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

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

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.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

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 Ca2+ 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 Ca2+ 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 Ca2+-dependent processes.

The free radical superoxide anion is an additional signaling molecule that may be produced in response to LTP-inducing 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 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 cell-impermeable 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
Abstract
Introduction
Methods
Results
Discussion
References

Materials

MnTBAP and MnTMPyP were purchased from Calbiochem (La Jolla, CA). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonopentanoic acid (D-AP5) were purchased from Tocris Cookson (St. Louis, MO). For the control experiments shown in Fig. 1C, 100× 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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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 = 6 for A and B). Closed squares are ensemble averages from slices given LTP-inducing high-frequency stimulation (HFS; arrow) with either 100 µM Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP; n = 6, A) or 25 µM Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP; n = 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 < 0.001, paired Student's t-test in A; P < 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 µM; open circles, n = 4) or light-inactivated MnTMPyP (25 µM; open squares, n = 4). D: representative field excitatory postsynaptic potentials (fEPSPs) 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.

Preparation of hippocampal slices

Hippocampi from male Sprague-Dawley rats (100-150 g) were removed and 400-µm 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 30°C. Responses to Schaffer collateral stimulation in area CA1 were monitored for >= 20 min before the delivery of LTP-inducing HFS. Test stimuli (50 µs) were given at a current (30-50 µA) that produced 50% of the maximum initial slope of the extracellular field excitatory postsynaptic potential (fEPSP). 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 µA) that elicited the maximum fEPSP. Responses to test stimuli were measured every 2.5 min as an average of four individual traces (0.1 Hz) for either 45 or 60 min after the final train of HFS. Post-HFS responses were elicited by the same test stimulation intensity as before HFS. LTP was defined as >= 20% increase in the initial slope of the fEPSP compared with pre-HFS control levels (within-slice 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 pre-HFS 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.

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 HFS-response 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
Abstract
Introduction
Methods
Results
Discussion
References

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 hippocampal slices perfused with 100 µM MnTBAP. LTP was absent in all six experiments [fEPSP slope = 103 ± 4% (mean ± SE) of control, n = 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 = 176 ± 16% of control, n = 6; Fig. 1A). These data suggest that superoxide is necessary for the induction of LTP.


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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 µM MnTBAP (square , n = 4) or 25 µM MnTMPyP (black-square, n = 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 µM 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 µM MnTBAP (square , n = 4) or 25 µM MnTMPyP (black-square, n = 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 µM D-2-amino-5-phosphonopentanoic acid (D-AP5). C: representative fEPSPs 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.


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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 µM MnTBAP (square , n = 4) or 25 µM MnTMPyP (black-square, n = 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.

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 µM; fEPSP slope = 157 ± 3% of control, n = 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.

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 µM MnTMPyP (fEPSP slope = 107 ± 3% of control, n = 6; Fig. 1B). In control experiments, all six slices exhibited LTP (fEPSP slope = 157 ± 3% of control, n = 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 µM; fEPSP slope = 152 ± 9% of control, n = 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 µM CNQX (Fig. 2B). Finally, neither porphyrin 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 = 6 for MnTBAP; 106 ± 7% of control, n = 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 = 6 for MnTBAP; 100 ± 2% of control, n = 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
Abstract
Introduction
Methods
Results
Discussion
References

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 (Gahatan 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 these two forms of synaptic plasticity suggests that blockade of LTP is not due to nonspecific effects of either compound. Second, these data indicate that superoxide is unlikely to be involved in the molecular mechanisms underlying either PTP or STP. The lack of an effect of the porphyrin compounds on either PTP or STP implies that the molecular actions of superoxide produced after HFS are unique to the biochemical signaling cascades involved in LTP.

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 long-term 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.

    ACKNOWLEDGEMENTS

  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.

    FOOTNOTES

  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.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society