Quantitative Dendritic Morphology Analysis

Images of GFP-transfected hippocampal neurons were captured with a 20× dry lens using Leica SP8 confocal microscopy systems. Five to eight serial optical sections (Z-interval: 1 μm) were collected. The maximum projection of the serial images and subsequent analyses were performed using ImageJ (version 1.52a) software. Dendritic morphology was manually traced using the Simple Neurite Tracer^{56 (link)} plugin embedded in ImageJ software followed by analyses of neurite number, neurite length, and dendritic tree complexity. Sholl analysis^{57 (link)} was performed individually for each neuron from all groups. In brief, the center of the cell soma was set as the center of a series of concentric circles with radii at 10-μm intervals. Then, the intersections between the dendritic tree and the concentric circles were counted. Dendritic morphogenesis from transgenic mouse cortical neurons as indicated by Golgi staining was captured using a Leica DM6000 B compound microscope. The three-dimensional structure of dendritic trees was reconstructed by manual tracing using the filament tool embedded in Imaris (version 8.3.0). Dendrite number and length were also determined using the filament tool.