Imaging of Mitochondrial and Cytosolic Ca2+ Signals in Cultured Astrocytes

Nannan Zhang1, Shinghua Ding2

1 Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri, 2 Department of Bioengineering, University of Missouri, Columbia, Missouri
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 2.29
DOI:  10.1002/cpns.42
Online Posting Date:  January, 2018
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This unit provides a step‐by‐step protocol for constructing cell type‐ and mitochondria‐targeted GCaMP genetically encoded Ca2+ indicators (GECIs) for mitochondrial Ca2+ imaging in astrocytes. Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Mitochondrial Ca2+ overload contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Live‐cell mitochondrial Ca2+ imaging is an important approach to understand mitochondrial Ca2+ dynamics and thus cell physiology and pathology. We implement astrocyte‐specific mitochondrial targeting of GCaMP5G/6s (mito‐GCaMP5G/6s). By loading X‐Rhod‐1 into astrocytes, we can simultaneously image mitochondrial and cytosolic Ca2+ signals. This protocol provides a novel approach to image mitochondrial Ca2+ dynamics as well as Ca2+ interplay between the endoplasmic reticulum and mitochondria. © 2018 by John Wiley & Sons, Inc.

Keywords: astrocyte; ATP; endoplasmic reticulum (ER); GCaMP5G/6s; mitochondrial Ca2+ uptake; mitochondrial matrix; X‐Rhod‐1

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Mitochondrial matrix (MM)‐targeting sequence (Rizzuto, Brini, Pizzo, Murgia, & Pozzan, )
  • AAV plasmid pZac2.1 containing astrocytic gfaABC 1D promoter (Lee et al., ; Li et al., ; Xie, Wang, Sun, & Ding, )
  • GCaMP5G/6s gene
  • Monomeric red fluorescent protein (mRFP) gene
  • EndoFree Plasmid Maxi Kit (Qiagen)
  • C57BL/6J mouse pup, 0 to 2 days old
  • Earle's Balanced Salt Solution (EBSS; Invitrogen), 4°C
  • 20 IU/ml papain (Sigma)
  • L‐cysteine
  • 10 mg/ml type II‐O trypsin inhibitor (Sigma)
  • Serum‐free Dulbecco's Modified Eagle's Medium (DMEM; Invitrogen), 37°C
  • α‐Modified Minimal Essential Medium (α‐MMEM; see recipe and Table 2.29.1), 37°C
  • DMEM containing 10% fetal bovine serum (FBS; HyClone), 37°C
  • Lipofectamine® 2000 Transfection Reagent (Thermo Fisher)
  • 1 mg/ml X‐Rhod‐1 (X14210, Thermo Fisher) stock solution (see recipe)
  • Artificial cerebrospinal fluid (ACSF; see recipe)
  • 10 mM ATP stock solution (see recipe)
  • 35‐mm‐diameter culture dish
  • 15‐ml conical tube
  • 37°C, 5% CO 2 incubator
  • Glass serological pipet
  • 25‐cm2 tissue culture flask
  • Orbital shaker
  • 12‐mm‐diameter glass coverslips (Fisher Scientific)
  • 24‐well plate
  • Microcentrifuge tubes
  • Perfusion system (ALA‐VM8, ALA Scientific), which can control solution change and drug application using pinch valves
  • Perfusion chamber (PH1, Warner Instruments)
  • Vacuum pump
  • 2‐P (Prairie Technologies) or confocal fluorescence microscope with imaging/acquisition system
  • ImageJ (NIH) or MetaMorph (Molecular Devices) software
  • Origin (OriginLab) or equivalent software
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.NOTE: Experiments involving PCR require extremely careful technique to prevent contamination; see unit 15.10 (Kramer & Coen, ).
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Literature Cited

Literature Cited
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