Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced superoxide production. Neurons in culture and in mouse hippocampus responded to NMDA with a rapid increase in superoxide production, followed by neuronal death. These events were blocked by the NADPH oxidase inhibitor apocynin and in neurons lacking the p47 phox subunit, which is required for NADPH oxidase assembly. Superoxide production was also blocked by inhibiting the hexose monophosphate shunt, which regenerates the NADPH substrate, and by inhibiting protein kinase C zeta, which activates the NADPH oxidase complex. These findings identify NADPH oxidase as the primary source of NMDA-induced superoxide production.Activation of the neuronal NMDAR initiates several downstream events, including cation influx, activation of nitric oxide synthase and formation of superoxide [1][2][3] . Superoxide functions as an inter-cellular messenger in long-term potentiation 4,5 and participates in redox inhibition of NMDAR channel function 6 ; however, superoxide can also promote neuronal death when NMDAR activation is sustained 1,7 . Notably, the primary source of superoxide induced by NMDAR activation remains unresolved.Initial studies suggested a mechanism in which Ca 2+ influx through NMDAR channels leads to mitochondrial depolarization 8,9 and subsequent mitochondrial production of superoxide 10,11 . However, a biochemical mechanism linking these events has not been identified and evidence supporting mitochondria as the primary source of neuronal superoxide production remains indirect 12,13 . Calcium was shown to induce superoxide production in isolated © 2009 Nature America, Inc. All rights reserved.Correspondence should be addressed to R.A.S. (Raymond.swanson@ucsf.edu). AUTHOR CONTRIBUTIONS A.M.B. carried out the cell culture studies and data analysis and prepared the manuscript drafts. S.W.S. supervised the mouse surgical studies and analyzed these data. S.J.W. performed mouse surgery studies and mouse brain histology. P.N. maintained the Sod2 + mouse colony and prepared the Sod2 + cell cultures. T.M.K. and Y.E. assisted with the p47 phox translocation studies and data analysis. H.L. assisted in the analysis of the cell culture ethidium fluorescence results. P.H.C. assisted with the studies involving Sod2 + neurons. R.A.S. organized the studies and prepared the final manuscript.Note: Supplementary information is available on the Nature Neuroscience website. 14 , but more recent studies indicate that this effect is highly dependent on experimental conditions, especially the presence of bovine serum albumin in the medium and succinate as a metabolic substrate 13 . The important question is w...

We show that beta forms of Neu differentiation factor (NDF), homologous to acetylcholine receptor-inducing activity, glial growth factor, and heregulin, prevent apoptotic death and stimulate DNA synthesis of the E14 Schwann cell precursor, an early cell in the rat Schwann cell lineage. When precursors are exposed to NDF in defined medium, they generate Schwann cells without the requirement for DNA synthesis and with a time course that is similar to that with which Schwann cells appear in embryonic nerves in vivo. Furthermore, a neuronal signal that also mediates precursor survival and maturation is blocked by the extracellular domain of the ErbB4 NDF receptor, a protein that specifically blocks the action of NDFs. These observations provide important evidence that NDF is one of the hitherto elusive neuron-glia signaling molecules long proposed to regulate development in the Schwann cell lineage.

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Although Schwann cell precursors from early embryonic nerves die in the absence of axonal signals, Schwann cells in older nerves can survive in the absence of axons in the distal stump of transected nerves. This is crucially important, because successful axonal regrowth in a damaged nerve depends on interactions with living Schwann cells in the denervated distal stump. Here we show that Schwann cells acquire the ability to survive without axons by establishing an autocrine survival loop. This mechanism is absent in precursors. We show that insulin-like growth factor, neurotrophin-3, and platelet-derived growth factor-BB are important components of this autocrine survival signal. The secretion of these factors by Schwann cells has significant implications for cellular communication in developing nerves, in view of their known ability to regulate survival and differentiation of other cells including neurons.

We examined mechanisms contributing to stimulus-evoked changes in NAD(P)H fluorescence as a marker of neuronal activation in area CA1 of murine hippocampal slices. Three types of stimuli (electrical, glutamate iontophoresis, bath-applied kainate) produced biphasic fluorescence changes composed of an initial transient decrease ("initial component," 1-3%), followed by a longer-lasting transient increase ("overshoot," 3-8%). These responses were matched by inverted biphasic flavin adenine dinucleotide (FAD) fluorescence transients, suggesting that these transients reflect mitochondrial function rather than optical artifacts. Both components of NAD(P)H transients were abolished by ionotropic glutamate receptor block, implicating postsynaptic neuronal activation as the primary event involved in generating the signals, and not presynaptic activity or reuptake of synaptically released glutamate. Spatial analysis of the evoked signals indicated that the peak of each component could arise in different locations in the slice, suggesting that there is not always obligatory coupling between the two components. The initial NAD(P)H response showed a strong temporal correspondence to intracellular Ca+ increases and mitochondrial depolarization. However, despite the fact that removal of extracellular Ca2+ abolished neuronal cytosolic Ca2+ transients to exogenous glutamate or kainate, this procedure did not reduce slice NAD(P)H responses evoked by either of these agonists, implying that mechanisms other than neuronal mitochondrial Ca2+ loading underlie slice NAD(P)H transients. These data show that, in contrast to previous proposals, slice NAD(P)H transients in mature slices do not reflect neuronal Ca2+ dynamics and demonstrate that these signals are sensitive indicators of both the spatial and temporal characteristics of postsynaptic neuronal activation in these preparations.