J Neurophysiol
87: 2896 –2903, 2002; 10.1152/jn.01011.2001.
NMDA Receptor Activation Mediates Hydrogen Peroxide–Induced
Pathophysiology in Rat Hippocampal Slices
MARAT V. AVSHALUMOV AND MARGARET E. RICE
Departments of Physiology and Neuroscience and Neurosurgery, New York University School of Medicine,
New York, New York 10016
Received 11 December 2001; accepted in final form 4 February 2002
The reactive oxygen species (ROS), H2O2, has been shown
previously to modulate synaptic transmission (e.g., Frantseva
et al. 1998; Pellmar 1986). Most studies of this modulation
have used the hippocampal slice preparation, in which H2O2
reversibly depresses the population spike (PS) evoked by stimulation of the Schaffer collaterals and recorded in CA1
(Avshalumov et al. 2000; Fowler 1997; Pellmar 1986, 1987).
Both pre- and postsynaptic sites of action may be involved,
since H2O2 can inhibit neurotransmitter release (Chen et al.
2001) and can cause hyperpolarization of CA1 pyramidal neurons (Seutin et al. 1995). Additional actions of H2O2 (and other
ROS) include modulation of synaptic plasticity (Auerbach and
Segal 1997; Klann et al. 1998), possibly mediated by alterations in intracellular signaling pathways, including activation
of certain kinases (Klann and Thiels 1999).
The reversibility of the effects of H2O2 on synaptic transmission and the demonstration that similar effects are seen with
endogenously generated, as well as exogenously added H2O2
(e.g., Auerbach and Segal 1997; Chen et al. 2001) implicate
H2O2 as an endogenous neuromodulator. This ROS is generated during normal mitochondrial respiration from dismutation
of the superoxide radical (·O⫺
2 ) (Boveris and Chance 1973;
Fridovich 1979; Halliwell 1992). Pathological consequences of
H2O2 elevation are normally prevented by the endogenous
antioxidant network, which includes the H2O2-scavenging enzymes glutathione (GSH) peroxidase and catalase (Cohen
1994; Desagher et al. 1996; Sokolova et al. 2001). It should be
noted, however, that modulatory, as well as pathological consequences of H2O2 are often mediated by the hydroxyl radical
(·OH) rather than by H2O2 per se (Avshalumov et al. 2000;
Halliwell 1992; Pellmar et al. 1989; Sah and Schwartz-Bloom
1999; Ying et al. 1999). Whereas the enzymes superoxide
dismutase (SOD), GSH peroxidase, and catalase regulate cellular levels of ·O⫺
2 and H2O2, there are no analogous enzymes
for the highly reactive ·OH. Instead, ·OH management depends
on the endogenous antioxidants ascorbate and GSH (Cohen
1994; Rice 2000). Consistent with this, normal extracellular
concentrations of ascorbate can prevent PS depression in the
presence of exogenous H2O2 (Avshalumov et al. 2000).
In addition to modulatory actions when H2O2 levels are
controlled, prolonged exposure to this ROS can also cause cell
death of both neurons and glia in vitro (Desagher et al. 1996;
Hoyt et al. 1997; Mailly et al. 1999; Sokolova et al. 2001).
Importantly, Mailly and colleagues (1999) reported that H2O2induced neuronal apoptosis was due to increased extracellular
glutamate concentration, N-methyl-D-aspartate (NMDA) receptor activation, and increased ROS production that occurred
during washout of H2O2. Transient exposure to H2O2 also
caused an increase in glutamate overflow from hippocampal
Address for reprint requests: M. E. Rice, Dept. of Physiology and Neuroscience, NYU School of Medicine, 550 First Ave., New York, NY 10016
(E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
INTRODUCTION
2896
0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
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Avshalumov, Marat V. and Margaret E. Rice. NMDA receptor
activation mediates hydrogen peroxide–induced pathophysiology
in rat hippocampal slices. J Neurophysiol 87: 2896 –2903, 2002;
10.1152/jn.01011.2001. Endogenous reactive oxygen species (ROS)
can act as modulators of neuronal activity, including synaptic transmission. Inherent in this process, however, is the potential for oxidative damage if the balance between ROS production and regulation
becomes disrupted. Here we report that inhibition of synaptic transmission in rat hippocampal slices by H2O2 can be followed by
electrical hyperexcitability when transmission returns during H2O2
washout. As in previous studies, H2O2 exposure (15 min) reversibly
depressed the extracellular population spike (PS) evoked by Schaffer
collateral stimulation. Recovery of PS amplitude, however, was typically accompanied by mild epileptiform activity. Inclusion of ascorbate (400 ␮M) during H2O2 washout prevented this pathophysiology.
No protection was seen with isoascorbate, which is a poor substrate
for the stereoselective ascorbate transporter and thus remains primarily extracellular. Epileptiform activity was also prevented by the
N-methyl-D-aspartate (NMDA) receptor antagonist, DL-2-amino-5phosphonopentanoic acid (AP5) during H2O2 washout. Once hyperexcitability was induced, however, AP5 did not reverse it. When
present during H2O2 exposure, AP5 did not alter PS depression by
H2O2 but did inhibit the recovery of PS amplitude seen during
pulse-train stimulation (10 Hz, 5 s) in H2O2. Inhibition of glutamate
uptake by l-trans-2,4-pyrrolidine dicarboxylate (PDC; 50 ␮M) during
H2O2 washout markedly enhanced epileptiform activity; coapplication of ascorbate with PDC prevented this. These data indicate that
H2O2 exposure can cause activation of normally silent NMDA receptors, possibly via inhibition of redox-sensitive glutamate uptake. When
synaptic transmission returns during H2O2 washout, enhanced NMDA
receptor activity leads to ROS generation and consequent oxidative
damage. These data reveal a pathological cycle that could contribute to
progressive degeneration in neurological disorders that involve oxidative
stress, including cerebral ischemia.
NMDA RECEPTORS AND H2O2-INDUCED PATHOPHYSIOLOGY
slices, which contributed to long-lasting damage to inhibitory
transmission (Sah and Schwarz-Bloom 1999). Increased glutamate levels in both of these studies suggests that an immediate consequence of H2O2 exposure might be inhibition of
redox-sensitive glutamate transporters (for review, see Trotti et
al. 1998), with further pathological consequences including
hyperexcitability and even cell death.
Here we report that recovery of the PS in rat hippocampal
slices after H2O2 exposure can be accompanied by pathological
electrical activity, indicated by an additional PS in response to
Schaffer-collateral stimulation in CA1. As in cultured neurons
(Mailly et al. 1999), this epileptiform activity appears to be a
consequence of enhanced NMDA receptor activation during
H2O2 washout and involves secondary NMDA receptor–mediated ROS production. Additional experiments tested the hypothesis that glutamate transporter inhibition might contribute
to this pathophysiology.
Hippocampal slice preparation
Hippocampal slices (400-␮m thickness) were prepared from young
adult (50- to 60-day-old) male Long-Evans rats. All animal experimentation was conducted following National Institutes of Health
guidelines and with approval by the NYU School of Medicine Institutional Animal Care and Use Committee. Before decapitation, rats
were deeply anesthetized with 50 mg/kg pentobarbital sodium. After
decapitation, the brain was rapidly removed and placed in ice-cold,
oxygenated artificial cerebrospinal fluid (ACSF) for about 1 min. The
brain was then bisected, blocked, and mounted on the stage of a
Vibratome (Ted Pella, St. Louis, MO), and transverse hippocampal
slices were cut in ice-cold ACSF, as described previously (Avshalumov et al. 2000). Normal ACSF contained (in mM) 120 NaCl, 5 KCl,
35 NaHCO3,1.25 NaH2PO4, 1.5 CaCl2, and 1.3 MgCl2, equilibrated
with 95% O2-5% CO2. Freshly prepared slices were maintained in
ACSF at room temperature for at least 1 h before recording.
Extracellular recording
For measurement of the evoked PS, slices were transferred to a
submersion-recording chamber (Warner Instrument, Hamden, CT),
where they were continuously superfused with ACSF at 1.5 ml/min at
31°C. Extracellular PS evoked by Schaffer-collateral stimulation were
recorded in the stratum pyramidale of CA1 with a standard glass
electrode (2- to 7-M⍀ tip resistance, backfilled with 1 M NaCl)
connected to an Axoprobe 1A amplifier (Axon Instruments, Foster
City, CA). Stimulation-pulse duration was 100 ␮s, with stimulus
intensity adjusted to the lowest potential (0.9 –1.8 mV) required to
evoke a PS of maximal amplitude (Avshalumov et al. 2000). The
stimulating electrode was a twisted bipolar electrode, made from
Teflon-insulated platinum-iridium wire. The evoked PS was elicited at
5-s intervals using an external timing circuit (Master-8; A.M.P.I.,
Jerusalem, Israel) and was monitored on a digital oscilloscope. Data
acquisition was controlled by Clampex 7.0 software (Axon Instruments, Foster City, CA), which imported PS records to a PC via a
DigiData 1200B D/A board (Axon Instruments) for averaging and
analysis. In some experiments, the behavior of the PS elicited by
pulse-train stimulation (10 Hz, 5 s) was also recorded; train stimulation was controlled by the Master-8, with the same pulse duration and
amplitude as in single-pulse stimulations.
For each slice, the evoked PS was monitored for 25–30 min in
normal ACSF to ascertain that the response was stable. Typical PS
amplitude in these submerged slices was 2 mV, as we have reported
previously (Avshalumov et al. 2000). Slices with PS amplitude lower
J Neurophysiol • VOL
than 1 mV were not tested further. For each experimental condition,
three evoked PS records were stored and averaged. The amplitude (in
mV) of PS records was measured from the mean of the positive peak
preceding and the positive peak following the negative PS.
Chemicals and solutions
ACS grade hydrogen peroxide solution, sodium ascorbate (L-ascorbate), isoascorbate (D-ascorbate), DL-2-amino-5-phosphonopentanoic
acid (AP5), and all components of ACSF were obtained from Sigma
(St. Louis, MO); l-trans-2,4-pyrrolidine dicarboxylate (PDC) was
from Tocris (Ballwin, MO). Test agents were added to ACSF immediately before use (Avshalumov et al. 2000).
Statistical analysis
Data are given as means ⫾ SE, normalized to percent of control PS
amplitude (100%) recorded before H2O2 exposure or pulse train
stimulation for a given slice. For statistical analysis, either Student’s
t-test or one-way ANOVA followed by Kruskal-Wallis post hoc
analysis test was used as appropriate. The statistical significance was
P ⬍ 0.05. All illustrated electrophysiological records represent the
averaged responses from 5–14 slices (as indicated).
RESULTS
Epileptiform activity after H2O2 washout
The mean amplitude of the evoked PS in submerged hippocampal slices under control conditions was 2.1 ⫾ 0.2 mV
(n ⫽ 14; Fig. 1A). Within this population, the minimum amplitude was 1.6 mV and the maximum was 2.3 mV, such that
the illustrated average responses well represent typical PS
behavior. We have shown previously that the effect of exogenous H2O2 on the evoked PS in rat hippocampal slices has a
sharp dependence on H2O2 concentration: 1.2 mM H2O2 does
not alter PS amplitude significantly, whereas a maximal effect
is seen at 1.5 mM H2O2 with no further depression at 2.0 mM
H2O2 (Avshalumov et al. 2000). As before, 15-min exposure to
1.5 mM H2O2 caused a decrease in PS amplitude to 19 ⫾ 2%
of control (n ⫽ 14; P ⬍ 0.001), with recovery to 83 ⫾ 11%
(n ⫽ 14; P ⬎ 0.05 vs. control) after 30 min H2O2 washout (Fig.
1A). Additionally, however, we report here that the recovery of
the PS after H2O2 washout can be accompanied by mild
epileptiform activity, indicated by an additional peak following
the primary PS (Fig. 1A; arrow).
In our experience, slices that have a stable, single PS during
the first 10 –15 min of recording do not develop additional
peaks (hyperexcitability) during normal recording for up to 3 h
(not illustrated). This indicates that repetitive stimulation at 5-s
intervals alone does not induce such hyperexcitability. To
examine whether stimulation during H2O2 exposure or washout might contribute to the H2O2-induced pathophysiology,
however, H2O2 was applied and washed out in the absence of
Schaffer-collateral stimulation (Fig. 1B). For this study, control
records were obtained with normal stimulation parameters,
then the stimulus turned off. Slices were then exposed to H2O2
for 15 min followed by 30-min washout, as usual, after which
the stimulus was turned on again to indicate PS behavior. The
same post-H2O2 hyperexcitability was seen under these conditions (Fig. 1B; n ⫽ 6) as when the stimulus was applied at 5-s
intervals during H2O2 exposure and withdrawal (Fig. 1A). Thus
evoked electrical activity and transmitter release did not contribute to the induction of this pathophysiology.
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METHODS
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M. V. AVSHALUMOV AND M. E. RICE
Protective effect of NMDA receptor antagonism
It was recently reported that neuronal toxicity following
H2O2 exposure could be prevented by NMDA receptor antag-
We then examined whether hyperexcitability could occur
following cessation of synaptic transmission alone. For this
experiment, slices were superfused for 15 min with nominally
Ca2⫹-free ACSF (no added Ca2⫹; n ⫽ 2) or normal ACSF (1.5
mM Ca2⫹) with 0.5 mM Mn2⫹ (n ⫽ 3), followed by 30-min
wash with normal ACSF. Stimulation of the Schaffer collaterals at 5-s intervals continued throughout. After superfusion for
⬃5 min with either medium, a PS could no longer be elicited
(Fig. 1C), which was similar to the time course of PS depression during H2O2 application. Under these conditions, PS
recovery was not accompanied by an additional peak (Fig. 1C;
n ⫽ 5), indicating that the pathophysiology following H2O2
exposure is not simply a consequence of rebound hyperexcitability following depression of synaptic transmission.
Involvement of intracellular ROS
We showed previously that the inhibitory effect of H2O2 on
the hippocampal PS can be prevented when H2O2 is applied in
the presence of the ROS scavenger, ascorbate, or its nonphysiological steroisomer, isoascorbate (D-ascorbate) (Avshalumov
et al. 2000). When this primary inhibitory effect of H2O2 was
prevented, no epileptiform activity was seen on H2O2 washout
(not illustrated). To investigate ROS involvement in the development of the pathophysiology, we examined the effect of
ascorbate and isoascorbate when either was present only during
washout. Ascorbate is a good substrate for the stereospecific
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FIG. 2. Protective effect of ascorbate but not isoascorbate on H2O2-induced
pathology. A: ascorbate (Asc, 400 ␮M) prevented the occurrence of the
additional PS during washout (⫹ Asc; n ⫽ 8). B: isoascorbate (Iso, 400 ␮M)
did not prevent H2O2-induced epileptiform activity (⫹ Iso, arrow; n ⫽ 6).
Illustrated PS records in A–C are the averaged responses from 6 – 8 slices, as
indicated. C: PS amplitude changes (as % control) after 15-min exposure to
H2O2 and following washout (N/D, not detectable). Data are means ⫾ SE;
* P ⬍ 0.05 and *** P ⬍ 0.001 indicate difference from pre-H2O2 control
amplitude (100%, indicated by dashed line).
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FIG. 1. Pathophysiology induced by H2O2 exposure. A: electrophysiological records showing the extracellular population spike (PS) evoked by stimulation of the Schaffer collaterals and recorded in CA1 before H2O2 exposure
(control), after 15-min superfusion with H2O2 (1.5 mM), and after 30-min
washout of H2O2 (wash). Recovery of the PS was accompanied by mild
epileptiform activity, indicated by an additional PS (Add’l PS) after washout
(arrow; n ⫽ 14). B: the appearance of an additional PS after H2O2 washout did
not depend on concurrent electrical stimulation. After control PS records were
taken, H2O2 application (15 min) and washout (30 min) were done in the same
sequence as in A, but with electrical stimulation off (stimulus off). Wash
records were taken when the stimulus was turned on again; the additional peak
(arrow) persisted throughout the recording period (up to 1 h; n ⫽ 6). C: loss
and recovery of synaptic transmission alone did not induce epileptiform
activity. After control PS records were taken, either normal artificial cerebrospinal fluid (ACSF) plus 0.5 mM Mn2⫹ (n ⫽ 3) or ACSF with 0 mM Ca2⫹
(n ⫽ 2) were superfused for 15 min; both caused complete suppression of the
evoked PS so that the data were pooled (Mn2⫹/0 mM Ca2⫹). No additional
peak was seen in the recovered PS after 30-min washout with normal ACSF
(wash). Illustrated PS records are the averaged responses for each condition
from 5–14 slices, as indicated.
neuronal ascorbate transporter, whereas isoascorbate is not
(Tsukaguchi et al. 1999), such that ascorbate is taken up into
the intracellular compartment, while isoascorbate remains predominantly extracellular (Brahma et al. 2000). Inclusion of
ascorbate in the washout medium at its normal physiological
concentration of 400 ␮M (see Rice 2000) completely prevented the appearance of an additional PS (Fig. 2A), which
implicated ROS involvement. By contrast, isoascorbate (400
␮M), did not prevent H2O2-induced pathology (Fig. 2B; arrow), suggesting that the site of ROS generation or action was
intracellular.
As noted above, PS amplitude of slices after H2O2 exposure
and washout was slightly, but not significantly different from
control levels (Figs. 1A and 2C). The additional PS was on
average, 33 ⫾ 3% of the control PS amplitude (n ⫽ 14; P ⬍
0.001; Fig. 2C). When ascorbate was present in the washout
medium, PS amplitude after H2O2 exposure also recovered to
control levels (93 ⫾ 3%; n ⫽ 8; P ⬎ 0.05 vs. control) with no
detectable second PS (Fig. 1C). By contrast, when H2O2 was
washed out with isoascorbate, the amplitude of the primary PS
did not fully recover (78 ⫾ 1%; n ⫽ 6; P ⬍ 0.05 vs. control)
and was accompanied by an additional PS that was 27 ⫾ 2%
of control (n ⫽ 6; Fig. 2C).
NMDA RECEPTORS AND H2O2-INDUCED PATHOPHYSIOLOGY
2899
followed by 15 min further wash with ACSF alone (Fig. 3C).
No additional peaks developed during AP5 washout periods of
up to 1.5 h (not illustrated), which indicated that the continued
presence of AP5 was not required to “suppress” epileptiform
activity. Moreover, once epileptiform activity was present,
application of AP5 did not alter it (not illustrated). This further
suggests that the increased excitability was not caused by
persistent activation of NMDA receptors. Together, these data
implicate transient NMDA receptor activation during H2O2
washout as a cause of irreversible pathophysiology.
Activation of NMDA receptors during H2O2 exposure
onists and by the nonspecific ROS scavenger DMSO; these
data implicate NMDA receptor–activated ROS generation in
H2O2-induced toxicity (Mailly et al. 1999). We therefore used
the NMDA receptor antagonist, AP5, to examine the possible
involvement of NMDA receptors in the generation of H2O2dependent pathophysiology in hippocampal slices (Fig. 3). As
expected, AP5 (50 ␮M) had no effect on PS amplitude compared with controls under normal conditions (not illustrated).
Coapplication of AP5 with H2O2 did not prevent PS depression
by H2O2 (to 18 ⫾ 4% of control; n ⫽ 8; P ⬍ 0.001 vs. control);
moreover, when AP5 was washed out with H2O2, the same
post-H2O2 hyperexcitability was seen as during normal H2O2
application and washout (compare Fig. 3, A and B).
When AP5 was present during H2O2 washout, however,
induction of epileptiform activity was prevented. The presence
of AP5 was required only during the initial washout period: a
single PS was seen after 15 min of H2O2 washout with AP5
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FIG. 4. AP5 inhibits PS recovery during pulse-train stimulation in H2O2. PS
amplitude changes during pulse-train stimulation (10 Hz, 5 s); stimulation
started at 0.1 s and ended at 5 s. Data are means ⫾ SE (n ⫽ 10) and given in
% of the PS amplitude evoked by single-pulse stimulation (100%). AP5 (50
␮M) significantly inhibited PS recovery during pulse train stimulation in H2O2
(n ⫽ 10, P ⬍ 0.05 for H2O2 vs. AP5 ⫹ H2O2). See RESULTS for further
description.
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FIG. 3. Protective effect of DL-2-amino-5-phosphonopentanoic acid (AP5)
during washout. A: evoked PS before H2O2 application (control), after 15-min
superfusion with H2O2, and after 30-min washout of H2O2 (wash; n ⫽ 14). B:
the presence of AP5 (50 ␮M) during H2O2 application only did not alter PS
depression (H2O2 ⫹ AP5), nor did it prevent epileptiform activity after H2O2
washout (wash, arrow; n ⫽ 8). C: inclusion of AP5 in the H2O2–washout
solution prevented epileptiform activity (n ⫽ 8); AP5 was superfused for 15
min, then washout with ACSF alone continued for 15 min longer (wash ⫹
AP5). Each record is the averaged response for each condition from 8 –14
slices, as indicated. D: PS amplitude changes (as % control) after 15-min
exposure to H2O2 and following washout (Add’l PS, additional PS; N/D, not
detectable). Data are means ⫾ SE, *** P ⬍ 0.001 indicate difference from
control (100%, indicated by dashed line).
The question then arises of whether enhanced NMDA receptor activation occurs during H2O2 exposure. We previously
reported that by the end of pulse-train stimulation (10 Hz, 5 s),
the amplitude of the depressed PS in the presence of H2O2
recovers to about 75% of that seen during train simulation in
ACSF alone (Avshalumov et al. 2000). We therefore tested the
hypothesis that increased NMDA receptor activation contributed to the recovery of the PS during prolonged stimulation in
the presence of H2O2. As before, PS amplitude during pulsetrain stimulation increased twofold during the first few stimulus pulses under control conditions, with an average increase to
227 ⫾ 17% of the single pulse control by the end of the train
(n ⫽ 10; P ⬍ 0.001 vs. single-pulse control). This pattern was
unaffected by AP5 (50 ␮M; Fig. 4).
In the presence of H2O2, PS amplitude was initially depressed during pulse-train stimulation, but began to return after
typically 25 pulses at 10 Hz, with an amplitude that was 157 ⫾
10% of single-pulse control by the end of the train (n ⫽ 10;
P ⬍ 0.05 vs. single-pulse control or the last pulse of the train
in ACSF alone). Recovery of PS amplitude during pulse-train
stimulation in H2O2 was inhibited by AP5. By the end of the
train in H2O2 plus AP5, PS amplitude had increased to only
94 ⫾ 7% of the single pulse control (n ⫽ 10; P ⬎ 0.05), which
2900
M. V. AVSHALUMOV AND M. E. RICE
was significantly lower than the last pulse in ACSF (P ⬍
0.001) or in H2O2 alone (P ⬍ 0.05; Fig. 4). These data show
that PS recovery during pulse-train stimulation in H2O2 involves activation of normally silent NMDA receptors.
NMDA receptor involvement in H2O2-induced
pathophysiology
Glutamate transport inhibition increased H2O2-dependent
pathology
DISCUSSION
The present results indicate that transient elevation of H2O2
in rat hippocampal slices not only can suppress synaptic transmission, but also can lead to secondary pathophysiology as
synaptic transmission returns during H2O2 withdrawal. The
induction of hippocampal hyperexcitability on H2O2 washout
could be prevented by ascorbate or AP5, but not by isoascorbate. These data implicate NMDA receptor activation with
FIG. 5. Glutamate transporter inhibition enhances H2O2-induced pathophysiology. A: evoked PS before H2O2 application (control), after 15-min
superfusion with H2O2, and after 20-min washout of H2O2 in the presence of
l-trans-2,4-pyrrolidine dicarboxylate (PDC; 50 ␮M) then 30-min PDC wash
with ACSF alone (wash post-PDC; n ⫽ 8). Note multiple additional peaks after
H2O2 washout with PDC (arrows). B: evoked PS under the same conditions as
in A, except that ascorbate (400 ␮M) was included with PDC during the first
20 min of H2O2 washout, followed by 30-min wash of PDC ⫹ Asc (wash
post-PDC ⫹ Asc). Each record is the average response for each condition from
8 slices.
A primary effect of H2O2 on synaptic transmission in hippocampal slices appears to be decreased synaptic release of
glutamate (Pellmar 1987). In support of inhibitory effects of
H2O2 on transmitter release, we have shown that H2O2 can
reversibly depress synaptic release of dopamine, which can
readily be monitored in brain tissue using voltammetric microelectrodes (Chen et al. 2001). Opposing the action of decreased
glutamate release, however, H2O2 can also reversibly inhibit
glutamate uptake by acting as an oxidant at sulfhydryl groups
in glutamate transport proteins (Trotti et al. 1997, 1998; Volterra et al. 1994). Increased glutamate spillover following
H2O2 exposure in both neuron-enriched cultures and hippocampal slices has been attributed to oxidative inhibition of
glutamate transport (Mailly et al. 1999; Sah and SchwartzBloom 1999). Enhanced glutamate levels and subsequent
NMDA receptor activation was found to be the initiating event
in H2O2-induced neuronal apoptosis (Mailly et al. 1999). Similarly, Sah and Schwartz-Bloom (1999) reported that H2O2
exposure can induce long-term disruption of Cl⫺ homeostasis
in hippocampal slices, which can lead to a long-term decrease
in the efficacy of inhibitory transmission; significantly, the
changes in ion homeostasis in that study could be inhibited by
glutamate receptor antagonists (Sah and Schwartz-Bloom
1999).
Loss of inhibitory transmission following transient H2O2
exposure is presumably an underlying factor in the irreversible
hyperexcitability seen in the present studies. The prevention of
this H2O2-induced pathophysiology by AP5 further implicates
similar initiating events, including decreased glutamate uptake
and increased NMDA receptor activation. Previous studies
have shown that uptake inhibition can increase the NMDA
receptor–mediated component of the evoked PS in hippocampal slices, even when glutamate release is depressed (el-Sherif
et al. 1999). Thus inhibition of glutamate transport during
H2O2 exposure could also contribute to the NMDA receptor–
dependent recovery of the evoked PS during pulse-train stimulation in the presence of H2O2 seen here (Fig. 4). If involved,
however, transport inhibition by H2O2 must be only partial,
since the effect could be amplified by pharmacological blockade using PDC (Fig. 5A). Additional actions of H2O2 that
might activate normally silent NMDA receptors include ROSdependent stimulation of tyrosine kinase activity (Klann and
Thiels 1999). Stimulation of this pathway can increase NMDA
receptor phosphorylation (Zheng et al. 1998), which potentiates NMDA receptor–mediated Ca2⫹ entry (Soderling and
Derckach 2000). It should be noted that post-H2O2 hyperexcitability is not a consequence of permanently activated
NMDA receptors, however, since AP5 could not inhibit epileptiform activity once it had appeared.
In addition, the actions of H2O2 in the present studies appear
to be independent of the redox modulatory site of NMDA
receptors. Sulfhydryl oxidation at this redox-sensitive site
causes a decrease in NMDA receptor activity (Aizenman et al.
1989, 1990); indeed, this may be an important site in regulating
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Elevated glutamate spillover in the presence of H2O2 has
been proposed to reflect decreased glutamate uptake (Mailly et
al. 1999; Sah and Schwartz-Bloom 1999) caused by oxidative
inhibition of redox-sensitive glutamate transport proteins
(Trotti et al. 1998; Volterra et al. 1994). To investigate whether
this might contribute to the pathophysiological consequences
of H2O2 in the present studies, we first examined the effect of
the nonselective transport inhibitor, PDC (50 –100 ␮M) on the
evoked PS. Superfusion with PDC alone did not induce epileptiform activity. Indeed, PDC caused a progressive decrease
in PS amplitude, especially at 100 ␮M (not illustrated), as has
been reported previously for other glutamate transport inhibitors (el-Sherif et al. 1999). When PDC (50 ␮M) was included
in the H2O2 washout medium, however, a marked enhancement in epileptiform activity was seen after PDC was also
washed out (Fig. 5A). This exacerbation of the H2O2-dependent pathophysiology shows that inhibition of glutamate transporters could be a contributing factor. Additionally, we examined whether the PDC-enhanced effect could also be prevented
by ascorbate. Strikingly, when ascorbate (400 ␮M) was included with PDC, the enhanced epileptiform activity was completely inhibited (Fig. 5B), which further implicates secondary,
glutamate-dependent ROS production during H2O2 washout.
J Neurophysiol • VOL
subsequent intracellular ROS generation as the cause of irreversible damage when glutamatergic transmission returns after
transient H2O2 exposure.
NMDA RECEPTORS AND H2O2-INDUCED PATHOPHYSIOLOGY
the severity of epileptiform activity under conditions in which
NMDA receptor involvement is enhanced (e.g., low Mg2⫹)
(Sanchez et al. 2000). This inhibitory effect contrasts sharply to
the H2O2-induced increase in NMDA receptor activity seen
here (e.g., Fig. 4) and in previous studies (Mailly et al. 1999).
Thus if H2O2 did have an inhibitory effect at the redoxmodulatory site in the present experimental paradigm, it was
overshadowed by this predominant activating action.
Protective role of ascorbate; involvement of secondary
ROS production
J Neurophysiol • VOL
which can be reversed by thiol reducing agents (Aizenman et
al. 1989, 1990). Thermodynamically, ascorbate cannot reduce
oxidized thiols (Buettner 1993), so that a “protective” effect of
ascorbate linked to redox modulation of NMDA receptors must
be the result of pro-oxidant action, as ascorbate itself oxidizes
(Rice 2000). The lack of equal “protection” by ascorbate and
isoascorbate in the present studies argues against this mechanism of action here; rather, the data are consistent with ROS
scavenging by ascorbate in the intracellular compartment.
It should be noted that previous reports of the effects of
H2O2 on hippocampal physiology did not describe epileptiform
activity after H2O2 exposure. Many of these studies, especially
those by Pellmar and colleagues (Pellmar 1986, 1987; Pellmar
et al. 1989) were made in guinea pig hippocampal slices. In
preliminary comparisons using the same experimental conditions as in the present studies with rat slices, we found that
guinea pig tissue is apparently resistant to H2O2–washout
pathology, with no epileptiform activity after H2O2 washout
(unpublished data). This may be a consequence of the higher
GSH content of guinea pig brain tissue compared with rat (Rice
et al. 1995), since Pellmar reported that the recovery of the PS
after H2O2 washout in guinea pig hippocampal slices is GSH
dependent and is significantly decreased by GSH-synthesis
inhibition (Pellmar et al. 1989).
Relevance for neuropathology
Together, these data confirm the importance of the antioxidant network, including ascorbate, in preventing oxidative
damage during normal cell signaling by ROS. Under conditions in which the antioxidant network is compromised, however, the sequence of events described here could contribute to
secondary pathology. For example, increased levels of H2O2,
·OH, and other ROS have been detected during reperfusion in
vivo after ischemic periods as brief as 5–30 min (Cao et al.
1988; Delbarre et al. 1992; Hyslop et al. 1995; Lei et al. 1997),
when the antioxidant network is relatively intact (Lyrer et al.
1991; Uemura et al. 1991). Increased ROS generation also
occurs in the penumbra surrounding the infarct (Solenski et al.
1997), which can contribute to progressive damage in penumbral tissue (Chan 1996; Love 1999).
During reperfusion after brief ischemia, extracellular H2O2
levels can reach 200 ␮M (Hyslop et al. 1995; Lei et al. 1997).
Assuming that the primary site of generation is the mitochondria, as during normal metabolism (Boveris and Chance 1973),
intracellular levels may be even higher. It should be noted that
under normal conditions, even low densities of neurons and
glia in culture have sufficient peroxidase activity to rapidly
metabolize 200-␮M exogenous H2O2 to below detectable levels (Desagher et al. 1996; Sokolova et al. 2001). Consequently,
actual tissue levels of H2O2 during exogenous application in
the present brain slice studies in are also expected to be
substantially lower than the applied concentration (1.5 mM)
required to depress the evoked PS.
Consistent with the cycle of progressive pathology proposed
here, glutamate receptor activation is an important contributing
factor to ischemia-induced ROS generation (Lipton 1999; Love
1999). Extracellular glutamate levels increase 6- to 30-fold
during ischemia, resulting in concentrations that can exceed 1
mM (Benveniste et al. 1984; Benveniste and Hüttemeier 1990;
Shimizu et al. 1993; Takagi et al. 1993). Moreover, ROS
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Increased NMDA receptor activation, regardless of mechanism, can initiate a neurotoxic cascade involving including
elevated intracellular Ca2⫹ (Lipton and Rosenberg 1994; Urushitani et al. 2001; Vergun et al. 2001) and increased mitochondrial ROS production (Bindokas et al. 1996; Dugan et
al.1995; Dykens 1994; Lafon-Cazal et al. 1993; Reynolds and
Hastings 1995; Urushitani et al. 2001; Vergun et al. 2001).
Increased ROS levels can lead to mitochondrial damage (Hoyt
et al. 1997), which can initiate a further cycle of increased
intracellular Ca2⫹, increased ROS production, and cyotoxicity
(Brouillet et al. 1995).
Ascorbate, by acting as an ROS scavenger to decrease levels
of intracellular ROS generated downstream from NMDA receptor activation, can break this pathological cycle and decrease NMDA-mediated excitoxicity (Atlante et al. 1997;
Ciani et al. 1996). Prevention of irreversible hyperexcitability
in the present studies by both AP5 and ascorbate implicates
NMDA receptor– dependent ROS generation as the initiating
event in secondary, H2O2-dependent pathology. This is further
supported by the ability of ascorbate to prevent the enhanced
pathophysiology caused by pharmacological inhibition of glutamate uptake by PDC during H2O2 withdrawal (Fig. 5B).
Significantly, isoascorbate was not protective against this
secondary pathology (Fig. 2B), indicating that the site of ROS
generation was intracellular. Isoascorbate has similar electrochemical properties to ascorbate (Iheanacho et al. 1995), but is
a poorly transported substrate for the stereoselective, neuronal
ascorbate transporter, SVCT2 (Spector and Lorenzo 1974;
Tsukaguchi et al. 1999). Thus isoascorbate does not readily
enter brain cells, whereas ascorbate does (Brahma et al. 2000;
Rice et al. 1994). We have previously used these isomers to
distinguish between intra- versus extracellular sites of action
for ascorbate neuroprotection (Avshalumov et al. 2000;
Brahma et al. 2000). Once in the intracellular compartment,
ascorbate is dynamically maintained in the reduced state by
endogenous thiols, including glutathione (GSH), by a GSHdependent dehydroascorbate reductase, and indirectly by other
components of the antioxidant network (Buettner 1993; Fornai
et al. 1999; Meister 1994; Rose 1993; Winkler et al. 1994).
Isoascorbate has limited access to this intracellular antioxidant
network and thus is more susceptible to oxidation in brain
tissue. Here, the incomplete recovery of PS amplitude during
H2O2 washout with isoascorbate (Fig. 2C) might reflect a slight
pro-oxidant effect of this agent in the extracellular compartment, as we have seen in other studies (Brahma et al. 2000).
Ascorbate has been suggested to be neuroprotective under
some conditions by decreasing NMDA receptor activity via the
redox modulatory site (Majewska and Bell 1990). As noted
above, NMDA receptor activity is decreased by oxidation,
2901
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M. V. AVSHALUMOV AND M. E. RICE
production during reperfusion is proportional to the increase in
extracellular glutamate during the ischemic period (Morimoto
et al. 1996).
Summary and implications
We are grateful to B. T. Chen for insightful comments on the manuscript.
These studies were supported by National Institute of Neurological Disorders and Stroke Grant NS-36362.
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On the basis of the present findings and earlier reports in the
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