in their drinking bottles [21]. Body weight and fluid intake were recorded regularly. The drug concentration was adjusted according to changes in water intake and body weight in order to reach the target dose of 10 mg/kg across the experiment. Vehicle groups received tap water during the same period. In order to control for water intake and thus fluoxetine dosage, a Plexiglas separator allowing visual and olfactory contact divided each housing cage into two parts. BrdU injections Rats were injected with the thymidine analog bromodeoxyur- idine (BrdU; 50 mg/kg, i.p.) (Sigma-Aldrich, St Louis, MO, USA) dissolved in a phosphate buffer (0.1 M, pH 8.4). Four injections were made, one every 12 hours over a period of two days, starting on day 21 of fluoxetine treatment [46]. Forced-swim Test One day after the last fluoxetine treatment, rats were submitted to a forced-swim test to evaluate depression-like behaviors [47,48]. Briefly, they were individually placed in a plastic beaker (25 cm diameter, 46 cm deep) containing 30 cm of water (2561uC) for 5 minutes. Even though this test is conventionally performed in two sessions, 24 hours apart, during which time spent in immobility is scored [49], we only performed one testing session. The reasons for applying a single forced swim session are the following: (i) The standard forced swimming test was developed for its sensitivity to evince behavioral effects of wide range of clinically active antidepressant drugs that, when injected on day 1 result in reduced immobility on the test performed on day 2 [49]. However, when the test has been used to assess whether chronic stress can affect behavioral ‘‘despair’’ and, hence, depression-like behavior, a single session has proven sensitive to evidence increased behavioral immobility in depressed animals [50,51]. (ii) The second important reason not to extend the protocol to a second forced swim test taking place on a subsequent day was related to the main goal of the study, aiming to compare the impact of different factors (notably, amygdala lesions and fluoxetine treatment) on hippo- campal cell proliferation. Since hippocampal cell proliferation and/or survival have been shown to be affected by prior stress [52,53], we reasoned that sacrificing animals immediately after a single stress session (forced swim) would not allow sufficient time for the stress-induced changes in cell proliferation to evolve. Behavior was recorded with a video camera and the time spent immobile (making only those movements necessary to keep the snout above the water), swimming, climbing or diving was quantified manually using a computer program (The Observer 5.0.25, Noldus IT, 2003). Histological procedures Fifteen minutes after the end of the forced-swim test, animals were anesthetized with isofluorane and transcardially perfused using 0.9% saline solution followed by a fixative solution of paraformaldehyde 4% in phosphate buffered saline (PBS, pH = 7.5). After perfusion-fixation, brains were removed and post-fixed in the same solution for four hours. Next, 50 mm coronal sections were cut on a vibratome (VT 1000S; Leica, Glattbrugg, Switzerland) and stored in 4uC PBS. Cell proliferation in the dentate gyrus was measured by assaying the expression of the endogenous marker, Ki67. Survival of proliferating cells was assayed by staining for BrdU. Alternate series of 1 in 10 sections were processed ‘‘free- floating’’ for immunohistochemical visualization of newborn neurons (Ki67) and mature neurons (NeuN), the latter helping determine the extent of the lesions. Briefly, sections were Amygdala and Neurogenesis incubated with 3% H2O2 in PBS for 10 minutes to block endogenous peroxidase activity. After washing in PBS (3610 minutes), the sections were treated for 1 hour with 5% normal donkey serum (NDS, Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS with 0.2% Triton-X100 (Sigma-Aldrich, St. Louis, MO). After washing in PBS, they were incubated overnight at room temperature with monoclonal mouse anti-Ki67 antibody (1:200; Novocastra) or monoclonal mouse anti-NeuN biotinylated antibody (1:200, Chemicon). PBS containing 0.2% Triton-X100 and 3% NDS (PBST) was used for primary and secondary antibodies dilution. After washing in PBS, sections for Ki67 were incubated for 120 minutes with donkey anti-mouse IgG, (1:250; Jackson ImmunoResearch Laboratories). Sections for NeuN did not require secondary antibody because the antibody used was biotinylated. After washing in PBS, sections were incubated during one hour with avidin-biotin-peroxidase complex (ABC; A 1:200, B 1:200; Vector Laboratories, Peterborough, UK), prepared 30 minutes before in PBS. And after washing in PBS, color development was achieved by incubating with 3,39 diaminobenzidine tetrahydrochloride (DAB; 0.5 mg/ml Sigma- Aldrich) for 15 minutes. For BrdU immunohistochemistry, a previous step of denatur- ation was performed: after blockade of the endogenous peroxidase, sections were incubated for 1 hour at 60uC, treated with 2 N HCl (30 min at 37uC) and then rinsed in borate buffer during 5 min (0.1 M; pH 8.4). After washing in PBS (3610 minutes), the sections were treated for 1 hour with 5% normal donkey serum (NDS, Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS with 0.2% Triton-X100 (Sigma-Aldrich, St. Louis, MO). After washing in PBS, they were incubated overnight at room temperature with a mouse monoclonal anti-BrdU antibody (1:500, Oxford). Following washes in PBS, sections were incubated for 120 minutes with donkey anti-rat IgG biotinilated antibody. Sections were processed in parallel, and immunoreactivities were visualized by the biotin–streptavidin technique (ABC kit; Vector Laborato- ries, Peterborough, UK) using 3,3-diaminobenzidine as chromo- gen (DAB; 0.5 mg/ml Sigma-Aldrich) [54]. Image analysis The total number of both Ki67 immunoreactive (IR) and BrdU- IR cells in the dentate gyrus was estimated using a modified version of the optical fractionator method on a systematic random sampling of every tenth section along the rostrocaudal axis of the hippocampal formation. Cell somata were identified and counted in both hemispheres, with a 40X objective using an Olympus BX51 light microscope linked to a computer. Cells appearing above and below the focal plane were omitted to prevent counting cell caps [54,55]. The extent of the BLA lesions was measured through images from NeuN stained serial sections. The border of the amygdala delineating the BLA and the border of the lesion were manually traced and the area determined with Scion Image software (available on the web at http://www.scioncorp.com). Measure- ments were taken bilaterally along the entire rostrocaudal axis of the BLA (6–9 sections for each brain) and the final values represent the left–right average [56,57]. Statistical analyses The SPSS 13.0 and Amos 17.0 (SPSS, Chicago, IL) statistical packages were used for the statistical analyses. Normality and homogeneity of variance was tested and adjusted statistics were used as required. Factorial analyses: Factorial analyses were applied to characterize animals according to their anxiety-like behaviors from the EPM. A PLoS ONE | www.plosone.org 3 January 2010 | Volume 5 | Issue 1 | e8618