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The Journal of Neuroscience, March 15, 1999, 19(6):1988-1997
Modification of Postsynaptic Densities after Transient Cerebral
Ischemia: A Quantitative and Three-Dimensional Ultrastructural
Study
Maryann E.
Martone1,
Ying Z.
Jones1,
Steve J.
Young1,
Mark H.
Ellisman1,
Justin A.
Zivin1, and
Bing-Ren
Hu2
1 Department of Neurosciences, National Center for
Microscopy and Imaging Research at San Diego, University of California,
San Diego, La Jolla, California 92093, and 2 Center for the
Study of Neurological Disease, Queen's Medical Center, Honolulu,
Hawaii 92813
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ABSTRACT |
Abnormal synaptic transmission has been hypothesized to be a cause
of neuronal death resulting from transient ischemia, although the
mechanisms are not fully understood. Here, we present evidence that
synapses are markedly modified in the hippocampus after transient cerebral ischemia. Using both conventional and high-voltage electron microscopy, we performed two- and three-dimensional analyses of synapses selectively stained with ethanolic phosphotungstic acid in the
hippocampus of rats subjected to 15 min of ischemia followed by various
periods of reperfusion. Postsynaptic densities (PSDs) from both area
CA1 and the dentate gyrus were thicker and fluffier in postischemic
hippocampus than in controls. Three-dimensional reconstructions of
selectively stained PSDs created using electron tomography indicated
that postsynaptic densities became more irregular and loosely
configured in postischemic brains compared with those in controls. A
quantitative study based on thin sections of the time course of PSD
modification indicated that the increase in thickness was both greater
and more long-lived in area CA1 than in dentate gyrus. Whereas the
magnitude of morphological change in dentate gyrus peaked at 4 hr of
reperfusion (140% of control values) and declined thereafter, changes
in area CA1 persisted and increased at 24 hr of reperfusion (191% of
control values). We hypothesize that the degenerative ultrastructural
alteration of PSDs may produce a toxic signal such as a greater calcium
influx, which is integrated from the thousands of excitatory synapses onto dendrites, and is propagated to the neuronal somata where it
causes or contributes to neuronal damage during the postischemic phase.
Key words:
postsynaptic density; hippocampus; cerebral ischemia; electron microscope; tomography; three-dimensional reconstruction; neuronal death
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INTRODUCTION |
Transient cerebral ischemia
selectively damages CA1 pyramidal neurons ~3 d after ischemia but
leaves CA3 and dentate gyrus neurons largely intact. During the 72 hr
preceding cell death, the neurons destined to die have a relatively
normal appearance when examined by light microscopy. However, some
ultrastructural abnormalities have been reported in postischemic
neurons, including disaggregation of polyribosomes and deposition of
dark substances (Kirino, 1982 ; Pulsinelli et al., 1982; Kirino et al.,
1984; Petito and Pulsinelli, 1984; Smith et al., 1984). Alterations of
synaptic transmission have also been reported during the postischemic
phase. A mild ischemic episode potentiates synaptic transmission
(Andiné et al., 1992; Miyazaki et al., 1993, 1994; Hammond et
al., 1994), whereas a more severe ischemia episode suppresses synaptic
neurotransmission and results in neurobehavioural deficits during the
postischemic phase (Volpe et al., 1984; Auer et al., 1989; Furukawa et
al., 1990; Xu, 1995; Dalkara et al., 1996).
Several lines of evidence suggest that excitotoxicity through
overactivation of glutamate receptors on synapses may be involved in
ischemic neuronal death. First, neurons can be killed by exposure to
glutamate (Choi, 1988). Second, extracellular glutamate is significantly increased after depolarization during ischemia
(Benveniste et al., 1984). Third, glutamate receptor blockade has been
shown to be neuroprotective (for review, see Lipton, 1994). However, the increase in extracellular glutamate present at the onset of ischemia rapidly returns to control values, whereas neuronal damage occurs at ~72 hr after an ischemic episode (Kirino, 1982; Pulsinelli et al., 1982). This paradox suggests the possibility that signals triggered during ischemia may propagate progressively from synapses to
the cell body to instigate delayed neuronal death during the postischemic phase.
In biochemical analyses of postsynaptic densities (PSDs), we recently
reported a dramatic accumulation of
N-ethylmaleimide-sensitive fusion protein, heat shock
cognate protein 70, calcium/calmodulin-dependent protein kinase II, and
protein kinase C in PSDs from the cerebral cortex of rats subjected to
15 min of transient ischemia followed by 4 hr of reperfusion (Hu et
al., 1995, 1998). These biochemical changes were accompanied by robust
morphological alterations in PSD structure. In this study, we conducted
a detailed examination of synaptic structure in the postischemic
hippocampus using quantitative and three-dimensional reconstruction
methods. Based on our earlier study in cortex, we wished to determine
(1) whether similar morphological alterations were observed in
hippocampal synapses after ischemia; (2) how PSD modification developed
over time; and (3) whether any differences existed in PSD alterations
between ischemic vulnerable neurons in area CA1 compared with the
relatively more resistant neurons in dentate gyrus. We demonstrate that
PSD ultrastructure in both two dimensional (2D) images and three
dimensional (3D) reconstructions is highly modified shortly after a
brief ischemic episode. The modification is severe and long-lasting in
hippocampal CA1 neurons but transient and less marked in dentate gyrus
granule cells. These long-term synaptic ultrastructural alterations may be involved in ischemia-induced alterations in synaptic transmission and may also be early events in the initiation of neuronal damage during the postischemic phase.
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MATERIALS AND METHODS |
Ischemia model. All experimental procedures were
approved by the Subcommittee on Animal Studies of the Veterans Affairs
Medical Center (San Diego, CA). Male Wistar rats (250-300 gm) were
fasted overnight. Anesthesia was induced with 3% halothane followed by maintenance with 1-2% halothane in an oxygen/nitrous oxide (30/70%) gas mixture. Catheters were inserted into the external jugular vein,
tail artery, and tail vein to allow blood sampling, arterial blood
pressure recording, and drug infusion. Both common carotid arteries
were exposed and encircled by loose ligatures. Fifteen minutes before
ischemia induction and 15 min after ischemia, blood gases were measured
and adjusted to PaO2 >90 mmHg and
PaCO2 35-45 mmHg, pH 7.35-7.45, by adjusting
tidal volume of the respirator. Bipolar EEG was recorded every 5-10
min before ischemia, continuously during the ischemic insult, and every
5 min after ischemia until the rat recovered from the anesthesia. At
the beginning of a 30 min steady-state period before induction of
ischemia, the inspired halothane concentration was decreased to 0.5%,
and 150 IU/kg heparin was administered intravenously. Blood was
withdrawn via the jugular catheter to produce a mean arterial blood
pressure of 50 mmHg, and ischemia was induced by clamping both carotid
arteries. Blood pressure was maintained at 50 mmHg during the ischemic
period by withdrawing or infusing blood through the jugular catheter. At the end of the ischemic period, the clamps were removed, and the
blood was reinfused through the jugular catheter, followed by 0.5 ml of
0.6 M sodium bicarbonate. In all experiments, temperature was maintained at 37°C before, during, and after ischemia (15 min of
reperfusion). Halothane was discontinued at the end of ischemia, and
all wounds were sutured. At 4 or 24 hr, 3 d, or 1 week after the
ischemic episode, the animals were reanesthetized, tracheotomized, and
artificially ventilated. For electron microscopic studies, the brains
were perfused with ice-cold 2% paraformaldehyde and 2.5%
glutaraldehyde in 0.1 M cacodylate buffer. Sham-operated control rats were subjected to the same surgical procedures but without
induction of ischemia.
Electron microscopic studies. Tissue sections from
experimental and control animals were stained either by 1% ethanolic
phosphotungstic acid (E-PTA) (Bloom and Aghajanian, 1966, 1968) or by
the conventional osmium-uranium-lead method. Briefly, coronal brain
sections were cut at a thickness of 200 µm with a Vibratome through
the level of the dorsal hippocampus and post-fixed for 1 hr with 4%
glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. For
conventional osmium-uranium-lead staining, sections were post-fixed
for 2 hr in 1% osmium tetroxide in 0.1 M cacodylate
buffer, rinsed in distilled water, and stained with 1% aqueous uranyl
acetate overnight. The tissue sections were then dehydrated in an
ascending series of ethanol to 100%, followed by dry acetone, and
embedded in Durupan ACM. Thin sections were counterstained with lead
citrate before examination in the electron microscope. For E-PTA
staining, sections were dehydrated in an ascending series of ethanol to
100% and stained for 1 hr with 1% PTA prepared by dissolving 0.1 gm
of PTA in 10 ml of 100% ethanol and adding four drops of 95%
ethanol. Sections were then embedded in Durcupan ACM.
EM tomography. E-PTA-stained thick sections (1 µm) were
examined by a JEOL 4000EX intermediate high-voltage electron microscope at an accelerating voltage of 400 keV. Before examination in the electron microscope, 10 nm colloidal gold particles were applied to the
section surface to serve as fiduciary cues for subsequent alignment of
images. Data for tomographic reconstruction was acquired using the
single-axis tilt method. A series of images was obtained as the
specimen was tilted in equal angular increments, about an axis
perpendicular to the optical axis of the microscope. Each of these
images represented a projection of densities in the specimen from a
particular angle of view, and the volume distribution was solved using
the R-weighted back-projection method. Images were typically obtained
over a range of ±60° at 2° increments using film. The micrographs
were digitized using a high-resolution 1000 × 1000 dpi CCD camera
(Photometrics Inc.). The digitized images were flat-fielded to correct
for any unevenness in the illumination, transformed appropriately to
achieve a measure of the projected density, and normalized. The tilt
axis was determined, and the images were rotationally and
translationally aligned using fiducial alignment and correlational
techniques implemented in the SUPRIM image-processing library
(Schroeter and Bretaudiere, 1996). The volume was reconstructed from
the tilt series using R-weighted back-projection implemented in SUPRIM.
Resulting volumes were viewed and manipulated using the program ANALYZE
(Biomedical Imaging Resource, Mayo Foundation, Rochester, MN).
Additional details of the tomographic method used can be found in
articles by Soto et al. (1994) and Perkins et al. (1997).
Collection of data. For both 2D and 3D analyses,
observations were obtained from a strip of tissue crossing the apical
dendritic region of CA1 and both limbs of the dentate gyrus. PSDs were
sampled from the stratum radiatum of area CA1, at a distance of ~200
µm above the pyramidal cell layer and from the upper limb of the dentate gyrus, within 70 µm of the granule cell layer. Distances were
measured by counting grid squares in 300 mesh grids (~73 µm/grid square).
Quantitative analyses of thin sections. Specimens were
selected for quantitative analysis based on the quality of E-PTA
staining and the degree of ultrastructural preservation, as determined from conventionally stained material from the same animals. Samples were analyzed from controls (n = 3) and from
postischemic brains at 4 hr of reperfusion (n = 4) and
24 hr of reperfusion (n = 3). Additional micrographs
were obtained from the dentate gyrus from postischemic brains after 1 week of reperfusion (n = 2). Tissue sections were cut
at a thickness of 100 nm and examined and photographed at 80 keV at a
magnification of 8300× with a JEOL 100CX electron microscope. For each
animal, five micrographs were obtained from both CA1 and the dentate
gyrus as described above. Each negative was digitized into a MacIntosh
computer. Using NIH Image 1.6, PSDs were first manually outlined, and
then the maximal thickness, minimum thickness, length, and total area
of each PSD were determined, as illustrated in Figure 3. All PSDs in
which the postsynaptic density, intracleft line, and presynaptic grid
(see Fig. 3) were clearly visible were chosen for analysis. PSDs were
analyzed for both dentate gyrus and CA1 in control and 4 and 24 hr
reperfusion conditions but only in dentate gyrus for 1 week of
reperfusion. CA1 was not analyzed at this time point, because most CA1
neurons had degenerated by 1 week after ischemia. No attempt was made to perform the analysis blindly, because the morphological changes were
so dramatic that the postischemic tissue was readily distinguishable from the control. The selection criterion resulted in analysis of
between 45 and 70 PSDs per animal for each region. The data for the
sham and 4 and 24 hr of reperfusion were analyzed using a 3 (control
and 4 and 24 hr) × 2 (CA1 vs dentate gyrus) ANOVA. Post hoc
analyses were conducted using Dunnett's test for comparison of the
reperfusion conditions with the control condition. A separate ANOVA was
conducted comparing the four conditions (sham, 4 and 24 hr, 3 d,
and 1 week of reperfusion) analyzed for the dentate gyrus. The
incidence of perforated PSDs was also determined from digitized images.
A PSD was classified as perforated if it contained a discontinuity (see
Fig. 7, 2D slices).
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RESULTS |
As has been reported previously for this ischemia model, 15 min of
transient cerebral ischemia followed by up to 24 hr of reperfusion did
not cause obvious abnormal morphology in hippocampal sections relative
to sham-operated controls, as determined by light microscopic analysis
of cresyl violet-stained sections (Fig. 1, Sham, 24h). However, a
frank neuronal degeneration occurred selectively in CA1 pyramidal
neurons ~3 d after the ischemic episode (Fig. 1, 3d),
whereas neurons in CA3 and the dentate gyrus remained largely intact
after the ischemic insult (Smith et al., 1984).
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Figure 1.
Low-power light micrographs of dorsal hippocampus
of control rats (Sham) and rats subjected to 15 min of
ischemia followed by 24 hr (24h) and 3 d
(3d) of reperfusion. Vibratome sections (50 µm) were
stained with cresyl fast violet. The majority of CA1 neurons at 24 hr
of reperfusion look normal. A frank neuronal death was apparent after
3 d of reperfusion (3d). so, Stratum
oriens; pcl, pyramidal cell layer; sr,
stratum radiatum.
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At the electron microscopic level, we found most neurons in the
conventionally stained material (osmium-uranium-lead) to possess relatively normal cell membranes and normal nuclei at 4 and 24 hr of
reperfusion, consistent with previous ultrastructural studies (Kirino
et al., 1984; Petito and Pulsinelli, 1984; Deshpande et al., 1992;
Rafols et al., 1995). No obvious breaks in the membranes of dendritic
shafts or spines were seen, and presynaptic terminals, presynaptic
vesicles, and PSDs were intact in the dendritic region of hippocampal
CA1 (Fig. 2, Sham, 4h, 24h) as
well as dentate gyrus (data not shown). Some PSDs from the postischemic
brain appeared slightly irregular in profile compared with those in controls.
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Figure 2.
Electron micrographs of
osmium-uranium-lead-stained synapses in the dorsal hippocampal CA1
areas from sham-operated rats (Sham) and rats subjected
to 15 min of ischemia followed by 4 hr (4h) and 24 hr
(24h) of reperfusion. The synapses
(arrows) were intact, and no obvious structural
alterations were seen in these osmium-uranium-lead-stained synapses
after ischemia. Scale bar, 0.5 µm.
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E-PTA resulted in selective staining of the postsynaptic density, the
presynaptic grid, and some material in the synaptic cleft. With the
exception of nuclear chromatin, other structures were not stained
(Bloom and Aghajanian, 1966, 1968). In contrast to the conventionally
stained material, robust alterations were apparent in postischemic
tissue stained with E-PTA (Fig. 3). After transient ischemia, postischemic PSDs were clearly thicker and fluffier
than those in controls (Fig. 3). Most control PSDs were more compact,
as evidenced by their generally greater electron density relative to
postischemic PSDs (Fig. 3). These changes were also present in the
dentate gyrus (data not shown). Both simple (top row) and
perforated (bottom row) PSDs were clearly more fluffy and
curved compared with control PSDs (Fig. 3). There was also a general
increase in the amount of E-PTA-stained material in the postischemic
brains compared with controls. This increase did not appear to be
attributable to an increase in the number of synapses in the
postischemic brain (see below) but appeared to represent flocculent
material that was loosely associated with or near to PSDs.
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Figure 3.
High-magnification electron micrographs of
E-PTA-stained synapses in hippocampal CA1 regions from sham-operated
rats (Sham) and rats subjected to 15 min of ischemia
followed by 24 hr (24h) of reperfusion. Examples of
simple PSDs are shown in the top row, and examples of
perforated PSDs are shown in the bottom row. In
comparison with the control synapses (Sham),
postischemic PSDs were generally curved and surrounded by diffuse
E-PTA-stained materials both in the single PSDs and perforated PSDs.
The inset in the top right panel
(large arrow) illustrates how PSD parameters were
measured (Table 1). The area of the PSD was calculated by manually
outlining the PSD (solid white line), whereas the
straight dotted lines indicate the minimum (small
arrow) and maximum thickness. Scale bar, 0.1 µm.
Pre-, Presynaptic specialization; Cleft,
synaptic cleft.
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To investigate these interesting alterations further, we conducted a
series of quantitative analyses of PSDs at different time points of
reperfusion. Because the most obvious change in PSD morphology was the
apparent change in thickness, we measured the maximum and minimum
thickness for individual PSDs from each animal as well as area and
length, as illustrated in Figure 3. Results of the analyses are
summarized in Table 1. A 2 × 3 ANOVA indicated a significant interaction between hippocampal area
[dentate gyrus (DG) vs CA1] and condition (sham vs 4 vs 24 hr) for
maximum thickness (F(2,14) = 4.28;
p < 0.05) and minimum thickness
(F(2,14) = 3.86; p < 0.05).
Post hoc comparisons using Dunnett's test indicated that
both the minimum and maximum thicknesses of postischemic PSDs were
greater than those of sham-operated controls in area CA1 at both 4 and
24 hr (p < 0.01, Dunnett's test) and in DG at 4 hr (p < 0.05) and 24 hr
(p < 0.05). The ANOVA also showed a significant
effect for area (p < 0.01), but the overall
interaction was not significant. However, Dunnett's test indicated a
significant increase in area relative to controls in CA1 at 4 hr
(p < 0.05) and 24 hr (p < 0.01) and in the dentate gyrus at 4 hr (p < 0.01) but not at 24 hr (p = 0.06). No
significant differences in length of PSDs were uncovered. No
significant differences compared with controls were observed in the
dentate gyrus for any measure at 1 week of reperfusion (Table 1).
The significant interaction between condition (sham vs 4 vs 24 hr) and
region for measures of PSD thickness indicates that the alteration in
PSD morphology was not equivalent between the dentate gyrus and area
CA1 in the three conditions. The magnitude of morphological alteration
in the dentate gyrus peaked at 4 hr of reperfusion but declined
thereafter, whereas the changes in area CA1 persisted and slightly
increased before cell death. This interaction is shown in graphic form
in Figure 4 for maximum thickness. In
comparison with controls, the increase in thickness in CA1 was 177% at
4 hr and 191% at 24 hr of reperfusion, whereas the increase in DG was
140% at 4 hr, 131% at 24 hr, and 118% at 7 d of reperfusion
(Fig. 4, Table 1). After 3 d of reperfusion, >95% of CA1 neurons
are degenerated (Fig. 1), and so no measurements were performed at
7 d of reperfusion for CA1. As can be seen in the frequency
histograms of PSD thickness in area CA1 and dentate gyrus shown in
Figure 5, nearly all synapses in CA1
showed increased thickness, whereas in the dentate gyrus, synapses on
the whole tended to be thicker, especially at early time points, but
there was considerable overlap in the populations from reperfused
versus control brain.
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Figure 4.
Maximum thickness of PSDs from control
(C) and postischemic brains after 4 hr
(4h), 24 hr (24h), and 1 week
(1w) of reperfusion, plotted as a percentage of
controls. Only dentate gyrus was sampled after 1 week of reperfusion,
because both pyramidal cell layer and neuropil of the CA1 region have
degenerated by this time point. The increase in thickness was greater
and more persistent in CA1 compared with dentate gyrus.
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Figure 5.
Frequency histogram of PSD thickness in
hippocampal CA1 (top panel) and dentate gyrus
(bottom panel) in control (sham)
and postischemic brains subjected to 4 hr (4hr), 24 hr
(24hr), or 1 week (1wk) of reperfusion.
The maximal thickness of most CA1 PSDs in controls was ~50 nm,
whereas the thickness of most postischemic PSDs ranged from ~80 nm at
4 hr to 100 nm at 24 hr of reperfusion. The peak of the curves for the
two postischemic conditions was shifted to the right compared with the
controls (top panel). In contrast, in the dentate
gyrus, considerable overlap was observed in the range of PSD
thicknesses from all conditions. The peak of the curve was shifted to
the right at 4 hr of reperfusion, although it occurred within the range
of control PSD thicknesses.
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The percentage of perforated PSDs as counted in randomly selected
sections showed a significant increase compared with controls in CA1
(p < 0.05, Dunnett's test) and an
insignificant increase in the dentate gyrus after transient cerebral
ischemia (Table 2).
To characterize further the structural alterations observed in thin
sections of E-PTA-stained material, we performed 3D reconstructions of
E-PTA-stained PSDs contained in 1-µm-thick sections using electron tomography. The use of selective staining with E-PTA facilitates high-voltage 3D electron microscopy examination of synaptic densities, because it allows observation of PSD structure from any view without interference by other subcellular structures (Igarashi et al., 1988).
We produced tomographic volume reconstructions of PSDs and presynaptic
grids in thick sections from CA1 and dentate gyrus from control brains
and postischemic brains at 4 and 24 hr of reperfusion. An example of a
tomographic reconstruction of PSDs from control brain is shown in
Figure 6. Each volume comprised ~2
µm3 of tissue and contained 5-20 synapses.
Approximately 30 synapses were analyzed for each condition in area CA1.
The density of synapses ranged from 2-9
synapses/µm3, and there was no significant
difference in synaptic density between control and postischemic brains.
The PSD volume was resectioned to produce 2D views similar to those in
individual thin sections, to correlate 2D and 3D data from the same PSD
(Fig. 6, bottom panel, 2D slice).
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Figure 6.
Tomographic volume reconstruction of E-PTA-stained
PSDs in a 1-µm-thick section from stratum radiatum of control CA1.
Only a portion of the volume is shown here. The contrast is reversed so
that stained PSDs appear bright relative to unstained structures. The
3D structure of PSDs in the volume can be easily visualized. Each PSD
in the volume can be viewed from any angle, as shown in the
bottom panel for the PSD indicated by an
arrow in the top panel. A 2D slice
through the volume at the position of a perforation
(arrow) is shown on the right, with the
contrast reversed again so that the stained PSD appears
black on white to facilitate comparison
with thin-section images in other figures. Scale bar, 0.5 µm.
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The tomographic volumes extended and supported observations made from
individual thin sections (Figs. 6-8). Resectioning of tomographic
volumes confirmed that the increase in PSD thickness observed in
individual thin sections was not attributable to a sectioning artifact
(compare 2D slices in Figs. 6, 8) and also showed that the increase in
thickness occurred in nearly every synapse visualized within the
postischemic volume in area CA1, suggesting that most E-PTA-stained
synapses were affected.
Three-dimensional reconstructions of PSDs revealed several additional
aspects of PSD alteration in postischemic brain not apparent from the
2D images, as seen in Figure 7. Each PSD
in Figure 7 was rotated to obtain an en face view of the entire PSD. Whereas both the 2D and 3D images indicated an increase in PSD thickness, the 3D reconstructions also showed that PSDs from
postischemic brain were more loosely configured, irregular in shape,
and fragmented than those from control brains (Fig. 7). PSDs from
control sections were thin, condensed, and disk-like structures,
whereas the PSDs from the postischemic hippocampus were less compact,
with ragged edges. This loosening of PSD structure was most apparent in
area CA1 at 24 hr of reperfusion. At 4 hr of reperfusion, PSD structure was intermediate between those of the control and 24 hr of reperfusion (Fig. 7). Three-dimensional views of PSDs also demonstrated a large
variation in PSD size within a given animal in both CA1 and dentate
gyrus. Nearly every large synapse in both control and 24 hr reperfusion
brains showed some sort of perforation; however, the perforations were
more clearly defined in control brains because of their more compact
structure. Perforations were not always in the center of the PSD. Small
PSDs generally were not perforated or contained one small perforation
but still exhibited the same loosening of PSD structure present in
large synapses.
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Figure 7.
Three-dimensional images of E-PTA-stained PSDs
from tomographic volumes of control (Sham), 4 hr
(4hr) and 24 hr (24hr) of reperfusion in
CA1. Six different PSDs are shown for each condition. Each PSD was
rotated individually to appear en face. The control PSDs were compact,
disk-like structures with generally round edges and discrete
perforations, whereas the postischemic PSDs were more loosely
configured structures with irregular edges and less well defined
perforations. PSDs at 4 hr of reperfusion appeared to be similar to
those in control brains. The hatching visible in some
PSDs, e.g., top left PSD in 24 hr, is attributable to
artifacts produced in by the limited angle reconstruction. Because the
specimen cannot be tilted in the electron microscope through a full
180°, resolution is degraded along the z-axis. Scale
bar, 0.5 µm.
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The 3D reconstructions also clarified the relationship between
additional E-PTA-stained material present in the postischemic tissue. As described above, this material was present only in postischemic brains and was not always obviously connected to a PSD in
individual thin sections. By resectioning the 3D reconstructions, the
flocculent stained material observed near the PSD was often observed to
be connected loosely to it (Fig. 8).
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Figure 8.
Three-dimensional volume reconstruction of an
individual PSD (top left, large arrow) with associated
E-PTA material loosely attached (arrowhead) from the
postischemic brain after 24 hr of reperfusion. The 3D volume is shown
in the left column, and 2D slices through the volume are
shown in the right column. This type of configuration
was not readily apparent from the 3D image alone but was easily
observed after resectioning of the volume along the planes indicated.
In the 2D slices, the postsynaptic density (large
arrow), the synaptic cleft (small arrow), and
the attached flocculent material (arrowhead) can be
resolved. Similarly, the association of the flocculent material with
the PSD could be missed in individual 2D sections if the plane of
section were not optimal. Scale bar, 0.5 µm.
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DISCUSSION |
In this study, we demonstrated 2D and 3D ultrastructural
modifications in hippocampal PSDs from CA1 and DG after a brief
ischemic episode. The ultrastructural alterations of PSDs were more
remarkable and persistent in CA1 than in the dentate gyrus and occurred
well ahead of the degeneration observed in CA1 neurons after ischemia. Although the type of synapse served by the modified PSDs cannot be
ascertained in the E-PTA material, both the 3D volume reconstructions and the frequency histograms (Fig. 5) indicated that most E-PTA-stained synapses showed an increase in thickness in area CA1. Because the
majority of synapses in the molecular layer of CA1 are glutamatergic onto dendritic spines, our data suggest that at least this class of
synapse is affected. We do not know whether inhibitory synapses are
also affected, and this question is currently under investigation. The
marked ultrastructural alterations in the synapses during the
postischemic phase could produce a toxic signal such as a greater
calcium influx that contributes to or causes delayed neuronal death.
Modification of PSDs
The finding of a robust ultrastructural modification of
postischemic PSDs stained with E-PTA was unexpected because several previous ultrastructural studies had not reported or found such noticeable changes in PSDs stained with osmium-heavy metal in tissue
from several transient ischemic models, including the model used in the
present study (von Lubitz and Diemer, 1983; Kirino et al., 1984; Petito
and Pulsinelli, 1984; Deshpande et al., 1992). These inconsistencies
may be attributable to the fact that general heavy metal staining
obscures the synaptic modifications occurring in postischemic tissue.
Alternatively, it is possible that E-PTA stains different components in
the PSD than osmium-uranium-lead methods. It has been known for a
long time that PSDs stained with E-PTA are shorter and probably wider
than those stained with the osmium-heavy metal method, although E-PTA
does not stain the plasma membranes adjacent to the PSDs (Jones et al.,
1974; Burry and Lasher, 1978). PTA, a heteropolyacid, preferentially
reacts with protein(s) rich in basic amino acid residues, including
lysine, arginine, and histidine, such as collagen or histones (Bloom
and Aghajanian, 1968), whereas conventional heavy metal staining
preferentially stains a wide array of lipids and cytoskeletal and
cytoplasmic structures (Burry and Lasher, 1978). The staining of
different components by these two methods may explain why such a robust ultrastructural alteration in postischemic PSDs stained with E-PTA has
not been previously noted in the studies using heavy metal-stained sections in postischemic brain tissues (von Lubitz and Diemer, 1983;
Kirino et al., 1984; Petito and Pulsinelli, 1984; Deshpande et al.,
1992; Tomimoto and Yanagihara, 1992; Rafols et al., 1995).
The use of electron tomography combined with EPTA staining proved to be
a useful method for observing the 3D morphology of individual PSDs and
presynaptic grids without having to resort to serial sectioning
techniques. Large numbers of synapses can be reconstructed concurrently
and because the entire PSD is contained within a single section, there
are minimal sectioning and alignment distortions. The combination of
selective staining and tomographic reconstruction produced gray scale
volumes that required no additional segmentation. Thus, the fine
structure of individual PSDs was clearly visible, and the uncondensed,
flocculent structure of PSDs was readily apparent in the postischemic
brain. This type of fragmented configuration was not as apparent in the
2D images of PSDs from thin sections. The resulting volume
reconstructions could also be sectioned in any arbitrary plane,
facilitating correlation of the 2D and 3D images.
PSDs are composed of a cytoskeletal frame, in which proteins, including
neurotransmitter receptors, ion channels, and adaptor proteins, are
anchored and targeted (Harris et al., 1992; Harris and Kater,
1994; Kennedy, 1994; Suzuki, 1994; Klauck and Scott, 1995). The
ischemia-induced increases in intracellular calcium and production of
free radicals activate proteases and result in partial degradation of
certain cross-linker proteins in PSDs such as spectrin and
microtubule-associated protein 2 (Yoshimi et al., 1991; Robert-Lewis et
al., 1994). In addition, unfolding proteins induced by ischemia may
aggregate and change the construction of PSDs. These may result in the
loosening of the PSD frame structure, rendering them more diffuse and
more irregular.
Transient cerebral ischemia induced a significant increase in
perforated synapses from 4.35 to 6.63% in the CA1 but not in dentate
gyrus at 24 hr of reperfusion. This finding suggests that the neurons
react to ischemia more severely in CA1 than in the dentate gyrus,
consistent with analysis of other PSD parameters in this study. It has
been suggested that perforated synapses are more efficient in synaptic
transmission than simple synapses (for review, see Edwards, 1995).
However, it is unclear that an increase of only ~2% of perforated
synapses without a change in total synapse number would contribute to
the neuronal vulnerability in CA1 neurons. It is possible that the
increase in synaptic perforation of synapse is a reactive process to
neuronal depolarization or the calcium influx induced by ischemia
(Siesjö, 1988). Alternatively, as observed in the 3D
reconstructions, PSDs from postischemic CA1 were more irregular in
profile and less condensed compared with their control counterparts.
The impression garnered from the 3D reconstructions was not that there
was an increase in discrete perforations in PSDs from postischemic
tissue, but that there was a general loosening of E-PTA-stained
material, leading to discontinuities in PSD structure. Thus this
fragmentation could lead to an apparent, rather than a real, increase
in the number of perforated synapses in a 2D analysis.
Significance of the synaptic changes
In our previous paper, we found that several protein kinases and
protein ATPases were translocated to the synapses in cortical tissue
after ischemia (Hu et al., 1998). Because only 5-20% of neocortical
neurons die after ischemia, we suggested that the changes in the
synapses might be attributable to a plastic synaptic response, i.e.,
kinase translocation to the PSDs to modulate receptor function. One
objective of the present investigation was to examine the possible
association between these synaptic changes and neuronal death after
transient cerebral ischemia. In the ischemic model used in this study,
CA1 neurons die after 2 d of reperfusion, whereas dentate granule
cells survive. Two questions are raised by our findings. First, do the
observed synaptic changes represent a pathological or physiological
phenomenon? Second, do the observed synaptic changes contribute to
neuronal cell death?
Consistent with the results of our previous study, there was a marked
increase in E-PTA-stained material in the PSDs from postischemic brain,
particularly in area CA1. However, from the 3D reconstructions of
synapses, we found that PSDs are not only increased in thickness, as
was seen in 2D views, but appear to be disintegrating in area CA1. This
type of alteration is much less noticeable and more transient in the
dentate gyrus as well as in the neocortex (data not shown). The
degenerative changes observed in CA1 synapses in this study suggest to
us that the translocation of protein ATPase and protein kinases
reported in our previous paper may also be related to degenerative
changes (Hu et al., 1998). Both heat-shock cognate protein 70 and
N-ethylmaleimide-sensitive fusion protein function as
protein chaperones to prevent protein missfolding during their
assembly. In addition, both protein kinase C and
calcium/calmodulin-dependent protein kinase II are inactive after translocation to the PSDs after ischemia (Hu et al., 1995). These
results suggest that accumulation of proteins in the PSDs may be
attributable to protein unfolding or denaturing to expose their
hydrophobic segments during ischemia, thereby causing interprotein aggregation in the PSDs after ischemia. The increased amount of material associated with PSDs in the postischemic brain could thus
represent denatured and aggregated proteins. Consistent with this
hypothesis, we recently found that PSD proteins from the CA1 region are
dramatically and persistently ubiquitinized, indicating that the
proteins in the PSDs are going to degrade (our unpublished data). Thus, transient ischemia may result in degradation of synaptic proteins by ubiquitin-dependent proteinases, leading to dysfunctional synaptic transmission at the altered synapse. Physiological studies suggest that synaptic transmission is depressed during the postischemic phase after 15 min of global ischemia (Volpe et al., 1984; Auer et al.,
1989; Furukawa et al., 1990; Xu, 1995; Dalkara et al., 1996), further
suggesting that the synaptic changes observed in the present study are
a pathological phenomenon.
The observations in the present study that the degenerative changes in
morphology are more robust and long-lived in vulnerable CA1 neurons
than in resistant dentate gyrus neurons and occur well before CA1
neurons degenerate raise the question of whether this synaptic
alteration may contribute to cell death. The morphological analyses
conducted in the present study indicate that the majority of synapses
in area CA1 are affected. There are ~10,000 dendritic spines per CA1
neuron, and >90% of excitatory synapses occur on dendritic spines
(Andersen et al., 1990; Harris and Kater, 1994). Thus, if alterations
in synaptic structure and biochemistry result in a toxic signal, e.g.,
greater calcium influx, from these changed synapses either because of
nonspecific leakage or altered glutamatergic transmission, the damage
signal could be integrated from several thousands of synapses and
propagate to the dendritic trunk and, eventually, the cell body to
cause cell death. This would be consistent with the observations by
many investigators that a damage signal propagates from the dendrites
to the somata of vulnerable neurons (Kirino et al., 1984; Petito and
Pulsinelli, 1984; Tomimoto and Yanagihara, 1992). This is also
consistent with the theory of an overload in intracellular calcium
(Choi, 1988; Siesjö, 1988). Because the changes in PSD structure
were observed to be greater and more prolonged in CA1 neurons compared
with dentate gyrus neurons, the cumulative effects of PSD damage would
be expected to be greater in CA1 than in dentate gyrus, thereby
contributing to their selective vulnerability.
| |
FOOTNOTES |
Received Sept. 24, 1998; revised Dec. 23, 1998; accepted Jan. 6, 1999.
This work was supported by National Institutes of Health Grants NS36810
(to B.R.H.), NS28121 (to J.A.Z.), and RR04050 (to M.H.E.).
Correspondence should be addressed to Dr. Bing-Ren Hu, Center for the
Study of Neurological Disease, The Queen's Medical Center, 1356 Lusitana Street, Eighth Floor, Honolulu, HI 96813.
| |
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Top
Abstract
Introduction
