Quantitative Analysis of Neuronal Density in Rat Brain

After the completion of all behavioral experiments, the rats were anesthetized with 5% isoflurane and i.p. somnopentyl (0.5 ml) on PND 70 and transcardially perfused with 4% paraformaldehyde. Brain tissues were collected and fixed with 4% paraformaldehyde, followed by 30% sucrose for cryoprotection. Tissues were cut into 50-μm sections using a microtome throughout the brain every 600 mm. To evaluate the hippocampal dentate gyrus and cerebral cortex, coronal sections of the brain were examined (bregma −1.4 mm to −8.6 mm). The sections were blocked with 0.6% H₂O₂ in phosphate buffered saline (PBS), then blocked using 3% normal donkey serum with 0.1% Triton-X100 in PBS for 30 min. Free-floating sections were stained with anti-neuronal nuclei (NeuN; product No. MAB377; dilution 1: 400; Merck Millipore) and secondary antibody (1:1,000; biotinylated donkey anti-mouse; Jackson ImmunoResearch Laboratories) (21 (link)). To evaluate the whole hippocampal dentate gyrus and cortex, the average numbers of tissue sections per brain were 8 and 12, respectively. Cell counts for positive cells per unit volume (mm³) were quantified using unbiased stereological counting techniques as previously described (Stereo Investigator version 10 stereology software, Micro Bright Field Europe EK, Magdeburg, Germany) (21 (link)).