Study of Calcium Signaling in Astrocytes with a novel Endoplasmic Reticulum Targeted GCaMP Sensor

Abstract

The endoplasmic reticulum (ER), the major organelle for the storage of Ca²⁺, maintains a concentration of Ca²⁺ much higher than the cytosol or other subcellular organelles such as the mitochondria. A variety of tools have been developed for measuring Ca²⁺ activity in neuronal and glial cells but most of these sensors target either the plasma membrane (PM) or the cytosol. Though these sensors are important in measuring Ca²⁺ transients they lack the capability to measure activity in the periphery of the endoplasmic reticulum or to measure low amplitude events resulting from Ca²⁺ exchange between the endoplasmic reticulum and other organelles such as the mitochondria. We recently developed an endoplasmic reticulum targeted GCaMP6f which is anchored to the cytosolic side of the endoplasmic reticulum and can measure minute calcium exchange occurring in this region. In this protocol, we discuss detailed methods to characterize the ER-GCaMP6f sensor, utilize it for calcium imaging in cultured astrocytes, and for its expression and calcium imaging in astrocytes in rodent brains.

Basic Protocol 1 : Expression and Characterization of ER-GCaMP6f

Support Protocol 1: ER-GCaMP6f Cell Line generation

Basic Protocol 2: In vitro Calcium Imaging with ER-GCaMP6f

Support Protocol 2: Imaging of Drug-induced Calcium Activity

Alternate Protocol 1: Transduction of astrocytes with ER-GCaMP6f AAV

Alternate Protocol 2: Calcium imaging of astrocytes with Fluo-4 AM

Basic Protocol 3: In vivo ER-GCaMP6f Expression and Slice Calcium Imaging

Support Protocol 3: Pharmacological studies with 2-APB in Brain Slices

INTRODUCTION:

Ca²⁺ is a major second messenger in intracellular signaling and regulates interactions between neurons and glial cells (Perea & Araque, 2005; Semyanov, 2019). Understanding Ca²⁺ activity is important to evaluating normal brain function as well as brain activity under pathological conditions such as neurodegenerative diseases and substance use disorders (S. P. Aryal et al., 2021; Galloway et al., 2018). One of the primary ways to study cellular calcium activity is through the use of calcium sensitive organic dyes (Russell, 2011). These dyes alter their fluorescent properties when bound with Ca²⁺. Some dyes such as fluo-4 have several fold increases in fluorescence intensity after binding with Ca²⁺ (Gee et al., 2000) while other dyes such as fura-2 have a shift in their fluorescence absorption spectra upon binding (Venkataraman, Holcomb, Harder, Knollmann, & Baudenbacher, 2012). The later class of compounds is typically used as a ratiometric sensor. Discovery of these dyes has advanced calcium imaging especially for in vitro or cell culture studies, but cell specific targeting and in vivo applications have been challenging with these types of biosensors.

To overcome these challenges, genetically encoded calcium sensors such as GCaMP were developed. GCaMP consists of a circularly permuted green fluorescent protein (cpeGFP), a calcium binding protein calmodulin and M13 light chain kinase (Nakai, Ohkura, & Imoto, 2001). These biosensors can be targeted genetically, and the use of cell specific promoters allows researchers to express the sensor in specific cell types to perform Ca²⁺ measurements in vivo. Although various GCaMP sensors have been developed to study astrocyte calcium activity, most of these sensors target either the plasma membrane or reside in the cytosol. Some localized sensors have been developed to target the lumen of the ER and have shown distinct dynamics for Ca²⁺ activity as compared to cytosolic sensors (Suzuki et al., 2014). We recently developed a GCaMP6f sensor which is anchored to the cytosolic side of the endoplasmic reticulum and can measure minute calcium changes occurring in astrocytes(S. P. Aryal et al., 2022). This sensor adds a new method for registering calcium activity in specific subcellular regions(S. P. Aryal et al., 2022).

This protocol provides detailed steps on the application of the ER-GCaMP6f sensor (Figure 1). This sensor is available in plasmid form to express it in vitro using transfection and in adeno associated viral vector form with an astrocyte specific promotor for expression in live animals as well as to specifically transduce astrocytes in vitro. The first part of the protocol discusses expression of the ER-GCaMP6f sensor in HEK-293 cells and its characterization to verify subcellular localization in the endoplasmic reticulum using fluorescence microscopy. The second protocol focuses on expression of ER-GCaMP6f in astrocytes for in vitro calcium measurements in astrocytes. The third protocol illustrates in vivo expression to perform calcium imaging in brain slices using the ER-GCaMP6f sensor. Alternatively, we also include protocols for transducing astrocytes in vitro and utilizing a calcium dye (Fluo-4 AM) for studying signaling in astrocytes in vitro.

Figure 1:

Schematic of the construction and expression of ER-GCaMP6f to image calcium activity in astrocyte in vitro and in slices.

CAUTION: Proper sterilization techniques should be employed to make all equipment and reagents sterile. All experiments discussed in this publication were conducted under the guidelines of National Institutes of Health approved by University of Kentucky's Institutional Animal Care and Use Committee

BASIC PROTOCOL 1 Expression and Characterization of ER-GCaMP6f

Introductory paragraph:

This protocol discusses processes to express and characterize ER-GCaMP6f in HEK-293 cells. HEK-293 cells are standard cell lines for plasmid expression and viral particle production(Chen, Keiser, & Davidson, 2018). ER-GCaMP6f is available with a CMV promotor which allows us to express it in HEK-293 cells as well as other mammalian cells(S. P. Aryal et al., 2022). The first part of the protocol discusses how to culture and maintain HEK-293 cells. The next step discusses transfection of HEK-293 cells to express the ER-GCaMP6f gene. Finally, we also discuss the steps for fluorescence imaging of ER-GCaMP6f expressing HEK-293 cells using epifluorescence, total internal reflection fluorescence (TIRF) and structured illumination microscopy (SIM).

Materials:

• HEK-293 cells (Catalog no. CRL-1573, ATCC)

• HEK-293 cell culture media (see Reagents and Solutions)

• 1% Cultrex (see Reagents and Solutions)

• T-75 Flask (Catalog no. BK369292, VWR International)

• Glass bottom dishes (35 mm, #1.5, MATTEK )

• 1X PBS ( Catalog no. 75800-988, VWR International)

• ER-GCaMP6f plasmid (Plasmid #182548, Addgene)

• Invitrogen™ Lipofectamine™ 2000 Transfection Reagent (Catalog no. 11668027, Fischer Scientific)

• ER-marker (ER-TrackerTM Red (BODIPY™ TR Glipalamide), ThermoFisher Scientific, E34250)

• Opti-MEM (Catalog no. 31-985-062, Fischer Scientific)

• 4% PFA (Catalog no. AAJ61899AP, Fischer Scientific)

• DMSO (Catalog no. 97061-250, VWR International)

• −20° C Freezer

• −80° C Freezer

• Cryogenic Freezer

• L-15 media (Gibco™ Leibovitz's L-15 Medium, no phenol red (Fischer Scientific, 21-083-027))

• TryplE (Gibco™ TrypLE™ Express Enzyme (1X), no phenol red (Fischer Scientific, 12-604-013))

• Hemocytometer (Catalog no. 15170-089, VWR International)

• Cryogenic vials (Catalog no. 10018-738, VWR International)

• Biosafety Cabinet

• Incubator

• A TIRF-capable microscope (Olympus, model: IX2-ZDC2, serial number: 0F83094)

• A structured illumination capable fluorescence microscope (Nikon, model: N-SIM-S)

Protocol steps with step annotations:

Culturing of HEK-293 Cells

1. Warm up 1% Cultrex, PBS and HEK-293 Cell culture media at 37° C for 1 hour.

2. Plate 1% Cultrex to a T75 flask and incubate for 20 minutes. Remove the Cultrex and rinse the flask with PBS 2 times.

   3-4 ml of 1% Cultrex can be added to a T75 flask.

3. Thaw a vial of HEK-293 cells using warm water and transfer it to the flask coated with 1% Cultrex. Add 10 ml of HEK-293 media and incubate it in the incubator.

   The cells should be checked every day with a microscope. The cell culture media should be changed every 3-days and splitting should be performed whenever cells are approximately 80% confluent.

Subculturing of HEK-293 cells

• 4.Prewarm TrypLE, PBS, 1% Cultrex and HEK-293 cell culture media.
• 5.Coat 1% Cultrex to the glass part of the glass bottom dish and incubate for 20 minutes. 200 μl of 1% Cultrex is enough to cover the glass portion of the dish. A larger volume of 1% Cultrex can be used if needed.
• 6.Detach the ~80% confluent HEK-293 cells from a T-75 flask using prewarmed TrypLE (3 ml per flask). Add 7 ml of HEK-293 cell culture media and count the cells using a hemocytometer.
• 7.Remove the glass bottom dishes from the incubator and rinse them with PBS 2 times. Plate approximately 50,000 cells in the glass portion of the glass bottom dish (~200 μl media just to cover the glass). Add 2 ml of media to the glass bottom dish after 6 hours.
• 8.Check the cells every day with a microscope. They become confluent in 1-2 days. After the cells reach ~70-80 % confluency, prepare reagents needed for transfection.

  Note: For transfection, follow step 14 directly after step 8. The freezing steps outlined in steps 9-13 should be done only when cells need to be frozen for future use.

Freezing of HEK-293 Cells

• 9.Count the number of cells using a hemocytometer.
• 10.Dilute the cell concentration to 10⁶-10⁷ cells/ml and add 10% DMSO. Note: If the cell concentration at the beginning is lower than 10⁶ cells/ml, concentrate the cells by removing the cell culture media after centrifugation.
• 11.Transfer the cells into cryogenic vials and incubate at −20° C for 2 hours.
• 12.Incubate the cells at −80° C for overnight.
• 13.Transfer the cell containing cryogenic vials into cryogenic freezer (liquid N2 container) and store until needed.

Transfection of HEK-293 Cells

• 14.Incubate Lipofectamine-2000 and the ER-GCaMP6f DNA (with CMV promotor) on ice. (Figure 2)
• 15.Incubate the HEK-293 cell culture media, PBS, and Opti-MEM at 37° C.
• 16.Replace the HEK-293 media in glass bottom dish with 1 ml of Opti-MEM after rinsing 2 times with 1 ml PBS.
• 17.Incubate the glass bottom dishes containing Opti-MEM in the incubator.
• 18.Transfer 250 μl of Opti-MEM to an Eppendorf tube and add 2 μl of Lipofetamine-2000. This volume is for single dish, increase the volume accordingly for more dishes.
• 19.Transfer 250 μl of Opti-MEM to another Eppendorf tube and add 500 ng of ER-GCaMP6f DNA. The volume and concentration provided are for single dish, please increase the volume accordingly for additional dishes.
• 20.Mix the DNA with Opti-MEM with gentle shaking . Do not vortex.
• 21.Mix the Lipofectamine-2000 with Opti-MEM with gentle shaking. Do not vortex.
• 22.After 5 minutes transfer the DNA in Opti-MEM to the tube containing Lipofectamine-2000 with Opti-MEM and mix them together with gentle shaking. Do not vortex. The mixture should stay there for exactly 25 minutes.
• 23.After 25 minutes, add the mixture to the center of the glass bottom dish containing Opti-MEM.
• 24.Put the dish back in incubator and replace the Opti-MEM media with HEK-293 cell culture media after 20-24 hours of transfection.
• 25.Check the cells every day to visually inspect whether there is any immune reaction or cell death. Wait 1-2 days for gene expression.
• 26.After 2 days of transfection, check the cells in a fluorescence microscope for transient ER-GCaMP6f expression.
• 27.Remove the media from glass bottom dishes. Rinse with PBS 2 times and add 1 μM of ER marker to the glass bottom dish.

  A 1 mM stock of ER marker solution can be made by dissolving the solid dye in 110 μl DMSO. 1 μl stock solution per ml of cell culture media can be used for labelling.

• 28.Incubate the cells at 37° C for 30 minutes.
• 29.Remove the ER marker solution and rinse the dishes with PBS 3 times.

Figure 2:

Genetic map of ER-GCaMP6f. A) Genetic map of ER-GCaMP6f containing CMV promotor. B) Genetic map of ER-GCaMP6f containing astrocyte specific GFAP promotor.

Epifluorescence Imaging of ER-GCaMP6f

• 30.Prewarm PBS and L-15 media at 37° C.
• 31.Remove the Opti-MEM media from the cells and rinse with PBS 2 times.
• 32.Add 1 ml of prewarmed L-15 media to the cells
• 33.Turn on the microscope and accompanying software.
• 34.Turn on the 488 nm laser (for ER-GCaMP6f) and 561 laser (for ER marker) and set up the microscope components to initiate the imaging process.
• 35.For oil objectives, put a drop of oil on top of the objective and place the glass bottom dish.
• 36.Find cells by focusing the microscope by progressively moving the objective closer to the glass bottom dish using bright field illumination. Be careful not to hit the objective against the sample dish.
• 37.After bringing the cells into focus, switch to fluorescence by turning the switch on. Use a 488 nm laser for ER-GCaMP6f and a 561 nm laser for a red emitting ER marker.
• 38.Make sure the camera temperature is ~−80° C and turn of the lights. This is specific for an EMCCD and specific cameras will have different settings.

  Imaging is done at room temperature, but the camera needs to be internally cooled at ~−80° C.

• 39.Direct the emission to the camera and go to live mode in the accompanying software.
• 40.Focus to get the best possible view, collect images and movies.

TIRF Imaging of ER-GCaMP6f

• 41.Turn the microscope on and set up as described above.

  Adjust the laser to the TIRF angle.

  For TIRF imaging, find the appropriate TIRF angle with respect to the incident laser light. Depending upon the microscope settings, TIRF can be achieved in different ways. One approach is to translate a tightly focused beam across the back aperture of the high NA objective which results in a change in the laser angle such that it can reach a critical angle beyond which total internal reflection is achieved. During this, the only portion of the cell that will be illuminated will be a small region within ~150 nm of the glass surface. Note: Depending on the microscope, the TIRF angle might be slightly different for 488 and 561 nm lasers, so it should be adjusted accordingly. Please follow the following publication for TIRF adjustment ((S. Aryal, Fu, Masud, Neupane, & Richards, 2021)

• 42.Focus the cells with the help of focus knob to get the best possible view.
• 43.Perform TIRF imaging of live cells.

  TIRF imaging of fixed cells can also be performed in a similar way.

• 44.Choose the appropriate power and exposure time.

  For a widefield set up it is reasonable to start with a laser power of 3 mW/cm² (for TIRF) and 1 mW/cm² for epifluorescence. Exposure time can be initially set to 100-200 ms and adjusted based on the brightness of the sample.

• 45.Collect images and construct a movie.

Fixation and Structured Illumination Microscopy (SIM)

• 46.Prewarm PBS and 4% PFA at 37° C
• 47.Remove the cell culture media from the glass bottom dish and rinse the dish with PBS 2 times.
• 48.Add 1 ml of 4% PFA and incubate at room temperature for 20 minutes.
• 49.Discard the PFA and rinse the dishes with PBS 3 times. Add 1 ml of PBS to each dish.
• 50.Turn on the SIM system and accompanying software. Make sure 488 nm and 561 nm lasers are on.
• 51.Put oil on the oil objective. 100X oil objective can be used for 2D and 3D SIM experiments.
• 52.Place the glass bottom dish containing the labelled cells and find focus using eyepiece.
• 53.Select range and wavelength for image collection. Approximately 15-20 μm range can be selected for 3D image collection.
• 54.Process the image by using the SIM processing module and save the file in a directory. Figure 3 illustrates an example of SIM image.

Figure 3:

Illustration of ER-GCaMP6f expression at the endoplasmic reticulum. 3-dimensional structured illumination microscopy image of HEK-293 cells transfected with ER-GCaMP6f and labelled with an ER marker showing A) ER-GCaMP6f fluorescence, B) ER marker fluorescence and C) merged image of A and B (scalebar = 10 μm).

Support Protocol 1 ER-GCaMP6f Cell Line Generation

Introductory paragraph

Transient transfection of HEK-293 cells can be utilized for protein studies with specific gene expression. However, most of the cells do not integrate the plasmid in their genomic DNA so the gene expression does not last for long periods of time(Büssow, 2015). It is believed that approximately 0.01% of the cells integrate the plasmid DNA to their genome during transfection (Mortensen, Chestnut, Hoeffler, & Kingston, 1997). Under selection, these cells continuously express the gene even after dividing. To make a stable cell line, the cells which do not express the DNA or transiently express the DNA should be selectively removed. A selection reagent can be used for generating a stable cell line. In this protocol, we discuss how to generate a stable cell line from transiently transfected cells by adding a selectable marker.

Materials

• HEK-293 cells( Catalog no. CRL-1573, ATCC)

• 1% Cultrex (See Reagents and Solutions)

• Geneticin/G418 (Catalog no. 97064-358, VWR International)

• 24-well plate (Catalog no. 30623-116, VWR International)

• 96-well plate (Catalog no. MSPP-P9615HN, VWR International)

• 6-well plate (Catalog no. 30623-114, VWR International)

• T-75 flask (Catalog no. BK369292, VWR International)

• TryplE (Gibco™ TrypLE™ Express Enzyme (1X), no phenol red (Fischer Scientific, 12-604-013)

• L-15 media (Gibco™ Leibovitz's L-15 Medium, no phenol red (Fischer Scientific, 21-083-027))

• DMSO (Catalog no. 97061-250, VWR International)

• −20° C Freezer

• −80° C Freezer

• Cryogenic Freezer

Protocol steps with step annotations:

Generation of kill curve

1. Prewarm 1% Cultrex, HEK-293 cell culture media, and PBS.

2. Coat a 24-well plate with 1% Cultrex and plate approximately 50,000 cells in each well in a 24-well plate with 0.5 ml of cell culture media.

3. Let the cells grow for 24 hours reaching confluency of around 60-70%.

4. Prepare a solution of the appropriate selection marker (e.g., G418 for pcDNA 3.1) in cell culture media with increasing concentration of the compound. For example, 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 μg/ml of G418 solution can be made and added to the 24-well plate.

5. Replace the selection media every 2 days with the same concentration. Visually inspect the cells during the media change and determine what minimum concentration is required for cell death.

6. Continue the media change and inspection for a week.

7. Determine the low, high, and optimal dose required for cell death on the 7^th day. The optimal dose is the minimum dose of the selection marker at which all the cells are dead after 7 days of treatment. The low dose is the dose which is lower than the optimal dose but is still able to kill a significant number of cells (~90%) on the 7^th day. The high dose is the dose which is well above the optimal dose and kills all the cells in 3-4 days of treatment.

Transient transfection and antibiotic selection

• 8.Prewarm all the required media.
• 9.Coat three T75 flasks with 1% Cultrex and plate approximately 10⁶ cells per flask.
• 10.Transfect the flask with 3 μg of plasmid per T75 flask and let the cells grow up to 3 days without adding any antibiotic.

  The ER-GCaMP6f plasmid has neomycin resistance and G418/Geneticin can be used as a selection marker.

• 11.After 3 days, add 3 different concentrations of antibiotic per flask (optimal, low, and high concentrations).
• 12.Observe the cell morphology/cell death every day. Change the media every 3 days. After 10-12 days of transfection (~7 days of antibiotic selection), all the untransfected or transiently transfected cells will be dead.

Generation of monoclonal cells

• 13.Coat a 96-well plate with 1% Cultrex.
• 14.Split the stably transfected cells and plate 1 cell per well in 96 well plates.
• 15.Check and count the number of cells per well after 18-24 hours of plating. Mark the wells which have only one cell and use them for further examination on a daily basis.
• 16.Observe the number of cells per plate after 2-3 days of culture.
• 17.When the cells become confluent which originally have only one cell, split the cells and culture them in a 24 well plate
• 18.Check the expression of gene by measuring fluorescence. All the cells should have GCaMP6f fluorescence. If some cells do not have GCaMP6f fluorescence, repeat the step 13-17 until all the cells express GCaMP6f.
• 19.Once the cells are monoclonal and confluent, plate them in 6 well plate.
• 20.Check the fluorescence from 6 well plates and if all the cells are fluorescent plate them in a T75 flask. If some cells are not fluorescent, split the cells and repeat step 13-17
• 21.Let the cells grow in T75 flask for 2-3 days and split the cells and freeze the cells using 10% DMSO. Perform serial freezing at −20° C and −80° C and transfer to cryogenic container.

Culture of monoclonal cells

• 22.Thaw a vial of ER-GCaMP6f stable cells.
• 23.Grow the cells in 1% Cultrex coated flask with HEK-293 media for 2 days.
• 24.Add low concentration of G418 and observe cell death.
• 25.Perform experiments. Occasional testing should be done to make sure no cells are growing without ER-GCaMP6f expression.

BASIC PROTOCOL 2 In Vitro Calcium Imaging with ER-GCaMP6f

Introductory Paragraph:

To express ER-GCaMP6f in cell culture, the plasmid containing a CMV promotor can be used while AAV viral particles generated from the viral construct can be used for in vivo expression in rodent astrocytes. The AAV particles can also be used to transduce astrocytes in culture for robust ER-GCaMP6f expression. In this protocol we discuss how to express ER-GCaMP6f in astrocytes, verify the expression, and measure the calcium activity. First, we discuss culturing of astrocytes to express ER-GCaMP6f gene using transfection. We then discuss how to perform imaging of ER-GCaMP6f expressing astrocytes using epifluorescence and total internal reflection fluorescence (TIRF) microscopy. Epifluorescence microscopy is used for whole cell imaging, but it suffers from high background. On the other hand, TIRF microscopy allows for the imaging of a highly localized region that encompasses the plasma membrane and the peripheral ER in close proximity to the plasma membrane. We further discuss how to study fixed cells labelled with ER-marker by using structured illumination microscopy (SIM) and TIRF microscopy.

Materials:

• Four P2-P4 pups from C57BL6 mice

• 70% ethanol

• Kim wipe (Product no. Z188964-1PAK, Sigma-Aldrich)

• Surgical scissors (Catalog no. MDS0838410, Medline)

• Smooth fine forceps (Catalog no. 72911-6, Electron Microscopy Sciences)Petri dish (Catalog no. 89090-270, VWR International)

• Cold Hanks Balanced Salt Solution HBSS (Catalog no. VWRL0121-0500, VWR International)

• Ice

• 2.5 % trypsin (Catalog no. 10128-782, VWR International)

• 0.1 % Poly-l-lysine (PLL) solution (Catalog no. 100496-610, VWR International)

• Astrocyte culture media (see Reagents and Solutions)

• T-75 Flask (Catalog no. BK369292, VWR International)

• Glass bottom dishes (35 mm, #1.5, MATTEK )

• TrypLE (Gibco™ TrypLE™ Express Enzyme (1X), no phenol red (Catalog no. 12-604-013, Fischer Scientific))

• 1X PBS (Catalog no. 75800-988, VWR International)

• Shaker/shaking incubator (Catalog no. 11-675-209, Fischer Scientific)

• ER-GCaMP6f plasmid (Plasmid #182548, Addgene)

• ER-GCaMP6f AAV (Plasmid #182501, Addgene)

• L-15 (Gibco™ Leibovitz's L-15 Medium, no phenol red (Fischer Scientific, 21-083-027))

• A TIRF-capable microscope (Olympus, model: IX2-ZDC2, serial number: 0F83094)

• A structured illumination capable fluorescence microscope (Nikon, model: N-SIM-S)

Protocol steps with step annotations:

Astrocyte Culture

1. Place Hanks Balanced Salt Solution (HBSS) on ice.

2. Collect four P2-P4 pups from C57BL6 mice.

   Four pups are generally enough to generate one T-75 flask of astrocytes.

3. Spray 70% ethanol around neck and wipe with Kim wipe.

4. Decapitate the heads and collect them in a Petri dish.

5. Remove the skin from the head.

6. Remove the skull and transfer the brain to a Petri dish containing cold HBSS solution.

7. Remove the midbrain and striatum from the brain and isolate the cortices from top. Discard the tissue containing the midbrain and striatum and collect cortical sections in a Petri dish containing cold HBSS.

   This article illustrates the isolation and culture of astrocytes ((Schildge, Bohrer, Beck, & Schachtrup, 2013)

8. Homogenize the cortices by chopping the tissue into small pieces with the help of forceps and/or small blade(Ioannou, Liu, & Lippincott-Schwartz, 2019). Bring the total volume to 22.5 ml by adding cold HBSS solution.

9. Add 2.5 ml of 2.5% trypsin and incubate at 37° C for 30 minutes.

10. Coat a T75 flask with 3 ml of poly-l-lysine (PLL) and incubate the flask in an incubator for 30 minutes. Remove the PLL and rinse the flask with PBS 2 times.

11. Pipette in and out the tissue trypsin homogenate several times to make a single cell suspension.

12. Centrifuge the suspension and discard the supernatant.

13. Transfer the cell suspension to the PLL coated flask and add 10 ml of astrocyte culture media.

14. Visualize the cells every day with the help of a microscope.

    After 5-6 days a mixed glial cell culture will be confluent.

15. Rotate the flask at 180 RPM for 30 minute and microglia will be detached. Remove the microglia and rinse the flask with PBS 3 times.

16. Add 10 ml of astrocyte culture media and rotate the flask at 240 RPM for 6 hours. Oligodendrocytes will be detached and remove them by washing with PBS 3-4 times.

17. Add 10 ml of astrocyte culture media and incubate the cells in an incubator.

18. After they become 70-80% confluent, plate them in glass bottom dishes as described in previous sections.

    The purity of astrocytes can be assessed using astrocyte and microglia specific antibodies such as anti-GFAP (for astrocytes) and anti-IBA1 (for microglia)

Subculturing of astrocytes

• 19.Prewarm TrypLE, PBS, 1% Cultrex and astrocyte cell culture media.
• 20.Coat 1% Cultrex to the glass part of the glass bottom dish and incubate for 20 minutes. 200 μl of 1% Cultrex is enough to cover the glass portion of the dish. A larger volume of 1% Cultrex can be used if needed.
• 21.Detach the approximately 70-80% confluent astrocytes from a T-75 flask using prewarmed TrypLE (3 ml per flask). Add 7 ml of astrocyte cell culture media and count the cells using hemocytometer.
• 22.Remove the glass bottom dishes from the incubator and rinse them with PBS 2 times. Plate approximately 50,000 astrocytes in the glass portion of the glass bottom dish (~200 μl media just to cover the glass). Add 2 ml of media to the glass bottom dish after 6 hours.
• 23.Check the cells every day with a microscope. They become confluent in 4-5 days. After the cells reach ~70-80 % confluency, prepare reagents needed for transfection.

Transfection of astrocytes

• 24.Incubate Lipofectamine-2000 and the ER-GCaMP6f DNA (with CMV promotor) in ice.
• 25.Incubate the astrocyte cell culture media, PBS, Opti-MEM at 37° C.
• 26.Replace the astrocyte media in the glass bottom dish with 500 μl of Opti-MEM after rinsing 2 times with Opti-MEM.
• 27.Incubate the glass bottom dishes containing Opti-MEM in the incubator.
• 28.Transfer 250 μl of Opti-MEM to an Eppendorf tube and add 2 μl of Lipofetamine-2000.
• 29.Transfer 250 μl of Opti-MEM to another Eppendorf tube and add 200 ng of ER-GCaMP6f DNA.
• 30.Mix the DNA with Opti-MEM with gentle shaking. Do not vortex.
• 31.Mix the Lipofectamine-2000 with Opti-MEM with gentle shaking. Do not vortex.
• 32.After 5 minutes transfer the DNA in Opti-MEM to the tube containing Lipofectamine-2000 and mix them together with gentle shaking. Do not vortex. The mixture should stay there for 25 minutes.
• 33.After 25 minutes, add the mixture to the glass bottom dish containing Opti-MEM.
• 34.Put the dish back in incubator and wait 1-2 days for gene expression.
• 35.Check the cells every day to visually inspect whether there is any immune reaction or cell death.
• 36.After 24-48 hours of transfection, check the cells in a fluorescence microscope for transient ER-GCaMP6f expression.

Calcium imaging of ER-GCamP6f in astrocytes

• 37.Prewarm L-15 media and replace the astrocyte cell culture media in the glass bottom dishes with L-15 media.
• 38.Turn on the microscope and accompanying software.
• 39.Put oil on 60X oil objective and place glass bottom dish on top.
• 40.Focus the cells using focus knob and use 488 nm laser to excite the GCaMP6f fluorescence.
• 41.Choose the laser power around 1 mw/cm^2.
• 42.Choose 200 ms exposure time and collect images at an interval of 0.5 seconds.
• 43.Stack the images using imageJ/FIJI to create movies.

  ImageJ/FIJI is an open-source software available at https://imagej.net/software/fiji/

  Figure 4 illustrates calcium activity in astrocytes measured using ER-GCaMP6f.

Figure 4:

ER-GCaMP6f calcium activity in astrocytes measured with an epifluorescence microscope. A) Sample calcium activity in astrocytic cell soma and processes expressing ER-GCaMP6 in different time interval from a widefield microscope. Scalebar = 40 um. B) Calcium activity in astrocytic cell soma C) Calcium activity in astrocytic processes. Reprinted with permission from {Aryal, S. P., Xia, M., Adindu, E., Davis, C., Ortinski, P. I., & Richards, C. I. (2022). ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes. Analytical Chemistry}. Copyright {2022} American Chemical Society.

TIRF microscopy of ER-GCaMP6f

• 44.For TIRF imaging, find the appropriate TIRF angle with respect to the incident laser light.

  Depending upon the microscope settings, TIRF can be achieved in different ways. One approach is to translate a tightly focused beam across the back aperture of the high NA objective which results in a change in the laser angle such that it can reach a critical angle beyond which total internal reflection is achieved. During this, the only portion of the cell that will be illuminated will be a small region within ~150 nm of the glass surface.

• 45.Choose the appropriate power and exposure time.

  For a widefield set up it is reasonable to start with a laser power of 3 mW/cm² (for TIRF) and 1 mW/cm² for epifluorescence. Exposure time can be initially set to 100-200 ms and adjusted based on the brightness of the sample.

• 46.Collect images and construct a movie.

  Figure 5 illustrates calcium activity in astrocytes measured by using ER-GCaMP6f.

  For data analysis, see the protocol below from step 58.

Figure 5:

Illustration of the measurement of low-amplitude calcium activity in astrocytic processes combining TIRF microscopy with ER-GCaMP6. A) Calcium activity measured in ER-GCaMP6f expressing astrocytes with widefield microscopy. B) Calcium activity measured in the same cell with TIRF microscopy. Reprinted with permission from {Aryal, S. P., Xia, M., Adindu, E., Davis, C., Ortinski, P. I., & Richards, C. I. (2022). ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes. Analytical Chemistry}. Copyright {2022} American Chemical Society.

Staining with ER marker

• 47.Add 1 μM of ER-marker dye on ER-GCaMP6f expressing astrocytes.
• 48.Incubate the cells at 37° C for 30 minutes.
• 49.Remove the ER-marker media and rinse astrocytes with PBS 3 times.
• 50.Add 1 ml of L-15 media and perform imaging.

Fixing of astrocytes and SIM imaging

• 51.Remove the cell culture media.
• 52.Rinse the cells with PBS three times.
• 53.Add 1 ml of 4% PFA and incubate the cells at room temperature for 20 minutes.
• 54.Remove the 4% PFA and rinse the cells with PBS three times.
• 55.Add 1 ml of PBS and store the cells in fridge at 4° C until imaging.
• 56.Image the cells using a super-resolution microscope.
• 57.Choose both 488 nm and 561 nm lasers for imaging both ER-GCaMP6f and ER-marker fluorescence.

  Here is a good reference on comparing different super resolution microscopy techniques (Thorley, Pike, & Rappoport, 2014).

Data analysis of ER-GCaMP6f calcium imaging data

• 58.Convert the movie files into “TIFF” file format. Original file should be kept in TIFF format as changing the file format can cause a loss of data.
• 59.Open the movie file with imageJ/FIJI software.
• 60.From the menu, select Image > Adjust > Brightness/Contrast.
• 61.Click “Auto” in the Brightness/Contrast (B&C) panel. Note: You can now go through the frame to visualize the activity of the cells.
• 62.Go to the ImageJ menu and select Image > Stacks > Z Projection. From the pull-down menu, choose start slice and stop slice numbers. Choose projection type as standard deviation. This will indicate which pixels have the most fluctuation in the movie.
• 63.Now go to Analyze> Tools and ROI manager.
• 64.Click freehand ROI shape.
• 65.Draw around the region of interest in the cells (Separate ROIs can be constructed from cell soma and processes if they are clearly distinguishable). Click add and continue this until all the cells/regions are registered. Make an ROI for the background as well.
• 66.Click “Show All” and save the ROIs when you are done.
• 67.Now go to menu and select Analyze>Set Measurements. Check “mean gray value” and uncheck all the other parameters.
• 68.Go to ROI manager and make sure “Show All” is checked. In ROI manager, choose More>Multi measure and click OK.
• 69.Now the results will appear in excel file and can be saved.
• 70.Subtract the background from the signal intensity in all the columns.
• 71.Normalize the data by using (F1-F0)/F0 where F1 is the maximum signal intensity and F0 is baseline signal intensity.
• 72.Determine the frequency of events by counting the events which have intensity higher than 3 times of standard deviation of baseline intensity
• 73.Determine the half-width by determining full width at half maxima.

Support Protocol 2: Imaging of Drug-induced Calcium Activity

Introduction

ER-GCaMP6f can be used to study drug induced activity in astrocytes. Calcium signaling in astrocytes occurs mainly through inositol triphosphate receptors (IP3R) and ryanodine receptors (RyR) (Haustein et al., 2014). Drug induced effects on synapses have been shown to be important (Rahmaninejad, Pace, Chun, & Kekenes-Huskey, 2022) in communication between neurons and astrocytes. Various blockers and activators of these receptors have been used for studying calcium activity. ATP is an activator of IP3Rs while 2-APB is a blocker of IP3Rs. Caffeine has been shown to activate RyRs but inactivate IP3Rs at the same time. In this support protocol, we discuss how to perform calcium imaging of astrocytes in vitro with ER-GCaMP6f during drug treatment.

Materials

• Primary astrocytes

• T-75 Flask (Catalog no. BK369292, VWR International)

• Glass bottom dishes (35 mm, #1.5, MATTEK )

• L-15 (Gibco™ Leibovitz's L-15 Medium, no phenol red (Fischer Scientific, 21-083-027))

• Drug of interest (e.g., ATP)

• A TIRF-capable microscope (Olympus, model: IX2-ZDC2, serial number: 0F83094)

Protocol steps with step annotations:

1. Culture the astrocyte in T75 flask as described above.

2. Subculture the astrocytes in glass bottom dishes and perform transfection as described above.

3. Replace the cell culture media with L-15 media. Add 2 ml of L-15 media.

4. Make the drug solution in L-15. The drug concentration should be made double of the required final concentration.

5. Put the glass bottom dish with L-15 solution at the microscope sample holder. Focus the objective and save the co-ordinates of 10 different fields of view.

6. Start timelapse imaging of each field of view so that each field is sequentially image for each time point and stop imaging after 3 minutes. This time interval can be changed based on the research objective.

7. Remove the 1 ml solution from the dish carefully without changing the position of the dish.

8. Add 1 ml of drug solution with double the concentration of drug.

9. Start time lapse imaging.

10. Save the time lapse images.

ALTERNATE PROTOCOL 1 Transduction of Astrocytes with ER-GCaMP6f AAV

Introductory paragraph:

Astrocytes can be transfected using the CMV version of ER-GCaMP6f plasmid, but transduction can also be performed using the version of ER-GCaMP6f with a GFAP or gfaABC1D promoter. This method of gene delivery is more targeted and specific to the astrocytes (Griffin et al., 2019). gfaABC1D is a truncated version of the astrocyte specific promotor GFAP. This AAV can also be used to specifically target astrocytes in a co-culture of neurons and astrocytes as well as glial culture where different glial cells (e.g., astrocytes), microglia and oligodendrocytes are present.

Materials:

• Primary astrocytes

• ER-GCaMP6f AAV ((Plasmid #182501, Addgene)

• PBS-MK (see Reagents and Solutions)

• Astrocyte culture media (see Reagents and Solutions)

• A TIRF-capable fluorescence microscope (Olympus, model: IX2-ZDC2, serial number: 0F83094)

• 5% sodium azide solution (Catalog no. BDH7465-2, VWR International)

• 0.1% sodium azide (see Reagents and Solutions)

Protocol steps with step annotations:

Astrocyte culture and maintenance

1. Culture and split astrocytes as described in previous section.

2. Plate approximately 10⁶ astrocytes in a flask and wait until the cells become 70-80% confluent.

Preparation of viral particles and transduction

• 3.Calculate the number of viral particles required for the ER-GCaMP6f AAV transduction.

  No of viral particles required = MOI ×number of cells

  Typically, an MOI value of 1000 to 10,000 is used for astrocyte transduction.

• 4.Remove the concentrated viral vector stock from the −80° C and dilute it using PBS-MK.

  The volume of vector should not exceed 10% of the total media volume and should not be less than 1%. Ideally, use the same stock titer for all concentrations.

• 5.Dilute it appropriately in the vector diluent.
• 6.Gently swirl the plates to evenly suspend the vector in media and place the plates back in the incubator.
• 7.After 3 days after transduction, add 100 μL of fresh media to the wells and return to the incubator.
• 8.After 5 days after transduction, remove 100 μL of media and add another 100 μL of fresh media to the wells and return to the incubator.

Gene expression and imaging

• 9.Change the cell culture media periodically and check for fluorescence every 3-4 days.

  It will take approximately 2 weeks for the protein expression to take place. You may need to split cells during this process. After the expression of GCaMP6f you can perform calcium imaging.

• 10.Remove the cell culture media and replace it with L-15.
• 11.Perform calcium imaging by using a fluorescence microscope.

  Cells can also be fixed with 4% PFA for 20 minutes and washed 3 times with PBS and stored in PBS + 0.1% sodium azide for long-term storage in fridge.

ALTERNATE PROTOCOL 2 : Calcium Imaging of Astrocytes with Fluo-4 AM

Introductory paragraph:

Although they are difficult to use for in vivo applications, organic dyes are well suited for in vitro calcium imaging. In this protocol we discuss how to use Fluo-4 AM for astrocyte calcium imaging (S. P. Aryal et al., 2021). Fluo-4 AM is a cell permeable fluorescence indicator which binds to Ca²⁺. Fluo-4 is the fluorescent part of this compound which absorbs at 494 nm and emits at 506 nm. When Fluo-4 AM enters cells the AM parts breaks down making it membrane impermeable and providing sufficient local concentration of fluo-4 to bind Ca²⁺. When it binds to Ca²⁺, the fluorescence increases significantly and can be detected using a fluorescence readout system such as fluorescence microscope.

Materials:

• PBS (see Reagents and Solutions)

• Astrocyte culture media (see Reagents and Solutions)

• Fluo-4 AM (Catalog no. 76483-080, VWR International)

• L-15 (Gibco™ Leibovitz's L-15 Medium, no phenol red (Fischer Scientific, 21-083-027))

• A TIRF-capable microscope (Olympus, model: IX2-ZDC2, serial number: 0F83094)

• 

Protocol steps with step annotations:

Astrocyte culture and Fluo-4 AM labeling

1. Subculture astrocytes in glass bottom dishes as described before.

2. After the cells reach ~70% confluency, replace the media with L-15 media.

3. Dissolve fluo-4 AM solution in L-15 medium.

4. Add 1 μM of fluo-4 AM solution to the cells.

5. Incubate the cells in incubator for 45 minutes

6. Perform calcium imaging using 488 nm laser. Calcium imaging can be performed in both epifluorescence and TIRF mode.

7. Save the time lapse images in sequences in TIFF format.

Data analysis

• 8.Convert the movie files into “TIFF” file format if they are in different file format.
• 9.Open the movie file with imageJ/FIJI software.
• 10.From the menu, select Image > Adjust > Brightness/Contrast.
• 11.Click “Auto” in the Brightness/Contrast (B&C) panel. Note: You can now go through the frame to visualize the activity of the cells.
• 12.Go to the ImageJ menu and select Image > Stacks > Z Projection. From the pull-down menu, choose start slice and stop slice numbers. Choose projection type as standard deviation. This will indicate which pixels have the most fluctuation in the movie.
• 13.Now go to Analyze> Tools and ROI manager.
• 14.Click freehand ROI shape.
• 15.Draw around the region of interest in the cells (Separate ROIs can be constructed from cell soma and processes if they are clearly distinguishable). Click add and continue this until all the cells/regions are registered. Make an ROI for the background as well.
• 16.Click “Show All” and save the ROIs when you are done.
• 17.Now go to menu and select Analyze>Set Measurements. Check “mean gray value” and uncheck all the other parameters.
• 18.Go to ROI manager and make sure “Show All” is checked. In ROI manager, choose More>Multi measure and click OK.
• 19.Now the results will appear in excel file and can be saved.
• 20.Subtract the background from the signal intensity in all the columns.
• 21.Normalize the data by using (F1-F0)/F0 where F1 is the maximum signal intensity and F0 is baseline signal intensity.
• 22.Determine the frequency of events by counting the events which have intensity higher than 3 times of standard deviation of baseline intensity
• 23.Determine the half-width by determining full width at half maxima.

BASIC PROTOCOL 3 : In Vivo ER-GCaMP6f Expression and Slice Calcium Imaging

Introductory paragraph:

This protocol discusses expression of ER-GCaMP6f in rat brain astrocytes in vivo to perform calcium imaging in acute brain slices. Stereotactic surgery is needed to inject the ER-GCaMP6f probe at the desired location. We targeted the nucleus accumbens shell at the following coordinates (mm, from Bregma): A/P +1.0, M/L ±1.0, D/V −7.0, but different locations can be chosen based on the research question. The Allen brain atlas is a very useful resource to locate the co-ordinates for a particular region of interest (Jones, Overly, & Sunkin, 2009). We have provided links for both mouse and rat brain atlases in the useful resources section. This protocol discusses how to perform stereotactic injections of ER-GCaMP6f in the rat brain. It also discusses how to perform brain slice preparation and calcium imaging. Stereotactic surgery should be done very carefully to minimize pain and distress to the animal. Depth of anesthesia, breathing, and body temperature should be continuously monitored during the surgery and palliative care administered during the surgery recovery period.

Materials:

• ER-GCaMP6f AAV (Plasmid #182501, Addgene)

• Stereotactic frame (Model 902 Dual Small Animal Stereotactic Instrument, Kopf instruments)

• 6 Sprague Dawley rats (2-3 months old)

• Heating pad (Catalog no. 470093-422, VWR International)

• 70 % ethanol (see Reagents and Solutions)

• Saline (see Reagents and Solutions)

• Isoflurane (Catalog no. 1182098, Henry Schein)

• Isoflurane box

• Ophthalmic ointment/artificial tears (Catalog no. MSPP-704001, VWR International)

• Animal hair clippers

• Scalpel (Catalog no. 76457-442, VWR International)

• Iodine solution (Catalog no. BDH7207-1, VWR International)

• Hemostat (Catalog no. 10806-116, VWR International)

• Cotton swab (Catalog no. 89031-270, VWR International)

• Meloxicam (NDC no. 11695-6936-2, Covetrus)

• Vibratome (Serial no. A13742, VetLand)

• VetBond (Serial no. 1469SB, 3M)

• Cutting solution (see Reagents and Solutions)

• Artificial cerebrospinal fluid (see Reagents and Solutions)

• Fluorescence microscope

Protocol steps with step annotations:

Virus dose and site selection

1. If frozen, thaw a vial of ER-GCaMP6f adeno associated virus (AAV) on ice.

2. Identify stereotactic coordinates for virus delivery.

   For the nucleus accumbens, virus can be injected at the following Stereotactic coordinates (in millimeters from bregma): A/P: +1.0, M/L: ±1.0, D/V: −7.0.

3. Titer the virus as appropriate for optimal expression in the target region.

   We used 2.0 μL ER-GCaMP6f at full titer (2.86 × 10¹³) for nucleus accumbens shell injections.

Preparation of rat for virus injection

• 4.Prepare the surgical area and isoflurane anesthesia set-up.
• 5.Set up stereotactic instruments, heating pad, and all the surgical tools.

  The tools must be sterilized before use.

• 6.Induce anesthesia at 5% isoflurane in oxygen (O₂ flow rate=1-1.5 L/min) until surgical plane of anesthesia is confirmed by absence of toe-pinch reflex. Maintain anesthesia at 1-3% isoflurane. Continue monitoring anesthesia depth, animal breathing, and body temperature. If the animal has difficulty breathing/gasping, decrease the isoflurane. If the animal starts responding to toe pinch, increase the isoflurane.
• 7.Secure the head in the stereotactic frame. The animal should be supplied with a steady flow of 1-3% of isoflurane.
• 8.Apply ophthalmic ointment to the eyes.
• 9.Shave the animal’s head with electric hair clippers. Start from the middle of the eyes to the ears. Move the razor around sidewise to effectively clean the fur.
• 10.Apply iodine solution around the shaved area of the head, being careful to avoid the eyes.
• 11.Use a sterile scalpel to perform an anterior/posterior incision starting just behind the eyes and to the middle of the ear.
• 12.Hold the skin open with hemostats on each side of the head. Brush off the skin with cotton swabs. Apply saline if necessary.
• 13.Secure the drill in the stereotactic frame and position it over the surgery site. Using the tip of the drill bit, verify that there is no vertical difference between bregma and lambda coordinates. Make sure that the head position is also flat in the horizontal direction by comparing the ventral coordinate difference to the left and right of the bregma. Adjust head position if necessary.
• 14.Drill two holes at the appropriate M/L coordinates. The drill should penetrate the thickness of the skull (~0.7 mm), without reaching the brain.
• 15.Secure a Hamilton syringe in the stereotactic frame, withdraw the appropriate volume of the virus and lower the syringe down to the desired D/V coordinate through the drilled hole.
• 16.Deliver the virus at the rate of 0.2 μl per minute. After infusion is finished, leave the syringe at injection site for 10 minutes to allow further passive diffusion. Slowly remove the syringe from the injection site.
• 17.Staple the skin and apply VetBond to skin edges to promote wound closure.
• 18.Inject 0.1 mg/kg meloxicam (subcutaneous) for analgesia and return the animal to its home cage. Monitor recovery from anesthesia until locomotion is regained.
• 19.Perform daily observation for post-surgical signs of infection and pain/distress.
• 20.If the animal shows signs of pain or distress, meloxicam (0.1-0.5 mg/Kg, subcutaneous) can be administered daily. For infection, antibiotic topical ointments can be administered or systemic treatment with penicillin (100,000 IU/Kg, intramuscular) can be performed every 12 hours for 4 days.
• 21.Expression of ER GCaMP6f can be observed 2-3 weeks after the surgery.

Brain slicing and imaging

• 22.Prepare cutting solution and ACSF calcium imaging solution.
• 23.Extract the rat brain and place it in a cutting solution oxygenated with 95 % O₂/5 % CO₂ gas mixture. The cutting solution should be oxygenated for at least 30 minutes before slice preparation.
• 24.Use the vibratome to cut 300 μm- thick slices, containing your area of interest.
• 25.Transfer the slices to a continuously oxygenated ACSF maintained at 32-34° C for 45 min in a dark container to prevent photobleaching.
• 26.Keep the slices oxygenated at room temperature thereafter until transfer to the imaging chamber.
• 27.Acquire and save calcium fluorescence videos using the desired imaging parameters.

  We typically image with a 40X water-immersible objective, at 512/512 pixels, 40 ms/frame to collect 1–2-minute videos.

Data analysis

• 28.Convert the movie files into “TIFF” file format.
• 29.Open the movie file with imageJ/FIJI software.
• 30.From the menu, select Image > Adjust > Brightness/Contrast.
• 31.Click “Auto” in the Brightness/Contrast (B&C) panel. Note: You can now go through the frame to visualize the activity of the cells.
• 32.Go to the ImageJ menu and select Image > Stacks > Z Projection. From the pull-down menu, choose start slice and stop slice numbers. Choose projection type as standard deviation. This will provide which pixels are most fluctuated in the movie.
• 33.Now go to Analyze> Tools and ROI manager.
• 34.Click freehand ROI shape.
• 35.Draw the region of interest in the cells (Separate ROIs can be constructed from cell soma and processes if they are clearly distinguishable). Click add and continue this until all the cells/regions are registered. Make a ROI for background as well.
• 36.Click “Show All” and save the ROIs when you are done.
• 37.Now go to menu and select Analyze>Set Measurements. Check “mean gray value” and uncheck all the other parameters.
• 38.Go to ROI manager and make sure “Show All” is checked. In ROI manager, choose More>Multi measure and click OK.
• 39.Now the results can be transferred to and saved in an Excel file.
• 40.Subtract the background from the signal intensity in all the columns.
• 41.Normalize the data by using (F1-F0)/F0 where F1 is the maximum signal intensity and F0 is baseline signal intensity.
• 42.Determine frequency of events by counting the events which have intensity higher than 3 times of standard deviation of baseline intensity.
• 43.Determine half-width by measuring the full width at half maxima. Figure 6 illustrates an example of calcium imaging data from rats injected with ER-GCaMP6f AAV.

Figure 6:

Measurement of calcium activity in brain slices by expressing ER-GCaMP6f in vivo. Comparison of calcium activity between ER-GCaMP6f and Lck-GCaMP6f (which is a membrane tethered GCaMP) in terms of A) number of ROI with and without 2-APB (which is an inhibitor of IP3 receptors), B) amplitude with and without 2-APB, C) number of events with and without 2-APB and D) half-width of events with and without 2-APB. Reprinted with permission from {Aryal, S. P., Xia, M., Adindu, E., Davis, C., Ortinski, P. I., & Richards, C. I. (2022). ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to Measure Calcium Activity in Astrocytic Processes. Analytical Chemistry}. Copyright {2022} American Chemical Society.

SUPPORT PROTOCOL 3 : Pharmacological Studies with 2-APB in Brain Slices

Introductory paragraph:

Pharmacological treatments can be applied to further study Ca²⁺ responses. Addition of the IP3 signaling blocker (2-APB) impacts Ca²⁺ events at both plasma and ER membrane sites. The frequency of Ca²⁺ events is partially blocked by 2-APB as reported by the plasma sensor, but not when using the ER sensor. The duration of Ca²⁺ events is partially blocked by 2APB as reported by both the plasma membrane and the ER sensor with the plasma membrane sensor reporting a larger effect. The amplitude of Ca²⁺ events is partially blocked by 2APB as reported by both the plasma membrane and the ER sensor (Figure 6).

Additional Materials:

1. 2-APB ( CAS no. 524-95-8, Tocris Bioscience)

Protocol steps with step annotations:

1. Set up and acquire calcium fluorescence videos using the desired imaging parameters as described in the previous section.

   We typically image with a 40X water-immersion objective, at 512/512 pixels, 40 ms/frame to collect 1–2-minute videos.

2. Record five one-minute videos with regular ACSF and then add ACSF containing 50 μM of 2-APB.

   We typically collect five one-minute videos of spontaneous activity with regular ACSF. Then, we bath-apply ACSF with 2-APB (50 μM). This concentration of 2APB, has been used widely to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca²⁺ signals (IC₅₀=42 μM). We typically wait for at least 5 minutes to allow for full exposure of the slice to 2APB, then acquire 5 one-minute videos of spontaneous activity using the same imaging parameters as used for ACSF alone.

   Note: To ensure that 2APB is fully rinsed between slices, we cleared the 2APB containing media out of the imaging chamber by vacuum suction and re-filled the chamber with regular ACSF. The chamber was then re-emptied and re-filled with ACSF two more times.

3. Perform analysis as described in the data analysis portion of the previous section.

REAGENTS AND SOLUTIONS:

1. Astrocyte cell culture media

   Make 500 ml of Astrocyte culture media by mixing 445 mL Dulbecco’s modified eagle medium(DMEM), 50 mL heat inactivated Fetal Bovine Serum(FBS) and 5 mL Penicillin and Streptomycin and store it at 4° C.

2. HEK-293 cell culture media

   Make 500 ml of HEK-293 cell culture media by mixing 445 mL Dulbecco’s modified eagle medium(DMEM), 50 mL Fetal Bovine Serum(FBS) and 5 mL Penicillin and Streptomycin and store it at 4° C

3. 1% Cultrex solution

   Mix 1 ml of Cultrex with 99 ml of DMEM and store it at 4° C

4. 70 % ethanol

   Mix 70% of ethanol with 30 % of DI water in a spray bottle

5. 0.001% Pluronic acid in PBS-MK

   Dissolve 1 μl of Pluronic acid to 100 ml of PBS-MgCl₂.

6. 0.1% sodium azide

   Dissolve 200 μl of 5% sodium azide in 9.8 ml of PBS. Use magnetic stirrer to mix well at store at 4°C.

7. Saline

   Dissolve 0.9% (w/v) of NaCl in DI water

8. Cutting solution

   3mM KCl, 1.25 mM NaH₂PO₄, 26mM NaHCO₃, 10mM Glucose, 2 mM CaCl₂, 1 mM MgCl2, 219 mM Sucrose, pH 7.2–7.4, when saturated with 95% O₂ and 5% CO₂

9. Artificial Cerebrospinal Fluid (ACSF)

   Make ACSF solution by mixing 130 mM NaCl, 3mM KCl, 1.25 mM NaHCO₃, 10 mM Glucose, 1 mM MgCl₂, 2 mM CaCl₂. Adjust pH between 7.2 to 7.4. Final osmolarity should be between 305 and 315 mOsm.

10. 1x PBS-MK buffer

    Make 1X PBS-MK solution by mixing 26.3 mg of MgCl₂, 14.91 mg of KCl in 1X PBS. Make the final volume of 100 ml and sterilize it by passing through a 0.22-μm filter and store at 4 °C.

COMMENTARY:

Background Information:

Astrocytes are a major class of glial cells widely distributed in the brain. Their function ranges from guiding synaptic processes to maintaining homeostasis and controlling the blood brain barrier and blood flow (Blackburn, Sargsyan, Monk, & Shaw, 2009; Leis, Bekar, & Walz, 2005). Astrocytes interact with synapses where astrocytic processes are involved in synaptic communication between neurons, and they regulate the formation and collapse of synapses. Processes from one astrocyte are believed to interact as many as ten thousand synapses (Kimelberg, 2007). Astrocytes alter their phenotypes based on (physio)pathological conditions which affects intra and inter cellular signaling processes such as calcium activity (Allen & Barres, 2009; Shane A Liddelow et al., 2017; S. A. Liddelow et al., 2017).

Calcium imaging is one of the major tools to study the effect of these phenotypes on physiological processes (Agulhon et al., 2012). Astrocytes in vitro have traditionally been studied with calcium sensitive dyes but their lack of specificity and inability to target subcellular regions have prohibited many researchers from understanding localized Ca²⁺ activity. Typical Ca²⁺ concentration at the extracellular space is in the range of 1-2 mM while the intracellular concentration of Ca²⁺ in the cytosol is approximately 100 nM. The endoplasmic reticulum is a major source of Ca²⁺ in cells and typically has a Ca²⁺ concentration in the range of several hundred micromolar. Ca²⁺ exchange takes place between the ER and other organelles. Many powerful tools have been developed to study Ca²⁺ in astrocytes but subcellular targeting has been challenging due to a lack of spatially localized sensors (Li et al., 2014; Niwa et al., 2016). ER-GCaMP6f targeted at the cytosolic side of the endoplasmic reticulum is able to measure calcium activity both in vitro and in vivo in astrocytes.

Critical Parameters:

Several parameters are critical in the implementation of this protocol. For transfection related experiments, the DNA quality is important, and it should be checked before the experiment. Concentration of DNA as well as purity should be measured.The absorbance ratio (A260/A280) gives purity of the DNA and a value ~1.8 suggests the DNA sample is pure. If it is much lower than 1.8, it should not be used for transfection.

For transduction, multiplicity of infection (MOI) is an important factor. It refers to the number of viral particles per cells to be used in transduction. Generally, a value of 1000 to 10,000 is optimal. The number of viral particles needed should be calculated by multiplying MOI with number of cells to be infected.

Confluency of the cells is another major factor during transfection and transduction. Cells should be ~70% confluent. If the cells are overconfluent or not sufficiently confluent, DNA delivery will be problematic. During transduction, the gene expression might take weeks so, cells should be split if they start becoming confluent.

Animal surgery for viral delivery is a critical step. Appropriate co-ordinates should be used, and the stereotactic apparatus should be handled very carefully. All the animal experiments should be performed according to the guidelines of IACUC and the institution guidelines.

In animal experiments, animals should be monitored every day after the viral injection for any distress or pain. Antibiotic should be given if the animal seems to have any such difficulties.

Photobleaching can be a problem in both cell culture and brain slice calcium imaging. Collecting time lapse images using limited laser exposure instead of movies with continuous laser exposure is one of the ways to minimize the effect of photobleaching.

Troubleshooting:

Table 1.

Troubleshooting Guide for ER-GCaMP6f Expression and Imaging

Problem	Possible Cause	Solution
ER-GCaMP6f is not expressed/Fluorescence is not observed in HEK-293 cells or astrocytes.	Plasmid concentration might have been low	Measure the concentration of plasmid and perform transfection with higher concentration of DNA
Too much background	Imaging media is not transparent	Replace the imaging media with L-15
Cell death is high	The plasmid DNA concentration might have been very high	Measure the concentration of plasmid and perform transfection with lower concentration of DNA
Transfection efficiency is low	Cells might have been overconfluent	Check the confluency of the cells and perform transfection when they are ~70% confluent
Cells are not focused on TIRF	It is probably because the dish is highly confluent, and cells are grown on top of each other	Check the cell confluency and make sure no cells are on top of each other before imaging
Animal is not recovered from anesthesia after surgery	The anesthesia dose might have been higher	Maintain ~2% isoflurane during the stereotactic surgery
No fluorescence is observed in slices	One reason might be not enough time for expression	Harvest slices after 3-4 weeks of ER-GCaMP6f expression and perform calcium imaging
Fluorescence decays very fast in cell culture or slice imaging	It might be because of high laser power or long exposure time	Decrease the laser power and/or exposure time
Movies/calcium images are oversaturated	The signal is too high	Decrease the laser power and/or exposure time

Understanding Results:

In the results section, we provide a cartoon which summarizes the protocol for ER-GCaMP6f expression in cell culture as well as in vivo (Figure 1). Figure 2 shows the genetic maps of ER-GCaMP6f with a CMV promotor and an astrocyte specific promotor. Figure 3 illustrates the localization of ER-GCaMP6f with an ER marker in a 3-dimensional image taken by using structured illumination microscopy (SIM). In figure 4 and 5, we showed in vitro astrocyte calcium activity in epifluorescence and TIRF mode as well as calcium activity plots associated with them. In figure 6, we showed in vivo calcium activity data from brain slices.

Time Considerations:

Cell culture, ER-GCaMP6f transfection and imaging can be finished in a week. Transduction of astrocytes with ER-GCaMP6f AAV and calcium imaging can be finished in 2-3 weeks. In vivo expression and imaging take approximately 4-5 weeks.

Supplementary Material

video

Movie 1: An example movie showing calcium activity measured in astrocytes using ER-GCaMP6f sensor

supinfo1

Supplementary information1: Genetic sequence of ER-GCaMP6f plasmid with CMV promotor

supinfo2

Supplementary information 2: Genetic sequence of ER-GCaMP6f plasmid with astrocyte specific GFAP promotor

Abbreviation

AAV

Adeno associated virus

ATP

Adenosine triphosphate

A/P

Anterior-posterior

ACSF

Artificial cerebrospinal fluid

CMV

Cytomegalovirus

D/V

Dorsal-ventral

ER

Endoplasmic Reticulum

GFAP

Glial fibrillary acidic protein

HEK-293

Human embryonic Kidney-293 cell line

MOI

Multiplicity of Infection

M/L

Medial-lateral

PLL

Poly-L-Lysine

PFA

Paraformaldehyde

PBS

Phosphate buffer saline

PBST

Phosphate buffer saline with 0.1% Triton X-100 (v/v)

PBST-20

Phosphate buffer saline with 0.2% Tween-20 (v/v)

ROI

Region of Interest

SIM

Structured Illumination Microscopy

TIRF

Total internal reflection fluorescence

DATA AVAILABILITY STATEMENT:

The data that support the findings of this study are available from the corresponding author upon reasonable request.