Figure 3

Treatment with fluoxetine boosts the generation and survival of newborn hippocampal neurons even after recurrent exposure to stress. (a and b) Quantification of the number of BrdU⁺ and BrdU⁺NeuN⁺ cells, per dentate gyrus (DG) area, revealed an increased production and survival of newborn neurons evoked by fluoxetine treatment. (c) Representative coronal section of the DG stained for BrdU (in red), NeuN (in green) and DAPI (in blue). (d) Density of newborn astroglial cells, identified as BrdU⁺GFAP⁺ cells, revealed no major effect of stress or ADs treatment. (e) Representative staining for BrdU (in red), GFAP (in green) and DAPI (in blue) in hippocampal DG. (f) Analysis of relative expression levels of STAT3 (upper panel) and BMP4 (lower panel) in the macrodissected DG corroborated the absence of major effects on gliogenesis in consequence of the recurrent stress exposure or AD treatment. (g and h) Quantitative analysis of Ki-67⁺ cells and amplifying progenitors, identified as Sox-2⁺Ki-67⁺ cells, revealed that fluoxetine leads to a depletion of DG progenitor cells. (i) Representative confocal image of Sox-2 (green) and Ki-67 (red) immunostaining. (j) Analysis of the number of DCX⁺Ki-67⁺ cells, representative of the neuroblasts population in the DG showed that animals subjected to repeated stress exposure, non-treated and treated with fluoxetine, presented a decreased number of neuroblasts. (k) Coronal section of the DG stained for DCX (in green), Ki-67 (in red) and DAPI (in blue). (l) Relative mRNA expression levels of NEUROD1 (upper panel) and DCX (lower panel) in the macrodissected DG revealed a decrease in the expression levels as a consequence of repeated stress exposure. In general, treatment with ADs restored the expression levels of both makers of neuronal maturation. Scale bars represent 30 μm. *Denotes the effect of unpredictable chronic mild stress (uCMS) analyzed by Student’s t-test; ^#denotes the effect of ADs, by comparison of treatment and SAL animals, analyzed by one-way analysis of variance (ANOVA). Data represented as mean±s.e.m. ^{*, #}P⩽0.05; n=± 4 animals per group. AD, antidepressant; CTRL, non-stressed animals; FLX, animals repeatedly exposed to uCMS and treated with fluoxetine; IMIP, animals repeatedly exposed to uCMS and treated with imipramine; SAL, animals repeatedly exposed to uCMS and non-treated.