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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02601
1 Instituto Cajal, Consejo Superior de Investigaciones Científicas
(CSIC), E-28002 Madrid, Spain.
2 Growth Factors in Vertebrate Development Group, Centro de Investigaciones
Biológicas, CSIC, Madrid, Spain.
3 Instituto de Neurociencias de Castilla y León, Universidad de
Salamanca, Salamanca, Spain.
4 Department of Immunology and Oncology, Centro Nacional de
Biotecnología/CSIC, Madrid, Spain.
* Author for correspondence (e-mail: cvicario{at}cajal.csic.es)
Accepted 30 August 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Olfactory bulb precursor cells, Interneurons, Dlx2, Gsh2, Pax6, Cell transplant, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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); it is nonetheless thought that many embryonic rodent
OB interneurons (as well as cortical interneurons) arise in the ganglionic
eminences (GE) (Marín and
Rubenstein, 2003). Dlx2/Gsh2/Er81-expressing precursor cells in
the lateral ganglionic eminence (LGE) are a source of mouse OB interneurons
(Stenman et al., 2003).
Supporting evidence includes the in vivo observation that migratory cells that
leave the GE reach the cortex and OB
(Anderson et al., 1997;
Anderson et al., 2001;
de Carlos et al., 1996;
Lavdas et al., 1999;
Pencea and Luskin, 2003), and
that GE precursor cells grafted in the walls of lateral ventricles migrate to
the cortex and the OB (Wichterle et al.,
1999; Wichterle et al.,
2001). In addition, mice lacking Dlx1/2, Gsh1/2, Arx and Sp8
transcription factors, expressed in the GE, show markedly decreased expression
of GABAergic and dopaminergic cell markers in the embryonic granule and
periglomerular layers of the OB (Anderson
et al., 1997; Bulfone et al.,
1998; Corbin et al.,
2000; Qiu et al.,
1995; Stenman et al.,
2003; Toresson and Campbell,
2001; Waclaw et al.,
2006; Yoshihara et al.,
2005; Yun et al.,
2003; Yun et al.,
2001). Other data suggest that, in addition to this exogenous
neuron source, interneurons can be generated intrinsically within the
embryonic OB. These include observations that GABAergic markers are restored
in the Gsh2 knockout mouse OB at late embryonic stages
(Corbin et al., 2000;
Toresson and Campbell, 2001;
Yun et al., 2003), that the
Gsh1/2 double null mutation reduces, but does not abolish, glutamic acid
decarboxylase 67 (GAD67; Gad1 - Mouse Genome Informatics) and Er81 expression
in the OB (Stenman et al.,
2003; Yun et al.,
2003), and that a considerable number of GABAergic neurons remains
in the OB of Arx mice (Yoshihara et al.,
2005). Finally, the Gsh2 null mutation does not affect OB neuron
generation and differentiation from cultured LGE cells
(Jensen et al., 2004).
Overall, these findings indicate that the LGE may be the primary source of
OB interneurons during the embryonic period
(Stenman et al., 2003;
Wichterle et al., 1999) but do
not rule out dopaminergic and GABAergic neuron generation from local OB
precursor cells, particularly before migratory LGE cells reach the OB.
According to previous studies (Pencea and
Luskin, 2003), the rostral migratory stream (RMS) reaches the rat
OB by embryonic day (E) 16.5; this embryonic age would correspond
approximately to mouse E14.5. A recent study using Arx to label migratory
cells in the mouse (Yoshihara et al.,
2005) corroborates the idea that the first GE-derived cells are
detected in the OB at E14.5.
Here we show efficient differentiation of local E13.5 OB precursor cells into GABAergic and dopaminergic interneurons, when transplanted into E13.5 OB slices or into P5-P7 OB in vivo, or when plated in short-term dissociated cultures. GABAergic neurons isolated from the rostral half of E13.5 OB and from E13.5 GE were distinguished by their distinct migration patterns following transplant into the OB, and by their different gene expression in short-term dissociated cultures. This suggests that the OB contains a distinct, endogenous pool of interneuron precursor cells. Our results also show that these precursors differentiate in vivo into morphologically mature neurons, suggesting that they could integrate into the OB circuit.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
OB cells and GE (both medial and lateral eminence) cells were mechanically
dissociated, then cultured for 6 or 17 days essentially as described
(Vicario-Abejón et al.,
1998). Cells were also isolated and cultured from the OB of E13.5
embryos from pregnant mice that were injected intraperitoneally (i.p.) with
5'-bromo-2-deoxy-uridine (BrdU; 100 µg/g) 4 or 14 hours before
embryos were removed.
Neural stem cell cultures
Olfactory bulb stem cells (OBSC) were prepared from E13.5-14.5 mice. In
some experiments, only the rostral half of the E13.5 OB was dissected. Cells
were plated and expanded with fibroblast growth factor 2 (Fgf2; PeproTech) and
epidermal growth factor (Egf; PeproTech). For cell differentiation assays,
mitogens were removed and cells were cultured for 15-20 days in Dulbecco's
modified Eagle medium (DMEM)/nutrient mixture F12 (F12)/insulin,
apotransferrin, putrescine, progesterone, sodium selenite (N2) plus 5% fetal
bovine serum (FBS). Some cultures were supplied with brain-derived
neurotrophic factor (Bdnf; PeproTech). Clonal analysis experiments of
E13.5-derived primary cell spheres were performed as reported
(Vicario-Abejón et al.,
2003).
Transplant of E13.5 OB precursor cells expressing EGFP into E13.5 OB slices
Cells were prepared from the rostral half of the OB from E13.5 transgenic
C57BL/6 mice expressing enhanced green fluorescent protein (EGFP)
(Okabe et al., 1997). Cells
were also prepared from E13.5 CD1 mouse OB, and subsequently infected with an
EGFP-expressing lentiviral vector (ViraPower Lentiviral Expression System;
Invitrogen) (provided by P. Tsoulfas, University of Miami). Both types of
labeled cells were suspended in Hank's balanced salt solution (HBSS), then
implanted into E13.5 OB slices. To prepare slices, E13.5 brains were embedded
in 4% low-melting-point agarose, and 250 µm-thick coronal sections were cut
on a vibratome. Sections were transferred to inserts containing a
polycarbonate culture membrane (8 µm pore size; Nunc; Corning Costar).
Using a pulled glass pipette tip with a 50-70 µm outer diameter connected
to a Hamilton syringe (mounted on a stereotaxic apparatus), 1 µl of a
suspension of 5x104 cells/µl was injected. Slices were
cultured for 2 days in DMEM/F12/N2 plus 10% FBS, then fixed with 4%
paraformaldehyde (PFA). Slices were immunostained with antibodies to GFP
(1:500; Molecular Probes), GABA (1:1000; Sigma), tyrosine hydroxylase (Th;
1:100; Chemicon) or Tbr1 (1:100, provided by M. Sheng, MIT; or 1:2000,
provided by R. Hevner, University of Washington).
Transplant of EGFP-expressing E13.5 OB and E13.5 LGE precursor cells into early postnatal OB
Cells were suspended in HBSS, then implanted into the subependymal zone
(SEZ) of postnatal day 5-7 (P5-P7) mouse OB, when hosts were at the peak of
maximal interneuron formation (Hinds,
1968a). Mice were anesthetized by placing them in ice for 1-3
minutes. Each mouse was then positioned in a miniaturized stereotaxic device
(Cunningham neonatal rat adaptor; Stoelting) fixed to a standard stereotaxic
apparatus (Kopf) (Vicario-Abejon et al.,
1995). Stereotaxic coordinates for implants into P5 OB were
anteroposterior to bregma (AP) +1.3 mm, lateral to midline (L) 0.6 mm, ventral
to dura (V) 0.5 mm, and for P7 OB were AP +1.6 mm, L 1.0 mm, V 0.8 mm.
Subsequently, 1.5-2 µl of a suspension of 105 cells/µl was
injected. Only round aggregates, possibly formed by macrophages and cell
debris, were found in control mice transplanted with freeze-thaw killed cells
(not shown).
At 1 to 10 weeks post-transplant, mice were anesthetized by i.p. ketamine/xylazine injection and perfused transcardially with 0.9% NaCl and 4% PFA. Brains were post-fixed, embedded in 3% agarose, and cut in serial 50 µm vibratome sections. Sections containing the OB were collected and examined under a confocal fluorescent microscope. Some sections were immunostained with antibodies to GABA, GAD (1:75), Th, calbindin (1:100; Swant), parvalbumin (1:1500; Swant) or synaptophysin (1:4; Zymed).
Immunostaining of cultured cells
Cells were incubated with antibodies to Dlx2 (1:2000; provided by D.
Eisenstat, University of Manitoba), Gsh2 (1:2000; provided by K. Campbell,
University of Cincinatti), Pax6 (1:300; Covance), GABA, Th, Tbr1,
ß-III-tubulin (TuJ1, 1:2000; Covance), synapsin I (1:750; provided by M.
Kennedy, Caltech), GAD, MAP2ab (1:150; Sigma), BrdU (1:1000) or SV2 (1:50).
GAD, BrdU (G3G4) and SV2 antibodies were developed by D. I. Gottlieb,
Washington University School of Medicine, S. J. Kaufman, University of
Illinois and K. M. Buckley, Harvard Medical School, respectively, and were
obtained from the Developmental Studies of Hybridoma Bank (University of Iowa
Department of Biological Sciences).
Immunostaining of anatomical cryostat sections
For histology, E12.5-13.5 embryo heads were fixed overnight in 4% PFA.
Alternatively, E13.5 embryos were first perfused transcardially. Heads were
immersed in 30% sucrose, then frozen at -70°C. Cryostat coronal or
sagittal sections (14 µm) were incubated with the antibodies mentioned
above. Postnatal mouse brain cryostat sections were double immunostained with
a rabbit antibody to the tyrosine kinase domain of TrkB [1:50; provided by S.
Feinstein and M. Radeke (University of California)
(Vicario-Abejón et al.,
1998)] and mouse anti-SV2.
Cell counts and statistical analysis
To determine the number of cultured cells expressing a specific antigen,
ten random fields were counted per coverslip using a 40x objective and
fluorescence filters
(Vicario-Abejón et al.,
2003). In transplantation experiments, the percentage of
GABA+ cells and Th+ cells were calculated relative to
the number of E13.5 GFP+ cells found in the slices. The percentage
of cells located in the different cell layers was calculated relative to
transplanted E13.5 GFP-positive OB or LGE cells found in the postnatal OB.
Morphological analysis was performed on 50 neurons derived from transplanted
E13.5 GFP+ OB cells, and on 50 neurons derived from E13.5
GFP+ LGE cells. Statistical analysis was performed using Student's
t-test.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Yuste, 2005).
Dlx/Gsh2/Er81-expressing precursors in the LGE are a source of mouse OB
interneurons (Stenman et al.,
2003); the newborn neurons then migrate to the OB. To determine
whether Gsh2, Dlx2 and interneuron markers are expressed in mouse E13.5 OB,
before the main wave of migration from the LGE to the OB, we immunostained
sections with anti-Gsh2 (Stenman et al.,
2003) and anti-Dlx2 antibodies
(de Melo et al., 2005). Gsh2
and Dlx2 expression were abundant in the medial ganglionic eminence (MGE) and
LGE (Fig. 1A-H), whereas E13.5
OB did not express Gsh2 and Dlx2 (Fig.
1I,J). These findings indicate that the RMS does not reach the
mouse OB at E13.5 but later, reportedly at E14.5 and after
(Pencea and Luskin, 2003;
Yoshihara et al., 2005). By
contrast, we found cells expressing interneuron markers such as GABA and Th in
the OB (Fig. 1K-M). As
predicted, we found cells expressing Tbr1, a marker of mitral/tufted neurons
(Bulfone et al., 1998), in the
OB (Fig. 1N). These data
indicate the presence of interneurons within the OB before LGE migratory cells
expressing Dlx2 or Gsh2 could be detected in the OB.
Transcription factor expression, BrdU labeling and interneuron generation in vivo and in short-term dissociated cultures of E13.5 OB and GE
To study the developmental expression of Gsh2 and Dlx2 and the generation
of interneurons in the OB independently of GE influence, we isolated cells
from the rostral half of the E13.5 OB and from the GE, and plated them in
short-term dissociated cultures for 6 days
(Fig. 2). GAD+
neurons were detected in both cultures as early as 6 hours post-plating (not
shown). At 6 days, 22.3 and 75.9% of total neurons (TuJ1+ cells)
were GAD+ in OB and GE cultures, respectively
(Fig. 2M). Double labeling of
GAD+ neurons with anti-Dlx2 and anti-Gsh2 antibodies revealed a
different expression pattern for these transcription factors in OB and GE
GABAergic cells. Of the total OB GAD+ neurons, none (0%) expressed
Gsh2 (Fig. 2A-C,N) and 53.3%
expressed Dlx2 (Fig. 2D-F,N),
whereas in GE cultures, 16.1 and 70.4% of GAD+ neurons expressed
Gsh2 (Fig. 2G-I,N) and Dlx2
(Fig. 2J-L,N), respectively.
Differences between OB and GE Dlx2+ and Gsh2+ cells were
best observed when proportions were calculated of double-labeled
Dlx2+GAD+ cells divided by the number of total
Dlx2+ cells, and of Gsh2+GAD+ cells divided
by total Gsh2+ cells (Fig.
2O). In OB cultures, 41.5% of the total Dlx2+ cells
were GAD+, whereas no Gsh2+ cells were GAD+.
By contrast, 75.5% of Dlx2+ cells and 49.1% of Gsh2+
cells in GE cultures co-labeled with GAD
(Fig. 2O). These results show a
significantly closer relationship in GE than in the OB between acquisition of
a GABAergic phenotype and Dlx2 and Gsh2 expression. The absence of
double-labeled Gsh2+ and GAD+ cells in OB cultures
strongly suggests that most GAD+ neurons derive from local OB and
not from GE precursor cells.
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) (Fig. 3A-C).
Pax6 labeled the entire OB neuroepithelial zone (NEZ) and cells distributed in
the mantle zone (MZ). Cultures of E13.5 OB cells showed 52.0±1.1%
(n=3) GAD+ neurons expressing Pax6
(Fig. 3D-F). Many
GABA+ cells located in the MZ were marked with anti-BrdU in
sections prepared from mice that received BrdU to label dividing cells 4 or 14
hours before sacrifice (Fig.
3G-I). In the OB cultures, 95.4±0.9% (n=3)
GABA+ neurons co-labeled with BrdU
(Fig. 3J-L), indicating that
the large majority of OB interneurons are descendants of E13.0-13.5 dividing
precursors.
Differentiation of E13.5 OB precursor cells transplanted into E13.5 OB slices
To analyze which neuron types are generated from E13.5 OB precursors within
the E13.5 OB, we transplanted EGFP-expressing cells into E13.5 OB slices
prepared from wild-type embryos. Precursor cells were transplanted immediately
after extraction from EGFP transgenic embryos, or were prepared from wild-type
embryos followed by immediate incubation with a lentiviral vector expressing
EGFP, and then transplanted. Precursor cells were thus injected with no
previous culture. Two days after injection, we found numerous GFP+
cells expressing interneuron markers in the slices
(Fig. 4). Of the total
GFP+ cells, 31.7% were GABA+
(Fig. 4A-C,G) and 12.1% were
Th+ (Fig. 4D-G).
Cells expressing Tbr1 were also found (not shown). These results indicate that
interneurons can be generated endogenously within the OB.
Migration and differentiation of E13.5 OB precursor cells in vivo and in short- and long-term dissociated cultures
Concurring with the considerable numbers of GAD+ cells found in
OB dissociated cultures, and with the fact that the majority of OB
dopaminergic neurons are also GABAergic
(Carleton et al., 2003;
Shipley and Ennis, 1996),
GABA+ and Th+ cells with clear neuron morphology were
abundant in these cultures (Fig.
5A,B). We then performed two types of experiments to determine
whether OB neurons can differentiate extensively and display mature
morphologies. E13.5 OB cell suspensions were cultured for 15-20 days to
promote neuron maturation (Fig.
5C-H). Cultures were double-stained with antibodies to MAP2ab (a
general neuron marker that labels dendrites specifically, in addition to cell
bodies) and to GABA, Th or Tbr1. The cultures had many MAP2ab+
neurons with large number of dendrites and dendritic branches
(Fig. 5C,E,G). We detected
GABAergic and dopaminergic interneurons
(Fig. 5D,F), as well as mitral
neurons (Fig. 5H), suggesting
that the major OB neuron types differentiate to attain mature morphological
features in this culture system.
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; Lledo and Saghatelyan,
2005; Shipley and Ennis,
1996). Fewer OB implanted cells migrated to the GL, where they
differentiated into periglomerular neurons
(Fig. 7B). Like host
periglomerular neurons (Fig. 7B
inset), some grafted cells oriented their cell bodies horizontally.
GFP+-transplanted cells were labeled with anti-GAD and anti-GABA
antibodies, which confirmed the GABAergic phenotype
(Fig. 7G-O). Some transplanted
GFP-expressing cells were Th+, indicating a dopaminergic phenotype
(Fig. 7P-R). These results
indicate that local E13.5 OB precursor cells can differentiate and mature in
vivo into the main types of OB interneurons, and that the cell body of these
neurons resides in the appropriate OB cell layers.
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;
Vicario-Abejón et al.,
2003). To test whether OB precursor cells having NSC features are
able to generate GABAergic, dopaminergic, and mitral/tufted neurons, we
expanded E13.5 and 14.5 OBSC and induced them to differentiate. A
characteristic of GABAergic neurons from various brain regions is that they
respond to Bdnf (Vicario-Abejón et
al., 2002). As this neurotrophin and its high-affinity TrkB
receptor are expressed in the OB (Nef et
al., 2001) (Fig.
10A-E), we tested the response of OBSC-derived neurons to
Bdnf. E13.5 OBSC that had differentiated for 15-20 days generated GAD+ neurons expressing Dlx2, although GAD+ and Dlx2- neurons were also found (Fig. 8A,B), as occurred in short-term dissociated OB precursor cells (Fig. 2). Double staining of control E13.5 and 14.5 OBSC-derived neurons with anti-MAP2ab and anti-GABA antibodies showed 64 and 57% GABA-positive neurons, respectively (Fig. 8C,D,G). Addition of Bdnf to the cultures produced no statistically significant changes in the number of GABAergic neurons (Fig. 8E-G). Some Bdnf-treated neurons showed more complex morphology than controls (Fig. 8C-F). To rule out possible detection of GABA+ neurons in E13.5 OB cultures derived from LGE extension into the caudal OB, we prepared cells from the rostral half of the E13.5 OB. In these conditions, 57.2±2.9% (n=4) of neurons were GABA+.
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We then analyzed whether OBSC can differentiate into dopaminergic neurons (Fig. 9A-E). Of the total MAP2ab+ neurons in E13.5 and 14.5 OBSC-derived cultures, 18 and 16%, respectively, were Th+ (Fig. 9E). As observed in vivo, the majority of cultured Th+ neurons expressed GAD (not shown). In this assay, Bdnf had no significant effect on Th+ cell numbers (Fig. 9C-E).
The OB excitatory neurons, mitral and tufted cells, express the Tbr1 transcription factor. In the previous assays (Fig. 8G, Fig. 9E) we observed no major differences in neuron differentiation between E13.5- and E14.5-derived OBSC; mitral/tufted neuron generation was thus only tested in E13.5 OBSC cultures (Fig. 9F-J). Of the total MAP2ab+ neurons in control and Bdnf-treated cultures, 9.2 and 11.6%, respectively, were Tbr1+ (Fig. 9F).
Unexpanded OB precursor cells generate morphologically mature GABAergic and
dopaminergic interneurons in culture (Fig.
5) and in vivo (Figs
6,
7). To further assess the
maturation stage of neurons arising from OB cells, we stained OBSC-derived
neurons with antibodies to synapsin I and SV2
(Fig. 10), two presynaptic
proteins involved in synaptogenesis and synapse function
(Vicario-Abejón et al.,
2002). We tested the effects of Bdnf on synaptic marker expression
by OB neurons, as staining with antibodies to TrkB revealed this neurotrophin
receptor in mitral (Fig. 10A)
and periglomerular neurons (Fig.
10B). Furthermore, double staining showed that TrkB was highly
expressed at SV2+ synaptic terminals in the glomeruli
(Fig. 10C-E). E13.5 OBSC
differentiated into neurons (Fig.
10F-Q), including GABAergic and Th+ cells that
expressed synapsin I and SV2 in a punctate pattern
(Fig. 10H-Q) characteristic of
mature neurons (Vicario-Abejón et
al., 1998). Neurons cultured with Bdnf had a more complex neuron
morphology, with more neurites and neuritic branches than control cells
(Fig. 10F,G). In these
conditions, Bdnf-treated neurons showed more synapsin I+ and
SV2+ boutons than control neurons
(Fig. 10; compare panels I
with H; L,M with J,K; P,Q with N,O).
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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;
Hinds, 1968b;
Nomura and Osumi, 2004;
Rakic, 1971). Interneuron
origin during the embryonic period nonetheless appears to be more diverse and
species-dependent. In humans, the main source of cortical GABAergic neurons
appears to be the cortical VZ (Letinic et
al., 2002). By contrast, it is thought that many embryonic mouse
OB interneurons (as well as cortical interneurons) are not generated locally
in the OB or in the cortical VZ, but arise in the GE
(Marín and Rubenstein,
2003; Stenman et al.,
2003), from which they migrate to their final destination
(Anderson et al., 1997;
de Carlos et al., 1996;
Lavdas et al., 1999;
Pencea and Luskin, 2003;
Wichterle et al., 1999;
Wichterle et al., 2001).
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;
Carleton et al., 2003;
Lemasson et al., 2005).
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;
Yoshihara et al., 2005). These
results, and the complete absence of Gsh2+/GAD+ neurons
in OB cultures, are consistent with the suggestion that the majority of
interneurons found in these cultures, in postnatal mice and E13.5 slices
grafted with E13.5 OB precursor cells, as well as in situ in the E13.5 OB,
arise from endogenous OB and not from GE precursor cells. The BrdU data
support the concept that most differentiated interneurons are born from
dividing precursors in the E13-13.5 OB.
Dlx2 and Gsh2 proteins were not detected in E13.5 OB sections, but were
expressed in E13.5 plated directly in short-term dissociated culture for 6
days. These findings suggest that these transcription factors are expressed by
endogenous OB precursor cells and that detectable levels of protein expression
are found later than E13.5. In support of this, Gsh2 mRNA was found
in the E13.5 OB by RT-PCR (not shown). Precursor cells expressing these
transcription factors appear to be distinct in the OB and the GE, based on GAD
colocalization; 41.5% of Dlx2+ and 0% of Gsh2+ cells
coexpressed GAD in OB cultures, whereas 75.5% of Dlx2+ and 49% of
Gsh2+ cells colocalized with GAD in GE cultures. Furthermore,
migration of transplanted OB and LGE precursors to the distinct postnatal OB
layers was dependent on the region of cell origin; OB cells migrated
preferentially to the GCL and the IPL-ML-EPL, whereas LGE cells were found
predominantly in the GCL and GL. OB and LGE cells also generated different
interneuron subtypes that can be distinguished by their morphology and
expression of calcium-binding proteins. These data indicate that the embryonic
OB contains a distinct local population of interneuron-generating precursor
cells, and suggest the importance of the spatial origin of interneuron
precursors in shaping their final phenotype, as proposed
(Butt et al., 2005;
Yuste, 2005). Specifically, a
considerable proportion of GAD+ neurons express the telencephalic
pallial domain marker Pax6 (Puelles et
al., 2000). Although Pax6 is expressed in the dorsal LGE, which is
also Gsh2+ (Kohwi et al.,
2005; Yun et al.,
2001), the absence of GAD+Gsh2+ cells in
cultures prepared from the rostral half of the OB suggests that the
GAD+Pax6+ neurons found in the OB cultures derived from
pallial OB Pax6+ precursors. In accordance with our results,
cortical NSC differentiated into Dlx+ and GAD+ neurons,
suggesting an endogenous subpopulation of cortical interneurons
(He et al., 2001;
Parmar et al., 2002). This
resembles data showing genesis of Dlx2+ cells
(Nery et al., 2003) and of
Dlx2+Mash1+-expressing GABAergic neurons in the dorsal
human and rodent cortical VZ (Bellion et
al., 2003; Letinic et al.,
2002). These findings support the concept that interneurons
originate in dorsal telencephalic VZ as well as in ventral telencephalic
zones, and that the quantitative contribution of each germinative zone to
total embryo interneuron number is species-dependent
(Gotz and Sommer, 2005).
The capacity of local OB precursors to give rise to interneurons in the
embryonic period appears to persist in the neonate
(Lemasson et al., 2005) and is
gradually lost in adulthood, although interneuron generation has been found in
the adult rodent and in human OB (Gritti
et al., 2002; Hoglinger et
al., 2004; Liu and Martin,
2003), in addition to the well-studied, predominant
postnatal-adult SVZ source of OB interneurons
(Kornack and Rakic, 2001;
Lois and Alvarez-Buylla,
1994).
Differentiation and maturation of OBSC-derived neurons
Here we show that a previously characterized OBSC population
(Vicario-Abejón et al.,
2003) not only generates mitral/tufted neurons, as predicted, but
that differentiation of OBSC gave rise to large numbers of GABAergic neurons.
It is very unlikely that OBSC originate in the GE rather than in the OB
neuroepithelium, as stem cell migration between the OB and the GE has not been
reported (Reid et al.,
1999).
To confirm that OB GABAergic neurons arise from NSC and not from committed GABA progenitor cells in OBSC cultures, we performed clonal analysis experiments, in which the majority of single cell-derived clones gave rise to GABA+ neurons (as well as to glial cells), indicating that many of these neurons are produced by multipotent cells with the ability to self-renew. As observed in the short-term OB dissociated cultures, a proportion of OBSC-derived GABAergic neurons were Dlx2+, suggesting that under these conditions, mitogen treatment that expands OBSC produces no obvious changes in the expression of genes that confer dorsoventral neural patterning. In support of this hypothesis, we also found neurons expressing Tbr1, a dorsal telencephalic marker in differentiating OBSC cultures, as well as Tbr-1 mRNA (not shown). GAD-negative/Gsh2+ cells were also found in OBSC differentiating cultures (not shown), as seen in the short-tem OB dissociated cultures.
E13.5 OB precursor cells plated in short-term dissociated cultures or
transplanted into the neonatal OB developed a mature morphology, including
dendritic spines in granule neurons. Consistent with these results,
OBSC-derived neurons differentiated extensively and expressed presynaptic
proteins in a punctate pattern characteristic of mature neurons. OBSC-derived
GABAergic and dopaminergic neurons cultured with Bdnf show more synapsin I and
SV2 expression than control cells, although this neurotrophin had no clear
effect on neuron survival. Bdnf nonetheless promotes the generation and/or
survival of new neurons in adult rostral SVZ and OB
(Nanobashvili et al., 2005;
Zigova et al., 1998).
OBSC-derived neuron responses to Bdnf, the abundance of TrkB at synaptic
sites, and the lack of Bdnf effects on the formation of new neurons from OBSC
(Vicario-Abejón et al.,
2003) suggest that Bdnf promotes morphological and synaptic
maturation of OB neurons. These findings concur with the action reported for
this neurotrophin in promoting synapse formation and stabilization
(Hu et al., 2005;
Martinez et al., 1998;
Nanobashvili et al., 2005;
Rico et al., 2002;
Vicario-Abejón et al.,
2002).
In summary, our results show efficient formation of mature GABAergic and dopaminergic neurons from endogenous embryonic OB precursor cells. These locally generated interneurons, along with the mitral and tufted neurons and the extrinsically generated OB interneurons, may contribute to the formation of the functional OB synaptic circuit.
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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