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。神经元对突触刺激的反应是胞内游离钙浓度([Ca~(2+)]_i)的升高，这种升高受细胞内钙库活性的强烈调节。我们早些时候已经证明，在青蛙的交感神经元中？研究细胞内钙动力学细节的优秀模型？去极化引起的[Ca+]i升高伴随着线粒体总钙浓度([Ca]m)的可逆性大幅升高。线粒体的这种钙转运活动引起了[Ca]m的空间梯度，因为它记录并记录了早期[Ca+]i的区域差异，而这反过来在时空塑造细胞内钙信号方面发挥了重要作用。我们现在已经确定了第二个主要的钙调节细胞器--内质网(ER)的功能，它的作用通常被认为是通过触发其内部存储的钙释放来放大引起的[钙]i升高，这一过程被称为“钙诱导钙释放”(CICR)。然而，我们发现，在低水平的Ca~(2+)进入(因此低[Ca~(2+)]i)，内质网实际上起到了Ca~(2+)缓冲器的作用，尽管它的强度被[Ca~(2+)]_i敏感的释放通路的分级激活所下调。理论模拟表明，CICR的这种“反向”模式是可以预期的；此外，随着[Ca~(2+)]i的升高，许多神经元应该表现出从缓冲Ca~(2+)到触发Ca~(2+)释放的渐进转变。这样的过渡？到经典的CICR？当[Ca~(2+)]i升高到约1uM。此外，这种钙释放优先定位于外周ER池，因此在同一细胞的不同区域，钙的摄取(中枢)和释放(外周)可以同时发生。最后，内质网Ca~(2+)转运的空间梯度与线粒体Ca~(2+)摄取的空间梯度是相反的，表明这些细胞器之间存在合作。在海马神经元中，诱发的[Ca~(2+)]i瞬变增强了钙调素(CaM)的核输入，这反过来又增强了重要转录因子CREB的磷酸化。这种产生磷酸化CREB(PCREB)的途径是突触诱发基因表达的中心，而突触诱发基因表达是长期记忆形成的基础。已有研究表明，超氧阴离子(O2-)可通过抑制pCREB的去磷酸化来增强其稳定性。利用定量免疫细胞化学和一组电子传递阻滞剂，我们现在已经评估了线粒体在连接细胞内钙进入和基因表达方面的作用。我们发现，在钙离子进入过程中，线粒体钙的积累主要发生在神经元的外周区域，导致ATP和O2-产生的增加。这反过来又增加了CaM的核进口。虽然O2-升高对CaM的运输在数量上是重要的，但它似乎对维持CREB的磷酸化是必不可少的。

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. Neurons respond to synaptic stimuli with a rise in cytosolic free Ca concentration ([Ca2+]i) that is strongly modulated by the activity of intracellular Ca stores. We had earlier shown that in frog sympathetic neurons ? an excellent model for studying intracellular details of Ca dynamics ? depolarization-induced increases in [Ca2+]i are accompanied by large, reversible elevations in total mitochondrial calcium concentration ([Ca]m). This mitochondrial Ca2+ transport activity gives rise to spatial gradients in [Ca]m because it registers and retains a record of early regional differences in [Ca2+]i, and this in turn plays an important role in spatio-temporally shaping cytosolic Ca signals. .We have now characterized the function of a second major Ca2+-regulating organelle, the endoplasmic reticulum (ER), whose role is generally thought to be amplification of evoked [Ca2+]i elevations by triggered Ca2+ release from its internal store, a process known as "calcium-induced calcium release" (CICR). We find, however, that at low levels of Ca2+ entry (and therefore low [Ca2+]i) the ER actually acts as a Ca2+ buffer, albeit one whose strength is down-regulated by graded activation of a [Ca2+]i-sensitive release pathway. Theoretical simulations show that such a "reverse" mode of CICR is expected; moreover, many neurons should exhibit a progressive transition from Ca2+ buffering to triggered Ca2+ release as [Ca2+]i increases. Such a transition ? to classical CICR ? was observed when [Ca2+]i increased above approx. 1uM. In addition, such Ca2+ release is preferentially localized to peripheral ER cisternae, so that both Ca2+ uptake (centrally) and release (peripherally) can occur at the same time in different regions of the same cell. Finally, the spatial gradient of ER Ca2+ transport is reciprocal to that of mitochondrial Ca2+ uptake, suggesting cooperation between these organelles. In hippocampal neurons, evoked [Ca2+]i transients enhance nuclear import of calmodulin (CaM), which in turn augments phosphorylation of the important transcription factor CREB. This pathway for phospho-CREB (pCREB) production is central to the synaptically-evoked gene expression that underlies long-term memory formation. It has been previously shown that superoxide (O2-) ions can enhance the stability of pCREB by inhibiting its dephosphorylation. Using quantitative immunocytochemistry and a panel of electron transport blockers, we have now evaluated the role of mitochondria in linking cytosolic Ca2+ entry to gene expression. We find that mitochondrial Ca accumulation, occurring predominantly in peripheral regions of neurons during Ca2+ entry, leads to increases in both ATP and O2- production. This, in turn, enhances nuclear import of CaM. While O2- elevation is quantitatively important for CaM transport, it appears to be essential for sustaining CREB phosphorylation.