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Structure –stability –function
relationships of dendritic spines
Haruo Kasai1, Masanori Matsuzaki1, Jun Noguchi1, Nobuaki Yasumatsu1 and
Hiroyuki Nakahara2
1
Department of Cell Physiology, National Institute for Physiological Sciences and The Graduate University for Advanced Studies
(SOKENDAI), Okazaki 444-8585, Japan
2
Laboratory for Mathematical Neuroscience, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan
Dendritic spines, which receive most of the excitatory
synaptic input in the cerebral cortex, are heterogeneous
with regard to their structure, stability and function.
Spines with large heads are stable, express large numbers of AMPA-type glutamate receptors, and contribute
to strong synaptic connections. By contrast, spines
with small heads are motile and unstable and contribute to weak or silent synaptic connections. Their structure –stability –function relationships suggest that large
and small spines are ‘memory spines’ and ‘learning
spines’, respectively. Given that turnover of glutamate
receptors is rapid, spine structure and the underlying
organization of the actin cytoskeleton are likely to be
major determinants of fast synaptic transmission and,
therefore, are likely to provide a physical basis for
memory in cortical neuronal networks. Characterization
of supramolecular complexes responsible for synaptic
memory and learning is key to the understanding of
brain function and disease.
A prominent feature of neurons is the huge number of
contacts that each forms with other neurons via axons and
dendrites. Such connections, known as synapses, have
long been considered the site of memory storage in the
brain [1,2]. Excitatory synapses in particular often form at
small appendages of dendrites known as spines. The
substantial structural diversity of dendritic spines has
attracted the attention of neuroscientists ever since these
structures were discovered in the 19th century [3]. It
is only recently, however, that modern biophysical techniques have begun to reveal the structure – function
relationships of dendritic spines. This review summarizes
the most recent progress in our understanding of such
relationships for pyramidal neurons of the cerebral cortex,
especially with regard to the insight obtained by twophoton excitation-induced uncaging of a caged-glutamate
compound. In addition, it addresses current working
hypotheses and missing links in this important field of
neuroscience.
Spine structure and stability
Many ultrastructural investigations have described morphological changes in neurons that accompany neuronal
Corresponding author: Haruo Kasai ([email protected]).
activity. In particular, increases in the number [4] or
volume [5] of dendritic spines or changes in spine shape [6]
have been observed. It is not possible, however, to study
the stability and function of such plasticity by electron
microscopy. Recent progress in two-photon excitation
imaging has, thus, been largely responsible for the
demonstration that the structure of central synapses is
plastic both during neuronal activity and at rest [7].
Dendritic spines can take various shapes and have been
classified on the basis of their structure as filopodial, thin,
stubby, fenestrated or mushroom-like [8]. As a first
approximation, in this review spines will be divided into
two groups, small spines (filopodial and thin spines)
and large spines (stubby, fenestrated, and mushroom
spines), representing spines with small and large
heads, respectively.
Small spines change their form rapidly [9– 11], either
disappearing or growing into large spines. New spines are
also generated and eliminated during the intense neuronal
activity that accompanies the induction of LTP [12,13].
These observations support the idea that structural
alterations of spines underlie adaptive and learning
processes. By contrast, large spines are relatively stable
in vitro [9] and survive for more than a month [10] or even
for a year [11] in the mouse neocortex in vivo. Close
examination reveals that even large spines show some
motility [14,15]. For example, such spines often extend
small spinules from the spine head [16]. However, large
spines remain within the category of large spines for long
periods of time even in vivo [9 – 11], suggesting that
memory is maintained in a structural form for extended
periods in the brain.
An important limitation of these studies is that they do
not directly address the correlation between spine structure and function. The most important function of
dendritic spines is to sense glutamate released from
presynaptic nerve terminals (Fig. 1a). The structure –
function relationships of spines were first addressed with
the use of immunogold labeling of AMPA-sensitive
glutamate receptors, which mediate the fast component
of glutamate-mediated synaptic transmission. This
approach revealed that spines with large postsynaptic
densities (PSDs) tended to exhibit a higher level of
AMPA-receptor immunoreactivity than did those with
smaller PSDs in both the hippocampus [17,18] and
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(a)
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Fig. 1. Two-photon excitation of methoxy-nitroindolino-glutamate (MNI-glutamate) in hippocampal neurons. (a,b) Schematic representations of glutamate release either
from a synaptic terminal (a) or by two-photon excitation of a caged-glutamate compound (b) at a single spine on a dendrite. The current traces (dotted lines) were recorded
from the postsynaptic neuron; (a) shows an average of ten miniature excitatory postsynaptic currents (mEPSCs) for a cell in which such currents were rapid and (b) shows
an average of 28 current traces obtained from the same cell in response to photolysis of MNI-glutamate (for 50 ms with 7 mW of light at a wavelength of 720 nm; indicated
by red shading). The solid lines were constructed as described [22]. (c) Recording configuration for mapping of glutamate sensitivity along a dendrite using two-photon
uncaging of the caged-glutamate compound. (d,e) Amplitudes of two-photon excitation-induced currents (green dots) along horizontal (d) or vertical (e) lines crossing the
center of an AMPA-receptor cluster, and fluorescence profiles of a 0.1-mm-diameter bead (black dots) along the horizontal (d) or vertical (e) axes. The black lines (fluorescence) represent Gaussian fitting of the data, and the green lines (current) are predicted from a theory described previously [22]. The full-width-at-half-maximal diameters
(FWHM) represent the spatial resolutions of the optical system (black) and of AMPA-receptor activation (green). Abbreviation: A.U., arbitrary units. (f,g) Fluorescence image
of a CA1 pyramidal neuron in a rat hippocampal slice preparation (f) and the corresponding map of glutamate sensitivity at the dendritic surface (g). Numbers in (f) indicate
the spines whose glutamate sensitivities are shown in Fig. 2m. Amplitudes of glutamate-induced currents are represented in (g) by the pseudo-color code shown at the
bottom of this panel; the map was smoothed by linear interpolation. Panels (d– g) are modified, with permission, from Ref. [22], q (2001) Nature Publishing Group
(http://www.nature.com/).
primary sensory cortex [19]. Given that the size of PSDs
is correlated with that of spine heads [20], these studies
suggested that large spines express a greater number of
glutamate receptors than do small spines. The relationship between AMPA-receptor immunoreactivity and spine
function, however, remained to be clarified.
Glutamate application with a femtosecond-pulse laser
Investigation of the function of individual spines requires
the systematic application of glutamate to identifiable
spines along a dendrite. Such studies have been impossible
with methodologies that rely only on microelectrodes
because brain tissue is too compact to allow the arbitrary
movement of an electrode with a resolution at the
micrometer level (Fig. 1c). The light-induced release of
glutamate from caged compounds was, therefore, applied
as a solution to this problem. The spatial resolution of this
approach with a conventional light source was, however,
limited (axial resolution ,20 mm) by activation of the
caged compound outside of the focal plane [21]. A
femtosecond-pulse laser was therefore applied to achieve
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the photolysis of caged-glutamate compounds by twophoton excitation within a small focal point of the objective
lens (Fig. 1b) by Matsuzaki, Ellis-Davies, Nemoto,
Miyashita, Iino and Kasai [22]. The major obstacle to
this approach was the lack of a caged-glutamate compound
with sufficient two-photon absorption. To address this
problem, Matsuzaki et al. synthesized a new compound,
methoxy-nitroindolino-glutamate (MNI-glutamate), that
exhibits a cross-section of two-photon action sufficiently
large for application to brain tissue; that is, MNIglutamate releases the neurotransmitter at a laser
power that does not damage neurons. The same compound
was independently synthesized for one-photon application
by Papageorgiou and Corrie [23] and later shown to
undergo photolysis within 1 ms [24]. MNI-glutamate is
now commercially available.
The currents induced by focal two-photon uncaging of
MNI-glutamate (Fig. 1b) were almost identical to miniature excitatory postsynaptic currents (mEPSCs) (Fig. 1a).
The laser-induced glutamate release also occurred within
a space restricted to 0.29 mm laterally (Fig. 1d) and
TRENDS in Neurosciences Vol.26 No.7 July 2003
0.89 mm axially (Fig. 1e), resulting in the focal activation of
AMPA receptors with a resolution of 0.45 mm laterally
(Fig. 1d) and 1.10 mm axially (Fig. 1e) (see following
discussion for further explanation). MNI-glutamate does
not manifest any antagonistic or agonistic effects at
glutamate receptors, even at concentrations as high as
10 mM [22]. Thus, two-photon uncaging of glutamate
provides a means to achieve rapid and fine 3D spatial
control of glutamate concentration, to mimic glutamate
release from presynaptic terminals.
Spine structure and function
Matsuzaki et al. attempted to measure glutamate sensitivity at the surface of dendrites of CA1 pyramidal neurons
in fresh slice preparations obtained from the rat hippocampus [22]. The two-photon uncaging of MNI-glutamate
was induced at 1064 points in a cubic region encompassing
a small portion of a dendrite (Fig. 1f). Sampling points
were pseudo-randomized to minimize receptor desensitization. The glutamate-induced currents were recorded at
the soma, and their peak amplitudes were displayed by
color-coding (Fig. 1g; for simplicity, only those data
obtained from photolysis at the surface of the dendrite
are shown).
Glutamate sensitivity was found to vary markedly
among individual spines: whereas some spines exhibited
pronounced glutamate sensitivity (spines 3 and 8 in
Fig. 1f), the sensitivity of others was relatively low (spines
1, 2 and 6). This was apparently the first experiment in
which synaptic function was systematically quantified
along the dendrites of central neurons. Glutamate
sensitivity was found to be highly correlated with spine
structure [22]. Data obtained from a total of nine dendrites
thus revealed that glutamate sensitivity was highest at
spines with the largest heads (Fig. 2), with thin spines and
filopodia exhibiting only a low sensitivity (Figs 1g, 2k).
Plots of glutamate sensitivity versus spine-head volume
yielded correlation coefficients of between 0.7 and 0.9
(mean ^ SD , 0.8 ^ 0.07; n ¼ 9 dendrites). The actual
correlation coefficient must be . 0.8, however, given the
stochastic variability in the number of open channels. This
relationship between glutamate sensitivity and spinehead volume was recently confirmed by another laboratory
[25], in a study in which currents were recorded from the
dendritic shaft rather than at the soma. These results
were also consistent with those of previous anatomical
studies [17,18], suggesting that immunoreactive AMPA
receptors in PSDs are functional.
The structure – function relationship suggests that, if
LTP is long-lasting at the level of the individual synapse,
then the affected spines should also be stable over the same
period. Long-term stability of large spines has recently
been supported by experimental evidence [10,11]. The
structure – function relationship also predicts that, if
postsynaptic glutamate sensitivity is increased by LTP,
this effect should be accompanied by spine enlargement, at
least in the long run. The latter prediction is likely to be
directly addressed by two-photon uncaging experiments
that induce synaptic plasticity.
Synapses on small spines and dendritic shafts [26] might
represent ‘silent synapses’ that express NMDA-sensitive
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(a)
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1
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(µm3)
0
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0.0 1.70 3.30 4.80 6.40 8.0
Mean distance (µm)
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Fig. 2. Spine structure and expression of functional AMPA receptors. (a –l) Fluorescence images (upper panels) and glutamate-sensitivity maps (lower panels) for
various dendritic spines of CA1 pyramidal neurons in rat hippocampal slices. The
former were obtained from stacked images containing the respective spine and
the latter were obtained from one x–y section (in which the maximal glutamate
sensitivity of the spine was detected) and were smoothed by linear interpolation.
White lines indicate the contours of the dendritic structures. Representative data
are from thin spines (a–e), large spines (f– j) and a filopodial spine (k) in slices prepared from 15–22-day-old animals and from a large spine in a 9-day-old animal (l).
Amplitudes of glutamate-induced currents are represented by the pseudo-color
code shown. (m) Correlation between spine-head volume and glutamate sensitivity for the dendrite shown in Fig. 1f; numbered points correspond to the numbered spines. (n) Spatial autocorrelation of glutamate sensitivity (blue) and of
spine-head volume (red) for the spines shown in Fig. 1f. The autocorrelaton coefficient is obtained from the equation
X
ðaðiÞ 2 aÞðaði þ jÞ 2 aÞ
i
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi
RðjÞ ¼ rX
X
ðaðiÞ 2 aÞ2
ðaði þ jÞ 2 aÞ2
i
i
where aðiÞ represents either the maximal amplitude of the glutamate-induced current or the spine-head volume of the ith spine, a is the mean value of aðiÞ for all
spines and j is the spine separation. The mean distances of two spines with the
spine separation of j are also shown. The correlations were obtained from nine
dendrites. Modified, with permission, from Ref. [22], q (2001) Nature Publishing
Group (http://www.nature.com/).
glutamate receptors rather than AMPA-sensitive ones and
that are preferential sites for the induction of LTP [27].
The proportion or even the existence of silent synapses in
the developing synaptic network, however, remains controversial [28]. This issue is also likely to be reinvestigated
with two-photon uncaging experiments that simultaneously reveal the expression of AMPA receptors and
NMDA receptors.
The results of Matsuzaki et al. [22] also suggest that the
decrease in the proportion of large spines apparent in
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TRENDS in Neurosciences Vol.26 No.7 July 2003
mentally retarded subjects [29] is likely to reflect a
decreased proportion of substantial and stable synaptic
connections. Large spines receive synaptic input from
presynaptic terminals with a larger diameter and greater
number of synaptic vesicles [20,30] compared with the
presynaptic terminals associated with small spines. These
observations suggest that the structure and function of
synapses are regulated coordinately in presynaptic and
postsynaptic components. The spatial distributions of
different types of synapse warrant detailed investigation
in future anatomical studies of the brain [29,31].
Memory density
Given that glutamate-mediated synaptic transmission
depends on a diffusible molecule, the length of glutamate
action is a crucial determinant of the practical density of
synaptic connections. The length of glutamate action is, in
turn, dependent on the gating properties of postsynaptic
AMPA receptors. If the activation of AMPA receptors is
slow, then glutamate released from a presynaptic terminal
will reach neighboring synapses before the corresponding
postsynaptic receptors are activated. Such a spillover
effect of glutamate on AMPA receptors has been mainly
addressed theoretically on the basis of kinetic values
obtained from macroscopic analysis [32]. With the use twophoton uncaging of glutamate, Matsuzaki et al. were able
to narrow the application of transmitter to a lateral region
as small as 0.29 mm (Fig. 1d) and to examine the spread of
AMPA-receptor activation. Such analysis yielded a value
of 0.45 mm for the lateral diameter of the region of
AMPA-receptor activation [22], which is consistent with
the theoretical prediction [32] and confirms that the area
of activation is crucially governed by the gating of AMPA
receptors. Given that spine density is , 1 – 3 mm21 in the
cerebral cortex [20,31,32], the gating of AMPA receptors is
marginally sufficient to confer independent sensing by
spines of the input from glutamatergic afferents. It has
been suggested that complex cognitive functions require
an increased spine density in the cortex [31].
With the use of statistical analysis of the glutamate
sensitivities of neighboring spines, Matsuzaki et al. next
investigated whether the expression of AMPA receptors is
controlled independently in such spines (Fig. 2n) [22].
They found that the statistical correlation between the
glutamate sensitivities of neighboring spines was weak or
nonexistent. This observation is, thus, consistent with a
tenet of spine function: that the narrow neck of spines
isolates their metabolism and allows independent control
of their functions [3,33]. LTP was shown to spread . 70 mm
[34] after stimulation of a bundle of presynaptic fibers,
with an overall diameter of 30 mm. It will thus be of
interest to determine the spread of LTP after stimulation
of individual spines.
Finally, Matsuzaki et al. determined how many AMPA
receptors were expressed in single spines. Two-photon
excitation allowed them to apply constant amounts of
glutamate repetitively to a postsynaptic membrane in slice
preparations and to determine the number of AMPA
receptors on the basis of non-stationary fluctuation
analysis [22]. Such analysis revealed that individual
spines in CA1 pyramidal neurons express at most 150
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363
AMPA receptors on the proximal dendrite [22]. Using the
same technique, Smith et al. reported that the actual
number of receptors depended on spine location on the
dendrite, being larger for distal spines (mean ¼ 171) than
for proximal spines (mean ¼ 91) [25], an observation that
might be related to the distance scaling of synaptic input to
CA1 pyramidal neurons. Given that AMPA-receptormediated currents can only summate within a spine, any
number of AMPA receptors between 0 and 150 is able to
code at most 7.2 (log2 150) bits of information. Neuronal
networks with all-or-nothing synaptic connections might
be nearly as efficient as those with analog connections
[35,36]. How the brain utilizes the memory capacity of
individual spines will be an important topic for future
investigation.
Currently available memory devices have memory
elements with a spatial dimension in the sub-micrometer
range, as is the case with cortical neuronal networks.
Photographic film, for example, stores memory in the form
of silver aggregates with a minimum spacing of ,1 mm21.
Large-capacity hard disks of computers store memory in
magnetic form at 10 bits mm21. And holographic memory
is able to store information with a resolution of 0.3 mm
in a cube, corresponding to a memory capacity of
27 Tbyte cm23. Holographic memory demonstrates that
data can be stored in a highly distributed manner in
memory devices as it can in the brain. Although algorithms
of storage and readout for functional sets of memories
differ markedly, memory capacity should ultimately
depend on the density of memory elements both in memory
devices and in the brain. The high density of synaptic
memory, akin to that of currently available memory
devices, and the absence of other similarly dense brain
structures support the classical notion that the synapse is
the major memory element in the cerebral cortex.
Implications of structure –stability –function
relationships of spines
In memory devices, memory storage and readout occur in
two different operational modes. New memory thus cannot
be stored in the readout mode. By contrast, storage and
readout of memory occur by inseparable processes in the
brain. This feature has been a major theme for the
operation of realistic neuronal networks and it has been
ascribed mainly to the highly distributed nature of
memory in neuronal networks and in the superposition
of memories, distributed over many synapses or ‘weights’,
as in holographic memory. One important question then
arises: how are both the robustness of readout and the
rapid acquisition of new memory achieved?
The structure – stability –function relationships of dendritic spines provide new insight into this important issue.
For simplicity, we consider only two categories of spines,
large and small. First, large spines are stable, or ‘writeprotected’, and maintain preexisting strong connections
with little interference from new memory formation. By
contrast, small spines are unstable, or ‘write-enabled’, and
are mostly responsible for the acquisition of memory,
which is possibly mediated by their transformation into
large spines. In this manner, the robustness of readout and
the rapid acquisition of new memory can be achieved
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TRENDS in Neurosciences Vol.26 No.7 July 2003
together. Second, large spines remain stable even during
rest or sleep, whereas small spines can be eliminated.
Thus, the lifetime of synaptic memory can vary widely
depending on spine structure, with large spines being the
structural basis for long-term memory. Finally, small
spines are generated during activity-dependent processes
or even at rest, providing an inexhaustible source of new
synapses and memory. Exploration of neuronal network
models that take into account these structure– stability –
function relationships of synapses should provide insight
into the operation of realistic neuronal networks that
experience various behavioral states in the long term. In
addition, the structure –stability – function relationships
of synapses should be further verified and specified
experimentally and their molecular basis elucidated.
Molecular basis of spine structure –stability– function
relationships
The trafficking and turnover of AMPA receptors are rapid
[24,37 – 40] and have been proposed to account for the
changes in synaptic strength during LTP [41] and LTD
[42]. By contrast, given that AMPA-receptor expression is
dynamically regulated in spines, the maintenance of
memory must depend on stable factors that regulate
AMPA-receptor expression; otherwise, the strength of
synaptic connections could not be maintained in the long
term. Such factors might include spine shape and the
underlying cytoskeletal organization, given that functional AMPA-receptor expression correlates with spinehead volume (Fig. 2m) and that large spines are stable.
Synaptic structure, including spine-head volume, is thus a
good candidate for storage of synaptic memory. It is still
possible that certain molecules might particularly contribute to memory storage. Phosphorylated Ca2þ/calmodulin-dependent protein-kinase II (CaMKII) is a candidate
to be such a molecule [43], although kinase inhibitors have
been found not to block the maintenance phase of LTP
[43,44]. Critical assessment of these possibilities should be
feasible after the supramolecular organization of synaptic
memory has been clarified further.
On the basis of the notion that synaptic structure
determines function and stability, the molecular control
of synaptic strength is likely to encompass the following
four aspects: (1) the genesis, motility, enlargement and
elimination of small spines, which underlie effective
acquisition of new memory; (2) the stability of large
spines, which is responsible for the maintenance of longterm memory; (3) the expression of AMPA receptors, which
determines the amplitude of rapid synaptic transmission;
and (4) the expression of NMDA receptors, which allow the
passage of Ca2þ and contribute to synaptic reorganization.
Spine instability
Small spines are motile, readily generated and eliminated,
and undergo enlargement into large spines. Various
molecular mechanisms might be responsible for such
instability. As predicted by Crick [45], cytoskeletal
elements are abundant in dendritic spines [46,47]. Indeed,
actin is more concentrated in spines than in other
neuronal structures [48]. Regulation of the actin cytoskeleton contributes to many cellular functions [49] and is
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fundamental to synaptogenesis and spine motility
[19,50 – 53]. Spontaneous spine motility requires actin
and is blocked by volatile anesthetics [54], suggesting that
it might play an important role in the organization of
neuronal networks. Actin organization is regulated by
small GTPases of the Rho family (Rho, Rac and Cdc42),
and these proteins also contribute to the determination of
spine morphology [55] (Fig. 3a). The protein encoded by
the gene mutated in individuals with fragile-X mental
retardation (FMRP) is thought to regulate Rac1 function,
and impairment of such regulation might be responsible
for the long, thin and tortuous dendritic spines characteristic of this disease [56]. In addition, LIMK-1, an inhibitor
of cofilin/ADF [a factor that induces depolymerization of
filamentous actin (F-actin)] is implicated in Williams
syndrome, which is characterized by profound cognitive
deficits [57]. Abnormalities of dendritic spines have also
been described in many other mental disorders [29],
although it is difficult to rule out the possibility that
they are the result, rather than the cause, of pathological
neuronal activity.
Ca2þ signaling is thought to play an important role in
the activity-dependent genesis and motility of small spines
[12,58], as well as in the induction of LTP. The potential
targets of Ca2þ in such signaling include calmodulin,
CaMKII, adenylate cyclase [39], Ras and Rap [42,59], and
mitogen-activated protein kinase (MAPK) pathways
[42,60] (Fig. 3a). The signaling mechanisms that underlie
the conversion of small spines to larger ones have not yet
been elucidated. It is possible that the level of Ca2þ
signaling is greater in small spines than in large spines, as
a result of the smaller head volume of the former, resulting
in greater instability. In addition, the small head volume
mechanically facilitates the instability of small spines.
Spine stability
Large spines remain within the category of large spines for
long periods of time [9– 11], even though all the molecules
that contribute to spine stability turn over within days or
weeks [61 – 63]. Several possible mechanisms might
underlie the stability of large spines.
First, existing clusters of F-actin might exert a positive
feedback effect on actin polymerization, cross-linking and
stability (Fig. 3b,A). Indeed, F-actin anchors many protein
complexes of PSDs, including the CaMKIIa– densin-180 –
a-actinin complex [64], the PSD95 – GKAP (a guanylatekinase-associated protein)–shank–cortactin complex [65],
and CaMKIIb [66]. Moreover, certain PSD proteins possess
enzymatic activities that affect F-actin organization,
including SPAR (a spine-associated GTPase-activating
protein for Rap) [67], kalirin (a guanine-nucleotideexchange factor for Rac1) [68] and spinophilin (which is
also called neurabinII and has actin-bundling activity)
[69]. Overexpression of SPAR [67], PSD95 [70] or shank
[65] in cultured neurons has also been shown to increase
spine volume. Thus, positive feedback mechanisms appear
to maintain a large spine size despite the potential rapid
turnover of actin [63]. Some actin filaments in F-actin
clusters are also capped and colocalize with other
structural proteins [51,71] such as drebrin [72]. The
small surface-to-volume ratio of large F-actin clusters
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365
TRENDS in Neurosciences Vol.26 No.7 July 2003
(a) Spine instability
(b) Spine stability
B
CaMK
A
NR
CaM
SPRCs
F-actin
Actin organizers
Scaffold proteins
Ca/CaM-binding proteins
NMDAR-binding scaffold
proteins
Adhesion molecules
GR
AMPARs
NR
NMDARs
(c) AMPAR expression
GR
GR
C
CaMK
Ribosome
Actin-capping proteins
PSD
(d) NMDAR expression
NR
NR
GR
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Fig. 3. Spine structure– stability– function relationships. (a) Spine instability. The cytoskeleton, various actin-organizing enzymes and Ca2þ/calmodulin (Ca/CaM)-binding
proteins contribute to spine genesis, motility, enlargement and elimination. (b) Spine stability. Hypothetical mechanisms of spine stability include the positive feedback
effects via filamentous actin (F-actin) organization within the spine (A), via spine interaction with the presynaptic terminal (B), and via protein synthesis inside or close to a
spine (C). (c) AMPA-receptor (AMPAR or GR) expression proportional to spine-head volume. The actin cytoskeleton and scaffold proteins might be major molecular determinants of AMPA-receptor expression. (d) NMDA-receptor (NMDAR or NR) expression is independent of F-actin. Although many scaffold proteins bind tightly to NMDA
receptors, NMDA-receptor expression is not proportional to postsynaptic density (PSD) size. White circles represent presynaptic vesicles of glutamate. Abbreviations:
CaMK, Ca2þ/calmodulin-dependent-kinase; SPRCs, synaptic polyribosome complexes.
might render them less sensitive to environmental
changes and prolong their life span.
Second, postsynaptic spines communicate with presynaptic terminals via adhesion molecules, possibly resulting
in mutual stabilization (Fig. 3b,B). Indeed, spines that are
attached to presynaptic terminals have been observed to
be more stable than those that are not [14]. The adhesion
molecules in spines, including cadherins [73] and neuoligin
(neurexin) [74], bind to PSDs via S-SCAM (a synaptic
scaffolding molecule) [75] or via PSD95 [76]. The presynaptic ligand ephrinB was shown to induce spine enlargement
by interaction with complexes of EphB–syndecan-2 [77],
intersectin – Cdc42 – N-WASP (neural Wiskott-Aldrich
syndrom protein)– actin [78], or kalirin – Rac1– PAK1
(p21-activated kinase 1)– actin [68].
Third, large spine heads might facilitate the incorporation of the cellular machinery for protein synthesis, and
proteins newly synthesized in situ could maintain spine
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size (Fig. 3b,C). Indeed, polyribosomes are preferentially
translocated to large spines during synaptic plasticity [79],
and synaptic polyribosome complexes [79,80] often
exhibit a rough-endoplasmic-reticulum-like configuration
(Fig. 3b). These complexes might allow the local synthesis of
CaMKII [80,81], FMRP [56], the cytoskeleton-associatedprotein Arc, and even transmembrane proteins [80]. This
possible mechanism of spine stability is consistent with the
fact that protein synthesis is required for the induction of
memory [82] and for late LTP [80,83]. Active protein
synthesis in spines might thus function as a synaptic tag
for transcription-dependent memory formation [84,85].
However, it remains to be demonstrated that protein
synthesis actually occurs within spines. Organelles in
spines might also help to maintain spine size independently of protein synthesis.
Finally, activity-dependent changes in large spines are
likely suppressed by attenuation of Ca2þ signaling caused
366
Review
TRENDS in Neurosciences Vol.26 No.7 July 2003
by the large volume of the spine head, contributing to the
stability of these spines.
AMPA-receptor expression
Consistent with the observation that AMPA-receptor
expression is proportional to spine-head volume
(Fig. 2m), several lines of evidence support the notion
that the expression of AMPA receptors is determined by
F-actin and PSD proteins (Fig. 3c), the distributions of
which are expected to correlate with spine-head volume.
Actin-depolymerizing agents have, thus, been shown to
induce redistribution of AMPA-receptor immunoreactivity
in cultured hippocampal neurons [86]. Such agents also
trigger a reduction in the number of functional AMPA
receptors [87] and prevent LTP [88,89]. Given that AMPA
recptors do not bind F-actin directly and are localized to
PSDs, their expression also requires certain PSD proteins
that link them to F-actin. Such interactions are mediated,
for example, by complexes of AMPA-receptor– SAP97–
protein 4.1 –F-actin [90] and AMPAR– stargazin– PSD95 –
GKAP–shank–cortactin–F-actin [91]. It will be important
in the future to determine how AMPA-receptor distribution
is regulated during structural plasticity of spines.
NMDA-receptor expression
The expression of NMDA receptors differs from that of
AMPA receptors. The distribution of NMDA-receptor
immunoreactivity is thus only weakly correlated with
PSD size in hippocampus [17,18]; the expression of AMPA
receptors, but not that of NMDA receptors, is modulated
during LTP [42]. Furthermore, in contrast to AMPA
receptors: (1) the expression of NMDA receptors does not
depend on F-actin [86,88]; (2) NMDA receptors bind tightly
to many PSD proteins, including a-actinin, phosphorylated CaMKII, PSD95 and S-SCAM (Fig. 3d) [39]. The
expression of these NMDA-receptor-binding proteins is
not necessarily proportional to PSD size; and (3) NMDAreceptor expression is modulated by protein kinases A [92]
and C [93,94] (Fig. 3d). The presence of NMDA receptors
in some small spines greatly promotes their activitydependent plasticity, whereas the lack of these receptors in
some large spines provides a basis for their structural and
functional stability and underlies the robustness of
memory readout. The regulation of NMDA-receptor
expression is, thus, important in the organization of
neuronal networks. Compared with AMPA-receptor
expression, however, little is known of the regulatory
mechanisms of NMDA-receptor expression at the level of
the individual spine.
Proteomic analysis has been performed on many of the
scaffold proteins discussed in this section [95], the results of
which are now available (http://www.anc.ed.ac.uk/mscs/PPID/).
Recapitulation
Recent progress in biophysical techniques and molecular
biology has provided insight into the structure– function
relationships of dendritic spines in the cerebral cortex, as
well as support for the century-old hypothesis that spine
structure is the basis for memory in the brain. The
structure – stability –function relationships of spines have
further suggested that small and large spines play distinct
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roles in learning and memory, enabling rapid acquisition of
new memory and robust readout, respectively. Clarification of such relationships will help to link molecular events
to higher-order brain functions, and to shed light on and
develop new treatments for mental disorders. Achievement of this goal will require a more complete characterization of the functional alterations associated with spine
structural plasticity, which will be facilitated by the
application of two-photon uncaging methodology, the
only approach available that allows stimulation of individual central synapses independently and systematically.
Progress in this field of research is rapid, and the time
could soon be ripe for construction of a material theory of
synaptic memory in brain tissue.
Acknowledgements
We thank Y. Hata for critical reading of the manuscript. Our work was
supported by Grants-in-Aid from the Japanese Ministry of Education,
Culture, Sports, Science and Technology and from the Japan Society for
the Promotion of Science, and by a research grant from the Human
Frontier Science Program Organization.
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