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• W2034991523 abstract "The induction of several forms of long-term potentiation (LTP) of synaptic transmission in the CA1 region of the mammalian hippocampus is dependent onN-methyl-d-aspartate receptor activation and the subsequent activation of protein kinase C (PKC), but the mechanisms that underlie the regulation of PKC in this context are largely unknown. It is known that reactive oxygen species, including superoxide, are produced byN-methyl-d-aspartate receptor activation in neurons, and recent studies have suggested that some reactive oxygen species can modulate PKC in vitro. Thus, we have investigated the role of superoxide in both the induction of LTP and the activation of PKC during LTP. We found that incubation of hippocampal slices with superoxide scavengers inhibited the induction of LTP. The effects of superoxide on LTP induction may involve PKC, as we observed that superoxide was required for appropriate modulation of PKC activation during the induction of LTP. In this respect, superoxide appears to work in conjunction with nitric oxide, which was required for a portion of the LTP-associated changes in PKC activity as well. Our observations indicate that superoxide and nitric oxide together regulate PKC in a physiologic context and that this type of regulation occurs during the induction of LTP in the hippocampus. The induction of several forms of long-term potentiation (LTP) of synaptic transmission in the CA1 region of the mammalian hippocampus is dependent onN-methyl-d-aspartate receptor activation and the subsequent activation of protein kinase C (PKC), but the mechanisms that underlie the regulation of PKC in this context are largely unknown. It is known that reactive oxygen species, including superoxide, are produced byN-methyl-d-aspartate receptor activation in neurons, and recent studies have suggested that some reactive oxygen species can modulate PKC in vitro. Thus, we have investigated the role of superoxide in both the induction of LTP and the activation of PKC during LTP. We found that incubation of hippocampal slices with superoxide scavengers inhibited the induction of LTP. The effects of superoxide on LTP induction may involve PKC, as we observed that superoxide was required for appropriate modulation of PKC activation during the induction of LTP. In this respect, superoxide appears to work in conjunction with nitric oxide, which was required for a portion of the LTP-associated changes in PKC activity as well. Our observations indicate that superoxide and nitric oxide together regulate PKC in a physiologic context and that this type of regulation occurs during the induction of LTP in the hippocampus. Hippocampal long-term potentiation (LTP) 1The abbreviations used are: LTP, long-term potentiation; HFS, high frequency stimulation; NMDA,N-methyl-d-aspartate; PKC, protein kinase C; ROS, reactive oxygen species; SOD, superoxide dismutase; DMPO, 5,5-dimethylpyrroline 1-oxide; EPSP, excitatory postsynaptic potential; NOS, nitric oxide synthase; NO, nitric oxide; NOArg,N-nitro-arginine. is a long-lasting synaptic enhancement that may be involved in certain types of mammalian learning and memory (1Bliss T.V.P. Collingridge G.L. Nature. 1993; 361: 31-39Crossref PubMed Scopus (9607) Google Scholar). It generally is agreed that in area CA1 of the hippocampus the induction of most forms of LTP induced by high frequency stimulation (HFS) is dependent on Ca2+ entry into the postsynaptic neuron triggered byN-methyl-d-aspartate (NMDA) receptor activation (2Collingridge G.L. Kehl S.J. McLennan H. J. Physiol. 1983; 334: 33-46Crossref PubMed Scopus (1794) Google Scholar, 3Lynch G. Larson J. Kelso S. Barrionuevo G. Schottler F. Nature. 1983; 305: 719-721Crossref PubMed Scopus (822) Google Scholar, 4Malenka R.C. Kauer J.A. Zucker R.S. Nicoll R.A. Science. 1988; 242: 81-84Crossref PubMed Scopus (606) Google Scholar). The exact biochemical sequence of events following Ca2+ influx into the postsynaptic cell is unknown, but it is clear that Ca2+-dependent protein kinases are involved in the induction of LTP (5Malinow R. Schulman H. Tsien R.W. Science. 1989; 245: 862-866Crossref PubMed Scopus (1041) Google Scholar, 6Malenka R.C. Kauer J.A. Perkel D.J. Mauk M.D. Kelly P.T. Nicoll R.A. Waxham M.N. Nature. 1989; 340: 554-557Crossref PubMed Scopus (778) Google Scholar). The activation of protein kinase C (PKC) is one of the requisite biochemical steps necessary for the induction of LTP, as selective PKC inhibitors can block induction of LTP (5Malinow R. Schulman H. Tsien R.W. Science. 1989; 245: 862-866Crossref PubMed Scopus (1041) Google Scholar, 7Wang J.-H. Feng D.-P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2576-2580Crossref PubMed Scopus (149) Google Scholar, 8Hvalby O. Hemmings Jr., H.C. Paulsen O. Czernik A.J. Nairn A.C. Godfraind J.-M. Jensen V. Raastad M. Storm J.F. Andersen P. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4761-4765Crossref PubMed Scopus (96) Google Scholar). Furthermore, both the second messenger-independent activity of PKC (autonomous activity; see Ref. 9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar) and total, cofactor-stimulated PKC activity (cofactor-dependent activity; see Refs. 9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar and 10Otani S. Ben-Ari Y. Roisin-Lallemand M.-P. Brain Res. 1993; 613: 1-9Crossref PubMed Scopus (57) Google Scholar) are increased shortly after LTP-inducing HFS. Mechanisms that have been proposed for the activation of PKC immediately following the induction of LTP (15 s to 2 min after the final HFS) include translocation of PKC to the membrane (11Suzuki T. Okumura-Noji K. Ogura A. Tanaka R. Nakamura K. Kudo Y. Biochem. Biophys. Res. Commun. 1992; 189: 1515-1520Crossref PubMed Scopus (43) Google Scholar, 12Sacktor T.C. Osten P. Valsamis H. Jiang X. Naik M.U. Sublette E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8342-8346Crossref PubMed Scopus (346) Google Scholar), conformational changes in PKC that result in the unmasking of activator sites (10Otani S. Ben-Ari Y. Roisin-Lallemand M.-P. Brain Res. 1993; 613: 1-9Crossref PubMed Scopus (57) Google Scholar), and decreases in protein phosphatase activity that might result in increased phosphorylation and activity of PKC (10Otani S. Ben-Ari Y. Roisin-Lallemand M.-P. Brain Res. 1993; 613: 1-9Crossref PubMed Scopus (57) Google Scholar). An additional mechanism that might be involved in the activation of PKC during the induction of LTP is an oxidative mechanism involving reactive oxygen species (ROS), particularly the superoxide anion (O·̄2). A variety of evidence suggest this possibility. First, levels of superoxide may increase during the induction of LTP, as it has recently been demonstrated that NMDA receptor activation in area CA1 of hippocampal slices results in the production of superoxide (13Bindokas V.P. Jordan J. Lee C.C. Miller R.J. J. Neurosci. 1996; 16: 1324-1336Crossref PubMed Google Scholar). Second, if superoxide levels increase, PKC is a possible target, given that treatment of several different types of cells and of hippocampal homogenates with superoxide or other ROS has been shown to increase both autonomous and cofactor-dependent PKC activity (14Gopalakrishna R. Anderson W.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6758-6762Crossref PubMed Scopus (371) Google Scholar, 15Larsson R. Cerutti P. Cancer Res. 1989; 49: 5627-5632PubMed Google Scholar, 16Palumbo E.J. Sweatt J.D. Chen S.-J. Klann E. Biochem. Biophys. Res. Commun. 1992; 187: 1439-1445Crossref PubMed Scopus (68) Google Scholar). Coupled with recent studies implicating superoxide dismutase (SOD), which catalyzes removal of superoxide, in neuronal function and the neuropathology of Alzheimer's disease (17Benzi G. Moretti A. Neurobiol. Aging. 1995; 16: 661-674Crossref PubMed Scopus (351) Google Scholar) and amyotrophic lateral sclerosis (18Rosen D.R. Siddique T. Patterson D. Figlewicz D.A. Sapp P. Hentati A. Donaldson D. Goto J. O'Regan J.P. Deng H.X. Rahmani Z. Krizus A. McKenna-Yasek D. Cayabyab A. Gaston S.M. Berger R. Tanzi R.E. Halperin J.J. Herzfeldt B. Van den Bergh R. Hung W.-Y. Bird T. Deng G. Mulder D.W. Smyth C. Laing N.G. Soriano E. Pericak-Vance M.A. Haines J. Rouleau G.A. Gusella J.S. Horvitz H.R. Brown Jr., R.H. Nature. 1993; 362: 59-62Crossref PubMed Scopus (5568) Google Scholar), these findings prompted us to determine whether superoxide was involved in the induction of LTP, and if so, to determine whether superoxide was involved in the regulation of PKC activity during LTP. 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 (124 mm NaCl, 4.4 mm KCl, 26 mm NaHCO3, 10 mmd-glucose, 2 mm CaCl2, 2 mm MgCl2, gassed with 95% O2, 5% CO2, pH 7.4) in an interface tissue slice chamber at 32 °C. After 1 h, baseline responses to Schaffer collateral stimulation in the CA1 region were monitored for at least 20 min before LTP-inducing HFS to assure a stable baseline. Test stimuli were given at a current that produced 50% of the maximum population EPSP (usually between 30 and 40 μA). Responses were measured every 2.5 min as an average of four individual traces. In our previous study (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar), LTP-inducing HFS consisted of three sets of stimuli, each set was delivered 5 min apart. Each set included two 1-s trains of stimuli (100 Hz), given 20 s apart at the current needed to elicit a maximum population EPSP. Using this stimulus paradigm, cofactor-dependent PKC activity increased 2 min after the final HFS train. However, because this paradigm spanned a period of 10 min, the LTP-associated increase in cofactor-dependent PKC activity was actually measured 12 min after the first HFS train. To determine changes in PKC activity that more accurately reflect changes during the induction of LTP, we modified our LTP-inducing HFS in this study to consist of three 1-s trains of stimuli (100 Hz), given 20 s apart at the current needed to elicit a maximal population EPSP (usually between 60 and 80 μA). In experiments in which PKC activity was assayed, slices were frozen 2 min after the final HFS train, dissected, and homogenized. In experiments in which LTP was examined, responses were measured every 2.5 min as an average of four individual traces for 1 h after the final HFS. Post-HFS responses were generated with the same test stimuli used prior to HFS. LTP was defined as an increase of 20% or greater in the initial EPSP slope. Slices were prepared as described above. After a 1-h incubation in standard saline solution, hippocampal slices were perfused with standard saline solution containing SOD (Calbiochem), SOD/catalase (Calbiochem), DMPO (OMRF Spin Trap Source), or NOArg (Sigma) for at least 30 min prior to LTP-inducing HFS. In more than 90% of the experiments, slices given HFS in the presence of SOD, SOD/catalase, DMPO, or NOArg were compared with slices from the same animal given HFS in the absence of the agents in an adjacent recording chamber (LTP controls). Autonomous and cofactor-dependent PKC activity in control and potentiated tissue from hippocampal area CA1 was determined using the selective PKC substrate NG(28–43), as described previously (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar). We defined autonomous PKC activity as activity assayed in the presence of 2 mm EGTA. Cofactor-dependent PKC activity was defined as activity assayed in the presence of 100 μmCaCl2, 320 μg/ml phosphatidylserine, and 30 μg/ml dioctonoylglycerol with the autonomous PKC activity subtracted (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar). Expressed in terms of absolute specific activities, autonomous NG(28–43) phosphorylation was 0.65 ± 0.10 pmol/min/μg in control slices and 1.10 ± 0.33 pmol/min/μg in slices given LTP-inducing HFS; cofactor-dependent NG(28–43) phosphorylation was 7.07 ± 0.63 pmol/min/μg protein in control slices and 11.27 ± 1.30 pmol/min/μg protein in slices given LTP-inducing HFS. The hypothesis that superoxide plays a role in the induction of LTP was investigated in area CA1 of rat hippocampal slices. LTP was induced with HFS consisting of three 1-s trains of 100-Hz stimulation delivered 20 s apart at a stimulus intensity that produced a maximum initial slope of the extracellular field excitatory postsynaptic potentials (EPSPs). This stimulus paradigm resulted in LTP in 96% (25 out of 26) of the control slices for the experiments shown in Fig. 1. The slope of the EPSPs was increased to 155.7 ± 4.0% (n = 26) of pre-HFS control levels (within-slice comparison) 60 min after the final HFS train (Fig. 1, A-C). To determine whether superoxide was involved in LTP induction, we gave slices HFS in the presence of the superoxide scavenger SOD. SOD is highly specific for superoxide and is not known to react enzymatically with other ROS (21Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Oxford University Press, New York1989Google Scholar). LTP was blocked in eight out of nine slices 60 min after the final HFS train (average slope of EPSPs = 101.0 ± 7.5% of control, n = 9) (Fig. 1 A). In contrast, slices from the same animal in an adjacent recording chamber given HFS without SOD exhibited LTP in all nine experiments (average slope of EPSPs = 158.7 ± 7.6% of control, n = 9) (Fig. 1 A). These data suggest that superoxide plays a role in the induction of LTP. An alternative explanation of the results in Fig. 1 A is that one of the products of the reaction between SOD and superoxide inhibits the induction of LTP. Specifically, SOD converts superoxide to H2O2 and O2 (22Halliwell B. J. Neurochem. 1992; 59: 1609-1623Crossref PubMed Scopus (2666) Google Scholar), and H2O2 has been shown to inhibit LTP (23Pellmar T.C. Hollinden G.E. Sarvey J.M. Neuroscience. 1991; 44: 353-359Crossref PubMed Scopus (81) Google Scholar). To address this possibility, slices were given HFS in the presence of bath-applied SOD along with catalase, an enzyme which scavenges H2O2 and catalyzes its conversion to O2 and water (22Halliwell B. J. Neurochem. 1992; 59: 1609-1623Crossref PubMed Scopus (2666) Google Scholar). In these experiments, LTP was inhibited in 10 out of 15 slices (average slope of EPSPs = 113.3 ± 4.7% of control for all 15 slices and 102.8 ± 2.4% of control for the 10 slices that did not meet our criteria for LTP) (Fig. 1 B). In control experiments, LTP was produced in 12 out of 13 slices (average slope of EPSPs = 150.4 ± 4.7% of control, n = 13) (Fig. 1 B). Interestingly, the five slices incubated with SOD and catalase that did exhibit LTP after HFS were potentiated to a lesser extent when compared with LTP control slices (average slope of EPSPs = 134.6 ± 7.6% of control, n = 5). Slices incubated with catalase alone exhibited LTP (average slope of EPSPs = 168.5 ± 12.5% of control, n = 3) similar to that observed in LTP control slices (average slope of EPSPs = 166.6 ± 7.4% of control,n = 3). Our interpretation of these data is that the effects of SOD on LTP induction are not solely a result of increased levels of H2O2. However, our findings do not exclude the possibility that H2O2 not removed by catalase may contribute to the observed effects of SOD on LTP. As mentioned, SOD is a specific enzyme not known to catalyze the removal of any ROS besides superoxide. However, SOD, like most other proteins, contains histidine, tryptophan, and methionine, each of which can react nonenzymatically with other ROS (21Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. 2nd Ed. Oxford University Press, New York1989Google Scholar). To demonstrate that the effects of SOD are not due to these nonenzymatic reactions with other ROS, we incubated slices with boiled SOD and catalase and then examined their effect on the induction of LTP. In slices incubated with the heat-inactivated enzymes, LTP was indistinguishable (average slope of EPSPs = 162.3 ± 11.3% of control, n = 4) (Fig. 1 C) from LTP in control slices (average slope of EPSPs = 154.4 ± 9.4% of control, n = 4) (Fig. 1 C). Thus, the effects of SOD and catalase on LTP are not attributable to nonenzymatic removal of ROS other than superoxide. Although the concentrations of SOD (121 units/ml) and catalase (260 units/ml) we used have been shown in previous studies to have no effect on baseline electrophysiological responses in area CA1 of guinea pig hippocampal slices (24Pellmar T.C. Lipinski D.L. Brain Res. 1992; 569: 189-198Crossref PubMed Scopus (11) Google Scholar), we performed a series of experiments to verify that the superoxide scavengers used in our studies did not have nonspecific effects on synaptic transmission. We observed no alteration of electrophysiological responses to test stimuli in area CA1 in rat hippocampal slices exposed to SOD and catalase (Fig. 2 A). Moreover, SOD and catalase had no effect on the NMDA component of the EPSP measured in the presence of 20 μm6-cyano-7-nitroquinoxaline-2,3-dione (Fig. 2 B). Finally, SOD and catalase had no effect on high-frequency synaptic transmission, as measured by either the total depolarization or the steady-state depolarization produced during HFS (Fig. 2 C). These results suggest that inhibition of LTP induction by superoxide scavengers is not due to effects on baseline synaptic transmission or NMDA receptor function. Overall, these data suggest that superoxide has a role in modulating the induction of LTP in area CA1. To demonstrate that a structurally and functionally distinct superoxide scavenger would also inhibit LTP induction, we incubated slices with 10 mm 5,5-dimethylpyrroline 1-oxide (DMPO), a superoxide and hydroxyl radical electron spin resonance (ESR) spin trap that previously has been shown to block NMDA-induced, superoxide-dependent neurotoxicity in cerebellar granule neurons (25Lafon-Cazal M. Pietri S. Culcasi M. Boeckart J. Nature. 1993; 364: 535-537Crossref PubMed Scopus (1106) Google Scholar). DMPO inhibited LTP in three out of five experiments and attenuated LTP in the other two experiments (average slope of EPSPs = 120.6 ± 4.8% of control, n = 5) (Fig. 3). In contrast, all five control slices exhibited LTP (average slope of EPSPs = 160.4 ± 7.8% of control, n = 5). In addition, DMPO had no effect on electrophysiological responses to test stimuli (data not shown). These results are consistent with results observed with SOD and catalase and provide further evidence of a role for superoxide in the induction of LTP. We conclude from this series of experiments that removing superoxide reduces the probability of LTP induction and the magnitude of LTP when potentiation occurs. Because neither SOD, catalase, nor DMPO can cross cell membranes, these data also suggest that superoxide may act as a transcellular messenger in LTP. The results of the experiments in Figs. 1 and 3 indicate that superoxide modulates the induction of LTP, but they do not address the possibility of a role for superoxide in the maintenance of LTP. To test this possibility, we induced LTP and added SOD and catalase 20 min after the final LTP-inducing HFS. Pre-established LTP was unaffected by addition of SOD and catalase when monitored up to 1 h after the addition of the enzymes (Fig. 4). These results indicate that superoxide modulates the induction, but not the maintenance, of LTP. We next sought to begin to elucidate the biochemical targets of superoxide in LTP. We began by testing the hypothesis that superoxide regulates PKC during the induction of LTP. We previously have shown that the induction of LTP is associated with increases in both autonomous (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar) and cofactor-dependent PKC activity (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar). To investigate a role for superoxide in regulating PKC activity during the induction of LTP, we first measured PKC activity after LTP-inducing HFS (see “Experimental Procedures”). Slices were frozen 2 min after the final train of HFS, dissected, and homogenized. Homogenates from control slices and slices given HFS were assayed for autonomous and cofactor-dependent PKC activity using the selective PKC substrate peptide NG(28–43) (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar, 26Chen S.-J. Klann E. Gower M.C. Powell C.M. Sessoms J.S. Sweatt J.D. Biochemistry. 1993; 32: 1032-1039Crossref PubMed Scopus (70) Google Scholar, 27Gonzalez A. Klann E. Sessoms J.S. Chen S.-J. Anal. Biochem. 1993; 215: 184-189Crossref PubMed Scopus (20) Google Scholar). In comparisons of slices given LTP-inducing HFS and control slices that received only test stimuli, we observed an increase in both autonomous and cofactor-dependent PKC activity 2 min after the HFS (183.4 ± 18.6 and 144.8 ± 11.6% of control, respectively,n = 12, p < 0.05) (Figs.5, A and B). These results agree with previous work demonstrating that HFS results in an increase in both autonomous (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar) and cofactor-dependent PKC activity (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar, 10Otani S. Ben-Ari Y. Roisin-Lallemand M.-P. Brain Res. 1993; 613: 1-9Crossref PubMed Scopus (57) Google Scholar), and are consistent with physiological studies showing that blockade of PKC inhibits induction of LTP (5Malinow R. Schulman H. Tsien R.W. Science. 1989; 245: 862-866Crossref PubMed Scopus (1041) Google Scholar, 7Wang J.-H. Feng D.-P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2576-2580Crossref PubMed Scopus (149) Google Scholar, 8Hvalby O. Hemmings Jr., H.C. Paulsen O. Czernik A.J. Nairn A.C. Godfraind J.-M. Jensen V. Raastad M. Storm J.F. Andersen P. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4761-4765Crossref PubMed Scopus (96) Google Scholar). To test the hypothesis that superoxide mediates the increases in autonomous and cofactor-dependent PKC activity associated with the induction of LTP, we assayed PKC activity after incubating slices with SOD/catalase and delivering LTP-inducing HFS to area CA1. We observed an attenuated but significant increase in autonomous PKC activity in slices given HFS in the presence of SOD/catalase (146.8 ± 20.1% of control, n = 8,p < 0.05) (Fig. 5 A). In contrast, the increase in cofactor-dependent PKC activity associated with the induction of LTP was blocked completely (91.4 ± 9.6% of control, n = 8) (Fig. 5 B). In control experiments, addition of SOD/catalase to both the slices after LTP-inducing HFS and to the homogenization buffer had no significant effect on the LTP-associated increases in either autonomous PKC activity (177.5 ± 23.4% of control, n = 3) or cofactor-dependent PKC activity (160.3 ± 15.7% of control, n = 3). These results suggest that the increase in cofactor-dependent PKC activity associated with the induction of LTP is mediated either directly or indirectly by superoxide. Furthermore, these data suggest that superoxide contributes to the generation of autonomously active PKC, consistent with the hypothesis that multiple mechanisms of PKC activation are associated with the induction of LTP (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar, 12Sacktor T.C. Osten P. Valsamis H. Jiang X. Naik M.U. Sublette E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8342-8346Crossref PubMed Scopus (346) Google Scholar, 28Osten P. Valsamis L. Harris A. Sacktor T.C. J. Neurosci. 1996; 16: 2444-2451Crossref PubMed Google Scholar). The results described above indicate that superoxide can modulate induction of LTP, possibly through interactions with PKC. In many systems, superoxide exerts its effects in conjunction with nitric oxide (NO) (see Ref. 22Halliwell B. J. Neurochem. 1992; 59: 1609-1623Crossref PubMed Scopus (2666) Google Scholar). Furthermore, it is known that inhibitors of nitric oxide synthase (NOS) modulate the induction of LTP induced by many stimulus paradigms (29Böhme G.A. Bon C. Stutzman J.-M. Doble A. Blanchard J.-C. Eur. J. Pharmacol. 1991; 199: 379-381Crossref PubMed Scopus (599) Google Scholar, 30Schuman E.M. Madison D.V. Science. 1991; 254: 1503-1506Crossref PubMed Scopus (994) Google Scholar, 31O'Dell T.J. Hawkins R.D. Kandel E.R. Arancio O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11285-11289Crossref PubMed Scopus (903) Google Scholar, 32Haley J.E. Wilcox G.L. Chapman P.F. Neuron. 1992; 8: 211-216Abstract Full Text PDF PubMed Scopus (445) Google Scholar, 33Chetkovich D.M. Klann E. Sweatt J.D. Neuroreport. 1993; 4: 919-922Crossref PubMed Scopus (116) Google Scholar, 34Schuman E.M. Madison D.V. Science. 1994; 263: 532-536Crossref PubMed Scopus (311) Google Scholar). In this study, slices given HFS in the presence of the NOS inhibitor N-nitro-arginine (NOArg, 100 μm) exhibited LTP (average slope of EPSPs = 135 ± 5% of control, n = 4). However, the slices were potentiated to a lesser extent (p < 0.05, paired Student's t test) when compared with LTP control slices from the same animal in an adjacent recording chamber (average slope of EPSPs = 166 ± 10% of control, n = 4). Therefore, we next sought to determine whether NO might be involved in the regulation of PKC activity during the induction of LTP. To examine interactions between NO and PKC during the induction of LTP, hippocampal slices were incubated with NOArg (100 μm), given LTP-inducing HFS, and assayed for changes in PKC activity. As with superoxide scavengers, in the presence of NOArg we observed an attenuated but significant increase in autonomous PKC activity (141.4 ± 20.5% of control, n = 10,p < 0.05) (Fig. 6 A), and complete inhibition of the increase in cofactor-dependent PKC activity (98.7 ± 14.6% of control, n = 10) (Fig. 6 B) associated with the induction of LTP. These results suggest that activation of NOS and production of NO play an important role in the activation of PKC during LTP induction. There is an alternative interpretation of these results, as NOS is known to produce superoxide as a by-product (35Pou S. Pou W.S. Bredt D.S. Snyder S.H. Rosen G.M. J. Biol. Chem. 1992; 267: 24173-24176Abstract Full Text PDF PubMed Google Scholar). If NOS were in fact the source of superoxide production during LTP induction, the requirement for NOS activity could reflect the need for superoxide, rather than a direct role for NO in activating PKC. To distinguish between these two possibilities, we tested whether hemoglobin, which binds NO avidly (36Gibson Q.H. Roughton F.J.W. J. Physiol. 1957; 136: 507-526Crossref PubMed Scopus (302) Google Scholar), but does not block effects of superoxide alone on PKC activity 2L. T. Knapp and E. Klann, unpublished observations. could block the changes in PKC activity associated with the induction of LTP. We delivered LTP-inducing HFS to hippocampal slices incubated with 20 μm hemoglobin and then assayed PKC activity. The increase in autonomous PKC activity associated with the induction of LTP was attenuated (146.7 ± 10.6% of control, n = 8,p < 0.05), as was the increase in cofactor-dependent PKC activity (120.4 ± 19.6% of control, n = 8, p > 0.05) in slices incubated with hemoglobin. We interpret these results to suggest that NO, like superoxide, plays an important role in PKC activation during the induction of LTP. However, it should be noted that hemoglobin can also bind other ROS, including superoxide, albeit with a lower affinity than NO. Therefore, we cannot completely rule out the possibility that hemoglobin is blocking effects of superoxide or another ROS on PKC activity. It is interesting to note that hemoglobin, like SOD/catalase and DMPO, is confined to the extracellular space; the results therefore are consistent with the hypothesis that transcellular signaling plays a role in PKC activation by superoxide and NO during LTP. The results in this report provide evidence of a role for superoxide in the induction of LTP. Our experiments with superoxide scavengers indicate that superoxide increases the probability of LTP induction and the magnitude of LTP when it occurs. Moreover, the demonstration of a superoxide-dependent change in PKC activity during the induction of LTP provides evidence that superoxide levels increase during the induction of LTP. Thus, we have shown that superoxide is likely to be produced during the induction of LTP, and that it has both biochemical and physiologic effects. The idea of a role for superoxide in synaptic plasticity is consistent with a number of findings from previous studies. First, NMDA receptor activation, a key step in most forms of LTP, results in the production of superoxide in area CA1 of hippocampal slices (13Bindokas V.P. Jordan J. Lee C.C. Miller R.J. J. Neurosci. 1996; 16: 1324-1336Crossref PubMed Google Scholar) and in other types of neurons (25Lafon-Cazal M. Pietri S. Culcasi M. Boeckart J. Nature. 1993; 364: 535-537Crossref PubMed Scopus (1106) Google Scholar). Second, activation of other glutamate receptors can produce superoxide through a number of biochemical pathways (37Coyle J.T. Puttfarcken P. Science. 1993; 262: 689-695Crossref PubMed Scopus (3538) Google Scholar). In addition, superoxide increases the release of glutamate in hippocampal slices (38Pellegrini-Giampietro D.E. Cherici G. Alesiani M. Carla V. Moroni F. J. Neurochem. 1988; 51: 1960-1963Crossref PubMed Scopus (197) Google Scholar), which suggests a means by which it might modulate synaptic efficacy. Finally, it is interesting to note that although the intracellular form of Cu/Zn-SOD, SOD1, is abundantly expressed in the dentate gyrus and area CA3 of the hippocampus, it is expressed to a much lesser degree in area CA1, the hippocampal subregion studied herein (39Pardo C.A. Xu Z. Borchelt D.R. Price D.L. Sisodia S.S. Cleveland D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 954-958Crossref PubMed Scopus (268) Google Scholar); thus, area CA1 is likely to have less capacity for removal of superoxide, consistent with a physiologic role for superoxide in this region. There are a variety of possible sources for superoxide production during LTP induction. First, NOS, which is known to be activated during the induction of LTP (33Chetkovich D.M. Klann E. Sweatt J.D. Neuroreport. 1993; 4: 919-922Crossref PubMed Scopus (116) Google Scholar), is a source of superoxide (35Pou S. Pou W.S. Bredt D.S. Snyder S.H. Rosen G.M. J. Biol. Chem. 1992; 267: 24173-24176Abstract Full Text PDF PubMed Google Scholar). The fact that NOS inhibitors and superoxide scavengers have similar effects is consistent with a role for NOS in producing superoxide, although it is difficult to dissect this role of NOS from its required role in producing NO. Another possible source of superoxide in LTP is arachidonic acid metabolism. Superoxide is produced by lipoxygenase, which initiates leukotriene synthesis from arachidonic acid, and prostaglandin endoperoxide synthase, which initiates prostaglandin synthesis from arachidonic acid (40Kukreja R.C. Kontos H.A. Hess M.L. Ellis E.F. Circ. Res. 1986; 59: 612-619Crossref PubMed Scopus (420) Google Scholar). Interestingly, arachidonic acid metabolism increases during LTP (41Lynch M.A. Errington M.L. Bliss T.V. Neuroscience. 1989; 30: 693-701Crossref PubMed Scopus (145) Google Scholar, 42Lynch M.A. Clements M.P. Voss K.L. Bramham C.R. Bliss T.V.P. Biochem. Soc. Trans. 1991; 19: 391-396Crossref PubMed Scopus (45) Google Scholar) and a lipoxygenase inhibitor, nordihydroguaiaretic acid, inhibits LTP induction (31O'Dell T.J. Hawkins R.D. Kandel E.R. Arancio O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11285-11289Crossref PubMed Scopus (903) Google Scholar, 41Lynch M.A. Errington M.L. Bliss T.V. Neuroscience. 1989; 30: 693-701Crossref PubMed Scopus (145) Google Scholar, 43Williams J.H. Bliss T.V.P. Neurosci. Lett. 1989; 107: 301-306Crossref PubMed Scopus (84) Google Scholar), consistent with a possible role of arachidonic acid metabolism in superoxide production during the induction of LTP. Other possible sources of superoxide include the mitochondrial electron transport chain (13Bindokas V.P. Jordan J. Lee C.C. Miller R.J. J. Neurosci. 1996; 16: 1324-1336Crossref PubMed Google Scholar, 44Dionsi D. Galeotti T. Terrehove T. Azzi A. Biochim. Biophys. Acta. 1975; 403: 292-300Crossref PubMed Scopus (234) Google Scholar) and xanthine oxidase, which could be produced by protease activation during LTP induction (45McCord J.M. N. Engl. J. Med. 1985; 312: 159-163Crossref PubMed Scopus (4999) Google Scholar). In addition to demonstrating a role for superoxide in modulating LTP induction, we have shown that activation of PKC may be a biochemical effect of superoxide production during the induction of LTP. Superoxide is required for increases in cofactor-dependent PKC activity and contributes to increases in autonomous PKC activity during LTP induction. In addition, because superoxide appears to work in conjunction with NO to mediate PKC activity during the induction of LTP, our studies provide evidence that PKC could be a biochemical effector of NO in LTP induction. Previous observations and our present studies suggest that superoxide and NO, and the subsequent increase in cofactor-dependent PKC activity (Figs. 5 B and 6 B), are likely to be modulatory rather than obligatory for the induction of LTP. First, although SOD/catalase blocked LTP in 10 out of 15 experiments, in five experiments an attenuated potentiation was observed (Fig. 1 B). Second, although SOD/catalase, NOArg, and hemoglobin blocked the HFS-induced increase in cofactor-dependent PKC activity, none completely blocked the LTP-associated increase in autonomous PKC activity (Figs. 5 A and 6 A). This last observation would suggest that there are multiple mechanisms of PKC activation after LTP-inducing stimulation (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar, 12Sacktor T.C. Osten P. Valsamis H. Jiang X. Naik M.U. Sublette E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8342-8346Crossref PubMed Scopus (346) Google Scholar,28Osten P. Valsamis L. Harris A. Sacktor T.C. J. Neurosci. 1996; 16: 2444-2451Crossref PubMed Google Scholar), 3J. D. Sweatt, C. M. Atkins, J. D. English, E. D. Roberson, and E. Klann, unpublished observations. which include activation of autonomous PKC that is obligatory (5Malinow R. Schulman H. Tsien R.W. Science. 1989; 245: 862-866Crossref PubMed Scopus (1041) Google Scholar, 7Wang J.-H. Feng D.-P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2576-2580Crossref PubMed Scopus (149) Google Scholar, 8Hvalby O. Hemmings Jr., H.C. Paulsen O. Czernik A.J. Nairn A.C. Godfraind J.-M. Jensen V. Raastad M. Storm J.F. Andersen P. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4761-4765Crossref PubMed Scopus (96) Google Scholar) and activation of cofactor-dependent PKC that is modulatory. Thus, it is tempting to speculate that the increase in cofactor-dependent PKC activity generated by superoxide and NO together, although transient, is involved in the generation of additional autonomously active PKC necessary for the maximum expression of LTP. For example, oxidation of PKC may modify the enzyme so that there is enhanced autophosphorylation, a mechanism that has been proposed to contribute to LTP (9Klann E. Chen S.-J. Sweatt J.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8337-8341Crossref PubMed Scopus (155) Google Scholar). How superoxide and NO might interact to regulate PKC activity is an interesting but unresolved question. Our data suggest that both superoxide and NO are necessary for the increase in PKC activity associated with the induction of LTP (Figs. 5 and 6). Superoxide and NO are known to react rapidly to form the highly reactive peroxynitrite intermediate, a potent oxidant that mediates oxidation of protein sulfhydryls (46Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar). Furthermore, peroxynitrite decomposes to form hydroxyl radical (22Halliwell B. J. Neurochem. 1992; 59: 1609-1623Crossref PubMed Scopus (2666) Google Scholar), which is likely able to activate PKC (16Palumbo E.J. Sweatt J.D. Chen S.-J. Klann E. Biochem. Biophys. Res. Commun. 1992; 187: 1439-1445Crossref PubMed Scopus (68) Google Scholar). The extent to which peroxynitrite and hydroxyl radical are involved in PKC activation by superoxide and NO during LTP is an area of active research. The cellular location of the superoxide- and NO-mediated changes in PKC activity is another interesting question. LTP is associated with enhanced phosphorylation of both pre- and postsynaptic PKC substrates (47Gianotti C. Nunzi M.G. Gispen W.H. Corradetti R. Neuron. 1992; 8: 843-848Abstract Full Text PDF PubMed Scopus (157) Google Scholar, 48Klann E. Chen S.-J. Sweatt J.D. J. Neurochem. 1992; 58: 1576-1579Crossref PubMed Scopus (67) Google Scholar, 49Ramakers G.M. de Graan P.N. Urban I.J. Kraay D. Tang T. Pasinelli P. Oestreicher A.B. Gispen W.H. J. Biol. Chem. 1995; 270: 13892-13898Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 50Chen S.-J. Sweatt J.D. Klann E. Brain Res. 1997; 749: 181-187Crossref PubMed Scopus (68) Google Scholar). In this regard, our results with SOD/catalase, DMPO, and hemoglobin are interesting, as all are confined to the extracellular space. The ability of these compounds to block the effects of superoxide and NO on PKC activity suggests that these species may serve as transcellular signals. It is well known that NO is a membrane-permeable molecule. In contrast, it is not clear how the negatively charged superoxide molecule would be able to cross membranes, although it has been proposed that superoxide can traverse cell membranes through anion channels. Consistent with this notion, it has been reported that NMDA receptor activation in cerebellar granule neurons results in the generation of superoxide that can be trapped extracellularly (25Lafon-Cazal M. Pietri S. Culcasi M. Boeckart J. Nature. 1993; 364: 535-537Crossref PubMed Scopus (1106) Google Scholar). An alternative explanation for our results is that the scavenging compounds could be acting as extracellular sinks to pull ROS out of the postsynaptic neurons, preventing activation of PKC postsynaptically. Although ROS traditionally have been thought of as toxic agents involved in cell injury, they recently have been speculated to act as cellular messengers. This is evidenced by reports showing that ROS increase: 1) the phosphorylation of the ribosomal S6 protein (51Larsson R. Cerutti P. J. Biol. Chem. 1988; 263: 17452-17458Abstract Full Text PDF PubMed Google Scholar); 2) PKC activity in mouse epidermal JB6 cells (15Larsson R. Cerutti P. Cancer Res. 1989; 49: 5627-5632PubMed Google Scholar); 3) tyrosine phosphorylation in human neutrophils (52Fialkow L. Chan C.K. Grinstein S. Downey G.P. J. Biol. Chem. 1993; 268: 17131-17137Abstract Full Text PDF PubMed Google Scholar); and 4) p21ras GTPase activity in PC12 cells (53Lander H.M. Ogiste J.S. Teng K.K. Novogrodsky A. J. Biol. Chem. 1995; 270: 21195-21198Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). In addition, it has been shown that generation of hydrogen peroxide is necessary for platelet-derived growth factor signal transduction (54Sunderesan M. Yu Z.-X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2322) Google Scholar) and may be involved in lysophosphatidic acid signal transduction (55Chen Q. Olashaw N. Wu J. J. Biol. Chem. 1995; 270: 28499-28502Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). To our knowledge, the results in this report are the first to demonstrate ROS regulation of protein kinase activity in the brain and are consistent with the idea that ROS act as normal cellular messengers under certain physiological conditions. Finally, our data are consistent with the hypothesis that ROS modulate the early expression of LTP via interactions with PKC. However, ROS also have been shown to inactivate protein phosphatases, including calcineurin (56Wang X. Culotta V.C. Klee C.B. Nautre. 1996; 383: 434-437Crossref PubMed Scopus (240) Google Scholar). Calcineurin is a calcium/calmodulin-dependent protein phosphatase that has been shown to be necessary for NMDA receptor-dependent hippocampal long-term depression (57Mulkey R.M. Endo S. Shenolikar S. Malenka R.C. Nature. 1994; 369: 486-488Crossref PubMed Scopus (907) Google Scholar), a use-dependentdecrease in synaptic strength. Thus, the production of ROS after induction of LTP, in addition to increasing PKC activity, might also serve to inactivate protein phosphatases such as calcineurin, thereby preventing long-term depression and favoring the expression of LTP. In closing, the data in this report indicate that superoxide modulates the induction of LTP, and that superoxide, in conjunction with NO, mediates increases in PKC activity associated with the induction of LTP. These findings present an interesting physiologic and biochemical context for the effects of ROS in the nervous system. We thank C. Wyatt and Dr. C. M. Powell for help with electrophysiological experiments and Drs. E. Aizenman and E. Thiels for critically reading previous versions of this manuscript." @default.
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• W2034991523 title "A Role for Superoxide in Protein Kinase C Activation and Induction of Long-term Potentiation" @default.
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