MF sprouting analysis by Timm staining

MF sprouting was analyzed as previously described^102,103. The extent of MF sprouting was assessed by estimating the fraction of the total volume of the granule cell layer plus molecular layer that was Timm⁺. To determine the Timm⁺ area in each section, a contour was drawn first around the granule cell layer plus inner molecular layer (Contour 1). The color images were then converted to black and white images to determine the Timm⁺ area within the Contour 1 by adjusting a darkness threshold tool. A Contour 2 was drawn to measure total granule cell layer + molecular layer area. The extent of MF sprouting in each section was then calculated as the % of Timm⁺-area within Contour 1 over the total granule cell layer + molecular layer area (Contour 2). For the entire hippocampus, the areas measured in each section were multiplied by six (1 every 6 sections were used) and 40 μm, and summed, to estimate volumes. Imaging processing and analysis was performed with ImageJ software.