The induction of several forms of long-term potentiation (LTP) of synaptic transmission in the CA1 region of the mammalian hippocampus is dependent on N-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 by N-methyl-Daspartate 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)1 is a long-lasting synaptic enhancement that may be involved in certain types of mammalian learning and memory (1). 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 Ca 2ϩ entry into the postsynaptic neuron triggered by N-methyl-D-aspartate (NMDA) receptor activation (2-4). The exact biochemical sequence of events following Ca 2ϩ influx into the postsynaptic cell is unknown, but it is clear that Ca 2ϩ -dependent protein kinases are involved in the induction of LTP (5, 6). 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 (5,7,8). Furthermore, both the second messenger-independent activity of PKC (autonomous activity; see Ref. 9) and total, cofactor-stimulated PKC activity (cofactor-dependent activity; see Refs. 9 and 10) 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 (11, 12), conformational changes in PKC that result in the unmasking of activator sites (10), and decreases in protein phosphatase activity that might result in increased phosphorylation and activity of PKC (10).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 in...

Recent evidence suggests that reactive oxygen species (ROS), including superoxide, are not only neurotoxic but function as small messenger molecules in normal neuronal processes such as synaptic plasticity. Consistent with this idea, we show that brief incubation of hippocampal slices with the superoxidegenerating system xanthine/xanthine oxidase (X/XO) produces a long-lasting potentiation of synaptic transmission in area CA1. We found that X/XO-induced potentiation was associated with a persistent superoxide-dependent increase in autonomous PKC activity that could be isolated via DEAE column chromatography. The X/XO-induced potentiation was blocked by the inhibition of PKC, indicating that the superoxidedependent increase in autonomous PKC activity was necessary for the potentiation. We also found that X/XO-induced potentiation and long-term potentiation (LTP) occluded one another, suggesting that these forms of plasticity share similar cellular mechanisms. In further support of this idea, we found that a persistent, superoxide-dependent increase in autonomous PKC activity isolated via DEAE column chromatography also was associated with LTP. Taken together, our findings indicate that X/XO-induced potentiation and LTP share similar cellular mechanisms, including superoxide-dependent increases in autonomous PKC activity. Finally, our findings suggest that superoxide, in addition to its well known role as a neurotoxin, also can be considered a small messenger molecule critical for normal neuronal signaling.

We investigated the effects of mild oxidation on protein kinase C (PKC) using the xanthine/xanthine oxidase system of generating superoxide. Exposure of various PKC preparations to superoxide stimulated the autonomous activity of PKC. Similarly, thiol oxidation increased autonomous PKC activity, consistent with the notion that superoxide stimulates PKC via thiol oxidation. The superoxide-induced stimulation of PKC activity was partially reversed by reducing agents, suggesting that disulfide bond formation contributed to the oxidative stimulation of PKC. In addition, superoxide increased the autonomous activity of the ␣, ␤ II , ⑀, and PKC isoforms, all of which contain at least one cysteinerich region. Taken together, our observations suggested that superoxide interacts with PKC at the cysteine-rich region, zinc finger motif of the enzyme. Therefore, we examined the effects of superoxide on this region by testing the hypothesis that superoxide stimulates PKC by promoting the release of zinc from PKC. We found that a zinc chelator stimulated the autonomous activity of PKC and that superoxide induced zinc release from an PKC-enriched enzyme preparation. In addition, oxidized PKC contained significantly less zinc than reduced PKC. Finally, we have isolated a persistent, autonomously active PKC by DEAE-cellulose column chromatography from hippocampal slices incubated with superoxide. Taken together, these data suggest that superoxide stimulates autonomous PKC activity via thiol oxidation and release of zinc from cysteine-rich region of PKC.

Reactive oxygen species (ROS) typically are characterized as molecules involved in neurotoxicity and neurodegeneration. However, recent evidence from both neuronal and nonneuronal cells suggests that ROS also function as small messenger molecules that are normal components of signal transduction cascades during physiological processes. Consistent with this idea, ROS have been shown to be critical for hippocampal long-term potentiation (LTP), a form of synaptic plasticity widely studied as a cellular substrate for learning and memory. On the other hand, ROS also have been shown to be involved in aging-related impairment of LTP. This review discusses the evidence supporting the notion that ROS both contribute to normal LTP and are involved in age-related impairment of LTP. We also discuss possible sources that might be responsible for the production of ROS after the induction of LTP. Finally, we propose a functional ROS continuum to help explain this dichotomy of ROS function in hippocampal LTP.