Visualization of Ca2+ dynamics within the endoplasmic reticulum for the study of astrocytic functions

[Speaker] Yohei Okubo:1
[Co-author] Kazunori Kanemaru:1, Masamitsu Iino:2
1:Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Japan, 2:Department of Cellular and Molecular Pharmacology, Nihon University School of Medicine, Japan

Astrocytes, a major type of glial cells in the brain, show a wide variety of intracellular Ca2+ dynamics spontaneously and in response to various stimuli. Accumulating evidence indicates that Ca2+ release from the endoplasmic reticulum (ER) in astrocytes may play a key role in physiological and pathophysiological events in the brain. Therefore, the development of techniques for sensitive detection of spatiotemporal features of ER-mediated Ca2+ dynamics in astrocytes is crucial to the understanding of such events. Although both synthetic and genetically encoded cytosolic Ca2+ indicators have been used to evaluate the Ca2+ release from the ER in astrocytes in situ, such method faces two major problems. Firstly, cytoplasmic Ca2+ transients are generated not only by release of Ca2+ from the ER but also by Ca2+ influx via the plasma membrane. Second, because cytoplasm has a strong Ca2+ buffering capacity, it is not always clear whether the sensitivity of the cytoplasmic Ca2+ indicators is sufficient to detect subtle Ca2+ release from the ER. We recently developed a genetically encoded ER Ca2+ indicator, G-CEPIA1er, to directly visualize the Ca2+ dynamics within the ER. Using G-CEPIA1er in astrocytes, we found large decreases in the ER Ca2+ concentration even when relatively small increases in the cytoplasmic Ca2+ concentration were observed. Thus, the ER Ca2+ visualization method allows us to detect ER-mediated Ca2+ signaling, which might have previously escaped detection. We also succeeded in detecting ER Ca2+ dynamics in astrocytes in vivo, which were associated with behavior of the animal. Collectively, the present ER Ca2+ visualization studies may provide an important new insight into ER Ca2+ dynamics and Ca2+-mediated functions in astrocytes.
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