Fluoxetine effects on molecular, cellular and behavioral endophenotypes of depression are driven by the living environment

Abstract

Selective serotonin reuptake inhibitors (SSRIs) represent the most common treatment for major depression. However, their efficacy is variable and incomplete. In order to elucidate the cause of such incomplete efficacy, we explored the hypothesis positing that SSRIs may not affect mood per se but, by enhancing neural plasticity, render the individual more susceptible to the influence of the environment. Consequently, SSRI administration in a favorable environment promotes a reduction of symptoms, whereas in a stressful environment leads to a worse prognosis. To test such hypothesis, we exposed C57BL/6 mice to chronic stress in order to induce a depression-like phenotype and, subsequently, to fluoxetine treatment (21 days), while being exposed to either an enriched or a stressful condition. We measured the most commonly investigated molecular, cellular and behavioral endophenotypes of depression and SSRI outcome, including depression-like behavior, neurogenesis, brain-derived neurotrophic factor levels, hypothalamic-pituitary-adrenal axis activity and long-term potentiation. Results showed that, in line with our hypothesis, the endophenotypes investigated were affected by the treatment according to the quality of the living environment. In particular, mice treated with fluoxetine in an enriched condition overall improved their depression-like phenotype compared with controls, whereas those treated in a stressful condition showed a distinct worsening. Our findings suggest that the effects of SSRI on the depression- like phenotype is not determined by the drug per se but is induced by the drug and driven by the environment. These findings may be helpful to explain variable effects of SSRI found in clinical practice and to device strategies aimed at enhancing their efficacy by means of controlling environmental conditions.

Figure 1

Experimental design and effects of fluoxetine (FLX) administered either in enriched or stressful condition. (a) FLX treatment in the enriched condition. (b) FLX treatment in the stressful condition. In both cases, before treatment, mice are exposed to a 14-day period of stress to induce a depression-like phenotype. (c) Liking-type anhedonia (saccharin preference). Although no difference was found between FLX and vehicle (VEH) mice exposed to an enriched condition, a significantly increase of anhedonia (that is, reduction of saccharin preference) was found in FLX mice at the end of treatment in the stressful condition. VEH, n=23; FLX, n=24. (d) Wanting-type anhedonia (progressive ratio). When treatment was administered in the enriched condition, FLX led to a reduction of anhedonia (that is, a higher break point) at week 1, whereas in the stressful condition mice showed an increase in anhedonia (that is, lower break point) both at week 1 and 3, compared with VEH mice. Enrich: n=8 in all groups; stress: VEH, n=10, FLX, n=11. (e) Cognitive bias. In an enriched condition, FLX mice displayed a significantly ‘more optimistic' attitude than VEH by responding significantly more often to the ambiguous stimulus. No difference was found in the stressful condition. *P<0.05 vs relative VEH group. VEH, n=6, FLX, n=5. Data are means±s.e.m.

Figure 2

Fluoxetine (FLX) treatment administered in stressful condition leads to a reduction of proliferation and hippocampal volume. (a) Representative sections of histological (Giemsa) and immunohistochemical (Ki67 and doublecortin (DCX) counterstained with hematoxylin) stainings in the mid region of the matrix-embedded, straightened hippocampal dentate gyrus. Scale bar: 250 μm, insert scale bar: 10 μm. (b) Ki67 cell number was not significantly affected by FLX administered in enriched condition, whereas it was significantly decreased in FLX compared with vehicle (VEH) mice when treatment was administered in the stressful condition, *P<0.05 vs VEH group. (c) DCX cell number was not significantly affected by FLX administered in both conditions. (d) Schematic drawing of the straightened hippocampus. Gray part represents CA1. The analysis has been performed independently in the septal, mid and temporal part of the hippocampus, because it has been reported that the effects of SSRIs and the environment are region specific. Boundaries of hippocampal fields are illustrated in the Giemsa-stained section of the mid region of the straightened hippocampus cut perpendicular to the septotemporal axis. S, septal; T, temporal. (e) Analysis of anatomically aligned data of volumetric measurements showed no significant differences between groups in enriched condition. However, septal CA1 volume was significantly reduced in FLX compared with VEH when treatment was administered in stressful condition. *P<0.05. n=8 in all groups. Data shown as mean±s.e.m.

Figure 3

Fluoxetine (FLX) treatment affects hippocampal signaling pathways and HPA axis activity according to the quality of the environment. (a) No significant treatment effect was found for pERK1/ERK1 and in pERK2/ERK2 ratios in the enriched condition. Whereas both ratios were reduced in the cytoplasmic, but not in the nuclear, fraction in FLX mice in the stressful condition. (b) No treatment effect on CREB phosphorylation was observed in the nuclear enriched fraction in both environmental conditions. No difference in the total hippocampal CREB protein levels was found in the enriched condition, but it was reduced by treatment in the stressful condition. (c) In enriched conditions, reduced proBDNF and increased mBDNF levels were found in FLX mice compared with vehicle (VEH) mice. No difference in the stressful condition. (d and e) Real-time PCR analysis revealed that BDNF and p11 mRNA levels were significantly increased by FLX in the enriched condition but were not affected in the stressful condition. (f) Representative western blottings are shown. (g) Corticosterone levels were significantly reduced by treatment in FLX compared with VEH mice in the enriched but not in the stressful condition. (h) Glucocorticoid receptor (GR) mRNA expression was reduced by treatment in the stressful condition. No difference for GR expression in the enriched condition and mineralocorticoid receptor (MR) expression in both condition was found. MR, mineralocorticoid receptor. Corticosterone analysis: VEH, n= 6; FLX, n=7. For all the other analyses, n=8 in all groups. Data shown as mean±s.e.m. *P<0.05 vs respective VEH group.

Figure 4

Fluoxetine (FLX) modifies molecular and cellular correlates of synaptic plasticity in an environment-dependent manner. (a) Hippocampal levels of AMPA receptor subunits GluR1 and GluR2, and (b) their phosphorylation measured by western blotting. FLX induced opposite effects on GluR1 phosphorylation at Ser 845 according to the quality of the environment. (c) Hippocampal levels of NMDA receptor subunits GluN1, GluN2A and GluN2B measured by western blotting. (d) Representative western blots. n=8 for all groups in western blot analyses. Data shown as mean±s.e.m. of the control. *P<0.05 vs respective vehicle (VEH) group. (e) FLX increased paired-pulse ratio in the stressful condition but had no effect in the enriched condition. VEH: n=18/5; FLX: n=15/4. (f) FLX affect CA1 plasticity in mice exposed to stress condition. In the enriched condition, both FLX and VEH mice developed a robust LTP 45 min after stimulation (single 100 Hz burst; VEH: n=12/5; FLX: n=11/5). In the stressful condition, FLX mice showed a remarkable increase in the LTP amplitude compared with VEH (VEH: n=14/6; FLX: n=10/5). Arrows indicate time of application of HFS. fEPSP, field excitatorypostsynaptic potential; PPR, paired-pulse ratio. Data shown as mean±s.e.m. *P<0.05 and **P<0.01 vs respective VEH group.