Nis elements imaging software 3

The average density of positive cells per animal was quantified. Thus, the estimation of the number of cells per section (30 μm deep) in both hemispheres was calculated. Each structure analyzed consisted of ~6 coronal sections, which resulted in one of every five equidistant sections (one representative section for each 180 μm) according to the rostro-caudal extent. To outline the area of study, the region of interest was drawn in each structure, the identification of which was performed at Bregma −1.58 to −2.46 mm in hippocampal and hypothalamic levels and at Bregma 1.42 to −0.10 mm in striatal levels according to a mouse brain atlas and cytoarchitectonic criteria [58 ]. Thus, the BrdU-immunoreactive (+) nuclei and IdU-immunoreactive (+) nuclei that came into focus were manually counted in the hypothalamus and the subventricular zone (SVZ) of the lateral ventricles. Regarding the hypothalamus, counting was performed in the ventromedial (VMH) and arcuate (ARC) nuclei of the hypothalamus and median eminence. Quantification was performed using a standard optical microscope with 40× objective (Nikon Instruments Europe B.V., Amstelveen, The Netherlands) coupled to NIS-Elements Imaging Software 3.00 (Nikon). The data were expressed as the number of positive cells per section.

Representative counting frames were obtained using a standard optical microscope equipped with the 40 × objective (Nikon Instruments Europe B.V., Amstelveen, The Netherlands) and coupled to the NIS-Elements Imaging Software 3.00 (Nikon). The brain regions of interest for this study were the following: PrL, Str, BLA, and the hippocampal areas CA1 and CA3 and the dentate gyrus. A minimum of eight coronal sections per brain region were analyzed, which resulted in one of every six equidistant sections (one representative section for each 180 µm) according to the rostro-caudal extent. Estimations of the number of IBA-1-immunoreactive ( +) cells per area (mm²) in both hemispheres were manually counted according to stereological methods and calculated based on the following formula (Rivera et al., 2018 (link)): $$Na=\mathrm{\Sigma }\left(Q-\right)/\mathrm{\Sigma }\left(\mathit{astr}\right),$$ where ΣQ − is the total number of positive ( +) cells counted per animal, and a_str is the area (mm²) of the structure analyzed. Cell number quantification was expressed as the average number of IBA-1 + cells per area (mm²) for each experimental group. For densitometric analysis, quantification of IBA1 immunoreactivity is determined using ImageJ software (NIH, USA) and expressed as arbitrary units of average intensity of the image field that corresponds to representative areas of the brain regions.