Golgi staining was done to study the neuronal morphologies (FD Rapid GolgiStain Kit, FD Neurotechnologies). Animals were anesthetized as described above. Brains were extracted and all protocols were performed according to the manufacturer's instructions. Three brains from each of the treated groups were dissected into 2-mm coronal pieces (Rodent Brain Slicer matrix, Zivic Instruments), mounted in freezing medium (TFM, Triangle Biomedical) and stored at −80 °C. Sections of 80–100 μm were cut using a cryostat and mounted on gelatin-coated microscope slides with solution C. We proceeded with the staining protocols by following the instructions included in the manual. Between 10 and 15 neurons of the hippocampus zone were analyzed from two animals from each group. Images of dendritic spines were acquired with a Zeiss Axio Observer Z1 with an Apotome microscope (magnification: Plan-APOCHROMAT; × 63 oil Dic NA. 1.4; Acquisition Software: ZEN 2011), and reconstructed in three dimensions using the NeuronStudio software for image processing to enable higher identification and to improve the quality of the spine analysis (Rodriguez et al, 2006 (link); Rodriguez et al, 2008 (link)). Dendritic spines were counted by Sholl analysis over a length of 20 μm with concentric circles of 10 μm (Sholl, 1953 (link)).