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

1

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

2

3

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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

10

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

19

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