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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Q., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-B-cell+colony-enhancing+factor+exerts+a+neuronal+protection+through+its+enzymatic+activity+and+the+reduction+of+mitochondrial+dysfunction+in+in+vitro+ischemic+models.">Pre-B-cell colony-enhancing factor exerts a neuronal protection through its enzymatic activity and the reduction of mitochondrial dysfunction in in vitro ischemic models.</a> Journal of Neurochemistry. 120 (2), 334-346 (2012).</li><li>Wang, X., Li, H., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-B-cell+colony-enhancing+factor+protects+against+apoptotic+neuronal+death+and+mitochondrial+damage+in+ischemia.">Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia.</a> Scientific Reports. 6, 32416 (2016).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+NAMPT-mediated+NAD++salvage+pathways+and+their+roles+in+bioenergetics+and+neuronal+protection+after+ischemic+injury.">Subcellular NAMPT-mediated NAD+ salvage pathways and their roles in bioenergetics and neuronal protection after ischemic injury.</a> Journal of Neurochemistry. 151 (6), 732-748 (2019).</li><li>Xie, Y., Chen, S., Wu, Y., Murphy, T. 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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 ...

Demonstrating the procedure will be graduate student Zhe Zhang, research specialist Nannan Zhang, professors Illker Ozden and Shinghua Ding. The overall goal of this procedure is to develop cell type specific pair astrocytes and the neurons and the mitochondrial targeting method for in vitro and in vivo mitochondrial counts of imaging using genetically encoded calcium indicator, GCaMP5G and 6s. For in vitro or in vivo imaging, we will construct DNA plasmids containing astrocyte or neurons specific promoters gfaABC1D or CaMKII and genetically encoded calcium indicators, GCaMP5G/6s with mitochondrial targeting sequences.For in vitro mitochondrial calcium imaging we transfected astrocytes neurons cultured on glass cover slips with the aforementioned DNA plasmids using the Lipofectamine method. Imaging can be started one to two days after transfection by transferring the glass cover slips to the profusion chamber under an epifluorescence or two-photon microscope. Here are examples of fluorescent signals from mitochondria and an astrocyte expressing GCaMP6s on the left, and for mitochondria in a neuron, expressing GCaMP5G on the right.For in vivo imaging, we prepared serotype 5 associated adenovirus for expression of mito-GCaMP5G in astrocytes and serotype 9 associated adenovirus for expression of mito-GCaMP6s in neurons. For stereotaxic virus injection surgeries, we first anesthetized the mouse with 3%isoflurane. Later during the surgery, the isoflurane levels is reduced to 2%After the proper level of anesthesia is reached, the hair over the scalp is shaved and the mouse is positioned on a stereotax surgical instrument.An ophthalmic ointment is applied to the eyes to protect them during the surgery. The injection surgeries are performed by using aseptic procedures, including sterile instruments and aseptic techniques. The surgical instruments are sterilized either by autoclaving or by a hot bead sterilizer.After the mouse is mounted on the stereotax, the scalp is cleaned with betadine and 70%ethanol three times. Then the skin is cut open along the Bregma Lambda axis and the vertical is made by a high-speed drill over the target location. In this work, we targeted either the motor cortex or the paracortex.A 33 gauge Hamilton syringe carrying an associated adenovirus vector is inserted through the hole into the brain and vectors are injected at the target area. For cortical delivery, we inject the virus solution at two depths in multiple steps. First, we insert the needle one millimeter deep into the cortex.After allowing five minutes for the brain to recover, we move the needle up to 700 micrometers deep, inject 500 nano liters of virus solution. After injection is completed, we wait for five minutes to allow the virus to diffuse in the brain. And then we move the needle up to the second injection location at 300 micrometers deep.Here, we inject another 500 nano liters of virus solution. After injection is completed, we wait for 10 minutes to allow the virus to diffuse and then we remove the needle from the brain and close the burr hole with bone wax or Kwik-Sil. Finally, the skin is sutured with Vetbond and the mouse is recovered on a heating pad before being sent back to the animal facility.The mouse will be ready for use for in vivo imaging in a minimum of three weeks, which is the timeframe in which GCaMP expression matures. Cranial window implantation surgeries are also done by using aseptic conditions as described for injection surgeries. The cranial window surgical procedures are identical to the injection surgical procedures until the point of skull exposure.Once the skull is exposed, quarters of a 2-3 mm diameter craniotomy over the virus injected area are marked with four shallow holes made by a high-speed drill attached to a manipulator. Then the section of the skull, at the gorges of the craniotomy are thinned by carefully and slowly drilling the bone around in a circle, connecting these four holes. Once the skull at the borders is thin enough, then the bone is lifted with sharp tweezers and removed.The exposed dura mater can be removed or kept intact for implantation of the cranial window. Our cranial window consists of a glass cover slip of 3-5 millimeters in diameter, creating a silicone disc about 300 microns thick at the center. The mounting of the cranial window is accomplished by placing it over the craniotomy.A piece of toothpick attached to a manipulator is used to push the cranial window gently onto the surface of the brain. Then, the edge of the cranial window is sealed with a small amount of silicone adhesive Kwik-Sil. Finally, the cranial window is fixed in the skull edges with dental acrylic.Care should be given for applying the dental acrylic slightly over the edges of the cranial window for strong bonding. As many other research groups have implemented in the past, a 1.2%agarose gel can be used between the cover glass and the brain as an alternative to the silicone disc. After the cranial window is securely installed, a metal plate is attached to the skull.This head plate will be used for fixing the head of the mouse on the stage of a two-photon microscope during imaging sessions. Mitochondrial calcium uptake in astrocytes can be elicited by ATD application while mitochondrial calcium uptake in neurons can be elicited by glutamate glycine application in artificial cerebral spinal fluid, with gravity through a perfusion system. Calcium uptake in individual mitochondria in astrocytes and neurons can then be observed.The following movies show mitochondrial calcium uptake in an astrocyte elicited by ATP on the left and in a neuron elicited by glutamate and glycine on the right. The imaging is done by collecting time-lapsed in vivo two-photon images of mitochondrial fluorescent signals in astrocytes and neurons with an Ultima two-photon microscopy system from Prairie Technologies, which is equipped with a million Ti Sapphire Ultra one laser from Coherent. We use the excitation wavelengths of 880 to 910 nanometers, which are optimal for in vivo imaging with GCaMP5G and 6s.Astrocytes and neuron-specific expression of mito-GCaMP5G and 6s and reconfirmed by co-localization of astrocyte-specific labeling of SR101 or neuron specific marker NeuN. Spontaneous mitochondrial calcium uptake in astrocytes and neurons in vivo can be observed by time-lapse imaging using two-photon microscopy. Images show spontaneous calcium increases in individual mitochondria in an astrocyte on the left and a neuron on the right.Our approaches are useful for cell type specific mitochondrial calcium imaging in vitro and in vivo. After watching this video, you should have a good understanding of how to image mitochondrial calcium uptake in astrocytes and in neurons.

Research

JoVE Journal

Neuroscience

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

In Situ Ca2+ Imaging of the Enteric Nervous System

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...