We and others have previously shown that overstimulation of NMDA receptors (NMDA-Rs) leads to excessive mitochondrial Ca2+ uptake, which is a key step in excitotoxic cell death. It is thought that different NMDA-R subunits play differential roles in this process. Specifically, only activation of NR2B-containing NMDA-Rs appears to promote cell death, while the activation of NR2A-containing NMDA-Rs conversely promotes cell survival. This year we have explored the hypothesis that the opposing effects of NR2A- and NR2B-containing NMDA-R's containing is mediated by different levels of intracellular calcium and therefore different degrees of mitochondrial damage. Exposure of cultured rat hippocampal neurons to 100 uM NMDA, which activates receptors containing both NR2A and NR2B subunits, normally leads to cell death, but the presence of the NR2B-selective antagonist Co101244 was strongly neuroprotective. It also reduced calcium accumulation in cytoplasm and mitochondria and prevented cytosolic Ca2+ deregulation. In contrast, selective activation of only synaptic NMDA-Rs induced TTX-sensitive, high-frequency Ca2+ spikes, the amplitude of which was low compared to NMDA-induced Ca2+ elevations. Co101244 minimally affected these evoked Ca2+ spikes, indicating a major role for NR2A-containing receptors with little input from synaptic NR2B subunits. Compared to global NMDA-R activation, which is characterized by strong calcium overload and severe mitochondrial damage, synaptic activity evoked very low calcium accumulation in both cytoplasm and mitochondria, and limited structural damage to mitochondria. Synaptic activity-induced changes in calcium concentrations were completely reversed within 30 min, while recovery from NMDA stimulation required much longer. These results indicate that NMDA-dependent calcium overload is mainly mediated by NR2B-containing, but not NR2A-containing, extrasynaptic receptors, thereby explaining the central role of NR2B in excitotoxicity.? ? Like mitochondria in intact neurons, isolated brain mitochondria are capable of accumulating large amounts of calcium. This calcium is stored in the matrix as phosphorus-rich precipitates, the chemical composition of which has been largely unknown. Also unknown is the fate of these precipitates when the inner mitochondrial membrane is breached by opening of the large conductance mitochondrial permeability transition (MPT) pore. In this study, we used inhibitors of MPT, in conjunction with a continuous cation infusion technique, in isolated rat brain (RBM) and liver (RLM) mitochondria to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial swelling (an indicator of MPT opening), precipitate composition and precipitate retention. In RBM, the endogenous MPT inhibitors ADP and ATP increased mitochondrial Ca2+ loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form, and probably its solubility, varies with the conditions of loading. Cyclosporin A, a pharmacological MPT inhibitor, similarly increased Ca2+ capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca2+ is rapid but incomplete; significant residual calcium, still in the form of precipitates, is retained in damaged mitochondria for prolonged periods. Since in general mitochondrial Ca2+ uptake and release significantly influences cell signaling, it seems likely that prolonging the release of precipitate Ca2+ will have a significant functional impact during post-stimulus recovery periods.? ? Slice cultures of hippocampus, which retain classical hippocampal organization and circuitry, represent an alternative model for studying neuronal tolerance, since pyramidal neurons of the CA1 region are quite sensitive to excitotoxic stimuli, while neurons in the CA3 region show a high level of endogenous neuroprotection. In an ongoing study to determine factors that underlie the selective vulnerability of CA1 neurons, we find that NMDA exposure induces high calcium elevations selectively in CA1, but not in CA3, neurons. Consistent with previous evidence that mitochondrial damage is a key event in determining excitotoxic vulnerability, mitochondrial calcium overload and damage were also much more severe in CA1. The NMDA antagonist MK-801 prevented CA1 calcium elevation and was neuroprotective. Considering results described above, we are currently testing the hypothesis that the known robust expression of NR2B-containing NMDA-Rs in CA1 relative to CA3 is responsible for calcium-dependent vulnerability of CA1 neurons.
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