Substantial migration of SVZ cells to the cortex results in the generation of new neurons in the excitotoxically damaged immature rat brain.
ABSTRACT Mammalian SVZ progenitors continuously generate new neurons in the olfactory bulb. After injury, changes in SVZ cell number suggest injury-induced migration. Studies that trace the migration of SVZ precursors into neurodegenerating areas are lacking. Previously, we showed a decrease in BrdU+SVZ cells following excitotoxic damage to the immature rat cortex. Here, we demonstrate that NMDA-induced injury forces endogenous Cell Tracker Green (CTG) labeled VZ/SVZ precursors out of the SVZ into the neurodegenerating cortex. CTG+/Nestin+/Filamin A+ precursors are closely associated with vimentin+/GFAP+/GLAST+ filaments and express both chemokine receptor CXCR4 and Robo1. In the cortex, SVZ-derived progenitors show a progressive expression of developing, migrating and mature neurons and glial markers. CTG+/GFAP+ astrocytes greatly outnumber CTG+/MAP2+/NeuN+ neurons. SVZ-derived progenitors differentiate into both tbr1+ cortical glutamatergic neurons and calretinin+ interneurons. But, there is little integration of these neurons into the existing circuitry, as seen by Fluorogold retrograde tracing from the internal capsule.
- SourceAvailable from: Jorge García-Marqués[Show abstract] [Hide abstract]
ABSTRACT: The rostral migratory stream (RMS) is a well defined migratory pathway for precursors of olfactory bulb (OB) interneurons. Throughout the RMS an intense astroglial matrix surrounds the migratory cells. However, it is not clear to what extent the astroglial matrix participates in migration. Here, we have analyzed the migratory behavior of neuroblasts cultured on monolayers of astrocytes isolated from areas that are permissive (RMS and OB) and nonpermissive (cortex and adjacent cortical areas) to migration. Our results demonstrate robust neuroblast migration when RMS-explants are cultured on OB or RMS-astrocytes, in contrast to their behavior on astroglia derived from nonpermissive areas. These differences, mediated by astrocyte-derived nonsoluble factors, are related to the overexpression of extracellular matrix and cell adhesion molecules, as revealed by real-time qRT-PCR. Our results show that astroglia heterogeneity could play a significant role in migration within the RMS and in cell detachment in the OB.Glia 08/2009; 58(2):218-30. · 5.07 Impact Factor
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ABSTRACT: The subventricular zone (SVZ) is the principal neurogenic niche present in the adult non-human mammalian brain. Neurons generated in the SVZ migrate along the rostral migratory stream to reach the olfactory bulb. Brain injuries stimulate SVZ neurogenesis and direct migration of new progenitors to the sites of injury. To date, cortical injury-induced adult SVZ neurogenesis in mice remains ambiguous and migration of neural progenitors to the site of injury has not been studied in detail. Here we report that aspiration lesion in the motor cortex induces a transient, but significant increase in the proliferation as well as neurogenesis in the SVZ. New neural progenitors migrate ectopically to the injured area with the assistance of blood vessels and reactive astrocytes. The SVZ origin of these progenitors was further confirmed using lentiviral transduction. In addition, we show that astrocyte-assisted ectopic migration is regulated by CXCR4/SDF-1 signaling pathway. Finally, upon reaching the lesion area, these progenitors differentiate mainly into glial cells and, to a lesser extent, mature neurons. These data provide a detailed account of the changes occurring in the SVZ and the cortex following lesion, and indicate the potential of the endogenous neural progenitors in cortical repair.Stem Cell Research 06/2013; 11(3):965-977. · 4.47 Impact Factor
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ABSTRACT: Neural precursor cells (NPCs) are activated in central nervous system injury. However, despite being multipotential, their progeny differentiates into astrocytes rather than neurons in situ. We have investigated the role of epidermal growth factor receptor (EGFR) in the generation of non-neurogenic conditions. Cultured mouse subventricular zone NPCs exposed to differentiating conditions for 4 days generated approximately 50% astrocytes and 30% neuroblasts. Inhibition of EGFR with 4-(3-chloroanilino)-6,7-dimethoxyquinazoline significantly increased the number of neuroblasts and decreased that of astrocytes. The same effects were observed upon treatment with the metalloprotease inhibitor galardin, N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001), which prevented endogenous transforming growth factor-α (TGF-α) release. These results suggested that metalloprotease-dependent EGFR-ligand shedding maintained EGFR activation and favored gliogenesis over neurogenesis. Using a disintegrin and metalloprotease 17 (ADAM-17) small interference RNAs transfection of NPCs, ADAM-17 was identified as the metalloprotease involved in cell differentiation in these cultures. In vivo experiments revealed a significant upregulation of ADAM-17 mRNA and de novo expression of ADAM-17 protein in areas of cortical injury in adult mice. Local NPCs, identified by nestin staining, expressed high levels of ADAM-17, as well as TGF-α and EGFR, the three molecules necessary to prevent neurogenesis and promote glial differentiation in vitro. Chronic local infusions of GM6001 resulted in a notable increase in the number of neuroblasts around the lesion. These results indicate that, in vivo, the activation of a metalloprotease, most probably ADAM-17, initiates EGFR-ligand shedding and EGFR activation in an autocrine manner, preventing the generation of new neurons from NPCs. Inhibition of ADAM-17, the limiting step in this sequence, may contribute to the generation of neurogenic niches in areas of brain damage.Stem Cells 08/2011; 29(10):1628-39. · 7.70 Impact Factor
Substantial migration of SVZ cells to the cortex results in the
generation of new neurons in the excitotoxically damaged
immature rat brain
Maryam Faiz,a,⁎Laia Acarin,a,⁎Sonia Villapol,aStefan Schulz,b
Bernardo Castellano,aand Berta Gonzaleza
aMedical Histology, Department of Cell Biology, Physiology and Immunology, Neuroscience Institute, Autonomous University of Barcelona, Spain
bInstitut für Pharmakologie und Toxikologie, Friedrich-Schiller-Universität Jena, Nonnenplan 4, D-07743 Jena, Germany
Received 18 September 2007; revised 5 February 2008; accepted 13 February 2008
Available online 4 March 2008
Mammalian SVZ progenitors continuously generate new neurons in the
olfactory bulb. After injury, changes in SVZ cell number suggest injury-
neurodegenerating areas are lacking. Previously, we showed a decrease in
neurodegenerating cortex. CTG+/Nestin+/Filamin A+ precursors are
closely associated with vimentin+/GFAP+/GLAST+ filaments and
express both chemokine receptor CXCR4 and Robo1. In the cortex,
SVZ-derived progenitors show a progressive expression of developing,
migrating and mature neurons and glial markers. CTG+/GFAP+
astrocytes greatly outnumber CTG+/MAP2+/NeuN+ neurons. SVZ-
derived progenitors differentiate into both tbr1+ cortical glutamatergic
neurons and calretinin+ interneurons. But, there is little integration of
these neurons into the existing circuitry, as seen by Fluorogold retrograde
tracing from the internal capsule.
© 2008 Elsevier Inc. All rights reserved.
Keywords: Neurogenesis; Gliogenesis; CNS injury; Differentiation;
The adult mammalian brain retains actively proliferating
progenitor cells and quiescent stem cells in the subventricular zone
(SVZ) of the lateral ventricles. In physiological conditions, these
cells generate new neurons that migrate through the rostral
migratory stream (RMS)to the olfactory bulb (OB) and differentiate
into interneurons (Altman, 1969; Alvarez-Buylla and Garcia-
Verdugo, 2002; Doetsch et al., 1997; Garcia-Verdugo et al., 1998;
Morshead et al., 1994). Brain damage induces neurogenesis in
various experimental paradigms in both the adult and developing
brain and raises the possibility of cell replacement from endogenous
neural stem cells (Arvidsson et al., 2002; Fagel et al., 2006; Magavi
et al., 2000; Ong et al., 2005; Parent et al., 2002; Plane et al., 2004;
Yang et al., 2007).
only have to include proliferation, but also differentiation, migration
and appropriate integration to provide functional and effective
regeneration. In the immature and adult brain, neural progenitor
transplantation studies have shown that cells are able to migrate
et al., 2002; Shin et al., 2000) and studies of endogenous progenitor
cells in the adult brain have shown new cells in the damaged areas
followingseveraltypesof injury (Jinetal.,2003,2006;Magavietal.,
2000; Parent et al., 2002). However, while the appearance of
proliferating cells showing immature neuronal markers such as
doublecortin (dcx) in non-neurogenic regions of the brain is sug-
gestive of migration and differentiation towards a neuronal fate, little
has been done to specifically characterize the migration of SVZ cells
to sites of damage and their integration into the existing neural
It is known that the early postnatal brain has a greater capacity for
uniquely to injury, showing increased plasticity and improved out-
come(Hagberg et al.,1997; Kolb et al.,1996; Vannucci and Hagberg,
Mol. Cell. Neurosci. 38 (2008) 170–182
⁎Corresponding authors. L. Acarin is to be contacted at Medical
Histology, Department of Cell Biology, Physiology and Immunology,
Autonomous University of Barcelona, 08193 Bellaterra, Spain. Fax: +34
935812392. M. Faiz, Department of Clinical Neuroscience and Rehabilita-
tion, Institute Neuroscience and Physiology, Göteborg University, SE-405-
30 Göteborg, Sweden. Fax: +46 314416108.
E-mail addresses: email@example.com (M. Faiz),
firstname.lastname@example.org (L. Acarin).
Available online on ScienceDirect (www.sciencedirect.com).
1044-7431/$ - see front matter © 2008 Elsevier Inc. All rights reserved.
2004). Surprisingly, the contribution of progenitor pools to recovery
from immature brain damage remains largely unknown. Only very
recently, a study of perinatal hypoxia/ischemia showed that there is a
2007). We have previously shown that postnatal cortical excitotoxi-
of proliferating cells (Bromodeoxyuridine, BrdU+) in the SVZ with a
later appearance of striatal groups of proliferating cells (Faiz et al.,
2005). The principal objective of the present study was to track the
migration of SVZ cells after cortical excitotoxic damage to the devel-
oping brain, to delineate the mechanisms used by migrating precursor
cells, to determine their phenotype at long survival times and to
evaluate if these cells are able to integrate into the damaged tissue.
Injection of NMDA into the right sensorimotor cortex of P9 rats
caused neuronal degeneration accompanied by a glial response in
the cortex at the level of the injection site and surrounding tissue
extending to the septum, striatum and rostral hippocampus. No
affectation was seen in the contralateral hemisphere, as previously
described in detail (Acarin et al., 1999a,b).
Excitotoxicity causes migration of VZ/SVZ cells to cortical layer VI
In order to trace the migration of VZ/SVZ cells, CTG was
injected into the right lateral ventricle. CTG is a non-transferable
lipophillic dye and injection into the ventricle resulted in the
labeling of all surrounding cells, both dividing and non-dividing,
such that it was used to track of SVZ cells (see Figs. 2a–d).
CTG and CTG–saline injected controls showed CTG labeling of
the ventricle walls (Figs. 2b–d), the OB (data not shown) and slight
labeling of the hippocampal fimbria until 14 days post-injection
(dpi; Fig. 2e). Very few CTG+ cells were found in the ipsilateral
cortex of control animals at 5 dpi (Table 2 Supplementary material)
group. NMDA-lesioned animals previously injected with CTG
showed no CTG+ cells in the contralateral hemisphere at any
survival time. In the ipsilateral hemisphere, CTG labeling was
(dpl), CTG+ SVZ cells had migrated into the lesioned striatum,
corpus callosum, layer VI of the cortex and the damaged upper
cortical layers (Figs. 1; 2g–p; 3). At 14 dpl CTG+ cells were still
present in layer VI and in the damaged upper cortex (Figs. 1 and 5)
and remained in these areas until the last time point studied, 49 dpl
(Figs. 1, 6, 7). Quantitative analysis showed that the number of
CTG+ cells seen along layer VI (zones 1–3,Fig. 1)and in the upper
damaged cortex (zone 4, Fig. 1) was significantly greater than the
number of CTG+ cells in either CTG-injected and CTG–saline-
injected control brains at all time points studied (Table 2 Sup-
plementary material; Fig. 1). There were no significant differences
between the total number of CTG cells in the lesioned cortex at the
differenttimepointsstudied (196±39 CTG+cellsat5dpl,226±45
CTG+ cells at 14 dpl, 186±60 CTG+ cells at 49 dpl; Table 2
P9 BrdU pulse-labeled SVZ CTG+ cells do proliferate en route to
To determine the relationship between proliferation and migration
of SVZ cells, BrdU pulse-labeled CTG-injected pups were analyzed
by immunohistochemistry for BrdU in order to analyze the
proliferative capacity of resident SVZ precursors immediately after
injury (Fig. 2f). CTG+ cells in the cortex did not colocalize with cells
that had incorporated BrdU on P9 (Fig. 2g). In addition, when the
proliferation marker Ki-67 at 5 dpl was used to analyze proliferation
cells expressing Ki67 were observed (data not shown).
Early after damage, at 5 dpl, CTG+ cells express markers of
progenitor and migrating cells and are found in contact with radial
glia-like elongated processes
To determine the phenotype of SVZ migrating cells, immunohis-
tochemically labeled sections of CTG-injected lesioned brains were
analyzed. CTG+ cells were found to express a number of proteins
demonstrative of progenitor cells and migrating cells. CTG+ cells
expressed the intermediate filament protein, nestin, characteristic of
A-interacting protein (FILIP) mechanism used by VZ/SVZ cells that
migrate out of the cortical ventricular zone early in development
(Nagano et al., 2002), was also detected (Fig. 2l). Most CTG+ cells
also expressed doublecortin (dcx), a marker for migrating immature
neurons (Figs. 2m–p). CTG+/nestin+ and CTG+/dcx+ cells had the
cortex (Figs. 2j, o). Tomato lectin binding was rarely seen in CTG+
Fig. 1. Excitotoxicity-induced migration of CTG+ cells mainly to damaged
cortex. (a) CTG+ cell counts were performed in 4 zones in the lesioned cortex
(zones 1–3 sampled layers Vand VI at different distances from midline, while
extension at 5 dpl is shown in yellow and area of CTG+ cells in green.
and 49 dpl, with no significant differences between survival times or zones.
Error bars represent the S.E.M.
171 M. Faiz et al. / Mol. Cell. Neurosci. 38 (2008) 170–182
Fig. 2. At 5 dpl, SVZ-derived CTG+ precursors are found in the cortex and show markers of progenitor cells and migrating immature neurons, but do not proliferate. Coronal and sagittal diagrams of rat brain show
the extension of the lesion (yellow) and the location of CTG+cells (green) at 5 dpl (a). In controls, CTG injection resulted in labeling of all cells in the ventricular wall 4 h post-injection (b, d) and slight labeling of the
hippocampal fimbria (e). Labeling was not seen in the cortex or corpus callosum (c) and at anterior levels to the injection site was restricted to the SVZ (b). After the lesion, although few CTG+ cells are found in the
ipsilateral striatum at 5 dpl (green square in a), most cells are found in the cortex (a, black boxes represent photographed area in g–p, corresponding to zone 2). BrdU staining show that P9 BrdU pulse-labeled CTG+
cells (injection paradigm is seen in f) do not proliferate at 5 dpl (g). CTG+ cells are double labeled with the immature progenitors marker nestin (h–k, k also shows orthogonal view), showing filament extensions.
Filamin A (l), a marker for migratory cells, is observed in all CTG+ cells. Dcx expression, a marker for immature neurons, is also seen in CTG+ cells (l–p; high magnification is seen in the insets, m–o and orthogonal
view in p). CTG (green); BrdU, nestin, Filamin A and dcx (red); DAPI (blue). Cc, corpus callosum; cx, cortex; vw, ventricular wall.
M. Faiz et al. / Mol. Cell. Neurosci. 38 (2008) 170–182
Fig. 3. Some SVZ-derived CTG+ cells precursors start differentiation programs for astrocytes and some are seen attached to radial-glia-like processes extending to the cortex at 5 dpl. CTG+ cells located in layer VI
(zones 2–3, a–h, m) and in the upper cortex (zone 4, i–l, n) show contact with vimentin+ (a–n), GFAP+ (a–h, m) and GLAST+ (i–l, n) glial processes. Vimentin and GFAP staining show elongated radial-glia-like
processes extending into the damaged cortex (b–h) and CTG+ cells in close association with glial filaments (arrowheads in e–h). GLAST immunohistochemistry is also seen in glial processes double labeled for
vimentin and associated to CTG+ cells (arrowheads in i–l; n), although some CTG+ cells show GLASTimmunolabeling (m). Semi-thin section analysis shows the presence of dark progenitors in contact with lightly
stained glial processes (arrowhead in o). CTG (green); GFAP (blue); vimentin (shown in red in b, d, f; shown in blue in k–l); GLAST (red). Cc, corpus callosum; cx, cortex; VI, cortical layer VI.
M. Faiz et al. / Mol. Cell. Neurosci. 38 (2008) 170–182
cells (data not shown). Confocal analysis of the expression of glial
GLAST/EAAT1 showed CTG+ cell somas in close proximity to
vimentin+/GFAP+ (Figs.3a–h, m) and vimentin+/GLAST+(Figs. 3i–
l, n) glial filaments. These glial processes were only seen projecting
towards the cortex and striatum at 5 dpl (Figs. 3a–d) and were not
observed in the contralateral hemisphere or CTG and CTG–saline
injected control brains. However, confocal analysis was unable to
confirm whether CTG+ cells were positive forthese glial markers orif
CTG+ cells were attached to the glial filaments. Toluidine blue semi-
thin section analysis showed that immature bipolar cells, with a dark
nucleiandcytoplasm andleadprocessextensionwere attachedtoglial
the SVZ and crossing the corpus callosum into cortical layers II to VI.
CXCR4 and Robo1 are expressed on migrating CTG+ SVZ
To elucidate possible guidance cues involved in the migration of
were analyzed in CTG-injected lesioned animals. Confocal analysis
CTG+ cells at both 5 and 14 dpl.
Two weeks after damage, CTG+ cells begin to express markers
associated with neuronal and glial differentiation
The study of immunolabeled sections at 14 dpl showed that
still doublepositivefor nestin (Figs.5a,e) andfilamin A (Figs. 5b, f).
migrating immature neurons and polysialylated neural adhesion
molecule (PSA-NCAM), a cell adhesion molecule important for
extended into upper cortical layers (Figs. 5d, l). When glial proteins
were examined, GFAP, vimentin and GLAST labeling showed a lack
of radial-glia-like elongated processes leaving the SVZ and heading
towards the cortex at this time point (Figs. 5g–j, o). Within the glial
with GFAP, vimentin and GLASTand showed astrocyte morphology
with thick processes emerging from the CTG+cellbody (Figs.5m–n,
q–t, p, u–w). Other CTG+ cells, however, still seemed to be
surrounded by GFAP+/Vimentin+ processes. When tomato lectin
(TL) labeling, a marker for microglia/macrophages, was analyzed,
some CTG+ cells were observed in contact with TL+ cellular pro-
corticallayers,suggestiveof macrophage phagocytosisofCTG+ cells
(data not shown).
At 49 days post injury, some CTG+ cells become mature neurons
but the majority become astrocytes
To investigate the long term fate of migrating SVZ cells to the
damaged cortex, immunohistochemistry for the mature neuronal
markers neuronal nuclei antigen (NeuN) and microtubule associated
protein (MAP2) was performed on CTG-injected lesioned animals.
GFAP was used to track glial fate. At 49 days post-lesion, CTG
staining was less intense than at earlier time points (Figs. 6a, e, i, m)
and was dotted within the cytoplasm or observed as a punctuate
staining in the parenchyma. Most CTG+ cells were found within the
GFAP+ cortical glial scar (Figs. 6a–d), where they colocalized with
GFAP (asterisks in Fig. 6d). Some TL+ microglia/macrophages
showed CTG+ labeling in the cytoplasm, suggesting phagocytosis of
CTG+ cells (Fig. 6l). In addition, CTG+ cells were also seen in the
(Figs. 6a–h). The few CTG+ mature neurons expressing NeuN and
MAP2 were seen in this area (Figs. 6a–h, u) and in cortical layer V
(Figs. 6i–k, q–s). However, CTG+ mature neurons were generally
smaller than the surrounding cortical neurons (Figs. 6e–h, asterisks
compared to arrowheads). Finally, in layer VI, CTG+ cells were still
seen (Fig. 6m) but they were not NeuN+ or MAP2+. Layer VI CTG+
and beta-III-tubulin (Figs. 6m–p, t, y–aa).
Some new mature neurons generated from SVZ cells express
calretinin or tbr1
To investigate whether CTG+ cells showed region-specific
calretinin, and the marker for early born cortical glutamatergic
neurons, tbr1, were analyzed in CTG-injected lesioned animals.
Calretinin expression was occasionally seen in CTG+/MAP2+ cells
at 49 dpl (Figs. 7a–h) in the cortex adjacent to the glial scar,
suggesting the generation of a number of interneurons from SVZ
progenitors. Trb1 was present in cortical neurons (Figs. 7i–l) at all
three survival times and in the contralateral hemisphere. However,
to the scar showing irregular lamination and in cortical layer V
(Figs. 7i–l), above CTG+ cells located in layer VI that did not
express tbr1 (Fig. 7i).
Fig. 4. SVZ-derived CTG+ precursors show expression of CXCR4 and
Robo1 receptors both at 5 and 14 dpl. In the damaged cortex, CTG+ cells are
labeled with the chemokine receptor CXCR4 (a, b; high magnification is
seen in inset, b) and the slit receptor Robo 1 (c, d; high magnification is seen
in inset, c) both at 5 and 14 dpl. CTG (green); CXCR4, Robo1 (red).
174M. Faiz et al. / Mol. Cell. Neurosci. 38 (2008) 170–182
Fig. 5. At 14 dpl, SVZ-derived CTG+ precursors show expression of neuronal and astroglial lineage markers, although some still show expression of immature cell markers. Nestin (a, orthogonal view in e) and
Filamin A (b, orthogonal view in f) expression is still present in some CTG+ cells at this time. The immature neuronal marker beta-III-tubulin is seen in some CTG+ (c, orthogonal view in k) and the marker for
migrating neuroblasts PSA-NCAM shows colocalization with CTG+ cells mainly in layer VI (d, orthogonal view in l; high magnification in inset, d). Glial protein immunostaining shows the absence of vimentin+/
GFAP+ (g–j) or GLAST+ (o) radial-glia-like processes extending from the ventricular wall crossing the corpus callosum towards the cortex (asterisks in g–j), as seen in earlier survival times (see Fig. 3). At 14 dpl,
some CTG+/vimentin+ (arrowhead in j, arrowhead in m, q, r–t), CTG+/GFAP+ (arrowhead in j, arrowhead in n, q–t), and CTG+/GLAST+ (arrowhead in p; u–w shown by arrowhead) cells were seen. CTG (green);
nestin, Filamin A, beta-tubulin III, PSA-NCAM, vimentin, GLAST (red); GFAP (blue). b-III-tub, beta-III-tubulin; vim, vimentin; cc, corpus callosum; cx, upper cortex; VI, cortical layer VI; vw, ventricular wall; les
cx, lesioned cortex.
M. Faiz et al. / Mol. Cell. Neurosci. 38 (2008) 170–182
Fig. 6. SVZ-derivedCTG+precursorsarerestrictedtocertaincorticalareasandexpressmarkersofmatureneuronsandgliaby49dpl.CTG+cellsareseenintheglial
scar(a–d), wheretheyshowdouble labelingwith GFAP (asterisksind),inthecortex adjacenttothe scarshowingirregularlamination(e–h),incorticallayer V(i–k)
(arrowheads in i–k; q–s). NeuN+/CTG+ cells (asterisks in e–h) are generally smaller that resident NeuN+ cortical neurons (arrowheads in e–h). CTG+ microglia/
macrophagesare restricted to theglial scar (arrowheadinl).No mature neurons are found incorticallayer VI,whereCTG+cellsare positivefor dcx(m–p, v–x) and
beta-III-tubulin (m–p, t, y–aa). CTG (green); NeuN, dcx, TL (red); MAP2 (red in i–k; blue in q–s); GFAP (blue), beta-tubulin III (blue in m–p, t; red in y–aa); adj.
cortex, cortex adjacent to the scar showing irregular lamination.
176M. Faiz et al. / Mol. Cell. Neurosci. 38 (2008) 170–182
Very few new neurons extend axons through the internal capsule
To investigate the putative extension of newly generated neuronal
projections to subcortical areas, Fluorogold, a retrograde tracer, was
injected into the ipsilateral internal capsule of CTG-injected animals
lamination was observed (Figs. 7l–q). These CTG+ cells were also
NeuN+ (Figs. 7q–v).
brain differentiate into neurons and glia following excitotoxic damage.
Our results show that although the majority of precursors differentiate
into glial cells, a number of regionally appropriate mature neurons that
axons through the internal capsule to subcortical structures.
Previous studies of this model have shown that excitoxicity
causes a depletion of the BrdU+ cell population in the SVZ with an
Fig. 7. SVZ-derived CTG+ mature neurons are both calretinin+ interneurons and tbr1+ glutamatergic neurons and a few of these new neurons are capable of axon
extension to the internal capsule. Some CTG+ mature neurons show MAP2 and are calretinin+ (a–g). CTG (e, h) and calretinin (g–h) are seen in the cytoplasm,
c) (blue in e, g); tbr1, GFAP (blue), Neun (red), MAP2 (red), fluorogold (blue in m–p; grey in q); cc, corpus callosum; VI, cortical layer VI; vw, ventricular wall.
177 M. Faiz et al. / Mol. Cell. Neurosci. 38 (2008) 170–182
appearance of striatal groups of proliferating cells at 5 dpl and a
concomitant decrease in BrdU+ cells in the RMS until 3 dpl (Faiz
et al., 2005). Here we demonstrate that the depletion in SVZ cells is
due to a redirection of migration out of the RMS and into areas of
neurodegeneration. The number of proliferating cells that decreased
in the SVZ after damage (Faiz et al., 2005) roughly matches the
number of cells traced with CTG out of the SVZ into the damaged
cortex (see Results). Although CTG labeling did show migration of
SVZ cells to the striatum, it was transient and only detectable at
5 dpl, while a more significant and sustained migration was seen at
SVZ precursors and Ki67 labeling showed very few proliferating
CTG+ SVZ cells at 5 dpl. The postnatal brain contains different
populations of progenitors that have different proliferative and
migratory abilities. It has been shown that the postnatal progenitors
in the SVZ are highly proliferative and migratory in comparison to
and olfactory bulb (Aguirre and Gallo, 2004; Aguirre et al., 2004;
Belachew et al., 2003). It is possible that the population of CTG
labeled (migratory) SVZ precursors divided so rapidly that at 5 dpl,
multiple divisions would have decreased BrdU. This observation
also implies that the levels of cortical neurogenesis seen in various
experimental paradigms may be understated when BrdU pulse
labeling is used for detecting the proliferation of precursor cells in
Differentiation and maturation of SVZ precursors after injury
In order to confirm that migrating CTG+ cells were SVZ pre-
cursors, nestin and Filamin A expression were examined. Nestin
expression alone, in regions outside of the germinative niches, is
insufficient as it is also upregulated in reactive astrocytes. However,
mechanism used in early development for migration out of the
as migratory progenitor cells. Following both neuronal and glial cell
differentiation potential of these cells. At early survival times, both
markers of immature neurons and glia were seen. In the last few
be sodistinct. Radial glia,in additiontotheir well establishedrole in
guiding neuronal migration, are neuronal precursors and can give
riseto corticalpyramidal neurons (Gotzet al.,2002; Malatestaet al.,
2003). Astroglial-like type B cells (Garcia-Verdugo et al., 1998) in
the neurogenic regions are also thought to have precursor prop-
erties. Early detection of many of these “glial” proteins could be a
reflection of a population of immature progenitor cells that will give
rise to mature neurons or glia at later survival times, especially
of new CTG+ glia generated was greater than the number of CTG+
mature neurons seen. At 49 dpl, CTG+ cells were found in upper
cortical layers and a number of newly generated astrocytes were
located within the glial scar. These findings are consistent with a
recent study indicating almost three fold greater astrocyte dif-
ferentiation than neuronal differentiation after perinatal hypoxia/
ischemia (Yang et al., 2007).
The transition fromimmature cellstomature neurons was seenby
identifying the neuronal protein expression of first, dcx (5 dpl), then,
beta-III-tubulin (14 dpl) and later NeuN and MAP2, general markers
of mature neuronal phenotypes. Using these markers, we found a
number of mature neurons generated by the late survival time, which
both the damaged adult and immature CNS (Arvidsson et al., 2002;
Fagel et al., 2006; Magavi et al., 2000; Ong et al., 2005; Parent et al.,
2002; Plane et al., 2004; Yang et al., 2007). Our results go a step
further in that we show the generation of both tbr1 and calretinin
expression was examined as it labels subpopulations of functional
classes of interneurons (Gabbott et al., 1997) including bipolar cells,
neurons resembling double-bouquet cells, multipolar cells, a type of
small basket neuron, and a distinct population of cells in layer I that
1994; Fonseca and Soriano, 1995; Gabbott and Bacon, 1996, 1997;
Glezer et al., 1992; Meskenaite, 1997). Tbr1 was chosen because it is
glutamatergic neuron determination (Hevner et al., 2006) and studies
from tbr1 knockout mice show that it regulates layer-related
differentiation, cell migration, and axon pathfinding in the neocortex
(Hevner et al., 2002, 2001). Both of these type specific markers were
seen to colocalize in CTG+ SVZ precursors after long survival times.
the SVZ that are able to follow diverse differentiation programs to
repopulate different types of neurons lost after injury.
The generation of new neurons from precursors that migrate to
layer VI also deserves comment. In models of immature brain
to injury (McQuillen et al., 2003). Of note, is that in tbr1 knockout
studies there is impaired differentiation of subplate and layer VI
the subplate (Hevner et al., 2006). After injury, an attempt to replace
cells especially vulnerable in the subplate and layer VI could explain
the dominant migration of SVZ precursors to this area. Expression of
tbr1 in CTG+ neurons may also suggest that there is an attempt at
retrograde labeling show that these newly generated “glutamatergic”
projection neurons also have the ability to extend axons to long
distance sites. The use of retrograde tracers is a first step in showing
pyramidal-projection neurons that are capable of extending axons to
far regions of the brain. However, electrophysiogical studies of
impulse conduction are required to demonstrate the efficacy of these
newly generated axons. Studies of perinatal hypoxic/ischemic injury
have shown similar results where neural stem cells transplanted on
scaffolds that aid regeneration can mature and extend axons through
the internal capsule and the corpus callosum into the contralateral
hemisphere (Park et al., 2002). Our study did not focus on con-
damage and that there is a subpopulation of callosally projecting
GABAergic interneurons in the cortex (Molnar and Cheung, 2006).
Migratory modes and migration cues used for precursor movement
to sites of injury
The dual potential of SVZ precursors to generate calretinin
positive interneurons and tbr1 positive pyramidal neurons could
correlate with developmental modes of migration. It is unclear if
newly formed interneurons are either regionally appropriate cortical
interneurons or misplaced olfactory interneurons. Previous work has
178 M. Faiz et al. / Mol. Cell. Neurosci. 38 (2008) 170–182
shown that transection of the olfactory bulb does not prevent
proliferation,migration and differentiationofnewlybornneuronsbut
instead, these cells leave the pathway and generate calretinin positive
interneurons in ectopic regions of the lesioned forebrain (Jankovski
et al., 1998). In either case, these interneurons are likely to use
must use their programmed mode of migration in order to integrate
and survive in different locations (Jankovski and Sotelo, 1996). Cor-
tical progenitors and olfactory granule neuron progenitors that use
radial migration cannot survive and integrate in the RMS, while
olfactory basket and stellate cell progenitors that use chain migration
can survive and migrate along the RMS. CTG+ precursors that
differentiate into interneurons are likely to use chain migration
strategies and as such, PSA-NCAM tracts. These tracts, normally
implicated in olfactory bulb migration, were observed at 5 dpl and
14 dpl extending from the SVZ into the lesioned cortex.
However, following their developmental program, newly gener-
ated tbr1 positive neurons may require radial guides to migrate to the
damaged cortex. We have shown that SVZ precursors are seen
attached to radial-glia-like, reflective of radial glial-guided migration
that aids neuroblasts in reaching their final destinations in the ap-
propriate cortical layers during development. Consistent with a recent
study byFageletal.where sublethal chronicperinatalhypoxiacaused
an increase in proliferating SVZ astroglial progenitor cells that show
markers of radial glial cells (Fagel et al., 2006), there was an
upregulation of radial glial like filaments (GFAP+, GLAST+,
vimentin+) after excitotoxicity and a notable production of newly
generatedglialcells.These filamentsmayaidin the migrationofSVZ
precursors to the damaged cortex; semi-thin sections showed
migrating precursor cells attached to glial filaments. It is unclear if
astrocytes that are produced from ongoing division of SVZ resident
precursor cells or if mature postnatal astrocytes dedifferentiate in
injury paradigms and aid migration of precursor cells to areas of
damage. These three possibilities remain to be investigated.
The OB normally regulates the pace of SVZ precursors migration
through the RMS (Jankovski et al., 1998) due to chemorepulsion and
chemottraction (Hu and Rutishauser, 1996; Lledo and Saghatelyan,
2005; Paratcha et al., 2006). It is possible that modifications in the
neurodegenerating environment, including molecular changes in
newly generated and resident cells and a combination of chemoat-
tractive and chemorepulsive cues in the damaged environment,
regulate the rapid deviation of cells destined for the olfactory bulb.
This suggests that an upregulation of gradients of diffusible guidance
cues in the damaged cortex and the concomitant expression of their
receptors on newly generated cells could direct migration. Similar to
the role of Slit/Robo in SVZ precursor migration in the RMS (Chen
et al., 2001; Wu et al., 1999), we suggest a role of Robo receptors in
Robo could position newly generated interneurons or direct axon
guidance of newly generated cortical or misplaced olfactory neurons,
the injury-induced involvement of Robo1 is required and more
specific conclusions could be drawn from the study of Robo1
the excitotoxically lesioned cortex suggests that signaling by SDF-1
through its receptor CXCR4 may be involved in attracting SVZ
precursors tothe lesioned area. Although studies of adult injury show
that SDF-1 is capable directing precursor recruitment (Imitola et al.,
2004),a recentstudy demonstrated an increase of CXCR4expression
on progenitor cells without a corresponding increase in SDF-1 in the
damaged cortex after perinatal hypoxia/ischemia and suggested that
other mechanisms may be more important for migration in the
postnatal cortex (Yang et al., 2007).
In summary, this study demonstrates that new neurons are
produced after immature brain damage and that these cells have the
ability to extend axons to subcortical areas. We have shown novel
aspects of the endogenous response to developmental brain injury,
such as production of both glutamatergic and interneuron and axon
extension. However, this study does not answer questions such as the
ability of these new neurons to conduct electric impulses or the
functionality of newly generated glia, which are important considera-
tions when defining neurogenesis or gliogenesis. The idea that the
generation of new neuronal cells underlies the better functional out-
come in younger animals is somewhat premature as a direct re-
lationship has yet to be established between correct neuronal
maturation, integration, function and communication of newly
generated cells and improved functional outcome. Furthermore, the
low percentage of newly generated neurons is unlikely to result in
CNS has the ability to induce gene expression and migratory patterns
that are used in normal neural development in order to attempt
regeneration after injury and when compared to studies after adult
brain damage it indicates that this response may be a common
mechanism used by the CNS to attempt to regenerate after injury in
order to restore lost connections.
Animal experimentation was conducted in accordance with the
Autonomous University of Barcelona Ethical Commission, in compliance
with Spanish legislation and European Union directives on the use of
animals in scientific research.
Cell Tracker Green injections
were used in this study after the litter size was adjusted to 10 pups. P9 pups
weighing between 16 and 18 g, were anesthetized with isoflurane and then
placedintoastereotaxicframe adaptedfor newborns(Kopf).Onemicroliterof
10% Cell Tracker Green (CTG; Invitrogen, C-2925) diluted in DMSO was
stereotaxically injected usinganautomatic microinjector ata rateof0.2 µl/min
0.5 mm lateral, 0.5 mm posterior and 1.5 mm deep. After CTG injection, pups
4 and 10 h post-injection and at 5, 14 and 49 days post-injection (dpi), CTG–
NMDA-injected lesioned pups (n=26) and CTG–saline-injected control pups
(n=10) as lesion controls.
Postnatal excitotoxicity model
Excitotoxicity was induced in P9 rats, 4 h after CTG injection, by
stereotaxic N-methyl-d-aspartate (NMDA) solution injection into the right
sensorimotor cortex (Acarin et al., 1999a; Acarin et al., 1999b). Briefly,
27 nmol of NMDA diluted in 0.15 µl of 0.9% NaCl was injected at a rate of
0.05 µl/min with a Hamilton syringe at Bregma coordinates 2 mm lateral and
0.5 mm deep. The incision was then sutured and animals were placed in an
incubatorfor2 h at 36°Cbeforebeing returnedto their mother. Lesionedpups
were left for survival times of 10 h (6 pups), 5 (8 pups), 14 (6 pups) and 49 (6
pups) days post-lesion (dpl) (Fig. 1a Supplementary material). As controls of
the lesion, animals were anesthetized and injected, 4 h after CTG, with 0.15 µl
of 0.9% NaCl (CTG+saline-injected control group) and left for survival times
of 10 h post-injection, 5, 14 and 49 dpi (n=2–4 pups per survival time).
179M. Faiz et al. / Mol. Cell. Neurosci. 38 (2008) 170–182
At 45 dpi, CTG–NMDA-injected, CTG–saline-injected and CTG
control animals were stereotaxically injected with the retrograde tracer
Fluorogold to label cortical neurons projecting axons through the internal
capsule. Briefly, 0.15 µl of Fluorogold (2% in dH2O) was injected using a
0.5 µl Hamilton syringe at Bregma coordinates 2 mm lateral, 4 mm
posterior and 5 mm deep. Rats were sacrificed 4 days later, at 49 dpi (Fig.
1b, Supplementary material).
5′Bromodeoxyuridine (BrdU) labeling
Additional pups injected with CTG only (n=6), CTG–saline (n=4) or
CTG–NMDA (n=8) received 5 intraperitoneal injections of BrdU (50 mg/
kg) every 2 h on P9 and sacrificed at 10 h or 5 days post-injection (Fig. 2f).
Histological tissue processing
Animals were anesthetized and then sacrificed by intracardiac perfusion
for 10 min with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).
Brains were removed, post-fixed in 4% paraformaldehyde for 2 h and
cryoprotected in30%sucrosein 0.1M phosphatebufferbeforefreezingwith
cryostat (Leitz CM 30503).
Antibodies, immunohistochemistry and histochemistry
Immunohistochemistry was performed on free-floating parallel cryostat
sections stored in Olmos anti-freeze buffer. Briefly, sections were washed in
incubated in blocking buffer (BB; 10% fetal calf serum in TBS-T) for 1 h
before incubation overnight at 4 °C and 1 h at room temperature (RT) with a
primary antibody diluted in BB (Table 1, Supplementary material). After
rinsing with TBS-T, sections were incubated for 1 h at RT with the
corresponding secondary antibody diluted in BB (Table 1, Supplementary
material). For double immunostaining, sections were rinsed again in TBS-T
and incubated overnight at 4 °C and 1 h at RTin the primary antibody for the
second labeling. After rinsing, sections were incubated for 1 h at RTwith the
corresponding secondary antibody. In the case of biotin-conjugated
secondary antibodies, sections were then incubated for 1 h at RT in
streptavidin-Cy5 or avidin-Cy3 (Table 1, Supplementary material). After the
last incubation, sections were rinsed in TBS (0.05 M Trizma base, 150 mM
NaCl, pH 7.4) and TB (0.05 M Trizma base, pH 7.4), mounted and
coverslipped with a fluorescent mounting medium (DAKO).
For BrdU immunostaining, DNA was denatured by first incubating
sectionsin0.082NHCl for10 minat4 °Candthenfor 30minin0.82NHCl
then incubated in BB before incubation with the primary and secondary
antibodies, as described above.
For tomato lectin histochemistry, sections were incubated in biotin-
labeled tomato lectin (1:150, Sigma, L0651) diluted in TBS-T overnight at
4 °C and then for 1 h at room temperature (RT). After rinsing with TBS-T,
sections were incubated for 1 h at RT with avidin-Cy3 (Table 1,
Data analysis and quantification
For colocalization studies, sections were analyzed with a Leica confocal
laser-scanning microscope (TCS SP2AOBS). Sequential Z sections were
taken and overlay projections were acquired using Leica software and
cropped and adjusted using Adobe Photoshop 7.0.
digital camera (Nikon Dxm1200) mounted on a Leitz optical fluorescence×
microscope (Nikon eclipse E600) and interfaced to a PC computer using ACT1
imagingsoftware. CTG-positive cellswerecounted in the ipsilateral hemisphere
VI right above the SVZ (zone 3) and in the lesioned cortex (zone 4) (Fig. 1a).
zone and as a total of all 4 zones. All countswere conducted every 4th coronal
section for a total of three sections per animal from at least 8 experimental
a Student unpaired t-test. All values are presented as Mean±Standard Error of
the Mean (s.e.m). Statistical significance was set at pb0.05. Because no
statistically significant differences in the amount of CTG-positive cells was
observed between the saline-injected and intact control animals, data from
these two groups was pooled to form a collective control group, which was
compared to NMDA-lesioned animals.
Tissue processing for semi-thin section analysis
for semi-thin-section analysis. Briefly, pups were anesthetized and sacrificed
0.1 M cacodylate buffer. Brains were removed, post-fixed for 2 h and then
using a vibratome (Leica VT1000S) and selected 1 mm2pieces were obtained
from the sections. Tissue pieces were post-fixed in 1% osmium tetraoxide,
washed, and dehydrated in graded ethanol before araldite embedding. One
micrometer semi-thin sections were cut using an ultramicrotome (Leica
and Science (BFU 2005-02783) and Marato TV3 061710. M. Faiz
and S.Villapolhold a fellowship fromthe AutonomousUniversityof
Barcelona. We thank Miguel A. Martil, Lola Mulero and Monica
Roldan for their outstanding technical support. We are especially
grateful to Dr. Eduardo Soriano, Dr. Oscar Marin and Dr. Robert F.
Hevner for their suggestions, Dr. Valentin Martin for the semi-thin
work and Dr. Catherine Rousset for the statistical analysis.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.mcn.2008.02.002.
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