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The Journal of Neuroscience, July 15, 2002, 22(14):6106-6113
Maturation and Death of Adult-Born Olfactory Bulb Granule
Neurons: Role of Olfaction
Leopoldo
Petreanu1 and
Arturo
Alvarez-Buylla2
1 The Rockefeller University, New York, New York
10021, and 2 Neurosurgery Research, University of
California, San Francisco, San Francisco, California 94134
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ABSTRACT |
Young neurons born in the subventricular zone (SVZ) of adult mice
migrate to the olfactory bulb (OB) where they differentiate into
granule cells (GCs) and periglomerular interneurons. Using retroviral labeling of precursors in the SVZ, we describe five stages
and the timing for the maturation of newly formed GCs: (1)
tangentially migrating neuroblasts (days 2-7); (2) radially migrating
young neurons (days 5-7); (3) GCs with a simple unbranched dendrite
that does not extend beyond the mitral cell layer (days 9-13); (4) GCs
with a nonspiny branched dendrite in the external plexiform layer (days
11-22); and (5) mature GCs (days 15-30). Using
[3H]thymidine, we show that the maximum number of
labeled GCs is observed around day 15 after injection. Interestingly,
between days 15 and 45 after birth, soon after the cells developed
spines, the number of [3H]thymidine-labeled GCs
declined by 50%. Using anosmic mice, we found that sensory input was
critical for the survival of GCs from day 15 to 45 after labeling.
However, the number and morphology of 15-d-old cells in the granule
cell layer was similar in anosmic and wild-type mice. We infer that the
lack of activity did not have an effect on the generation, migration,
and early differentiation of granule cells. Soon after young GCs
matured, and presumably became synaptically connected, their survival
depended on the level of activity that they received. This selection
mechanism might allow the construction of specific OB circuits based on olfactory experience and suggests possible functions of OB cell replacement.
Key words:
olfactory bulb; granule cells; neurogenesis; new neurons; cell death; olfaction
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INTRODUCTION |
Neurogenesis persists in two regions
of the adult mammalian brain: the hippocampus and the olfactory bulb
(OB) (Altman, 1969 ; Lois and Alvarez-Buylla, 1994; Gage, 2000). The
adult-generated neurons of the OB are born in the subventricular zone
(SVZ) (Luskin, 1993; Lois and Alvarez-Buylla, 1994) and migrate along a
network of pathways (Doetsch and Alvarez-Buylla, 1996) into the rostral migratory stream (RMS) and into the OB where they differentiate into
granule cells (GCs) and periglomerular cells. Most of the OB granule
neurons are generated postnatally and continue to be added in adulthood
(Kaplan and Hinds, 1977; Bayer, 1983).
Because apoptosis is observed in the layers where the newly generated
cells are incorporated (Najbauer and Leon, 1995; Fiske and Brunjes,
2001) and the volume of the OB does not increase in adult mice (Pomeroy
et al., 1990), newly generated GCs are thought to replace older cells.
Less than 10% of the GCs born in young adult rats survive 21 months,
suggesting that many of these cells have been replaced (Kaplan et al.,
1985).
Granule cell bodies cluster, forming sheets in the granule cell layer
(GCL). They extend a few short neurites toward deeper parts of the GCL
and one large dendrite toward the external plexiform layer (EPL)
(Shepherd, 1972; Greer, 1987; Shipley and Ennis, 1996) where it
branches extensively. Granule neurons have no axons, and their output
is mediated by bidirectional dendrodendritic synapses located in spines
(Jahr and Nicoll, 1982; Woolf et al., 1991). GCs are thought to
modulate the activity of mitral (M) and tufted (T) cells, optimizing
olfactory function by reducing overlap for odor representation by M and
T cells (Mori and Shepherd, 1994; Yokoi et al., 1995; Hildebrand and
Shepherd, 1997). The function of olfactory granule neuron replacement
in the adult brain is not known. It has been suggested that this
process may be used for plasticity and olfactory discrimination (Gheusi
et al., 2000; Cecchi et al., 2001).
Although newly generated cells in adult mice are known to populate the
GCL at ~12 d after they are born in the SVZ (Lois and Alvarez-Buylla,
1994), little is known about the timing of differentiation and death of
the newly formed neurons. Defining the different stages of maturation
and times when newly formed olfactory interneurons die is required to
understand how neuronal replacement in the OB is regulated. Changes in
the morphology of the cells as they migrate along the RMS and invade
the bulb have been described using the Golgi method (Kishi, 1987), but
the time course of this process in the adult OB is not known. Using
retroviral labeling of the newly born neurons in the SVZ, we describe
the stages of maturation of newly generated GCs in adult mice. We also
identify an early wave of cell death that occurs shortly after the
newly formed cells develop their dendritic arbors and synaptic
gemmules. Using anosmic mice, we show that GC survival during this
period of fast cell death is dependent on incoming activity from the olfactory epithelium.
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MATERIALS AND METHODS |
Animals. Two-month-old CD-1 male mice were obtained
from Charles River Laboratories (Wilmington, MA). Animals were housed in groups of five per cage, under a 12 hr light/dark cycle (lights on
at 8:00 A.M.) with food and water available ad libitum.
Olfactory cyclic nucleotide gated channel knock-out mice (Brunet et
al., 1996) were bred in-house. Heterozygous females were bred with
C57BL/6 males to obtain hemizygous mutant males. Most of the mutants
died of starvation soon after birth; however, by reducing litters to
four pups 24 hr after birth, 4 of 60 mutants managed to survive to
adulthood (Baker et al., 1999; Zheng et al., 2000). DNA was extracted
from the animals' tails and screened for the mutation by PCR as
described (Zheng et al., 2000). Control animals were male littermates.
All of the animal procedures were approved by The Rockefeller
University's Animal Care and Use Committee.
3H-thymidine and bromodeoxyuridine labeling and
tissue processing. Forty-five CD-1 animals were injected
intraperitoneally with 50 µl of
[3H]thymidine (6.7 Ci/mmol, 1 mCi/ml;
New England Nuclear, Boston, MA) every 24 hr for 4 consecutive days. At
each of the different survival times, five of the animals were killed
by an overdose of pentobarbital (140 mg/kg body weight, i.p.).
Olfactory cyclic nucleotide gated channel knock-out mice and control
littermates were injected once daily for 2 consecutives days with
bromodeoxyuridine (BrdU; 50 mg/kg) (Sigma, St. Louis, MO). One month
later they received a single injection of 50 µl of
[3H]thymidine (6.7 Ci/mmol, 1 mCi/ml),
and 15 d later were killed by an overdose of pentobarbital. All of
the animals received the injections between 2 and 4 P.M. Animals were
intracardially perfused with 0.9% NaCl, followed by 3.0%
paraformaldehyde (PFA), and the brains were postfixed overnight in the
same fixative. Brains were embedded in polyethyleneglycol (PEG) and
sectioned coronally at 6 µm (Alvarez-Buylla et al., 1988). Serial
sections 120 µm apart from the whole olfactory bulb were mounted.
Olfactory bulbs from CD-1 mice were processed for Nissl staining or
NeuN immunochemistry followed by autoradiography. Sections from
anosmic mice and control littermates were processed for
autoradiography, terminal deoxynucleotidyl transferase-mediated
biotinylated UTP nick end labeling (TUNEL), BrdU, and Nissl staining.
NeuN immunochemistry. PEG sections (6 µm) were incubated
with 3% H2O2 in methanol
for 15 min and then for 48 hr at 4°C with NeuN monoclonal antibody
(1:200; Chemicon, Temecula, CA). Slides were then incubated in
biotinylated mouse secondary antisera (1:200; Vector Laboratories,
Burlingame, CA) for 60 min (room temperature), rinsed, incubated
in avidin-biotin-horseradish peroxidase (AB; 1:200; Vector
Laboratories) for 30 min, and in 0.02% diaminobenzidine (DAB) with
0.003% H2O2 for 5 min.
TUNEL technique staining. Apoptotic cell death was detected
in 6 µm PEG sections using the TUNEL staining method (Gavrieli et
al., 1992; Holcomb et al., 1995) and visualized using AB (1:200; Vector
Laboratories) for 30 min followed by 5 min in 0.02% DAB with 0.003%
H2O2.
Retrovirus microinjection and tissue processing.
Replication-incompetent retroviruses encoding the marker gene human
placental alkaline phosphatase (DAP) were harvested from the psi2 DAP
cell line (American Type Cell Culture 1949-CRL) (Fields-Berry et
al., 1992), concentrated, titered, and tested for helper virus (Cepko, 1992). The titer was 108 colony-forming
units/ml.
Each animal was injected stereotaxically with 0.2 µl of retrovirus in
two coordinates for each hemisphere (relative to bregma: anterior, 1;
lateral, ±1; depth, 2.2; and anterior, 0; lateral, ±1.4;
depth, 1.6). At different survival times, the animals were killed by an
overdose of anesthesia (pentobarbital, 140 mg/kg). Animals were
perfused intracardially with 30 ml of 0.9% saline followed by 30 ml
3% PFA. The brain was extracted and fixed overnight in the same
fixative. The olfactory bulbs were cut horizontally in 60 µm serial
sections using a vibratome and processed for alkaline phosphatase
histochemistry as described (Fields-Berry et al., 1992).
Image analysis and quantification. Cell counts and area
measurements were performed using a computer-based mapping microscope (Alvarez-Buylla and Vicario, 1988). For each OB section, the area of
the granule cell layer was measured by subtracting the area of the RMS
and the accessory olfactory bulb from the area delimited by the mitral
cell layer. The area of the RMS in coronal sections, from the rostral
tip of the lateral ventricle to the rostral end of the OB, was measured
in Nissl-stained sections. The RMS is easily distinguishable from
surrounding tissue because of its higher cell density. Areas were
measured in 6-µm-thick serial sections at 120 µm intervals. The
volumes of the granule cell layer and the RMS were approximated with
the equation:
where A equals the area of the i-th section,
d is the distance between traced sections, and n
is the total number of measured sections. The density of NeuN-labeled
cells per unit area was measured in five fields (400× magnification)
in the granule cell layer. The number of
[3H]thymidine-, BrdU-, or TUNEL-labeled
nuclei was counted in the entire GCL of two sections, 120 and 600 µm
rostral to the accessory olfactory bulb. Neurons were considered to be
labeled if they had over their nuclei 10×, or greater, the
number of exposed silver grains compared with background. In anosmic
and control littermates, the number of silver grains was counted on 200 [3H]thymidine-labeled cells for each group.
Total numbers of [3H]thymidine- and
BrdU-labeled cells in the OB were estimated counting nuclear profiles.
Given the size range of granule cell nuclei, it is not necessary to
correct for cell splitting between 6 µm sections for
[3H]thymidine-labeled nuclei (Clark et
al., 1990). Therefore, the total number of
[3H]thymidine-labeled cells was
estimated using the formula: T = (N × V)/t, where T is the total
number of cells, N is the number granule cell nuclei per
unit area, V is the volume of the granule cell layer, and
t is the section thickness. To derive total numbers of
BrdU-labeled cells, a cell splitting correction factor based on the
Abercrombie method (Guillery and Herrup, 1997) was used. The total
number of BrdU cells was calculated using the formula T = (N × V)/(t + D), where D is the average diameter of
BrdU-labeled nuclei. Granule cell diameters from 250 cells per group
were measured from photomicrographs of the GCL using NIH Image. No
difference in cell diameter was found between anosmic and control mice.
Note that absolute estimates of number of cells in the OB allow for comparisons between experimental groups within this study, but that
these numbers are limited by the sampling methods and assumptions of
the Abercrombie correction.
[3H]thymidine autoradiography in
6-µm-thick sections allows for high histological resolution, but
unbiased stereological methods are not possible in this material (Clark
et al., 1990).
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RESULTS |
Maturation of adult-generated granule neurons
To study the time course of the development of newly generated
granule neurons, we injected the SVZ with a replication incompetent retrovirus encoding the human placental alkaline phosphatase
(Fields-Berry et al., 1992). Five days after injection,
DAP-labeled cells were localized along the RMS and in the core of the
olfactory bulb. Staining was seen over the entire surface of the
membrane of each cell, revealing its morphology with fine detail (Fig.
1) (Doetsch et al., 1999).
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Figure 1.
Alkaline phosphatase staining of virus-infected
newly generated cells in the GCL of the olfactory bulb.
Photomicrographs (left and center
columns) and camera lucida drawings (right
column) show examples of stained developing granule neurons at
different maturation stages as defined in Results: class 1 (A-C); class 2 (D-F); class 3 (G-I); class 4 (J-L); class 5 (M-O). Scale bars, 25 µm.
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Five and 7 d after viral injection, the majority of cells had the
typical morphology of migrating neuroblasts (class 1) (Fig. 1A-C). This morphology has been described
in detail previously (Kishi, 1987; Lois et al., 1996; Wichterle et al.,
1997). Class 1 cells had a round or elongated cell body with a
prominent leading process and growth cone, and occasionally a small
trailing process. These cells were found only in the RMS and its
rostral extension in the core of the OB. Cells with their growth cone
oriented either rostrally (58 ± 5%; n = 2; 91 cells) or caudally (42 ± 5%) were found in the core of the OB.
This suggests that migrating precursors in the core of the OB move in
both directions. With longer survivals, many of the young migrating
neurons were found farther away from the OB, migrating radially toward
more superficial layers. These cells maintained their elongated
morphology, but their leading and trailing processes were longer. The
leading process was frequently bifurcated with multiple, small
ramifications (class 2) (Fig. 1D-F). The growth cone of class 2 cells was less prominent than that of class 1 cells. Beginning at day 9 after injection, a third class of DAP-labeled cells was observed. These
cells had a larger, rounder cell body, with a prominent single process
that extended toward the EPL, but did not cross the mitral cell layer
(class 3) (Fig. 1G-I). We infer that this
process corresponds to the growing apical dendrite. The smooth trailing
process observed in class 1 and 2 cells was not observed in class 3 cells. Instead, these cells had neurites developing on the basal side,
extending into the neighboring GCL, probably corresponding to the
developing basal dendrites. In contrast to the smooth contours of class
1 and 2 cells, class 3 cells had irregular borders with varicosities around the cell body and in the developing basal dendrites (Fig. 1G-I). The cell bodies of class 3 cells
were aligned with clusters of other resident granule neurons. The
morphology and location of class 3 cells suggested that they had
completed their migration, and their cell bodies had become settled in
a particular location next to other granule neurons.
At later time points, cells grew dendrites that extended beyond the
mitral cell layer and branched in the EPL. We subdivided these cells
into two classes (class 4 and class 5). Class 4 cells had elaborate
branched apical dendrites (Fig. 1J-L),
but had few, if any, spines (Fig.
2A). The size and shape
of the basal processes and soma were similar to that of class 3 cells.
Class 5 cells (Fig. 1M-O), in addition to
the elaborate branched apical dendrites, had many spines (gemmules) at
high density on the apical dendrites (Fig. 2B).
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Figure 2.
Alkaline phosphatase staining of the dendritic
arbors of virus-infected newly generated cells in EPL of the olfactory
bulb. Representative photomicrographs show the dendritic arborization
of developing granule neurons at early stages of maturation
(A) and at later stages, after developing
dendritic spines (B). Scale bars, 25 µm.
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To determine the order of appearance of the different cell types as
defined above, mice were killed at eight different survival points, and
the number of cells of each type was quantified (Fig. 3). Class 1 cells were found in the core
of the OB between days 5 and 9, and thereafter their number decreased.
This indicates that the progeny of virally infected cells in the SVZ
arrived at the bulb as a bolus of young neurons, and that by day 9 most of the new neurons had left the RMS. This is consistent with previous work indicating that most of the cells labeled by a single injection of
an S phase marker correspond to the young neuroblasts and the actively
dividing transit amplifying precursors, not the stem cells (Doetsch et
al., 1997). By day 9, a few cells had already developed elaborate
dendritic arbors (class 4), but most of the cells at this time were
migrating radially (class 2). Within the next 4 d (days 9-13),
there was a very rapid maturation of the dendritic fields, but cells
had relatively few spines. By day 13 the majority of labeled cells
corresponded to class 4. This changed dramatically in the subsequent
2 d. By day 15 most of the labeled cells had developed many
spines, and by day 22 the majority of the labeled cells corresponded to
class 5. This suggests that the majority of synaptic contacts on
spines of the newly formed neurons developed between days 13 and 22. By
day 30 all of the labeled cells had developed branched apical
dendrites, and most of them had many spines. This indicates that within
a week the cells moved from the core of the OB to the superficial layer
and developed their mature morphology. No cells with other morphologies
besides those described above were observed in the RMS and
the GCL.
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Figure 3.
Time course of the development of adult-generated
olfactory bulb granule neurons. Percentage of alkaline
phosphatase-labeled cells in the GCL of the olfactory bulb belonging to
each of the developing stages defined in Results (Fig. 1) at
different survival times. Top diagram indicates an
example of each morphological class. EPL, External
plexiform layer; MCL, mitral cell layer;
GCL, granule cell layer; RMS, rostral
migratory stream.
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Population dynamics of the incorporation of adult generated
granule neurons
Although the viral injections allowed us to determine the
morphology of the newly formed cells in the OB, it did not allow for a
quantitative estimation of the absolute number of new neurons recruited
into the bulb. To determine total new granule cells incorporated into
the adult olfactory bulb, mice were injected with
[3H]thymidine, and labeled cells in the
GCL were quantified at different survival times. As seen in Figure
4B, the number of
labeled cells in the granule cell layer increased nonsignificantly from
days 10 to 15 after injection (Newman-Keuls test; p > 0.25) and declined thereafter. Labeled cells were seen up to 1 year
later, showing that some newly generated cells can live at least this
long in the OB. Interestingly, however, between days 15 and 45 after
injection there was a sharp decline in the number of newly formed
neurons observed in the granule cell layer, with the number of labeled cells reduced by one-half (Newman-Keuls test; p < 0.001). After 45 d, the number of labeled cells stabilized (Fig.
4B), and their number did not differ significantly
during the following 3 months (Newman-Keuls test; p > 0.05)). After 1 year, one-third of these surviving cells were still
found in the GCL. These results identify an early wave of cell death
soon after newly generated cells mature and develop synaptic spines.
The more gradual decline that occurs 3 months after cell birth might be
related to the rate of neuronal replacement in the GC layer. Very few
cells were seen in the RMS after 15 d (data no shown). It is
therefore unlikely that the arrival of new cohorts of labeled cells
from secondary stem cell division contributes to the plateau of cell
survival. This is consistent with evidence that very few long-term
renewing stem cells get labeled in the SVZ after proliferation marker
injections (Morshead et al., 1994; Doetsch et al., 1997). As seen in
Table 1, double labeling of the newly
generated cells with the neuronal marker NeuN showed that at least 84%
of the new cells become neurons. The remaining 16% could correspond to
astrocytes or microglia, or neurons that express NeuN at levels below
the sensitivity of the method. The four injections of
[3H]thymidine given to the animals
resulted in 2.2% labeled NeuN-positive granule neurons at 15 d
and 1.5% at 45 d (Table 1).
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Figure 4.
Labeled cells in the GCL of the olfactory bulb at
different survival points after [3H]thymidine
injection. A, Photomicrograph of a
[3H]thymidine-positive, NeuN-positive cell in the
GCL. Scale bar, 20 µm. B, The number of
[3H]thymidine-labeled cells in the GCL at
different survival times. C, The volume of the GCL at
different survival times. Values indicate mean ± SE.
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Granule cell incorporation and survival in anosmic mice
The above analysis indicated that between 15 and 22 d after
birth, newly generated granule cells show a mature morphology. These
results suggest that during the early wave of cell death, granule
neurons already receive connections from the M and T cells. Activity
through M and T cells may regulate the survival of newly incorporated
neurons during this period. To test whether olfactory input has a role
in the regulation of granule cell death, we analyzed the dynamics of
granule cell incorporation in adult anosmic mice. Animals that have a
mutation in the olfactory cyclic nucleotide gated channel are not able
to transduce the signal from olfactory receptors in olfactory receptor
neurons. These animals lack all electrical activity in the olfactory
epithelium and do not have electrical input into the olfactory bulb
(Brunet et al., 1996; Zhao and Reed, 2001). These mice have small
olfactory bulbs (Baker et al., 1999), suggesting that granule cells
might be dying. In coronal sections of the OB, the area occupied by the
rostral end of the RMS in the core of the OB was larger than that seen
in wild-type mice (Fig.
5A,B).
However, because the OB is much smaller in the mutant mice, the volume
of the RMS measured from the frontal tip of the lateral ventricles to
the rostral end of the OB did not differ significantly (Student's
t test; p > 0.2) in the two groups
(anosmic: 0.035 ± 0.011 mm3;
control: 0.044 ± 0.007 mm3). These
results indicate that despite the lack of activity and the smaller GCL,
the RMS is not significantly affected, suggesting that many new neurons
migrate along the RMS and arrive in the core of the OB of the mutant
mice.
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Figure 5.
Morphology and cell death in the olfactory
bulbs of anosmic mice. Photomicrographs of a Nissl-stained coronal
section of the olfactory bulb of an anosmic mouse
(A) or a control littermate
(B). Dotted lines outline the
contour of the RMS. Scale bars, 0.5 mm. C, Volume of the
GCL in anosmic (n = 3) and control mice
(n = 4). Representative photomicrographs show
TUNEL-stained apoptotic cells in the GCL of anosmic
(D) and control mice (E).
Scale bars, 50 µm. E, Quantification of TUNEL staining
in the GCL of anosmic (KO) (n = 3)
and control mice (wt) (n = 4).
Values indicate mean ± SE. Statistical analysis was conducted by
unpaired Student's t test. Significance was established
at **p < 0.01.
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Using TUNEL staining, we detected significantly higher levels of
apoptotic cell death in the granule cell layer in the anosmic mice
compared with littermate controls (Fig.
5D-F). To test whether granule cells in
anosmic animals were dying before or after they developed their mature
morphology, animals were injected with BrdU at day 0 and with
[3H]thymidine at day 30 and were
perfused at day 45. BrdU immunostaining and
[3H]thymidine autoradiography
revealed granule cells that were 45 and 15 d old, respectively.
The number of 15-d-old cells in the granule cell layer was slightly
decreased in mutant mice but not significantly different when compared
with control mice (Fig. 6D). This resulted in a
twofold higher density of 15-d-old
[3H]thymidine-labeled cells in the GCL
(data not shown) of the mutant mice compared with controls because the
volume of this layer is reduced by half in the mutants. However, the
total number of 45-day-old granule cells was reduced four times in
mutant mice compared with controls (Fig. 6D), showing
that most of the granule cells of the anosmic animals died between 15 and 45 d after birth.
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Figure 6.
Morphology and cell survival of newly generated
cells in the olfactory bulb of anosmic mice. A,
Photomicrograph of a class 4 cell in the GCL of an anosmic mouse.
Several focal planes were combined in Photoshop to reconstruct the
cell. Scale bar, 50 µm. B, Photomicrograph of the
dendritic tree of a class 5 cell in the EPL of an anosmic mouse. Scale
bar, 20 µm. C, The percentage of cells belonging to
each of the five morphological classes in anosmic (KO)
and control littermates (wt). D, Total number of
[3H]thymidine- and BrdU-labeled cells in the GCL
in control (n = 4) or anosmic mice
(n = 3). Statistical analysis was conducted by
unpaired Student's t test. Significance was established
at *p < 0.05. Time line represents
the injection protocol for the experiment. Arrows
indicate injection of either BrdU or
[3H]thymidine. X indicates time of
perfusion. Values indicate mean ± SE.
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To determine whether cell division during migration (Lois and
Alvarez-Buylla, 1994; Menezes et al., 1995) was affected by incoming
activity, we counted the number of silver grains in the [3H]thymidine-labeled cells from both
groups. We found no significant difference (Student's t
test; p > 0.5) between the anosmic (mean 21.52 ± 14.32 grains per labeled cells) and the control littermates (22.4 ± 15 grains per labeled cells). It was possible that the new granule
cells in the anosmic animals failed to mature normally and that this
resulted in lower survival. Retroviral injections in mutant mice
revealed that as with control mice, most of the 15-d-old granule cells
in the anosmic mice had dendritic arborization beyond the EPL (classes
4 and 5) (Fig. 6A-C), and many of these cells had dendritic spines (Fig. 6B). We conclude
that in the anosmic mice, granule cells are produced, migrate, and
mature in a manner very similar to those of the wild-type mice.
However, after this initial period of maturation, many more granule
cells die when olfactory activity is not present.
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DISCUSSION |
By using retroviral labeling methods, we were able to describe and
time the development and maturation of adult-generated GCs. Most of the
adult-generated GCs attain a fully mature morphology between 15 and
30 d after birth in the SVZ. The morphology of all the cells in
the GCL that originated in the SVZ, as determined by the viral
injections, corresponded to that of granule neurons. This suggests that
cells born in the SVZ that end up in the GCL become only granule
neurons. The small proportion (16%) of NeuN-negative cells in the
[3H]thymidine experiment (Table 1) are
either glia that are generated locally or neurons that express NeuN at
levels below the sensitivity of the technique used here. The sequence
of stages for the development of GCs that we describe here is similar
to those observed in earlier postnatal animals using Golgi staining
(Kishi, 1987), suggesting that developing granule cells follow a
similar maturation process in juvenile and adult animals. In agreement
with previous studies in the adult mice (Bayer, 1983; Lois and
Alvarez-Buylla, 1994), we also observed a small proportion of
periglomerular neurons being formed. Given the proportionally smaller
number of periglomerular cells that formed, we did not study this
population in detail here.
Fifteen days after four daily injections of
[3H]thymidine, 2.2% of the
NeuN-positive GCs were found labeled. By 45 d after [3H]thymidine injection, 1.5% were
found labeled. We estimate that 15 d after 1 month of
neurogenesis, ~14% of the cells in the GCL will be new. This is
probably an underestimation because it assumes that with each daily
injection all of the neurons born that day were labeled. This is
unlikely given that the estimated cell cycle in the SVZ is likely <24
hr (Morshead and Van der Kooy, 1992). Such a rate of neuronal
replacement might allow for considerable circuit plasticity in the OB,
potentially changing the way it processes olfactory information over a
relatively short period of time.
We found a sharp decrease in the number of
[3H]thymidine-labeled granule cells
between 15 and 45 d after
[3H]thymidine injection, during which
approximately one-half of the newly generated cells are lost. After
this time point, the number of labeled cells remained constant for
several months. The reduction in
[3H]thymidine-labeled cells is probably
caused by cell death and not migration to the glomerular layer, because
we have shown with retroviral labeling that during this time window
labeled cells have a postmigratory morphology. Because the volume of
the GCL (Fig. 4B) and the GC density (data not shown)
were found not to change significantly (ANOVA; F = 0.76; df = 8, 30; p > 0.6) during the period
studied, the reduction in
[3H]thymidine-labeled cells cannot be
caused by a decrease in the density of labeled cells as a result of an
increase in the total number of cells in the GCL. Interestingly, a
similar profile of cell death is observed for the adult-generated
neurons of the high vocal center of canaries (Kirn et al., 1999), where
one-half of the labeled cells died shortly after their arrival into the nucleus.
Cell death seems to be a prominent factor regulating adult neuronal
incorporation, perhaps a mechanism similar to that observed during
earlier development (Linden, 1994; Katz and Shatz, 1996; Voyvodic,
1996).
Cell death in the embryo is generally thought to function as a
mechanism to adjust the size of a neuronal pool to that of its input
and target (Oppenheim, 1991). During development, the size of the pool
of cells providing the input or serving as the target changes with
growth. In the adult olfactory bulb it is relatively stable, because
the number of M and T cells does not change significantly with time
(Hinds and McNelly, 1977). Cell death in the adult may be closely
linked to activity and to the functional contribution of specific
neurons. The retroviral labeling experiment showed that during this
early wave of cell death, between days 15 and 45 after neuronal birth,
most of the dying cells had already matured and developed dendritic
arborizations. This suggests that before undergoing apoptosis, these
new cells became connected to M and T cells. It will be important to
compare the specific functional characteristics of cells that become
connected in the OB environment but will not survive versus those that
will survive for the following 90 d. Our work provides the time
window to study the selection process of newly incorporated neurons.
Using anosmic mice, we have shown that during the period of fast cell
death, survival is dependent on incoming activity. We have seen that
the lack of electrical activity in mutant mice dramatically reduced the
survival of the newly generated neurons during this period.
Interestingly, the number of labeled cells in the GCL at 15 d
after injection is not significantly reduced in anosmic compared with
wild-type mice. Their overall morphology, 15 d after injection,
was also not affected in anosmic mice, suggesting that olfactory
activity is not important for the early differentiation of newly
generated GCs. In agreement with the present observations, naris
closure experiments in postnatal animals have shown that neither GC
morphology (Frazier-Cierpial and Brunjes, 1989b) nor cell proliferation
(Frazier-Cierpial and Brunjes, 1989a; Cummings et al., 1997) is
affected by sensory deprivation. Our results showed that the lack of
activity dramatically reduces survival after the cells have become
lodged in the GCL, have grown dendritic arborizations covered by
spines, and probably have made connections. These results suggest that
olfaction plays a critical role only in the survival of cells once they
have attained a mature morphology and not in the production, migration,
or initial recruitment. As during development, neuronal incorporation
and synapse formation might be mediated by an activity-independent
mechanism (Ziv and Garner, 2001) that is regulated by endogenous
programs within the young neurons. This may be followed by an
activity-dependent mechanism in which many cells are eliminated by
apoptosis (Verhage et al., 2000).
The fact that the newly generated granule cells are dependent on
afferent activity for their survival once they have developed their
dendritic arborization and are likely to be connected has interesting
consequences for the processing of olfactory information in the
olfactory bulb circuitry. GCs may be selected for survival in an
activity-dependent manner according to the specific connection that
they make with M and T cells. The ones that connect active M and T
cells may be selected to survive and those that connect to inactive
cells may be lost. Previous modeling experiments have shown that such
an activity-dependent mechanism could potentially redistribute the
representation of odorants in the mitral cell layer to maximize the
differences between similar odors and therefore increase olfactory
discrimination (Cecchi et al., 2001). Granule cells are known to
extensively shape mitral cell response to odors (Yokoi et al., 1995),
and there is evidence that the OB circuitry does maximize differences
in odor representations (Friedrich and Laurent, 2001). The
representation of odorants in the olfactory bulb can thus be
renormalized and kept optimal for a changing olfactory environment
using such an activity-dependent mechanism. The observation that a
reduction in cell migration to the OB hampers olfactory discrimination
is consistent with this hypothesis (Gheusi et al., 2000). It is likely
that a similar mechanism may be operating since birth. Ninety percent
of the granule cells are incorporated postnatally, and their survival
is also dependent on incoming activity (Rosselli-Austin and Williams,
1990; Brunjes, 1994). The development of olfactory discrimination and
the maintenance of this sensory modality in the adult may depend
critically on the selective survival of new neurons on the basis of
their level of activity.
It remains a mystery why the olfactory bulb might need cell replacement
as a mechanism of plasticity, which in principle seems much more
drastic and costly than mechanisms of synaptic modification (Hebb,
1949; Kandel, 1997). This might be related to the unique characteristics of the olfactory system. The OB is the first processing stage of olfactory information, similar to the retina for the visual
system (DeVries and Baylor, 1993; Nakanishi, 1995). In the retina, a
given photoreceptor activates inhibitory circuits that influence cells
around it, producing the center-surround properties of the ganglion
cell receptive fields. Unlike vision, olfaction has intrinsic high
dimensionality (Hopfield, 1991). As a consequence, in the OB, a given M
and T neuron needs an inhibitory network that influences other M and T
cells, not necessarily physically around them but in the more complex
olfactory space (Galizia and Menzel, 2000; Sachse and Galizia, 2002).
That usually means that groups of M and T cells far away from each
other may need to be linked by inhibitory connections. Coactivation of
glomeruli that respond to a single odor cannot be predicted on the
basis of the physical position of one with respect to the other (Kauer
and White, 2001). Thus, unlike other sensory systems, it might not be
feasible to preassemble a unique inhibitory circuit that processes olfactory information optimally for all the possible odors that an
animal might experience during development and in adult life. Therefore, it might be necessary to build the inhibitory circuit of the
OB according to experience by the activity-dependent survival mechanism
described here.
By building the inhibitory circuit postnatally, an optimal OB can be
assembled for the initial olfactory environment of the animal. Because
this circuitry is custom built for a given olfactory experience, it
might become suboptimal as the odor environment changes. The
continuation of interneuron generation and replacement in adult life
could reflect the necessity to readapt the OB to ongoing environmental
changes to maintain maximal discrimination.
| |
FOOTNOTES |
Received Nov. 28, 2001; revised April 15, 2002; accepted April 19, 2002.
This work was supported by National Institutes of Health Grant HD32116.
We thank B. Haripal for technical assistance, D. Vicario for advice on
statistical analysis, F. Doetsch, B. Alvarez-Borda, and L. Wilbrecht
for helpful comments on this manuscript, A. Newman for assistance in
the preparation of this manuscript, and F. Nottebohm for inspiring
discussions and support.
Correspondence should be addressed to Arturo Alvarez-Buylla,
Neurosurgery Research, Box 0520, 10 Kirkham Street K-27, University of
California, San Francisco, San Francisco, CA 94134. E-mail: abuylla{at}itsa.ucsf.edu.
| |
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W. R. Kim, Y. Kim, B. Eun, O.-h. Park, H. Kim, K. Kim, C.-H. Park, S. Vinsant, R. W. Oppenheim, and W. Sun
Impaired Migration in the Rostral Migratory Stream But Spared Olfactory Function after the Elimination of Programmed Cell Death in Bax Knock-Out Mice
J. Neurosci.,
December 26, 2007;
27(52):
14392 - 14403.
[Abstract]
[Full Text]
[PDF]
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H.T. Ghashghaei, J. M. Weimer, R. S. Schmid, Y. Yokota, K. D. McCarthy, B. Popko, and E.S. Anton
Reinduction of ErbB2 in astrocytes promotes radial glial progenitor identity in adult cerebral cortex
Genes & Dev.,
December 15, 2007;
21(24):
3258 - 3271.
[Abstract]
[Full Text]
[PDF]
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J. Ninkovic, T. Mori, and M. Gotz
Distinct Modes of Neuron Addition in Adult Mouse Neurogenesis
J. Neurosci.,
October 3, 2007;
27(40):
10906 - 10911.
[Abstract]
[Full Text]
[PDF]
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M. C. Whitman and C. A. Greer
Synaptic Integration of Adult-Generated Olfactory Bulb Granule Cells: Basal Axodendritic Centrifugal Input Precedes Apical Dendrodendritic Local Circuits
J. Neurosci.,
September 12, 2007;
27(37):
9951 - 9961.
[Abstract]
[Full Text]
[PDF]
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M. Kohwi, M. A. Petryniak, J. E. Long, M. Ekker, K. Obata, Y. Yanagawa, J. L. R. Rubenstein, and A. Alvarez-Buylla
A Subpopulation of Olfactory Bulb GABAergic Interneurons Is Derived from Emx1- and Dlx5/6-Expressing Progenitors
J. Neurosci.,
June 27, 2007;
27(26):
6878 - 6891.
[Abstract]
[Full Text]
[PDF]
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I. Fishbein and M. Segal
Miniature Synaptic Currents Become Neurotoxic to Chronically Silenced Neurons
Cereb Cortex,
June 1, 2007;
17(6):
1292 - 1306.
[Abstract]
[Full Text]
[PDF]
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R. Balu, R. T. Pressler, and B. W. Strowbridge
Multiple Modes of Synaptic Excitation of Olfactory Bulb Granule Cells
J. Neurosci.,
May 23, 2007;
27(21):
5621 - 5632.
[Abstract]
[Full Text]
[PDF]
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N. Andrade, V. Komnenovic, S. M. Blake, Y. Jossin, B. Howell, A. Goffinet, W. J. Schneider, and J. Nimpf
ApoER2/VLDL receptor and Dab1 in the rostral migratory stream function in postnatal neuronal migration independently of Reelin
PNAS,
May 15, 2007;
104(20):
8508 - 8513.
[Abstract]
[Full Text]
[PDF]
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G. Gheusi and P.-M. Lledo
Control of Early Events in Olfactory Processing by Adult Neurogenesis
Chem Senses,
May 1, 2007;
32(4):
397 - 409.
[Abstract]
[Full Text]
[PDF]
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S. De Marchis, S. Bovetti, B. Carletti, Y.-C. Hsieh, D. Garzotto, P. Peretto, A. Fasolo, A. C. Puche, and F. Rossi
Generation of Distinct Types of Periglomerular Olfactory Bulb Interneurons during Development and in Adult Mice: Implication for Intrinsic Properties of the Subventricular Zone Progenitor Population
J. Neurosci.,
January 17, 2007;
27(3):
657 - 664.
[Abstract]
[Full Text]
[PDF]
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M. Alonso, C. Viollet, M.-M. Gabellec, V. Meas-Yedid, J.-C. Olivo-Marin, and P.-M. Lledo
Olfactory Discrimination Learning Increases the Survival of Adult-Born Neurons in the Olfactory Bulb
J. Neurosci.,
October 11, 2006;
26(41):
10508 - 10513.
[Abstract]
[Full Text]
[PDF]
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N. Kaneko, H. Okano, and K. Sawamoto
Role of the cholinergic system in regulating survival of newborn neurons in the adult mouse dentate gyrus and olfactory bulb.
Genes Cells,
October 1, 2006;
11(10):
1145 - 1159.
[Abstract]
[Full Text]
[PDF]
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U Shivraj Sohur, J. G Emsley, B. D Mitchell, and J. D Macklis
Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells
Phil Trans R Soc B,
September 29, 2006;
361(1473):
1477 - 1497.
[Abstract]
[Full Text]
[PDF]
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A. Mizrahi, J. Lu, R. Irving, G. Feng, and L. C. Katz
In vivo imaging of juxtaglomerular neuron turnover in the mouse olfactory bulb
PNAS,
February 7, 2006;
103(6):
1912 - 1917.
[Abstract]
[Full Text]
[PDF]
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F. Luzzati, S. De Marchis, A. Fasolo, and P. Peretto
Neurogenesis in the Caudate Nucleus of the Adult Rabbit
J. Neurosci.,
January 11, 2006;
26(2):
609 - 621.
[Abstract]
[Full Text]
[PDF]
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S. S. P. Magavi, B. D. Mitchell, O. Szentirmai, B. S. Carter, and J. D. Macklis
Adult-Born and Preexisting Olfactory Granule Neurons Undergo Distinct Experience-Dependent Modifications of their Olfactory Responses In Vivo
J. Neurosci.,
November 16, 2005;
25(46):
10729 - 10739.
[Abstract]
[Full Text]
[PDF]
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C. Giachino, S. De Marchis, C. Giampietro, R. Parlato, I. Perroteau, G. Schutz, A. Fasolo, and P. Peretto
cAMP Response Element-Binding Protein Regulates Differentiation and Survival of Newborn Neurons in the Olfactory Bulb
J. Neurosci.,
November 2, 2005;
25(44):
10105 - 10118.
[Abstract]
[Full Text]
[PDF]
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M. Kohwi, N. Osumi, J. L. R. Rubenstein, and A. Alvarez-Buylla
Pax6 Is Required for Making Specific Subpopulations of Granule and Periglomerular Neurons in the Olfactory Bulb
J. Neurosci.,
July 27, 2005;
25(30):
6997 - 7003.
[Abstract]
[Full Text]
[PDF]
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M. Lemasson, A. Saghatelyan, J.-C. Olivo-Marin, and P.-M. Lledo
Neonatal and Adult Neurogenesis Provide Two Distinct Populations of Newborn Neurons to the Mouse Olfactory Bulb
J. Neurosci.,
July 20, 2005;
25(29):
6816 - 6825.
[Abstract]
[Full Text]
[PDF]
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M. Yamaguchi and K. Mori
Critical period for sensory experience-dependent survival of newly generated granule cells in the adult mouse olfactory bulb
PNAS,
July 5, 2005;
102(27):
9697 - 9702.
[Abstract]
[Full Text]
[PDF]
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T.-W. Wang, H. Zhang, and J. M. Parent
Retinoic acid regulates postnatal neurogenesis in the murine subventricular zone-olfactory bulb pathway
Development,
June 15, 2005;
132(12):
2721 - 2732.
[Abstract]
[Full Text]
[PDF]
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N. Miwa and D. R. Storm
Odorant-Induced Activation of Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase in the Olfactory Bulb Promotes Survival of Newly Formed Granule Cells
J. Neurosci.,
June 1, 2005;
25(22):
5404 - 5412.
[Abstract]
[Full Text]
[PDF]
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A. J. Lombardino, X.-C. Li, M. Hertel, and F. Nottebohm
Replaceable neurons and neurodegenerative disease share depressed UCHL1 levels
PNAS,
May 31, 2005;
102(22):
8036 - 8041.
[Abstract]
[Full Text]
[PDF]
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V. Egger, K. Svoboda, and Z. F. Mainen
Dendrodendritic Synaptic Signals in Olfactory Bulb Granule Cells: Local Spine Boost and Global Low-Threshold Spike
J. Neurosci.,
April 6, 2005;
25(14):
3521 - 3530.
[Abstract]
[Full Text]
[PDF]
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D. N. Abrous, M. Koehl, and M. Le Moal
Adult Neurogenesis: From Precursors to Network and Physiology
Physiol Rev,
April 1, 2005;
85(2):
523 - 569.
[Abstract]
[Full Text]
[PDF]
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P.-M. Lledo, G. Gheusi, and J.-D. Vincent
Information Processing in the Mammalian Olfactory System
Physiol Rev,
January 1, 2005;
85(1):
281 - 317.
[Abstract]
[Full Text]
[PDF]
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H. G. Kuhn, C. Cooper-Kuhn, P. Eriksson, and M. Nilsson
Signals Regulating Neurogenesis in the Adult Olfactory Bulb
Chem Senses,
January 1, 2005;
30(suppl_1):
i109 - i110.
[Full Text]
[PDF]
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M. Yamaguchi
Analysis of neurogenesis using transgenic mice expressing GFP with nestin gene regulatory regions
Chem Senses,
January 1, 2005;
30(suppl_1):
i117 - i118.
[Full Text]
[PDF]
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A. Pignatelli, M. Benedusi, J. Ackman, J. J. LoTurco, and O. Belluzzi
Functional Properties of Adult-born Juxtaglomerular Cells in the Mammalian Olfactory Bulb
Chem Senses,
January 1, 2005;
30(suppl_1):
i119 - i120.
[Full Text]
[PDF]
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W. Sun, A. Winseck, S. Vinsant, O.-h. Park, H. Kim, and R. W. Oppenheim
Programmed Cell Death of Adult-Generated Hippocampal Neurons Is Mediated by the Proapoptotic Gene Bax
J. Neurosci.,
December 8, 2004;
24(49):
11205 - 11213.
[Abstract]
[Full Text]
[PDF]
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J. Chen, S. S. P. Magavi, and J. D. Macklis
Neurogenesis of corticospinal motor neurons extending spinal projections in adult mice
PNAS,
November 16, 2004;
101(46):
16357 - 16362.
[Abstract]
[Full Text]
[PDF]
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A. Consiglio, A. Gritti, D. Dolcetta, A. Follenzi, C. Bordignon, F. H. Gage, A. L. Vescovi, and L. Naldini
From the Cover: Robust in vivo gene transfer into adult mammalian neural stem cells by lentiviral vectors
PNAS,
October 12, 2004;
101(41):
14835 - 14840.
[Abstract]
[Full Text]
[PDF]
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A. Nicot, T. Otto, P. Brabet, and E. M. DiCicco-Bloom
Altered Social Behavior in Pituitary Adenylate Cyclase-Activating Polypeptide Type I Receptor-Deficient Mice
J. Neurosci.,
October 6, 2004;
24(40):
8786 - 8795.
[Abstract]
[Full Text]
[PDF]
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A. F. Schinder and F. H. Gage
A Hypothesis About the Role of Adult Neurogenesis in Hippocampal Function
Physiology,
October 1, 2004;
19(5):
253 - 261.
[Abstract]
[Full Text]
[PDF]
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E. Enwere, T. Shingo, C. Gregg, H. Fujikawa, S. Ohta, and S. Weiss
Aging Results in Reduced Epidermal Growth Factor Receptor Signaling, Diminished Olfactory Neurogenesis, and Deficits in Fine Olfactory Discrimination
J. Neurosci.,
September 22, 2004;
24(38):
8354 - 8365.
[Abstract]
[Full Text]
[PDF]
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P.-M. Lledo, A. Saghatelyan, and M. Lemasson
Inhibitory Interneurons in the Olfactory Bulb: From Development to Function
Neuroscientist,
August 1, 2004;
10(4):
292 - 303.
[Abstract]
[PDF]
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N. Mechawar, A. Saghatelyan, R. Grailhe, L. Scoriels, G. Gheusi, M.-M. Gabellec, P.-M. Lledo, and J.-P. Changeux
Nicotinic receptors regulate the survival of newborn neurons in the adult olfactory bulb
PNAS,
June 29, 2004;
101(26):
9822 - 9826.
[Abstract]
[Full Text]
[PDF]
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B. Alvarez-Borda, B. Haripal, and F. Nottebohm
Timing of brain-derived neurotrophic factor exposure affects life expectancy of new neurons
PNAS,
March 16, 2004;
101(11):
3957 - 3961.
[Abstract]
[Full Text]
[PDF]
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K. T. Nguyen-Ba-Charvet, N. Picard-Riera, M. Tessier-Lavigne, A. Baron-Van Evercooren, C. Sotelo, and A. Chedotal
Multiple Roles for Slits in the Control of Cell Migration in the Rostral Migratory Stream
J. Neurosci.,
February 11, 2004;
24(6):
1497 - 1506.
[Abstract]
[Full Text]
[PDF]
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O. Belluzzi, M. Benedusi, J. Ackman, and J. J. LoTurco
Electrophysiological Differentiation of New Neurons in the Olfactory Bulb
J. Neurosci.,
November 12, 2003;
23(32):
10411 - 10418.
[Abstract]
[Full Text]
[PDF]
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D. Ehninger and G. Kempermann
Regional Effects of Wheel Running and Environmental Enrichment on Cell Genesis and Microglia Proliferation in the Adult Murine Neocortex
Cereb Cortex,
August 1, 2003;
13(8):
845 - 851.
[Abstract]
[Full Text]
[PDF]
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R. C. Murray, D. Navi, J. Fesenko, A. D. Lander, and A. L. Calof
Widespread Defects in the Primary Olfactory Pathway Caused by Loss of Mash1 Function
J. Neurosci.,
March 1, 2003;
23(5):
1769 - 1780.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION
