Ghrelin stimulates proliferation, migration and differentiation of neural progenitors from the subventricular zone in the adult mice

Experimental Neurology 252 (2014) 75–84

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Ghrelin stimulates proliferation, migration and differentiation of neural progenitors from the subventricular zone in the adult mice Endan Li a, Yumi Kim a, Sehee Kim a, Takahiro Sato b, Masayasu Kojima b, Seungjoon Park a,⁎ a b

Department of Pharmacology and Medical Research Center for Bioreaction to ROS and Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul, Republic of Korea Department of Molecular Genetics, Institute of Life Science, Kurume University, Hyakunen-kohen 1-1, Kurume, Fukuoka, Japan

a r t i c l e

i n f o

Article history: Received 5 June 2013 Revised 21 November 2013 Accepted 22 November 2013 Available online 1 December 2013 Keywords: Ghrelin Adult neurogenesis Subventricular zone Rostral migratory system Olfactory bulb

a b s t r a c t Ghrelin has been shown to regulate neurogenesis in the hippocampus. The aim of this study was to investigate the possible influence of ghrelin on cell proliferation and neuroblast formation in the subventricular zone (SVZ) and rostral migratory system (RMS) and generation of interneurons in the olfactory bulb (OB). We found that ghrelin receptors were expressed in the SVZ–RMS–OB system. Ghrelin knockout (GKO) mice have fewer proliferating neural progenitor cells and neuroblasts in the SVZ, while ghrelin administration attenuated these changes. We also found that not only the number of BrdU-labeled cells but also the fraction of migratory neuroblasts in the RMS was decreased in the GKO mice compared with controls. Treatment of GKO mice with ghrelin restored these numbers to the wild-type control values. Far fewer BrdU/NeuN double-labeled cells were found in the OB of GKO mice than in wild-type mice 4 weeks after labeling, which were increased by ghrelin replacement. GKO mice showed less numbers of BrdU/calbindin, BrdU/calretinin and BrdU/tyrosine hydroxylase double-labeled cells in the periglomerular layer of the OB. However, these numbers were increased to wild-type values after ghrelin administration. Finally, in the GH-deficient spontaneous dwarf rats, ghrelin increased the number of progenitor cells and neuroblasts in the SVZ, without significant effect on the differentiation in the OB. These findings suggest that ghrelin is involved in the regulation of proliferation of progenitor cells in the SVZ, the number of migratory neuroblasts in the SVZ, and the differentiation of interneurons in the OB. © 2013 Elsevier Inc. All rights reserved.

Introduction It is now well known that adult neurogenesis occurs in at least two regions of the healthy mammalian brain: the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) and the anterior subventricular zone (SVZ) along the walls of the lateral ventricles (Ming and Song, 2005; Zhao et al., 2008). In the SVZ, there are three types of precursor cells: type B glial fibrillary acidic protein (GFAP)-expressing neural progenitor cells, type C transit amplifying cells and type A migrating neuroblasts (Zhao et al., 2008). Type B cells, primary precursors in the SVZ, give rise to type C cells, which rapidly divide to produce doublecortin (DCX)-expressing neuroblasts or type A cells. These newly generated neuroblasts migrate over a long distance through the rostral migratory stream (RMS) to the olfactory bulb (OB), where they become granular and periglomerular interneurons (Zhao et al., 2008). Neurogenesis in the SVZ has been shown to be modulated by several endogenous factors, such as neurotransmitters (serotonin, neuropeptide Y and pituitary adenylate cyclase-activating peptide), hormones (growth hormone (GH) and insulin-like growth factor (IGF)-1) and ⁎ Corresponding author at: Department of Pharmacology and Medical Research Center for Bioreaction to ROS and Biomedical Science Institute, School of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea. Fax: +82 2 967 0534. E-mail address: [email protected] (S. Park). 0014-4886/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.11.021

neurotrophic factors (epidermal growth factor, vascular endothelial growth factor and fibroblast growth factor-2) (Aberg et al., 2009; Bath and Lee, 2010; Decressac et al., 2009; Hurtado-Chong et al., 2009; Ming and Song, 2005; Stanic et al., 2008). However, the endogenous factors that regulate the neurogenesis of neural progenitor cells in the adult SVZ need to be further clarified. Ghrelin, an endogenous ligand of GH secretagogue receptor (GHS-R) 1a, is a novel GH-releasing acylated peptide that is principally synthesized and released from the stomach (Date et al., 2000). In addition to its effects on GH release and energy homeostasis (Kojima et al., 1999; Peino et al., 2000), ghrelin also exerts numerous peripheral effects (Ghigo et al., 2005; Kojima and Kangawa, 2005; Van der Lely et al., 2004). Moreover, recent evidence indicated that ghrelin acts in the central nervous system to control neuronal functions and subsequently influence diverse brain functions (Andrews, 2011). We previously reported that systemic administration of ghrelin stimulated proliferation of newly generating cells in the hippocampus of adult mice and immunoneutralization of ghrelin by using anti-ghrelin antiserum reduced proliferation of hippocampal progenitor cells in the SGZ (Moon et al., 2009). Furthermore, in our recent report (Li et al., 2013a), we found that mice with targeted deletion of the ghrelin gene contain a reduced number of progenitor cells in the DG of the hippocampus, while ghrelin administration restored progenitor cell numbers to those of wild-type controls. Ghrelin-induced proliferative

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effect on hippocampal progenitor cells appears to be mediated through the activation of GHS-R1a because treatment with a receptor-specific antagonist completely blocked the effect of ghrelin in vitro (Chung et al., 2013). Taken together, these findings suggest that endogenous ghrelin may play an important role in adult hippocampal neurogenesis. However, no study thus far has reported the expression of GHS-R1a in the SVZ and the consequences of in vivo ghrelin administration on neurogenesis in the adult SVZ–RMS–OB system. Therefore, we investigated whether endogenous ghrelin influences cell proliferation and neuroblast formation in the SVZ and RMS and affects interneuron generation in the OB of the brain of adult mice by using ghrelin knockout (GKO) mice. We also investigated the effects of intraperitoneal administration of ghrelin on neurogenesis in the SVZ–RMS–OB system in GKO mice. We report that these mice had less proliferating cells, migratory neuroblasts and OB interneurons than wild-type mice, and that this reduction was reversed by ghrelin administration. In addition, to exclude the possibility that the effect of ghrelin on neurogenesis might be mediated via indirect action of ghrelin on the GH/IGF-1 axis, we assessed the impact of ghrelin in the spontaneous dwarf rats (SDRs), a dwarf strain with a mutation of the GH gene resulting in the total loss of GH and decreased IGF-1 levels (Nogami et al., 1989a,b; Takeuchi et al., 1990). Materials and methods Animals To determine the role of ghrelin on adult neurogenesis in the SVZ–RMS–OB system, we used 8–9 week-old male GKO mice (Sato et al., 2008) and age-matched wild-type C57BL/6J mice. The GKO mice we used have been backcrossed at least ten times onto a C57BL/6J background and were obtained from homozygous breeding pairs. Therefore, control mice were not the littermates but were agematched wild-type C57BL/6J mice. To study the effect of ghrelin in the absence of GH, 8–9 week-old male SDRs were used. The animals were housed under controlled environmental conditions (12-h light and 12-h dark) with free access to food and water. They were habituated to the housing conditions for 7–10 days prior to the beginning of the experimental procedures. All experiments were approved by the Kyung Hee University Animal Care Committee and conducted according to the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U. S. National Institutes of Health. All efforts were made to minimize animal suffering and to reduce the number of animals used. Ghrelin treatment and BrdU injection Acylated ghrelin (Peptides International, Inc., Louisville, KY) was dissolved in 0.9% saline and administered intraperitoneally once daily (between 09:00 and 10:00) for 8 consecutive days into the GKO mice or the SDRs at a pharmacological dose of 80 μg/kg. This dose of ghrelin was chosen on the basis of our previous studies demonstrating its effectiveness on hippocampal neurogenesis in adult mice (Li et al., 2013a; Moon et al., 2009) and SDRs (Li et al., 2013b). Control animals were given saline at corresponding times. BrdU (50 mg/kg; SigmaAldrich, St. Louis, MO) was given twice daily at 8-h intervals during the last 3 days of ghrelin treatment, and mice were transcardially perfused 1, 7, or 28 days after the last BrdU administration (for the evaluation of BrdU-labeled cells). Tissue preparation Animals were anesthetized with xylazine and ketamine and then perfused transcardially with a freshly prepared solution of 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were removed and post-fixed overnight in the same fixative before

being immersed in a solution of 30% sucrose in PBS. Serial 30 (for mice) or 40 (for SDRs)-μm-thick coronal tissue sections were cut using a microtome and stored in cryoprotectant (25% ethylene glycol, 25% glycerol, 0.05 M PB; pH 7.4) at − 20 °C for later immunohistochemistry procedures. Immunohistochemistry and immunofluorescence studies In order to minimize staining variations, the brain sections from the same experiment were processed for immunohistochemical staining simultaneously. Free-floating tissue sections were then incubated overnight at 4 °C with primary antibodies (rat anti-BrdU antibody, 1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; mouse anti-BrdU antibody, 1:1000, Roche, Mannheim, Germany; mouse anti-proliferating cell nuclear antigen (PCNA) antibody, 1:500, DakoCytomation, Denmark; goat anti-DCX antibody, 1:1000, Santa Cruz Biotechnology, Inc.). The sections were incubated with appropriate biotinylated secondary antibody (1:200, Vector Laboratories, Burlingame, CA) and then visualized using the avidin–biotin– peroxidase complex method with diaminobenzidine tetrahydrochloride (DAB) as the chromogen. To ensure the detection of BrdU-labeled nuclei, we denatured the DNA before incubation with BrdU antibody because BrdU is incorporated into the DNA. DNA denaturation was performed in the following manner: tissue was incubated in 50% formamide and 2× SSC (1× SSC, 0.3 M NaCl and 0.03 M sodium citrate) for 2 h at 65 °C, rinsed for 15 min in 2× SSC, incubated again for 10 min in 0.1 M boric acid at pH 8.5. For immunodetection of BrdU, the DAB-nickel enhancement technique was used. Rabbit anti-Ki-67 (1:1000, Abcam, Cambridge, UK), mouse antiMash1 (1:500, BD Biosciences, San Jose, CA) and mouse anti-polysialylated neural cell adhesion molecule (PSA-NCAM) (1:500, Chemicon, Temecula, CA) antibodies were also used. After incubations in Ki-67, Mash1 or PSA-NCAM primary antisera, sections were incubated at RT with Cy3-goat anti-rabbit IgG (for Ki-67) or Cy3-goat anti-mouse IgG (for Mash1 and PSA-NCAM) (1:400, Jackson ImmunoResearch, West Grove, PA) for 1.5 h. We routinely checked for aberrant background staining for secondary antibodies by omitting the primary antibodies. To determine the phenotypes of BrdU-labeled cells, brain sections were processed for BrdU and DCX, neuronal nuclei (NeuN), GFAP, calbindin (CB), calretinin (CR) or tyrosine hydroxylase (TH) fluorescent double labeling. Briefly, free-floating brain sections were first pretreated for DNA denaturation as described above, and then incubated overnight at 4 °C with rat anti-BrdU antibody (1:1000) and goat anti-DCX (1:1000), mouse anti-NeuN (1:1000, Millipore, Billerica, MA), rabbit anti-GFAP (1:1000, DakoCytomation), rabbit anti-CB (1:500, Chemicon), rabbit anti-CR (1:1000, Abcam) or rabbit anti-TH (1:2000, Millipore). After washing in 0.1 M PBS, sections were incubated for 1.5 h with fluorescent secondary antibodies: Alexa Fluor 488 goat anti-rat IgG (for BrdU), Cy3-conjugated donkey anti-goat IgG (for DCX), Cy3-conjugated goat anti-mouse IgG (for NeuN) or Cy3conjugated goat anti-rabbit IgG (for GFAP, CB, CR and TH) (1:400, Jackson ImmunoResearch). Then, the sections were washed in PBS and mounted onto coated glass slides, and coversliped with fluorescent mounting medium. Detection of GHS-R1a expression To determine if GHS-R1a was expressed in proliferating progenitor cells, early neuronal cells and astroglial cells in the SVZ–RMS–OB pathway, double immunofluorescence staining was performed. Freefloating sections were incubated with 5- (and 6-) carboxyfluorescein labeled rabbit anti-GHS-R1a antibody (1:500, Phoenix Pharmaceuticals, Burlingame, CA) and rabbit anti-Ki-67 (1:1000), goat anti-DCX (1:1000) or rabbit anti-GFAP (1:1000) overnight at 4 °C. After washing, the sections were incubated with Cy3-conjugated secondary antibodies at room temperature for 1.5 h. Sections were counterstained with DAPI

E. Li et al. / Experimental Neurology 252 (2014) 75–84

before mounting. The specificity of the GHS-R1a antibody used in this study was validated previously (Chung et al., 2013). The sections incubated without the primary antibody for GHS-R1a were also included as negative controls.

Image processing Sections were examined by using a computer-assisted image analysis system consisting of a Zeiss Axioscope-2 microscope equipped with a computer-controlled motorized stage, a video camera, and Stereo Investigator software (MicroBrightField, Williston, VT). For confocal analysis, sections were examined with a Carl Zeiss LSM 700 Meta (Oberkochen, Germany) confocal microscope equipped with appropriate objectives and excitation and emission filters. Digital images from the microscopy were slightly modified to optimize for image resolution, brightness and contrast using Adobe Photoshop CS5 software (Adobe Systems, San Jose, CA).

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Quantification All BrdU-, PCNA-, Ki-67-, Mash1-, DCX-, and PSA-NCAM-labeled cells along the wall of the lateral ventricle were counted manually in five evenly spaced coronal sections per animal (n = 4 to 5 per group), which equaled to every sixth section through the SVZ, from coordinates 0.02 to 0.63 relative to bregma, as defined previously (Franklin and Paxinos, 2001). We have selected the serial sections in which SVZ was clearly observed, based on an anatomical structure. To avoid double counting we did not analyze adjacent sections. The areas of the SVZ were measured on the Nissl-stained sections with Stereo Investigator software. The number of labeled cells (per square millimeter) in the SVZ was calculated and converted per cubic millimeter by taking into account the section thickness. The numbers of double labeled cells for BrdU and DCX were quantified in five evenly spaced coronal sections of RMS per animal (n = 4 to 5 per group), which equaled to every sixth section through this region, from coordinates 2.58 to 3.08 relative to bregma, using

A SVZ

DAPI

Ki-67

GHS-R1a

Merged

DCX

GHS-R1a

Merged

B SVZ

DAPI

C

D

RMS

Granular layer

DAPI/GHS-R1a/DCX

F SVZ

DAPI/GHS-R1a/GFAP

E Periglomerular layer

DAPI/GHS-R1a/DCX

G RMS

DAPI/GHS-R1a/GFAP

H Granular layer

DAPI/GHS-R1a/GFAP

DAPI/GHS-R1a/DCX

I Periglomerular layer

DAPI/GHS-R1a/GFAP

Fig. 1. Expression of GHS-R1a in SVZ–RMS–OB pathway in adult mice. A and B, Confocal microscopic images show that SVZ progenitor cells stained with Ki-67 (red) (A) or neuroblasts stained with DCX (red) (B) are positive for immunoreactivity of GHS-R1a (green). Scale bars represent 5 μm. C–E, Confocal images of colocalization between GHS-R1a (green) and DCX (red) in the RMS (C), OB granular layer (D), and OB periglomerular layer (E). Scale bars represent 10 μm. F–I, Double immunostaining of GHS-R1a (green) and GFAP (red). Cells in the SVZ (F), RMS (G), OB granular layer (H), and OB periglomerular layer (I) showing no certain colocalization between GHS-R1a and GFAP. Scale bars represent 5 μm. DAPI staining revealed nuclei of cells.

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Wild-type

GKO+ Vehicle

GKO+ Ghrelin

Wild-type

GKO+ Vehicle

GKO+ Ghrelin

D

A

PCNA

BrdU

B

E

Ki-67

DCX

C

F

Mash1

PSA-NCAM

Fig. 2. Effect of ghrelin on neural progenitor cells and neuroblasts in the SVZ of adult mice. Animals were injected intraperitoneally with vehicle or ghrelin (80 μg/kg) once daily for 8 consecutive days. BrdU (50 mg/kg, ip) was administered twice daily during the last 3 days of ghrelin treatment and transcardially perfused 1 day after the last BrdU injection. The numbers of PCNA-, Ki-67-, Mash1-, BrdU-, DCX- and PSA-NCAM-labeled cells were counted along the wall of lateral ventricle. Representative microscopic imaging showing PCNA (A), Ki-67 (B), Mash1 (C), BrdU (D), DCX (E) and PSA-NCAM (F). Scale bars represent 100 μm.

confocal microscopy. Co-localization of BrdU and NeuN, GFAP, CB, CR or TH was counted in six evenly spaced coronal sections of OB per animal (n = 4 to 5 per group), which equaled to every sixth section through this area, from coordinates 3.56 to 4.28 relative to bregma, using confocal microscopy. Z-series of focal planes were used to determine co-expression of BrdU and cell-specific markers in the three dimensions. The average number of double-labeled cells per section was determined for each animal, and data were expressed as the mean per group. Investigators were blind to genotype and treatment history for all quantifications. Statistical analysis Data are presented as mean ± SEM. Statistical analysis of the effects of ghrelin in the GKO mice was performed using 1-way ANOVA and

Table 1 Number of cells counted in the SVZ and percentage change from wild-type in parentheses. Data are expressed as the average number of labeled cells per cubic millimeter ± SEM.

PCNA-labeled cells Ki-67-labeled cells Mash1-labeled cells BrdU-labeled cells DCX-labeled cells PSA-NCAM-labeled cells

Wild-type

GKO + vehicle

GKO + ghrelin

36,864 ± 4040 (100 ± 11) 46,058 ± 2663 (100 ± 6) 19,065 ± 562 (100 ± 3) 135,293 ± 3243 (100 ± 2) 34,205 ± 2463 (100 ± 7) 31,516 ± 2169 (100 ± 7)

21,792 ± 4553⁎ (59 ± 12)⁎ 30,524 ± 3243⁎ (66 ± 7)⁎ 12,984 ± 1122⁎ (68 ± 6)⁎ 89,939 ± 6202⁎ (67 ± 5)⁎ 21,183 ± 3101⁎ (62 ± 9)⁎ 22,137 ± 520⁎ (70 ± 2)⁎

30,977 ± 2832⁎⁎ (84 ± 8)⁎⁎ 38,000 ± 2473⁎⁎ (83 ± 5)⁎⁎ 16,331 ± 1225⁎⁎ (86 ± 6)⁎⁎ 117,289 ± 6074⁎⁎ (87 ± 5)⁎⁎ 28,004 ± 2272⁎⁎ (82 ± 7)⁎⁎ 28,065 ± 1412⁎⁎ (89 ± 5)⁎⁎

⁎ P b 0.05 vs. wild-type mice. ⁎⁎ P b 0.05 vs. vehicle-treated GKO mice.

Holm–Sidak method for multiple comparisons using SigmaStat for Windows Version 3.10 (Systat Software, Inc. Point Richmond, CA). The effects of ghrelin in the SDRs were determined by Student's t test. P b 0.05 was considered statistically significant. Results Ghrelin receptors are expressed in SVZ–RMS–OB pathway To examine whether SVZ progenitor cells in adult mice express ghrelin receptors, we carried out double immunohistochemistry with GHS-R1a and Ki-67 (a marker for proliferating cells) antibodies. As shown in Fig. 1A, GHS-R1a immunoreactivities were detected in the SVZ. Moreover, we found that Ki-67 immunoreactivities were colocalized with GHS-R1a immunoreactivities. These results indicate that ghrelin receptors are expressed in Ki-67-positive SVZ progenitor cells in adult mice. We next determined if DCX-positive neuroblasts expressed ghrelin receptors by performing double immunohistochemical staining for GHS-R1a and DCX. Confocal microscopic analysis through the SVZ revealed that GHS-R1a immunoreactivities were present in DCX-positive neuroblasts (Fig. 1B). GHS-R1a/DCX doublelabeled cells were also found in RMS and OB (Figs. 1C-E). GHS-R1a did not colocalize with GFAP, a marker of astrocytes (Figs. 1F–I). Effect of ghrelin on SVZ neural progenitor cells To investigate the function of endogenous ghrelin on adult SVZ neural progenitor cells, we counted the number of cycling cells in the SVZ by using PCNA, a cellular proliferation marker (Ming and Song, 2005), in GKO mice and their wild-type control mice. We detected significantly lower number of newly generating PCNA-labeled cells in GKO mice compared to wild-type controls (Fig. 2A, Table 1). To determine whether exogenous ghrelin can alter the number of

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Effect of ghrelin on migration of newly generated neuroblasts in the RMS Newly generated cells from the SVZ have been known to reach the OB via the RMS (Zhao et al., 2008). To determine whether endogenous ghrelin affects neuroblast migration we injected BrdU twice daily over a period of 3 days and sacrificed mice 1 week after the last BrdU injection. We compared the number of BrdU/DCX double-labeled cells in the RMS in GKO mice and wild-type controls and found significant decreases (59% of wild-type controls) in the number of these cells in GKO mice (Figs. 3A and B). The number of BrdU-labeled cells was also significantly decreased in the RMS of GKO mice compared with wild-type controls (data not shown), suggesting that dividing RMS cells were decreased in these mice. Ghrelin treatment in GKO mice restored the number of BrdU/DCX double-labeled cells in the RMS to the wild-type control values (Figs. 3A and B). Effect of ghrelin on differentiation of newly generated cells in the OB RMS neuroblasts further migrate to the granular and periglomerular layer of the OB, where they differentiate into interneurons called granule and periglomerular cells (Zhao et al., 2008). They gradually lose DCX and express NeuN, a neuronal marker, as they differentiate within the OB (Brown et al., 2003). The number of BrdU/NeuN double-labeled cells in the granular layer (Fig. 4A) was significantly decreased to 57% of wild-type controls in GKO mice 4 weeks after the last BrdU administration (Fig. 4B), suggesting decrease in the number of new neurons in the OB of GKO mice. However, this decrement was significantly attenuated when GKO mice were treated with ghrelin (Fig. 4B). We also determined whether endogenous ghrelin affected the number of newly generated neurons in the periglomerular layer of the OB (Fig. 4C). The number of BrdU/NeuN double-labeled cells in this layer was significantly decreased to 50% of wild-type controls in GKO mice, whereas treatment of GKO mice with ghrelin restored this change (Fig. 4D). In contrast, the numbers of BrdU/GFAP double-labeled cells in both layers were not altered either in GKO mice or by ghrelin treatment (Figs. 4E-H).

GKO+Vehicle

GKO+Ghrelin

BrdU/DCX

B

20

† 15

*

10

in el +G KO G

+V KO G

hr

eh

-ty

ic

pe

0

le

5

ild

In order to determine whether endogenous ghrelin also affects migratory neuroblasts in the SVZ, we measured the number of cells labeled with DCX, a well-known marker for neuroblasts (Gleeson et al., 1999). The number of DCX-labeled cells in the GKO mice was significantly lower than in the wild-type mice, which was reversed by ghrelin treatment (Fig. 2E, Table 1). We also investigated the changes in the number of PSA-NCAM-labeled cells, another marker for SVZ neuroblasts (Bonfanti and Theodosis, 1994), and found similar results (Fig. 2F, Table 1).

WT

W

Effect of ghrelin on SVZ neuroblasts

A

BrdU+/DCX+ cells (mean number)

PCNA-labeled cells in the SVZ of GKO mice, we assessed this parameter after treatment of GKO mice with pharmacological doses of ghrelin (80 μg/kg, ip, once daily for 8 consecutive days). This ghrelin treatment resulted in a shift in the number of PCNA-labeled cells of GKO mice toward the wild-type mice values (Fig. 2A, Table 1). We also examined the changes in the number of Ki-67-labeled cells and found similar results (Fig. 2B, Table 1). We then examined the numbers of type C cells by counting the Mash1-labeled cells in the SVZ (Fig. 2C, Table 1). There were significantly lower numbers of Mash1-labeled cells in the SVZ of the GKO mice than in their wild-type controls. Furthermore, ghrelin administration in the GKO mice significantly increased the numbers of Mash1-labeled cells. Consistent with these findings, we found a similar pattern of changes in the number of BrdU-labeled cells in the SVZ 1 day after the last BrdU injection (Fig. 2D, Table 1). We also counted the numbers of TUNEL- and activated caspase 3-positive cells in the SVZ and found no significant differences between groups (data not shown).

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Fig. 3. Effect of ghrelin on migration of newly generated neuroblasts in the RMS of adult mice. Animals were injected intraperitoneally with vehicle or ghrelin (80 μg/kg) once daily for 8 consecutive days. BrdU (50 mg/kg, ip) was administered twice daily during the last 3 days of ghrelin treatment and transcardially perfused 7 days after the last BrdU injection. A, Representative confocal microscopic images of sagittal sections showing co-localization of BrdU (green) with DCX (red) in the RMS. Scale bar represents 50 μm. B, Quantitative analysis of the mean number of BrdU-labeled cells double-labeled for DCX. The numbers of double-labeled cells for BrdU and DCX were counted in coronal sections of RMS. The data expressed as the mean ± SEM (n = 4–5/group). *P b 0.05 vs. wildtype, †P b 0.05 vs. vehicle-treated GKO mice.

Interneurons in both granular and periglomerular layers express specific neurochemical markers, such as the calcium binding proteins CB and CR (Rogers, 1992) or the dopamine synthesizing enzyme TH (Betarbet et al., 1996). To determine whether decreases in cell proliferation and migratory neuroblasts observed in the SVZ and RMS of GKO mice led to changes in the generation of OB interneurons, we quantified the number of CB-, CR-, or TH-expressing interneurons in the periglomerular layer of the OB in wild-type and GKO mice. The numbers of BrdU/CB (Figs. 5A and B), BrdU/CR (Figs. 5C and D), and BrdU/TH (Figs. 5E and F) double-labeled cells in the periglomerular layer were significantly decreased to 52, 52, and 53% of wild-type controls, respectively, in GKO mice (Figs. 5A–F). However, in ghrelintreated GKO mice, these numbers were increased to wild-type values (Figs. 5A–F). The decreased number of newly generated neurons in the OB of GKO mice could be due to increased cell death. However, the numbers of TUNEL- and activated caspase 3-positive cells in the granular layer and the periglomerular layer did not differ between groups (data not shown). Effect of ghrelin in the GH-deficient SDRs Our previous study showed that ghrelin receptor was expressed in the progenitor cells (Ki-67-positive cells) in the DG of the SDRs (Li et al., 2013b). To examine whether adult SVZ progenitor cells in the SDRs express GHS-R1a, we performed double immunohistochemical staining for GHS-R1a and Ki-67. Shown in Figs. 6A and B, GHS-R1a immunoreactivity was observed in the SVZ. Subsets of cells expressing GHS-R1a were positive for Ki-67 (Fig. 6A). We also found that

E. Li et al. / Experimental Neurology 252 (2014) 75–84

A

B

BrdU

BrdU+/NeuN+ cells (mean number)

80

Merge

C

D

BrdU

NeuN

Merge

F

GFAP

Merge

G

200 Periglomerular layer 150

† 100

*

50

7 Granular layer 6 5 4 3 2 1 0

GFAP

Merge

16 Periglomerular layer 14 12 10 8 6 4 2 0 W

ild -ty pe G KO +V eh ic le G KO +G hr el in

BrdU

*

50

H BrdU+/GFAP+ cells (mean number)

BrdU

†

100

0

BrdU+/GFAP+ cells (mean number)

E

Granular layer

150

0

BrdU+/NeuN+ cells (mean number)

NeuN

200

Fig. 4. Effect of ghrelin on the number of new neurons and astrocytes in the OB of adult mice. Animals were injected intraperitoneally with vehicle or ghrelin (80 μg/kg) once daily for 8 consecutive days. BrdU (50 mg/kg, ip) was administered twice daily during the last 3 days of ghrelin treatment and transcardially perfused 28 days after the last BrdU injection. A, C, E and G, Confocal microscopic images showing co-localization of BrdU (green) with NeuN (red) or GFAP (red) in the OB granular (A and E) and periglomerular (C and G) layer. Scale bars represent 50 μm. B, D, F and H, Quantitative analysis of the mean number of BrdU-labeled cells double-labeled for NeuN or GFAP in the OB granular (B and F) and periglomerular (D and H) layer. The data expressed as the mean ± SEM (n = 4–5/group). *P b 0.05 vs. wild-type, †P b 0.05 vs. vehicle-treated GKO mice.

DCX-positive cells colocalized with GHS-R1a immunoreactivity, indicating that neuroblasts in the SVZ of the SDRs express ghrelin receptors (Fig. 6B). To examine if ghrelin induces SVZ progenitor cell proliferation through the indirect action of ghrelin on the GH/IGF-1 axis, we tested whether treatment of GH-deficient SDRs with ghrelin increased neurogenesis in the SVZ. As shown in Figs. 6C–H and Table 2, we found that ghrelin treatment significantly increased the number of PCNA-, Ki-67, Mash1-, and BrdU-labeled cells to 148%, 146%, 136%, and 129% of vehicle-treated controls, respectively, in the SVZ. DCX- and PSA-NCAM-labeled cells in the SVZ were also increased by ghrelin treatment in these animals (Figs. 6G, H and Table 2). However, there were no significant differences in the numbers of BrdU/NeuN double-labeled cells in the granular (vehicle, 29.1 ± 1.1 vs. ghrelin, 24.6 ± 2.2) and periglomerular layers (vehicle, 4.5 ± 0.8 vs. ghrelin, 4.2 ± 0.7) of the OB after 28 days of BrdU injection. In addition, the numbers of BrdU/GFAP double-labeled cells were not altered by ghrelin in the granular (vehicle, 2.6 ± 0.3 vs. ghrelin,

2.2 ± 0.2) and periglomerular layers (vehicle, 2.8 ± 0.3 vs. ghrelin, 2.6 ± 0.2) of the OB. We also examined the effect of ghrelin treatment on the generation of periglomerular interneurons in the OB, and we found no changes in the numbers of BrdU/CB, BrdU/CR, and BrdU/TH double-labeled cells by ghrelin in the absence of GH (data not shown).

Discussion In the current study, using mice with targeted disruption of the ghrelin gene, we provide evidence that endogenous ghrelin can regulate neurogenesis in the adult mouse SVZ–RMS–OB pathway. Specifically, mice lacking the ghrelin gene have fewer neural progenitor cells in the SVZ, reduced migratory neuroblasts in the RMS, and less OB interneurons. In contrast, ghrelin administration restored these changes to those of wild-type controls. Our data also show that ghrelin receptors are expressed in the adult SVZ and along the normal route of neuroblast migration. To the best of our knowledge, this is the first report

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eh ic le G KO +G hr el in

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Fig. 5. Effect of ghrelin on the number of new CB-, CR- and TH-labeled interneurons in the OB of adult mice. Animals were injected intraperitoneally with vehicle or ghrelin (80 μg/kg) once daily for 8 consecutive days. BrdU (50 mg/kg, ip) was administered twice daily during the last 3 days of ghrelin treatment and transcardially perfused 28 days after the last BrdU injection. A, Confocal microscopic images showing co-localization of BrdU (green) with CB (upper panel), CR (middle panel) and TH (lower panel) (red) in the OB periglomerular layer. Scale bars represent 50 μm. B–D, Quantitative analysis of the mean number of BrdU-labeled cells double-labeled for CB (B), CR (C) and TH (D) in the OB periglomerular layer. The data expressed as the mean ± SEM (n = 4–5/group). *P b 0.05 vs. wild-type, †P b 0.05 vs. vehicle-treated GKO mice.

demonstrating in vivo regulatory effects of ghrelin on proliferation, migration, and differentiation of neural progenitors from the SVZ. Ghrelin is known to stimulate proliferation of cells in the dorsal motor nucleus of the vagus (DMNV) (Zhang et al., 2004) and nucleus of the solitary tract (NTS) (Zhang et al., 2005) in adult rats with cervical vagotomy but not in the sham-operated rats. They also found that exposure of cultured DMNV and NTS neurons to ghrelin increased the BrdU incorporation. In addition, proliferative effects of ghrelin were observed in primary cultures of rat fetal spinal cord (Sato et al., 2006). We have recently reported that ghrelin also promotes proliferation of adult hippocampal progenitor cells in mice (Li et al., 2013a). Thus, in the current study, we investigated whether ghrelin modulates cell proliferation in the adult SVZ. Indeed, targeted deletion of the ghrelin gene resulted in reduced numbers of progenitor cells in the SVZ, which were attenuated by replacement of pharmacological doses of ghrelin. These results suggest that endogenous ghrelin regulates adult SVZ neurogenesis and peripherally administered ghrelin directly stimulates proliferation of SVZ progenitors. This notion is supported by the following findings: i) decreased neuronal cell death and increased neurogenesis are observed in SVZ of dietary restricted rats (Kumar et al., 2009), where circulating ghrelin levels are increased (Lutter et al., 2008), ii) ghrelin is able to cross the blood–brain barrier and enters the brain parenchyma (Diano et al., 2006), iii) ghrelin has a direct proliferative effect on hippocampal progenitor cells in vitro (Chung et al., 2013), and iv) our present results showed that GHS-R1a is expressed in SVZ progenitors. GHS-R1a is the only functional receptor through which acylated ghrelin exerts its effects. This receptor is a member of G-protein-coupled receptors operating via the Gq-phospholipase C signaling pathways and has been first cloned from the pituitary

gland and hypothalamus (Howard et al., 1996). GHS-R1a-mediated regulation of cell proliferation by ghrelin has been reported in cultured adult hippocampal neural stem cells (Chung et al., 2013) and NTS neuronal cells (Zhang et al., 2005). Receptor-mediated effects of ghrelin were also observed in neuronal cells exposed to oxygen–glucose deprivation (Chung et al., 2007, 2008). Ghrelin potently stimulates GH secretion from the anterior pituitary gland (Kojima et al., 1999) and consequently increases IGF-1. Given that the GKO mice show a preservation of GH/IGF-1 axis (Pfluger et al., 2008; Sun et al., 2003; Wortley et al., 2005), there is a possibility that the action of ghrelin on SVZ neurogenesis could be, at least in part, due to the ability of ghrelin to stimulate the somatotropic axis. Supporting evidence of this assumption is the fact that long-term GH treatment has been shown to increase SVZ neurogenesis (Aberg et al., 2009). In order to further investigate the direct effect of ghrelin on SVZ neurogenesis, we examined the effect of ghrelin in the SDRs, a dwarf strain with a mutation of the GH gene resulting in total loss of GH (Nogami et al., 1989a,b; Takeuchi et al., 1990). In the pituitary of the SDR, neither GH cells nor GH protein was detected by immunologic methods. Furthermore, neither accumulation nor secretion of immunoreactive GH was detected from SDR pituitaries stimulated by GHRH or db-cAMP (Takeuchi et al., 1990). In the SDRs, plasma IGF-1 and insulin levels are also reduced, compared to levels in Sprague–Dawley rats (Kuramoto et al., 2010). Taken together, these findings suggest that exogenous administration of ghrelin into SDRs does not stimulate GH and IGF-1 secretion. Therefore, the SDRs have been widely used for the study of GH-independent effects of ghrelin (Fukushima et al., 2005; Sangiao-Alvarellos et al., 2009, 2010; Shintani et al., 2001). In addition, we have reported that the number of newly generated cells

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Table 2 Number of cells counted in the SVZ and percentage change from vehicle-treated SDRs in parentheses. Data are expressed as the average number of labeled cells per cubic millimeter ± SEM.

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20,551 ± 2689 (100 ± 13) 22,946 ± 1771 (100 ± 8) 6243 ± 446 (100 ± 7) 90,918 ± 4470 (100 ± 5) 7587 ± 862 (100 ± 11) 6738 ± 835 (100 ± 12)

30,391 ± 2590⁎ (148 ± 13)⁎ 33,560 ± 1290⁎ (146 ± 6)⁎ 8471 ± 837⁎ (136 ± 13)⁎

116,884 ± 6178⁎ (129 ± 7)⁎ 13,297 ± 855⁎ (175 ± 11)⁎ 10,929 ± 759⁎ (162 ± 11)⁎

⁎ P b 0.05 vs. vehicle-treated SDRs.

PCNA

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Fig. 6. Expression of GHS-R1a in SVZ progenitor cells and neuroblasts (A and B) and effect of ghrelin on neurogenesis in the GH-deficient spontaneous dwarf rats (SDRs) (C–H). A and B, Confocal microscopic images show that SVZ progenitor cells stained with Ki-67 (red) (A) or neuroblasts stained with DCX (red) (B) are positive for immunoreactivity of GHS-R1a (green). DAPI staining revealed nuclei of SVZ cells in the lateral ventricle. Scale bars represent 5 μm. C–H, SDRs were injected intraperitoneally with vehicle or ghrelin (80 μg/kg) once daily for 8 consecutive days. BrdU (50 mg/kg, ip) was administered twice daily during the last 3 days of ghrelin treatment and transcardially perfused 1 day after the last BrdU injection. The numbers of PCNA-, Ki-67-, Mash1-, BrdU-, DCX- and PSA-NCAM-labeled cells were counted along the wall of lateral ventricle. Representative microscopic imaging showing PCNA (C), Ki-67 (D), Mash1 (E), BrdU (F), DCX (G) and PSA-NCAM (H). Scale bars represent 100 μm (C, D and F) and 50 μm (E, G and H). The data expressed as the mean ± SEM (n = 4–5/group). *P b 0.05 vs. vehicle-treated control group.

in the SGZ of the hippocampus in the SDRs was significantly lower than in normal Sprague–Dawley rats (Li et al., 2011). In the current study, we found that ghrelin augmented proliferation of SVZ progenitor cells in the GH-deficient SDRs. Our findings suggest that ghrelin promotes proliferation of SVZ newborn cells, at least in part, in a GH-independent fashion. Indeed, we recently reported that ghrelin increased proliferation of hippocampal progenitor cells in the SDRs, suggesting that ghrelin-induced hippocampal neurogenesis is mediated independently of GH/IGF-1 axis (Li et al., 2013b). In addition, it should be noted that the activation of vagus nerve might have significant role in conveying systemically administered ghrelin signals to the brain. In fact, blockade of the gastric vagal afferent system by vagotomy abolished peripheral ghrelin-induced feeding, reduced ghrelin-induced GH secretion

(Asakawa et al., 2001; Date et al., 2002) and blunted neuroprotective effects of ghrelin in focal cerebral ischemia (Cheyuo et al., 2011). In agreement with our recent study reporting fewer neuroblasts in the SGZ of GKO mice (Li et al., 2013a), we found that mice with targeted deletion of the ghrelin gene had less migratory DCX- or PSA-NCAMpositive neuroblasts in the SVZ and RMS than wild-type controls, which were recovered by ghrelin administration. The reduction of neuroblasts in the GKO mice is likely a consequence of the reduced number of proliferating cells observed in these animals. It is unclear whether ghrelin directly and acutely affects SVZ migration from the findings obtained from this study. However, it should be noted that ghrelin is known to stimulate migration of other cell types, including cardiac microvascular endothelial cells, glioma cells and endothelial progenitor cells (Chen et al., 2011, 2013; Wang et al., 2012). It remains to be determined whether ghrelin directly regulates neuroblast migration from the SVZ. In the current study, we found fewer number of newborn neurons in the granular and periglomerular layer of the OB in GKO mice 4 weeks after the last BrdU administration, suggesting that this reduction appears to be due to decreased number of proliferating progenitors in the SVZ. We also have shown that there was a reduced number of BrdU-labeled cells coexpressing CB, CR or TH in the periglomerular layer of the OB, suggesting another role of ghrelin in the differentiation and final placement of newly derived interneurons in the OB. Considering that animals with decreased number of OB interneurons, such as NCAM knockout mice, show deficits in olfactory discrimination (Gheusi et al., 2000; Zhao et al., 2008), these findings suggest that endogenous ghrelin may play a role in olfactory behavior. Supporting evidence of this notion is that fasting enhances olfactory sensitivity (Aime et al., 2007) and elevates circulating ghrelin (Patterson et al., 2011). Moreover, Tong et al. (2011) reported that ghrelin administration decreased odor detection threshold and enhanced sniffing frequency in rats and humans. It remains to be resolved whether endogenous ghrelin is directly associated with olfactory discrimination. However, in the GH-deficient SDRs, the number of BrdU/NeuN double-labeled cells 4 weeks after the last BrdU administration as well as the numbers of BrdU/CB, BrdU/CR, and BrdU/TH double-labeled cells did not differ between vehicle-treated and ghrelin-treated groups, suggesting that the effect of ghrelin on the differentiation and final placement of newly derived interneurons in the OB appears to require GH and/or IGF-1 increased by ghrelin treatment. In conclusion, our data suggest that ghrelin is involved in the regulation of progenitor cell proliferation and migration in the SVZ and RMS. Our findings also suggest that ghrelin may influence their differentiation into distinct subsets of interneurons in the OB. Our results obtained from the SDRs suggest that ghrelin-induced

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proliferation of SVZ progenitor cells and neuroblasts appears to be mediated independently of the somatotropic axis. In contrast, the effect of ghrelin on the differentiation of newly generated OB interneurons might be mediated, at least in part, by indirect action of ghrelin on the GH/IGF-1 axis. These findings may further explain how neurogenesis is regulated in the adult brain and may help to lead to future development of regenerative therapies for neurodegenerative diseases. Disclosure summary The authors have nothing to disclose. Acknowledgments This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. 20120009383 and NRF-2012R1A1A2039867). References Aberg, N.D., Johansson, I., Aberg, M.A., Lind, J., Johansson, U.E., Cooper-Kuhn, C.M., Kuhn, H.G., Isgaard, J., 2009. Peripheral administration of GH induces cell proliferation in the brain of adult hypophysectomized rats. J. Endocrinol. 201, 141–150. Aime, P., Duchamp-Viret, P., Chaput, M.A., Savigner, A., Mahfouz, M., Julliard, A.K., 2007. Fasting increases and satiation decreases olfactory detection for a neutral odor in rats. Behav. Brain Res. 179, 258–264. Andrews, Z.B., 2011. The extra-hypothalamic actions of ghrelin on neuronal function. Trends Neurosci. 34, 31–40. Asakawa, A., Inui, A., Kaga, T., Yuzuriha, H., Nagata, T., Ueno, N., Makino, S., Fujimiya, M., Niijima, A., Fujino, M.A., Kasuga, M., 2001. Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology 120, 337–345. Bath, K.G., Lee, F.S., 2010. Neurotrophic factor control of adult SVZ neurogenesis. Dev. Neurobiol. 70, 339–349. Betarbet, R., Zigova, T., Bakay, R.A., Luskin, M.B., 1996. Dopaminergic and GABAergic interneurons of the olfactory bulb are derived from the neonatal subventricular zone. Int. J. Dev. Neurosci. 14, 921–930. Bonfanti, L., Theodosis, D.T., 1994. Expression of polysialylated neural cell adhesion molecule by proliferating cells in the subependymal layer of the adult rat, in its rostral extension and in the olfactory bulb. Neuroscience 62, 291–305. Brown, J.P., Couillard-Despres, S., Cooper-Kuhn, C.M., Winkler, J., Aigner, L., Kuhn, H.G., 2003. Transient expression of doublecortin during adult neurogenesis. J. Comp. Neurol. 467, 1–10. Chen, J.H., Huang, S.M., Chen, C.C., Tsai, C.F., Yeh, W.L., Chou, S.J., Hsieh, W.T., Lu, D.Y., 2011. Ghrelin induces cell migration through GHS-R, CaMKII, AMPK, and NF-kappaB signaling pathway in glioma cells. J. Cell. Biochem. 112, 2931–2941. Chen, X., Chen, Q., Wang, L., Li, G., 2013. Ghrelin induces cell migration through GHSR1amediated PI3K/Akt/eNOS/NO signaling pathway in endothelial progenitor cells. Metabolism 62, 743–752. Cheyuo, C., Wu, R., Zhou, M., Jacob, A., Coppa, G., Wang, P., 2011. Ghrelin suppresses inflammation and neuronal nitric oxide synthase in focal cerebral ischemia via the vagus nerve. Shock 35, 258–265. Chung, H., Kim, E., Lee, D.H., Seo, S., Ju, S., Lee, D., Kim, H., Park, S., 2007. Ghrelin inhibits apoptosis in hypothalamic neuronal cells during oxygen–glucose deprivation. Endocrinology 148, 148–159. Chung, H., Seo, S., Moon, M., Park, S., 2008. Phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3 beta and ERK1/2 pathways mediate protective effects of acylated and unacylated ghrelin against oxygen–glucose deprivation-induced apoptosis in primary rat cortical neuronal cells. J. Endocrinol. 198, 511–521. Chung, H., Li, E., Kim, Y., Kim, S., Park, S., 2013. Multiple signaling pathways mediate ghrelin-induced proliferation of hippocampal neural stem cells. J. Endocrinol. 218, 49–59. Date, Y., Kojima, M., Hosoda, H., Sawaguchi, A., Mondal, M.S., Suganuma, T., Matsukura, S., Kangawa, K., Nakazato, M., 2000. Ghrelin, a novel growth hormone-relasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 141, 4255–4261. Date, Y., Murakami, N., Toshinai, K., Matsukura, S., Niijima, A., Matsuo, H., Kangawa, K., Nakazato, M., 2002. The role of the gastric afferent vagal nerve in ghrelin-induced feeding and growth hormone secretion in rats. Gastroenterology 123, 1120–1128. Decressac, M., Prestoz, L., Veran, J., Cantereau, A., Jaber, M., Gaillard, A., 2009. Neuropeptide Y stimulates proliferation, migration and differentiation of neural precursors from the subventricular zone in adult mice. Neurobiol. Dis. 34, 441–449. Diano, S., Farr, S.A., Benoit, S.C., McNay, E.C., da Silva, I., Horvath, B., Gaskin, F.S., Nonaka, N., Jaeger, L.B., Banks, W.A., Morley, J.E., Pinto, S., Sherwin, R.S., Xu, L., Yamada, K.A., Sleeman, M.W., Tschop, M.H., Horvath, T.L., 2006. Ghrelin controls hippocampal spine synapse density and memory performance. Nat. Neurosci. 9, 381–388. Franklin, K.B.J., Paxinos, G., 2001. In: Franklin, K.B.J.P.G. (Ed.), The Mouse Brain in Stereotaxic Coordinates. Academic Press, San Diego.

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