Dendritic Morphology Analysis Using Confocal Microscopy

For assessment of dendrites and dendritic spine morphology, a Z-stack of the optical section was captured with a confocal microscope (Thorlabs, USA). For dendritic analysis 2048 × 2048 pixel images with 0.25 μm/pixel resolution were captured with Z interval of 1 μm using a 20× objective lens (NA = 0.85, UPlanSApo; Olympus, Japan). For dendritic spine analysis, 2048 × 2048 pixel images with 0.025 μm/pixel resolution were captured with Z interval of 0.1 μm using a 100× objective lens (NA = 1.4, UPlanSApo; Olympus, Japan). Quantitative analysis of dendritic length and branching was performed with the help of the Sholl analysis module of a freely available Neurostudio software package [56 (link)]. Quantitative analysis of dendritic spines, including measurements of dendritic spine head area, neck length, and neck length/dendritic spine length ratio, was performed using SpineJ software [41 (link)]. Before analysis, dendritic protrusions in cultured primary hippocampal neurons were classified as headed spines, which have clearly defined head and neck, filopodia—An extremely long protrusions and stubby, relatively short protrusions without neck. Quantitative analysis was performed only on headed spines. At least 18 transfected neurons from three independent experiments were used for quantitative analysis.