Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

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 conditions^1,2,3,4. 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.......

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.......

Materials

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