Associations between antibiotic prescriptions and recurrent urinary tract infections in female college students.

Urinary tract infections (UTIs) are common among college-aged women and often recur. Some antibiotics recommended to treat UTIs trigger dysbiosis of intestinal and vaginal microbiomes - where uropathogens originate, though few studies have investigated associations between these therapies with recurrent infections. We retrospectively analysed the electronic medical records of 6651 college-aged women diagnosed with a UTI at a US university student health centre between 2006 and 2014. Women were followed for 6 months for incidence of a recurrent infection. In a secondary analysis, associations in women whose experienced UTI recurrence within 2 weeks were also considered for potential infection relapse. Logistic regression was used to assess associations between infection recurrence or relapse and antibiotics prescribed, in addition to baseline patient characteristics including age, race/ethnicity, region of origin, year of encounter, presence of symptomology, pyelonephritis, vaginal coinfection and birth control consultation. There were 1051 instances of infection recurrence among the 6620 patients, indicating a prevalence of 16%. In the analysis of patient characteristics, Asian women were statistically more likely to experience infection recurrence whereas African American were less likely. No significant associations were identified between the antibiotic administered at the initial infection and the risk of infection recurrence after multivariable adjustment. Treatment with trimethoprim-sulphamethoxazole and being born outside of the USA were significantly associated with increased odds of infection relapse in the multivariate analysis. The results of the analyses suggest that treatment with trimethoprim-sulphamethoxazole may lead to an increased risk of UTI relapse, warranting further study.

Activation of a novel p70 S6 kinase 1-dependent intracellular cascade in the basolateral nucleus of the amygdala is required for the acquisition of extinction memory.

Repeated presentations of a previously conditioned stimulus lead to a new form of learning known as extinction, which temporarily alters the response to the original stimulus. Previous studies have shown that the consolidation of extinction memory requires de novo protein synthesis. However, the role of specific nodes of translational control in extinction is unknown. Using auditory threat conditioning in mice, we investigated the role of mechanistic target of rapamycin complex 1 (mTORC1) and its effector p70 S6 kinase 1 (S6K1) in the extinction of auditory threat conditioning. We found that rapamycin attenuated the consolidation of extinction memory. In contrast, genetic deletion and pharmacological inhibition of S6K1, a downstream effector of mTORC1, blocked within-session extinction, indicating a role for S6K1 independent of protein synthesis. Indeed, the activation of S6K1 during extinction required extracellular signal-regulated kinase (ERK) activation in the basolateral nucleus of the amygdala (BLA) and was necessary for increased phosphorylation of the GluA1 (Thr840) subunit of the AMPA receptor following extinction training. Mice exposed to brief uncontrollable stress showed impaired within-session extinction as well as a downregulation of ERK and S6K1 signaling in the amygdala. Finally, using fiber photometry we were able to record calcium signals in vivo, and we found that inhibition of S6K1 reduces extinction-induced changes in neuronal activity of the BLA. These results implicate a novel ERK-S6K1-GluA1 signaling cascade critically involved in extinction.

Altered mTOR signaling and enhanced CYFIP2 expression levels in subjects with fragile X syndrome.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism. The protein (FMRP) encoded by the fragile X mental retardation gene (FMR1), is an RNA-binding protein linked to translational control. Recently, in the Fmr1 knockout mouse model of FXS, dysregulated translation initiation signaling was observed. To investigate whether an altered signaling was also a feature of subjects with FXS compared to typical developing controls, we isolated total RNA and translational control proteins from lymphocytes of subjects from both groups (38 FXS and 14 TD). Although we did not observe any difference in the expression level of messenger RNAs (mRNAs) for translational initiation control proteins isolated from participant with FXS, we found increased phosphorylation of the mammalian target of rapamycin (mTOR) substrate, p70 ribosomal subunit 6 kinase1 (S6K1) and of the mTOR regulator, the serine/threonine protein kinase (Akt), in their protein lysates. In addition, we observed increased phosphorylation of the cap binding protein eukaryotic initiation factor 4E (eIF4E) suggesting that protein synthesis is upregulated in FXS. Similar to the findings in lymphocytes, we observed increased phosphorylation of S6K1 in brain tissue from patients with FXS (n = 4) compared to normal age-matched controls (n = 4). Finally, we detected increased expression of the cytoplasmic FMR1-interacting protein 2 (CYFIP2), a known FMRP interactor. This data verify and extend previous findings using lymphocytes for studies of neuropsychiatric disorders and provide evidence that misregulation of mTOR signaling observed in the FXS mouse model also occurs in human FXS and may provide useful biomarkers for designing targeted treatments in FXS.

Peroxynitrite-induced tyrosine nitration and inhibition of protein kinase C.

Protein kinase C (PKC) is an important intracellular signaling molecule whose activity is essential for a number of aspects of neuronal function including synaptic plasticity. We investigated the regulation of PKC activity by reactive nitrogen species in order to examine whether such species regulate PKC in neurons. Neither autonomous nor cofactor-dependent PKC activity was altered when either hippocampal homogenates or rat brain purified PKC were incubated briefly with three different nitric oxide donor compounds. However, brief incubation of either hippocampal homogenates or purified PKC with peroxynitrite (ONOO(-)) inhibited cofactor-dependent PKC activity in a manner that correlated with the nitration of tyrosine residues on PKC, suggesting that this modification was responsible for the inhibition of PKC. Consistent with this idea, reducing agents had no effect on the inhibition of PKC activity caused by ONOO(-). Because there are numerous PKC isoforms that differ in the composition of the regulatory domain, we studied the effect of ONOO(-) on various PKC isoforms. ONOO(-) inhibited the cofactor-dependent activity of the alpha, betaII, epsilon, and zeta isoforms, indicating that inhibition of enzymatic activity by ONOO(-) was not PKC isoform-specific. We also were able to isolate nitrated PKCalpha and PKCbetaII from ONOO(-)-treated hippocampal homogenates via immunoprecipitation. Collectively, our findings support the hypothesis that ONOO(-) inhibits PKC activity via tyrosine nitration in neurons.

Extracellular signal-regulated kinase, synaptic plasticity, and memory.

The mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), has been studied extensively in recent years for its involvement in synaptic plasticity and memory function. Activation of ERK is coupled to stimulation of cell-surface proteins via several different upstream signaling pathways, and contributes to the regulation of diverse cellular processes, ranging from cell excitability to gene expression. We herein review evidence for ERK's role in different forms of synaptic plasticity and different types of learning paradigms, drawing on examples from different systems in molluscs as well as the mammalian brain. The picture that emerges is that ERK activation in response to conditions that give rise to synaptic and behavioral modification contributes to that modification in a multitude of functionally distinct ways. The functional diversity is likely to be achieved by the operation of multiple, parallel ERK cascades that differ with respect to the subcellular compartments in which ERK exerts its effects and the temporal windows during which the effects are manifested. We conclude that our understanding of the mechanisms by which ERK contributes to synaptic plasticity and memory has much to gain by further study of the signaling events up- and downstream of ERK activation and the spatiotemporal characteristics of ERK activation in association with activity-dependent synaptic modification and information processing.

Impairment of long-term potentiation and associative memory in mice that overexpress extracellular superoxide dismutase.

Reactive oxygen species, including superoxide, generally are considered neurotoxic molecules whose effects can be alleviated by antioxidants. Different from this view, we show that scavenging of superoxide with an antioxidant enzyme is associated with deficits in hippocampal long-term potentiation (LTP), a putative neural substrate of memory, and hippocampal-mediated memory function. Using transgenic mice that overexpress extracellular superoxide dismutase (EC-SOD), a superoxide scavenger, we found that LTP was impaired in hippocampal area CA1 despite normal LTP in area CA3. The LTP impairment in area CA1 could be reversed by inhibition of EC-SOD. In addition, we found that EC-SOD transgenic mice exhibited impaired long-term memory of fear conditioning to contextual cues despite exhibiting normal short-term memory of the conditioning experience. These findings strongly suggest that superoxide, rather than being considered exclusively a neurotoxic molecule, should also be considered a signaling molecule necessary for normal neuronal function.

Protein phosphatase-mediated regulation of protein kinase C during long-term depression in the adult hippocampus in vivo.

The neural substrates of learning and memory are thought to involve use-dependent long-term changes in synaptic function, including long-term depression (LTD) of synaptic strength. One biochemical event hypothesized to contribute to the maintenance and expression of LTD is decreased protein phosphorylation, caused by a decrease in protein kinase activity and/or an increase in protein phosphatase activity. We tested whether the activity of protein kinase C (PKC) decreases after the induction of LTD in area CA1 of the adult hippocampus in vivo, and then investigated the mechanism responsible for the LTD-associated alteration in PKC activity. We found that LTD was associated with a significant decrease in both autonomous and cofactor-dependent PKC activity. The decrease in PKC activity was prevented by NMDA receptor blockade and was not accompanied by a decrease in the level of either PKCalpha, beta, gamma, or zeta. Western blot analysis with phosphospecific antibodies revealed that phosphorylation of Ser-657 on the catalytic domain of PKCalpha (Ser-660 on PKCbetaII) was decreased significantly after the induction of LTD, and that this dephosphorylation was prevented by the protein phosphatase inhibitor okadaic acid. The decrease in autonomous and cofactor-dependent PKC activity likewise was prevented by okadaic acid. These findings suggest that LTD in the adult hippocampus in vivo involves a decrease in PKC activity that is mediated, at least in part, by dephosphorylation of the catalytic domain of PKC by protein phosphatases activated after LTD-inducing stimulation. Our findings are consistent with the idea that protein dephosphorylation contributes to the expression of LTD.

Hypothermia differentially increases extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun terminal kinase activation in the hippocampus during reperfusion after asphyxial cardiac arrest.

Mitogen-activated protein kinases are signal transduction mediators that have been implicated in cell survival and cell death. This study characterized the activation of pathways in the hippocampus during reperfusion after global cerebral ischemia, as well as the influence of a regimen of hypothermia that reduces ischemic cell death in the hippocampus. Circulatory arrest was induced in rats by 8 min of asphyxia. Relative levels of phosphorylated and total extracellular signal-regulated kinase, stress-activated protein kinase/c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were measured in the hippocampus after 6, 12 or 24h of reperfusion using immunoblotting. Asphyxia induced a progressive increase in phosphorylated extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase, but no change in phosphorylated p38 mitogen-activated protein kinase. Induction of mild hypothermia (33 degrees C) during reperfusion increased extracellular signal-regulated kinase phosphorylation and produced a smaller increase in stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation at 24h. Hypothermia did not alter extracellular signal-regulated kinase activation in rats not subjected to ischemia. Extracellular signal-regulated kinase activation was associated with an increase in phosphorylation of the mitogen-activated protein kinase kinase 1/2, and was inhibited by administration of the specific mitogen-activated protein kinase kinase 1/2 inhibitor SL327. Immunohistochemical staining showed an increase in active extracellular signal-regulated kinase in the CA1, CA2, CA3 and dentate gyrus regions of the hippocampus after ischemia and reperfusion. In contrast, active stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity was most intense in the CA3 and dentate gyrus regions. These data demonstrate that both extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase pathways are activated during the first 24h of reperfusion after global cerebral ischemia, and that hypothermia increases the activation of extracellular signal-regulated kinase relative to stress-activated protein kinase/c-Jun N-terminal kinase. Thus, an increase in extracellular signal-regulated kinase activation may be associated with improved neuronal survival after ischemic injury.

Superoxide-induced stimulation of protein kinase C via thiol modification and modulation of zinc content.

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 alpha, beta(II), epsilon, and zeta PKC isoforms, all of which contain at least one cysteine-rich 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.

Persistent activation of ERK contributes to glutamate-induced oxidative toxicity in a neuronal cell line and primary cortical neuron cultures.

Oxidative stress can trigger neuronal cell death and has been implicated in several chronic neurological diseases and in acute neurological injury. Oxidative toxicity can be induced by glutamate treatment in cells that lack ionotrophic glutamate receptors, such as the immortalized HT22 hippocampal cell line and immature primary cortical neurons. Previously, we found that neuroprotective effects of geldanamycin, a benzoquinone ansamycin, in HT22 cells were associated with a down-regulation of c-Raf-1, an upstream activator of the extracellular signal-regulated protein kinases (ERKs). ERK activation, although often attributed strictly to neuronal cell survival and proliferation, can also be associated with neuronal cell death that occurs in response to specific insults. In this report we show that delayed and persistent activation of ERKs is associated with glutamate-induced oxidative toxicity in HT22 cells and immature primary cortical neuron cultures. Furthermore, we find that U0126, a specific inhibitor of the ERK-activating kinase, MEK-1/2, protects both HT22 cells and immature primary cortical neuron cultures from glutamate toxicity. Glutamate-induced ERK activation requires the production of specific arachidonic acid metabolites and appears to be downstream of a burst of reactive oxygen species (ROS) accumulation characteristic of oxidative stress in HT22 cells. However, inhibition of ERK activation reduces glutamate-induced intracellular Ca(2+) accumulation. We hypothesize that the precise kinetics and duration of ERK activation may determine whether downstream targets are mobilized to enhance neuronal cell survival or ensure cellular demise.

The extracellular signal-regulated kinase cascade is required for NMDA receptor-independent LTP in area CA1 but not area CA3 of the hippocampus.

Activation of extracellular signal-regulated kinase (ERK) has been shown to be necessary for NMDA receptor-dependent long-term potentiation (LTP). We studied the role of ERK in three forms of NMDA receptor-independent LTP: LTP induced by very high-frequency stimulation (200 Hz-LTP), LTP induced by the K(+) channel blocker tetraethylammonium (TEA) (TEA-LTP), and mossy fiber (MF) LTP (MF-LTP). We found that ERK was activated in area CA1 after the induction of both 200 Hz-LTP and TEA-LTP and that this activation required the influx of Ca(2+) through voltage-gated Ca(2+) channels. Inhibition of the ERK signaling cascade with either PD 098059 or U0126 prevented the induction of both 200 Hz-LTP and TEA-LTP in area CA1. In contrast, neither PD 098059 nor U0126 prevented MF-LTP in area CA3 induced by either brief or long trains of high-frequency stimulation. U0126 also did not prevent forskolin-induced potentiation in area CA3. However, incubation of slices with forskolin, an activator of the cAMP-dependent protein kinase (PKA) cascade, did result in increases in active ERK and cAMP response element-binding protein (CREB) phosphorylation in area CA3. The forskolin-induced increase in active ERK was inhibited by U0126, whereas the increase in CREB phosphorylation was not, which suggests that in area CA3 the PKA cascade is not coupled to CREB phosphorylation via ERK. Overall, our observations indicate that activation of the ERK signaling cascade is necessary for NMDA receptor-independent LTP in area CA1 but not in area CA3 and suggest a divergence in the signaling cascades underlying NMDA receptor-independent LTP in these hippocampal subregions.

Long-term depression in the hippocampus in vivo is associated with protein phosphatase-dependent alterations in extracellular signal-regulated kinase.

There is growing evidence that activation of either protein kinases or protein phosphatases determines the type of plasticity observed after different patterns of hippocampal stimulation. Because activation of the extracellular signal-regulated kinase (ERK) has been shown to be necessary for long-term potentiation, we investigated the regulation of ERK in long-term depression (LTD) in the adult hippocampus in vivo. We found that ERK immunoreactivity was decreased following the induction of LTD and that this decrease required NMDA receptor activation. The LTD-associated decrease in ERK immunoreactivity could be simulated in vitro via incubation of either purified ERK2 or hippocampal homogenates with either protein phosphatase 1 or protein phosphatase 2A. The protein phosphatase-dependent decrease in ERK immunoreactivity was inhibited by microcystin. Intrahippocampal administration of the protein phosphatase inhibitor okadaic acid blocked the LTD-associated decrease in ERK2, but not ERK1, immunoreactivity. Collectively, these data demonstrate that protein phosphatases can decrease ERK immunoreactivity and that such a decrease occurs with ERK2 during LTD. These observations provide the first demonstration of a biochemical alteration of ERK in LTD.

Modulation of protein kinases and protein phosphatases by reactive oxygen species: implications for hippocampal synaptic plasticity.

1. Reactive oxygen species are known for their role in neurotoxicity. However, recent studies indicate that reactive oxygen species also play a role in cell function under physiological conditions. 2. Both superoxide and hydrogen peroxide alter the activity of various protein kinases and protein phosphatases, some of which are involved in hippocampal synaptic plasticity. Specifically, the activity of protein kinase C, extracellular-regulated kinase 2, and a protein tyrosine kinase(s) is increased in the presence of these reactive oxygen species, whereas the activity of protein phosphatases 2A and 2B, and a protein tyrosine phosphatase(s) is decreased. 3. Protein kinase C, extracellular-regulated kinase 2, and protein tyrosine kinases critically participate in the induction and/or early expression of long-term potentiation at glutamatergic synapses in hippocampus. Protein phosphatases 2A and 2B participate in the induction and/or early expression of long-term depression at these synapses. 4. Treatment of hippocampal slices with scavengers of either superoxide or hydrogen peroxide prevents the full expression of long-term potentiation. Long-term potentiation in hippocampus also is attenuated in transgenic mice that overexpress Cu/Zn superoxide dismutase. 5. The link between reactive oxygen species and long-term potentiation may be the activating effect on protein kinases. The inhibiting effect of reactive oxygen species on protein phosphatases may also contribute to long-term potentiation. 6. The authors hypothesize that reactive oxygen species play a critical role in hippocampal long-term potentiation by favoring the activation of a protein kinase over a protein phosphatase signaling cascade.

A nitric oxide/cyclic GMP-dependent protein kinase pathway alters transmitter release and inhibition by somatostatin at a site downstream of calcium entry.

We have examined the somatostatin-mediated modulation of acetylcholine release from intact chick embryo choroid tissue and compared these data with those obtained using acutely dissociated neuronal cell bodies from the chick ciliary ganglion. Acetylcholine release, evoked in a calcium-dependent manner by a high potassium (55 mM KCI) stimulation in both preparations, was inhibited almost completely by 100 nM somatostatin. Measurement of intracellular calcium in these neurons revealed that somatostatin blocked the large calcium transient that was observed in control neurons following KCI exposure. The modulatory effect of somatostatin on transmitter release was significantly attenuated by pre-treatment with pharmacologic agents that selectively block cyclic GMP (cGMP)-dependent protein kinase (PKG) or nitric oxide (NO) synthase. It is interesting that this prevention of somatostatin-mediated acetylcholine release inhibition occurred without reversal of the somatostatin-mediated block of the KCl-evoked calcium transient. Furthermore, a NO donor or cGMP analogue could block KCI-evoked acetylcholine release, but only cGMP could reduce the KCI-evoked calcium transient. Although cGMP could reduce the KCI-evoked calcium transient, a cGMP analogue was shown to reduce calcium ionophore-evoked transmitter release. Thus, somatostatin reduces acetylcholine release by modulating calcium influx, but the NO-PKG pathway can inhibit acetylcholine release, and alter somatostatin-mediated inhibition, by affecting transmitter release at some point after calcium entry.

Localization of extracellular superoxide dismutase in adult mouse brain.

Extracellular superoxide dismutase (EC-SOD) is one of three mammalian SOD isozymes. Although there is knowledge of the functional role of EC-SOD in arteries, little is known about the function of EC-SOD in other tissues, including the brain. As a first step toward improving our understanding of EC-SOD in the brain, we studied the localization of EC-SOD in the central nervous system of the adult mouse using immunohistochemistry. We detected EC-SOD staining in a subpopulation of neurons throughout the brain as well as in tanycytes in the mediobasal hypothalamus. Particularly prominent EC-SOD staining was observed in neurons of the hilar region of the hippocampus, the lateral habenular nucleus of the thalamus, and the suprachiasmatic nuclei of the hypothalamus. Substantial numbers of neurons were distributed throughout the striatum and cortex; the morphology and distribution of these cells was similar to neurons previously shown to contain the neuronal isoform of nitric oxide synthase. In contrast to other regions with prominent EC-SOD immunoreactivity, EC-SOD localization in tanycytes occurred in a region lacking a blood-brain barrier. The high levels of EC-SOD present in discrete populations of cells in these regions suggest that EC-SOD plays an important, specialized role in the physiology and/or pathology in the brain.

Transient and persistent increases in protein phosphatase activity during long-term depression in the adult hippocampus in vivo.

The neural substrates of learning and memory most likely involve activity-dependent long-term changes in synaptic strength, including long-term potentiation and long-term depression. A critical element in the cascade of events hypothesized to underlie such changes in synaptic function is modification of protein phosphorylation. Long-term depression is thought to involve decreases in protein phosphorylation, which could result from reduction in protein kinase activity and/or enhancement in protein phosphatase activity. We present here direct evidence that long-term depression in the hippocampus in vivo is associated with an increase in the activity of the serine/threonine phosphatases 1 and 2A. The increase in activity of phosphatase 1 was transient, whereas that of phosphatase 2A lasted > 65 min after the induction of long-term depression. Blockade of long-term depression prevented the observed increases in phosphatase activity, as did selective inhibition of phosphatase 1 and 2A. Induction of long-term depression had no effect on the level of either phosphatase, which suggests that our results reflect increases in the intrinsic activity of these two enzymes. Our findings are consistent with a model of synaptic plasticity that implicates protein dephosphorylation by serine/threonine phosphatases in the early maintenance and/or expression of long-term depression of synaptic strength.

Protected-site phosphorylation of protein kinase C in hippocampal long-term potentiation.

One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the alpha and betaII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a "protected site" model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.

Cell-permeable scavengers of superoxide prevent long-term potentiation in hippocampal area CA1.

Long-term potentiation (LTP) in hippocampal area CA1 is generally dependent on N-methyl--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.

Stimulation of p42 and p44 mitogen-activated protein kinases by reactive oxygen species and nitric oxide in hippocampus.

Reactive oxygen species (ROS) have been suggested to act as cellular messengers that mediate signal transduction cascades in various cell types. However, little is known about their role in this capacity in the nervous system. We have begun to investigate the role of ROS, and that of nitric oxide (NO), in mediating mitogen-activated protein kinase (MAPK) signaling in rat hippocampal slices. Our studies have revealed that direct exposure of hippocampal slices to hydrogen peroxide, xanthine/xanthine oxidase (a superoxide-generating system), sodium nitroprusside (an NO donor compound), S-nitroso-N-acetylpenicillamine (an NO donor compound), or 3-morpholinosydnonimine (a compound that produces NO and superoxide) results in an enhancement in tyrosine phosphorylation of several proteins, including proteins with apparent molecular masses of 42 and 44 kDa. We investigated the possibility that these proteins correspond to the active forms of p42 MAPK and p44 MAPK. Hippocampal slices exposed to various ROS and NO donors resulted in increases in levels of the active forms of both p42 MAPK and p44 MAPK. The ROS- and NO-enhanced tyrosine phosphorylation and activation of p42 MAPK and p44 MAPK were inhibited by pretreatment with the antioxidant N-acetyl-L-cysteine. Our observations indicate that ROS and NO can mediate protein tyrosine phosphorylation and MAPK signaling in the hippocampus via a redox-sensitive mechanism and suggest a potential cellular mechanism for their effects in the nervous system.

A role for superoxide in protein kinase C activation and induction of long-term potentiation.

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-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.