Computational protocol: AII amacrine cells discriminate between heterocellular and homocellular locations when assembling connexin36-containing gap junctions

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Protocol publication

[…] Retinas from the same experimental group were prepared, incubated and scanned with a Leica TCS SP2 or SP5 (Leica, Nussloch, Germany) under identical conditions. Unless stated otherwise, images are presented either as maximum or sum projections of the collapsed confocal stack and adjusted for brightness and contrast for presentation purposes. For quantitative analyses, we used the ImageJ software (NIH, Bethesda, MD). Analyzed images were background-subtracted and noise-reduced when relevant, and intensity and size thresholds were either set automatically or adjusted manually to best reflect visible clusters; threshold settings were kept constant for a particular experimental group. Unless stated otherwise, 12 independent scanned images were evaluated per mouse.To evaluate densities, clusters were counted using the ‘analyze particle’ plugin in ImageJ and colocalization analysis was performed using the ‘colocalization highlighter’; three-color colocalization was carried out sequentially. Binary images of colocalized pixels are displayed (; ) or shown as dots representing their centroids () in some of the figures. For determining Cx36–EGFP cluster size, colocalized clusters were identified as above and their size was determined in Metamorph 4D viewer (Molecular Devices, Sunnyvale, CA). The density of glycine-positive cells was determined from confocal stacks comprising the entire INL of glycine-immunolabeled whole-mount retinas (two mice per genotype). The ‘cell counter’ plugin was used to determine glycine-positive cell numbers from substacks comprising the proximal INL for amacrine cells or distal INL for bipolar cells.The dendritic areas of the AII cells injected with the tracer were measured by drawing a convex polygon around the outermost tips of the dendrites, as revealed in a flattened stack. The average area for all injected AII cells was then used to define a circle for the plots in . For the distributions of coupled AII and ON cone bipolar cells relative to the injected AII cell for all WT and KO-Cx36-EGFP tracer-injection experiments, the x,y positions of the soma center (injected-AII and coupled cells) were determined from confocal stacks. The area of all tracer-coupled AII somata was defined as a circle whose radius was the distance from the farthest tracer-coupled cell to the injected AII. Similarly for cone bipolar cells, the centroid of all tracer-coupled cone bipolar cells was determined and the area of tracer-coupled cone bipolar somata was defined as a circle whose radius is the distance of the farthest cell from the respective centroid. This correction was necessary as the area of coupled cone bipolar cells was not always centered on the injected AII cell (for example, see ). The distances of all cone bipolar cells from the centroid are plotted (). Although the plot retains the relative location of coupled cone bipolar cells among each other, it randomly allocates the different sets of coupled cone bipolar cells around the center of the injected AII. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens