1
ADULT NEUROGENESIS IN THE FOUR-STRIPED
MOUSE (RHABDOMYS PUMILIO) AND COMMON
MOLE RAT (CRYPTOMYS HOTTENTOTUS)
By:
Olatunbosun Oriyomi Olaleye (BSc. Hons)
A dissertation submitted to Faculty of Science, University of the
Witwatersrand, in fulfillment of the requirements for the degree
of Master of Science.
Supervisor(s): Dr Amadi Ogonda Ihunwo
Co- Supervisor: Professor Paul Manger
Johannesburg, 2010
2
Contents Page
DECLARATION v
ABSTRACT vi
ACKNOWLEDGEMENTS vii
DEDICATION viii
LIST OF FIGURES ix
LIST OF TABLES xi
ABREVIATIONS xii
CHAPTER 1- INTRODUCTION 1
1.1 Introduction 1
1.2 Objectives of the study 2
1.3 Literature review 3
1.2.1. Active neurogenic sites in the brain 6
1.2.2. Other neurogenic sites with neurogenic potential 8
1.2.3. Non-neurogenic regions with neurogenic potential 9
CHAPTER 2- MATERIALS AND METHODS 11
2.1 Experimental animals 11
2.1.1 Four-striped mouse (Rhabdomys pumilio) 11
2.1.2 Common mole rat (Cryptomys hottentotus) 13
2.2 Experimental groups 16
2.3 Markers of proliferation 17
2.3.1 Bromodeoxyuridine (BrdU) administration 17
3
2.3.2 Ki-67 18
2.3.3 Doublecortin (DCX) 19
2.4 Tissue processing 19
2.5 Bromodeoxyuridine immunohistochemistry 20
2.5.1 Pre- incubation 20
2.5.2 Primary antibody incubation 20
2.5.3 Secondary antibody incubation 21
2.5.4 Avidin-biotin-complex method 21
2.5.5 3, 3?-diaminobenzidine tetrahydochloride (DAB) staining 21
2.6 Ki-67 immunohistochemical staining 22
2.7 Doublecortin (DCX) immunohistochemical staining 23
2.8 Data analysis 24
CHAPTER 3- RESULTS 25
3.1 General observations 25
3.2 Immunohistochemical findings in the four-striped mouse 27
3.2.1 BrdU positive cells in the proliferating and survival groups 27
3.2.2 Ki-67 positive cells 32
3.2.3 Doublecortin (DCX) positive cells 41
3.3 Immunohistochemical findings in the common mole rat 49
3.3.1 BrdU positive cells in proliferative and survival groups 49
3.3.2 Ki-67 positive cells 54
3.3.3 Doublecortin (DCX) positive cells 63
4
Chapter 4 Discussion 76
4.1. Adult neurogenesis in the established active neurogenic sites 76
4.2. Adult neurogenesis in the potential neurogenic sites 79
Chapter 5 Conclusion and Further Studies 83
5.1. Conclusion 83
5.2. Further studies 84
Conference Presentations 86
REFERENCES 87
5
DECLARATION
I declared that this dissertation is my own unaided work. It is being submitted for
the Degree of Master of Science in the University of the Witwatersrand,
Johannesburg. It has not been submitted before for any degree or examination in
any other University.
Signature of candidate:
Date:
6
ABSTRACT
Adult neurogenesis was investigated in captive bred four-striped mouse
(Rhabdomys pumilio) and wild caught common mole-rat (Cryptomys hottentotus).
Eight individuals per species were used in the study. The animals were
anaesthetized and transcardially perfused with saline followed by 4%
paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Brains were removed
and post fixed in the same fixative overnight. Following equilibration in 30%
sucrose in PB, 50 ?m frozen sections were cut in the saggital planes followed by
BrdU immunohistochemistry. Ki-67 and doublecortin (DCX) staining was
undertaken as an additional confirmatory staining for cell proliferation.
BrdU, Ki-67 and DCX immunostainings confirmed adult neurogenesis in
the subventricular zone (SVZ) of the lateral ventricle and dentate gyrus (DG) of
the hippocampus of the four-striped mouse and common mole rat. The existence
of adult neurogenesis was observed in potential sites namely, the striatum in the
four-striped mouse and substantia nigra in both species. Immature neurons were
observed in the cerebral cortex only in the common mole rat.
7
ACKNOWLEDGEMENTS
My sincere appreciation and thanks to Dr. Amadi Ogonda Ihunwo for his
supervision of this work. His support and contribution is greatly appreciated and
invaluable through my Master?s degree, contributing intellectual guidance,
invaluable moral support and for always looking for various ways to relieve me,
including helping me to find part-time job.
I also would like to offer my thanks to my co- supervisor, Professor Paul
Manger for his availability and encouragement to pursue my area of research
interest. To Dr. Virginia Meskenaite and Mrs Ali, thank you for your kindness and
support towards the completion of my laboratory work.
The support from the Faculty of Health Sciences, University of the
Witwatersrand for the Individual Faculty Research Grant in the course of this
work. Professor Neville Pillay is greatly acknowledged for providing the four-
striped mice used.
I acknowledged the contribution of the International Brain Research
Organization awarding travel grant to participate in an international workshop and
be exposed to different research facet in the field of neuroscience. My
appreciation also goes to the Switzerland South Africa Joint Research Project of
Dr. Amadi Ihunwo for travel grant to the Conference of the Society of
Neuroscientist of Africa (SONA) in Sharm El Sheikh Egypt where part of this
study was presented.
Finally, I extend my gratitude to my mother, Mrs Abosede Tosin Olaleye
and my siblings as well as Dr. Rosie McNeil and her family for the love, support
and believing in me during my study. Thank you.
8
DEDICATION
TO THE GENTLE VOICE THAT ASSURES ME.
THE THICK CLOUD THAT SHIELDS ME
MY DAYSPRING AND COMPANION IN LONLELY NIGHTS
MY WISDOM, STRENGTH AND STAFF
BLESSED JESUS,
UNTO YOU I SUBMIT THIS CROWN
AND IN LOVING MEMORY OF MY LATE FATHER, SAMUEL ABIODUN
OLALEYE
9
LISTS OF FIGURES
Figure 1: A schematic diagram of the saggital section of the rat brain showing the
different regions of the brain 6
Figure 2.1: Photograph of the four-striped mouse (Rhabdomys pumilio) 13
Figure 2.2: Photograph of the common mole rat (Cryptomys hottenttotus) 16
Figure 3.1: Representative photomicrograph showing BrdU positive cells in the
DG of the hippocampus of the four-striped mouse 28
Figure 3.2: Representative photomicrograph showing BrdU positive cells in the
wall of the SVZ of the four-striped mouse 30
Figure 3.3: Representative photomicrograph showing Ki-67 positive cells in the
DG of the hippocampus of the four-striped mouse 33
Figure 3.4: Representative photomicrograph showing Ki-67 positive cells in the
wall of the SVZ of the four-striped mouse 35
Figure 3.5: Representative photomicrograph showing Ki-67 positive cells in the
rostral migratory stream/ olfactory bulb of the four-striped mouse 37
Figure 3.6: Representative photomicrograph showing Ki-67 positive cells in the
substantia nigra of the four-striped mouse 39
Figure 3.7: Representative photomicrograph showing DCX positive cells in the
dentate gyrus of the hippocampus of the four-striped mouse 42
Figure 3.8: Representative photomicrograph showing DCX positive cells in the
wall of the subventricular zone of the four-striped mouse 44
Figure 3.9: Representative photomicrograph showing DCX positive cells in the
rostral migratory stream/ olfactory bulb of the four-striped mouse 46
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Figure 3.10: Representative photomicrograph showing BrdU positive cells in the
dentate gyrus of the hippocampus of the common mole rat 50
Figure 3.11: Representative photomicrograph showing BrdU positive cells in the
wall of the subventricular zone of the common mole rat 52
Figure 3.12: Representative photomicrograph showing Ki-67 positive cells in the
dentate gyrus of the hippocampus of the common mole rat 55
Figure 3.13: Representative photomicrograph showing Ki-67 positive cells in the
wall of the subventricular zone of the common mole rat 57
Figure 3.14: Representative photomicrograph showing Ki-67 positive cells in the
rostral migratory stream/ olfactory bulb of the common mole rat 59
Figure 3.15: Representative photomicrograph showing Ki-67 positive cells in the
substantia nigra of the common mole rat 61
Figure 3.16: Representative photomicrograph showing DCX positive cells in the
dentate gyrus of the hippocampus of the common mole rat 65
Figure 3.17: Representative photomicrograph showing DCX positive cells in the
wall of the subventricular zone of the common mole rat 67
Figure 3.18: Representative photomicrograph showing DCX positive cells in the
rostral migratory stream/ olfactory bulb of the common mole rat 69
Figure 3.19: Representative photomicrograph showing DCX positive cells in the
striatum of the common mole rat 71
Figure 3.20: Representative photomicrograph showing DCX positive cells in the
cerebral cortex of the common mole rat 73
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LIST OF TABLES
Table 1: List of some strains of rats and mice investigated to date 5
Table 2: Reported sites of neurogenesis in adult mammal brains (Ihunwo and
Pillay, 2007) 10
Table 3: Distribution of animals into groups 17
Table 4: Summary of active and potential neurogenic sites in the four-striped mouse.
48
Table 5: Summary of active and potential neurogenic sites in the common mole rat.
75
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2.9. Abbreviations
ABC- avidin biotin complex
AMG- amygdala
BrdU- bromodeoxyuridine
BSA- bovine serum albumin
DAB- 3, 3?-diaminobenzidine tetrahyrochloride
DCX- doublecortin
DG- dentate gyrus of hippocampus
H2O2- hydrogen peroxide
NGS- normal goat serum
NRbS- normal rabbit serum
OB- olfactory bulb
PB- phosphate buffer
PBS- phosphate buffer saline
RMS- rostral migratory stream
SC- spinal cord
SN- substantia nigra
STR- striatum
SVZ- subventricular zone
TB- tris buffer
TBS- tris buffer saline
TBST- tris buffer saline triton-X
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CHAPTER 1: INTRODUCTION
1.1 Introduction
One of the great achievements of the past three decades is the discovery
that the mammalian brain has the capacity to generate new neurons throughout its
lifespan (See review in Ihunwo and Pillay, 2007). Neurogenesis has been defined
as the ability of brain cells to regenerate themselves (Gage, 2000). Brain cells that
possess regenerative abilities are called neural stem cells (Okano, et al., 2002).
The concept of adult neurogenesis began as early as 1912 when scientists
discovered that mitotically active cells reside in the mammalian central nervous
system throughout life (Watts et al., 2005; Rakic, 2002). In the 1930s and 1940s,
cytological investigations revealed the presence of these cells in the postnatal and
adult rodent brain (Alvarez-Buylla and Garcia-Verdugo, 2002; Lennington, et al.,
2003). In 1965, Joseph Altman, in a similar study reported that adult neurogenesis
occurs in discrete areas like the wall of the subventricular zone and dentate gyrus
of the hippocampus of the adult brain in rodents (Watts et al., 2005). With the
advent of new methods for labelling and identifying dividing cells such as
Bromodeoxyuridine (BrdU) labelling, retroviral labelling and confocal
microscopy, studies have confirmed the findings of Joseph Altman (1965). Adult
neural stem cells are self renewing, multipotent cells that normally generate the
main phenotypic cells of the nervous system namely, neurons, astrocytes and
oligodendrocytes (Taupin, 2006a). However, neurons behave as non renewable
epithelia unlike most somatic cells that are continuously renewed or can be
regenerated (Peterson & Leblond, 1964). Adult neurogenesis has been
investigated in several mammalian brains from rodents to human. Details of these
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are presented under the literature review section. Adult neurogenesis is regarded
as an exciting area in the field of neuroscience despite the controversies in the
evidences in adult neurogenesis occurring in human and experimental animals.
This research area however, is believed to provide clues to the treatment of
various neurological disorders (Rackic, 2002; Kempermann & Kronenberg, 2003;
Duman, 2004; Feldmann et al., 2007; Vollmayr et al., 2007). It is a dynamic
process that can be modulated by various exogenous stimuli (Siwak- Tapp et al.,
2007). So the need for the study of adult neurogenesis in the captive bred four-
striped mouse and wild caught common mole-rat is important to have a broad
understanding of adult cell proliferation not only in laboratory rodent but in wild
rodents as well which are readily available. And also, exposure to more stimuli
results in different/ more pronounced neurogenesis in captive-bred and wild
caught rodents compared to laboratory rodents.
1.2. Objective of the study
The main objective of the study is to provide and describe qualitative evidence
of adult neurogenesis in the four-striped mouse (Rhabdomys pumilio) and
common mole-rat (Cryptomys hottentotus). The specific objectives of this study
are:
To establish whether adult neurogenesis occurs in the active neurogenic sites;
subventricular zone and dentate gyrus in the four-striped mouse and common
mole-rat.
To establish whether adult neurogenesis occurs in other reported potential
sites such as the striatum, substantial nigra, ependymal wall of the third
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ventricle, amygdala, cerebral cortex and olfactory bulb as this is not a
common occurrence in all species.
Describe the morphology of the proliferating cells as revealed by
bromodeoxyuridine (BrdU), Ki-67 and doublecortin (DCX)
immunohistochemistry at the different locations.
1.3. Literature review
Adult neurogenesis is a complex process involving the proliferation,
survival, differentiation and functional integration of new cells into the
hippocampus (Warne-Schmidt and Duman, 2006). Neurogenesis has been
reported in the mammalian brain of rats, mice, tree shrews, guinea pigs, rabbits,
cats, monkeys and humans (Khun et al., 1996; Kempermann et al., 1997; Gould et
al., 1997; Altman & Das, 1967; Gueneau et al., 1982; Wyss & Sripanidkulchai,
1985; Gould et al., 1998; Gould et al., 1999; Rakic & Nowakowski, 1981;
Eriksson et al., 1998). It is low or absent in bats due to spatial behaviour (Amrein
et al., 2004). Though, cellular and molecular mechanisms that regulate adult
neurogenesis remain unclear (Schauwecker, 2006), the process can be modulated
by a variety of factors including glutamate receptor activation (Cameron et al.,
1995, 1998; Gould et al., 1997; Bernabeu and Sharp, 2000), dietary restriction
(Lee et al., 2002a,b), growth factors ( Scharfman et al., 2005; Palmer et al., 1995),
stress (Brunson et al., 2005; Nichols et al., 2005) and neuronal injury (Parent,
2003; Cooper-Kuhn et al., 2004). Enriched environments (Kempermann et al.,
1997), running wheel exercise (van Praag et al., 1999), hippocampal-dependent
learning (Gould et al., 1999), and dietary restriction (Lee, Seroogy, & Mattson,
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2002), all increase neurogenesis in the adult hippocampus, while stress (Gould et
al., 1997, 1998) and social isolation (Lu et al., 2003, Lievajova et al., 2010)
reduces neurogenesis. Even though estradiol alters; and neurogenesis does not. A
decrease in hippocampal neurogenesis does not always correlate with the
development of learned helplessness in male rats (Pawluski & Galea, 2006;
Vollmayr et al., 2003).
Likewise adult neurogenesis also occurs in all non rodents mammals
studied to date (Taupin, 2006a). The concept of adult neurogenesis is now widely
accepted in the scientific community (e.g. Kaplan and Bell, 1984; Kaplan and
Hinds, 1997; Alvarez-Buylla, et al, 2002; Zhao et al., 2003; Lennington et al,
2003; Watts et al., 2005; Takemura, 2005; Luzzati et al., 2006) and Table 1 below
shows a list of some strains of rats and mice investigated to date.
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TABLE 2: List of some strains of rats and mice investigated to date.
Rodent/ Strains Site Reference
Rats
Sprague- Dawley III VEN, SN Xu et al., 2005;
Frielingdorf et al., 2004.
Wistar DG, CTX Seaberg and Kooy, 2002;
Takemura, 2005
Fisher 344 SEP/STR Palmer et al., 1995.
Pine vole SGZ, DG Amrein et al., 2004
Transgenic Tg2576 SGZ, DG Ihunwo & Schliebs, 2010
Mice
CD1 DG, SC Seaberg & Kooy, 2002;
Weiss et al, 1996;
Kemperman et al., 1997.
C57BL/ 6 SN, DG, SGZ Zhao et al, 2003;
Schauwecker, 2006;
Kemperman et al., 1997.
A/J SGZ Kempermann and Gage,
2002
BALB/ c SGZ Kemperman et al., 1997
C3H/ H3J SGZ Kemperman et al., 1997
DBA/ 2J SGZ Kemperman et al., 1997
129/ SVJ DG Kemperman et al., 1997
Albino OB Gritti et al, 2002.
FVB/NJ DG Schauwecker, 2006
Wood mouse DG Hauser et al., 2009
Key: OB, olfactory bulb; DG, Dentate gyrus; SN, Substantia nigra; SC, Spinal
cord; SEP\STR, Septum and Striatum; CTX, Cerebral Cortex; III VEN, Third
ventricle; SGZ, Subgranular zone.
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1.3.1. Active neurogenic sites in the brain
Adult neurogenesis occurs predominantly in two active sites of the brain,
the rostral subventricular zone of the lateral ventricle and the subgranular zone of
the dentate gyrus of the hippocampus (Kaplan and Bell, 1984; Kaplan and Hinds,
1997; Ihunwo and Pillay, 2007; Figure 1).
Figure 1: A schematic diagram of the saggital section of the rat brain showing the
different regions of the brain. OB-olfactory bulb, DG- dentate gyrus, Hipp-
hippocampus, SVZ-subventricular zone, SN-substantia nigra. Drawing modified
from Paxinos and Watsons, (2006) 5th ed.
The subventricular zone has two precise layers of cells: the first is a
monolayer of multiciliated cells lining the lateral ventricle called the ependymal
layer; and the second layer is a 2-3 cell layer of thick area adjacent to the
ependymal layer called the subependymal layer. The subventricular zone
neuroblast cells travel a long distance to the olfactory bulb through a network of
interconnecting pathways that become confluent at the rostral margin of the lateral
ventricular wall to form the rostral migratory stream (RMS) (Watts et al., 2005).
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The olfactory bulb is continuously being supplied with newly generated
neurons from the subventricular zone (Hind, 1968; Altman, 1969; Bayer, 1983;
Corotto et al., 1993). Cells in the subventricular zone and rostral migratory stream
move rapidly by chain migration (Doetsch et al., 1997; Jankovski et al., 1996;
Lois and Alvarez-Buylla, 1993; Kirschenbaum et al., 1994). Studies have shown
that SVZ has been a source for cortical and subcortical neurons (Watts et al.,
2005; Alvarez-Buylla and Gracia-Verdugo, 2002). The subventricular zone is
considered the largest active neurogenic site in the brain (Schauwecker, 2006).
There is persistence of neurogenic activities in the DG of the hippocampus which
generates neural precursor cells that exhibit stem cell properties (Eriksson et al.,
1998; Taupin, 2006a; Ihunwo and Pillay, 2007). The dentate gyrus is
characterized by sparse and powerful unidirectional projections to CA3 pyramidal
cells; the mossy fibre cells (Treves et al., 2008). In the rodent dentate gyrus as
many as 9000 neuronal cells are produced every day, contributing 0.01% of the
granule cell population per day. Most of these cells undergo apoptosis with only a
restricted number of cells that go on to mature. The matured cells can survive for
an extended amount of time, and this may lead to a permanent replacement of
cells (Taupin, 2006b). The dentate gyrus in the mammalian lineage is a strikingly
well conserved part of the cortex with a trilaminar structure (Stephan, 1975). The
outermost layer called the molecular layer is relatively cell free which comprises
the dendrites of the dentate principal cells (Treves et al., 2008). Also, it contains
axons that originate in a limited number of sources. Hippocampal neurogenesis
occurs over the lifetime of a mammal and it appears to maintain normal
hippocampal function of brain tissues (Eriksson et al., 1998). The four-striped
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mouse and common mole-rat were selected for this study because of their
availability.
1.3.2. Other neurogenic sites with neurogenic potential
Adult neurogenesis does not only occur in the subventricular zone and the
dentate gyrus but also in other areas of the brain (Reynolds and Weiss, 1992;
Ihunwo and Pillay, 2007). Fibroblast growth factor (FGF) was also reported to
stimulate proliferation of neuronal progenitors in the septum and striatum in
rodents (Palmer et al., 1995). The hippocampus and subventricular zone yield
more established colonies and a larger number of progenitors than the septum and
striatum. The substantia nigra pars compacta is another region where adult
neurogenesis is said to be present as evidenced by the slow turnover of
dopaminergic projection neurons in the adult rodent brain (Zhao et al., 2003).
However, Frielingdorf and colleagues (2004), found no new dopaminergic
neurons in the adult mammalian substantia nigra pars compacta.
Evidence provided by Bernier et al., (2002) indicated that neurogenesis is
present in the amygdala and surrounding cortex of adult monkeys with the
occurrence of a Temporal Migratory Stream (TMS) which is similar to RMS.
From reports of multipotent stem cells in more caudal regions of the neuroaxis
such as the spinal cord, it has become clear that stem cells are present in all parts
of the central nervous system (Temple and Alvarez, 1999). Prior to this time,
Weiss and colleagues (1996) isolated neural stem cells from the thoracic and
lumbo-sacral segments of the spinal cord of adult mice.
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1.3.3. Non- neurogenic regions with neurogenic potential
Adult neural stem cells reside in regions considered to be non-neurogenic,
for example cerebral cortex and olfactory bulb (Gritti et al., 2002; Pagano et al.,
2000). Takemura (2005) provided evidence that active neurogenesis persists
within the white matter beneath the temporal neocortex when he explored
previously overlooked neurogenic region in the adult rat brain and detected the
evidence of neuron production within the subcortical white matter. From his
results, it was suggested that cell genesis, death and migration persists in a
restricted sub-region of the adult white matter. Arsnijevic et al., (2001)
demonstrated the existence of multipotent precursor cells in the adult human
cerebral cortex and Moyse et al., (2006) confirmed neurogenesis in the dorsal
vagal complex of the brain stem (a major centre for autonomic reflexes). Ihunwo
and Pillay (2007) provided a detail review of active and potential neurogenic sites
in the adult mammalian brain (Table 2).
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TABLE 2: Reported sites of neurogenesis in adult mammal brains (Ihunwo and
Pillay, 2007).
Site SVZ DG SEP\
STR
SN III
VEN
SC AMG CTX OB\
RMS
DVC
Rodents
Rats + + + - + - + + +
Mice + + + + + + - - -
Rabbits + + + - - - -
Human + + - - - + + -
Squirrel
Monkeys
+ + - - - + - - -
Macaque
monkey
+ + - - - + + - -
Key: SVZ, subventricular zone; DG, Dentate gyrus; SEP/ STR, Septum and
Striatum; SN, Substantia nigra; III VEN, Third Ventricle; SC, Spinal Cord; AMG,
Amygdala; CTX, Cerebral cortex; OB/ RMS, Olfactory bulb/ Rostral migratory
stream, DVC, Dorsal Vagal Complex ( + indicates presence; - indicates absence).
Kempermann et al., (1997) investigated different strains of laboratory mice
and concluded that adult hippocampal neurogenesis is differentially influenced by
the genetic background of the species. Amrein et al., (2004) investigated different
strains of wild rodents and observed differences in cell proliferation. No
significant differences in the relative ratio of neurogenesis and gliogenesis were
observed in two strains of laboratory mice. With differences in both laboratory
and wild rodents, it becomes imperative to document the pattern of adult
neurogenesis in the four-striped mouse and common mole-rat which have not
been reported but readily available in the Southern African region.
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CHAPTER 2: MATERIALS AND METHOD
2.1. Experimental Animals
Eight adult specimens from each species were used. The four-striped mice
were captive reared at the Central Animal Service (CAS) unit of the University of
Witwatersrand, Johannesburg, South Africa but had wild caught ancestors. The
common mole-rat were wild caught from a golf course in Pretoria, South Africa
and transported to the CAS at the medical school of the University of
Witwatesrand. All animals used were adults based on their body weight, dentition
and sexual maturity. They were kept under standard laboratory conditions with a
14: 10 hourly light-dark cycle with lights on at 6 am for the four-striped mouse
and 12 hourly light and dark for the common mole-rat. Room temperature was
between 20-24 ?C and 30 % - 60 % relative humidity. A 40 X 12 X 25 cm (length,
height and width) lab-o-tec cages (Labotec, Halfway House, South Africa) with
saw dust or wood waste shavings as litter and hay as nesting material was used to
house this animal. The animals were treated and used according to the guidelines
of the University of the Witwatersrand Animal Ethics and Screening Committee
(AESC Clearance No: 2007/45/03).
2.1.1. Four-striped mouse (Rhabdomys pumilio)
The four-striped mouse (Rhabdomys pumilio) belongs to the, Muridae,
Rodentia. They are widely distributed in Southern Africa, occurring in different
habitats, such as grassland, marsh, forests, semi- deserts and deserts (Skinner and
Chimimba, 2005; Figure 2). The four striped mice, Rhabdomys pumilio, are small
diurnal murid rodent. They do not usually live in colonies. In fact very few
populations live as groups, and most are solitary living. They demonstrate bi-
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parental care. It is easily identified by the four distinct dark longitudinal stripes
running the length of the back (Figure 2.1). Unlike most rodents, the four-striped
mouse displays a diurnal bimodal activity pattern with its activities mainly in the
mornings and evenings (Schumann et al., 2005). It has a reduced activity in the
afternoon or midday period. It is an omnivorous animal. Its diet contains a
minimum of 15% water (Wilan and Meester, 1989). It has an extreme plasticity in
habitat preference which gives the reason for it widespread distribution
throughout Southern Africa (Skinner and Chimimba, 2005). Colour of the stripe
varies from dark brown to gray-white. The four-striped mice have a body mass
ranging from 40- 80g (Schradin and Pillay, 2004; Maini, 2003) and a small brain
with an average mass of about 0.64 g (Bhagwandin et al., 2006).
It breeds seasonally usually from spring to autumn (Schradin and Pillay,
2003). Its gestation period is 22-23 days. Their females, that are free- living, give
birth to approximately five pups: captive females have slightly larger litter 6-7
(Pillay, 2000). Their pups begin to consume solid food at ten days after birth and
leave their nest from twelfth day after gestation. Weaning starts at around the
sixteenth day after birth. Sexual maturity is reached at around fifth to sixth week
of life which depends on environmental and social factors as well as its
development status (Pillay, 2000).
Four-striped mouse has got a flexible social organization and mating
system which is controlled majorly by resource availability and population
density. In the arid habitat, they can be described as a territorial, group-living,
solitary forager that displays bi-parental care (Schradin and Pillay, 2005a). In
grasslands, females maintain intra-sexually exclusive territories, and males?
25
territories overlap with those of other males and females (Schradin and Pillay,
2005a). Both sexes maintain their territory which overlaps their opponent?s
(Schradin and Pillay, 2005a). In captivity, males from both mesic and xeric
populations display paternal care. Four-striped mouse from the southwestern
regions of southern Africa are slightly larger than the northern regions (Yom Tov,
1993).
Figure 2.1: Photograph of the four-striped mouse (Rhabdomys pumilio) by
Selvakumar. Internet accessed 12 June, 2010.
2.1.2. Common mole-rat (Cryptomys hottentotus)
The common mole-rat, Cryptomys hottentotus, (Figure 2.2) belongs to the
order of the Rodentia. Its family is Bathyergidae and genus is Cryptomys. The
common mole-rat is a burrowing rodent that is found in Africa, mainly in
26
southwest Cape Province in South Africa. Also found in other parts of Africa like
Lesotho, Malawi, Mozambique, Swaziland, Tanzania, Zambia and Zimbabwe.
They have a reduced visual function. They have an average body length of 10.5-
16.5 cm with tail length of 1.2-3.8 cm. It has a thick fur with many different
colours. Their body shape is cylindrical with short limbs. They have a chisel-like
incisor that is used for digging. They have an average body mass of 120.5 g, and a
brain mass that on average is 1.26 g (Genelly, 1965). They dwell in small colonies
(up to 14 individuals) that are comprised of a breeding female, her consorts, and
their non-breeding offspring (the workers). They have a unique characteristic of
having one reproductive pair, consisting of the largest female and male in one
group. Mating occurs between the months of September and October They breed
seasonally (October?January), the gestation period is about 81 days with 2-5
litters. Common mole-rat reaches sexuality at about 450 days. Females maintain
reproductive function during non-reproductive months.
Common mole-rats are fossorial mammals that can live in wide range of
substrates. They are herbivorous. They are wide spread and they show a sign of
localization due to soil requirements. Their pattern of borrowing optimizes their
access to food whereas it has a negative economic impact to man in that it
damages properties but also improves soil drainage and turnover as a positive
view.
In comparison to the naked mole-rat, the common mole-rats have the
ability to generate their own heat and keep their body temperature above ambient
temperature which gives them an added advantage to survive any weather. They
have lower individual body masses in arid environment that helps with energy
27
conservation. They also have long sensory hairs, vibrissae, which stand out from
their fur covering their body.
Common mole-rats are social creatures that live in family units of up to 14
per group. They are widely distributed across the whole of the southern African
region which is found in all sorts of habitats. The fur of the common mole-rat is
silky, soft and short. Its colour though depends on the colour of the soil it is
burrowing in however, they are usually brown in colour and lacks patches on its
nose, ear, eye and throat. It has a keen sense of hearing and a very poor eye-sight.
Common mole-rats are able to survive underground by exhibiting many
thermoregulatory and metabolic adaptations to living in burrows. This is also
possible because of their reduced resting metabolic rate and lower core body
temperature with a higher thermal conductance (Bennett et al., 1992; Bennett et
al., 1994; Bennett et al., 2009). They exhibit specialized behaviour and
cooperative care of the young. The younger ones are like to-be workers and older
ones may be casual workers. The worker for the most part burrows and forage.
The oldest are breeders. The main predator species of common mole-rat are barn
owls, eagle owls and mole snakes. If caught out in the open, the cunning leopard
will also prey upon the common mole-rat.
28
Figure 2.2: Photograph of the common mole-rat (Cryptomys hottenttotus) Piechl,
(2004). Internet accessed on 12 June, 2010.
2.2. Experimental groups
The grouping of the experimental animals was based on two time points; a
proliferation time point, 2 hours post BrdU- injection and a survival time point, 4
weeks post BrdU- injection (Lagace et al., 2007). In the proliferation time point,
BrdU is administered to the animal 2 hours before sacrifice to check for
proliferating cells that are BrdU positive. Likewise in the 4 weeks post BrdU
injected animal group, they are injected once and sacrificed after 28 days with
animal under observation every day.
29
TABLE 3: Distribution of animals into groups is as shown below.
Four-striped mouse Common mole-rat
2 hrs post BrdU injection to evaluate
proliferating cells.
3 3
4 weeks post BrdU injection to evaluate
surviving cells.
3 3
Number of controls (No BrdU injection) 2 2
Total 8 8
2.3. Markers of cell Proliferation
There are two main classes of markers used to label proliferating cells:
exogenous and endogenous markers. Endogenous markers are molecules that the
cell expresses during the progression of the cell cycle, which correlates with the
duplication of its DNA or with the mitotic division while exogenous markers are
injected then binds to DNA in vivo and may produce DNA mutations. These types
of marker have been widely used in the study of adult neurogenesis. There are
three markers used in this study, bromodeoxyuridine (BrdU), Ki-67 and
doublecortin.
2.3.1. Bromodeoxyuridine (BrdU) Administration
Evidence for neurogenesis is obtained by the use of the thymidine analog
bromodeoxyuridine (BrdU) (Cameron et al., 1998), which incorporates into DNA
during the S phase of cell cycle (Nowakowski et al., 1989). BrdU labelling is used
to reveal neural stem cells and has proven to be a valid marker for studying adult
neurogenesis (Cameron et at., 1998; Kempermann et al., 1997; Taupin, 2007).
BrdU crosses the blood- brain barrier (del Rio & Soriano, 1989). Advantages of
BrdU to H-thymidine (H-dT) is that its immunohistochemical detection is more
30
easily combined with that of various cell class-specific markers to determine cell
phenotype in small neurons such as granule cells that are difficult to distinguish
from astrocytes (Rackic, 2002). It also allows for the analysis of changes in the
production of specific cell types under various experimental conditions (Rackic,
2002). With BrdU, it is particularly useful for the detection of newly generated
cells that were difficult to identify with the autoradiographic method (Rackic,
2002). However, the immunohistochemical approach can also lead to false
conclusion if potential technical problems are ignored (Rackic, 2002; Hayes &
Nowakowski, 2002). There are no established criteria for the use of BrdU as a
marker of neuronal birth date at the moment, though it is not a marker for cell
division, but a marker for DNA synthesis (Rackic, 2002). BrdU
immunohistochemistry is not stoichiometric in contrast to H-dT autoradiography
(Hayes & Nowakowski, 2002). However, BrdU is also considered to be a
mutagen (Morris, 1991). BrdU was dissolved in saline and administered
intraperitoneally for the proliferation and survival groups. A dose of 50 mg /kg
body weight was administered (Mitra et al., 2006). The disadvantages of BrdU are
that it has to be injected into the animal and may require multiple injections.
2.3.2. Ki-67
Ki-67 is an endogenous proliferation marker which reacts with a nuclear
antigen. Ki-67 is expressed in all proliferating cells which are in active phases of
cell cycle but absent in resting cells. It is a nuclear protein present in all the phases
of the cell cycle except in the G0 (Gil-Perotin et al., 2006). In G1, it is
predominantly localized in the perinucleolar region and also found in nuclear
matrix in the later phases (Gerdes, 1990). It is thought to be involved in the
31
maintenance of cell proliferation however its exact mechanism for function is
unknown. Due to these facts, it is considered an important marker for evaluation
of tumour diagnosis and prognosis. It is a nuclear protein present in all the phases
of the cell cycle except in the G0 (Gil-Perotin et al., 2006). Ki-67 labels the nuclei
of the proliferation cells.
2.3.3. Doublecortin (DCX)
DCX is expressed in migrating neurons throughout the central and
peripheral nervous system during embryonic and postnatal development (Gleeson
et al., 1999). DCX co-assembles with brain microtubules, and recombinant DCX
stimulates the polymerization of purified tubulin. Over expression of DCX in
heterologous cells leads to a dramatic microtubule phenotype that is resistant to
depolymerization. Therefore, DCX likely directs neuronal migration by regulating
the organization and stability of microtubules (Gleeson et al., 1999). Doublecortin
is a phenotypic marker which stains the cytoplasm of immature cells (Lu et al.,
2005).
2.4. Tissue processing
The animals were euthanized with sodium pentobarbital (Euthanaze, i.p.
80 mg/kg) and transcardially perfused with 0. 9% cold saline (4 ?C) followed by
4% paraformaldehyde in 0.1M phosphate buffer (PB). Brain tissues were then
carefully removed from the skull, weighed and post-fixed in 4%
paraformaldehyde in 0.1 M PB, then allowed to equilibrate in 30% sucrose in 0.1
M PBS. Brain tissues were then kept frozen in dry ice and sectioned using a
32
sliding microtome in the saggital plane at 50 ?m section thickness covering the
complete brain. Subsequently, sections were placed in vials containing PBS.
A one in five series of sections were each stained for BrdU, Ki-67 and
doublecortin; the remaining two sections of the series were stored in
cryoprotectant solution (CPX) in a freezer at -20 ?C for future experimental
purpose. This provided a section of the brain stained at every 250 ?m throughout
the brain.
2.5. Bromodeoxyuridine immunohistochemistry
2.5.1 Pre- incubation:
Sections were rinsed 3 times for 10 minutes in 0.1M PBS, under gentle
shaking at room temperature. Tissues were then pre-incubated, for 2 hours at
room temperature, in a solution containing 3 % normal goat serum (NGS;
Chemicon Int.), 2 % bovine serum albumin (BSA; Sigma) and 0.25 % Triton
X100 (Merck) in 0.1M PB. Triton X100 is a detergent necessary to create
micropores in the membranes of the cells, allowing the antibodies and the
reactives to penetrate the tissue. It also removes the antibodies from its antigens.
The normal serum and BSA, which are proteins, are used to avoid unspecific
staining. They help to prevent the primary and secondary antibodies to join to
unspecific places that have a similar structure.
2.5.2. Primary Antibody Incubation:
The sections were then placed in 0.25 % Triton X100, 3 % NGS, 2% BSA,
and primary mouse anti-BrdU monoclonal antibody (Millipore International, MA,
33
USA, 1:1000, Mitra et al., 2006) for four-striped mouse and rat anti-BrdU
antibody (Millipore International, MA, USA 1:1000) for common mole-rat
overnight at 4 ?C under a gentle shaking. These antibodies are species specific.
Subsequently, tissues were rinsed three times for 10 minutes in 0.1M PB.
2.5.3. Secondary Antibody Incubation:
The tissues were incubated in a secondary antibody solution which
contained 1:500 dilution of biotinylated-goat-anti-mouse or goat-anti-rat IgG
(Dako, Denmark, 1:100), 3 % normal goat serum, 2 % BSA, in 0.1 M PB, for two
hours at room temperature.
2.5.4. Avidin-Biotin-Complex-method:
Sections were rinsed three times for 10 minutes in 0.1 M PB, then incubated for 1
hour in AB solution (the ratio that were used for A and B reactive were 40 ?l each
in 5000 ?l of 0.1 M PB, Vector Labs), and again rinsed three times in 0.1M PB
under gentle shaking at room temperature, each for 10 minutes.
2.5.5. 3, 3?-diaminobenzidine tetrahyrochloride (DAB) staining:
Sections were treated in a solution containing DAB in 0.1M PB for 5
minutes. Thereafter, 3 ?l of 30 % H2O2 per 0.5 ml of solution was added. A low
power stereomicroscope was used to follow up the development of the reaction.
Once an appropriate level of background staining was observed, the reaction was
stopped by placing sections in 0.1 M PBS. The sections were then mounted on 0.5
% gel coated glass slides and air-dried overnight. They were then dehydrated in a
graded series of alcohols, cleared in xylene and cover slipped with Entelan. Two
controls were used in the immunostainings, one which did not contain primary
34
antibody and the other which omitted the secondary antibody in selected sections
to test whether the anti-bodies worked.
2.6. Ki-67 immunohistochemical staining
Sections were washed 2 times for 10 minutes in PBS and then rinsed with
tris buffer saline triton-X (TBST) once for 5 minutes under gentle shaking at room
temperature. The sections were then treated for 40 minutes at 94 ?C (in water
bath) in citrate buffer pH 6.1 diluted with distilled water (1:10) for anti-gene
retrieval. Sections were then allowed to cool down on the bench to room
temperature for 20 minutes.
Sections were then washed in TBST 2 times for 5 minutes under gentle
shaking and transferred into blocking solution, 5 % NGS in TBST for 30 minutes.
Tissues were then transferred into primary antibody, NCL-Ki-67 (Novocastra,
Wetzlar, Germany; 1:5000) in TBST supplemented with 2 % BSA and 2 % NGS
overnight under gentle shaking at 4? C.
The following day, the tissues were removed from the fridge and allowed
for 30 minutes to equilibrate to room temperature under gentle shaking. The
tissues were then washed 3 times for ten minutes in TBST under gentle shaking at
room temperature. Secondary antibody was then applied, biotinylated goat- anti-
rabbit (Vector lab, CA, USA; 1:250) in TBST supplemented with 2 % NGS for 60
minutes at room temperature. Tissues were then washed 3 times for 10 minutes in
TBST. ABC reagent (Vector lab, CA, USA; 1:100, A and B) was then applied for
40 minutes at room temperature under gentle shaking. After that, sections were
then washed in TBS 2 times for 15 minutes under gentle shaking at room
35
temperature. Tissues were then washed in TB pH 7.6 two times for 10 minutes
under gentle shaking at room temperature. They were then pre-incubated in the
dark for 30 minutes with DAB solution, 0.5 mg/ml, in TB 7.6 under gentle
shaking at room temperature using 2 ml per vial. 35 ?l of 0.5 % H2O2 was then
added to each vial and mixed very well to develop under visual guidance until a
strong nuclear staining was observed. Reaction was then stopped by washing
sections TB pH 7.6 3 times for 10 minutes under gentle shaking at room
temperature. Tissues were then washed in PB and mounted onto 0.5 % gelatinized
slides and air dried overnight. They were then dehydrated in a graded ascending
series of alcohols, cleared in xylene, and coverslipped with Entelan.
2.7. Doublecortin (DCX) immunohistochemical staining
Sections were washed 2 times for 10 minutes in PBS and then rinsed with
TBST once for 5 minutes under gentle shaking at room temperature. Sections
were then treated with blocking solution, 5 % normal rabbit serum (NRbS) in
TBST for 30 minutes.
Tissues were then transferred into primary antibody DCX (Santa Cruz biotech,
CA, USA; 1:400) in TBST supplemented with 2 % BSA and 2 % NRbS overnight
at 4 ?C under gentle shaking.
On the following day, tissues were removed from the fridge and left on the
shaker to equilibrate at room temperature followed by a 3 times 10 minutes wash
in TBST under gentle shaking at room temperature. Secondary antibody,
biotinylated rabbit-anti-goat (Vector lab, CA, USA; 1:250) in TBS supplemented
with 2 % NRbS for 60 minutes at room temperature under gentle shaking was
36
then applied. After that, sections were washed 3 times for 10 minutes each under
gentle shaking at room temperature. Thirty minutes prior to the time of use, ABC
reagent in TBS (1:100, A plus B) was applied to the tissues for 40 minutes at
room temperature under gentle shaking. Sections were then washed in TBS twice
at 15 minutes each under gentle shaking at room temperature followed by washing
in TB (pH 7.6) two times for 10 minutes each.
Section were then pre-incubated in DAB solution, 0.5 mg/ml in TB (pH
7.6) by using 2 ml per vial for 30 minutes in the dark under gentle shaking at
room temperature. 35 ?l of 0.5 % H2O2 was added to each vial and mixed very
well and allowed to develop under visual guidance until strong nuclear staining
appears within the rostral migratory stream. The reaction was stopped by washing
sections in TB pH 7.6, 3 times for 10 minutes each under gentle shaking at room
temperature. Tissues were then washed in PB and mounted onto 0.5 % gelatinized
slides and air dried overnight. They were then dehydrated in an ascending graded
series of alcohols, cleared in xylene and cover slipped with Entelan.
2.8. Data analysis
Sections of the brains were analysed with a light microscope using the
Zeiss Axioskop 2 plus microscope, Germany. The immunostained sections were
compared with previous report studies (Lagace et al., 2007; Amrein et al., 2004;
Lu et al., 2005; Plumpe et al., 2006). Photomicrographs were taken at different
magnification with the aid of the Zeiss Axioskop 2 plus microscope with a fitted
camera, model AxioCam HRc Zeiss camera (Germany).
37
CHAPTER THREE: RESULTS
3.1 General observations
The main objective of this study was to identify proliferative cells in the
regions of the adult brain of non captive South African rodents. The distribution
of the immunohistochemically reactive brain tissues to the BrdU, Ki-67 and DCX
were found to be different between the four-striped mouse and common mole-rat.
BrdU labelled the nuclei of proliferating cells which appeared dark and
clustered in the two hours post BrdU injected group. For the four weeks post
BrdU injection group the BrdU positive cells appeared more singly with darkly
stained nuclei and more rounded in the two active neurogenic sites for both the
four-striped mouse and common mole-rat. BrdU positive cells were absent in
other potential sites in both the four-striped mouse and common mole-rat.
Ki-69, an intrinsic marker for proliferating cells, labelled the nuclei of the
proliferating cells which appeared in clusters in almost all the region that was
observed. More new neurons were stained appearing in clusters and in some areas
in isolated form. Ki-67 labels proliferating cells in the two active neurogenic sites,
subventricular zone and dentate gyrus of the hippocampus of the two experimental
animals. In the potential neurogenic sites, Ki-67 positive cells were observed in
the substantia nigra, striatum and olfactory bulb of the four-striped mouse while in
the common mole-rat, Ki-67 positive cells were present in the substantia nigra and
olfactory bulb but absent in the striatum.
DCX is an intrinsic marker for immature neurons, labelled the cytoplasm of
the immature cells along with their processes. They appeared in clusters but varied
38
in shape. The DCX positive cells were categorized according to the shape and
presence of apical dendrites as described earlier by Plumpe et al., (2006):
Category A and B DCX positive cells were with very short or no processes
respectively. The processes were less than one nucleus wide in the DCX
positive cells of category B.
Category C and D DCX positive cells had processes of intermediate length
and immature morphology. The processes were longer in DCX positive
cells in category C compared to processes of DCX positive cells in
category B whereby the processes reached the granule cell layer but did
not reach the molecular layer.
In DCX positive cells in category D, the processes reached the molecular
layer.
Category E and F DCX positive cells had a more matured appearance. In
the DCX positive cells for category E, they had a one thick dendrite that
reached into the molecular layer and displayed a comparatively sparse
branching in the molecular layer. The DCX positive cells of category F
had a dendritic tree which showed delicate branching and few major
branches close to the soma or within the granule cell layer.
DCX positive cells were observed in the two neurogenic sites of the
experimental animals. Likewise in the potential neurogenic sites, they were
observed in the striatum and olfactory bulb of the four-striped mouse but
absent in their substantial nigra. In the common mole-rat, DCX positive cells
were present in the striatum, substantia nigra and olfactory bulb. In addition to
39
these potential neurogenic sites in the common mole-rat, DCX positive cells
were observed in the cerebral cortex of the common mole-rat.
3.2. Immunohistochemical findings in the four-striped mouse
3.2.1. BrdU positive cells in the proliferating and survival group
Proliferative cells were observed in the dentate gyrus of the hippocampus
(Figure 3.1). The cells appeared in clusters and irregular in shape in the 2 hours
post BrdU injected group (Figure 3.1 B) while the 4 weeks positive cells appeared
singly, more rounded with darkly stained nuclei (Figure 3.1 C). These cells were
found in the granular cell layer and subgranular layer of the dentate gyrus of the
hippocampus and in the hilus.
BrdU positive cells were observed in the subventricular zone of the lateral
ventricle. The nuclei were darkly stained appeared in clusters along the wall of the
subventricular zone of this region of the brain (Figure 3.2, A-C). Under high
magnification, these cells appeared darkly stained, irregular in shape and in
clusters in the 2 hours post BrdU injected group (Figure 3.2 B) while in the 4
weeks group, they appeared more regular in shape with a darkly stained nuclei
(Figure 3.2 C). BrdU positive cells in the two hours and four weeks post BrdU
groups were distributed along the wall subventricular zone area.
40
Figure 3.1: Representative photomicrograph showing BrdU positive cells in the
dentate gyrus of the hippocampus of the four-striped mouse. The two hours
proliferating groups (A and B) show the BrdU positive cells at different
magnification. Figure 3.1 C, for the four weeks group. The arrows indicate
positive BrdU immunostained cells. GCL-granule cell layer and SGL-subgranular
layer. Scale bar; A =10 ?m, B= 2.5?m and C= 1 ?m.
41
42
Figure 3.2: Representative photomicrograph showing BrdU positive cells in the
wall of the subventricular zone of the four-striped mouse. In the two hours group
(A and B), the proliferative cells appear in clusters and in the four weeks (C), the
immunopositive cell appear singly and more rounded with a darkly stained
nucleus. The arrows indicate positive BrdU immunostained cells. SVZ-
subventricular zone and LV- lateral ventricle. Scale bar; A= 10 ?m, B and C= 1
?m.
43
44
3.2.2. Ki-67 positive cells
The Ki-67 positive cells were observed to be distributed along the length
of the granular cell layer of the dentate gyrus of the hippocampus (Figure 3.3, A &
B). The nuclei of the Ki-67 positive cells were darkly stained and distributed in
the dentate gyrus of the hippocampus (Figure 3.3 B). The Ki-67 positive cells in
the subventricular zone appeared in clusters and widely distributed (Figure 3.4, A-
C). These immunopositive cells had centrally located darkly stained nuclei (Figure
3.4 C).
Ki-67 positive cells appear darkly stained and in clusters and observed
along the rostral migratory stream which is enroute towards the olfactory bulb
(Figure 3.5, A & B).
The proliferative Ki-67 positive cells in the substantia nigra appeared darkly
stained with a regular shape and in clusters (Figure 3.6, A & B). There were no
positive Ki-67 cells in the cerebral cortex in the four-striped mouse.
45
Figure 3.3: Representative photomicrograph showing Ki-67 positive cells in the
dentate gyrus of the hippocampus in the four-striped mouse (A and B). Majority
of the cells are located in the subgranular layer (arrows). The cells appear darkly
stained and in clusters. GCL-granule cell layer and SGL-subgranular layer. Scale
bar; A =10 ?m, B=1 ?m.
46
47
Figure 3.4: Representative photomicrograph showing Ki-67 positive cells in the
subventricular zone of the four-striped mouse. Majority of the cells are located in
the subventricular wall at different magnifications (A, B and C). The cells appear
darkly stained and in clusters 9 (arrows). DG- dentate gyrus, Hipp- hippocampus,
SVZ- subventricular zone and Str- striatum. Scale bar; A= 20 ?m, B=2.5 ?m, C=1
?m.
48
49
Figure 3.5: Representative photomicrograph showing Ki-67 positive cells in the
rostral migratory stream/ olfactory bulb of the four-striped mouse. Ki-67 positive
cells are migrating towards the olfactory bulb (A). Figure B is a higher
magnification of the rostral migratory stream.. OB- olfactory bulb and RMS-
rostral migratory stream. Scale bar; A= 20 ?m and B=1 ?m.
50
51
Figure 3.6: Representative photomicrograph showing Ki-67 positive cells in the
substantia nigra of the four-striped mouse. Ki-67 positive cells are more rounded
with darkly stained nuclei. Figure B is a higher magnification of the positive cells
(arrows). InfC- inferior colliculus, SupC- superior colliculus and SN- substantia
nigra. Scale bar; A= 20 ?m, B=2.5 ?m.
52
.
53
3.2.4. Doublecortin (DCX) positive cells
DCX positive cells were observed in the dentate gyrus of the hippocampus
as immature neurons along with their processes (Figure 3.7, A-C). The cell bodies
lined the subgranular layer of the dentate gyrus of the hippocampus (Figure 3.7
B). Majority of the cells are bipolar with an ovoid soma and fall under category E
and F (see section 3.1). Their processes extended into the granular and molecular
layers (Figure 3.7, B & C).
The DCX positive cells in the subventricular zone appeared in a web-like
fashion in the wall of the subventricular zone at low and high power magnification
together with their processes. The cytoplasm of the immature cells stained brown
with short or long processes and a well developed cell body (Figure 3.8, A & B).
Their soma is ovoid with a prominent nucleus (Figure 3.8 C).
The immature neurons were observed in the olfactory bulb in clusters
(Figure 3.9, A-C). The soma of the DCX positive cells are stained brown along
with their processes. The soma was ovoid in shape with a more prominent nucleus
(Figure 3.9 B) in the olfactory bulb. They migrate from the subventricular zone to
the olfactory bulb via the rostral migratory stream with their processes extending
into the striatum (Figure 3.9 C).
54
Figure 3.7: Representative photomicrograph showing DCX positive cells in the
dentate gyrus (DG) of the four-striped mouse. DCX positive cells are in cluster (B
and C) with their soma and processes (C, arrows). The soma lies in the
subgranular layer with their processes projecting as far as the molecular layer.
Hipp- hippocampus, DG- dentate gyrus, SGL subgranular layer and GCL- granule
cell layer. Scale bar; A= 20 ?m, B=2.5 ?m, C=1 ?m.
55
56
Figure 3.8: Representative photomicrograph showing DCX positive cells in the
subventricular zone (SVZ) of the four-striped mouse. DCX positive cells are seen
in the photomicrograph in cluster (A and B) with their soma and processes. The
soma can be seen on the luminar surface of the SVZ with their processes
projecting towards the striatum. RMS- rostral migratory stream, SVZ-
subventricular zone, LV- laterals ventricle and Str- striatum. Scale bar; A=10 ?m
and B=1 ?m.
57
58
Figure 3.9: Representative photomicrograph showing DCX
immunohistochemistry in the RMS-OB of the four-striped mouse. Figure 3.9 B,
shows the six layers of the olfactory bulb as follows; ONL-olfactory nerve layer,
GL- glomerular layer, EPL- external plexiform layer, MCL- mitral cell layer, IPL-
internal plexiform layer and GCL- granule cell layer. DCX positive cells in cluster
(B) with their soma in the granule cell layer and their processes extend as far as
the glomerular layer. OB- olfactory bulb and RMS- rostral migratory stream.
Scale bar; A= 20 ?m, B=10 ?m, C and D =2.5 ?m.
59
60
Table 4: Summary of active and potential neurogenic sites in the four-striped
mouse.
Site SVZ DG SEP\
STR
SN CTX OB\
RMS
Cell
proliferation
(2hrs)
+ + - - - +/-
Cell Survival
(4weeks)
+ + - - - +/-
Ki-67 ++ ++ +/- +/- - +++
DCX +++ +++ +/- - - ++++
Key: SVZ, subventricular zone; DG, Dentate gyrus; SEP/ STR, Septum and
Striatum; SN, Substantia nigra; CTX, Cerebral cortex; OB/ RMS, Olfactory bulb/
Rostral migratory stream, (+ indicates presence; - indicates absence, +/- indicates
indecisive and more than one ?+? indicates increasing level of immunopositive
staining).
61
3.3. Immunohistochemical findings in the common mole-rat
3.3.1. BrdU positive cells in proliferative and survival groups
BrdU positive cells were observed in the dentate gyrus of the hippocampus
of common mole-rat. In the 2 hours post BrdU injected group, the cells appeared
in cluster and irregular in shape with darkly stained nuclei (Figure 3.10, A-C). In
the 4 weeks post BrdU injected group, BrdU positive cells appeared isolated and
more rounded with darkly stained nuclei (Figure 3.10 C).
In the subventricular zone, BrdU positive cells in the 2 hours group
appeared in clusters and irregular in shape with darkly stained nuclei (Figure
3.11). While in the 4 weeks group the BrdU positive cells appeared singly and
more rounded in shape with darkly stained nucleus (Figure 3.11 B).
In the olfactory bulb and rostral migratory stream, BrdU positive cells in
the 2 hours group appeared in cluster and irregular in shape with darkly stained
nuclei (Figure 3.12, A & B). While in the 4 weeks group the BrdU positive cells
appeared singly and more rounded in shape with darkly stained nuclei (Figure
3.12B).
62
Figure 3.10: Representative photomicrograph showing BrdU positive cells in the
dentate gyrus of the hippocampus of the common mole-rat in the two time points.
In the two hours group, the proliferative cells are darkly stained nuclei in clusters
(Figure 3.10 B, arrow) while in the 4 weeks group (Figure 3.10 C), the matured
cells appear more rounded and singly. The arrows indicate positive BrdU
immunostained cells. SGL- subgranular layer and GCL- granule cell layer. Scale
bar; A= 10 ?m, B= 2.5 ?m, C 1 ?m.
63
64
Figure 3.11: Representative photomicrograph showing BrdU positive cells in the
common mole-rat (Figure 3.11, A-B) while in the four weeks (C), they appear
more rounded with darkly stained nuclei. The arrows indicate positive BrdU
immunostained cells. SVZ- subventricular zone, LV- lateral ventricle, Str-
striatum, Hipp- hippocampus and OB- olfactory bulb. Scale bar; A=10 ?m, B, C =
1 ?m.
65
66
3.3.2. Ki-67 positive cells
Ki-67 positive cells were present in the subgranular layer of the dentate
gyrus of the hippocampus (Figure 3.12, A & B). The Ki-67 positive cells were
distributed along the subgranular layer of the dentate gyrus of the hippocampus
with darkly stained nuclei (Figure 3.12 B).
The new neurons were found in the subventricular zone, more of which are
on the lateral ventricle connecting to the rostral migratory stream (Figure 3.13, A
& B). They appeared in clusters with darkly stained nucleus (Figure 3.13 B).
The Ki-67 positive cells in the olfactory bulb and rostral migratory stream
appeared in clusters (Figure 3.14, A-C). The ki-67 positive cells were observed
from the rostral migratory stream enroute the olfactory bulb (Figure 3.14 A). The
Ki-67 positive cells were also observed lining the rostral migratory stream coming
from the subventricular zone (Figures 3.14 C).
In the substantia nigra, the Ki-67 positive cells that were observed
appeared in clusters and irregular in shape with less prominent nuclei (Figure 3.
15, A & B). Ki-67 positive cells were absent in the striatum and cerebral cortex.
67
Figure 3.12: Representative photomicrograph showing Ki-67 positive cells in the
dentate gyrus of the common mole-rat. Ki-67 positive cells are in cluster and more
rounded with darkly stained nuclei (Figure 3.12 B). SGL- subgranular layer and
GCL- granule cell layer. Scale bar; A= 10 ?m and B=1 ?m.
68
69
Figure 3.13: Representative photomicrograph showing Ki-67 positive cells in the
subventricular zone of the common mole-rat. Ki-67 positive cells are in cluster
and more rounded with darkly stained nuclei (B). SVZ- subventricular zone, Str-
striatum, LV lateral ventricle and RMS- rostral migratory stream. Scale bar; A=
10 ?m, B=1 ?m.
70
71
Figure 3.14: Representative photomicrograph showing Ki-67 positive cells in the
common mole-rat. Ki-67 positive cells in the rostral migratory stream (A, arrows
indicate the direction of migration).Figure B is a higher magnification of A.
Figure C shows positive Ki-67 cells in the olfactory bulb. RMS- rostral migratory
stream and OB- olfactory bulb. Scale bar; A= 10 ?m, B=2.5 ?m, C=1 ?m.
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73
Figure 3.15: Representative photomicrograph showing Ki-67 positive cells in the
substantia nigra of the common mole-rat. Ki-67 positive cells appear in cluster
(arrows) and more rounded (A, and B at higher magnification). InfC- inferior
colliculus, SupC- superior colliculus and SN- substantia nigra. Scale bar; A= 10
?m and B =1 ?m
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75
3.3.3. Doublecortin (DCX) positive cells
The DCX positive cells in the dentate gyrus of the hippocampus were
observed in the subgranular layer of the dentate gyrus of the hippocampus (Figure
3.116, A-C). The soma of these cells appeared ovoid in shape with their dendrites
reaching as far as the molecular layer of the dentate gyrus of the hippocampus and
some dendrites only ending in the granular layer and could be classified as E and
F categories of DCX positive cells (Figure 3.16, B & C).
The DCX positive cells in the subventricular zone appeared in a web-like
fashion with their processes. At high magnification, the DCX positive cells
presented ovoid shaped soma and branched processes in multi-directional way
(Figure 17 B).
DCX positive cells were observed in the olfactory bulb-rostral migratory
stream (Figure 3.18, A-C). These cells appeared in clusters in a uniformed
direction. The soma of the DCX positive cells were meshed within processes and
located in the granule cell layer of the olfactory bulb with their processes
extending as far as the external plexiform layer of the olfactory bulb (Figure 3.18
B). They continued from the subventricular zone via the rostral migratory stream
enroute the olfactory bulb (Figure 3.18).
DCX positive cells were observed in the striatum (Figure 3.19, A). These
cells appeared isolated with an ovoid soma (Figure 3.19, C & D). The processes
are bi-directional with branched dendrites (Figure 3.19, C & D).
There were DCX positive cells present in the cerebral cortex (Figure 3.20,
A-C). The majority of the DCX positive cells were observed in the second layer of
the somatosensory, entorhinal and piriform cortices. The DCX positive neuron in
76
layer II had bipolar and ovoid shaped soma (Figure 3.20 C). Small number of the
DCX positive cells has an ovoid shaped soma with apical dendrites which
bifurcates (Figure 3.20, B & C). Some DCX positive cells were observed in layer
III with bipolar and some pyramidal in shape (Figure 3.20 A, red arrow).
77
Figure 3.16: Representative photomicrograph showing DCX positive cells in the
dentate gyrus of the common mole-rat. DCX positive cells appear in cluster (A)
with their soma and definite processes which is category B can C respectively (B
and C). The soma can be seen in the SGL with their processes projecting as far as
into the molecular layer. SGL- subgranular layer and GCL- granule cell layer.
Scale bar; A=10 ?m, B and C=1 ?m.
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79
Figure 3.17: Representative photomicrograph showing DCX positive cells in the
subventricular zone of the common mole-rat. DCX positive cells are in cluster (B)
with their soma in the wall of the SVZ and processes projecting into the striatum.
The soma can be seen on the wall of the SVZ with their processes projecting as
far as into the Striatum. LV- lateral ventricle, SVZ- subventricular zone, Hipp-
hippocampus and Str- striatum. Scale bar; A=20 ?m, B=1 ?m.
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81
Figure 3.18: Representative photomicrograph showing DCX positive cells in the
rostral migratory stream/ olfactory bulb of the common mole-rat. DCX positive
cells are in cluster (B and C) with their soma and processes (C). Figure 3.18 A,
shows the six layers of the olfactory bulb as follows; ONL-olfactory nerve layer,
GL- glomerular layer, EPL- external plexiform layer, MCL- mitral cell layer, IPL-
internal plexiform layer and GCL- granule cell layer. The soma can be seen in the
granule cell layer with their processes projecting into the glomerular layer. OB-
olfactory bulb and RMS- rostral migratory stream. Scale bar; A=20 ?m, B and
C=2.5 ?m.
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83
Figure 3.19: Representative photomicrograph showing DCX positive cells in the
striatum of the common mole-rat. DCX positive cells appearing more singly (B-
D) with their soma and processes (C and D). The soma can be seen with
bifurcated processes projecting out. SVZ- subventricular zone, RMS- rostral
migratory stream and Str- striatum. Scale bar; A =20 ?m, B=2.5 ?m, C and D=1
?m.
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85
Figure 3.20: Representative photomicrograph showing DCX positive cells in the
cerebral cortex of the common mole-rat. DCX positive cells appear in clusters (C)
with their soma and processes (C). The DCX positive cells are more numerous in
layer II (B) and in layer III (A, red arrows). Scale bar; A =10 ?m, B=2.5 ?m, C=1
?m.
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87
Table 5: Summary of active and potential neurogenic sites in the common mole-
rat.
Site SVZ DG SEP\
STR
SN CTX OB\
RMS
Cell
proliferation
(2hrs)
+ + - - - +/-
Cell Survival
(4weeks)
+ + - - - +/-
Ki-67 ++ ++ - +/- - +++
DCX +++ +++ +/- + ++++ ++++
Key: SVZ, subventricular zone; DG, Dentate gyrus; SEP/ STR, Septum and
Striatum; SN, Substantia nigra; CTX, Cerebral cortex; OB/ RMS, Olfactory bulb/
Rostral migratory stream, (+ indicates presence; - indicates absence, +/- indicates
indecisive and more than one ?+? indicates increasing level of immunopositive
staining).
88
CHAPTER FOUR: DISCUSSION
This study was to provide a descriptive evidence of the existence of adult
neurogenesis in the brain of two South African rodents; the four-striped mouse
(Rhabdomys pumilio), a captive bred animal originating from wild caught parents
and the common mole-rat (Cryptomys hottentotus), wild caught animals compared
to inbred, laboratory strains of rodents that were studied previously (Nacher et al.,
2001; Kee et al., 2002; McDonald and Wojtwicz, 2005; Plumpe et al., 2006;
Zhang et al., 2009).
A combination of BrdU, Ki-67 and DCX immunohistochemistry has
provided evidence of adult neurogenesis in the active sites and in some potential
sites in the four-striped mouse and common mole-rat. There were variations based
on the different markers and animal species.
4.1. Adult neurogenesis in the established active neurogenic sites
In the adult brain, the subventricular zone and dentate gyrus of the
hippocampus remain the active neurogenic sites in adult neurogenesis; these have
been assessed by different cell markers for cell proliferation (Eriksson et al.,
1998; Nacher et al., 2001; Pham et al., 2003; Schauwecker, 2006; Plumpe et al.,
2006; Lagace et al., 2007; Kim et al., 2009; Hauser et al., 2009). BrdU is cleared
from the rat brain within a short phase of 2 hours (Cameron and McKay, 2007),
endogenous Ki-67 is altered during active cell cycle (Hayes and Nowakowski,
2002) and DCX stains immature neurons (Lu et al., 2005). The subventricular
zone has two precise layers of cells: the first is a monolayer of multiciliated cells
lining the lateral ventricle called the ependymal layer; and the second layer is a 2-
89
3 cell layer thick area adjacent to the ependymal layer called the subependymal
layer (Alvarez-Buylla and Garcia-Verdugo, 2002). Although the exact identity of
the cell lineage in the subventricular zone was not part of this study, it is evident
that adult neurogenesis do occur at this site in the four-striped mouse and common
mole-rat. The subventricular zone neuroblast cells travel a long distance to the
olfactory bulb through a network of interconnecting pathways that become
confluent at the rostral margin of the lateral ventricular wall to form the rostral
migratory stream (Watts et al., 2005). This was also present in the four-striped
mouse and common mole-rat.
In the hippocampus, few BrdU positive cells were also observed compared
to previous work in which the same type of markers were used in rats (Lagace et
al., 2007). This could have been as a result of the limitation in the cell cycle stage
at which the BrdU stains the proliferating cells. BrdU staining is limited to the S-
phase of the cell cycle while Ki-67 stains all the phases except the resting (Go)
phase (Alvarez-Buylla and Garcia-Verdugo, 2002). There are reports of the use of
repeated doses of BrdU to enhance cell proliferation (Nacher et al., 2001; Kee et
al., 2002) with the associated toxicity (Taupin, 2007). Consequently, a single dose
of BrdU was administered in this study and may have contributed to the sparse
distribution and identification of the positive cells but with less toxicity in the
four-striped mouse and common mole-rat.
In addition to the question of dose and toxicity of BrdU, the procedure of
its injection has been proposed to induce an element of stress which could also
decrease neurogenesis. Although Gould et al., (1997) reported a decrease in adult
neurogenesis in the tree shrew following psychosocial stress, the reduced staining
90
of BrdU positive cells in this study cannot be linked to stress as this was minimal
during the injection of BrdU. The blood-brain barrier that limits the penetration of
BrdU and its non specificity for labelling dividing cells (Taupin, 2007) may also
be associated with less staining. Apoptosis (Biebl et al., 2000) may also contribute
to the low turn-out of BrdU positive cells in these sites but this was not tested in
this investigation. A similar experiment was performed on different strains of
adult laboratory animals, BALB/ c, C3H/ H3J, DBA/ 2J, 129/ SVJ and CD1, by
Kempermann et al., (1997) showed some BrdU positive cells were identified
although in varying amounts. This led Kempermann et al., (1997) to conclude that
this might be as a result of strain differences which may reflect genetic differences
of the laboratory animal that were used.
Ki-67 immunostaining was used as a confirmatory study to the BrdU
staining and the results indicated adult neurogenesis in the four-striped mouse and
common mole-rat. Hauser et al., (2009), confirmed in the wood mouse that the
subventricular zone was a site of cell proliferation not only in laboratory animals
but also in wild non captive animals. The report correlated with the result from
this study that adult neurogenesis is present in the subventricular zone and dentate
gyrus of the hippocampus captive reared four-striped mouse and wild caught
common mole-rat using the Ki-67 marker.
Ki-67 positive cells are numerous in the subventricular zone and they can
be seen throughout the wall of the subventricular zone forming a kind of
confluence which then continues as the rostral migratory stream. Reports have
indicated a low rate of BrdU positive cells in the dentate gyrus of the
hippocampus in Sprague-Dawley rats (Nacher et al., 2001). Likewise in
91
previously studied adult animals like laboratory rodents, dogs and monkeys, it is
suggested that the migration of newly generated cells in the hippocampus might
be hindered in these experimental animals (Siwak-Tapp et al., 2007; Zhang et al.,
2009). However in this study, DCX immunohistochemistry showed DCX positive
cells in the dentate gyrus of the hippocampus with processes projecting from the
subgranular layer to the molecular layer as confirmed by previous studies (Plumpe
et al., 2006; Hauser et al., 2009; Kim et al., 2009; Zhang et al., 2009). The cells
were observed in the subgranular layer of the dentate gyrus of the hippocampus
and the processes extending as far as the molecular layer where synaptic contacts
are expected to be established. With this outcome, it confirms that the
subventricular zone and the dentate gyrus of the hippocampus is a continuous
adult neurogenic site as reported (Amrein et al., 2004; Ihunwo and Pillay, 2007;
Hauser et al., 2009). From the result of Ki-67 and DCX immunohistochemistry,
adult neurogenesis does occur in the four-striped mouse and common mole-rat.
However, results of the BrdU do indicate some technical shortcomings.
4.2. Adult neurogenesis in the potential neurogenic sites
Other regions of the brain investigated for adult neurogenesis were the
olfactory bulb, rostral migratory stream, substantia nigra, cortex and striatum. The
result showed that cells from the subventricular zone migrated to the olfactory
bulb via the rostral migratory stream in the four-striped mouse and common mole-
rat. However, there were no BrdU positive cells in the substantia nigra (Zhao et
al., 2003), cortex (Takemura, 2005), striatum (Luzzati et al., 2007) and amygdala
(Bernier et al., 2002) as against the findings in the laboratory rodents.
92
Although, it was reported that adult neurogenesis may be restricted to a
particular region of the brain (Palmer et al., 1995), potential adult neurogenic sites
were confirmed using additional markers namely, Ki-67 and DCX markers for
immature cells, that stained more cell stages since the BrdU marker used in this
experiment though did not stain other neurogenic sites in the brains of the four-
striped mouse and common mole-rat. In the four-striped mouse, Ki-67 positive
cells were observed in striatum as darkly stained cells which could have been as a
result of migrating cells from the rostral migrating stream or they even deviated
and incorporated into the striatum. The converse was true for the common mole-
rat as no Ki-67 positive cells were observed in the striatum.
Most of the DCX positive cells in the subventricular zone are believed to
migrate to the olfactory bulb and incorporate there (Bartkowska et al., 2010)
while some may deviate from this track and move inferiorly towards the striatum
or superiorly towards the frontal cortex between layers II and III. These cells on
reaching the striatum are incorporated depending on the state of the cell or might
undergo apoptosis (Biebl et al. 2000). Though absent in the cerebral cortex of the
four-striped mouse, DCX positive cells were observed in the somatosensory
cortex of the brain of the common mole-rat. Luzzati et al., (2007) proposed that
neurogenesis in these potential neurogenic regions can be due to progenitors
derived from the subventricular germinal zone and/ or local parenchyma
progenitor. However, the presence of immature neurons in the somatosensory and
entorhinal cortices of the common mole-rat may be associated with the social
lifestyle of the common mole-rat with its continuous burrowing which indicate a
motor function of the brain. Mole rats have keen burrowing habits especially at
93
nights and produce different compartments with tunnels of about one kilometre
and 15 centimetres to 20 centimetres below the ground surface. Eloff (1952) cited
from Jarvis and Sale, (1971) observed that the foot vibration of Cryptomys during
digging last about one second and the vibrations are between 25 to 30 times. The
presence of cell proliferation in the entorhinal and piriform cortices associated
with memory may provide the explanation for the common mole-rat navigation of
each compartment with the burrows.
Adult neurogenesis remains a complex process involving the proliferation,
survival, differentiation and functional integration of new cells in the brain. The
cellular and molecular mechanisms that regulate adult neurogenesis remain
unclear (Schauwecker, 2006) but can be modulated by a variety of factors
including glutamate receptor activation (Cameron et al., 1995; Gould et al., 1997;
Bernabeu and Sharp, 2000), dietary restriction (Lee et al., 2002a, b), growth
factors (Scharfman et al., 2005; Palmer et al., 1995), stress (Brunson et al., 2005;
Nichols, et al., 2005) and neuronal injury (Parent, 2003; Cooper-Kuhn et al.,
2004). Enriched environments (Kempermann et al., 1997), running wheel exercise
(van Praag et al., 1999; Hauser et al., 2009), hippocampal-dependent learning
(Gould et al., 1996) and dietary restriction (Lee et al., 2002a), all these increase
adult neurogenesis in the adult hippocampus. It was reported that estradiol alter
adult hippocampal neurogenesis in female rodents (Galea et al., 2006) but
decreases in hippocampal neurogenesis do not always correlate with the
development of learned helplessness in male rats (Vollmayr et al., 2003).
With the different environment from which the four-striped mouse and
common mole-rat were obtained, it might be of interest to further investigate the
94
possibility that these might influence the rate of cell proliferation in these animal
models in terms of an enriched and survival instinct of these animals in the wild.
95
CHAPTER FIVE: CONCLUSION AND FURTHER STUDIES
5.1. Conclusion
This study has provided evidence of adult neurogenesis in the four-striped
mouse (Rhabdomys pumilio) and common mole-rat (Cryptomys hottentotus) using
the BrdU, Ki-67 and DCX markers for cell proliferation. The results showed adult
neurogenesis in the two active sites; subventricular zone of the lateral ventricle
and the dentate gyrus of the hippocampus.
BrdU labelled the nuclei of proliferating cells which appeared dark and
clustered in the two hours post BrdU injected group. For the four weeks post
BrdU injection (survival) group the BrdU positive cells appeared more singly with
darkly stained nuclei and more rounded in the two active neurogenic sites for both
the four-striped mouse and common mole-rat. BrdU positive cells were absent in
other potential sites in both the four-striped mouse and common mole-rat.
Ki-69, an intrinsic marker for proliferating cells, labelled the nuclei of the
proliferating cells which appeared in clusters in almost all the region that was
observed. More new neurons were stained appearing in clusters and in some areas
in isolated forms. Ki-67 labels proliferating cells in the two active neurogenic
sites, subventricular zone and dentate gyrus of the hippocampus of the two
experimental animals. In the potential neurogenic sites, Ki-67 positive cells were
observed in the substantia nigra, striatum and olfactory bulb of the four-striped
mouse while in the common mole-rat, Ki-67 positive cells were present in the
substantia nigra and olfactory bulb but absent in the striatum.
96
DCX, an intrinsic marker for immature neurons, labelled the cytoplasm of the
immature cells along with their processes with most falling under the category D,
processes reaching molecular layer of the dentate gyrus of the hippocampus. They
appeared in clusters but varied in shape. DCX positive cells were observed in the
two neurogenic sites of the experimental animals. In the potential neurogenic
sites, they were observed in the striatum and olfactory bulb of the four-striped
mouse but absent in their substantial nigra. In the common mole-rat, DCX
positive cells were present in the striatum, substantia nigra and olfactory bulb. In
addition to these potential neurogenic sites in the common mole-rat, DCX positive
cells were observed in the cerebral in the somatosensory and entorhinal cortices of
the cerebral cortex but absent in the four-striped mouse.
Such cell proliferation in the cerebral cortex opens up the potential sites for
further investigation in the light of reports on cerebral cortex as a reactive site.
The substantial nigra, in both the four-striped mouse and common mole-rat
indicate it is a potential site which must be further investigated for adult
neurogenesis.
5.2. Further studies
With the descriptive qualitative results obtained so far, it becomes imperative
for further investigation to be conducted in the area of quantification of newly
formed cells and the cell numbers of the dentate gyrus of the hippocampus.
Further research will therefore focus on:
1. Cell counting of the Ki-67 positive cells in the dentate gyrus of the
hippocampus for possible correlation with pyknotic cells.
97
2. Plastic embedding of the second half of the cerebral hemispheres followed
by sectioning at 20 ?m and staining with Giemsa staining for identification
of pyknotic cells and correlation of dentate gyrus for quantification.
3. Cell counting of pyknotic cells in the dentate gyrus of the hippocampus for
correlation with Ki-67 proliferating cells.
4. Total granule cell count of the dentate gyrus of the hippocampus using the
optical fractionator stereology which will be performed at the University
of Zurich, Switzerland.
98
Conference Presentations
2009 International Federation of Association of Anatomists (IFAA), Cape
Town South Africa
Detail citation:
Title: Adult Neurogenesis in the adult four-striped mouse and common mole-rat
Authors: Olatunbosun Olaleye, Moyosore Ajao, Paul Manger and Amadi O.
Ihunwo
Venue: International Conference Center, Cape Town, South Africa
Date: 16th- 19th August, 2009.
2009 Society of Neuroscientists of Africa (SONA) Sharma El Sheikh, Egypt
Detail citation:
Title: Adult Neurogenesis in the common mole rat, Cryptomys hottentotus, and
greater cane rat, Thryonomys swinderianus.
Authors: Olatunbosun Olaleye, Moyosore Ajao, Virginia Meskenaite, Paul
Manger and Amadi O. Ihunwo
Venue: Hotel Pyramisa, Sharma El Sheikh, Egypt
Date: 8th- 13th December, 2009.
99
REFERENCE
1. Altman, J. & Das, G.D. (1965). Autoradiographic and histological evidence
of postnatal hippocampal neurogenesis in rats. J.Comp. neurol. 124:139-335.
2. Altman, J. & Das, G.D. (1967). Postnatal neurogenesis in the guinea-pig.
Nature, 214(5093), 1098-1101.
3. Altman, J. (1969). Autoradiographic and histological studies of post natal
neurogenesis. IV. Cell proliferation and migration in anterior forebrain, with
special reference to persisting neurogenesis in the olfactory bulb. J. Comp.
Neurol. 137:433-458.
4. Alvarez-Buylla, A. And Garcia-Verdugo, J.M. (2002). Neurogenesis in adult
subventricular zone. J. Neurosci. 22(3):629-634.
5. Amrein, I., Slomianka, L., Poletaeva, I.I., Bologova, N.V. and Hans-Peter, L.
(2004). Marked species and age-dependent differences in cell proliferation
and neurogenesis in the hippocampus of wild-living rodents. J.Hipp.
14:1000-1010.
6. Arsnijevic, Y., Villemure, J.G., Brunet, J.F., Bloch, J.J., Deglon, N., Kostic,
C., Zum, A. and Aebischer, P. (2001). Isolation of multipotent neural
precursors residing in the cortex of the adult human brain. Exp. Neurol., 170:
48-62.
7. Bartkowska, K., Turlejski, K., Grabiec, M., Ghazaryan, A., Yavruoyan, E
and Djavadian, R.L. (2010). Adult neurogenesis in the Hedgehog (Erinaceus
concolor) and Mole (Talpa euroapaea). Brain Behav Evol, 76:128-143.
8. Bayer, S.A. (1983). 3H-thymidine-radiographic studies of neurogenesis in
the rat olfactory bulb. Exp. Brain Res. 50:329-340.
100
9. Bennett, N.C. (2009). African mole-rats (family Bathyergidae): models for
studies in animal physiology. African Zoology 44(2): 263-270.
10. Bennett, N.C., Aguilar, G.H., Jarvis, J.U.M. & Faulkes, C.G. 1994. Thermo-
regulation in three species of Afrotropical subterranean mole-rats (Rodentia:
Bathyergidae) from Zambia and Angola and scaling within the genus Cryp-
tomys. ecologia 97:222?228.
11. Bennett, N.C., Clarke, B.C. and Jarvis, J.U.M. 1992. A comparison of meta-
bolic acclimation in two species of social mole-rats (Rodentia: Bathyergidae)
in Southern Africa. Journal of Arid Environments 22: 189?198.
12. Bernabeu, R. and Sharp, F.R. (2002). NMDA and AMPA/kainite glutamate
receptors modulate dentate neurogenesis and CA3 synapsin-1 in normal and
ischemic hippocampus. J. Cereb. Blood Flow Metab. 20: 1669-1680.
13. Bernier P.J., Bedard A., Vinet J., Mevesque, M. and Parent, A. (2002).
Newly generated neurons in the Amygdala and adjoining Cortex of Adult
Primates: Proceedings of the national academy of sciences of the United
States of America. 99(10): 11464- 11469.
14. Biebl, M., Cooper, C.M., Winkler, J. and Kuhn, G.H. (2000) Analysis of
neurogenesis and programmed cell death reveals a self renewing capacity in
the adult rat brain. Neursci. lett. 291: 17-20.
15. Brunson, K.L., Kramar, E., Lin, B., Colgin, L.L., Yanagihara, T.K., Lynch,
G. and Baram, T.Z. (2005).Mechanism of late-onset cognitive decline after
early-life stress. J. Neurosci. 25(42):9328-9338.
101
16. Cameron, H.A., McEwen, B.S., Gould, E. (1995). Regulation of adult
neurogenesis by excitatory input and NMDA receptor activation in the
dentate gyrus. J. Neurosci. 15:4687-4692.
17. Cameron, H.A., Tanapat, P., Gould, E. (1998). Adrenal steroids and N-
methyl-D-aspartate receptor activation regulate neurogenesis in the dentate
gyrus of adult rats through a common pathway. Neuroscience 82: 349-354.
18. Cooper-Kuhn, C.M., Winkler, J., Kuhn, H.G. (2004). Decreased
neurogenesis after cholinergic forebrain lesion in the adult rat. J. Neurosci.
Res. 77: 155-165.
19. Corotto, F.S., Henega, J.A. and Maruniak, J.A. (1993). Neurogenesis persists
in the subependymal layer of the adult mouse brain. Neurosci. Lett. 149:
111-114.
20. del Rio, J.O. and Soriano, E. (1989). Immunocytochemical detection of 5-
bromodeoxyuridine incorporation in the central nervous system of the
mouse. Brain Res. 49: 311-317.
21. Doetsch, F. (2003). The glia identity of neural stem cells. Nat Neurosci.
6:1127-1134.
22. Doetsch, F., Alvarez-Buylla, A. and Garcia-Verdugo, J.M. (1999).
Regeneration of a germinal layer in the adult mammalian brain. PNAS. 96:
11619-11624.
23. Duman, R.S. (2004) Depression: a case of neuronal life and death? Biol.
Psychiatry 56, 140-145.
24. Eloff, G. (1952). Sielkundige aangepastheid van die mol aan onderaardse
leefwyse en sielkundige konvergensie. Tydskr. Wet. Kuns. 12: 210-225.
102
25. Eriksson, P.S., perfilieva, E., Bjork-Eriksson, T., Alborn, A.M., Nordborg, C
and Peterson, D.A. (1998). Neurogenesis in the adult human hippocampus.
Nat. Med. 4:1313-1317.
26. Feldmann Jr., R.E., Sawa, A., Seidler, G.H. (2007). Casualty of stem cell
based neurogenesis and depression- to be or not to be is that the question? J.
Psychaiatr. Res. 41, 713-723.
27. Frielingsdorf, H., Schwarz, K., Brundin, P. and Mohapel, P. (2004). No
evidence for new dopaminergic neurons in the adult mammalian substantia
nigra. Proc. Natl. Acad. Sci., USA, 101: 10177-10182.
28. Gage, F.H. (2000) Structural plasticity: cause, result, or correlate of
depression. Biol. Psychiatry 48, 713-714.
29. Galea, L.A., Spritzer, M.D., Barker, J.M. and Pawluski, J.L. (2006) Gonadal
hormone modulation of hippocampal neurogenesis in the adult. J.
Hippocamp 16:225-232.
30. Genelly, R.E. (1965). Ecology of the Common mole-rat (Cryptomys
hottentotus) in Rhodesia. J. Mammology. 46(4):647-665.
31. Gerdes, J. Semin Cancer. (1990). Biol. 1(3):199-206.
32. Gil-Perotin, S., Marin-Husstege, M., Li, J., Soriano-Navariro, M., Roussel,
F., Garcia-Verdugo, M.F. and Casaccia-Bonnefil, P. (2006). Loss of p53
induces changes in the behaviour of subventricular cells: implications for the
genesis of glia tumors. J. Neurosci. 26(4): 1107-1116.
33. Gleeson, J.G., Lin, P.T., Flanagan, L.A. and Walsh, C.A. Doublecortin is a
microtubule-associated protein and is expressed widely by migrating
neurons. (1999). Neurons. 23(2):257-271.
103
34. Gould, E., Beylin, P., Tanapat, P., Reeves, A. and Shors, T.J. (1999).
Learning enhances adult neurogenesis in the hippocampal formation. Nat.
Neurosci. 2:260-265.
35. Gould, E., McEwen, B.S., Tanapat, P., Galea, L.A.M. and Fuchs, E. (1997).
Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by
psychosocial stress and NMDA receptor activation. J. Neurosci. 17:2492-
2498.
36. Gould, E., Tanapat, P., McEwen, B.S., Flugge, G. and Fuchs, E. (1997a)
Proliferation of granule cell prescursors in the dentate gyrus of adult
monkeys is diminished by stress. Proc. Natl. Acad. Sci. USA. 95:3168-3171.
37. Gould, E., Tanapat, P., McEwen, B.S., Flugge, G., & Fuchs, E. (1998).
Proliferation of granule cell precursors in the dentate gyrus of adult monkeys
is diminished by stress. Proceedings of the National Academy of Sciences of
the United States of America, 95, 3168-3171.
38. Gritti, A., Bonfanti, L., Doetsch, F., Callie, I., Alvarez-Buylla, A., Lim,
D.A., Galli, R., Garcia Verdugo, J.M., Herrera, D.G. and Vescovi, A.L.
(2002). Multipotent neural stem cells reside into the rostral extension and
olfactory bulb of adult rodents. J. Neurosci., 22: 437-445.
39. Gueneau, G., Privat, A., Drouet, J. and Court, L. (1982). Subgranular Zone
of the Dentate Gyrus of Young Rabbits As a Secondary Matrix.
Developmental Neursci. 5:345-358.
40. Hauser T., Klaus F., Lipp H. and Amrein I. (2009) No effect of running and
laboratory housing on adult hippocampal neurogenesis in wild caught long-
tailed wood mouse. BMC Neursci, 2009, 10:43, 1471-2202.
104
41. Hayes, N.L. and Nowakowski, R.S. (2002). Dynamics of cell proliferation in
the adult dentate gyrus of two inbred strains of mice. Dev. Brain Research.
134: 77-85.
42. Hinds, J.W. (1968). Autoradiography study of histogenesis in the mouse
olfactory bulb. I. Time of origin of neurons and neuroglia. J. Comp. Neurol.
287-304.
43. Ihunwo, A.O. and Pillay, S. (2007). Neurogenic sites in the Adult
Mammalian central nervous system. Research Journal of Biological
Sciences. 2(2): 170- 177.
44. Ihunwo, A.O. and Schliebs, R. (2010). Cell proliferation and total granule
cell number in dentate gyrus of transgenic Tg2576 mouse. Acta neurobiol
Exp. 70: 362-369.
45. Jankovski, A., Rossi, F. and Sotelo, C. (1996). Neuronal precursors in the
post natal mouse cerebellum is fully committed cells: evidence from
heterochronic transplantation. Eur. J. Neurosci. 8: 2308-2320.
46. Jarvi, J.U.M. and Sale, J.B. (1971). Burrowing and burrow and patterns of
East African mole-rats Tachyoryctes, Heliophobius and Heterocephalus. J.
Zool. Lond. 163: 451-479.
47. Kaplan, M.S. and Bell, D.H. (1984). Mitotic neuroblasts in the 9-day old and
11 months old rodents? hippocampus. J. Neurosci. 4: 1429-1441.
48. Kaplan, M.S. and Hinds, J.W. (1997). Neurogenesis in the adult rat. Electron
microscopic analysis of light auto- radiographs. Science 1997: 1092-1095.
105
49. Kee, N., Sivalingam, S., Boonstra, R. and Wojtowicz, J.M. (2002). The
utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. J.
Neurosci. 115:97-105.
50. Kempermann, G. and Gage, F.H. (2002). Genetic influence on phenotypic
differentiation in adult hippocampal neurogenesis. Brain Res. Dev. 134(1-
2):1-12.
51. Kempermann, G. and Kronenberg, G. (2003). Depressed new neurons?
Adult hippocampal neurogenesis and a cellular plasticity hypothesis of major
depression. Biol. Psych. 54(5):499-503.
52. Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M., Gage, F.
(2003). Early determination and long-term persistence of adult generated
new neurons in the hippocampus of mice. Development 130: 391-399.
53. Kempermann, G., H. Georg Kuhn, and Fred H.G. (1997). Genetic Influence
on neuron genesis in the dentate gyrus of adult mice. Proc Natl Acad Sci
USA. 10409- 10414.
54. Kempermann, G., Jessberger, S., Steiner, B., Kronenberg, G. (2004).
Milestones of neuronal development in the adult hippocampus, TINS 27:
446-452 (Abstact).
55. Kim, J.G., Jung, J., Lee, H.J., Kim, J.C., Wang, H., Kim, S.H., Shin, T. and
Moon, C. (2009). Differences in immunoreactivities of Ki-67 and
doublecortin in the adult hippocampus in three strains of mice. Acta
histochemica. 111: 150-156.
56. Kirschenbaum, B., Nedergaard, M., Preuss, A., Barami, K., Fraser, R.A. and
Goldman, S.A. (1994). In vitro neuronal production and differentiation by
106
precursor cells derived from the adult human forebrain. Cereb. Cortex 4:
576-589.
57. Kuhn H.G., Dickson-Anson H., Gage F.H. (1996). Neurogenesis in the
dentate of the adult rat: age-related decrease of neuronal progenitor
proliferation. J Neursci 16:2027-2033.
58. Lagace, D.C., Noonan, M.A and Eisch, A.J. (2007). Hippocampal
neurogenesis: A matter of survival. American Journal of Psychiatry. 164:
205.
59. Lee, J., Duan, W., Mattson, M.P. (2002b). Evidence that brain-derived
neurotrophic factor is required for basal neurogenesis and mediates, in part,
the enhancement of neurogenesis by dietary restriction in the hippocampus
of adult mice. J. Neurochem. 82: 1367-1375.
60. Lee, J., Seroogy, K.B., Mattson, M.P. (2002a). Dietary restriction enhances
neurotrophin expression and neurogenesis in the hippocampus of adult mice.
J. Neurochem. 80: 539-547.
61. Lennington, J.B., Yang, Z. and Conover, J.C. (2003). Neural stem cells and
the regulation of adult neurogenesis (review). Reproductive biol. and
endocrinol. 1: 99.
62. Lievajova, K., Martoncikova, M., Blasko, J., Orendacova, J., Almasiova, V.
and Racekova, E. (2010). Early stress affects neurogenesis in the rats rostral
migratory stream. Cent. Eur. J. Biol.
63. Lois, C. And Alvarez-Buylla, A. (1993). Proliferating subventricular zone
cells in the adult mammalian forebrain can differentiate into neurons and
glia. Proc. Natl. Acad Sci. USA. 90:2074-2077.
107
64. Lu, C.S., Hodge, J.J., Mehren, J., Sun, X.X. and Griffith, L.C. (2003).
Regulation of the Ca2+/CaM- responsive pool of CaMKII scaffold-
dependent autophosphorylation. Neuron 40(6): 1185-1197.
65. Lu, G., Wai, S.M., Poon, W.S. and Yew, D.T. (2005). Ki-67 and
doublecortin positive cells in the human prefrontal cortices of normal aging
and vascular dementia. Microscopy Research and Technique, 68:255-257.
66. Luzzati, F., De Marchis, S., Fasola, A., Panetto, P. (2007). Adult
neurogenesis and local neuronal progenitors in the striatum. Neurodegener.
Dis. 4(4):322-327.
67. Luzzati, F., Marchis, S.D. and Peretto, P. (2006). Neurogenesis in the
caudate nucleus of the adult rabbit. J. Neurosci. 26(2): 609-621.
68. Maini, P.K. (2003). How the mouse got its stripes. Centre for Mathematical
Biology, Mathematical institute. PNAS 100(17): 9656- 9657.
69. McDonald, H.Y. and Wojtowicz, J.M. (2005). Dyanmics of neurogenesis in
the dentate gyrus of adult rats. Neurosc. Lett. 385: 70-75.
70. Miller, M.W. and Nowakowski, R.S. (1988). Use of bromodeoxyuridine
Immunohistochemistry to examine the proliferation, migration and time
origin of cells in the central nervous system. Brain Res. 457: 44-52.
71. Mitra, R., Sundlass, K., Parker, K.J., Schatzberg, A.F. and Lyons, D.M.
(2006). Social stress related behaviour affects hippocampal cell proliferation
in mice. Physiology and Behaviour. 89: 123-127.
72. Moyse, E., Baner, S., Charrier, C., Coronas, V., Krantic, S. and Jean, A.
(2006). Neurogenesis and neuronal stem cells in the dorsal vagal complex of
108
adult rat brain; New vistas about autonomic regulations- a review.
Autonomic neuroscience: Basic and clinical. 126-127: 50- 58.
73. Nacher, J., Rosell, D., Alonso-Llosa, G. And McEwen, B.S. (2001). NMDA
receptor antagonist treatment induces a long-lasting increase in the number
of proliferating cells, PSA-NCAM-immunoreactive granule neurons and
radial glia in the adult rat dentate gyrus. Europ. J. Neursci. 13:512-520.
74. Nichols, N.R., Agolley, D., Zieba, M., Bye, N. (2005). Glucocorticoid
regulation of glia responses during hippocampal neurodegeneration and
regeneration. Brain Res. Rev. 48: 287-301.
75. Nowakowski, R.S., Lewin, S.B. and Miller, M.W. (1998).
Bromodeoxyuridine immunohistochemical determination of the lengths of
the cell cycle and the DNA-synthetic phase for an anatomically defined
population. J. NeuroCytol. 18: 311-318.
76. Okano, H., Islam, M.O., Kanemura, Y., Tajira, J., Mori, H., Kobayashi, S.,
Masayuki, H., Yamasaki, M. and Miyake, J. (2002). Functional expression
of ABCG2 transport in human neural stem/ progenitor cells. Neuroscience
Research. 52(1): 75-82.
77. Pagano, S.F., Impagnatiello, F., Girelli, M., Cova, L., Grioni, E, Onofri, M,
Cavallaro, M., Etteri, S., Vitello, F., Giombini, S., Solero, C.L. and Parati,
E.A. (2000). Isolation and characterization of neural stem cells from the
adult human olfactory bulb. Stem Cells. 18: 295-300.
78. Palmer, T.D., Ray, J. and Gage, F.H. (1995). FGF-2 responsive neural
progenitors reside in proliferative and quiescent regions of the adult rodent
brain. Mol. Cell. Neurosci. 6: 474-486.
109
79. Parent, J.M. (2003). Injury-induced neurogenesis in the adult mammalian
brain. Neuroscientist. 9: 261-272.
80. Pawluski, J.L. and Galea, L.A.M. (2006). Hippocampal morphology is
differentially affected by reproductive experience. J. of Neurobiol. 66: 71-81.
81. Paxinos, G. and Watson, C. (2006). The rat brain in stereotaxic coordinates.
6th ed. Elsevier, Amsterdam.
82. Peterson M. & Leblond C.P. (1964) Synthesis of complex carboydrates in
the Golgi region, as shown by radioautography after injection of labeled
glucose. J. Cell Biol. 21:143?148.
83. Pham, K., Nacher, J., Hof, P.R. and McEwen, B.S. (2003) Repeated restraint
stress suppresses neurogenesis PSA-NCAM expression in adult rat dentate
gyrus. Europ. J. of Neurosci. 17: 879-886.
84. Piechl, L. (2004).Too good eyes for living below ground. Max Planck
society. New B (19).
85. Pillay, N. (2000). Female mate preference and reproductive isolation in
populations of the striped-mouse, Rhabdomys pumilio. J. of Behaviour.
137:1431-1441.
86. Plumpe, T., Ehninger, D., Steiner, B., Klempin, F., Jessberger, S., Brandt,
M., Romer, B., Rodriguez, G.R., Kronenberg, G. and Kempermann, G.
(2006). Variability of doublecortin-associated dendrite maturation in adult
hippocampal neurogenesis is independent of the regulation of precursor cell
proliferation. BMC Neurosc. 7(77): 1-14.
87. Rakic P. (2002). Neurogenesis in adult primate neocortex: an evaluation of
the evidence. Nat Rev Neurosci 3:65-71.
110
88. Rakic P. and Nowakowski R.S. (1981). The time of origin of neurons in the
hippocampal region of the rhesus monkey. J Comp Neurol 196: 99-128.
89. Reynolds, B.A. and Weiss, S. (1992). Generation of neurons and astrocytes
from isolated cells of the adult mammalian CNS. Science, 255: 1707-1710.
90. Scharfman, H., Goodman, J., Macleod, A., Phani, S., Antonelli, C., Croll, S.
(2005). Increased neurogenesis and the ectopic granule cells after
intrahippocampal BDNF infusion in adult rats. Exp. Neurol. 192: 248-256.
91. Schauwecker, P.E. (2006). Genetic influence on neurogenesis in the dentate
gyrus of two strains of adult mice. Brain Research. 1120: 83-92.
92. Schradin, C. and Pillay, N. (2004). The striped mice (Rhabdomys pumilio)
from the succulent Karoo of South Africa: A territorial group living solitary
forager with communal breeding and helpers at the nest. Journal of
Comparative Psychology. 118: 37-47.
93. Schradin, C. And Pillay, N. (2005a). Intraspecific variation in the spatial and
social organization of the African striped-mouse. J. Of Mammol. 86:99-107.
94. Schradin, C. And Pillay, N. (2005b). The influence on the father on offspring
development in the striped-mouse. Behavioural Ecology. 16:450-455.
95. Seaberg, R.M. and Van der Kooy, D. (2002). Adult rodent neurogenic
regions: The ventricular subependymal contains neural stem cells, but the
dentate gyrus contains restricted progenitors. J. Neurosci., 22: 1784-1793.
96. Selvakumar, C.R. Four-striped grass mouse.
www.thewebsiteofeverything.com
97. Siwak-Tapp C.T., Head E., Muggenburg B.A., Milgram N.W. and Cotman
C.W. (2007). Neurogenesis decreases with age in the canine hippocampus
111
and correlates with cognitive function. Neurobiol Learn Mem. 88(2):249-
59.Epub 2007 Jun 22.
98. Skinner, J.D. and Chimimba, C.T. (2005) Mammals of the Southern African
Sub-region. Cambridge University Press, Cambridge.
99. Takemura, N.U. (2005). Evidence for neurogenesis within the white matter
beneath the temporal neocortex of the adult rat brain. Neurosci. 134: 121-
132.
100. Taupin P. (2007). BrdU Immunohistochemistry for Studying Adult
Neurogenesis: paradigms, pitfalls, limitations, and validation. Brain
Research Reviews, 53 (1), p.198-214.
101. Taupin, P. (2006a). Neurogenesis in the adult central nervous system. C.R.
Biology. 134: 121-132.
102. Taupin, P. (2006b). The therapeutic potential of the adult neural stem
cells: Current opinion in Molecular Therapeutic 8(3): 225- 231.
103. Temple, S. and Alvarez-Buylla, A. (1999). Stem cells in the adult
mammalian central nevous system. Current Opinion in Neurogiol. 9: 135-
141.
104. Treves, A., Tashiro, A., Witter, E.M. and Moser, E.I. (2008). What is the
mammalian dentate gyrus good for? Neuroscience. doi.
10:1016/j.neuroscience.2008.04.073.
105. van Praag, H., Kempermann, G. & Gage, F. H. (1999). Running increases
cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nature
Neurosci. 2, 266?-270.
112
106. Vollmayr, B., Mahlstedt, M.M., Henn, F.A. (2007). Neurogenesis and
depression: what animal models tell us about the link? Eur. Arch. Psychiatry
Clin. Neurosci. 257, 300-303.
107. Vollmayr, B., Simonis, C., Weber, S., Gass, P., Henn, F. (2003). Reduced
cell proliferation in the dentate gyrus is not correlated with the development
of learned helplessness. Biol. Psychiatry 54, 1035-1040.
108. Warne-Schmidt, J.L., Duman, R.S. (2006). Hippocampal neurogenesis:
opposing effects of stress and antidepressant treatment. Hippocampus 16,
239-249.
109. Watts, C., McConkey H., Anderson, L. and Caldwell, M. (2005).
Anatomical perspectives on adult neural stem cells. J. Anat. 207: 197-208.
110. Weiss, S., Dunne, C., Hewson, J., Wohl, H., Wheatley, M., Peterson, A.C.
and Reynolds, B.A. (1996). Multipotent CNS stem cells are present in the
adult mammalian spinal cord and ventricular neuroaxis. J. Neurosci. 16:
7599-7609.
111. Willan, K. And Meester, J. (1989). Food deprivation and drinking in two
African rodents, Mastomys natalensis and Phadomys pumilio. South African
of Zoology. 22:190-194.
112. Wyss, J.M. and Sripanidkulchai, B. (1985). Localization of renal -
adrenoceptors receptors in the rat kidney. (Abstract) J.Cell Biol. 101:103.
113. Xu, Y., Tamamaki, N., Noda, T., Kimura, K., Itokazu, Y., Matsumoto, N.,
Dezawa, C. and Ide, M. (2005). Neurogenesis in the ependymal layer of the
adult rat 3rd ventricle. Exp. Neurol., 192: 251-264.
113
114. Yom-Tov, Y. (2003). Body sizes of carnivores commensal with humans
have increase over the past 50 years. Functional Ecology. 17: 323-327.
115. Zhang, X., Cai, Y., Chu, Y., Chen, E., Feng, J., Luo, X., Xiong, K.,
Struble, R.G., Clough, R.W., Patrylo, P.R., Kordower, J.H. and Yan, X.
(2009). Doublecortin-expressing cells in the associative cerebral cortex and
amygdala in aged nonhuman primates. Frontiers in Neuroanat. 3(17):1-12.
116. Zhao M.S., Momma S., Delfani K., Carlen M. and Casidy R.M. (2003).
Evidence for neurogenesis in the adult mammalian substantia nigra. Proc.
Natl. Acad. Sci., USA. 100: 7925-7930.