Quiescent neuronal progenitors are activated in the juvenile guinea pig lateral striatum and give rise to transient neurons

Abstract

In the adult brain, active stem cells are a subset of astrocytes residing in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. Whether quiescent neuronal progenitors occur in other brain regions is unclear. Here, we describe a novel neurogenic system in the external capsule and lateral striatum (EC-LS) of the juvenile guinea pig that is quiescent at birth but becomes active around weaning. Activation of neurogenesis in this region was accompanied by the emergence of a neurogenic-like niche in the ventral EC characterized by chains of neuroblasts, intermediate-like progenitors and glial cells expressing markers of immature astrocytes. Like neurogenic astrocytes of the SVZ and DG, these latter cells showed a slow rate of proliferation and retained BrdU labeling for up to 65 days, suggesting that they are the primary progenitors of the EC-LS neurogenic system. Injections of GFP-tagged lentiviral vectors into the SVZ and the EC-LS of newborn animals confirmed that new LS neuroblasts originate from the activation of local progenitors and further supported their astroglial nature. Newborn EC-LS neurons existed transiently and did not contribute to neuronal addition or replacement. Nevertheless, they expressed Sp8 and showed strong tropism for white matter tracts, wherein they acquired complex morphologies. For these reasons, we propose that EC-LS neuroblasts represent a novel striatal cell type, possibly related to those populations of transient interneurons that regulate the development of fiber tracts during embryonic life.

Full text

RESEARCH ARTICLE STEM CELLS AND REGENERATION
Quiescent neuronal progenitors are activated in the juvenile
guinea pig lateral striatum and give rise to transient neurons
Federico Luzzati
1,2,
*, Giulia Nato
1,2
, Livio Oboti
1,2
, Elisa Vigna
3
, Chiara Rolando
2,4
, Maria Armentano
2,5
,
Luca Bonfanti
2,5
, Aldo Fasolo
1,2
and Paolo Peretto
1,2,
*
ABSTRACT
In the adult brain, active stem cells are a subset of astrocytes residing
in the subventricular zone (SVZ) and the dentate gyrus (DG) of the
hippocampus. Whether quiescent neuronal progenitors occur in other
brain regions is unclear. Here, we describe a novel neurogenic
system in the external capsule and lateral striatum (EC-LS) of the
juvenile guinea pig that is quiescent at birth but becomes active
around weaning. Activation of neurogenesis in this region was
accompanied by the emergence of a neurogenic-like niche in the
ventral EC characterized by chains of neuroblasts, intermediate-like
progenitors and glial cells expressing markers of immature
astrocytes. Like neurogenic astrocytes of the SVZ and DG, these
latter cells showed a slow rate of proliferation and retained BrdU
labeling for up to 65 days, suggesting that they are the primary
progenitors of the EC-LS neurogenic system. Injections of GFP-
tagged lentiviral vectors into the SVZ and the EC-LS of newborn
animals confirmed that new LS neuroblasts originate from the
activation of local progenitors and further supported their astroglial
nature. Newborn EC-LS neurons existed transiently and did not
contribute to neuronal addition or replacement. Nevertheless, they
expressed Sp8 and showed strong tropism for white matter tracts,
wherein they acquired complex morphologies. For these reasons, we
propose that EC-LS neuroblasts represent a novel striatal cell type,
possibly related to those populations of transient interneurons that
regulate the development of fiber tracts during embryonic life.
KEY WORDS: Neural stem cells, Parenchymal progenitors, Postnatal
neurogenesis, Internal capsule, Striatum, Transient neurons
INTRODUCTION
In the mammalian brain, a subset of neuronal progenitors (NPs)
continues to produce neurons during postnatal and adult life. These
cells are astrocytes residing in the subventricular zone (SVZ) and
subgranular zone (SGZ) neurogenic niches, and give rise to
olfactory bulb (OB) interneurons and dentate gyrus (DG) granule
cells, respectively (Fuentealba et al., 2012; Ming and Song, 2011).
The presence of quiescent NPs within other regions of the adult
telencephalon has long been hypothesized (Emsley et al., 2005;
Ourednik et al., 2001). NPs have been isolated in vitro from several
non-neurogenic areas, particularly after invasive injuries (Buffo
et al., 2008; Palmer et al., 1999), but it is unclear whether these
progenitors only represent in vitro artifacts. Indeed, NPs generate
mainly glial cells when transplanted into the mature brain
parenchyma, thus leading to the idea that this environment is not
permissive for neurogenesis during adulthood (Lim et al., 2000;
Shihabuddin et al., 2000; Ninkovic and Götz, 2013).
However, low-level neurogenesis has been reported in several brain
regions, such as the neocortex and striatum, of some mammalian
species (Bonfanti and Peretto, 2011), and neurodegeneration strongly
enhances such activity in rodents (Kernie and Parent, 2010). Newborn
cortical and striatal neurons often exhibit a transient existence and,
with few exceptions (Le Magueresse et al., 2011), their fate remains
unclear or debated (Gould et al., 2001; Arvidsson et al., 2002; Chen
et al., 2004; Dayer et al., 2005; Liu et al., 2009; Luzzati et al., 2011a;
Ohira et al., 2010). Interestingly, in some cases these cells are
generated by local parenchymal NPs. In the neocortex of adult rats,
NPs are activated in layer I after mild ischemia (Ohira et al., 2010),
and cortical GFAP
+
astrocytes have been shown to produce new
neurons locally in perinatal mice under hypoxia/ischemia (Bi et al.,
2011). In the striatum, we have identified local NPs generating new
neurons under normal condition in rabbits, and during progressive
neurodegeneration in mice (Luzzati et al., 2006, 2011a). In both mice
and rabbits, striatal NPs showed features of intermediate NPs.
Although these results support the contention that the mature striatal
parenchyma can be permissive for the genesis of neurons, it remainsto
be clarified whether the observed NPs originate from the SVZ or from
the activation of a local quiescent element.
Here, we demonstrate that the external capsule and lateral
striatum (EC-LS) of the guinea pig contain local NPs that are
quiescent at birth and become activated during postnatal
development, peaking at weaning. Newborn striatal neurons in the
guinea pig exist transiently and do not express markers of striatal
neurons, yet they might be involved in a transient form of plasticity.
RESULTS
DCX
+
neuroblasts in the juvenile guinea pig EC-LS
In the juvenile guinea pig a population of DCX-labeled cells is visible
in the ventral half of the EC and the surrounding parenchyma of the
LS (Fig. 1A,D) (Luzzati et al., 2011b). All the DCX
+
cells in the EC-
LS expressed the neuronal marker Tuj1 (Fig. 1E,F), but not the
oligodendrocyte markers Olig2 and SOX10 (not shown), or the
astrocyte marker GFAP (Fig. 2C), indicating they belong to the
neuronal lineage. At p18, the distribution ofthe DCX
+
cells spans ∼4-
5 mm along the EC-LS, rostral to the decussation of the anterior
commissure. This system is always anatomically separated from the
SVZ (Fig. 2A). In the more ventral part of the EC, the DCX
+
cells
were mostly organized in large chains, oriented parallel to the EC
fibers and closely associated to blood vessels (Fig. 1C; supplementary
material Fig. S1). Here, we will refer to this ventral part of the EC as
the ventral pallial-subpallial boundary (vPSB; Fig. 1D). In contrast to
Received 17 January 2014; Accepted 27 August 2014
1
Department of Life Sciences and Systems Biology (DBIOS), University of Turin,
Turin 10123, Italy.
2
Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano
10010, Italy.
3
Department of Oncology, University of Turin, c/o Institute for Cancer
Research and Treatment (IRCC), Candiolo 10060, Italy.
4
Department of
Neuroscience, University of Turin, Turin 10126, Italy.
5
Department of Veterinary
Sciences, University of Turin, Grugliasco 10095, Italy.
*Authors for correspondence ( federico.luzzati@unito.it; paolo.peretto@unito.it)
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the vPSB, in the dorsal EC and in the LS DCX
+
cells were exclusively
represented by individual elements, often located within the white
matter tracts of EC and internal capsule (IC; Fig. 1B). Interestingly,
the vPSB was highly enriched in astrocytes that, like neurogenic
astrocytes of the embryonic and adult neurogenic niches, expressed
GFAP (Doetsch et al., 1999; Garcia et al., 2004), BLBP (Anthony
et al., 2004; Giachino et al., 2013), Vimentin (Doetsch et al., 1999;
Schnitzer et al., 1981) and PAX6 (Götz et al., 1998; Maekawa et al.,
2005) (Fig. 2B,C; supplementary material Fig. S2; data not shown).
Ultrastructural analyses of the vPSB at postnatal day ( p) 18 confirmed
the presence of clusters of small cells showing characteristics typical
of neuroblasts: immunoelectrondense cytoplasm filled with free
ribosomes, scarce endoplasmic reticulum and darkly stained nuclei
(Fig. 2D). These cells form chain-like structures that are in contact
with unmyelinated and myelinated axons, abundant astrocytic
processes and, in some cases, blood vessels.
Overall, these data support the occurrence of an SVZ-independent
neurogenic niche in the vPSB of the juvenile guinea pig. Similar to
the SVZ-OB system, the vPSB chains might represent early stages of
the neurogenic process, which are followed by the detachment of
individual cells migrating toward the dorsal EC and LS.
Development of the EC-LS neurogenic system
To better characterize the EC-LS neurogenic system, we followed its
postnatal development. Note that, in contrast to mouse, rat and
rabbit, the guinea pig is a highly precocial mammal. Newborns have
teeth, fully grown fur and well developed sensory and locomotor
abilities (Künkele and Trillmich, 1997).
There were very few DCX
+
cells in the EC-LS at birth (176±46
cells, n=3) and their number was not significantly changed at p7
(460±129 cells, n=3; one-way ANOVA, F=35,09; Tukey’s post-hoc
p1 versus p7, P=0.99; Fig. 3A,E,H). By contrast, these cells increased
abruptly between p7 and p18 (5440±1355 cells at p18, n=3; p7 versus
p18, P<0.0001; Fig. 3A,F,I), then declined gradually, being reduced
to about half by p50 (p18 versus p50, P=0.003) and almost absent at
8 months of age (p18 versus p240, P<0.0001; Fig. 3A,G,J).
The proportion of DCX
+
cells organized in chains or as
individual elements was similar at all ages, with the exception of
p1 animals, in which no chains were visible (Fig. 3A,E,H),
supporting a direct relationship between chains and individual
elements, at least by p7. At all ages, strong DCX immunolabeling
was found in the SVZ and DG indicating that the trend in DCX
expression found in the EC-LS is not due to age-dependent
variations in immunoreactivity for DCX (Fig. 3B-D).
To further analyze the differential abundance of neuroblasts
during postnatal development, we cultured explants of the SVZ and
EC-LS from p1 and p18 animals (supplementary material Fig. S3).
Consistent with previous studies in other species (Lois and Alvarez-
Buylla, 1993; Luzzati et al., 2006), numerous DCX
+
cells migrated
out of both p1 and p18 SVZ explants after 3 days in vitro
(supplementary material Fig. S3A,B). By contrast, the EC-LS
explants were surrounded by very few DCX
+
cells at p1, whereas at
p18 the number of DCX
+
neuroblasts increased ∼20-fold (52±55
DCX
+
cells at p1 versus 1022±286 DCX
+
cells at p18, n=4 animals
per group; t-test, P=0.001; supplementary material Fig. S3C,D,F).
Collectively, these data indicate that the EC-LS DCX
+
cells are
mostly associated with a specific developmental window that spans
from the weaning period ( p18) to the peri-pubertal age ( p50).
EC-LS neuroblasts are mostly generated between p7 and p18
To establish the rate of genesis of the DCX
+
cells in the EC-LS
during postnatal development, BrdU was given at p1, p7 and p18,
Fig. 1. DCX
+
neuroblasts in the EC-LS of the juvenile guinea pig. (A,A′) Coronal section stained for myelin associated glycoprotein (MAG; magenta), DCX
(green) and with nuclear stain (Cytox Green, CYG; white). (B-C′) Higher magnifications of boxed regions in A showing a cell body (arrow) and processes of
individual DCX
+
cells within an IC bundle (B), and a chain of DCX
+
neuroblasts running along a blood vessel in the vPSB; lower panel in B,B′shows an x,zview
of the stack. (D) Schematic of the guinea pig striatum (coronal view). (E-F′) Both individual DCX
+
(green) cells (E) and chains (F) express the neuronal marker
Tuj1 (magenta). BV, blood vessel; CC, corpus callosum; LS, lateral striatum; EC, external capsule; d/vPSB, dorsal/ventral pallial-subpallial border; STR, striatum;
SVZ, subventricular zone. Scale bars: 40 μm in A,A′;15μm in B,B′;10μm in C,C′,E-F′.
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and the animals sacrificed 11 days after the injection (n=4 animals
per group; Fig. 4G). In p1 and p7 injected animals, only very few
DCX
+
/BrdU
+
colabeled elements were found (p1, 44±12 cells; p7,
222±149 cells; one-way ANOVA, F=16,3, P=0.004; Tukey’s post-
hoc, P=0.658), whereas in animals treated at p18 these cells were
dramatically increased (p18, 1096±390 cells; Tukey’s post-hoc p7
versus p18, P=0.004; n=4; Fig. 4A-E,G). This indicates that EC-LS
neuroblasts are mostly generated between p7 and p18.
At all survival times considered, the DCX
+
/BrdU
+
cells were
found both as individual elements, homogeneously distributed
among the EC and LS (Fig. 4A-C), and in vPSB chains (Fig. 4D,E).
Note that although the density of BrdU
+
cells in the LS increased
between p1 and p18 (t-test, P=0.020), this increase involved only
the DCX
+
cells (P=0.021), since no variation in cell density was
found for BrdU
+
/SOX10
+
oligodendrocytes (P=0.111), or in those
BrdU
+
cells not expressing DCX or SOX10 (P=0.195; Fig. 4F,H).
Thus, in contrast to the SVZ and DG, in which neurogenesis begins
during embryonic development (Kriegstein and Alvarez-Buylla,
2009), in the EC-LS of the guinea pig neurogenesis is activated
during postnatal development.
Induction of neurogenesis in the vPSB is associated with the
appearance of primary and intermediate-like NPs
The presence of chains of neuroblasts surrounded by cells showing
features of neurogenic astrocytes (Fig. 2B-D) is consistent with the
occurrence of a neurogenic niche in the vPSB. To test this
possibility, we first analyzed the density and distribution of EC-LS
cells expressing the marker of proliferation Ki67 (Scholzen and
Gerdes, 2000). Between p1 and p18 the density of Ki67
+
cells
remained constant in the LS (t-test, P=0.333) and dorsal EC
(P=0.125), whereas it strongly increased in the vPSB (P=0.004;
n=3; Fig. 5A,B,I). This increase was mostly due to the appearance of
Ki67
+
cells organized in clusters (Fig. 5C), which represent
68±11% of all the Ki67
+
cells in the vPSB at p18. 60.9±4.1% of
the clustered Ki67
+
cells expressed DCX, and 89.3±5.3% of these
double-labeled cells were part of a DCX
+
chain (n=3). These results
show that the Ki67
+
clusters and the DCX
+
chains are closely
associated and partly overlapping structures. A 2 h pulse of BrdU
confirmed that the clustered Ki67
+
cells of the vPSB were actually
proliferating (Fig. 5E).
Among the Ki67
+
clusters containing at least one DCX
+
cell,
24.32±6.61% of the cells did notexpress DCX (n=3). We refer to these
cells as Ki67
+
cells associated with proliferating neuroblasts, or KaD
cells (Fig. 5D). The clustered arrangement, together with the lack of
DCX expression suggest that the KaD cells might represent
intermediate NPs (Fuentealba et al., 2012). Accordingly, in contrast
to the postmitotic (DCX
+
/Ki67
−
)neuroblaststheDCX
+
/Ki67
+
and
KaD cells strongly expressed PAX6 (Fig. 5F,H) and SOX9 (not
shown), two markers that characterize the SVZ intermediate NPs
(Cheng et al., 2009; Hack et al., 2005; Kohwi et al., 2005).
Fig. 2. Anatomical and cellular organization of the EC-LS neurogenic system. (A) Frontal (left) and lateral (right) views of a 3D model obtained from DCX-
stained coronal sections. The location of the reconstructed region is outlined in the central model. DCX
+
chains (red) are mostly restricted to the vPSB and in
close contact with blood vessels (violet). (B-C‴) Coronal section at the level of the vPSB labeled with GFAP (green), BLBP (magenta), DCX (orange) and DAPI
(blue in C). (C) A single confocal plane showing GFAP/BLBP co-expression (arrowhead). As BLBP labels mainly the cell body, whereas GFAP labels the
processes, colocalization is often ambiguous (arrow). The asterisk marks a DCX+ but GFAP–and BLBP–neuroblast. (D) Ultrastructural analysis of vPSB. Small
cells are observed with the typical feature of neuroblasts forming a chain (Ch) and contacting axons (Ax), astrocytes (Ast) and astrocytic processes (recognizable
by their white cytoplasm). CA, anterior commissure; RMS, rostral migratory stream. Scale bars: 1 mm in A; 300 μm in B-C‴;3μminD.
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Interestingly, the appearance of intermediate-like NPs between p1 and
p18 was accompanied by a steep increase in BLBP
+
cells that
expressed Ki67 in the vPSB (P=0.003; Fig. 5G,J). The astrocytic
nature of the vPSB BLBP
+
cells was consistent with their predominant
co-expression of GFAP, but not DCX (Fig. 2C) or SOX10 (Fig. 5G).
By contrast, the Ki67
+
/SOX10
+
oligodendrocyte progenitors (Fig. 5G)
showed only a small tendencyto increase in thevPSB at p18 (P=0.077;
Fig. 5K). Like the primary progenitors of other neurogenic niches
(Ponti et al., 2013), vPSB BLBP
+
cells were rarely proliferating,
representing only 3.7±1.8% of all Ki67
+
cells at p18 (n=3). To
determine whether these are label-retaining cells, we injected BrdU for
5 consecutive days from p18 and analyzed the animals either 15 (15 d)
or 65 (65 d) days after the first injection. Although the total number
of BrdU
+
cells dropped between 15 d and 65 d (15 d, 1745±150
cells/mm
2
; 65 d, 265±40 cells/mm
2
;t-test, P=0.002; n=3), those
expressing BLBPshowed a more moderate decrease (15 d, 80±7 cells/
mm
2
; 65 d, 45±8 cells/mm
2
;P=0.003). Accordingly, only 4.4±0.3%
of BrdU
+
cells expressed BLBP in the vPSB at 15 d, but this value
increased to 16.8±1.8% at 65 d (P=0.007). At both time points the
BLBP
+
/BrdU
+
cells were at least in part colabeled for GFAP (Fig. 6;
data not shown), confirming their astrocytic nature. Thus, by p18,
vPSB astrocytes become proliferative and retain BrdU labeling long-
term, further supporting the contention that they include primary NPs.
Overall, thesedata indicate that theoccurrence ofimmatureneuroblasts
in the EC-LS correlates with the appearance of cells showing features
of both primary and intermediate NPs.
To further confirm the occurrence of local NPs in the EC-LS,
after a 2 h BrdU pulse in vivo we performed tissue explants of the
EC-LS and SVZ from p18 animals. Double staining for DCX and
BrdU showed several colabeled cells surrounding both the SVZ and
EC-LS explants (n=3; Fig. 7). Interestingly, DCX
+
/BrdU
+
cells
were also identified in EC-LS and SVZ cultures when BrdU was
added to the culture medium for 24 and 48 h starting from the
second day in vitro (n=2; Fig. 7C). These data indicate that, at p18,
the EC-LS of the guinea pig contains active NPs.
EC-LS NPs derive from the activation of local cells
Two non-mutually exclusive hypotheses can be drawn regarding the
origin of the vPSB NPs: (1) they migrate from already active
neurogenic niches (i.e. the SVZ); (2) they are local cells that become
active around weaning. To investigate this, we injected newborn
animals with a GFP-tagged lentiviral vector in the SVZ or in the EC-
LS. In addition, to determine whether infected cells behave as NPs
later in development, a group of animals was treated with BrdU at
p18 for 5 consecutive days (50 mg/kg, one intraperitoneal injection/
day) and left to survive for a further 15 days until p33 (Fig. 8A).
Fig. 3. Timecourse analysis of the DCX
+
cells in the EC-LS
during postnatal development. (A) Stereological estimates
of the mean±s.d. number of DCX
+
cells (blue line) in the EC-
LS at different postnatal ages. Red and cyan lines indicate
cells organized in chains or as individual elements. (B-J) DCX
expression in SVZ (B-D) and EC-LS (E-J) at p1 (B,E,H), p18
(C,F,I) and p150 (D,G,J). (H-J) Higher magnifications of the
boxed regions in E-G. Dashed line (E-G) indicates the
border between LS and EC. Scale bars: 100 μm in E-G; 25 μm
B-D,H-J.
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When the VSVG-GFP virus was injected in the lateral ventricle,
GFP-labeled cells were observed over the entire SVZ up to the
olfactory ventricle. Many of these cells had an astrocytic-like
morphology (Fig. 9A; data not shown). In addition, GFP labeling
was also found in the migrating neuroblasts of the RMS, often
colabeled with BrdU (Fig. 9B), as well as in the deep and superficial
granules and periglomerular cells of both main (Fig. 9B-D) and
accessory OB, indicating that VSVG-GFP targeted primary SVZ
progenitors over multiple domains (Merkle et al. 2007). In the six
analyzed hemispheres we estimated atotal of 3588 GFP
+
cells in the
OB, whereas we never observed any GFP-labeled cells in the EC-
LS, suggesting that the SVZ does not contribute to the EC-LS
neurogenic system.
In order to label the vPSB, we took advantage of the fact that this
region is supplied by numerous large veins that drain the striatum
(Fig. 2A); thus, we injected VSVG-GFP in the dorsolateral caudate
putamen (Fig. 10A,B). At 3 days post-infection a very strong GFP
labelingwas evident at both theinjection site and in thevPSB, whereas
only rare GFP
+
cells were visible in the LS (Fig. 10C) and dorsal EC
(n=7 hemispheres from five animals). Surprisingly, at this time GFP
expression in the vPSB cellswas found mainly associated with BLBP
+
astrocytes (82.3±11.2%), and to a much lesser extent with NeuN
+
neurons (8.2±3.9%) and SOX10
+
oligodendrocytes (4.6±2.2%). At
p33, a considerable proportion of DCX
+
cells of the LS and dorsal EC
expressed GFP [19.6-39.7%; mean±s.d. of DCX
+
/GFP
+
cells among
DCX
+
cells: 30.8±7.3% (187 DCX
+
/GFP
+
among 602 DCX
+
cells
counted in four animals; number of D CX
+
/GFP
+
cells ranged from 30
to 83 per animal)]. The morphology and distribution of GFP
+
and
GFP
−
neuroblasts in the EC and LS were not evidently different
(Fig. 10E). 33.8±7.3% of DCX
+
/GFP
+
cells incorporated BrdU (63
DCX
+
/GFP
+
/BrdU
+
cells in four animals; Fig. 10F). Notably, this
percentage was the same as that of BrdU
+
cells among DCX
+
/GFP
−
cells (27.1±6.3%; t-test, P=0.266). Thus, vPSB cells infectedat p1 can
behave as NPs at p18, giving rise to a progeny that is virtually identical
to that of uninfected progenitors. These data indicate that the EC-LS
neurogenic system involves the activation of local quiescent NPs and
further suggest that these cells are BLBP
+
astrocytes.
Neuroblasts generated in the EC-LS exist transiently and
belong to a novel neuronal type
To investigate the fate of DCX
+
neuroblasts, we performed a BrdU
timecourse analysis in animals that received BrdU for 5 consecutive
days, starting at p18 (Fig. 8B). The number of BrdU
+
/DCX
+
cells in
the LS and EC dropped between 15 and 35 days from the first BrdU
injection (t-test, P=0.01), and these cells disappeared by 65 days
(Fig. 11A). At all survival times, BrdU
+
cells did not express markers
of known striatal neurons [DARPP-32, Calbindin, Parvalbumin,
Calretinin, nNOS, ChAT (Kawaguchi, 1997)] or transcription factors
involved in their differentiation [Isl1, Nkx2.1 (Marin et al., 2000)].
Thus, newborn DCX
+
cells of the EC-LS may either die or
differentiate into a neuronal type that does not express conventional
striatal markers. To analyze this possibility, we traced the vPSB cells
by injection of a GFP lentiviral vector at p14. Between p18 and p26,
the animals were treated with six BrdU injections, and then sacrificed
30 days after the last injection (Fig. 8C; n=4). In the LS and dorsal
EC of these animals (one in three sections/animal) we identified 56
GFP
+
/BrdU
+
cells (Fig. 11B) and onlytwo of themwere negative for
DCX (3.6% of all GFP
+
/BrdU
+
; not shown). Notably, in the vPSB
we always found some GFP
+
cells showing morphologic features of
astrocytes and that expressed BLBP and were BrdU
+
(Fig. 11E-H),
indicating that VSVG-GFP-infected vPSB BLBP
+
cells were able to
proliferate and retain BrdU labeling long-term. Except for the
Fig. 4. Rate of cell genesis in the EC-LS during postnatal development.(A) Section of EC-LS stained for DCX (green) and BrdU (magenta) in a p29 animal
injected with BrdU at p18 (p18+11d). (B-D′) Higher magnifications of the boxed regions in A. In B, single confocal planes of the cells marked by asterisk and
asterisk with prime are shown in the insets. (E,E′) Single confocal plane of the chains in D. (F) Coronal section from a p12 animal injected with BrdU at p1 ( p1+11)
showing BrdU
+
cells (magenta) that are either SOX10
+
(arrowhead) or SOX10
−
(arrow). (G) Number of DCX/BrdU colabeled cells in the EC-LS at different
postnatal ages (blue line). The red lines indicate the survival time. (H) Density of total BrdU
+
cells and of BrdU
+
cells that are DCX
+
, SOX10
+
or DCX
−
/SOX10
−
in the EC-LS between p1 and p18. Error bars indicate s.d. *P<0.05, **P<0.005 (G, one-way ANOVA; H, t-test). Scale bars: 75 μm in A,F; 10 μm in B-E′.
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injection site in the dorsal striatum, BrdU
+
/GFP
+
cells were not
observed in other regions of the striatum or of the IC and EC.
Collectively, these data indicate that most newborn DCX
+
cells in the
EC-LS are transient elements that retain the expression of DCX
throughout their existence.
Interestingly, 72±12% of the DCX staining in the EC-LS was
located within the white matter tract of IC or EC. Conversely, a 3D
reconstruction of 23 randomly chosen IC bundles located within
300 μm of the EC in a p18 animal indicated that 16 (70%) of them
contained DCX
+
processes for more than 80% of their length. Serial
section 3D reconstructions of DCX
+
cells in these IC bundles
revealed that most of these cells have complex morphologies with
some of their processes extending over a millimeter (Fig. 11I-L;
supplementary material Movie 1). In general, the morphology of the
DCX
+
cells in the EC, LS and IC did not match that of any known
striatal cell type, supporting the proposal that these cells represent
previously undescribed cell types, the function of which remains to
be investigated. Interestingly, almost all EC-LS individual DCX
+
cells expressed Sp8 (Fig. 11D), a transcription factor that is
associated with distinct lateral and caudal ganglionic eminence-
derived interneurons (Ma et al., 2012; Waclaw et al., 2006).
DISCUSSION
Here we demonstrate that the LS of the guinea pig contains a
population of NPs that is quiescent at birth and becomes transiently
activated during juvenile life, giving rise to neuroblasts that are
characterized by a transient existence.
Activation of quiescent NPs
The occurrence of quiescent NPs within the adult brain parenchyma
has long been hypothesized (Emsley et al., 2005; Ourednik et al.,
2001; Palmer et al., 1999). Here, we provide clear evidence that such
cells can actually occur in vivo and can be activated under normal
conditions. By coupling a viral lineage tracing with BrdU labeling,
we directly proved that new EC-LS neuroblasts are generated locally
from progenitors that already reside in the EC-LS at birth but become
activated only later in postnatal development. Intraventricular
injections of the VSVG-GFP did not reveal any contribution of the
SVZ to the EC-LS neurogenic system. Thus, although we cannot
exclude the possibility that a small population of spatially restricted
SVZ progenitors was spared from our viral injections, these results
support the notion that local NPs are the major, if not unique, source
of EC-LS neuroblasts.
Our analyses strongly suggest that EC-LS NPs are located in the
vPSB, where, around weaning, we observed the appearance of a
germinative-like layer showing chains of neuroblasts closely
associated with clusters of intermediate-like progenitors. The vPSB
was also enriched in astrocytes that, as the primary NPs of constitutive
neurogenic niches, express immature markers, proliferate at a low rate
and retain BrdU labeling in the long-term (Doetsch et al., 1999;
Lugert et al., 2010; Ponti et al., 2013). These cells were also
preferentially labeled by VSVG-GFP at birth, further supporting that
the primary NPs of the vPSB are BLBP
+
astrocytes. This idea is
consistent with the fact that both embryonic and adult primary NPs
are within the astroglial lineage (Doetsch et al., 1999; Anthony et al.,
Fig. 5. Ki67
+
cells in the EC-LS during postnatal development. (A,B) Coronal sections of the EC-LS from p1 (A) and p18 (B) animals stained for BLBP
(magenta) and Ki67 (green). (C) Clusters of Ki67
+
cells (green) closely associated with DCX
+
chains (orange). (D-D″) Single confocal plane showing Ki67
+
cells
that in part express DCX (KD arrowheads) and in part do not (KaD arrow). (E,E′) Ki67
+
cells that express (arrowheads) or do not express (arrow) DCX are labeled
by a 2 h BrdU (violet) pulse in the vPSB at p18. (F-F‴) Single confocal plane showing the colocalization of PAX6 (violet) and Ki67 in both KaD (arrow) and KD
(arrowhead) cells in the vPSB at p18. (G-G‴) Colabeling between Ki67 and BLBP (magenta, asterisk) or SOX10 (orange, asterisk with prime) in the vPSB at p18.
A single confocal plane and a reslice ( yellow outline) along the yellow line in G is shown for both cells. (H-K) The percentage of KaD, KD and D cells co-expressing
PAX6 (H) and the density of Ki67
+
(I), BLBP
+
/Ki67
+
(J) and SOX10
+
/Ki67
+
(K) cells in the vPSB, EC and LS, between p1 and p18. Error bars indicate s.d.
**P<0.005 (t-test). Scale bars: 100 μm in A,B; 20 μm in C,E,E′,G-G‴;10μm in F-F‴;5μm in D-D″and insets in G.
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DEVELOPMENT

2004). Although parenchymal astrocytes have not yet been shown to
produce neurons in the adult brain, acute lesions can stimulate a
neurogenic potential in these cells, at least in vitro (Buffo et al., 2008;
Sirko et al., 2013). Together with our results, one interpretation of
these data is that some parenchymal astrocytes possess a
physiological neurogenic potential that requires specific cues in
order to be activated. It is of note that, in respect to other EC-LS
regions, the vPSB of newborn animals showed a relatively high level
of proliferation and contained a few BLBP/Ki67
+
cells. Accordingly,
BrdU injections at birth resulted in a few BrdU-labeled neuroblasts at
p12. This might indicate that the vPSB is not entirely quiescent at
birth, but rather that some rare EC-LS progenitors are either already
activated or undergo non-neurogenic cell division.
The ability to switch between active and quiescent states is a
fundamental feature of adult stem cells, including the neurogenic
astrocytes of the SVZ and DG (Cheung and Rando, 2013; Wang et al.,
2011). These cells operatewithin neurogenic niches that are set during
embryonic development and are thought to play an active role in
regulating their quiescence and their capacity to produce neurons
(Fuentealba et al., 2012; Kriegstein and Alvarez-Buylla, 2009). In this
respect, the activation of vPSB progenitors is particularly surprising
as it occurs within a tissue that is classically considered strongly
gliogenic (Emsley et al., 2005; Ninkovic and Götz, 2013). This latter
concept, however, is mainly derived from heterotopic transplantation
of NPs (Eriksson et al., 2003; Lim et al., 2000; Shihabuddin et al.,
2000) and it should not be generalized. Accordingly, a few cases of
parenchymal NPs are emerging (Bi et al., 2011; Luzzati et al., 2006,
2011a; Ohira et al., 2010; Ponti et al., 2008) and here we provide an
unequivocal example that such cells can eventually give rise to a
germinative-like layer. The next step will be to understand how
quiescent parenchymal NPs are activated in the adult brain as well as
the cues that sustain their ability to produce neurons in vivo.
Fate of the newly generated neuroblasts
Although quiescent parenchymal NPs represent an attractive new
source of neurons for endogenous repair, understanding their
specification remains a fundamental issue. Newborn DCX
+
cells in
the EC-LS exist transiently and do not express markers of either
mature or immature striatal cell types. Nonetheless, these cells often
show differentiated morphologies. It is noteworthy that an elevated
death rate of newly generated neurons is a common feature in
different studies of striatal and cortical neurogenesis, in both normal
and pathologic conditions (Arvidsson et al., 2002; Chen et al., 2004;
Gould et al., 2001; Luzzati et al., 2006, 2011a). The death of new
striatal and cortical neurons has often been attributed to a strong
selection exerted by a non-permissive environment (Kernie and
Parent, 2010). This hypothesis is not, however, consistent with data
showing that transplanted embryonic precursors can survive and
differentiate within the adult striatum and neocortex (Luzzati et al.,
2011a; Shin et al., 2000). Rather, transient neurons have been
described during embryonic development and early postnatal stages
(Niquille et al., 2009; Teissier et al., 2012) and, at least in the
neocortex, it has been suggested that their death is regulated by
intrinsically defined mechanisms (Southwell et al., 2012).
Fig. 6. BLBP
+
cells are label-retaining cells.(A) z-projection of the vPSB in a
p65 animal showing two label-retaining cells (marked by asterisk and asterisk
with prime) stained for BLBP (magenta) and BrdU (green), and surrounded by
GFAP
+
processes. (B-E) Single confocal plane along the lines in A (B,C)
and reslice (D,E) showing a clear colocalization with GFAP (asterisk) and a
more ambiguous colocalization (asterisk with prime). Scale bars: 20 μminA;
10 μm in B-E.
Fig. 7. Tissue explants from BrdU-treated animals. (A,B) SVZ (A) and
EC-LS (B) tissue explants grown in culture for 3 days were obtained from a
p18 animal that received a BrdU injection 2 h before sacrifice. Explants are
labeled for DCX (green) and BrdU (magenta). Insets show higher
magnifications. (C) The percentage of DCX
+
cells labeled by BrdU in the SVZ
or EC-LS when the BrdU is given in vivo (2 h) or in vitro between 24 and 48 h or
24 and 72 h after plating. Error bars indicate s.d. Scale bars: 20 μm.
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Interestingly, these cells are often directed to white matter tracts,
where they contribute to the guidance of growing axons (López-
Bendito et al., 2006; Niquille et al., 2009, 2013; Sato et al., 1998).
Moreover, these cells originate, in part, from the caudal ganglionic
eminence, a major source of Sp8
+
interneurons (Ma et al., 2012;
Niquille et al., 2013). These observations raise the possibility that
the transient EC-LS neuroblasts belong to a broader population of
cells involved in transient forms of plasticity, possibly related to the
remodeling of long-range connections. Additional analyses will be
required to test this hypothesis.
Similarities between EC-LS and lesion-induced neurogenic
systems
Striatal neurogenesis has been described in different mammalian
species in both normal and pathologic conditions (Bédard et al.,
2002; Luzzati et al., 2006, 2011a; Tattersfield et al., 2004). The
organization of the vPSB is consistently different from the
scattered and PAX6-negative clusters of proliferating cells that we
observed in the striatal gray matter of the rabbit and of a mice
Fig. 10. Viral lineage tracing of EC-LS
progenitors. (A) Frontal views of a 3D
model of GFP staining in the striatum of a p4
animal injected with VSVG-GFP at p1. The
needle track is in black; GFP staining is dark
green within the striatum and light green
outside. (B) GFP staining (green) in a
section at the level of the injection site.
(C,C′) The vPSB at p4 stained for GFP
(green), BLBP (magenta), SOX10 (orange)
and with DAPI (blue). Arrowhead indicates a
GFP
+
cell in the LS. (D) z-projection and
reslice (asterisk) of the BLBP
+
/GFP
+
cell
indicated by an arrow in C. (E-F‴) Dorsal EC-
LS of a p33 animal labeled for GFP (green),
DCX (white), BrdU (red) and NeuN
(magenta). The inset in E-E″shows a DCX
+
/
BrdU
+
but GFP
−
cell. (F-F‴) Higher
magnification of the area outlined in E-E″
showing three cells triple labeled for DCX,
BrdU and GFP (one cell is magnified in the
inset). Scale bars: 200 μminB;50μmin
E-E″;40μm in C,C′;20μminF.
Fig. 9. Viral lineage tracing of SVZ progenitors. (A) GFP (green) and DCX
(magenta) staining in the SVZ at the level of the lateral ventricle of a p33
animal. (B) BrdU
+
(magenta) and GFP
+
(green) cells in the deep granule cell
layer and RMS of a p33 animal. Insets show a higher magnification and a
reslice of a BrdU
+
/GFP
+
cell. Dotted line indicates the border between RMS
and GCL. (C,D) GFP-labeled periglomerular (C) and upper (D) granule cell.
CC, corpus callosum; GCL, granule cell layer; GL, glomerular layer; LV, lateral
ventricle; STR, striatum; RMS, rostral migratory stream. Scale bars. 400 μmin
A; 100 μminB;50μm in C,D.
Fig. 8. Experimental design for analysis of the origin and fate of EC-LS
neurons. (A) Lineage tracing of EC-LS and SVZ cells before the activation of
neurogenesis. (B) Timecourse of newborn neuron survival and differentiation.
(C) Fate of virally tagged newborn neurons. Red lines indicate survival time.
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model of progressive degeneration (Luzzati et al., 2006). Rather,
the vPSB is more closely related to the SVZ and, in particular,
with its lateral extension along the dorsal pallial subpallial
boundary (dPSB) (Luzzati et al., 2011a). We previously showed
that the mouse dPSB is strongly expanded during progressive
degeneration and is the main source of neuroblasts directed to the
striatum. These cells share several features with the EC-LS
neuroblasts, including the expression of Sp8, a transient existence
and the tropism for IC bundles. Since DCX
+
cells are not observed
in the EC-LS of rabbits and mice (F.L., unpublished observation),
we cannot exclude the possibility that the postnatal EC-LS
neurogenic system is specific to the guinea pig, possibly related to
the highly precocial nature of this species. Nonetheless, its precise
spatial and temporal regulation makes the vPSB an ideal model
with which to study the activation of quiescent progenitors as well
as the role of parenchymal neurogenesis in the striatum. In this
respect, this model might also shed new light on the role of lesion-
induced neurogenesis.
MATERIALS AND METHODS
Animals, BrdU injections and tissue preparation
All experimental procedures were in accordance with the European
Communities Council Directive of 24 November 1986 (86 609 EEC), the
Italian law for the care and use of experimental animals (DL116 92) and
approved by the Italian Ministry of Health and the Bioethical Committee of
the University of Turin. All experiments were designed to minimize the
numbers of animals used and their discomfort.
Experiments were performed on 88 male and female Hartley guinea pigs
(Cavia porcellus) ranging from birth to 8 months of age. Forty-one animals
were intraperitoneally injected with 5-bromo-2-deoxyuridine (BrdU;
Sigma; 50 mg/kg body weight in 0.1 M Tris pH 7.6). Animals were
deeply anesthetized with a ketamine/xylazine solution (100 and 33 mg/kg,
respectively) and transcardially perfused with an ice-cold saline solution
(0.9% NaCl), followed by a solution of 4% paraformaldehyde (PFA) plus
2% picric acid in 0.1 M sodium phosphate buffer. Brains were then
postfixed overnight, cryoprotected, frozen at −80°C, and cut on a cryostat in
series of 40 or 50 μm thick coronal sections.
Electron microscopy
p18 guinea pigs were perfused intracardially with an heparinized saline
solution followed by 2% glutaraldehyde plus 1% PFA in 0.1 M sodium
phosphate buffer pH 7.4. Brains were postfixed for 2 h and cut coronally
with a Vibratome (300 μm slices). Sections were fixed in osmium
ferrocyanide for 1 h, stained with 1% uranyl acetate, dehydrated and
embedded in Araldite. Ultra-thin sections were examined under a JEM-1010
transmission electron microscope (Jeol) equipped with a side-mounted
CCD camera (Mega View III; Soft Imaging Systems) for photography.
Lentiviral vectors
Vector stocks were produced by transient transfection of the transfer plasmid
expressing eGFP under the control of the CMV promoter, the packaging
plasmids pMDLg/pRRE and pRSV.REV, and the VSV envelope plasmid
pMD2.VSV-Gin 293T cells as described (Follenzi et al., 2000). Viralparticles
were purified and concentrated by ultracentrifugation as described (Dull et al.,
1998). Vectortiter on HeLa cells was 2×10
9
TU/ml. The virus wasthen diluted
1/20 in PBS containing 0.6% glucose and frozen at −80°C.
Fig. 11. Fate of the newly generated EC-LS neuroblasts. (A) Timecourse of the number of DCX/BrdU double-labeled cells in the EC-LS. Error bars indicate s.d.
(B,B′) One cell in the LS of an animal treated as shown in Fig. 8C, triple labeled for DCX (white), GFP (green) and BrdU (red). (C) Higher magnification of the
cell body of the cell in B; single and double asterisks are reslices along the indicated planes. (D,D′) DCX
+
cells (white) in the LS expressing Sp8 (green).
(E-H) z-projection (E), single slice (F) and reslices (G,H) of a cell labeled for GFP (green), BLBP (magenta) and BrdU (violet) in an animal treated as shown
in Fig. 8C. (I) z-projection of a 3D reconstructed tract of 750 μm of the dorsal EC-LS stained for DCX. (J-L) Reconstruction of an IC bundle (traced on the basis of
the MAG staining; yellow outline in I, grayshade in J,K), and of all the DCX
+
cells and processes contacting it (colored lines). Some cells have the cell body outside
the IC bundle (blue, magenta and light green). The small yellow spheres indicate processes exiting outside the reconstructed volume (blue and light green).
(L) Individual views of the reconstructed cells. Scale bars: 100 μm in I-L; 20 μm in B,B′,D-E; 10 μm in C,F-H.
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DEVELOPMENT

Stereotaxic injections
Animals were anesthetized by intraperitoneal injection of ketamine (30 mg/kg;
Ketavet; Bayer) supplemented with xylazine (3 mg/kg; Rompun; Bayer) and
positioned in a stereotaxic apparatus (Stoelting). Using a glass micropipette and
a pneumatic pressure injection apparatus (Picospritzer II; General Valve Corp),
3 µl of VSVG-GFP was bilaterally injected at stereotaxic coordinates 2 mm
AP, ±2 mm ML a nd −4 mm DV;2 µlof theVSVG-GFP virus were injectedat
stereotaxic coordinates 1.5 mm AP, ±5 mm ML and −5mmDV.
Organotypic cultures
Tissue explant cultures were performed as described by Garzotto et al.
(2008). Briefly, brains were dissected out and cut by Vibratome into 250 μm
thick coronal slices. Tissues from SVZ and vPSB were trimmed into pieces
of ∼300 μm. Explants were subsequently embedded in 75% Matrigel
growth factor reduced (BD Biosciences). Explants were maintained 3 days
in vitro in 5% CO
2
at 37°C in Neurobasal medium (Invitrogen)
supplemented with 1× N-2 (Invitrogen), 25 μg/ml gentamicin (Invitrogen)
and 0.5 mM glutamine (Invitrogen). BrdU (10 μM) was added to the culture
medium of some explants on the first or second day in vitro.
Immunohistochemistry
Immunohistochemical reactions were performed on sections incubated for
24-48 h at 4°C in a solution of 0.01 M PBS ( pH 7.4) containing 0.5-1%
Triton X-100, normal serum and primary antibodies (supplementary
material Table S1). For BrdU staining, DNA was denatured in 2 N HCl
for 30 min at 37°C. Sections were then rinsed in 0.1 M borate buffer (pH
8.5). Sections were then incubated with appropriate solutions of secondary
antisera (supplementary material Table S1). Slides were coverslipped with
antifade mounting medium with DABCO (Sigma) and analyzed with a laser
scanning TCS SP5 (Leica Microsystems). Images were processed using
ImageJ (NIH) and Photoshop 7.0 (Adobe Systems). Only whole-image
adjustments to color, contrast and brightness were made.
Light and confocal 3D reconstructions
3D reconstructions were performed as described by Luzzati et al. (2011a,b).
Briefly, images from each section were stitched in Fiji (http://fiji.sc/
Image_Stitching), aligned, traced and rendered in Reconstruct 1.1 (http://
synapses.clm.utexas.edu/tools/reconstruct/reconstruct.stm). The entire EC-
LS 3D reconstruction at p18 was performed on 165 40 μm thick coronal
sections (voxel size 0.76×0.76×40 µm). The reconstruction of the vPSB
chains, IC bundles (voxel size 0.76×0.76×2 µm) and DCX
+
cells in an IC
bundle (voxel size 0.12×0.12×0.8 µm) was performed on 15 consecutive
50 μm thick sections. For reconstruction of DCX
+
cells, the IC was traced in
Reconstruct and DCX
+
cells in Neuronstudio 1.6 (Darren Myatt, University
of Reading, UK). The two reconstructions were fused in Blender 2.6
(Blender Foundation, Amsterdam, The Netherlands). Texturing and
rendering of the 3D models was performed in Blender.
Quantifications and statistical analyses
The total number of cells was evaluated stereologically either with
Neurolucida 7.0 (MBF Bioscience) (DCX
+
and DCX
+
/Ki67
+
)orby
confocal microscope (DCX
+
/BrdU
+
) in a one-in-six series of sections
(40 µm thick).
The density of BrdU
+
and Ki67
+
cells was evaluated in ImageJ in three to
five confocal sections (n=3; voxel size 0.38×0.38×1 µm). For the LS we
considered the area located within the first 300 μm lateral to the EC. Cells
counted per animal ranged 38-110 for BrdU
+
, 108-363 for Ki67
+
, 0-12 for
BLBP
+
/Ki67
+
and 5-12 for BLBP
+
/BrdU
+
cells in the vPSB.
The percentage of cells in KI67
+
clusters (defined as a group of at least
four cells with closely contacting cell bodies) that expressed DCX was
evaluated by counting 137-170 cells per animal using a confocal microscope
(voxel size 0.38×0.38×1 µm). The percentage of DCX
+
cells in Ki67
+
clusters was counted on 24 clusters per animal (n=3). Percentage of KaD,
Ki67
+
/DCX
+
and Ki67
−
/DCX
+
cells colabeled for PAX6 and SOX9 was
evaluated in 80-126 cells per animal for each cell type.
The percentage of DCX
+
cells colabeled for GFP and BrdU in the LS and
dorsal EC was counted in Fiji in 50 µm thick sections spaced 300 µm apart,
acquired at the confocal microscope (voxel size 0.38×0.38×1 µm). For the
lineage tracing of vPSB progenitors, in each animal a minimum of three
sections and 30 GFP
+
/DCX
+
cells were counted. For the fate of virally
tagged newborn neurons, all sections were counted.
The total number of cells that had migrated from in vitro explants was
manually counted with Neurolucida 7.0.
Statistical analyses were performed using Statistical Package for the
Social Sciences 14.0 (SPSS). Analysis of variance (ANOVA) was followed
by Tukey’s post-hoc test when appropriate.
Acknowledgements
We thank Andrea Moscato and Monica Masciavèfor their important contribution to
the preliminary set of experiments of this work. This work is dedicatedto the memory
of Professor Maria Fosca Franzoni.
Competing interests
The authors declare no competing financial interests.
Author contributions
F.L. designed and performed the experiments, analyzed the data and wrote the
paper. P.P. assisted in experiment design, data analysis and writing. A.F. assisted in
the experimental design. M.A. and L.B. performed the electron microscopy. E.V. and
C.R. produced the viral vector. G.N. and L.O. assisted in the acquisition and analysis
of data.
Funding
This work was supported by PRIN-Peretto 2010-2011.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.107987/-/DC1
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RESEARCH ARTICLE Development (2014) 141, 4065-4075 doi:10.1242/dev.107987
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