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Effects of hindlimb unloading on neurogenesis in the hippocampus of newly weaned rats
Neuroscience Letters 509 (2012) 76–81
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Effects of hindlimb unloading on neurogenesis in the hippocampus of newly weaned rats Sachiko Nomura a , Katsuya Kami a , Fuminori Kawano a , Yoshihiko Oke a , Naoya Nakai a , Takashi Ohira b , Ryo Fujita a , Masahiro Terada b , Kazuhiko Imaizumi c , Yoshinobu Ohira a,b,∗ a
Graduate School of Medicine, Osaka University, Toyonaka City, Osaka 560-0043, Japan Frontier Biosciences, Osaka University, Toyonaka City, Osaka 560-0043, Japan c Graduate School of Human Sciences, Waseda University, Tokorozawa City, Saitama 359-1192, Japan b
a r t i c l e
i n f o
Article history: Received 4 June 2011 Received in revised form 29 November 2011 Accepted 12 December 2011 Keywords: Hindlimb suspension Newly weaned rat Hippocampal neurogenesis Doublecortin-positive neurons
a b s t r a c t Effects of hindlimb suspension (HS) and ambulation recovery on hippocampal neurogenesis of newly weaned rats were studied by using immunohistochemical techniques. The number of proliferating cell nuclear antigen-positive (PCNA+ ) cells in the subgranular zone (SGZ) markedly decreased during normal growth. However, neither HS nor subsequent recovery caused additional changes in the number of PCNA+ cells. The number of doublecortin-positive (DCX+ ) neurons decreased gradually during normal growth. HS resulted in a further decrease in these neurons. However, DCX+ cell numbers became identical to the levels in age-matched controls after 14 days of recovery. PCNA and DCX-double positive cells in the SGZ were also observed, and their cell numbers were not affected by HS and 14-day ambulation. Thus, HS suppressed the generation of DCX+ neurons without affecting PCNA+ cells in the SGZ of weaned rats. Taken together, hippocampal neurogenesis in weaned rats was not severely affected by HS while it decreased significantly as they had grown. © 2011 Elsevier Ireland Ltd. All rights reserved.
Introduction Unlike most neurons in the adult brain, granule neurons continue to be produced in the dentate gyrus (DG) of the hippocampus throughout life. Hippocampal neurogenesis is a multi-step process that is followed by the formation of postmitotic functionally integrated neurons in the granular cell layer (GCL) of the DG. Markers that are expressed at different stages of hippocampal neurogenesis have been investigated to monitor newly generated cells in the subgranular zone (SGZ) of the DG. Proliferating cell nuclear antigen (PCNA), a mitotic marker, labels all proliferating cells in the DG; thus they do not allow newly formed neurons and glia to be distinguished from one another. To determine whether changes in PCNA-positive (PCNA+ ) cells are indeed related to altered neurogenesis, this labeling must be combined with other immunohistochemical markers that label newly formed neurons at later stages of neurogenesis. Doublecortin (DCX) expression is specific for newly generated neurons, since virtually all DCX-positive (DCX+ ) cells express early neuronal antigens but lack antigens specific to glia [20]. Therefore, PCNA and DCX
∗ Corresponding author at: Section of Applied Physiology, Graduate School of Medicine, Osaka University, 1-17 Machikaneyama-cho, Toyonaka City, Osaka 5600043, Japan. Tel.: +81 6 6850 6032; fax: +81 6 6850 6032. E-mail address: [email protected] (Y. Ohira). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.12.022
double-positive (PCNA+ /DCX+ ) cells in the SGZ can be regarded as mitotic neuronal precursors. Exercise enhances neurogenesis in the DG [2,4,23–25] and improves cognitive performance [1]. Increased numbers of PCNA+ cells, DCX+ cells and PCNA+ /DCX+ cells were observed in the SGZ of rats that performed chronic treadmill running [23]. The effects of exercise contrast the effects of stress, which is one of the most potent environmental parameters; both physiological and psychosocial stressors inhibit one or more processes of hippocampal neurogenesis [19]. For example, maternal separation is currently drawing attention as a well-characterized model of stress in rodents, and maternally deprived rats exhibit suppression of hippocampal neurogenesis later in life [14], more anxiety, and diminished spatial learning [12]. These studies indicate that a lasting reduction in hippocampal neurogenesis tends to follow stress exposure at early stages in life. Hindlimb suspension (HS) of rats, which inhibits the antigravity activity of hindlimb muscles, was initially proposed for studying spaceflight-associated inhibition of bone formation [15]. Thereafter, investigations on the effects of HS on hindlimb muscles [8,9,18,26] have been performed. A recent study [27] reported that the HS model was a useful tool to study how hippocampal neurogenesis can be modulated by withholding physical stimulation, thereby providing novel information that shows the exercisemediated enhancement of hippocampal neurogenesis [24,25]. Although the effects of HS on hippocampal neurogenesis in adult
S. Nomura et al. / Neuroscience Letters 509 (2012) 76–81
rats have been reported [27], its effects in earlier stages of life have not yet been examined. It was hypothesized that HS during early life in rats might suppress the hippocampal neurogenesis more severely because promotion or inhibition of hippocampal neurogenesis depends largely on experiences during the early postnatal period rather than experiences after maturity [14]. To further understand the regulatory systems that control neurogenesis in the DG, it is important to establish whether neurogenesis is severely affected by withholding physical stimulation in weaned rats. Therefore, the effects of HS on neurogenesis in the DG of the hippocampus and responses to ambulation recovery from HS in weaned rats were investigated in the present study. Materials and methods All experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23; Revised 1996). The study was also approved by the Animal Use Committee of Osaka University. Male Wistar Hannover rats (3 weeks old) were used. Each rat was individually housed in a cage. Water and a commercial solid diet (CE-2, Nihon CLEA, Tokyo, Japan) were supplied ad libitum. A total of 56 rats were randomly divided into the following five groups; pre-experimental group (pre, n = 12); normal cage housing for 14 days (day 14-control, n = 12); HS for 14 days (day 14-HS, n = 12); normal cage housing for 28 days (day 28-control, n = 10); and 14 days of HS followed by 14-day ambulation recovery (day 28-HS, n = 10). HS of rats was performed as described previously [9]. At the end of each paradigm, half of the rats in each group (n = 5 or 6) were anesthetized and sacrificed by transcardial perfusion of 0.85% NaCl prior to perfusion with 4% paraformaldehyde in 0.1 M phosphate-buffer (PB, pH 7.4). The whole brain was removed, postfixed in 4% paraformaldehyde in PB for 24 h at 4 ◦ C, and immersed in 30% sucrose in PB at 4 ◦ C. The brain was then frozen in liquid nitrogen-cooled isopentane and stored at −80 ◦ C until analysis. The frozen cerebral region including the hippocampus was cut into 20-m-thick sections by using a cryostat at −20 ◦ C. Immunohistochemical analysis was performed using a modified method described by Uda and colleagues [23]. Briefly, sections were immersed in 0.01 M citrate buffer (pH 6.0) and boiled for antigen retrieval. After blocking, the sections were incubated for 48 h at 4 ◦ C with a mixture of two primary antibodies: a mouse anti-PCNA antibody (1:50; DAKO, Carpinteria, CA) and a goat anti-DCX antibody (1:200; Santa Cruz Biotech., Santa Cruz, CA). Subsequently, the sections were incubated overnight at 4 ◦ C with secondary antibodies: Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:400; Molecular Probes, Eugene, OR) and Alexa Fluor 594-conjugated anti-goat IgG antibody (1:400). Finally, the sections were mounted in Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) with 4 ,6-diamidino-2-phenylidole (DAPI). Quantitative analyses for immune-stained cells were performed with an Olympus BX-51 fluorescent microscope (Olympus, Tokyo, Japan) and a Leica TCS SP5 Confocal Laser Scanning Microscope (Leica, Wetzlar, Germany). All of the PCNA+ and DCX+ cells in the SGZ were manually counted in the whole GCL of each section. In addition, PCNA+ /DCX+ cells in the SGZ were also manually counted as mitotic neuronal precursors according to the previously described method [23]. The results were reported as the number of labeled cells per area of whole GCL (mm2 ) that was calculated in each Nissl-stained section, because the mean area of the GCL in the DG was not different among the groups (data not shown). Several growth factors and hormones play important roles in the regulation of hippocampal neurogenesis in adult rats [11]. Therefore, the levels of these factors in the plasma and
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hippocampus were measured. Blood samples were collected from the right atrium, before transcardiac perfusion, in all rats by using heparinized syringes. The blood samples were immediately centrifuged for 10 min (1200 × g, 4 ◦ C) for enzyme-linked immunosorbent assay (ELISA). The remaining rats in all five groups (n = 5 or 6) were anesthetized and sacrificed by blood removal. Each brain was quickly removed, and the hippocampal formation was freshly isolated. The tissue was homogenized by sonication in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) with protease inhibitor cocktail (Sigma–Aldrich Co., St. Louis, MO), and centrifuged for 20 min (15,000 rpm, 4 ◦ C). The supernatant was used for ELISA. Total protein concentrations of these hippocampus samples were determined by the bicinchoninic acid method. In addition to the hippocampus, unfixed soleus muscles were removed from these rats, and their wet weights were measured as an indicator of antigravity muscle atrophy. Commercial ELISA systems were used to determine plasma and hippocampal levels of insulin-like growth factor-1 (IGF-1; R&D Systems, Inc., Minneapolis, MN, USA), vascular endothelial growth factor (VEGF; RayBiotech Inc., Norcross, GA), corticosterone (Endocrine Technologies, Inc., Newark, CA) and brain-derived neurotrophic factor (BDNF; Promega Corp., Madison, WI), according to the manufacturer’s protocols. The hippocampal level of each protein was expressed as the ratio to total protein content of the tissue sample. All data are expressed as the mean ± SEM. The results were evaluated by using one-way factorial analysis of variance (ANOVA) followed by Scheffe’s post hoc test. Statistical significance was accepted at p < 0.05. Results and discussion General growth Growth-associated increases in body weight were seen in the control groups at day 14 and day 28 (Table 1). Even though significant gain of body weight was also seen in the HS group, the mean weight was slightly less than the age-matched controls. However, HS and the subsequent recovery did not change the body weight significantly in weaned rats. Both the absolute and relative (to body weight) weights of the soleus were lower in the HS group at day 14 than in the age-matched control group (Table 1). Compared with the HS group at day 14, the mean absolute weight was increased by 440% within 14 days of recovery at day 28, but was still lower than in the age-matched control group. The mean relative weights showed a similar response (Table 1). These results clearly show that hindlimb unloading induced atrophy of soleus muscles in the present study, which is consistent with our previous report [26]. Neurogenesis in the DG of the hippocampus In a previous study with adult rats [27], 5-bromodeoxyuridine (BrdU) was used as a mitotic marker in the DG. However, it was reported that BrdU has teratological effects on the body, brain and behavior of fetal and infant rats [10]. Therefore, we used PCNA as a mitotic marker (Fig. 1, arrow b and c in B, arrow d in F) according to the previous study [23]. Mitotic neuronal precursors (PCNA+ /DCX+ cells) and newly generated neurons (DCX+ cells) in the DG of weaned rats were also immunolabeled as shown in Fig. 1. In 5-week-old control rats (day14-control, Fig. 1E–H), the morphological features of DCX+ cells were in agreement with those in adult rats [23], and the processes growing from DCX+ somata penetrated through the GCL into the molecular layer (Fig. 1G, arrow e). However, in the 3-week-old group (pre, Fig. 1A–D), DCX+ cells protruded
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S. Nomura et al. / Neuroscience Letters 509 (2012) 76–81
Table 1 Effects of hindlimb suspension on body weight and soleus weight. Pre
Body weight (g) Soleus weight Absolute weight (mg) Relative weight (mg/g body weight)
88.3 ± 2.75 32.6 ± 0.78 0.67 ± 0.017
Day 14
Day 28
Control
HS
Control
HS
164.0 ± 2.88*
150.5 ± 6.68*
256.4 ± 5.27* , †
240.4 ± 8.95* , †
160.3 ± 6.73* , † 0.73 ± 0.011
116.5 ± 3.24* , † , § 0.62 ± 0.008§
89.8 ± 1.70* 0.70 ± 0.020
26.2 ± 2.75† 0.24 ± 0.018* , †
The rats were randomly divided into five groups as follows: pre group (pre-experimental group), day 14-control group (normal cage housing for 14 days), day 14-HS group (HS for 14 days), day 28-control group (normal cage housing for 28 days), and day 28-HS group (14 days of HS followed by 14-day ambulation recovery). All data are indicated as the mean ± SEM. * p < 0.05 vs. pre group. † p < 0.05 vs. day 14-control group. §
p < 0.05 vs. day 28-control group.
many fine processes (Fig. 1C, arrow a and b) that were not observed at 5 weeks of age. Despite the morphological differences between the 3- and 5-week-old groups, DCX+ cells and PCNA+ /DCX+ cells (Fig. 1D, arrow b) were localized only in the SGZ within the DG in both groups (Fig. 2A and B). Moreover, HS and subsequent recovery did not alter the localization of these cells (Fig. 2).
Quantitative comparisons of PCNA+ , DCX+ and PCNA+ /DCX+ cells in the SGZ are shown in Fig. 2. The number of PCNA+ cells in the SGZ was significantly less in the control groups at days 14 and 28 than in the pre-experimental group, indicating that the number of proliferating cells in the SGZ decreased after the lactation period (Fig. 2F). The developmental reduction in the number of PCNA+ cells was
Fig. 1. Photomicrographs showing proliferating cells, mitotic neuronal precursors and newly generated neurons in the SGZ. Brain sections were prepared from rats in the pre-experimental group (A–D) and after 14 days of cage housing (E–F). Triple fluorescence staining was performed with DAPI (in blue, A and E), anti-PCNA antibody (in green, B and F) and anti-DCX antibody (in red, C and G). The merged images are also shown (D and H). Arrows in panels indicate DCX+ newly generated neurons (arrow a, b and e), PCNA+ /DCX+ mitotic neuronal precursors (arrow b), and PCNA+ proliferating cells (arrow b, c and d) in the SGZ. Bars = 10 m.
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Fig. 2. Effects of HS on cell proliferation and neuron generation in the SGZ. PCNA and DCX immunoreactivities were observed in the SGZs before initiation of the experiment (A), after 14 days of cage housing (B), after 14 days of HS (C), after 28 days of cage housing (D), and after 14 days of ambulation recovery from 14-day HS (E). The number of PCNA+ cells (F), DCX+ neurons (G), and PCNA+ /DCX+ mitotic neuronal precursors (H) were counted. Results are shown as the mean ± SEM per mm2 of granular cell layer. Bar = 100 m (A–E). * and #p < 0.05 vs. pre and day 14-control, respectively.
not influenced by HS and ambulation recovery. The total number of DCX+ cells decreased gradually following the normal growth of control rats (Fig. 2G). The number of DCX+ cells in the HS group at day 14 was significantly less than in the age-matched control group. However, the number of DCX+ cells was stable even after 14 days of ambulation, and the number of DCX+ cells in the HS group at day 28 was identical to the number in the age-matched controls. Meanwhile, the numbers of PCNA+ /DCX+ cells in the SGZ in all groups at days 14 and 28 were significantly less than in the preexperimental group (Fig. 2H). There were no effects of HS and a 14-day ambulation period on the numbers of PCNA+ /DCX+ cells. The previous study with adult rats [27] showed that the numbers of proliferating cells in the SGZ were decreased by 14 days of HS. Thus, hippocampal neurogenesis in weaned rats thought to be less susceptible to the suppressive effects of HS than in adult animals. However, the difference of the experimental system for newly weaned rat, PCNA labeling, in the present study from that for adult rats, BrdU injection, in the previous reports [27] obstructs this statement. An earlier study has warned against the substitution of BrdU labeling with PCNA staining in rat peripheral tissues [16]. Although there are some cases where the PCNA staining well correlates with BrdU labeling in the SGZ [7,28], these immunohistochemical data cannot prove that newly weaned rats are less susceptible to the HS-treatment than adult rats. Neuronal precursors in the SGZ can shift their fate toward cell death during the processes of hippocampal neurogenesis [6]. Therefore, we evaluated caspase 3-dependent apoptosis and DNA fragmentation of newly generated neurons in the SGZ. Active caspase 3-positive cells were detected in the SGZ of 3-week-old rats (data not shown). However, no immunoreactivity for active caspase 3 was observed in the SGZ of 5-week-old control rats. Similar to the age-matched control group, no active caspase 3 expression was evident in the SGZ in the HS group at 5 weeks of
age. Then DNA fragmentation of these neurons was detected by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method. Although TUNEL-positive cells were observed in the subventricular zone of 3-week-old rats, no immunoreactivities were observed in the SGZ of 3- and 5-week-old control rats, which is consistent with a previous report [5]. Besides control groups, no TUNEL-positive cells were detected in the SGZ in the HS group (data not shown). In the present study, there was no evidence that HS induced apoptosis in newly generated neurons in the SGZ. Therefore, we conclude that the HS-induced decrease in newly born neurons does not result from increased cell death. Effects of growth factors and hormones We also checked the plasma and hippocampal levels of candidate factors affecting hippocampal neurogenesis. Trejo et al. showed that peripheral IGF-1 crosses the blood–brain barrier and stimulates hippocampal neurogenesis [22]. In the present study, the plasma levels of IGF-1 increased during the normal growth of the rats, but HS did not cause a significant decrease (Table 2). VEGF, another neurotrophic factor, was not detected in plasma in weaned rats (Table 2), unlike previously reported results with adult rats [27]. Moreover, recent evidence suggests that the elevation of plasma glucocorticoid levels by various stimuli plays a critical role in suppressing hippocampal neurogenesis [13]. However, our data indicate that HS did not cause a significant increase in the plasma level of corticosterone (Table 2). Meanwhile, the BDNF level in the hippocampus was significantly decreased in the HS group at day 14 as compared to the pre-experimental group, but it did not change in the age-matched controls (Table 2). There was also a slight difference in the BDNF levels between the HS and control groups at day 14, although the difference was not statistically significant (p = 0.09). HS did not cause significant changes in the IGF-1 level in
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Table 2 Effects of hindlimb suspension on plasma and hippocampal levels of hormones and neurotrophic factors. Pre
Day 14
Day 28
Control
HS
Control
HS
Plasma levels IGF-1 (g/ml) Corticosterone (ng/ml)
0.63 ± 0.025 80.4 ± 7.37
1.20 ± 0.059* 106.4 ± 10.71
1.03 ± 0.101* 122.0 ± 10.67
1.64 ± 0.039* , † 112.4 ± 8.31
1.41 ± 0.039* 131.4 ± 15.46
Hippocampal levels BDNF (pg/mg protein) IGF-1 (pg/mg protein) VEGF (pg/mg protein)
12.8 ± 0.56 37.8 ± 2.17 27.6 ± 3.23
11.6 ± 0.42 40.9 ± 1.29 28.4 ± 5.33
9.8 ± 0.26 44.3 ± 2.79 33.0 ± 8.19
13.4 ± 0.36 40.2 ± 1.87 124.6 ± 10.53* , †
12.7 ± 0.34 39.7 ± 1.36 111.6 ± 7.38* , †
The rats were randomly divided into five groups as follows; pre group (pre-experimental group), day 14-control group (normal cage housing for 14 days), day 14-HS group (HS for 14 days), day 28- control group (normal cage housing for 28 days), and day 28-HS group (14 days of HS followed by 14-day ambulation recovery). At the end of each paradigm, blood and hippocampal formation were collected. The levels of insulin-like growth factor-1 (IGF-1), corticosterone, brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were measured with ELISA systems. All data are indicated as the mean ± SEM. * p < 0.05 vs. pre group. † p < 0.05 vs. day 14-control group.
the hippocampus. The VEGF level in the hippocampus was significantly increased in the control group at day 28 as compared to day 14, but the developmental increase in the VEGF level was not influenced by HS and ambulation recovery (Table 2). Thus, there were no significant differences in the plasma and hippocampal levels of candidate factors regulating hippocampal neurogenesis between the HS and control groups at day 14. It is possible that HS did not cause serious damage to the hippocampal neurogenesis of weaned rats because the rats have high adaptability to environmental changes at that stage of life. In addition to withholding physical stimulation, HS seems to be a strong stressor associated with neurogenesis. Indeed, in some reports, the lack of exercise achieved by HS is accompanied by chronic stress [3] and depression, which in turn increases the glucocorticoid levels [21]. Restraint stress also suppresses hippocampal neurogenesis [19], and decreased body weights and increased plasma corticosterone levels were observed in rats subjected to restraint stress [17]. However, the present HS model did not alter body weight and only produced an incremental trend in the plasma corticosterone levels of rats. Taken together, relatively modest hormonal stress accompanied HS, indicating that the generation of neurons in the DG was likely suppressed as a function of lack of exercise in weaned rats. Similar results were obtained from a study of adult rats [27].
Conclusion HS induced a transient reduction in the total number of newly generated neurons without any changes in the number of proliferating cells or mitotic neuronal precursors in the SGZ. Contrary to our expectation, these findings suggest that HS did not cause serious damage to the process of neurogenesis in the DG of weaned rat.
Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research S (19100009) from the Japan Society for the Promotion of Science and a Grant-in-Aid for Young Scientists B (21700654) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Effects of hindlimb unloading on neurogenesis in the hippocampus of newly weaned rats Sachiko Nomura a , Katsuya Kami a , Fuminori Kawano a , Yoshihiko Oke a , Naoya Nakai a , Takashi Ohira b , Ryo Fujita a , Masahiro Terada b , Kazuhiko Imaizumi c , Yoshinobu Ohira a,b,∗ a
Graduate School of Medicine, Osaka University, Toyonaka City, Osaka 560-0043, Japan Frontier Biosciences, Osaka University, Toyonaka City, Osaka 560-0043, Japan c Graduate School of Human Sciences, Waseda University, Tokorozawa City, Saitama 359-1192, Japan b
a r t i c l e
i n f o
Article history: Received 4 June 2011 Received in revised form 29 November 2011 Accepted 12 December 2011 Keywords: Hindlimb suspension Newly weaned rat Hippocampal neurogenesis Doublecortin-positive neurons
a b s t r a c t Effects of hindlimb suspension (HS) and ambulation recovery on hippocampal neurogenesis of newly weaned rats were studied by using immunohistochemical techniques. The number of proliferating cell nuclear antigen-positive (PCNA+ ) cells in the subgranular zone (SGZ) markedly decreased during normal growth. However, neither HS nor subsequent recovery caused additional changes in the number of PCNA+ cells. The number of doublecortin-positive (DCX+ ) neurons decreased gradually during normal growth. HS resulted in a further decrease in these neurons. However, DCX+ cell numbers became identical to the levels in age-matched controls after 14 days of recovery. PCNA and DCX-double positive cells in the SGZ were also observed, and their cell numbers were not affected by HS and 14-day ambulation. Thus, HS suppressed the generation of DCX+ neurons without affecting PCNA+ cells in the SGZ of weaned rats. Taken together, hippocampal neurogenesis in weaned rats was not severely affected by HS while it decreased significantly as they had grown. © 2011 Elsevier Ireland Ltd. All rights reserved.
Introduction Unlike most neurons in the adult brain, granule neurons continue to be produced in the dentate gyrus (DG) of the hippocampus throughout life. Hippocampal neurogenesis is a multi-step process that is followed by the formation of postmitotic functionally integrated neurons in the granular cell layer (GCL) of the DG. Markers that are expressed at different stages of hippocampal neurogenesis have been investigated to monitor newly generated cells in the subgranular zone (SGZ) of the DG. Proliferating cell nuclear antigen (PCNA), a mitotic marker, labels all proliferating cells in the DG; thus they do not allow newly formed neurons and glia to be distinguished from one another. To determine whether changes in PCNA-positive (PCNA+ ) cells are indeed related to altered neurogenesis, this labeling must be combined with other immunohistochemical markers that label newly formed neurons at later stages of neurogenesis. Doublecortin (DCX) expression is specific for newly generated neurons, since virtually all DCX-positive (DCX+ ) cells express early neuronal antigens but lack antigens specific to glia [20]. Therefore, PCNA and DCX
∗ Corresponding author at: Section of Applied Physiology, Graduate School of Medicine, Osaka University, 1-17 Machikaneyama-cho, Toyonaka City, Osaka 5600043, Japan. Tel.: +81 6 6850 6032; fax: +81 6 6850 6032. E-mail address: [email protected] (Y. Ohira). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.12.022
double-positive (PCNA+ /DCX+ ) cells in the SGZ can be regarded as mitotic neuronal precursors. Exercise enhances neurogenesis in the DG [2,4,23–25] and improves cognitive performance [1]. Increased numbers of PCNA+ cells, DCX+ cells and PCNA+ /DCX+ cells were observed in the SGZ of rats that performed chronic treadmill running [23]. The effects of exercise contrast the effects of stress, which is one of the most potent environmental parameters; both physiological and psychosocial stressors inhibit one or more processes of hippocampal neurogenesis [19]. For example, maternal separation is currently drawing attention as a well-characterized model of stress in rodents, and maternally deprived rats exhibit suppression of hippocampal neurogenesis later in life [14], more anxiety, and diminished spatial learning [12]. These studies indicate that a lasting reduction in hippocampal neurogenesis tends to follow stress exposure at early stages in life. Hindlimb suspension (HS) of rats, which inhibits the antigravity activity of hindlimb muscles, was initially proposed for studying spaceflight-associated inhibition of bone formation [15]. Thereafter, investigations on the effects of HS on hindlimb muscles [8,9,18,26] have been performed. A recent study [27] reported that the HS model was a useful tool to study how hippocampal neurogenesis can be modulated by withholding physical stimulation, thereby providing novel information that shows the exercisemediated enhancement of hippocampal neurogenesis [24,25]. Although the effects of HS on hippocampal neurogenesis in adult
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rats have been reported [27], its effects in earlier stages of life have not yet been examined. It was hypothesized that HS during early life in rats might suppress the hippocampal neurogenesis more severely because promotion or inhibition of hippocampal neurogenesis depends largely on experiences during the early postnatal period rather than experiences after maturity [14]. To further understand the regulatory systems that control neurogenesis in the DG, it is important to establish whether neurogenesis is severely affected by withholding physical stimulation in weaned rats. Therefore, the effects of HS on neurogenesis in the DG of the hippocampus and responses to ambulation recovery from HS in weaned rats were investigated in the present study. Materials and methods All experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23; Revised 1996). The study was also approved by the Animal Use Committee of Osaka University. Male Wistar Hannover rats (3 weeks old) were used. Each rat was individually housed in a cage. Water and a commercial solid diet (CE-2, Nihon CLEA, Tokyo, Japan) were supplied ad libitum. A total of 56 rats were randomly divided into the following five groups; pre-experimental group (pre, n = 12); normal cage housing for 14 days (day 14-control, n = 12); HS for 14 days (day 14-HS, n = 12); normal cage housing for 28 days (day 28-control, n = 10); and 14 days of HS followed by 14-day ambulation recovery (day 28-HS, n = 10). HS of rats was performed as described previously [9]. At the end of each paradigm, half of the rats in each group (n = 5 or 6) were anesthetized and sacrificed by transcardial perfusion of 0.85% NaCl prior to perfusion with 4% paraformaldehyde in 0.1 M phosphate-buffer (PB, pH 7.4). The whole brain was removed, postfixed in 4% paraformaldehyde in PB for 24 h at 4 ◦ C, and immersed in 30% sucrose in PB at 4 ◦ C. The brain was then frozen in liquid nitrogen-cooled isopentane and stored at −80 ◦ C until analysis. The frozen cerebral region including the hippocampus was cut into 20-m-thick sections by using a cryostat at −20 ◦ C. Immunohistochemical analysis was performed using a modified method described by Uda and colleagues [23]. Briefly, sections were immersed in 0.01 M citrate buffer (pH 6.0) and boiled for antigen retrieval. After blocking, the sections were incubated for 48 h at 4 ◦ C with a mixture of two primary antibodies: a mouse anti-PCNA antibody (1:50; DAKO, Carpinteria, CA) and a goat anti-DCX antibody (1:200; Santa Cruz Biotech., Santa Cruz, CA). Subsequently, the sections were incubated overnight at 4 ◦ C with secondary antibodies: Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:400; Molecular Probes, Eugene, OR) and Alexa Fluor 594-conjugated anti-goat IgG antibody (1:400). Finally, the sections were mounted in Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) with 4 ,6-diamidino-2-phenylidole (DAPI). Quantitative analyses for immune-stained cells were performed with an Olympus BX-51 fluorescent microscope (Olympus, Tokyo, Japan) and a Leica TCS SP5 Confocal Laser Scanning Microscope (Leica, Wetzlar, Germany). All of the PCNA+ and DCX+ cells in the SGZ were manually counted in the whole GCL of each section. In addition, PCNA+ /DCX+ cells in the SGZ were also manually counted as mitotic neuronal precursors according to the previously described method [23]. The results were reported as the number of labeled cells per area of whole GCL (mm2 ) that was calculated in each Nissl-stained section, because the mean area of the GCL in the DG was not different among the groups (data not shown). Several growth factors and hormones play important roles in the regulation of hippocampal neurogenesis in adult rats [11]. Therefore, the levels of these factors in the plasma and
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hippocampus were measured. Blood samples were collected from the right atrium, before transcardiac perfusion, in all rats by using heparinized syringes. The blood samples were immediately centrifuged for 10 min (1200 × g, 4 ◦ C) for enzyme-linked immunosorbent assay (ELISA). The remaining rats in all five groups (n = 5 or 6) were anesthetized and sacrificed by blood removal. Each brain was quickly removed, and the hippocampal formation was freshly isolated. The tissue was homogenized by sonication in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) with protease inhibitor cocktail (Sigma–Aldrich Co., St. Louis, MO), and centrifuged for 20 min (15,000 rpm, 4 ◦ C). The supernatant was used for ELISA. Total protein concentrations of these hippocampus samples were determined by the bicinchoninic acid method. In addition to the hippocampus, unfixed soleus muscles were removed from these rats, and their wet weights were measured as an indicator of antigravity muscle atrophy. Commercial ELISA systems were used to determine plasma and hippocampal levels of insulin-like growth factor-1 (IGF-1; R&D Systems, Inc., Minneapolis, MN, USA), vascular endothelial growth factor (VEGF; RayBiotech Inc., Norcross, GA), corticosterone (Endocrine Technologies, Inc., Newark, CA) and brain-derived neurotrophic factor (BDNF; Promega Corp., Madison, WI), according to the manufacturer’s protocols. The hippocampal level of each protein was expressed as the ratio to total protein content of the tissue sample. All data are expressed as the mean ± SEM. The results were evaluated by using one-way factorial analysis of variance (ANOVA) followed by Scheffe’s post hoc test. Statistical significance was accepted at p < 0.05. Results and discussion General growth Growth-associated increases in body weight were seen in the control groups at day 14 and day 28 (Table 1). Even though significant gain of body weight was also seen in the HS group, the mean weight was slightly less than the age-matched controls. However, HS and the subsequent recovery did not change the body weight significantly in weaned rats. Both the absolute and relative (to body weight) weights of the soleus were lower in the HS group at day 14 than in the age-matched control group (Table 1). Compared with the HS group at day 14, the mean absolute weight was increased by 440% within 14 days of recovery at day 28, but was still lower than in the age-matched control group. The mean relative weights showed a similar response (Table 1). These results clearly show that hindlimb unloading induced atrophy of soleus muscles in the present study, which is consistent with our previous report [26]. Neurogenesis in the DG of the hippocampus In a previous study with adult rats [27], 5-bromodeoxyuridine (BrdU) was used as a mitotic marker in the DG. However, it was reported that BrdU has teratological effects on the body, brain and behavior of fetal and infant rats [10]. Therefore, we used PCNA as a mitotic marker (Fig. 1, arrow b and c in B, arrow d in F) according to the previous study [23]. Mitotic neuronal precursors (PCNA+ /DCX+ cells) and newly generated neurons (DCX+ cells) in the DG of weaned rats were also immunolabeled as shown in Fig. 1. In 5-week-old control rats (day14-control, Fig. 1E–H), the morphological features of DCX+ cells were in agreement with those in adult rats [23], and the processes growing from DCX+ somata penetrated through the GCL into the molecular layer (Fig. 1G, arrow e). However, in the 3-week-old group (pre, Fig. 1A–D), DCX+ cells protruded
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Table 1 Effects of hindlimb suspension on body weight and soleus weight. Pre
Body weight (g) Soleus weight Absolute weight (mg) Relative weight (mg/g body weight)
88.3 ± 2.75 32.6 ± 0.78 0.67 ± 0.017
Day 14
Day 28
Control
HS
Control
HS
164.0 ± 2.88*
150.5 ± 6.68*
256.4 ± 5.27* , †
240.4 ± 8.95* , †
160.3 ± 6.73* , † 0.73 ± 0.011
116.5 ± 3.24* , † , § 0.62 ± 0.008§
89.8 ± 1.70* 0.70 ± 0.020
26.2 ± 2.75† 0.24 ± 0.018* , †
The rats were randomly divided into five groups as follows: pre group (pre-experimental group), day 14-control group (normal cage housing for 14 days), day 14-HS group (HS for 14 days), day 28-control group (normal cage housing for 28 days), and day 28-HS group (14 days of HS followed by 14-day ambulation recovery). All data are indicated as the mean ± SEM. * p < 0.05 vs. pre group. † p < 0.05 vs. day 14-control group. §
p < 0.05 vs. day 28-control group.
many fine processes (Fig. 1C, arrow a and b) that were not observed at 5 weeks of age. Despite the morphological differences between the 3- and 5-week-old groups, DCX+ cells and PCNA+ /DCX+ cells (Fig. 1D, arrow b) were localized only in the SGZ within the DG in both groups (Fig. 2A and B). Moreover, HS and subsequent recovery did not alter the localization of these cells (Fig. 2).
Quantitative comparisons of PCNA+ , DCX+ and PCNA+ /DCX+ cells in the SGZ are shown in Fig. 2. The number of PCNA+ cells in the SGZ was significantly less in the control groups at days 14 and 28 than in the pre-experimental group, indicating that the number of proliferating cells in the SGZ decreased after the lactation period (Fig. 2F). The developmental reduction in the number of PCNA+ cells was
Fig. 1. Photomicrographs showing proliferating cells, mitotic neuronal precursors and newly generated neurons in the SGZ. Brain sections were prepared from rats in the pre-experimental group (A–D) and after 14 days of cage housing (E–F). Triple fluorescence staining was performed with DAPI (in blue, A and E), anti-PCNA antibody (in green, B and F) and anti-DCX antibody (in red, C and G). The merged images are also shown (D and H). Arrows in panels indicate DCX+ newly generated neurons (arrow a, b and e), PCNA+ /DCX+ mitotic neuronal precursors (arrow b), and PCNA+ proliferating cells (arrow b, c and d) in the SGZ. Bars = 10 m.
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Fig. 2. Effects of HS on cell proliferation and neuron generation in the SGZ. PCNA and DCX immunoreactivities were observed in the SGZs before initiation of the experiment (A), after 14 days of cage housing (B), after 14 days of HS (C), after 28 days of cage housing (D), and after 14 days of ambulation recovery from 14-day HS (E). The number of PCNA+ cells (F), DCX+ neurons (G), and PCNA+ /DCX+ mitotic neuronal precursors (H) were counted. Results are shown as the mean ± SEM per mm2 of granular cell layer. Bar = 100 m (A–E). * and #p < 0.05 vs. pre and day 14-control, respectively.
not influenced by HS and ambulation recovery. The total number of DCX+ cells decreased gradually following the normal growth of control rats (Fig. 2G). The number of DCX+ cells in the HS group at day 14 was significantly less than in the age-matched control group. However, the number of DCX+ cells was stable even after 14 days of ambulation, and the number of DCX+ cells in the HS group at day 28 was identical to the number in the age-matched controls. Meanwhile, the numbers of PCNA+ /DCX+ cells in the SGZ in all groups at days 14 and 28 were significantly less than in the preexperimental group (Fig. 2H). There were no effects of HS and a 14-day ambulation period on the numbers of PCNA+ /DCX+ cells. The previous study with adult rats [27] showed that the numbers of proliferating cells in the SGZ were decreased by 14 days of HS. Thus, hippocampal neurogenesis in weaned rats thought to be less susceptible to the suppressive effects of HS than in adult animals. However, the difference of the experimental system for newly weaned rat, PCNA labeling, in the present study from that for adult rats, BrdU injection, in the previous reports [27] obstructs this statement. An earlier study has warned against the substitution of BrdU labeling with PCNA staining in rat peripheral tissues [16]. Although there are some cases where the PCNA staining well correlates with BrdU labeling in the SGZ [7,28], these immunohistochemical data cannot prove that newly weaned rats are less susceptible to the HS-treatment than adult rats. Neuronal precursors in the SGZ can shift their fate toward cell death during the processes of hippocampal neurogenesis [6]. Therefore, we evaluated caspase 3-dependent apoptosis and DNA fragmentation of newly generated neurons in the SGZ. Active caspase 3-positive cells were detected in the SGZ of 3-week-old rats (data not shown). However, no immunoreactivity for active caspase 3 was observed in the SGZ of 5-week-old control rats. Similar to the age-matched control group, no active caspase 3 expression was evident in the SGZ in the HS group at 5 weeks of
age. Then DNA fragmentation of these neurons was detected by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method. Although TUNEL-positive cells were observed in the subventricular zone of 3-week-old rats, no immunoreactivities were observed in the SGZ of 3- and 5-week-old control rats, which is consistent with a previous report [5]. Besides control groups, no TUNEL-positive cells were detected in the SGZ in the HS group (data not shown). In the present study, there was no evidence that HS induced apoptosis in newly generated neurons in the SGZ. Therefore, we conclude that the HS-induced decrease in newly born neurons does not result from increased cell death. Effects of growth factors and hormones We also checked the plasma and hippocampal levels of candidate factors affecting hippocampal neurogenesis. Trejo et al. showed that peripheral IGF-1 crosses the blood–brain barrier and stimulates hippocampal neurogenesis [22]. In the present study, the plasma levels of IGF-1 increased during the normal growth of the rats, but HS did not cause a significant decrease (Table 2). VEGF, another neurotrophic factor, was not detected in plasma in weaned rats (Table 2), unlike previously reported results with adult rats [27]. Moreover, recent evidence suggests that the elevation of plasma glucocorticoid levels by various stimuli plays a critical role in suppressing hippocampal neurogenesis [13]. However, our data indicate that HS did not cause a significant increase in the plasma level of corticosterone (Table 2). Meanwhile, the BDNF level in the hippocampus was significantly decreased in the HS group at day 14 as compared to the pre-experimental group, but it did not change in the age-matched controls (Table 2). There was also a slight difference in the BDNF levels between the HS and control groups at day 14, although the difference was not statistically significant (p = 0.09). HS did not cause significant changes in the IGF-1 level in
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Table 2 Effects of hindlimb suspension on plasma and hippocampal levels of hormones and neurotrophic factors. Pre
Day 14
Day 28
Control
HS
Control
HS
Plasma levels IGF-1 (g/ml) Corticosterone (ng/ml)
0.63 ± 0.025 80.4 ± 7.37
1.20 ± 0.059* 106.4 ± 10.71
1.03 ± 0.101* 122.0 ± 10.67
1.64 ± 0.039* , † 112.4 ± 8.31
1.41 ± 0.039* 131.4 ± 15.46
Hippocampal levels BDNF (pg/mg protein) IGF-1 (pg/mg protein) VEGF (pg/mg protein)
12.8 ± 0.56 37.8 ± 2.17 27.6 ± 3.23
11.6 ± 0.42 40.9 ± 1.29 28.4 ± 5.33
9.8 ± 0.26 44.3 ± 2.79 33.0 ± 8.19
13.4 ± 0.36 40.2 ± 1.87 124.6 ± 10.53* , †
12.7 ± 0.34 39.7 ± 1.36 111.6 ± 7.38* , †
The rats were randomly divided into five groups as follows; pre group (pre-experimental group), day 14-control group (normal cage housing for 14 days), day 14-HS group (HS for 14 days), day 28- control group (normal cage housing for 28 days), and day 28-HS group (14 days of HS followed by 14-day ambulation recovery). At the end of each paradigm, blood and hippocampal formation were collected. The levels of insulin-like growth factor-1 (IGF-1), corticosterone, brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were measured with ELISA systems. All data are indicated as the mean ± SEM. * p < 0.05 vs. pre group. † p < 0.05 vs. day 14-control group.
the hippocampus. The VEGF level in the hippocampus was significantly increased in the control group at day 28 as compared to day 14, but the developmental increase in the VEGF level was not influenced by HS and ambulation recovery (Table 2). Thus, there were no significant differences in the plasma and hippocampal levels of candidate factors regulating hippocampal neurogenesis between the HS and control groups at day 14. It is possible that HS did not cause serious damage to the hippocampal neurogenesis of weaned rats because the rats have high adaptability to environmental changes at that stage of life. In addition to withholding physical stimulation, HS seems to be a strong stressor associated with neurogenesis. Indeed, in some reports, the lack of exercise achieved by HS is accompanied by chronic stress [3] and depression, which in turn increases the glucocorticoid levels [21]. Restraint stress also suppresses hippocampal neurogenesis [19], and decreased body weights and increased plasma corticosterone levels were observed in rats subjected to restraint stress [17]. However, the present HS model did not alter body weight and only produced an incremental trend in the plasma corticosterone levels of rats. Taken together, relatively modest hormonal stress accompanied HS, indicating that the generation of neurons in the DG was likely suppressed as a function of lack of exercise in weaned rats. Similar results were obtained from a study of adult rats [27].
Conclusion HS induced a transient reduction in the total number of newly generated neurons without any changes in the number of proliferating cells or mitotic neuronal precursors in the SGZ. Contrary to our expectation, these findings suggest that HS did not cause serious damage to the process of neurogenesis in the DG of weaned rat.
Acknowledgments This study was supported by a Grant-in-Aid for Scientific Research S (19100009) from the Japan Society for the Promotion of Science and a Grant-in-Aid for Young Scientists B (21700654) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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