Computational protocol: Motor-Skill Learning Is Dependent on Astrocytic Activity

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Protocol publication

[…] Slices were loaded at room temperature with fluo-4 AM (5 μM, Molecular Probes), 0.04% Pluronic F-127, and SR101 (10 μM, Sigma-Aldrich) for 40 minutes and then transferred to dye free ACSF for at least 30 minutes. Simultaneous monitoring of multiple layer 1 astrocytes was carried out in frame scan mode at 1 Hz at image resolution of 256 × 256. Time-lapse imaging was carried out; image series consisting of 300 frames were collected.In order to assess the Ca2+ activity of astrocytes in the presence of FC, spontaneous Ca2+ changes before and after perfusion of FC (100 μM) in the slices were compared. Following baseline imaging in regular ACSF, slices were perfused for 30 minutes with FC and then the same positions were reimaged in presence of FC.To assess the efficiency of the Cre-mediated recombination, we performed Ca2+ imaging on bulk loaded coronal slices containing the M1 from controls (vehicle control and tamoxifen controls) and mutant IP3R2 mice. Time-lapse imaging was carried out to monitor spontaneous Ca2+ oscillations followed by reimaging of some of the same positions with perfusion of ATP (100 μM).We imaged with a two-photon microscope (Moving Objective Microscope, Sutter) attached to a Ti : sapphire laser (Chameleon Vision II, Coherent), using a 60x water immersion objective (1.0 NA, Nikon). Excitation wavelength of 800 nm with power measured at the back aperture at 10–30 mW was used. Two-channel detection of emission wavelength was achieved by using a 565 nm dichroic mirror and two external photomultiplier tubes. A 535/50 bandpass filter was used to detect fluo-4 AM emission wavelength, and a 610/75bandpass filter was used to detect SR101. For imaging, we used ScanImage software [] written in MATLAB (The MathWorks). [...] Imaging data was analyzed using ImageJ software. Regions of interest (ROIs) were placed around astrocyte soma. Fluorescence was averaged over ROIs placed and expressed as relative fluorescence changes (ΔF/F) after subtraction of background fluorescence from a neighboring region. Ca2+ transients of astrocyte were detected when fluorescence intensity reached higher than 2 SD value of baseline fluorescence intensity.The following parameters were analyzed to assess the Ca2+ activity in the presence of FC: percent active astrocytes, the mean number of spontaneous events per astrocyte, and the average peak value of the Ca2+ oscillations. We analyzed the percent active cells and percentage of cells responding to ATP to assess the spontaneous Ca2+ changes and ATP-induced Ca2+ signals in control and mutant IP3R2 mice. For astrocyte soma analysis, an astrocyte that exhibited at least one spontaneous Ca2+ oscillation during the imaging session was referred to as an active astrocyte. The ratio of the number of active astrocytes and SR101 labeled cells in every imaging field was used to analyze the percent active astrocytes based on soma Ca2+ measurements. Analysis was performed on raw unprocessed images and for presentation purposes images were despeckled. […]

Pipeline specifications

Software tools ScanImage, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Chemicals Calcium, alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid, Glutamic Acid