ELEMENTAL AND STRUCTURAL ORGANIZATION OF NEURONS AND GLIA

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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.