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John Gobran, Delaney CM Henderson, Balwantray C Chauhan; Effects of 3D stratification of Retinal Ganglion Cells in Sholl Analysis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4822. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Sholl analysis is an analytical technique used to quantify the complexity of dendritic arbors of neurons. Previous studies have compared 2D and 3D Sholl analysis in CNS neurons, demonstrating a significant difference between 2D and 3D Sholl profiles due to extensive stratification of dendrites. Retinal ganglion cell (RGC) dendrites also stratify, to varying degrees, in the inner plexiform layer. The purpose of the present study was to compare 2D and 3D Sholl analysis parameters and determine the implications of RGC dendrite stratification in Sholl analysis.
Retinas from transgenic mice, with less than 0.5% of RGCs expressing yellow fluorescent protein (YFP; Thy-1 YFP line H; Jackson Laboratories, ME) were processed for immunohistochemistry and fluorescence imaging (z-stacks, Zeiss Apotome). Dendritic arbors were traced with the simple neurite tracer plugin (Fiji) and Sholl analysis (Bitmap plugin, Fiji) was performed on each z-stack (3D Sholl) and maximum projected image of the z-stack (2D Sholl) for each RGC. Sholl profiles were plotted for each cell and area under the curve (AUC) calculated for each method (2D vs. 3D Sholl). AUC provides a measure of dendritic arbor complexity. To determine whether estimates of dendritic complexity were altered based on measuring 2D vs. 3D RGC reconstructions; AUC was calculated for each cell, along with the maximum number of intersections.
AUC calculated from 2D Sholl profiles was, on average, 13.6% higher compared to those from the 3D images (Figure; mean (SD): 5889 (1738) vs. 6770 (1761), 3D vs. 2D; n=10, P < 0.01). The maximum number of intersections was also slightly higher in 2D images (by 10.1%) compared to 3D Sholl profiles (27.7 (7.0) vs. 30.9 (7.6), 3D vs. 2D; P = 0.01). The average arbour radius was 449 (67) microns away from the cell soma and the mean arbor thickness (depth of stratification) was 13.7 (5.1) microns. Therefore, the results of the present study show that the difference in Sholl profiles generated from 2D and 3D RGC reconstructions are not negligible.
Statistical analysis performed on 2D and 3D Sholl analysis suggests that the dendritic stratification of RGCs has an impact on Sholl analysis parameters.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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