Chronic fluoxetine stimulates maturation and synaptic plasticity of adult-born hippocampal granule cells

Abstract

Chronic treatments with selective serotonin reuptake inhibitors (SSRIs) have been shown to increase hippocampal neurogenesis. However, it is not known whether SSRIs impact the maturation and functional integration of newborn neurons. Here we examined the effects of subchronic and chronic fluoxetine on the structural and physiological properties of young granule cells. Our results show that doublecortin-positive immature neurons displayed increased dendritic arborization after chronic fluoxetine treatment. In addition, chronic but not subchronic fluoxetine elicited a decrease in the number of newborn neurons expressing immature markers and a corresponding increase in those expressing mature markers. These results suggest that chronic fluoxetine accelerates the maturation of immature neurons. We also investigated the effects of fluoxetine on a form of neurogenesis-dependent long-term potentiation (LTP) in the dentate gyrus. This form of LTP was enhanced by chronic fluoxetine, and ablation of neurogenesis with x-irradiation completely blocked the effects of chronic fluoxetine on LTP. Finally, we demonstrated that the behavioral effect of fluoxetine in the novelty-suppressed feeding test requires chronic administration and is blocked by x-irradiation. These results show that the effects of fluoxetine on LTP and behavior both require neurogenesis and follow a similar delayed time course. The effects of chronic fluoxetine on the maturation and functional properties of young neurons may therefore be necessary for its anxiolytic/antidepressant activity and contribute to its delayed onset of therapeutic efficacy.

Figure 1.

Chronic but not subchronic fluoxetine treatment increases cell proliferation but not the number of DCX⁺ immature granule cells in the dentate gyrus. A, Schematic diagram of BrdU administration protocol to examine cell proliferation (n = 5–6 per group). Mice were treated with vehicle (Veh), 5 d of fluoxetine (5d Flx), or 28 d of fluexeinte (28d Flx). BrdU (150 mg/kg) was given 2 h before they were killed (Sac). B, The number of BrdU⁺ cells increased significantly after chronic (28d Flx) but not subchronic (5d Flx) fluoxetine treatment compared with vehicle-treated animals (ANOVA, F _(2,12) = 4.11, p = 0.043 for treatment). Fisher's post hoc analysis revealed significant differences between the chronic-treated group and both vehicle- and subchronic-treated groups (p < 0.05). The results are mean ± SEM of BrdU⁺ cells in the dentate gyrus. C, The total number of DCX⁺ cells did not change after subchronic and chronic fluoxetine treatment (ANOVA, F _(2,12) = 0.69, p = 0.52 for treatment). The results are mean ± SEM of DCX⁺ cells. D–G, Images of BrdU (D, E) and DCX (F, G) immunohistochemistry after chronic fluoxetine treatment. Images were taken at 20× magnification. D, F, Vehicle-treated groups. E, G, Chronic fluoxetine-treated groups.

Figure 2.

Chronic but not subchronic fluoxetine stimulates dendritic maturation of DCX⁺ cells. A, B, Categorization of DCX⁺ immature cells. We categorized DCX⁺ cells according to their dendritic morphology into DCX⁺ cells without tertiary dendrites (A) and DCX⁺ cells with tertiary dendrites (B) (n = 5–6 mice per group). C, Chronic [28 d of fluoxetine (28d Flx)] but not subchronic fluoxetine [5 d of fluoxetine (5d Flx)] increased the number of DCX⁺ cells with tertiary dendrites compared with vehicle (Veh)-treated animals (ANOVA, F _(2,12) = 7.31, p = 0.008 for treatment). Fisher's post hoc analysis revealed significant differences between vehicle- and chronic-treated groups (p = 0.006), as well as subchronic- and chronic-treated groups (p = 0.007). The results are mean ± SEM of DCX⁺ cells with tertiary dendrites. D, Neither chronic nor subchronic fluoxetine changed the number of DCX⁺ cells without tertiary dendrites (ANOVA, F _(2,12) = 0.98, p = 0.40 for treatment).

Figure 3.

Chronic but not subchronic fluoxetine enhances dendritic complexity of DCX⁺ cells. A, Representative image and traces from Sholl analyses of DCX⁺ cells with tertiary branches after vehicle (Veh), subchronic fluoxetine (5d Flx), and chronic fluoxetine (28d Flx) (n = 5 mice per group, 10–12 cells per mouse). B, Chronic but not subchronic fluoxetine increased dendritic length (ANOVA, F _(2,12) = 10.11, p = 0.003 for treatment). We also detected a treatment × radius interaction (F _(38,228) = 2.17, p < 0.001). Fisher's post hoc analysis revealed significant difference between vehicle- and chronic-treated groups (*p < 0.05). C, Chronic but not subchronic fluoxetine increased the number of intersections of DCX⁺ cells (ANOVA, F _(2,12) = 9.13, p = 0.004 for treatment). We also detected a treatment × radius interaction (F _(38,228) = 1.48, p < 0.001). Fisher's post hoc analysis revealed significant difference between vehicle- and chronic-treated groups (*p < 0.05). D–G, Representing images and traces from Sholl analysis of 3-week-old DCX⁺BrdU⁺ cells after 3 weeks of fluoxetine treatment. Hippocampal sections were double stained for DCX (D) and BrdU (E), and double-positive cells (F) were chosen to perform Sholl analysis on (n = 5 mice per group, 4–8 cells per mouse). H, Three weeks of fluoxetine (21d Flx) increased dendritic length compared with the vehicle group (Veh) (ANOVA, F _(1,8) = 17.68, p = 0.003). We also detected a treatment × radius interaction (F _(1,18) = 2.68, p = 0.0006). Fisher's post hoc analysis revealed significant difference between vehicle- and chronic-treated groups (*p < 0.05). I, Chronic fluoxetine also increased the number of intersections (ANOVA, F _(1,8) = 21.68, p = 0.002). We also detected a treatment × radius interaction (F _(1,18) = 2.34, p = 0.003). Fisher's post hoc analysis revealed significant difference between vehicle- and chronic-treated groups (*p < 0.05).

Figure 4.

Chronic fluoxetine facilitates the maturation of newborn granule cells. A, Schematic diagram of BrdU administration protocol to examine survival of newborn cells (n = 5–6 per group). Mice were given four BrdU injections (75 mg/kg) over 8 h on day 0. Vehicle (Veh) or fluoxetine (Flx) treatment began on day 1, 24 h after the last BrdU injection. Mice were killed 3 or 4 weeks later (Sac). B, C, Confocal images of BrdU (green), DCX (red), and NeuN (blue) immunohistochemistry. D, Chronic fluoxetine increased the number of total BrdU⁺ cells 3 and 4 weeks later compared with vehicle-treated groups (ANOVA, F _(1,16) = 12.63, *p = 0.003 for treatment; F _(1,16) = 24.50, p < 0.0001 for time). E, Chronic fluoxetine increased the number of BrdU⁺NeuN⁺ cells 3 and 4 weeks later (ANOVA, F _(1,16) = 8.89, *p = 0.01 for treatment; F _(1,16) = 30.12, p < 0.0001 for time). F, Chronic fluoxetine increased the number of BrdU⁺DCX⁻NeuN⁺ cells (ANOVA, F _(1,14) = 30.65, *p < 0.0001 for treatment; F _(1,14) = 2.38, p = 0.14 for time) but not the number of BrdU⁺DCX⁺NeuN⁺ cells (ANOVA, F _(1,14) = 1.47 × 10⁻⁴, p = 0.99 for treatment; F _1,14 = 62.52, p < 0.0001 for time). G, Chronic fluoxetine decreased the proportion of BrdU⁺NeuN⁺ cells that are DCX⁺ (percentage of BrdU cells) but increased the proportion that are DCX⁻ (ANOVA, F _(1,14) = 18.98, *p = 0.0007 for treatment; F _(1,14) = 132.64, p < 0.0001 for time).

Figure 5.

Effects of subchronic and chronic fluoxetine on hippocampal synaptic plasticity. A, B, Chronic fluoxetine (28d Flx; B) but not subchronic fluoxetine (5d Flx; A) reduces paired-pulse depression in both sham (Sham) and x-irradiated (x-ray) animals at stimulation intensity that elicited one-third of the maximal response compared with the vehicle group (Veh) (ANOVA, F _(1,29) = 9.05, *p = 0.005 for chronic treatment; F _(1,29) = 0.95, p = 0.34 for irradiation; F _(1,23) = 0.17, p = 0.68 for subchronic treatment; F _(1,23) = 0.31, p = 0.58 for irradiation). Inset, Representative traces of first response (1) and second response (2) (PPR, paired-pulse ratio, second response/first response). C, D, Both subchronic (C) and chronic (D) fluoxetine increased input–output relationships in both sham and x-irradiated animals (ANOVA, F _(1,36) = 11.46, p = 0.0017 in subchronic group for treatment; F _(1,36) = 0.62, p = 0.44 for irradiation; F _(1,27) = 16.72, p = 0.0003 in chronic group for treatment; F _(1,27) = 0.23, p = 0.63 for irradiation). Curves are fitted with a four-parameter logistic formula (McNaughton, 1980). E, Subchronic fluoxetine suppressed ACSF–LTP, and x-irradiation completely eliminates ACSF–LTP. F, ANOVA performed on the last 10 min of LTP recording revealed a significant main effect of irradiation (F _(1,25) = 7.28, p = 0.012) as well as a main effect of subchronic fluoxetine (F _(1,25) = 4.84, p = 0.037) (F). S, Sham; X, x-irradiation, V, vehicle; F, fluoxetine; Fisher's post hoc analysis were performed between individual groups (*p < 0.05). G, Chronic fluoxetine enhanced ACSF–LTP, and x-irradiation completely blocked LTP. Insets show averages of five consecutive fEPSPs at baseline (1) and in the last 10 min of LTP recordings (2). H, ANOVA performed on the last 10 min of LTP recording revealed a significant main effect of irradiation (F _(1,27) = 63.01, p < 0.0001), a main effect of chronic fluoxetine (F _(1,27) = 4.61, p = 0.041), as well as an irradiation × treatment interaction (F _(1,27) = 6.21, p = 0.019). Fisher's post hoc analysis were performed between individual groups (*p < 0.05).

Figure 6.

Behavioral effects of fluoxetine depend on adult neurogenesis. Novelty-suppressed feeding test on day 5 (A, B) and day 28 (C, D) of vehicle (Veh) or fluoxetine treatment. A, Five days of fluoxetine (5d Flx) did not reduce latency to feed in sham (Sham) or x-irradiated (x-ray) animals (Cum. Survival, cumulative survival, percentage of animals that have not eaten) (Kaplan–Meier survival analysis, Mantel–Cox log-rank test, p > 0.05). B, Box plot of latency to feed after 5 d of vehicle or fluoxetine. C, Twenty-eight days of fluoxetine (28d Flx) reduced latency to feed in sham but not x-ray animals (Kaplan–Meier survival analysis, Mantel–Cox log-rank test, p = 0.038 for treatment; *p < 0.05 between sham fluoxetine and the other three groups; p > 0.05 for all other groups). D, Box plot of latency to feed after 28 d of fluoxetine treatment. The box plot displays 10, 25, 50, 75, and 90% percentiles.

Figure 7.

Chronic fluoxetine stimulates dendritic maturation and synaptic plasticity of newborn granule cells, a possible mechanism for antidepressant action. A and B, from left to right, shows anatomical and functional stages during neuronal differentiation and maturation, including quiescent, radial glia-like progenitors (green), rapidly amplifying neural progenitors (light green), immature granule cells (red), and mature granule cells. Bottom panels show immunohistochemical markers for each stage. We can conclude from this study and others that fluoxetine stimulate adult neurogenesis in a multifold manner. Chronic fluoxetine treatment: first, increases proliferation of neural progenitors; second, stimulates dendritic branching as well as facilitates maturation; third, enhances survival of immature granule cells; fourth, enables young neurons to functionally integrate into the local hippocampal circuit, resulting in an enhancement of long-term synaptic plasticity. Finally, these synergistic actions lead to an improved behavior outcome. (Malberg et al., 2000a; Encinas et al., 2006).