RAPID COMMUNICATION
Cell-Permeable Scavengers of Superoxide Prevent Long-Term
Potentiation in Hippocampal Area CA1
ERIC KLANN
Department of Neuroscience, Center for the Neural Basis of Cognition, University of Pittsburgh,
Pittsburgh, Pennsylvania 15260
INTRODUCTION
Long-term potentiation (LTP) of synaptic transmission in
the hippocampus is a widely studied form of synaptic plasticity that has been hypothesized to be a cellular substrate
for learning and memory formation (Bliss and Collingridge
1993). Induction of LTP by high-frequency stimulation
(HFS) in the CA1 region of the hippocampus is generally
dependent on postsynaptic Ca 2/ influx after the activation
of N-methyl-D-aspartate (NMDA) receptors (Collingridge
et al. 1983; Lynch et al. 1983; Malenka et al. 1988). One
function of Ca 2/ after LTP-inducing HFS is activation of
enzymes that produce signaling molecules (Roberson et al.
1996). For example, LTP-inducing HFS results in increased
production of cAMP (Chetkovich et al. 1991), nitric oxide
(Chetkovich et al. 1993), and arachidonic acid (Lynch et
al. 1989), all of which are produced via Ca 2/ -dependent
processes.
The free radical superoxide anion is an additional signaling molecule that may be produced in response to LTPinducing HFS. Consistent with this possibility, it was
shown that NMDA receptor activation in area CA1 of
hippocampal slices results in the production of superoxide
( Bindokas et al. 1996 ) . Furthermore, it recently was
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shown that extracellular application of superoxide dismutase ( SOD ) and catalase, enzymes that catalyze the removal of superoxide and hydrogen peroxide, respectively,
attenuates LTP induction in area CA1 ( Klann et al. 1998 ) .
In agreement with these findings, transgenic mice that
overexpress Cu / Zn SOD have impaired LTP that can be
rescued partially by catalase ( Gahatan et al. 1998 ) . Finally, incubation of hippocampal slices with 5,5 dimethyl
pyrolline 1-oxide ( DMPO ) , an electron spin resonance
spin trap reagent that is capable of removing superoxide,
also attenuates LTP induction ( Klann et al. 1998 ) .
Extracellular application of cell-impermeable compounds, such as SOD, catalase, and DMPO, to slices likely
results in the scavenging of superoxide extracellularly.
The observation that these compounds attenuate and do
not completely block LTP ( Klann et al. 1998 ) could be
because of intracellular actions of superoxide that are unaffected by extracellular scavengers after LTP-inducing
HFS. Therefore intracellular actions of superoxide might
be crucial for the full expression of LTP. The characterization of cell-permeable manganese porphyrin compounds
that either mimic SOD or scavenge superoxide ( Faulkner
et al. 1994; Gardner et al. 1996 ) has permitted the investigation of processes requiring intracellular superoxide
( Day et al. 1995; Patel et al. 1996 ) . In this study two
cell-permeable manganese porphyrin compounds, Mn(III)
tetrakis (4-benzoic acid) porphyrin (MnTBAP) and Mn(III)
tetrakis (1-methyl-4-pyridyl porphyrin (MnTMPyP), were
employed to determine whether or not superoxide is necessary for LTP. In contrast to the previous studies with cellimpermeable superoxide scavengers, LTP was completely
blocked by either of the cell-permeable superoxide scavengers. These results indicate that superoxide should be added
to the list of signaling molecules necessary for the induction
of LTP.
METHODS
Materials
MnTBAP and MnTMPyP were purchased from Calbiochem (La
Jolla, CA). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D2-amino-5-phosphonopentanoic acid ( D-AP5) were purchased
from Tocris Cookson (St. Louis, MO). For the control experiments
shown in Fig. 1C, 1001 solutions of MnTBAP and MnTMPyP
were made and were light-inactivated before being diluted in standard saline solution for use in LTP experiments.
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Klann, Eric. Cell-permeable scavengers of superoxide prevent
long-term potentiation in hippocampal area CA1. J. Neurophysiol.
80: 452–457, 1998. Long-term potentiation (LTP) in hippocampal
area CA1 is generally dependent on N-methyl-D-aspartate
(NMDA) receptor activation. Reactive oxygen species (ROS),
including superoxide, are produced in response to NMDA receptor
activation in a number of brain regions, including the hipppocampus. In this study, two cell-permeable manganese porphyrin compounds that mimic superoxide dismutase (SOD) were used to determine whether production of superoxide is required for the induction of LTP in area CA1 of rat hippocampal slices. Incubation of
hippocampal slices with either Mn(III) tetrakis (4-benzoic acid)
porphyrin (MnTBAP) or Mn(III) tetrakis (1-methyl-4-pyridyl)
porphyrin (MnTMPyP) prevented the induction of LTP. Incubation
of slices with either light-inactivated MnTBAP or light-inactivated
MnTMPyP had no effect on induction of LTP. Neither MnTBAP
nor MnTMPyP was able to reverse preestablished LTP. These
observations suggest that production of superoxide occurs in response to LTP-inducing stimulation and that superoxide is necessary for the induction of LTP.
SCAVENGERS OF SUPEROXIDE BLOCK LTP
453
Preparation of hippocampal slices
Hippocampi from male Sprague-Dawley rats (100–150 g) were
removed and 400- mm slices were prepared with a McIlwain tissue
chopper. The slices were perfused for 1 h with a standard saline
solution containing (in mM) 124 NaCl, 4.4 KCl, 26 NaHCO3 , 10
D-glucose, 2 CaCl2 , 2 MgCl2 ; gassed with 95% O2-5% CO2 , pH
7.4 in an interface tissue slice chamber at 307C. Responses to
Schaffer collateral stimulation in area CA1 were monitored for
¢20 min before the delivery of LTP-inducing HFS. Test stimuli
(50 ms) were given at a current (30–50 m A) that produced 50%
of the maximum initial slope of the extracellular field excitatory
postsynaptic potential ( f EPSP). Responses to test stimuli were
measured every 2.5 min as an average of four individual traces
(0.1 Hz).
Induction of LTP
LTP-inducing HFS consisted of three 1-s trains of stimuli (100
Hz), given 20 s apart with the use of a current (60–100 m A)
that elicited the maximum f EPSP. Responses to test stimuli were
measured every 2.5 min as an average of four individual traces
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(0.1 Hz) for either 45 or 60 min after the final train of HFS. PostHFS responses were elicited by the same test stimulation intensity
as before HFS. LTP was defined as ¢20% increase in the initial
slope of the f EPSP compared with pre-HFS control levels (withinslice comparison).
Application of porphyrin compounds to hippocampal
slices
After incubating the slices in standard saline solution for 1 h,
baseline responses were monitored for 10 min to ensure a stable
baseline. The perfusate then was changed to standard saline solution containing either MnTBAP, MnTMPyP, light-inactivated
MnTBAP, or light-inactivated MnTMPyP for 20 min (10 min preHFS and 10 min post-HFS).
To determine whether the porphyrin compounds exerted an effect on normal and/or NMDA receptor-mediated synaptic transmission, baseline responses were monitored for 20 min to ensure
a stable baseline. Responses were monitored for 20 min while the
slices were perfused with either MnTBAP or MnTMPyP and for
an additional 20 min after washing out the porphyrin compounds.
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FIG . 1. Effect of cell-permeable superoxide scavengers on the induction of long-term potentiation (LTP) in hippocampal
area CA1. A and B: open squares are ensemble averages from control LTP experiments (n Å 6 for A and B). Closed squares
are ensemble averages from slices given LTP-inducing high-frequency stimulation (HFS; arrow) with either 100 m M Mn(III)
tetrakis (4-benzoic acid) porphyrin (MnTBAP; n Å 6, A) or 25 m M Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin
(MnTMPyP; n Å 6, B) in the perfusing solution (horizontal bar). Responses recorded from slices given LTP-inducing HFS
in the presence of either MnTBAP or MnTMPyP always were compared with responses recorded from a control slice from
the same animal (in an adjacent recording chamber) that received LTP-inducing HFS in the absence of the compound. In
the presence of the manganese porphyrin compounds, LTP was significantly blocked ( P õ 0.001, paired Student’s t-test in
A; P õ 0.00001, paired Student’s t-test in B). C: ensemble averages from slices given LTP-inducing HFS in the presence
of either light-inactivated MnTBAP (100 m M; open circles, n Å 4) or light-inactivated MnTMPyP (25 m M; open squares,
n Å 4). D: representative field excitatory postsynaptic potentials ( f EPSPs) taken before and 45 min after HFS from either
control LTP slices, slices given HFS in the presence of MnTBAP, or slices given HFS in the presence of MnTMPyP.
Calibration bars are 2 mV and 3 ms.
454
E. KLANN
To determine whether or not the porphyrin compounds exerted
an effect on high-frequency synaptic transmission, the responses
to the first HFS were analyzed by both integrating the entire HFSresponse trace (integral) and measuring the level of steady-state
depolarization during HFS (averaged over the last 50 ms of the
HFS). Measurements in all slices were normalized to the amplitude
of the fEPSP produced by a single pulse at the stimulus intensity
used for the HFS. Data from slices incubated with either MnTBAP
or MnTMPyP slices were compared with control slices and expressed as percent of control.
RESULTS
Effects of porphyrin compounds on the induction of LTP
To test the hypothesis that superoxide is necessary for
the induction of LTP in hippocampal area CA1, HFS was
delivered to Schaffer collateral-commissural fibers of hippo-
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campal slices perfused with 100 m M MnTBAP. LTP was
absent in all six experiments [fEPSP slope Å 103 { 4%
(mean { SE) of control, n Å 6; Fig. 1A]. In contrast, when
HFS was delivered to control slices in an adjacent recording
chamber, LTP was observed in all six experiments (fEPSP
slope Å 176 { 16% of control, n Å 6; Fig. 1A). These data
suggest that superoxide is necessary for the induction
of LTP.
To address the possibility that MnTBAP prevented the
induction of LTP via a nonspecific effect, control experiments were conducted with light-inactivated MnTBAP. LTP
was observed in all slices perfused with light-inactivated
MnTBAP (100 m M; fEPSP slope Å 157 { 3% of control,
n Å 4; Fig. 1C). These data suggest that blockade of LTP
by MnTBAP is due to the capacity of this compound to act
as either a SOD mimetic or a superoxide scavenger.
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FIG . 2. Effect of MnTBAP and MnTMPyP on baseline synaptic transmission and the N-methyl-D-aspartate (NMDA)
receptor-mediated component of the fEPSP. A: baseline responses were recorded for 20 min before slices were perfused for
20 min with either 100 m M MnTBAP ( h, n Å 4) or 25 m M MnTMPyP ( j, n Å 5). Responses were recorded for an
additional 20 min after washout of each compound. B: NMDA receptor-mediated fEPSPs were isolated by adding to the
bath solution 20 m M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 0 mM MgCl2 , and 4 mM CaCl2 . Baseline responses
were recorded for 20 min before slices were perfused for 20 min with either 100 m M MnTBAP ( h, n Å 4) or 25 m M
MnTMPyP ( j, n Å 4). Responses were recorded for an additional 20 min after washout of each porphyrin compound. After
the washout period, it was determined that the fEPSPs recorded in the presence of CNQX under these conditions were
NMDA receptor-dependent, because they were blocked completely by 50 m M D-2-amino-5-phosphonopentanoic acid ( DAP5). C: representative f EPSPs taken (a) before and (b) 20 min after washout of either MnTBAP or MnTMPyP. Calibration
bars are 2 mV and 3 ms. D: representative NMDA receptor-mediated fEPSPs taken (a) before application of the porphyrin
compounds, (b) 20 min after washout of either MnTBAP or MnTMPyP, and (c) after application of D-AP5. Calibration
bars are 2 mV and 3 ms.
SCAVENGERS OF SUPEROXIDE BLOCK LTP
455
Dismutation of superoxide by MnTBAP, a SOD mimetic,
would result in the production of hydrogen peroxide. However, hydrogen peroxide has been shown to prevent induction of LTP (Auerbach and Segal 1997; Pellmar et al. 1991).
Therefore the effects of MnTMPyP on LTP were examined.
In contrast to MnTBAP, MnTMPyP was reported to act
as a superoxide scavenger but not as a SOD mimetic in
mammalian cells (Gardner et al. 1996). LTP was prevented
when slices were perfused with 25 m M MnTMPyP (fEPSP
slope Å 107 { 3% of control, n Å 6; Fig. 1B). In control
experiments, all six slices exhibited LTP (fEPSP slope Å
157 { 3% of control, n Å 6). These results are consistent
with the idea that removal of superoxide prevents the induction of LTP.
Additional control experiments were performed with
light-inactivated MnTMPyP. LTP was observed in all slices
perfused with light-inactivated MnTMPyP (25 m M; fEPSP
slope Å 152 { 9% of control, n Å 4; Fig. 1C). The results
of these experiments suggest that blockade of LTP is not
due to a nonspecifc effect of MnTMPyP, but occurs because
of the scavenging of superoxide by MnTMPyP.
Effects of manganese porphyrin compounds on synaptic
transmission and NMDA receptor function
Experiments were performed to ensure that the manganese
porphyrin compounds used in the experiments described in
Fig. 1 did not have nonspecific effects on synaptic transmission. No changes in responses to test stimuli were observed
in area CA1 in slices exposed to either MnTBAP or
MnTMPyP (Fig. 2A). Similarly, neither MnTBAP nor
MnTMPyP caused a significant alteration of the NMDA receptor-mediated component of the fEPSP measured in the
presence of 20 m M CNQX (Fig. 2B). Finally, neither por-
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phyrin compound had a significant effect on high-frequency
synaptic transmission, measured as either total depolarization (integrating the entire HFS response; 105 { 4% of
control, n Å 6 for MnTBAP; 106 { 7% of control, n Å 6
for MnTMPyP) or the steady-state depolarization produced
during HFS (averaged over the last 50 ms of the HFS;
104 { 3% of control, n Å 6 for MnTBAP; 100 { 2% of
control, n Å 6). Taken together, these results suggest that
blockade of LTP by the manganese porphyrin compounds
is not because of effects on baseline synaptic responses or
NMDA receptor function.
Effects of manganese porphyrin compounds on
preestablished LTP
The results presented in Fig. 1 show that superoxide is
necessary for the induction of LTP. To test for a possible role
of superoxide in the maintenance of LTP, either MnTBAP or
MnTMPyP was added to slices 20 min after the final HFS
and perfused for 20 min before being washed out. Preestablished LTP was not affected by either manganese porphyrin
compound (Fig. 3), which suggests that superoxide is not
necessary for the maintenance of LTP.
DISCUSSION
The findings described in this report show that superoxide
is required for induction of LTP. Previously it was shown
that when slices were incubated with either SOD and catalase
or DMPO the probability of LTP induction was less likely
and LTP was attenuated when potentiation was observed
(Klann et al. 1998). In a different study using slices from
transgenic mice that overexpress Cu/Zn SOD, LTP was only
attenuated when slices were incubated with catalase (Gaha-
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FIG . 3. Effect of MnTBAP and MnTMPyP on the maintenance of LTP in hippocampal area CA1. A: baseline responses
were recorded for 20 min before delivery of HFS (indicated by arrow). Twenty minutes after delivery of HFS, slices were
perfused for 20 min with either 100 m M MnTBAP ( h, n Å 4) or 25 m M MnTMPyP ( j, n Å 4). Responses were recorded
for an additional 20 min after washout of each compound. B: representative fEPSPs taken (a) before HFS, (b) 20 min after
the last train of HFS (immediately before application of the porphyrin compounds), and (c) 20 min after washout of either
MnTBAP or MnTMPyP. Calibration bars are 2 mV and 3 ms.
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E. KLANN
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The author thanks Dr. Edda Thiels, L. T. Knapp, E. D. Norman, and B. I.
Kanterewicz for thoughtful comments on the manuscript.
This work was supported by National Institute of Neurological Disorders
and Stroke Grant NS-34007 and by the Winters Foundation.
Address for reprint requests: Dept. of Neuroscience, University of Pittsburgh, 446 Crawford Hall, Pittsburgh, PA 15260.
Received 10 March 1998; accepted in final form 7 April 1998.
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tan et al. 1998). In contrast, in this study blockade of LTP
was observed in all experiments when slices were incubated
with either MnTBAP or MnTMPyP (Fig. 1), both of which
are cell-permeable superoxide scavengers. These results suggest that superoxide is necessary for LTP and that the reason
that cell-impermeable scavengers only attenuate LTP is because these compounds do not intracellularly scavenge superoxide produced after HFS.
In contrast to LTP, both posttetanic potentiation (PTP)
and short-term potentiation (STP) were insensitive to treatment with either MnTBAP or MnTMPyP (Fig. 1, A and B).
This observation is significant for two reasons. First, the lack
of an effect of the cell-permeable superoxide scavengers on
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The origin of the superoxide produced after LTP-inducing HFS has not been investigated, although results from
previous studies provide a number of intriguing possibilities. For example, superoxide can be produced via the
actions of lipoxygenase on arachidonic acid ( Kukreja et
al. 1986 ) . Consistent with this possibility, a lipoxygenase
inhibitor was shown to prevent the induction of LTP
( Lynch et al. 1989 ) . In addition, nitric oxide synthase,
which was shown to be activated after HFS ( Chetkovich
et al. 1993 ) , is capable of producing superoxide under the
appropriate conditions ( Pou et al. 1992 ) . Thus superoxide
might be produced either as a result of, or in conjunction
with, other small signaling molecules that are necessary
for LTP.
If superoxide is produced in response to HFS to serve
as a cellular signaling molecule in LTP, then on what
enzymes might it act? One candidate enzyme is protein
kinase C, which was shown to be activated by superoxide
( Larsson and Cerutti 1989 ) as well as after LTP-inducing
HFS ( Klann et al. 1991, 1993, 1998 ) , and is necessary
for the induction of LTP ( Malinow et al. 1989 ) . Similarly,
in hippocampal slices, p42 mitogen-activated protein kinase was shown to be activated by superoxide ( Kanterewicz et al. 1998 ) as well as after LTP-inducing HFS ( English and Sweatt 1996 ) and to be necessary for the induction of LTP ( English and Sweatt 1997 ) . In addition,
superoxide can inactivate calcineurin ( Wang et al. 1996 ) ,
a calcium/ calmodulin-dependent protein phosphatase that
was shown to be necessary for the induction of longterm depression in hippocampal area CA1 ( Mulkey et al.
1994 ) . Thus, in addition to enhancing the activity of protein kinases, superoxide might inactivate protein phosphatases as a means of enhancing the phosphorylation of critical substrates after the induction of LTP.
The results presented in this study are consistent with the
idea that superoxide is produced after HFS and plays an
important role in the induction of LTP. Additional studies
are needed to determine how superoxide is produced and
what it acts on in the context of LTP.
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