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⁺ overloading is also a determinant for cell death, including necrosis and apoptosis in various brain disorders^5,6,7. 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 K_d), 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 see^10,11). 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 GECIs^{12,13,14,15,16}. 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.

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 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 promoter^18,19,20.
2. 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 neurons^20,21 (Figure 1A).
3. 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).

2. In vitro mitochondrial Ca²⁺ imaging in astrocytes and neurons

1. Prepare primary astrocytes from the cortex of P1 neonatal mice and primary neurons from the cortex of E15-16 embryos^18,22,23,24, and culture them on 12 mm diameter glass coverslips in 24-well plates using Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), and neuronal basal medium (NBM) containing 2% B27, respectively.
2. 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 neurons^18,20,21. Transfect cells in each well with 0.5 µg of DNA, and change the medium 6 h later.
   NOTE: The astrocytes and neurons are ready for imaging 1-2 days after transfection.
3. Perform in vitro mitochondrial Ca²⁺ imaging 1-2 days after transfection.
   1. Transfer the glass coverslips cultured with astrocytes or neurons to the PH-1 perfusion chamber under an epifluorescence or two photon microscope.
   2. Stimulate astrocytic mitochondrial Ca²⁺ uptake with 100 μM ATP in ACSF, or stimulate neuronal mitochondria with 100 μM glutamate/10 μM glycine^20,25 (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.

3. In vivo mitochondrial Ca²⁺ imaging in astrocytes and neurons

1. AAV preparations.
   1. 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.
2. Stereotaxic AAV injection.
   1. Anesthetize the mouse with 3% isoflurane.
      NOTE: Later during the surgery, the isoflurane levels are reduced to 2%.
   2. 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.
   3. 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 °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.
   4. 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.
   5. 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.
   6. 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]) to inject up to 1 µL 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 and allow 5 min for the brain to recover. Then, move the needle up to ~700 µm 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 µm. Here, inject an additional 500 nL of the virus solution. Wait for 10 min to allow the virus to diffuse into the brain.
   7. 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.
3. Cranial-window intstallation and in vivo 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 cortex^26,27,28,29. Carprofen (10 mg/kg) is injected 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.
   1. 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.
   2. 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 °C throughout the surgery.
      NOTE: All surgical tools need to be sterilized.
   3. 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.
   4. 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.
   5. 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.
   6. 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).
   7. Add 0.5 mL of ACSF solution over the coverslip on the cranial window for water immersion during imaging.
   8. 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.
   9. Label astrocytes in vivo with sulforhodamine 101 (SR101) when it is necessary to determine colocalization.
      1. At the end of step 3.3.4, apply 100 µL of 100 µM SR101 in ACSF on the cortical surface for 1-5 min.
      2. 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).

Results

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...

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