Elemental And Structural Organization Of Neurons And Gli

该项目研究神经元钙信号的细胞和生理方面，长期侧重于中枢神经系统神经元的突触后反应。神经元对突触刺激的反应是胞内游离钙离子浓度的升高，这种升高受到细胞内钙储存细胞器转运活性的强烈调节。这种转运活动在时空上塑造调控基因表达和突触可塑性等下游过程的信号方面发挥着重要作用。早些时候，包括我们在内的几个实验室已经在各种神经元上表明，刺激引起的胞浆游离钙浓度升高会导致线粒体内钙浓度的大幅可逆增加，这反过来又对生理和病理生理过程产生重要影响。今年，我们继续探索诱发的线粒体钙摄取的后果，重点是了解缺血预适应(PC)的机制，这是一种重要但鲜为人知的现象，即神经元在接受类似但更温和的非致命性挑战后，对通常致命的伤害变得耐受。 以往的研究表明，NMDA过度刺激导致兴奋性迟发性细胞死亡(DCD)与线粒体强烈的钙摄取有关。这种线粒体活动在空间上是异质性的，这已被证明是重要的，因为受损线粒体的数量和位置决定了细胞？S对过量NMDA的脆弱性。今年，我们探索了一种假设，即线粒体功能障碍的减少是缺血预适应的神经保护作用的原因。几种标准预适应方案的结果表明，在细胞水平上，这些处理通过诱导细胞内钙离子的重新分配和线粒体的瞬时钙负荷和去极化，定性地概括了NMDA的作用。然而，这些影响较弱，完全可逆，并未导致DCD。某些方案比其他方案诱导了更强的神经保护，但无论效果如何，PC神经保护与兴奋性毒性NMDA暴露后线粒体损伤的减少是平行的。此外，较强的PC也显著减少了钙离子的内流。这些结果表明，PC通过提高线粒体对大钙负荷的耐受性而发挥保护作用，从而减轻线粒体功能障碍。最有效的PC协议似乎招募了额外的、附加的机制。今年发现的一种这样的机制涉及到表面表达的、因此是活跃的NMDA受体的显著下调。 海马片培养是研究神经元耐受的另一种模型，因为CA1区的锥体神经元对兴奋性毒性刺激非常敏感，而CA3区的神经元表现出高水平的内源性神经保护。与先前的证据一致的是，线粒体损伤是确定兴奋性毒性易损性的关键事件，在应用兴奋性NMDA后，CA1树突中肿胀、受损的线粒体比例基本上为100%，而在CA3中，只有线粒体的一部分肿胀，其余的保持其正常的杆状形状。在这两个区域的细胞体中，都有许多线粒体受损，但在CA1细胞中，线粒体的数量更多。结果表明，线粒体损伤是CA1神经元对兴奋性毒性损伤的选择性易损性的主要因素，这反过来又是它们对缺血损伤的敏感性的基础，并证明了进一步研究CA3的线粒体与CA1的不同是合理的。

This project studies cellular and physiological aspects of neuronal calcium (Ca2+) signaling, with long-range emphasis on postsynaptic responses in central nervous system neurons. Neurons respond to synaptic stimuli with a rise in cytosolic free Ca2+ concentration that is strongly modulated by the transport activity of intracellular calcium storage organelles. This transport activity plays an important role in spatio-temporally shaping the signals that regulate downstream processes like gene expression and synaptic plasticity. Several labs, including ours, had earlier shown in a variety of neurons that stimulus-evoked cytosolic free Ca2+ elevations induce large, reversible increases in the concentration of calcium within mitochondria, which in turn has important effects on physiological and pathophysiological processes. This year we continued to explore the consequences of evoked mitochondrial Ca2+ uptake, with emphasis on understanding the mechanisms of ischemic preconditioning (PC), an important but poorly understood phenomenon whereby neurons become tolerant to a normally lethal insult after pretreatment with a similar but milder, non-lethal challenge. It was previously shown that NMDA overstimulation that leads to excitotoxic delayed cell death (DCD) is associated with strong mitochondrial Ca2+ uptake. This mitochondrial activity is spatially heterogeneous, which has proven to be important because the number and location of damaged mitochondria determine a cell?s vulnerability to excessive NMDA. This year we have explored the hypothesis that the reduced mitochondrial dysfunction accounts for the neuroprotective effects of ischemic preconditioning. The results from several standard preconditioning protocols showed that at the cellular level these treatments qualitatively recapitulate the effects of NMDA by inducing the redistribution of intracellular Ca2+ and the transient Ca2+ loading and depolarization of mitochondria. However, these effects were weaker, fully reversible and did not lead to DCD. Certain protocols induced stronger neuroprotection than others but, regardless of efficacy, PC neuroprotection was paralleled by reduced mitochondrial injury after excitotoxic NMDA exposure. In addition, stronger PC also substantially reduced Ca2+ entry. These results indicate that PC exerts a protective effect by increasing mitochondrial tolerance for large Ca loads, thereby attenuating mitochondrial dysfunction. The most effective PC protocols appear to recruit additional, additive mechanisms. One such mechanism uncovered this year involves a significant down-regulation of surface-expressed, and therefore active, NMDA receptors. Slice cultures of hippocampus 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. Consistent with previous evidence that mitochondrial damage is a key event in determining excitotoxic vulnerability, the fraction of swollen, damaged mitochondria in CA1 dendrites following application of excitotoxic NMDA was essentially 100%, while in CA3 only a subset of mitochondria was swollen, the rest retaining their normal, rod-like shape. In cell bodies of both regions, many mitochondria were damaged, but again these were more numerous in CA1 cells. Results suggest that mitochondrial damage is a major factor in the selective vulnerability of CA1 neurons to excitotoxic insult, which in turn underlies their susceptibility to ischemic injury, and justify further investigation into how the mitochondria of CA3 differ from those of CA1.