hippocampal long-term potentiation (LTP) and spatial memory in rats [36,37]. Furthermore, animal studies have also shown that acute electrical stimulation of the amygdala (i.e., kindling) increases hippocampal neurogenesis [38,39,40,41]. Here, we investigated whether the amygdala and anxiety contribute to the effects of antidepressants on hippocampal cell proliferation and survival, and on a behavioral index of depression-like behavior (the forced swim test). We used a lesion approach targeting the basolateral amygdala together with a chronic treatment with the SSRI antidepressant fluoxetine. Anxiety (as indexed by animal’s anxiety levels before the onset of antidepressant treatment) was included in the analyses given its important modulatory role in both vulnerability and recovery from depression [42]. Moreover, given the interest for understanding how indices of both cell proliferation and survival, as well as of depression-like behavior relate to each other [3,8,9,43], the relationship between these factors was also explored. We applied parametric statistics followed by structural equation modeling (SEM) to test for unique and shared variance of the factors on depression-like behavior, as well as to assess how cell proliferation and survival are engaged by the interacting effects of fluoxetine, BLA lesions, and anxiety, in relation to the selected index of depressive-like behavior. Materials and Methods Subjects Adult male Sprague-Dawley rats (Charles River Laboratories, France) were used (250–275 g of body weight at the beginning of the experiment, approximately three months old). Rats were housed two per cage and maintained with ad libitum access to food under light (12 hours light/dark cycle; lights on at 7:00 AM) and temperature (2262uC) controlled conditions. Experimental pro- cedures were approved by the Cantonal Veterinary Authorities (Vaud, Switzerland). General Experimental Procedures Rats were submitted to stereotaxic surgery for Sham (n = 38) or BLA lesions (n = 43). After recovery from surgery (11 days), rats were handled again for 3 days and tested in the elevated plus-maze (EPM) to determine individual anxiety levels. Three days after testing in the EPM, Sham and BLA lesion groups were each divided into two subgroups that were matched according to anxiety levels and body weight, and assigned to the drug treatment ‘vehicle’ or ‘fluoxetine’. Therefore, the study comprised four groups: Sham lesion/Vehicle (Sh/Vh), Sham lesion/Fluoxetine (Sh/Flx), BLA lesion/Vehicle (BLA/Vh), and BLA lesion/Fluoxetine (BLA/Flx). Fluoxetine treatment was given daily during 5 weeks. Since the number of animals in each group was corrected after histological Amygdala and Neurogenesis verification of the extent of the lesions, the sample size for each group differed from the initial group size and is reported in the corresponding Results section. Bromodeoxyuridine (BrdU) injec- tions to evaluate survival of proliferating cells were given on days 21 and 22 of fluoxetine treatment. One day after the end of fluoxetine treatment, rats were tested in the forced-swim test and sacrificed 15 min after the test (see Figure 1). Elevated Plus Maze Anxiety levels were evaluated using the EPM test [44]. Briefly, the test consists of two opposing open arms (45610 cm) perpendicular to two enclosed arms (45610650 cm) that extend from a central platform (10610 cm), elevated 65 cm above the floor. The rats were placed individually on the central platform and allowed to explore the maze for five minutes. Their behavior was monitored using a video camera and analyzed with a computerized tracking system (Ethovision 3.1.16, Noldus IT, The Netherlands). The time spent in the open and closed arms, distance moved and transitions between the different arms were recorded. Surgery Rats were anesthetized using an intraperitoneal injection of a combination of 75 mg/kg of ketamine chlorhydrate (2-(2-chlorophe´- nyl)-2-me´thylamino-cyclohexan-1-one; Graeub, Bern, Switzerland) and 10 mg/kg of xylazine chlorhydrate (N-(2,6-dimethylphenyl)-5,6- dihydro-4H-1,3-thiazin-2-amine; Graeub, Bern, Switzerland). Rats were then placed into a stereotaxic apparatus (Stoelting, USA), the scalp was incised and retracted, the head position was adjusted to place Bregma and Lambda in the same horizontal plane and small burr holes were drilled in the skull above the basolateral complex of the amygdala to accommodate a 28 gauge infusion cannula (2.5 mm posterior to bregma, +/2 4.5 mm lateral to bregma, 8.2 mm ventral to the skull surface). A 10 ml Hamilton syringe mounted on an infusion pump (Harvard Apparatus, South Natick, MA) and connected to the cannula with polyethylene tubing was used to deliver infusions. BLA lesions were made by injecting bilaterally 0.4 ml of NMDA (N-methyl-D-aspartic acid) (20 mg/ml; Sigma, St. Louis, MO) in 100 mM PBS, pH 7.4, at a rate of 0.2 ml/min. Sham lesion rats underwent an identical procedure, except that the cannula was filled with vehicle, lowered and no infusions were made [45]. After surgery, animals were returned to their home cages and allowed 11 days of recovery followed by three days of handling before behavioral testing. Chronic fluoxetine treatment Fluoxetine hydrochloride ((7)-N-methyl-g-(4-[trifluoromethyl]- phenoxy)-benzenepropanamine) (Mepha, Aesch, Switzerland) was administered daily during 35 days in the rats’ tap water (10 mg/kg) Figure 1. Schematic plan of experimental procedures and timetable. After handling (H), rats were submitted to stereotaxic surgery for BLA or sham lesions. After recovery, animals were handled again and levels of anxiety were characterized using the Elevated Plus Maze (EPM). Next, chronic fluoxetine treatment was initiated and BrdU was injected after twenty one days of drug administration. After interruption of fluoxetine treatment, depression-like behavior was evaluated using the Forced Swim Test (FST) and then sacrificed. doi:10.1371/journal.pone.0008618.g001 PLoS ONE | www.plosone.org 2 January 2010 | Volume 5 | Issue 1 | e8618