Computational protocol: The Role of Activity in Synaptic Degeneration in a Protein Misfolding Disease, Prion Disease

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Protocol publication

[…] Electron microscopy images were taken from the stratum radiatum at a magnification of x20,000 or x40,000. The images were sampled in a quasi-random fashion as previously described but avoiding blood vessels, the major dendrites and rare cell somas within the stratum radiatum. Measurements of the PSD and pre-synaptic terminal areas were performed on asymmetric synapses at 2, 4 and 8 weeks after injection with BoNT/A (or vehicle) on images taken at x20,000 magnification. The area of a PSD was measured only if synaptic vesicles were present in the same section and typical membrane apposition between pre-synaptic and post-synaptic element was present. A total of 115–636 PSDs and pre-synaptic terminals were measured for each time point from three to six different sections at least ten sections apart for both BoNT/A and vehicle injected animals. The areas of pre-synaptic terminals containing synaptic vesicles and PSDs were included only if synaptic terminal profiles were clearly visible. In a subset of the fields taken at x40,000 the size (equivalent diameter), circularity and density of synaptic vesicles was measured in asymmetric synapses from BoNT/A-treated and control animals at 4 weeks after injection. Density of vesicles was measured in 30 synaptic terminals per group from 3 vehicle- and 3 BoNT/A-injected mice. Circularity and vesicle diameter were determined from 1962 and 927 synaptic vesicles from BoNT/A- and vehicle-treated terminals. Measurement of PSD areas, presynaptic terminal areas, vesicle area and circularity were performed using ImageJ software (U.S. National Institutes of Health, http://rsb.info.nih.gov/ij/download.html). The identities of images were coded and only revealed to the observer after the data analysis was complete.To examine synaptic degeneration, images from prion animals were taken from the stratum radiatum at a magnification of x20,000. The analysis was conducted in ME7-only animals (10, 12, 16 and 18 weeks after injection; n = 3 per time point) and ME7 mice treated with vehicle (ME7+vehicle; n = 4) or BoNT/A (ME7+BoNT/A; n = 4). For each image (52–136 images per condition) we determined the number of degenerating and healthy synapses. We scored as degenerating terminals those boutons with a typical dark appearance and a curved PSD . Results were expressed either as the percentage of degenerating/healthy synapses in each condition or as the mean number of degenerating/healthy synapses per image.The density of Iba-1-positive cells was quantified by means of the Stereo Investigator software (MicroBrightField, USA), using three-dimensional counting boxes (100 µm×100 µm×30 µm) positioned in the stratum radiatum of the CA1 region. For each animal, at least five sections were examined with the operator blind to experimental treatment. [...] Statistical analysis was performed with SigmaPlot (version 11). Differences between two groups were assessed with Student's t-test for data normally distributed, and with Mann-Whitney rank sum test for data non-normally distributed. Differences between four groups were evaluated with one way analysis of variance (ANOVA) followed by Holm-Sidak test or Dunn's test. Normality of distributions was assessed with a Kolmogorov-Smirnov test. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, cryo-EM, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Parkinson Disease, Prion Diseases, Neurodegenerative Diseases