Computational protocol: Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors

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Protocol publication

[…] Banker cultures were transfected at 11 days in vitro (DIV) using Effectene (Qiagen N.V., Venlo, Netherlands). Doxycycline (1 μg/ml) was added to the culture medium 24-48 hours before experiments using inducible GluA2 constructs. Coverslips of transfected neurons (14-16 DIV) were mounted in a Ludin chamber (Life Imaging Services) and transferred to an inverted microscope (Leica, DMI 6000B) maintained near-physiological temperature with a microscope temperature control system (Life Imaging Services, Cube 2). The chamber was perfused at 1 mg/ml (Gilson MiniPuls3) with extracellular solution containing (in mM): NaCl (145), KCl (3.5), MgCl2 (2), CaCl2 (2), d-glucose (10), HEPES (10) (pH 7.4, ~300 mOsm) and the preparation was observed through a 63x oil objective (Leica, HCX PL APO CS, Iris NA 1.4-0.6). Transfected cells were identified under epifluorescence (Leica EL6000) and illuminated with 473 nm laser light using a high-speed spinning disk confocal scanner unit (Yokogawa CSU22) for acquisition. Emission was captured with an electron multiplying charge-coupled device (EMCCD) camera (Photometrics Quantem 512SC) and data was stored on the hard disk of a personal computer (DELL Precision, Precision PWS690). Hardware was controlled with MetaMorph software (Molecular Devices, v7.1.7) and ILAS system software (Roper).Images of SEP-GluA1 expressing neurons were acquired before and after perfusion of 2 ml of fluorescently-labelled biotin-binding proteins and then washed for 5 minutes with Tyrode’s solution. Following the wash, using an exposure time of 0.5 seconds, the protocol consisted of: 1) acquiring 11 images at 0.67 s intervals; 2) photobleaching the regions of interest (ROI), then 3) acquiring 40 images at 0.67 s intervals and a further 55 images at 5 s intervals. For bleaching, we used a 5 ms pulse of 488 nm laser light (Coherent Sapphire CDRH 100 mW) set to ~3.7 mW laser power at the back of the objective, which was sufficient to reduce fluorescence by ~50 %. A FRAP head (Roper/Errol) was used to scan ROIs (~0.690 μm diameter) with the bleaching laser. ROIs were positioned over a small number of independent spines across the field of view. Finally, at the end of the experiment, we obtained an image before and after application of membrane impermeant GFP quencher (5 mM Trypan red) to confirm that most of the SEP fluorescence was on the cell surface. Time-lapse images were corrected for XY-drift observational and bleach-pulse photobleaching using macros in either MetaMorph (Molecular Devices) or ImageJ (http://imagej.nih.gov/ij/). ROIs of fixed diameter (10 pixels, eq. to 2.3 μm) were then carefully positioned over each bleached spine head and the integrated or mean fluorescence intensity was measured for every image. The fluorescence intensity for each ROI was subjected to full scale normalization by subtracting the average fluorescence intensity of the pre-bleach images and dividing the result by the fluorescence of the first post-bleach image. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Microscopic phenotype analysis, FRAP
Organisms Mus musculus
Chemicals alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid, Glutamic Acid