ELEMENTAL AND STRUCTURAL ORGANIZATION OF NEURONS AND GLIA

该项目研究神经元钙信号的生理和细胞方面，长期侧重于中枢神经系统神经元的树突和树突棘。最近的工作集中在牛蛙交感神经元的钙调节上，因为这些大的球形细胞是揭示细胞内钙运动细节的优秀模型。交感神经元对去极化刺激的反应是胞内游离钙浓度([Ca~(2+)]_i)升高，这种升高是由电压门控通道中的Ca~(2+)进入引起的，但也受细胞内钙储存库活动的调节。我们现在已经研究了一个主要的细胞内存储，线粒体，在确定膜去极化过程中和之后的时间进程中所起的作用。我们早些时候已经证明，在这些神经元中，去极化诱导的[Ca+]i增加伴随着线粒体总钙浓度([Ca]m)的大幅、渐进性和可逆性升高。有趣的是，这些增加在个体线粒体之间和内部都是空间异质性的。因此，我们使用一种新的高分辨率技术--电子能量损失光谱成像技术来表征线粒体内钙的分布和化学形态，以高于10 nm的分辨率绘制细胞内钙的空间分布。在去极化细胞的单个线粒体内，钙集中在小的(&lt；10 nm)富磷包裹体中；其高的钙含量表明它们是准晶体。这些包裹体可能反映了线粒体内钙隔离的高容量机制。除了细胞器内的异质性外，外周线粒体中包涵体的数量和大小都高于内部线粒体，因此去极化引起的[Ca]m升高表现出与质膜距离的径向依赖关系。[Ca]m的分布与我们基于已建立的钙离子传输模型的扩散模拟是一致的。在维持的去极化过程中，[Ca]m梯度的持续意味着线粒体有记忆，在自由离子梯度消失后很长一段时间内，仍保留着[Ca+]i早期空间差异的记录。目前正在研究第二个主要的钙调节细胞器--内质网的作用，并确定其与线粒体钙库的时空相互作用。-钙/海马体/树突/交感神经元/内质网/线粒体/突触/X射线显微分析/电子能量损失谱

This project studies physiological and cellular aspects of neuronal calcium signaling, with long-range emphasis on dendrites and dendritic spines of central nervous system neurons. Recent work has focused on Ca regulation in bullfrog sympathetic neurons because these large, spherical cells are excellent models for revealing intracellular details of Ca movements. Sympathetic neurons respond to depolarizing stimuli with a rise in cytosolic free Ca concentration ([Ca2+]i) that is initiated by Ca2+ entry through voltage-gated channels but also modulated by the activity of intracellular Ca stores. We have now studied the role of one major intracellular store, the mitochondrion, in defining the time course of [Ca2+]i during and after membrane depolarization. We had earlier shown that in these neurons depolarization-induced increases in [Ca2+]i are accompanied by large, graded, and reversible elevations in total mitochondrial calcium concentration ([Ca]m). Interestingly, these increases were spatially heterogeneous both among and within individual mitochondria. Therefore, we have characterized the distribution and chemical form of intramitochondrial Ca by using a novel high-resolution technique, spectrum imaging by electron energy loss spectroscopy, to map, at better than 10-nm resolution, the spatial distribution of intracellular Ca. Within single mitochondria of depolarized cells, Ca was concentrated in small (<10 nm) phosphorus-rich inclusions; their high Ca content suggests that they are quasi-crystalline. These inclusions likely reflect a high-capacity mechanism of intramitochondrial Ca sequestration. In addition to intra-organelle heterogeneity, the number and size of inclusions was higher in peripheral than in internal mitochondria, so that depolarization-evoked elevations in [Ca]m showed a radial dependence on distance from the plasma membrane. The distribution of [Ca]m is consistent with our diffusion simulations based on established models of Ca2+ transport. Persistence of the [Ca]m gradient during maintained depolarization implies that mitochondria have a memory, retaining a record of early spatial differences in [Ca2+]i long after free ion gradients have dissipated. Work is now underway to characterize the role of a second major Ca-regulating organelle, the endoplasmic reticulum, and to define its spatio-temporal interactions with the mitochondrial Ca pool. - calcium/ hippocampus/ dendrites/ sympathetic neuron/ endoplasmic reticulum/ mitochondria/ synapse/ x-ray microanalysis/ electron energy loss spectroscopy