Spontaneous Calcium Imaging for Compartment-Specific Dynamics in Astrocytes

Abstract

Source: Bannai, H., et. al., Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators. J. Vis. Exp. (2019)

The video demonstrates fluorescence imaging to observe compartment-specific spontaneous calcium dynamics in astrocytes by sequentially monitoring calcium-sensitive proteins localized on the plasma membrane and endoplasmic reticulum.

Protocol

1. Ca²⁺ Imaging

1. Recording spontaneous activities of astrocytes expressing Lck-RCaMP2 and OER-GCaMP6f/
   NOTE: Without image-splitting optics, the Ca²⁺ signals at the plasma membrane and those around the endoplasmic reticulum (ER) can be monitored in the same cell. Here, the sequential recording of Lck-RCaMP2 (Plasma membrane-targeted genetically encoded Ca²⁺ indicator) and OER-GCaMP6f (Outer endoplasmic reticulum membrane-targeted genetically encoded Ca²⁺ indicator) in the same astrocytes is described. An oil-immersion objective with a numerical aperture larger than 1.3 is highly recommended for spontaneous Ca²⁺ activity.
   1. Turn on the microscope, camera, light source, and the microscope heating chamber for at least 30 min before recording.
   2. Mount the coverslip containing the cells transfected with Lck-RCaMP2 and OER-GCaMP6f in the recording chamber and add 400 µL of the imaging medium. Place a lid on top of the chamber.
   3. Choose the filter set for GCaMP6f and the light source (blue excitation light, e.g., 470– 490 nm; see Table of Materials). Locate the astrocytes expressing OER-GCaMP6f.
   4. Choose a filter set and the light source for RCaMP2 (green excitation light, e.g., 510–560 nm; see Table of Materials) and confirm whether Lck-RCaMP2 is expressed in the same astrocytes. Avoid long exposure to the light source to prevent photobleaching.
   5. Record time-lapse images of Lck-RCaMP2 at 2 Hz for 2 min. Save the imaging data on the HDD.
   6. Change the filter set to that for GCaMP6f. Record time-lapse images of OER-GCaMP6f in the same field of view at 2 Hz for 2 min. Save the data on the HDD.
   7. Analyze the data using the image analysis software.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
18-mm diameter circular coverslips	Karl Hecht "Assistent"	#41001118	Thickness 1, 18-mm diameter circular coverslips; alternative coverslips can be used.
Camera for microscopic image recording			The following cameras were available for use: cooled-CCD camera (e.g., Hamamatsu Photonics, OECA-ER), EM-CCD camera (e.g., Hamamatsu Photonics, ImagEM; Andor, iXon) or CMOS camera (e.g., Hamamatsu Photonics ORCA-Flash4.0)
Image analysis software			Such as Metamorph (Molecular Devices), Image J (NIH), and TI Workbench14 (custom made)
Imaging medium and buffer			Use optimal medium or buffer for the experiment. When medium is used, medium without phenol red is desirable to reduce background fluorescence. Add 20 mM HEPES to maintain pH outside of carbon dioxide incubator.
Inverted fluorescence microscope			Such as IX73 (Olympus) or Eclipse TI (Nikon Instech)
Microscope filter set for GCaMP6f imaging			Appropriate filter for GFP (excitation, 480 ± 10 nm; emission, 530 ± 20 nm)
Microscope filter set for RCaMP2 imaging			Appropriate filter for RFP (excitation, 535 ± 50 nm; emission, 590 nm long pass)
Microscope heating system			A heating system to maintain cells at 37°C during the imaging. To avoid drift caused by thermal expansion, heating systems covering the entire microscope itself (e.g., Tokai Hit, Thermobox) are recommended.
Microscope light source for excitation			Mercury lamp (100 W), xenon lamp (75 W), Light-emitting diode (LED) illumination system (e.g., CoolLED Ltd., precisExcite; Thorlabs Inc., 4-Wavelength LED Source; Lumencor, SPECTRA X light engine). In case of mercury lump and xenon lamp, use ND filter to reduce the excitation intensity.
Microscope objective lens			Plan-Apochromat oil immersion objective with numerical aperture higher than 1.3 is highly recommended for the recording of spontaneous Ca²⁺ activity in neurons and astrocytes.
Recording chamber	Elveflow	Ludin Chamber	This recording chamber is for 18 mm diameter round coverslips.
Stereomicroscope			Used to dissect hippocampi. Olympus SZ60 or equivalent stereomicroscopes are available.