Brain Sectioning and Neuron Density Quantification

The coronal sections of the brain containing the dorsal hippocampus were prepared at 10 μm thick via a cryostat (Thermo Scientific™ HM525 Cryostat). In brief, the brains were removed and fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) in 0.1 M phosphate buffer pH 7.4, 4°C overnight. Then, they were infiltrated with 30% sucrose (Merck, Germany) solution for 48–72 hours. Following the mentioned process, the serial sections of the brains were prepared at 10 μm thick and placed on slides coated with 0.3% aqueous solution of gelatin containing 0.05% aluminum potassium sulfate (Sigma-Aldrich, USA). Then, they were stained with 0.25% cresyl violet (Sigma-Aldrich, USA), dehydrated through graded alcohols (70, 95, 100%; 2x) (RCI Labscan, Thailand), placed in xylene (Merck, Germany), and mounted using a DPX mounting reagent (Merck, Germany). The assessment of neuron density in the prefrontal cortex and CA1, CA2, CA3 and the dentate gyrus of the hippocampus was performed under an Olympus light microscope model BH-2 (Japan) at 40x magnification. Counts were performed in three adjacent fields, and the mean number was calculated. The results were expressed as the density of neurons per 255 μm² [23 (link)].