Computational protocol: Amyloid plaque formation precedes dendritic spine loss

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Protocol publication

[…] All images were deconvolved using the adaptive blind 3D deconvolution algorithm of AutoDeblur with ten iterations (Version x2.0.1, Media Cybernetics Inc., Bethesda, MD, USA). The images were maximum intensity projected (Imaris 6.1, Bitplane, Zurich, Switzerland). In some figures, distracting neighboring dendritic elements were removed. Spines were counted in z-stacks by manually scrolling through the images of subsequent time points of the same position. The spine scoring method has previously been described [, , ]. Spine densities refer to the amount of spines per dendrite length in μm from which they protrude. Spine densities investigated on dendrites closer than 50 μm from a plaque were only analyzed on the segment that is located within the 50 μm radius from the plaque border. Spine morphology classification was performed by using the filament tracer module for 3D reconstruction in Imaris software (Version 7.4.2, Bitplane, Zurich, Switzerland). The volume measurements were done with the following parameters: small diameter 0.112 μm, large diameter 2 μm, and contrast threshold 0.3. Dendritic spines were classified due to their morphology into thin, stubby, and mushroom spines []. Filopodia spines were not found in the investigated brain region at 18–19 months of age. The geometrical classification rules from Harris et al. were interpreted by the following hierarchical expressions in the Imaris XT spine classification module: mushroom spines = “max_width(head)/min_width(neck) >1.4 and max_width(head) >0.5 and min_width(neck) >0”; stubby spines = “length(spine)/mean_width(neck) ≤3 or min_width(neck) = 0 or min_width(neck) >0.5”; thin spines = “length(spine)/mean_width(neck) >3” []. The volume of amyloid plaques was automatically calculated using Imaris software (Version 6.2, Bitplane, Zurich, Switzerland) as previously described []. The size of new born plaques was directly presented as volume (Fig. ), whereas for pre-existing plaques that were already present at the first imaging time point the radius was calculated assuming a spherical plaque shape []. All data are presented as mean ± SD or ±SEM or 95 % confidence intervals (95 % CI). Error types are stated where appropriate. Statistical differences in measurements over time were determined using repeated-measures ANOVA while statistical comparison between two groups was performed with Student’s t test. Multiple group comparison was done by one-way ANOVA followed by Tukey–Kramer post hoc test. The slope from a linear regression was tested for statistical difference from zero by F test. Distributions of morphological subtypes of dendritic spines were tested for differences by Chi-square test. All statistical analysis and graphs were done using Prism 5.04 (GraphPad Software Inc., La Jolla, CA, USA). Figures were arranged using Adobe Illustrator CS4. […]

Pipeline specifications

Software tools Imaris, Adobe Illustrator
Application Miscellaneous
Organisms Mus musculus
Diseases Alzheimer Disease