Axiophot light microscope

An even representation of the hippocampus over the rostral-caudal axis was obtained by selecting 6 coronal, bilateral sections of the hippocampus with 300 μm intersection distance, therewith including three sections rostral of Bregma −2.30 mm and three sections caudal of Bregma −2.30 mm. All quantifications were performed by a researcher blind to the experimental conditions.
BrdU+ cells in the sub granular zone (SGZ) of the dentate gyrus (DG) were counted manually on a Leica CTR5500 microscope (40× objective) using the Leica MM AF program (MetaMorph version 1.6.0, Nashville, TN, USA). DCX+ cells were manually counted in the SGZ and granular cell layer (GCL) of the DG on a Zeiss Axiophot light microscope (40× objective) with Microfire camera using StereoInvestigator software (MBF Bioscience, Williston, VT, USA). DCX+ cells were further classified for DCX+ cell maturation based on the morphological appearance; type I, horizontal cells without a process reflected immature, mitotic cells, type II cells with an apical process into the GCL reflected an intermediate stage and type III cells with a dendritic tree reaching to the molecular layer reflected the immature neuronal stage (Hoeijmakers et al., 2018 (link)).

For quantification, DCX+ cells were counted across 8 coronal, bilateral sections throughout the hippocampus on a Zeiss Axiophot light microscope (40× objective) with a Microfire camera using StereoInvestigator software (MBF Bioscience, Williston, VT, USA). 4 sections rostral and 4 sections caudal to bregma −2.30 mm (i.e. ± 300 μm intersection distance) were analyzed to obtain an even representation of the hippocampus over the rostral-caudal axis. All quantification procedures were performed by a researcher blind to the experimental conditions.
DCX-immunoreactive cell numbers in the granular cell layer (GCL) and subgranular zone (SGZ) of the hippocampus were quantified manually on a Nikon Eclipse Ni-E light microscope equipped with a Nikon DS-Ri2 Camera (Nikon, Japan). DCX+ cells were classified into three types based on morphology to reflect their relative maturity, namely 1) Proliferative, or horizontal cells without a process, 2) Intermediate, or cells with an apical process into the GCL, and 3) Postmitotic, or cells with a dendritic tree reaching the molecular layer (Hoeijmakers et al., 2018 (link)). The Cavalieri principle was used in order to estimate GCL, SGZ, and DG volume as previously described (Hoeijmakers et al, 2017 (link), 2018b (link)), with the GCL and SGZ volume used to calculate DCX+ cell density.