Neuroscience 238 (2013) 270–279
ADULT NEUROGENESIS IN A GIANT OTTER SHREW (POTAMOGALEVELOX)
N. PATZKE, a* C. KASWERA, b E. GILISSEN, c,d,e
A. O. IHUNWO a AND P. R. MANGER a
aSchool of Anatomical Sciences, Faculty of Health
Sciences, University of the Witwatersrand, 7 York Road,
Parktown, 2193 Johannesburg, South Africa
bFaculte des Sciences, University of Kisangani, B.P. 1232,
Kisangani, Democratic Republic of the Congo
cDepartment of African Zoology, Royal Museum for Central Africa,
Leuvensesteenweg 13, B-3080 Tervuren, BelgiumdLaboratory of Histology and Neuropathology, Universite Libre de
Bruxelles, 1070 Brussels, Belgium
eDepartment of Anthropology, University of Arkansas,
Fayetteville, AR 72701, USA
Abstract—Adult neurogenesis in mammals is typically
observed in the subgranular zone of the hippocampal den-
tate gyrus and the subventricular zone. We investigated
adult neurogenesis in the brain of a giant otter shrew (Pot-
amogale velox), a semi-aquatic, central African rainforest
mammal of the family Tenrecidae that belongs to the super-
order Afrotheria. We examined neurogenesis immunohisto-
chemically, using the endogenous marker doublecortin
(DCX), which stains neuronal precursor cells and immature
neurons. Our results revealed densely packed DCX-positive
cells in the entire extent of the subventricular zone from
where cells migrated along the rostral migratory stream to
the olfactory bulb. In the olfactory bulb, DCX-expressing
cells were primarily present in the granular cell layer with
radially orientated dendrites and in the glomerular layer
representing periglomerular cells. In the hippocampus,
DCX-positive cells were identified in the subgranular and
granular layers of the dentate gyrus and strongly labelled
DCX-positive processes, presumably dendrites and axons
of the newly generated granular cells, were observed in
the CA3 regions. In addition, DCX immunoreactive cells
were present in the olfactory tubercle, the piriform cortex
and the endopiriform nucleus. While DCX-positive fibres
have been previously observed in the anterior commissure
0306-4522/13 $36.00 � 2013 IBRO. Published by Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.neuroscience.2013.02.025
*Corresponding author. Tel: +27-(0)11-7172137; fax: +27-(0)86-7655101.
E-mail address: [email protected] (N. Patzke).Abbreviations: 3V, third ventricle; ac, anterior commissure; BSA,bovine serum albumin; C, caudate nucleus; CA, cornu ammonis; CMS,caudoventral migratory stream; DAB, diaminobenzidine; DCX,doublecortin; DG, dentate gyrus; DT, dorsal thalamus; EN,endopiriform nucleus; EPL, external plexiform layer of olfactory bulb;f, fornix; GCL, granular cell layer of olfactory bulb; GL, glomerular layerof olfactory bulb; Hbm, medial habenular nucleus; ICj, islands ofCalleja; ICjM, major island of Calleja; LV, lateral ventricle; MCL, mitralcell layer of olfactory bulb; N.Acc, nucleus accumbens; NRS, normalrabbit serum; OB, olfactory bulb; ON, olfactory nerve; PB, phosphatebuffer; PIR, piriform cortex; PVL, periventricular layer of olfactory bulb;RMS, rostral migratory stream; SGZ, subgranular zone; SVZ,subventricular zone; TOL, olfactory tubercle.
270
of the hedgehog and mole, we were able to demonstrate
the presence of DCX-positive cells presumably migrating
across the anterior commissure. Taken together, the giant
otter shrew reveals patterns of neurogenesis similar to that
seen in other mammals; however, the appearance of possi-
ble neuronal precursor cells in the anterior commissure is a
novel observation. � 2013 IBRO. Published by Elsevier Ltd.
All rights reserved.
Key words: adult neurogenesis, Afrotheria, anterior commis-
sure, doublecortin, immunohistochemistry.
INTRODUCTION
The generation of new neurons in the adult brain across
vertebrate species is a widely accepted phenomenon
(Lindsey and Tropepe, 2006; Barker et al., 2011). In
mammals, the generation of new neurons is most
commonly observed in the subgranular zone of the
hippocampal dentate gyrus, and the subventricular
zone from where cells migrate along the rostral
migratory stream to the olfactory bulb (Ming and Song,
2011). In addition, there is evidence for the existence of
newly generated neurons in several regions, including
the neocortex, piriform cortex, striatum, amygdala,
substantia nigra, and hypothalamus; however, the
results are inconsistent and difficult to replicate
consistently (Gould, 2007). A major problem is that
studies on mammalian adult neurogenesis are for the
most part conducted on a few species of laboratory
rodents (Bonfanti et al., 2011) and therefore probably do
not reflect the whole spectrum of neurogenetic potential
in the adult mammalian brain, limiting our view to the
hippocampus and the olfactory bulb (Barnea, 2010). To
date adult neurogenesis has been examined only in a
few species of mammals (Kempermann, 2012) relative
to the total number of mammalian species. Therefore it
is likely that different mammalian species will show a
greater diversity of neurogenetic origins and end sites in
different brain regions.
The functional significance of neurogenesis in the
healthy adult mammalian brain is largely unknown, but
clues augmenting our growing understanding of this
phenomenon may be obtained by examining a broader
range of species. A set of detailed studies would reveal
not only similarities, but also differences among species.
These differences are likely to be particularly useful to
correlate with differences in ecology or behaviour of the
animals, thus providing a powerful tool to approach a
functional understanding of adult neurogenesis. Such a
d.
Fig. 1. Photomicrographs showing the dentate gyrus (DG) of the P.velox. (A) Nissl staining of the DG. (B) DCX immunoreactivity revealed
a very high number of DCX-positive cells in the subgranular zone and
the granular layer of theDG.DCX-positive processeswere observed in
the hilus and inferior to the stratum pyramidale of the cornu ammonis
(CA3). (C) High-power photomicrograph of DCX-positive cells located
in the subgranular zone and the granular layer of the DG. Dorsal
thalamus (DT), medial habenula (Hbm), third ventricle (3V). Scale bar
in B = 500 lm and applies to A. Scale bar in C = 100 lm.
N. Patzke et al. / Neuroscience 238 (2013) 270–279 271
comparative approach can further provide clues leading
to a better understanding of the role, dynamics, and
mechanisms of adult neurogenesis.
Under this rationale we investigated the occurrence,
extent and topography of adult neurogenesis in the brain
of a natural living giant otter shrew (Potamogale velox),which is the single species of the genus Potamogale
belonging to the family Tenrecidae, order Afroscoricida,
which is part of the Afrotherian clade (Tabuce et al.,
2008). The giant otter shrew is a semi-aquatic mammal
that lives in the main rainforest block of Central Africa
along fast-flowing rivers and streams, sluggish lowland
streams and forest pools (Nowak, 1999; Kingdon, 2004).
The giant otter shrew is solitary, becomes active in the
late afternoon and continues activity into the night
(vespertine/nocturnal) and feeds mainly on crabs, fish
and amphibians (Nowak, 1999). Since environmental
conditions have been demonstrated to have an impact on
neurogenesis (Kempermann, 2011), P. velox, with its
diverse (both land and water), variable, information rich
habitats makes it an interesting object of study.
EXPERIMENTAL PROCEDURES
Specimen and tissue preparation
In the current study we used only a single male giant otter shrew
(P. velox), which was captured in the Yoko rainforest, near
Kisangani in the Democratic Republic of the Congo. The animal
was captured using a Fyke net with the cod-end collection
funnel suspended above the water allowing the captured
animal to breathe. We were only able to capture one specimen
of this species during the field season, and due to the small
number of previous reports detailing aspects of the brain of this
species, and the likelihood that few will appear in the future, we
felt that the current report was warranted. As the specimen was
caught from the wild it is difficult to assess its age precisely;
however, as we are interested in adult neurogenesis, it is
important to know, at the very least, if the animal is fully mature
and can be considered an adult. In order to assess the
developmental status of this individual, we compared the body
mass and length of our specimen with data obtained from
previously published literature. The body mass of our specimen
was 540 g, with a brain mass of 3.46 g. In the literature two
body mass estimates for adult P. velox have been provided,
and these vary from between 340–397 g (Nowak, 1999) and
300–950 g (Kingdon, 2004). According to body mass, our P.velox specimen was fully mature and hence an adult. This was
also confirmed by the condition of dental eruption. The body
length of our specimen was 36.5 cm, which compared well with
the available body lengths of adult P. velox in the literature,
which are given as 29–35 cm (Nowak, 1999; Kingdon, 2004). In
contrast to this, the published data on the adult tail length of P.velox are inconsistent, where Nowak (1999) reports a length of
245–290 mm and Kingdon (2004) a length of 4.5–9 cm. Our
specimen had a tail length of 15 cm. Thus, in all three
comparable aspects of body size, it would appear that our
specimen is clearly an adult animal. The harvesting and use of
the specimen was approved by the University of the
Witwatersrand Animal Ethics Committee (2008/36/1).
To minimise any external influences on adult neurogenesis,
the animal was immediately anaesthetised following capture and
perfused through the left ventricle with 0.9% saline, followed by
4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4).
The brain was removed and post-fixed in 4% paraformaldehyde
overnight, cryoprotected in 30% sucrose in 0.1 M PB at 4 �C and
stored in an antifreeze at �20 �C until sectioning. Before
sectioning, the tissue was allowed to equilibrate in 30% sucrose
272 N. Patzke et al. / Neuroscience 238 (2013) 270–279
in 0.1 M PB at 4 �C. The specimen was frozen in crushed dry ice
and sectioned in the frontal plane into 50-lm-thick sections on a
sliding microtome.
Tissue staining and immunohistochemistry
To examine adult neurogenesis in the brain of the giant otter
shrew, we used immunohistochemistry to the endogenous
marker doublecortin (DCX) and Ki-67. DCX is a microtubule-
associated phosphoprotein, which is expressed in actively
dividing neuronal precursor cells and their neuronal daughter
cells for up to 2–3 weeks. The expression of DCX is down
regulated after approximately 2 weeks, with the onset of the
expression of NeuN, a marker for mature neurons (Brown
et al., 2003; Rao and Shetty, 2004). Ki-67, an endogenous
protein expressed in dividing cells during late G1, S, G2 and M
phases of the cell cycles in all mammalian species is typically
used to identify proliferating cells (Scholzen and Gerdes, 2000).
The advantage of using DCX and Ki-67 to localise adult
neurogenesis is that no pre-handling of the animal is needed,
reducing potential confounding influences. The visualisation of
DCX-positive cell also provides an average of the rate of
expression of new neurons in natural conditions prior to capture
of the animal (Bartkowska et al., 2010). To visualise DCX we
used the goat-anti DCX C-18 primary antibody from Santa Cruz
Biotechnology, Dallas, Texas, USA since this antibody has
been demonstrated to provide a more distinct and intense
labelling both in rodents (Brown et al., 2003) and humans (Liu
et al., 2008) than other commercially available antibodies. To
attempt to visualise Ki-67 positive cells we used the rabbit anti-
Ki-67 antibody (NCL-Ki-67 P, Dako), unfortunately, no reactivity
to the Ki-67 antibody was observed in the current study, and in
our experience, Ki-67 immunoreactivity using this antibody is
limited to rodents, megachiropterans and primates, with
species falling within the Afrotheria not demonstrating any
specific staining (Ngwenya et al., 2011).
The entire brain was sectioned into a 1 in 10 series. The first
section of each series was mounted on 0.5% gelatine-coated
slides, dried overnight, cleared in a 1:1 mixture of 100%
ethanol and 100% chloroform and stained with 1% Cresyl
Violet. The second section of each series was mounted on 1%
gelatine-coated slides, dried and then stained with a modified
silver stain to reveal myelinated structures (Gallyas, 1979).
Every 10th section of the series was used for free-floating DCX
immunohistochemistry. The sections were incubated in a 1.6%
H2O2, 49.2% methanol, 49.2% 0.1 M PB solution, for 30 min to
reduce endogenous peroxidase activity, which was followed by
three 10-min rinses in 0.1 M PB. To block unspecific binding
sites the sections were then pre-incubated for 2 h, at room
temperature, in blocking buffer (3% normal rabbit serum, NRS,
2% bovine serum albumin, BSA, and 0.25% Triton X-100 in
0.1 M PB). Thereafter, the sections were incubated for 48 h at
4 �C in the primary antibody solution (1:300, goat anti-
doublecortin, DCX, SC-18 Santa Cruz Biotech, Dallas, Texas,
USA) under gentle agitation. The primary antibody incubation
was followed by three 10-min rinses in 0.1 M PB and the
sections were then incubated in a secondary antibody solution
(1:1000 dilution of biotinylated anti-goat IgG, BA 5000, Vector
Labs, Burlingame, California, USA in 3% NRS and 2% BSA in
0.1 M PB) for 2 h at room temperature. This was followed by
three 10-min rinses in 0.1 M PB, after which sections were
incubated for 1 h in an avidin–biotin solution (1:125; Vector
Labs, Burlingame, California, USA), followed by three 10-min
rinses in 0.1 M PB. Sections were then placed in a solution
containing 0.05% diaminobenzidine (DAB) in 0.1 M PB for
5 min, followed by the addition of 3.3 ll of 30% hydrogen
peroxide per 1 ml of DAB solution. Chromatic precipitation was
visually monitored under a low power stereomicroscope.
Staining continued until such time as the background stain
was at a level that would allow for accurate architectonic
matching to the Nissl and myelin sections without obscuring the
immunopositive structures. Development was arrested by
placing sections in 0.1 M PB for 10 min, followed by two more
rinses in this solution. Sections were then mounted on 0.5%
gelatine-coated glass slides, dried overnight, dehydrated in a
graded series of alcohols, cleared in xylene and cover-
slipped with Depex. To ensure non-specific staining of the
immunohistochemical protocol, we ran tests on sections where
we omitted the primary antibody, and sections where we
omitted the secondary antibody. In both cases no staining was
observed. The observed immunostaining patterns support the
specificity of the antibodies for the antigens in P. velox as they
are compatible with observations made in other species (e.g.
Shapiro et al., 2007; Alpar et al., 2010; Bartkowska et al.,
2010). It was not possible to undertake Western blot control
testing in the P. velox material. Digital photomicrographs were
captured using Zeiss Axioshop and Axiovision software. No
pixilation adjustments, or manipulation of the captured images
was undertaken, except for the adjustment of contrast,
brightness, and levels using Adobe Photoshop 7.
RESULTS
In the present study we examined neurogenesis
immunohistochemically, using the endogenous marker
DCX in P. velox. Our staining revealed the two
commonly known neurogenic areas, the subgranular
zone of the dentate gyrus in the hippocampal formation,
and the subventricular zone of the lateral ventricle that
gives rise to the rostral migratory stream ending in the
olfactory bulb. Furthermore the presence of DCX-
positive cells provided evidence of migrating and
immature neurons in several other brain regions.
DCX immunostaining in the hippocampal formation
In the hippocampal formation a seemingly very large
number of DCX-positive neurons were identified at the
base of the granular layers (GL) and some DCX-positive
cells were observed in the subgranular zone (SGZ) of
the dentate gyrus (DG) (Fig. 1). The DCX positive cells
in the GL were characterised by large somata with long
apical dendrites extending to and ramifying within the
molecular layer (Fig. 1c). In the hilus of the DG, loosely
packed DCX-positive fibres were found. In addition,
densely packed DCX-positive processes were observed
inferior to the stratum pyramidale of the cornu ammonis
(CA3), presumably mossy fibres of the newly generated
granular cells (Fig. 1b).
DCX immunostaining in the subventricular zone ofthe lateral ventricle
DCX-immunopositive cells were present in the
subventricular zone (SVZ) along the entire rostrocaudal
extent of the lateral ventricle; however, the density of
DCX-positive cells was low at the caudal portion and
increased towards the rostral end. In the caudal part of
the lateral ventricle a few clusters and single DCX-
positive cells were predominantly found in the lateral
wall in the SVZ with the highest density at the ventral
part of the lateral ventricle. The density of DCX-positive
cell increased gradually towards the rostral end of the
lateral ventricle. At the plane of the anterior pole of the
hippocampus, the complete SVZ of the lateral ventricle
Fig. 2. Photomicrographs showing the olfactory bulb (OB) of the P. velox. (A) Nissl stained sections showing the layers of the OB. (B) DCX-positive
cells were observed in the periventricular layer (PVL) the granule cell layer (GCL) and the glomerular layer (GL), external plexiform layer (EPL),
mitral cell layer (MCL), olfactory nerve (ON). Scale bar in B = 500 lm and applies to A.
N. Patzke et al. / Neuroscience 238 (2013) 270–279 273
was lined with densely packed clusters of DCX-positive
cells, here again with the highest density in the ventral
part of the lateral ventricle.
Rostral migratory stream to the olfactory bulb
From the SVZ of the lateral ventricle two main streams of
DCX cells could be observed, the rostral migratory stream
(RMS) and the caudoventral migratory stream (CMS –
see below). In the rostral portion of the lateral ventricle
clusters of densely packed DCX-labelled cells appeared
to migrate from the SVZ along the RMS towards the
olfactory bulb (OB). The RMS consisted of a single
stream formed by densely packed DCX-expressing
neuroblasts. DCX-positive cells and processes could be
observed in almost all layers of the OB with different
expression patterns. In the most inner layers of the OB,
the ependymal layer and the periventricular layer, that
surround the olfactory ventricle, densely packed and
tangentially orientated DCX-expressing cells and
processes were seen (Fig. 2). DCX immunopositive
radially orientated dendrites were primarily present in
the GCL layer. Only a few DCX-positive cells were
observed in the CGL, probably due to the loss of DCX
expression, as a result of their maturation. Some DXC-
immunopositive cells were observed in the glomerular
layer, representing periglomerular cells; however, a few
neuronal precursor cells were observed in all olfactory
bulb layers (Fig. 2).
DCX immunostaining in the caudoventral migratorystream
At the level of the caudal third of the piriform cortex (PIR),
loosely packed or single DCX-positive cells were
observed from the SVZ of the caudal portion of the
lateral ventricle along the CMS (Fig. 3D). These cells
appear to migrate through white and grey matter
towards the endopiriform nucleus (EN) and the piriform
cortex, where DCX-positive cells and processes were
present (Fig. 3B). The CMS ceased rostral to the
decussation of the anterior commissure, and further
rostral to this level no DCX-positive cells could be
observed in the EN or PIR. In the EN a few loosely
packed DCX-positive cells were observed, and these
were characterised by large, almost oval, cell bodies
with a loosely arranged network of long dendrites
(Fig. 3B). DCX-expressing cells in the EN were bipolar
or multipolar. In the PIR DCX-positive cells were
predominantly present in layer II, but a few cells were
observed in layer III. As in the EN these cells showed
large, almost oval, cell bodies; however, they were more
densely packed, in cluster-like structures, with a dense
network of dendrites (Fig. 3B). Some of the DCX-
positive dendrites extended into layer I. DCX-expressing
cells in the PIR were mostly bipolar or multi-polar, but
occasionally unipolar cells were observed.
Additional migratory streams originating from theSVZ of the lateral ventricle and their destinations
In addition to the two main migratory streams, three small
streams appear to originate from the SVZ of the lateral
ventricle. Anterior to the decussation of the anterior
commissure (ac) three small streams of DCX cells could
be observed. The mediorostral stream runs from the
ventrolateral tip of the lateral ventricle, laterally through
the white matter and the putamen towards the olfactory
tubercle (TOL) and the anterior commissure. The
mediomedial stream appears to also originate from the
ventrolateral tip of the lateral ventricle; however, here
the DCX-positive cells appear to migrate through
the internal capsule towards the ac and the OT. The
medioventral migratory stream runs from the SVZ of the
ventral tip of the lateral ventricle through the white
Fig. 3. Photomicrographs showing the piriform cortex (PIR) and the nucleus endopiriformis (EN) of the P. velox. (A) Nissl staining of the PIR and the
EN and (C) the lateral ventricle (LV). (B) In PIR and EN DCX-positive cells were present. (C) These cells appear to migrate through white and grey
matter towards the nucleus endopiriformis (EN) or the PIR along the caudoventral migratory stream (CMS). Scale bar in D = 500 lm and applies to
A, B and C.
274 N. Patzke et al. / Neuroscience 238 (2013) 270–279
matter medial the caudate nucleus (C) and lateral to the
nucleus accumbens (N.Acc) towards the TOL. At the
level of islands of Calleja (ICj) and its major (ICjM)
subdivision a branch of DCX-positives cell splits to an
additional stream that presumably migrates medially to
the nucleus accumbens towards the ICj, ICjM and the
TOL, and ceases at the most rostral end of the ICjM.
Further rostral, where the lateral ventricle ends and
the RMS begins, all three streams appear to persist;
however, the presumably migratory cells are supplied by
the RMS and not the SVZ (Fig. 4). The streams cease
at the most rostral level of the TOL.
In the TOL and the ICjM very few DCX-expressing
cells and processes were present. In both areas the
DCX-positive cells were either bipolar or unipolar with
fusiform-shaped cell bodies.
In the ac DCX-positive cells were observed (Fig. 5).
These DCX-positive cells and processes were only present
rostral to the level of the decussation of the ac. No cells or
processes were observed caudal to this level. These DCX
cells had the typical fusiform morphology of migrating cells
with trailing and/or leading processes (Fig. 5C). The DCX-
positive cells and fibres were predominantly present in the
lateral portion of the ac (Fig. 4B).
DISCUSSION
In the current study we observed the two commonly
identified regions of adult neurogenesis in mammals,
these being the subgranular zone of the dentate gyrus
in the hippocampal formation and the subventricular
zone of the lateral ventricle that gives rise to the rostral
Fig. 4. Photomicrographs showing the rostral migratory stream (RMS) of the P. velox. (A) Nissl staining of the RMS. DCX immunoreactivity
revealed additional migratory stream branching from the RMS: the mediorostral migratory stream running towards the anterior commissure (AC);
the mediomedial migratory stream and the medioventral migratory stream running around the nucleus caudate (C) towards the olfactory tubercle
(TOL). Scale bar in B = 500 lm and applies to A.
N. Patzke et al. / Neuroscience 238 (2013) 270–279 275
migratory stream which ends in the olfactory bulb. In
addition to this, the immunohistochemical staining for
DCX provided evidence of presumably migrating and
maturing neurons in several other brain regions
including the caudoventral migratory stream ending in
the piriform cortex and the endopiriform nucleus, a
mediorostral and a mediomedial stream that decussated
through the anterior commissure, and a medioventral
stream that terminated in the islands of Calleja and the
olfactory tubercle. No Ki-67-immunopositive cells were
observed in the present study; however, this appears to
be related to the phylogenetic specificity of the antibody,
as our previously published (Ngwenya et al., 2011) and
unpublished studies have shown that immunoreactivity
to the DAKO Ki-67 antibody (NCL-Ki-67 P) is limited to
rodents, megachiropterans and primates, and does not
react with this molecule in the Afrotherians.
Neurogenesis in the hippocampal formation
The mammalian dentate gyrus of the hippocampal
formation is known to be continuously invested with
newly generated neurons throughout life. These
neurons are generated in the subgranular zone of the
DG, migrate from there into the granular layer and
become functionally integrated. Several studies on
captive-bred laboratory rodents demonstrated that an
enriched environment as well as exercise up-regulates
neurogenesis (Kempermann et al., 1997a,b; van Praag
et al., 1999a,b; Olson et al., 2006; Snyder et al., 2009),
whereas stress (Gould and Cameron, 1996; Gould
et al., 1998; Pham et al., 2003; Warner-Schmidt and
Duman, 2006) and impaired environmental and social
conditions (Lu et al., 2003) reduce neurogenesis in the
hippocampus. Moreover, it was demonstrated that adult
neurogenesis in the DG of laboratory rodents varies with
age and the strain of the animals (Kuhn et al., 1996;
Kempermann et al., 1997b; Knoth et al., 2010). To date
only few studies have been carried out on wild living
animals (Bonfanti et al., 2011). In line with the results
from laboratory animals, studies on wild-living mice and
rats also demonstrated a negative correlation of ageing
and neurogenesis (Amrein et al., 2004; Epp et al.,
2009). In addition, the neurogenetic rate in the adult
hippocampus seems to be species-specific. For
example, two species of wood mouse had proliferation
rates two times greater than bank and pine voles
(Amrein et al., 2004), whereas chipmunks showed a
lower proliferation rate than squirrels (Barker et al.,
2005). On the other hand, one comparative study did
not demonstrate any significant difference of the
proliferation rate in the adult hippocampus between one
strain of wild-living rats and three strains of captive bred
rats (Epp et al., 2009). Besides the negative ageing
effect on adult hippocampal neurogenesis in rodents,
neither the effect of environmental conditions nor
exercise could be demonstrated in one strain of wild
living mice (Hauser et al., 2009), indicating that, at least
in part, the results from studies on laboratory mice
cannot be easily translated to other animals, including
humans, and must be interpreted with caution.
To date only few studies have been conducted on other
non-laboratory mammals with varying results. Adult
hippocampal neurogenesis was observed in the order of
Chiroptera; whereby one species of megachiropteran
revealed a low level of proliferating cells (Gatom et al.,
2010) and nine of 12 microchiropteran species examined
did not reveal any neurogenesis (Amrein et al., 2007).
Adult hippocampal neurogenesis was also observed in
the order Eulipotyphla: hedgehog and mole (Bartkowska
et al., 2010) and in the Sorex shrews (Bartkowska et al.,
2008); in the order Lagomorpha: rabbit (Zhu et al., 2003);
in the order Artiodactyla: pig and sheep (Zhu et al., 2003;
Guidi et al., 2011); in the order Carnivora: the red fox
Fig. 5. Photomicrographs showing the anterior commissure (AC) of
the P. velox. (A) Nissl staining of the AC. (B) DCX immunoreactivity
revealed immature neurons migrating along the AC to the contralat-
eral hemisphere. (C) High-power photomicrograph of DCX-positive
cells in the AC. These DCX cells revealed a typical fusiform
morphology of migrating immature neurons with trailing and/or
leading processes (see arrows). Scale bar in B = 500 lm and
applies to A. Scale bar in C = 100 lm.
276 N. Patzke et al. / Neuroscience 238 (2013) 270–279
(Amrein and Slomianka, 2010) and the domestic dog
(Siwak-Tapp et al., 2007); in the order Afrosoricida;
hedgehog tenrec (Alpar et al., 2010); and in the order
Scandentia: tree shrews (Gould et al., 1997).
Furthermore, adult hippocampal neurogenesis was
observed in New World monkeys (Gould et al., 1998;
Leuner et al., 2007), Old World primates (Gould et al.,
1999; Kornack and Rakic, 1999), and humans (Eriksson
et al., 1998; Knoth et al. 2010), as well as in two
marsupial species, the fat-tailed dunnart (Harman et al.,
2003) and the Monodelphis opossum (Grabiec et al.,
2009). Even if some of the listed mammalian species
show a low rate of hippocampal neurogenesis, with the
exception of some microchiropteran species where no
neurogenesis could be observed, it appears that adult
hippocampal neurogenesis is a trait that appears to be
present in many mammalian species, but examining a
broader range of species may provide clues leading to a
better understanding of the role of neurogenesis in the
hippocampus.
With this study we are adding another animal to the
short list of species, outside the order Rodentia, analysed
to date. Interestingly the wild-living P. velox revealed a
very high number of DCX-positive cells in the DG where
even the mossy fibres, the axons of the newly generated
granular cells, were DCX immunopositive. Although we
cannot state the exact age of this individual, the mass,
the length, and the dental eruption of the animal strongly
suggest that it was an adult. The functional relevance of
these newly generated neurons in the DG is largely
unknown (Leuner et al., 2006); however, some studies
provide evidence that they might be important for learning
and memory formation, especially spatial memory
(Snyder et al., 2005; Dupret et al., 2008). Moreover, the
high rate of neurogenesis in P. velox might be positively
correlated with its complex environment. First, P. velox is
a semi-aquatic animal living on both land and water. This
lifestyle might require a strong ability for spatial
navigation, resulting in a higher demand/survival of newly
generated granule cells in the DG. Second, the high rate
of neurogenesis in the DG might be a result of the
constantly altering environmental complexity between
underwater and terrestrial environments, as an enriched
environment was demonstrated to increase the
neurogenetic rate.
Neurogenesis in the olfactory areas
It is well known that the olfactory bulb continually
incorporates new neurons during adulthood. The
precursor cells of these neurons are generated in the
SVZ of the lateral ventricle and migrate along the RMS
to the olfactory bulb from where they radially migrate to
the granular and glomerular layers to become
functionally integrated in the olfactory bulb circuitry
(Peretto et al., 1997; Bedard and Parent, 2004; Lledo
et al., 2006). This continuous repopulation of the OB
with newly generated neurons throughout life has been
demonstrated to occur in every single species studied to
date (e.g. Pencea et al., 2001; Bedard et al., 2002;
Bedard and Parent, 2004; Curtis et al., 2007; Ngwenya
et al., 2011); however, the occurrence of newly
generated cells in the human adult OB is still under
debate (Bergmann et al., 2012). In P. velox densely
packed DCX positive neuroblasts were observed to
N. Patzke et al. / Neuroscience 238 (2013) 270–279 277
migrate from the SVZ along the RMS to the OB; where
DCX-positive cells were predominantly found in the
granule cell layer.
Besides the OB, DCX-expressing cells in P. veloxwere also found in several secondary olfactory
structures: in layer II of the piriform cortex, in the
endopiriform nucleus and in the olfactory tubercle. The
occurrence of DCX cell in the PIR is in line with
previous data in mice (Shapiro et al., 2007), rat (Pekcec
et al., 2006; Shapiro et al., 2007), the hedgehog tenrec
(Alpar et al., 2010), moles and hedgehogs (Bartkowska
et al., 2010), and primates (Gould et al., 1999; Bernier
et al., 2002). In P. velox the newly generated neurons
appear to emanate from the SVZ of the caudoventral
portion of the lateral ventricle and migrate along the
CMS to the piriform cortex. A similar migration was
observed in rodents (Shapiro et al., 2007) and in non
human primates (Bernier et al., 2002). Both studies
report a migration of cells from the subventricular zone,
also referred to as the subependymal zone, to the
piriform cortex. Whereas Shapiro et al. (2007) report
that newly generated cells migrating to the rostral
piriform cortex traverse the ventrolateral migratory
stream, which splits from the RMS, an analogous
stream was not observed in P. velox. Shapiro et al.
(2007) also proposed the possibility of a second
migratory stream, where newly generated neurons
emanate from the caudal portion of the lateral ventricle,
and migrate along the caudoventral migratory stream to
the caudal PIR. This is in accordance with our results. A
similar migratory stream was also reported in primates
(Bernier et al., 2002), where cells emanate from the
temporal horn of the lateral ventricle and migrate along
the temporal stream to the piriform cortex.
In P. velox, the caudoventral migratory stream also
appears to supply the endopiriform nucleus with newly
generated cells, but a comparable migration has not yet
been reported in other mammals; however, the
presence of the polysialylated neuronal cell adhesion
molecule (PSA-NCAM), which is considered a marker of
developing and migrating neurons and of
synaptogenesis, in cells of the rat endopiriform nucleus
(Varea et al., 2009) provides supporting evidence that
these might be newly generated cells in P. velox.
A few DCX-positive cells were also observed in the
olfactory tubercle of P. velox. The occurrence of newly
generated neurons in the olfactory tubercle was
previously demonstrated in rodents and primates
(Bedard et al., 2002; Shapiro et al., 2007).
Neurogenesis in the anterior commissure
In P. velox we observed DCX-positive cells, presumably
immature neurons migrating across the anterior
commissure to the contralateral hemisphere. In a
previous study on the hedgehog and mole, DCX-positive
processes were observed to cross the anterior
commissure, but no migratory neuroblasts were
reported (Bartkowska et al., 2010). Bartkowska et al.
(2010) suggested that these processes might originate
from either the piriform cortex or the OB, or both, since
both structures are reciprocally interconnected with their
contralateral homologous structures, providing evidence
that newly generated neurons might be able to send
long-range projections. In our study we demonstrated
these DCX-positive cells might presumably migrate from
the SVZ and the RMS via the anterior commissure to
the contralateral hemisphere; however these cells could
also be generated in the ac. Since DCX is expressed for
a short time period in newly generated and young
neurons, we can only speculate to which specific area
the cells might migrate and possibly become functionally
integrated. The anterior commissure is subdivided into
two parts, the anterior and the posterior limb, where the
anterior limb for the most part consists of decussating
fibres of the olfactory system (Fox and Schmitz, 1943;
Fox et al., 1948; Bennett, 1968). Since DCX-positive
cells were exclusively present in the anterior limb, we
might assume that the newly generated neurons
possibly migrate from the SVZ across the anterior limb
of the anterior commissure towards the contralateral
olfactory areas where they may differentiate into mature
neurons and become functionally integrated into the
olfactory circuitry.
Acknowledgements—This work was supported by funding from
the South African National Research Foundation (P.R.M.), the
Swiss-South African Joint Research Programme (A.O.I. and
P.R.M.), the Belgian cooperation service at the Royal Museum
for Central Africa (E.G.) and by a fellowship within the Postdoc-
Programme of the German Academic Exchange Service, DAAD
(N.P.).
REFERENCES
Alpar A, Kunzle H, Gartner U, Popkova Y, Bauer U, Grosche J,
Reichenbach A, Hartig W (2010) Slow age-dependent decline of
doublecortin expression and BrdU labeling in the forebrain from
lesser hedgehog tenrecs. Brain Res 1330:9–19.
Amrein I, Slomianka L (2010) A morphologically distinct granule cell
type in the dentate gyrus of the red fox correlates with adult
hippocampal neurogenesis. Brain Res 1328:12–24.
Amrein I, Slomianka L, Poletaeva II, Bologova NV, Lipp HP (2004)
Marked species and age-dependent differences in cell
proliferation and neurogenesis in the hippocampus of wild-living
rodents. Hippocampus 14:1000–1010.
Amrein I, Dechmann DK, Winter Y, Lipp HP (2007) Absent or low rate
of adult neurogenesis in the hippocampus of bats (Chiroptera).
PLoS One 2:e455.
Barker J, Boonstra R, Wojtowicz J (2005) Where is my dinner? Adult
neurogenesis in free-living food-storing rodents. Genes Brain
Behav 4:89–98.
Barker JM, Boonstra R, Wojtowicz JM (2011) From pattern to
purpose: how comparative studies contribute to understanding
the function of adult neurogenesis. Eur J Neurosci 34:963–977.
Barnea A (2010) Wild neurogenesis. Brain Behav Evol 75(2):86–87.
Bartkowska K, Djavadian RL, Taylor JRE, Turlejski K (2008)
Generation recruitment and death of brain cells throughout the
life cycle of Sorex shrews (Lipotyphla). Eur J Neurosci
27:1710–1721.
Bartkowska K, Turlejski K, Grabiec M, Ghazaryan A, Yavruoyan E,
Djavadian RL (2010) Adult neurogenesis in the hedgehog
(Erinaceus concolor) and mole (Talpa europaea). Brain Behav
Evol 76(2):128–143.
Bedard A, Parent A (2004) Evidence of newly generated neurons in
the human olfactory bulb. Dev Brain Res 151:159–168.
278 N. Patzke et al. / Neuroscience 238 (2013) 270–279
Bedard A, Levesque M, Bernierm PJ, Parent A (2002) The rostral
migratory stream in adult squirrel monkeys: contribution of new
neurons to the olfactory tubercle and involvement of the
antiapoptotic protein Bcl-2. Eur J Neurosci 16:1917–1924.
Bennett MH (1968) The role of the anterior limb of the anterior
commissure in olfaction. Physiol Behav 3:507–515.
Bergmann O, Liebl J, Bernard S, Alkass K, Yeung MSY, Steier P,
Kutschera W, Johnson L, Landen M, Druid H, Spalding KL, Frisen
J (2012) The age of olfactory bulb neurons in humans. Neuron
74:634–639.
Bernier PJ, Bedard A, Vinet J, Levesque M, Parent A (2002) Newly
generated neurons in the amygdala and adjoining cortex of adult
primates. Proc Natl Acad Sci USA 99:11464–11469.
Bonfanti L, Rossi F, Zupanc GK (2011) Towards a comparative
understanding of adult neurogenesis. Eur J Neurosci 34:845–846.
Brown JP, Couillard-Despres S, Cooper-Kuhn CM, Winkler J, Aigner
L, Kuhn HG (2003) Transient expression of doublecortin during
adult neurogenesis. J Comp Neurol 467:1–10.
Curtis MA, Kam M, Nannmark U, Anderson MF, Axell MZ, Wikkelso
C, Holtas S, van Roon-Mom WM, Bjork-Eriksson T, Nordborg C,
Frisen J, Dragunow M, Faull RL, Eriksson PS (2007) Human
neuroblasts migrate to the olfactory bulb via a lateral ventricular
extension. Science 315:1243–1249.
Dupret D, Revest JM, Koehl M, Ichas F, De Giorgi F, Costet P,
Abrous DN, Piazza PV (2008) Spatial relational memory requires
hippocampal adult neurogenesis. PLoS One 3:e1959.
Epp JR, Barker JM, Galea LAM (2009) Running wild: neurogenesis in
the hippocampus across the lifespan in wild and laboratory-bred
Norway rats. Hippocampus 19:1040–1049.
Eriksson PS, Perfilieva E, Eriksson TB, Alborn AM, Nordberg C,
Peterson DA, Gage FH (1998) Neurogenesis in the adult
hippocampus. Nat Med 4:1313–1317.
Fox CA, Schmitz JT (1943) A Marchi study of the distribution of the
anterior commissure in the cat. J Comp Neurol 79:297–314.
Fox CA, Fischer RR, Desalva SJ (1948) The distribution of the
anterior commissure in the monkey, Macaca mulatta. J Comp
Neurol 89:245–277.
Gallyas F (1979) Silver staining of myelin by means of physical
development. Neurol Res 1:203–209.
Gatom CW, Mwangi DK, Lipp HP, Amrein I (2010) Hippocampal
neurogenesis and cortical cellular plasticity in Wahlberg’s
epauletted fruit bat: a qualitative and quantitative study. Brain
Behav Evol 76:116–127.
Gould E (2007) How widespread is adult neurogenesis in mammals?
Nat Rev Neurosci 8:481–488.
Gould E, Cameron HS (1996) Regulation of neuronal birth, migration
and death in the rat dentate gyrus. Dev Neurosci 18:22–35.
Gould E, McEwen BS, Tanapat P, Galea LA, 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(7):2492–2498.
Gould E, Tanapat P, McEwen BS, Flugge G, Fuch E (1998)
Proliferation of granule cell precursors in the dentate gyrus of
adult monkeys is diminished by stress. Proc Natl Acad Sci USA
9:3168–3171.
Gould E, Reeves AJ, Graziano MS, Gross CG (1999) Neurogenesis
in the neocortex of adult primates. Science 286:548–552.
Grabiec M, Turlejski K, Djavadian RL (2009) The partial 5-HT1A
receptor agonist buspirone enhances neurogenesis in the
opossum (Monodelphis domestica). Eur Neuropsychopharmacol
19(6):431–439.
Guidi S, Bianchi P, Alstrup AK, Henningsen K, Smith DF, Bartesaghi
R (2011) Postnatal neurogenesis in the hippocampal dentate
gyrus and subventricular zone of the Gottingen minipig. Brain Res
Bull 85(3–4):169–179.
Harman A, Meyer P, Ahmat A (2003) Neurogenesis in the
hippocampus of an adult marsupial. Brain Behav Evol 62:1–12.
Hauser T, Klaus F, Lipp HP, Amrein I (2009) No effect of running and
laboratory housing on adult hippocampal neurogenesis in wild
caught long-tailed wood mouse. BMC Neurosci 10:43.
Kempermann G (2011) Seven principles in the regulation of adult
neurogenesis. Eur J Neurosci 33(6):1018–1024.
Kempermann G (2012) New neurons for ‘survival of the fittest’. Nat
Rev Neurosci 13(10):727–736.
Kempermann G, Kuhn HG, Gage FH (1997a) More hippocampal
neurons in adult mice living in an enriched environment. Nature
386:493–495.
Kempermann G, Kuhn HG, Gage FH (1997b) Genetic influence on
neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad
Sci USA 94(19):10409–10414.
Kingdon J (2004) The Kingdon pocket guide to African
mammals. Princeton University Press. pp. 183–184.
Knoth R, Singec I, Ditterm M, Pantazis G, Capetian P, Meyer RP,
Horvat V, Volk B, Kempermann G (2010) Murine features of
neurogenesis in the human hippocampus across the lifespan from
0 to 100 years. PLoS One 5(1):e8809.
Kornack DR, Rakic P (1999) Continuation of neurogenesis in the
hippocampus of the adult macaque monkey. Proc Natl Acad Sci
USA 96:5768–5773.
Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis in the
dentate gyrus of the adult rat: age-related decrease of neuronal
progenitor proliferation. J Neurosci 16:2027–2033.
Leuner B, Gould E, Shors TJ (2006) Is there a link between adult
neurogenesis and learning? Hippocampus 16(3):216–224.
Leuner B, Kozorovitskiy Y, Gross CG, Gould E (2007) Diminished
adult neurogenesis in the marmoset brain precedes old age. Proc
Natl Acad Sci USA 104(43):17169–17173.
Lindsey BW, Tropepe V (2006) A comparative framework for
understanding the biological principles of adult neurogenesis.
Prog Neurobiol 80:281–307.
Liu YW, Curtis MA, Gibbons HM, Mee EW, Bergin PS, Teoh HH,
Connor B, Dragunow M, Faull RL (2008) Doublecortin expression
in the normal and epileptic adult human brain. Eur J Neurosci
28:2254–2265.
Lledo P, Alonso M, Grubb S (2006) Adult neurogenesis and functional
plasticity in neuronal circuits. Nat Rev Neurosci 7:179–193.
Lu L, Bao G, Chen H, Xia P, Fan X, Zhang J, Pei G, Ma L (2003)
Modification of hippocampal neurogenesis and neuroplasticity by
social environments. Exp Neurol 183:600–609.
Ming GL, Song H (2011) Adult neurogenesis in the mammalian brain:
significant answers and significant questions. Neuron
70:687–702.
Ngwenya A, Patzke N, Ihunwo AO, Manger PR (2011) Organisation
and chemical neuroanatomy of the African elephant (Loxodonta
africana) olfactory bulb. Brain Struct Funct 216(4):403–416.
Nowak RM (1999). Walker’s mammals of the world, vol. I. Baltimore
and London: Johns Hopkins University Press. p. 187.
Olson AK, Eadie BD, Ernst C, Christie BR (2006) Environmental
enrichment and voluntary exercise massively increase
neurogenesis in the adult hippocampus via dissociable
pathways. Hippocampus 16:250–260.
Pekcec A, Loscher W, Potschka H (2006) Neurogenesis in the adult
rat piriform cortex. Neuroreport 17(6):571–574.
Pencea V, Bingaman KD, Freedman LJ, Luskin MB (2001)
Neurogenesis in the subventricular zone and rostral migratory
stream of the neonatal and adult primate forebrain. Exp Neurol
172:1–16.
Peretto P, Merighi A, Fasolo A, Bonfanti L (1997) Glial tubes in the
rostral migratory stream of the adult rat. Brain Res Bull
42(1):9–21.
Pham K, Nacher J, Hof PR, McEwen BS (2003) Repeated restraint
stress suppresses neurogenesis and induces biphasic PSA-
NCAM expression in the adult rat dentate gyrus. Eur J Neurosci
17:879–886.
Rao MS, Shetty AK (2004) Efficacy of doublecortin as a marker to
analyse the absolute number and dendritic growth of newly
generated neurons in the adult dentate gyrus. Eur J Neurosci
19:234–246.
Scholzen T, Gerdes J (2000) The Ki-67 protein: from the known and
the unknown. J Cell Physiol 182(3):311–322.
N. Patzke et al. / Neuroscience 238 (2013) 270–279 279
Shapiro A, Ng KL, Kinyamu R, Whitaker-Azmitia P, Geisert EE,
Blurton-Jones M, Zhou QY, Ribak CE (2007) Origin, migration
and fate of newly generated neurons in the adult rodent piriform
cortex. Brain Struct Funct 212:133–148.
Siwak-Tapp CT, Head E, Muggenburg BA, Milgram NW, Cotman CW
(2007) Neurogenesis decreases with age in the canine
hippocampus and correlates with cognitive function. Neurobiol
Learn Mem 88:249–259.
Snyder JS, Hong NS, McDonald RJ, Wojtowicz JM (2005) A role for
adult neurogenesis in spatial long-term memory. Neuroscience
130:843–852.
Snyder JS, Glover LR, Sanzone KM, Kamhi JF, Cameron HA (2009)
The effects of exercise and stress on the survival and maturation
of adult-generated granule cells. Hippocampus 19(10):898–906.
Tabuce R, Asher RJ, Lehmann T (2008) Afrotherian mammals: a
review of current data. Mammalia 72:2–14.
van Praag H, Christie BR, Sejnowski TJ, Gage FH (1999a) Running
enhances neurogenesis, learning, and long-term potentiation in
mice. Proc Natl Acad Sci USA 96(23):13427–13431.
van Praag H, Kempermann G, Gage FH (1999b) Running increases
cell proliferation and neurogenesis in the adult mouse dentate
gyrus. Nat Neurosci 2(3):266–270.
Varea E, Castillo-Gomez E, Gomez-Climent MA, Guirado R, Blasco-
Ibanez JM, Crespo C, Martınez-Guijarro FJ, Nacher J (2009)
Differential evolution of PSA-NCAM expression during aging of
the rat telencephalon. Neurobiol Aging 30(5):808–818.
Warner-Schmidt JL, Duman RS (2006) Hippocampal neurogenesis:
opposing effects of stress and antidepressant treatment.
Hippocampus 16(3):239–249.
Zhu H, Wang ZY, Hansson HA (2003) Visualization of proliferating
cells in the adult mammalian brain with the aid of ribonucleotide
reductase. Brain Res 977:180–189.
(Accepted 17 February 2013)(Available online 26 February 2013)
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