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The Journal of Neuroscience, April 15, 2002, 22(8):3108-3116
Remodeling of Hippocampal Synaptic Networks by a Brief
Anoxia-Hypoglycemia
Pascal
Jourdain*,
Irina
Nikonenko*,
Stefano
Alberi, and
Dominique
Muller
Neuropharmacology, University Medical Center, 1211 Geneva 4, Switzerland
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ABSTRACT |
Cerebral ischemia is a major cause of brain dysfunction. Using a
model of delayed death induced by a brief, transient oxygen and glucose
deprivation, we studied here how this affected the structural
organization of hippocampal synaptic networks. We report that brief
anoxic-hypoglycemic episodes rapidly modified the structure of
synapses. This was characterized, at the electron microscopic level, by
a transient increase in the proportion of perforated synapses, followed
after 2 hr by an increase in images of multiple synapse boutons.
These changes were considerable because 10-20% of all synapses were
affected. This structural remodeling was correlated by three kinds of
modifications observed using two-photon confocal microscopy: the growth
of filopodia, occurring shortly (5-20 min) after anoxia-hypoglycemia,
enlargements of existing spines, and formation of new spines, both seen
mainly 20-60 min after the insult. All of these structural changes
were calcium and NMDA receptor dependent and thus reproduced, to a
larger scale, those associated with synaptic plasticity. Concomitantly
and related to the severity of anoxia-hypoglycemia, we could also
observe spine loss and images of spine, dendrite, or presynaptic
terminal swellings that evolved up to membrane disruption. These
changes were also calcium dependent and reduced by NMDA receptor
antagonists. Thus, short anoxic-hypoglycemic episodes, through NMDA
receptor activation and calcium influx, resulted in a profound
structural remodeling of synaptic networks, through growth, formation,
and elimination of spines and synapses.
Key words:
ischemia; synaptic plasticity; spines; morphology; synaptogenesis; anoxic LTP
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INTRODUCTION |
Reduced supply of oxygen and glucose
such as occurs during failure of blood circulation is a primary cause
of brain damage. A central mechanism responsible for the neuronal death
that occurs under these conditions is a massive release of glutamate
(Lee et al., 1999 ) by excitatory terminals and glial cells. This
process appears, at least partly, to be mediated by a reverse
functioning of cellular uptake systems (Rossi et al., 2000; Jabaudon et
al., 2001). Glutamate then activates excitatory amino acid receptors and probably leads to toxicity through excessive calcium influx and
calcium overload of neurons (Lee et al., 1999). When ischemia is
important, excitotoxicity results in immediate cell death. However, if
the insult is less severe, cell death is considerably reduced and is
usually delayed. Because recovery of function critically depends on
protection of these less affected regions, it is important to
understand the regulatory mechanisms activated in these areas by ischemia.
To this end, we examined here how production of a short, transient
period of anoxia-hypoglycemia affected synaptic connectivity within
the network. Much recent evidence has revealed that synapses express a
high degree of functional and structural plasticity (Luscher et al.,
2000). Modifications of synaptic efficacy, such as long-term
potentiation (LTP), are believed to underlie important functions of the
brain and have been reported in association with anoxia (anoxic LTP;
Crepel et al., 1993; Hammond et al., 1994; Hsu and Huang, 1997). Also,
recent studies suggest that this functional plasticity is associated
with a structural remodeling of synapses (Luscher et al., 2000).
Formation of filopodia (Maletic-Savatic et al., 1999), possible
precursors of spines (Ziv and Smith, 1996; Fiala et al., 1998),
formation of new spines, or new types of synapses (Engert and
Bonhoeffer, 1999; Toni et al., 1999) have been observed in association
with induction of LTP. Also, modulation of the intracellular calcium
concentration in spines appears to generate changes in the shape of
spines (Segal et al., 2000). Finally, some evidence suggests that
lasting interference with the function of excitatory amino acid
receptors affects spine number (Kirov and Harris, 1999; McKinney et
al., 1999). Thus, anoxia could represent a condition in which the
structural organization of synapses is modified. Previous work has
indeed shown that interruption of cerebral blood flow or anoxia results
in appearance of focal dendritic swellings (Hsu and Buzsaki, 1993; Park
et al., 1996; Werth et al., 1998) or disappearance of dendritic spines
(Hori and Carpenter, 1994; Park et al., 1996; Hasbani et al., 2001). There is also strong evidence that swelling and spine loss are caused
by activation of excitatory amino acid receptors (Park et al., 1996;
Smart and Halpain, 2000). Transient ischemic episodes were therefore
likely to affect synaptic networks. We examined here this issue using
electron and confocal microscopy and provide evidence that an intense
process of synaptic growth and remodeling, similar to that associated
with plasticity, occurs concomitantly with swelling, bursting, and
elimination of spines, thereby markedly affecting connectivity within
the network.
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MATERIALS AND METHODS |
Preparation of cultures and anoxia-hypoglycemia
experiments. Hippocampal organotypic slice cultures were prepared
from 7-d-old rats and maintained 10-15 d in culture as described
previously (Stoppini et al., 1991). Electrophysiology was performed in
an interface-type chamber, with slice cultures continuously perfused with a medium containing (in mM): 124 NaCl, 1.6 KCl, 2.5 CaCl2, 1.5 MgCl2,
24 NaHCO3, 1.2 KH2PO4, 10 glucose, and 2 ascorbic acid, pH 7.4 (temperature of 33°C). EPSPs were
recorded in the stratum radiatum of the CA1 area and evoked by
stimulation of a group of CA3 neurons. For two-photon confocal
microscopy experiments, slice cultures were placed on an infrapatch
setup (Luigs and Neuman, Ratingen, Germany) under continuous
perfusion (4 ml/min). In both cases, anoxia-hypoglycemia was produced
by replacing oxygen with N2 and switching to a
medium containing sucrose instead of glucose.
Two-photon confocal microscopy. CA1 pyramidal neurons were
patched using electrodes filled with a medium containing (in
mM): 130 K-gluconate, 4 NaCl, 5 EGTA, 20 HEPES, 1 CaCl2, 1 MgCl2, 0.2 Na3GTP, and 2 Na2ATP, pH
7.2-7.4 (290 mOsm, 0.04-0.06% sulforhodamine). Imaging of dendritic
spines was performed through a 40× water immersion objective using a
Bio-Rad (Hercules, CA) MRC1024 scan head and a Mira 900 laser (Coherent
Inc., Santa Clara, CA) set at 830 nm wavelength. Analyses were
performed by focusing on small dendritic segments (100-200 µm in
length), located on secondary and tertiary dendrites, and taking images
every 2-5 min over a 1-2 hr period. The size of the dendritic
segments under analysis remained constant throughout the experiments,
as indicated by measurements made between specific markers (spines and
branch points) easily identifiable on those segments. Filopodia were defined as thin, dynamic dendritic protrusions of over 2 µm in length. Spine enlargements were considered when the spine head diameter
increased by a factor of two.
Electron microscopy processing. At different times (15 min,
30 min, and 2 hr) after anoxia, slice cultures were fixed and processed
for electron microscopy as described previously (Toni et al., 2001).
Briefly, cultures were fixed overnight at 4°C in 3% glutaraldehyde,
rinsed in 0.1 M phosphate buffer (pH 7.4), and
post-fixed in a fresh solution of 1% osmium tetroxide
(OsO4) with 1.5% potassium chromium trisoxalate
(K3Cr(C2O4))3
(Aldrich, Milwaukee, WI), pH 9.5, for 2 hr. After a 5 min rinse in
distilled water adjusted to pH 9.5 with KOH, the samples were
dehydrated in ethanol and propylene oxide and embedded in Epon (Fluka,
Buchs, Switzerland). For light microscopy, sections were stained with methylene blue. For serial electron microscopy, ribbons of up to 60 sections were cut in the middle portion of the apical arborization of
CA1 pyramidal neurons (ultratome Ultracut-E; Leica, Deerfield, IL) and collected on single-slotted Formvar-coated grids.
Sections were stained for 20 min in 5% uranyl acetate and 30 sec in
lead citrate and analyzed on a Philips CM10 electron microscope at a
magnification of 8900× to 28,500×.
Morphological analyses. Synapses were defined by a clear
postsynaptic density facing at least three presynaptic vesicles, perforated synapses by the presence of a discontinuity in the postsynaptic density (Geinisman et al., 1987), and multiple synapse boutons (MSBs) by the presence of two independent dendritic spines contacting the same axon terminal (Sorra and Harris, 1993). The proportion of perforated synapses and MSBs was determined by taking random pictures of synapses in the middle one-third of the stratum radiatum and defining those corresponding to the above criteria.
For stereological analyses, five to six serial sections per culture
were examined, and synapses present in a volume of 31.3-71.2 µm3 were analyzed. The dissector
procedure was performed as described by Geinisman et al. (1996). For
three-dimensional analyses, 45 MSBs were reconstructed out of seven
hippocampal slice cultures. They were selected on the test section
based on the presence of two clearly recognizable spines contacting the
same terminal. The profiles were then photographed serially at a
magnification of at least 15,500× and three dimensionally
reconstructed using a software developed by J. C. Fiala and
K. M. Harris (Boston University, Boston, MA).
Data are presented as a mean ± SEM, with n indicating
the number of synapses or slice cultures analyzed, as indicated.
Statistical analyses were performed using the Student's t test.
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RESULTS |
Using a model of hippocampal organotypic slice cultures, we
investigated the effects of brief, transient episodes (2-10 min) of
anoxia-hypoglycemia on spine morphology. These short episodes produced
only minimal damage to the tissue and enhanced synaptic transmission.
In accordance with previous reports (Crepel et al., 1993; Hsu and
Huang, 1997), a lasting increase in the size of EPSPs was recorded in
the CA1 area of hippocampal slice cultures (Fig.
1A) by stimulation of a
group of CA3 neurons. This effect was NMDA receptor dependent, because
it could be prevented by treatment of slice cultures with 100 µM D-AP-5 (Fig.
1A). These short anoxic-hypoglycemic episodes did
not produce acute cell death. As shown on semithin sections, the
morphology of pyramidal neurons in the CA1 area was still preserved
2-6 hr after the anoxia-hypoglycemia (Fig. 1B).
Electron microscopic analyses further confirmed that there were no
obvious signs of cell death or gross morphological alterations at those
time points (Fig. 1C). Propidium iodide experiments revealed
only rare cases of labeled neurons up to 6 hr after the anoxia (Fig.
1D). However, staining increased significantly after 24-48 hr, thereby indicating a process of delayed cell death (Laake et
al., 1999) (Fig. 1E).
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Figure 1.
Enhancement of excitatory transmission and delayed
cell death induced by short anoxic-hypoglycemic episodes.
A, Changes in EPSP slope produced by a short
anoxia-hypoglycemia (black bar indicates
10 min) in hippocampal organotypic slice cultures in the absence
(filled circles; n = 5) or
presence (open circles; n = 4) of
the NMDA receptor antagonist D-AP-5 (100 µM).
Data are mean ± SEM. B, Semithin section of the
CA1 area of a slice culture fixed 6 hr after a short (10 min)
anoxia hypoglycemia. Scale bar, 100 µm. C, Electron
microscopic image of the stratum radiatum of a slice culture fixed 6 hr
after a short (10 min) anoxia-hypoglycemia. Scale bar, 1 µm.
D, E, Propidium iodide staining of slice
cultures showing only a limited, nonspecific cell death 6 hr
(D) after a short anoxia-hypoglycemia but a more
intense delayed cell death 48 hr after the injury
(E), Scale bar, 500 µm.
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A more careful analysis of the morphology of excitatory synapses in
these challenged cultures revealed, however, several changes taking
place during the first 2 hr after the anoxia-hypoglycemia. A first
observation was a marked increase in the proportion of synapses with
perforated postsynaptic densities in slice cultures fixed 15-30 min
after the anoxic episode (Fig.
2A,C).
This change was detectable by analyzing the entire population of spine
profiles. It was significant using both single-section analysis applied to a large number of spine profiles (30 ± 2.0 vs 14 ± 3.5%; 2557 synapses analyzed; 11 slice cultures; p < 0.01) and unbiased stereological methods applied to a smaller group of
serial sections (35.0 ± 3.7 vs 16.3 ± 0.7; 667 synapses
analyzed; 10 slice cultures; p < 0.01) (Fig.
2C). The increase in perforated synapses could be prevented
by treatment of slice cultures with 100 µM
D-AP-5 applied during the anoxic episode (Fig.
2C). Also, the change was only transient, because the
proportion of perforated synapses was back to control values after 2 hr. At that time, however, another modification could be observed,
consisting of an increase in the proportion of MSBs (Fig.
2B). The increase in MSBs was also highly significant and confirmed using both single-section analysis (15.0 ± 0.4 vs 5.0 ± 0.4%; 2383 synapses analyzed; 11 slice cultures;
p < 0.01) and unbiased stereological methods
(21.4 ± 1.4 vs 6.4 ± 0.7; 444 synapses analyzed; 11 slice
cultures; p < 0.01) (Fig. 2D).
Application of D-AP-5 (100 µM) during the anoxic episode also completely
prevented the increase in MSB proportion (Fig. 2D).
These changes thus reproduced those reported after LTP induction (Toni
et al., 1999).
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Figure 2.
Anoxia-hypoglycemia induced morphological
remodeling of spine synapses. A, Illustration of a
synapse with a perforated postsynaptic density (arrow).
Scale bar, 1 µm. B, Illustration of an MSB in
which three individual spines contact the same terminal.
C, Increase in the proportion of synapses with a
perforated PSD observed 15-30 min after a short (10 min)
anoxia-hypoglycemia. Data are mean ± SEM, obtained through
analysis of 10 slice cultures using unbiased stereological methods (667 synapses analyzed). Note that the change in perforated synapses is
prevented by treatment with the NMDA receptor antagonist
D-AP-5. D, Increase in the proportion of
MSBs observed 2 hr after a short anoxia-hypoglycemia. Data are
mean ± SEM, obtained through analysis of 11 slice cultures using
unbiased stereological methods (444 synapses analyzed).
E, Illustration of an MSB in which the two spines arise
from the same dendrite in a slice culture fixed 2 hr after
anoxia-hypoglycemia. Scale bar, 1 µm. F, Proportion
of MSBs with two spines arising from the same dendrite
(gray columns) or different dendrites
(black column) under control conditions and 2 hr after
anoxia-hypoglycemia.
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To investigate the origin of the spines in MSBs, we proceeded to
three-dimensional reconstruction of cases observed under control
conditions or in slice cultures fixed 2 hr after anoxia-hypoglycemia (Fig. 2E). Two of 30 MSBs reconstructed under control
conditions had spines originating from the same dendrite (6.7%) (Fig.
2F). For comparison, 4 of 15 MSBs reconstructed 2 hr
after anoxia exhibited adjacent spines contacting the same terminal
(26.7%). When expressed as a ratio of the proportion of MSBs observed
under the two conditions (Fig. 2F), the results
suggested that both MSBs with spines originating from the same dendrite
and MSBs with spines from different dendrites increased after
anoxia-hypoglycemia.
Because these observations suggested a process of synapse formation
similar to that reported after LTP induction, we investigated the
changes in spine morphology that could be detected using two-photon confocal microscopy. To visualize dendrites and spines, CA1 pyramidal neurons were patched under visual control using an electrode filled with sulforhodamine (0.04-0.06%). Within 10-15 min of whole-cell access, the entire dendritic arborization could be visualized, and the
changes in the morphology of spines could be analyzed repetitively
(every 2-5 min) before and after a short (2-10 min) anoxic-hypoglycemic episode. Several modifications of spines were identified. As illustrated in Figure 3, a
first change triggered by anoxia was the appearance of thin filopodia.
Under control conditions, filopodia were occasionally observed (1 of 11 experiments with 60-80 min of repetitive analysis). After an
anoxic-hypoglycemic episode, their occurrence markedly increased (17 of 24 experiments). When expressed as number of filopodia observed per
100 µm of dendritic length, the increase was statistically
significant (p < 0.05) (Fig. 3B).
Most filopodia initially appeared 5-20 min after the onset of anoxia
(Fig. 3C). Then, they grew in size and retracted, although
usually not completely, over the time course of the next hour. The
triggering of filopodia by anoxia-hypoglycemia was calcium dependent,
because both intracellular injection of 10 mM
BAPTA, a calcium chelator (n = 11), or reduction of the
extracellular calcium concentration (0.1 mM
Ca2+ and 10 mM
Mg2+; n = 10) prevented
their occurrence. Furthermore, the induction of filopodia was blocked
by treatment of slice cultures with the NMDA receptor antagonist
(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine
maleate (MK-801) (40 µM;
n = 12).
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Figure 3.
Anoxia-hypoglycemia induced formation of
filopodia. A, Time sequence illustrating the formation
and modifications of a filopodia induced after a short (10 min)
anoxia-hypoglycemia. Scale bar, 5 µm. B, Quantitative
analysis of the number of filopodia per 100 µm dendritic length
observed over periods of 80 min of observation under control conditions
(white column), after a short anoxia-hypoglycemia
(black column), after an anoxia-hypoglycemia but with
10 mM BAPTA in the pipette solution (gray
column), or a medium containing low calcium (0.1 mM), high magnesium (10 mM), or MK-801 (40 µM; dashed column). Data are mean ± SEM of 11-24 experiments. *p < 0.05. C, Time interval at which filopodia were seen to appear
after anoxia-hypoglycemia. Data are expressed as percentage of the
total number of events.
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A second type of changes induced by anoxia were modifications of the
morphology of dendritic spines. Figure
4A illustrates three
examples of enlargements of the spine head observed after an
anoxic-hypoglycemic episode. Similar cases were seen in 13 of 24 experiments, whereas only 1 of 11 experiments showed such changes under
control conditions. When expressed per unit length of dendrite, the
occurrence of these spine enlargements significantly increased
(p < 0.05) (Fig. 4C, black
columns). These changes appeared to be spine specific, because
neighboring spines were not affected. As illustrated in Figure 4,
C and D, they mainly occurred between 20 and 40 min after the anoxia, and they were prevented by intracellular injection of 10 mM BAPTA by reducing the
extracellular calcium concentration (0.1 mM
Ca2+ and 10 mM
Mg2+), as well as by blocking NMDA
receptors with 40 µM MK-801.
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Figure 4.
Anoxia-hypoglycemia induced enlargement of spines
and formation of new spines. A, Representative examples
of shape changes and enlargement of spine heads observed after an
anoxic-hypoglycemic episode (asterisks; 2 min anoxia).
Scale bars, 5 µm. B, Representative examples of
formation of a new spine after anoxia-hypoglycemia
(asterisks; 2 and 10 min anoxia). Scale bars, 5 µm.
C, Quantitative analysis of the number of spine
enlargements (black columns) and new spines
(dashed columns) observed per 100 µm dendritic length
under control conditions, after a short (2-10 min)
anoxia-hypoglycemia, after an anoxia-hypoglycemia but with either 10 mM BAPTA in the pipette solution or a medium containing low
calcium (0.1 mM), high magnesium (10 mM), or
with MK-801 (40 µM). Data are mean ± SEM of 10-24
experiments. *p < 0.05. C, Time
interval at which spine enlargements and new spines were seen to appear
after anoxia-hypoglycemia. Data are expressed as percentage of the
total number of events.
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Associated with the spine enlargements, we also observed frequently a
process of new spine formation. In 13 of 24 experiments, new spines
were seen to appear on the dendritic segment under analysis. Two
typical examples are illustrated in Figure 4B. Images from sections below and above the labeled dendrite were systematically recorded, allowing in this way to exclude that the new spines could
have been mistaken because initially present on a different plane of
section. Formation of new spines rarely occurred under control
conditions (1 of 11 experiments). When expressed per unit length of
dendrite, this mechanism was markedly enhanced by anoxia-hypoglycemia (p < 0.05) (Fig. 4C, dashed
columns). This effect was calcium dependent, as well as NMDA
receptor dependent. Curiously, BAPTA was less efficient in preventing
the phenomenon. These new spines usually started to be detectable
between 30 and 60 min after the anoxic episode and persisted until the
end of the experiment, up to 90 min in some cases.
Although all of the changes reported above suggested mechanisms of
synaptic growth, there were also modifications that lead to a decrease
of synaptic connectivity. Figure 5
illustrates a process referred to as dendritic swelling (Hsu and
Buzsaki, 1993; Park et al., 1996). This phenomenon was observed in 17 of 28 experiments, whereas it occurred only once in 11 experiments
under control conditions. Interestingly, Figure 5A reveals
that these dendritic swellings could evolve within 10-30 min into a
complete disruption of the dendritic structure through a bursting
phenomenon. Of 31 cases, 17 (55%) evolved to bursting within 30 min.
This phenomenon was seen with both single- and two-photon confocal
microscopy and occurred even without repetitive illumination of the
labeled cell. They were thus not produced by laser illumination. The
frequency of these dendritic swellings correlated well with the
severity (duration) of the anoxic-hypoglycemic episode (Fig.
5B). They were calcium dependent and could be prevented by
intracellular injection of BAPTA or by application of MK-801 (Fig.
5B). The swellings were seen mainly between 20 and 40 min
after the anoxia and tended to occur faster when the anoxia was more
prolonged. Associated with the swellings, there was usually a process
of spine loss. Figure 5A shows an example in which swellings
were accompanied by a smoothing of the dendrite and disappearance of spines. In 11 of 17 experiments in which swellings occurred, spine loss
was also observed, whereas spine loss without swellings was observed
only twice. Also, swellings were usually preceded by a few minutes of
spine loss (5.1 ± 1.3 min; n = 11). As for
swellings, spine loss was calcium dependent and reduced by blockade of
NMDA receptors. Interestingly, the structures from which these
swellings originated could be spines and dendrites but also axonal
varicosities. Figure 6 illustrates
confocal and electron microscopic images showing swellings at the level
of a postsynaptic spine (Fig.
6A,B), as well as at the level of a
DiI-labeled axonal varicosity (Fig. 6C) and a presynaptic
terminal (Fig. 6D). Thus, swelling is a mechanism
that concerned both presynaptic and postsynaptic structures.
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Figure 5.
Anoxia-hypoglycemia-induced swelling and spine
loss. A, Time sequence illustrating the formation of
dendritic and spine swellings after a short (10 min) anoxia. Note that
the formation of dendritic swellings is associated with a smoothing of
the dendrite and the disappearance of spines
(asterisks). These swellings progressively increased in
size over a time course of 10-30 min and finally burst out, disrupting
the cellular membrane (arrows). Scale bar, 10 µm.
B, Quantitative analysis of the number of swellings
(black columns) and spine loss (dashed
columns) observed per 100 µm dendritic length under control
conditions, after a short anoxia-hypoglycemia (2-5 min,
n = 12; 10 min, n = 16), or
after an anoxia-hypoglycemia but with either 10 mM BAPTA
in the pipette solution (n = 11) or a medium
containing low calcium (0.1 mM), high magnesium (10 mM; n = 10), or MK-801 (40 µM; n = 12). Data are mean ± SEM. *p < 0.05; **p < 0.01. C, Time interval at which swellings and spine loss were
seen to occur after anoxia-hypoglycemia. Data are expressed as
percentage of the total number of events.
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Figure 6.
Anoxia-hypoglycemia-induced swelling may occur at
the level of postsynaptic spines, as well as presynaptic terminals.
A, Illustration of a case of swelling initiated at the
level of a spine head. Scale bar, 5 µm. B, Electron
microscopic image showing a similar case in which the swollen structure
still receives a synaptic contact. C, Confocal
microscopy of a DiI-labeled axonal fiber illustrating swelling at the
level of one of the varicosities. Scale bar, 5 µm. D,
Electron microscopic image showing swelling at the level of a
presynaptic terminal. Scale bar, 1 µm.
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DISCUSSION |
The present study shows that brief periods of
anoxia-hypoglycemia, which produce minimal damage to the tissue and do
not result in acute cell death, are associated within the first 2 hr
with an intense remodeling of synaptic connections, both through a process of growth and formation of new spines and new synapses and
through spine loss, swelling, and destruction of existing synapses,
axon terminals, and dendrites. The two processes appeared to occur
independently because they were sometimes seen concomitantly on the
same dendritic branches. They were both calcium and NMDA receptor
dependent and could contribute to the early pathological events leading
to delayed death.
A particularly interesting aspect of the present results is that the
growth mechanisms reported here after anoxia show a strong analogy with
the changes that have been described in association with synaptic
plasticity and LTP induction (Luscher et al., 2000). In both cases,
changes in synaptic efficacy take place. This is well documented for
LTP, but this has also been reported for anoxia (Crepel et al., 1993;
Hammond et al., 1994; Hsu and Huang, 1997). As illustrated here, this
anoxic potentiation, similar to LTP, involves a long-lasting, NMDA
receptor-dependent increase of synaptic responses. Furthermore,
evidence suggests that, under both conditions, changes in the
expression of glutamate receptor subunits may take place (Shi et al.,
1999, 2001; Kobayashi and Millhorn, 2001; Optiz et al., 2001). The
present study shows in addition that there is also a remarkable analogy
in terms of the structural modifications of spines. Growth of
filopodia, changes of the ultrastructure of spines, and formation of
new spines and synapses occur under both conditions. Most interesting,
these modifications take place with a very similar time sequence. As
reported by Maletic-Savatic et al. (1999) after LTP, the first changes
that we observed here after anoxia-hypoglycemia were the growth of
filopodia. They occurred 5-20 min after the anoxic episode, and they
were calcium and NMDA receptor dependent. Furthermore, we could detect
formation of new spines 20-60 min after anoxia, similar to what has
been reported by Engert and Bonhoeffer (1999) after LTP. Associated
with this process of spine formation, we could also observe many cases
of spine enlargements that were not observed previously in confocal studies of LTP. Similar images, however, were seen recently using confocal microscopic analyses of spines labeled with GFP-tagged postsynaptic density 95 (PSD-95) (Marrs et al., 2001). This
study clearly indicated a highly dynamic nature of the synaptic
membrane and PSD that is likely to reflect some of the changes reported using electron microscopy (Toni et al., 2001). Both here after anoxia
and after LTP induction, electron microscopic analyses reveal major
differences in the ultrastructural characteristics of spines. In
particular, we found a marked but transient increase in the proportion
of spines with perforated PSDs ~30 min after anoxia, a phenomenon
that is followed 2 hr by an increase in the proportion of MSBs. This
sequence of changes thus fully corresponds to that reported after LTP
or in association with associative learning (Geinisman, 1993; Toni et
al., 1999; Geinisman et al., 2001). It is also interesting to note that
the synapses with perforated PSDs seen after LTP are usually
particularly large and exhibit features that suggest an important
remodeling of synaptic membranes (Toni et al., 2001). They could thus
correspond to the spine enlargements seen here with confocal microscopy
and the dynamic changes reported recently at the level of the PSD
(Marrs et al., 2001). Although the physiological and functional
significance of these morphological changes remains unclear, an obvious
interpretation is that they reflect a calcium- and NMDA
receptor-dependent process of growth and synapse formation. The
evidence suggesting an activation of proteases and the involvement of
actin filament or other elements of the cytoskeleton during anoxia
supports this interpretation (Friedman et al., 1998; Brana et al.,
1999; Smart and Halpain, 2000).
One important characteristic of this growth process in the case of
anoxia is that it is widespread and takes place at a very large scale.
The number of filopodia, of newly formed spines or of spines that
became enlarged, increased by almost an order of magnitude.
Interestingly, the frequency of these events did not seem to vary
between 2 and 10 min after anoxia-hypoglycemia, in contrast to the
number of swellings (Fig. 5). Overall, the number of spines that
significantly changed shape or were newly formed after anoxia
represented between 2 and 5% of all visible spines. At the
ultrastructural level, the number of synapses that showed changes at
the level of their PSD was even larger and represented 15-20% of all
spine profiles. Accordingly, the magnitude of these changes may be
expected to significantly alter excitability properties and information
processing within the network. In this respect, these data are
consistent with the observation of marked differences in the density of
spines on CA1 neurons after preparing acute slices, a process that
involves exposure to a short ischemia (Kirov et al., 1999). Thus,
through the release of glutamate, ischemia could activate mechanisms
analogous to those implicated in synaptic plasticity but to a larger
scale. Consistent with this, the changes reported here, like those
described after LTP, are calcium and NMDA receptor dependent. This
phenomenon could very well have deleterious effects by negatively
affecting the functioning of the network and, through increased
excitability, initiate intracellular cascades responsible for delayed
cell death (Obrenovitch and Urenjak, 1997).
An additional mechanism susceptible to lead to tissue damage is that
responsible for swelling and bursting of neuronal structures. The two
mechanisms are probably distinct and unrelated, although they sometimes
occurred concomitantly. Swelling and bursting were more pronounced with
more severe anoxia and they were calcium dependent (Abdel-Hamid et al.,
1997) but less NMDA receptor sensitive than growth mechanisms (Park et
al., 1996; Smart and Halpain, 2000). Also, swelling was probably
causally related to spine loss, because the two phenomena were usually
associated with swelling slightly preceding spine loss. Whereas spine
loss has been reported recently to be only transient (Hasbani et al.,
2001), swelling, and particularly bursting, appears to be a potentially
very damaging process, disrupting the membrane continuity. It is very
interesting in this respect that the phenomenon may in fact occur on
dendrites, spines, as well as presynaptic terminals, being therefore
not structure specific.
In conclusion, the present study shows that a short episode of
anoxia-hypoglycemia markedly affects the organization of synaptic networks: it modifies the structure and function of synapses and triggers the growth of new spines, while, at the same time, other spines, dendrites and axonal varicosities are being destroyed. These
changes are thus likely to contribute to the functional impairment that
characterizes tissue injured by a transient ischemia.
| |
FOOTNOTES |
Received Nov. 7, 2001; revised Jan. 28, 2002; accepted Feb. 5, 2002.
*
P.J. and I.N. contributed equally to this work.
This work was supported by Swiss National Science Foundation Grant
31-56852.99 and Program Scientific Co-operation between Eastern Europe
and Switzerland Grant 7UKPJ062401. We thank M. Moosmayer and L. Parisi for excellent technical support and Fred Pillonel for
photographic work.
Correspondence should be addressed to Prof. Dominique Muller,
Neuropharmacology, University Medical Center, 1211 Geneva 4, Switzerland. E-mail: dominique.muller{at}medecine.unige.ch.
| |
REFERENCES |
-
Abdel-Hamid KM,
Tymianski M
(1997)
Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins.
J Neurosci
17:3538-3553[Abstract/Free Full Text].
-
Brana C,
Benham CD,
Sundstrom LE
(1999)
Calpain activation and inhibition in organotypic rat hippocampal slice cultures deprived of oxygen and glucose.
Eur J Neurosci
11:2375-2384[Web of Science][Medline].
-
Crepel V,
Hammond C,
Chinestra P,
Diabira D,
Ben-Ari Y
(1993)
A selective LTP of NMDA receptor-mediated currents induced by anoxia in CA1 hippocampal neurons.
J Neurophysiol
70:2045-2055[Abstract/Free Full Text].
-
Engert F,
Bonhoeffer T
(1999)
Dendritic spine changes associated with hippocampal long-term synaptic plasticity.
Nature
399:66-70[Medline].
-
Fiala JC,
Feinberg M,
Popov V,
Harris KM
(1998)
Synaptogenesis via dendritic filopodia in developing hippocampal area CA1.
J Neurosci
18:8900-8911[Abstract/Free Full Text].
-
Friedman JE,
Chow EJ,
Haddad GG
(1998)
State of actin filaments is changed by anoxia in cultured rat neocortical neurons.
Neuroscience
82:421-427[Medline].
-
Geinisman Y
(1993)
Perforated axospinous synapses with multiple, completely partitioned transmission zones: probable structural intermediates in synaptic plasticity.
Hippocampus
3:417-434[Web of Science][Medline].
-
Geinisman Y,
Morrell F,
de Toledo-Morrell L
(1987)
Axospinous synapses with segmented postsynaptic densities: a morphologically distinct synaptic subtype contributing to the number of profiles of "perforated" synapses visualized in random sections.
Brain Res
423:179-188[Web of Science][Medline].
-
Geinisman Y,
Gundersen HJ,
van der Zee E,
West MJ
(1996)
Unbiased stereological estimation of the total number of synapses in a brain region.
J Neurocytol
25:805-819[Web of Science][Medline].
-
Geinisman Y,
Berry RW,
Disterhoft JF,
Power JM,
van der Zee EA
(2001)
Associative learning elicits the formation of multiple-synapse boutons.
J Neurosci
21:5568-5573[Abstract/Free Full Text].
-
Hammond C,
Crepel V,
Gozlan H,
Ben-Ari Y
(1994)
Anoxic LTP sheds light on the multiple facets of NMDA receptors.
Trends Neurosci
17:497-503[Web of Science][Medline].
-
Hasbani MJ,
Schlief ML,
Fisher DA,
Goldberg MP
(2001)
Dendritic spines lost during glutamate receptor activation reemerge at original sites of synaptic contact.
J Neurosci
21:2393-2403[Abstract/Free Full Text].
-
Hori N,
Carpenter DO
(1994)
Functional and morphological changes induced by transient in vivo ischemia.
Exp Neurol
129:279-289[Web of Science][Medline].
-
Hsu KS,
Huang CC
(1997)
Characterization of the anoxia-induced long-term synaptic potentiation in area CA1 of the rat hippocampus.
Br J Pharmacol
122:671-681[Web of Science][Medline].
-
Hsu M,
Buzsaki G
(1993)
Vulnerability of mossy fiber targets in the rat hippocampus to forebrain ischemia.
J Neurosci
13:3964-3979[Abstract].
-
Jabaudon D,
Scanziani M,
Gahwiler BH,
Gerber U
(2001)
Acute decrease in net glutamate uptake during energy deprivation.
Proc Natl Acad Sci USA
97:5610-5615[Abstract/Free Full Text].
-
Kirov SA,
Harris KM
(1999)
Dendrites are more spiny on mature hippocampal neurons when synapses are inactivated.
Nat Neurosci
2:878-883[Web of Science][Medline].
-
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[Abstract/Free Full Text].
-
Kobayashi S,
Millhorn DE
(2001)
Regulation of N-methyl-D-aspartate receptor expression and N-methyl-D-aspartate-induced cellular response during chronic hypoxia in differentiated rat PC12 cells.
Neuroscience
101:1153-1162.
-
Laake JH,
Haug FM,
Wieloch T,
Ottersen OP
(1999)
A simple in vitro model of ischemia based on hippocampal slice cultures and propidium iodide fluorescence.
Brain Res Brain Res Protoc
4:173-184[Medline].
-
Lee JM,
Zipfel GJ,
Choi DW
(1999)
The changing landscape of ischaemic brain injury mechanisms.
Nature
399:A7-A14[Medline].
-
Luscher C,
Nicoll RA,
Malenka RC,
Muller D
(2000)
Synaptic plasticity and dynamic modulation of the postsynaptic membrane.
Nat Neurosci
3:545-550[Web of Science][Medline].
-
Maletic-Savatic M,
Malinow R,
Svoboda K
(1999)
Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity.
Science
283:1923-1927[Abstract/Free Full Text].
-
Marrs GS,
Green SH,
Dailey ME
(2001)
Rapid formation and remodeling of postsynaptic densities in developing dendrites.
Nat Neurosci
4:1006-1013[Web of Science][Medline].
-
McKinney RA,
Capogna M,
Durr R,
Gahwiler BH,
Thompson SM
(1999)
Miniature synaptic events maintain dendritic spines via AMPA receptor activation.
Nat Neurosci
2:44-49[Web of Science][Medline].
-
Obrenovitch TP,
Urenjak J
(1997)
Altered glutamatergic transmission in neurological disorders: from high extracellular glutamate to excessive synaptic efficacy.
Prog Neurobiol
51:39-87[Web of Science][Medline].
-
Optiz T,
Grooms SY,
Bennett MV,
Zukin RS
(2001)
Remodeling of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia.
Proc Natl Acad Sci USA
97:13360-13365[Abstract/Free Full Text].
-
Park JS,
Bateman MC,
Goldberg MP
(1996)
Rapid alterations in dendrite morphology during sublethal hypoxia or glutamate receptor activation.
Neurobiol Dis
3:215-227[Web of Science][Medline].
-
Rossi DJ,
Oshima T,
Attwell D
(2000)
Glutamate release in severe brain ischaemia is mainly by reversed uptake.
Nature
403:316-321[Medline].
-
Segal I,
Korkotian I,
Murphy DD
(2000)
Dendritic spine formation and pruning: common cellular mechanisms?
Trends Neurosci
23:53-57[Web of Science][Medline].
-
Shi S,
Hayashi Y,
Esteban JA,
Malinow R
(2001)
Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons.
Cell
105:331-343[Web of Science][Medline].
-
Shi SH,
Hayashi Y,
Petralia RS,
Zaman SH,
Wenthold RJ,
Svoboda K,
Malinow R
(1999)
Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation.
Science
284:1811-1816[Abstract/Free Full Text].
-
Smart FM,
Halpain S
(2000)
Regulation of dendritic spine stability.
Hippocampus
10:542-554[Medline].
-
Sorra KE,
Harris KM
(1993)
Occurrence and three-dimensional structure of multiple synapses between individual radiatum axons and their target pyramidal cells in hippocampal area CA1.
J Neurosci
13:3736-3748[Abstract].
-
Stoppini L,
Buchs P-A,
Muller D
(1991)
A simple method for organotypic cultures of nervous tissue.
J Neurosci Methods
37:173-182[Web of Science][Medline].
-
Toni N,
Buchs PA,
Nikonenko I,
Bron CR,
Muller D
(1999)
LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite.
Nature
402:421-425[Medline].
-
Toni N,
Buchs PA,
Nikonenko I,
Povilaitite P,
Parisi L,
Muller D
(2001)
Remodeling of synaptic membranes after induction of long-term potentiation.
J Neurosci
21:6245-6251[Abstract/Free Full Text].
-
Werth JL,
Park TS,
Silbergeld DL,
Rothman SM
(1998)
Excitotoxic swelling occurs in oxygen and glucose deprived human cortical slices.
Brain Res
782:248-254[Medline].
-
Ziv NE,
Smith SJ
(1996)
Evidence for a role of dendritic filopodia in synaptogenesis and spine formation.
Neuron
17:91-102[Web of Science][Medline].
Copyright © 2002 Society for Neuroscience 0270-6474/02/2283108-09$05.00/0
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TOP
ABSTRACT
INTRODUCTION
