5-HT1A receptors on mature dentate gyrus granule cells are critical for the antidepressant response

Abstract

Selective serotonin reuptake inhibitors (SSRIs) are widely used antidepressants, but the mechanisms by which they influence behavior are only partially resolved. Adult hippocampal neurogenesis is necessary for some of the responses to SSRIs, but it is not known whether mature dentate gyrus granule cells (DG GCs) also contribute. We deleted the serotonin 1A receptor (5HT1AR, a receptor required for the SSRI response) specifically from DG GCs and found that the effects of the SSRI fluoxetine on behavior and the hypothalamic-pituitary-adrenal (HPA) axis were abolished. By contrast, mice lacking 5HT1ARs only in young adult-born GCs (abGCs) showed normal fluoxetine responses. Notably, 5HT1AR-deficient mice engineered to express functional 5HT1ARs only in DG GCs responded to fluoxetine, indicating that 5HT1ARs in DG GCs are sufficient to mediate an antidepressant response. Taken together, these data indicate that both mature DG GCs and young abGCs must be engaged for an antidepressant response.

Figure 1

5HT1ARs in DG GCs are required for the behavioral effects of fluoxetine. a) Floxed 1A mice were crossed with POMC-Cre mice. White triangles indicate loxP sites. Htr1A p.: 5HT1AR promoter; Htr1A e1: 5HT1AR exon; pA: polyadenylation signal. Timeline is for panels b–c. POMC-Cre/fl1A mice were sacrificed at 4 or 8 weeks and compared to control littermates sacrificed at 8 weeks. n=5 per group. b) I-125 MPPI labeling. Sections are from ventral dentate gyrus or dorsal raphe nucleus. c) I-125 MPPI quantification in DG. One-Way ANOVA: F_(2,12)=324.2, p<.0001. *** indicates p<.0001 (Tukey’s). n=5 per group. d) I-125 MPPI quantification in raphe nucleus. One-Way ANOVA: F(2,13)=.280, p=.7600. n=5–6 per group. e) Timeline for panels f–h. Control or POMC-Cre/fl1A mice were administered fluoxetine (18 mg/kg/day) or vehicle starting at 8 weeks of age. Behavior started three weeks after initiation of fluoxetine. n=19–27 per group. f) NSF results. Both bar graphs (left) and survival curves (right) indicating latency to eat are shown. *** indicates p<.0001 (Kaplan-Meier Survival Analysis with Bonferroni correction and Mantel-Cox p-values). g) EPM results. Open arm entries (left) and open arm duration (right) are shown. Both open arm entries (F_(1,89)=8.120, p=.0054) and open arm duration (F_(1,89)=6.435, p=.0129) were analyzed by Two-Way ANOVA. In the left panel, *** indicates p<.0001 (Tukey’s). In the right panel, *** indicates p=.0003 (Tukey’s). h) FST results. Immobility duration (F_(1,86)=9.769, p=.0024) was analyzed by Two-Way ANOVA. *** indicates p<.0001 (Tukey’s). Mean lines and error bars throughout the figure represent mean ± SEM. V: Vehicle. F: Fluoxetine.

Figure 2

Viral-mediated deletion shows that 5HT1ARs in DG GCs are required for the behavioral effects of fluoxetine. a) Floxed 1A mice were injected with either AAV8-CamKII-Cre or AAV8-CamKII-GFP virus. Timeline is for panels e–g. fl1A mice were injected with virus at 4 weeks of age and were then administered fluoxetine (18 mg/kg/day) or vehicle starting at 8 weeks of age. Behavior started three weeks after initiation of fluoxetine. n=14–15 per group for behavior. b) I-125 MPPI labeling. Sections are from ventral dentate gyrus or dorsal CA1. In top left panel, arrow indicates dentate gyrus. In bottom panels, arrows indicate CA1. c) I-125 MPPI quantification in DG. ** indicates p=.0010 (Student’s t-test with Welch’s correction). n=5 per group. d) I-125 MPPI quantification in CA1. p=.7593 (Student’s t-test). n=5 per group. e) NSF results. Both bar graphs (left) and survival curves (right) indicating latency to eat are shown. *** indicates p=.0004 (Kaplan-Meier Survival Analysis with Bonferroni correction and Mantel-Cox p-values). f) EPM results. Open arm entries (left) and open arm duration (right) are shown. Both open arm entries (F_(1,53)=13.95, p=.0005) and open arm duration (F_(1,53)=13.00, p=.0007) were analyzed by Two-Way ANOVA. *** indicates p<.0001 for open arm entries and p=.0004 for open arm duration (Tukey’s). g) FST results. Immobility duration (F_(1,54)=5.385, p=.0241) was analyzed by Two-Way ANOVA. * indicates p=.0142 (Tukey’s). Mean lines and error bars throughout the figure represent mean ± SEM. V: Vehicle. F: Fluoxetine.

Figure 3

5HT1ARs in young abGCs are not required for the behavioral response to antidepressants. a) Floxed 1A mice were crossed with Nestin-CreERT2 mice. Timelines are for panels b–d. Nestin-CreER/fl1A mice were pretreated with 200mg/kg tamoxifen or vehicle (three days, twice per day). Daily fluoxetine (18 mg/kg) or vehicle treatment began either when the mice were 8 weeks old (left, concurrent with the tamoxifen) or when they were 11 weeks old (right). Behavior commenced three weeks after initiation of fluoxetine treatment. n=15 per group. b) NSF results. Both bar graphs (left) and survival curves (right) indicating latency to eat are shown. *** indicates p<.0001 or p=.0002 (only for Vehicle Vehicle vs Vehicle Fluoxetine (11 wks)) (Kaplan-Meier Survival Analysis with Bonferroni correction and Mantel-Cox p-values). c) EPM results. Open arm entries (left) and open arm duration (right) are shown. *** indicates p<.0001 (Two-Way ANOVA; treatment effect only). d) FST results. *** indicates p<.0001 (Two-Way ANOVA; treatment effect only). Mean lines and error bars throughout the figure represent mean ± SEM. V: Vehicle. F(8): Fluoxetine (8 wks). F(11): Fluoxetine (11 wks).

Figure 4

5HT1ARs in DG GCs are sufficient for the behavioral effects of fluoxetine. a) DG-1A+ mice were generated and crossed with 5HT1AR deficient mice (1A KO). Timeline is for panel b. 1A KO and DG-1A+ mice were sacrificed at 8 weeks. n=5 per group. b) I-125 MPPI labeling. Sections are from ventral dentate gyrus or dorsal raphe nucleus. c) Timeline for panels d–f. DG-1A+ or 1A KO mice were administered fluoxetine (18 mg/kg/day) or vehicle starting at 8 weeks of age. Behavior started three weeks after initiation of fluoxetine. n=13–17 per group. d) NSF results. Both bar graphs (left) and survival curves (right) indicating latency to eat are shown. ** indicates p=.0079 (Kaplan-Meier Survival Analysis with Bonferroni correction and Mantel-Cox p-values). e) EPM results. Open arm entries (left) and open arm duration (right) are shown. Both open arm entries (F_(1,58)=7.204, p=.0095) and open arm duration (F_(1,58)=6.773, p=.0117) were analyzed by Two-Way ANOVA. ** indicates p=.0022 in left panel and p=.0086 in right panel (Tukey’s). f) FST results. Immobility duration (F_(1,58)=4.848, p=.0317) was analyzed by Two-Way ANOVA. *** indicates p=.0001 (Tukey’s). Mean lines and error bars throughout the figure represent mean ± SEM. V: Vehicle. F: Fluoxetine.

Figure 5

5HT1ARs in DG GCs are necessary and sufficient for the neurogenic effects of fluoxetine. a) Mice used for panels b–d were randomly chosen from behavioral cohort in Figure 1. n=8 per group. b) Proliferation results. The number of BrdU-positive cells was analyzed by Two-Way ANOVA (F_(1,28)=10.66, p=.0029). *** indicates p<.0001 for Control Vehicle vs Fluoxetine and POMC-Cre/fl1A Vehicle vs Fluoxetine and p=.0007 for Control Fluoxetine vs POMC-Cre/fl1A Fluoxetine (Tukey’s). c) The number of young abGCs. The number of Dcx-positive cells was analyzed by Two-Way ANOVA (F_(1,28)=5.292, p=.0291). *** indicates p<.0001, and * indicates p=.0247 for Control Fluoxetine vs POMC-Cre/fl1A Fluoxetine (Tukey’s). d) The number of young abGCs with tertiary dendrites. The number of Dcx-positive cells with tertiary dendrites was analyzed by Two-Way ANOVA (F_(1,28)=4.954, p=.0343). *** indicates p<.0001, ** indicates p=.0015, and * indicates p=.0212 (Tukey’s). e) Mice used for panels f–h were randomly chosen from behavioral cohort in Figure 3. n=8 per group. f) Proliferation results. *** indicates p<.0001 (Two-Way ANOVA; treatment effect only) g) The number of young abGCs. *** indicates p<.0001 (Two-Way ANOVA; treatment effect only). h) The number of young abGCs with tertiary dendrites. *** indicates p<.0001 (Two-Way ANOVA; treatment effect only). i) Mice used for panels f–h were randomly chosen from behavioral cohort in Figure 4. n=8 per group. j) Proliferation results. The number of BrdU-positive cells was analyzed by Two-Way ANOVA (F_(1,28)=23.7, p<.0001). *** indicates p<.0001 (Tukey’s). k) The number of young abGCs. The number of Dcx-positive cells was analyzed by Two-Way ANOVA (F_(1,28)=6.311, p=.0180). ** indicates p=.0014 (Tukey’s). l) The number of young abGCs with tertiary dendrites. The number of Dcx-positive cells with tertiary dendrites was analyzed by Two-Way ANOVA (F_(1,28)=8.031, p=.0084). ** indicates p=.0075 (Tukey’s). Mean lines and error bars throughout the figure represent mean ± SEM. V: Vehicle. F: Fluoxetine. F(8): Fluoxetine (8 wks). F(11): Fluoxetine (11 wks).

Figure 6

5HT1ARs in DG GCs are required for the neuroendocrine response to fluoxetine. a) Timeline for panels b–c. Control or POMC-Cre/fl1A mice were administered daily fluoxetine (18 mg/kg) or vehicle starting at 8 weeks of age. Blood was collected from mice in their home cage three weeks after the start of fluoxetine treatment and then again from the same mice one week later 45 minutes after EPM exposure. n=6 per group. b) Home cage plasma corticosterone levels. There were no differences (Two-Way ANOVA). c) Plasma corticosterone levels after EPM exposure. Corticosterone levels were analyzed by Two-Way ANOVA (F_(1,20)=5.585, p=.0284). * indicates p=.0117 (Tukey’s). d) Timeline for panels e–f. Nestin-CreER/fl1A mice were pretreated with 200mg/kg tamoxifen or vehicle (three days, twice per day). Daily fluoxetine (18 mg/kg) or vehicle treatment began either when the mice were 8 weeks old (left, concurrent with the tamoxifen) or when they were 11 weeks old (right). Blood was collected from mice in their home cage three weeks after the start of fluoxetine treatment and then again from the same mice one week later 45 minutes after EPM exposure. n=6 per group. e) Home cage plasma corticosterone levels. There were no differences (Two-Way ANOVA). f) Plasma corticosterone levels after EPM exposure. *** indicates p<.0001 (Two-Way ANOVA; treatment effect only). Bars and error bars throughout the figure represent mean ± SEM. g) Timeline for panels h–i. 1A KO or DG-1A+ mice were administred daily fluoxetine (18 mg/kg) or vehicle starting at 8 weeks of age. Blood was collected from mice in their home cage three weeks after the start of fluoxetine treatment and then again from the same mice one week later 45 minutes after EPM exposure. n=6 per group. h) Home cage plasma corticosterone levels. There were no differences (Two-Way ANOVA). i) Plasma corticosterone levels after EPM exposure. Corticosterone levels were analyzed by Two-Way ANOVA (F_(1,20)=8.878, p=.0074). ** indicates p=.0079 (Tukey’s). Mean lines and error bars throughout the figure represent mean ± SEM. V: Vehicle. F: Fluoxetine. F(8): Fluoxetine (8 wks). F(11): Fluoxetine (11 wks).

Figure 7

Fluoxetine-induced increases in BDNF and VEGF in the DG are attenuated in mice lacking 5HT1ARs in DG GCs. a) Timeline for panels b–c. Control or POMC-Cre/fl1A mice were administered daily fluoxetine (18 mg/kg) or vehicle starting at 8 weeks of age. DG was dissected and RNA was prepared three weeks after the start of fluoxetine treatment. n=3 per group. b) DG BDNF RNA expression levels were analyzed by Two-Way ANOVA (F_(1,8)=10.68, p=.0114). *** indicates p=.0004 (Tukey’s). c) DG VEGF RNA expression levels were analyzed by Two-Way ANOVA (F_(1,8)=6.749, p=.0317). *** indicates p=.0003, * indicates p=.0210 for POMC-Cre/fl1A Vehicle vs Fluoxetine, and p=.0266 for Control Fluoxetine vs POMC-Cre/fl1A Fluoxetine (Tukey’s). Mean lines and error bars throughout the figure represent mean ± SEM. V: Vehicle. F: Fluoxetine.