behavioral effects displayed by chronic antidepressant treatment [9,69,90]. Our SEM analyses suggest that, in relation to the attenuation of depression-like behavior, reduced BLA output and low anxiety are critical factors determining the engagement of newborn cells—differentially affecting proliferative potential and survival. These findings might help explain existing discrepancies in the literature with regards to the dependence of behavioral effects of antidepressants on hippocampal neurogenesis [3]. In fact, antidepressant-like behavioral effects of chronic fluoxetine treatment were blocked by ablation of neurogenesis in the low anxious strain of mice, C57BL/6 [9], but not in the highly anxious strain BABL/c [91]. Although neurogenesis is frequently found to be unnecessary for fluoxetine effects in the forced-swim test in mice [8,91], it was shown to be critical in rats [92], supporting the specific link observed in our study. However, it is important to note that a recent study showed that antidepressants (including fluoxetine) retain their therapeutic efficacy in reducing both several indices of depression-like behavior (including the forced swim test) even when neurogenesis is pharmacologically blocked implicating, instead neuronal remodeling as the mechanism associated with the antidepressant activity [93]. Anxiety seems to be central to the converging effects of antidepressant treatment and BLA lesions. It emerged that modulation of cell proliferation by fluoxetine was particularly strong with a normally functioning BLA. Critically, the antide- pressant treatment was found to suppress the inhibitory indirect effect of the BLA by reducing the impact of anxiety on cell proliferation (Fig. 6). However, the relationship between anxiety and neurogenesis is still far from being clearly understood. A recent report showed that administration of the anxiolytic diazepam simultaneously to fluoxetine treatment completely blocked the SSRI-induced increase in both neurogenesis and cell survival [94]. Taken together, our observations would suggest that the behavioral effects of antidepressants seem to be antagonized by a normally functioning BLA. Furthermore, antidepressants and normal BLA function exert opposite, interacting influences over cell proliferation and survival that is largely associated with the resulting impact of anxiety (Fig. 3). In summary, our study highlights an important role for the amygdala on fluoxetine-stimulated cell proliferation and survival, as well as on the establishment of a link between cell proliferation and depression-like behavior. It also highlights an important role for anxiety in mediating the effects of basolateral amygdala and antidepressant activity on cell proliferation and survival. The application of structural equation modeling in the investigation of a complex, multidimensional phenomenon allowed us to capture important contributing factors operating together to determine the behavioral and neurobiological outcomes of fluoxetine treatment. Given the multiple targets of antidepressants effects in the brain [95,96], our study emphasizes the importance of considering indirect actions involved in their modulatory effects on adult hippocampal neurogenesis. Supporting Information Figure S1 BLA histochemical lesions with NMDA. A. Recon- struction of the minimal (black) and maximal (gray) extents of lesion. B. NeuN immunohistochemistry for representative BLA lesions. C. NeuN immunohistochemistry for representative sham References Amygdala and Neurogenesis lesion. Coordinates of the coronal sections are indicated with reference to Bregma. Plates are adapted from the atlas of Paxinos and Watson (Paxinos and Watson, 1997). Found at: doi:10.1371/journal.pone.0008618.s001 (1.80 MB TIF) Figure S2 Effect of BLA neurochemical lesion on anxiety levels. Values show the mean + SEM. **p,0.01. Found at: doi:10.1371/journal.pone.0008618.s002 (0.37 MB TIF) Figure S3 Levels of anxiety after classification of animals into the dichotomized categories of low and high anxiety. Values show the mean + SEM. **p,0.01. Found at: doi:10.1371/journal.pone.0008618.s003 (0.43 MB TIF) Table S1 Factor analysis for the Elevated Plus Maze. Found at: doi:10.1371/journal.pone.0008618.s004 (0.03 DOC) MB Table S2 Correlation matrix for anxiety levels, cell proliferation, cell survival, and depression-like behavior in the total sample and by groups of lesion and drug treatment. Found at: doi:10.1371/journal.pone.0008618.s005 (0.05 MB DOC) Table S3 Total and indirect effects for the general model in Figure 3. Found at: doi:10.1371/journal.pone.0008618.s006 (0.03 MB DOC) Table S4 Multiple-sample structural equation model analyses as shown in Figure 4. Found at: doi:10.1371/journal.pone.0008618.s007 (0.04 MB DOC) Table S5 Total and indirect effects for models in Figure 4. Found at: doi:10.1371/journal.pone.0008618.s008 (0.04 DOC) Table S6 Multiple-sample structural equation model analyses as shown in Figure 5. Found at: doi:10.1371/journal.pone.0008618.s009 (0.03 MB DOC) Table S7 Total and indirect effects for models in Figure 5. Found at: doi:10.1371/journal.pone.0008618.s010 (0.03 DOC) Table S8 Multiple-sample structural equation model analyses as shown in Figure 6. Found at: doi:10.1371/journal.pone.0008618.s011 (0.04 MB DOC) Table S9 Total and indirect effects for models in Figure 6. Found at: doi:10.1371/journal.pone.0008618.s012 (0.04 DOC) MB MB MB Acknowledgements We thank Coralie Siegmund, Alex Procter, Nicolas Perret, and Filip Vercruysse for their excellent technical assistance. Author Contributions Conceived and designed the experiments: JC EV CM MIC CS. Performed the experiments: JC EV. Analyzed the data: JC GP. Contributed reagents/ materials/analysis tools: CM MIC. Wrote the paper: JC GP CS. 1. Dranovsky A, Hen R (2006) Hippocampal neurogenesis: regulation by stress and antidepressants. Biol Psychiatry 59: 1136–1143. 2. Duman RS, Malberg J, Thome J (1999) Neural plasticity to stress and antidepressant treatment. Biol Psychiatry 46: 1181–1191. PLoS ONE | www.plosone.org 9 January 2010 | Volume 5 | Issue 1 | e8618