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The Journal of Neuroscience, August 15, 2001, 21(16):6245-6251
Remodeling of Synaptic Membranes after Induction of Long-Term
Potentiation
Nicolas
Toni,
Pierre-Alain
Buchs,
Irina
Nikonenko,
Patrisia
Povilaitite,
Lorena
Parisi, and
Dominique
Muller
Neuropharmacology, University Medical Center, 1211 Geneva 4, Switzerland
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ABSTRACT |
Several morphological changes of synapses have been reported to be
associated with the induction of long-term potentiation (LTP) in the
CA1 hippocampus, including an transient increase in the proportion of
synapses with perforated postsynaptic densities (PSDs) and a later
occurrence of multiple spine boutons (MSBs) in which the two spines
arise from the same dendrite. To investigate the functional
significance of these modifications, we analyzed single sections and
reconstructed 134 synapses labeled via activity using a calcium
precipitation approach. Analyses of labeled spine profiles showed
changes of the spine head area, PSD length, and proportion of spine
profiles containing a coated vesicle that reflected variations in the
relative proportion of different types of synapses. Three-dimensional
reconstruction indicated that the increase of perforated spine profiles
observed 30 min after LTP induction essentially resulted from synapses
exhibiting segmented, completely partitioned PSDs. These synapses had
spine head and PSD areas approximately three times larger than those of
simple synapses. They contained coated vesicles in a much higher
proportion than that of any other type of synapse and exhibited large
spinules associated with the PSD. Also the MSBs with two spines arising from the same dendrite that were observed 1-2 hr after LTP induction included a spine that was smaller and a PSD that was smaller than those
of simple synapses. These results support the idea that LTP induction
is associated with an enhanced recycling of synaptic membrane and that
this process could underlie the formation of synapses with segmented
PSDs and eventually result in the formation of a new, immature spine.
Key words:
morphology; postsynaptic density; coated vesicles; plasticity; hippocampus; rat
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INTRODUCTION |
An interesting finding of recent
years is that synapses are extremely dynamic structures that may change
not only their functioning with activity but also their morphology.
Analyses of spine dynamics using confocal microscopy showed that these
small dendritic protrusions are characterized by continuous shape
changes (Fischer et al., 1998 ) that may depend on neuronal activity
(Fischer et al., 2000; Lüscher et al., 2000; Matus, 2000). There
is also evidence that increases in intracellular calcium may affect
spine morphology (Korkotian and Segal, 1999; Segal et al.,
2000). Another interesting possibility is that synaptic activity
results in modifications of spine number. Agonist or antagonist
modulation of excitatory transmission or even just preparation of
hippocampal slices was reported to affect spine density (Kirov and
Harris, 1999; Kirov et al., 1999; McKinney et al., 1999). Furthermore,
application of high-frequency trains that induce long-term potentiation
(LTP) trigger the growth of filopodia (Maletic-Savatic et al., 1999) or
even dendritic spines-like structures (Engert and Bonhoeffer, 1999). In
agreement with these reports, we found, using an electron microscopic
analysis of synapses likely to be activated, that LTP induction
resulted in a sequence of morphological changes with a transient
increase in synapses with perforated postsynaptic densities (PSDs),
followed by multiple spine boutons (MSBs) in which the two spines arise
from the same dendrite (Toni et al., 1999).
Whether and how these morphological changes relate to the increase in
synaptic efficacy remain, however, unknown. Among possibilities, one
suggestion has been that the synapses with a perforated PSD that were
observed initially could represent a morphological correlate of the
mechanisms of receptor recycling proposed to contribute to LTP
(Lüscher et al., 2000). Evidence from several studies indicates
that addition of postsynaptic AMPA receptors to the postsynaptic
density is likely to play an important role in changes of synaptic
efficacy (Lüscher et al., 2000). Activity modifies the insertion
of new receptors at the membrane (Shi et al., 1999; Zhu et al. 2000),
and interference via peptides or toxins with the mechanisms of
exocytosis or endocytosis affects LTP or LTD induction and AMPA
receptor-mediated synaptic responses (Lledo et al., 1998;
Nishimune et al., 1998; Lüscher et al., 1999). Thus postsynaptic
exocytotic or endocytotic mechanisms (Maletic-Savatic and Malinow,
1998) could contribute to receptor turnover and changes in synaptic
function. Furthermore they could mediate the remodeling of synaptic
membranes that underlies the formation of synapses with a perforated
PSD. An additional possibility concerning synapses with a perforated
PSD is that they represent an intermediary step in a process of spine
duplication (Nieto-Sampedro et al., 1982; Carlin and Siekevitz,
1983; but see Sorra et al., 1998).
To understand better the functional implication of the different types
of synapses observed after LTP induction and to investigate the
possibility that an increased recycling of synaptic membrane takes
place, we used electron microscopy and analyzed the properties of 134 reconstructed, labeled synapses. The results are consistent with the
idea that LTP induction promotes a remodeling of synaptic membranes
that may account for the reported changes in the different types of synapses.
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MATERIALS AND METHODS |
Preparation and stimulation of cultures. Hippocampal
organotypic slice cultures were prepared from 7-d-old rats and
maintained 12 d in culture as described by Stoppini et al.
(1991) before testing in an interface recording chamber. A
stimulation electrode made of twisted nichrome wires was placed in the
CA3 area, and evoked field potentials were recorded in the CA1. LTP was
induced using  burst stimulation consisting of five bursts at 5 Hz
with each burst composed of four pulses at 100 Hz. This pattern was applied twice at 10 sec intervals and once again, 5 min before fixation, to relabel the same synapses. All slice cultures processed for EM came from different animals.
Electron microscopy processing. At different times after LTP
induction, cultures were fixed and processed for electron microscopy as
described previously (Buchs et al., 1994). Briefly, cultures were fixed
overnight at 4°C in 3% glutaraldehyde, rinsed in 0.1 M phosphate buffer, pH 7.4, and post-fixed 2 hr in a fresh solution of 1% osmium tetroxide
(OsO4) with 1.5% potassium chromium trisoxalate [(K3Cr(C2O4))3;
Aldrich, Milwaukee, WI], pH 9.5. After a 5 min rinse in KOH, pH 9.5, the samples were classically dehydrated in ethanol and embedded in Epon
(Fluka, Buchs, Switzerland). For serial EM, ribbons of up to 60 sections were cut in the middle portion of the apical arborization of
CA1 pyramidal neurons (ultratome Ultracut-E; Reichert-Jung) and
collected on single-slotted Formvar-coated grids (TAAB Laboratories,
Aldermaston, UK). Sections were stained for 45 min in 0.5%
uranyl acetate and 45 sec in lead citrate and analyzed on a Philips
CM10 electron microscope at a magnification of 11,000-29,100×.
Morphological analyses. Synapses were defined by the
presence of a clear postsynaptic density facing at least three
presynaptic vesicles. Perforated synapses were defined by the presence
of a discontinuity in the postsynaptic density (Geinisman et al., 1987), and MSBs were defined by the presence of two independent dendritic spines contacting the same axon terminal (Sorra and Harris, 1993).
For two-dimensional morphometrical studies, five to six sections per
culture were examined, and a total of 200-1519 synapses out of 4-12
hippocampal slice cultures per time point were analyzed. For each
section, all labeled synapses observed in an area corresponding to the
middle third of the CA1 stratum radiatum and clearly identified by the
presence of a PSD were considered for statistical measurements. Negatives were digitized, and PSD length and cross-sectional area were
measured using a software developed by D. Smithies on AVS.
For three-dimensional experiments, a total of 134 synapses were
reconstructed out of eight hippocampal slice cultures. Synapses were
selected on the test section on the basis of the presence of a
precipitate in the postsynaptic spine and the possibility to identify
easily the PSD as simple or perforated on one of the serial sections.
The profiles were then photographed serially at a magnification of at
least 16,000× and analyzed using a software developed by J. C. Fiala and K. M. Harris. The small-fold procedure was used for
determination of section thickness, and an average value of 42 nm was
used for reconstruction. This value and the measurements on serial
sections were used for calculating the surface area of PSDs and spine heads.
Data are presented as a mean ± SEM, with n indicating
the number of synapses analyzed. Statistical analyses were performed using the Student's t test or the
 2 test, when stated.
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RESULTS |
Organotypic slice cultures prepared from 7-d-old neonate rats were
stimulated using burst-patterned stimulation and fixed at different
times after LTP induction using a protocol that reveals, in the form of
a fine precipitate, the presence of calcium accumulated in subcellular
structures. Previous work showed that this approach makes it possible
to identify a subset of labeled spine profiles (~12-15% of all
profiles) likely to represent stimulated synapses (Buchs and Muller,
1996; Toni et al., 1999). In the present study, we analyzed labeled
synapses observed 15-30 min and 1-2 hr after LTP induction and
compared them with labeled synapses observed 5 min after LTP induction,
a time at which morphological modifications are not yet detected, or
with labeled synapses obtained from slices stimulated in the presence
of KN-93, a calcium/calmodulin-dependent protein kinase II antagonist
that prevents LTP induction (Toni et al., 1999).
As illustrated in Figure 1, the
morphological changes detected after LTP induction consisted of changes
in the relative proportion of three distinct synaptic types referred to
as simple synapses (synapses with a unique and continuous PSD; Fig.
1A, left), synapses with perforated PSDs
(synapses exhibiting a discontinuity of the PSD; Fig.
1A, middle), and MSBs (defined by the
presence of two spines contacting the same presynaptic terminal; Fig.
1A, right) (Sorra and Harris, 1993). Under
control conditions, simple synapses represented the majority of labeled
spine profiles (70.4 ± 2.3%; n = 14; 1519 profiles analyzed; Fig. 1B). Their proportion,
however, decreased markedly 30 min after LTP induction (46.9 ± 2.8%; n = 4; 358 profiles analyzed; p < 0.01), because of an increase in the proportion of synapses with
perforated PSDs (from 22.4 ± 2.3 to 45.8 ± 2.8%;
n = 4; 358 profiles analyzed; p < 0.01; Fig. 1B). The proportion of synapses with
perforated PSDs completely recovered 1-2 hr after LTP induction, and
these were replaced, to some extent, by images of MSBs (from 7.1 ± 1.2 to 14.6 ± 1.5%; n = 10; 755 profiles
analyzed; p < 0.01; Fig. 1B).
Previous work performed using three-dimensional reconstruction provided
evidence that the increase in the proportion of MSBs observed 1-2 hr
after LTP induction mainly represented cases in which the two spines arose from the same dendrite, cases that will be referred to as duplicated spines (Toni et al., 1999).
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Figure 1.
Changes in the proportion of different types of
synapses and their characteristics during LTP. A,
Illustration of a simple synapse with a single PSD
(left), a synapse with a perforated PSD
(middle), and an MSB (right). Scale bar,
0.5 µm. B, Proportion of the three types of synapses
under control conditions and at 30 and 45-120 min after LTP induction
(n = 4-14 hippocampal slice cultures and 358-1519
synaptic profiles analyzed; *p < 0.01).
C, Changes in PSD length (left), spine
head profile area (middle), and the proportion of spine
profiles containing coated vesicles (right) determined
via single-section analysis of the entire population of labeled
synapses (n = 4-9 hippocampal slice cultures and
360-660 synaptic profiles; *p < 0.01).
Ctrl, Control.
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Single-section analyses of parameters of these labeled synapses, such
as the spine profile area, PSD length, and presence of coated vesicles,
revealed changes as a function of time after LTP induction. As shown on
Figure 1C, all of these parameters increased 30 min after
LTP induction and recovered to control values or close to control
values after 1-2 hr. These changes, however, were not caused by
modifications in the characteristics of synapses but essentially
reflected modifications in the relative proportion of the three types
of synapses. Comparison of the spine profile area or PSD length of
simple synapses revealed no statistically significant differences over
time (0.26 ± 0.01 vs 0.29 ± 0.01 µm2 and 0.38 ± 0.01 vs 0.37 ± 0.01 µm, respectively; n = 2-3; 131 and 163 profiles analyzed for spine area and PSD length, under control
conditions and at 30 min, respectively). Similarly, the characteristics
of spine profiles of synapses with perforated PSDs were comparable
under control conditions and at 30 min (0.42 ± 0.02 vs 0.43 ± 0.02 µm2 and 0.51 ± 0.02 vs
0.50 ± 0.01 µm; n = 101 and 163). Thus
two-dimensional analyses did not reveal particular changes in the
characteristics of these types of synapses but rather revealed
modifications in the proportion of the different types of synapses.
We therefore continued by analyzing their properties using
three-dimensional reconstruction on a group of 134 labeled synapses. In
Figure 2, we compared the surface area of
the spine head, the surface area of the PSD, and the proportion of
spines exhibiting coated vesicles in a set of 31 simple synapses and 42 synapses with perforated PSDs. As expected from previous work, synapses with perforated PSDs were characterized by a much larger spine head, a
larger PSD area, but also a much higher proportion of spines containing
one or several coated vesicles (1.58 ± 0.19 vs 0.62 ± 0.06 µm2; 0.17 ± 0.02 vs 0.07 ± 0.02 µm; 44 vs 13%; n = 6-8 slice cultures, 31 and
42 reconstructed synapses). The ratio of these parameters between
synapses with perforated PSDs and simple synapses was 2.9, 3.1, and
3.4, respectively. Interestingly, the ratio of the PSD area versus the
spine membrane area remains constant between simple synapses and
synapses with perforated PSDs (10.7 vs 11.3%, respectively), despite a
marked difference in the size of the spines.
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Figure 2.
Characteristics of three-dimensionally
reconstructed simple synapses and synapses with perforated PSDs
observed 30 min after LTP induction. A, Illustration of
the spine head of and PSD of a reconstructed simple synapse
(left) and a synapse with a perforated PSD
(right). Scale bar, 0.5 µm.
B, Values of spine head surface area
(left), PSD surface area (middle), and
the proportion of spines containing coated vesicles for simple synapses
(Ctrl) and synapses with perforated PSDs
(Perforated). Data are the mean ± SEM of
measurements made on 31 reconstructed simple synapses and 42 synapses
with perforated PSDs (*p < 0.01).
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We then investigated whether the characteristics of the synapses with
perforated PSDs seen 30 min after LTP induction were similar to those
of synapses with perforated PSDs observed under control conditions. For
this, we compared the parameters of 20 and 23 synapses with perforated
PSDs reconstructed from serial sections of slices fixed either 30 or 5 and 60 min after LTP induction. The 5 and 60 min time points were used
as the control, because the slice cultures have received the same
stimulation protocol, but the changes in the proportion of perforated
synapses are not detected at those moments. Consistent with the results
obtained with two-dimensional analyses, the dimensions of synapses with perforated PSDs (spine area and PSD area) did not vary with time after
LTP induction (data not shown). However, as illustrated in Figure
3, the synapses reconstructed 30 min
after LTP induction differed from the others in several ways. First,
there were differences in the shape of the PSD. Synapses with
perforated PSDs have been classified in three major types depending on
the organization of the PSD (Fig. 3): fenestrated (with a central,
macular zone lacking PSD), horseshoe-shaped (when the perforation
reaches the edges of the PSD), or segmented (when the PSD is completely
partitioned into two distinct zones). As shown on Figure 3B,
synapses with perforated PSDs under control condition were mostly of
the fenestrated or horseshoe type (83%). In contrast however, synapses
with perforated PSDs 30 min after LTP induction were mainly of the
segmented type (Fig. 3B; 45 vs 17%; p < 0.05; 2). When considering the relative
proportion of these different types of synapses with perforated PSDs
and the increase observed after LTP induction, it appears that the
changes are essentially caused by synapses with segmented, completely
partitioned PSDs (Fig. 3C).
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Figure 3.
Increase in the proportion of synapses with
segmented PSDs 30 min after LTP induction. A,
Illustration of three types of reconstructed synapses with perforated
PSDs. From left to right, synapses with
fenestrated, horseshoe-shaped, and segmented PSDs are shown. Scale bar,
0.5 µm. B, Proportion of the three types of synapses
with perforated PSDs under control conditions and 30 min after LTP
(n = 20 and 23, respectively). The two
distributions are statistically significantly different
(p < 0.05, 2).
C, Changes in the proportion of synapses with perforated
PSDs calculated for synapses with segmented (white
column) and nonsegmented (black column) PSDs
according to the three-dimensional reconstruction. Data are the
mean ± SEM.
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A second interesting characteristic of the synapses with perforated,
segmented PSDs seen at 30 min is that they contained in a great
proportion one or several coated vesicles (Fig.
4D). In 69% of
synapses with segmented PSDs, coated vesicles could be observed,
whereas only 27% of synapses with perforated, nonsegmented PSDs
exhibited such vesicles (n = 13 and 30;
p < 0.05). Note also that the proportion of
reconstructed, simple synapses that contained coated vesicles was even
lower (13%; n = 31; p < 0.01). To
examine the location within spines of coated vesicles, we then analyzed 452 spine profiles taken at different times after LTP induction. As
illustrated in Figure 4, these vesicles were usually located in the
center of the spine head (50% of cases), but they could also be seen
in contact with or emerging from the spine apparatus (6% of cases),
closed to or fusing with the synaptic membrane (44% of cases),
including the area of the PSD (12% of cases). No significant changes
in the distribution of coated vesicles could be detected as a function
of time after LTP induction. Thus an important characteristic of the
synapses with segmented PSDs observed 30 min after LTP induction was
the presence of an increased proportion of coated vesicles.
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Figure 4.
Increased proportion of coated vesicles in
synapses with segmented PSDs observed 30 min after LTP induction.
A, Example of a coated vesicle emerging from or fusing
with the spine apparatus. B, Coated vesicle within the
spine head. C, Example of a coated vesicle seen fusing
with the synaptic membrane at the level of the PSD. Scale bar:
A-C, 0.5 µm. D, Proportion of synapses
with fenestrated (gray column), horseshoe-type
(white column), and segmented (black
column) PSDs that contained one or several coated vesicles in a
population of 15, 15, and 13 reconstructed synapses, respectively
(*p < 0.05); arrows in A-C
point to examples of coated vesicles.
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A third characteristic concerned the presence and size of spinules in
association with the PSD. Spinules are small finger-like protrusions of
the postsynaptic membrane into the presynaptic ending (Fig.
5A,B). The size of these
protrusions was determined by measuring their maximal length, and they
were classified as small when <0.2 µm or large for sizes >0.2 µm.
As shown in Figure 5C, the majority of synapses with
fenestrated PSDs did not contain spinules, and if present, they were
almost systematically of a small size. In contrast, most of the
synapses with segmented PSDs, in addition to having clearly separated
PSDs, usually also exhibited a spinule in between, which in many cases
was characterized by a large size. Synapses with horseshoe-type PSDs
somehow represented an intermediary stage, because most of them
contained a spinule of small size. The three distributions of the three
types of synapses with perforated PSDs are statistically significantly
different (p < 0.05;
2). The synapses with perforated PSDs
observed 30 min after LTP were thus particular in that they mostly
exhibited segmented PSDs and a higher proportion of them contained
coated vesicles and large spinules associated with the PSD.
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Figure 5.
Presence of large spinules associated with the PSD
of synapses with segmented PSDs. A, Illustration of a
spine profile with a large spinule emerging between the two
parts of the PSD. B, Illustration of a spinule on
another reconstructed spine; arrows in A and
B point to large spinules. Scale bar, 1 µm.
C, Proportion of synapses with fenestrated,
horseshoe-type, and segmented PSDs that exhibited no spinules
(gray column) or spinules of small (<0.2 µm;
white column) or large (>0.2 µm; black
column) size in a population of 15, 15, and 13 reconstructed
synapses, respectively.
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We then also analyzed the characteristics of MSBs, the third type of
synapse found to appear 1-2 hr after LTP induction. Previous work
performed using three-dimensional reconstruction showed that the spines
contacting such MSBs 1-2 hr after LTP induction originated from the
same dendrite and exhibited features of mature synapses (Toni et al.,
1999). Comparison of 30 reconstructed duplicated synapses with 30 reconstructed simple synapses revealed, however, some differences.
First, duplicated synapses are characterized by smaller PSDs than
are simple synapses. In average, the PSD of a duplicated synapse
represented ~76% of the size of the PSD of a simple synapse (Fig.
6). Interestingly, this difference was related to the fact that duplicated synapses were constituted of two
spines with distinct characteristics. The largest of the two spines
exhibited a spine head area and PSD area indistinguishable from those
of simple synapses (Fig. 6B, 0.65 ± 0.05 vs 0.61 ± 0.04 µm2;
n = 15 and 31, for spine head area; 0.070 ± 0.009 vs 0.066 ± 0.06 µm2 for PSD area).
In contrast, however, the smallest of the two spines exhibited a spine
head area and PSD area significantly smaller than those of simple
synapses (0.48 ± 0.05 vs 0.61 ± 0.04 µm2; n = 15 and 31;
p < 0.05 for spine head area; 0.039 ± 0.006 vs 0.066 ± 0.006 µm2;
p < 0.01 for PSD area). Interestingly, duplicated
synapses very rarely exhibited perforated PSDs. The proportion of
perforated PSDs among duplicated synapses was 10% of the total number
of spines (n = 30). This value is lower than that of
control synapses (22.4 ± 2.2%; n = 2158) or even
lower than that of MSBs formed by spines arising from different
dendrites (23%; n = 30). These characteristics of
duplicated synapses suggest therefore that one of the two spines is
still characterized by features of immaturity.
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Figure 6.
MSBs with two spines arising from the same
dendrite include one immature spine with a small PSD. A,
Illustration of a reconstructed simple synapse (left)
and an MSB with duplicated spines (right). Scale
bar, 1 µm. B, Left, Size of the PSD area
measured in a population of 31 reconstructed simple synapses
(gray column) and 30 MSBs with duplicated spines
(black column). Data are the mean ± SEM
(*p < 0.05). Right, Comparison of
the PSD area of the larger (gray column) and
smaller (black column) spines of the reconstructed MSBs
with duplicated spines. Note that the larger spine has a PSD area
comparable with that of a simple synapse (B, left;
gray column), whereas the PSD area of the smaller spine
is almost one-half that of a simple synapse (*p < 0.01).
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DISCUSSION |
The aim of the present study was to analyze the morphological
characteristics of the three types of synapses the proportion of which
has been found to change after LTP induction to investigate their
possible functional role with regard to LTP mechanisms. These analyses
point to three main observations. First, the transient increase in
synapses with perforated PSDs found to occur 30 min after LTP induction
essentially results from an increased proportion of synapses with
segmented, fully partitioned PSDs. Second, these synapses are
particular in that a much higher proportion of them contain coated
vesicles and exhibit large spinules associated with the PSD, two
features that strongly suggest a process of synaptic membrane
remodeling. Third, the MSBs formed by duplicated spines and found 1-2
hr after LTP induction consist of two spines, one that is
indistinguishable from simple synapses and one that is smaller and has
a smaller PSD and is thus likely to be immature and newly formed. Taken
together, these data support the hypothesis that LTP is associated with
an increased recycling of synaptic membranes and proteins and that this
process may underlie the formation of synapses with perforated PSDs and
eventually lead to the formation of a new, immature spine.
The characteristics of perforated synapses have been examined in
several previous studies using either single section, stereological analyses or three-dimensional reconstruction (Calverley and Jones, 1987, 1990; Geinisman et al., 1987, 1989, 1991, 1993; Harris and Stevens, 1989; Jones and Calverley, 1991; Harris et al., 1992; Itarat and Jones, 1992; Harris and Kater, 1994). Despite some variability, these studies showed that synapses with perforated PSDs
exhibit spine heads and PSDs considerably larger than those of simple
synapses, a result confirmed here. From a quantitative point of view,
our data in slice cultures perfectly coincide with those reported
previously by Harris et al. (1992) in acutely prepared hippocampal
slices. They indicate that synapses with perforated PSDs have a spine
head area and PSD area approximately three times greater than those of
simple synapses. From the morphology of the PSD, three different types
of synapses with perforated PSDs have been described and referred to as
synapses with fenestrated, horseshoe-shaped, or segmented PSDs
(Geinisman et al., 1989, 1991; Harris and Stevens, 1989; Harris
et al., 1992). The size of the spine head or PSD area is not different
between these three types of synapses; only the organization of the PSD
changes is different. Interestingly, we found that the increase in the
proportion of synapses with perforated PSDs observed 30 min after LTP
induction essentially results from an increase in synapses with
segmented, fully partitioned PSDs. This result is thus very consistent
with the observations made previously by Geinisman et al. (1991, 1993) who also proposed that this type of synapse could be implicated in LTP. These synapses are particularly interesting for several reasons. They have large PSDs and thus probably also more receptors (Desmond and Weinberg, 1998). They exhibit two distinct PSDs that could
face release sites with their own independent release probability. As
reported previously (Harris and Stevens, 1989), we also observed that distinct pools of synaptic vesicles accumulate in front of each
PSD. Thus an increased proportion of synapses with segmented PSDs could
be equivalent to increasing the number of release sites, a mechanism
that has been proposed to contribute to LTP (Geinisman et al., 1993;
Edwards, 1995).
A second interesting characteristic of the synapses with segmented PSDs
observed 30 min after LTP induction is that they very often contained
coated vesicles; the proportion of synapses with coated vesicles was
three to five times higher than that in any other type of synapse. This
result was obtained using three-dimensional reconstruction of a small
number of spines with perforated PSDs, but it is also consistent with
the single-section analyses of a large number of spine profiles (Fig.
1C). Also the distribution of coated vesicles within spines
suggests that they are involved in a transfer of membrane and proteins
between the spine apparatus and the synaptic membrane (Spacek and
Harris, 1997). Coated vesicles were often seen fusing with the synaptic
membrane, even at the level of the PSD. The frequent observation of
coated vesicles in synapses with segmented PSDs is thus likely to be
indicative of a process of synaptic membrane recycling and could even
represent a morphological correlate of the mechanisms that lead to the
incorporation of new receptors to the synaptic membrane. Strong recent
evidence indeed supports the idea that expression of new receptors at
the synapse plays an important role in LTP mechanisms (Lüscher et al., 2000). Coated vesicles could thus be part of the cargo system for
adding or retrieving receptors to or from the synaptic membrane. Immunolabeling for glutamate receptors has indeed been associated with
coated vesicles (Nusser et al., 1998). In addition to this, the
recycling process evidenced by the presence of coated vesicles could
contribute to the membrane expansion and increase in PSD size that
probably underlie the formation of synapses with perforated PSDs.
Another observation in agreement with this interpretation is the
presence of large spinules associated with the segmented PSDs of the
synapses observed 30 min after LTP induction. The role of these
spinules is also unclear, and their presence is not necessarily
associated with synapses with perforated PSDs (Sorra et al., 1998).
However, they may resemble in some ways the thin protrusions called
filopodia that are now believed to represent precursors of spine
formation (Fiala et al., 1998). Thus, spinules could also somehow
reflect the existence of a process of growth and remodeling of synaptic
membranes or an increased motility of spines (Matus, 2000). Together,
the fact that synapses with segmented PSDs occur transiently, that they
have a large spine head and large PSD, and that they contain more
coated vesicles and large spinules than do the other types of synapses
suggests that they represent an unstable synaptic structure undergoing a morphological remodeling. This, of course, raises the possibility that synapses with segmented PSDs could be further transformed, such
as, for example, into MSBs with duplicated spines. The characteristics of MSBs with duplicated spines and particularly the fact that they
include one spine with a PSD significantly smaller than that of a
simple synapse suggest that the smaller spine could be immature and
newly formed. Also, the striking temporal coincidence between the
decrease in the proportion of synapses with segmented PSDs and the
increase in the proportion of MSBs with duplicated spines suggests that
the two types of synapses could evolve from one to the other (Toni et
al., 1999).
It is tempting therefore to propose that segmented perforated synapses
represent a stage of membrane expansion produced as a result of LTP
induction and that this process is reversible and could evolve back
either to a simple spine or, in some cases, to an MSB with duplicated
spines. From a functional point of view, these morphological changes
could be directly related to the increase in synaptic strength, because
the process of membrane expansion that characterizes synapses with
segmented PSDs includes an enlargement of the PSD area and thus
probably also insertion of new receptors in the synaptic membrane
(Lüscher et al., 2000). Furthermore, the formation of segmented,
fully partitioned PSDs may result in the creation of two independent
release sites, with their own release probabilities (Geinisman et al.,
1993; Edwards, 1995). Finally this phenomenon could then be stabilized
by a transformation into MSBs with duplicated spines.
In summary, the quantitative data presented here are consistent with
the hypothesis that the changes in synaptic types observed after LTP
reflect a process of synaptic membrane remodeling and that coated
vesicles contribute to this process. Continuous monitoring of spine
shape changes during LTP will be important eventually to test more
directly this hypothesis.
| |
FOOTNOTES |
Received Jan. 31, 2001; revised May 21, 2001; accepted May 31, 2001.
This work was performed with the support of Grant 31-56852.99 from the
Swiss National Science Foundation. We thank M. Moosmayer for excellent
technical support, Fred Pillonel for photographic work, and D. Smithies, K. M. Harris, and J. C. Fiala for providing image
analysis software.
Correspondence should be addressed to Dr. D. Muller,
Neuropharmacologie, Centre Médical Universitaire, 1211 Geneva 4, Switzerland. E-mail: Dominique.Muller{at}medecine.unige.ch
| |
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TOP
ABSTRACT
INTRODUCTION
