= 6) and WT littermate male mice at 6 months of age were used. Mice were treated with donepezil at 1 mg*kg. Donepezil hydrochloride was dissolved in water with 1.8% 2-hydroxypropyl-β-cyclodextrin and administered through drinking water. During treatment, donepezil was freshly dissolved in water each week. Prior to treatment and after 4 weeks of treatment, ABR and DPOAE tests were performed to evaluate auditory function, following the same procedure described in Section 2.2.
Sections containing the brain nuclei of interest were selected by comparison with the Mouse Brain Atlas . Specifically, sections containing the subiculum and CA1 region of the hippocampus were selected as positive controls for amyloid deposition.
For each animal, 3–4 coronal tissue sections that included the Sub, CA1, AC, MGB, IC, CN, SOC, and MNTB were imaged with a Leica Stellaris 5 Inverted Confocal Microscope using a 5× or 10× objective lens as indicated in the figure legends. Imaging parameters were kept constant across all sections for each set of immunofluorescent labels. Experimenters were blinded to the genotypes and ages of the animals.
For the plaque coverage analysis, regions of interest outlining the above-mentioned structures were drawn on maximum z-projections of the acquired 6E10 images and the corresponding masks were generated with a custom ImageJ plugin. Images were subsequently thresholded and binarized using the minimum cross-entropy thresholding algorithm embedded in CellProfiler. The plaque coverage was calculated as the ratio between the number of pixels thresholded over all pixels in the ROIs.