Eutasil

In the last day of behavioral testing, animals were given single intraperitoneal injection of Bromo-deoxyuridine (BrdU, Sigma-Aldrich, 100 mg.kg⁻¹; thymidine analog that incorporates into DNA during the S-phase of the mitotic process). Twenty-four hours after the injection, animals were deeply anaesthetized with sodium pentobarbital (20%; Eutasil, Sanofi) and were transcardially perfused with cold 4% paraformaldehyde (PFA). Brains were removed and post-fixed in 4% PFA. Serial coronal cryosections (20 μm) were cut and stained for BrdU (1:50; Dako). Secondary antibody Alexa Fluor® 488 (Molecular Probes) was used for detection. Nuclei were counterstained using DAPI. Proliferation densities were estimated in the subgranular zone (SGZ; defined as a two-cell layer-thick zone on the inner side of the granule cell layer of the dentate gyrus), using a confocal microscope (Olympus FV1000).

For the three-dimensional morphometric analysis, four animals from each group were deeply anaesthetized with sodium pentobarbital (20%; Eutasil, Sanofi), transcardially perfused with 0.9% saline and processed. Briefly, brains were immersed in Golgi-Cox solution for 21 days; transferred to a 30% sucrose solution and cut on a vibratome. Coronal sections (200 μm) were collected in 6% sucrose and blotted dry onto gelatin-coated microscope slides. They were subsequently alkalinized in 18.7% ammonia, developed in Dektol (Kodak), fixed in Kodak Rapid Fix, dehydrated and xylene cleared before coverslipping. Dendritic arborization was analyzed in the dentate gyrus. For each selected neuron, all branches of the dendritic tree were reconstructed at 1000x (oil) magnification using a motorized microscope (BX51, Olympus) and Neurolucida software (Microbrightfield). A three-dimensional analysis of the reconstructed neurons was performed using NeuroExplorer software (Microbrightfield). For each animal, 10 neurons were studied and total dendritic length was determined.

All rats were killed by decapitation under intraperitoneal sodium pentobarbital (20% Eutasil®, Sanofi, Gentilly, France) anesthesia at the end of the study. After decapitation, the brain was rapidly removed and placed on an ice-chilled Petri plate. The whole amygdala was dissected from the brains (Paxinos and Watson, 2007 ) and stored at – 80 °C until further analyses. A necropsy was performed, and the adrenal glands were taken out, cleaned out of the surrounding tissues, and weighed (PR503, Mettler Toledo). The weight of the adrenal glands was divided by the weight of the rat to assess “relative weight,” and these data were used for later analyses.