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 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.
Subscription Required. Please recommend JoVE to your librarian.
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.
- 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.
- 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).
- 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
- 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's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), and neuronal basal medium (NBM) containing 2% B27, respectively.
- 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 µg of DNA, and change the medium 6 h later.
NOTE: The astrocytes and neurons are ready for imaging 1-2 days after transfection. - Perform in vitro mitochondrial Ca2+ imaging 1-2 days after transfection.
- Transfer the glass coverslips cultured with astrocytes or neurons to the PH-1 perfusion chamber under an epifluorescence or two photon microscope.
- Stimulate astrocytic mitochondrial Ca2+ uptake with 100 μM ATP in ACSF, or stimulate neuronal mitochondria with 100 μM glutamate/10 μM 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.
3. In vivo mitochondrial Ca2+ imaging in astrocytes and neurons
- AAV preparations.
- 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.
- 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.
- Stereotaxic AAV injection.
- Anesthetize the mouse with 3% isoflurane.
NOTE: Later during the surgery, the isoflurane levels are reduced to 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.
- 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. - 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.
- 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.
- 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. - 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.
- Anesthetize the mouse with 3% isoflurane.
- 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 cortex26,27,28,29. 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.- 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. - 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. - 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.
- 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.
- 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. - 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). - Add 0.5 mL of ACSF solution over the coverslip on the cranial window for water immersion during imaging.
- 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. - Label astrocytes in vivo with sulforhodamine 101 (SR101) when it is necessary to determine colocalization.
- 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.
- 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).
- Anesthetize the mouse with 3% isoflurane.
Subscription Required. Please recommend JoVE to your librarian.
Representative 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 astrocytes can be elicited by ATP application, and mitochondrial Ca2+ uptake in neurons can be elicited by glutamate and glycine application through a perfusion system. Figure 2 and Figure 3 show GCaMP6s is expressed in cultured astrocytes and GCaMP5G in neurons, respectively. Mitochondrial Ca2+ uptake in astrocytes was elicited by 100 μM ATP with single mitochondrial resolution (Figure 2B-D). Mitochondrial Ca2+ uptakes in neurons was elicited by 100 μM glutamate and 10 μM glycine with single mitochondrial resolution (Figure 3B-D).
In vivo 2-P imaging of mitochondrial expression of GCaMP5G or 6S in astrocytes and neurons
The imaging is done by collecting time-lapse in vivo 2-P imaging of mitochondrial fluorescence signals in astrocytes and neurons with an Ultima 2-P microscope system. We use excitation wavelength 880-910 nm. Figure 4 shows expression of GCaMP5G in mitochondria in astrocytes in the mouse cortex. The astrocyte-specific expression of GCaMP5G was confirmed by colocalization of SR101 with GCaMP5G with single mitochondrial resolution (Figure 4A), and spontaneous Ca2+ changes in individual mitochondria can be observed (Figure 4B-E). Figure 5A shows neuron-specific expression of mito-GCaMP6s colocalized with neuronal marker NeuN. The fluorescence of mito-GCaMP6s in neurons shows mitochondrial morphology in dendrites (Figure 5B). Spontaneous mitochondrial Ca2+ increases in dendrites can be observed (Figure 5C-F).
Analysis of mitochondrial Ca2+ signals
Quantify the fluorescent signals by calculating the mean pixel intensities of the cell body or individual mitochondria in astrocytes and neurons using an image analysis software. Ca2+ changes overtime (t) are expressed as F/Fo (t) values versus time, where Fo is the background subtracted baseline fluorescence and F is the baseline subtracted fluorescence change20,21. Use the peak F/Fo values to compare the amplitude of Ca2+ signals.
Figure 1: DNA constructs for astrocyte- and neuron-specific mitochondria-targeting transgene expression, and in vivo 2-P imaging. (A) DNA constructs of genetically encoded Ca2+ indicator GCaMP5G or GcaMP6s in pZac2.1 plasmid with gfaABC1D (up) and CaMKII (low) promoters for delivery to the astrocytic and neuronal mitochondrial matrix, respectively. Mitochondria-targeting is achieved using a mitochondrial matrix (MM) specific sequence (mito) appended to the N-terminus of the fluorescent proteins. (B) A craniotomy over the cortex of a mouse. (C) The skull of a mouse is attached to a metal plate connected to the post fixed on the stage of 2-P microscope. The inset shows the cranial window with a metal plate attached to the skull. Please click here to view a larger version of this figure.
Figure 2: Mitochondrial Ca2+ imaging of cultured astrocytes. (A-B) A 2-P image of an astrocyte expressing mito-GCaMP6s (A) and its response to the stimulation of 100 µM ATP at the indicated time (B). (C) Images of mito-GCaMP6s in the four individual mitochondria (in A, circles) at the different times after ATP stimulation. (D) The time courses of mito-GCaMP6s fluorescence changes, plotted as ΔF/Fo, in the four individual mitochondria after ATP stimulation. The red arrow indicates the starting time of imaging. The pseudocolor scale is a linear representation of the fluorescence intensity in this and other figures. Please click here to view a larger version of this figure.
Figure 3: Mitochondrial Ca2+ imaging of cultured neurons. (A-B) A 2-P image of mito-GCaMP5G expressing neuron (A) and its response to 100 µM glutamate and 10 µM glycine at the indicated time (B). (C) Images of mito-GCaMP5G in the four individual mitochondria (in A, circles) at different times after glutamate stimulation. (D) The time courses of mito-GCaMP5G fluorescence changes in the four individual mitochondria. Please click here to view a larger version of this figure.
Figure 4: In vivo 2-P imaging of mitochondrial Ca2+ signaling in astrocytes. (A) 2-P images of mito-GCaMP5G expressing astrocytes colocalized with SR101. (B-C) 2-P image of an astrocyte expressing mito-GCaMP5G for analysis of spontaneous Ca2+ increase. (C-E) Images of mito-GCaMP5G in the four individual mitochondria in the astrocyte (in B, circles) at the different times (C) and the time courses of mito-GCaMP5G fluorescence changes, plotted as ΔF/Fo, in the four individual mitochondria (E). Please click here to view a larger version of this figure.
Figure 5: In vivo 2-P imaging of mitochondrial Ca2+ signaling in neurons. (A) Colocalization of GCaMP6s (upper) with neuronal marker NeuN (middle) in the brain. (B) High resolution images of dendrites expressing GCaMP6s with mitochondrial morphology (indicated by *s). (C) GCaMP6s expressed in neuronal mitochondria. (D-F) Analysis of spontaneous mitochondrial Ca2+ increase in neurons. Pseudocolor 2-P image of the mitochondria expressing mito-GCaMP6s in C at different times (D). Images of mito-GCaMP6s in the four individual mitochondria in C (circles) at the different times (E-F) and the time courses of mito-GCaMP6s fluorescence changes, plotted as ΔF/Fo, in the four individual mitochondria (F). Please click here to view a larger version of this figure.
Movie: Mitochondrial Ca2+ increases based on GCaMP5G fluorescence changes in response to 100 μM ATP in cultured astrocytes. Please click here to download this Movie.
Subscription Required. Please recommend JoVE to your librarian.
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 expression of GCaMP5G/6s in astrocytes and neurons can be confirmed by SR101 labeling in astrocytes and immunostaining of neurons with NeuN. From our data, these strategies provide a reliable cell type specific approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vivo.
One potential problem for GECI expression is that it might cause Ca2+ buffering since it may reduce free Ca2+ by Ca2+ binding. Another problem that might be paid attention to is the amount of virus injected. Individual cells expressing GECI may not be identified if an excessive virus is injected. These problems can be effectively ameliorated by reducing the titer of AAV. Photobleaching might also be an issue. Theoretically, all fluorescent indicators are subject to photobleaching. GCaMP5G/6s are quite stable, but they will bleach under continual exposure to excitation light. One general practice to avoid photobleaching is to reduce the exposure time of tissue to laser light while ensuring enough fluorescence is collected. This can be achieved if high sensitivity PMTs and high light transmission objectives are used. Photobleaching can also be reduced by closing the shutter between images.
In our results of in vivo 2-P imaging, spontaneous mitochondrial increases can be observed both in astrocytes and neurons. Notably, these mitochondrial Ca2+ transients have long durations (Figure 4E and Figure 5E), consistent with a recent report by Gobel et al.30. The underlying mechanism of this phenomenon is not clear but is worth being pursued further. For cytosolic Ca2+ increase, astrocytes and neurons have different mechanisms. G-protein receptor stimulations cause Ca2+ increase in ER in astrocytes while the activations of voltage gated Ca2+ channels or glutamate receptors cause cytosolic Ca2+ increase in neurons, which can be uptaken by mitochondria. In our previous study20, we found that when mito-GCaMP5G was cotransfected with IP3 5-phosphatase (5ppase) cultured astrocytes, ATP-induced mitochondrial Ca2+ increase could be largely abolished. However, the two mutants of 5ppase, i.e., R343A and R343A/R350A 5ppase, which lack enzymatic activity, did not affect the mitochondrial Ca2+ increase after ATP stimulation. These results indicate that cytosolic and mitochondrial Ca2+ levels are highly coupled, likely because of the intimate physical connection between the ER and mitochondria in astrocytes, with the cytosol serving as an intermediary conduit for Ca2+ delivery. We also found that glutamate stimulation caused mitochondrial Ca2+ increase in neurons, suggesting glutamate receptors play a role in Ca2+ entry from extracellular space. In the future, it will be interesting to study sensory-driven mitochondrial Ca2+ increases in astrocytes and neurons.
Our approach can be used to simultaneously image cytosolic and mitochondrial Ca2+ signals in the same cell type when two GECIs of different fluorescence wavelengths are expressed, e.g., a red florescence GECI RCaMP in cytoplasm and GCaMP in mitochondria, or vice versa31. This approach can also be used for the in vivo study of astrocyte-neuron interactions in physiology and pathology with GCaMP expressed in astrocytes and RCaMP in neurons, or vice versa.
GCaMP is a GFP-based single wavelength fluorophore GECI. Currently, GCaMPs are the most preferred Ca2+ indicators because of their high signal-to-noise ratio (SNR) and large dynamic ranges (DR). Recently, jGCaMP7 sensors, the optimized version of GCaMP6, were reported with improved sensitivity to individual spikes16. GCaMP7 sensors can be easily subcloned in our plasmids for mitochondrial Ca2+ imaging. In summary, the strategies we presented here can be used to image mitochondrial Ca2+ uptake and handling in neurons and astrocytes with sufficient sensitivity to resolve Ca2+ changes at single mitochondrial level in vivo. This protocol represents a useful means to study cytosolic and mitochondrial Ca2+ signaling in astrocytes and neurons, as well as astrocyte-neuron interactions.
Subscription Required. Please recommend JoVE to your librarian.
Disclosures
The authors have nothing to disclose.
Acknowledgments
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.
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 |
References
- Griffiths, E. J., Rutter, G. A. Mitochondrial calcium as a key regulator of mitochondrial ATP production in mammalian cells. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1787 (11), 1324-1333 (2009).
- Pizzo, P., Drago, I., Filadi, R., Pozzan, T. Mitochondrial Ca2+ homeostasis: mechanism, role, and tissue specificities. Pflugers Archiv - European Journal of Physiology. 464 (1), 3-17 (2012).
- Llorente-Folch, I., et al. Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons. The Journal of Neuroscience. 33 (35), 13957-13971 (2013).
- Burkeen, J. F., Womac, A. D., Earnest, D. J., Zoran, M. J. Mitochondrial calcium signaling mediates rhythmic extracellular ATP accumulation in suprachiasmatic nucleus astrocytes. The Journal of Neuroscience. 31 (23), 8432-8440 (2011).
- Duchen, M. Mitochondria, calcium-dependent neuronal death and neurodegenerative disease. Pflugers Archiv - European Journal of Physiology. 464 (1), 111-121 (2012).
- Gouriou, Y., Demaurex, N., Bijlenga, P., De Marchi, U. Mitochondrial calcium handling during ischemia-induced cell death in neurons. Biochimie. 93 (12), 2060-2067 (2011).
- Qiu, J., et al. Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals. Nature Communications. 4, 2034 (2013).
- Filadi, R., Greotti, E. The yin and yang of mitochondrial Ca2+ signaling in cell physiology and pathology. Cell Calcium. 93, 102321 (2021).
- Finkel, T., et al. The ins and outs of mitochondrial calcium. Circulation Research. 116 (11), 1810-1819 (2015).
- Paredes, R. M., Etzler, J. C., Watts, L. T., Zheng, W., Lechleiter, J. D. Chemical calcium indicators. Methods. 46 (3), 143-151 (2008).
- Contreras, L., Drago, I., Zampese, E., Pozzan, T. Mitochondria: The calcium connection. Biochimica et Biophysica Acta (BBA. 1797 (6-7), 607-618 (2010).
- Tian, L., et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nature Methods. 6 (12), 875-881 (2009).
- Yamada, Y., et al. Quantitative comparison of genetically encoded Ca2+ indicators in cortical pyramidal cells and cerebellar Purkinje cells. Frontiers in Cellular Neuroscience. 5, 18 (2011).
- Akerboom, J., et al. Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging. The Journal of Neuroscience. 32 (40), 13819-13840 (2012).
- Chen, T. W., et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. 499 (7458), 295-300 (2013).
- Dana, H., et al. High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nature Methods. 16 (7), 649-657 (2019).
- Rizzuto, R., Brini, M., Pizzo, P., Murgia, M., Pozzan, T. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Current Biology: CB. 5 (6), 635-642 (1995).
- Xie, Y., Wang, T., Sun, G. Y., Ding, S. Specific disruption of astrocytic Ca2+ signaling pathway in vivo by adeno-associated viral transduction. Neuroscience. 170 (4), 992-1003 (2010).
- Lee, Y., et al. GFAP promoter elements required for region-specific and astrocyte-specific expression. Glia. 56 (5), 481-493 (2008).
- Li, H., et al. Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: Mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store. Cell Calcium. 56 (6), 457-466 (2014).
- Zhang, N., Ding, S. Imaging of mitochondrial and cytosolic Ca2+ signals in cultured astrocytes. Current Protocols in Neuroscience. 82, 1-11 (2018).
- Bi, J., Li, H., Ye, S. Q., Ding, S. 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. Journal of Neurochemistry. 120 (2), 334-346 (2012).
- Wang, X., Li, H., Ding, S. Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia. Scientific Reports. 6, 32416 (2016).
- Wang, X., et al. Subcellular NAMPT-mediated NAD+ salvage pathways and their roles in bioenergetics and neuronal protection after ischemic injury. Journal of Neurochemistry. 151 (6), 732-748 (2019).
- Xie, Y., Chen, S., Wu, Y., Murphy, T. H. Prolonged deficits in parvalbumin neuron stimulation-evoked network activity despite recovery of dendritic structure and excitability in the somatosensory cortex following global ischemia in mice. The Journal of Neuroscience. 34 (45), 14890-14900 (2014).
- Ding, S. In vivo imaging of Ca2+ signaling in astrocytes using two-photon laser scanning fluorescent microscopy in Astrocytes. Milner, R. , Humana Press. 545-554 (2012).
- Li, H., et al. Disruption of IP3R2-mediated Ca2+ signaling pathway in astrocytes ameliorates neuronal death and brain damage while reducing behavioral deficits after focal ischemic stroke. Cell Calcium. 58 (6), 565-576 (2015).
- Ding, S., et al. Photothrombosis ischemia stimulates a sustained astrocytic Ca2+ signaling in vivo. Glia. 57 (7), 767-776 (2009).
- Ding, S., et al. Enhanced astrocytic Ca2+ signals contribute to neuronal excitotoxicity after status epilepticus. The Journal of Neuroscience. 27 (40), 10674-10684 (2007).
- Gobel, J., et al. Mitochondria-endoplasmic reticulum contacts in reactive astrocytes promote vascular remodeling. Cell Metabolism. 31 (4), 791-808 (2020).
- Diaz-Garcia, C. M., et al. The distinct roles of calcium in rapid control of neuronal glycolysis and the tricarboxylic acid cycle. eLife. 10, 64821 (2021).
