Elemental And Structural Organization Of Neurons And Glia

我们和其他人先前已经表明，NMDA受体（NMDA-Rs）的过度刺激导致过度的线粒体Ca 2+摄取，这是兴奋性毒性细胞死亡的关键步骤。据认为，不同的NMDA-R亚基在这一过程中发挥不同的作用。具体而言，只有含NR 2B的NMDA-R的活化似乎促进细胞死亡，而含NR 2A的NMDA-R的活化相反地促进细胞存活。今年，我们已经探索了一种假设，即含有NR 2A和NR 2B的NMDA-R的相反作用是由不同水平的细胞内钙介导的，因此线粒体损伤程度不同。将培养的大鼠海马神经元暴露于100 μ M NMDA（其激活含有NR 2A和NR 2B亚基的受体）通常导致细胞死亡，但NR 2B选择性拮抗剂Co 101244的存在具有强烈的神经保护作用。它还减少了细胞质和线粒体中的钙积累，并防止胞质Ca 2+失调。与此相反，选择性激活突触NMDA-Rs诱导TTX敏感的，高频率的Ca 2+尖峰，其幅度较低相比，NMDA诱导的Ca 2+升高。Co 101244对这些诱发的Ca 2+尖峰的影响最小，表明含NR 2A的受体具有重要作用，而突触NR 2B亚基的输入很少。与以强烈的钙超载和严重的线粒体损伤为特征的整体NMDA-R激活相比，突触活动引起细胞质和线粒体中非常低的钙积累，并且对线粒体的结构损伤有限。突触活动引起的钙离子浓度的变化在30分钟内完全逆转，而从NMDA刺激的恢复需要更长的时间。这些结果表明，NMDA依赖的钙超载主要是由含有NR 2B，但不含NR 2A，突触外受体介导的，从而解释了兴奋性毒性的核心作用NR 2B。 像完整神经元中的线粒体一样，分离的脑线粒体能够积累大量的钙。这种钙以富磷沉淀物的形式储存在基质中，其化学成分在很大程度上是未知的。还未知的是，当线粒体内膜被大电导线粒体渗透性转换（MPT）孔打开而破裂时，这些沉淀物的命运。在这项研究中，我们使用MPT的抑制剂，结合连续阳离子输注技术，在离体大鼠脑（RBM）和肝（RLM）线粒体，以确定线粒体钙摄取的量和速率如何与线粒体肿胀（MPT开放的指标），沉淀物组成和沉淀物保留。在RBM中，内源性MPT抑制剂ADP和ATP增加线粒体Ca 2+负载能力，并促进沉淀物的形成。在ADP的存在下，Ca/P比接近1.5，而ATP或降低的输注速率使该比降低至1.0，表明沉淀物的化学形式，以及可能其溶解度，随装载条件而变化。环孢菌素A，一种药理学MPT抑制剂，类似地增加Ca 2+容量和沉淀Ca/P比。MPT和/或去极化后，累积的Ca 2+的释放迅速但不完全;大量残留的钙仍以沉淀物形式长时间保留在受损的线粒体中。由于在一般线粒体Ca 2+的摄取和释放显着影响细胞信号传导，它似乎可能是延长沉淀Ca 2+的释放将有显着的功能影响在刺激后恢复期。 海马切片文化，保留经典的海马组织和电路，代表了一种替代模型，用于研究神经元的耐受性，因为锥体神经元的CA 1区是相当敏感的兴奋性刺激，而在CA 3区的神经元显示出高水平的内源性神经保护。在一项正在进行的研究，以确定因素的基础上的选择性脆弱性的CA 1神经元，我们发现，NMDA暴露诱导高钙升高选择性CA 1，但不是在CA 3，神经元。与先前的证据一致，线粒体损伤是确定兴奋性毒性脆弱性的关键事件，线粒体钙超载和损伤在CA 1中也更为严重。NMDA拮抗剂MK-801可防止CA 1钙升高，并具有神经保护作用。考虑到上述结果，我们目前正在测试的假设，即已知的强大的表达NR 2B-含有NMDA-R在CA 1相对于CA 3是负责钙依赖性的脆弱性的CA 1神经元。

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.