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: nina.patzke@wits.ac.za (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.).

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(Accepted 17 February 2013)(Available online 26 February 2013)