In the SGZ (Bregma −1.8 to −5.6 mm), BrdU-IR-positive cells were counted at 60× (oil immersion objective), while Ki67-IR and DCX-IR were counted at 40× magnification. Cell counts were obtained as number of IR positive cells/mm2 using the Bioquant Life Science Image Analysis System (USA). It has previously been established under similar conditions that cell counts obtained by this methodology result in estimates that are highly concordant with those obtained using stereology (Crews et al. 2004). In the SVZ (Bregma +1.0 to +0.2 mm), cell counts were obtained for Ki67-IR cells, while the densities of BrdU-, DCX- and SOX2-positive cells were too high to lend themselves to cell counting. We therefore first established, in a subset of sections, that optical density measurements were highly correlated with cell counts (r=0.92, p<0.0001, n=24), in agreement with a published comparison of these two measures within the dentate gyrus (Crews et al. 2004). Densitometry was then performed bilaterally, in 40× magnification, in 2×6 squares (25×25 μm) per section over Bregma levels +1.6 to −0.4 mm, and yielded mean integrated optical densities (IOD). Exposed and control animals sacrificed at their respective time-points were always processed in parallel.
