Name: Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs) (Video) | JoVE Journal
Uploaded: 2022-01-22T14:00:00.000+00:00
Duration: 7 min 46 s
Description: Learn to image mitochondrial calcium uptake in astrocytes and neurons using genetically encoded calcium indicators GCaMP5G and GCaMP6s. This protocol enables cell-type specific in vitro and in vivo studies.

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5 minutes

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 CCGTCCTGAC GCCGCTGCTG CTGCGGGGCT TGACAGGCTC GGCCCGGCGG CTCCCAGTGC CGCGCGCCAA GATCCATTCG TTG17 into the cloning sites EcoRI and BamHI in the backbone of adeno-associated virus (AAV) plasmid pZac2.1 to obtain plasmids containing astrocytic gfaABC1D promoter or neuronal CaMKII promoter18,19,20.</li>
	<li>Subclone GCaMP5G/6s into the cloning sites BamH I and Not 1 in the above plasmids to obtain new plasmids pZac-gfaABC1D-mito-GCaMP5G/6s and pZac-CaMKII-mito-GCaMP5G/6s that target transgene expression in mitochondria in astrocytes and neurons20,21 (Figure 1A).</li>
	<li>Prepare pZac-gfaABC1D-mito-GCaMP5G and pZac-CaMKII-mito-GCaMP6s DNA plasmids for transfection for in vitro study (Section 2). Produce AAV vectors with serotype 5 for astrocytes and serotype 9 for neurons for in vivo study18 (Section 3).</li>
</ol>
2. In vitro mitochondrial Ca2+ imaging in astrocytes and neurons
<ol>
	<li>Prepare primary astrocytes from the cortex of P1 neonatal mice and primary neurons from the cortex of E15-16 embryos18,22,23,24, and culture them on 12 mm diameter glass coverslips in 24-well plates using Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), and neuronal basal medium (NBM) containing 2% B27, respectively.</li>
	<li>Transfect mature astrocytes and neurons with pZac-gfaABC1D-GCaMP6s and pZac-CaMKII-GCaMP5G plasmids using lipid based transfection reagent to express GCaMP6s in the mitochondria of astrocytes and GCaMP5G in the mitochondria of neurons18,20,21. Transfect cells in each well with 0.5 &#181;g of DNA, and change the medium 6 h later. 
	NOTE: The astrocytes and neurons are ready for imaging 1-2 days after transfection.</li>
	<li>Perform in vitro mitochondrial Ca2+ imaging 1-2 days after transfection.
	<ol>
		<li>Transfer the glass coverslips cultured with astrocytes or neurons to the PH-1 perfusion chamber under an epifluorescence or two photon microscope.</li>
		<li>Stimulate astrocytic mitochondrial Ca2+ uptake with 100 &#956;M ATP in ACSF, or stimulate neuronal mitochondria with 100 &#956;M glutamate/10 &#956;M glycine20,25 (Figure 2 and Figure 3). 
		NOTE: Solution changes from ACSF to ATP- and glutamate/glycine-containing ACSF are controlled by an ALA-VM8 perfusion system21. The speed of the solution change is controlled at 1-2 mL/min by adjusting a valve.</li>
	</ol>
	</li>
</ol>
3. In vivo mitochondrial Ca2+ imaging in astrocytes and neurons
<ol>
	<li>AAV preparations.
	<ol>
		<li>Prepare the following recombinant adeno-associated virus (rAAV) vectors using the DNA plasmids prepared in section 1: rAAV2/5-gfaABC1D-mito-GCaMP5G and rAAV2/9-CaMKII-mito-GCaMP6s vectors. 
		NOTE: In this experiment, rAAV vectors of serotype 5 were prepared to express GCaMP5G in mitochondria in astrocytes and rAAV vectors of serotype 9 were prepared to express GCaMP6s in mitochondria in neurons.</li>
	</ol>
	</li>
	<li>Stereotaxic AAV injection.
	<ol>
		<li>Anesthetize the mouse with 3% isoflurane. 
		NOTE: Later during the surgery, the isoflurane levels are reduced to 2%.</li>
		<li>After the mouse reaches a surgical level of anesthesia, as determined by tail and toe pinch, shave the hair over the surgery site, motor or somatosensory cortex, with a hair trimmer.</li>
		<li>Position the mouse on the mouse stereotaxic device and fix the head with ear bars. Apply ophthalmic ointment to the eyes to protect them during the surgery. Use a heating pad to keep the body temperature of the mouse at 37 &#176;C throughout the surgery. 
		NOTE: Perform surgery using aseptic procedures. All surgical tools need to be sterilized either by autoclaving or a hot bead sterilizer.</li>
		<li>After the mouse is mounted on the stereotaxic device, sterilize the scalp with alternating iodine based scrub and 70% ethanol three times. Make an incision in the midline of the scalp to expose the injection site.</li>
		<li>Cut open the skin in the bregma-lamda axis and a create a ~1 mm diameter burr hole with a high speed drill at the intended injection location of the motor or somatosensory cortex.</li>
		<li>Use a 33 G Hamilton syringe containing adeno-associated virus (rAAV2/5-gfaABC1D-mito-GCaMP5G vectors [1 x 1011 GC] or rAAV2/9-CaMKII-mito-GCaMP6s vectors [1 x 1011 GC])&#160;to inject up to 1 &#181;L of virus at the target area. 
		NOTE: For example, for cortical viral delivery, inject the virus solution at two depths in multiple steps. First, insert the needle to a depth of 1 mm from the dura&#160;and allow 5 min for the brain to recover. Then, move the needle up to ~700 &#181;m depth and inject 500 nL of the virus solution at an injection speed of 10 nL/s using a hamilton syringe controlled by a microsyringe pump controller. After the injection is completed, wait for 5 min to allow the virus to diffuse into the brain. Then, move the needle up to the second injection location at a depth of 300 &#181;m. Here, inject an additional 500 nL of the virus solution. Wait for 10 min to allow the virus to diffuse into the brain.</li>
		<li>Close the scalp and the skin using a tissue adhesive. Let the mice recover on the heating pad. Send mice back to the animal facility after recovery.</li>
	</ol>
	</li>
	<li>Cranial-window intstallation and in vivo&#160;two-photon (2-P) imaging of mitochondrial Ca2+ signals. 
	NOTE: Cranial window implantation is done 3 weeks post AAV injection over the motor or somatosensory cortex26,27,28,29. Carprofen (10 mg/kg) is injected&#160;subcutaneously to provide relief from potential pain before surgery. The cranial window surgical procedures are identical to the AAV injection surgical procedures and are performed under aseptic conditions.
	<ol>
		<li>Anesthetize the mouse with 3% isoflurane. 
		NOTE: This is the initial dose and reduce to 2% for the surgery later on. During imaging, an intraperitoneal (IP) injection of 130 mg ketamine/10 mg xylazine/kg body weight dissolved in ACSF is used for anesthesia.</li>
		<li>Position the mouse on the mouse stereotaxic device and fix the head with ear bars. Apply ophthalmic ointment to eyes. Use a heating pad to keep the body temperature of the mouse at 37 &#176;C throughout the surgery. 
		NOTE: All surgical tools need to be sterilized.</li>
		<li>Make an incision of 5-8 mm long in the midline of the scalp and remove a flap of skin using a pair of scissors.</li>
		<li>After the skull is exposed, perform 2.0-3.0 mm diameter craniotomy using a high-speed drill over the virus injected area (i.e., motor cortex or somatosensory cortex, Figure 1B). First, make four small holes, and then drill along in a circle connecting the holes. Then, lift and remove the bone with sharp scissors and remove. The exposed dura mater can be removed or kept intact for impantation of the cranial window.</li>
		<li>Place a glass coverslip of 3-5 mm in diameter carrying a transparent silicone over the craniotomy. Use a toothpick to push the cranial window gently onto the surface of the brain. Then, seal the edge with a small amount of silicone adhesive. 
		NOTES: Alternatively, instead of silicone disk, 1.2% low melting point agarose gel can be used between the cover glass and the brain tissue.</li>
		<li>Finally, seal the edges of the coverslip with dental cement. Take care to apply the cement slightly at the edge of the cranial window for strong bonding. Attach a custom-made metal head plate to the skull with dental cement. 
		NOTE: The metal plate is used to fix the head of the mouse to the stage of 2-P microscope during the imaging session (Figure 1C).</li>
		<li>Add 0.5 mL of ACSF solution over the coverslip on the cranial window for water immersion during imaging.</li>
		<li>Perform time-lapse in vivo 2-P imaging of mito-GCaMP5G in astrocytes and mito-GCaMP6s in neurons with 910 nm wavelength through the cranial window (Figure 1C)18,20,27. 
		NOTE: During imaging, mice are on the heating pad to maintain physiological temperature. The mice will be sacrificed immediately after the imaging session is completed.</li>
		<li>Label astrocytes in vivo with sulforhodamine 101 (SR101) when it is necessary to determine colocalization.
		<ol>
			<li>At the end of step 3.3.4, apply 100 &#181;L of 100 &#181;M SR101 in ACSF on the cortical surface for 1-5 min.</li>
			<li>Rinse the surface with ACSF to wash away the unbound SR101. Using 2-P imaging, co-labeling of mito-GCaMP5G and SR101 in astrocytes can be observed 45-60 min later (Figure 4A).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

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.

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

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 astr...

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 ...

imaging-mitochondrial-ca2-uptake-astrocytes-neurons-using-genetically

Research

JoVE Journal

Neuroscience

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described.  The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

This video describes a confocal microscopy imaging-based assay to measure mitochondrial calcium uptake in cultured cells. The assay uses the calcium-sensitive fluorescent dye Rhod-2/AM to measure the mitochondrial calcium in permeabilized cultured cells.

Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

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

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

This protocol describes in vivo GCaMP calcium imaging of intact trigeminal ganglion (TG) neurons. This description includes TG exposure surgery, in vivo confocal microscopy imaging of TG neurons, application of somatic stimuli during TG neurons microscopic imaging, and analysis of in vivo GCaMP calcium imaging data.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Trigeminal Ganglia

This protocol provides a comprehensive description of a method for the simultaneous recording of calcium signals from neurons and astrocytes across multiple memory-related brain regions and demonstrates its application during a learning and memory behavior paradigm.

A Dual-Color Fiber Photometry Method for Recording Astrocyte-Neuron Activity Across Multiple Brain Regions During Learning and Memory Behaviors

Learn to image mitochondrial calcium uptake in astrocytes and neurons using genetically encoded calcium indicators GCaMP5G and GCaMP6s. This protocol enables cell-type specific in vitro and in vivo studies.

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

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