Fluoxetine enhances behavior, neuronal number and activity

To define the involvement of neurogenesis and newborn neuronal activity in antidepressant action, first we treated C57Bl/6 mice with fluoxetine for 14 days and performed the tail suspension test (TST), the zero maze (ZM) and open-field tests (OFTs) to evaluate depression- and anxiety-like behaviors together with an analysis of locomotion (Supplementary Fig. 1). As expected, fluoxetine treated animals showed less immobility in the TST, spent more time in the open arms of the ZM, and also a greater proportion of distance in the center of the OFT chamber, indicating a reduction in depression- and anxiety-like behavioral phenotypes (Supplementary Fig. 1a–c). Additionally, there were no significant changes in the locomotor activity of these animals as assessed by the total distance traveled in the OFT (Supplementary Fig. 1d). Fluoxetine treatment also significantly increased the total number of dividing cells that are labeled with 5-bromo-2’-deoxyuridine (BrdU) or express Ki67 + in the SGZ of DG (Supplementary Fig. 1e–i), reproducing the well described effects of the antidepressants on neurogenesis1,2,12,16,18,32. We also examined expression of the immediate early gene c-Fos as a marker of neuronal activity. The number of c-Fos + cells was significantly increased in the DG after 14 days of fluoxetine treatment (Supplementary Fig. 1g, j). Taken together, these data indicate that fluoxetine treatment improves affective behavior in parallel with increases in neurogenesis and in neuronal activity in the DG.

Effects of silencing newborn neurons on behavior

To investigate whether newly generated neurons are required for the antidepressant effects of fluoxetine, we used a chemogenetic approach in combination with transgenic mouse models33. The designer receptors exclusively activated by designer drugs (DREADDs), hM4Di and hM3Dq, are synthetic variants of human muscarinic receptors, coupled to Gi and Gq proteins, respectively34,35. They are exclusively activated by the exogenous ligand clozapine-N-oxide (CNO) with high efficacy34. First, we expressed the inhibitory DREADD, hM4Di, in newly generated granule neurons by crossing a transgenic mouse line with a tamoxifen-inducible Cre recombinase under the control of the Ascl1 gene promoter36 to mice expressing STOP-floxed hM4Di (Ascl1-CreERTM;R26LSL−hM4Di double-transgenic line referred to as + hM4Di mice, see Methods). + hM4Di mice express HA-Tagged hM4Di in Type 2 transit amplifying neural progenitor cells (NPCs) and their progeny, as well as a small subset of Type 1 neural stem cells (NSCs) after the administration of tamoxifen37,38. Upon the binding of CNO, hM4Di induces the canonical Gi pathway resulting in hyperpolarization of the neurons35. This provided a way of specifically and exclusively suppressing excitability of newborn neurons after they have integrated into the hippocampal circuitry. We have already shown the specificity of Ascl1-CreERTM line, where high levels of recombination are seen in the SGZ of the DG32,36. To address the recombination efficacy of the hM4Di transgenes, we immunohistochemically analyzed HA-Tag expression in NPCs marked by SRY-box 2 (Sox2) and neuroblasts marked by doublecortin (Dcx) 2 days after 5 days of tamoxifen injection in adult animals. 69.4% ± 1.93% of HA-Tag + cells were colabeled with Sox2, while 27.4% ± 1.93% colocalized with Dcx, which showed that nearly all of HA-Tag-positive cells are initially precursors to the DG granule neurons. Subsequently, we examined expression of HA-Tagged hM4Di in the DG immunohistochemically after the recombined cells were allowed to mature for 21 days (Supplementary Fig. 2a) and found selective colocalization of the HA-Tag with granule cell markers. 86.6% ± 3.6% of the HA-Tag + cells were detected in NeuN + neurons in the granule cell layer, while only 2% ± 0.9 of them expressed the mature granule cell marker Calbindin, indicating that the majority of hM4Di + recombined cells were young granule neurons 3 weeks after tamoxifen injection (Supplementary Fig. 2b). Next, we confirmed CNO’s effect on newly generated DG neurons by performing patch clamp recordings from ±hM4Di neurons, which express YFP in the presence of hM4Di. We found that CNO decreased the number of action potentials elicited by depolarizing current injection in these neurons (Supplementary Fig. 2c, d), demonstrating that CNO mediated activation of hM4Di had a potent effect on the excitability of the neurons39. Additionally, expression of hM4Di without CNO treatment or exposing wild type adult-born DG neurons to CNO did not affect action potential firing (Supplementary Fig. 2c, d), which confirmed that both the hM4Di receptor and CNO are inert elements by themselves.

We then examined whether chronic chemogenetic in vivo inhibition of newly generated neurons alters the behavioral effects of fluoxetine. Transgenic mice received 3 weeks of daily intraperitoneal injection of fluoxetine or saline with or without chronic CNO supplementation in the drinking water (Fig. 1a). Inhibiting the activity of newborn neurons by CNO administration blocked the ameliorative effects of fluoxetine on affective behavior (Fig. 1b, c). Specifically, fluoxetine treatment decreased immobility in the TST in the absence of both hM4Di (-hM4Di) and CNO. This effect was reversed by CNO treatment to mice expressing hM4Di ( + hM4Di) (Fig. 1b). Interestingly, inhibiting activity of newborn neurons in the absence of fluoxetine ( + hM4Di + CNO in saline treated mice) increased immobility in the TST, indicating that the baseline activity of new neurons regulates behavior of mice in this test. Next, anxiety-like behavior was evaluated by the measurement of the distance traveled in the center of the OFT chamber (Fig. 1c). The findings paralleled the results with the TST. Fluoxetine treated −hM4Di and + hM4Di (−CNO) animals preferred to explore the center of the arena significantly more than saline treated animals, indicative of the anti-anxiety effects of fluoxetine. CNO treatment of mice expressing hM4Di blocked the anti-anxiety effects of fluoxetine, as indicated by the reduction in distance traveled in the center of the testing chamber compared to the other fluoxetine treated animals. Further, CNO treatment of + hM4Di animals in the absence of fluoxetine reduced the distance traveled in the center of the chamber, providing additional evidence that the baseline activity of new neurons regulates behavior even in the absence of fluoxetine. There were no significant differences among groups in terms of locomotor activity (Fig. 1d).

Fig. 1 Silencing newborn dentate gyrus neurons prevents the effects of fluoxetine and alters baseline affective behavior. a Timeline representing the experimental design for determining whether silencing of newborn neurons inhibits the effects of fluoxetine treatment in Ascl1-CreERTM;R26LSL−hM4Di mice. b Total time-spent immobile in tail suspension test, a measure of depression-like behavior (Fluoxetine F 1,60 = 23.11, ***P < 0.0001; hM4Di F 1,60 = 6.92, *P = 0.011; CNO F 1,60 = 15.60, ***P < 0.0001; hM4Di*CNO F 1,60 = 5.08, *P = 0.028; Tukey posthoc test + hM4Di + CNO Saline treatment versus other saline treatment groups *P < 0.05; + hM4Di + CNO Fluoxetine treatment versus other fluoxetine treatment groups *P < 0.05). c Distance spent in center exploration in open-field test, a measure of anxiety-like behavior (Fluoxetine F 1,60 = 42.32, ***P < 0.0001; hM4Di F 1,60 = 28.59, ***P < 0.0001; CNO F 1,60 = 5.27, *P = 0.025; hM4Di*CNO F 1,60 = 44.46, ***P < 0.0001; Tukey posthoc test + hM4Di + CNO Saline treatment versus other saline treatment groups **P < 0.01; + hM4Di + CNO Fluoxetine treatment versus other fluoxetine treatment groups ***P < 0.0001). d Total distance in open-field test, measure of locomotor activity (Fluoxetine F 1,60 = 0.002, P = 0.965; hM4Di F 1,60 = 1.297, P = 0.259; CNO F 1,60 = 0.138, P = 0.711; hM4Di*CNO F 1,60 = 0.030, P = 0.862). Data are presented as means ± s.e.m. and analyzed by three-way ANOVA Full size image

When we analyzed the distribution of the HA-Tagged hM4Di + cells in the DG, we found that fluoxetine treatment increased the number of HA-Tag + cells regardless of CNO treatment (Fig. 2a), however it did not have a significant effect on the maturation of these adult-born neurons; as assessed by double labeling of HA-Tag + cell with neuronal maturity markers, Sox2 (Saline: 3.71% and Fluoxetine: 3.45%), Dcx (Saline: 16.24% and Fluoxetine: 16.81%), NeuN (Saline: 77.73% and Fluoxetine: 76.51%), and Calbindin (Saline: 2.32% and Fluoxetine: 3.23%) (Supplementary Fig. 3). As expected, fluoxetine increased neurogenesis measured by an increased number of Ki67 + cells in the DG (Fig. 2b). Neither hM4Di nor CNO alone or together altered the level of neurogenesis (Fig. 2b) and neuronal maturation (Supplementary Fig. 3).

Fig. 2 CNO silencing does not alter the increase in neurogenesis after fluoxetine treatment. a Representative images and quantification of HA-Tag + dentate gyrus cells (By two-way ANOVA: Fluoxetine F 1,13 = 16.15, **P = 0.0015; CNO F 1,13 = 0.2021, P = 0.660; Interaction F 1,13 = 0.0009, P = 0.976). b Representative images and quantification of ki67-positive dentate gyrus cells (by three-way ANOVA: Fluoxetine F 1,32 = 59.52, ***P < 0.0001; hM4Di F 1,32 = 0.130, P = 0.721; CNO F 1,32 = 0.143, P = 0.708; hM4Di*CNO F 1,32 = 0.001, P = 0.980). Data are presented as means ± s.e.m. Scale bars 50 μm Full size image

To verify silencing effects of CNO on activity of newborn neurons in vivo, we examined the expression of immediate early genes Early Growth Response 1 (Egr1) and c-Fos, which are upregulated by neuronal activity40 (Fig. 3). Fluoxetine treatment increased the number of HA-Tag + Egr1 + double-labeled cells in the DG indicating an increase in newborn neuron activity, while CNO administration suppressed newborn neuronal activity in both Saline and Fluoxetine groups, as measured by decreased total of HA-Tag + Egr1 + cells (Fig. 3a, b). These findings confirmed that the inhibition of activity observed in CNO treated + hM4Di neurons in vitro (Supplementary Fig. 2) also occurred in vivo. Similarly, fluoxetine treatment increased the number of c-Fos immunoreactive cells in the DG with a representation of a more global increase in the DG activity (Fig. 3c, d). However, CNO treatment of + hM4Di mice abolished this effect of fluoxetine on c-Fos expression. CNO treatment of + hM4Di animals in the absence of fluoxetine also reduced the number of c-Fos + cells below baseline levels (Fig. 3c, d), correlating with the behavioral findings in the TST and OFT (Fig. 1), while CNO treatment of -hM4Di mice had no effect. Taken together, these findings indicate that the activity of newly generated neurons in the DG exerts profound effects on behavior. Further, activity of newly generated neurons is necessary for the behavioral effects of fluoxetine. While many prior studies have demonstrated the parallel effects of antidepressants on neurogenesis and behavior, our findings demonstrate that there is a causal relationship between the increased number and activity of newly generated neurons and the behavioral phenotypes.

Fig. 3 CNO prevents the fluoxetine-induced increase in activity of dentate gyrus neurons in vivo. a Representative images of HA-Tag + Egr1 + double-labeled dentate gyrus cells. b Quantification of HA-Tag + Egr1 + cells in the dentate gyrus (by two-way ANOVA: Fluoxetine F 1,16 = 4.288, P = 0.0549; CNO F 1,16 = 26.16, ***P = 0.0001; Interaction F 1,16 = 1.184, P = 0.293). c Representative images of c-Fos-positive dentate gyrus cells. d Quantification of c-Fos expression in the dentate gyrus. (by three-way ANOVA: Fluoxetine F 1,32 = 21.55, ***P < 0.0001; hM4Di F 1,32 = 9.01, **P = 0.005; CNO F 1,32 = 10.85, **P = 0.002; hM4Di*CNO F 1,32 = 6.14, *P = 0.019; Tukey posthoc test + hM4Di + CNO Saline treatment versus other saline treatment groups *P < 0.05; + hM4Di + CNO Fluoxetine treatment versus other fluoxetine treatment groups *P < 0.05). Data are presented as means ± s.e.m. Scale bars 50 μm Full size image

Activating newborn neurons leads to antidepressant effect

The findings described above demonstrated that the baseline level of activity of newly generated neurons regulates behavior. Next, we asked whether enhancing excitability of newly generated neurons without an increase in newborn neuron numbers would be sufficient to induce effects similar to those associated with fluoxetine treatment and increased neurogenesis. To explore this, we produced an Ascl1-CreERTM;R26LSL−hM3Dq double-transgenic line (referred as + hM3Dq mice), where HA-Tagged hM3Dq is expressed in NPCs and their progeny after administration of tamoxifen, similar to hM4Di expression. Upon the binding of CNO, hM3Dq induces the canonical Gq pathway resulting in increased firing of the neurons35. Three weeks after tamoxifen induced recombination, we tested the effects of acute activation of adult-born DG neurons by injecting CNO 2 h before behavioral testing (Fig. 4a), as plasma levels of CNO peak within 30 min and decline over the following 2 h41. Acute activation of newly generated adult DG neurons with CNO led to behavioral phenotypes similar to fluoxetine treatment. + hM3Dq CNO treated animals showed decreased total immobility in TST compared to Vehicle and –hM3Dq CNO groups (Fig. 4b), representing a reduction in depression-like behavior. At the same time, these animals were less anxious as measured by a significantly increased exploration in the center of OFT chamber (Fig. 4c). No significant differences were found in locomotor activity, assessed by OFT total distance (Fig. 4d). At the cellular level, acute hM3Dq activation significantly increased numbers of HA-Tag + Egr1 + double-labeled newborn granule neurons (Fig. 4e) and c-Fos + neurons globally in the DG (Fig. 4f). The number of newborn neurons and cell proliferation, as measured by the number of HA-Tag + and Ki67 + cells in the DG (Supplementary Fig. 5a, b) were not changed by either hM3Dq expression alone or by acute activation of hM3Dq expressing neurons by CNO. Thus, enhancing the excitability of small cohort of newborn DG granule neurons in the absence of changes in hippocampal neurogenesis leads to an antidepressant effect, further substantiating the role of newly generated neurons in regulating depression- and anxiety-like behaviors.

Fig. 4 Acute activation of newborn dentate gyrus neurons results in antidepressive effects. a Timeline representing experimental design for determining whether enhancing the activity of newborn neurons induces an antidepressant effect in Ascl1-CreERTM;R26LSL−hM3Dq mice. b Total time spent immobile in tail suspension test, a measure of depression-like behavior (hM3Dq F 1,20 = 5.86, *P = 0.025; CNO F 1,20 = 7.16, *P = 0.015; Interaction F 1,20 = 4.72, *P = 0.042; Tukey posthoc test + hM3Dq + CNO versus other groups *P < 0.05). c Center exploration distance in open-field test, a measure of anxiety-like behavior (hM3Dq F 1,20 = 4.42, *P = 0.048; CNO F 1,20 = 4.65, *P = 0.044; Interaction F 1,20 = 4.98, *P = 0.037; Tukey posthoc test + hM3Dq + CNO versus other groups *P < 0.05). d Total distance in open-field test, measure of locomotor activity (hM3Dq F 1,20 = 0.005, P = 0.945; CNO F 1,20 = 0.418, P = 0.525; Interaction F 1,20 = 0.927, P = 0.347). e Representative images and quantification of HA-Tag + Egr1 + double-positive cells in the dentate gyrus (t 6 = 2.459, *P = 0.049). f Representative images and quantification of c-Fos expression in the dentate gyrus (hM3Dq F 1,16 = 10.11, **P = 0.006; CNO F 1,16 = 5.31, *P = 0.035; Interaction F 1,16 = 13.79, **P = 0.002; Tukey posthoc test + hM3Dq + CNO versus other groups **P < 0.01). Data are presented as means ± s.e.m. and analyzed by two-way ANOVA (b, c, d, and f) or two-tailed Student’s t-test (e). Scale bar 50 μm Full size image

We tested whether increasing newborn neuronal activity is also sufficient to avert the adverse effects of unpredictable chronic mild stress (uCMS), which is known to elicit behavioral phenotypes associated with MDD that are reversed by fluoxetine treatment42. Ascl1-CreERTM;R26LSL−hM3Dq ( + hM3Dq) mice were exposed to 3 weeks of uCMS (Supplementary Table 1) and then evaluated behaviorally with or without acute CNO treatment to assess the effects of excitation of newborn neurons via hM3Dq receptor activation (Fig. 5a). Exposure to stress produced robust depression-like (Fig. 5b) and anxiety-like (Fig. 5c) behaviors as measured by increased total immobility in the TST and decreased center distance in the OFT, respectively, compared to the control vehicle group. By contrast, CNO-treated uCMS mice exhibited control levels of total immobility in the TST and center exploration in the OFT (Fig. 5b, c), indicating that increased newborn neuronal activity is sufficient to promote stress resilience. There were no locomotor activity differences among the experimental groups in the OFT (Fig. 5d). Parallel to behavioral changes, CNO administration significantly increased the number of c-Fos + cells in the DG in both control and uCMS conditions (Fig. 5e), though there was a significant reduction of c-Fos expression in uCMS exposed animals compared to controls. We also quantified the number of HA-Tag + cells in the DG and found that neither uCMS nor acute CNO treatment changed the numbers of HA-Tagged hM3Dq expressing cells (Supplementary Fig. 5), further validating the influence of newborn neuronal activity on behavioral phenotypes without changes of the number of neurons. The maturation of these newborn neurons was also not affected by these variables as assessed by double labeling of HA-Tag + cell with neuronal maturity markers; Sox2, Dcx, NeuN, and Calbindin (Supplementary Fig. 6). Thus, increased activation of newly generated neurons is sufficient to promote stress resilience.

Fig. 5 Acute activation of newborn dentate gyrus neurons reverses the behavioral effects of unpredictable chronic mild stress (uCMS). a Timeline representing the experimental design for determining whether acutely enhancing the activity of newborn neurons in adult hippocampus reverses the behavioral effects of unpredictable chronic mild stress (uCMS) in Ascl1-CreERTM;R26LSL−hM3Dq mice. b Total time-spent immobile in tail suspension test, a measure of depression-like behavior (uCMS F 1,27 = 11.23, **P = 0.002; CNO F 1,27 = 19.93, ***P = 0.0001; Interaction F 1,27 = 0.313, P = 0.581; Tukey posthoc test Control + Vehicle versus uCMS + Vehicle *P < 0.05). c Center exploration distance in open-field test, a measure of anxiety-like behavior (uCMS F 1,27 = 12. 1, **P = 0.002; CNO F 1,27 = 17.04, ***P = 0.0003; Interaction F 1,27 = 0.564, P = 0.459; Tukey posthoc test Control + Vehicle versus uCMS + Vehicle *P < 0.05). d Total distance in open-field test, measure of locomotor activity (uCMS F 1,27 = 0.242, P = 0.627; CNO F 1,27 = 0.267, P = 0.609; Interaction F 1,27 = 0.008, P = 0.930). e Representative images and quantification of c-Fos expression in the dentate gyrus (uCMS F 1,12 = 19.19, ***P = 0.0009; CNO F 1,12 = 22.01, ***P = 0.0005; Interaction F 1,12 = 0.088, P = 0.771; Tukey posthoc test Control + Vehicle versus uCMS + Vehicle *P < 0.05). Data are presented as means ± s.e.m. and analyzed by two-way ANOVA. Scale bar 50 μm Full size image

Our results demonstrate that activity of adult-born neurons in the DG of the hippocampus is both necessary and sufficient for antidepressant action of fluoxetine. Cell autonomous restriction of our experimental manipulations, without drug or radiation induced cell death43, and activating and silencing DREADD receptors specific to the population of newly generated neurons enabled us to establish this causal relationship. The pathophysiology of depression and anxiety is still poorly understood, and current medications are almost exclusively based on modifications of first-generation antidepressants, which were developed decades ago. Almost a third of patients fail to respond to any of the currently available medications, and treatment with these medications is associated with many significant complications and side effects44. Our results suggest that strategies that target both neurogenesis and activity of newborn neurons may lead to more effective antidepressants.