Address correspondence and reprint requests to Per Svenningsson, MD, PhD, Translational Neuropharmacology, Department of Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institute, 171 76 Stockholm, Sweden. E-mail: email@example.com
Dopamine plays an important role in cellular processes controlling the functional and structural plasticity of neurons, as well as their generation and proliferation, both in the developing and the adult brain. The precise roles of individual dopamine receptors subtypes in adult neurogenesis remain poorly defined, although D3 receptors are known to be involved in neurogenesis in the subventricular zone. By contrast, very few studies have addressed the influence of dopamine and D3 receptors upon neurogenesis in the subgranular zone of the hippocampus, an issue addressed herein employing constitutive D3 receptor knockout mice, or chronic exposure to the preferential D3 receptor antagonist, S33138. D3 receptor knockout mice revealed increased baseline levels of cell proliferation and ongoing neurogenesis, as measured both using Ki-67 and doublecortin, whereas there was no difference in cell survival as measured by BrdU (5-bromo-2′-deoxyuridine). Chronic administration of S33138 was shown to be functionally active in enhancing levels of the plasticity-related molecule, delta-FosB, in the D3 receptor-rich nucleus accumbens. In accordance with the stimulated neurogenesis seen in D3 receptor knockout mice, S33138 increased proliferation in wild-type mice. These observations suggest that D3 receptors exert a tonic, constitutive inhibitory influence upon adult hippocampal neurogenesis.
Adult neurogenesis is a process through which cells are born, mature, and develop into neurons, which fully integrate into the circuitry of the adult brain (see Hagg 2009 for review). Current data suggest that the dopamine system has a direct role in adult neurogenesis as a result of dopamine innervation and dopamine receptor expression in cells undergoing various stages of neurogenesis both in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus (DG) (Hoglinger et al. 2004; Borta and Hoglinger 2007; Mu et al. 2011). Specifically, there are abundant data supporting the observation that in the SVZ, dopamine, acts as a strong regulator of cell proliferation (Hoglinger et al. 2004; Kippin et al. 2005; Freundlieb et al. 2006; O'Keeffe et al. 2009). However, dopaminergic modulation of adult neurogenesis in the SGZ has been much less studied than in the SVZ and data thus far come largely from dopaminergic denervation experiments, which have been shown to have potent effects on levels of cell proliferation in the SGZ, indicating that dopaminergic modulation in the DG might also be of critical importance (Hoglinger et al. 2004; Peng et al. 2008; Park and Enikolopov 2010). Results from these studies of dopaminergic modulation of cell proliferation in both the SVZ and SGZ reveal numerous discrepancies as some studies have reported that dopaminergic denervation inhibits cell proliferation in the SVZ and SGZ (Baker et al. 2004; Hoglinger et al. 2004; Freundlieb et al. 2006; Yang et al. 2008), whereas others report a stimulatory effect (Peng et al. 2008; Park and Enikolopov 2010).
These contradictory results indicate a complex regulation by dopamine, suggesting the involvement of multiple dopamine receptors on aspects of adult neurogenesis. Technical limitations, particularly the limited access of effective antibodies, have posed challenges in deciphering which specific dopamine receptors are involved, in which cells they are expressed and what their exact roles in adult neurogenesis are. Cell-specific expression of dopamine receptors in the SVZ has been studied on a limited scale where in one study, Hoglinger et al. (2004) used immune-electron microscopy and showed that both D1- and D2-like receptors are expressed in cells undergoing neurogenesis in the rat. Similarly, Kim et al. (2010) showed, using hGFAP-Green Fluorescent Protein (GFP) and DCX-GFP mice and combined FACS sorting followed by RT-PCR, that in the SVZ of the mouse, adult-born neuroblasts express multiple dopamine receptors (D1, D2, and D5), but do not express the D3R (D3R). However, D3R expression was found in the rapidly dividing transit amplifying progenitor cells, indicating a direct involvement of this receptor in cell proliferation (Kim et al. 2010). Indeed, previous experiments in the SVZ indicate that D3R stimulation in this niche increases cell proliferation as exemplified by D3R-specific agonists, which increase cell proliferation in the rat SVZ (Van Kampen et al. 2004) whereas, correspondingly, specific D3R antagonists decrease cell proliferation in the mouse SVZ (Kim et al. 2010). In the SGZ, however, the specific distribution of the dopamine receptors is largely unknown, although qPCR data demonstrated the presence of all dopamine receptor subtypes in the DG (Mu et al. 2011). Technical advancements, particularly the development of bacterial artificial chromosome (BAC) transgenic GFP reporter mice have allowed the visualization of gene expression patterns. The publicly available gene expression atlas, GENSAT (www.gensat.org), has mapped many proteins including the D3R using this technique. Confocal data from these mice show the expression of the D3R in distinct cells in the area of the DG corresponding to the SGZ (www.gensat.org Heintz 2004). This novel finding of expression of D3Rs in this subregion combined with previous experiments describing dopaminergic modulation of cell proliferation indicate a possible relevance of the D3R in aspects of adult neurogenesis in the SGZ.
Given this background, the relevance of D3Rs in the SGZ was therefore investigated using pharmacological and genetic strategies. Inactivation of D3Rs was achieved either with a genetic deletion using D3R knockout (D3R KO) mice or with pharmacological blockade using a preferential D3R antagonist. D3R KO mice have been examined with regards to several aspects of behavior (Micale et al. 2010; Chourbaji et al. 2008; Le Foll et al. 2005; Leggio et al. 2008). However, potential changes in adult hippocampal neurogenesis have not yet been examined in these mice. The pharmacological blockade of D3Rs was achieved with a novel compound, S33138—a preferential D3 versus D2 receptor antagonist (Millan et al. 2008b). Besides its interest as a pharmacological tool, this compound is also of clinical interest as it has proven pharmacological activity in animal models of schizophrenia and therefore has potential as an atypical antipsychotic (Millan et al. 2008b). The effects of acute treatment with S33138 on the immediate early gene c-Fos have been used to investigate modulation of neuroplasticity and showed specifically that acute administration induces a c-Fos pattern reminiscent of an atypical antipsychotic with high expression in the nucleus accumbens (Millan et al. 2008b). Delta-FosB is another inducible Fos member, which is highly up-regulated during chronic treatment paradigms, but also in D3R KO mice (Robertson et al. 2004). Therefore, the levels of delta-FosB in the nucleus accumbens and ventral striatum were used as marker of chronic activation to guide decisions on relevant and effective dosing of S33138 for neurogenesis studies. A combination of a pharmacological blockade and genetic deletion via treatment of D3R KO mice with S33138 enabled further testing of pharmacological effects of S33138, which do not involve the D3R. D3R KO and WT mice were therefore treated chronically with vehicle and S33138, after which several aspects of hippocampal neurogenesis were examined including cell proliferation, ongoing neurogenesis, as well as cell survival.
Materials and methods
In a first experiment, normal male C57Bl6 mice (Charles River, Stockholm) of 8 weeks of age were administered with saline and increasing doses of S33138. In a second experiment, male WT or D3R KO mice (C57bl6/129SV background; The Jackson Laboratory, Bar Harbor, ME, USA) were used. Mice were of 6 weeks at the time of first injections. All animals were group housed in air-conditioned rooms (12-hour dark/light cycle) at 20°C with humidity of 53%. Experiments were performed in agreement with the European Communities Council (86/609/ECC) and efforts were made to minimize pain or discomfort. They were approved by the ethical committee at Karolinska Institute (N351/08).
Experimental design, drug treatments, and fixation
In the first experiment, WT mice were divided into groups which were injected subcutaneously (s.c.) for 21 days with either 33138 (0.16, 0.64, or 2.5 mg/kg), Haloperidol (2 mg/kg), or vehicle (saline) (Fig. 1). In the second experiment, WT and D3R KO mice were injected subcutaneously (s.c.) for 21 days with a single concentration of S33138 (0.64 mg/kg) or vehicle (saline) (Fig. 1). To measure cell survival, BrdU (Sigma, Stockholm, Sweden) was injected intraperitoneally (i.p.) twice daily (75 mg/kg) for three consecutive days before commencing chronic treatment with S33138 (Fig. 1). On the final day of injections, brains were fixed via transcardial perfusion. Briefly, mice were anesthetized with Ketamine/Xylazine (80/10 mg/kg; Intervet/Bayer, Stockholm, Sweden) and then perfused with cold phosphate-buffered saline (PBS) (0.1 M; pH 7.4) followed by 4% paraformaldehyde. Mouse brains were then removed, post-fixed overnight at 4°C, and cryoprotected in 30% sucrose for several days. Brains were snap frozen and sectioned at 40μm using a cryostat (Leica CM1850; Sollentuna, Sweden) and stored at 4°C in 0.1 M PBS containing sodium azide (Sigma) until stained.
Original immunohistochemistry stainings to visualize the dose response of delta-FosB expression were performed using 3,3-diaminobenzidine. Briefly, sections were permeabilized (15 min in 1% Triton X-100/0.1 M PBS) and blocked against endogenous peroxidases (20 min with 3% H2O2/0.1 M PBS). Sections were then incubated with blocking solution for 1 h with serum followed by the primary antibody, rabbit anti-delta-FosB (1 : 500; Santa Cruz Biotechnology; Santa Cruz,CA, USA; overnight at 4°C), and the secondary biotinylated antibody (goat anti-rabbit, 1 : 200, Vector Laboratories, Burlingame, CA, USA; 2 h at 20°C). A signal amplification step was then performed by incubation in ABC reagent (ABC kit Vector Laboratories; for 1 h) followed by a reaction with 0.05% 3,3-diaminobenzidine-tetrahydrochloride/0.01% hydrogen peroxide in phosphate buffer. Finally, sections were dehydrated and mounted on Polylysine-coated slides (Histolab), dried, and coverslipped using VECTASHIELD® HardSet™ anti-fade mounting medium (Vector Laboratories). For improved confocal visualization, subsequent examination of delta-FosB in WT and KO mice were performed using fluorescent labeling as described below.
For fluorescent immunohistochemical staining, sections were incubated for 1 h in blocking solution (3% Bovine Serum Albumin (Sigma), 0.3% Triton X-100 (Sigma) in 0.1 M PBS) followed by incubation overnight in primary antibody. For BrdU staining, DNA in sections was denatured with HCl (15 min in 2 M HCl (Sigma) at 37°C) before incubation with primary antibodies. The primary antibodies used were rat anti-BrdU (1 : 500; Accurate Chemical; Trichem AB, Skandeborg, Denmark); rabbit anti-Ki-67 (1 : 1000; Novocastra, Newcastle upon Tyne, UK), rabbit anti-DCX (1 : 500; Cell Signaling Technology; In vitro AB, Stockholm, Sweden), mouse anti-NeuN (1 : 1000; Chemicon; Millipore AB, Stockholm, Sweden), and rabbit anti-delta-FosB(1 : 500; Santa Cruz Biotechnology). Sections were then incubated for 2 h with respective fluorescent secondary antibodies: anti-rat IgG Alexa Fluor™ -488, anti-rabbit IgG Alexa Fluor™-568, anti-mouse IgG Alexa Fluor™ -488 or 568 (1 : 200–500; Invitrogen, Lidingö, Sweden). For double immunostainings, sections were incubated simultaneously in two primary antibodies followed by simultaneous incubation in two secondary antibodies. For stainings with 4′,6-diamidino-2-phenylindole (DAPI), sections were also incubated for 20 min in 300 nM DAPI (Invitrogen). Sections were finally mounted on Poly-Lysine-coated slides (Histolab, Stockholm, Sweden), dried overnight, and mounted using FluorSave mounting medium (Calbiochem, San Diego, CA, USA). Confocal images were obtained using a Zeiss 710LSM laser scanning microscope (Carl Zeiss HB, Stockholm, Sweden) using a 40 × W lens and Zen 2009 software (Carl Zeiss HB).
For quantifications of delta-FosB, six striatal sections from each mouse were quantified. For each region, an image was captured from both hemispheres of each section using a Nikon Eclipse E600 light microscope with appropriate fluorescent filters connected to a Nikon digital sight DS-U1 camera with the NIS-Elements F 2.20 software (NIS-Elements; Melville, New York). The number of delta-FosB-positive cells for each image was then quantified using Image J. Briefly, the image was converted into an 8-bit image, and thereafter the intensity threshold was restricted (lower threshold 20, upper threshold 75). To differentiate adjacent touching nuclei, an Image J watershed function was used. Finally, the nucleus counter function (plug-in from WCIF- http://www.uhnresearch.ca/facilities/wcif/fdownload.html) was used with a specific threshold for size (min: 40, max: 1000). The resulting values from the quantifications were transferred to MS office EXCEL and the total number of positive cells averaged for each region in each brain. For quantification of neurogenesis, immunostainings were quantified by an observer blind to experimental conditions using a modified stereological method (Malberg et al. 2000). Briefly, analysis of each parameter was performed on a set of sections consisting of every sixth section throughout the hippocampus (roughly six to eight sections per set), which was then stained with the appropriate antibody. Labeled cells were manually quantified in the SGZ along the granule cell layer of the hippocampus using a 40 × W lens. To estimate the total number of cells in the entire dentate gyrus, the number of positive cells was divided by the number of sections counted to get an average and then multiplied by 60, the average number of sections in the hippocampus.
Animals with different treatments and genotypes were analyzed with two-way anova followed by Student's t-test for pairwise comparisons. Statistical significance was defined as p < 0.05. Statistical analyses of the data were carried out using GraphPad Prism 5(GraphPad Software Inc., San Diego, CA, USA).
Dose response of chronic treatment with S33138 on delta-FosB levels
D3Rs are strongly expressed in the ventral striatum and nucleus accumbens (Bouthenet et al. 1991). Delta-FosB levels are increased by chronic antipsychotic treatment as well as in D3R KO mice in the above mentioned regions, and this feature was therefore used to find an optimal and relevant dose of S33138 for chronic treatment (Robertson et al. 2004). Immunohistochemistry was used to examine delta-FosB-positive cells in the dorsal striatum and nucleus accumbens, and low levels were found in both regions in vehicle-treated animals (Figure S1a and b). Following treatment with S33138 at 0.16 mg/kg, there was a trend for increased delta-FosB levels in the shell of nucleus accumbens. Following treatment with the intermediate concentration of S33138, 0.64 mg/kg, there were significant increases in delta-FosB levels in the nucleus accumbens core and shell (p < 0.05). The higher concentration of S33138, 2.5 mg/kg, also significantly increased delta-FosB in nucleus accumbens as well as in the dorsomedial and dorsolateral striatum (p < 0.01). The positive control, haloperidol (2 mg/kg), significantly increased delta-FosB in all studied regions (p < 0.01). Thus, significant effects were found in the nucleus accumbens in doses including 0.64 mg/kg and above. However, in the striatum, 2.5 mg/kg produced significant increases of delta-FosB in the dorsolateral striatum. As increased expression of delta-FosB in this region is associated with extrapyramidal side effects, the optimal and relevant dose of S33138 was determined to be 0.64 mg/kg.
Delta-FosB levels in the nucleus accumbens and striatum are affected by both a genetic and pharmacological blockade of D3 receptors
To further establish the potential modulation of delta-FosB expression by D3Rs, 0.64 mg/kg of S33138 was given to WT or D3R KO mice, and analyses of delta-FosB in nucleus accumbens and striatum were made (Fig. 2 and 3). In the nucleus accumbens shell, results from a two-way anova revealed significance in treatment (F(1,19)=9.043; p = 0.0072), although not in genotype (F(1,19)=1.412; p = 0.2493) or interaction (F(1,19)=1.975; p = 0.1760) (Fig. 2a and b). In WT mice, t-test analysis showed that S33138 treatment significantly increased delta-FosB levels (p = 0.014). In D3R KO mice, S33138 treatment did not further increase expression. In the nucleus accumbens core, results from a two-way anova revealed significance in treatment (F(1,19) = 4.948; p = 0.0378) and interaction (F(1,19) = 4.943; p = 0.0379), although not in genotype (F(1,19) = 1.151; p = 0.2961) (Fig. 2c and d). Subsequent t-test analysis of WT mice revealed that S33138 treatment significantly increased delta-FosB expression (p= 0.023). Analysis of D3R KO mice further revealed a significant baseline increase in the levels of delta-FosB expression (p = 0.038), although S33138 treatment had no added effect in KO mice.
Levels of delta-FosB in WT or D3R KO mice treated with S33138 were also examined in the dorsal striatum. In the dorsomedial striatum, results from a two-way anova revealed a significant difference in genotype (F(1,20) = 5.466; p = 0.0299), but no interaction (F(1,20) = 1.844; p = 0.1896) (Fig. 3a–d). Despite a trend for an increase in delta-FosB in WT mice upon treatment with S33138, this did not reach significance in two-way anova analysis (F(1,20) = 3.834; p = 0.0643), likely as a result of opposite effects between genotypes (Fig. 3a and b). Results from Student's t-test analysis revealed that in the WT mice, S33138 treatment significantly increased delta-FosB levels (p = 0.043). However, in D3R KO mice, S33138 had no effect. No baseline difference of delta-FosB levels were found between WT versus D3R KO mice. The effects seen in the dorsomedial striatum were not seen in the dorsolateral striatum as a two-way anova revealed no significance in either genotype (F(1,20) = 0.9616; p = 0.3391), treatment (F(1,20) = 1.106; p = 0.3062), or interaction (F(1,20) = 1.337; p = 0.2619) (Fig. 3c and d).
Genetic and pharmacological blockade of D3 receptors increases cell proliferation
In an attempt to find a potential role of the D3R in the regulation of adult neurogenesis in the dentate gyrus, experiments were designed to investigate possible changes in stages of adult neurogenesis in mice with a genetic and/or pharmacological blockade of D3R (Fig. 1). D3R KO mice were used to obtain a genetic deletion of D3R, whereas the preferential D3R antagonist S33138 was used for pharmacological blockade. Cell proliferation, an early stage of neurogenesis, was examined using an antibody against Ki-67, a cell cycle marker which has been demonstrated to effectively measure this aspect of neurogenesis (Fig. 4a) (Kee et al. 2002). D3R KO mice or WT littermates were thus treated chronically with either S33138 or vehicle. Results from a two-way anova comparison of numbers of Ki-67-positive cells in these mice revealed a significant difference in Ki-67-positive cells between genotypes (F(1,23) = 5.64; p = 0.0263). A t-test analysis of vehicle-treated mice demonstrated a significantly higher baseline levels of Ki-67-positive cells in the D3R KO compared with WT mice representing a highly significant increase in baseline cell proliferation (p = 0.0053) (Fig. 4b). Despite a 24% induction in Ki-67-positive cells as a result of treatment in WT mice, the overall effect of treatment alone was not significant in a two-way anova analysis (F(1,23) = 0.3559; p = 0.556) (Fig. 4a, b, and c). However, when the treatment effect was studied together with genotype, there was a highly significant interaction (F(1,23) = 13.00; p = 0.0015) (Fig. 4b). This treatment × genotype interaction is represented by the effect of S33138 treatment, which resulted in an up-regulation of Ki-67-positive cells in the WT mice, but an opposing 22% reduction in D3R KO mice, with a significant difference between induction levels (p < 0.001) (Fig. 4c). Because of this interaction, t-test analysis was used to statistically evaluate the treatment-induced changes and revealed that the induction in S33138-treated WT mice was significant (p = 0.045), whereas S33138-treated D3R KO mice exhibited a significant decrease in cell proliferation (p = 0.015). In summary, cell proliferation is robustly increased in the hippocampus of D3R KO mice and, correspondingly, the preferential D3R antagonist S33138 also increased cell proliferation in WT mice. These results, overall, demonstrate that both a genetic and pharmacological blockade of D3R increase cell proliferation in the SGZ.
Genetic disruption of D3 receptors increases ongoing neurogenesis
Doublecortin (DCX) is an endogenous protein expressed by immature neurons, which is commonly used to measure the level of ongoing neurogenesis (Couillard-Despres et al. 2005). To investigate a possible role of the D3R in the regulation of ongoing neurogenesis, DCX-positive cells in the DG were quantified from the mice with a genetic and/or pharmacological D3R blockade. Two-way anova analysis of DCX-positive cells from these mice demonstrated a highly significant difference in genotype (F(1,20) = 31.12; p < 0.0001), but no significance in treatment or a treatment × genotype interaction (F(1,20) = 1.564; p = 0.2255 and F(1,20)=2.413; p = 0.1360, respectively) (Fig. 5a and b). Post hoc analysis of baseline levels of ongoing neurogenesis shows that the increase in DCX-positive cells in the KO mice is highly significant, representing an increase in ongoing neurogenesis in D3R KO mice (p < 0.001). Treatment with S33138 in WT mice caused an approximate 28% induction in cell proliferation, whereas in the D3R KO mice no effect (−2%) was seen (p < 0.05) (Fig. 5c). These results demonstrate that deletion of the D3R increases ongoing neurogenesis in the SGZ and further supports a role of D3R in the modulation of adult neurogenesis in the hippocampus.
Genetic disruption of D3 receptors does not affect cell survival
An important measure of neurogenesis is the number of cells which proliferate and become mature neurons expressing neuronal markers, a measure of cell survival. This measure is currently only obtainable via the injection of exogenous substance such as BrdU, which label DNA and can be detected at a later time. As described, cell proliferation and ongoing neurogenesis were increased by blockade of D3R. Therefore, BrdU was used to assess if these changes translated into changes in cell survival. Quantification of BrdU-labeled cells at 3 weeks after injection revealed numerous cells found in the dentate gyrus, which expressed the mature neuronal marker NeuN (Fig. 6a–c). Two-way anova analysis revealed no significant difference in BrdU-positive cell numbers in either genotype (F(1,23) = 0.2774; p = 0.767) or treatment (F(1,23) = 3.434; p = 0.6034) groups representing no changes in cell survival as a result of either genetically or pharmacologically disrupting D3R signaling. Significance was found, however, in a treatment × genotype interaction (F(1,23) = 5.67; p = 0.0259); a post hoc t-test analysis revealed a significant decrease of BrdU labeling in D3R KO mice upon treatment with S33138 (p = 0.017)(Fig. 6b). Accordingly, when compared in terms of induction, there was a highly significant difference in genotypes with a 6% increase in induction in WT mice, but a 40% reduction in D3R KO mice (p < 0.01) (Fig. 6c). In summary, it can be concluded that increases in cell proliferation and ongoing neurogenesis induced by D3R blockade with either genetic or pharmacological methods do not translate into significant changes in cell survival.
In the hippocampus, baseline levels of both cell proliferation and ongoing neurogenesis were robustly increased in D3R KO mice. Furthermore, the preferential D3R antagonist S33138 had a comparable effect on cell proliferation in WT mice. These data indicate an involvement of the D3R in the regulation of neurogenesis in the SGZ and, contrary to the situation in the SVZ, suggest that D3R inhibits aspects of neurogenesis in this neurogenic niche.
S33138, a preferential D3R versus D2R antagonist with clinical potential
S33138 was chosen as the pharmacological agent to block D3R, as it has been shown to behave as a pure and preferential antagonist D3R versus D2R in vitro and in vivo (Millan et al. 2008a,b). D3R blockade is thought (in contrast to D2R blockade) to promote cognitive function (Laszy et al. 2005; Lacroix et al. 2006; Micale et al. 2010; Millan et al. 2010; Xing et al. 2010). Hence, S33138 was designed to treat schizophrenia and in particular to counter the cognitive symptoms of this disorder (Millan et al. 2008c), which are currently poorly treated (Millan et al. 2012). However, data are limited as regards its long-term effects on cellular events related to cognition processing in the brain, notably hippocampal processes of cellular proliferation and neurogenesis in adult animals (Deng et al. 2010). This is an important issue as such mechanisms may be important to the influence of D3R upon certain domains of cognition (Laszy et al. 2005; Lacroix et al. 2006; Millan and Brocco 2008; Micale et al. 2010; Millan et al. 2012).
D3R antagonism increase Delta-FosB
Delta-FosB is a well-studied biochemical marker of receptor-mediated adaptations in neurons (McClung et al. 2004). In particular, chronic treatment with clozapine and D3R KO up-regulates delta-FosB levels (Robertson et al. 2004). In fact, previous findings indicated that D3R blockade is involved in clozapine-induced increases in delta-FosB and also suggest that D3R antagonism may contribute to the unique therapeutic profile of this atypical antipsychotic. Initial experiments therefore established a relevant concentration at which chronic administration of S33138 increases delta-FosB. The results showed that S33138 at 0.64 mg/kg up-regulated delta-FosB in the nucleus accumbens and also confirmed a constitutive increase in delta-FosB activity in D3R KO mice in this region (Robertson et al. 2004). In the dorsolateral striatum, the dose of 0.64 mg/kg did not have any effects in the WT mice, however, in the D3R KO mice there was a non-significant trend toward an increase in delta-FosB levels. S33138 has previously been described to have a higher (25-fold) affinity for the D3 receptors versus D2 receptors (Millan et al. 2008a,b,c). However, in the absence of D3 receptors, S33138 might exert a somewhat more marked pharmacological activity on D2 receptors following putative adaptive changes upon constitutive deletion of D3 receptors (see below; Le Foll et al. 2005; Sokoloff et al. 2006). A stimulation of delta-FosB in dorsolateral striatum, an area particularly rich in D2 receptors, may therefore be a result of S33138 acting on D2 receptors in the D3R KO mice to a larger extent than in WT mice. The present data indicate that S33138, at 0.64 mg/kg, has a pharmacological profile on this parameter that resembles that of clozapine. The molecular mechanisms, whereby D3R antagonism stimulates delta-FosB could involve changes in several signaling pathways including cAMP/Protein kinase A (PKA)/cAMP-response element-binding protein (CREB), calcium/calmodulin, and Akt/GSK-3 pathways (Mannoury la Cour et al. 2011). Indeed, D3R antagonism stimulates the cAMP/PKA/CREB pathway and it has been shown that antisense toward CREB reduces delta-FosB induction in the striatum (Andersson et al. 2001). Moreover, a recent study showed that the cAMP/PKA/CREB pathway acts in cooperation with additional pathways, such as the calcium/calmodulin II pathway, to stimulate delta-FosB in nucleus accumbens (Vialou et al. 2012).
Dopaminergic modulation of adult cell proliferation in the SGZ is multifaceted
As described in the introduction, dopaminergic regulation of hippocampal neurogenesis, and specifically the question of potential D3 receptor-mediated control, has been only marginally addressed, primarily in several dopaminergic denervation studies. Initial studies reported that dopaminergic denervation decreases proliferating cells in the SGZ, indicating that dopamine acts to stimulate proliferation in this niche (Hoglinger et al. 2004). In contrast, two subsequent studies report that denervation increases cell proliferation in the SGZ indicating, in agreement with our studies, that dopamine can act to inhibit cell proliferation (Peng et al. 2008; Park and Enikolopov 2010). Several differing factors between these studies might explain the conflicting results and it should be noted that a dopaminergic denervation also impedes on other possible neurogenic regulators, such as neuropeptides, neurotrophic factors, and sonic hedgehog (Kottmann et al. 2011), known to be expressed in dopaminergic neurons. Taken together, it appears that the dopaminergic modulation of adult neurogenesis in the SGZ is multifaceted. In particular, individual dopamine receptors may have distinct roles.
D3R antagonism stimulates adult cell proliferation in the SGZ
A previous study has reported that quinpirole, a dopamine D2/3/4R agonist, stimulated cell proliferation in the SGZ indicating that dopamine stimulates rather than inhibits proliferation (Yang et al. 2008). This study described in detail the dependence on the D2R for these effects. Specifically, it demonstrated that this proliferative effect of dopamine is mediated by ciliary neurotrophic factor (CNTF) whose expression is dependent on specific D2R stimulation. Although data regarding the cellular localization of D2Rs in the SGZ were not investigated by the authors in this study, CNTF was found to be expressed exclusively by astrocytes in the SGZ (as well as in the SVZ), indicating that dopamine acts indirectly via D2Rs found on astrocytes rather than directly on possible D2Rs found on neurogenic cells themselves. The roles of D3Rs and D4Rs were not examined. Given the stimulatory properties of the D2R on cell proliferation in the SGZ, the dopaminergic-mediated inhibition of cell proliferation in SGZ previously described (Peng et al. 2008; Park and Enikolopov 2010) is likely to involve a dopamine receptor other than the D2R. The fact that the D3R according to GENSAT (Heintz 2004) appears to be expressed by cells in the neurogenic niche of the SGZ indicates that the D3R might be involved in the neurogenic process in this region. Indeed, our present data indicate an important role of D3Rs in the SGZ. Somewhat surprisingly, and opposite to the reported D2R-mediated actions on neurogenesis in the SGZ, both D3R KO mice and wild-type mice treated chronically with 0.64 mg/kg of S33138 exhibited increased cell proliferation. D3R KO mice also showed increased ongoing neurogenesis. Thus, both genetic and pharmacological strategies points at a previously undescribed role of D3R in aspects of adult hippocampal neurogenesis. The opposing regulation by D2Rs and D3Rs on hippocampal cell proliferation might also help to explain the decrease in cell proliferation in D3R KO mice treated with S33138, which as described may, in the absence of D3 receptors, have increased activity at D2 receptors, and therefore may actually decrease cell proliferation in D3RKO mice as a result of D2R antagonism (Millan et al. 2008a). The opposing regulation of proliferation by D3 versus D2 receptors is also of interest in light of their contrasting influences on cognition, in the regulation of which the hippocampus (and processes of proliferation and neurogenesis) are implicated (Deng et al. 2010; Laszy et al. 2005; Lacroix et al. 2006; Xing et al. 2010; Millan et al. 2010).
D3R antagonism stimulates cell proliferation and neurogenesis, but not cell survival, in the SGZ
The present neurogenesis and previous data regarding the cell-stage expression of D3Rs indicate that it affects solely cell proliferation and that changes in other aspects of neurogenesis are likely a downstream consequence of this. Our data indicate that increases in cell proliferation translated into changes in ongoing neurogenesis by D3R blockade, but did not translate into an increase in cell survival. In fact, S33138 actually decreased cell survival in D3R KO mice via potential D2R antagonism in the absence of D3 receptors. Somewhat surprising was the fact that despite S33138 decreased cell proliferation and survival in D3R KO mice, there was no difference in ongoing neurogenesis potentially as a result of the experimental design, which may not have allowed time for the decreases in cell proliferation to translate into changes in ongoing neurogenesis. Alternatively, D2 antagonism may decrease the maturation rate (Mu et al. 2011) creating a larger DCX-expressing window, potentially masking any decreases in cell number. Despite a slight increase in cell survival in D3R KO mice, this did not reach significance. Although this study did not evaluate apoptosis, an explanation for this lack of effect may simply be that as the rate of cell proliferation increased in these otherwise healthy mice, the rate of cell death of the these new cells also increased and they undergo apoptosis before reaching maturity—no longer detectable within the cell survival experiments. Indeed, in a normal physiological situation, an increase in cell proliferation is balanced by an increase in cell death (Egeland et al. 2010; Sairanen et al. 2005). Alternatively, it cannot be excluded that as a result of the dilution of BrdU through several generations, the 3-week experimental design may be underestimating the full effect on survival. This lack of an increase in cell survival in these healthy mice does not mean, however, that there is no benefit to increasing proliferation as immature neurons are also thought to be important in mediating specific plastic changes of hippocampal function (Bruel-Jungerman et al. 2007, 2011). Indeed, literature indicates an increased plasticity of hippocampal function in D3R KO mice as these mice have an enhanced cognitive performance in the hippocampal-dependent passive avoidance test (Micale et al. 2010). Furthermore, a D3-mediated increase in cell proliferation may be of benefit in situations where neurogenesis is decreased, for example during stress. Indeed, stress an important regulator of neurogenesis (Lucassen et al. 2010; Petrik et al. 2012), and therefore dopamine modulation may counteract the stress-induced decreases in cell proliferation and cell survival. Although no non-handled control was included in this study, it could be speculated that the stress induced by handling and injections maybe affecting neurogenesis in these mice and perhaps masking even greater effects on cell proliferation or, alternatively, that the increased cell proliferation may render the mice more resilient to the effects of stress.
Indirect mechanisms possibly involved in D3R-mediated actions on cell proliferation in the SGZ
Another aspect that must be taken into consideration in this study is how the modulation D3Rs may affect other aspects of the dopamine system including dopamine levels and expression of other dopamine receptors. Studies examining D3 KO mice have indicated that these mice have an elevated level of extracellular dopamine in the striatum, which is suggested to be the result of diminished autoreceptor function on dopaminergic neurons themselves (Koeltzow et al. 1998; Joseph et al. 2002), although this finding is not without controversy (Zapata et al. 2001; see Le Foll et al. 2005; : Sokoloff et al. 2006). However, no changes in either dopamine D1 or D2 receptor expression levels were found in these mice when measured in striatum (Le Foll et al. 2005). However, this remains to be confirmed in other areas and it is therefore possible that expression of other dopamine receptors thought to be involved in the modulation of adult neurogenesis, such as the D2 receptor, may be altered in the dentate gyrus. Although the possibility that potential changes in the dopamine system, particularly a hyperdopaminergic tone, may modify aspects of neurogenesis in D3 KO mice, the combined pharmacological and genetic strategy employed herein, in parallel with our knowledge of D3 receptor distribution in the dentate gyrus, strongly supports the argument that changes in cell proliferation observed mainly reflect events occurring directly at D3 versus D2 receptors. To circumvent a potential involvement of extra-hippocampal D3Rs, such as autoreceptors, future experiments could employ local hippocampal infusion of drugs or anatomically restricted and conditional local knockout strategies to more specifically characterize the role of hippocampal D3 receptors alone.
Contrasting regulation of cell proliferation by D3R in the SGZ and SVZ
In contrast to the SGZ, numerous articles have addressed the role of the D3R in the SVZ, which generally describe a proliferative effect upon D3R stimulation in this niche (Baker et al. 2004; Coronas et al. 2004; Kim et al. 2010; Van Kampen et al. 2004 Joyce and Millan 2007). This provides an intriguing contrast to our present data from the SGZ describing an inhibitory effect of the D3R on cell proliferation. One possible explanation is that regulation of cell proliferation differs between these niches. Another explanation is that the role of the D3R in the regulation of adult neurogenesis differs among cellular populations. Knowledge of the localization of D3Rs is of crucial importance to decipher these potential differential roles. As mentioned in the introduction, D3Rs have been reported to be expressed not only in transit amplifying progenitor cells, but also in niche astrocytes (Kim et al. 2010). Although the authors of this study suggest that D3R has a direct stimulatory effect on transit amplifying progenitors, their anatomical data indicate that D3R could also stimulate cell proliferation in the SVZ via CNTF release from astrocytes (Yang et al. 2008). Moreover, expression of D3Rs in transit amplifying progenitor cells in SVZ is interesting as it suggests the possibility that the D3R-positive cells described in the SGZ could be the SGZ equivalent of these progenitors: the amplifying neural progenitor cells. Another possibility is that the D3R-positive cells in the SGZ are quiescent neural progenitors. Future studies need to better characterize D3R-positive cells in the SGZ, perhaps taking advantage of the availability of D3R-BAC GFP mice. In any event, these observations further underline the importance of defining the roles of individual populations of receptors like D3R, as generalizations from one tissue to another in the absence of direct evidence may turn out to be misleading.
Conclusions and Perspectives
The present studies suggest a role of D3R in the regulation of adult neurogenesis in the SGZ and provide a functional counterpart to previous observations that D3R-GFP BAC transgenic mice show high expression of D3R in cells located in the neurogenic niche of the SGZ. D3R KO mice display increased levels of cell proliferation and ongoing neurogenesis, suggesting that D3Rs controlling these cells normally inhibit cell proliferation. This finding is underpinned by observations with the novel D3R preferential antagonist, S33138, for which chronic treatment likewise increased cell proliferation. These data, which contrast to those reported from the subventricular zone, underscore the notion of distinct functional outcomes on neurogenesis mediated by individual populations of dopamine receptors. Finally, the present observations may be related to accumulating evidence that blockade of D3 receptors favors cognitive function. As such, they may be of significance to the pathogenesis and potential treatment of various disorders in which D3Rs are implicated, including schizophrenia, depression, Parkinson's disease, as well as drug addiction.
We would like to thank Servier for donating S33138, the D3R KO mice as well as for partial financial support. This study was funded by Vetenskapsrådet and Karolinska Institutet. Dr. Martin Egeland was involved in the conception and design, acquisition, analysis, interpretation of the data, and writing of the article. Dr. Xiaoqun Zhang was involved in the acquisition and analysis of data. Drs. Mark J Millan and Elisabeth Mocaer were involved in the interpretation of the data and writing of the article. Dr. Per Svenningsson was involved in the conception and design, interpretation of the data, and writing of the article. Drs. Mark J Millan and Elisabeth Mocaer are full-time employees of Institut de Recherches Servier. Drs Martin Egeland, Xiaoqun Zhang, and Per Svenningsson declare no conflict of interest in this work.