Neuronal differentiation process of astrocyte-like …Neuronal differentiation process of...
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Neuronal differentiation process of astrocyte-like
progenitor cells in the postnatal hippocampus
生後海馬に存在するアストロサイト様神経前駆細胞のニューロン分化過程の解析
2006 年 3 月
難波 隆志
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Neuronal differentiation process of astrocyte-like
progenitor cells in the postnatal hippocampus
生後海馬に存在するアストロサイト様神経前駆細胞のニューロン分化過程の解析
2006 年 3 月
早稲田大学大学院理工学研究科
生命理工学専攻・生体制御研究
難波 隆志
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1: ABSTRACT
In the dentate gyrus neurons continue to be generated from late embryonic to adult stage.
Recent extensive studies have unveiled several key aspects of the adult neurogenesis,
but only few attempts have so far been made on the analysis of the early postnatal
neurogenenesis, a transition state between the embryonic and adult neurogenesis. Here
we focus on the early postnatal neurogenesis and examine the nature and development
of neural progenitor cells. Immunohistochemistry for Ki67, a cell cycle marker, and
5-bromo-2-deoxyuridine (BrdU) labeling show that cell proliferation occurs mainly in
the hilus and partly in the subgranular zone. A majority of the proliferating cells express
S100β and GLAST and the subpopulation are also positive for GFAP and nestin.
Tracing with BrdU and our modified retrovirus vector carrying enhanced green
fluorescent protein indicate that a substantial population of the proliferating cells
differentiate into proliferative neuroblasts and immature neurons in the hilus, which
then migrate to the granule cell layer (66.8%), leaving a long axon-like process behind
in the hilus, and the others mainly become star-shaped astrocytes (12.0%) and radial
glia-like cells (4.7%) in the subgranular zone. These results suggest that the progenitors
of the granule cells expressing astrocytic and radial glial markers proliferate and
differentiate into neurons mainly in the hilus during the early postnatal period.
Furthermore, in the basis of the in vivo data, we established the slice culture methods
and assessed its utility for studying neurogenesis by comparing the neuronal
differentiation and migration between slice culture and in vivo.
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2: INTRODUCTION
The formation of the granule cell layer (GCL) can be divided roughly into two
stages. In the first stage, progenitor cells proliferate within the periventricular zone of
the medial part of the embryonic cerebral cortex, and then the neural progenitor cells
and neuroblasts migrate to the prospective dentate region during the perinatal periods
(Stensaas, 1967; Eckenhoff & Rakic, 1984; Rickmann et al., 1987; Altman & Bayer,
1990b; Sievers et al., 1992; Nakahira & Yuasa, 2005). In rats, the migrating cells form
the outer shell of the GCL by P5, as well as inhabiting the proliferative zone within the
hilus (Altman & Bayer, 1990a). In the second stage, the newborn cells generated in the
hilus and subgranular zone (SGZ) are added to the inner part of the GCL and form the
inner shell of the GCL(Altman & Bayer, 1990a). Thus, more than half of the granule
cells are born postnatally (Angevine, 1965; Schlessinger et al., 1975; Bayer, 1980) and
neurogenesis continues into adulthood only in the SGZ (Altman & Das, 1965; Kaplan &
Hinds, 1977; Bayer et al., 1982; Cameron et al., 1993; Seki & Arai, 1993; 1995; Kuhn
et al., 1996; Eriksson et al., 1998; Gould et al., 1998; Kornack & Rakic, 1999). The
adult neurogenesis in the hippocampus has been extensively studied by
5-bromo-2-deoxyuridine (BrdU) labeling and immunohistochemistry based on neuronal
and glial markers (Seki & Arai, 1993; Kuhn et al., 1996; Parent et al., 1997; Palmer et
al., 2000; Gould & Gross, 2002; Seki, 2002). Recent studies have indicated that
neuronal progenitors in the adult hippocampus are glial fibrillary acidic protein
(GFAP)-expressing cells, and they give rise to neurons (Seri et al., 2001;
Alvarez-Buylla et al., 2002; Garcia et al., 2004). However, in early postnatal
neurogenesis, it has not been made clear as yet what types of proliferative cells are the
neuronal progenitors, or how these progenitors differentiate into neurons. The analysis
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of postnatal neurogenesis, a transition state between embryonic and adult neurogenesis
in the hippocampus is an important issue, because it should provide key information
about how neurogenesis continue in this special region from the embryonic to adult
stages.
Additionally, the postnatal hippocampus is generally used in slice culture which should
be powerful tool to analyze hippocampal neurogenesis in vitro (Kamada et al., 2004;
Raineteau et al., 2004). Organotypic slice cultures of the hippocampus are a popular ex
vivo model and have several advantages for investigating the physiology, pharmacology
and pathology of hippocampus formation (Stoppini et al., 1991; Gahwiler et al., 1997;
Sakaguchi et al., 1997). Cultured hippocampal slices maintain normal tissue
organization and physiological membrane properties (Stoppini et al., 1991; Okada et al.,
1995; Gahwiler et al., 1997) and can be directly observed under a fluorescent
microscope or confocal laser scanning microscope together with live cell labeling
techniques. Furthermore, the fact that postnatal neurogenesis requires
microenvironments surrounding precursors (Palmer et al., 2000; Seki, 2002; 2003)
suggests that a slice culture containing various neural and non-neural elements is a more
suitable ex vivo model for postnatal neurogenesis than neurosphere culture. However,
application of the hippocampal organotypic slice cultures for postnatal neurogenesis is
relatively rare (Kamada et al., 2004; Raineteau et al., 2004; Laskowski et al., 2005;
Poulsen et al., 2005). Furthermore, neurogenesis in organotypic cultures has not been
precisely assessed by comparison with in vivo neurogenesis of age-matched rats.
To obtain the basic knowledge about the postnatal neurogenesis of the hippocampus,
we have examined the nature of the proliferative cells in the postnatal hilus and traced
the fate of the proliferative cells by use of BrdU and green fluorescent protein
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(GFP)-retrovirus labelings. The present results show that the postnatal hilus is
transiently filled with proliferative cells expressing astrocytic and radial glial markers
and immature neurons, and suggest that a substantial population of these hilar
proliferative cells generates proliferative neuronal precursor cells and immature neurons
in the hilus, which then migrate to the granule cell layer and become granule cells.
Furthermore, on the basis of the basic in vivo data, we analyzed in vitro neurogenesis in
an organotypic hippocampal slice culture. Consequently, we found a useful labeling
method for investigating neural development of neural precursor cells to allow efficient
neuronal production similar to in vivo postnatal neurogenesis.
In addition, here we developed the slice culture and time-lapse imaging methods to
observe the mode of progenitor cell division and follow the fate of the daughter cell
directly. To observe the primary progenitor cell division and their neuronal
differentiation, we traced enhanced green fluorescent protein (GFP) positive cells from
transgenic mice that express GFP driven under the mouse GFAP promoter
(mGFAPp-GFP Tg mouse) (Suzuki). In the present work, we directly show the neuronal
differentiation of GFP-positive astrocyte-like progenitor cells under the time-lapse
imaging observation. Furthermore, we found that both asymmetric cell divisions are
involved in the postnatal hippocampal neurogenesis.
3: MATERIALS AND METHODS
3-1: in vivo experiments
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Animals and Tissue Preparation
All of the animal treatments were approved by the institutional animal care and use
committee at Juntendo University. Wister rats of 5 - 14 days of age were deeply
anesthetized with sodium pentobarbital, and perfused intracardially first with 0.01 M
phosphate-buffed saline (PBS), pH 7.4, followed by 4% paraformaldehyde in 0.1 M
phosphate buffer (PB), pH 7.4, at room temperature. The brains were removed from the
skull and postfixed overnight in the same solution at 4°C. The fixed brains were washed
three times with PBS and immersed in 10% and then 20% sucrose in PBS over 2 days.
The cerebral cortices containing the hippocampal formation were dissected away from
the remaining brain structure. Next, 1- to 2-mm-thick slices were cut from the medial
part of the hippocampus in a plane perpendicular to the septo-temporal axis of the
hippocampal formation, embedded in OTC compound and stored at –80°C. The samples
were thawed and washed in PBS, embedded in 5% agarose in PBS and were sectioned
by a vibratome into sections 50 µm thick.
BrdU administration
Rats on postnatal day 5 (P5) were given an i.p. injection of BrdU dissolved in 0.9%
NaCl (50 mg/kg body weight). Thirty minutes (P5), 1 (P6), 3 (P8), 7 (P12) and 14 (P19)
days after the BrdU injection, the rats were perfused with fixative as described above.
Retroviral injections
To trace the proliferative cells, we used our modified retrovirus vector, GCDNsap-EGFP.
Details of construction of the expression vector were described previously (Suzuki et al.
2002). P5 rats anesthetized on ice were stereotactically injected with 0.5 µl of
GCDNsap-EGFP retrovirus (Suzuki et al., 2002; Tanaka et al., 2004; Yamada et al.,
2004) into the dentate gyrus of the hippocampus in certain locations (anteroposterior =
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1.2 mm from bregma; lateral = 2.1 mm; ventral = 2 mm). Three (P8) and 14 (P19) days
after the retroviral injection, the rats were perfused with fixative as described above.
Antibodies and Immunofluorescent staining
The antibodies, concentrations and vendors used for this work are listed in Table 1. The
primary antibodies were diluted with PBS containing 1% Bovine serum albumin (BSA)
and 1% normal donkey serum, and the secondary antibodies were diluted with PBS
containing 1% BSA. Vibratome sections of the hippocampal formation were washed
with PBS. All subsequent incubations were carried out with free-floating sections in
10-ml vials using a rotator. Each of the following steps was followed by PBS washing.
The sections were treated with PBS containing 1% BSA and 1% normal donkey serum
at room temperature for 30 min, and were incubated with the following combinations of
primary antibodies diluted in PBS at 4°C for 24 hr. The sections were then incubated at
room temperature for 1-2 hr with a mixture of secondary antibodies. In the case of BrdU
analysis, the sections were subsequently treated with 2N HCl at 37°C for 35 min and
neutralized with 0.1M borate buffer (pH 8.5). Next the sections were incubated with a
rat monoclonal anti-BrdU at 4°C overnight and then incubated with Cy3-conjugated
anti-rat IgG. Finally the specimens were mounted on slide glasses. The samples were
viewed through a Zeiss confocal laser-scanning microscope (LSM510; Germany) with
20X, 40X and 100X objectives. Stacks of optical sections (4.2 µm for 20X-objective,
1.8 µm for 40X-objective and 0.7µm for 100X-objective in thickness) were obtained at
2.1 µm increments in the z-axis for the 20X-objective, 0.9 µm for the 40X-objective and
0.4µm for the 100X-objective analysis. The images were corrected for brightness and
contrast using Zeiss LSM image Browser, Adobe Illustrator 9.0 (Adobe systems inc.,
CA, USA) and Adobe Photoshop 7.0 (Adobe systems inc). When the primary antibodies
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were omitted in immunofluorescent staining, no immunoreactivity was detected.
3-2: in vitro experiments
Slice culture and tissue processing
Hippocampal slices were prepared from postnatal day 5 (P5) Wistar rats and cultured as
the standard interface method with few modifications (Stoppini et al., 1991; Sakaguchi
et al., 1997; Kamada et al., 2004). Rats were briefly anesthetized with Diethyl Ether and
then deeply anesthetized on ice. Their heads were cut and the brains removed. The
hippocampi were dissected in Minimum Essential Medium (MEM: Invitrogen, CA,
USA) supplemented with 25 mM HEPES (Sigma, MO, USA). The whole hippocampi
were sliced into 350-µm-thick slices using the Mcllwain tissue chopper (The Mickle
Laboratory Engineering, UK). The slices were randomly chosen from the hippocampus
except for regions near the septal and temporal poles and transferred onto a porous
membrane (Millicell-CM: PICM03050, Millipore, MA, USA) and maintained in an
incubator at 34 °C with a 5% CO2-enriched atmosphere. The culture medium was 50%
MEM (Invitrogen), 25% heat inactivated horse serum (Invitrogen) and 25% Hank's
balanced salt solution (Invitrogen) supplemented with
Penicillin-Streptomycin-Glutamine (Invitrogen) and glucose (final concentration, 6.5
mg/ml). The medium was changed twice a week. Two weeks after BrdU or RV
treatment, the slices were fixed by 4% paraformaldehyde in 0.1 M phosphate buffer
(PB), pH 7.4, for 8h at 4 °C. The fixed slices were embedded in 5% agar, washed three
times with PBS and immersed in 10% and then 20% sucrose in PBS over 2 days. Next,
the slices were embedded in OTC compound, frozen in liquid nitrogen and stored at
–80°C. The slices were sectioned with a cryostat into 30-µm-thick sections.
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5-bromo-2-deoxyuridine (BrdU) and retrovirus (RV) treatment
Newly generated cells were labeled by the following three methods: (1) an i.p injection
of bromodeoxyuridine (BrdU) (Sigma) dissolved in 0.9% NaCl (50 mg/kg body weight)
into P5 rats 30 min before slice preparation, (2) incubation with 1 µM BrdU-containing
culture medium for 30 min from the beginning of culture (3) or for 1 day from 7 days in
vitro (DIV). To visualize the newly generated cells, we used our modified retrovirus
vector, GCDNsap-EGFP. Details of the construction and titer of this vector were
described previously (Suzuki et al., 2002). Drops of GCDNsap-EGFP retrovirus
solution were put on to the cultured slices at the beginning of culture (0.5 µl per one
slice). In case of time-lapse imaging, retrovirus-vector (0.5 µl) were stereotactically
injected into the hilus of P5 rats (posterior = 1.2 mm from bregma; lateral = 2.1 mm;
ventral = 2 mm), as described previously (Namba et al., 2005).
Time lapse imaging
Three days after the retroviral injection (P8), hippocampal slices at a thickness of 350
µm were cultured as described above. Time-lapse recording was performed manually
using an inverted confocal laser-scanning microscope (LSM510META; Zeiss,
Germany). To follow the movements of the labeled cells, stacks of images were
collected in the z-plane every day using a 20X-objective. Between the time points, the
slices were kept in an incubator at 37°C and 5% CO2.
Antibodies and Immunofluorescent staining
The antibodies, concentrations and vendors used for this work are listed in Table 1.
Tissue processing and immunostaining were done as described above.
Cell counting
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To determine the number of BrdU-, astrocyte-specific glutamate transporter (GLAST),
Hu-, NeuN, PSA-NCAM, Ki67 and S100β-positive cells in the granule cell layer (GCL)
including subgranular zone, and hilus of rats, one of 5 sections per cultured slice was
used and each experimental group consisted of 7-21 cultured slices from 3-4
independent rats. For the in vivo analysis, an average of 5 sections per rat was used and
each experimental group consisted of 3 - 5 rats. Adjacent sections were not used for the
cell counting to avoid double counting. All of the counting was performed under the
confocal laser-scanning microscope and using 40x-objective in stacks of 5 optical
sections. Data were analyzed statistically using one-way analysis of variance followed
by post-hoc Scheffe’s F-test. All values are given as mean ± SEM.
Morphological analysis
To analyze the morphological characters of GFP+/Hu+ cells, the Z-series of images
was obtained under the Zeiss confocal laser-scanning microscope (LSM510 and
LSM510 META) using a 40x objective. The number of dendritic branching points and
the dendrite length were measured three-dimensionally using the Imaris 4 (Zeiss) and
the Imaris Measurement Pro (Zeiss). Data were analyzed statistically using one-way
analysis of variance followed by the post-hoc Scheffe’s F-test. All values are given as
mean ± SEM.
3-3: Time-lapse imaging of astrocyte-like progenitor cells
Slice culture preparation
To detect the astrocyte-like progenitors in living tissues, we used mGFAPp-EGFP
transgenic mouse. The mice were deeply anesthetized on ice. Then the hippocampal
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slices were prepared as the standard method (Stoppini et al., 1991; Sakaguchi et al., NR
20: 157-164). The hippocampal slices (350µm in thickness) were transferred onto a
collagen-coated grass bottom dish. The culture medium was a mixture of 50% MEM
(Invitrogen, Carlsbad, CA, USA), 25% heat inactivated horse serum (Invitrogen) and
25% Hank's balanced salt solution (Invitrogen) supplemented with
Penicillin-Streptomycin-Glutamine (Invitrogen). Glucose was added to reach a final
concentration (6.5mg/ml).
Time-lapse confocal imaging
Time-lapse recording was performed manually using inverted confocal laser-scanning
microscope (LSM510 META; Zeiss, Germany) and done using minimal laser exitation
(typically 1 % of a Argon 488 laser) to prevent photodamage and photobleaching. DIC
images were obtained to confirm the granule cell layer. To follow the
movements of cells, stacks of images were collected in z-plane every 2 hr by using
40X-objective. Between time points, slices were kept in a water-jacketed
incubator at 34 °C, 5% CO , 5% O . After the time-lapse imaging, cultured
slices were fixed
2 2
overnight in the4% PFA solution at 4°C. Time-lapse sequences
arranged using Photoshop (Adobe Systems) and Quick Time pro (Apple).
Antibodies and Immunofluorescent staining
The antibodies, concentrations and vendors used for this work are listed in Table 1. The
primary antibodies were diluted with PBS containing 1% Bovine serum albumin (BSA),
0.2% Triton X-100 and 10% normal donkey serum, and the secondary antibodies were
diluted with PBS containing 1% BSA, 0.1% Triton X-100 and 1% normal donkey serum.
Fixed slices were washed with PBS. All subsequent incubations were carried out with
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free-floating sections in 10-ml vials using a rotator. Each of the following steps was
followed by PBS washing. The slices were incubated 3 overnights with a mixture of
primary antibodies diluted in same solution at 4°C. The sections were then incubated at
room temperature for 3 hr with a mixture of secondary antibodies. Then the slices were
incubated at room temperature for 3 hr with streptoavidin-Alexa 405 (1:400). Finally
the specimens were mounted on slide glasses. The samples were viewed through a Zeiss
confocal laser-scanning microscope (LSM510; Germany) with 20X and 63X objectives.
Stacks of optical sections (2.3 µm for 20X-objective and 1.1µm for 63X-objective in
thickness) were obtained at 1.15 µm increments in the z-axis for the 20X-objective and
0.55µm for the 63X-objective analysis. The images were corrected for brightness and
contrast using Zeiss LSM image Browser, Adobe Illustrator 9.0 (Adobe systems inc.,
CA, USA) and Adobe Photoshop 7.0 (Adobe systems inc). When the primary antibodies
were omitted in immunofluorescent staining, no immunoreactivity was detected.
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Table 1. Antibodies
Marker Species, isotype Label Working dilution Vendor
Primary antibodies
BrdU Rat IgG none 1:200 ImmunologicalsDirect.com, UK
GFAP Mouse IgG none 1:2000 Sigma, MO, USA
GFP Mouse IgG none 1:400 Sigma
GFP Rabbit IgG none 1:200 Gift from Dr. N. Tamamaki
GLAST Rabbit IgG none 1:400 CovalAb, France
Hu Human IgG none 1:2000 Gift from Dr. HJ. Okano
Hu Mouse IgG none 1:100 Molecular Probes, OR, USA
Ki67 Mouse IgG none 1:100 Novocastra Laboratories, UK
MASH1* Mouse IgG none 1:400 BD Bioscience, CA, USA
Nestin Mouse IgG none 1:2000 BD Bioscience
NeuN Mouse IgG none 1:200 Chemicon Interenational, CA, USA
S100β Mouse IgG none 1:2000 Sigma
S100β Rabbit IgG none 1:5000 Swant, switzerland
Secondary antibodies
Anti-human IgG Donkey IgG Cy3 1:200 Jackson, PA, USA
Anti-mouse IgG Donkey IgG Cy2 1:200 Jackson
Anti-mouse IgG Donkey IgG Cy5 1:200 Jackson
Anti-mouse IgG Goat IgG Cy5 1:200 Jackson
Anti-mouse IgG* Horse IgG Biotin 1:200 Vector, CA, USA
Anti-rabbit IgG Donkey IgG Cy2 1:200 Jackson
Anti-rabbit IgG Donkey IgG Cy5 1:200 Jackson
Anti-rabbit IgG Donkey IgG FITC 1:200 Jackson
Anti-rabbit IgG** Goat IgG Biotin 1:200 Vector
Anti-rat IgG Donkey IgG Cy3 1:200 Jackson
*To detect a mammalian achaete-acute homolog-1 (MASH-1), biotinylated horse anti-mouse
IgG and Alexa 488-conjugated streptavidin (Molecular probes, 1: 400) were used.
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4: RESULTS
4-1: Neurogenesis in in vivo
Cellular components of the hilar proliferative zone during the postnatal period
Previous studies have described the proliferative zone in the postnatal hilus by
3H-thymidine-uptake into dividing cells (Altman & Bayer, 1990a). We first re-examined
the distribution pattern of proliferative cells in rats from P5 to P19 by
immunohistochemistry for Ki67 (Cooper-Kuhn & Kuhn, 2002; Kee et al., 2002), a
proliferative marker, which visualizes all of the cells during late G1, S, M and G2
phases of the cell cycle. A dense population of Ki67-positive cells was found mainly in
the hilus and partly in the subgranular zone and inner region of the granule cell layer
from P5 to P8, although Ki67-positive cells were also distributed sparsely throughout
the entire dentate gyrus (Fig. 1A1, 2). Thereafter, the distribution pattern of the
Ki67-positive cells gradually changed. By P19 the dense population of Ki67-positive
cells had almost disappeared from the hilus and Ki67-positive cells were confined
mainly to the SGZ and most inner part of the granule cell layer (Fig. 1A3). The manner
of the neurogenesis seen on P19 is nearly identical to that of adult neurogenesis (Altman
& Das, 1965; Seki & Arai, 1993; Kuhn et al., 1996; Gould & Gross, 2002).
Next, we examined the cellular components of the hilar proliferative zone using
immunohistochemistry for S100β, an astrocytic marker (Ogata & Kosaka, 2002), and
Hu, a neuronal marker (Okano & Darnell, 1997). Although S100β is reported to be
expressed by neurons in the brain stem and there is controversy over the S100β
expression in the forebrain (Rickmann & Wolff, 1995; Yang et al., 1996; Vives et al.,
2003), we found no S100β expression in typical mature neurons in the postnatal and
adult hippocampus in agreement with the previous data (Yang et al., 1996). The hilus
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contained many S100β or Hu positive cells between P5 and P8 (Fig. 1 B1, 2, C1, 2), a
period in which many Ki67-positive proliferative cells were observed. Most of the
Hu-positive cells were relatively small in size and large Hu-positive cells resembled the
large hilar cells seen in adults (Fig. 1E1, 2) (Amaral, 1978). As the developmental
stages proceeded, the number of the S100β-positive cells and small Hu-postitive cells
decreased in the hilus, but star-shaped S100β-positive cells and Hu-positive large cells
remained on P19 (Fig. 1B1-3, C1-3, D1, 2, E1, 2). Double immunostaining for Ki67 and
S100β or Hu revealed that the majority of the Ki67-positive cells expressed S100β in
the hilus at P5 (62.2±1.0%, total cells counted = 2167, n = 3; Fig. 4A, 5A) and the
minority expressed Hu (15.2±3.5%, total cells counted = 2298, n = 3).
Tracing newly generated cells: BrdU analysis
To follow the fate of proliferating cells, BrdU was injected into rats at P5, a point
when the hilar region contains many proliferative cells, as described above. Thirty
minutes after the BrdU injection, a small number of the BrdU-labeled cells had been
distributed sparsely throughout the entire hippocampal formation (Fig. 2A). At 1 (P6)
and 3 (P8) days after the BrdU injection, a dense population of the BrdU-labeled cells
was found in the hilus (Fig. 2B,C). Seven (P12) and fourteen (P19) days after the
injection, a majority of the BrdU-labeled cells were detected in the inner half of the
granule cell layer (Fig. 2D,E). These results suggest that newborn cells were generated
in the hilus and migrate to the GCL during their development.
To ascertain the proliferation and migration of the BrdU-labeled cells, a
quantitative analysis was performed (Fig. 3). The total number of BrdU-labeled cells
(hilus + GCL) increased from 30 min to 7 days after the BrdU injection. The increase of
the BrdU-labeled cells may be due to the further incorporation of BrdU into dividing
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cells during the period of the relatively high circulating BrdU level, and re-division of
BrdU-labeled cells, since the length of the cell cycle of the granule cells are extimated
to be 16-25 hours (Nowakowski et al., 1989; Cameron & McKay, 2001). During this
period, the number of BrdU-labeled cells was larger in the hilus than the GCL. At 7 and
14 days after the injection, inversely, the number of BrdU-labeled cells in the hilus
became smaller than that in the GCL. These data support the notion that proliferative
cells can divide several times in the hilus and migrate into the GCL.
We next characterized the BrdU-labeled cells at certain time points after the BrdU
injection. Here antibodies to S100β and GLAST (Lehre et al., 1995; Shibata et al.,
1997) were used to detect astrocytes, since BrdU-positive nuclei can be detected clearly
within the somata of these immunoreactive cells. On the other hand, in the GFAP
immunostaining most commonly used, the association between the BrdU-labeled nuclei
and GFAP-positive cytoskeleton is frequently not evident. We used an antibody for
GFAP only to visualize the astrocytic processes. In addition, antibodies to Hu and NeuN
were used for the detection of neuronal markers.
A majority of BrdU-labeled cells in the hilus exhibited S100β immunoreactivity at
30 minutes after the BrdU injection (Fig. 4B-G, 5B, 7). BrdU- and S100β double
positive cells were fusiform in appearance or had radial processes that extended to the
granule cell layer. A majority of BrdU-labeled cells also expressed GLAST (80.6±2.1%,
n = 3; Fig. 4D, 5B) and most S100β-/BrdU-double positive cells expressed GLAST
(93.5±0.28%, n = 3; Fig. 4D, 5B). A subpopulation of the S100β-/BrdU-double positive
cells appeared to have GFAP- or Nestin-immunoreactive filaments (Fig. 4B,C). These
results suggest that a majority of the proliferating cells in the hilus possess astrocytic
features in the S-phase of the cell cycle and a minority are neuroblast-like cells that can
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proliferate as those in the subventribular zone of the forebrain (Rousselot et al., 1995;
Luzzati et al., 2003). A small number of the BrdU-labeled cells were positive for Hu
(6.16±0.98%, n = 5, Fig. 4E, F, 7). Additionally a very small number of the
BrdU-labeled cells expressed both S100β- and Hu (1.65±0.17%, n = 5, Fig. 4F, 7). To
confirm simultaneous expression of neuronal and astrocytic markers in proliferating
cells, we examined the expression of proneural gene, Mammalian achaete-schute
Homolog-1 (MASH-1), which is reported to be expressed by neuronal precursor cells
(Torii et al., 1999; Pleasure et al., 2000; Yun et al., 2002). As shown in Fig. 4G,
BrdU-labeled proliferating cells were double positive for S100β and MASH-1. In
addition, we found MASH-1-, Hu- and S100β-triple positive cells. The rest of cells were
negative for both S100β and Hu (Fig. 4E, 7).
From 1 to 3 days after BrdU injection, a majority of BrdU-labeled cells still was
positive for S100β and a minority was positive for Hu (Fig. 6A, B, 7). At times, the
S100β- and the Hu-positive BrdU-labeled cells were found to make contact with large
Hu-positive cells and formed a cluster (Fig.6A, B).
Fourteen days after BrdU injection, most BrdU-labeled cells were found in the
granule cell layer, as mentioned above (Fig. 2E), and expressed Hu (Fig. 6C, 7) as well
as NeuN (Fig. 6D). The Hu-/BrdU- and NeuN-/BrdU-positive cells were distributed
mainly in the inner half of the granule cell layer. In the hilus, a very small number of
NeuN- or Hu-/BrdU-double positive cells were present. The S100β-/BrdU-double
positive cells were detected in both the granule cell layer and hilus. However, the
number of the S100β/BrdU- double positive cells was fewer than that of the
Hu/BrdU-double positive cells. The S100β-/ BrdU-double positive cells were divided
into two groups by shape and position (Fig. 6E). One population of these cells was
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located in the inner granule cell layer and subgranular zone, and sometimes extended
GFAP-positive radial fibers through the granule cell layer. Another population was
present in the hilus and had multipolar processes. No radial glia-like
BrdU-/S100β-positive cells were seen in the hilus.
Using confocal microscopy, we quantified the number of BrdU-labeled cells that
were positive for Hu or S100β, and those that were negative for both markers in the
granule cell layer and hilus at 30 minutes (P5), 1 (P6), 3 (P8), 7 (P12) and 14 (P19) days
after the BrdU injection. The results are shown in Figure 7. Thirty minutes (P5) after
BrdU injection, 64.9±3.4% of the total BrdU-labeled cells were positive for S100β.
Thereafter, the percentage of the S100β-/BrdU-expressing cells gradually decreased and
fell to 21.7±1.9% 14 days (P19) after the injection. By contrast, the percentage of the
Hu-/ BrdU-expressing cells was very low 30 min (P5; 6.16±0.98%), but then gradually
increased and reached 67.7±1.0% at 14 days (P19) after the injection. Together with the
above data, these quantitative results suggest that a substantial population of the
proliferative cells expressing S100β in the hilus at P5 became neurons in the granule
cell layer at P19.
Morphology of newly generated cells: retroviral analysis
We further used our modified retroviral vector carrying an enhanced green
fluorescent protein (GFP) that labels only dividing cells and which can supply a more
complete image of newly generated cells (Tanaka et al., 2004; Yamada et al., 2004). We
injected the retroviral vector directly into the dentate gyrus at P5.
Three days after the injection, a point at which newly generated cells are assumed
to be migrating on the basis of the present BrdU analysis, many retroviral labeled
21
(GFP-positive) cells were found in the hilus and SGZ (Fig. 8A1). More than one third of
the GFP-positive cells in the hilus expressed S100β (37.4%, total GFP+ cells counted =
267). These S100β-/GFP-positive cells are divisible into two categories: multipolar cells
(85%, total S100β+/GFP+ cells counted = 100; Fig. 8A1-5) and radial glia-like cells
(5%, total S100β+/GFP+ cells counted = 100; Fig. 8B1-5). In the radial glia-like cells,
their processes reached the GCL (Fig. 8B1). In addition to the S100β-/GFP-positive
cells, about half of the GFP-positive cells in the hilus expressed Hu (48.7%, total cells
counted = 267; Fig. 8C1-7, D1-4). Hu-/GFP-positive cells were divided roughly into
two types: elongated cells (65.4%, total Hu+/GFP+ cells counted = 130, Fig. 8C1-7)
and polygonal cells (18.5%, total Hu+/GFP+ cells counted = 130, Fig. 8D1-4). The
elongated cells had an appearance of migrating immature neurons with branched
leading and single trailing processes (Fig. 8C1-7). Sometimes, they appear to have
presumptive leading processes with a small bulge at the tip that resembled a growth
cone, and a very long presumptive trailing process that possessed varicosities and were
branched (Fig. 8C1) (Kishi, 1987; Tamamaki et al., 1997; Hatanaka & Murakami, 2002).
The polygonal cells gave rise to several fine short processes. The polygonal cells were
similar in appearance to multipolar migrating cells as reported in the embryonic
neocortex (Fig. 8D) (Tamamaki et al., 2001; Tabata & Nakajima, 2003; Kriegstein &
Noctor, 2004).
Fourteen days after the retroviral injection (P19), many GFP-positive cells were
found in the GCL and hilus (Fig. 9A1). We did not find any differences in the
distribution of GFP-positive cells between supra- and infrapyramidal granule cell layer.
In the granule cell layer, the majority of the GFP-positive cells expressed Hu and
possessed the typical morphology of granule cells (Fig. 9B1-4, 66.8%, total GFP+ cells
22
counted = 274). These Hu-/GFP- positive cells exhibited a few spines in the dendrites
(Fig. 9B5) and extended axons with large boutons toward the CA3 region of the
pyramidal cell layer (Fig. 9A1-2), suggesting that the newly generated neurons had
functionally incorporated into hippocampal circuit. Additionally, there were a small
number of other types of Hu-/GFP-double positive cells (less than 2% of GFP-positive
cells), some of which possessed multipolar processes. In the hilus, however, we could
not find the Hu-/GFP-positive elongated cells as we had observed at P8. This implies
that they were only transiently present in the hilus during the early postnatal period. The
subpopulation of the GFP-positive cells in the GCL were radial glia-like cells (4.7%,
total GFP+ cells counted = 274) or astrocytic cells which expressed S100β (Fig. 9C1-3,
12.0%, total GFP+ cells counted = 274). Typical type of the radial glia-like cells had a
triangular soma that gave rise to an apical process and many fine short processes, and
resembled putative nestin-positive neural stem cells as has been reported by Filippov et
al. (2003) and Fukuda et al. (2003). S100β-/GFP- positive multipolar cells were also
found in the hilus and the GCL. However, S100β-/GFP-positive radial glia-like cells
were not found in the hilus. Additionally, we found GFP-positive cells with no
immunoreactivity for either Hu and S100β in the GCL.
Figure 1
23
Figure 2
24
Figure 3
25
26Figure 4
Figure 5
27
Figure 6
28
Figure 7
29
Figure 8
30
Figure 9
31
32
Figure Legends
Fig. 1. Changes in the distribution pattern of the Ki67-, S100β- and Hu-positive cells in
the dentate gyrus during postnatal development. A relatively dense population of
Ki67-positive proliferating cells is visible in the hilus at P5 and 8 (A1-2), whereas at
P19 the Ki67-positive cells of the hilus decrease in number and are confined to the
subgranular zone, the border of the GCL and the hilus (A3). S100β-positive astrocytic
cells (B1-2) and Hu-positive neurons (C1-2) are also evidently dense in the hilus at P5
and 8. The hilus contains S100β-positive cells (arrow; D1), and small (arrowheads; E1)
and large (arrows; E1) Hu-positive cells. At P19, however, the numbers of the
S100β-positive cells and small Hu-positive cells decrease in the hilus (B3, C3, D2, E2).
Note that only the S100β-positive star-shaped astrocytes (D2, arrows) and large
Hu-positive cells (E2, arrows) are sparsely scattered in the hilus at P19. Scale bar:
50µm.
Fig. 2. Changes in the distribution pattern of BrdU-labeled cells in rats injected with
BrdU on P5 and fixed 30 min (P5), 1 (P6), 3 (P8), 7(P12), 14 (P19) days after the
injection. The dotted lines indicate GCL and the CA3 field of the pyramidal cell layer.
BrdU-labeled cells are found throughout the entirety of the dentate gyrus 30 min after
the injection (A). Between P5 and P8, the number of BrdU-labeled cells can be seen to
increase more in the hilus than the molecular layer (A, B, C), suggesting that cell
proliferation occurs mainly in the hilus. Thereafter, the majority of the BrdU-labeled
cells appears to migrate into the granule cell layer (D, E) where the BrdU labeled cells
mainly become granule cells at P19 (see also Fig 6, 7). Scale bar: 50µm.
33
Fig. 3. Quantitative analysis of BrdU-positive cells in the granule cell layer and hilus in
rats injected with BrdU on P5 and fixed 30 minutes (P5), 1 (P6), 3 (P8), 7 (P12) and 14
(P19) days after the injection. The values show the number of BrdU-positive cells per
optical section (1.8µm in thickness). The total number of the BrdU-positive cells
increases between P5 and 8 (solid circles), but then does not change between P8 and 19.
The Open and Solid bars indicate the number of the BrdU-labeled cells in the hilus and
GCL, respectively. In the hilus, the number of the BrdU-labeled cells increases between
P5 and 8, but then decreases between P8 and 19. In the granule cell layer, the number of
the BrdU-labeled cells gradually increased between P5 and P19. These results suggest
that cells generated in the hilus migrate into the GCL. Each experimental group consists
of five independent rats. Error bars indicate the S.E of the mean.
Fig. 4. Phenotypic analysis of Ki67-positive or BrdU-labeled cells in rats injected with
BrdU on P5 and fixed 30 minutes after the injection. The S100β-positive cells indicated
by arrows in B1, C1, D1, E1, F1 and G1 are shown at middle magnification in B2
(arrows) and at high magnification in B3-5, C2-5, D2-5, E2-5, F2-5 and G2-5 (arrows).
A: A majority of Ki67-positive cells expresses S100β (arrows and double arrows).
Orthogonal images of a cell indicated by double arrow in A1 show co-localization of
Ki67 and S100β (A2-4). B: An S100β-/BrdU-positive cell expresses GFAP (arrow;
B3-5). The triple positive cell has a radial process extending toward the GCL
(arrowheads; B2-5). C: Two BrdU-labeled cells express S100β strongly (white arrow)
or weakly (black arrow) and also have nestin-positive processes (arrowheads, C2-5). D:
An S100β-/BrdU-positive cell expresses GLAST (arrows, D2-5). E: A BrdU-labeled
cell is positive for Hu (arrowheads, E2-5). Additionally, the cell negative for both S100β
34
and Hu is visible in the hilus (black arrows, E2-5). F: A BrdU-labeled cell expresses
both S100β and Hu (arrows, F2-5). Another BrdU-labeled cell is positive only for Hu
(arrowheads, F2-5). G: A BrdU-labeled cell expresses both S100β and MASH-1 (arrows,
G2-5). Five optical sections (4.2 µm in thickness) were projected in B1, C1, D1, E1, F1
and G1, and 17 optical sections (0.7 µm in thickness) in B2. Scale bar: 50µm for B1, C1,
D1, E1, F1 and G1, 20µm for A1, 10µm for B2-3, C2, D2, E2, F2 and G2.
Fig. 5. Composition of proliferating cells in the granule cell layer and hilus at P5. A:
Proliferating cells were detected by immunohistochemistry for Ki67, a cell cycle marker.
More than two thirds of Ki67 positive cells express S100β. B: Proliferating cells were
labeled with BrdU which was injected 30 minutes before tissue fixation. The bulk of
BrdU-labeled cells express GLAST and S100β. These data indicate that a majority of
proliferating cells possess astrocytic features in the early postnatal dentate gyrus. The
number of immunoreactive cells was counted in five serial optical sections (1.8 µm in
thickness) that were obtained at 0.9 µm increments on the z-axis.
Fig. 6. Phenotypic analysis of BrdU-positive cells in rats injected with BrdU on P5
and fixed 1 (P6) 3 (P8) and 14 (P19) days after the injection. The higher magnification
images of boxed areas in A1, B1, C1, D1 and E1 are shown in A2-5, B2-5, C2-5, D2-5
and E2-5, respectively. A: One day after BrdU injection (P6), some BrdU-labeled cells
in the hilus express S100β (arrowheads). Note that two S100β-/BrdU-positive cells are
in contact with Hu-positive large hilar cells (asterisks). B: Three days after BrdU
injection (P8), BrdU-positive cells expressing Hu, a neuronal marker (arrows) are seen
in the hilus. A Hu-positive cell makes contact with a Hu-positive large hilar cell
35
(asterisk). C: Fourteen days after BrdU injection (P19), most BrdU-labeled cells are
present in the granule cell layer and have Hu immunoreactivity (arrows). D:
BrdU-positive cells in the granule cell layer are also positive for NeuN (arrows), a
mature neuronal marker at P19. E: An S100β-/BrdU-positive cell (large arrowheads)
extending GFAP-positive radial processes (small arrowheads) is found in the inner
granule cell layer. In the hilus, an S100β-/GFAP-/BrdU- positive cell appears to be a
star-shaped astrocyte (double arrowheads). Scale bar: 20µm.
Fig. 7. Percentage of BrdU-labeled cells that co-express neural (Hu) and astrocytic
(S100β) markers in the granule cell layer and hilus in rats injected with BrdU on P5 and
fixed 30 minutes (P5), 1 (P6), 3 (P8), 7 (P12) and 14 (P19) days after the injection. The
percentage of S100β-expressing cells decreases gradually between P5 and P19. In
contrast, the percentage of Hu-expressing cells increases linearly. The reciprocal change
suggests the possibility that S100β-expressing cells are converted into Hu expressing
cells. The number of immunoreactive cells was counted in five serial optical sections
(1.8 µm in thickness) that were obtained at 0.9 µm increments on the z-axis. Each
experimental groups consist five independent rats.
Fig. 8. Morphology and phenotype of retrovirus-labeled cells in the hilus. A retroviral
vector bearing the GFP gene was injected into the dentate gyrus on P5 and the rats were
fixed 3 days (P8) after the injection. A: S100β-/GFP-positive cells with multipolar
processes. The GFP-labeled cells indicated by the arrowhead in A1 are shown at higher
magnification in A2-5. Fifteen optical sections (4.2−µm in thickness) and 19 optical
sections (0.7 µm in thickness) are projected in A1 and A2, respectively. B: An
36
S100β-/GFP-positive cell is located in the hilus and extends radial fiber reaching the
GCL (small arrowheads, B1-5). The GFP-labeled cells indicated by the arrowhead in B1
are shown at higher magnification in B2-5. Four optical sections (1.8−µm in thickness)
are projected in B1. C: Hu-/GFP-positive elongated immature neurons with branched
leading and trailing processes (arrows, C1). The trailing processes are very long and
possess varicosities (arrowhead, C1). The GFP-positive cells indicated by C2 and C5
arrows in Fig. C1 correspond to the GFP-positive cells in the separate images of C2-4
and C5-7, respectively. Twenty-five optical sections (1.8−µm in thickness) are projected
in C1. C2 and C5 are merged images of C3-4 and C6-7, respectively. D:
Hu-/GFP-positive polygonal immature neurons with several fine, short processes
(arrowheads). Thirty-four optical sections (0.7 µm in thickness) are projected in D1.
The GFP-positive cell in D1 corresponds to the GFP-positive cells in the separate
images of D2-4. The dotted lines in B1 and C1 indicate the boundary between the GCL
and hilus. Scale bar: 50µm for A1; 20µm for B1-2 and C1; 10µm for A2-3, C2, 5 and
D2; 5µm for D1.
Fig. 9. Retrovirus-labeled cells differentiate into neurons and astrocytes 14 days after
the injection. Retrovirus bearing the GFP gene was injected into the dentate gyrus of P5
rats and the rats were fixed 14 days later (P19). A: Low magnification image of the
hippocampus showing many GFP-labeled cells. A majority of the GFP-labeled cells are
located in the granule cell layer (GCL) and appear to be granule cells. The GFP-labeled
cells extend apical dendrites into the molecular layer and give rise to the axons that run
above the CA3 pyramidal cell layer (CA3). The boxed area in A1 is enlarged in A2.
Note that the GFP-positive fibers have large boutons, which are a typical feature of the
37
mossy fibers (A2). Astrocytic GFP-positive cells also are visible mainly in the hilus and
molecular layer. B: Example of a GFP-positive cell with typical morphology of the
granule cells. The GFP-positive cell (arrow) in B1 corresponds to that indicated by the
arrow in A1, and is shown at higher magnification in B1-5. The GFP-labeled cells are
positive for Hu, a neuronal marker (B2-4), and extend apical processes that possess
spines (arrows, B1, 5). The boxed area in B1 is enlarged in B5. Thirty-one optical
sections (1.8 µm) and 6 optical sections (0.7 µm) are projected in B1 and B5,
respectively. C: An S100β-/GFP-positive cell in the subgranular zone has a radial
process which branches in the inner part of the molecular layer. Additionally, many fine
processes arise from the cell body of the S100β-/GFP-positive cell. The
S100β-/GFP-positive cell (arrowhead) in C1 corresponds to the GFP-positive cell
(arrowhead) in A1. Scale bar: 50µm for A1, 20µm for A2, B1 and C1, 10µm for B2,
5µm for B5.
38
4-2: Neurogenesis in hippocampal slice culture
Late in vitro-labeling cultures
In the initial experiments, since cultured slices are generally used in experiments after
1-2 weeks in culture (Okada et al., 1995), hippocampal slices from P5-P6 rats were
treated with BrdU from DIV7 to DIV8 and fixed 14 days after BrdU treatment (DIV21;
Fig. 10A). Triple immunostaining revealed that a small proportion of BrdU-positive
(BrdU+) cells expressed a neuronal marker, Hu (9.40±2.1%) and the large proportion
were S100β-positve (16.0±1.8%) and double-negative cells (74.6±2.9%, n = 8 slices
from 3 rats, Fig11A, 12A). Most of the double-negative cells should be microglial cells,
because approximately half of BrdU-labeled cells expressed microglial marker Iba-1
(data not shown). The numbers of BrdU/RIP-double positive oligodendrocytes and
BrdU/Ki67-double positive proliferating cells were very low (data not shown). Since it
has been reported that about two-thirds of precursor cells differentiate into neurons
under in vivo conditions in the early postnatal period (Namba et al., 2005) and in the
adult hippocampus (Kempermann et al., 2003), the results indicate that the activity of
the neuronal production is very low in proliferative neural precursor cells after 7 days in
culture.
In vivo-labeling and early in vitro-labeling cultures
To search for suitable labeling methods of neural precursor cells that lead to efficient
neuronal production, we attempted to use the following two cultures with different
BrdU-labeling methods: in vivo-labeling cultures (Fig. 10B) and early in vitro-labeling
(Fig. 10C) cultures. Further, to precisely ascertain the efficiency of the neuronal
39
production, we compared the results of these two cultures with those of living
age-matched rats.
In the first in vivo-labeling cultures, to determine whether or not the in vitro
condition itself affects the capacity for neuronal differentiation, proliferative neural
precursor cells were labeled in vivo and then hippocampal slices were cultured to allow
the in vivo-labeled precursors to differentiate into neurons in vitro. P5 rats were injected
with (BrdU) at 30 min before slice preparation (Fig. 10B) and hippocampal slices were
cultured for 14 days. At the end of the cultures, the majority of BrdU-labeled cells were
located in the GCL (56.9±3.3%, n=21 slices form 3 rats, Fig. 4). More than half of the
BrdU-labeled cells in the GCL expressed immature and mature neuronal marker Hu
(60.1±3.7%) and the others were S100β-positive (27.7±3.0%) and double-negative cells
(12.8±2.3%, n=21 slices form 3 rats, Fig. 11C, Fig. 12B). In the living age-matched
rats that were injected with BrdU at P5 and fixed at P19, most of these BrdU-labeled
newly generated cells were located in the GCL at P19 (Fig. 4). These BrdU-labeled cells
in the GCL were mainly positive for Hu (79.1±0.8%) and a small proportion was
positive for S100β (13.0±1.5%) or double-negative cells (7.4±1.1%, n=5 rats, Fig. 11G,
Fig. 12B). Although the proportion of Hu-positive neuronal cells in the early in
vitro-slice culture is smaller by 19% than that in age-matched rats (P<0.05), the
proportion was 7-fold higher than that of the late in vitro culture (P<0.001). These
results suggest that neural precursor cells labeled in vivo can differentiate into neurons
efficiently in cultured slices when compared with the late-in vitro labeling culture, and
that the in vitro condition itself is not the cause of the low rate of neuronal
differentiation.
In the second early in vitro-labeling cultures, to determine whether precursor cells
40
labeled early in the culture can differentiate into neurons, the hippocampal slices were
labeled in vitro with BrdU for 30 min at the beginning of the culture and were cultured
for 14 days to allow the in vitro-labeled precursors to differentiate to neurons in vitro.
Similar to the in vivo-labeling cultures, the majority of BrdU-labeled cells were located
in the GCL at DIV 14 (62.6±3.8%, n=10 slices form 4 rats, Fig. 13). More than
two-thirds of the BrdU-labeled cells in the GCL expressed immature and mature
neuronal marker Hu (58.5±3.1%), and the others were S100β-positve (26.5±2.4%) and
double-negative cells (12.8±2.3%, n=10 slices form 4 rats, Fig. 11B, E, Fig. 12B). The
results suggest that neural precursors labeled early in cultures can differentiate into
neurons efficiently.
Further, the neuronal maturation of the BrdU-labeled cells was assessed by a
mature neuronal marker, NeuN, and an immature neuronal marker, PSA-NCAM (Seki
& Arai, 1993; Seki, 2002). In the in vivo-labeling cultures, most of the BrdU-labeled
cells in GCL were double-positive for PSA-NCAM and NeuN (43.7±3.2% n=10 slices
from 3 rats), and the minority was single positive for PSA-NCAM (3.2±1.1%) or for
NeuN (12.7±3.6%; n=10 slices from 3 rats, Fig. 11D, Fig. 12C). Similarly, in the early
in vitro-labeling cultures, most of the BrdU-labeled cells were double-positive for
PSA-NCAM and NeuN (40.5±3.7%), and the minority was single positive for
PSA-NCAM (6.6±1.5%) or for NeuN (15.7±3.9%; n=10 slices from 3 rats, Fig. 11F, Fig.
12C). There was almost no difference in the efficiency of neuronal production between
the two labeling methods performed just before or soon after cultures. However, the
proportion of NeuN-positive mature neurons in these two cultures was significantly
smaller than that in the living age-matched rats (54.5±7.1%) that were labeled with
BrdU at P5 and fixed at P19. Conversely, the proportion of immature neurons in these
41
two cultures was higher than that in the age-matched rats (16.5±2.2%,
BrdU+/PSA+/NeuN+ cells and 10.9±3.7%, BrdU+/PSA+ cells, n = 3 rats, Fig. 11H, Fig.
12C). These results suggest that neural precursor cells labeled early in culture can
differentiate into neurons efficiently under culture conditions, although the maturation
of differentiated neurons is somewhat delayed in the two cultures.
Retrovirus-EGFP labeling
To examine whether newly generated cells develop into normal-shaped granule cells,
we labeled dividing cells with our modified high-titer retroviral vector (RV) carrying an
enhanced green fluorescent protein (GFP) (Namba et al., 2005). Since BrdU labeling
experiments showed that the early in vitro labeling induced efficient neuronal
differentiation of precursor cells, the retrovirus labeling was performed at the beginning
of slice cultures.
Similar to BrdU labeled cells, most of the retroviral-labeled cells (GFP-positive
cells) were located in GCL (75.1%, n = 489 cells, Fig. 14A) at DIV 14, and these cells
expressed Hu (70.8%, n = 367 cells, Fig. 14B). Other GFP-labeled cells in the GCL
were positive for S100β (17.2%) or devoid of the two markers (12.0%, Fig. 14C, D).
The retrovirus labeling clearly demonstrated that GFP-/Hu-positive neuronal cells
extended dendrites with a few spines (Fig. 14B, E, F), and also gave rise to axons with
boutons toward the CA3 region of the pyramidal cell layer (Fig. 14A, G). To assess the
in vitro maturation of GFP-/Hu-double positive newly generated cells in detail, the
length and branching points of the dendrites were examined and compared with those in
the in vivo maturation in living rats. In the GFP-labeled granule cells raised in vitro, the
mean total length of the dendrite was 301.6±15.7 µm and the mean number of
42
branching points was 5.45±0.31 (n = 11 cells). On the other hand, these values were
higher in GFP-labeled granule cells raised in living animals that were labeled with
retrovirus-GFP at P5 and fixed at P19 (614.3±14.8 µm in length and 7.36±0.20 at the
branching points, n = 11 cells). These results suggest that new cells generated in the
slice cultures develop into normal dentate granule cells, but their maturation could be
somewhat delayed when compared with in vivo maturation.
Time-lapse imaging
One of the advantages of slice culture experiments is to be able to observe the migration
of labeled cells using the same slices. Here, we tried to examine whether the migration
of neural precursors suggested by BrdU experiments (Fig. 13) can be observed in the
present culture system. Retrovirus-EGFP was injected into the hilus of P5 rats.
Hippocampal slices (n=14 slices from 10 rats) were cultured 3 days after the retroviral
injection at P5, and were observed every day. Small population of EGFP-labeled cells
was distributed in the hilus at DIV 0 (Fig. 13E1) and thereafter, the labeled cells were
increased in number from DIV 1 to 5. Simultaneously, the distinct population of the
labeled cells appeared in the subgranular zone and granule cell layer, and the numbers
of these labeled cells were increased (Fig. 13E2-5). Finally the labeled cells developed
apical dendrites at DIV 5 (Fig. 13E6). The time-lapse imaging directly demonstrates
that the neural precursors migrate from the hilus to the granule cell layer.
Figure 10
43
Figure 11
44
Figure 12
45
46Figure 13
Figure 14
47
48
Figure Legends
Fig. 10. Schematic drawing of BrdU-labeling time course. A, B, C: For slice culture
experiments, newly generated cells were labeled by the following three methods: (A)
incubation in culture medium containing 1 µM BrdU for 1 day from the time point of 7
days in vitro culture (DIV), (B) an i.p injection of bromodeoxyuridine (BrdU) into P5
rats at the time point of 30 min before slice preparation, or (C) incubation in culture
medium containing 1 µM BrdU for 30 min from the beginning of culture. Fourteen days
after the BrdU treatment, the cultured slices were fixed. D: For the in vivo experiments,
rats on postnatal day 5 (P5) were given an i.p. injection of BrdU and fixed 14 days after
the injection (P19).
Fig. 11. Phenotypic analysis of BrdU-positive cells in a cultured slice that was treated
with BrdU for 1 day from the time point of 7 days in vitro culture (A), for 30 min before
(in vivo BrdU treatment, C and D) or after (in vitro BrdU treatment, B, E and F) the
beginning of culture and fixed 14 days after treatment, and rats injected with BrdU on
P5 and fixed 14 (P19, G and H) days after the injection. A, B: Most BrdU-labeled cells
were negative for neuronal marker Hu when newly generated cells were labeled during
DIV7-8 (A). However, the newly generated cells labeled at the beginning of culture
were positive for Hu (B). C, D, E, F: In the cultured slices, more than half of the
BrdU-positive cells in the GCL are positive for neuronal markers such as Hu (C and E,
arrows), PSA-NCAM/NeuN (D and F, arrows) and NeuN (D, arrowheads). A small
population of the BrdU-positive cells in the GCL is positive for astrocytic marker
S100β (C and E, arrowhead). G, H: Fourteen days after the BrdU injection in vivo,
about two-thirds of the BrdU-positive cells in the GCL are positive for neuronal markers
49
such as Hu (G, arrows) and NeuN (H, arrowheads). Scale bar: 10 µm applies to B in A,
to D1-H1 in C1, and to D2-H2 in C2.
Fig. 12. Quantitative analysis of neurogenesis in the slice culture and in vivo. A: In the
late in vitro labeling culture, the newly generated cells were labeled by BrdU during
DIV7-8. Only about 10% of these BrdU-labeled cells are positive for neuronal marker
Hu. B, C: Comparison of the neurogenic ability in the cultured slices and in vivo.
Cultured slices were treated with BrdU before (in vivo labeling, Fig. 1B) or after (in
vitro labeling, Fig. 1C) the beginning of culture. For the in vivo experiments, P5 rats
were injected BrdU and fixed 14 days after the injection (Fig. 1D). B: In the cultured
slices, about 60% of the BrdU-positive cells in the GCL express Hu and about 30% of
the BrdU-labeled cells are positive for S100β. C: To clarify the degree of neuronal
maturation, we quantify the percentage of BrdU-labeled cells that co-express immature
(PSA-NCAM) and/or mature (NeuN) neuronal markers in the granule cell layer and
hilus. The percentage of the mature neuron (NeuN+/BrdU+) in the cultured slices is
lower than that in vivo. However, the percentage of immature neurons
(PSA+/NeuN+/BrdU+ and PSA+/NeuN-/BrdU+) is higher than that in vivo.
Fig. 13. Distribution of the Ki67-positive (P5, A) and BrdU-labeled cells in rats injected
with BrdU on P5 and fixed 14 (P19, D) days after the injection and in the cultured slices
treated with BrdU for 30 min before (in vivo BrdU treatment, B) or after (in vitro BrdU
treatment, C) the beginning of culture and fixed 14 days after treatment. The dotted
lines indicate GCL and the CA3 field of the pyramidal cell layer. A: Ki67-positive
proliferating cells are found throughout the entirety of the dentate gyrus at P5. B, C, D:
50
The majority of the newly generated (BrdU-labeled) cells are located in the granule cell
layer. This suggests that the newly generated cells migrate toward the granule cell layer
in the cultured slices (B, C) and in vivo (D). E: Time-lapse imaging showing the
migration of hilar precursor cells to the granle cell layer. Retrovirus vector bearing
EGFP gene was directly injected into the hilus. The red lines passing through the
hippocampal crest and the CA3 pyramidal cell layer indicate the border of the
suprapyramidal and infrapyramidal regions. Scale bar: 100 µm in A applies to B, C, D,
50 µm in E1.
Fig. 14. Phenotypic and morphological analysis of RV-positive cells (GFP-positive
cells) in cultured slices treated with RV at the beginning of culture and fixed 14 days
after treatment.
A: Most of the GFP+ cells are located in the GCL and extend axons, and a mossy fiber
(asterisk). B: In the GCL, most of the GFP-positive cells express Hu, a neuronal marker
(70.8% of all GFP-positive cells in the GCL). These cells extend apical dendrites
(arrow) and some basal dendrites or axons (arrowhead). It suggests that these
Hu-/GFP-positive cells possess the features of typical immature granule cells. C: About
one-fifth of the GFP-positive cells are positive for astrocytic marker S100β (arrow).
These possess highly lamified processes. D: A small population of the GFP-positive
cells in the GCL is negative for both Hu and S100β. The density of the ramified process
of GFP single-positive cells is lower than that of the S100β-/GFP- positive cells. E: The
GFP-positive cell in the GCL expresses Hu (arrows in inset) and extends apical
dendrites. F: In the apical dendrite (arrowhead in E), there are a few spines
(arrowheads). G: The GFP-positive axons (mossy fibers) are found in the CA3 region of
51
the PCL. There are some boutons (arrowheads). sr; Stratum radiatum, so; Stratum oriens.
B2, C2 and D2 are merged images of B3-4, C3-4 and D3-4, respectively. Scale bar: 20
µm for E, G, 10 µm for B1, C1, D1, F 5 µm for B2-4, C2-4, D2-4.
52
4-3: Direct observation of neuronal differentiation of astrocyte-like progenitor
cells.
Previous in vivo experiments suggest that the astrocyte-like progenitor cells
differentiate into neurons. However, the process of neuronal differentiation is still
unclear. In this part, we monitored the fates of GFP+ daughter cells in the slices from
mGFAPp-EGFP transgenic mice at P4-P9.
Phenotypic analysis of the GFP+ progenitor cells
We first examined the cell character of the GFP+ dividing cells from P5 GFAP-EGFP
transgenic mouse. The GFP+ daughter cell pairs in anaphase and telophase were
detected by the Ki67 immunohistochemistry and judged their cell characters by
astrocytic marker GFAP and neuronal marker Hu. Most of the GFP+ daughter cell pairs
in the GCL and hilus expressed GFAP and negative for Hu (82.5%, Fig. 15A).
Minorities were GFAP/Hu-double positive cells (10.0%, Fig. 15B) and Hu-single
positive cells (7.5%, Fig. 15C). All of the GFP+ daughter cell pairs possessed
symmetric feature. These results showed the majority of the proliferating cells were
astrocyte-like cells and re-confirmed the previous in vivo data.
Tracing the GFP+ daughter cells under the time-lapse imaging
We next follow these GFP+ daughter cells under the CLSM up to 30hr. We monitored
the fates of 51 daughter cells generated from GFAP-EGFP positive dividing cells at
P4-P9. We divided the GFP+ daughter cells into two groups by the length of the time
after the cell division, within 10 hours (0-10hr) and over 12 hours (12hr-). The results
are shown in table 1. Over 12 hr after the cell division, about half of the GFP+ daughter
53
cells were expressed GFAP and Hu, less than half of the GFP+ daughter cells were
single positive for GFAP (51.9% and 46.2%, respectively). In contrast, most of the
GFP+ daughter cells in anaphase and telophase and within 10 hrs after the cell division
were single positive for GFAP. It suggests that the GFP+ cells were differentiate from
GFAP+ astrocyte-like cells to GFAP+/Hu+ neuronal lineage-committed intermediate
cells.
Direct evidence for neuronal differentiation of astrocyte-like progenitor cells
As described above, there are three possibilities about the nature of the GFP+
progenitor cells, astrocyte-like cells, neuronal lineage-committed intermediate cells,
neuroblasts. To directly show the neuronal differentiation of astrocyte-like progenitor
cells, it needs to show the asymmetric GFP+ daughter cell pairs that consist of
astrocyte-like cells (GFAP+) and neuronal cells (Hu+). We found a few asymmetric
GFP+ daughter cell pairs that consist of GFAP+ cell and GFAP/Hu+ cell (Fig. 16).
These types of pairs shown that the GFAP+ primary progenitors asymmetrically
produce the neuronal lineage-committed cells.
Symmetric progenitor cell divisions were principal cell division mode during the
postnatal neurogenesis
To understand the process of the neurogenesis, we focused on the types of the
progenitor cell divisions. The results are shown in table 2. Within 10 hours after the
division, most of the GFP+ daughter cell pairs possessed astrocytic symmetric feature
(G-G, 76.0%) and minority was symmetric neuronal lineage-committed intermediate
cell pairs (8.0%). In contrast, over 12 hours after the division, about half of the GFP+
54
daughter cell pairs were symmetric neuronal lineage-committed intermediate cell pairs
(Fig. 17, 46.2%) and symmetric astrocytic pairs (42.3%). This reciprocal change
suggests that symmetric astrocytic daughter cell pairs symmetrically differentiate into
neuronal cells. Furthermore, the asymmetric daughter cell pairs were also found. Most
of the asymmetric pairs were GFAP+ cell and GFAP/Hu+ cell. These results suggest
that the both symmetric progenitor cell divisions were principal type during the
neuronal differentiation of the astrocyte-like progenitors.
Table 2
in vivo in vitro (time-lapse imaging)
% telophase 0-10 hr 12 hr -
G 82.5 82.0 46.2 (P<0.01)
G/N 10.0 16.0 51.9 (P<0.01)
N 7.5 2.0 1.9
Table 3
in vivo in vitro (time-lapse imaging)
% telophase 0-10 hr 12 hr -
Symmetric 100 84.0 88.5
G-G 82.5 76.0 42.3
G/N-G/N 10 8.0 46.2
Asymmetric 0 16.0 11.5
G-N 0 0.0 0.0
G-G/N 0 12.0 7.7
G/N-N 0 4.8 3.8
Figure 15
55
Figure 16
Figure 17
56
57
Figure Legends
Table 2. Percentage of mGFAPp-GFP+ cells that co-express neural (Hu) and astrocytic
(GFAP) markers in telophase, 0-10 hours and 12-26 hours after the cell division. The
percentage of GFAP-expressing cells decreases gradually between telophase and 12-26
hr after the cell division. In contrast, the percentage of Hu-expressing cells increases
linearly. The reciprocal change suggests that GFAP-expressing cells are converted into
Hu expressing neuronal lineage-committed cells.
Table 3. Percentage of symmetric and asymmetric cell fate of mGFAPp-GFP+ daughter
cell pairs in telophase, 0-10 hours and 12-26 hours after the cell division. The
percentage of GFAP-expressing symmetric pairs decreases gradually between telophase
and 12-26 hr after the cell division. In contrast, the percentage of Hu-/GFAP-expressing
symmetric pairs increases linearly. The reciprocal change suggests that
GFAP-expressing daughter cells are symmetrically differentiated into Hu expressing
neuronal lineage-committed cells. In addition, there are a few asymmetric daughter cell
pairs.
Fig. 15. Phenotypic analysis of anaphase and telophase dividing pairs in the P5
GFAP-EGFP Tg mouse dente gyrus. GFP-positive dividing cells (green, A3, B3, C3) in
anaphase and telophase are detected by morphology of Ki67-positive chromosomes
(arrowheads, purple, A4, B4, C4). All of the GFP-positive dividing pairs possess
symmetric features. A: Symmetric astrocytic division (G-G). Both of the
GFP/Ki67-positive cells express astrocytic marker GFAP (white, A2) but do not express
neuronal marker Hu(blue, A5). B: Symmetric intermediate progenitor division
58
(G/N-G/N). Two daughter cells symmetrically express both GFAP and Hu. C:
Symmetric neuronal division (N-N). Scale bar, 10um.
Fig. 16. Asymmetric production of radial type astrocytic cell and intermediate
progenitor cell. One GFP+ daughter cell has radial type astrocytic cell feature (GFAP+).
Another GFP+ daughter cell possesses intermediate progenitor cell feature
(GFAP+/Hu+). Slice are prepared from P6 GFAP-EGFP Tg mouse. These daughter cells
are located in the GCL. A: Time-lapse imaging of GFP+ cells. Division may have
occurred by 2 hr. B, C, D: At the end of time-lapse imaging (t = 12 hr), slice was fixed
and then processed for immunohistochemistry. The GFP-positive cells (arrowhead-C,
-D) in B correspond to that indicated by arrowheads in C and D, respectively. One
GFP+ daughter cell has radial process and astrocytic cell marker (GFAP+). Another
GFP+ daughter cell expresses GFAP and neuronal marker, Hu. Scale bar, 10um.
Fig. 17. Symmetrical neuronal fate of GFP-positive daughter cells. Both of GFP+
daughter cells possess intermediate progenitor cell feature (GFAP+/Hu+). Slice are
prepared from P6 GFAP-EGFP Tg mouse. These daughter cells are located in the GCL.
A: Time-lapse imaging of GFP+ cells. Division may have occurred by 0 hr. B, C, D: At
the end of time-lapse imaging (t = 12 hr), slice was fixed and then processed for
immunohistochemistry. The GFP-positive cells (arrowhead-C, -D) in B correspond to
that indicated by arrowheads in C and D, respectively. Both of these daughter cells
possess GFAP and Hu immunoreactivity (C, D). Scale bar, 10um.
59
5: DISCUSSION
5-1: Developmental process of the newlygenerated neurons in the postnatal
hippocampus; in vivo and time-lapse imaging analysis
The present study has revealed the nature of postnatal progenitors of dentate granule
cells and their developmental pattern. The hilar progenitors are those that principally
express astrocytic and radial glial markers. They proliferate and differentiate in the hilus
mainly into immature neurons via neuronal lineage-committed intermediate progenitors,
and partly into star-shaped astrocytes and radial glial cells, the latter of which are
putative neural progenitors in the SGZ. Finally, the immature neurons migrate to the
GCL, leaving their long axon-like trailing processes behind in the hilus where the axons
of the granule cells pass through. Using time-lapse imaging system, we found that both
of symmetric and asymmetric cell divisions are involved in this developmental process.
Furthermore, most notably, the present results directly show the neuronal differentiation
of mGFAPp-GFP+ astrocyte-like progenitor cells by the time-lapse imaging
observation.
The nature and fate of proliferative cells
The present Ki67 and BrdU analysis shows that cell proliferation occurs mainly in
the hilus during early postnatal period in good agreement with the previous study using
3H-thymidine autoradiography (Altman & Bayer, 1990a). We further found that a
majority of the hilar proliferating cells express S100β, GFAP and GLAST, and at least
the subpopulation exhibit nestin immunoreactivity. These results indicate that hilar
proliferating cells have features of astrocytes and radial glia (Levitt & Rakic, 1980;
Shibata et al., 1997; Kriegstein & Gotz, 2003). However, a majority of the hilar
60
proliferating cells did not possess highly branched multipolar processes like typical
star-shaped astrocytes. Additionally not all proliferating cells extend a radial process.
Therefore, the hilar proliferating cells have some characteristics differ from typical
astrocytes and radial glia, and could be considered as a distinct type of cell population.
In our preliminary experiments, we also examined NG-2 expression in hilar
proliferating cells, since hippocampal inhibitory neurons have been reported to be
derived from NG-2 positive progenitors (Belachew et al., 2003). Our results show that
the number of NG2- and Ki67-double positive cells were very small and few NG2-,
S100β- double positive cells were detected in P5 rats (data not shown). This suggests
that most of the present proliferating cells differ from NG-2 positive progenitors.
In the development of newly generated cells labeled with BrdU at P5, as
S100β-/BrdU-positive cells decreased in number, Hu-/BrdU-positive immature neurons
increased, suggesting that proliferating cells expressing an astrocytic marker generate
immature neurons. This interpretation is strongly supported by present time-lapse
imaging data that have directly shown that GFAP-expressing astrocyte-like progenitor
cells differentiate into neuronal cells. Furthermore, this interpretation is also supported
by the recent studies which have demonstrated that GFAP-expressing cells indeed do
generate neurons in the hippocampus (Seri et al., 2001; Garcia et al., 2004) and the SVZ
of the forebrain in the adult (Doetsch et al., 1999; Garcia-Verdugo et al., 2002).
Similarly, radial glial cells have been shown to produce neurons in the embryonic
neocortex, (Miyata et al., 2001; Noctor et al., 2001; Tamamaki et al., 2001; Malatesta et
al., 2003; Anthony et al., 2004). Additionally, GFAP-expressing cells from postnatal
and adult forebrain have been shown to give rise to neurons in culture (Laywell et al.,
2000; Malatesta et al., 2000; Imura et al., 2003). Together, the present results show that
61
during the early postnatal stages, proliferative cells with astrocytic and radial glial
features generate neurons.
We found proliferative cells expressing neuronal markers such as Hu and MASH-1
in the postnatal hilus, suggesting that the subset of the proliferative cells have already
committed to neuronal lineage (Marusich et al., 1994; Okano & Darnell, 1997; Torii et
al., 1999; Pleasure et al., 2000; Yun et al., 2002). It is thus possible that the proliferative
cells expressing astrocytic and radial glial markers differentiate into proliferative
neuroblasts and then become immature neurons. The notion is supported by the recent
studies using nestin-GFP mice that have suggested that proliferative progenitors
expressing nestin are converted from astrocytic to neuronal cells (Filippov et al., 2003;
Fukuda et al., 2003; Kronenberg et al., 2003). Further, we detected proliferative cells
expressing both neuronal and astrocytic markers shortly after BrdU injection. This
suggests that the differentiation from the progenitors to proliferative neuroblasts could
occur via transitional intermediate precursor cells expressing both astrocytic and
neuronal markers.
Several previous studies have described the presence of GFAP-positive
proliferative glioblasts in the postnatal hilar region (Eckenhoff & Rakic, 1984;
Rickmann et al., 1987; Sievers et al., 1992; Yuasa, 2001) and suggest that the glioblasts
are integrated into the SGZ and become radial glial cells. In this respect, our BrdU- and
retrovirus-EGFP-labeling experiments suggest that although a small population of the
hilar proliferating cells differentiate into radial glial cells in the SGZ, a majority of the
proliferative cells similar to glioblasts give rise to neurons. Since the radial glia cells in
the adult dentate granule cell layer are presently considered to be neural progenitors
(Seri et al., 2001; Filippov et al., 2003; Fukuda et al., 2003; Seri et al., 2004), the hilar
62
proliferative zone could be a source of the adult neural progenitors as well as granule
cells.
In this study of postnatal rats, proliferative cells were positive for S100β. In adult
mice, however, GFAP-expressing neural progenitors have been shown to be negative for
S100β (Filippov et al., 2003). In this respect, we found that the distribution of
S100β-positive cells in the hilus was much different in rats and mice (unpublished data).
It has also been reported that in rats and mice, newly generated cells exhibit different
expression patterns for calretinin, a member of the EF-hand Ca2+–biding proteins family
to which S100β belongs (Schafer & Heizmann, 1996; Murakawa & Kosaka, 1999).
Therefore, there may be species differences in S100β expression on proliferative cells
between rats and mice.
The neuronal differentiation process of astrocyte-like progenitor cells
We found that most of the GFP+ daughter cell pairs possessed symmetric feature.
During the neocortex development, in general, symmetrical progenitor cell divisions
expand the number of neuronal cells or progenitor cells and asymmetrical progenitor
cell divisions produce neuronal cell and self-renewed progenitor cell (Noctor, Rakic,
Gotz). Most of the symmetrical divisions are occurred in the SVZ and produce neuronal
pair (Miyata, Noctor). In contrast, asymmetric divisions are mainly occurred in the VZ
where the primary progenitors located. In this context, the neonatal dentate gyrus is
thought to be as SVZ regarding their roles in neurogenesis and differ from the VZ. This
concept is supported by the developmental process of the dentate gyrus. The progenitor
cohorts are migrate from the hippocampal VZ and make a neurogenic region in the DG.
Thus the progenitors in the DG possess similar feature of the progenitors in the
63
embryonic SVZ.
We also found the asymmetric GFP+ daughter cell pairs that consist of astrocyte-like
cell and neuronal cell. The astrocyte-like cells possess GFAP and radial-like process. It
suggests the possibility that the asymmetric produced astrocyte-like cells are progenitor
cells and made by self-renewing.
The migration of proliferative cells
Most Hu-/GFP-double positive small cells were transiently present in the hilus during
the early postnatal period and disappeared at P19. Further, they had the typical features
of migrating cells in the hilus at 3 days after the injection (Kishi, 1987; Tamamaki et al.,
1997; Hatanaka & Murakami, 2002). Therefore, neuronal differentiation mainly occurs
in the hilus during the early postnatal period, and the immature neurons migrate from
the hilus into the granule cell layer. One of the interesting features of the
Hu-/GFP-double positive cells is that they have a very long trailing process with
varicosities. Similar long trailing processes of migrating cells have been reported in the
developing neocortex (Schwartz et al., 1991; Hatanaka & Murakami, 2002). These
reports suggest that the long trailing processes of the migrating cells become axons.
Taken together, Hu-/GFP-double positive cells could migrate, probably extending a
trailing process or axon so that the tip of the process is left near the CA3c pyramidal
cells which is a target of the granule cell axons.
Conclusions
Finally, we propose a model for the postnatal neurogenesis of the dentate granule
cells (Fig. 18). During the early postnatal period, cell proliferation occurs mainly in the
64
hilus and partly in the subgranular zone and inner part of the granule cell layer. The
majority of the proliferating cells in the hilus express astrocytic and radial glial markers
such as S100β, GFAP and GLAST. A substantial population of these cells should thus
differentiate into proliferative neuroblasts and immature neurons within the hilus, via
transitional intermediate progenitor cells expressing both astrocytic and neuronal
markers. This differentiation process is mostly symmetric, however, asymmetric process
is also existed. The neuroblasts and immature neurons move to the GCL. When the
immature neurons migrate, they are extending a trailing process whose tip is left behind
near the CA3c pyramidal cells. Additionally a subpopulation of the proliferating cells
move to the SGZ and become radial glia-like cells which are considered to be putative
neural stem cells (Seri et al., 2001; Alvarez-Buylla et al., 2002; Filippov et al., 2003;
Fukuda et al., 2003; Garcia et al., 2004) or star-shaped astrocytes in the hilus and GCL.
The data presented here have revealed the nature of the hilar progenitor cells and their
distinctive developmental pattern, and have shown that early postnatal hilus is a unique
neurogenic region where cell proliferation, neuronal differentiation and cell migration
occur. Additionally, the present results indicate that the early postnatal hilar progenitors
share a similar nature with adult progenitors. This implies that the early postnatal
hippocampus, often used in slice culture (Kamada et al., 2004; Raineteau et al., 2004),
is suitable for a model for adult neurogenesis, particularly in terms of the differentiation
of neural progenitors expressing astrocytic markers into neurons.
5-2: Neurogenesis in hippocampal slice culture
In research on postnatal neurogenesis using organotypic hippocampal slice cultures, it is
important to know the extent to which cultured slices possess neurogenic capacity. In
65
the present study, we have shown that at the beginning of culture, neural precursor cells
had a high neurogenic capacity that was similar to that in age-matched rats. However,
after 7 days in culture, the neural precursor cells lost their high neurogenic capacity,
although they still exhibited a low rate of neuronal production. This indicates that
although cultured hippocampal slices are generally used in electrophysiological and
pharmacological studies after 1 - 2 weeks in culture (Okada et al., 1995), the late in
vitro-labeling of proliferative precursors has the disadvantage of requiring a high
proliferating activity of neural precursors for analysis. On the other hand, since in vivo
and early in vitro-labelings of proliferative neural precursors allow efficient neuronal
differentiation of labeled precursors, these labelings are suitable methods for studies that
need more chance to observe neuronal production in organotypic hippocampal slice
cultures.
Late in vitro labeling culture
The findings of the present experiments indicate that in late in vitro-labeling, only a
small number of proliferative neuronal precursors differentiated into neurons, with most
becoming non-neuronal cells. In this regard, previous reports have shown that
non-neuronal cells such as astrocytes, microglia and fibroblasts continue to proliferate
in organotypic hippocampal slice cultures (del Rio et al., 1991; Gahwiler et al., 1997;
Raineteau et al., 2004). This suggests that the cellular composition of cells proliferating
in cultured hippocampal slices is considerably different from that of in vivo. The
proliferative activity is also reported to be reduced over the first 7 DIV (Hajos et al.,
1994; Sadgrove et al., 2006). Furthermore, there are discrepancies among published
reports showing the capacity for neuronal production after 1 – 2 weeks in organotypic
66
hippocampal slice cultures. Raineteau et al. (2004) have shown that although more than
80% of proliferating cells labeled with BrdU at 14 DIV are GFAP-positive, a small
proportion of proliferating cells can differentiate into neurons, and the rate of the
neuronal differentiation is enhanced by the serum-free condition and EGF. Similarly,
Kamada et al. (2004) have indicated that among proliferative cells labeled with
retrovirus-EGFP at 14 DIV, one-quarter of the EGFP positive cells expressed NeuN and
Tuj1 2 weeks after infection. Poulsen et al. (2005) have reported that dividing cells
labeled with BrdU at 12 – 16 DIV did not give rise to TUC-4 positive neuronal cells.
Laskowski et al. (2005) revealed that bFGF and EGF stimulate the proliferation of cells
labeled at 7 – 9 DIV, but not neurogenesis. Taken together, these findings suggest that
although neuronal production occurs under certain culture conditions in late in vitro
labeling cultures, the rate of neuronal production is relatively low. Late in vitro labeling
cultures could be useful for examining a small number of newly generated cells to
differentiate into neurons.
In vivo and early in vitro labeling culture
Our previous report showed that proliferative precursor cells at P5 are mainly in the
hilus and express astrocytic markers (Namba et al., 2005). During their developmental
period, they migrate to the granule cell layer and become granule neurons. In the in vivo
and early in vitro slice culture experiments, most of the proliferating cells labeled in
vivo at P5 or early in vitro were found at 14 DIV in the granule cell layer and the
efficiency of neuronal production was much higher than in the late in vitro labeling
cultures. Furthermore, retrovirus-EGFP labeling and time-lapse imaging indicate that
the hilar neural precursor cells migrated to the granule cell layer and finally
67
differentiated into normal granule cells. Therefore, the in vivo-like capacity of neural
precursor cells for neuronal production could persist in hippocampal slices in the early
period of culture, and thereafter, during the culture period, the capacity for migration
and differentiation could also be maintained under culture conditions. On the other hand,
during the culture period, the capacity of neural precursors for neuronal production
would be reduced. It should also be noted that, generally, in experiments using
embryonic neocortical slices, cell proliferation, differentiation and migration are
examined during the early culture period (Miyata et al., 2001; Noctor et al., 2001).
Collectively, these results show that in vivo and early in vitro-labeling cultures are
useful for studying the developmental dynamics of the hippocampus.
Application and limits of Hippocampal slice culture for postnatal and adult
neurogenesis model
As in hippocampal slice cultures, hippocampal slices are taken from early postnatal rats,
the development of newly generated cells in slice cultures represents postnatal
neurogenesis. However, the results of hippocampal slice cultures could be able to
provide some information on the developmental mechanism of adult neurogenesis,
because there are some similarities between early postnatal and adult neurogenesis. In
the adult, it has been demonstrated that the proliferation of neural precursors occurs in
the subgranular zone (Altman & Das, 1965; Seki, 2002), and the precursor cells
expressing GFAP give rise to neurons (Seri et al., 2001). Similarly, in the postnatal
hippocampus, proliferating precursors are found in the subgranular zone, although they
are predominantly present in the hilus (Altman & Bayer, 1990a; Namba et al., 2005),
and the precursor cells positive for astrocytic markers produce neurons (Namba et al.,
68
2005). Thus it is probable that adult-like neuronal differentiation from astrocytic
precursors can be examined in the hilus and subgranular zone of the postnatal
hippocampus, and that adult-like neuronal migration and neurite formation can be
observed in the subgranular zone. Future studies are required to define the extent to
which neurogenesis in the hippocampal organotypic slice culture represents adult
neurogenesis.
Additionally, it should also be noted that new cells raised in vitro exhibited a
delay in neuronal maturation. The underlying reason for the delay in immature neuron
maturation remains obscure. Maturation of the newly generated neurons may require
some growth factors or input from extrahippocampal regions such as the entorhinal
cortex and septum. However, the retrovirus labeling indicated that the newly generated
neurons extended axons with large boutons exhibiting the typical features of mossy
fibers, and dendrites with spines. This suggests that the functional incorporation of
newly generated neurons to the hippocampal network. In this respect, Raineteau et al.
(2006) have demonstrated electrophysiologically that new granule cells arising in
organotypic hippocampal slice cultures mature and integrate normally into the
hippocampal circuitry (Raineteau et al., 2006). Therefore, the early in vitro labeling
cultures used in the present study could provide a useful ex vivo model to use in the
search for the mechanism of granule cell maturation.
Figure 18
Figure Legends
Fig. 18. A Model of postnatal neurogenesis in the dentate gyrus. The hilus contains two
types of neural progenitors: the majority are non-radial astrocyte-like cells (nA) and the
minority are radial astrocyte-like cells (rA). They can proliferate and produce three
types of cells: granule neurons (gN), star-shaped astrocyte (sA) and radial glia-like cells
(rG). In the neurogenic course, proliferative astrocyte-like cells (nA or rA) differentiate
into immature neurons (iN) via proliferative neuroblasts (pNb) which express neuronal
marker and/or transient intermediate cells (iC) which express both neuronal and
astrocytic markers. Then they move to GCL extending their trailing process, future axon,
and finally become granule cells. In the other course, proliferative astrocyte-like cells
(nA or rA) differentiate into star-shaped astrocyte (sA) and radial glia-like cells (rG).
The latter cells move to SGZ and become presumptive neural progenitor cells which
give rise to neurons in the late postnatal and adult dentate gyrus (dashed arrow).
69
70
6:Acknowledgements:
This study was done under the strong leadership of Dr. Tatsunori Seki (Juntendo
University). I really appreciate his instruction. I also express deep gratitude to Dr. Hideo
Namiki (Waseda University) for his lenient treatments.
I very thank Drs Hideki Mochizuki (Juntendo University) for retrovirus vector, Seiji
Shioda and Ryusuke Suzuki (Showa University) for mGFAPp-EGFP transgenic mouse,
Hirotaka J. Okano (Keio University) and Robert B. Darnell (The Rockefeller
University) for anti-Hu antibody, Nobuaki Tamamaki (Kumamoto University) for the
anti-GFP antibody and Kazunori Toida (The University of Tokushima Graduate School)
for useful information about the MASH1 antibody. I appreciate the review of the
manuscript prior to submission by Pacific Edit.
This study was supported by a Grant-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (17500238) and in part by a High Technology
Research Center Grant from the Japanese Ministry of Education, Culture, Sports and
Science.
7:Abbreviations
BrdU: 5-bromo-2-deoxyuridine
BSA: Bovine serum albumin
GCL: granule cell layer
GFAP: glial fibrillary acidic protein
GFP: green fluorescent protein
GLAST: astrocyte-specific glutamate transporter
MASH-1: Mammalian achaete-schute Homolog-1
71
P: postnatal day
PB: phosphate buffer
PBS: phosphate-buffed saline
SGZ: subgranular zone
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9:Achievements
9-1:Original papers
○Namba T, Mochizuki H, Onodera M, Namiki H, Seki T. Postnatal neurogenesis in
hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to
efficient neuronal production. J Neurosci Res. In press.
Seki T, Namba T, Mochizuki H, Onodera M. Clustering, migration and neurite
formation of neural precursor cells in the adult rat hippocampus. J Comp Neurol. In
press.
Liu J, Suzuki T, Seki T, Namba T, Tanimura A, Arai H. Effects of repeated
phencyclidine administration on adult hippocampal neurogenesis in the rat. Synapse.
60(1):56-68, 2006.
○Namba T, Mochizuki H, Onodera M, Mizuno Y, Namiki H, Seki T. The fate of neural
progenitor cells expressing astrocytic and radial glial markers in the postnatal rat
dentate gyrus. Eur J Neurosci. 22(8):1928-41, 2005.
Tozuka Y, Fukuda S, Namba T, Seki T, Hisatsune T. GABAergic excitation promotes
neuronal differentiation in adult hippocampal progenitor cells. Neuron. 47(6):803-15,
2005.
Yamaguchi M, Suzuki T, Seki T, Namba T, Liu J, Arai H, Hori T, Shiga T. Decreased
cell proliferation in the dentate gyrus of rats after repeated administration of
cocaine. Synapse. 58(2):63-71, 2005.
Yamaguchi M, Suzuki T, Seki T, Namba T, Juan R, Arai H, Hori T, Asada T. Repetitive
cocaine administration decreases neurogenesis in adult rat hippocampus. Ann N Y
Acad Sci. 1025:351-62, 2004.
82
9-2: Oral presentations
難波隆志、並木秀男、石龍徳. 生後海馬で起こる神経細胞新生のTime-lapse
imaging法を用いた解析. 平成 18 年度神経発生討論会, 岡崎, (2006 年 12 月)
Seki T, Namba T, Mochizuki H. Clustering, migration and neurite formation of neural
precursor cells in the adult rat hippocampus. Soc Neurosci, 517.10, Atlanta, USA,
(Oct. 2006).
Namba T, Namiki H, Seki T. New migration pattern in the postnatal neurogenesis of
the dentate gyrus. NEUROSCIENCE RESEARCH 55: S242-S242 Suppl. 1, (2006)
難波隆志、並木秀男、石龍徳. Developmental process of granule cells in the
hippocampus. 神経組織の成長・再生・移植研究会 第 21 回学術集会, P12, 東京,
(2006 年 5 月).
石龍徳、難波隆志. 成体海馬のニューロン新生:ニューロブラストの移動と突起形成.
第 111 回日本解剖学会総会, P15-08, 神奈川, (2006 年 3 月).
難波隆志、並木秀男、石龍徳. 海馬切片培養法を用いたニューロン新生の解析. 第
111 回日本解剖学会総会, P15-08, 神奈川, (2006 年 3 月).
Namba T, Namiki H, Seki T. Neurogenesis in hippocampal slice cultures. 第28回日本
神経科学大会, P1-173, 横浜, (2005 年 7 月).
Tozuka Y, Fukuda S, Seki T, Namba T, Hisatsune T. Neurogenesis in hippocampal
slice cultures. 第 28 回日本神経科学大会, P1-174, 横浜, (2005 年 7 月).
Namba T, Namiki H, Seki T. Nature, migration and fate of hilar proliferating cells in
the postnatal rat dentate gyrus. Soc Neurosci, 31.20, SanDiego, USA, (Nov. 2004).
Namba T, Namiki H, Seki T. Nature, cell-cell interaction and fate of hilar proliferating
cells in the postnatal rat dentate gyrus. 生理研カンファレンス・未来開拓シンポジウ
83
ム“Adult neurogenesis in normal and pathological conditions”, P1, 岡崎, (2004 年 11
月).
Namba T, Namiki H, Seki T. Nature, migration and fate of hilar proliferating cells in
the postnatal rat dentate gyrus. 第 27 回日本神経科学大会, 31.20, 大阪, (2004 年 9
月).
Namba T, Namiki H, Seki T. Development of newly generated cells into granule cells
in the postnatal rat hippocampus. 第 26 回日本神経科学大会, S124, 名古屋, (2003
年 7 月).
難波隆志、並木秀男、石龍徳. 生後の海馬歯状回でおこるニューロン新生の解析.
第 55 回日本動物学会関東支部大会, P48, 東京, (2003 年 3 月).
難波隆志、石龍徳. 海馬切片培養における神経細胞新生の解析. 第 54 回日本動物
学会関東支部大会, P20, 東京, (2002 年 3 月).
84