To determine the density of choline acetyltranferase (ChAT) immuno-reactive (IR) neurons, a technique using Bioquant imaging software was used by a blinded investigator with three to six sections per mouse (Crews et al., 2004). The region of the basal forebrain that had the greatest volumetric reduction as measured by MRI, (bregma +0.14 to −0.22) was identified on the sections using the adult mouse atlas (Franklin and Paxinos, 2001). This has been designated previously in rodents as the Ch4 region (Mesulam et al., 1983). Sections from three bregma locations were analyzed (+0.14,−0.10, and −0.22). These bregma locations were identified reliably using major landmarks in the mouse atlas (+0.14-the decussation of the anterior commissure, −0.10 shape of the posterior region of the anterior commissure and the triangular configuration of the fornices and dorsal third ventricle, −0.22 reduced size of the posterior region of the anterior commissure and the third ventricle). ChAT+IR neurons per immunohistochemical area of the region including the horizontal diagonal band, substantia innominata, and ventral pallidum was determined. The number of ChAT+IR neurons was counted manually using bioquant, and the
