Neuroscience 327 (2016) 6478

CHANGES IN SYNAPTIC PLASTICITY AND EXPRESSION OF

GLUTAMATE RECEPTOR SUBUNITS IN THE CA1 AND CA3 AREAS OF
THE HIPPOCAMPUS AFTER TRANSIENT GLOBAL ISCHEMIA
XIN-JIA HAN, a,b ZHONG-SHAN SHI, a,b LUO-XING XIA, a
LI-HUI ZHU, a LING ZENG, a JUN-HUA NIE, a
ZAO-CHENG XU c AND YI-WEN RUAN a,b,d,e,f*

receptors in the CA1 and CA3 hippocampal regions of rats

after ischemia. Our results demonstrated that the NR2B/
NR2A ratio became larger after ischemia although the
expression of both the NR2B subunit (activation of apoptotic pathway) and NR2A subunit (activation of survival
pathway) decreased in the CA1 area from 6 h to 48 h after
reperfusion. Furthermore, expression of the GluR2 subunit
(calcium impermeable) of the AMPA receptor class
signicantly decreased while the GluR1 subunit (calcium
permeable) remained unchanged at the same examined
reperfusion times, which subsequently caused an increase
in the GluR1/GluR2 ratio. Despite these notable dierences
in subunit expression, there were no obvious changes in
the density of synapses or expression of NMDAR and
AMPAR subunits in the CA3 area after ischemia. These
results suggest that delayed CA1 neuronal death may be
related to the dramatic uctuation in the synaptic structure
and relative upregulation of NR2B and GluR1 subunits
induced by transient global ischemia. 2016 Published by
Elsevier Ltd on behalf of IBRO.

a
GHM Institute of CNS Regeneration (GHMICR), Jinan
University, Guangzhou, Guangdong 510632, China
b
Department of Anatomy, Jinan University School of Medicine,
Guangzhou, Guangdong 510632, China
c

Department of Anatomy and Cell Biology, Indiana University

School of Medicine, Indianapolis, IN 46202, USA
d
Co-innovation Center of Neuroregeneration, Nantong
University, Nantong, Jiangsu 226019, China
e

GHM Collaboration and Innovation Center for Tissue

Regeneration and Repair, Jinan University, Guangzhou,
Guangdong 510623, China
f
Ministry of Education CNS Regeneration International
Collaborative Laboratory, Jinan University, Guangzhou,
Guangdong 510623, China

AbstractExcess glutamate release from the presynaptic

membrane has been thought to be the major cause of
ischemic neuronal death. Although both CA1 and CA3 pyramidal neurons receive presynaptic glutamate input, transient cerebral ischemia induces CA1 neurons to die while
CA3 neurons remain relatively intact. This suggests that
changes in the properties of pyramidal cells may be the
main cause related to ischemic neuronal death. Our
previous studies have shown that the densities of dendritic
spines and asymmetric synapses in the CA1 area are
increased at 12 h and 24 h after ischemia. In the present
study, we investigated changes in synaptic structures in
the CA3 area and compared the expression of glutamate

Key words: transient global ischemia, hippocampus, glutamate

receptor, synapse, ultrastructure, protein expression.

INTRODUCTION
Glutamate receptors play crucial roles in synaptic
transmission, synaptic plasticity and excitatory signaling
pathways regulating various physiological conditions
(Kohr, 2006; Zorumski and Izumi, 2012) and excitotoxicity
under pathological conditions (Martin et al., 1998; Vizi
et al., 2013; Zhou and Sheng, 2013). NMDA
(N-methyl-D-aspartate) receptors (NMDARs) and AMPA
(a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)
receptors (AMPARs) are two major groups of glutamate
receptors in the brain. There are three subtypes of
NMDAR subunits including NR1, NR2 (A-D) and NR3
(A, B). NR1 contains an extracellular binding site for
glycine while NR2 links an extracellular binding site for
glutamate (Mayer and Armstrong, 2004; Paoletti, 2011).
In the adult brain, NR2A is widely expressed, NR2B is
restricted to the forebrain, and NR2C is highly enriched
in the cerebellum (Kohr, 2006). It has been identied that
NMDAR subunits form heterotetramers that composed of
NR1/NR2A or NR1/NR2B or NR1/NR2A/NR2B subunits,
which play dierent biophysical and pharmacological
functions (Cull-Candy and Leszkiewicz, 2004; Furukawa
et al., 2005; Chen and Wyllie, 2006). NR2A subunits are

*Correspondence to: Y.-W. Ruan, GHM Institute of CNS Regeneration, Jinan University, 601 West Huangpu Avenue, The 2nd Science
& Technology Building, 803, Guangzhou, Guangdong 510632, China.
Tel: +86-20-85227086; fax: +86-20-85223563.
E-mail addresses: tyiwen@jnu.edu.cn, yiwenruan@yahoo.com
(Y.-W. Ruan).
Abbreviations: AAD, asymmetric axodendritic synapses; AAS,
asymmetric axospinous synapses; AMPA, a-amino-3-hydroxy-5-meth
yl-4-isoxazolepropionic acid; AMPARs, a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptors; BDNF, brain-derived neurotrophic
factor; CAMKII, calcium/calmodulin-dependent protein kinase II; CCA,
common carotid arteries; CREB, cAMP response element binding
protein; DC, direct current; DSR, distal stratum radium; FD,
uorescence intensity; ID, ischemic depolarization; NMDA, N-methylD-aspartate; NMDARs, N-methyl-D-aspartate receptors; PL, pyramidal
layer; PSD, postsynaptic density; PSD95, postsynaptic density-95;
PSR, proximal stratum radiatum; SAD, symmetric axodendritic
synapses; SAS, symmetric axospinous synapses; SO, stratum
oriens; SP, stratum pyramidal cell layer; TAS, total asymmetric
synapses; TSS, total symmetric axospinous synapses.
http://dx.doi.org/10.1016/j.neuroscience.2016.04.011
0306-4522/ 2016 Published by Elsevier Ltd on behalf of IBRO.
64

X.-J. Han et al. / Neuroscience 327 (2016) 6478

mainly located at synaptic sites, whereas NR2B subunits

are mainly located at extrasynaptic sites (Stocca and
Vicini, 1998; Rumbaugh and Vicini, 1999; Tovar and
Westbrook, 1999; Traynelis et al., 2010). Recent studies
have shown that activation of the synaptic NR2A subunit
can induce survival signaling pathways as evidenced by
the expression of cAMP response element binding protein
(CREB) and brain-derived neurotrophic factor (BDNF)
genes along with death suppresser genes such as Puma
and FOXO. Conversely, activation of the extrasynaptic
NR2B subunit can trigger apoptotic pathways subsequently increasing ROS expression, shutting o CREB
and ERK expression, and inhibiting FOXO expression
(Leveille et al., 2008; Hardingham and Bading, 2010;
Gladding and Raymond, 2011). It has been addressed
that many neurodegenerative diseases are related to
the activation of extrasynaptic NMDAR subunits including
stroke and traumatic brain injury (TBI), Alzheimers
disease (AD), depression, schizophrenia and pain (Vizi
et al., 2013; Zhou and Sheng, 2013).
Another major group of glutamate receptors are the
AMPA receptors. Four subunits in this group have been
identied, GluR1, GluR2, GluR3, and GluR4 (Shi et al.,
1999; Song and Huganir, 2002). AMPAR subunits are
homo- or hetero-oligomeric assemblies that have dierent
permeability to calcium, sodium and potassium
(Nakanishi et al., 1990). AMPA receptors that contain
GluR2 subunit will almost render the channel impermeable to calcium, whereas the channel will be permeable
to calcium, sodium and potassium if it lacks a GluR2 subunit. (Nakanishi et al., 1990; Stein et al., 1992). AMPA
receptors are also activated by glutamate, which will
repulse the Mg2+ cation from the pore of NMDA receptors
and thus in turn cause ion channels (Na+ and Ca2+ channel) of NMDARs to open (Arundine and Tymianski, 2003).
The Ca2+ inux further stimulates upregulation of
AMPARs to the cellular membrane and induces longlasting increases in the excitatory post synaptic potential
(EPSP) size underlying LTP (Whitlock et al., 2006). It
has been identied that AMPARs can be tracked from
perisynaptic regions to the synaptic region, from intracellular vesicles to the postsynaptic density (PSD) (Malenka,
2003). Therefore, AMPARs participate in LTP formation,
synaptic plasticity and excitotoxicity (Silverman et al.,
2007; Kessels and Malinow, 2009; Rogawski, 2013).
Under pathological conditions, AMPA receptors exert
excitotoxicity as demonstrated in a number of neurodegeneration diseases. For example, it has been reported
that epilepsy is predominantly mediated by AMPA receptors inducing fast synaptic excitation in the brain
(Rogawski, 2013). Neuronal death following ischemia is
mainly related to the downregulation of GluR2 mRNA
and increases in AMPA receptor-mediated Ca+ inux
(Gorter et al., 1997; Liu et al., 2004).
Glutamate receptors are inserted into cellular
membranes and changes in the expression and
distribution of glutamate receptors will ultimately aect
the number and size of synapses. It has been reported
that all Schaer collateralcommissural (SCC) synapses
in the CA1 area of the hippocampus in adult rats
express NMDA receptors, yet only 75% of these

65

synapses are inserted with AMPARs. And the ratio of

AMPA to NMDA receptors will determine the PSD area
and function (Takumi et al., 1999). A large number of
AMPARs were found in mushroom spines, whereas little
or no AMPARs were seen in thin spines and lopodia
(Fiala et al., 1998; Matsuzaki et al., 2001). It has also
been reported that transient cerebral ischemia induces
the postsynaptic densities of CA1 neurons to become
thicker and wider (Martone et al., 2000; Kovalenko
et al., 2006).
Transient global ischemia induces selectively
neuronal death in the CA1 area, whereas neurons in the
CA3 area remain relative intact (Kirino, 1982; Petito and
Pulsinelli, 1984). Our previous studies also proved these
ndings and have shown that transient global cerebral
ischemia induces increases in the number of dendrites,
the thickness of PSDs and the number of asymmetric
synapses in the CA1 area (the vulnerable region to ischemia) 2 days after ischemia before neurons die (Ruan
et al., 2006, 2012). Whether these changes are related
to the neuronal death in the CA1 area after ischemia
remains unclear. Here, we hypothesized that these alterations reect the vulnerability of CA1 neurons, most probably are related to ischemic neuronal injury. To test this
hypothesis, we investigated whether the number of
synapses and the thickness of PSD will change in the
CA3 area (the area resistant to ischemia) in the present
study. We also examined changes in expression of
NMDAR and AMPAR subunits in the CA1 and CA3
regions and analyzed the relationship between changes
in synaptic structures and changes in expressions of
glutamate receptors.

EXPERIMENTAL PROCEDURES
Animals
Male adult Wistar rats (weighing 200300 g, obtained
from Experimental Animal Center of Zhongshan
University, Guangzhou, China) were used in the present
study. Experimental protocols were approved by
Competent Ethics Committees of Jinan University and
the Institutional Animal Care and Use Committee
(IACUC) of Indiana University School of Medicine in
accordance with the NIH Guidelines (NIH Publications
No. 8023, revised 1978) for the Care and Use of
Laboratory Animals. All eorts were made to minimize
the number of animals used as well as their suering.
Animals were grouped as the following: For the
protein detection by immunouorescence and Western
blotting, rats were randomly divided into ve
experimental groups (57 rats in each group): sham
group (the same operating process except ischemia), Is
6-h group (rats were sacriced at 6 h after reperfusion),
Is 12-h group (12 h after reperfusion), Is 24-h group
(24 h after reperfusion), Is 48-h group (48 h after
reperfusion). For the ultrastructure observation and
analysis by electron microscopy, rats were randomly
allocated into four experimental groups (34 rats in each
group): sham group, Is 12-h group, Is 24-h group and Is
48-h group.

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X.-J. Han et al. / Neuroscience 327 (2016) 6478

Transient forebrain ischemia

Quantitative analysis of synapses in the CA3 area

Transient forebrain ischemia was induced using the

four-vessel occlusion method (Pulsinelli et al., 1982) with
modications (Xu et al., 1999). Briey, rats were fasted
overnight (812 h) to produce uniform blood glucose
levels. For surgical procedures, the animals were anesthetized with 12% halothane mixed with 33% O2 and
66% N2. A silicone tube was placed loosely around each
carotid artery to allow subsequent occlusion of the vessel.
After the rats were placed in a stereotaxic frame, the vertebral arteries were electrocauterized. Then, a small temperature probe (0.8 mm outside diameter) was inserted
beneath the skull in the extradural space and the brain
temperature was maintained at 37 C via a heating lamp
enabled with a temperature control system (Physitemp
BAT-10, Clifton, NJ, USA). This process took approximately 10 min. Under a stable temperature condition, a
microelectrode lled with 2 M KCl was inserted into the
hippocampus (2.2 mm below the brain surface, 2.2 mm
lateral from the middle line, 4.3 mm posterior from the
bregma) to record ischemic depolarization (ID) with an
amplier (Neuroprobe 1600, A-M System, Carlsborg,
MA, USA). Transient forebrain ischemia was induced by
occluding both common carotid arteries (CCA). The duration of ID was determined by measuring the period from
the beginning of the extracellular direct current (DC)
potential reaching  20 mV to the point where the potential returned to zero after recirculation. The DC potential
shifted from zero to approximately 20 mV, which occurs
only when the blood ow reduces to 10% of the control
levels (Gao et al., 1998). The ID period was set for 10 min.
Due to it usually took 2 min to shift the potential to
20 mV after occlusion of bilateral CCA and took
2 min to return to zero (repolarization) after releasing
the occlusion of CCA, the real ID period was measured
as 13 min.

In order to understand better changes in the entire

neuropil of the CA3 area after ischemia, photos were
captured from 3 neuropil areas of the CA3 region, which
include an area in the stratum oriens (SO) at
30130 lm from the stratum pyramidal cell layer (SP),
an area in the proximal stratum radium (PSR) at
30130 lm from the SP, and another area in the distal
stratum radium (DSR) at 200300 lm from the SP
(Fig. 1). To avoid overlap, micrographs were taken
under the observation eld from left to right; the area
with large dendrites or more than three myelinated
axons was skipped. Approximately, 2035 images were
taken from each group in the SO, PSR and DSR areas
at a magnication of 17,500 times. The number of
synapses in single images (measured area of 32 lm2)
was accounted. A presynaptic terminal is determined by
docked synaptic vesicles in the active zone facing the
postsynaptic membrane. The total measured area of
6401120 lm2 was analyzed in each group. For
analysis of the thickness of synapses, we measured the
thickest part of the PSD from the postsynaptic
membrane to the interface toward the cytoplasm using
UTHSCSA Image Tool program.

Tissue preparation for electron microscopy

Rats were deeply anesthetized with Ketamine (80 mg/kg),
and perfused with xative containing 2% glutaraldehyde
and 2% paraformaldehyde in 0.15 M phosphate buer
(PB), pH 7.4. Brains were removed and kept in the
same xative overnight at 4 C. Coronal sections
(50 lm) were cut through the hippocampus on a
vibratome. Sections were post-xed with 0.5%osmium
tetroxide for 1 h, dehydrated in a graded ethanol series
(30507095%) for 5 min each, and 100% ethanol for
3  10 min, propylene oxide for 3  15 min, and
embedded with EMbed 812 (Electron Microscope
Sciences) at 60 C for 48 h. Small pieces of samples
(approximate 1 mm2 in size) containing the CA3 region
were cut from embedded sections and glued onto resin
blocks with cryze glue. Observation areas were
determined by semi-thin sections (1 lm) which were rst
cut on an ultramicrotome and stained with toluidine blue.
Ultrathin sections of light yellow/gray color were
collected on 200 mesh grids, and stained with 3%
uranyl acetate for (30 min) and 2.66% lead citrate for
15 min. Images of the CA3 neuropil were then captured
with a Philips 400 transmission electron microscope.

Immunouorescent staining
After survived for 6 h, 12 h, 24 h or 48 h following
reperfusion, rats were sacriced respectively. Under
anesthesia, rats were perfused transcardially with saline
followed by xative containing 0.15% glutaraldehyde
and 4% paraformaldehyde in 0.1MPB. The brains was
subsequently dissected out and post-xed overnight at
4 C. Coronal sections (40 lm) were cut with a
vibratome. Sections were incubated with blocking serum
(5% goat serum, 0.1% Triton X-100 in PBS) for 2 h, and
then incubated with primary antibodies (NR2B, 1:100,
Cat#06-600, Millipore; NR2A, 1:100, Cat#05432,
Millipore; GluR1, 1:1000, Cat#04-855, Millipore; GluR2,
1:500, Cat#556341, BD) at 4 C overnight. The sections
were washed with 0.01PBS for 3  5 min, and then
incubated with secondary antibodies (488 nm FITClabeled goat anti-rabbit IgG and 546 nm TRITC-labeled
goat anti-mouse IgG, 1:1000) for 2 h at room
temperature. Immunostaining sections were examined
and photographed under a uorescent microscope
(Leica DM1000, Germany).

Measurement of uorescence intensity

To detect the immunoreactivity of NMDA receptor
subunits and AMPA receptor subunits, 20 sections from
each group (4 sections per animal) were used for the
measurement of uorescence intensity. Images were
captured from the CA1 and CA3 area under the same
light exposure time and magnication (10 & 40). The
uorescence intensity (FD) of the pyramidal layer (PL)
of these areas was measured in 10 images by NIH
Image J. The mean ratio was determined by FD of
PL/FD of background and compared between groups.

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X.-J. Han et al. / Neuroscience 327 (2016) 6478

Fig. 1. Photomicrographs showing three neuropil regions in quadrangle boxes in which synaptic morphology was investigated under electron
microscope in the CA3 area: the stratum oriens (SO) was terminated at 30130 lm from the stratum pyramidal cell layer (SP); the proximal stratum
radium (PSR) was limited at 30130 lm from SP; and the distal stratum radium (DSR) were detected at 200300 lm from SP. Scale bar = 500 lm.

Western blotting
Under anesthesia, rats were transcardially perfused with
saline for 23 min until the liver became a light yellow
color. Immediately after perfusion, the CA1 and CA3
areas of the hippocampus were separately dissected
from the fresh brain tissue placed on ice under a
dissecting microscope (Zeiss, Stemi 305). The CA1 or
CA3 tissues were homogenized by an ultrasonic wave
machine (Xing Zhi Biotechnology Research Institute,
Shanghai, China). Samples were centrifuged at 12,000g
for 15 min at 4 C. The supernatant was collected and
stored at
20 C until required for use. Protein
quantication was performed to keep the same total
protein amount (40 lg) for loading. The loading volume
was 20 ll in each lane. The proteins were separated on
8% SDSPAGE gels and transferred in 0.45 lm PVDF,
and then the membranes were rinsed in 0.1% TBST
and incubated in 5% skim milk in 0.1% TBST at room
temperature for 2 h. The membranes were rinsed again
and incubated in primary antibodies in 0.1% TBST
containing 5% BSA overnight at 4 C. The primary
antibodies were used as follows: NR2A (1:500,
Cat#05432, Millipore), NR2B (1:500, Cat#06-600,
Millipore), GluR1 (1:2000, Cat# 04-855, Millipore),
GluR2 (1:250, Cat#556341, BD) and Tubulin (1:1000,
Cat#2128, CST). After being washed in 0.1% TBST, the
membranes were incubated in secondary antibodies in
5% BSA and 0.1% TBST solution for 2 h. Targeted
protein bands on the membranes were detected with an
ECL Western blotting Detection Kit (WBKLS001000,
Millipore) then further developed and xed in an X-ray
lm (Kodak XBT-1, Xiamen, China). Films were

scanned into images by MICROTEK ScanMaker i2000

(Shanghai, China) and the densitometry of protein
bands in the images were analyzed with the NIH
ImageJ analysis software.
Statistical analysis
Data were analyzed using StatView statistical analysis
software using unpaired t-test (Stat View 5.0, SAS
Institute, Cary, NC, USA). Values were presented as
Mean SE and statistical signicance was denoted as
P < 0.05.

RESULTS
Similarity in ultrastructural structures of synapses in
the CA3 neuropil after ischemia
In the present study, we observed changes in the synaptic
morphology of the CA3 area using an electron
microscope (Philips 400) under the same ischemic
conditions as previously described (Ruan et al., 2012).
We selected three areas, including the stratum oriens
(SO), proximal stratum radiatum (PSR) and distal stratum
radiatum (DSR) of the CA3 region (Fig. 1), and typical
synaptic structures in the intact CA3 neuropil were found.
These included the presynaptic membrane (Fig. 2, black
arrows), synaptic cleft, and postsynaptic membrane
(Fig. 2, white arrows). Most presynaptic structures were
from axonal terminals (a) with round vesicles and most
postsynaptic densities (PSD) attached to the postsynaptic
membrane of spines (s) (Fig. 2, white arrows). Mitochondria (m) were mainly distributed in dendrites and axons

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X.-J. Han et al. / Neuroscience 327 (2016) 6478

Fig. 2. Electron micrographs showing ultrastructural changes in synaptic morphology at neuropil regions of the SO, PSR and DSR of the CA3 area
after ischemia. In the normal CA3 area, typical synaptic structures were clearly seen which consisted of presynaptic membrane (dark arrows),
synaptic cleft, and postsynaptic membrane (white arrows) (AC). Typical postsynaptic densities (PSD, white arrows) of asymmetric synapses were
connected by axons (a) with round vesicles which were distributed into the presynaptic terminals. Mitochondria (m) occurred mainly in axons (a) and
dendrites, but rarely appeared in dendritic spines (s). Although fewer vesicles were found in many terminals at 48 h after reperfusion, there were no
obvious changes in PSD morphology after ischemia (DL). Scale bar = 500 nm.

(a), but rarely appeared in dendritic spines. After ischemia, the structures of synapses did not show obvious
structural changes at dierent observed reperfusion times
(Fig. 2).
Percentage of each type of synapse in the intact CA3
area
In order to reveal whether ischemia would cause changes
in the number of synapses, rst we analyzed dierent
types of synapses of the intact CA3 area. There were
mainly 4 types of synapses identied in the intact CA3
area including asymmetric axospinous synapses (AAS,
Fig. 3A) characterized by a thicker PSD in the
postsynaptic membrane of the spine, symmetric
axospinous synapses (SAS) without obvious PSDs in
the postsynaptic membrane of the spine (Fig. 3B),
asymmetric axodendritic synapses (AAD) with a thicker
PSDs in the postsynaptic membrane of the dendrite
(Fig. 3C), and symmetric axodendritic synapses (SAD)
without obvious PSDs in postsynaptic membrane of the
dendrite (Fig. 3D). Quantitative analysis demonstrated
that the number of total asymmetric synapses (TAS)
accounted for 95% 0.7 of the total synapses in the

SO (Table 1), which covered a number of AAS and

AAD, whereas the number of total symmetric
axospinous synapses (TSS) in the SO was 5% 0.7,
including a number of SAD and SAD. The number of
TAS in the PSR and DSR areas accounted for 94%
1.0 and 94% 0.4 separately, whereas the number
of TSS in the PSR and DSR areas was 6% 0.9 and
6% 0.4, respectively.

No signicant dierence in the number of synapses

in the CA3 area after ischemia
After we obtained the percentage of each synapse type in
the intact CA3, we further analyzed changes in the
numbers of these synapses in the SO, PSR and DSR of
the CA3 region after ischemia. Fig. 4 shows the
changes in the number of asymmetric and symmetric
synapses at dierent reperfusion times after ischemia.
Quantitative analysis revealed that the numbers of both
asymmetric and symmetric synapses were not
signicantly dierent in SO, PSR and DSR areas before
and after ischemia at dierent reperfusion times, all
P > 0.05 compared to sham (Fig. 4AC).

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X.-J. Han et al. / Neuroscience 327 (2016) 6478

Fig. 3. Electron micrographs showing 4 synapse types in the CA3 neuropil. (A) A representative image of asymmetric axospinous synapses (AAS)
is the connection between an axonal terminal (a) and a spine (s). There is a thicker PSD in postsynaptic membrane of the spine (white arrow). (B) A
representative image of symmetric axospinous synapses (SAS) shows no obvious PSD in the postsynaptic membrane of the spine. The synapse is
connected between the presynaptic membrane of an axon (a) and postsynaptic membrane of a spine (s) at two slightly thicker parts (black arrows).
(C) A representative image of asymmetric axodendritic synapses (AAD) is the connection between an axonal terminal (a) and a dendrite (d). There
is also a thicker PSD in postsynaptic membrane of the dendrite (white arrow). (D) A representative of symmetric axodendritic synapses (SAD)
shows no obvious PSD in postsynaptic membranes of the dendrite. The synapse is connected between the presynaptic membrane of an axon (a)
and postsynaptic membrane of a dendrite (d) at two slightly thicker parts (black arrows). Scale bar = 500 nm.

Table 1. Percentage of each synaptic type in the normal CA3 area

Region

TAS

AAS

AAD

TSS

SAS

SAD

SO
PSR
DSR

95% 0.7
94% 1.0
94% 0.4

82% 1.7
72% 2.0
79% 0.8

13% 1.7
22% 1.8
15% 0.7

5% 0.7
6% 0.9
6% 0.4

1% 0.4
1% 0.5
1% 0.1

4% 0.6
5% 0.9
5% 0.4

No obvious changes in the PSD thickness of the CA3

area after ischemia
The postsynaptic density mainly consists of NMDA
receptors, AMPA receptors, postsynaptic density-95
(PSD95), and calcium/calmodulin-dependent protein
kinase II (CAMKII) (Sessoms-Sikes et al., 2005; Chen
et al., 2011; Farley et al., 2015). Changes in PSD thickness may reect changes in any of these components.
Therefore, we further investigated the PSD thickness in
the SO, PSR and DSR neuropil of the CA3 area.
Approximately, 300350 synapses in each group were
randomly selected for measurement of PSD thickness.
Quantitative analysis demonstrated that the thickness of
the PDS in the SO, PSR and DSR neuropil of the CA3 area
did not signicantly change before and after ischemia, all
P > 0.05 compared to the sham (Table 2).
Changes in immunoreactivities of NMDA receptor
subunits in CA1 and CA3 pyramidal neurons after
ischemia
Glutamate receptors are important components of the
PSD. Changes in the thickness of the PSD may aect

expression of glutamate receptors, or vice versa.

Therefore, we further investigated the expression of
dierent subunits of glutamate receptors. First we
evaluated the immunoreactivities of NMDAR subunits
(including NR2A and NR2B subunits) before and after
ischemia. The immunoreactivity of NR2A and NR2B
subunits in CA1 and CA3 pyramidal neurons were
seen in Fig. 5(AJ and A-1 to J-1). Quantitative
analysis demonstrated that the uorescent intensity of
NR2A subunits was reduced to 69.6% 1.91 at 6 h,
69.2% 2.44 at 12 h, 69.8% 3.03 at 24 h, and
68.6% 1.65 at 48 h after ischemia in the CA1 area,
P < 0.01 compared to sham (Fig. 5K). The uorescent
intensity of NR2B subunit was reduced to 77.7%
2.53 at 6 h, 79.9% 2.24 at 12 h, 82.3% 2.55 at
24 h, and 78.9% 3.36 at 48 h, P < 0.01 compared
to sham (Fig. 5K-1). However, in the CA3 area, both
NR2A
and NR2B
immunoreactivities
did
not
signicantly change before or after ischemia (Fig. 5A-1
to J-1). There was also no signicant dierence in the
statistical data of NR2A and NR2B uorescent
intensities before or after ischemia (Fig. 5K-1), all
P > 0.05.

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X.-J. Han et al. / Neuroscience 327 (2016) 6478

Fig. 4. Histograms showing changes in the number of asymmetric and symmetric synapses in the CA3 area. The results showed that there were no
signicant dierences among the total number of synapses (TS), the total number of asymmetric axospinous synapses (TAS), numbers of AAS and
AAD, the total number of symmetric synapses (TSS), or numbers of SAS and SAD in the SO (A), PSR (B) and DSR (C) of the CA3 region. All
P > 0.05 compared with the sham group.

Table 2. Changes in the PSD thickness of the CA3 area after ischemia
Region

Sham

IS12h

IS24h

IS48h

SO
PSR
DSR

38.59 0.68
36.18 0.92
36.82 0.96

36.53 0.53
33.16 0.82
36.18 0.61

36.87 0.72
36.39 0.78
36.08 0.60

37.70 0.56
33.94 0.72
38.27 0.49

Changes in immunoreactivities of AMPA receptor

subunits in CA1 and CA3 pyramidal neurons after
ischemia
Next we detected the immunoreactivities of AMPAR
subunits, both GluR1 and GluR2 before and after
ischemia. In the CA1 area, immunouorescent labeling

showed that pyramidal neurons did not observe

changes in uorescence intensity of GluR1 subunit
before and after ischemia (Fig. 6A, C, E, G, I).
Fluorescence intensity of GluR2 subunit did become
weak however, from 12 h to 48 h (Fig. 6F, H, J)
compared to the sham and 6 h after ischemia groups
(Fig. 6B, D). Quantitative analysis demonstrated that the

X.-J. Han et al. / Neuroscience 327 (2016) 6478

71

Fig. 5. Changes in NMDAR receptor subunits (NR2A and NR2B) in the CA1 and CA3 pyramidal neurons after ischemia were detected by
immunouorescent labeling. Immunouorescent micrographs showing changes in uorescence intensity of NR2A and NR2B subunits in CA1
pyramidal neurons (AJ) and CA3 pyramidal neurons (A-1 to J-1). Quantitative analysis revealed that uorescence intensity of NR2A in CA1
pyramidal neurons decreased from 6 h to 48 h after ischemia (K, #P < 0.01 vs sham). The uorescence intensity of NR2B also decreased from 6 h
to 48 h after ischemia (K, #P < 0.01 vs sham). However, there were no signicant changes in uorescence intensity of NR2A or NR2B in CA3
pyramidal neurons after ischemia between 6 h and 48 h (K-1, P > 0.05 vs sham). Scale bar = 50 lm.

level of GluR1 subunit in the normal, 6-h, 12-h, 24-h, and

48-h groups were 100% 5.55, 91.3% 4.86, 92.1%
3.87, 90.9% 4.30 and 90.8% 3.18 (Fig. 6K),
respectively. There was no signicant dierence across
groups, (P > 0.05) however, expression of the GluR2
subunit was reduced at 6 h (86.7% 3.58, P < 0.05),

12 h (82.5% 2.65, P < 0.05), 24 h (78.0% 3.27,

P < 0.01), and 48 h (75.5% 3.52, P < 0.01) after
ischemia compared to the sham (Fig. 6K). In the CA3
area, both immunoreactivities of GluR1 and GluR2
subunits of pyramidal neurons did not obviously change
before or after ischemia (Fig. 6A-1 to J-1). The

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X.-J. Han et al. / Neuroscience 327 (2016) 6478

Fig. 6. Changes in AMPAR receptor subunits (GluR1 and GluR2) in the CA1 and CA3 pyramidal neurons after ischemia were detected by
immunouorescent labeling. Immunouorescent micrographs showing changes in uorescence intensity of GluR1 and GluR2 in CA1 pyramidal
neurons (AJ) and CA3 pyramidal neurons (A-1 to J-1). Quantitative analysis showed that uorescence intensity of GluR1 in CA1 pyramidal
neurons did not change from 6 h to 48 h after ischemia (K, P > 0.05 vs sham) whereas uorescence intensity of GluR2 decreased from 6 h to 48 h
after ischemia (K, *P < 0.05, #P < 0.01 vs sham). In the CA3 pyramidal neurons, there was no signicant change in either uorescence intensity of
GluR1 and GluR2 after ischemia from 6 h to 48 h (K-1, P > 0.05 vs sham). Scale bar = 50 lm.

uorescent intensity of GluR1 was 92.3% 2.96 at 6 h,

91.2% 5.42 at 12 h, 93.4% 3.66 at 24 h, and
96.0% 7.55 at 48 h, all P > 0.05 compared with
sham (Fig. 6K-1). The uorescent intensity of GluR2
was 100.9% 4.39 at 6 h, 104.7% 3.58 at 12 h,
101.4% 4.25 at 24 h, and 104.9% 7.65 at 48 h
(Fig. 6K-1), all P > 0.05 compared with sham.

Changes in protein levels of NMDAR receptor

subunits in the CA1 and CA3 areas after ischemia
In order to verify the immunouorescence results, we
used western blotting to detect the protein levels of the
NR2A and NR2B subunits before and after ischemia. All
data were normalized to endogenous control protein
(b-tubulin, as 100%). The expression pattern of the

X.-J. Han et al. / Neuroscience 327 (2016) 6478

NR2A and NR2B protein levels was similar to that

observed in the uorescent intensity analysis of the CA1
and CA3 areas (Fig. 7). In the CA1 area, the protein
level of the NR2A subunit signicantly decreased from
0.66 0.05 at 6 h to 0.66 0.04 at 12 h, 0.66 0.05
at 24 h and 0.65 0.05 at 48 h after ischemia, all
P < 0.05 vs sham (Fig. 7A, B). The protein level of
NR2B subunit decreased from 0.90 0.03 (6 h), to
0.89 0.02 (12 h), 0.87 0.05 (24 h) and 0.88 0.03
(Is 48 h) after ischemia, (*P < 0.05, #P < 0.01 vs sham)
(Fig. 7A, B). Because the reduction of NR2A protein
was much larger than the reduction of the NR2B
protein, the NR2B/NR2A ratio was signicantly
increased at dierent reperfusion times observed after
ischemia. It was 0.849 0.06 in sham group, 1.212
0 at 6 h, 1.222 0.09 at 12 h, 1.203 0.09 at 24 h,
1.191 0.105 and at 48 h after ischemia, all P < 0.05
vs sham (Fig. 7C). In the CA3 area, there were no

73

signicant changes in either NR2A or NR2B protein

levels before or after ischemia (P > 0.05) (Fig. 7D, E).
Due to the relatively stable expression of the NR2B and
NR2A subunits, theNR2B/NR2A ratio did not change
signicantly after ischemia; it was 0.98 0.11 in the
sham group, 1.03 0.223 at 6 h, 1.00 0.01 at 12 h,
1.09 0.23 at 24 h and 0.99 0.09 at 48 h groups
after ischemia (Fig. 7F).
Changes in protein levels of AMPA receptor subunits
in the CA1 and CA3 areas after ischemia
Next, we used western blotting to detect the protein levels
of the GluR1 and GluR2 subunits before and after
ischemia. All data were normalized to an endogenous
protein (b-tubulin, as 100%). The expression pattern of
the GluR1 and GluR2 proteins was similar to that
observed in the uorescent intensity analysis of the CA1

Fig. 7. Changes in protein levels of NMDAR receptor subunits (NR2B and NR2A) in the CA1 and CA3 areas after ischemia were detected by
Western Blotting method. (A) Western blotting bands of NR2B, NR2A and tubulin in the CA1 area before and after ischemia. (B) Quantitative
analysis showed that NR2B protein levels in the CA1 area declined signicantly from 6 h to 48 h after ischemia (*P < 0.05 at 6 h, 24 h and 48 h,
#
P < 0.01 at 12 h vs sham). NR2A protein levels dramatically decreased to 65% after ischemia (P < 0.01 at 6 h, 12 h, 24 h and 48 h vs sham).
Therefore, the ratio of NR2B/NR2A at each time point after ischemia was higher compared to the normal group, P < 0.05 (C). However, In the CA3
area, NR2B and NR2A protein levels did not signicantly change at dierent time points after ischemia, P > 0.05 vs sham, (D, E). Additionally, there
was no signicant dierence in the ratio of NR2B/NR2A before or after ischemia (F), P > 0.05 vs sham.

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X.-J. Han et al. / Neuroscience 327 (2016) 6478

and CA3 areas (Fig. 8). In the CA1 area, the GluR1 protein
level was 1 0.06 in the sham group, 0.99 0.05 at 6-h,
0.99 0.08 at 12-h, 0.98 0.04 at 24-h and 0.98 0.04
at 48-h groups (Fig. 8A, B). There was no signicant
dierence observed before and after ischemia
(P > 0.05). However GluR2 protein level signicantly
decreased to 0.77 0.02 (6 h), 0.74 0.03 (12 h), 0.73
0.04 (24 h) and 0.73 0.06 (48 h) after ischemia, all
P < 0.05 compared to the sham (1 0.07). Therefore,
the GluR1/GluR2 ratio signicantly increased to 1.306
0.029 at 6 h, 1.354 0.058 at 12 h, 1.352 0.012 at
24 h, and 1.375 0.054 at 48 h, all P < 0.05 compared
to sham (1.018 0.016) (Fig. 8C). However, in the CA3
area, both GluR1 and GluR2 protein levels did not
signicantly change before or after ischemia (Fig. 8D, E).
There was also no signicant dierence observed in the
GluR1/GluR2 ratio before or after ischemia (Fig. 8F), all
P > 0.05.

DISCUSSION
The present study revealed that transient global ischemia
was able to induce an increase in the ratio of NR2B/NR2A
subunits as well as the ratio of GluR1/GluR2 subunits in
the CA1 region of the hippocampus within 2 days after
reperfusion. Despite this, there were no detectable
changes in the expression of NR2B, NR2A, GluR1 and
GluR2 subunits in the CA3 region after ischemia. The
number of synapses and their PSD thickness at the
CA3 region also remained unchanged.
Dierent changes in expressions of glutamate
receptor subunits in the CA1 and CA3 areas after
transient global ischemia
In the present study we demonstrated that both the NR2A
and NR2B subunit protein level in the CA1 area is
decreased within 6 h to 24 h after ischemia. However,

Fig. 8. Changes in protein levels of AMPAR receptor subunits (GluR1 and GluR2) in the CA1 and CA3 areas after ischemia detected by Western
Blotting method. (A) Western blot bands of GluR1, GluR2 and tubulin in the CA1 area before and after ischemia. (B) Quantitative analysis showed
that GluR1 protein levels in the CA1 area did not signicantly change at either time point evaluated after ischemia (all P > 0.05 vs sham). However,
GluR2 protein level signicantly decreased at dierent time points after ischemia (all P < 0.05 vs sham). Overall, the ratio of GluR1/GluR2 was
higher at 6 h (P < 0.05), 12 h (P < 0.05), 24 h (P < 0.01), and 48 h (P < 0.05) after ischemia compared to the normal group (C). However, In the
CA3 area, GluR1 and GluR2 protein levels did not signicantly change at dierent time points after ischemia, P > 0.05 vs sham (D, E). Additionally,
there was also no signicant dierence detected in the ratio of GluR1/GluR2 before or after ischemia, P > 0.05 vs sham (F).

X.-J. Han et al. / Neuroscience 327 (2016) 6478

75

the ratio of NR2B /NR2A is signicantly increased overall

because NR2A protein levels appear much lower than
NR2B protein levels. In the CA3 region, NR2B and
NR2A protein level was not signicantly changed when
observed at the same periods after ischemia. These
results are consistent with observed changes in the
mRNA level of these receptor subunits in the CA1 area
which have been previously induced by the same
ischemic model (Zhang et al., 1997; Hsu et al., 1998;
Dos-Anjos et al., 2009b). As far as AMPA receptor
subunits, our results showed that GluR2 protein level is
signicantly decreased in the CA1 area between 6 h
and 48 h after ischemia. GluR1 protein level, on the other
hand, remained unchanged which resulted in an increase
in the ratio of GluR1/GluR2 subunits. As with NMDA
receptors in the CA3 area, the protein levels of AMPA
receptor subunits in the CA3 area did not change when
observed at the same periods after ischemia. These
results are also consistent with changes in expression of
GluR2 and GluR1 mRNA in the CA1 and CA3 regions
of previous studies (Pellegrini-Giampietro et al., 1994;
Dos-Anjos et al., 2009a).
Alterations in expression of NMDA and AMPA
receptor subunits in the CA1 area and CA3 area may
contribute to dierential vulnerability to ischemic injury.
Many studies have shown that excess calcium inux is
the major cause of CA1 neuronal death after ischemia.
Overexpression of calcium permeable AMPA receptor
subunits as well as knockdown of GluR2 expression has
been shown to induce ischemic neuronal death in the
CA1 region (Oguro et al., 1999; Opitz et al., 2000; Anzai
et al., 2003). Here, our results also support the idea that
ischemia may induce selective downregulation of the
GluR2 subunit without aecting GluR1 subunit, an event
which can lead to neuronal death in the CA1. Furthermore, ischemia was also shown to dramatically inhibit
the expression of NR2A (which has been linked to activation of survival pathways), and subsequently induce activation of the NR2B subunit which has been implicated in
neuronal apoptosis. It has been reported that stimulation
of NR2A induces activation of CREB and CREB-evoked
BDNF, a mediator of neuroprotective action (Leveille
et al., 2008). However, stimulating extrasynaptic NR2B
can trigger the CREB shut-o pathway and activate
apoptotic pathway, prompting the interaction with deathassociated protein kinase 1 (DAPK1), Ca2+/calmodulin
(CaM)-dependent protein kinase II (CaMKII) and protein
kinase A (PKA), ultimately culminating in neuronal death
(Wahl et al., 2009; Tu et al., 2010; Vizi et al., 2013).

play a crucial role in the regulation of the number and morphology of postsynaptic membranes on dendritic spines
via interaction with CaMKII and actin (Barria and
Malinow, 2005; Bourne and Harris, 2008; Okamoto
et al., 2009; Foster et al., 2010; Shonesy et al., 2014),
Therefore, changes in expression of glutamate receptors
will aect the synaptic morphology and plasticity. In our
previous study, we found that transient global ischemia
induced increases in the number of dendritic spines and
excitatory synapses 12 h and 24 h after reperfusion, and
an increase in the thickness of the PSD (Ruan et al.,
2009, 2012). Postischemic enlargement of PSD has also
been found in cortical neurons and CA1 neurons (Ito
et al., 2006; Kovalenko et al., 2006). In the present study,
we further demonstrate that expression of both NMDARs
and AMPARs is down-regulated in the CA1 area during
these post-ischemic periods. The increase in the number
of dendritic spines and excitatory synapses may be due to
a feedback stimulation which lowers the levels of
NMDARs and AMPARs after ischemia. The increase in
the thickness of the PSD appears not directly related to
the expression of glutamate receptor subunits since both
NMDAR and AMPAR subunits are down regulated. However, a relative increase of NR2B after ischemia may
enhance the binding of CaMKII. It has been reported that
oxidation of calmodulin results in increased CaMKII binding to the NR2B subunit (Robison et al., 2007) and causes
aggregation of CaMKII in the PSD (Shetty et al., 2008).
This may be one possible reason for the observed
increased thickness of the PSD after ischemia. Ischemia
also enhances the interaction between NR2BPSD95
nNOS (Aarts et al., 2002), which may be another reason
for the thickening of the PSD after ischemia. In addition,
ischemia also stimulates the dramatic accumulation of
N-ethylmaleimide-sensitive fusion protein, heat shock
cognate protein 70, and protein kinase C in the cortex
(Martone et al., 1999). This may also contribute to the
increased thickness of PSD. Excess accumulation of
these proteins in the PSD may damage synaptic plasticity
and ultimately lesion neurons. In comparison with the CA1
area, neither NMDAR nor AMPAR protein levels nor the
number of excitatory synapses in the CA3 area were
notably increased within 2 days of evaluation after reperfusion. This evidently implies that ischemia induces
dramatic changes in synaptic number and structure and
glutamate receptor expression in the CA1 area, such
changes which will ultimately enhance the vulnerability
of neurons and eventually lead to CA1 pyramidal neuron
death.

Dierent changes in the postsynaptic density in the

CA1 and CA3 regions after ischemia

Dierent response to ischemia between CA1 and CA3

neurons

The postsynaptic density is an area where postsynaptic

membrane proteins are intensively specialized. The
PSD is mainly formed by glutamate receptors (AMPA
and NMDA receptor), skeleton protein (PSD95),
cytosolic proteins, and signaling proteins (PKC and
CaMKII) (Farley et al., 2015). Proteins of the postsynaptic
density vary in response to synaptic activities, which in
turn may lead to structural plasticity (Dosemeci et al.,
2001; Meyer et al., 2014). AMPA and NMDA receptors

Between our previous and present studies, we have

demonstrated that ischemia induces changes in the
expression of glutamate receptors and synaptic
morphology of CA1 neurons but not CA3 neurons (Ruan
et al., 2012) before neuronal death occurs in the CA1
region. One possibility is that the presynaptic input,
though mainly received from excitatory aerents in both
CA1 and CA3, CA3 neurons receive more GABA innervation than CA1 neurons (Yao et al., 1996). Consequently,

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X.-J. Han et al. / Neuroscience 327 (2016) 6478

CA3 neurons have strong inhibitory ability against

ischemic excitotoxicity. Another possibility may be related
to the expression of glutamate receptors in the postsynaptic membrane. Under normal physiological conditions,
although there are no dierences in NR2A levels between
CA1 and CA3 regions, expressions of NMDA receptor
subunits (NR2B and NR1) and AMPA receptor subunits
(GluR1 and GluR2) are higher in the CA1 region than in
the CA3 area (Coultrap et al., 2005; Butler et al., 2010).
This may be one possible pathway for the increased sensitivity of CA1 neurons to ischemic excitotoxicity. In recent
years, it has also been reported that ischemia selectively
induces the expression of hamartin in CA3 neurons
(Papadakis et al., 2013). Hamartin is an endogenous neuroprotective factor which can confer resistance to
ischemic injury by increasing autophagy through an
mTorC1-dependent mechanism (Papadakis et al.,
2013). Collectively there is ample evidence, including differences in presynaptic input, expression of glutamate
receptor subunits and endogenous neuroprotection, that
CA1 neurons are more vulnerable to ischemic injury than
CA3 neurons. Therefore, try to nd a way to maintain the
balance in synaptic plasticity, expression of glutamate
receptor subunits and interruption of the accumulation of
abnormal protein in the PSD may be one of better treatment strategies for ischemic injury.
AcknowledgmentsThis study was supported by the National
Natural Science Foundation of China (NNSFC, 30971530);
National Program on Key Basic Research Project of China (973
Program, 2011CB707501); the Program of Introducing Talents
of Discipline to Universities from China (B14036), Science and
Technology programs of Guangdong Province (20140504), and
0070048 (AHA). We thank Clarity Manuscript Consultants LLC
for assistance editing the manuscript.

REFERENCES
Aarts M, Liu Y, Liu L, Besshoh S, Arundine M, Gurd JW, Wang YT,
Salter MW, Tymianski M (2002) Treatment of ischemic brain
damage by perturbing NMDA receptor-PSD-95 protein
interactions. Science 298:846850.
Anzai T, Tsuzuki K, Yamada N, Hayashi T, Iwakuma M, Inada K,
Kameyama K, Hoka S, Saji M (2003) Overexpression of Ca2+permeable AMPA receptor promotes delayed cell death of
hippocampal CA1 neurons following transient forebrain
ischemia. Neurosci Res 46:4151.
Arundine M, Tymianski M (2003) Molecular mechanisms of calciumdependent neurodegeneration in excitotoxicity. Cell Calcium
34:325337.
Barria A, Malinow R (2005) NMDA receptor subunit composition
controls synaptic plasticity by regulating binding to CaMKII.
Neuron 48:289301.
Bourne JN, Harris KM (2008) Balancing structure and function at
hippocampal dendritic spines. Annu Rev Neurosci 31:4767.
Butler TR, Self RL, Smith KJ, Sharrett-Field LJ, Berry JN, Littleton
JM, Pauly JR, Mulholland PJ, Prendergast MA (2010) Selective
vulnerability of hippocampal cornu ammonis 1 pyramidal cells to
excitotoxic insult is associated with the expression of polyaminesensitive
N-methyl-D-aspartate-type
glutamate
receptors.
Neuroscience 165:525534.
Chen PE, Wyllie DJ (2006) Pharmacological insights obtained from
structure-function studies of ionotropic glutamate receptors. Br J
Pharmacol 147:839853.

Chen X, Nelson CD, Li X, Winters CA, Azzam R, Sousa AA, Leapman

RD, Gainer H, Sheng M, Reese TS (2011) PSD-95 is required to
sustain the molecular organization of the postsynaptic density. J
Neurosci 31:63296338.
Coultrap SJ, Nixon KM, Alvestad RM, Valenzuela CF, Browning MD
(2005) Dierential expression of NMDA receptor subunits and
splice variants among the CA1, CA3 and dentate gyrus of the
adult rat. Brain Res Mol Brain Res 135:104111.
Cull-Candy SG, Leszkiewicz DN (2004) Role of distinct NMDA
receptor subtypes at central synapses. Sci STKE 2004:re16.
Dos-Anjos S, Martinez-Villayandre B, Montori S, Regueiro-Purrinos
MM, Gonzalo-Orden JM, Fernandez-Lopez A (2009a) Global
ischemia-induced modications in the expression of AMPA
receptors and inammation in rat brain. Brain Res 1287:2027.
Dos-Anjos S, Martinez-Villayandre B, Montori S, Regueiro-Purrinos
MM, Gonzalo-Orden JM, Fernandez-Lopez A (2009b) Transient
global ischemia in rat brain promotes dierent NMDA receptor
regulation depending on the brain structure studied. Neurochem
Int 54:180185.
Dosemeci A, Tao-Cheng JH, Vinade L, Winters CA, Pozzo-Miller L,
Reese TS (2001) Glutamate-induced transient modication of the
postsynaptic density. Proc Natl Acad Sci U S A 98:1042810432.
Farley MM, Swulius MT, Waxham MN (2015) Electron tomographic
structure and protein composition of isolated rat cerebellar,
hippocampal and cortical postsynaptic densities. Neuroscience
304:286301.
Fiala JC, Feinberg M, Popov V, Harris KM (1998) Synaptogenesis via
dendritic lopodia in developing hippocampal area CA1. J
Neurosci 18:89008911.
Foster KA, McLaughlin N, Edbauer D, Phillips M, Bolton A,
Constantine-Paton M, Sheng M (2010) Distinct roles of NR2A
and NR2B cytoplasmic tails in long-term potentiation. J Neurosci
30:26762685.
Furukawa H, Singh SK, Mancusso R, Gouaux E (2005) Subunit
arrangement and function in NMDA receptors. Nature
438:185192.
Gao TM, Howard EM, Xu ZC (1998) Transient neurophysiological
changes in CA3 neurons and dentate granule cells after severe
forebrain ischemia in vivo. J Neurophysiol 80:28602869.
Gladding CM, Raymond LA (2011) Mechanisms underlying NMDA
receptor synaptic/extrasynaptic distribution and function. Mol Cell
Neurosci 48:308320.
Gorter JA, Petrozzino JJ, Aronica EM, Rosenbaum DM, Opitz T,
Bennett MV, Connor JA, Zukin RS (1997) Global ischemia
induces downregulation of Glur2 mRNA and increases AMPA
receptor-mediated Ca2+ inux in hippocampal CA1 neurons of
gerbil. J Neurosci 17:61796188.
Hardingham GE, Bading H (2010) Synaptic versus extrasynaptic
NMDA receptor signalling: implications for neurodegenerative
disorders. Nat Rev Neurosci 11:682696.
Hsu JC, Zhang Y, Takagi N, Gurd JW, Wallace MC, Zhang L,
Eubanks JH (1998) Decreased expression and functionality of
NMDA receptor complexes persist in the CA1, but not in the
dentate gyrus after transient cerebral ischemia. J Cereb Blood
Flow Metabol 18:768775.
Ito U, Kawakami E, Nagasao J, Kuroiwa T, Nakano I, Oyanagi K
(2006) Restitution of ischemic injuries in penumbra of cerebral
cortex after temporary ischemia. Acta Neurochir Suppl
96:239243.
Kessels HW, Malinow R (2009) Synaptic AMPA receptor plasticity
and behavior. Neuron 61:340350.
Kirino T (1982) Delayed neuronal death in the gerbil hippocampus
following ischemia. Brain Res 239:5769.
Kohr G (2006) NMDA receptor function: subunit composition versus
spatial distribution. Cell Tissue Res 326:439446.
Kovalenko T, Osadchenko I, Nikonenko A, Lushnikova I, Voronin K,
Nikonenko I, Muller D, Skibo G (2006) Ischemia-induced
modications in hippocampal CA1 stratum radiatum excitatory
synapses. Hippocampus 16:814825.
Leveille F, El Gaamouch F, Gouix E, Lecocq M, Lobner D, Nicole O,
Buisson A (2008) Neuronal viability is controlled by a functional

X.-J. Han et al. / Neuroscience 327 (2016) 6478

relation between synaptic and extrasynaptic NMDA receptors.
FASEB J 22:42584271.
Liu S, Lau L, Wei J, Zhu D, Zou S, Sun HS, Fu Y, Liu F, Lu Y (2004)
Expression of Ca(2+)-permeable AMPA receptor channels
primes cell death in transient forebrain ischemia. Neuron 43:
4355.
Malenka RC (2003) Synaptic plasticity and AMPA receptor tracking.
Ann N Y Acad Sci 1003:111.
Martin LJ, Al-Abdulla NA, Brambrink AM, Kirsch JR, Sieber FE,
Portera-Cailliau C (1998) Neurodegeneration in excitotoxicity,
global cerebral ischemia, and target deprivation: a perspective on
the contributions of apoptosis and necrosis. Brain Res Bull
46:281309.
Martone ME, Hu BR, Ellisman MH (2000) Alterations of hippocampal
postsynaptic densities following transient ischemia. Hippocampus
10:610616.
Martone ME, Jones YZ, Young SJ, Ellisman MH, Zivin JA, Hu BR
(1999) Modication of postsynaptic densities after transient
cerebral ischemia: a quantitative and three-dimensional
ultrastructural study. J Neurosci 19:19881997.
Matsuzaki M, Ellis-Davies GC, Nemoto T, Miyashita Y, Iino M, Kasai
H (2001) Dendritic spine geometry is critical for AMPA receptor
expression in hippocampal CA1 pyramidal neurons. Nat Neurosci
4:10861092.
Mayer ML, Armstrong N (2004) Structure and function of glutamate
receptor ion channels. Annu Rev Physiol 66:161181.
Meyer D, Bonhoeer T, Scheuss V (2014) Balance and stability of
synaptic structures during synaptic plasticity. Neuron 82:430443.
Nakanishi N, Shneider NA, Axel R (1990) A family of glutamate
receptor genes: evidence for the formation of heteromultimeric
receptors with distinct channel properties. Neuron 5:569581.
Oguro K, Oguro N, Kojima T, Grooms SY, Calderone A, Zheng X,
Bennett MV, Zukin RS (1999) Knockdown of AMPA receptor
GluR2 expression causes delayed neurodegeneration and
increases damage by sublethal ischemia in hippocampal CA1
and CA3 neurons. J Neurosci 19:92189227.
Okamoto K, Bosch M, Hayashi Y (2009) The roles of CaMKII and Factin in the structural plasticity of dendritic spines: a potential
molecular identity of a synaptic tag? Physiology (Bethesda)
24:357366.
Opitz T, Grooms SY, Bennett MV, Zukin RS (2000) 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 U S A 97:1336013365.
Paoletti P (2011) Molecular basis of NMDA receptor functional
diversity. Eur J Neurosci 33:13511365.
Papadakis M, Hadley G, Xilouri M, Hoyte LC, Nagel S, McMenamin
MM, Tsaknakis G, Watt SM, Drakesmith CW, Chen R, Wood MJ,
Zhao Z, Kessler B, Vekrellis K, Buchan AM (2013) Tsc1
(hamartin) confers neuroprotection against ischemia by inducing
autophagy. Nat Med 19:351357.
Pellegrini-Giampietro DE, Pulsinelli WA, Zukin RS (1994) NMDA and
non-NMDA receptor gene expression following global brain
ischemia in rats: eect of NMDA and non-NMDA receptor
antagonists. J Neurochem 62:10671073.
Petito CK, Pulsinelli WA (1984) Delayed neuronal recovery and
neuronal death in rat hippocampus following severe cerebral
ischemia: possible relationship to abnormalities in neuronal
processes. J Cereb Blood Flow Metabol 4:194205.
Pulsinelli WA, Brierley JB, Plum F (1982) Temporal prole of neuronal
damage in a model of transient forebrain ischemia. Ann Neurol
11:491498.
Robison AJ, Winder DG, Colbran RJ, Bartlett RK (2007) Oxidation of
calmodulin alters activation and regulation of CaMKII. Biochem
Biophys Res Commun 356:97101.
Rogawski MA (2013) AMPA receptors as a molecular target in
epilepsy therapy. Acta Neurol Scand Suppl:918.
Ruan YW, Han XJ, Shi ZS, Lei ZG, Xu ZC (2012) Remodeling of
synapses in the CA1 area of the hippocampus after transient
global ischemia. Neuroscience 218:268277.

77

Ruan YW, Lei Z, Fan Y, Zou B, Xu ZC (2009) Diversity and uctuation

of spine morphology in CA1 pyramidal neurons after transient
global ischemia. J Neurosci Res 87:6168.
Ruan YW, Zou B, Fan Y, Li Y, Lin N, Zeng YS, Gao TM, Yao Z, Xu ZC
(2006) Dendritic plasticity of CA1 pyramidal neurons after
transient global ischemia. Neuroscience 140:191201.
Rumbaugh G, Vicini S (1999) Distinct synaptic and extrasynaptic
NMDA receptors in developing cerebellar granule neurons. J
Neurosci 19:1060310610.
Sessoms-Sikes S, Honse Y, Lovinger DM, Colbran RJ (2005)
CaMKIIalpha enhances the desensitization of NR2B-containing
NMDA receptors by an autophosphorylation-dependent
mechanism. Mol Cell Neurosci 29:139147.
Shetty PK, Huang FL, Huang KP (2008) Ischemia-elicited oxidative
modulation of Ca2+/calmodulin-dependent protein kinase II. J
Biol Chem 283:53895401.
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 284:18111816.
Shonesy BC, Jalan-Sakrikar N, Cavener VS, Colbran RJ (2014)
CaMKII: a molecular substrate for synaptic plasticity and memory.
Prog Mol Biol Transl Sci 122:6187.
Silverman JB, Restituito S, Lu W, Lee-Edwards L, Khatri L, Zi EB
(2007) Synaptic anchorage of AMPA receptors by cadherins
through neural plakophilin-related arm protein AMPA receptorbinding protein complexes. J Neurosci 27:85058516.
Song I, Huganir RL (2002) Regulation of AMPA receptors during
synaptic plasticity. Trends Neurosci 25:578588.
Stein E, Cox JA, Seeburg PH, Verdoorn TA (1992) Complex
pharmacological properties of recombinant alpha-amino-3hydroxy-5-methyl-4-isoxazole propionate receptor subtypes. Mol
Pharmacol 42:864871.
Stocca G, Vicini S (1998) Increased contribution of NR2A subunit to
synaptic NMDA receptors in developing rat cortical neurons. J
Physiol 507(Pt 1):1324.
Takumi Y, Ramirez-Leon V, Laake P, Rinvik E, Ottersen OP (1999)
Dierent modes of expression of AMPA and NMDA receptors in
hippocampal synapses. Nat Neurosci 2:618624.
Tovar KR, Westbrook GL (1999) The incorporation of NMDA
receptors with a distinct subunit composition at nascent
hippocampal synapses in vitro. J Neurosci 19:41804188.
Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM,
Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R (2010)
Glutamate receptor ion channels: structure, regulation, and
function. Pharmacol Rev 62:405496.
Tu W, Xu X, Peng L, Zhong X, Zhang W, Soundarapandian MM, Balel
C, Wang M, Jia N, Zhang W, Lew F, Chan SL, Chen Y, Lu Y
(2010) DAPK1 interaction with NMDA receptor NR2B subunits
mediates brain damage in stroke. Cell 140:222234.
Vizi ES, Kisfali M, Lorincz T (2013) Role of nonsynaptic GluN2Bcontaining NMDA receptors in excitotoxicity: evidence that
uoxetine selectively inhibits these receptors and may have
neuroprotective eects. Brain Res Bull 93:3238.
Wahl AS, Buchthal B, Rode F, Bomholt SF, Freitag HE, Hardingham
GE, Ronn LC, Bading H (2009) Hypoxic/ischemic conditions
induce expression of the putative pro-death gene Clca1 via
activation of extrasynaptic N-methyl-D-aspartate receptors.
Neuroscience 158:344352.
Whitlock JR, Heynen AJ, Shuler MG, Bear MF (2006) Learning
induces long-term potentiation in the hippocampus. Science
313:10931097.
Xu ZC, Gao TM, Ren Y (1999) Neurophysiological changes
associated with selective neuronal damage in hippocampus
following transient forebrain ischemia. Biol Signals Recept 8:
294308.
Yao ZB, Li X, Xu ZC (1996) GABAergic and asymmetrical synapses
on somata of GABAergic neurons in CA1 and CA3 regions of rat
hippocampus. A quantitative electron microscopic analysis.
Stroke 27:14111415. discussion 14151416.

78

X.-J. Han et al. / Neuroscience 327 (2016) 6478

Zhang L, Hsu JC, Takagi N, Gurd JW, Wallace MC, Eubanks JH (1997)
Transient global ischemia alters NMDA receptor expression in rat
hippocampus: correlation with decreased immunoreactive protein
levels of the NR2A/2B subunits, and an altered NMDA receptor
functionality. J Neurochem 69:19831994.

Zhou Q, Sheng M (2013) NMDA receptors in nervous system

diseases. Neuropharmacology 74:6975.
Zorumski CF, Izumi Y (2012) NMDA receptors and metaplasticity:
mechanisms and possible roles in neuropsychiatric disorders.
Neurosci Biobehav Rev 36:9891000.

(Accepted 8 April 2016)

(Available online 16 April 2016)