International Journal of Neuroscience

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Proliferation, Migration, and Neuronal

Differentiation of the Endogenous Neural
Progenitors in Hippocampus after Fimbria Fornix
Transection

Linqing Zou, Guohua Jin, Xinhua Zhang, Jianbing Qin, Huixia Zhu, Meiling
Tian & Xuefeng Tan

To cite this article: Linqing Zou, Guohua Jin, Xinhua Zhang, Jianbing Qin, Huixia Zhu,
Meiling Tian & Xuefeng Tan (2010) Proliferation, Migration, and Neuronal Differentiation
of the Endogenous Neural Progenitors in Hippocampus after Fimbria Fornix Transection,
International Journal of Neuroscience, 120:3, 192-200, DOI: 10.3109/00207450903464579

To link to this article: https://doi.org/10.3109/00207450903464579

Published online: 08 Apr 2010.

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International Journal of Neuroscience, 120, 192–200, 2010
Copyright
C 2010 Informa Healthcare USA, Inc.
ISSN: 0020-7454 print / 1543-5245 online
DOI: 10.3109/00207450903464579

Proliferation, Migration, and Neuronal Differentiation

of the Endogenous Neural Progenitors in Hippocampus
after Fimbria Fornix Transection
Linqing Zou,1 Guohua Jin,1 Xinhua Zhang,1 Jianbing Qin,1
Huixia Zhu,2 Meiling Tian,1 and Xuefeng Tan1
1
Department of Anatomy and Neurobiology, Jiangsu Key Laboratory of Neuroregeneration,
Nantong University, Nantong, China
2
Department of Biochemistry, Nantong University, Nantong, China

A B STRACT
Neurogenesis in the hippocampus continues throughout adult life and can be regulated by the local microenvi-
ronment. To determine whether denervation stimulates neurogenesis in hippocampus, proliferation, migration,
and differentiation of local neural stem cells (NSCs) in dentate gyrus was investigated after fimbria fornix transec-
tion. In the denervated hippocampus, NSCs proliferated markedly and migrated along the subgranular layer, and
more newborn cells differentiated into neurons or astrocytes. After denervation, more newborn cells in the deaf-
ferented hippocampus expressed Brn-4 and differentiated into β-Tubulin III positive neurons. It is concluded that
the local NSCs in hippocampus may proliferate and migrate into granule cell layer, in which changes in the deaf-
ferented hippocampus provided a suitable microenvironment for hippocampal neurogenesis and the increased
Brn-4 in denervated hippocampus may be involved in this process.
KEYWORDS: Brn-4, dentate gyrus, fimbria fornix, hippocampus, neural stem cell, neurogenesis

INTRODUCTION 2005). NSCs in SVZ were found proliferating and

Properties of neural stem cells (NSCs), such as self-
renewal, multipotential differentiation, and prolifer-
ation have fuelled expectations for the clinical ex-
ploitation of NSCs in restorative therapies for acute
brain trauma, brain ischemia, and neurodegenera-
tive disease (Grote & Hannan, 2007; Wu et al.,
2008; Yamasaki et al., 2007). Subventricular zone
(SVZ) and subgranular zone (SGZ) in dentate gyrus
(DG) are proven to be areas of neurogenesis in
the adult mammalian brain (Jin & Galvan, 2007;
Munoz, Stoutenger, Robinson, Spees, & Prockop,
Received 18 May 2009.
This work was supported by a grant from National Natural Science
Foundation of China (NO. 30670648), Nature Foundation of Jiangsu (NO.
BK2006057) and College Nature Foundation of Jiangsu (NO. 04KJB180111),
People’s Republic of China.
Corresponding author: Guohua Jin, Department of Anatomy and
Neurobiology, Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu,
China. E-mail: jguohua@ntu.edu.cn
migrating along the rostral migratory stream (RMS)
into the olfactory bulb where they differentiate into
several classes of interneurons (Bovetti, Bovolin, Per-
roteau, & Puche, 2007). Meanwhile NSCs in SGZ
developed into granular cells and anchored at the
granular layer, and even established synaptic connec-
tion with the local nervous cells (McDonald & Woj-
towicz, 2005). In response to hippocampal ischemic
injury, the local progenitors proliferated and differ-
entiated into mature neurons and glia cells, and ap-
peared to integrate into the granule cell layer (GCL)
(Leker, 2006; Miles & Kernie, 2008). In our early
studies (Zhang, Jin, Tian, Qin, & Huang, 2007),
NSCs grafted into adult deafferented hippocampus
well survived, migrated, and differentiated into neu-
rons. These results suggest that differentiation of
NSCs partly responds to environmental cues in the
host.
Brn-4, POU domain transcription factor brain 4,
not only is related with the characteristic of inner
ear normalities, but also participates in the regulation

192

of neural development. Recent researches demon-
strated the role of Brn-4 in neuronal differentiation,
regeneration, and restoration of striatum after injury
(Shimazaki, Arsenijevic, Ryan, Rosenfeld, & Weiss,
1999). Recently we have observed that the expression
of Brn-4 mRNA and protein increased markedly after
fimbria fornix (FF) transection (Zhang et al., 2009).
This study first determined the neurogenesis of
hippocampus subjected with mechanical deaffer-
ented injury by transecting FF. It is showed that there
was a marked increase in neurogenesis in the DG
of hippocampus after FF transection. The majority
of newborn cells migrated along the SGZ into the
GCL gradually. Furthermore, Brn-4 expressed in-
creasingly preceding the process of neuronal differ-
entiation. The results indicated that the local NSCs
in DG may give rise to neurogenesis in response to FF
transection and the expression of Brn-4 in hippocam-
pus may be related with neuronal differentiation of
NSCs.
MATERIALS AND METHODS
Animals and operation
Adult female Sprague-Dawley rats weighing 200–220
g from experimental animal center of Nantong Uni-
versity were kept in an environment with a controlled
temperature (23 ± 2◦ C) and 12-hr light/12-hr dark
cycle and caged in an approved facility with free
access to food and water ad libitum. All animal
experiments were conducted in accordance with the
United States National Institutes of Health Guide
for the care and use of laboratory animals.
Transection of the FF was performed stereo-
taxically with the aid of a wire-knife as described
previously (Hefti, 1986). After anesthetization, an
aperture in the skull were drilled between AP = 1.4,
L = 1.0 and AP = 1.4, L = 4.0 according to atlas
of Paxinos and Watson (Paxinos & Watson, 1986).
A wire-knife was lowered to a depth 5.4 mm ventral
to the dura and shifted laterally in the aperture for
three times. Wire-knife was slowly withdrawn from
the brain.
Brdu labeling
To label the proliferating progenitors in hippocam-
pus, 5-Bromo-2-deoxy-uridine (BrdU, Roche,
Mannheim, Germay) was injected intraperitoneally
(single dose 50 mg/kg) by two injection programs.
One is that animals were injected at 8-hr intervals for
three times on day 3, 5, 7, 14, 21, and 28 after FF le-
sion and 8 hr after the last injection of BrdU animals
were sacrificed to assess maximal neurogenesis. The


C 2010 Informa Healthcare USA, Inc.
Neurogenesis in Denervated Hippocampus 193
other is that BrdU was administrated from day 2 after
FF transcetion once a day for 5 consecutive days and
animals were sacrificed to investigate migration and
differentiation of the newborn cells.
Immunofluorescence assays
Coronal sections of 20-µm thickness through
hippocampus (−2.8 to −4.4 mm from bregma)
were prepared by using a cryostat (Leica CM1900,
Germany). To detect BrdU, sections were pretreated
in 2-N HCl at 37◦ C for 20 min and neutralized
with sodium borate buffer (0.1 M, pH 8.5) at
room temperature for 10 min. After washing with
0.01-M PBS, sections were incubated with mouse
monoclonal anti-BrdU (Sigma, St. Louis, MO),
rat monoclonal anti-BrdU (Abcam, UK), mouse
monoclonal anti-Nestin (Millipore, USA), rabbit
polyclonal anti-NF-200, anti-GFAP or chicken
anti-β-Tubulin III (Chemicon, USA) antibodies
and then with Alex Fluor 568-conjugated goat
antimouse, rat or chicken IgG (Molecular Probes,
USA), FITC-conjugated goat antirabbit or mouse
IgG (Chemicon, USA) or aminomethylcoumarin
(ACMA)-conjugated goat antimouse IgG (Chemi-
con, USA). Immunofluorescence signals were
visualized at excitation/emission wavelengths of
578/603 nm (Alex Fluor 568), 495/520 nm (FITC),
and 450/350 nm (ACMA) respectively.
Image processing and statistics analysis
All counts were performed using a 200× objective
lens on a fluorescence microscope (Leica DMR, Ger-
many). The number of positive cells at each time
point or group was analyzed using the Kruskal–Wallis
ANOVA on ranks and SNK test, followed by posthoc
tests using Dunn’s method (SigmaStat; Jandel Scien-
tific, San Rafael, CA). Differences were considered
significant when p < .05.
RESULTS
Nissl staining
At different study points after FF transection Nissl
staining (0.1% cresyl violet) showed that the right FF
was still disconnected and the left was not affected at
all (Figure 1), indicating model of FF transection was
successful and the following studies were reliable.
Proliferation in the DG after FF Transection
To determine the neurogenesis in denervated hip-
pocampus, BrdU was injected intraperitoneally as the
first injection program described. In DG of normal
194 L. Zou et al.
FIGURE 1 Morphological image stained by cresyl violet 14 days af-
ter unilateral FF transection. As circled, the right hippocampal FF
(transected) is still disconnected completely, while the left (normal)
was intact.
side there were only a few BrdU positive cells and no
distinctive difference in the number of BrdU positive
cells between the time points, demonstrating that uni-
lateral FF lesion did not promote contralateral pro-
liferation in DG (Figure 2a). However, in the den-
ervated hippocampus, BrdU positive cells increased
gradually from 3 to 7 days after FF transection (Fig-
ure 2b). Cell proliferation at seven days was about six-
folds over the normal side and reached the peak. The
number of BrdU positive cells decreased gradually at
days 14 and 21, and was still greater than normal side.
While at day 28, the number of BrdU positive cells
returned to the level of normal side (Figures 2b and
c). These results indicated that the FF lesion indeed
pronouncedly triggered the local cell proliferation in
hippocampus and the high stage of neurogenesis was
at about seven days after FF lesion.
Response of neural progenitors
in hippocampus
rdU labeling assay have revealed the proliferation
in hippocampus, but it is still not known whether
neural progenitors in hippocampus were activated by
denervation. Nestin immunohistochemistry was used
to determine the neural progenitors in hippocampus.
Nestin expression in deafferented hippocampus
transiently increased and the Nestin positive cells
mostly located in the hillus of DG. At the first day
after FF lesion about 81.1 ± 14.6% of BrdU positive
cells were positive to Nestin while at the 2nd day the
ratio decreased to 40.6 ± 10.1%. At the 3rd day, not
only the number of Nestin positive cells continuously
reduced but also the immunoreactive intensity weak-
ened obviously. Till the 7th day, the Nestin positive
cells almost were not found in the hippocampus (Fig-
ures 3a and c). At the first and second day, there were
also some Nestin immunoreactive cells which did not
integrate BrdU (Figure 3a). The reasons may be that
BrdU did not reach these cells in limited time or that
these cells were activated but did not enter into pro-
liferation. Compared the number of Nestin positive
cells, at the first day after FF lesion, the number of
Nestin positive cells in the deafferented hippocampus
were about seven-folds over the normal side (Figure
3b). These results indicated that the neural pro-
genitors in hippocampus were activated in the early
stage by the denervation lesion and then the neural
progenitor marker Nestin began to decrease and even
disappear.
Migration and differentiation of local NSCs
To determine the migration and differentiation of the
newborn cells in hippocampus, BrdU were adminis-
trated as the second injection program described. As
shown in Figure 4(b), the BrdU-labeled cells mostly
distributed in the hilus of DG at day three after FF
transection. At day five, there was a slight increase in
the number of BrdU-labeled cells, and some of them
migrated into the SGZ. At day seven, the number of
BrdU positive cells increased markedly and majority
of them were located in the SGZ or the edge of the
GCL as a strip. At day 14, many of the cells were still
in the SGZ, but some of them were found throughout
the GCL. At day 28, more newborn cells were found
in the GCL and maintained till day 42. However, in
the DG of normal side only a few BrdU-labeled cells
were found in the hilus of DG and the SGZ, and the
number and location was not significantly different at
various time points (Figure 4a).
GFAP and NF-200 immunohistochemistry
demonstrated that the newborn cells can differenti-
ate into astrocytes and neurons (Figures 5a and b).
International Journal of Neuroscience
 Neurogenesis in Denervated Hippocampus 195

FIGURE 2 FF transection increases cell proliferation in the dentate gyrus (DG). BrdU was
administrated on day 3, 5, 7, 14, 21, and 28 after right FF transection and 24 hr later animals
are killed for BrdU immunofluorescence. (a) BrdU-labeled cells (showed by arrows) in DG
of normal sides at series of time points. (b) BrdU-labeled cells (showed by arrows) in DG
of transection sides at series of time points after FF transection. Scale bar, 50 µm. (c) Sta-
tistically analysis on the number of BrdU positive cells in DG at different time points. The
number of BrdU positive cells in transection sides is significantly more than that in normal
sides at almost all the studied pointed, except for day 28. ∗ p < .05 and ∗∗ p < .01 compared
with the normal sides at respective time points by using variance analysis (ANOVA) (n = 6).

In the deafferented hippocampus, there were some population in the DG, about 7% were NF-200
BrdU/NF-200 positive cells at day 7 and the number positive neurons, and about 70% were GFAP pos-
increased gradually and reached the peak at day itive astrocytes at day 28. In the normal DG there
28 (Figures 5a and c). Among the BrdU-labeled were only several BrdU/GFAP positive cells, but

C 2010 Informa Healthcare USA, Inc.
196 L. Zou et al.

FIGURE 3 Expression of Nestin in hippocampus after FF transection. Animals were injected with BrdU imme-
diately after right FF transection and 1, 2, 3, and 7 days later sections through hippocampus were subjected to
Nestin and BrdU immunofluorescence. (a) Nestin (green) or BrdU (red) positive cells in the hillus of DG at day
1, 2, 3, and 7 after FF lesion. Left, the deafferented side; Right, the normal side. Arrow indicates the Nestin and
BrdU double positive cells; Arrow head indicates the Nestin positive cells. Scale bar, 20 µm. (b) Quantitative
analysis of the Nestin positive neural progenitors in the hillus of DG at the first day after FF lesion in (a). Data
are mean ± SEM, ∗∗p < .01 compared with normal. (c) Quantitative analysis of the Nestin and BrdU double
positive cells in the hillus of DG of the deafferented side in (a). Data are mean ± SEM, ∗ p < .05 and ∗∗ p < .01
compared with day 1.

no BrdU/NF-200 double-labeled neurons at any Relationship between Brn-4 expression

time points. These evidences together suggested that and neuronal differentiation
the microenvironment in denervated hippocampus
Our previous study has showed that FF transec-
benefits the migration and neuronal differentiation
tion increased significantly Brn-4 in deafferented
of local neural progenitors.
hippocampus and Brn-4 was involved in neuronal

International Journal of Neuroscience

 Neurogenesis in Denervated Hippocampus 197

FIGURE 4 Distribution of BrdU positive cells in DG after FF transection. From the day
2 after FF transection BrdU was given once daily for 5 consecutive days, and the animals
were then killed for BrdU immunofluorescence at day 3, day 5, day 7, day 14, day 28, and
day 42 after FF operation. (a) BrdU positive cells (showed by arrows) in DG of normal
sides. There is no significant difference in the location of the newly divided cells between
the different times. Scale bar, 50 µm. (b) BrdU positive cells (showed by arrows) in DG of
the transection sides, the newly divided cells migrated along the subgranular zone (SGZ)
with time elapse, and some cells entering into granular cell layer (GCL). Scale bar, 50 µm.

differentiation of hippocampal NSCs (Zhang et al., DISCUSSION

2009). In this study we determined the in vivo rela-
tionship between Brn-4 and neuronal differentiation
in DG. Immunofluorescence showed that in hilus
and SGZ of DG with hippocampus denervated some
proliferated cells labeled with BrdU were positive to
Brn-4 (Figure 6a) and the number of BrdU/Brn-4
double positive cells increased significantly at day
3 after transection and reached peak at day 14,
and then kept relatively higher level (Figure 6b).
At day 14, BrdU/Brn-4 double positive cells partly
differentiated into β-Tubulin III positive neurons,
the number of which increased and reached the peak
at day 28 (Figure 6c). These data suggested that FF
transection stimulated Brn-4 expression in DG and
subsequently the neuronal differentiation of newborn
cells, indicating the possible relationship between
Brn-4 and neuronal differentiation of hippocampal
neural progenitors.
Activation of endogenous neural progenitors is con-
sidered as an ideal strategy for neurodegenera-
tion and brain injury. However, how to induce
NSCs differentiating into region-specific neurons
is still unknown. The published reports indicated
that the local microenvironmental cues may di-
rect the migration and differentiation of NSCs
(Johe, Hazel, Muller, Dugich-Djordjevic, & McKay,
1996). Lesion-induced alternation of microenviron-
ment arouses the proliferation, migration, differ-
entiation, and reconfiguration of local progenitors.
However, Cooper-kuhn (Cooper-Kuhn, Winkler, &
Kuhn, 2004) demonstrated that neurogenesis de-
creased after cholinergic forebrain lesion induced by
immunotoxin 192IgG-saporin infuse. The toxicity of
192IgG-saporin on the endogenous NSCs may be re-
sponsible for the decreased neurogenesis. So in this

C 2010 Informa Healthcare USA, Inc.
198 L. Zou et al.

FIGURE 5 Newborn BrdU positive cells can be induced to differentiate into NF-200 posi-
tive neurons in deafferented hippocampus. (a) Microscope images of sections through deaf-
ferented hippocampus stained by BrdU and NF-2 immunofluorescence on day 7, 14, 28, and
42 after transection. Some newborn cells labeled with BrdU (red) differentiate into NF-200
positive (green) neurons. Colocalization of BrdU with NF-200 is shown in cells with green
cytoplasm surrounding red nuclei (arrows). Higher magnification view of the rectangular part
shows BrdU/NF-200 double-labeled neurons, respectively. Scale bar, 50 µm. (b) Microscope
image of BrdU positive (red) and GFAP positive (green) astrocytes in right hippocampus 28
days after transection. Arrow showed the double-labeled positive astrocytes. Higher magnifica-
tion view of the rectangular part in the corresponding image shows BrdU/GFAP double-labeled
astrocytes. Scale bar, 50 µm. (c) Quantitative analysis of BrdU/NF-200 double labeled cells
in DG of the normal and deafferented (in a) hippocampus at different time points. (Data are
mean ± SEM, ∗ p < .05, ∗∗ p < .01).

International Journal of Neuroscience

 Neurogenesis in Denervated Hippocampus 199

FIGURE 6 Brn-4 expression increases rapidly and precedes neuronal differentiation in vivo. (a) Indirect triple
immunofluorescence micrographs of anti-BrdU (blue), anti-Brn-4 (green), and anti-β-tubulin III (red) in coro-
nal sections of the hippocampus at day 7, 14, 21, 28, and 35 after operation. Higher magnification view of the
rectangular part shows the triple-labeled neurons, respectively. Arrows show the cells that were immunore-
active for BrdU, Brn-4 and β-tubulin III. Scale bar, 50 µm. (b) Quantitative analysis of BrdU/Brn-4 double
labeled cells. The number of BrdU/Brn-4 double positive cells increases significantly at day 3, reachs peak at
day 14 and then keep higher level. (c) BrdU/Brn-4/β-tubulin III triple-labeled cells in hippocampus after FF
transection. The number of triple-labeled neurons increases significantly with time lapse. Data are mean ±
SEM. ∗ p < .05 and ∗∗ p < .01 compared with normal side.

C 2010 Informa Healthcare USA, Inc.
200 L. Zou et al.
study the FF was singly transected to generate a den-
ervated hippocampus model to well determine the
neurogenesis in hippocampus. Depletion of cholin-
ergic innervation by transecting FF, which carries
cholinergic fibers from septal area to hippocampus,
elevates the APP level and Aβ accumulation and
deposition which are associated with the pathology
of Alzheimer’s disease (AD) (Leanza, 1998). As the
published reports (Liu et al., 2002) described, the
hemisphere FF-damaged animals were used to model
AD. In such a deafferented microenvironment, the lo-
cal proliferation in DG was activated and the new-
born cells migrated into the SGZ and differentiated
into neurons. The neural progenitors were activated
quickly in the early stage and soon entered into dif-
ferentiation and lost their embryonic marker. It is in-
dicated that the environment changes in the deaffer-
ented hippocampus provided an optimal soil for the
proliferation, migration, and neuronal differentiation
of the local progenitors, which may be due to the up-
regulation of some factors related with neuronal de-
velopment after FF transection.
Studies have shown that Brn-4 was not only im-
plicated in neuronal differentiation of the developing
striatum, but also correlated with neural regenera-
tion and restoration of adult mammal striatum after
the dopaminergic denervation (Le Moine & Young,
1992; Shimazaki et al., 1999). Our previous studies
(Zhang et al., 2009) have shown that Brn-4 mRNA
and protein expression levels in hippocampus in-
creased significantly after FF transection. Moreover,
the number of Brn-4-expressing cells in the hilus and
SGZ of the deafferented hippocampus were increased
markedly. In this study, some BrdU-labeled cells dif-
ferentiated into neurons and more of them in the
deafferented hippocampus expressed Brn-4 protein
than that in the normal side. During time course Brn-
4 expression was preceding the neuronal differentia-
tion, which appeared to imply the good correlation
between neuronal differentiation of the local NSCs
and Brn-4 expression. So it is concluded that the local
NSCs in DG are activated after denervation lesion of
hippocampus and proliferate and migrate into GCL,
in which changes provided a suitable microenviron-
ment for hippocampal neurogenesis and it is implied
that the increased Brn-4 in denervated hippocampus
may be involved in neurogenesis process.
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International Journal of Neuroscience