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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.

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Figure 1: 5HT1ARs in DG GCs are required for the behavioral effects of fluoxetine.
Figure 2: Viral-mediated deletion shows that 5HT1ARs in DG GCs are required for the behavioral effects of fluoxetine.
Figure 3: 5HT1ARs in young abGCs are not required for the behavioral response to antidepressants.
Figure 4: 5HT1ARs in DG GCs are sufficient for the behavioral effects of fluoxetine.
Figure 5: 5HT1ARs in DG GCs are necessary and sufficient for the neurogenic effects of fluoxetine.
Figure 6: 5HT1ARs in DG GCs are required for the neuroendocrine response to fluoxetine.
Figure 7: Fluoxetine-induced increases in BDNF and VEGF in the DG are attenuated in mice lacking 5HT1ARs in DG GCs.

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Acknowledgements

The authors thank K. Win and D. Tora for technical support, and M. Kheirbek and D. Leonardo for discussions. This work was supported by NIMH R37MH068542 (R.H.), NIMH R01MH083862 (R.H.), HDRF MPPN8883 (R.H.), NYSTEM C029157 (R.H.), NIMH K01MH098188 (B.A.S.), BBRF NARSAD Young Investigator 19658 (B.A.S.), a Charles H. Revson fellowship (B.A.S.), a German Research Foundation (DFG) postdoctoral fellowship (C.A.), NIMH T32MH015144 (Z.R.D.), NIMH R01MH01844 (A.D.), funds from EMBL (C.T.G.), and an EC Marie Curie Fellowship (N.M.).

Author information

Authors and Affiliations

Authors

Contributions

B.A.S. conceived and performed the experiments, analyzed the results, and wrote the manuscript. C.A. contributed to virus injection experiments. A.H. and M.R.L. performed mouse husbandry and contributed to most of the experiments. A.P., Z.R.D. and L.J.D. contributed to experiments and analysis. T.T., N.M. and C.T.G. performed mouse husbandry for 1A KO and DG-1A+ mice and carried out the experiments shown in Supplementary Figure 6. A.D. performed mouse husbandry for Nestin-CreER mice and contributed to analysis. K.F.T. made the fl1A mice and performed the experiments shown in Supplementary Figure 3. Most experiments were performed in the laboratory of R.H., who also conceived the experiments and contributed to the analysis and writing of the manuscript.

Corresponding author

Correspondence to René Hen.

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René Hen receives compensation as a consultant for Lundbeck and Roche.

Integrated supplementary information

Supplementary Figure 1 Wild-type and homozygous fl1A mice have similar behavioral and neurogenic responses to fluoxetine treatment.

a) Floxed 1A mice were generated and compared to wild-type littermates. White triangles indicates loxP sites. Htr1A p.: 5HT1AR promoter; Htr1A e1: 5HT1AR exon; pA: polyadenylation signal. Timeline is for panels b-h. Wild-type (WT) or homozygous fl1A (fl1A (Control)) mice were treated with daily fluoxetine (18 mg/kg) or vehicle administration that began when the mice were 8 weeks old. Behavior commenced three weeks after the start of fluoxetine treatment, and mice were administered BrdU (150 mg/kg) six weeks after the start of fluoxetine treatment and 2 hours before sacrifice. For behavior, n = 16-17 per group. For neurogenesis, n=8 per group randomly chosen from the behavioral cohort. b) NSF results. *** indicates p = .0001 for WT Vehicle vs Fluoxetine and p<.0001 for fl1A Vehicle vs Fluoxetine. Latencies were analyzed using Kaplan-Meier Survival Analysis with Bonferroni correction and Mantel-Cox p-values. c) Percentage weight loss and home cage consumption controls for the NSF experiment. The percentage of weight lost during the deprivation period and the amount of food consumed over 5 min when the mice were placed back into their home cage immediately after NSF exposure were calculated. No significant differences were seen among genotype and treatment groups (Two-Way ANOVA). d) EPM results. Bar graphs indicating open arm entries (left) and open arm duration (right) are shown. For both open arm entries (left) and open arm duration (right) there were only significant main effects of treatment (Two-Way ANOVA assessing genotype/treatment, *** indicates p<.0001 treatment effect for both open arm entries and open arm duration). e) FST results. For immobility duration there were only significant main effects of treatment (Two-Way ANOVA assessing genotype/treatment, *** indicates p<.0001 treatment effect). f) Proliferation results. For the number of BrdU-positive cells, there were only significant main effects of treatment (Two-Way ANOVA assessing genotype/treatment, *** indicates p<.0001 treatment effect). g) The number of abGCs. For the number of Dcx-positive cells, there were only significant main effects of treatment (Two-Way ANOVA assessing genotype/treatment, *** indicates p<.0001 treatment effect). h) The number of abGCs with tertiary dendrites. For the number of Dcx-positive cells with tertiary dendrites, there were only significant main effects of treatment (Two-Way ANOVA assessing genotype/treatment, *** indicates p<.0001 treatment effect). Bars and error bars throughout the figure represent mean ± SEM.

Supplementary Figure 2 NSF control experiments for Figures 14.

a) NSF controls for POMC-Cre/fl1A and Control mice in Figure 1. b) Percentage weight loss and home cage consumption controls for the NSF experiment. The percentage of weight lost during the deprivation period and the amount of food consumed over 5 min when the mice were placed back into their home cage immediately after NSF exposure were calculated. No significant differences were seen among genotype and treatment groups (Two-Way ANOVA). c) NSF controls for fl1A mice injected with AAV8-CamKII-GFP or AAV8-CamKII-Cre in Figure 2. d) Percentage weight loss and home cage consumption controls for the NSF experiment. The percentage of weight lost during the deprivation period and the amount of food consumed over 5 min when the mice were placed back into their home cage immediately after NSF exposure were calculated. No significant differences were seen among virus and treatment groups (Two-Way ANOVA). e) NSF controls for Nestin-CreER/fl1A mice in Figure 3. f) Percentage weight loss and home cage consumption controls for the NSF experiment. The percentage of weight lost during the deprivation period and the amount of food consumed over 5 min when the mice were placed back into their home cage immediately after NSF exposure were calculated. No significant differences were seen among genotype and treatment groups (Two-Way ANOVA). g) NSF controls for DG-1A+ and 1A KO mice in Figure 4. h) Percentage weight loss and home cage consumption controls for the Novelty Suppressed Feeding experiment. The percentage of weight lost during the deprivation period and the amount of food consumed over 5 min when the mice were placed back into their home cage immediately after NSF exposure were calculated. No significant differences were seen among genotype and treatment groups (Two-Way ANOVA). Bars and error bars throughout the figure represent mean ± SEM.

Supplementary Figure 3 In situ hybridizations for 5HT1AR and POMC in arcuate nucleus.

Top left) Complete section through dorsal DG and arcuate nucleus showing 5HT1AR in situ hybridization. Top right) Complete section through dorsal DG and arcuate nucleus showing POMC in situ hybridization. Bottom left) Close up of boxed region in top left showing 5HT1AR in situ hybridization in arcuate nucleus. Bottom right) Close up of boxed region in top right showing POMC in situ hybridization in arcuate nucleus.

Supplementary Figure 4 Effects of fluoxetine in Control mice for Figure 3.

All mice in this figure are homozygous fl1A (Control) littermates of the Nestin-Cre/fl1A mice shown in Figure 3. a) Timelines are for panels b-h. Control 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. For behavior, n = 15 mice per group. For neurogenesis, n = 8 mice per group that were randomly chosen from the behavioral cohort. b) NSF results. *** indicates significant effect of treatment and that p<.0001. Latencies were analyzed using Kaplan-Meier Survival Analysis with Bonferroni correction and Mantel-Cox p-values. c) Percentage weight loss and home cage consumption controls for the NSF experiment. The percentage of weight lost during the deprivation period and the amount of food consumed over 5 min when the mice were placed back into their home cage immediately after NSF exposure were calculated. No significant differences were seen among genotype and treatment groups (Two-Way ANOVA). d) EPM results. Bar graphs indicating open arm entries (left) and open arm duration (right) are shown. For both open arm entries (left) and open arm duration (right) there were only significant main effects of treatment (Two-Way ANOVA assessing pretreatment/treatment, *** indicates p<.0001 treatment effect for both open arm entries and open arm duration). e) FST results. For immobility duration there were only significant main effects of treatment (Two-Way ANOVA assessing pretreatment/treatment, *** indicates p<.0001 treatment effect). f) Proliferation results. For the number of BrdU-positive cells, there were only significant main effects of treatment (Two-Way ANOVA assessing pretreatment/treatment, *** indicates p<.0001 treatment effect). g) The number of abGCs. For the number of Dcx-positive cells, there were only significant main effects of treatment (Two-Way ANOVA assessing pretreatment/treatment, *** indicates p<.0001 treatment effect). h) The number of abGCs with tertiary dendrites. For the number of Dcx-positive cells with tertiary dendrites, there were only significant main effects of treatment (Two-Way ANOVA assessing pretreatment/treatment, *** indicates p<.0001 treatment effect). Bars and error bars throughout the figure represent mean ± SEM.

Supplementary Figure 5 5HT1AR expression in abGCs.

a) Construction of Nestin-CreER/fl1A mice and timeline of experiment in b. Floxed 1A mice were generated and crossed with Nestin-CreERT2 mice. Upon Cre-mediated excision, a yellow fluorescent protein (YPet) was expressed under control of the 5HT1AR promoter and marked the cells where 5HT1AR had been deleted. Timeline indicates pretreatment protocol used in experiment shown in b. Nestin-CreER/fl1A mice were gavaged twice per day for three days with 200mg/kg tamoxifen when they were 54, 55, and 56 days old. Mice were then sacrificed 2 days, 1 week (9 weeks old), 2 weeks (10 weeks old), 3 weeks (11 weeks old), and 4 weeks (12 weeks old) after the final tamoxifen administration. n = 6 per time point. b) Representative sections from Nestin-CreER/fl1A mice treated with tamoxifen when they were 8 weeks old and then sacrificed 3 weeks (11 weeks old; left) or 4 weeks later (12 weeks old, right). The hilus and dentate gyrus granule cell layer (GCL) are indicated. Scale bar = 60 μm.

Supplementary Figure 6 Developmental timecourse of 5HT1AR expression in dentate gyrus of DG-1A+ mice.

I-125 MPPI labeling. Sections are from dorsal or ventral dentate gyrus as indicated on left, and age E18, P7, or P15 mice as indicated on top. Arrows in P15 panels indicate dentate gyrus.

Supplementary Figure 7 Effects of fluoxetine in 5HT1AR-deficient mice.

a) Construction of 1A KO mice and timeline of experiments in b-h. 5HT1AR-deficient (1A KO) mice were previously described7,29. Timeline is for panels b-h. Wild-type (WT) or 1A KO mice were treated with daily fluoxetine (18 mg/kg) or vehicle administration that began when the mice were 8 weeks old. Behavior commenced three weeks after the start of fluoxetine treatment, and mice were administered BrdU (150 mg/kg) six weeks after the start of fluoxetine treatment and 2 hours before sacrifice. For the behavior, n = 15 per group. For neurogenesis, n = 8 mice per group that were randomly chosen from the behavioral cohort. b) NSF results. ** indicates p = .0036 (Kaplan-Meier Survival Analysis with Bonferroni correction and Mantel-Cox p-values). c) Percentage weight loss and home cage consumption controls for the NSF experiment. The percentage of weight lost during the deprivation period and the amount of food consumed over 5 min when the mice were placed back into their home cage immediately after NSF exposure were calculated. No significant differences were seen among genotype and treatment groups (Two-Way ANOVA). d) EPM results. Both open arm entries (F(1,56) = 10.21, p = .0023) and open arm duration (F(1,56) = 39.92, p<.0001) were analyzed by Two-Way ANOVA assessing genotype/treatment. *** indicates p = .0003 for Open Arm Entries and p<.0001 for Open Arm Duration (Tukey’s). e) FST results. Immobility duration (F(1,56) = 7.246, p = .0094) was analyzed by Two-Way ANOVA assessing genotype/treatment. *** indicates p<.0001 (Tukey’s). f) Proliferation results. The number of BrdU-positive cells was analyzed by Two-Way ANOVA (F(1,28) = 23.70, p<.0001) assessing genotype/treatment. *** indicates p<.0001 (Tukey’s). g) The number of abGCs. The number of Dcx-positive cells was analyzed by Two-Way ANOVA (F(1,28) = 79.56, p<.0001) assessing genotype/treatment. *** indicates p<.0001 (Tukey’s). h) The number of abGCs with tertiary dendrites. The number of Dcx-positive cells with tertiary dendrites was analyzed by Two-Way ANOVA (F(1,28) = 78.04, p<.0001) assessing genotype/treatment. *** indicates p<.0001 (Tukey’s). Bars and error bars throughout the figure represent mean ± SEM.

Supplementary Figure 8 Serum fluoxetine levels.

a) Serum fluoxetine levels in Control and POMC-Cre/fl1A mice. Control or POMC-Cre/fl1A mice were administered fluoxetine (18 mg/kg/day) starting at 8 weeks of age. Blood was collected three weeks after initiation of fluoxetine. There were no differences (Student’s t-test). n = 6 per group. b) Serum fluoxetine levels in Control and Nestin-CreER/fl1A mice. Control or Nestin-CreER/fl1A (8 wks) mice were administered fluoxetine (18 mg/kg/day) starting at 8 weeks of age. Nestin-CreER/fl1A (11 wks) mice were administered fluoxetine starting at 11 weeks of age. Blood was collected three weeks after initiation of fluoxetine. There were no differences (One-Way ANOVA). n = 6 per group. c) Serum fluoxetine levels in 1A KO and DG-1A+ mice. 1A KO or DG-1A+ mice were administered fluoxetine (18 mg/kg/day) starting at 8 weeks of age. Blood was collected three weeks after initiation of fluoxetine. There were no differences (Student’s t-test). n = 6 per group.

Supplementary Figure 9 The interaction between mature DG GCs and young abGCs during the antidepressant response.

SSRIs increase serotonin (5-HT) levels throughout the brain. Serotonin binds to somatodendritic 5HT1ARs (1A; red and blue cylinders) on mature DG GCs (blue cell) in order to trigger an antidepressant response. The mature DG GCs release growth factors such as BDNF and VEGF that enhance proliferation of neural progenitors and maturation of young abGCs (green cell). young abGCs may then modulate the mature DG GCs by acting on the local microcircuit. The resulting combined activity of the mature DG GCs and young abGCs defines the output of the dentate gyrus into a circuit that mediates the antidepressant response. The borders of the granule cell layer (GCL) with the molecular layer (ML) and Hilus are indicated with a dashed orange line.

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Samuels, B., Anacker, C., Hu, A. et al. 5-HT1A receptors on mature dentate gyrus granule cells are critical for the antidepressant response. Nat Neurosci 18, 1606–1616 (2015). https://doi.org/10.1038/nn.4116

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