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The Journal of Clinical Investigation ReseaRch aRticle
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Introduction
Brain ischemia is the fourth cause of death and the leading cause
of long-term disability in industrialized countries . Relatively
short periods of blood ow interruption in the brain can pro-
duce irreversible neuronal damage . Energy failure and ox-
ygen deprivation that occur in ischemic episodes induce a loss
of membrane potential in neurons and glia, a process known
as anoxic depolarization AD, which spreads across suscepti-
ble brain tissue as a self-propagating wave-like depolarization
, and can be initiated by factors that release K and gluta-
mate . Recordings from neurons in hippocampal and cerebel-
lar slices have shown that this AD is associated with a large gluta-
mate-evoked inward current, which can be blocked by a cocktail
of agents blocking ionotropic glutamate receptors , . In par-
ticular, activation of NMDA receptors NMDARs plays a crucial
role in neuronal cell death .
NMDAR-mediated signaling can be either benecial or
deleterious, and this dichotomous behavior has been proposed
to be related to its localization within or outside the synapse
, . Activation of NMDARs in synapses provides plasticity and
cell survival signals, whereas extrasynaptic NMDARs trigger
neurodegeneration refs. ; but see also refs. , . These
opposing signals are transduced to and discriminated by the nu-
cleus on the basis of the dierential phosphorylation state of the
Jacob protein messenger . However, most of these data have
been obtained in vitro, and the role of synaptic or extrasynaptic
NMDARs in ischemic neuronal damage has not been studied in
a more intact preparation.
The functional dichotomy of NMDAR signaling would also
depend on the location of the glutamate source. A major source
of extrasynaptic glutamate is the cystine/glutamate antiporter
, also known as system xc–, a solute carrier identied as the
main source of nonsynaptic glutamate in the brain .
This transport system is a membrane-bound, Cl–-dependent,
Na-independent antiporter that mediates the cellular uptake of
cystine in exchange for glutamate at a : ratio . Struc-
turally, it is a heterodimer composed of a heavy-chain subunit,
Fhc, and a light-chain–specic subunit, xCT . System
xc– is an important source of cystine, which is intracellularly
converted to cysteine, the rate-limiting substrate in glutathione
synthesis . The high rate of oxygen consumption in the brain
renders this antiporter vital to antioxidant defense , and its
expression is rapidly upregulated in vitro under conditions of
oxidative stress , . Nonetheless, the obligate exchange of
During brain ischemia, an excessive release of glutamate triggers neuronal death through the overactivation of NMDA
receptors (NMDARs); however, the underlying pathways that alter glutamate homeostasis and whether synaptic or
extrasynaptic sites are responsible for excess glutamate remain controversial. Here, we monitored ischemia-gated currents
in pyramidal cortical neurons in brain slices from rodents in response to oxygen and glucose deprivation (OGD) as a real-time
glutamate sensor to identify the source of glutamate release and determined the extent of neuronal damage. Blockade of
excitatory amino acid transporters or vesicular glutamate release did not inhibit ischemia-gated currents or neuronal damage
after OGD. In contrast, pharmacological inhibition of the cystine/glutamate antiporter dramatically attenuated ischemia-gated
currents and cell death after OGD. Compared with control animals, mice lacking a functional cystine/glutamate antiporter
exhibited reduced anoxic depolarization and neuronal death in response to OGD. Furthermore, glutamate released by the
cystine/glutamate antiporter activated extrasynaptic, but not synaptic, NMDARs, and blockade of extrasynaptic NMDARs
reduced ischemia-gated currents and cell damage after OGD. Finally, PET imaging showed increased cystine/glutamate
antiporter function in ischemic rats. Altogether, these data suggest that cystine/glutamate antiporter function is increased
in ischemia, contributing to elevated extracellular glutamate concentration, overactivation of extrasynaptic NMDARs, and
ischemic neuronal death.
Extrasynaptic glutamate release through
cystine/glutamate antiporter contributes
to ischemic damage
Federico N. Soria,1 Alberto Pérez-Samartín,1 Abraham Martin,2 Kiran Babu Gona,3 Jordi Llop,3 Boguslaw Szczupak,2
Juan Carlos Chara,1 Carlos Matute,1 and María Domercq1
1Centro de Investigaciones Biomédicas en Red (CIBERNED), Achucarro Basque Center for Neuroscience and Departamento de Neurociencias, Universidad del País Vasco (UPV/EHU), Leioa, Spain.
2Molecular Imaging Unit and 3Radiochemistry Department, CIC biomaGUNE, San Sebastián, Spain.
Related Commentary: p. 3279
Conflict of interest: The authors have declared that no conflict of interest exists.
Submitted: July 9, 2013; Accepted: May 21, 2014.
Reference information: J Clin Invest. 2014;124(8):3645–3655. doi:10.1172/JCI71886.
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accumulation. Although these results do not rule out that OGD
partially reduces glutamate uptake or that a minor population of
EAATs could indeed be working in reverse-uptake mode, they
strongly suggest that another source of glutamate contributes to
the alteration of glutamate homeostasis in ischemia.
We also tested the eect of EAAT blockade in cell death using
organotypic cortical slices . TBOA exacerbated cell death af-
ter OGD and reoxygenation, as assessed by lactic dehydrogenase
LDH release assays Figure D, and see also in Supplemental
Figure the eect of TBOA in normoxia; supplemental material
available online with this article; doi:./JCIDS. Paral-
lel staining with propidium iodide revealed cell death in the cortex,
which was proportional to the damage quantied by LDH release
Figure E. TBOA-mediated exacerbation in excitotoxic damage in
hypoxia/ischemia has also been observed in cultured neurons
and organotypic cortical slices , conrming the primary physi-
ological role of EAATs in the clearance of extracellular glutamate.
Inhibition of exocytosis has no eect on ischemia-gated currents
or cell death. An increase in vesicular synaptic glutamate release
and in miniature excitatory postsynaptic currents mEPSCs oc-
curs at early phases of ischemia and before AD , , . We
therefore tested whether vesicular release was contributing to
the increase in AD currents in acute slices by using balomycin
A Baf A; μM, a vacuolar type H-ATPase inhibitor, to block
neurotransmitter loading into synaptic vesicles. Baf A practically
abolished the mEPSCs Supplemental Figure , thus indicating
an eective inhibition of spontaneous vesicular glutamate re-
lease. In contrast, we found that neither the latency nor the ampli-
tude of the anoxic current was changed when Baf A was present
at the onset of OGD Figure , A and B. Accordingly, neuronal
damage was not signicantly dierent in Baf A–treated μM
or tetanus toxin–treated TeTN-treated; μg/ml slices compared
with OGD alone Figure , C and D. These results suggest that
vesicular release after OGD does not signicantly contribute to
AD currents and toxicity and are consistent with an extrasynaptic
source of glutamate in ischemia.
Blockade of the cystine/glutamate exchanger reduces ischemia-
gated currents and neuronal damage after OGD. The cystine/
glutamate antiporter reportedly regulates extracellular gluta-
mate concentration in physiological , and pathological
conditions , , . We next analyzed whether ischemia-
induced glutamate release might be caused as a result of cystine
exchange by system xc–. To analyze the possible contribution
of the cystine/glutamate antiporter to glutamate release dur-
ing OGD, we applied S-carboxyphenylglycine CPG; μM
Figure A, a nonsubstrate inhibitor of the antiporter refs. ,
, and see also Supplemental Figure . We found that although
CPG did not change latency to AD, it signicantly reduced its
amplitude Figure , A and B. We further assessed the eect of
CPG on extracellular direct-current DC eld potentials
and showed that CPG signicantly reduced the ΔVo amplitude
after OGD Figure C. Because CPG is also a competitive in-
hibitor of group I metabotropic glutamate receptors mGluR
, we also tested sulfasalazine SAS; μM, another non-
substrate inhibitor of the cystine/glutamate antiporter ,
with no anity for glutamate receptors, and obtained a similar
attenuation of the AD current Figure , A and B. In contrast,
glutamate, which is released into the extracellular space, could
be deleterious to neuronal cells and other tissues that are sus-
ceptible to excitotoxic damage. Accordingly, the cystine/gluta-
mate antiporter is implicated in glutamate-associated disorders
such as glioma-derived epileptic seizures , oxidative gluta-
mate toxicity , and excitotoxic oligodendroglial death .
Although ischemia is a disorder that occurs in an environ-
ment of oxidative stress and lack of nutrients, which are induc-
ers of the cystine/glutamate antiporter , its contribution to
glutamate homeostasis alteration and neuronal cell death after
ischemia has not been explored before. Here, we analyze the
contribution of dierent mechanisms of glutamate release, in-
cluding the cystine/glutamate antiporter, and the role of synap-
tic versus extrasynaptic NMDARs in ischemia-gated currents
and neuronal damage.
Results
Inhibition of glutamate transporters shortens the latency to AD and
exacerbates neuronal damage in OGD. Na-dependent excitatory
amino acid transporters EAATs could contribute to alter gluta-
mate homeostasis during ischemic insults by mechanisms: a
decreased glutamate uptake, b reverse transport, and c hete-
roexchange. Severe chemical ischemic conditions decrease net
glutamate uptake within to minutes , and at later stages,
ischemia promotes reverse glutamate transport , . We there-
fore tested the eect of a broad-spectrum nonsubstrate antagonist
of EAATs, DL-threo-β-benzyloxyaspartic acid TBOA; μM on
OGD-gated currents in pyramidal cells from acute cortical slices,
and its eect on neuronal cell death in organotypic cortical slices.
OGD activated a large inward current within minutes Figure
, A and B, which corresponds to AD. This current was mainly
mediated by glutamate receptors, since a cocktail of ionotropic
glutamate receptor antagonists μM AP plus μM CNQX
applied concomitantly with OGD greatly reduced OGD-gated
currents Figure , A and B. Reduction of AD was not complete,
suggesting that other channels, such as the acid-sensitive anion
channel , the PX receptor , , and pannexin , also
contribute to the OGD current.
Application of TBOA during OGD did not decrease the am-
plitude of the OGD-activated current Figure , A and B. Fur-
thermore, TBOA reduced the latency to onset of AD in min-
utes Figure B, as previously observed by others . This
implies that in this paradigm, most transporters are still active
and work in direct uptake mode at the beginning of ischemia,
since its chronic blockade during OGD results in increased con-
centrations of glutamate and reduced latencies to AD. Indeed,
we found that OGD-activated currents in the presence of TBOA
were similarly blocked by glutamate receptor antagonists Fig-
ure , A and B. EAATs reverse as a result of alterations in the
transmembrane gradients once AD has been initiated . Thus,
to ensure that we were blocking EAATs when they are mostly in
reverse-uptake mode, we applied TBOA at the peak of the AD.
Blockade of EAATs after the onset of AD still potentiated the
amplitude of AD current Figure C, suggesting that, despite
the strongly reduced transmembrane sodium and potassium
concentration gradients, EAAT still takes up glutamate during
AD, and its blockade further enhances extracellular glutamate
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Next, we investigated whether
pharmacological inhibition of the
cystine/glutamate antiporter re-
duced ischemic damage in cerebral
cortex organotypic slices. Inhi-
bition of system xc– with CPG
and SAS μM signicantly
reduced neuronal damage after
OGD Figure , E and F in a way
similar to the neuroprotection ob-
served with NMDAR antagonist
MK μM; Figure , E and
F. In contrast, no inhibition of
OGD-induced neuronal damage
was detected with the mGluR an-
tagonist AIDA μM; Figure ,
E and F. To further conrm the
role played by the cystine/gluta-
mate antiporter, we performed
additional experiments in mutant
mice lacking a functional system
xc– Slcasut. These mice
have a large spontaneous deletion
from intron to the adjacent sut
region of the Slca gene, leading
to a truncated and nonfunctional
xCT protein. As observed with
the system xc– inhibitors, sut mice
showed a signicant reduction in
anoxic currents Figure , A and
B, DC extracellular potential
shifts Figure C, and neuronal
damage after OGD Figure , D
and E. Altogether, these data sug-
gest that the cystine/glutamate
antiporter is a relevant source
of glutamate release during is-
chemia and that its inhibition is
neuroprotective against ischemic
neuronal damage.
Cystine/glutamate antiporter
activates extrasynaptic NMDARs
in cortical neurons. Although the
cystine/glutamate antiporter has been proposed as an important
source of nonsynaptic glutamate , , , there is no direct
link between glutamate release by this transport system and the
activation of extrasynaptic glutamate receptors. Previous data
showed that glutamate release by the cystine/glutamate anti-
porter evokes glutamate receptor–mediated inward currents in
Purkinje cells upon acute exposure to cystine . Hence, we next
analyzed whether glutamate release induced by acute cystine ap-
plication could activate synaptic and/or extrasynaptic NMDARs.
Cortical neurons were clamped at – mV to remove Mg block
from NMDARs, and cystine mM was applied concomitantly
with glycine μM. Cystine evoked an inward current in most
cells, with a mean value pA, while glycine alone only in-
duced small inward currents Figure . Cystine-evoked currents
AIDA μM, a selective inhibitor of mGluR, did not aect
the OGD-activated current Figure , A and B, excluding any
role of these receptors in the eect observed with CPG. Interest-
ingly, we did not observe an increase in the amplitude of OGD
currents in TBOA-treated slices in the presence of CPG com-
pare Figure , A and B with Figure , B and D, which suggests
that blockade of glutamate uptake by EAATs does not increase
glutamate extracellular levels when the source of glutamate,
cystine/glutamate antiporter, is inhibited. These data are in ac-
cordance with data in normoxic conditions and reinforce
the important role played by the cystine/glutamate antiporter in
glutamate homeostasis during ischemia. It should be stressed,
however, that the latency to AD was still reduced when TBOA
was present along with CPG Figure B and see also Figure B.
Figure 1. Blockade of EAATs during OGD contributes to extracellular glutamate accumulation in acute slices
and organotypic cultures. (A) Voltage-clamp recording of cortical neurons at 30 mV in acute slices demon-
strates activation of a large current after OGD (n = 32). Ionotropic glutamate receptor antagonists AP5
(100 μM) plus CNQX (30 μM) significantly inhibited the OGD-activated current (n = 9). Nonsubstrate
broad-spectrum EAAT inhibitor DL-TBOA (100 μM) did not significantly change (but tended to increase) the
amplitude of the OGD-induced current and shortened the onset of AD in cortical neurons (n = 12), an eect
abolished when applied concomitantly with AP5 plus CNQX (n = 7). (B) Histograms showing the average am-
plitude (pA ± SEM) and latency (minutes ± SEM) of the OGD-induced current for each condition. Latency was
significantly reduced in the presence of TBOA. *P < 0.05 versus OGD. (C) TBOA increased the OGD-activated
current when applied after the onset of AD (n = 11). (D) Inhibition of EAATs exacerbated OGD-induced cell death
in organotypic slices, as demonstrated by increased LDH release in cultures subjected to 45 minutes of OGD and
24 hours of reoxygenation in the presence or absence of TBOA (100 μM). Data are expressed as the mean ± SEM
(n = 3–5). **P < 0.01 and *P < 0.05 versus OGD. (E) Representative fields demonstrate propidium iodide labeling
in cortical layers of slices treated as in D. Scale bar: 1 mm.
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trasynaptic receptors without interfering with synaptic activity ,
, also reduced the anoxic current Figure , A and C. None of
the antagonists, independently of their subunit specicity, altered
the latency to the anoxic current Figure C. These results suggest
that NRB-containing receptors play a role in AD in ischemia.
Finally, we checked the contribution of synaptic and extra-
synaptic NMDARs to neuronal damage after OGD in organotypic
cortical cultures. The NRA antagonists NVPAAM ,
and TCN did not reduce cell death signicantly Figure
, D and E. It should be noted, however, that high concentra-
tions of glycine in culture medium during reperfusion may reduce
TCN eectiveness . In contrast, memantine μM and
RO μM produced a massive reduction in OGD-induced
cell death Figure , D and E. Neuroprotection with memantine
was similar to that obtained with the NMDAR antagonist MK
μM; Figure , D and E. Altogether, these data indicate that
activation of extrasynaptic NMDARs contributes substantially to
excitotoxic death during OGD in organotypic cortical cultures and
suggest that these receptors are likely activated by extrasynaptic
tonic release of glutamate from the cystine/glutamate antiporter.
xCT expression and function are increased after ischemia in vitro
and in vivo. The excessive release of glutamate that causes neu-
ronal death during brain ischemia takes place in an environment of
oxidative stress , a condition that favors an increase in the ex-
pression and function of the cystine/glutamate antiporter , .
Therefore, we next analyzed the expression levels of the cata-
lytic subunit of the cystine/glutamate antiporter xCT in cortical
neuron cultures exposed to chemical ischemia in vitro, ischemic
conditions that induced massive cell death . . and
reproduced metabolic failure in the ischemic core. Real-time
quantitative PCR qPCR analysis demonstrated that xCT mRNA
was signicantly more abundant in neurons after hour
of chemical ischemia Figure A. To examine xCT levels by im-
munoblotting, total protein was extracted hours after chemical
ischemia to allow the translational machinery to produce xCT.
were completely abolished in the presence of the NMDA antago-
nist AP μM; Figure . To exclude a direct action of cystine
plus glycine, μM on NMDARs, we applied cystine in the pres-
ence of the cystine/glutamate antiporter blocker CPG μM.
Cystine-evoked currents were signicantly inhibited by CPG
Figure , suggesting that cystine evokes glutamate release and
indirectly activates NMDARs.
According to their localization in the cell membrane, NMDARs
dier in their subunit composition. Thus, synaptic NMDARs are
enriched in NRA subunits, while NRB subunits are located
mainly in extrasynaptic NMDARs , . This feature allows
for pharmacological blockade of either subtype by using subun-
it-specic antagonists. RO μM, an NRB antagonist
, almost totally blocked cystine-evoked inward currents Fig-
ure . In contrast, NVPAAM nM, which preferentially
inhibits NRA-containing receptors , did not change the am-
plitude of cystine-evoked inward currents Figure . These data
suggest that glutamate release in exchange with cystine activates
preferentially extrasynaptic NMDARs.
Antagonists of extrasynaptic, but not of synaptic, NMDARs at-
tenuate ischemia-gated currents and cell death after OGD. If the
cystine/glutamate exchanger regulates extrasynaptic glutamate
levels as previously demonstrated Figure , then glutamate re-
leased during OGD would favor the activation of extrasynaptic
NMDARs over synaptic ones. Hence, we analyzed the contri-
bution of synaptic and extrasynaptic NMDARs to ischemic in-
ward currents and neuronal damage. We observed a signicant
reduction in OGD-activated currents in the presence of ifen-
prodil μM; Figure , A and C, a known NRB antagonist .
RO μM, a more potent antagonist of NRB , pro-
duced a -fold reduction in the amplitude of the OGD-activated
current Figure , A and C. In contrast, the NRA antagonists
NVPAAM nM and TCN μM , did not
change the amplitude of OGD-activated currents Figure , B and
C. Finally, memantine, which at μM preferentially blocks ex-
Figure 2. Inhibition of vesicular glutamate release does not
change anoxic current or cell death in OGD. (A) OGD-activated
currents (recorded at 30 mV) in the absence or presence of
vesicular fusion inhibitor Baf A1 (μM). (B) Histograms show the
average amplitude (pA ± SEM) and latency (minutes ± SEM) of
the OGD-induced current for each condition. Baf did not change
the amplitude or latency of the OGD-activated current (n = 7). (C)
Cell death measured by LDH release in cultures subjected to 45
minutes of OGD and 24 hours of reperfusion in the presence or
absence of the exocytosis inhibitors Baf (1 μM) and TeNT (1 μg/ml).
Data are expressed as the mean ± SEM (n = 3–5). *P < 0.001 versus
OGD. (D) Representative fields showing propidium iodide labeling
of organotypic slices treated as in C. Scale bar: 1 mm.
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of Cl–. Specicity was further
assessed using the system xc–
competitive inhibitor aminoad-
ipic acid AAA; μM, which
signicantly blocked C
L-cystine uptake. In accordance
with Western blot data, cystine
uptake by the antiporter was in-
creased in neurons subjected to
chemical ischemia Figure C.
Altogether, these data reveal
an increase in neuronal xCT
expression and function after in
vitro ischemia.
Next we analyzed the func-
tion of cystine/glutamate anti-
porter short times minutes
and hours after inducing
transient focal ischemia via
middle cerebral artery occlu-
sion MCAO in rats. In order to
measure xCT function in vivo,
we took advantage of the re-
cently developed radioligand
S---Ffluoropropyl-
L-glutamate FFSPG to im-
age xCT transporter activity
by PET . As previously de-
scribed , the brain showed
low FFSPG uptake. However,
we observed tracer accumu-
lation as a result of higher ac-
tivity of the cystine/glutamate
antiporter in the ipsilateral
hemisphere immediately after
initiation of reperfusion PET
imaging could not be performed
during MCAO, and this accu-
mulation peaked at the striatum
at hours Figure , D and E.
This is in agreement with the fact
that MCAO induces early striatal
infarction hours but delayed
necrosis in the cortex hours
. Altogether, these data indi-
cate that the cystine/glutamate
antiporter increases its expression and activity following ischemia
and further support the relevance of this antiporter in glutamate
homeostasis alteration in stroke.
Discussion
While glutamate-induced excitotoxicity is widely accepted as a
critical event that leads to neuronal death during ischemia, several
other questions have yet to be answered. For instance, the mecha-
nisms behind the alteration in glutamate homeostasis and the bal-
ance between the release and clearance of glutamate in ischemia
are not yet completely understood, and the location of the gluta-
Western blot analysis revealed a -fold increase in xCT protein in
neurons exposed to chemical ischemia Figure B. This increase
in xCT levels at hours of reoxygenation is consistent with the
fact that reactive oxygen species are generated not only during
ischemia, but also during the reoxygenation period as a result of
the activation of NADPH oxidase . To conrm these nd-
ings, we next examined the cystine transport activity of system
xc– by measuring C L-cystine uptake in neurons exposed to
hour of chemical ischemia plus hours of reoxygenation. Spe-
cic Cl–-dependent cystine uptake by system xc– was dened
as the dierential cystine uptake in the presence and absence
Figure 3. Inhibition of the cystine/glutamate antiporter reduces the OGD-induced current, DC depolarization,
and cell death. (A) Voltage-clamp recording of OGD-activated currents in the absence or presence of system xc–
inhibitors CPG (250 μM; n = 12) and SAS (250 μM; n = 10). Both treatments significantly inhibited the OGD-acti-
vated current (n = 25). The mGluR1 inhibitor AIDA (500 μM; n = 6) had no eect on anoxic current amplitude or
on latency. (B) Histograms show the average OGD-induced current amplitude (pA ± SEM) and latency (minutes ±
SEM) for each condition. **P < 0.01 and *P < 0.05 versus OGD. (C) AD was recorded as a negative DC field voltage
shift in acute cortical slices subjected to OGD in the absence or presence of the ionotropic glutamate receptor
antagonists AP5 and CNQX or of the cystine/glutamate antiporter inhibitor CPG. Histogram shows the average DC
potential shift (mV ± SEM) for each condition. **P < 0.01 and *P < 0.05 versus OGD. (D) TBOA applied concomi-
tantly with CPG did not enhance the CPG-induced decrease of the OGD current amplitude, but reduced the latency
to AD (n = 6). (E) Cell death measured by LDH release in cultures subjected to 45 minutes of OGD applying system
xc– inhibitors CPG (250 μM; n = 5) and SAS (250 μM; n = 5), NMDAR antagonist MK-801 (50 μM, n = 5), and mGluR1
antagonist AIDA (500 μM; n = 3). LDH was measured after 24 hours of reoxygenation, and data are expressed as
the mean ± SEM. *P < 0.05 and **P < 0.01 versus OGD. (F) Representative fields demonstrate propidium iodide
labeling of organotypic slices treated as in E. Scale bar: 1 mm.
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mate receptors that trigger the death signal in ischemia is still con-
troversial. The data reported here provide evidence that glutamate
release by the cystine/glutamate antiporter contributes to ische-
mia-induced anoxic currents and neuronal damage. Electrophys-
iology data suggest that glutamate release by system xc– activates
mainly extrasynaptic NMDARs. Accordingly, blocking extrasyn-
aptic, but not synaptic, NMDARs reduces ischemia-induced cur-
rents and cell damage. Finally, in vivo PET imaging indicated that
system xc– function was robustly increased during ischemia and
reperfusion, suggesting a further contribution to glutamate re-
lease and cell death. To our knowledge, this is the rst report of
the contribution of the cystine/glutamate antiporter to glutamate
homeostasis alteration and subsequent cell death in ischemia.
Activation of the cell death program in neurons appears to be
linked to the overstimulation of extrasynaptic NMDARs , .
Our data suggest that extrasynaptic NRB-containing receptors
are the main contributors to anoxic currents and neuronal damage
in OGD. Accordingly, previous data showed that cerebral ischemia
recruits death-associated protein kinase DAPK to the NRB
complex at extrasynaptic sites, and this interaction functions as a
central mediator of stroke damage . In contrast, preblocking
synaptic NMDARs with MK has been reported to protect cul-
tured hippocampal neurons against hypoxic damage . The role
of synaptic and extrasynaptic receptors in neuronal cell death is
currently a matter of intense debate . It is known that NRB
subunits segregate outside the synapses , , whereas NRA
subunits are conned to the postsynaptic membrane . The dif-
ferent availability of endogenous coagonists D-serine and glycine,
which show activation selectivity for NRA and NRB, respec-
tively, also contributes to segregate NMDARs at specic locations
. However, it is possible that such segregation is an oversimpli-
cation , and additional tools to specically block each recep-
tor will be necessary in the future.
The role played by synaptic and extrasynaptic NMDARs in
neuronal cell death in neurological diseases would depend on
the source of glutamate. Glutamate levels outside the synaptic
cleft are maintained by nonvesicular release through system xc–
, and glutamate released by the cystine/glutamate anti-
porter is sucient to activate glutamate receptors , , .
We observed that glutamate release by system xc– preferentially
activated extrasynaptic NRB receptors. Moreover, nonsubstrate
inhibitors of system xc– signicantly attenuated the anoxic cur-
rents and ischemic damage, results conrmed in mutant sut mice,
in which xCT is nonfunctional P .. This strongly suggests
a role for the cystine/glutamate antiporter as a source of gluta-
mate in ischemia. We thus propose that an important source of
glutamate in this pathology is extrasynaptic, a nding further sup-
ported by our observations that vesicular inhibitor Baf A did not
aect ischemic currents or neuronal damage.
It should be noted that latency to AD was still reduced when
CPG was applied concomitantly with TBOA therefore, glutamate
clearance was inhibited, suggesting that another source of gluta-
mate contributes to altering glutamate homeostasis. Severe ische-
mic conditions may induce the reversion of glutamate transporters
. However, glial glutamate transporters do not readily reverse
and remain functional during ischemia , . In accordance,
Figure 4. AD and OGD-induced cell death are reduced in Slc7a11sut mice, which lack a functional cystine/glutamate antiporter. (A) Voltage-clamp record-
ing of OGD-activated currents in mouse acute slices. The anoxic current amplitude was reduced in the xCT-deficient (sut/sut; n = 13) mice when compared
with that in wild-type mice (+/+; n = 14). However, the latency to AD was unchanged. (B) Histograms show the average amplitude (pA ± SEM) and latency
(minutes ± SEM) of the OGD-induced current for each genotype. *P < 0.05. (C) Simultaneous extracellular DC field potential recording in acute slices from
wild-type and sut/sut mice subjected to OGD. Histogram shows the average DC field potential (mV ± SEM) for each genotype. *P < 0.05. (D) Cell death
measured by LDH release in organotypic cultures from wild-type and sut/sut mice exposed to 45 minutes of OGD and 24 hours of reoxygenation. Data are
expressed as the mean ± SEM (n = 3). (E) Representative fields demonstrate propidium iodide labeling of organotypic slices treated as in D. Scale bar: 1 mm.
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we found that blocking glutamate transporters shortened latency
to AD, amplied the anoxic current at the peak of AD, and, conse-
quently, increased neuronal damage see also refs. , . These
data do not exclude the possibility that a small proportion of gluta-
mate transporters i.e., neuronal EAAT function in reverse mode,
though inhibition of EAATs mostly in astrocytes leads to glutamate
accumulation. It is worth noting that the latency to AD was signi-
cantly increased in Eaac–/– , but not in Eaat–/–, mice .
Another possible source of glutamate is vesicular release. An in-
crease in mEPSCs has been described previously at early phases of
ischemia, before AD , , . The increase in mEPSC frequency
was Ca independent and caused by ischemia-induced actin la-
ment depolymerization . However, the increased spontaneous
vesicular transmitter release caused by energy depletion only leads
to extracellular glutamate accumulation when glial glutamate uptake
is blocked . Accordingly, we did not detect any change in anoxic
currents or damage in the presence of Baf A or TeTN. In conclusion,
this work does not challenge the current notion that exocytosis or
EAATs are key contributors to the release of ischemic glutamate, but
suggests that other sources of neurotransmitters and in particular
the cystine/glutamate antiporter are contributing to the process.
Several reports of glutamate release in pathological condi-
tions by this antiporter have been published recently, and a link
between glutamate release by system xc– and some CNS diseases
has been established. Thus, xCT silencing diminished glutamate
secretion from gliomas and alleviated neurodegeneration or
epileptic seizures . Similarly, inhibition of cystine/glutamate
antiporter–mediated glutamate release secondary to either mi-
croglial activation or Parkinson-inducing toxin -hydroxydo-
pamine prevented glial and neuronal damage. On the other
hand, expression of the catalytic subunit xCT is augmented in
processes that involve oxidative stress such as inammation ,
viral infection , and tumor proliferation . We also detected
an increase in xCT expression and function in primary cortical
neurons. Furthermore, we have used for the rst time a tracer spe-
cic for the system xc– in brain in a live animal to monitor cystine/
glutamate antiporter activity during ischemia and reperfusion. We
found that ischemia induced a rapid increase in system xc– activity
in vivo. This augmented function after ischemia would contribute
to further alter glutamate homeostasis during reperfusion.
A better understanding of the alterations of glutamate homeo-
stasis in ischemia is a key challenge for the eld. Protecting neu-
rons from ischemic excitotoxic damage by inhibiting ionotropic
glutamate receptors has been a useful strategy that has rendered
promising results both in vitro and in vivo. However, clinical trials
did not provide satisfactory results, because the use of glutamate
receptor antagonists has many secondary eects that are detri-
mental to patients. Therefore, blocking the source of glutamate
rather than the site of action may be a relevant therapeutic inter-
vention for the prevention of ischemic brain injury.
Methods
Reagents and chemicals. Calcein-AM and all cell culture supplies
were purchased from Invitrogen. The cytotoxicity assay for the
quantication of LDH release was acquired from Roche. L-cystine,
NVPAAM, propidium iodide, SAS, iodoacetate, and TeNT were
obtained from Sigma-Aldrich. Baf A, bicuculine, DAP, CNQX, ifen-
prodil, MK, memantine, RO , CPG, DLTBOA, AIDA, and
TCN were purchased from Tocris.
Mice and genotyping. Mutant Slcasut mice lacking the cystine/
glutamate antiporter and wild-type CH/HeSnJ mice were pur-
chased from The Jackson Laboratory. We used littermates for all
experiments. For PCR genotyping, sets of primers were designed:
Rv-E ′-CAAGGCCTCAAGCCCCTG′, which matches to exon
of the Slca gene only in / mice this region is deleted in sut
mice, and Rv-E′ ′-ATTTGACCACAATCTTTGAGACCA′,
which hybridizes an alternative exon E′ located within ampli-
cation range only in sut mice. The forward primer used in both
cases was Fw-E ′-TGAAACATGGAAACCGAAATCAC′, which
hybridizes to exon of Slca.
Electrophysiolog. Cortical slices -μm from P to P
Sprague-Dawley rats except for NMDA antagonist experiments, in
which P to P rats were used and from P to P CH/HeSnJ mice
were prepared in articial cerebrospinal uid aCSF; pH . that con-
tained mM NaCl, mM NaHCO, mM NaHPO, . mM KCl,
. mM CaCl, mM MgCl, and mM D-glucose constantly bubbled
with O and CO. Slices were allowed to recover for at least
hour and were then transferred to a °C chamber with continuous
ow ml/minute with aCSF plus bicuculline μM to block GABA
receptors and cystine μM. Pyramidal cells of layer V of the cortex
Figure 5. Cystine-evoked (1 mM) inward currents
in pyramidal cortical neurons (n = 21). Currents
were blocked in the presence of CPG (50 μM; n = 5),
the NMDAR antagonist AP5 (50 μM; n = 5), and the
NR2B antagonist RO-256981 (1 μM; n = 6), but not in
the presence of the NR2A antagonist NVP-AAM077
(250 nM; n = 6). *P < 0.05; **P < 0.01.
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3652 jci.org Volume 124 Number 8 August 2014
Millicell CM culture inserts Millipore and maintained in neu-
robasal-B, inactivated horse serum, HBSS all media from
Invitrogen, . mM glucose, mM L-glutamine, and X antibiotic-an-
timycotic U/ml penicillin, μg/ml streptomycin, and . μg/ml
amphotericin B; Life Technologies at °C and CO. Slices were
used after to days in vitro.
OGD minutes was achieved by incubating slices in an anaer-
obic chamber, in which O was replaced with N and external glucose
mM with sucrose in an extracellular solution containing mM
NaCl, . mM KCl, . mM CaCl, mM NaHCO, . mM MgCl,
. mM NaHPO, and μM L-cystine pH .. Organotypic corti-
cal slices were washed with this solution minus glucose for min-
utes prior to OGD to deplete the remaining glucose from extracellular
spaces. After minutes of OGD, extracellular solution was replaced
with medium and O supply restored. Antagonists were present during
the preincubation minutes, OGD minutes, and reoxygenation
hours periods. Cell death was determined hours after OGD by
measuring the release of LDH into the medium, using a colorimetric
assay Cytotoxicity Detection Kit; Roche Diagnostics according to the
manufacturer’s instructions. To evaluate the damaged region within
the slice, cultures were labeled with propidium iodide, the staining for
which correlates with LDH release in models of excitotoxicity and
hypoxia . Briey, after collecting media for the LDH assay, slices
were incubated in propidium iodide–containing medium μM for
hour at °C. Slices were washed times with culture medium and
photographed using a Nikon AZ uorescence microscope.
were identied visually using infrared dierential interference contrast
DIC microscopy Leica DM LFSA. Voltage-clamp recording pipettes
M Ω were lled with a solution containing mM CsCl, mM NaCl,
. mM CaCl, mM BAPTA, mM HEPES, mM Mg-ATP, and .
mM Na-GTP pH . . Access resistance and holding current were mon-
itored throughout the experiment. To simulate ischemia, glucose was
replaced with mM sucrose, and O/ CO was replaced with
N/ CO. Cells were held at – mV to facilitate the sensing of ische-
mia-evoked currents through NMDARs. All antagonists were applied
concomitantly with ischemia stimulation. For cystine pus, a microp-
erfusion manifold World Precision Instruments containing cystine
mM plus glycine μM diluted with bathing solution was connected
to an air-pressured system and placed into layer V of the cortex at a dis-
tance of μm from the cell soma. Simultaneously with voltage-clamp
recording, AD was recorded as negative extracellular DC potential shift
ΔVo induced by OGD. The DC potential is an extracellular recording
that is considered to provide an index of the polarization of cells sur-
rounding the tip of the glass electrode .. MΩ lled with M NaCl
see ref. . For mEPSC recordings, refer to Supplemental Methods.
Organotypic slice cultures. Cultures were prepared from coronal
cerebral sections -μm of brains from -day-old Sprague-Dawley
rat pups using the method described by Plenz and Kitai with minor
modications . Cortex was sliced using a McIllwain tissue chopper
Mickle Laboratory Engineering Co.. Slices containing cortex and
striatum but not hippocampus were selected under a microscope and
dissected to eliminate the corpus callosum. Slices were plated onto
Figure 6. Blockade of extrasynaptic NMDARs attenuates OGD-induced currents and cell death. (A and B) Representative voltage-clamped recordings of
cortical neurons at 30 mV in acute slices. OGD-activated currents were attenuated by the NR2B antagonists ifenprodil (3 μM; n = 9), memantine (10 μM;
n = 11), and RO-256981 (1 μM; n = 7). NR2A antagonists TCN-201 (10 μM; n = 6) and NVP-AAM077 (250 nM; n = 11) failed to reduce OGD-gated currents. (C)
Histograms showing the average amplitude (pA ± SEM) and latency (minutes ± SEM) of OGD-induced currents for each condition. *P < 0.05 versus OGD.
(D) Cell death measured by LDH release in organotypic cultures subjected to 45 minutes of OGD plus 24 hours of reoxygenation in the presence of the NR2B
antagonists memantine (10 μM; n = 4) and RO-256981 (1 μM; n = 3) or of the NR2A antagonists TCN-201 (10 μM; n = 3) and NVP-AAM077 (250 nM; n = 4).
MK801 (50 μM; n = 5) was also used to corroborate NMDA-mediated excitotoxicity. Data are expressed as the mean ± SEM. **P < 0.01 and ***P < 0.001
versus OGD. (E) Representative fields demonstrate propidium iodide labeling of organotypic slices treated as in D. Scale bar: 1 mm.
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groups were analyzed using an unpaired, -tailed Student’s t test.
Comparisons among multiple groups were analyzed by -way ANOVA
followed by Bonferroni’s multiple comparison tests for post hoc analy-
sis. Statistical signicance was considered at P ..
Study approval. Organotypic cultures, primary cultures, and elec-
trophysiology protocols using rats and wild-type and Slcasut mice
were approved by the Comité de Ética y Bienestar Animal Animal Eth-
ics and Welfare Committee of the UPV/EHU. MCAO and PET studies
were approved by the animal ethics committee of CIC biomaGUNE
and by local authorities and were conducted in accordance with the
directives of the European Union on animal ethics and welfare.
Acknowledgments
The authors would like to thank to H. Gómez, S. Marcos, and S.
Martín for technical assistance with cultures and electrophysiol-
ogy studies; V. Gómez-Vallejo, M. González, and A. Leukona for
technical support with radiosynthesis; and A. Cano, A. Arrieta,
and M. Errasti for technical assistance in the PET studies. This
work was supported by the Instituto de Salud Carlos III fellow-
ship to F.N. Soria; the Fundación Koplowitz; the Spanish Ministry
of Education and Science SAF; the Basque Govern-
ment; the UPV/EHU; and the CIBERNED.
Address correspondence to: Carlos Matute or Maria
Domercq, Dpto. Neurociencias, Universidad del País Vasco,
E Leioa, Spain. Phone: .; E-mail:
carlos.matute@ehu.es C. Matute. Phone: .;
E-mail: maria.domercq@ehu.es M. Domercq.
Primary cortical neuron cultures. Primary neurons were obtained
from the cortical lobes of E Sprague-Dawley rat embryos . Cells
were resuspended in B-supplemented neurobasal medium Life
Technologies plus FBS and then seeded onto poly-l-ornithine–
coated glass coverslips at a density of . × cells/cm. The medium
was replaced with serum-free, B-supplemented neurobasal medium
hours later. Cultures were essentially free of astrocytes and micro-
glia and were maintained at °C and CO. Cultures were used
after days in vitro. See Supplemental Methods for xCT expression
and function assays after in vitro ischemia and glutamine synthetase
assay using primary cortical neurons.
Transient focal ischemia. Transient focal ischemia was produced
by a -hour intraluminal occlusion of the MCA followed by reper-
fusion in adult -week-old male Sprague-Dawley rats obtained
from Janvier g body weight; n , as described elsewhere
. Briey, rats were anesthetized with isourane for to
minutes in O, and a .-cm length of monolament
nylon suture was introduced into the right external carotid artery up
to the level where the MCA branches out, and animals were sutured
and placed in their cages with ad libitum access to food and water.
After hours, the animals were reanesthetized, and the lament
was removed to allow reperfusion and PET image acquisition. Ani-
mals were studied at minutes n and at hours n following
the ischemic episode. See Supplemental Methods for details about
the PET assay and radiosynthesis.
Statistics. Data are presented as the mean SEM unless other-
wise indicated from at least independent experiments, in which all
conditions were assayed at least in triplicate. Comparisons between
Figure 7. Cystine/glutamate antiporter expression and function are increased in cortical neurons in vitro after chemical ischemia and in vivo after transient
focal ischemia. (A) xCT mRNA levels in neurons exposed to chemical ischemia (1 hour), as measured by qPCR. Data are expressed as the mean ± SEM (n = 4).
(B) xCT protein levels after chemical ischemia (1 hour) plus a 4-hour reoxygenation in cortical neurons. Data were normalized to actin and are expressed as
the mean ± SEM (n = 4). Control and OGD lanes were run on the same gel but were noncontiguous. Full, uncut gels are shown in the Supplemental Material.
(C) Cl–-dependent [14C] L-cystine uptake by neurons after 1 hour of chemical ischemia plus 4 hours of reoxygenation. Note that cystine/glutamate antagonist
AAA (250 μM) almost completely inhibited uptake. Data were normalized to protein concentration and are expressed as the mean ± SEM (n = 3). (D) 18[F]FSPG
uptake study by PET imaging in control rats and after MCAO (n = 5 per group). Representative images of control rats and images at 5 minutes and at 5 hours
of reperfusion (RPF) following MCAO. (E) Histogram shows 18[F]FSPG signal expressed as the percentage of injected dose per gram (% ID/g) and normalized
to the contralateral hemisphere in the total infarct area or in the volumes of interest (VOIs) defined in the striatum and cortex at 5 minutes or at 5 hours after
reperfusion. Data are expressed as the mean ± SEM (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001 versus control.
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