Zhi-Gang Xiong

A Key Role for TRPM7 Channels in Anoxic Neuronal Death

Excitotoxicity in brain ischemia triggers neuronal death and neurological disability, and yet these are not prevented by antiexcitotoxic therapy (AET) in humans. Here, we show that in neurons subjected to prolonged oxygen glucose deprivation (OGD), AET unmasks a dominant death mechanism perpetuated by a Ca2+-permeable nonselective cation conductance (IOGD). IOGD was activated by reactive oxygen/nitrogen species (ROS), and permitted neuronal Ca2+ overload and further ROS production despite AET. IOGD currents corresponded to those evoked in HEK-293 cells expressing the nonselective cation conductance TRPM7. In cortical neurons, blocking IOGD or suppressing TRPM7 expression blocked TRPM7 currents, anoxic 45Ca2+ uptake, ROS production, and anoxic death. TRPM7 suppression eliminated the need for AET to rescue anoxic neurons and permitted the survival of neurons previously destined to die from prolonged anoxia. Thus, excitotoxicity is a subset of a greater overall anoxic cell death mechanism, in which TRPM7 channels play a key role.

Enhanced LTP in Mice Deficient in the AMPA Receptor GluR2

AMPA receptors (AMPARs) are not thought to be involved in the induction of long-term potentiation (LTP), but may be involved in its expression via second messenger pathways. However, one subunit of the AMPARs, GluR2, is also known to control Ca2+ influx. To test whether GluR2 plays any role in the induction of LTP, we generated mice that lacked this subunit. In GluR2 mutants, LTP in the CA1 region of hippocampal slices was markedly enhanced (2-fold) and nonsaturating, whereas neuronal excitability and paired-pulse facilitation were normal. The 9-fold increase in Ca2+ permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Mutant mice exhibited increased mortality, and those surviving showed reduced exploration and impaired motor coordination. These results suggest an important role for GluR2 in regulating synaptic plasticity and behavior.

Platelet-derived Growth Factor Induces a Long-term Inhibition of N-Methyl-D-aspartate Receptor Function

Platelet-derived growth factor (PDGF) is a multifunctional protein that plays important roles in many tissues, including the mammalian central nervous system. PDGF and PDGF receptors (PDGFRs) are expressed in virtually every region of the central nervous system where they are involved in the development, survival, growth, and differentiation of both neuronal and glial cells. We now report that a brief activation of PDGFRs produced a long-lasting inhibition of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents in CA1 pyramidal neurons in rat hippocampal slices. PDGF also inhibited NMDA receptors (NMDA-Rs) in cultured hippocampal neurons by a mechanism that involves a decrease in single channel open probability. Non-NMDA receptor function was not affected by PDGF in hippocampal neurons. Experiments with mutant PDGFRs and chelation of intracellular Ca2+ in Xenopus oocytes indicate that this inhibition depends on a phospholipase C-γ-induced elevation of intracellular Ca2+ levels. The PDGF-induced inhibition of NMDA-Rs is produced by a mechanism different than the well characterized phenomenon of Ca2+-dependent NMDA-R run down because the effect of PDGF was blocked by the phosphatase inhibitor, calyculin A, and was not affected by the microtubule polymerizing agent, phalloidin. Because elevations of PDGF levels are associated with neurological trauma or disease, we propose that PDGF can exert neuroprotective effects by inhibiting NMDA-R-dependent excitotoxicity.

Blockade of Glutamate Receptors and Barbiturate Anesthesia Increased Sensitivity to Pentobarbital-induced Anesthesia Despite Reduced Inhibition of AMPA Receptors in GluR2 Null Mutant Mice

BACKGROUNDnBarbiturates enhance gamma-aminobutyric acid type A (GABA(A)) receptor function and also inhibit the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor. The relative contribution of these actions to the behavioral properties of barbiturates is not certain. Because AMPA receptor complexes that lack the GluR2 subunit are relatively insensitive to pentobarbital inhibition, GluR2 null mutant mice provide a novel tool to investigate the importance of AMPA receptor inhibition to the anesthetic effects of barbiturates.nnnMETHODSnGluR2 null allele (-/-), heterozygous (+/-), and wild-type (+/+) mice were injected with pentobarbital (30 and 35 mg/kg intraperitoneally). Sensitivity to anesthetics was assessed by measuring the latency to loss of righting reflex, sleep time, and the loss of corneal, pineal, and toe-pinch withdrawal reflexes. In addition, patch-clamp recordings of acutely dissociated CA1 hippocampal pyramidal neurons from (-/-) and (+/+) mice were undertaken to investigate the effects of barbiturates on kainate-activated AMPA receptors and GABA-activated GABA(A) receptors.nnnRESULTSnBehavioral tests indicate that sensitivity to pentobarbital was increased in (-/-) mice. In contrast, AMPA receptors from (-/-) neurons were less sensitive to inhibition by pentobarbital (concentrations that produced 50% of the maximal inhibition [IC50], 301 vs. 51 microM), thiopental (IC50, 153 vs. 34 microM), and phenobarbital (IC50, 930 vs. 205 microM) compared with wild-type controls, respectively. In addition, the potency of kainate was greater in (-/-) neurons, whereas no differences were observed for the potentiation of GABA(A) receptors by pentobarbital.nnnCONCLUSIONSnThe GluR2 null mutant mice were more sensitive to pentobarbital anesthesia despite a reduced sensitivity of GluR2-deficient AMPA receptors to barbiturate blockade. Our results indicate that the inhibition of AMPA receptors does not correlate with the anesthetic effects of barbiturates in this animal model. We postulate that the increase in the sensitivity to anesthetics results from a global suppression of excitatory neurotransmission in GluR2-deficient mice.

Platelet-derived Growth Factor Receptor-induced Feed-forward Inhibition of Excitatory Transmission between Hippocampal Pyramidal Neurons

Growth factor receptors provide a major mechanism for the activation of the nonreceptor tyrosine kinase c-Src, and this kinase in turn up-regulates the activity ofN-methyl-d-aspartate (NMDA) receptors in CA1 hippocampal neurons (1). Unexpectedly, applications of platelet-derived growth factor (PDGF)-BB to cultured and isolated CA1 hippocampal neurons depressed NMDA-evoked currents. The PDGF-induced depression was blocked by a PDGF-selective tyrosine kinase inhibitor, by a selective inhibitor of phospholipase C-γ, and by blocking the intracellular release of Ca2+. Inhibitors of cAMP-dependent protein kinase (PKA) also eliminated the PDGF-induced depression, whereas a phosphodiesterase inhibitor enhanced it. The NMDA receptor-mediated component of excitatory synaptic currents was also inhibited by PDGF, and this inhibition was prevented by co-application of a PKA inhibitor. Src inhibitors also prevented this depression. In recordings from inside-out patches, the catalytic fragment of PKA did not itself alter NMDA single channel activity, but it blocked the up-regulation of these channels by a Src activator peptide. Thus, PDGF receptors depress NMDA channels through a Ca2+- and PKA-dependent inhibition of their modulation by c-Src.

Acidosis decreases low Ca2+ ‐induced neuronal excitation by inhibiting the activity of calcium‐sensing cation channels in cultured mouse hippocampal neurons

The effects of extracellular pH (pHo) on calcium‐sensing non‐selective cation (csNSC) channels in cultured mouse hippocampal neurons were investigated using whole‐cell voltage‐clamp and current‐clamp recordings. Decreasing extracellular Ca2+ concentrations ([Ca2+]o) activated slow and sustained inward currents through the csNSC channels. Decreasing pHo activated amiloride‐sensitive transient proton‐gated currents which decayed to baseline in several seconds. With proton‐gated channels inactivated by pre‐perfusion with low pH solution or blocked by amiloride, decreasing pHo to 6.5 inhibited the csNSC currents with a leftward shift of the Ca2+ dose–inhibition curve. Increasing pH to 8.5, on the other hand, caused a rightward shift of the Ca2+ dose–inhibition curve and potentiated the csNSC currents. Intracellular alkalinization following bath perfusion of quinine mimicked the potentiation of the csNSC currents by increasing pHo, while intracellular acidification by addition and subsequent withdrawal of NH4Cl mimicked the inhibition of the csNSC currents by decreasing pHo. Intracellular pH (pHi) imaging demonstrated that decreasing pHo induced a corresponding decrease in pHi. Including 30 mM Hepes in the pipette solution eliminated the effects of quinine and NH4Cl on the csNSC currents, but only partially reduced the effect of lowering pHo. In current‐clamp recordings, decreasing [Ca2+]o induced sustained membrane depolarization and excitation of hippocampal neurons. Decreasing pHo to 6.5 inhibited the low [Ca2+]o‐induced csNSC channel‐mediated membrane depolarization and the excitation of neurons. Our results indicate that acidosis may inhibit low [Ca2+]o‐induced neuronal excitation by inhibiting the activity of the csNSC channels. Both the extracellular and the intracellular sites are involved in the proton modulation of the csNSC channels.

Multiple sites of action of neomycin, Mg2+ and spermine on the NMDA receptors of rat hippocampal CA1 pyramidal neurones

1 The effects of neomycin on NMDA‐evoked currents in isolated CA1 hippocampal pyramidal neurones were investigated and single channel activity was examined in outside‐out patches taken from cultured hippocampal neurones. The effects of neomycin on two combinations of NMDA receptor subunits (NR1a‐NR2A and NR1a‐NR2B) expressed in human embryonic kidney (HEK293) cells were also studied. 2 Neomycin (0.01–1 mm) caused a potentiation of NMDA‐activated currents in all neurones examined. No evidence of a voltage‐dependent depression was observed in whole‐cell recordings. 3 In outside‐out patch recordings relatively low concentrations (30 and 100 μm) of neomycin caused a voltage‐dependent reduction in single channel current amplitude as well as a large increase in the frequency of channel opening. 4 In saturating concentrations of glycine, neomycin enhanced NMDA‐activated currents and this glycine‐independent enhancement was confirmed using recombinant NR1a‐NR2B receptors. Neomycin substantially increased the potency of glycine for the receptor by reducing the rate of dissociation of glycine from the receptor. Neomycin demonstrated a glycine‐dependent enhancement of currents mediated by the NR1a‐NR2A combination of subunits but a paradoxical depression was observed in saturating concentrations of glycine. 5 Neomycin increased the rate of deactivation of glutamate‐activated currents consistent with neomycin causing a reduction in the affinity of the receptor for agonist. 6 These results indicate that neomycin has multiple and complex effects on NMDA receptors.