Effects of 3D Stratification of Retinal Ganglion Cells in Sholl Analysis

Background: Sholl analysis is used to quantify the dendritic complexity of neurons. Differences between two-dimensional (2D) and three-dimensional (3D) Sholl analysis can exist in neurons with extensive axial stratification of dendrites, however, in retinal ganglion cells (RGCs), only 2D analysis is typically reported despite varying degrees of stratification within the retinal inner plexiform layer. We determined the impact of this stratification by comparing 2D and 3D analysis of the same RGCs. New Method: Twelve retinas of mice expressing yellow fluorescent protein in RGCs under the control of the Thy1 promotor were whole-mounted. The entire dendritic arbor of 120 RGCs was traced, after which 2D and 3 Sholl analysis was performed. Two parameters describing dendritic complexity; area under the curve (AUC) and peak number of intersections (PNI) were then derived and analyzed. Results and Comparison with Existing Methods: The AUC and PNI were significantly higher with 3D analysis compared to 2D analysis with medians of 2805 and 2508 units, and 31 and 27, respectively (P < 0.01). Both 2D and 3D AUC increased with arbor thickness. The discrepancy in AUC between the two methods depended on mean AUC (with every 1 unit increase in mean AUC resulting in a discrepancy of 0.1 unit), but not arbor thickness. Conclusion: In RGCs imaged in vitro, there is a difference in AUC and PNI derived with 2D and 3D Sholl analysis. Where possible, 3D Sholl analysis of RGCs should be performed for more accurate quantitative analysis of dendritic structure.