HIPPOCAMPUS 10:587–595 (2000)
Structure-Function Relations in Dendritic Spines:
Is Size Important?
Eduard Korkotian and Menahem Segal*
Department of Neurobiology, The Weizmann Institute,
Rehovot, Israel
ABSTRACT:
The recent use of novel high-resolution imaging methods
of living neurons in vitro has led to a change in the view of the dendritic
spine, from a stable, long-term memory storage device to that of a
dynamic structure, which can undergo fast morphological changes over
periods of hours and even minutes. While the functional significance of
these changes in spine dimensions is still obscure, we have obtained
evidence to indicate that the length of the spine has a critical role in
determining the degree of interaction between the spine head and the
parent dendrite, such that longer spines are more independent of the
parent dendrite than the short ones. We have now studied the role of
intracellular calcium stores in determining the magnitude and time course
of spine responses to a calcium surge evoked in response to glutamate,
which causes an influx of calcium, and the results indicate that spine
morphology has an important role in determining the involvement of the
stores in calcium responses. Since spines can change their length over a
rather short time, these results indicate that changes in spine length serve
to fine-tune the interaction between the spine head and the parent dendrite on a continuous basis. Hippocampus 2000;10:587–595.
©
2000 Wiley-Liss, Inc.
KEY WORDS:
development
calcium stores; tissue culture; plasticity; hippocampus;
INTRODUCTION
The great variety of dendritic spine shapes, sizes, and density distribution on the parent dendrites of a single neuron and the apparent
persistence of spines throughout the life of the neurons underlie the
assertion that the spine is the unitary locus of memory formation and
storage. While this intuitive view prevailed throughout the 20th century,
evidence for it is rather scarce. This is due to the small size of the
dendritic spine, which is prohibitive to a systematic electrophysiological
analysis. Thus, it is not entirely obvious that the morphological heterogeneity seen among spines has any functional relevance. Based on EM
measurements of spine dimensions, early modeling studies concluded
that the spine neck cannot constitute a barrier for transfer of charge
between the spine head, where the synapse is made with a presynaptic
terminal, to the parent dendrite (Shepherd, 1996). More recent studies,
using high spatial and temporal resolution imaging methods of spines
and dendrites, concluded that the spine is actually a unique calcium
Grant sponsor: US-Israel Binational Foundation; Grant number: 97/230.
*Correspondence to: Menahem Segal, Department of Neurobiology, The
Weizmann Institute, Rehovot, 76100 Israel.
E-mail: menahem.segal@weizmann.ac.il
Accepted for publication 13 June 2000
©
2000 WILEY-LISS, INC.
compartment, in that an afferent stimulation that is
subthreshold for generation of an action potential can
evoke changes in intracellular calcium concentration
([Ca2⫹]i) that can be restricted to single spines. Synaptic stimulation, which causes release of glutamate
from presynaptic nerve endings, causes activation of
the postsynaptic glutamate receptors located on dendritic spines. Activation of the glutamate receptors can
cause influx of calcium into the spine via several
routes. The main one is probably the voltage/glutamate/glycine-gated NMDA channel (Kovalchuk et al.,
2000; Segal, 1995a). Voltage-gated calcium channels,
known to exist in dendritic spines (Yuste and Denk,
1995), can also be activated by the depolarization of
the spine, and the possibility that the observed changes
in [Ca2⫹]i result from release of calcium from stores
has also been suggested (Emptage et al., 1999). Calcium stores are associated with smooth endoplasmic
reticulum, which has been studied extensively in dendritic spines of hippocampal neurons by Spacek and
Harris (1997). Functionally, stores can be caused to
release bound calcium into the intracellular space by
two distinct receptors, the ryanodine receptor and the
IP-3 receptor. The former has been found in dendritic
spines of cultured hippocampal neurons, and is known
to be activated directly by caffeine (Hernandez-Cruz et
al., 1995). Caffeine-evoked calcium transients have
been described in the hippocampus (Garaschuk et al.,
1997), and the machinery for caffeine-induced calcium release from dendritic spines has been studied by
Korkotian and Segal (1998). Thus, the spine is a
unique chemical compartment in that it may allow a
local rise of [Ca2⫹]i needed for activation of plasticityrelated signaling molecules. This realization facilitated
renewed interest in the role of spines in memory functions. Some of the emerging issues, still not resolved,
include the functional significance of the great heterogeneity of spine morphologies, or, simply stated, do
spine shape and size affect its function? We obtained
preliminary data by manipulating calcium released
from intracellular stores using caffeine, suggesting that
spine neck length correlates with mechanisms that link
calcium flux in the spine head with the parent dendrite
(Korkotian and Segal, 1998; Volfovski et al., 1999).
588
KORKOTIAN AND SEGAL
These studies, conducted with fast calcium imaging in a confocal laser scanning microscope, were now extended to include
responses of spines and parent dendrites to glutamate. The
results suggest that spine length has an important role in spine/
dendrite communication.
METHODS
Cultures
Hippocampal cultures were prepared as described (Papa et
al., 1995). Briefly, dissociated hippocampi of 19-day-old embryos were plated onto poly-l-lysine-coated 12-mm glass coverslips in Eagle’s MEM containing 10% heat-inactivated horse
serum and 5% fetal calf serum. Two to three-week-old cultures
were used for the imaging experiments. A coverslip was transferred into the recording chamber, and placed in a confocal
laser-scanning microscope (CLSM, Leica, Heidelberg, Germany) where it was perfused with recording medium containing (in mM) NaCl 129, KCl 4, MgCl2 1, CaCl2 2, glucose 4.2,
and HEPES 10; pH was adjusted to 7.4 with NaOH, and
osmolarity to 320 mOsm with sucrose. The recording medium
was perfused at a rate of 3–5 ml/min at room temperature. In
calcium-free medium, calcium was replaced by equimolar
magnesium. Drugs were prepared in the recording medium
from frozen stocks before use. Glutamate (0.1 mM) was dissolved in the recording medium and loaded in a pressure pipette
with a tip diameter of 2 ␮m, which was placed some 25 ␮m
from the dendrite. Caffeine (1 mM) was applied in the perfusion medium. Presynaptic terminals were stained with the fluorescent dye FM4-64 (Molecular Probes, CA) in a medium
containing 50 mM KCl, replacing equimolar NaCl, which
was superfused into the culture for 1 min, followed by extensive wash in calcium-free medium. Ryanodine receptor immunoreactivity in spines was assessed as before (Korkotian and
Segal, 1998).
Imaging
The confocal laser scanning microscope (CLSM) is equipped
with an argon-ion laser for excitation at a wavelength of 488
nm, and a HeNe laser for excitation at 543 nm. Individual cells
were impaled with micropipettes containing 10 mM Oregon
Green-1. The dye was iontophoresed into the cell for 1 min,
and was allowed to equilibrate in the cell for about 1 h before
experiments commenced, to assure equal distribution of the dye
in the different cellular compartments. For each experiment, a
fresh spine/dendritic segment in a new dish was used. Images of
256 ⫻ 256 pixels were taken with a 63⫻ water immersion
objective. Three-dimensional (3D) reconstruction of the dendrites was made from successive 0.1-␮m optical sections. An
apparent nonuniformity in staining of the dendrites was occasionally seen due to the different number of sections used in the
particular reconstructed dendrite. A line was scanned through
the center of a dendrite/spine pair (about 0.8 ms per line) to
reveal fast changes in fluorescence during a response to drug
application (Fig. 1). Laser light was reduced to 1–3% of nominal intensity. Using this setting, we were able to stimulate the
same spine/dendrite pair every 5–10 min for 2–3 h with no
significant loss of reactivity due to dye bleaching or photodynamic damage. Also, baseline fluorescence did not change
across the 2–3-h observation time. An effort was made to scan
single lines through spine/dendrite segments having the same
initial fluorescence intensity, so as to obtain similar baseline
values for calculating the response value DF/F. Fluorescence
was quantified using Leica analysis software and Adobe Photoshop (Adobe Systems, CA). Autofluorescence, measured in
BAPTA-AM-loaded cells, under identical conditions as used
here, was negligible (Korkotian and Segal, 1998).
RESULTS
Glutamate-Evoked Calcium Signals in Short and
Long Dendritic Spines
High-resolution imaging of Oregon-Green-loaded cells can
easily distinguish between spines of different lengths, residing
side by side on the same dendrite (Fig. 1). A brief application of
glutamate near the spine/dendrite pair caused a fast-onset slow
decay of [Ca2⫹]i surge, detected with the fast line scan mode of
the CLSM. The exact latency of the response to glutamate could
be measured in both the spine and the adjacent parent dendrite
(Fig. 1A2,B2). Responses to glutamate could be evoked repetitively from the same spine/dendrite pair without fatigue for
several hours. For the analysis, we grouped together spines with
short necks (0.1– 0.4 ␮m neck length) and compared them to
spines with long necks (⬎0.5 ␮m). In all the analyzed cases, it
was verified, using 3D reconstruction of the spine and parent
dendrite, that we have visual access to the entire length of the
spine. Since we (Segal, 1995c; Korkotian and Segal, 1999b) and
others (Halpain et al., 1998) have shown that exposure to glutamate can cause shrinkage of dendritic spines, we were careful
to include only spines that did not change length across the
experiment. In fact, in the conditions that we used in the
FIGURE 1.
Dendritic spines respond to glutamate by a fast rise in
[Ca2ⴙ]i. A, B: Short and long spines, respectively. 1: Images of the two
spine/dendrite segments, reconstructed in 3D. In each, a line was
scanned between the spine head and the parent dendrite (arrows).
Some fluorescence was seen through the spine neck. 2: Typical line
scans depicting the beginning of the fast response to glutamate, expressed as a rise in basal fluorescence level. The image is composed of
256 lines scanned at a rate of about 0.8 ms per line, from top to
bottom, going through the line seen in A, arrow, which crosses the
spine head and parent dendrite. 3: Time course of the change in
fluorescence seen in the region of interests comprising the spine head
and the parent dendrite. Note that the spine head (light grey) of the
long spine starts to increase fluorescence before the change is seen in
the parent dendrite (dark grey).
Figure 1
590
KORKOTIAN AND SEGAL
present experiment, i.e., the absence of extracellular calcium,
there were minimal if any changes in spine length following
exposure to glutamate (Korkotian, unpublished observations).
Significantly, there was a difference in the spine compartment,
relative to the parent dendrite, between the short and long
spines, in that the response to glutamate had a shorter latency in
the long spines than in their parent dendrite, relative to the case
of the short spines, which were similar to their parent dendrites.
This distinction was evident when an averaged response of 20
short spines was compared to 18 long ones (Fig. 2). In addition,
there was a clear difference in the magnitude of the responses
between the short and the long spines, with the long ones expressing a larger peak response to glutamate compared to their
parent dendrites, as seen before (Segal, 1995a; Korkotian and
Segal, 1998).
Intracellular Calcium Determines Responses to
Glutamate
In previous studies we explored the role of intracellular calcium stores in the calcium-handling ability of spines, and proposed that dendritic spines contain caffeine-sensitive stores
(Korkotian and Segal, 1998). Since there is an unsettled debate
as to the role of calcium stores in synaptically evoked calcium
variations (see Kovalchuk et al., 2000; Emptage et al., 1999) we
focused in the present analysis on calcium variations in response
to glutamate, in the presence and absence of nominal extracellular calcium (Fig. 3). Removing extracellular calcium
([Ca2⫹]o), while leaving normal calcium levels only in the glutamate pressure pipette, caused a slow depletion of calcium
stores, which maintain equilibrium with free [Ca2⫹]i, such that
the remaining [Ca2⫹]i rise was due to influx of calcium in
association with the applied glutamate. The removal of
[Ca2⫹]o, which happened within 5 min of medium replacement, did not affect the initial response to glutamate in the
short spines, indicating that calcium entering the cell via the
glutamate pressure pipette is sufficient to cause a rise in [Ca2⫹]i
(compare Fig. 2A to Fig. 3A, left). The fact that the calcium in
the glutamate pipette was sufficient to cause a rise in [Ca2⫹]i
was tested in experiments where the glutamate pipette did not
contain calcium. In such cases, there was no response to glutamate in the absence of [Ca2⫹]o (data not shown).
Short-term incubation with calcium-free medium was sufficient
to annihilate the spine/dendrite disparity in the peak response of
the long spines (compare Fig. 2B and Fig. 3A, right). The longer
incubation time in [Ca2⫹]o-free medium shortened the decay time
constant of recovery from glutamate response, but a substantial
and stable response to glutamate was still seen (Fig. 3B). Only
following exposure to a low concentration of caffeine (1 mM for 5
min) was there a marked reduction in calcium response, down to
about 40% of control values (Fig. 3C). These effects are summarized in Figure 4.
The rise-time of [Ca2⫹]i responses to glutamate in the control condition can be described by a sum of two exponents, with
a short time constant of 90 –120 ms, and a longer time constant
of 450 – 600 ms. These two time constants were characteristic of
FIGURE 2.
Averaged responses to glutamate of 20 short spines
(A; note the symbol of short spines, here and in the following figures)
and 18 long ones (B; note symbol). The averages are derived from line
scans, as seen in Figure 1(3). In each of these and the following
average plots, the responses in the spines are depicted in gray, and the
traces of dendrites are depicted in black. Arrow indicates time of
glutamate application.
the long spines (Fig. 5B). The reduction in response following
exposure to [Ca2⫹]o-free medium was associated with disappearance of the fast time constant of the calcium response (Fig.
5A,B). Here again, there was a large difference between the
short and the long spines, with the former maintaining a similarity to the parent dendrite (Fig. 5A), whereas the long spines
were progressively dissimilar from their parent dendrites (Fig.
5B). These experiments indicate that the long spines are more
dependent on their own calcium stores to maintain a viable
response to glutamate.
The kinetic differences between the short and long spines can
reflect several alternative processes. First, it is possible that the
long spines are more immature than the short ones (this has
been shown in young cultures, where spines are longer, on
____________________________________________
SPINE MORPHOLOGY AFFECTS SPINE FUNCTION
FIGURE 3.
Averaged responses to glutamate application in the
absence of [Ca2ⴙ]o. Averages of 15 short spines, at left, summing 55
responses, and 16 long ones, at right, summing 43 responses, are
presented, at different times after onset of perfusion with [Ca2ⴙ]ofree medium. A: 5 min after onset of o[Ca2ⴙ]o medium. B: 20 min in
591
[Ca2ⴙ]-free medium. Between B and C, cultures were superfused for
5 min with 1 mM caffeine, which facilitated the reduction in response
to glutamate. Caffeine by itself did not cause a persistent change in
[Ca2ⴙ]i (not shown).
592
KORKOTIAN AND SEGAL
sponses to glutamate. Thus, spine morphology seems to play an
important role in regulation of the link between the spine head and
the parent dendrite, especially when calcium is removed, and stores
are depleted.
FIGURE 4.
Summary diagram of the responses to glutamate at
various times after perfusion with a calcium-free medium, in short
and long spines and their parent dendrites. Ct, control medium with
normal calcium concentration; 5ⴕ, 20ⴕ, and 40ⴕ are time in minutes
after removal of [Ca2ⴙ]o.
average, than in older cultured neurons; see Papa et al., 1995).
If indeed they are immature, they may not be innervated by
afferent fibers, and they may also contain fewer calcium stores
compared to the short ones. This is not the case in the current
study. First, while we did not quantify the density of innervation by presynaptic terminals, there was no distinct difference
between the short and the long spines in presynaptic innervation (Fig. 6C). Likewise, although short and long spines may
contain different amounts of endoplasmic reticulum (Spacek
and Harris, 1997), both spine types produce similar responses
to caffeine (Korkotian and Segal, 1998), and they seem to contain similar densities of calcium stores, labeled with an antibody
to ryanodine receptors (Fig. 6D). Furthermore, both the short
and the long spines express fast and transient spontaneous calcium fluctuations (Fig. 6A,B). These could be divided into large
events, caused probably by backpropagated action potentials,
and smaller events, caused by synaptic activation of the spine
(Fig. 6A,B; see also Volfovsky et al., 1999). These events peaked
within10 –20 ms of their spontaneous onset, and lasted about
200 – 400 ms. The spine [Ca2⫹]i changes were larger and had a
faster rise time than the dendritic [Ca2⫹]i changes, as seen in
the responses to glutamate. There was no apparent difference in
the frequency of synaptic events, indicating that both short and
long spines are likely to be innervated.
Finally, it is possible that the long spines tend to lose glutamate
receptors faster than the short ones in a process of desensitization
(Lissin et al., 1999), regardless of the presence of calcium in stores.
While this is a viable possibility, we found a rapid restoration of
responses following replenishment of normal [Ca2⫹]o (data not
shown), indicating that even if some desensitization occurs, it may
contribute only minimally to the asymmetric reduction in re-
FIGURE 5.
Expanded time scale of the initial part of the response
to glutamate in the short (A) and long (B) spines, taken at the beginning and end of the experiment shown in the top and bottom traces of
Figure 3. For both A and B, the top two traces represent the time
course at the beginning of the perfusion time, for both spines and
dendrites, and the bottom two traces are the responses after 40 min of
incubation with calcium-free medium. While in the short spines the
time course is the same for the spines and their parent dendrite, there
is a large disparity in the long spines between the spine and the
dendritic calcium responses. The top traces in B were fit with a double
exponent, with time constants of 90 ms and 600 ms. The bottom two
traces in B were fit with a single exponent.
____________________________________________
SPINE MORPHOLOGY AFFECTS SPINE FUNCTION
DISCUSSION
The present results demonstrate that spine neck length may have
an important role in regulating the interaction between afferent
input impinging on the spine head and the parent dendrite, such
that the shorter the spine neck, the higher the similarity in intracellular calcium dynamics between the spine head and the parent
dendrite. This can be seen both under control conditions, and
when intracellular calcium is depleted. Spine length is proposed to
fine-tune the interaction between spine head and parent dendrite.
The fact that both the time course of calcium rise as well as calcium
clearance mechanisms are affected by spine length has been alluded
to in other recent studies as well (Majewska et al., 2000).
Our results do not suggest that the charge transferred by the excitatory postsynaptic potential (EPSP) is in any way affected by the
length of the spine, but that subsequent, probably longer-term
changes, associated with calcium-regulated “processes,” may be affected by spine length. These may include activation of kinases and
phosphatases, or mobilization of glutamate receptors to the postsynaptic membrane (Lissin et al., 1999). In addition, these results suggest
that stores can contribute under certain conditions to the calcium
surge seen in response to the topical application of glutamate, and that
the stores may have a differential effect in the short and long spines.
The current results may reflect two opposite modes of involvement
of calcium stores in the response to glutamate: one possibility is that
when the stores are empty, influx of calcium leads to fast refilling of the
stores, leading to a subsequent smaller and shorter duration of a rise of
free [Ca2⫹]i. Alternatively, the increase in [Ca2⫹]i may be partly due
to release of calcium from stores, in a calcium-induced calcium release
process, and in the absence of calcium in the stores, the observed
responses are caused only by influx of calcium, and are therefore
smaller than in the control condition. While we do not present evidence to support either of these two possibilities, recent studies indicate that store antagonists block the [Ca2⫹]i transients in response to
glutamate and to synaptic input (Emptage et al., 1999).
Regardless of the mechanism, it is apparent that long spines are
more dependent on stores than short ones, as their response to
glutamate is lower than that of their short counterparts when the
stores are depleted.
A possible cause for the difference between the short and long
spines is that the short spines contain calcium stores that are continuous with the dendritic ones, and are replenished following each
response to glutamate, whereas the stores in the long ones are not
fed by the parent dendrite, and thus have a limited volume which
tends to deplete faster. A morphological difference in smooth endoplasmic reticulum has been described (Spacek and Harris, 1997)
which may underlie this difference, except that a functional assay
of the calcium stores, i.e., caffeine-induced calcium transients, does
not show a distinct difference between the two (Korkotian and
Segal, 1998). These results were analyzed in a model of a spine/
dendrite (Volfovsky et al., 1999), which assumes only a morphological difference between short and long spines.
While [Ca2⫹]i transients in spines and dendrites have been studied extensively in recent years, there is no clear concept of what the
significance is of these transients. Unlike the case of presynaptic
593
terminals, where a rise in [Ca2⫹]i is important for transmitter
release, the individual postsynaptic [Ca2⫹]i transients are not assumed to play a major role in the fast depolarization caused by the
incoming afferent. By comparison, [Ca2⫹]i transients are important in the dendrites, where they underlie calcium spikes, which
travel to the soma and contribute to the generation of sodium
action potentials. On the other hand, a sustained rise in [Ca2⫹]i
has been suggested to underlie long-term processes including calcium-dependent activation of kinases and phosphatases, leading to
activation/insertion/deletion of glutamate receptor subunits, proposed to underlie long-term potentiation and depression (Bear, 1995;
Bliss and Collingridge, 1993). Altogether, the spine is proposed to
restrict transient changes in [Ca2⫹]i, and thus protect the rest of the
dendrite from the adverse effects of an excess [Ca2⫹]i (Segal, 1995b).
The role of calcium stores in the regulation of calcium transients
evoked by afferent stimulation has been debated recently; while
one study suggests that most, if not all, of the [Ca2⫹]i change
evoked by afferent stimulation is caused by release of calcium from
stores (Emptage et al., 1999), another recent study could not confirm this observation and proposed, instead, that synaptically
evoked calcium variations are independent of release of calcium
from stores (Kovalchuk et al., 2000). In our previous work, we
found that dendritic spines do contain calcium stores, which can
be triggered to release calcium by caffeine. We now find that these
stores are also instrumental in supporting the transient rise in
[Ca2⫹]i evoked by glutamate, which causes primarily an influx of
calcium from the extracellular space. Thus, in the absence of stored
calcium, the calcium transient is smaller and shorter.
Possible clues to functions of dendritic spines can be found in
the time course of changes in spines following an environmental
challenge. If indeed spines are made to store information for long
periods of time, one can expect them to react slowly to environmental changes, and once they are made, to remain unchanged for
a long time. This is not the case: A reduction in spine density and
dendritic morphology in hibernating ground squirrels can be completely restored within 2 h of arousal from torpor (Popov et al.,
1992). Likewise, changes in dendritic spine density vary quite precisely across the estrus cycle, with large variations seen over periods
of 24 h (Woolley and McEwen, 1993). By the same token, cutting of
brain slices can produce marked changes in spine density in less than
1 h (Kirov et al., 1999). The newly described short time constants of
spine changes in vivo as well as the ability to label individual neurons
and follow changes in spines in real time have popularized the use of
simpler, in vitro preparations for the study of spine motility.
A major advantage of the in vitro preparation is that it allows for the
labeling of individual cells and for following changes in their morphology over extended periods of time. This circumvents the need to
compare large independent populations, and allows a precise timing of
the observed changes in dendritic spines. This ability has prompted
the detection of rapid changes in spine morphology, on a minute time
scale, including fluctuations around the same length (Okabe et al.,
1999), and fast responses to glutamate (Segal, 1995c; Halpain et al.,
1998). These fluctuations appear not to depend on action potential
discharges, but are developmentally regulated, and involve rapid
changes in actin polimerization (Fischer et al., 1998). Recent studies
of cultured slices, which combines the advantages of in vitro cells
Figure 6
____________________________________________
SPINE MORPHOLOGY AFFECTS SPINE FUNCTION
FIGURE 6.
Lack of differences between short and long spines. A, B:
Long and short spines, respectively (A was illustrated in Fig. 1). 1: 3-Dreconstructed images of spine-dendrite segments. 2: Summed line scans
to show the responses to glutamate, left, and spontaneous events occurring later in time, at right. In both cases, the spontaneous events reflect
excitatory postsynaptic potentials likely to have been evoked in the recorded spines. They do not reflect backpropagating action potentials,
which are larger and longer-lasting (not shown). 3: Expansion of traces
indicated in 2 by asterisks to illustrate the time course of spontaneous
events. C: Image of Calcium Green-labeled dendrites and spines (green)
and FM4-64-labeled presynaptic varicosities (red). Note that both the
long spine (left) and the short spine (right) possess a presynaptic terminal. Several other presynaptic terminals are seen attached to the dendrite,
and probably to other dendrites in the field. D: Short (top) and long
(bottom) spines stained with an antibody for the ryanodine receptor
(from the experiment detailed in Korkotian and Segal, 1998). d, dendrite. Scale bars in B–D, 2 ␮m.
(follow-up of individual spines) with the advantages of in vivo cells
(spatial organization of a cell and its afferents) have already yielded
preliminary observations of novel spines produced following expression of LTP in pyramidal neurons (Engert and Bonhoeffer,
1999) as well as rapid growth of filopodia in response to electrical
stimulation (Maletic-Savatic et al., 1999). In the dissociated culture, formation of novel spines in response to release of calcium
from stores (Korkotian and Segal, 1999a) or the retraction/extension of existing spines in response to glutamate (Korkotian and
Segal, 1999b) have also been reported.
These results, obtained primarily with in vitro preparations, paint a
very different picture of spine motility and stability than ever before
appreciated. The spine appears as a dynamic structure, which can
change continuously as a function of synaptic input. The current
reliance on in vitro systems may bias the view of the role of spines in
long-term neuronal functions; after all, most of the studies cited herein
deal with the immature brain, and may not reflect processes that take
place in the adult one. Thus, rapid motility may be a process by which
a spine searches and finds its target, but this motility may be lost weeks
or months after the connection is stable. Given these limitations, it
appears that the demand that changes the spine at such a fast rate has
to do with the need to restrict changes in [Ca2⫹]i in the area of the
activated synapse. This indicates that spine shape may have an important role in regulating the interaction between the synapse and the
parent dendrite, and the ability of the dendrite to control the efficacy
of the synapse and its modifiability.
Acknowledgments
We thank Ms. V. Greenberger for preparation of cultures.
REFERENCES
Bear M. 1995. Mechanism for a sliding synaptic modification threshold.
Neuron 15:1– 4.
Bliss TVP, Collingridge GL. 1993. A synaptic model of memory: long
term potentiation in the hippocampus. Nature 361:31–39.
Emptage N, Bliss TV, Fine A. 1999. Single synaptic events evoke NMDA
receptor mediated release of calcium from internal stores in hippocampal dendritic spines. Neuron 22:115–124.
595
Engert F, Bonhoeffer T. 1999. Dendritic spine changes associated with
hippocampal long-term synaptic plasticity. Nature 399:66 –70.
Fischer M, Kaech S, Knutti D, Matus A. 1998. Rapid actin-based plasticity in dendritic spines. Neuron 20:847– 854.
Garaschuck O, Yaari Y, Konnerth A. 1997. Release and sequestration of
calcium by ryanodine-sensitive stores in rat hippocampal neurons.
J Physiol [Lond] 502:13–30.
Halpain S, Hipolito A, Saffer L. 1998. Regulation of F-actin stability in
dendritic spines by glutamate receptors and calcineurin. J Neurosci
18:9835–9844.
Hernandez-Cruz A, Diaz-Munoz M, Gomez-Chavarin M, Canedo-Merino R, Protti DA, Escobar AL, Sierralta J, Suarez-Isla BA. 1995 Properties of the ryanodine-sensitive release channels that underlie caffeine
induced Ca2⫹ mobilization from intracellular stores in mammalian
sympathetic neurons. Eur J Neurosci 7:1684 –1699.
Kirov SA, Sorra KE, Harris KM. 1999. Slices have more synapses than
perfusion-fixed hippocampus from both young and mature rats.
J Neurosci 19:2876 –2886.
Korkotian E, Segal M. 1998. Fast confocal imaging of calcium released
from stores in dendritic spines. Eur J Neurosci 10:2076 –2084.
Korkotian E, Segal M. 1999a. Release of calcium from stores alters the
morphology of dendritic spines in cultured hippocampal neurons.
Proc Natl Acad Sci USA 96:12068 –12072.
Korkotian E, Segal M. 1999b. Bidirectional regulation of dendritic spine
dimensions by glutamate receptors Neuroreport 10:2875–2877.
Kovalchuk Y, Eilers J, Lisman J, Konnerth A. 2000. NMDA receptormediated subthreshold Ca2⫹ signas in spines of hippocampal neurons. J Neurosci 20:1791–1799.
Lissin DV, Carroll RC, Nicoll RA, Malenka RC, von Zastrow M. 1999.
Rapid, activation-induced redistribution of ionotropic glutamate receptors in cultured hippocampal neurons. J Neurosci 19:1263–1272.
Majewska A, Brown E, Ross J, Yuste R. 2000. Mechanisms of calcium
decay kinetics in hippocampal spines: role of spine calcium pumps and
calcium diffusion through the spine neck in biochemical compartmentalization. J Neurosci 20:1722–1734.
Maletic-Savatic M, Malinow R, Svoboda K. 1999. Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 283:1923–1927.
Okabe S, Kim HD, Miwa A, Kuriu T, Okado H. 1999. Continual remodeling of postsynaptic density and its regulation by synaptic activity. Nat Neurosci 2:804 – 811.
Papa M, Bundman MC, Greenberger V, Segal M. 1995. Morphological
analysis of the development of dendritic spines in primary cultures of
hippocampal neurons. J Neurosci 15:1–11.
Popov VI, Bocharova LS, Bragin AG. 1992. Repeated changes of dendritic
morphology in the hippocampus of ground squirrels in the course of
hibernation. Neuroscience 48:45–51.
Segal M. 1995a. Imaging of calcium variations in dendritic spines of
cultured hippocampal neurons. J Physiol [Lond] 486, 285–296.
Segal M. 1995b. Dendritic spines for neuroprotection. Trends Neurosci
11:468 – 471.
Segal M. 1995c. Morphological alternations in dendritic spines of rat
hippocampal neurons exposed to NMDA. Neurosci Lett 193:73–75.
Shepherd GM. 1996. The dendritic spine: a multifunctional integrative
unit. J Neurophysiol 75:2197–2210.
Spacek J, Harris KM. 1997 Three-dimensional organization of smooth
endoplasmic reticulum in hippocampal CA1 dendrites and dendritic
spines of the immature and mature rat. J Neurosci 17:190 –203.
Volfovsky N, Parnas H, Segal M, Korkotian E. 1999. Geometry of dendritic spines affects calcium dynamics In hippocampal neurons: theory
and experiments. J Neurophysiol 82:450 – 462.
Woolley CS, McEwen BS. 1993. Roles of estradiol and progesterone in
regulation of hippocampal dendritic spine density during the estrous
cycle in the rat. J Comp Neurol 336:293–306.
Yuste R, Denk W. 1995. Dendritic spines as basic functional units of
neuronal integration. Nature 375:682– 684.