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This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.
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.
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+
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.
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.......
In this article, we provide a method and protocol for imaging mitochondrial Ca2+Â 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.......
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.
....Name | Company | Catalog Number | Comments |
Artificial tears ointment | Rugby | NDC-0536-6550-91 | 83% white petrolatum |
Cyanoacrylate glue | World Precision Instruments | 3M Vetbond Adhesive | |
Dissecting stereomicroscope | Nikon | SMZ 2B | Surgery |
Dumont forceps with fine tip | Fine Science Tools | 11255-20 | for removal of dura |
Glass cover slips, 0.13-0.17 mm thick | Fisher Scientific | 12-542A | for cranial window cover |
High speed micro drill | Fine Science Tools | 18000-17 | with bone polishing drill bit |
Injection syringe | Hamilton | 2.5 ml | for viral injection |
Ketamine | VEDCO | NDC-50989-996-06 | 100 mg/kg body weight |
Low melting point agarose | Sigma-Aldrich | A9793 | reducing movement artifacts |
Metal frame | Custom-made | see Fig 1 | for brain attachment to microscope stage |
MicroSyringe Pump Controller | World Precision Instruments | UMP3 | Injection speed controller |
Mouse stereotaxic device | Stoelting | 51725 | for holding mice |
Perfusion chamber | Warner Instruments | 64-0284 | |
Persfusion system | ALA Scientific Instruments | ALA-VM8 | |
Self-regulating heating pad | Fine Science Tools | 21061 | to prevent hypothermia of mice |
Sulforhodamine 101 | Invitrogen | S-359 | red fluorescent dye to label astrocytes |
Surgical scissors, 12 cm | Fine Science Tools | 14002-12 | for dissection |
Trephine | Fine Science Tools | 18004-23 | for clearing of material |
Xylazine | VEDCO | NDC-50989-234-11 | 10 mg/kg body weight |
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