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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

Abstract

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Introduction

Mitochondria are dynamic subcellular organelles and are considered as the cell powerhouses for energy production. On the other hand, mitochondria can take up Ca2+ to the matrix in response to local or cytosolic Ca2+ rises. Mitochondrial Ca2+ uptake affects mitochondrial function, including metabolic processes such as reactions in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, and regulates Ca2+-sensitive proteins under physiological conditions1,2,3,4. Mitochondrial Ca2+

Protocol

Procedures involving animals have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Missouri-Columbia.

1. Construction of DNA plasmids

NOTE: For in vitro and in vivo imaging, DNA plasmids encoding GCaMP5G/6s with astrocyte- and neuron-specific promoters and mitochondrial targeting sequences are constructed.

  1. Insert mitochondrial matrix (MM)-targeting sequence (mito-) ATGT CCGTC.......

Representative Results

The aim of this study was to provide a methodology to image mitochondrial Ca2+ signals using GECIs in astrocytes and neurons in vitro and in vivo. Results of both in vitro and in vivo mitochondrial Ca2+ imaging are presented here.

In vitro mitochondrial Ca2+ signaling in cultured astrocytes and neurons
Mitochondrial Ca2+ uptake in as.......

Discussion

In this article, we provide a method and protocol for imaging mitochondrial Ca2+ signals in astrocytes and neurons. We implemented mitochondria-targeting and cell type-specific strategies to express GECI GCaMP5G/6s. To target GCaMP5G/6s in mitochondria, we included a mitochondria-targeting sequence in the plasmids. To express GCaMP5G/6s in astrocytes and neurons in vivo, we inserted an astrocyte-specific promoter gfaABC1D and neuron-specific promoter CaMKII into the plasmids. Cell-type specific e.......

Acknowledgements

This work was supported by the National Institute of Health National Institute of Neurological Disorders and Stroke (NINDS) grants R01NS069726 and R01NS094539 to SD. We thank Erica DeMers for the audio recording.

....

Materials

NameCompanyCatalog NumberComments
Artificial tears ointmentRugbyNDC-0536-6550-9183% white petrolatum
Cyanoacrylate glueWorld Precision Instruments3M Vetbond Adhesive
Dissecting stereomicroscopeNikonSMZ 2BSurgery
Dumont forceps with fine tipFine Science Tools11255-20for removal of dura
Glass cover slips, 0.13-0.17 mm thickFisher Scientific12-542Afor cranial window cover
High speed micro drillFine Science Tools18000-17with bone polishing drill bit
Injection syringeHamilton2.5 mlfor viral injection
KetamineVEDCONDC-50989-996-06100 mg/kg body weight
Low melting point agaroseSigma-AldrichA9793reducing movement artifacts
Metal frameCustom-madesee Fig 1for brain attachment to microscope stage
MicroSyringe Pump ControllerWorld Precision InstrumentsUMP3Injection speed controller
Mouse stereotaxic deviceStoelting51725for holding mice
Perfusion chamberWarner Instruments64-0284
Persfusion systemALA Scientific InstrumentsALA-VM8
Self-regulating heating padFine Science Tools21061to prevent hypothermia of mice
Sulforhodamine 101InvitrogenS-359red fluorescent dye to label astrocytes
Surgical scissors, 12 cmFine Science Tools14002-12for dissection
TrephineFine Science Tools18004-23for clearing of material
XylazineVEDCONDC-50989-234-1110 mg/kg body weight

References

  1. Griffiths, E. J., Rutter, G. A. Mitochondrial calcium as a key regulator of mitochondrial ATP production in mammalian cells. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1787 (11), 1324-1333 (2009).
  2. Pizzo, P., Drago, I., Filadi, R., Pozzan, T.

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