Eclipse e600 microscope

Stereological cell counts were performed using StereoInvestigator (MBF Bioscience) on a computer interfaced with a Nikon Eclipse E600 microscope and MBF Bioscience CX9000 camera. Every third section (45 μm thick) was used for analysis for a total of four sections, and the amygdala, prefrontal cortex, nucleus accumbens, and paraventricular nucleus of both hemispheres were quantified. The boundaries of each region were drawn with a 4x objective, referencing a neonatal rat atlas (Ashwell and Paxinos, 2008 ), and the optical fractionator method was used to quantify microglia and BrdU+ cells at 40x magnification, using a 100 μm x 100 μm counting grid with a 250 μm x 250 μm sampling grid for microglia and a 50 μm x 50 μm counting grid with a 250 μm x 250 μm sampling grid for BrdU+ cells. The optical dissector height was set to 12 μm with a 2 μm guard zone on the top and bottom for both quantifications. Microglia were counted based on the presence of an observable cell body within the designated counting region and determined to be phagocytic if the microglia contained an observable phagocytic cup that was distinctly identifiable from the cell body. BrdU+ cells were counted if the nuclear staining was uniformly dark and was within the designated counting region.

Quantification of the number of BrdU+ cells across the four MeA subregions was performed using Neurolucida software (MBF Bioscience, RRID:SCR_001775) on a computer interfaced with a Nikon Eclipse E600 microscope and MBF Bioscience CX9000 camera. Contours outlining the boundaries of each subregion were drawn at 4x magnification, and the area of each subregion was recorded. Sections from three alternate series were used to quantify the total number of BrdU+ cells and the number colocalized with either GFAP, NeuN or Iba1. Cells in each subregion were counted at 20x magnification across both hemispheres of the MeA. Additionally, the total number of GFAP+ cells was quantified only in the MePD. The data were normalized to the area of the subregion to account for any volumetric differences in the subregions between the sexes and averaged across hemispheres to generate a density estimate. In all cases, BrdU+ cells were counted if the nuclear staining was uniformly dark and present within the boundaries of the designated subregion. BrdU+ cells were counted as colocalized if a well-defined BrdU+ nucleus was associated with an immunopositive (i.e., GFAP+, NeuN+, Iba1+) cell body. For a subset of samples, colocalization criteria was confirmed by confocal microscopy.