermore, gephyrin is
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Presynaptic influences: glutamatergic synapses
Individual neurons can establish a distinct complement of glutamate receptor subtypes opposite different glutamatergic afferents (Fig. 1d). Such selective targeting can occur between
dendrites or between parts of a single dendrite. A classic example
is the composition and properties of mossy fiber versus commissural/associational synapses on hippocampal CA3 pyramidal
neurons. Whereas commissural/associational-CA3 synapses
exhibit LTP that is dependent on activation of postsynaptic
NMDA receptors, LTP at mossy fiber-CA3 synapses has a presynaptic locus and is independent of NMDA receptor activity70.
Furthermore, the NMDA receptor-mediated conductance is less
at mossy fiber-CA3 synapses than at commissural/associationalCA3 synapses71. The basis for this difference can be accounted
for by differences in postsynaptic levels of NMDA receptor protein observed with the sensitive method of antigen retrieval
immunohistochemistry72,73. NR1 and NR2A subunits are of low
abundance at synapses postsynaptic to mossy fibers, and NR2B
almost undetectable, whereas all are abundant at synapses postsynaptic to commissural/associational fibers. These differences
could be visualized by light microscopy due to the laminar distribution of inputs. We suspect that such heterogeneity in postsynaptic glutamate receptor content may be widespread, but
perhaps not so readily visualized in regions where inputs are not
so highly segregated. Indeed, single hippocampal interneurons
can express functional GluR2-containing AMPA receptors opposite inputs from CA3 neurons and GluR2-lacking AMPA receptors opposite some inputs from mossy fibers74; the sites of input
have not been mapped but may well be intermingled.
Two general cellular mechanisms could underlie such selective postsynaptic targeting; local receptor clustering could be triggered by specific innervation during a limited developmental
stage, or be continuously dependent on an anterograde signal.
The developmental stage of the postsynaptic neuron can be a limiting factor in synaptogenesis. Neurons from different embryonic stages grown in culture for identical periods have different
synaptogenic potential75,76, as do neurons from a single embryonic stage grown in culture for different periods77. Postsynaptic
protein expression restricted to the time of contact by one input
could lead to a localized response that is then propagated by other
means. However, this is not the only possible mechanism for
establishing differential synaptic composition, and indeed evidence indicates that some glutamatergic synapses initially
develop with a similar composition and later diverge. Specifically, in mature Purkinje cells, delta-type glutamate receptors are
present at parallel fiber but not climbing fiber synapses, even
though these receptors are present at both glutamatergic synapse
types earlier in development78. Moreover, a simpler possible
mechanism than differential responsiveness on the part of the
postsynaptic neuron is a differential signal from the presynaptic
afferent. Such a signal could be either differential patterned activnature neuroscience • volume 4 no 6 • june 2001
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ity by the two inputs or expression of different cell surface or
secreted molecular signals independent of activity.
Regardless of the nature of the afferent-specific inducing signal, establishment and maintenance of differential receptor localization presents an interesting challenge for the postsynaptic
neuron. Targeting of postsynaptic receptors presumably involves
synthesis in the soma and sorting into vesicles from the Golgi
network, vesicle transport, vesicle fusion with the plasma membrane, and anchoring and possibly trapping of the receptors in
the plasma membrane (Fig. 3). Questions abound regarding all of
these steps. For example, are glutamate versus GABA receptors,
and even different glutamate receptor types, transported in common or separate vesicles? Which molecular motors79 transport
each class of vesicle? A molecular linkage mediated by the PDZ
domain proteins mLin2, mLin7 and mLin10 connects NMDA
receptors and KIF17, a novel dendritic (+) end-directed microtubule motor80. It is not yet known whether these KIF17-driven
vesicles also carry other neurotransmitter receptors. Immunogold localization revealed segregation of GluR4 and mGluR1α
to synapses and to intracellular membranes of basal dendrites
relative to apical dendrites in fusiform cells of the dorsal cochlear
nucleus81, suggesting the possibility of selective transport to different dendrites. Additional such molecular and anatomical studies combined with live imaging of dendritic vesicles following
the GFP-based visualization of polarized vesicle transport82 may
serve to define classes and routes of dendritic vesicle trafficking.
An equally challenging question is to determine the sites of
fusion of receptor-bearing vesicles with the plasma membrane
and the behavior of receptors once in the plasma membrane. It
seems unlikely that vesicle fusion occurs within the postsynaptic density, but whether it is localized near the appropriate synapse
or occurs over the entire dendrite surface is not known. Trapping and anchoring in the membrane, and endocytosis, further
sorting, and reinsertion or degradation may also have major roles
in selective receptor localization. Any of these processes could be
influenced by a local afferent-derived signal.
NMDA receptors are maintained at synapses
independent of F-actin26, possibly by anchora
Fig. 4. Selective localization of mGluR7a receptors
to presynaptic sites formed onto GABAergic
interneurons. (a) Immunogold localization of
mGluR7a receptors in the CA3 area of the hippocampus shows a differential expression of
mGluR7a depending on the target cell. A single
nerve terminal (T) contains two types of synapses:
the one heavily labeled for mGluR7 is facing a dendritic shaft (D), characteristic of GABAergic
interneurons, whereas the other one lacking any
detectable mGluR7a immunoreactivity is facing a
dendritic spine (S), characteristic of pyramidal cells.
Scale bar, 0.2 µm. (b) Target-signal-dependent
presynaptic localization of mGluR7 as evidenced in
the hippocampus. (c) The target-specific localization
of mGluR7a onto GABAergic interneurons is also
observed in cultured hippocampal neurons.
Cultured hippocampal neurons at 18 days in vitro
were immunolabeled for mGluR7a and glutamic acid
decarboxylase (GAD), as a marker for GABAergic
cells (*). mGluR7a immunoreactivity is selectively
more intense in axon boutons innervating
GABAergic interneurons (arrows) than pyramidal
cells (arrowheads). Scale bar, 50 µm. Panels (a) and
(b) provided by P. Somogyi and colleagues89; published with permission from Nature, copyright 1996.
nature neuroscience • volume 4 no 6 • june 2001
ing to PDZ domain proteins such as the PSD-95 family83, and
do not undergo rapid cycles of endo/exocytosis84. In contrast,
AMPA receptor maintenance is dependent on F-actin26 and
involves rapid recycling of surface and intracellular receptors84,85.
Examples of changes in anchoring or endo/exocytosis exist for
activity regulation of individual synapses (see below), but not
yet for afferent cell-type specific regulation. Yet another possible
form of afferent-specific response could be through regulation
of local protein synthesis. However, this would only seem possible for those few components with moderately abundant levels
of dendritic mRNA86, such as CaMKIIα, the NMDA receptor
NR1 subunit87, and the glycine receptor alpha subunits88.
Similar cellular trafficking events as those described above and
outlined in Fig. 3 in the context of differential targeting of postsynaptic glutamate receptors are also presumed to underlie the
transmitter-specific clustering of glutamate versus GABA receptors
(previous section) as well as the target-specific presynaptic clustering of glutamate receptors presented in the next section.
Postsynaptic influences
Not only can two postsynaptic sites on a single dendrite exhibit
heterogeneity, but two boutons of a single axon can have a different molecular composition and functional properties
(Fig. 1e). Such a phenomenon must involve target-specific retrograde signals. One of the most striking examples of presynaptic heterogeneity is the selective localization of mGluR7a and
other group III metabotropic glutamate receptors 89–91
(Fig. 4). mGluR7a density in a single hippocampal CA3 axon is
∼10-fold higher at terminals opposite a subset of interneurons
compared with terminals opposite CA1 pyramidal cells89. This
target-specific selective localization is reflected in functional properties of transmission. Glutamate release from a single CA3 cell
was inhibited by group III mGluR antagonists at synapses onto
interneurons but not onto CA1 cells92. The selective localization
of mGluR7a is reproduced in dissociated neuron cultures
b
c
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a
b
(Fig. 4), a system that may be amenable to mechanistic analysis.
Several other instances of differential target-dependent signaling
by a single axon have been reported, although in most cases the
molecular basis for this difference is not known. For example,
high-frequency stimulation of single neocortical neurons leads
to a facilitating response in some target cells but a depressing
response in others93,94, and BDNF-induced potentiation of transmission from single glutamatergic hippocampal neurons is also
target-cell specific95. The locus of these differences in signaling
is thought to be presynaptic in all of these cases, suggesting that
postsynaptic contact during development results in differences
in molecular composition and thus release properties, and possibly also structural characteristics, of these different terminals
of single axons.
It may be common for particular synaptic properties to result
only from interdependent afferent and target-derived signals. For
instance, hippocampal dentate granule–CA3 contacts are characterized by large zinc-rich mossy fiber boutons onto branched
postsynaptic spines96. The same axons form simple bouton contacts with interneurons, and the same dendrites form simple
spine contacts with other pyramidal cells, suggesting that joint
anterograde and retrograde signaling is involved in determining
the complex synaptic form. In this case, postsynaptic domainspecific factors may also have a role, because these complex
synapses form on the proximal dendrites of the CA3 neurons,
forming the stratum lucidum.
Regulation by cellular domain
Intrinsic features of neuronal geometry can specify synapse type
and molecular composition (Fig. 1f). Certain subcellular domains
can be permissive for specific synapse types and even selective
for particular receptors. A prime example of such a selective cellular domain is the axon initial segment. This region of hippocampal pyramidal cells is permissive (or perhaps instructive)
for formation of GABAergic but not glutamatergic postsynaptic
sites8. Furthermore, GABAergic synapses on the axon initial segment have a higher density of the GABAA receptor α2 subunit
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Fig. 5. Model for the trafficking of glutamate AMPA receptors at postsynaptic sites. (a) Both AMPA and NMDA receptor activation induce a
dynamin-dependent AMPA receptor endocytosis into intracellular compartments without affecting NMDA receptor distribution. The PDZ
domain-containing protein PICK1 might participate in this process,
whereas GRIP might be primarily involved in the postsynaptic stabilization of AMPA receptors. NMDA receptor activation leads to a complete
recycling of AMPA receptors to the cell surface, whereas upon AMPA
receptor activation, a fraction of internalized AMPA receptors is
degraded. The intracellular pathway of AMPA receptor trafficking is
depicted in red when induced by AMPA receptor activation and in blue
when induced by NMDA receptor activation. (b) Hypothesis of the
involvement of AMPA receptor cycling in LTP and LTD. Under basal conditions, exocytosis and endocytosis occur at the same rate. Specific
stimuli, by decoupling exo- and endocytosis, could induce a net gain
(LTP) or loss (LTD) of membrane-associated synaptic AMPA receptors.
than do GABA synapses on dendrites of the same cell, whereas
the α1 subunit is more uniformly targeted to both somatodendritic and axon initial segment GABAergic synapses97. This selective targeting of the α2 subunit may be in part induced by the
specific innervation of the axon initial segment by the axo-axonic class of GABAergic neuron. However, the postsynaptic neuron
seems to have an active role in the formation of GABAergic
synapses on this domain, as the GABAA but not glutamate receptor clusters form on this domain even in pyramidal neurons
grown in the absence of GABAergic input (A. Rao and A.M.C.,
unpublished data). GABAergic synapses also form preferentially
on somata by an activity-independent mechanism98.
Another intriguing example of how geometry influences
synaptic composition and function is the evidence for gradients of
increasing synaptic size and current amplitude along a single dendrite with increasing distance from the soma. In hippocampal
pyramidal neurons, synaptic conductance increases with distance
from the soma, thus counterbalancing dendritic filtering effects
and resulting in location-independent responses at the soma99.
The data are consistent with a mechanism involving a gradient in
synapse size. For inhibitory glycinergic synapses, somatodendritic gradients in synapse size, or immunofluorescence cluster size
of glycine receptors100 or gephyrin28, has been observed, potentially also to compensate for greater electrotonic attenuation of
synaptic responses with distance. Exactly how proximo-distal position is sensed to establish such a gradient presents an interesting
puzzle. Another study101 reported that postsynaptic responsiveness varies inversely with the density of synaptic input onto hippocampal neurons in culture, perhaps a mechanism to adjust the
dynamic range of the spike-firing decision.
Heterogeneity within one synapse
Considering only one class of synapse (a single presynaptic cell
type making synapses onto a single postsynaptic cell type), a surprising heterogeneity of features still exists (Fig. 1g). This diversity
may be in large part determined by the activity of individual
synapses. One aspect of this heterogeneity that has received much
recent attention is the variability in postsynaptic AMPA type glutamate receptor content in hippocampal pyramidal neurons.
Quantitative electron microscopy studies of CA3 Schaeffer collateral synapses onto CA1 neurons found 19% of synapses strongly immunoreactive, 67% moderately immunoreactive and 17%
immunonegative for AMPA receptors23. Immunonegative synapses were exclusively the smaller synapses31. Because every Schaeffer
collateral synapse onto CA1 pyramidal cell spines contains
immunoreactive NMDA receptors31,32, a subset of these synapses
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review
express only NMDA receptors and could correspond to the
anatomical substrate for the electrophysiologically defined ‘silent
synapses.’ These observations suggest that the targeting mechanisms and the regulation of cell surface expression of these two
types of glutamate receptors follow different rules. Indeed, recent
studies converge to indicate that, unlike NMDA receptors, AMPA
receptors are highly mobile in a short time scale and that these
dynamic properties are relevant for the observed heterogeneity in
AMPA receptor synapse composition and could have an important
part in mediating different forms of synaptic plasticity.
Based on imaging and biochemical approaches, several reports
indicate that all AMPA receptors subunits can be rapidly removed
from and inserted into the postsynaptic membrane (Fig. 5). The
selective labeling of cell-surface AMPA receptors by antibodies
against extracellular epitopes or cell surface biotinylation assays
in cultured hippocampal neurons showed a rapid constitutive
internalization of AMPA receptors but not NMDA receptors under
basal conditions84,85,102. Intriguingly, blocking clathrin-mediated internalization of AMPA receptors affected neither the total
number of AMPA receptors expressed at the cell surface nor
AMPA mEPSCs84,102,103 (but see ref. 104). Ehlers84 directly showed
that internalization and reinsertion of the internalized receptors to
the plasma membrane was achieved at the same rate, and that an
increase in endocytosis induced by picrotoxin directly correlated
with an increase in receptor recycling to the plasma membrane.
These results suggest that there is an unknown mechanism tightly coupling the constitutive endocytosis and insertion of AMPA
receptors to maintain a stable amount of synaptic AMPA receptor.
This mechanism seems to involve interactions of AMPA receptor
subunits with the PDZ domain proteins GRIP or ABP105 and
interactions of GluR2 with the chaperone protein NSF106.
The rates of AMPA receptor endocytosis and exocytosis can
be uncoupled by synaptic activity, depending on the extent and
balance of AMPA and NMDA receptor stimulation, to effect a
net change in surface AMPA receptor levels. Treatment of cultured neurons with high levels of glutamate produces a massive
internalization of AMPA receptors, again without affecting
NMDA receptor distribution107. Treatment with either AMPA or
NMDA induces AMPA receptor endocytosis at a similar rate84.
However, the mechanisms, the intracellular sorting and the subsequent fate of internalized AMPA receptors are different upon
activation of each receptor type. NMDA induced only a transient
decrease in the total level of surface AMPA receptors, whereas
AMPA treatment induced late endosomal targeting and lysosomal
degradation of a fraction of internalized AMPA receptors84. This
results in a loss of membrane-associated AMPA receptors even
after a long time of recovery84,85, which parallels a decrease in
the frequency of mEPSCs107.
The hypothesis that activity-dependent redistribution of
AMPA receptors to and from the postsynaptic plasma membrane
is important in NMDA receptor-dependent LTP and LTD has
been strengthened over the past few years. Tetanic stimulation
generating LTP induced the appearance of electrophysiologically tagged GluR1 at synapses in hippocampal slices108. This synaptic recruitment of GluR1 observed upon LTP expression is
dependent on a PDZ domain-containing protein because this
effect required an intact C-terminus of GluR1 (ref. 20). Several
studies report that LTD involves net endocytosis of synaptic
AMPA receptors102,109, possibly through protein kinase C phosphorylation of the receptor, binding to the PDZ domain protein
PICK1, and inhibition of binding to GRIP and ABP110–112. Trafficking of different AMPA receptor subunits can also occur independently, as evidenced by another form of plasticity in cerebellar
nature neuroscience • volume 4 no 6 • june 2001
stellate cells involving a rapid activity-dependent switch in the
subunit composition of synaptic AMPA receptors113.
Unlike AMPA receptors, short-term manipulation of the level
of activity does not change the subcellular distribution of NMDA
receptors, but chronic blockade in hippocampal cultures induces
a large increase in synaptic NMDA receptor immunoreactivity114
and NMDA receptor-mediated excitotoxicity115. This effect is
mediated by redistribution of existing receptors to the synapse
by a mechanism requiring activation of cAMP-dependent protein kinase115. AMPA receptors also exhibit long-term homeostatic regulation of synaptic density, in most studies apparently
independently of NMDA receptors114,116,117 (but see ref. 118).
The localization of synaptic signaling proteins can also be
rapidly modified in response to changes in synaptic activity. The
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a signaling protein involved in synaptic plasticity: CaMKIIα mutant
mice show impaired hippocampal LTP and behavioral defects in
spatial learning and memory119. Live imaging studies of a GFPtagged CaMKII expressed in cultured hippocampal neurons
showed that NMDA receptor stimulation, through Ca2+ influx,
led to a rapid redistribution of CaMKII from dendritic cytosol
to postsynaptic sites120. This synaptic translocation required
binding to calmodulin, and the stabilization at synapses for several minutes required the autophosphorylation of the
kinase120–122. The transient localization of CaMKII at synapses
could modulate the function of various synaptic proteins, such as
AMPA receptors, by a direct phosphorylation21,22 or by phosphorylation of intermediate molecules involved in GluR1 synaptic delivery during LTP20.
Heterogeneity of postsynaptic receptor composition also exists
at inhibitory synapses. Cerebellar granule cells receive GABAergic input only from Golgi cells and thus represent an anatomically homogeneous class of synapses. All Golgi synapses were
found to contain similar levels of GABAA receptor β2/3 subunits,
but to be highly heterogeneous with regard to their α subunit
content. Quantitative electron microscopy indicated that three
populations of synapses were found according to their α subunit
content: one expressing only or mostly α1 subunit, one expressing only or mostly α6 subunit and one expressing both subunits
at similar levels39,123. This variability in α1/α6 subunit ratio could
account for the existence of fast and slow decays of spontaneous
IPSCs observed in granule cells124. The insertion of different α
subunits might be regulated by the activity of pre- and postsynaptic elements to adapt the postsynaptic composition of GABAA
receptors to changing needs of the system.
The basis for synaptic heterogeneity within one class of
synapse can also originate from some variability at presynaptic
sites. Measures of the release probability of neurotransmitter at
Schaeffer collateral/commissural fibers making synapses onto
CA1 neurons indicated that among these glutamatergic
synapses, two distinct classes of synapses with a sixfold difference in the release probability are resolved125. Synapses with low
release probability contribute over half of the transmission and
are more sensitive to drugs enhancing transmitter release. This
nonuniformity in release probability could rely on structural factors, such as the size of the release site, the number of available
vesicles and possible differences in the Ca2+ channel density126.
Autaptic heterogeneity
Even in the case of autaptic synapses formed in isolated neurons,
much evidence suggests that all synapses are not equivalent
(Fig. 1h). For instance, there is a wide variability in the level of
immunostaining for several synaptic vesicle-associated proteins,
575
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review
such as synaptophysin, synapsin I, and SV2, which correlates with
the extent of FM1-43 labeling at synapses127. Thus, differential
expression of synaptic vesicle-associated proteins can occur at
different boutons of the same neuron. The existence of two classes of synapses with different release probability described above
was also demonstrated in single cultured hippocampal neurons128. Similarly, the existence of a heterogeneity in the presynaptic maturation of synaptic vesicles in glutamatergic
hippocampal neurons was recently demonstrated by the generation of null mutant mice for Munc 13-1, a brain-specific presynaptic phorbol ester receptor 129. Recordings performed on
cultures from Munc 13-1-deficient mice showed that most
synapses of any given single excitatory neuron did not release
neurotransmitter, whereas transmitter release from a small subpopulation of synapses of the same cell was not affected and was
therefore independent of Munc 13-1. It is not yet clear whether
these differences in release properties are related to synapse size,
nor whether they are somehow determined by geometrical considerations such as the distance between the release site and the
soma or whether they result from a more random process. Postsynaptic receptor composition also varies widely among autaptic
sites of isolated neurons, as described above, resulting in both
matched appositions of terminals with the appropriate receptors
and mismatched appositions with chemically inappropriate
receptors36. In this case, sites of different receptor composition
have been observed intermingled along a single dendrite.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Conclusions
The molecular composition and functional properties of individual synapses, even among limited afferent and target cell types,
are surprisingly heterogeneous. Synaptic diversity results from
selective cellular expression and selective subcellular targeting of
synaptic components regulated by combinations of afferentderived and target-derived signals. Synaptic activity is one major
afferent-derived signal. Additional intrinsic factors such as cellular geometry and perhaps some element of randomness are
also required to explain the diversity even among autapses made
by a single neuron. Synaptic variation is manifest as differences in
release and response characteristics, ultrastructural features, and
molecular composition; future studies combining all three types
of assays will be important to understand the properties of individual synapses.
ACKNOWLEDGEMENTS
We thank A. Rao, K. Tovar and R. Wong for comments. Research in the authors’
laboratory is supported by NIH grants NS33184, NS39286, NS34448 and the
Pew Charitable Trust.
21.
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RECEIVED 28 FEBRUARY; ACCEPTED 9 APRIL 2001
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nature neuroscience • volume 4 no 6 • june 2001