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The Journal of Neuroscience, January 1, 2001, 21(1):186-193
Functional Plasticity Triggers Formation and Pruning of Dendritic
Spines in Cultured Hippocampal Networks
Miri
Goldin,
Menahem
Segal, and
Elena
Avignone
Department of Neurobiology, The Weizmann Institute, Rehovot 76100, Israel
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ABSTRACT |
Despite widespread interest in dendritic spines, little is known
about the mechanisms responsible for spine formation, retraction, or
stabilization. We have now found that a brief exposure of cultured hippocampal neurons to a conditioning medium that favors activation of
the NMDA receptor produces long-term modification of their spontaneous
network activity. The conditioning protocol enhances correlated
activity of neurons in the culture, in a process requiring an increase
in [Ca2+]i and is associated with both
formation of novel dendritic spines and pruning of others. The novel
spines are likely to be touched by a presynaptic terminal, labeled with
FM4-64 dye, whereas the absence of such terminals increases the
likelihood of spine pruning. These results indicate that long-term
functional changes are correlated with morphological modifications of
dendritic spines of neurons in a network.
Key words:
dendritic spines; hippocampus; NMDA; FM4-64; calcium; synapse
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INTRODUCTION |
Novel methods of high-resolution
imaging of living dendrites and spines contributed to a radical shift
in the century-old view of the spine, from a stable storage site for
long-term memory, to a dynamic structure, which can undergo rapid
changes in shape and function (Fischer et al., 1998 ; Segal et al.
2000). The spine is now perceived both as a stable and a plastic
structure, and thus it becomes imperative to define the conditions that
change spine morphology in association with long-term functional
plasticity and dissociate them from ongoing transient variations in
spine shape. Dendritic spines in young neurons express considerable motility in different time domains, ranging from small and fast vibrations to slow expansion and retraction (Ziv and Smith, 1996; Fischer et al., 1998; McKinney et al., 1999). These spontaneous movements are reduced in the mature neuron (Dunaevsky et al., 1999),
which can still undergo lasting changes in spine shape and density
after functional changes. Time lapse photography allows the detection
of changes in existing spines or formation of novel ones after exposure
of a cultured slice of the hippocampus to a long-term potentiation
(LTP) protocol (Engert and Bonhoeffer, 1999), yet there is no
evidence that these novel spines are in any way functional, i.e., that
they are innervated by afferent fibers and contribute to the enhanced
synaptic response. The dissociated culture provides an easy, stable
optical access to both the presynaptic and postsynaptic components of
the synapse, so that rapid changes in spines can be easily detected
(Korkotian and Segal, 1999) in connection with changes in presynaptic
elements (Vardinon-Friedman et al., 2000). Unfortunately, except where
LTP is recorded between pairs of cells (Tao et al., 2000), there are
few established procedures for inducing a reliable and robust LTP in
dissociated cultures (Malgaroli and Tsien, 1992; Luscher et al., 2000).
A conventional LTP induction protocol involves an intensive but
transient activation of a small set of synapses, which may not favor a
large scale formation of dendritic spines. In contrast, continuous
activation of a neuronal network, which involves many more synapses,
may maximize the likelihood of detection of morphological changes in
the cells involved. Taking advantage of the ability to grow neurons in
controlled conditions (Segal and Furshpan, 1990), we have developed a
protocol in which one can produce a persistent modification of network
activity in cultured neurons and monitor simultaneously morphological
changes in dendritic spines of these neurons.
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MATERIALS AND METHODS |
Culture preparation. Rat pups on day of birth were
decapitated, and their brains were removed and placed in a chilled
(4°C), oxygenated Leibovitz L15 medium (Biological Industries, Beit
Haemek, Israel) enriched with 0.6% glucose and Gentamicin (20 µg/ml; Sigma, St. Louis, MO). Bilateral hippocampi of 8-10 pups in
each experiment were dissected out and collected in the same medium.
Tissue was mechanically dissociated with a fire-polished pasteur
pipette and passed to the plating medium consisting of 5% heat
inactivated horse serum (HS), 5% fetal calf serum (FCS), prepared in
MEM-Earl salts (Biological Industries), enriched with 0.6% glucose,
Gentamicin, and 2 mM glutamax. Approximately
0.3-0.5 × 106 cells in 1 ml of
medium were plated in each well of a 24 well plate, onto
polylysine-coated round (13 mm) glass coverslips. A monolayer of glia
was grown on the glass for 1-2 weeks before the plating of the neurons
(Papa et al., 1995; Murphy and Segal, 1996). Cells were left to grow in
the incubator at 37°C, 5% CO2, for 4 d,
then medium was changed to 10% HS in enriched MEM, plus a mixture of
5'-fluoro-2-deoxyuridine-uridine (20 mg and 50 mg/ml, respectively; Sigma) to block glial proliferation. Four days later the
medium was changed once again to 10% HS in enriched MEM, and no
further changes were made. In some glasses of the plate, 50 µM
DL-2-amino-7-phosphonovalerate (APV; Tocris,
Bristol, UK) was added to the medium every other day, and in a few
glasses, 3 mM Mg was also added to the medium.
There was no clear additional effect of the increased Mg concentration.
Electrophysiological recording. The cultures were
transferred to the recording chamber placed in a Zeiss Axioscope
equipped with Nomarski optics, a water immersion lens, and an infrared camera. Single or double whole-cell patch recordings from hippocampal neurons were performed using micropipettes containing (in
mM): K-gluconate 140, NaCl 2, HEPES 10, EGTA 0.2, Na-GTP 0.3, Mg-ATP 2, and phosphocreatine 10, pH 7.4, having a
resistance in the range of 6-12 M . QX314 (2 mM) was added in the pipette solution in
voltage-clamp recording. Current and voltage-clamp recording were
performed using Axoclamp 2 and Axopatch 200A (Axon Instruments, Foster
City, CA), respectively. Simultaneous recording from two cells in
current and voltage clamp were conducted to maximize the chance to
detect changes in synaptic and intrinsic currents and also to detect
possible changes in firing rates and in the appearance of sustained
depolarizations. Voltage-clamped neurons were held at  60 mV, and
signals were filtered at 10 kHz. Cells in current-clamp were held at
approximately 65 mV. Signals were stored in an IBM personal computer
using Axon Instruments software. The culture was perfused at a rate of
1 ml/min at room temperature with standard recording medium containing
(in mM): NaCl 129, KCl 4, MgCl2 1, CaCl2 2, glucose
10, and HEPES 10, pH was adjusted to 7.4 with NaOH, and osmolarity to
320 mOsm with sucrose. Perfusion velocity was increased to 4 ml/min for
3 min during the change of medium to speed up the exchange. During
recording from APV-treated cultures, 50 µM APV
was added to the recording medium. The conditioning medium (CM) was
similar to the standard recording medium, except that
Mg2+ and APV were omitted, and glycine (1 µM) was added to the medium. Activity of cells
was compared before and after perfusion with the CM, which was perfused
for 2-10 min.
Data were analyzed off-line using pClamp6 (Axon Instruments) and
software written in Matlab. The Wilcoxon test was applied for
nonparametric statistical analysis to compare data before and after the
treatment, and the median of value was considered as representative
value of a single cell. The percentage change was calculated as
100*(data after treatment/data before treatment 1). Data were
averaged between different experiments and presented as mean ± SEM, and t test was applied.
Morphological analysis. Control or APV-treated
cultures were washed with the standard recording medium and placed in a
recording chamber. Individual cells were injected with calcein (20 mM) using a sharp micropipette backfilled with
K-acetate. The cells were allowed to rest for 30 min, and the chamber
was transferred to the stage of a confocal laser-scanning microscope
(Zeiss 510). The cultures were perfused with the same recording medium
as above. Three-dimensional (3D) images of selective dendrites were
taken at 30 min intervals. If no significant movement of spines and filopodia was seen over 30-60 min, the cultures were exposed to the CM
for 4 min, followed by wash with the standard recording medium. The
same set of dendrites was repeatedly imaged for up to 3 hr after the
application of the CM. Image analysis was conducted off-line.
Protrusions were categorized into "spines with head", not longer
than 3 µm, "spines with no heads", "stubby spines", 0.5 µm
or shorter without a spine neck, and "filopodia", thin, longer than
3 µm protrusions. The number of spines of all forms was
counted in successive dendritic segments of 100-200 µm in length.
The reliability of the measurements was assessed by a double-blind
procedure in which two independent observers analyzed the same subset
of images. The two observers yielded highly correlated results. Further
analysis of the morphological data were therefore conducted by one
trained observer.
When presynaptic terminals were labeled, FM4-64 (2 µM;
Molecular Probes, Eugene, OR) was applied in a medium containing
90 mM KCl, replacing equimolar NaCl, for 1 min, followed by
an extensive wash in a Ca-free medium, followed by wash in regular
medium. FM4-64 labeled puncta were easily detected. These puncta were maintained after extensive wash in Ca-free medium, but disappeared within minutes after wash in normal medium. When FM4-64-labeled particles were imaged, the two-channel option of the confocal microscope was used to take simultaneous images with excitation wavelengths of 488 and 543 nm.
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RESULTS |
Transient activation of the culture induces long-lasting changes in
network activity
Hippocampal neurons obtained from postnatal day 0 rat hippocampus
and grown in drug-free dissociated culture conditions express spontaneous network activity, consisting of mixed excitatory and inhibitory synaptic currents that develop over 7-8 d into coordinated rhythmic bursts of activity, as seen elsewhere (Verderio et al., 1999).
Cells grown in a medium containing the NMDA antagonist APV showed
higher frequency of large bursts and small interburst synaptic events
(0.72 ± 0.08 and 10.05 ± 0.97 Hz, respectively) by
comparison to cells in control cultures (0.26 ± 0.05 and 4.8 ± 0.75 Hz). This difference was attributable to the growth of the
culture in APV and not to the presence of the drug in the recording
medium, because exposure of control cultures to APV did not change
their activity patterns (data not shown). Exposure to a CM (APV- and
Mg-free) for 3 min induced a lasting change in activity in the
APV-grown cultures; synaptic events in the burst became larger (Fig.
1) and more organized: enhancement of network activity persisted for as long as recording was possible (up to
1 hr, data not shown). Such long-term changes were not seen in control
cultures (Fig. 2). The change in network
activity could be expressed as an increase of mean synaptic current,
increase in amplitude, and/or frequency of bursts and an increase of
cross-correlation between cells. A significant (50.8 ± 10.7%)
increase of mean synaptic current in APV-grown cultures was observed
between 3 and 20 min after wash in 14 of 17 cells tested. In contrast,
only one of nine cells showed an increase in mean current in control
culture, and three of nine showed a decrease (on average, 9.9 ± 7.6%).
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Figure 1.
Activity of cells grown in APV-containing medium
undergoes long-term modification after brief exposure to a conditioning
medium (CM) that favors activation of NMDA
receptors. 1-4, Examples of spontaneous activity
recorded in APV-treated cultures, before (1),
during (2), and at two times after exposure to
the CM. Cell was voltage-clamped at 60 mV. Bottom,
Continuous recording of burst peaks, normalized to the mean of control
periods, during the experiment. A persistent increase in burst size is
evident as long as recording could be made from this cell.
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Figure 2.
Persistent modification of network activity is
dependent on growth conditions of the cultures. a,
Sample records taken (from left to right)
before, within 10 min, and 20 min after exposure to the CM.
Top, Cell grown in control conditions;
bottom, cell grown in APV-containing growth medium.
b, c, Summary of cells analyzed between 3 and 10 min and 10 and 20 min after the CM (control, open
symbols, n = 5; APV-grown, filled
symbols, n = 9). The mean synaptic current
and burst amplitude (circle) increased after treatment
only in APV-treated culture. Despite the increase in burst amplitude,
the amplitude of interburst events did not change
(triangles).
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To determine the temporal course of changes, mean synaptic currents per
15 sec intervals and amplitude of bursts were measured between 3 and 10 min and between 10 and 20 min after replacement of the CM by the
standard recording medium (n = 9 and 5 in APV-grown and
control culture, respectively; Fig. 2). The mean current in APV-treated
culture increased by 64.9 ± 14.1% and by 45.8 ± 11.2% between 3 and 10 min and 10 and 20 min, respectively, whereas in
control culture, only one cell showed a change, and the average was
4.3 ± 4.3% and 4 ± 4%, respectively (Fig. 2).
Measuring the burst peak amplitude in the same cells showed an increase of 46.8 ± 9.9% and 37.2 ± 14.1% in APV-grown culture,
whereas on average control culture showed a change of 1.3 ± 8.4% and 5.9 ± 8.7 (Fig. 2c). In contrast, the
amplitude of the interburst events (small individual synaptic events
probably representing single EPSCs) did not change at any time after
exposure to the CM (Fig. 2c), indicating that the network
change may not be reflected in the properties of individual synapses.
One of the striking consequences of exposure to the CM was that
activity in APV-grown culture was not only larger, but also became more
synchronized. To investigate the degree of synchronicity, we performed
double patch recording from adjacent cells in the culture, with one
cell in voltage-clamp mode and a second in current-clamp mode. Usually
the activity consists of both synchronous and asynchronous events (Fig.
3). After the treatment, besides an
increase in their amplitude, all the events became synchronized (Fig.
3b,c). In general cells that were already organized and
synchronous before the exposure to the CM did not show any change,
independently from the condition in which they grown (Fig.
3d).
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Figure 3.
CM increases the correlation between cells
proportionally to their initial correlation value. a,
b, Voltage-clamp (top trace, 1) and
current-clamp (bottom trace, 2) recording from two
adjacent cells and their current versus voltage plot with the linear
fit (3), before (a) and
after (b) treatment. After the treatment, besides
increasing its amplitude, the signal became less "noisy", and there
is an increase in correlation coefficient of the linear fit between the
two cells from 0.77 to 0.88 (the median of the experiment is from
r = 0.72-0.84). c,
Cross-correlation between the two traces, normalized to their area so
that the increase in the amplitude does not affect it, became narrower
and bigger after the treatment (thicker line).
d, Plot of percentage change of correlation coefficient
r versus initial value of r of all double
recordings: control (open circle), APV-treated cells
(closed circle), and APV-treated cells plus Mg
(triangle). r before and after the
treatment were calculated as a median of different trace sections (from
3 to 13 different sections; 9.8 sec duration each), and the
significance of the difference was tested (Wilcoxon test;
p < 0.001). The linear regression is of
r = 0.94.
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We then explored some parameters that may affect the change in network
activity. The duration of the treatment beyond 3 min did not affect the
magnitude of change, but when the protocol was applied for <3 min, no
consistent effect was detected (n = 3). To investigate
the role of activation of the NMDA receptor, a Mg-free, CM-containing
100 µM APV was applied. In none of three cases
tested with this CM was there a persistent change in activity. In
another set of experiments, APV-free, Mg-containing CM was applied. In
three of six cells a small effect was detected. In one of these three
cells the entire protocol was later applied with the Mg-APV-free CM,
and a further enhancement was observed. Thus, activation of the NMDA
receptor is necessary for inducing the network change.
We then tested the involvement of changes in
[Ca2+]i in the production of the
long-term change in network activity. In three cultures pre-exposed to
BAPTA-AM for 70 min (20 µM; which did not affect
spontaneous network activity because this was similar to nonincubated
culture), subsequent exposure to the CM failed to produce the long-term
modification of the network activity (Fig.
4).
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Figure 4.
Buffering of intracellular calcium prevents an
increase in the activity and in the correlation between cells.
a, b, Double recording from adjacent
cells in a culture incubated for 70 min with BAPTA-AM (20 µM) before (a) and after
(b) CM exposure for 7 min. Cells (voltage-clamp,
top trace; current-clamp, bottom trace)
still show network activity with both synchronous and asynchronous
events. No change in activity or in the correlation coefficient was
detected after perfusion with the CM.
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Morphological analysis: postsynaptic changes
Randomly selected, individual calcein-labeled neurons grown for at
least 11 d in culture show little spontaneous motility or
formation of novel spines, by comparison to younger cells (Papa et al.,
1995; Ziv and Smith, 1996). The density of dendritic spines at this age
was 45 ± 5.3 spines per 100 µm dendrites in the control conditions, and 29.2 ± 2.5 spines per 100 µm dendrites in the APV-grown cultures. These values are similar to densities of dendritic spines of cultured hippocampal neurons reported elsewhere (Papa et al.,
1995; Murphy and Segal, 1996). A reduction in spine density in the
presence of APV has also been seen before in young neurons (Collin et
al., 1997). After exposure to the CM, which, in sister cultures
produced long-term changes in network activity, cells began to express
new protrusions, and modify existing ones, in a time-dependent manner.
In an early series of experiments (our unpublished
observations), we found that the presence of a robust electrophysiological change after exposure to the CM is a good predictor to a subsequent change in the morphology of sister cultures, and whenever such changes were not found, there were no changes in
dendritic spines. Thus, although we did not systematically quantify the
parallel changes in electrophysiology and morphology, further
morphological experiments were conducted after verification that the
culture can express long-term electrophysiological changes in network activity.
The new protrusions were divided into new filopodia, headless,
mushroom, and stubby spines. Spines of all categories showed a
significant increase in number over control values already at 1 hr
after exposure to the CM and continued to be formed within the
following 2 hr of observation (Fig. 5).
Altogether, in 25 APV-treated cells (3892 µm length of dendrites)
there were 27.0 new spines (with or without heads) per millimeter of
dendrite, compared with 2.9 new spines per millimeter in a total of 13 control cells (2070 µm total length of dendrites). There were 20.0 additional new stubby spines per millimeter of dendrite in the
APV-treated cells compared with 0.48 new stubby spines per millimeter
of dendrite in the control cells. These differences were highly
significant statistically. The number of new filopodia was small and
not different between the treated and controls (Fig. 5d).
The functionality of the novel spines was examined in a subset of
neurons by labeling presynaptic terminals, at the end of the
observation period (Fig. 5c). In total, 56 of 64 novel
spines were adjacent to FM4-64-labeled terminals. This indicates that
the novel spines are likely to be functional and receive input from
viable terminals.
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Figure 5.
Exposure to a conditioning medium causes formation
of novel dendritic spines in cultured hippocampal neurons.
a illustrates a dendritic spine, 3D-reconstructed at 30 min intervals, before (1, 2) and after (3, 4, 5) exposure to the CM. b, Two series of
0.75-µm-thick optical sections of a dendritic segment used for the
reconstruction of the images presented in c,
illustrating the images shown in c2, top, and c3,
bottom. A novel spine, not seen in B, top, is
clearly seen in B, bottom. c illustrates
a time-lapse, 3D image of a dendrite from a neuron in a culture that
was exposed at the end of the imaging sequence to a medium containing
FM4-64, to label presynaptic terminals (c5). Note that
after exposure to the FM4-64-containing medium (high K, depolarizing
medium) the spine head shrinks. Cells were grown in APV, injected with
calcein, and exposed to the CM in the recording chamber for 4 min,
followed by an extensive wash, as detailed above. Scale bars:
A, C, 1 µm (2.5 µm in
B). D, Summary diagram of the formation
of novel dendritic spines in the experimental (left) and
control groups (right) depicting different types of
spines, including headed spines, spines with no head, stubby spines,
and filopodia. Spines of the first three categories are formed within 2 hr of exposure to the CM, whereas no similar change was seen in the
control cultures, exposed to the same CM. The difference between
control and experimental groups of neurons is highly significant
(t test for comparison between control and APV-treated
cells yielded p < 0.01.
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Pruning of dendritic spines
In addition to the formation of novel spines, we found quite a
number of cases in which existing spines disappeared after exposure to
the CM (Fig. 6). Altogether, 22.5% of
the total counted spines disappeared after exposure to the CM in the
experimental group, by comparison to only 8.8% of the spines in the
control neurons. This was further analyzed below.
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Figure 6.
Selective spine pruning after exposure to the CM.
a, A sequence of 3D images of a dendrite taken at
various times before and after exposure to the CM (at time 0). Many,
but not all the spines on this dendrite disappear in the course of the
experiment, after exposure to the CM. b, A summary
comparison between control (Ct) and APV-treated
cultures, illustrating that the latter group has a significantly higher
proportion of disappearing spines of the total population sampled, than
control groups. After exposure to the CM, 22.5% of the spines
disappear in the APV-treated culture, compared with only 8.8% of the
spines in the control cultures (t test,
p < 0.003). Scale bar, 1 µm.
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Morphological analysis: postsynaptic changes correlated with
presynaptic activity
In an attempt to trace down the origin of the novel innervated
spines and the fate of the existing ones, we labeled presynaptic terminals at the beginning and the end of the experiment (Fig. 7a,b). The goal of these
experiments was to determine whether presence of a presynaptic terminal
either on the shaft or on the spine affects the ability of the spine to
change its morphology as a result of exposure to the CM. As opposed to
the previous study, in which only new spines were counted, in the
present study all spines were counted and categorized into spines that
were maintained or disappeared after exposure to the CM. The results are summarized in Figure 7c, comprising a total of 16 cells
(3543 nm total dendritic length) exposed to the CM. Strikingly, spines that were not innervated before had a significantly higher tendency to
disappear after the exposure to the CM by comparison to innervated spines, which tended to maintain their shape. Thus, exposure to a CM
promotes the pruning of noninnervated spines. This is by no means an
all-or- none effect, because there are many spines not associated with
an FM4-64 terminal that were maintained across the measurement time.
Finally, a detailed analysis of the novel spines indicated that in most
cases studied, novel spines were formed where previously there was an
FM4-64 bouton adjacent to a dendritic shaft, (n = 232)
compared with cases in which a novel spine was formed where there was
no detectable FM4-64 bouton beforehand (n = 119). Thus,
the presence of a putative shaft synapse facilitates formation of a
novel spine.
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Figure 7.
Dynamic changes in dendritic spines are predicted
by the presence of active presynaptic terminals. a,
b, Two illustrations of dendrites and spines stained
with FM4-64 before and after exposure to the CM. In a,
several FM-labeled puncta are seen near the 3D-reconstructed dendrite,
which, before the start of the experiment, did not contain any clear
spines. Within 90 min after exposure to the CM, several spines are
formed and are eventually associated 30 min later, with FM4-64-stained
particles. In b, existing spines are seen already at the
start of the experiment, some of which are associated with an
FM4-64-labeled particles. After to the CM two spines, not associated
with FM, disappear (yellow arrows) and the rest
(white arrows) are maintained. c, The
maintenance of dendritic spines is correlated with the presence of FM
particles. The population of dendritic spines was divided into spines
linked and not linked to FM. Most of those linked to FM particles (FM+)
were maintained (16.3 spines/100 µm dendrites maintained compared
with 1.77 spines disappearing), whereas a large proportion of spines
not associated with FM (FM) disappeared during the experiment (8.36 spines/100 µm were maintained, compared with 5.41 spines/100 µm
dendrites disappearing). The difference between the labeled and
nonlabeled spines was highly significant (t test;
p < 0.001). Scale bars: a, 2 µm;
b, 1 µm.
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DISCUSSION |
The present results demonstrate that an acute exposure of cultured
hippocampal neurons, grown in presence of an NMDA receptor antagonist,
to a conditioning medium, which favors activation of the NMDA receptor,
can produce a lasting change in their network activity. This change is
expressed as an increase in the size of AMPA receptor-mediated EPSCs as
well as in the synchronization among cells in the culture. The network
changes are followed by morphological plasticity, including formation
of novel dendritic spines that are in close proximity to viable
presynaptic terminals and pruning of existing spines. Thus, a
correlation between functional and morphological plasticity can be
demonstrated in dissociated hippocampal neurons in culture, which
encourages further experiments on the mechanisms of morphological plasticity.
The cellular basis of the long-term change in network activity is not
clear as yet. It involves activation of the NMDA receptor, as it was
not found when the culture was exposed to Mg-free, APV-containing medium. It involved a rise of intracellular calcium concentration and
was blocked by BAPTA, as seen with classical LTP, but whether the
change is at the synapse, involving presynaptic (Ma et al., 1999) or
postsynaptic (Liao et al., 1995) changes or changes in excitability of
the neurons is not entirely clear. In preliminary experiments we could
not find lasting changes in miniature synaptic currents recorded in
cells that expressed the change in network activity, but this result
may just indicate that only a subset of synapses are changed, and the
change is diluted among many active synapses. Likewise, we did not find
a change in firing threshold (our unpublished observations), but
the possibility of a change in excitability of the cells cannot be
ruled out. Finally, although we do observe changes in the AMPA-mediated
excitatory synaptic currents, recorded in presence of APV, we do not
exclude the possibility that inhibitory synaptic currents are also
enhanced by the CM. We did not specifically address this issue in the
present study, but it is likely that an enhanced network activity will also involve a rise in activation of the inhibitory neurons in the
network. A change in network may thus reflect small changes in many
participating neurons, which may not be detected otherwise, but will
result in an amplified reverberating activity of the entire network.
This reverberating, ongoing activity will enhance detection of
morphological changes in neurons of the network.
A major issue in the understanding of the role of dendritic spine in
neuronal plasticity involves the rules governing formation and pruning
of dendritic spines and the relevance of these processes to the
functioning of the synapse. Although there is evidence to indicate that
spines are formed from filopodia, after formation of a synapse with
axon terminal in vitro (Jontes and Smith, 2000), this may
not be the only or even the main mechanism for formation of a spine.
Fiala et al. (1998) suggested that filopodia may be involved in drawing
presynaptic terminals to dendritic shaft, to cause formation of shaft
synapses, which will eventually form spines, as suggested here.
Clearly, in the mature neuron, where filopodia are not abundant, spine
density can vary by 35% across the estrus cycle (Woolley and McEwen,
1993), indicating that novel spines may evolve from a shaft synapse. In
few cases in the present study, we observed spine head branching (Figs.
6a, 7b), which may constitute another mode of
formation of novel spines (Sorra et al., 1998; Toni et al., 1999), but
most of them seem to grow off the dendritic shaft. This growth may be
facilitated by the presence of an active synapse on the shaft, but the
signal for the formation of a spine from a shaft synapse over several
minutes is not known. Likewise, it is not entirely clear what might be the functional significance of the formation of a spine from a shaft
synapse. Unlike previous studies (Engert and Bonhoeffer, 1999), we were
able to show that novel spines are associated with FM4-64-labeled
terminals and that there is a clear relation between the fate of a
spine and its association with a viable terminal. Still, the fact that
synaptic activity, recorded in the soma, increases after a conditioning
protocol does not necessarily mean that the novel spine actually
contributed to this enhanced activity, which can be performed by
existing spine-shaft synapses just the same. The difference between
the impact produced by shaft and spine synapses on the electrical
response recorded in the soma is still unknown. A role of spine neck
length in regulation of calcium responses in the spine head relative to
the parent dendritic shaft has been proposed recently (Volfovsky et
al., 1999), and changes in spine length may contribute to the unique
features of a spine relative to a shaft synapse.
The enhanced network activity is associated with both formation of
novel spines and pruning of existing ones. Whereas formation of novel
spines has long been associated with enhanced synaptic activity (Harris
and Kater, 1994), pruning of existing spines has only recently been
associated with enhanced excitation both in vivo and
in vitro, as indicated by its blockade by APV (Bock and
Braun, 1999). In fact, a very large enhancement of activity in a
hippocampal network, associated with an epileptic seizure, can lead to
a net pruning of dendritic spines (Jiang et al., 1998), as suggested
recently (Segal et al. 2000). Thus, the simultaneous formation of novel
spines, and pruning of existing ones, as is the case with a moderate
increase in activity, may lead to little or no apparent change in the
total number of spines. Instead, an increase in the number of spines
associated with an active presynaptic terminal can be seen. As
suggested elsewhere (Ma et al., 1999), it is possible that the FM4-64
only stains a subset of active synapses, and that this subset can grow
in size after a conditioning protocol. In preliminary experiments, we
immunostained the cultures with anti-synaptophysin antibodies, and we
found that indeed most if not all of the FM4-64 particles are stained for synaptophysin, but that a sizable fraction of synaptophysin particles are not stained with FM4-64. The reason for this discrepancy is not known, but it may explain the persistence of spines in the
absence of viable FM4-64-stained terminals. Further experiments are
needed to analyze this possibility.
The pruning seen in the present experiments, in spines not associated
with an active presynaptic terminal, can still result from a rise of
intracellular calcium, caused by backpropagating action potentials and
synaptic current spread from adjacent synapses. We (Volfovsky et al.,
1999) have shown that a calcium wave coming from the soma can invade
the spine, especially if it is a short one. Thus, spine pruning may
reflect a Hebbian rule, predicting that a spine may disappear if
postsynaptic activity in it is not correlated with presynaptic activity.
The time course of formation and pruning of dendritic spines seen in
the present study corresponds to a similar time course of spine
formation seen in a cultured slice (Engert and Bonhoeffer, 1999), and
to the time course of formation of novel synapses (Vardinon-Friedman et
al., 2000). Thus, whereas the morphological change cannot account for
the initial electrophysiological elevation of network activity, it can
certainly underlie the late maintenance of the enhanced activity. In a
way, the CM-enhanced network activity can be visualized as a sped-up
maturation process because older cultures maintain a more organized
network activity and a higher density of mature dendritic spines (Papa
et al., 1995), supporting the general proposal that a maturation
process uses similar rules as those found after an LTP protocol
(Ben-Ari et al., 1997).
The induction of long-term changes in patterned synaptic activity seen
here was selective to the APV-treated cultures, although both cultures
were exposed to the same medium, which enhances activation of the NMDA
receptor. One possible explanation for this selective effect is that
the growth of the cells in the presence of blocked NMDA receptors may
result in upregulation of the NMDA receptors, to allow much more
calcium influx into the affected cells compared with controls. This
possibility has been alluded to before (Segal and Furshpan, 1990). As
seen elsewhere (Collin et al., 1997), growth of cultured hippocampal
slices in APV reduces spine density, but when APV is washed away, LTP
is enhanced in these slices compared with controls. The opposite
possibility, that dendritic spines actually are formed in the presence
of APV, has been suggested before (Rocha and Sur, 1995), but this may involve a unique type of sparsely spiny thalamic neurons studied there.
At any rate, although the NMDA receptor does not play a crucial role in
formation of synaptic networks, it does affect maturation of these
networks, as predicted by others (Liao et al., 1995; Ben-Ari et al.,
1997)
The relation between production of LTP and an increase in network
activity is not entirely clear. Whereas most of the studies on LTP are
focusing on the first EPSP produced by single pulse stimulation, there
is little evidence that an increase in single EPSP is relevant to the
ongoing activity in a network (Markram and Tsodyks, 1996). In fact,
enhanced synaptic activity may cause scaling down of the network
activity (Turrigiano and Nelson, 2000). Thus, a tetanic stimulation
evoking LTP may not be the most efficient stimulation to evoke a
network change. Our results indicate that network activity can be
changed, and the rules governing these changes, i.e., dependence on
activation of the NMDA receptor, and on influx of calcium, are similar
to those studied in the isolated EPSP case. Thus, the long-term
enhancement of the behavior of the network, and not necessarily of the
individual synapse, is associated with both formation of novel
functional spines and pruning of existing ones.
| |
FOOTNOTES |
Received Aug. 17, 2000; revised Oct. 16, 2000; accepted Oct. 19, 2000.
This work was supported by Grant 97/230 from the United States-Israel
Binational Science Foundation. We thank Ms. V. Greenberger for the
production and maintenance of the cultures and Dr. E. Korkotian for
help with the imaging data collection and analysis.
Correspondence should be addressed to Dr. Menahem Segal,
Department of Neurobiology, The Weizmann Institute, Rehovot 76100, Israel. E-mail: menahem.segal{at}weizmann.ac.il.
| |
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TOP
ABSTRACT
INTRODUCTION
