Elemental And Structural Organization Of Neurons And Gli

本项目研究神经元钙（Ca2+）信号的细胞和生理方面，长期强调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质游离Ca2+浓度的升高，这是由胞内钙储存细胞器的运输活性强烈调节的。这种转运活动在时空上对调控下游过程如基因表达和突触可塑性的信号形成起着重要作用。包括我们在内的几个实验室早先已经在各种神经元中表明，刺激诱发的胞质游离Ca2+升高诱导线粒体内钙浓度的大幅可逆增加，这反过来对生理和病理生理过程产生重要影响。今年，我们继续探索引起线粒体Ca2+摄取的后果，重点是了解缺血预处理（PC）的机制，这是一个重要但鲜为人知的现象，即神经元在预处理类似但较温和的非致命挑战后，对通常致命的损伤变得耐受。

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.