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Cerebral Cortex November 2010;20:2583--2591
doi:10.1093/cercor/bhq006
Advance Access publication February 12, 2010
Generation of Glutamatergic Neurons from Postnatal and Adult Subventricular Zone with
Pyramidal-Like Morphology
Eduardo B. Sequerra1,2,3, Leo M. Miyakoshi1,2,3, Maira M. Fróes1, João R. L. Menezes*,1 and Cecilia Hedin-Pereira*,1,2
1
Laboratório de Neuroanatomia Celular, Programa de Anatomia, Instituto de Ciências Biomédicas, 2Instituto de Biofı́sica Carlos
Chagas Filho, Universidade Federal do Rio de Janeiro and 3Programa de Pós-Graduacxão em Ciências Biológicas (Biofı́sica), Instituto
de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21940-590 Rio de Janeiro, Brazil
*These authors contributed equally to this work.
Address correspondence to Cecilia Hedin-Pereira, Av. Rio de Janeiro, s/no, UFRJ/CCS, sala F1-025, 21940-590 Rio de Janeiro, Brazil.
Email: [email protected].
The mammalian subventricular zone (SVZ) contains progenitors
derived from cerebral cortex radial glia cells, which give rise
to glutamatergic pyramidal neurons during embryogenesis.
However, during postnatal life, SVZ generates neurons that
migrate and differentiate into olfactory bulb g-aminobutyric acid
(GABA)ergic interneurons. In this work, we tested if SVZ cells are
able to produce glutamatergic neurons if confronted with the
embryonic cortical ventricular zone environment. Different from
typical SVZ chain migration, cells from P9--P11 SVZ explants
migrate into embryonic cortical slices individually, many of which
radially oriented. An average of 82.5% of green fluorescent
protein--positive cells were immunolabeled for neuronal marker
class III b-tubulin. Invading cells differentiate into multiple
morphologies, including a pyramidal-like morphotype. A subset
of these cells are GABAergic; however, about 28% of SVZ-derived
cells are immunoreactive for glutamate. Adult SVZ explants also
give rise to glutamatergic neurons in these conditions. Taken
together, our results indicate that SVZ can be a source of
glutamatergic cortical neurons when submitted to proper
environmental cues.
Keywords: cerebral cortex, GABAergic, neurogenesis, olfactory bulb,
plasticity
Introduction
The subventricular zone (SVZ) is a continuous source of new
neurons in postnatal and adult mammals (for review, see Gage
2000). The newly generated neurons migrate to the ipsilateral
olfactory bulb (OB; Altman 1969; Luskin 1993) where they
differentiate into granular and periglomerular neurons (Luskin
1993). A known feature of these cells is that they differentiate
into c-aminobutyric acid (GABA)ergic neurons (Betarbet et al.
1996). It was recently described that a subpopulation of
periglomerular neurons could be devoid of GABA (GutièrrezMecinas et al. 2005). However, recent evidence shows that all OB
periglomerular neurons activate the promoter of the gene for 1 of
the 2 forms of glutamic acid decarboxilase, 67KD form of glutamic
acid decarboxilase (GAD67), as detected by green fluorescent
protein (GFP) expression in transgenic mice (Panzanelli et al.
2007). In addition, a subpopulation of periglomerular neurons
(Kosaka et al. 1985; Betarbet et al. 1996; Hack et al. 2005) and
superficial granular neurons (Kohwi et al. 2005) are double
labeled for GABA and tyrosine hydroxylase.
It is suggested that the telencephalic embryonic germinal
layer, the ventricular zone (VZ), gives rise to different
neurochemical phenotypes in a spatial-dependent fashion
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(Marin and Rubenstein 2001). In the telencephalon, glutamatergic neurons are generated by the dorsal VZ (Schuurmans
et al. 2004) and GABAergic neurons from its ventral domain
(Anderson et al. 1997). At the end of embryonic neurogenesis,
radial glia (RG), the main progenitors of the VZ (Noctor et al. 2001),
start to transform into other cell types, including SVZ astrocytes
(Alves et al. 2002; Tramontin et al. 2003) and ependymal cells
(Spassky et al. 2005). Postnatal progenitors, derived from RG, are
also known to give rise to inhibitory interneurons of the OB,
irrespective of dorsoventral position (Merkle et al. 2007). These
observations lead to the question of whether dorsal progenitors, which initially make glutamatergic neurons, respecify
their program for neurotransmitter choice after embryonic
neurogenesis ends. An alternative possibility is that dorsal
telencephalic progenitors are still capable of giving rise to
glutamatergic neurons, and the external signals present in the
postnatal brain do not allow or stimulate this phenotype. SVZ
neuroblasts already express GABA in route to the OB (Bolteus
and Bordey 2004), suggesting an early commitment by these
cells with a GABAergic destiny.
Recent data suggest that most GABA-immunoreactive neuroblasts of the SVZ are not endowed with the GABA synthetic
machinery typical of differentiated neurons (De Marchis et al.
2004; Sequerra et al. 2007). Instead, an alternative synthetic
pathway has been identified, which converts putrescine into
GABA (Sequerra et al. 2007). Therefore, neuroblasts in the SVZ
may not be irreversibly committed to a GABAergic destiny. The
question herein addressed is whether the dorsal embryonic VZ
environment could drive postnatal and adult SVZ progenitors
to a glutamatergic phenotype.
Materials and Methods
In this work, we used wild-type postnatal and embryonic mice
(Mus musculus, Swiss), transgenic mice with the expression of
enhanced green fluorescent protein (EGFP) driven by the chicken
b-actin promoter and cytomegalovirus enhancer (TgEGFP+ BCF1
background; Okabe et al. 1997). Procedures for the use of animals
were in accordance with the Committee for Ethics in the Use of
Animals for Research of the Instituto de Biofı́sica Carlos Chagas Filho of
the Universidade Federal do Rio de Janeiro and follow National
Institutes of Health’s guidelines for animal research.
To obtain embryonic slices, pregnant mothers received an ip
injection of chloropentobarbital (60 mg/mL sodic pentobarbital and
0.042 mg/mL chloral hydrate) of 3.5 mL/kg of body weight, with 30%
more volume added after 5 min. Embryos were removed by C-section
and decapitated. For postnatal explants, animals were anesthetized
with ether inhalation before decapitation. For adult explants, animals
were anesthetized with ether inhalation and euthanized by cervical
dislocation.
Briefly, embryonic and postnatal brains were removed from crania in
cold (4 °C) Gey’s basal salt solution in sterile conditions. The brains were
coronally sliced using a tissue chopper (McIlwain, O’Fallon, MO) at 350
lm thickness. Slices and explants were plated on membranes permeable
to gases (Petriperm; Sigma, Saint Louis, MO) coated with Poly-L-lysine
(Sigma; 10 mg/mL) and covered with culture medium containing 60%
Dulbecco’s Modified Eagles Medium (GIBCO, Grand Island, NY), 30%
Hank’s solution (GIBCO), 10% fetal calf serum (GIBCO), 1% solution of
penicillin (10 000 U/L) + streptomycin (10 mg/mL) (GIBCO), 1%
Fungizone (GIBCO), and 1% glucose. Slices were cultivated at 37 °C for
up to 5 days in a 5% CO2/95% air incubator (Heraeus Instruments, Hanau,
Germany), and culture medium was entirely changed after 12 and 72 h.
Coculture Models
We performed 2 coculture groups (Supplementary Fig. 1): 1) Heterochronic and heterotopic cocultures: Postnatal or adult SVZ explants
were cocultured with telencephalic embryonic coronal slices. In this
culture model, SVZ explants obtained from P9--P11 (birthday—P0) or 3month-old TgEGFP+ mice were plated inside the ventricular lumen of
E15 telencephalic coronal slices touching its dorsal ventricular surface.
Slices used represent the rostral 2/3 of the embryonic telencephalon.
SVZ explants were dissected from the ventricular wall adjacent to the
striatum and from the SVZ rostral projection present in OB coronal
slices (see also Sequerra et al. 2007). 2) Homotopic and homochronic
cocultures: Postnatal SVZ explants were cocultured with postnatal OB
slices; P9 OB coronal slices were plated after removal of the SVZ. Then,
TgEGFP+ SVZ explants were dissected from OB coronal slices and used
as replacement for the homotopic SVZ that was removed. The GFP+ SVZ
explant was plated touching the internal face of the granular layer
exposed by the excision of the original SVZ.
In Vitro and In Situ Immunohistochemistry
For immunohistochemistry, cultures were prefixed in formaldehyde
vapor by placing the Petriperm culture plates without their lids into
a chamber containing paper towel embedded with formaldehyde for
10 min. Subsequently, medium was removed and cultures were
immersed in buffered 4% paraformaldehyde for 15 min. Cultures
processed for glutamate immunohistochemistry were immersed in
buffered 4% paraformaldehyde/1% glutaraldehyde. After several washes
in phosphate-buffered saline (PBS; pH 7.4), cocultures were incubated
for 72 h at 4 °C with primary antibodies diluted in PBS with 0.3% TritonX 100 (Reagen, Curitiba, PR, Brazil) and 5% normal goat serum
(Invitrogen, Carlsbad, CA). Primary antibodies were the following: antiGABA (rabbit 1:2000; Sigma), anti-glutamate (rabbit 1:250; Chemicon,
Temecula, CA; antibody made against vesicular glutamate), anti--class III
b-tubulin (Tuj1, mouse 1:500; Covance, Emeryville, CA), anti-glial
fibrillary acidic protein (GFAP; rabbit 1:400; Dako, Carpinteria, CA),
anti-S100b (rabbit 1:500; Sigma), anti-Tbr1 (1:200; Chemicon), antiCtip2 (rat 1:500; Abcam, Cambridge, MA), anti-Satb2 (mouse 1:200; Abcam),
and anti-PSD-95 (rabbit 1:100; Santa Cruz, Santa Cruz, CA). Secondary
antibodies were incubated for 2 h at room temperature. Secondary
antibodies were the following: goat anti-rabbit IgG conjugated with
Cy3 (1:800; Jackson ImmunoResearch, West Grove, PA), goat antimouse IgG conjugated with Cy3 (1:800; Jackson ImmunoResearch),
and goat anti-rabbit conjugated with Cy5 (1:500; Jackson ImmunoResearch). Pieces of Petriperm membrane were then mounted on
glass slides and coverslipped using gel-mounting medium (Biomeda,
Foster City, CA).
For in situ immunohistochemistry, P11 Swiss mice (2 animals) were
ether anaesthetized and intracardially perfused with 4% paraformaldehyde and 1% glutaraldehyde in phosphate buffer. Brains were removed
and sectioned with a vibratome (Vibratome 3000; Pelco, Redding, CA)
at 50 lm. Free-floating sections were processed as described above for
cocultures. Sections were mounted on glass slides and coverslipped
using gel-mounting medium (Biomeda).
Microscopy and Quantification
Conventional fluorescence microscopy was performed in an Eclipse
TE200 inverted microscope (Nikon, Tokyo, Japan), equipped with
2584 Glutamatergic Neurons from Postnatal and Adult SVZ
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Sequerra et al.
a CoolSNAP-Pro cf CCD camera (Media Cybernetics, Silver Spring, MD;
monochrome). Images were acquired with the aid of Image-Pro
Express software (version 4.5.1.3) and edited with Photoshop CS2
(Adobe, San Jose, CA). In one particular figure (Supplementary Fig.
3A,B), a Zeiss Inverted Fluorescent Microscope (Axiovert 200M)
equipped with an ApoTome structural microscopy module (Zeiss,
Gottingen, Germany) was used. Confocal microscopy was performed
in a Zeiss Axiovert 200 microscope equipped with LSM 510 Meta NLO
confocal system. Images were collected and processed with the LSM
Image Browser software (Zeiss). Neurons were classified into 5
morphotypes (pyramidal, horizontal bipolar, multipolar, periglomerular like, and nondescript) based on dendritic patterns and the
existence of a thin process identifiable as an axon. These morphotypes were counted as a percentage of total EGFP-positive differentiated cells (n = 11 slices in 3 independent experiments, 30 animals,
76 cells, and field visualization at 363 objective—oil). Typical
migratory morphologies were excluded from the quantification. We
also counted the percentage of EGFP cells that were positive for the
neuronal marker class III b-tubulin under 340 objectives (n = 11 slices
in 2 independent experiments, 19 animals used, and 273 cells) and
the percentage of EGFP cells that were labeled with anti-glutamate
antibody (n = 8 slices in 2 independent experiments, 18 animals used,
312 cells, and field visualization at 363 objective—oil). In all cases, all
labeled cells were counted within focal depths that included
immunolabeling. Numbers are expressed as mean ± SEM.
Results
SVZ Cells Invade Embryonic Telencephalic Slices and
Differentiate into Multiple Morphotypes
To test possible effects of the dorsal VZ environment on the
differentiation of SVZ progenitors, we cocultured telencephalic slices with SVZ explants derived from constitutively
expressing GFP transgenic mice at P9--P11 (Okabe et al. 1997;
Supplementary Fig. 1). After 2 days in vitro (DIV), SVZ-derived
cells had entered the slice and showed bipolar morphologies
typical of migrating cells (Fig. 1A). These cells were oriented
both radially (arrows in Fig. 1A) and tangentially (arrowhead in
Fig. 1A) and were found dispersed throughout the cortical
depth (Fig. 1B,C, arrows) without any particular laminar pattern.
An average of 82.5 ± 3.8% of GFP-positive cells (11 slices in 2
independent experiments) were identified as immature neurons since they were immunolabeled for class III b-tubulin
(Fig. 2E, Tuj1 antibody; Menezes and Luskin 1994).
After 3 DIV, SVZ cells derived from EGFP mice started to
differentiate within the wild-type cortical plate (CP) into
multiple morphotypes, some of them resembling cortical
(Fig. 1B--F) rather than OB neurons. Analysis of differentiated
cell types at 5 DIV revealed pyramidal-like cells (Fig. 1C; 15.5 ±
4.7% of EGFP cells, 11 slices, and 2 independent experiments),
characterized by a pyramidal-shaped cell body, a prominent
apical dendrite oriented to the pia, basal dendrites, and a thin
axon running in the opposite direction, toward the white
matter (Fig. 1C; Peters and Kara 1985). Another characteristic
of these cells is the presence of small processes sprouting from
the dendrites, some of which are filopodia arising from the
dendritic shafts and some shorter possibly emerging dendritic
spines (Fig. 1D). Other cell types were found, such as
horizontal bipolar neurons (Fig. 1E; 5.68 ± 1.96%), multipolar
cells with highly branched neurites radiating in all directions
(Fig. 1F; 55.03 ± 7.81%), morphologically nondescript (Fig. 1G;
1.30 ± 1.30%), and cells resembling OB periglomerular neurons
(Fig. 1H; 23.03 ± 8.51%). The latter subtype is characterized by
absence of an axon and presence of a single thick process that
Figure 1. A) GFP-positive cells from SVZ explants invade and migrate into the dorsal telencephalic tissue. After 2 DIV, cells with migratory morphology are found both
radially (arrows) and tangentially (arrowhead) oriented relative to the pial surface. (B) After 3 DIV, SVZ cells start to differentiate into the CP displaying multiple
morphotypes. Dashed line indicates the pial surface. (C--G) At 5 DIV, cells differentiate into wide spectrum of morphotypes classified solely by morphological criteria. The
pyramidal neuron morphology (C) is characterized by a pyramidal-shaped cell body (white arrow), basal dendrites (white arrowhead), a spiny apical dendrite oriented to the
pia (black arrowhead), and a thin axon oriented to the VZ (black arrow). (D) A detail of another pyramidal neuron apical dendrite showing dendritic spines (arrowheads).
Other morphologies were bipolar (E), multipolar (F), an undetermined (G), and periglomerular like (H). The periglomerular-like morphology (H) is characterized by the
absence of an axon and the extension of a thick process (white arrowhead) that ramifies into multiple dendrites (black arrowhead). Scale bars: A, 100 lm; B, 50 lm; C--G,
10 lm; and H, 5 lm.
Figure 2. A) GFAPþ cells from SVZ explants (Exp) do not invade the telencephalic slice (Sl) but spreads over the culture surface (Pl). Arrowheads indicate GFPþ cells that
migrated into the slice and are not labeled with GFAP. (B) A rare example of a coculture showing GFAPþ cells within the slice. When this occurred, GFAPþ cells were limited to
the first few micrometers of the slice. Dashed line indicates the ventricular surface. (C) Thin confocal optical slice (0.44 lm) confirming the double labeling of the cell shown in
(B). (D) S100b immunohistochemistry showing that GFP cells were not labeled with this marker. (E) GFPþ neuroblast labeled with the Tuj1 antibody (Z slice 0.38 lm). Scale
bars: A, 100 lm; B, 20 lm; D and E, 10 lm.
branches into multiple thinner putative dendrites, arising from
the soma (Schneider and Macrides 1978).
Glial SVZ Cells Do Not Migrate into the Embryonic
Telencephalic Tissue
We observed SVZ-derived cells differentiated into multipolar
morphological types that were compatible with neuronal and
astrocytic phenotypes. To test if SVZ-derived cells were also
present as astrocytes in the embryonic dorsal telencephalic slice,
we performed immunohistochemistry for GFAP and S100b,
astrocytic lineage markers. SVZ explants contained many GFAP+
cells, and these were able to migrate centrifugally out of the
explant into the culture plate (Fig. 2A). However, GFP+ cells in
the dorsal telencephalic tissue did not express GFAP (Fig. 2A,
arrowheads) or S100b (Fig. 2D). The few GFP+/GFAP+ cells
found within the slice (Fig. 2B,C, 4 GFP+ cells out of 118 in 3
slices from independent experiments) were restricted to the
presumptive VZ. Absence of labeling by poor antibody penetration has been ruled out since GFAP+ cells from the host were
found within the slice (GFAP+/GFP–, data not shown).
SVZ-Derived Pyramidal-Like Neurons Are Glutamatergic
and Express PSD-95 in Short Filopodia Arising from
Dendritic Shafts
We have analyzed GABA and glutamate expression by SVZderived neurons within the embryonic telencephalic tissue. We
found multiple SVZ-derived cells expressing GABA in the CP
(Fig. 3A), some presenting a typical multipolar morphology
(Fig. 3A). Double labeling was confirmed with confocal
reconstruction (Fig. 3B,B#).
To search for a possible glutamatergic phenotype, we
immunolabeled cultured slices with an antibody that recognizes synaptic vesicle glutamate fixed with glutaraldehyde.
Glutamate immunolabeling was restricted to the CP (Fig. 3C) as
expected for normal cortical distribution of glutamatergic
neurons. SVZ-derived cells that disperse tangentially underneath the CP in the intermediate zone and SVZ are not labeled
with glutamate (Fig. 3C, arrow). After 5 DIV, GFP+ pyramidallike neurons were found labeled with glutamate within the CP
(Fig. 3D,E). A mean of 27.6 ± 11.6% (n = 5) of the GFP-positive
SVZ-derived cells were double labeled for glutamate. These
double-labeled cells include cells with immature morphology
Cerebral Cortex November 2010, V 20 N 11 2585
cortical layer neurons, respectively (Arlotta et al. 2005;
Britanova et al. 2005). We found no double labeling (Supplementary Fig. 2), although both antibodies labeled neighboring
cells in the cortical tissue.
An important feature of our morphological characterization of
the pyramidal-like neurons is the presence of small protrusions
on the dendrites. These resemble short dendritic filopodia as
most of them lack the morphological features of more mature
dendritic spines, such as bulbous heads and necks (Yuste and
Bonhoeffer 2004). However, their position on the dendritic
shafts and the spacing of these filopodia were compatible with
that of nascent spines rather than dendritic branches appearing
in more or less 10-lm intervals. To further characterize these
structures, 5-DIV slices were submitted to immunohistochemistry for PSD-95, a protein found in the postsynaptic density of
glutamatergic synapses (Cho et al. 1992). The expression of PSD95 was also detected in GFP+-derived SVZ cells. Although some
cells present a diffuse somatodendritic immunostaining pattern,
frequent clusters of PSD-95 were present along dendrites
(Fig. 3G,H, arrows). Through confocal microscopy optical
sectioning, it is possible to demonstrate that some of these
clusters are located within the protrusions that occur along the
dendrites (inset in Fig. 3H).
Figure 3. (A) Three GABA (red)-expressing GFPþ (green) within the CP after 5 DIV.
(B and B#) An optical slice of the cell boxed in (A) showing double labeling with GFP
and GABA. (C) Glutamate expression of an embryonic slice 5 DIV. Note that labeling is
restricted to the CP as expected. Arrowheads point to explant (Exp)-derived GFPþ
cells that reached the CP, and many are still located in the presumptive intermediate
and SVZ (arrow). (D) Higher magnification confocal stack of a glutamate-labeled
pyramidal-like neuron also labeled with GFP (E). (F) A stack of 6 nonconsecutive
optical slices (0.5 lm) of a pyramidal cell not labeled with the antibody for Tbr1 (red).
(G) Two GFP+ cells (green) labeled with PSD-95 (red). (H) Higher magnification of
the pyramidal-like neuron inside the rectangle in (G). Arrows indicate shaft filopodia.
Inset shows an orthogonal view of a PSD-95--immunolabeled filopodia obtained from
0.43-lm-thick optical slices. LV, lateral ventricle. Scale bars: A, F, and G, 10 lm; C,
50 lm; E, 20 lm.
that could not be classified. All the pyramidal-like cells in these
slices were glutamate positive, and no other morphological
type was found labeled. Therefore, the postnatal SVZ contains
progenitors capable of differentiating into glutamatergic
neurons of the cerebral cortex. We have also tested Tbr1
immunoreactivity, a marker for a subpopulation of cortical
glutamatergic neurons (Hevner et al. 2001) in pyramidalshaped GFP+ cells, with no observable immunostaining (Fig. 3F,
19 slices in 2 independent experiments). Additionally, we
tested if these cells are immunoreactive for Ctip2 or Satb2
since these markers are expressed in deep and superficial
2586 Glutamatergic Neurons from Postnatal and Adult SVZ
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Sequerra et al.
Rare SVZ-Derived Cells Express Glutamate in the OB
Granular Layer
Since postnatal SVZ generates glutamatergic neurons when
confronted with the embryonic telencephalon environment, we
investigated if SVZ-derived cells are also capable of expressing
this neurotransmitter when cocultivated with their normal
target tissue, the OB, of the same age. This approach tests for the
alternative of an SVZ progenitor capable of generating glutamatergic neurons destined to the OB, which could have been
overlooked in previous studies. SVZ explants from coronal OB
slices were dissected from P9--P11 mice and cocultured with
age-matched SVZ explants derived from GFP transgenic mice,
touching the internal face of the granule cell layer. After 5 DIV,
glutamate immunolabeling is present in the glomerular layer
(Fig. 4A), probably reflecting the presence of neuropil and short
axon cell bodies (Aungst et al. 2003). GFP+ cells in this region
were devoid of glutamate immunolabeling (Fig. 4A).
We found glutamate+ cells in the granular layer (Fig. 4B);
however, rare GFP-positive cells were colabeled (Fig. 4C).
When that was the case, labeled cells presented a smooth
unbranched radial bipolar morphology (Fig. 4D). Although GFPpositive cells invaded the whole extension of the granular layer
of the OB slice, glutamate-labeled cells (both from the explants
and from the slice) were restricted to a small extension of this
structure (data not shown). Since it is not expected to observe
glutamate-positive neurons in the OB granular layer (Shepherd
et al. 2007), we performed double-labeling immunohistochemistry in OB sections of P11 mice. Rare glutamate-positive cells
were found in the granular layer, but these were not labeled
with the neuronal specific marker, class III b-tubulin (Fig. 4E,
Tuj1 antibody). This indicates a non-neuronal or immature
nature of glutamate-expressing cells within the granule layer.
Adult SVZ Generates Glutamatergic Progenitors in
Embryonic Telencephalic Slices
To observe if SVZ capacity to generate glutamatergic pyramidallike neurons postnatally is a transitory characteristic, we
Figure 4. A) SVZ-derived cells that reach the glomerular layer never express glutamate. (B) Glutamate-expressing cells are present within the granular layer. (C) A subset of the
SVZ/GFPþ-invading cells also expresses glutamate (white arrows). White arrowheads point to glutamate() GFPþ cells within the granular layer, and yellow arrows point to
glutamateþ-only cells. (D) A double-labeled cell showing its bipolar morphology close to the explant/slice border. (E) Confocal orthogonal view showing a glutamate-positive cell
(red) devoid of labeling with Tuj1 antibody (green) in the postnatal granular layer. Scale bars: A and C, 20 lm; D and E, 10 lm.
cocultured SVZ explants from 3-month-old mice with embryonic
telencephalic slices (as shown in Supplementary Fig. 1) for 5--7
DIV. We observed GFP-positive cells within cortical slices, most
of which presented a polarized morphology (Fig. 5A--D). These
immature pyramidal morphologies were also found coexpressing
class III b-tubulin and glutamate within the CP (Fig. 5E--K).
Confocal orthogonal sectioning showed these putative pyramidal
neurons to be colabeled with glutamate immunolabeling. These
data show that the SVZ potential for generating glutamatergic
neurons is a long-lasting phenomenon.
Discussion
Our present results show that postnatal SVZ explants give rise
to glutamatergic neurons and multiple cortical neuronal
morphotypes when cocultured with embryonic telencephalon
slices. These include glutamatergic neurons with morphologies
coherent with those of immature pyramidal neurons. In fact,
around 28% of the SVZ-derived invading cells were identified as
glutamatergic. Some of these glutamate immunoreactive cells
displayed immature profiles, with dendrites beginning to
extend apical processes. Among more differentiated morphotypes, an average of 16% pyramidal-like neurons was identified.
PSD-95 immunohistochemistry revealed labeled clusters in
dendritic protrusions supporting the existence of dendritic
filopodia with putative dendritic spine characteristics in
pyramidal-like neurons. Coherently, the recruitment of PSD95 to filopodia was previously described as a step toward their
stabilization into mature spines (Prange and Murphy 2001). We
recognize that the time constraints in our culture model
obligate us to characterize very immature phases in pyramidal
cell differentiation. However, we have no reason to believe that
these refer to another cell type since all cells were bipolar with
an apical dendrite directed toward the pia and coherently we
never observed multipolar cells (also with their morphologies
quite clear) expressing glutamate for example. In addition, we
show that the adult SVZ also generates glutamatergic neurons,
indicating that the progenitors capable of giving rise to
glutamatergic cortical neurons remain in the SVZ during the
whole life span of the animal.
Since neuroblasts, RG, astrocytes, and possibly intermediate
progenitors are present in postnatal SVZ (Alves et al. 2002;
Peretto et al. 2005), all 4 could be candidates for the generation
of neurons in the embryonic cortex. We observed that the
great majority of the SVZ cells migrating into the slices were
neuroblasts, as revealed by class III b-tubulin immunolabeling.
Why the cells labeled for GFAP do not migrate into E15 slices
remains unknown. It appears that embryonic cortex may be
nonpermissive or even repulsive to astrocytes at this age. In
a previous attempt of heterochronic transplantation of postnatal SVZ cells to the embryonic brain, Lim et al. (1997) did not
find any SVZ cells invading the cortical wall. However, when
cells did cross the ventricular surface in other regions and
incorporated into the host brain parenchyma, they were class
III b-tubulin--positive neuroblasts. Coherently, the embryonic
CP is largely devoid of astrocytes, and astrocyte invasion into
the cortical parenchyma only starts at later embryonic stages
(Choi 1989; Levers et al. 2001).
In the SVZ, neuroblasts migrate in chains using each other as
substrates (Lois et al. 1996). In contrast, postnatal SVZ-derived
cells that enter the slice migrate individually. Their orientations
are compatible with radial and tangential migration modes,
typically displayed by glutamatergic and GABAergic cells,
respectively (Marin and Rubenstein 2001), adding to previously
mentioned features of neurons generated in the embryonic
telencephalon. It has been shown that migrating neuroblasts of
the SVZ retain proliferative capacity (Menezes et al. 1995);
therefore, one possibility is that invading cells reenter the cycle
within the VZ before migrating to the CP or, even later, within
the CP. Nevertheless, no evidence of GFP+ mitotic figures was
found in the embryonic slices.
Recent demonstration that cell fusion can occur spontaneously between blood-borne cells and neurons (Alvarez-Dolado
et al. 2003; Weimann et al. 2003) makes it possible that our
results could arise from cell fusion between cells of donor
explants and host brain slices. Although we have not
thoroughly excluded this possibility, we have reasons to
believe that cell fusion does not play a significant role on the
appearance of glutamatergic pyramidal-like cells derived from
our explants. First, we have not observed the occurrence of
double-nucleated cells any time during the culture period
(Supplementary Fig. 3). Second, our observation that explantderived cells go through several steps of maturation, such as
migratory, immature, and mature profiles as time of culture
increases, excludes the possibility that non-neuronal GFPpositive cells like microglia would fuse with more mature cells
within the embryonic slice. Third, we have not at any time seen
satellite cells closely apposed to GFP-positive pyramidal cells,
such as the microglia/pyramidal cell pairs described in Ackman
et al. (2006). We could not exclude that cell fusion may occur
at the VZ between progenitor cells; however, this is also highly
unlikely. First, several groups that used modified neural stem
Cerebral Cortex November 2010, V 20 N 11 2587
Figure 5. Adult SVZ generates glutamatergic neurons when in contact with the embryonic slice. (A--D) Confocal image showing double labeling of a GFPþ cell (green) with
immunolabeling for vesicular glutamate (red) and Tuj1 antibody (blue). Arrows point to the cell body in all images (B), and small arrows points to same principal dendrite of the
same cell. The same field showing GFP labeling only. (C) Double-labeled cell with GFP and anti-glutamate. (D) Immunolabeling for glutamate only. (E) Another SVZ-derived GFPpositive neurons with triple labeling for Tuj1, anti-glutamate, and GFP inside inset. (F) Higher power view of the triple-labeled neuron in the inset. In (G), same view showing GFP
labeling only. (H) Labeling for Tuj1 only. (I) Glutamate immunolabeling only. In (J), another double-labeled cell for GFP and glutamate. (K) Only the anti-glutamate labeling. (L)The
orthogonal views of a stack of confocal images of GFP (green) and anti-glutamate labeling (red). Scale bars: A--D, 20 lm; E--I, 10 lm; J--L, 10 lm.
cells for transplantation have excluded the occurrence of cell
fusion (i.e., Muotri et al. 2005) between these cells. Second,
given that heterokarya may be unstable (Nern et al. 2009), the
loss of donor nuclei would give rise to decreased GFP
expression that we have not detected. The lack of evidence
for cell fusion in our model is in agreement with the low
incidence of this phenomenon reported in other brain regions
outside the cerebellum (Alvarez-Dolado et al. 2003).
Upon coculturing SVZ explants with postnatal OB, glutamate-positive cells were unexpectedly found in the granular
layer derived from both host OB and donor SVZ. Several lines of
evidence suggest that these cells are non-neuronal: 1)
Endogenous glutamate-positive cells of the OB granular layer
are not labeled for Tuj1 antibody, 2) extensive characterization
of OB granular neurons has shown that they are all GABAergic
(Shepherd et al. 2007), and 3) it has been shown that SVZ
astrocytes can be labeled by the glutamate immunohistochemistry (Platel et al. 2007). This further supports the hypothesis
that the neuronal glutamatergic phenotype triggered in SVZ
2588 Glutamatergic Neurons from Postnatal and Adult SVZ
d
Sequerra et al.
progenitors requires signals specific to the embryonic telencephalon. We could not, however, rule out the possibility of
existence of cells with a yet undefined morphology.
Data shown here indicate that the postnatal SVZ is capable of
generating multiple neuronal types with a broader neurotransmitter repertoire than previously thought. Why would this
potential be hidden in vivo? Two hypotheses are postulated:
The first one is that progenitors with a broader potential would
be capable of generating glutamatergic neurons, besides
GABAergic, when exposed to proper signals. The second
possibility is that restricted glutamatergic progenitors remain
quiescent until recruited. Recent data favor the hypothesis that
neurotransmitter bipotent progenitors may be present in the
SVZ since dorsal RG, which are known to give rise to cortical
pyramidal glutamatergic neurons early on, originate OB
GABAergic neurons postnatally (Merkle et al. 2007; Ventura
and Goldman 2007). This suggests that signals present in the
embryonic environment may reinstruct SVZ progenitors to
a differentiation pathway that is normally inactive in postnatal
ages. In our experiments, explants from different regions of the
SVZ, striatal, and OB SVZ generated glutamatergic neurons. For
this reason, we believe that glutamatergic progenitors in our
explants are in route migratory neuroblasts. We cannot, however,
rule out the possibility that quiescent restricted progenitors are
present throughout the rostral migratory stream. In support of
the environmental influence hypothesis, it has been shown that
neurotransmitter specification can be changed by environmental
factors, such as glutamate and GABA signaling in Xenopus laevis
(Root et al. 2008). This signaling changes calcium-spiking profiles
in progenitors and consequently neurotransmitter specification
(Borodinsky et al. 2004). The choice of SVZ neuroblasts to
synthesize GABA from putrescine instead of using GAD (Sequerra
et al. 2007) favors the idea that these progenitors maintain
neurochemical plasticity during their migration. Our present
results add up to the latter ones in showing neurotransmitter
phenotype plasticity regulated by environmental factors.
Whether bipotent or plastic, SVZ progenitors give rise to
glutamatergic neurons triggered by environmental factors.
In contrast, Merkle et al. (2007) have suggested by means of
heterotopic transplantation of SVZ cells in vivo that specification
of neuronal type would be cell autonomous and environment
independent. In contrast, our data suggest that the heterochronic
transplantation to the embryonic dorsal VZ environment provides
SVZ cells with signals that could release a suppressed program or
directly stimulate the pathway that leads to the pyramidal neuron
phenotype. In light of our data, a possible interpretation of the
results in Merkle et al. (2007) is that the positional identity
endows progenitors with a restricted spectrum of possible
phenotypic destinies. We suggest that heterotopic transplantation
within the SVZ would not be sufficient to uncover other
phenotypes because putative cues present along the rostral
migratory stream must be the same for all neuroblasts since it is
a common pathway for all migratory cells destined to the OB.
Macklis and collaborators have shown that endogenous
telencephalic progenitors are able to partly replace cortical
projection neurons lost by targeted apoptotic cell death
(Magavi et al. 2000; Chen et al. 2004). The source of these
endogenous progenitors is still unclear. The local parenchyma
may contain putative progenitor cells capable of cell
replacement (Palmer et al. 1999). In addition, the marginal
zone/layer I was recently shown to give rise to both neurons
and glia in the embryo, and it is still unclear if it can become
neurogenic in response to neural injury in postnatal or mature
brains (Costa et al. 2007). Macklis and collaborators suggest
that a major candidate for this cell replacement is the
underlying postnatal SVZ since they found BrdU-labeled
doublecortin-positive cells in the white matter, midways
between SVZ, and the cerebral cortex, shortly after the
photolytic lesion (Magavi et al. 2000; Chen et al. 2004). In
accordance to this idea, Fagel et al. (2009) have shown that,
after perinatal chronic hypoxia, Tbr1+ cells are generated
in the postnatal cerebral cortex. This phenomenon is correlated with an increase in SVZ proliferation, and both events are
FGFr1 dependent. Our work directly supports the hypothesis
that the postnatal SVZ is a potential source for replacement of
pyramidal neurons in postnatal and adult cerebral cortex.
We analyzed GFP-positive cells for Tbr1, Ctip2, and Satb2
expression since these are considered pyramidal cell markers of
different cortical layers (Hevner et al. 2001; Arlotta et al. 2005;
Britanova et al. 2005). However, GFP-positive pyramidal neurons
do not express any of these markers in our embryonic slice
preparation. During development, cortical neurons from different layers are generated at different times (Angevine and Sidman
1961) and are sequentially specified (McConnell 1988). However, after embryogenesis, progenitors may acquire phenotypic
determination via an alternative specification pathway.
During the review process of this paper, another article
appeared showing that dorsal SVZ progenitors that expressed
Tbr1 and Tbr2 gave rise to glutamatergic interneurons of the
OB glomerular layer (Brill et al. 2009). We have not identified
glutamatergic neurons in the glomerular layer with our
coculture study. This could be due to the short survival period
of our cultures allied to the low frequency of occurrence of
these cells described by Brill et al. (2009). The transitory
expression of Tbr1 shown by those authors in glutamatergic
neuronal progenitors of the SVZ opens the possibility that the
glutamatergic neurons we detected could also have expressed
Tbr1 transiently. An important point to be discussed here is
whether the glutamatergic phenotype is intrinsic or could be
influenced by extracellular signals. Tbr-positive progenitors
were found to be restricted to the dorsal portion of the SVZ
(Brill et al. 2009); however, these authors do not show if the
generation of these progenitors is intrinsic to this region or if it
is being induced by signals present in the dorsal adult
telencephalon. Therefore, as we are observing the generation
of glutamatergic neurons derived by ventral SVZ (striatal), it is
possible that the extracellular dorsal environment is inducing
the expression of this phenotype. Since the proportion of
glutamatergic cells in the model herein presented seems to be
higher than the one found by Brill et al. (2009) in vitro studies,
we suggest that this phenotype is at least partially controlled by
environmental cues. Another possibility is that the embryonic
CP is selecting glutamatergic neurons, thereby increasing its
proportions in relation to what would normally be expected.
Again, our data establish direct evidence that the postnatal SVZ
and adult SVZ are sources for glutamatergic neurons capable to
differentiate into cortical morphotypes.
Taken together, our results indicate that progenitors capable
of generating pyramidal neurons are continuously present in
the proximity of the lateral ventricles postnatally and until
adulthood. This potential is revealed by stimulation with signals
present in embryonic telencephalon. The knowledge of the
external factors that direct SVZ cells to a glutamatergic
phenotype will be an important next step for achieving
successful cell replacement therapies in cerebral cortex and
other central nervous system regions.
Supplementary Material
Supplementary material can be found at: http://www.cercor
.oxfordjournals.org/.
Funding
Fundac
xão de Amparo à Pesquisa do Estado do Rio de Janeiro,
Conselho Nacional de Pesquisa e Desenvolvimento, CNPq/
PRONEX, FINEP research grant ‘‘Rede Instituto Brasileiro de
Neurociência (IBN-Net)’’ 01.06.0842-00.
Notes
The authors thank Adiel Batista do Nascimento for animal care, Carla
Moreira Furtado, MSc, and Elizabeth Cunha Penna de Moraes, MSc, for
technical assistance, and Marcos Romualdo Costa for helpful comments
on the text. E.B.S. and L.M.M. were recipients of DSc and Post Doctoral
Cerebral Cortex November 2010, V 20 N 11 2589
fellowships from CNPq and PROCAD/CAPES respectively. Conflict of
Interest : None declared.
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Cerebral Cortex November 2010, V 20 N 11 2591