Adult neurogenesis in the four-striped mice (Rhabdomys pumilio)

Olatunbosun O. Olaleye, Amadi O. Ihunwo

1 Department of Oral Biological Sciences, School of Oral Health Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

2 School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

Adult neurogenesis in the four-striped mice (Rhabdomys pumilio)

Olatunbosun O. Olaleye1,2, Amadi O. Ihunwo2

1 Department of Oral Biological Sciences, School of Oral Health Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

2 School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa

In this study, we investigated non-captive four-striped mice (Rhabdomys pumilio) for evidence that adult neurogenesis occurs in the adult brain of animal models in natural environment. Ki-67 (a marker for cell proliferation) and doublecortin (a marker for immature neurons) immunostaining con fi rmed that adult neurogenesis occurs in the active sites of subventricular zone of the lateral ventricle with the migratory stream to the olfactory bulb, and the subgranular zone of the dentate gyrus of the hippocampus. No Ki-67 proliferating cells were observed in the striatum substantia nigra, amygdala, cerebral cortex or dorsal vagal complex. Doublecortin-immunoreactive cells were observed in the striatum, third ventricle, cerebral cortex, amygdala, olfactory bulb and along the rostral migratory stream but absent in the substantia nigra and dorsal vagal complex. The potential neurogenic sites in the four-striped mouse species could invariably lead to increased neural plasticity.

nerve regeneration; adult neurogenesis; four-striped mouse; Ki-67; doublecortin; subventricular zone; dentate gyrus; olfactory bulb; potential neurogenic site; neural plasticity; neural regeneration

Funding:This project was supported by Individual Faculty Research Grant and Swiss-South Africa Joint Research Progamme (SSAJRP).

Olaleye OO, Ihunwo AO. Adult neurogenesis in the four-striped mice (Rhabdomys pumilio). Neural Regen Res. 2014;9(21)：1907-1911.

Introduction

Neurogenesis has been de fi ned as the ability of brain cells to regenerate themselves (Gage, 2000). This process has been known to occur in the adult mammalian brain for over 50 years (Altman, 1962; Altman and Das, 1965). 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, 2006). Neurogenesis has been reported in the mammalian brain of rats (Kuhn, et al., 1996), mice (Kempermann, et al., 1997), tree shrews (Gould, et al., 1997), guinea pigs (Altman and Das, 1967), rabbits (Gueneau, et al., 1982), cats (Wyss and Sripanidkulchai, 1985), monkeys (Rakic and Nowakowski, 1981; Gould, et al., 1998, 1999), humans (Eriksson et al., 1998) and the hippocampi of several other species (Patzke et al., 2013).

New neurons are continually produced in the adult mammalian brain from progenitor cells located in specific regions, including the subgranular zone of the dentate gyrus of the hippocampus (Siwak-Tapp, 2007). The mammalian brain consists of two types of neurogenic sites, active and potential neurogenic sites (Ihunwo and Pillay, 2007). The two active neurogenic regions of the brain are the rostral subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus of the hippocampus (Kaplan and Hinds, 1977; Kaplan and Bell, 1984). The subventricular zone is considered the largest active neurogenic site in the brain (Schauwecker, 2006) and it has been shown to be a source for cortical and subcortical neurons (Alvarez-Buylla and Garcia-Verdugo, 2002; Watts et al., 2005). Other areas of the brain where adult neurogenesis has been reported include the septum, substantia nigra, ependymal wall of the third ventricle, cerebral cortex, olfactory bulb and dorsal vagal complex (Reynolds and Weiss, 1992; Ihunwo and Pillay, 2007). Evidence 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, which is similar to rostral migratory stream.

Animals in their natural environment experience a complex interactions compared to the laboratory animals that are limited in their exposures. The four-striped mouse is widespread and found in habitats such as grasslands, deserts and forests of Southern Africa and parts of the northern Africa (Schradin and Pillay, 2004) and usually in colonies with very few population living as a group. They demonstrate bi-parental care and cover long distances in foraging for food. It has a body mass that ranges from 40 to 80 g (Maini, 2003; Schradin and Pillay, 2004) and a small brain with an average mass of about 0.64 g (Schradin et al., 2014). Exploring effects of environmental in fl uences, animal behavior andsocial interactions on adult neurogenesis is becoming a major initiative in the field and collectively, these factors constitute a number of combined forces acting on brain plasticity, and ultimately, the rate and location of neurogenesis in adulthood (Boonstra et al., 2001). The aim of this study therefore is to explore adult neurogenesis occurrence in the active neurogenic sites: subventricular zone and dentate gyrus, as well as other reported potential sites in the fourstriped mice using immunohistochemistry.

Materials and Methods

Experimental animals

Four adult male four-striped mice were used with an average age of 100 days. The animals were captive-reared at the Central Animal Service Unit of the University of the Witwatersrand, South Africa. Their ancestors were of a de fi ned colony typical of the description under introduction and captured with cage traps. The animals were treated and used according to the guidelines of the University of the Witwatersrand Animal Ethics and Screening Committee, which parallel those set down by the National Institute of Health (NIH) for use of animals in scienti fi c experiments. The fourstriped mice are widely distributed in Southern Africa occurring in different habitats, such as grassland, marsh, forests, semi-deserts and deserts (Skinner and Chimimba, 2005). Four-striped mice are small diurnal murid rodents that are solitary in their living. They demonstrate bi-parental care with a very flexible social organization and mating system which is controlled mainly by resource availability and population density (Schradin and Pillay, 2005). They are very unique by the four distinct dark longitudinal stripes running the length of their back.

Tissue processing

The animals were euthanized intraperitoneally with sodium pentobarbital (80 mg/kg) and transcardially perfused with 0.9% cold saline followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer. Brains were carefully removed, weighed and post- fi xed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer, then allowed to equilibrate in 30% sucrose in 0.1 mol/L PBS. Brains were then kept frozen in dry ice and sectioned using a sliding microtome in the sagittal plane at 50 μm section thickness covering the entire brain. One in fi ve series of sections was stained for a cell proliferation marker Ki-67 and an immature neuronal marker doublecortin (DCX).

Immunohistochemistry for Ki-67

The Ki-67 is a chromosome-associated protein present during division (G1, S, G2, and M, but absent from cells at rest, G0). Free floating sections were incubated for epitope retrieval in citrate buffer, pH 6.0, at 90°C for 40 minutes, followed by incubation in endogenous peroxidase blocking reagent, 0.6% H2O2in Tris-buffered saline (TBS)-Triton (0.05% Triton X-100 in TBS, pH 7.4) for 30 minutes at room temperature. Thereafter, sections were pre-incubated in 2% serum (normal goat serum) + 0.1% bovine serum albumin (BSA) + 0.25% Triton in TBS, for 60 minutes at room temperature. Afterwards, sections were incubated with polyclonal rabbit-anti-lyophilized-Ki-67p antibody (Novocastra, Newcastle, UK; 1:5,000 in preincubation solution) overnight at 4°C. Incubation with biotinylated goat anti-rabbit IgG (1:1,000 + 2% normal goat serum + 0.1% BSA in TBS; Vector lab, CA, USA; 1:250) was performed for 2 hours at room temperature followed by incubation with streptavidin-biotin complex (Vectastain Elite ABC kit) and stained with 3,3′-diaminobenzidine (DAB) as chromogen. Until incubation with primary antibody, all rinses in between incubations were made with TBS-Triton, afterwards with TBS alone.

Immunohistochemistry for DCX

DCX is a microtubule associated protein expressed specifically in newly generated neuronal precursors, immature & migrating neurons. Sections were washed three times for 10 minutes in PBS and then rinsed with Tris-buffered saline and Tween 20 (TBST) once for 5 minutes under gentle shaking at room temperature. Thereafter, sections were then treated with blocking solution, 5 % normal rabbit serum in TBST for 30 minutes. Tissues were transferred into primary antibody goat anti-mouse DCX antibody (Santa Cruz Biotechnology, CA, USA; 1:400) in TBST supplemented with 2 % BSA and 2 % normal rabbit serum overnight at 4°C under gentle shaking. Incubation with secondary antibodies (rabbit anti-goat IgG (1:250) + 2 % normal rabbit serum + 0.1% BSA in TBS) was performed for 2 hours followed by incubation with streptavidin-biotin complex (Vectastain Elite ABC kit) and stained with DAB as chromogen.

Microscopic analysis

Sections of the brains were analyzed and photomicrographs were taken with the aid of Zeiss Axioskop light microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) using variable objective lenses and Axiovision software (Carl Zeiss Microscopy GmbH).

Results

Ki-67-immunoreactive cells

Ki-67-immunoreactive cells appeared darkly stained and were easily seen in various sites of the brain (Figure 1A-C). The Ki-67-immunoreactive cells appeared more numerous in the subventricular layer of the dentate gyrus. These cells were present in the two established neurogenic sites; subventricular zone and the dentate gyrus (Figure 1A, B). Also, Ki-67-immunoreactive neurons were visible in the rostral migratory stream and olfactory bulb (Figure 1C). No Ki-67-immunoreactive cells were observed in the striatum, amygdala, cerebral cortex or dorsal vagal complex.

DCX-immunoreactive cells

DCX-immunoreactive cells presented visible bipolar neurons with ovoid cell bodies and axonal and dendritic processes, an evidence of immature neurons. The dendrites extended towards the outer molecular layer of the dentate gyrus. In addition to the two active neurogenic sites, DCX-immunoreactive cells were observed in the striatum, third ventricle, cerebral cortex, amygdala, olfactory bulb and along the ros-tral migratory stream. They were however absent in the substantia nigra and dorsal vagal complex. The staining intensity of the DCX in the dentate gyrus of the hippocampus and the rostral migratory stream were quiet conspicuous (Figure 2). A summary of sites where adult neurogenesis was either present or absent in the four-striped mice using immunohistochemistry for Ki-67 and DCX are shown in Table 1.

Figure 1 Representative photomicrograph of Ki-67 immunohistochemical staining in the various brain regions of four-striped mice.

Figure 2 Representative photomicrograph of doublecortin (DCX) immunohistochemical staining in the brain of four-striped mice.

Discussion

We assessed adult neurogenesis using two different cell markers, the Ki-67 and DCX for cell proliferation and immature neurons respectively. 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, 1998; Gould et al., 1997; Bernabeu and Sharp, 2000), dietary restriction (Lee et al., 2002), growth factors (Palmer et al., 1995; Scharfman et al., 2005), 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., 1999), and dietary restriction (Lee, et al., 2000, 2002), all these increase neurogenesis in the adult hippocampus. Stress (Gouldet al., 1997, 1998) and social isolation (Lu et al., 2003; Chetty et al., 2009) reduces hippocampal neurogenesis. However, Kannangara et al. (2009) reported that social isolation does not affect adult neurogenesis. It was reported that estradiol alter hippocampal neurogenesis in the adult female rodents (Galea et al., 2006) even though a decrease in hippocampal neurogenesis does not always correlate with the development of learned helplessness in male rats (Vollmayr et al., 2003). According to Boonstra et al. (2001), both the daily behaviour and the immediate environment of species are intimately related with addition of new neurons throughout adulthood which provides a basis for investigating animals in the wild for increased neural plasticity and possible factors for thismechanism. In all the immunostaining of the brain of fourstriped mice, new neurogenic cells were observed either in clustered or isolated form. These differences were evident in relation to the neurogenic regions, active or potential sites. In the hippocampus, more Ki-67 and DCX-immunoreactive cells were observed. DCX-immunoreactive cells were also found in the striatum of the brain but sparsely distributed compared to the subventricular zone and dentate gyrus.

Table 1 A summary of the neurogenic sites observed in four-striped mice

The predominant DCX-immunoreactive cells appeared in clusters but varied in shape according to Plumpe et al. (2006). The somas of DCX-immunoreactive cells were primarily located in the subgranular layer and were bipolar with an ovoid soma. Their dendrites reach as far as the molecular layer of the dentate gyrus while some dendrites only end in the granular layer.

The subventricular zone has two precise layers of cells: the fi rst is a monolayer of multi-ciliated cells lining the lateral ventricle called the ependymal layer; and the second layer is a 2-3 cell layer thick area adjacent to the ependymal layer called the subependymal layer. The neuroblasts from the subventricular zone travel a long distance to the olfactory bulb through a network of interconnecting pathways that become confluent at the rostral margin of the lateral ventricle wall to form the rostral migratory stream (Watts et al., 2005). In mammals, the generation of new neurons is mostcommonly 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). This is true in the four-striped mice. This study therefore adds the fourstriped mice to the growing list of wild-caught mammalian species with persistent adult neurogenesis. In conclusion, a combination of Ki-67 and DCX immunohistochemistry has provided evidence of adult neurogenesis in the active and some potential sites in four-striped mice.

Acknowledgments:We expressed our thanks to Professor Pillay N, School of Animal, Plant and Environmental Sciences Wits University, South Africa for providing four-striped mice, to the Wits Central Animal Services, South Africa for excellent care of animals, and to Ali H and Meskenaite V for assistance with laboratory techniques.

Author contributions:Olaleye OO was responsible for conception of research protocol, data collection, tissue processing, immunostaining and drafted the manuscript. Ihunwo AO designed this study, reviewed the results and revised the manuscript. Both of these two authors approved the final version of this paper.

Con fl icts of interest:None declared.

Altman J (1962) Are new neurons formed in the brains of adult mammals? Science 135:1127-1128.

Altman J, Das GD (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124:319-335.

Altman J, Das GD (1967) Postnatal neurogenesis in the guinea-pig. Nature 214:1098-1101.

Alvarez-Buylla A, Garcia-Verdugo JM (2002) Neurogenesis in adultsubventricular zone. J Neurosci 22:629-634.

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 U S A 99:11464-11469.

Bernabeu R, Sharp FR (2000) NMDA and AMPA/kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus. J Cereb Blood Flow Metab 20:1669-1680.

Boonstra R, Galea L, Matthews S, Wojtowicz JM (2001) Adult neurogenesis in natural populations. Can J Physiol Pharmacol 79:297-302.

Brunson KL, Kramár E, Lin B, Chen Y, Colgin LL, Yanagihara TK, Lynch G, Baram TZ (2005) Mechanisms of late-onset cognitive decline after early-life stress. J Neurosci 25:9328-9338.

Cameron HA, Woolley CS, McEwen BS, Gould E (1993) Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience 56:337-344.

Cameron HA, McEwen BS, Gould E (1995) Regulation of adult neurogenesis by excitatory input and NMDA receptor activation in the dentate gyrus. J Neurosci 15:4687-4692.

Cameron HA, 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.

Cooper-Kuhn CM, Winkler J, Kuhn HG (2004) Decreased neurogenesis after cholinergic forebrain lesion in the adult rat. J Neurosci Res 77:155-165.

Chetty T, Ajao MS, Manger PR, Ihunwo AO (2009) Behavioural patterns and corticosterone levels induced by chronic psychosocial stress in the four-striped mouse (Rhabdomys pumilio). J Animal Vet Adv 8:459-463.

Eriksson PS, Per fi lieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in the adult human hippocampus. Nat Med 4:1313-1317.

Galea LA, Spritzer MD, Barker JM, Pawluski JL (2006) Gonadal hormone modulation of hippocampal neurogenesis in the adult. Hippocampus 16:225-232.

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 17:2492-2498.

Gould E, Tanapat P, McEwen BS, Flugge G, Fuchs E (1998) Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. Proc Natl Acad Sci U S A 95:3168-3171.

Gould E, Reeves AJ, Graziano MS, Gross CG (1999) Neurogenesis in the neocortex of adult primates. Science 286:548-552.

Gueneau G, Privat A, Drouet J, Court L (1982) Subgranular zone of the dentate gyrus of young rabbits as a secondary matrix.A high-resolution autoradiographic study. Dev Neurosci 5:345-358.

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.

Ihunwo AO, Pillay S (2007) Neurogenic sites in the adult mammalian central nervous system. Res J Biol Sci 2:170-177.

Kannangara TS, Webber A, Gil-Mohapel J, Christie BR (2009) Stress differentially regulates the effects of voluntary exercise on cell proliferation in the dentate gyrus of mice. Hippocampus 19:889-897.

Kaplan MS, Bell DH (1984) Mitotic neuroblasts in the 9-day-old and 11-month-old rodent hippocampus. J Neurosci 4:1429-1441.

Kaplan MS, Hinds JW (1977) Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs. Science 197:1092-1094.

Kempermann G, Kuhn HG, Gage FH (1997) Genetic in fl uence on neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad Sci U S A 94:10409-10414.

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.

Lee J, Duan W, Long JM, Ingram DK, Mattson MP (2000) Dietary restriction increases the number of newly generated neural cells, and induces BDNF expression, in the dentate gyrus of rats. J Mol Neurosci 15:99-108.

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Lee J, Seroogy KB, Mattson MP (2002) Dietary restriction enhances neurotrophin expression and neurogenesis in the hippocampus of adult mice. J Neurochem 80:539-547.

Lu L, Bao G, Chen H, Xia P, Fan X, Zhang J, Pei G, Ma L (2003) Modifi cation of hippocampal neurogenesis and neuroplasticity by social environments. Exp Neurol 183:600-609.

Maini PK (2003) How the mouse got its stripes. Proc Natl Acad Sci U S A 100:9656-9657.

Ming G, Song H (2011) Adult neurogenesis in the mammalian brain: Signi fi cant answers and signi fi cant questions. Neuron 70:687-702.

Nichols NR, Agolley D, Zieba M, Bye N (2005) Glucocorticoid regulation of glial responses during hippocampal neurodegeneration and regeneration. Brain Res Rev 48:287-301.

Palmer TD, Ray J, Gage FH (1995) FGF-2 responsive neural progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci 6:474-486.

Parent JM (2003) Injury-induced neurogenesis in the adult mammalian brain. Neuroscientist 9:261-272.

Patzke N, Spocter MA, Karlsson KA, Bertelsen MF, Haagensen M, Chawana R, Streicher S, Kaswera C, Gilissen E, Alagaili AN, Mohammed OB, Reep RL, Bennett NC, Siegel JM, Ihunwo AO, Manger PR (2013) In contrast to many other mammals, cetaceans have relatively small hippocampi that appear to lack adult neurogenesis. Brain Struct Funct DOI: 10.1007/s00429-013-0660-1.

Plumpe T, Ehninger D, Steiner B, Klempin F, Jessberger S, Brandt M, Romer B, Rodriguez GR, Kronenberg G, Kempermann G (2006) Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation. BMC Neurosci 7:77.

Rakic P, Nowakowski RS (1981) The time of origin of neurons in the hippocampal region of the rhesus monkey. J Comp Neurol 196:99-128.

Reynolds BA,Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707-1910.

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 192:348-356.

Schauwecker PE (2006) Genetic in fl uence on neurogenesis in the dentate gyrus of two strains of adult mice. Brain Res 1120:83-92.

Schradin C, Pillay N (2005) The influence of the father on offspring development in the striped mouse. Behav Ecol 16:450-455.

Schradin C, Larke RH, Bales KL (2014) Growing up in the family or growing up alone in fl uences behavior and hormones, but not arginine vasopressin receptor 1a expression in male African striped mice. Physiol Behav 129:205-213.

Skinner JD, Chimimba CT (2005) Mammals of the Southern African Sub-region. Cambridge University Press, Cambridge.

Siwak-Tapp CT, Head E, Muggenburg BA, Milgram NW, Cotman CW (2008) Region speci fi c neuron loss in the aged canine hippocampus is reduced by enrichment. Neurobiol Aging 29:39-50.

Taupin P (2006) Neurogenesis in the adult central nervous system. CR Biol 329:465-475.

Van Praag H, Kempermann G, Gage FH (1999) Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 2:266-270.

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.

Watts C, McConkey H, Anderson L, Caldwell M (2005) Anatomical perspectives on adult neural stem cells. J Anat 207:197-208.

Wyss JM, Sripanidkulchai B (1985) The development of Ammon’s horn and the fascia dentata in the cat: a [3H]thymidine analysis. Brain Res 350:185-198.

Copyedited by J. Gil-Mohapel, B. Lang, Li CH, Song LP, Zhao M

Amadi O. Ihunwo, School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193, Johannesburg, South Africa, Amadi.Ihunwo@wits.ac.za.

10.4103/1673-5374.143435

http://www.nrronline.org/

Accepted: 2014-07-04