Introduction

Neural stem/progenitor cells (NSCs) located in the subgranular layer of the dentate gyrus of the hippocampus continuously produce primary projection neurons called dentate granule cells (DGCs) and these adult-born DGCs incorporate into the preexisting hippocampal neural circuits1,2,3,4. This hippocampal neurogenesis in the adult brain provides plasticity that has been shown to play a key role in learning and memory5. In addition to the role of adult-born DGCs in cognition, it has become clear that hippocampal neurogenesis is also required for the control of emotional status6,7. Previous seminal studies showed that fluoxetine, a selective serotonin reuptake inhibitor (SSRI), functions as an antidepressant by acting on hippocampal NSCs and thus enhancing neurogenesis8,9, while the blockage of neurogenesis abolishes the antidepressant function of fluoxetine9.

The distinct roles of hippocampal neurogenesis in cognition and emotion have raised an interesting possibility that adult-born DGCs may be functionally heterogeneous. This view has been supported by recent studies suggesting that the hippocampus is anatomically and functionally dissociated along the dorsoventral (DV) or septotemporal axis10,11,12,13. Selective ablation of the hippocampal sub-regions followed by behavioral tests, gene expression profiling, and functional imaging analysis strongly suggested that the dorsal (septal pole) hippocampus is involved in spatial learning, navigation, and memory while the ventral (temporal pole) hippocampus may mediate anxiety-related behaviors14,15,16,17. Furthermore, regional-specific blockage of neurogenesis by focal x-irradiation supported the possibility that the role of adult-born DGCs in different hippocampus-dependent functions is determined by the position of NSCs along the DV axis: adult-born DGCs in the dorsal hippocampus are required for acquisition of contextual discrimination whereas adult-born DGCs in the ventral hippocampus are necessary for the anxiolytic function of fluoxetine in “non-depressed” mice18.

This regional-specific requirement of adult-born DGCs for fluoxetine-mediated antidepressant function raised the possibility that NSCs may differentially respond to fluoxetine depending upon their location along the DV axis of the dentate gyrus of the hippocampus19. In this study, proliferation of NSCs in response to fluoxetine was quantitatively analyzed along the DV axis. Our approach showed that fluoxetine specifically increased proliferation of NSCs located in the ventral portion of the hippocampus, but not in the dorsal hippocampus, revealing a positional effect. Within the ventral portion of the hippocampus, fluoxetine specifically induced proliferation of type II NSCs and neuroblasts while mitotic activity of type I NSCs was unaltered. Moreover, epistatic analysis with pharmacological reagents demonstrated that serotonin receptor 1A (5-HTR1A) is a key downstream molecule that mediates the effect of fluoxetine on proliferation of type II NSCs and neuroblasts specifically in the ventral hippocampus. This positional effect on fluoxetine-induced NSC proliferation may be attributed to the contribution of the ventral hippocampus to emotional control.

Results

Regional-specific proliferation and survival of newborn cells in response to fluoxetine along the DV axis

We divided the whole hippocampus into dorsal and ventral segments along the dorsoventral (DV) axis20,35. A previous study showed that fluoxetine increased division of type II cells, but the effect of fluoxetine on proliferation of NSCs along the DV axis has not been investigated22. Our study showed that fluoxetine specifically increased proliferation of type II cells and neuroblasts, but not type I cells in the ventral part of the hippocampus. This is also consistent with running-induced neurogenesis, which showed an increased proportion of dividing type II and neuroblast cell populations while the overall number of type I and type II NSCs remained unaltered23. Thus, while running and fluoxetine target NSCs located in different positions along the DV axis, they appear to target identical types of NSCs.

Previous studies showed that 5-HTR1A mRNA is expressed in the hippocampus, building a dorsal-low to ventral-high concentration gradient36 and that 5-HTR1A plays both a necessary and sufficient role in mediating fluoxetine-induced behavior and neurogenic response9,37. This observation suggested that the action of 5-HTR1A may contribute to a regional-specific and cell-type-dependent response of NSCs to fluoxetine. Indeed, by using pharmacological reagents, we identified 5-HTR1A as a key downstream molecule that mediates the effect of fluoxetine on proliferation of specific NSC types in a regional-specific manner. Administration of a potent activator of 5HTR1A, 8-OH-DPAT, was sufficient to increase proliferation of NSCs. This increase in proliferation is specific to the ventral hippocampus while the mitotic index in the dorsal hippocampus was not affected. Moreover, treatment with a potent inhibitor of 5HTR1A, NAN-190, effectively abolished fluoxetine-mediated proliferation of NSCs in the ventral hippocampus. The action of NAN-190 also normalized the increased cycling type II and neuroblast populations that were induced by fluoxetine, collectively revealing the essential role of 5-HTR1A in mediating fluoxetine-induced proliferation specifically in the ventral hippocampus. Is the function of 5-HTR1A autonomous to NSCs or does 5-HTR1A in other cell types influence the behavior of NSCs? It has been demonstrated that 5-HTR1A is expressed in DGCs; however, the co-expression of 5-HTR1A in NSCs has not been convincingly confirmed. A recent genetic study has provided indirect evidence that the promoter of the 5- HTR1A gene is inactive in NSCs as well as immature newborn DGCs and becomes active when DGCs become mature38. Moreover, this study clearly demonstrated that 5-HTR1A in mature DGCs but not NSCs is necessary and sufficient for the behavioral and neurogenic effects of fluoxetine38. Thus, the function of 5-HTR1A in the regional-specific and cell-type-dependent response of NSCs to fluoxetine is likely to occur in a non-cell autonomous manner. Although 8-OH-DPAT and NAN-190 have been widely used as an agonist and antagonist for 5-HTR1A27,28, we should note that the affinity of 8-OH-DPAT and NAN-190 to additional receptors also has been reported39,40,41. A genetic deletion of 5-Htr1a will be needed to unambiguously confirm the role of 5-HTR1A in the regional-specific and cell-type-specific response of NSCs to fluoxetine.

One important question remains: can our observation that fluoxetine affects proliferation of NSCs in a regional-specific and cell type-dependent manner extrapolate to mice in “depressed” conditions? Recent studies strongly suggest that the effect of fluoxetine on behavior and neurogenic response is dependent upon the state of the animal18,42. When fluoxetine was applied to two independent animal groups, including “non-depressed” control mice and “depressed” mice, signatures of gene expression in the dentate gyrus between these two experimental groups were distinct42. Moreover, addition of a small number of newborn DGCs into the dorsal or ventral hippocampus appears to be sufficient to contribute to cognitive enhancement or fluoxetine-mediated anxiolytic/antidepressant-related behavioral effects in “non-depressed” control mice. However, in a case where animals were situated in a more challenged status such as “depressed conditions”, functional dissociation along the DV axis of the hippocampus appeared to be abrogated and newborn DGCs throughout the entire dentate gyrus were required to mediate fluoxetine-induced behavioral response18. These results collectively suggest that the regional-specific and cell-type-dependent effect of fluoxetine on proliferation of NSCs may also be determined by the state of the animal. Future study will be needed to understand the differential response of NSCs to fluoxetine in a state-dependent manner, which will provide an insight to understanding and develo** novel antidepressants.

Methods

Subjects

All animal procedures were approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic and Nan**g Medical University. All experiments were performed in accordance with relevant guidelines and regulations of the Cleveland Clinic and Nan**g Medical University. Eight - ten weeks old female C57BL/6 mice (purchased from The Jackson Laboratory) were used. Eight - ten weeks old female Nestin-GFP (green fluorescent protein) mice in which the Nestin gene regulatory elements drive the expression of a GFP gene were used in this study22. This mouse line has been backcrossed to C57BL7/6 more than 10 generations. Mice were housed in a temperature- and humidity-controlled environment with an alternating 12 hour light and 12 hour dark cycle.

Treatment with pharmacological reagents

Fluoxetine, (±)−8-Hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT), and 1-(2-Methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN-190) were purchased from Sigma-Aldrich (St Louis, MO, USA) and were dissolved in saline. Fluoxetine (10 mg/kg/d, Sigma-Aldrich, St Louis, MO, USA) was intraperitoneally injected between 10:00 AM-12:00 PM for 14 days. 8-OH-DPAT (0.1 mg/kg/d) and NAN-190 (0.3 mg/kg/d) was intraperitoneally injected. NAN-190 was injected 30 minutes before fluoxetine injection.

Immunohistochemistry (IHC)

The mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) and perfused transcardially with saline followed by 4% paraformaldehyde (PFA). Brains were removed and postfixed overnight in the same solution. To identify the cell types, labeling was carried out on 40-μm free-floating sections as described43. Primary antibodies: 5-bromo-2′-deoxyuridine (BrdU) (rat, 1:200; Accurate Chemical & Scientific Corporation, NY), GFP (chicken, 1:100, Aves Labs, OR), glial fibrillary acidic protein (GFAP) (rabbit, 1:200, Dako, CA) were diluted in 0.1 M TBS with 3% normal donkey serum and 0.25% Triton X-100 (TBST) and binding was visualized with a Cy3-conjugated secondary antibody (1:200; Thermo Fisher Scientific Inc, MA). Nuclei were visualized with 4′-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St.Louis, MO). Antigen retrieval steps were used for BrdU staining. Sections were treated with DAPI and fixed in 4% PFA for 10 minutes. Later, sections were treated with 2N HCl at 37 °C, neutralized with 0.1 M boric acid, and blocked. Subsequent steps are identical to those described above (except DAPI treatment). In addition, brain slices also were stained for BrdU with the peroxidase method (ABC system, with biotinylated horse anti-mouse antibodies and diaminobenzedine chromogen; Vector Laboratories). Coronal brain sections (40 μm in thickness) through the entire dentate gyrus were maintained in the serial order. Every sixth section throughout the hippocampus was processed for immunohistochemistry and counting.

Quantification

Quantification of BrdU+ cells in C57BL/6 mice was achieved by counting the number of positively labeled cells in the right side of the dentate gyrus using a 40x objective of an upright microscope (Leica, Germany). One section in each block was counted and reported as a total number of cells in each block by multiplying by 6. Only BrdU+ cells in the subgranular zone were counted. For quantification of BrdU+ cells in Nestin-GFP mice, fluorescent images of the dentate gyrus were acquired by using a confocal microscope (SP5; Leica, Germany), BrdU+ cells located in the subgranular zone were counted, and type I, type II, and neuroblasts were scored based upon the morphology and different expression of markers.

Measurement of Area

The area of the dentate gyrus in which we quantified cells was measured by using by ImageProPlus5 software (Media Cybernetics, Inc., USA). The area of the dentate gyrus of fluoxetine treated mice was normalized to that of control mice.

Statistics

Comparisons among multiple groups were performed using two-way ANOVA. Sidak’s multiple comparison method was used for post-hoc analysis. Data are presented as mean ± SEM; p < 0.05 was considered statistically significant.

Additional Information

How to cite this article: Zhou, Q.-G. et al. Regional-specific effect of fluoxetine on rapidly dividing progenitors along the dorsoventral axis of the hippocampus. Sci. Rep. 6, 35572; doi: 10.1038/srep35572 (2016).