treatments was the way that, compared to vehicle, treatment with fluoxetine reduced the relationship from anxiety to FST immobility (p,0.05). Additionally, an increase in the negative relationship of BLA lesion to FST immobility approached significance (p = 0.066). Finally, as the beta weights show in Figure 6, specifically in the Vehicle group, the BLA lesion indirectly increased Ki67 levels by down-regulating anxiety (Table S9). Overall, in addition to the confirmation of the result obtained with the univariate analyses that fluoxetine treatment had a positive effect on BrdU-IR only when the BLA was lesioned, the main findings of the path analyses, that could not have been predicted from the univariate analyses, are the revelation that i) anxiety was critically related to BrdU levels, and this relationship varied with the lesion condition, being negative in sham animals and positive in BLA lesioned ones; and (ii) BLA lesions and low anxiety were critical factors to facilitate a negative relationship between cell proliferation and depression-like behavior. Discussion Our goal here was to investigate whether the amygdala plays a key role on fluoxetine modulation of the proliferation and survival of progenitor cells in the dentate gyrus of the adult hippocampus, and on its impact on an index of depression-like behavior. A lesion approach was applied to specifically study the contribution of the BLA. Since BLA lesions affected animals’ anxiety, and emerging evidence indicates that anxiety trait is linked to hippocampal neurogenesis [43,51,63], structural equation modeling (SEM) was applied to assess the relative contribution of both BLA lesions and anxiety on fluoxetine effects. In addition, we explored the rela- tionship of indices of cell proliferation and survival to the selected index of depression-like behavior (i.e., immobility in the forced swim test). We found evidence in sham-lesioned rats of a facilitating effect of chronic fluoxetine on cell proliferation, but not on cell survival, as recently reported in adult male rats [64]. The increase in cell proliferation [4,65,66] but see and lack of effect on cell survival [67,68] are in agreement with previous findings in the literature. See [69] for negative effects on cell proliferation and [4] for positive effects on cell survival. Both parametric statistics and SEM confirmed a key role for BLA lesions in enabling a facilitating effect of fluoxetine on the survival of recently generated cells, suggesting that under non lesion conditions, the BLA restrains the positive effects of the antidepressant on cell survival (Fig. 4). As a note of caution, it is important to notice that although in all cases amygdala lesions included at least 40% of the BLA, in some cases the maximum extension included adjacent basal and/or medial areas whose potential role in the present observations cannot be excluded. Interestingly, these results implicating the involvement of the BLA on hippocampal cell survival stimulated by fluoxetine, but not under basal conditions, are reminiscent of the BLA involvement in the modulation of memory consolidation in other brain regions (notably the hippocampus) induced by stress and emotions without affecting memory formation in itself [32,36,70]. A potential role for the amygdala in these results is supported by several examples of inhibitory effects of SSRIs on amygdala activity in animals [19,22] and humans as well [23], although activating effects were also reported [71], as evidence indicating that enhanced amygdala activity as induced by seizures associated with rapid electrical amygdala kindling increases the addition of newly generated granule cell neurons to the granular cell layer of the dentate gyrus [72]. Alternatively, the BLA might be exerting a modulatory influence on the hippocampal milieu that is relevant for fluoxetine- Amygdala and Neurogenesis stimulated cell survival. This concept is linked to the view that the amygdala is able to affect both the induction of plasticity [73,74] and meta-plasticity [35] in the dentate gyrus. Translated into the current framework, amygdala influences on cell survival might be achieved by BLA modulation of fluoxetine’s molecular and network targets in the hippocampus. The stacked model (Fig. 4) revealed a surprising relationship between anxiety and cell survival that differed upon the lesion condition: while in Sham animals anxiety levels negatively predicted cell survival, this relationship was positive in BLA lesioned rats. Given the strong anxiolytic effects induced by BLA lesions, these results reveal the existence of an inverted-U shape relationship between anxiety and cell survival, with both low and high extremes in anxiety being related to lower levels of cell survival than intermediate levels of anxiety. Although intriguing given its novelty in the field of neurogenesis, the existence of an inverted-U shape between emotionality and hippocampal function is not surprising. There are many examples in the literature for complex effects of stress on cognition [75,76,77,78,79] and hippocampal plasticity [80,81] showing similar biphasic relationships. Further studies should explore to what extent this observation for an inverted-U shape between anxiety and neurogenesis is applicable to the range of normal variation in anxiety trait or related, for the lower extreme of the anxiety spectrum to pathological conditions linked to amygdala damage. As for cell proliferation, our analyses in contrast revealed that BLA lesions seem to indirectly influence its rate through their effects in anxiety: by reducing anxiety levels, which are negatively related to cell proliferation, BLA lesions seem to have a positive impact in cell proliferation. In fact, anxiety on its own was a highly predictive factor of cell proliferation. This finding is in agreement with emerg- ing evidence supporting a link between anxiety and hippocampal neurogenesis. Individual differences in rats’ trait anxiety were related to the negative and positive impact, respectively, of chronic stress and chronic treatment with a corticotrophin releasing factor receptor 1 (CRFR1) antagonist on hippocampal cell proliferation [51]. Transgenic mice, in which hippocampal neurogenesis was specifi- cally impaired, exhibited a striking increase in anxiety-related behaviors [43]. It should also be noted that several studies did not find any impact of inhibiting neurogenesis on anxiety- or depression- related behaviors [9,82,83,84]. We also found that BLA lesions exerted antidepressive behavioral effects irrespective of fluoxetine treatment. These results are in agreement with previous observations showing attenuated anxiety- and depression-related behaviors following bilateral BLA lesions in animals [85] and humans [86], while anxiogenic-like effects after amygdala stimulation through kindling protocols [87]. Viral-induced increase in the molecular expression of the transcription factor CREB in the BLA in rats was also shown to result in changes in anxiety- and depression-like behaviors [88]. Moreover, anxiety here largely explained (partly via Ki67, and also via other unspecified factors) differences in depression-like behavior in vehicle treated animals. Note that anxiety was evaluated before drug treatment and that anxiety levels have been reported to be changed by fluoxetine [18,89]. Interestingly, these results are also in line with a large body of literature in humans indicating close links between high anxiety trait, depression and the propensity of the amygdala to show hyperactivation [42]. A specific goal of this study was to explore whether BLA lesions and anxiety might influence the link from hippocampal cell proliferation and survival to depression-like behavior, a topic that has received a great deal of attention in recent years. A view is emerging that pre-existing anxiety might determine the range of PLoS ONE | www.plosone.org 8 January 2010 | Volume 5 | Issue 1 | e8618