Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression

Abstract

Understanding the physiopathology of affective disorders and their treatment relies on the availability of experimental models that accurately mimic aspects of the disease. Here we describe a mouse model of an anxiety/depressive-like state induced by chronic corticosterone treatment. Furthermore, chronic antidepressant treatment reversed the behavioral dysfunctions and the inhibition of hippocampal neurogenesis induced by corticosterone treatment. In corticosterone-treated mice where hippocampal neurogenesis is abolished by X-irradiation, the efficacy of fluoxetine is blocked in some, but not all, behavioral paradigms, suggesting both neurogenesis-dependent and -independent mechanisms of antidepressant action. Finally, we identified a number of candidate genes, the expression of which is decreased by chronic corticosterone and normalized by chronic fluoxetine treatment selectively in the hypothalamus. Importantly, mice deficient in one of these genes, beta-arrestin 2, displayed a reduced response to fluoxetine in multiple tasks, suggesting that beta-arrestin signaling is necessary for the antidepressant effects of fluoxetine.

Figure 1. Chronic antidepressant treatment following corticosterone-induced behavioral changes

(A–B) Effects of 3 weeks of antidepressant treatment (IMI: imipramine; FLX: fluoxetine), started after 4 weeks of corticosterone (35 ug/ml/day), on anxiety behaviors in the Open-Field. Anxiety is measured as mean of the total time spent in the center in seconds (A). Locomotor activity measured as total ambulatory distance traveled (B). Values plotted are mean±SEM (n=10–12 per group). **p<0.01; #p<0.05, ##p<0.01, versus control group and corticosterone/vehicle group respectively. (C) Effects of chronic antidepressant treatment after 7 weeks of corticosterone, on anxiety- and depression-like behaviors in the Novelty Suppressed Feeding paradigm. Results are expressed as mean of latency to feed in seconds. Values plotted are mean±SEM (n=10–12 per group). **p<0.01, ##p<0.01, versus control group and corticosterone/vehicle group respectively. (D) Effects of chronic antidepressant treatment after 7 weeks of corticosterone in the mouse Forced Swim Test. Results are expressed as mean of mobility duration in seconds. Values plotted are mean±SEM (n=10–12 per group). **p<0.01, ##p<0.01, versus control group and corticosterone/vehicle group respectively. (E) Effects of chronic antidepressant treatment on corticosterone induced deterioration of the coat state. Results are expressed as the total resulting from the sum of the score of five different body parts. Values plotted are mean±SEM (n=10–12 per group). **p<0.01; #p<0.05 versus vehicle group and corticosterone/vehicle group respectively. (F) Effects of chronic antidepressant treatment on corticosterone induced anxiety- and depression related behaviors in the splash test. Results are expressed as mean frequency of grooming after squirting a 10% sucrose solution on the mouse’s snout. Values plotted are mean±SEM (n=10–12 per group). **p<0.01, ##p<0.01, versus control group and corticosterone/vehicle group respectively. (G) The effects of 3 weeks of antidepressant treatment (reboxetine 20 mg/kg/day; fluoxetine, 18 mg/kg/day), started after 4-weeks of corticosterone (35 ug/ml/day), on anxiety behaviors in the Elevated plus. Anxiety is expressed as mean total entries in the open arms. Values plotted are mean±SEM (n=12–15 per group). **p<0.01, versus corticosterone/vehicle group. (H) Effects of chronic antidepressant treatment on corticosterone induced behavior in the Tail Suspension test. Results are expressed as mean of mobility duration in seconds. Values plotted are mean±SEM (n=12–15 per group). *p<0.05, **p<0.01, versus corticosterone/vehicle group.

Figure 2. Fluoxetine stimulates cell proliferation, survival and dendritic maturation of young neurons in the dentate gyrus of the adult hippocampus

(A) BrdU (150 mg/kg) was given 2 hours before sacrifice to examine the effects of 7 weeks of corticosterone (35 ug/ml/day) ± fluoxetine (FLX, 18 mg/kg/day) during the last 3 weeks. Data are the mean±SEM of the BrdU-positive cell counts from 3–4 animals per treatment group for the SGZ and adjacent zone defined as a two-cell body wide zone along the hilar border (40X magnification). *p<0.05; ##p<0.01; §p<0.05 versus vehicle group, corticosterone/vehicle group and fluoxetine/vehicle group respectively. (B) BrdU was given twice a day for 3 days prior to drug treatment to examine the effects of 7 weeks of corticosterone ± fluoxetine during the last 3 weeks. Data are the mean±SEM of the BrdU-positive cells from 5–6 animals per treatment group. *p<0.05; #p<0.05 versus vehicle group and corticosterone/vehicle group respectively. (C–F) Images of doublecortin staining following corticosterone for 7 weeks ± chronic fluoxetine treatment for the last 3 weeks. 10x magnification and 20x for the inset. Left panels (D, F) are vehicle and right panels (E, G) are fluoxetine treated groups. (G) Effects of fluoxetine treatment on total number of DCX+ cells; mean±SEM (n=4 per group) were measured after 7 weeks of corticosterone. **p<0.01; #p<0.05; p<0.05§ versus vehicle group; corticosterone/vehicle group and fluoxetine group respectively. (H–I) DCX+ cells were categorized as to whether or not they exhibited tertiary dendrites. Effects of fluoxetine treatment on the DCX+ cells with tertiary dendrites (H) and maturation (I) of newborn granule cells were measured after 7 weeks of corticosterone. Values are mean±SEM (n=5 per group). **p<0.01; ##p<0.01; p<0.05§ versus vehicle group, corticosterone/vehicle group, fluoxetine/vehicle group respectively.

Figure 3. Neurogenesis-dependent and independent effects of chronic fluoxetine on corticosterone-induced behavioral changes

(A–D) The effects of fluoxetine (FLX, 18 mg/kg/day) treatment after focal X-irradiation of the mouse hippocampus on corticosterone (35 ug/ml/day) induced anxiety-like behaviors in the Open-Field. Anxiety is expressed as mean total of the time spent in seconds for each 5 min period (A), for the entire session (B) and also for the number of entries (C). Locomotor activity is reported as percentage ambulatory distance in the center over total ambulatory distance traveled (D). Values are mean±SEM (n=10–12 per group). *p<0.05, **p<0.01; #p<0.05, versus control group and corticosterone/vehicle group respectively. (EG) Effects of fluoxetine treatment after focal X-irradiation on corticosterone induced anxiety- and depression related behaviors in the Novelty Suppressed Feeding paradigm. Results are mean of latency to feed in seconds (E) or cumulative survival of animals that have not eaten over 10 minutes (G). Feeding drive was assessed by returning the animals to their home cage and measuring food consumed over a period of 5 min (mg/g of mouse) (F). Values are mean±SEM (n=10–12 per group). **p<0.01 versus SHAM corticosterone/vehicle group. (H) Effects of 3 weeks of fluoxetine treatment in 7 weeks corticosterone treated animals after X-irradiation on behavior in the Forced Swim Test. Results are mean of mobility duration in seconds. Values are mean±SEM (n=10–12 per group). **p<0.01 versus control group.

Figure 4. Effects of chronic fluoxetine treatment on corticosterone-induced changes in β-arrestin 1, β-arrestin 2 and Giα2 gene expression in mouse hypothalamus, amygdala and hippocampus

(A–C) Effects of fluoxetine (FLX, 18 mg/kg/day) treatment in corticosterone (35 ug/ml/day) treated animals on the mean β-arrestin 1, β-arrestin 2 and Giα2 gene expression (in % normalized to cyclophilin and GAPDH gene expression)±SEM (n=10–12 per group) in the mouse hypothalamus. *p<0.05; #p<0.05 versus control group and corticosterone/vehicle group respectively. (D–F) Same as above performed for the mouse amygdala. *p<0.05 versus control group. (G–I) Same as above performed for the mouse hippocampus. *p<0.05 versus control group.

Figure 5. The role of β-arrestin 2 in mediating the behavioral effects of chronic fluoxetine

(A–B) Effects of 4 weeks of fluoxetine treatment (18 mg/kg/day) in β-arrestin 2 knockout mice (βArr2-KO) and littermates on anxiety behaviors in the Open Field. Anxiety is expressed as mean time in the center (A). Locomotor activity is reported as ambulatory distance traveled for the entire session (B). Values are mean±SEM (n=15–18 per group). §p<0.05, versus fluoxetine treated wild-type mice. (C–D) Effects of chronic fluoxetine in β-arrestin 2 knockout mice and littermates in the light/dark paradigm. Results are mean total entries into the light (C). Locomotor activity is reported as ambulatory distance traveled in the dark (D). Values plotted are mean±SEM (n=9–10 per group). §p<0.05, versus fluoxetine treated wild-type mice. (E–F) Effects of chronic fluoxetine in β-arrestin 2 knockout mice and littermates in Novelty Suppressed Feeding. Results are mean of latency to feed in seconds (E) Feeding drive was assessed by returning the animals to their home cage after the test and measuring food consumed over 5 min (mg/g of mouse) (F). Values are mean±SEM (n=15–18 per group). *p<0.05, versus control group and fluoxetine treated wild-type mice respectively. (G) Effects of chronic fluoxetine in β-arrestin 2 knockout mice and littermates in the Forced Swim Test. Results are mean of mobility duration in seconds. Values are mean±SEM (n=15–18 per group). *p<0.05; #p<0.05, significant difference versus vehicle wild-type or β-arrestin 2 knockout animals respectively. (H) Effects of chronic fluoxetine in the splash test. Results are mean frequency of grooming after squirting a 10% sucrose solution on the mouse’s snout. Values are mean±SEM (n=9–10 per group). *p<0.05; §§p<0.01, versus vehicle wild-type group or fluoxetine treated wild-type respectively.