Computational protocol: The MK2/3 cascade regulates AMPAR trafficking and cognitive flexibility

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Protocol publication

[…] For morphological analysis, the experimenter was blinded to the cDNA constructs transfected into the tissue, and animal genotype. Immunofluorescence was observed using a × 63 oil-immersion lens on an inverted Leica SP5 confocal microscope (Leica Microsystems). Fluorophores were excited with 488 or 568 nm wavelengths and emission from one single plane was detected through 505–530 nm band-pass or 560 nm long-pass filters. For imaging acquisition, the physical parameters such as laser intensity, gain and magnification were set up for control conditions; the settings were saved and used throughout the same experiment. Z-stacks were obtained at 0.2 μm steps and processed using Leica Microsystem LAS software (Version 2.6.0, Leica). The image calibration and the quantification of pixel intensity were performed as previously described. Briefly, the TIFF images were imported into ImageJ software ( http://imagej.nih.gov/ij/) and scaled according to the physical parameters used for the images acquisition. For morphological analysis, a random primary or secondary process was selected and a line was drawn along the selected process and electronically measured. Spines along this process were manually counted. The neck length was measured from the base of the spine on the process to the base of the spine head, which was kept consistent throughout the analysis. ImageJ software was used to manually select each of the visible and measurable spine necks with the line tool. The line drawn along the spine neck was measured electronically to give an accurate measurement. To measure spine volume and spine head diameter, TIFF images were exported as compressed Z-stacks using ImageJ, opened and calibrated in Neuronstudio software and processes were automatically selected and spines identified manually. Co-localization of pre- and postsynaptic markers was evaluated using ImageJ software. Briefly, correlation between green and red fluorescence was calculated as intensity correlation quotient (ICQ) based on the relative differences from the mean for each pixel (PDM); the pseudo-colour PDM images were generated as an output of ImageJ analysis routine. The theoretical limits for ICQ are ±1; random staining should show ICQ 0, positive values are characteristic for dependent staining. [...] Brains were removed from P30-P79 mice, washed briefly with cold PBS and treated according to the vendor instructions for the FD Rapid GolgistainTM Kit (FD NeuroTechnologies, Inc.). Following the initial impregnation process, 200 μm parasagittal slices of the hippocampus were prepared using a vibratome (Microm, HM650V). The slices were mounted onto gelatin-coated microscope slides and left overnight at room temperature. The following day, the slices were further processed using the FD Rapid GolgistainTM Kit and coverslip mounted with Mowiol. The slices were then visualized using a × 100 oil-immersion lens on a Zeiss Axioskop FS transmission light microscope and imaged using a Nikon DXM1200F digital camera. The images were captured from Golgi-impregnated pyramidal neurons of the CA1 region of the hippocampus. The TIFF files were imported into ImageJ and Z-stacks compressed. Images were then calibrated accordingly to the acquisition parameters and the length of the process measured electronically as described previously, and spine numbers from primary and secondary processes were measured for both genotypes. Morphological analysis was performed with the experimenter blinded to the genotype of the animals. Spines neck length and head diameter were measured using Neurolucida software (MicroBrightField) on a Nikon 80i microscope (Nikon) equipped with a × 100, 1.25 numerical aperture oil-immersion lenses. [...] Data were analysed using Prism (Version 5.04, GraphPad) and SPSS statistics 21 (IBM) software. Unpaired t-tests, Mann–Whitney t-tests, Kolmogorov–Smirnov test, one-way analysis of variance and the corresponding post-hoc tests (Turkey or Dunn’s) were performed as appropriate. […]

Pipeline specifications

Software tools ImageJ, NeuronStudio, Neurolucida, SPSS
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Chemicals Glutamic Acid