Study of Calcium Signaling in Astrocytes with a Novel Endoplasmic Reticulum–Targeted GCaMP Sensor

Surya P. Aryal, Mengfan Xia, Pavel I. Ortinski, Christopher I. Richards

Research output: Contribution to journalArticlepeer-review

Abstract

The endoplasmic reticulum (ER), the major organelle for the storage of Ca2+, maintains a concentration of Ca2+ much higher than in the cytosol or other subcellular organelles, such as the mitochondria. A variety of tools have been developed for measuring Ca2+ activity in neuronal and glial cells, but most of these sensors target either the plasma membrane (PM) or the cytosol. Though these sensors are important for measuring Ca2+ transients, they lack the capability to measure activity in the periphery of the ER or to measure low-amplitude events resulting from Ca2+ exchange between the ER and other organelles, such as the mitochondria. We recently developed an ER-targeted GCaMP6f anchored to the cytosolic side of the ER that can measure minute calcium exchange occurring in this region. In this article, we discuss detailed methods to characterize the ER-GCaMP6f sensor, utilize it for calcium imaging in cultured astrocytes, and assess its expression and calcium imaging in astrocytes in rodent brains.

Original languageEnglish
Article numbere491
JournalCurrent Protocols
Volume2
Issue number8
DOIs
StatePublished - Aug 2022

Bibliographical note

Publisher Copyright:
© 2022 Wiley Periodicals LLC.

Funding

The authors acknowledge the Genetic Technologies Core at the Center of Molecular Medicine and Light Microscopy Core, University of Kentucky, for use of their facilities. CIR acknowledges support from the National Institutes of Health (DA038817). Cartoons were constructed with BioRender (biorender.com). Chemical structures were drawn with the help of ChemSketch.

FundersFunder number
National Institutes of Health (NIH)DA038817
National Institute on Drug AbuseR01DA041513

    Keywords

    • AAV
    • ER-GCaMP6f
    • astrocyte
    • calcium
    • fluorescence

    ASJC Scopus subject areas

    • General Immunology and Microbiology
    • General Pharmacology, Toxicology and Pharmaceutics
    • Medical Laboratory Technology
    • General Biochemistry, Genetics and Molecular Biology
    • Health Informatics
    • General Neuroscience

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