Name: imaging mitochondrial ca2+ uptake in astrocytes and neurons using genetically encoded ca2+ indicators (gecis)
Uploaded: 2022-01-22T14:00:00
Description: Watch this Scientific Journal Video about Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs at JoVE.com

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.

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.
<ol>
	<li>Insert mitochondrial matrix (MM)-targeting sequence (mito-) ATGT CCGTC...

<ol>
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In this article, we provide a method and protocol for imaging mitochondrial Ca2+&#160;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...

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+ overloading is also a determinant for cell death, including necrosis and apoptosis in various brain disorders5,6,7. It causes the opening of mitochondrial permeability transition pores (mPTPs) and the release of caspase cofactor, which initiate apoptotic cell death. Therefore, it is important to study mitochondrial Ca2+ dynamics and handling in living cells to understand cellular physiology and pathology better.
Mitochondria maintain matrix Ca2+ homeostasis through a balance between Ca2+ uptake and efflux. Mitochondrial Ca2+ uptake is mainly mediated by mitochondrial Ca2+ uniporters (MCUs), while mitochondrial Ca2+ efflux is mediated by the Na+-Ca2+-Li+ exchangers (NCLXs) and the H+/Ca2+ exchangers (mHCXs)8. The balance can be perturbed through the stimulation of G-protein coupled receptors (GPCRs)9. Mitochondrial Ca2+ homeostasis is also affected by mitochondrial buffering by the formation of insoluble xCa2+-xPO4x-xOH complexes8.
Intracellular and mitochondrial changes in Ca2+ concentration ([Ca2+]) can be evaluated by fluorescent or luminescent Ca2+ indicators. Ca2+ binding to indicators causes spectral modifications, allowing to recording of free cellular [Ca2+] in real-time in live cells. Two types of probes are currently available to monitor Ca2+ changes in cells: organic chemical indicators and genetically-encoded Ca2+ indicators (GECIs). Generally, different variants with different Ca2+ affinities (based on Kd), spectral properties (excitation and emission wavelengths), dynamic ranges, and sensitivities are available for the biological questions under investigation. Although many synthetic organic Ca2+ indicators have been used for cytosolic Ca2+ imaging, only a few can be selectively loaded in the mitochondrial matrix for mitochondrial Ca2+ imaging, with Rhod-2 being the most widely used (for reviews see10,11). However, Rhod-2 has a major drawback of leakage during long time-course experiments; in addition, it is partitioned between mitochondria, other organelles and the cytosol, making absolute measurements in different subcompartments difficult. In contrast, by using cell-type specific promoters and subcellular compartment targeting sequences, GECIs can be expressed in different cell types and subcellular compartments for cell- and compartment-specific Ca2+ imaging in vitro or in vivo. Single-wavelength fluorescence intensity-based GCaMP Ca2+ indicators have recently emerged as major GECIs12,13,14,15,16. In this article, we provide a protocol for mitochondria-targeting and cell-type specific expression of GCaMP5G and GCaMP6s (GCaMP5G/6s) in astrocytes and neurons, and imaging mitochondrial Ca2+ uptake in these cell types. Using this protocol, the expression of GCaMP6G/6s in individual mitochondria can be revealed, and Ca2+ uptake in single mitochondrial resolution can be achieved in astrocytes and neurons in vitro and in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>Artificial tears ointment</td>
			<td>Rugby</td>
			<td>NDC-0536-6550-91</td>
			<td>83% white petrolatum</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>World Precision Instruments</td>
			<td></td>
			<td>3M Vetbond Adhesive</td>
		</tr>
		<tr>
			<td>Dissecting stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ 2B</td>
			<td>Surgery</td>
		</tr>
		<tr>
			<td>Dumont forceps with fine tip</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td>for removal of dura</td>
		</tr>
		<tr>
			<td>Glass cover slips, 0.13-0.17 mm thick</td>
			<td>Fisher Scientific</td>
			<td>12-542A</td>
			<td>for cranial window cover</td>
		</tr>
		<tr>
			<td>High speed micro drill</td>
			<td>Fine Science Tools</td>
			<td>18000-17</td>
			<td>with bone polishing drill bit</td>
		</tr>
		<tr>
			<td>Injection syringe</td>
			<td>Hamilton</td>
			<td>2.5 ml</td>
			<td>for viral injection</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>VEDCO</td>
			<td>NDC-50989-996-06</td>
			<td>100 mg/kg body weight</td>
		</tr>
		<tr>
			<td>Low melting point agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9793</td>
			<td>reducing movement artifacts</td>
		</tr>
		<tr>
			<td>Metal frame</td>
			<td>Custom-made</td>
			<td>see Fig 1</td>
			<td>for brain attachment to microscope stage</td>
		</tr>
		<tr>
			<td>MicroSyringe Pump Controller</td>
			<td>World Precision Instruments</td>
			<td>UMP3</td>
			<td>Injection speed controller</td>
		</tr>
		<tr>
			<td>Mouse stereotaxic device</td>
			<td>Stoelting</td>
			<td>51725</td>
			<td>for holding mice</td>
		</tr>
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			<td>Perfusion chamber</td>
			<td>Warner Instruments</td>
			<td>64-0284</td>
			<td></td>
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			<td>Persfusion system</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA-VM8</td>
			<td></td>
		</tr>
		<tr>
			<td>Self-regulating heating pad</td>
			<td>Fine Science Tools</td>
			<td>21061</td>
			<td>to prevent hypothermia of mice</td>
		</tr>
		<tr>
			<td>Sulforhodamine 101</td>
			<td>Invitrogen</td>
			<td>S-359</td>
			<td>red fluorescent dye to label astrocytes</td>
		</tr>
		<tr>
			<td>Surgical scissors, 12 cm</td>
			<td>Fine Science Tools</td>
			<td>14002-12</td>
			<td>for dissection</td>
		</tr>
		<tr>
			<td>Trephine</td>
			<td>Fine Science Tools</td>
			<td>18004-23</td>
			<td>for clearing of material</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>VEDCO</td>
			<td>NDC-50989-234-11</td>
			<td>10 mg/kg body weight</td>
		</tr>
	</tbody>
</table>

imaging mitochondrial ca2+ uptake in astrocytes and neurons using genetically encoded ca2+ indicators (gecis)

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.

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...

Watch this Scientific Journal Video about Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs at JoVE.com

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

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

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

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

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