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Proliferation zones in adult medaka (Oryzias latipes) brain
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Proliferation zones in adult medaka (Oryzias latipes) brain Yoshiko Kuroyanagi a,1 , Teruhiro Okuyamaa,1 , Yuji Suehiroa , Haruka Imadaa , Atsuko Shimadaa , Kiyoshi Naruse b , Hiroyuki Takedaa , Takeo Kuboa , Hideaki Takeuchia,⁎ a
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan Laboratory of BioResource, National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 444-8585, Japan
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Cell proliferation in the adult mammalian brain is maintained at a low rate, but cell
Accepted 20 January 2010
proliferation in the adult fish brain is prominent. To compare the distribution of
Available online 28 January 2010
proliferating cells among fish species, mutants, and under different growing environments, we mapped the zones of cell proliferation in the adult medaka (Oryzias
Keywords:
latipes) brain and identified 17 proliferation zones in both male and female brains. These
Social environment
zones were distributed in the telencephalon (4 zones), preoptic area (2 zones), pineal body
Adult neurogenesis
(1 zone), hypophysis (1 zone), habenular nucleus (1 zone), optic tectum (2 zones), third
Brain development
ventricular zone (1 zone), ventromedial nucleus (1 zone), hypothalamus (1 zone), and
Medaka mutant
cerebellum (3 zones). Of the 17 zones, 16 corresponded to brain regions where cells proliferate in the zebrafish brain, suggesting that the persistence of the generation of new cells, at least in these zones, might be conserved among some fish species. We then compared the distribution of proliferation zones using two body-color mutant medaka, the T5 and Quintet, the latter of which is an albino mutant that completely lacks pigmentation. There was no apparent difference in the distribution pattern among these mutant strains. Finally, we compared these proliferation zones in the brains of isolated- and group-reared fish and detected no significant difference between the two groups. These findings demonstrate that there is persistent cell proliferation in at least these 16 zones of the adult medaka brain, irrespective of sex, body-color, and growth environment, suggesting that proliferation capacity in the 16 zones is maintained robustly in the adult medaka brain. © 2010 Elsevier B.V. All rights reserved.
1.
Introduction
In mammals, the capacity to produce new cells in the intact brain is severely limited. Adult neurogenesis is restricted to only two brain regions: the anterior part of the subventricular zone where newly born neurons migrate into the olfactory bulb, and the subgranular zone of the dentate gyrus, where new neurons migrate into the granule cell layer of the hippocampus (Bayer et al., 1982; Kaplan et al., 1985). In contrast, in the adult brain of
teleosts, most of the proliferating cells are observed in small, welldefined areas of the brain (proliferation zones) (Chapouton et al., 2007). A large number of proliferation zones are present throughout the whole brain in three teleosts, zebrafish (Adolf et al., 2006; Grandel et al., 2006; Zupanc et al., 2005), gymnotiform fish (Zupanc and Horschke, 1995), and three-spined stickleback (Ekstrom et al., 2001), and many of these zones are situated at or near the surfaces of ventricles or related systems. It has been unknown, however, whether the distribution pattern of these
⁎ Corresponding author. Fax: +81 3 5841 4449. E-mail addresses: [email protected] (T. Kubo), [email protected] (H. Takeuchi). 1 These two authors contributed equally to this work. 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.01.045
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proliferation zones is dependent on genetic factors or the growth environment. In the present study, to examine whether the distribution is affected by the genetic background and/or growth environment, we newly mapped proliferation zones in the adult medaka brain. The medaka (Oryzias latipes) is a freshwater teleost fish native to East Asia that has long been a favorite pet in Japan. The medaka is a model organism for a wide range of biologic studies, such as developmental genetics (FurutaniSeiki and Wittbrodt, 2004) and sexual differentiation (Kondo et al., 2002; Kurokawa et al., 2007). Efficient methods of generating both transgenic medaka (Nakamura et al., 2008) and knockout strains (Taniguchi et al., 2006) are now available. We have focused on the medaka for analysis of the mechanisms underlying adult neurogenesis using molecular and genetic techniques. As the medaka is relatively robust compared with tropical fish such as zebrafish, medaka fish can be kept easily under various environmental conditions (FurutaniSeiki and Wittbrodt, 2004). Furthermore, over 10 inbred medaka strains have been made by over 20 generations of sibling mating. An intraspecific genetic variation in the gross brain morphology exists between wild-type and body-color mutant inbred strains (Hi3, albino mutant [i-3/i-3] and HO5, orange-red fish [b/b]) (Ishikawa et al., 1999). The volumetric proportion of the olfactory bulb to that of the total brain in the HO medaka (2.1%) is higher than that in wild-type strains (approximately 1.8%), whereas that of the cerebellum in the Hi3 medaka (8.8%) is higher than that in wild-type strains (approximately 6.5%). A comprehensive medaka brain atlas is available and most brain regions of the medaka correspond to those in the zebrafish brain (Anken and Bourrat, 1998). Thus, using medaka, we can compare proliferation zones among species and strains, as well as among fish raised under different environments. In the present study, we identified 17 proliferation zones in the adult medaka brain and demonstrated that the proliferation capacity is robustly maintained, at least in these 16 zones, irrespective of genetic background and growth environment.
2.
Result
2.1. Identification of 17 proliferation zones in the medaka adult brain First, we mapped proliferation zones in the medaka brain using wild-type female and male medaka (drR strain). In the present study, cell proliferation was detected based on the incorporation of the thymidine analogue 5-bromo-29-deoxyuridine (BrdU) into newly synthesized DNA during cell mitosis. Cells that take up BrdU were detected by anti-BrdU immunohistochemistry after 4 h. Vibratome sections (130 μm) cut from dissected whole brains were stained with diaminopimelic acid (DAPI) and used for immunohistochemistry. Based on the distribution of DAPI staining and the medaka brain atlas (Anken and Bourrat, 1998), we identified the locations of the sections in the whole brain. We mapped BrdU-positive cells in a total of 10 brains and identified 17 proliferation zones (zones-A to -Q). These proliferation zones were detected in both male and female brains (male n = 3, female n = 2) and we observed no sex-specific proliferation zone. The zones were mapped in the telencephalon (zones-A to -D),
preoptic area (zones-E and -F), pineal body (zone-G), habenular nucleus (zone-H), ventromedial nucleus (zone-I), optic tectum (zones-J and -K), marginal zone of the third ventricular zone (zone -L), hypophysis (zone-M), hypothalamus (zone-N), and cerebellum (zones-O, -P and -Q; Figs. 1 and 2). In the anterior part of the telencephalic areas, a large number of mitotic cells were located near the surfaces of the dorsolateral region (zones-A and -B; Supplemental Movie 1). In some medaka brains, a few mitotic cells were detected in the olfactory bulb. A high concentration of mitotic cells was also detected near the surfaces of the medial region (zone-C). In the anterior part of the optic tectum, mitotic cells were mainly detected in the preoptic area (zones-E and -F). In the dorsal anterior part of the optic tectum, there was a welldefined cluster of mitotic cells (zone-J) in the dorsal areas, whereas signals were restricted to the ventral zones in the posterior part (zone-K). In the anterior part of the cerebellum, signals were detected in the two areas (zones-O and -P). In contrast, in the posterior part of the cerebellum, signals were detected mainly in the top of stratum granulare (zone-Q).
2.2. Quantitative analysis of proliferating cells the medaka adult brain We also performed a quantitative analysis of medaka adult brains. First, we prepared a series of paraffin sections from the whole brain and counted anti-BrdU immunopositive cells using three fish (1 male, 2 females). The total number of positive cells ranged from approximately 6000 to 7000 cells per brain (Supplemental Table 1). Approximately 26%, 38%, and 18% of all mitotic cells in the adult brain were observed in the telencephalon, optic tectum, and cerebellum, respectively. We performed comparisons of some regions (telencephalon, preoptic area, pineal body, and habenula) between sexes using a total of 10 medaka fish (5 males, 5 females), and demonstrated no significant quantitative difference between sexes (Fig. 3).
2.3. Maintenance of 16 proliferation zones in the medaka body-color mutant An intraspecific genetic variation in gross brain morphology exists between wild-type and body-color mutant strains (Hi3, albino mutant [i-3/i-3] and HO5, orange-red fish [b/b]), therefore we examined whether the distribution pattern of proliferating cells was affected by some genes that are necessary for pigmentation. We mapped the proliferation zones of two medaka inbred strains, T5 and Quintet, which each have a number of mutant alleles related to body-color. The T5 strain is homozygous for five recessive pigmentation mutations (b/b, lf/lf, gu/gu, ib/ib, wl/wl) derived from a southern Japanese population of wild-type medaka (Shimada and Shima, 2001). The Quintet mutant strain is an albino mutant (i/i or i-3/i-3, b/b, r/r, lf/lf, gu/gu; Shimada, personal communication). 16 proliferation zones were detected in the two strains (T5, n= 2; Quintet, n =2). We could not compare proliferative patterns in the hypophysis (region M) among the different conditions because the hypophysis often detached from the rest of the brain during the fixation step of the immunoreaction procedure. Thus we observed no mutantselective proliferation zones (Supplemental Fig. 2). These findings suggest that the distribution of proliferation zones was not altered by the loss of these body-color genes.
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Fig. 1 – Map of the proliferation zones in the adult medaka braina. Schematic drawing of the transverse sections of the medaka brain. The positions of sections (1–9) are indicated by lines. b. Schematic representation of the distribution of the 17 proliferation zones. Red dots indicate proliferating cells. Zone-A: Marginal zones of the anterior part of the telencephalon. Zone-B: Marginal zones of the dorsolateral part of the telencephalon. Zone-C: Medial zones of the telencephalon. Zone-D: Dorsolateral part of the posterior part of the telencephalon. Zonec. Anti-BrdU immunohistochemistry of vibratome sections from wild-type adult medaka (the drR strain). Nuclei were stained with DAPI (Blue). Mitotic cells are indicated by the red color. All 16 proliferation zones except hypophysis (zone-M) were observed in all brains (arrows). Numbers in the panels correspond to section numbers shown in A. Scale bars indicate 100 μm. Te: telencephalon, Cb: cerebellum, OT: optic tectum. Proliferation zones were determined according to the medaka fish brain atlas (Supplemental Fig. 1; Anken and Bourrat, 1998).
2.4. Maintenance of 16 proliferation zones between group- and isolated-reared medaka Under natural conditions, medaka fish form groups and exhibit prominent shoaling behavior (Nakamura, 1952; Tsu-
bokawa et al., 2009). To examine whether these social interactions affect the distribution of the proliferation zones, we compared the distribution of proliferation zones between group- and isolation-reared medaka. For the group-reared medaka, five medaka were kept in a circular tank (8 cm in
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Fig. 2 – Magnified view of the proliferation zones in the optic tectum, third ventricular zone, and hypothalamus (zones-J, -L, and -N). Scale bars indicate 100 μm.
diameter), and for the isolation-reared medaka (n = 5), each medaka was kept in a smaller circular tank (3.5 cm in diameter). In both groups, the 16 proliferation zones were observed 45 days after hatching and there were no differences in the distribution of proliferation zones between groups (Fig. 4). These findings suggest that cell proliferation ability is maintained, even under isolation-reared conditions.
3.
Discussion
3.1. Maintenance of distribution of the proliferating zone in the adult medaka brain We performed a comprehensive mapping of the proliferating cells in the medaka whole brain. The use of thick vibratome sections (130-μm) rather than thin paraffin sections enabled us to efficiently identify the proliferation zones. Based on antiBrdU immunohistochemistry, we identified 17 proliferation
zones in the medaka brain and the results indicated that cell proliferation in at least these 16 zones of the adult medaka brain was maintained, irrespective of sex, body-color mutant, and growth environment. We detected no proliferation zones that appeared in a condition-specific manner. In the present study, it was difficult to count the number of positive cells precisely, due to the overlapping BrdU-positive signals. A qualitative difference in mitotic cells in the area that differs between sexes has been reported in the brain of the adult gilt headsea bream teleost (Sparus aurata: Sparidae), although no differences are observed in the distribution pattern of the proliferation zones (Zikopoulos et al., 2000). In rodent, environmental enrichment increases adult hippocampal neurogenesis and alters hippocampal-dependent behavior (Meshi et al., 2006). Although our quantitative analysis revealed no significant differences between sexes in some brain regions (telencephalon, pineal body, and habenula), we cannot exclude the possibility that the number of proliferating cells and/or the fate of newborn neurons/glial cells/oligodendrocytes differ in
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Fig. 3 – Quantitative comparison of proliferating cells in the telencephalon (zones-A to -D), preoptic area (zones-E and -F), pineal body (zone-G), and habenular nucleus (zone-H) between sexes using a series of paraffin sections.
some other brain regions depending on sex, body-color mutation, and growth environment. The findings of the present study, however, strongly suggest that the 16 proliferation zones in the adult medaka brain do not disappear in the adult medaka and that the cell proliferation capacity is robustly maintained in the proliferation zones.
3.2.
Comparison of proliferating zones among fish species
We mapped 17 proliferation zones in the adult medaka brain. In the telencephalic area , a similar distribution has been reported in zebrafish (Adolf et al., 2006; Grandel et al., 2006; Zupanc et al., 2005), gymnotiform fish (Zupanc and Horschke, 1995), and three-spined stickleback (Ekstrom et al., 2001). The comparatively high capacity of proliferating cells in the dorsal telencephalon (zones-A, -B, -C, and -D) between species is remarkable. The dorsolateral part of the telencephalon is thought to be homologous to the mammalian hippocampus (Broglio et al., 2005; Butler, 2000; Chapouton et al., 2007; Portavella et al., 2004; Rodriguez et al., 2002; Saito and
Watanabe, 2006). In the preoptic area (zones-E and -F), a high concentration of proliferating cells was also observed in the three types of fish (zebrafish, gymnotiform fish, and threespined stickleback). In the pineal body (zone-G), a small number of mitotic cells (3–13 cells) were detected (Supplemental Table 1). Although there is no previous report of cell proliferation in the pineal body of zebrafish, three-spined stickleback, or gymnotiform fish, it has been observed in the brain of the immature rainbow trout (Oncorhynchus mykiss) (Omura, 2007). In the habenular nucleus (zone-H) and ventromedial thalamic nucleus (zone-I), approximately 30 to 60 and 60 to 140 mitotic cells were observed, respectively (Supplemental Table 1); mitotic cells are also found in these two brain regions in the zebrafish (Zupanc et al., 2005). In the optic tectum, two well-defined clusters of proliferating cells (zones-J, and -K) were detected in the two marginal zones. In threespined stickleback, a similar distribution is observed (Ekstrom et al., 2001), while in zebrafish and gymnotiform fish new cells are generated predominantly at the caudal end of the periventricular gray zone (Ekstrom et al., 2001; Zupanc and
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Fig. 4 – Comparison of the proliferation zones between isolated-reared and group-reared medaka. Nuclei were stained with DAPI (Blue). Mitotic cells are indicated by the red color. All of the 16 proliferation zones except hypophysis (zone-M) were observed in the two groups (arrows). Numbers in the panels correspond to section numbers shown in Fig. 1a. Scale bars indicate 100 μm.
Horschke, 1995; Zupanc et al., 2005). The third ventricular zone (zone-L) in the medaka brain is homologous to the diencephalic ventricle, the marginal zones of which have cell proliferation capacities in the three types of fish (zebrafish, gymnotiform fish, and three-spined stickleback). Approximately 7% and 18% of all new cells were detected in the hypothalamus and cerebellum, respectively, in the medaka brain and a high concentration of mitotic cells was observed in
the two brain regions in the three types of fish (zebrafish, gymnotiform fish, and three-spined stickleback). Approximately 75% of all new cells produced in the adult brain are observed in the cerebellum in the gymnotiform fish (Zupanc and Horschke, 1995). A similar pattern of cell proliferation in the cerebellum is also observed in other teleosts, including zebrafish (Zupanc et al., 2005), three-spined stickleback (Ekstrom et al., 2001), and gilthead sea bream (Zikopoulos
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et al., 2000). Furthermore, Candal et al. compared the pattern of PCNA immunoreactivity in the cerebellum throughout development between trout and medaka and reported that there were no significant differences between the two fish (Candal et al., 2005). Thus, the proliferation pattern in the cerebellum might be well conserved among fish. All the zones except the pineal body (zone-G) correspond to proliferation zones in the zebrafish brain and proliferating cells in the pineal body (zone-G) are also observed in rainbow trout. These findings indicate that the capacity for the production of new cells, at least in these 17 proliferation zones, is conserved among some fish species. Thus, the distribution of proliferation zones in the adult brain might be important for the maintenance and development of the fundamental structure of fish brains throughout the adult stage.
4.
Experimental procedures
4.1.
Animals
Medaka (O. latipes) of a wild-type strain (drR strain, more than 3 months after hatching), T5 strain (more than 3 months after hatching), and the Quintet mutant strain (more than 3 months after hatching) were maintained in like groups in plastic aquariums (12-cm × 13-cm × 19-cm). The medaka T5 strain is homozygous for five recessive pigmentation mutations (b: colorless melanophore [melanophore], lf: leucophore free [leucophore], gu: guanineless [iridophore], ib [delayed melanization], wl: leucophore free [leucophore]) derived from a southern Japanese population of wild-type medaka (Shimada and Shima, 2001). The Quintet mutant strain is homozygous for five recessive pigmentation mutations (i/i3: albino [melanophore], b: colorless melanophore [melanophore], r: colorless xanthophore [xanthophore], lf: leucophore free [leucophore], gu: guanineless [iridophore]), and are generated by crossing an albino mutant and bodycolor mutant (AA2; personal communication from A. Shimada, The University of Tokyo). Because the original albino medaka were obtained from a pet shop, the Quintet genotype is uncertain, especially for the albino locus. All groups were maintained at a temperature of 25 °C and under a 14 h:10 h light:dark cycle. To compare the proliferation zones in the brains of isolation- and group-housed medaka, eggs were placed separately in a 24-well microplate (IWAKI Glass Co, Ltd., Chiba, Japan). The walls of the wells were covered with white opaque paper to avoid visual interindividual interaction after hatching. Within 24 h after hatching, for the “isolation” test, each larval medaka was transferred to a 3.5cm diameter glass dish with white opaque walls. For the “group” test, five larval medaka were transferred to an 8-cm diameter glass dish. These glass dishes were maintained at 28 °C and under a 14 h:10 h light:dark cycle until the BrdU experiment. The sections were examined using a Zeiss Axio imager.Z1 microscope, equipped with 10×/NA 0.45 and 20×/ NA 0.8 optics (Plan-Apochromat, Zeiss) and Apotome apparatus, coupled to a computer-driven Zeiss AxioCAM digital camera (MRm), using Zeiss Axio Vision (4.6.3) software. Optical sections were obtained at resolutions of 1024 × 1024
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using a confocal microscope (FV500, Olympus), equipped with 40×/NA 1.15 optics (UApo/340, Olympus) and Fluoview (5.0) software.
4.2.
Detection of mitotic cells in the adult medaka brain
Four hours after exposure to water containing 1 g/L BrdU (Sigma Aldrich Japan), cells that took up BrdU were detected by anti-BrdU immunohistochemistry. The fish were anesthetized on ice and the whole brains were dissected. The brains were fixed overnight with formaldehyde in phosphate-buffered saline (PBS; pH 7.2; 3.7%) and embedded with 4% low-melting agarose (SeaPlaque GTG agarose, Cambrex) or paraffin for vibratome or microtome sectioning, respectively. Serial coronal sections, 130-μm or 10-μm thick, were cut with a Vibratome (VT1000S, Leica) or microtome (LR-85, Yamato Kohki), respectively. Immunostaining was performed following standard procedures. For the analysis of BrdU incorporation, sections were treated with 2 M HCl for 30 min at room temperature, and washed with PBS containing 0.2% Triton X100 (PBS-TritonX). The sections were incubated with the primary antibodies at 4 °C in PBS-TritonX and 3% bovine serum albumin overnight. After thorough washing with PBSTritonX, secondary antibodies were incubated overnight in the same solution at room temperature. The antibodies used were as follows: mouse monoclonal antibody to BrdU (PharMingen, 1:200); and Alexa Fluor 555 conjugated goat anti-mouse IgG (Invitrogen, 1:1000). After washing with PBS-TritonX, the sections were stained with DAPI.
Acknowledgments We thank the Medaka National BioResource Project for providing the medaka strains. This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology, Scientific Research on Priority Areas (Area no. 454, Mobiligence Project). We thank Dr. Tetsuaki Kimura, and Ms. Yasuko Ozawa for using Quintet medaka line.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.brainres.2010.01.045.
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Research Report
Proliferation zones in adult medaka (Oryzias latipes) brain Yoshiko Kuroyanagi a,1 , Teruhiro Okuyamaa,1 , Yuji Suehiroa , Haruka Imadaa , Atsuko Shimadaa , Kiyoshi Naruse b , Hiroyuki Takedaa , Takeo Kuboa , Hideaki Takeuchia,⁎ a
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan Laboratory of BioResource, National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 444-8585, Japan
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Cell proliferation in the adult mammalian brain is maintained at a low rate, but cell
Accepted 20 January 2010
proliferation in the adult fish brain is prominent. To compare the distribution of
Available online 28 January 2010
proliferating cells among fish species, mutants, and under different growing environments, we mapped the zones of cell proliferation in the adult medaka (Oryzias
Keywords:
latipes) brain and identified 17 proliferation zones in both male and female brains. These
Social environment
zones were distributed in the telencephalon (4 zones), preoptic area (2 zones), pineal body
Adult neurogenesis
(1 zone), hypophysis (1 zone), habenular nucleus (1 zone), optic tectum (2 zones), third
Brain development
ventricular zone (1 zone), ventromedial nucleus (1 zone), hypothalamus (1 zone), and
Medaka mutant
cerebellum (3 zones). Of the 17 zones, 16 corresponded to brain regions where cells proliferate in the zebrafish brain, suggesting that the persistence of the generation of new cells, at least in these zones, might be conserved among some fish species. We then compared the distribution of proliferation zones using two body-color mutant medaka, the T5 and Quintet, the latter of which is an albino mutant that completely lacks pigmentation. There was no apparent difference in the distribution pattern among these mutant strains. Finally, we compared these proliferation zones in the brains of isolated- and group-reared fish and detected no significant difference between the two groups. These findings demonstrate that there is persistent cell proliferation in at least these 16 zones of the adult medaka brain, irrespective of sex, body-color, and growth environment, suggesting that proliferation capacity in the 16 zones is maintained robustly in the adult medaka brain. © 2010 Elsevier B.V. All rights reserved.
1.
Introduction
In mammals, the capacity to produce new cells in the intact brain is severely limited. Adult neurogenesis is restricted to only two brain regions: the anterior part of the subventricular zone where newly born neurons migrate into the olfactory bulb, and the subgranular zone of the dentate gyrus, where new neurons migrate into the granule cell layer of the hippocampus (Bayer et al., 1982; Kaplan et al., 1985). In contrast, in the adult brain of
teleosts, most of the proliferating cells are observed in small, welldefined areas of the brain (proliferation zones) (Chapouton et al., 2007). A large number of proliferation zones are present throughout the whole brain in three teleosts, zebrafish (Adolf et al., 2006; Grandel et al., 2006; Zupanc et al., 2005), gymnotiform fish (Zupanc and Horschke, 1995), and three-spined stickleback (Ekstrom et al., 2001), and many of these zones are situated at or near the surfaces of ventricles or related systems. It has been unknown, however, whether the distribution pattern of these
⁎ Corresponding author. Fax: +81 3 5841 4449. E-mail addresses: [email protected] (T. Kubo), [email protected] (H. Takeuchi). 1 These two authors contributed equally to this work. 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.01.045
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proliferation zones is dependent on genetic factors or the growth environment. In the present study, to examine whether the distribution is affected by the genetic background and/or growth environment, we newly mapped proliferation zones in the adult medaka brain. The medaka (Oryzias latipes) is a freshwater teleost fish native to East Asia that has long been a favorite pet in Japan. The medaka is a model organism for a wide range of biologic studies, such as developmental genetics (FurutaniSeiki and Wittbrodt, 2004) and sexual differentiation (Kondo et al., 2002; Kurokawa et al., 2007). Efficient methods of generating both transgenic medaka (Nakamura et al., 2008) and knockout strains (Taniguchi et al., 2006) are now available. We have focused on the medaka for analysis of the mechanisms underlying adult neurogenesis using molecular and genetic techniques. As the medaka is relatively robust compared with tropical fish such as zebrafish, medaka fish can be kept easily under various environmental conditions (FurutaniSeiki and Wittbrodt, 2004). Furthermore, over 10 inbred medaka strains have been made by over 20 generations of sibling mating. An intraspecific genetic variation in the gross brain morphology exists between wild-type and body-color mutant inbred strains (Hi3, albino mutant [i-3/i-3] and HO5, orange-red fish [b/b]) (Ishikawa et al., 1999). The volumetric proportion of the olfactory bulb to that of the total brain in the HO medaka (2.1%) is higher than that in wild-type strains (approximately 1.8%), whereas that of the cerebellum in the Hi3 medaka (8.8%) is higher than that in wild-type strains (approximately 6.5%). A comprehensive medaka brain atlas is available and most brain regions of the medaka correspond to those in the zebrafish brain (Anken and Bourrat, 1998). Thus, using medaka, we can compare proliferation zones among species and strains, as well as among fish raised under different environments. In the present study, we identified 17 proliferation zones in the adult medaka brain and demonstrated that the proliferation capacity is robustly maintained, at least in these 16 zones, irrespective of genetic background and growth environment.
2.
Result
2.1. Identification of 17 proliferation zones in the medaka adult brain First, we mapped proliferation zones in the medaka brain using wild-type female and male medaka (drR strain). In the present study, cell proliferation was detected based on the incorporation of the thymidine analogue 5-bromo-29-deoxyuridine (BrdU) into newly synthesized DNA during cell mitosis. Cells that take up BrdU were detected by anti-BrdU immunohistochemistry after 4 h. Vibratome sections (130 μm) cut from dissected whole brains were stained with diaminopimelic acid (DAPI) and used for immunohistochemistry. Based on the distribution of DAPI staining and the medaka brain atlas (Anken and Bourrat, 1998), we identified the locations of the sections in the whole brain. We mapped BrdU-positive cells in a total of 10 brains and identified 17 proliferation zones (zones-A to -Q). These proliferation zones were detected in both male and female brains (male n = 3, female n = 2) and we observed no sex-specific proliferation zone. The zones were mapped in the telencephalon (zones-A to -D),
preoptic area (zones-E and -F), pineal body (zone-G), habenular nucleus (zone-H), ventromedial nucleus (zone-I), optic tectum (zones-J and -K), marginal zone of the third ventricular zone (zone -L), hypophysis (zone-M), hypothalamus (zone-N), and cerebellum (zones-O, -P and -Q; Figs. 1 and 2). In the anterior part of the telencephalic areas, a large number of mitotic cells were located near the surfaces of the dorsolateral region (zones-A and -B; Supplemental Movie 1). In some medaka brains, a few mitotic cells were detected in the olfactory bulb. A high concentration of mitotic cells was also detected near the surfaces of the medial region (zone-C). In the anterior part of the optic tectum, mitotic cells were mainly detected in the preoptic area (zones-E and -F). In the dorsal anterior part of the optic tectum, there was a welldefined cluster of mitotic cells (zone-J) in the dorsal areas, whereas signals were restricted to the ventral zones in the posterior part (zone-K). In the anterior part of the cerebellum, signals were detected in the two areas (zones-O and -P). In contrast, in the posterior part of the cerebellum, signals were detected mainly in the top of stratum granulare (zone-Q).
2.2. Quantitative analysis of proliferating cells the medaka adult brain We also performed a quantitative analysis of medaka adult brains. First, we prepared a series of paraffin sections from the whole brain and counted anti-BrdU immunopositive cells using three fish (1 male, 2 females). The total number of positive cells ranged from approximately 6000 to 7000 cells per brain (Supplemental Table 1). Approximately 26%, 38%, and 18% of all mitotic cells in the adult brain were observed in the telencephalon, optic tectum, and cerebellum, respectively. We performed comparisons of some regions (telencephalon, preoptic area, pineal body, and habenula) between sexes using a total of 10 medaka fish (5 males, 5 females), and demonstrated no significant quantitative difference between sexes (Fig. 3).
2.3. Maintenance of 16 proliferation zones in the medaka body-color mutant An intraspecific genetic variation in gross brain morphology exists between wild-type and body-color mutant strains (Hi3, albino mutant [i-3/i-3] and HO5, orange-red fish [b/b]), therefore we examined whether the distribution pattern of proliferating cells was affected by some genes that are necessary for pigmentation. We mapped the proliferation zones of two medaka inbred strains, T5 and Quintet, which each have a number of mutant alleles related to body-color. The T5 strain is homozygous for five recessive pigmentation mutations (b/b, lf/lf, gu/gu, ib/ib, wl/wl) derived from a southern Japanese population of wild-type medaka (Shimada and Shima, 2001). The Quintet mutant strain is an albino mutant (i/i or i-3/i-3, b/b, r/r, lf/lf, gu/gu; Shimada, personal communication). 16 proliferation zones were detected in the two strains (T5, n= 2; Quintet, n =2). We could not compare proliferative patterns in the hypophysis (region M) among the different conditions because the hypophysis often detached from the rest of the brain during the fixation step of the immunoreaction procedure. Thus we observed no mutantselective proliferation zones (Supplemental Fig. 2). These findings suggest that the distribution of proliferation zones was not altered by the loss of these body-color genes.
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Fig. 1 – Map of the proliferation zones in the adult medaka braina. Schematic drawing of the transverse sections of the medaka brain. The positions of sections (1–9) are indicated by lines. b. Schematic representation of the distribution of the 17 proliferation zones. Red dots indicate proliferating cells. Zone-A: Marginal zones of the anterior part of the telencephalon. Zone-B: Marginal zones of the dorsolateral part of the telencephalon. Zone-C: Medial zones of the telencephalon. Zone-D: Dorsolateral part of the posterior part of the telencephalon. Zonec. Anti-BrdU immunohistochemistry of vibratome sections from wild-type adult medaka (the drR strain). Nuclei were stained with DAPI (Blue). Mitotic cells are indicated by the red color. All 16 proliferation zones except hypophysis (zone-M) were observed in all brains (arrows). Numbers in the panels correspond to section numbers shown in A. Scale bars indicate 100 μm. Te: telencephalon, Cb: cerebellum, OT: optic tectum. Proliferation zones were determined according to the medaka fish brain atlas (Supplemental Fig. 1; Anken and Bourrat, 1998).
2.4. Maintenance of 16 proliferation zones between group- and isolated-reared medaka Under natural conditions, medaka fish form groups and exhibit prominent shoaling behavior (Nakamura, 1952; Tsu-
bokawa et al., 2009). To examine whether these social interactions affect the distribution of the proliferation zones, we compared the distribution of proliferation zones between group- and isolation-reared medaka. For the group-reared medaka, five medaka were kept in a circular tank (8 cm in
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Fig. 2 – Magnified view of the proliferation zones in the optic tectum, third ventricular zone, and hypothalamus (zones-J, -L, and -N). Scale bars indicate 100 μm.
diameter), and for the isolation-reared medaka (n = 5), each medaka was kept in a smaller circular tank (3.5 cm in diameter). In both groups, the 16 proliferation zones were observed 45 days after hatching and there were no differences in the distribution of proliferation zones between groups (Fig. 4). These findings suggest that cell proliferation ability is maintained, even under isolation-reared conditions.
3.
Discussion
3.1. Maintenance of distribution of the proliferating zone in the adult medaka brain We performed a comprehensive mapping of the proliferating cells in the medaka whole brain. The use of thick vibratome sections (130-μm) rather than thin paraffin sections enabled us to efficiently identify the proliferation zones. Based on antiBrdU immunohistochemistry, we identified 17 proliferation
zones in the medaka brain and the results indicated that cell proliferation in at least these 16 zones of the adult medaka brain was maintained, irrespective of sex, body-color mutant, and growth environment. We detected no proliferation zones that appeared in a condition-specific manner. In the present study, it was difficult to count the number of positive cells precisely, due to the overlapping BrdU-positive signals. A qualitative difference in mitotic cells in the area that differs between sexes has been reported in the brain of the adult gilt headsea bream teleost (Sparus aurata: Sparidae), although no differences are observed in the distribution pattern of the proliferation zones (Zikopoulos et al., 2000). In rodent, environmental enrichment increases adult hippocampal neurogenesis and alters hippocampal-dependent behavior (Meshi et al., 2006). Although our quantitative analysis revealed no significant differences between sexes in some brain regions (telencephalon, pineal body, and habenula), we cannot exclude the possibility that the number of proliferating cells and/or the fate of newborn neurons/glial cells/oligodendrocytes differ in
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Fig. 3 – Quantitative comparison of proliferating cells in the telencephalon (zones-A to -D), preoptic area (zones-E and -F), pineal body (zone-G), and habenular nucleus (zone-H) between sexes using a series of paraffin sections.
some other brain regions depending on sex, body-color mutation, and growth environment. The findings of the present study, however, strongly suggest that the 16 proliferation zones in the adult medaka brain do not disappear in the adult medaka and that the cell proliferation capacity is robustly maintained in the proliferation zones.
3.2.
Comparison of proliferating zones among fish species
We mapped 17 proliferation zones in the adult medaka brain. In the telencephalic area , a similar distribution has been reported in zebrafish (Adolf et al., 2006; Grandel et al., 2006; Zupanc et al., 2005), gymnotiform fish (Zupanc and Horschke, 1995), and three-spined stickleback (Ekstrom et al., 2001). The comparatively high capacity of proliferating cells in the dorsal telencephalon (zones-A, -B, -C, and -D) between species is remarkable. The dorsolateral part of the telencephalon is thought to be homologous to the mammalian hippocampus (Broglio et al., 2005; Butler, 2000; Chapouton et al., 2007; Portavella et al., 2004; Rodriguez et al., 2002; Saito and
Watanabe, 2006). In the preoptic area (zones-E and -F), a high concentration of proliferating cells was also observed in the three types of fish (zebrafish, gymnotiform fish, and threespined stickleback). In the pineal body (zone-G), a small number of mitotic cells (3–13 cells) were detected (Supplemental Table 1). Although there is no previous report of cell proliferation in the pineal body of zebrafish, three-spined stickleback, or gymnotiform fish, it has been observed in the brain of the immature rainbow trout (Oncorhynchus mykiss) (Omura, 2007). In the habenular nucleus (zone-H) and ventromedial thalamic nucleus (zone-I), approximately 30 to 60 and 60 to 140 mitotic cells were observed, respectively (Supplemental Table 1); mitotic cells are also found in these two brain regions in the zebrafish (Zupanc et al., 2005). In the optic tectum, two well-defined clusters of proliferating cells (zones-J, and -K) were detected in the two marginal zones. In threespined stickleback, a similar distribution is observed (Ekstrom et al., 2001), while in zebrafish and gymnotiform fish new cells are generated predominantly at the caudal end of the periventricular gray zone (Ekstrom et al., 2001; Zupanc and
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Fig. 4 – Comparison of the proliferation zones between isolated-reared and group-reared medaka. Nuclei were stained with DAPI (Blue). Mitotic cells are indicated by the red color. All of the 16 proliferation zones except hypophysis (zone-M) were observed in the two groups (arrows). Numbers in the panels correspond to section numbers shown in Fig. 1a. Scale bars indicate 100 μm.
Horschke, 1995; Zupanc et al., 2005). The third ventricular zone (zone-L) in the medaka brain is homologous to the diencephalic ventricle, the marginal zones of which have cell proliferation capacities in the three types of fish (zebrafish, gymnotiform fish, and three-spined stickleback). Approximately 7% and 18% of all new cells were detected in the hypothalamus and cerebellum, respectively, in the medaka brain and a high concentration of mitotic cells was observed in
the two brain regions in the three types of fish (zebrafish, gymnotiform fish, and three-spined stickleback). Approximately 75% of all new cells produced in the adult brain are observed in the cerebellum in the gymnotiform fish (Zupanc and Horschke, 1995). A similar pattern of cell proliferation in the cerebellum is also observed in other teleosts, including zebrafish (Zupanc et al., 2005), three-spined stickleback (Ekstrom et al., 2001), and gilthead sea bream (Zikopoulos
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et al., 2000). Furthermore, Candal et al. compared the pattern of PCNA immunoreactivity in the cerebellum throughout development between trout and medaka and reported that there were no significant differences between the two fish (Candal et al., 2005). Thus, the proliferation pattern in the cerebellum might be well conserved among fish. All the zones except the pineal body (zone-G) correspond to proliferation zones in the zebrafish brain and proliferating cells in the pineal body (zone-G) are also observed in rainbow trout. These findings indicate that the capacity for the production of new cells, at least in these 17 proliferation zones, is conserved among some fish species. Thus, the distribution of proliferation zones in the adult brain might be important for the maintenance and development of the fundamental structure of fish brains throughout the adult stage.
4.
Experimental procedures
4.1.
Animals
Medaka (O. latipes) of a wild-type strain (drR strain, more than 3 months after hatching), T5 strain (more than 3 months after hatching), and the Quintet mutant strain (more than 3 months after hatching) were maintained in like groups in plastic aquariums (12-cm × 13-cm × 19-cm). The medaka T5 strain is homozygous for five recessive pigmentation mutations (b: colorless melanophore [melanophore], lf: leucophore free [leucophore], gu: guanineless [iridophore], ib [delayed melanization], wl: leucophore free [leucophore]) derived from a southern Japanese population of wild-type medaka (Shimada and Shima, 2001). The Quintet mutant strain is homozygous for five recessive pigmentation mutations (i/i3: albino [melanophore], b: colorless melanophore [melanophore], r: colorless xanthophore [xanthophore], lf: leucophore free [leucophore], gu: guanineless [iridophore]), and are generated by crossing an albino mutant and bodycolor mutant (AA2; personal communication from A. Shimada, The University of Tokyo). Because the original albino medaka were obtained from a pet shop, the Quintet genotype is uncertain, especially for the albino locus. All groups were maintained at a temperature of 25 °C and under a 14 h:10 h light:dark cycle. To compare the proliferation zones in the brains of isolation- and group-housed medaka, eggs were placed separately in a 24-well microplate (IWAKI Glass Co, Ltd., Chiba, Japan). The walls of the wells were covered with white opaque paper to avoid visual interindividual interaction after hatching. Within 24 h after hatching, for the “isolation” test, each larval medaka was transferred to a 3.5cm diameter glass dish with white opaque walls. For the “group” test, five larval medaka were transferred to an 8-cm diameter glass dish. These glass dishes were maintained at 28 °C and under a 14 h:10 h light:dark cycle until the BrdU experiment. The sections were examined using a Zeiss Axio imager.Z1 microscope, equipped with 10×/NA 0.45 and 20×/ NA 0.8 optics (Plan-Apochromat, Zeiss) and Apotome apparatus, coupled to a computer-driven Zeiss AxioCAM digital camera (MRm), using Zeiss Axio Vision (4.6.3) software. Optical sections were obtained at resolutions of 1024 × 1024
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using a confocal microscope (FV500, Olympus), equipped with 40×/NA 1.15 optics (UApo/340, Olympus) and Fluoview (5.0) software.
4.2.
Detection of mitotic cells in the adult medaka brain
Four hours after exposure to water containing 1 g/L BrdU (Sigma Aldrich Japan), cells that took up BrdU were detected by anti-BrdU immunohistochemistry. The fish were anesthetized on ice and the whole brains were dissected. The brains were fixed overnight with formaldehyde in phosphate-buffered saline (PBS; pH 7.2; 3.7%) and embedded with 4% low-melting agarose (SeaPlaque GTG agarose, Cambrex) or paraffin for vibratome or microtome sectioning, respectively. Serial coronal sections, 130-μm or 10-μm thick, were cut with a Vibratome (VT1000S, Leica) or microtome (LR-85, Yamato Kohki), respectively. Immunostaining was performed following standard procedures. For the analysis of BrdU incorporation, sections were treated with 2 M HCl for 30 min at room temperature, and washed with PBS containing 0.2% Triton X100 (PBS-TritonX). The sections were incubated with the primary antibodies at 4 °C in PBS-TritonX and 3% bovine serum albumin overnight. After thorough washing with PBSTritonX, secondary antibodies were incubated overnight in the same solution at room temperature. The antibodies used were as follows: mouse monoclonal antibody to BrdU (PharMingen, 1:200); and Alexa Fluor 555 conjugated goat anti-mouse IgG (Invitrogen, 1:1000). After washing with PBS-TritonX, the sections were stained with DAPI.
Acknowledgments We thank the Medaka National BioResource Project for providing the medaka strains. This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology, Scientific Research on Priority Areas (Area no. 454, Mobiligence Project). We thank Dr. Tetsuaki Kimura, and Ms. Yasuko Ozawa for using Quintet medaka line.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.brainres.2010.01.045.
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