Neurogenesis of corticospinal motor neurons
extending spinal projections in adult mice
Jinhui Chen*, Sanjay S. P. Magavi, and Jeffrey D. Macklis†
Departments of Neurosurgery and Neurology and Program in Neuroscience, Massachusetts General Hospital–Harvard Medical School Center for Nervous
System Repair, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114
Communicated by Richard L. Sidman, Harvard Medical School, Boston, MA, September 15, 2004 (received for review March 17, 2004)
The adult mammalian CNS shows a very limited capacity to regen-
erate after injury. However, endogenous precursors, or stem cells,
provide a potential source of new neurons in the adult brain. Here,
we induce the birth of new corticospinal motor neurons (CSMN),
the CNS neurons that die in motor neuron degenerative diseases,
including amyotrophic lateral sclerosis, and that cause loss of
motor function in spinal cord injury. We induced synchronous
apoptotic degeneration of CSMN and examined the fates of new-
born cells arising from endogenous precursors, using markers for
DNA replication, neuroblast migration, and progressive neuronal
differentiation, combined with retrograde labeling from the spinal
cord. We observed neuroblasts entering the neocortex and pro-
layer V. We found 20–30 new neurons per mm3in experimental
axons into the spinal cord and survived >56 weeks. These results
demonstrate that endogenous precursors can differentiate into
even highly complex long-projection CSMN in the adult mamma-
lian brain and send new projections to spinal cord targets, sug-
gesting that molecular manipulation of endogenous neural pre-
cursors in situ may offer future therapeutic possibilities for motor
neuron degenerative disease and spinal cord injury.
future repair of neuronal loss from neurodegenerative diseases or
trauma or stroke (1–5). In the adult brain, new neurons are
continuously generated, but such neurogenesis is normally re-
stricted to two evolutionarily primitive regions: the olfactory bulb,
from precursors in the subventricular zone (SVZ) (6), and the
hippocampal dentate gyrus (1, 7), from local precursors in the
A variety of factors, ranging from genetics (8) to environmental
(9), environmental enrichment (10), pregnancy (11), and even
seizure activity (12, 13) promote neurogenesis, whereas depression
factors that control adult neurogenesis could yield new approaches
to replacing neurons lost to injury or disease.
Recently, our laboratory and those of others have demon-
strated that neurogenesis can be induced from endogenous
precursors by manipulating the microenvironment in regions of
the adult CNS that are normally nonneurogenic (16–21). Selec-
tive neuronal death due to targeted apoptosis or ischemia
induces endogenous precursors to divide and differentiate into
neurons in mammalian neocortical layer VI (16), hippocampal
region CA1 (17), striatum (18, 19), substantia nigra (20), and the
high vocal center in songbirds (21). These neurons express
neuron-specific proteins and adopt appropriate morphologies,
and in some cases their recruitment correlates with restoration
of function (17, 21). However, questions with significant clinical
implications remain: Can neurogenesis be induced in regions of
the brain further from the precursor-rich SVZ? Is it possible to
induce the birth of very complex corticospinal motor neurons
(CSMN) from endogenous precursors in adults, and can they
he identification of populations of neural precursors, or stem
cells, in the adult mammalian CNS raises the possibility of
reform and maintain extremely long-distance corticospinal projec-
tions affected by spinal cord injury and CSMN degenerations?
in the adult mouse cortex were targeted for apoptotic cell death via
chromophore photoactivation. Then, mice were administered Br-
dUrd for the 2 weeks after the induction of apoptosis, and the fate
survival times by using morphological and immunocytochemical
markers of progressive neuronal and glial differentiation. We
examined projections formed by newborn neurons using the ret-
15, and 55 weeks, and examined 1 week later.
Targeted Neuronal Degeneration. Details of chlorin e6injection and
23). In summary, a suspension of chlorin e6-conjugated fluorescent
nanospheres was injected bilaterally into the dorsal spinal cord at
the cervical C5–C6 level of 4-week-old female mice, from which
they were retrogradely transported to the somata of layer V CSMN
in the motor cortex, where they remain inert until photoactivated.
The nanospheres fluoresce in the same range as FITC, allowing
unambiguous identification of targeted neurons (Fig. 1 E–I). Two
weeks later, the motor cortex was exposed to 674-nm long-
wavelength light through intact dura, photoactivating the chlorin e6
to produce singlet oxygen and induce synchronous apoptosis (24)
exclusively in nanosphere-containing motor neurons in the light-
exposed region. Surrounding glia and unlabeled neurons remain
intact. Degeneration of ?10–20% of targeted projection neurons
occurred, with neuronal degeneration confirmed in previous stud-
ies by loss of retrogradely labeled neurons compared with adjacent
control regions and by the presence of terminal deoxynucleotidyl-
transferase-mediated dUTP nick end labeling-positive nuclei in
projection neurons, confirming apoptotic death.
BrdUrd Labeling of Dividing Cells.Wegaveexperimentalandcontrol
mice drinking water containing BrdUrd (2.5 mg?ml) for 2 weeks
after induction of apoptosis (approximate dose, 180 mg?kg per
day). BrdUrd (Sigma) incorporates into dividing cells during S
Immunocytochemistry. Four percent paraformaldehyde-fixed brain
sections were washed in PBS and incubated with 2 M HCl for 2 h
at room temperature. Then, the sections were blocked in PBS
containing 4% goat serum, 0.3% BSA, and 0.3% Triton X-100 and
incubated with primary antibodies overnight and with secondary
antibodies for 2 h in blocking solution. We incubated the sections
Abbreviations: CSMN, corticospinal motor neuron(s); SVZ, subventricular zone; FG, Fluoro-
Gold; Dcx, Doublecortin; GFAP, glial fibrillary acidic protein.
Research Center, University of Kentucky, Lexington, KY 40506.
© 2004 by The National Academy of Sciences of the USA
November 16, 2004 ?
vol. 101 ?
no. 46 ?
in rat anti-BrdUrd (1:100, Harlan Breeders, Indianapolis) and the
following primary antibodies: rabbit anti-Doublecortin (anti-Dcx)
[1:100, courtesy of J. G. Gleeson (University of California, San
Diego) and C. A. Walsh (Harvard Institutes of Medicine, Boston];
mouse anti-NeuN antibody (1:100, Chemicon); rabbit antiglial
fibrillary acidic protein (anti-GFAP) (1:100, Sigma). We used
(Molecular Probes) secondary antibodies to avoid crossreactivity.
Microscopy. We performed fluorescence microscopy using a Zeiss
Axioplan microscope with high-numerical-aperture immersion ob-
jective lenses. We performed confocal microscopy using Noran
confocal microscopes and INTERVISION (Noran Instruments,
Middleton, WI) and ZEISS 3D analysis software. We produced 3D
digital reconstructions from a series of confocal images taken at
0.5-?m intervals through the regions of interest.
Quantification. We quantified the number of BrdUrd??NeuN?
neurons in experimental and control regions by sampling every
sixth section, using a modified version of the fractionator method
(16, 25). We quantified the number of BrdUrd??FG? neurons in
experimental and control regions by examining all of the sections
spanning the motor cortex, 40–50 sections per mouse. To avoid
false identification of nuclei of closely apposed newborn glial cells
reconstructions to modified stereological methods (see ref. 16 and
Fig. 5, which is published as supporting information on the PNAS
web site). We confirmed the identity of adult-born neurons using
confocal microscopy and omitted all cells that had been sectioned
to eliminate false positives. These conservative methods likely lead
to an underestimate of the true number of neurons generated. The
experimental regions of the cortex extended from the dorsal cortex
to the medial bank and spanned the thickness of cortical layer V.
We compared numbers of new neurons in experimental mice to
control mice using the unpaired t test with Welch correction, with
P values ?0.001 interpreted as demonstrating a significant differ-
ence between groups.
FG Injections. We microinjected 200 nl of 3% FG solution dissolved
level at 7, 11, 15, and 55 weeks after induction of targeted CSMN
apoptosis. We examined projections formed by newborn neurons 1
week after FG injection.
To examine whether adult endogenous neural precursors can
differentiate into new neurons that extend long-distance projec-
tions to the spinal cord, we induced targeted apoptosis of CSMN
and examined the progressive differentiation of endogenous pre-
cursors. The adult neocortex undergoing targeted apoptosis reex-
presses developmental signals that direct transplanted or endoge-
nous precursors to differentiate into neurons (16). We induced
synchronous apoptotic degeneration of CSMN in 6-week-old mice
via chromophore targeting (Fig. 1) (22, 23, 26, 27). Approximately
10–20% of CSMN underwent apoptosis after successful chro-
to the mice for 2 weeks in their drinking water immediately after
initiation of neuronal apoptosis and examined the phenotype of
newly generated BrdUrd? cells 2, 4, 8, 12, 16, and 56 weeks after
induction of apoptotic degeneration. We examined the differenti-
ation of these cells morphologically and by using markers of
GFAP. We also retrogradely labeled CSMN from the cervical
spinal cord with FG to investigate whether newborn cortical
neurons can project long-distance axons to the spinal cord in the
We first examined the early differentiation of adult-born cells
using antibodies to Dcx, a protein expressed exclusively in neurons
(16, 28, 29). The BrdUrd allowed us to establish that the cells were
recently born, and the Dcx allowed us to investigate their potential
differentiation into neurons. We found adult-born BrdUrd? cells
expressing Dcx only in or underlying the regions of the cortex
undergoing targeted CSMN degeneration. In contrast, we did not
find any such cells in the cortex of sham-operated control mice.
fluorescent nanospheres carrying chlorin e6were microinjected into the dorsal spinal cord at the cervical 5–6 level in 4-week-old mice. The nanospheres were
retrogradely transported to the somata of layer V CSMN via the corticospinal tract. (B) Two weeks later, in mice at 6 weeks of age, we exposed the motor cortex
through intact dura to 674-nm-wavelength light collimated at layer V. Photoactivated chlorin e6produced singlet oxygen within neuronal lysosomes, inducing
apoptosis exclusively in nanosphere-containing motor neurons. (C) CSMN that both contain nanospheres and are exposed to light undergo selective apoptosis.
Surrounding neurons and glia are undamaged. (D) Oblique coherent contrast image of a coronal section of anterior brain (Nikon SMZ 1500), indicating location
of the CSMN were targeted by green fluorescent nanospheres carrying chlorin e6. (F) Enlarged view of nanosphere-labeled motor neuron in layer V with
pyramidal morphology typical of CSMN. Due to the light dosimetry used, degeneration of ?10–20% of targeted projection neurons occurred. (G–I) Targeted
CSMN developed pyknotic and fragmented nuclei (arrows), one indication that they were undergoing apoptotic neuronal death. Arrowheads indicate normal
nuclei of surrounding healthy neurons. (G) CSMN selectively labeled by green photoactive nanospheres 8 days after photoactivation. (H) Targeted CSMN
developed pyknotic and fragmented nuclei, labeled with Hoechst 33258 in blue (arrows). (I) Merged image of G and H.
Targeting and induction of CSMN apoptosis. (A–C) Schematic of sequential targeting and photoactivation steps to induce CSMN apoptosis. (A) Green
www.pnas.org?cgi?doi?10.1073?pnas.0406795101 Chen et al.
expected; however, in the cortex, these cells differentiated into
neurons only in regions undergoing targeted apoptosis. No differ-
ences were observed in the number of BrdUrd? cells between
control and experimental mice in the cortex or in the SVZ,
consistent with previous results (16). Control mice were injected
with conjugated nanospheres and underwent a craniotomy and
sham exposure to long-wavelength light. Control mice had no
BrdUrd? neurons in the cortex, demonstrating that BrdUrd was
not incorporated into mature neurons because of toxicity. In
addition, it is extremely unlikely that the BrdUrd? neurons we
observed incorporated BrdUrd while undergoing apoptosis, be-
cause (i) the BrdUrd? newborn neurons were never labeled by the
original nanospheres; (ii) surviving neurons were BrdUrd?; (iii)
punctate as seen with DNA repair; and (iv) the neurons survived
in the neocortex for 1 year after the end of the BrdUrd adminis-
tration. Furthermore, neurons that had integrated BrdUrd during
Neurons labeled with lower levels of the chlorin e6-conjugated
nanospheres survive the photoactivating process. These surviving
neurons, having undergone sublethal levels of damage, still do not
integrate BrdUrd (n ? 5,192 cells examined in five mice), demon-
strating that this model of apoptosis does not induce BrdUrd
integration into preexisting neurons. Furthermore, this model of
targeted apoptosis occurs without inducing an inflammatory re-
sponse (22, 24), gliosis (22–24, 30), or activated cytokine-release-
stage microglia (22, 24), as assessed by routine histology, GFAP
staining, and F4-80 microglial staining, further confirming that the
cellular injury is limited to targeted neurons. As expected, Brd-
Urd??Dcx? cells were present in the SVZ and rostral migratory
stream of both experimental and control mice. The BrdUrd??
Dcx? cells in the cortex of experimental mice extended leading
processes, adopting morphologies typical of migrating neurons in
the developing brain (Fig. 2). In experimental mice, the migratory
morphology and orientation of Dcx? newborn neurons suggested
their migration from the SVZ, through the corpus callosum and
neocortical layer VI, and into layer V of the cortex (Fig. 2). No
Dcx? neurons were found in more superficial cortical layers or in
the cortex outside the regions undergoing CSMN apoptosis.
To confirm the newborn identity of these Dcx? neurons and to
investigate the unlikely possibility that adult-born cells were merely
closely apposed to Dcx? neurons with migratory morphology, we
used laser-scanning confocal microscopy to generate 3D recon-
structions of these neurons (Fig. 2 E, E?, and E?). We confirmed
that the newborn BrdUrd? nuclei belong to newborn Dcx?
neurons with migratory morphology. Consistent with the role of
Dcx in migration and early differentiation, BrdUrd??Dcx? neu-
rons were present in the cortex only soon after the induction of
apoptosis at 2 weeks. As the adult-born neurons matured, Dcx
expression and migratory morphology were replaced by expression
of the mature neuronal marker NeuN and increasingly mature
time points, typical of immature migrating and differentiating
neurons, demonstrates the appropriate developmental progression
of these neurons, further confirming that BrdUrd is appropriately
indicating newborn neurons. Together, these results demonstrate
that targeted apoptosis of CSMN leads to microenvironmental
change that recruits immature neurons to the cortex in a spatially
and temporally specific manner.
To determine whether the migratory neuroblasts differentiate
into mature neurons in the adult neocortex, we examined the
to NeuN, a transcription factor that is expressed in the nucleus and
cytoplasm only of mature neurons (31). Newborn BrdUrd??
NeuN? neurons were found exclusively in regions of layer V of the
cortex undergoing apoptotic degeneration of CSMN (Fig. 3 A–C;
n ? 27). We found no adult-born neurons in control mice under-
going craniotomies and sham light exposure (n ? 12) or in regions
of the cortex that were not targeted to undergo apoptosis, yielding
a highly statistically significant difference between control and
BrdUrd??Dcx? adult-born neurons were located between the corpus callosum and layer 5, appearing to migrate from the SVZ, through the corpus callosum
[A–C, E, E?, and E?? and layer 6 (F–H) and into experimental regions of cortical layer 5 (I and K)]. (D) A drawing of the coronal section for A–C shows the position
of many Dcx? newborn neurons in corpus callosum (A–C), layer 6 (F–H), and layer 5 (I–K). Adult-born neurons in experimental cortical layer 6 and underlying
corpus callosum exhibit migratory morphology with leading and trailing processes (arrowheads in B, C, G, and H). No BrdUrd??Dcx? cells were found in corpus
callosum or the cortex of control mice. Dcx is preferentially expressed by immature and migratory neurons (26, 27); Dcx staining does not overlap with A2B5,
images. (E) Confocal analysis confirms that a subset of newborn cells expresses Dcx, a marker of immature, migrating neurons. Laser-scanning confocal images
were combined to produce 3D reconstructions of the newborn neurons. Viewing this example, the BrdUrd??Dcx? newborn neuron along the x (E?), y (E?), and
with larger, rounder cell bodies and early extension of dendritic processes typical of pyramidal CSMN. (Bars, 10 ?m.)
Newborn cells in the cortex adopt a migratory morphology and express the migratory neuronal marker Dcx 2 weeks after induction of apoptosis. Many
Chen et al. PNAS ?
November 16, 2004 ?
vol. 101 ?
no. 46 ?
experimental mice (P ? 0.001, unpaired t test with Welch correc-
tion). To confirm that the adult-born BrdUrd? cells were truly
expressing NeuN, we imaged them using laser-scanning confocal
microscopy and produced 3D digital reconstructions (Fig. 3 E, E?,
and E?). The results confirm that the adult-born cells are neurons
truly expressing NeuN and are not merely newborn glia that are
closely apposed to preexisting neurons (see Fig. 5 for an example
of such a closely apposed newborn glial cell identified by 3D
confocal reconstruction). Some newborn neurons had pyramidal
morphology (large 10- to 15-?m-diameter somata with apical
process), characteristic of projection neurons, which extend long-
distance axonal projections. These results demonstrate that endog-
enous neural precursors can be induced in situ to differentiate into
mature neurons in layer V.
To examine the possibility that these new cells were inappropri-
ately expressing markers of multiple neural phenotypes, we triple-
labeled sections with antibodies against the astroglial marker
GFAP, in addition to BrdUrd and NeuN. BrdUrd??NeuN?
neurons never expressed GFAP, confirming that they had differ-
entiated specifically into mature neurons.
We found the greatest number of adult-born neurons 2–4 weeks
NeuN? newborn neurons per mm3in layer V 2 weeks after the
induction of apoptosis (n ? 5) and 34 ? 19 newborn neurons per
mm34 weeks after induction (n ? 5) (Fig. 3F). Especially at later
quite variable among experimental animals. We observed 7–15
adult-born neurons per mm3in three of five experimental animals
at 8 weeks after induction and seven adult-born neurons per mm3
in three of five experimental animals at 12 weeks after induction
(Fig. 3F). Mature adult-born neurons with long-distance projec-
tions were still present 56 weeks after induction of apoptosis. These
results demonstrate that newborn neurons can be recruited from
endogenous neural precursors in situ, and that some of these
adult-born neurons can survive for ?1 year in the cortex. We
observed the greatest number of newborn neurons soon after
induction of apoptosis and fewer newborn neurons at later times.
These results are consistent with patterns of both developmental
neurogenesis and adult neurogenesis; more neurons are generated
than eventually survive. For example, in the case of olfactory
neurogenesis, over half of adult-generated neurons undergo apo-
ptosis in the first 2–3 months after their generation (32, 33). The
partially due to the technical challenges involved in injecting
nanospheres into the extremely small murine corticospinal tract.
Pilot experiments showed that only ?60% of experimental mice
underwent sufficient nanosphere transport to the cortex to induce
targeted CSMN apoptosis. We included all experimental mice in
our analyses, contributing to the variability in the number of
projection neurons induces endogenous precursors to form mature
NeuN-expressing neurons in adult cortex.
A subset of newborn neurons displayed very large pyramidal
morphologies, characteristic of CSMN. To further define the
whether these adult-born neurons could reestablish long-distance
projections to the spinal cord, we injected the retrograde tracer FG
into the cervical spinal cord at level C2–C3 (above the level of the
initial chromophore targeting injection) weeks and months after
neuronal recruitment. The FG was retrogradely transported and
labeled both newborn and original CSMN in layer V of the
neocortex (Fig. 4A). We investigated potential projections of new-
born neurons 8, 12, 16, and 56 weeks after induced synchronous
apoptosis of CSMN. At 8 weeks, no projections by newborn CSMN
projections takes longer than 8 weeks, consistent with prior results
with transplanted neuroblasts (26, 27, 34). At 12 and 16 weeks after
neuronal recruitment, we observed a subpopulation of newborn
BrdUrd? neurons that were retrogradely labeled with FG (Fig. 4
B–G; n ? 7), demonstrating that newly recruited adult-born neu-
rons can form axonal projections to the cervical spinal cord. At 56
weeks, one to seven adult-born neurons per mm3(n ? 5) main-
We confirmed these results using laser-scanning confocal micros-
copy and 3D digital reconstructions (Fig. 4 H, H?, and H?, and see
Fig. 6, which is published as supporting information on the PNAS
web site), demonstrating that the BrdUrd? nuclei were located
completely within the FG? neuronal cell bodies. The absence of
FG? adult-born neurons at times earlier than 12 weeks further
confirms that the BrdUrd is not simply integrating into preexisting
neurons; only adult-born neurons that have had enough time to
form long-distance projections are both BrdUrd? and FG?.
migratory neuroblasts to mature NeuN? neurons without spinal
cord projections and finally to mature neurons with projections to
the spinal cord. Taken together, these results demonstrate that
endogenous neural precursors can be induced to differentiate into
mature neurons that form and maintain extremely long-distance
degeneration. (A–C) Four weeks after induction of apoptosis, a subset of
newborn cells with nuclei labeled with BrdUrd (red; arrow), expressed NeuN
(green), a mature neuronal marker. BrdUrd??NeuN? neurons had large
nuclei typical of mature neurons. Preexisting neurons were not labeled with
BrdUrd (arrowhead). (Bar, 10 ?m.) (C) Merged image. No BrdUrd??NeuN?
neurons contained nanospheres, further confirming that they are not preex-
isting targeted neurons, and that they did not simply integrate BrdUrd be-
layer V of the motor cortex, only in the region that had undergone targeted
CSMN apoptosis (blue). (E) Laser-scanning confocal images were combined to
produce 3D reconstructions of the newborn neurons. Viewing the BrdUrd??
NeuN? neurons along the x (E??), x (E?), and z (E) axes unequivocally demon-
strated the colocalization of BrdUrd and NeuN. (F) Quantification of Br-
dUrd??NeuN? adult-born neurons 2, 4, 8, and 12 weeks after induction of
CSMN apoptosis. Each point indicates the number of adult-born BrdUrd??
Newly generated BrdUrd? cells can be induced to differentiate into
www.pnas.org?cgi?doi?10.1073?pnas.0406795101Chen et al.
projections from the motor cortex to the spinal cord over many
To further examine the theoretical possibility that preexisting
developmentally generated neurons had integrated BrdUrd either
because of injury or cell fusion, we labeled existing CSMN before
inducing cell death. We injected FG into the cervical spinal cord,
retrogradely prelabeling essentially all CSMN. We then injected
(additionally labeling preexisting neurons) and, 1 week later, ex-
of the targeted neurons. Mice were administered BrdUrd and were
killed 3 weeks after induction of apoptosis. We could identify
essentially all preexisting developmentally generated neurons via
FG and the green fluorescent nanospheres. We examined FG- and
nanosphere-labeled cells in every 12th section through the motor
cortex of five mice. None of the FG-labeled (n ? 4,371 cells) or
nanosphere-labeled (n ? 5,192 cells) neurons contained BrdUrd,
unequivocally demonstrating that the BrdUrd? neurons we iden-
There is substantial precedent for neuronal death modifying the
fate of immature precursor cells, but until relatively recently, only
in regions of the vertebrate brain that have ongoing neurogenesis
(35). Recent evidence (16–21) suggests that neuronal death can
trigger increased neuron addition in regions of adult CNS that are
of the adult mouse neocortex undergoing synchronous apoptotic
interneurons, up-regulate the expression of a specific set of devel-
neuron differentiation (30). Immature neurons or multipotent
neural precursors that are transplanted into these regions migrate
selectively to layers of the cortex where projection neurons are
neurons (26, 27), receive afferent synapses (26, 27), and reform
appropriate long-distance connections to the original contralateral
targets of the degenerating neurons (26). Similarly, induction of
targeted neuronal death in projection neurons of the song circuitry
in the avian forebrain causes increased neuron replacement from
endogenous neural precursors in a system already undergoing
low-level neurogenesis (21). In more recent work from our lab, we
manipulated endogenous precursors in situ to undergo neurogen-
esis and reformation of corticothalamic connections de novo in the
adult mouse cortex, where it does not normally occur, by first
inducing synchronous apoptotic degeneration of corticothalamic
neurons (16). These previous results, regarding both transplanta-
tion of immature precursors and induction of adult neocortical
neurogenesis, provided support for the hypothesis that manipula-
tion of the local microenvironment by targeted apoptosis of CSMN
could induce neurogenesis of this important neuronal population.
Here, our results demonstrate that adult-born CSMN can be
generated from endogenous neural precursors and can reform
long-distance connections extending from the motor cortex to the
spinal cord. Only in regions of the cortex undergoing targeted
apoptosis do endogenous precursors, labeled by BrdUrd, enter as
migratory neuroblasts and progressively mature into neurons. In
our experiments, we gave experimental and control mice drinking
water containing BrdUrd after induction of apoptosis. BrdUrd
cells. The newborn neurons first express the immature neuronal
marker Dcx with migratory morphology, then express the mature
neuronal marker NeuN with mature pyramidal morphology, and
finally form long-distance projections to the spinal cord over a
There are reports that damaged or dying cells can also integrate
small amounts of BrdUrd during DNA repair, and that cell fusion
with adult neurons can occur (36, 37), suggesting that the simple
presence of low-level punctate BrdUrd labeling alone does not
unequivocally confirm that a cell is adult-born. Several lines of
evidence demonstrate that the neurons we observed were indeed
generated during adulthood. First, developmentally generated
CSMN labeled with FG before the induction of death do not
integrate BrdUrd after cell death. Second, because preexisting
projection neurons are also labeled by the fluorescent nanospheres
cervical spinal cord. (A) Both newborn and original layer V CSMN were
retrogradely labeled by FG (blue). (B) Field expanded in C–F showing a
BrdUrd??NeuN??FG? triple-labeled adult-born neuron (arrow). (Bar, 10
within this neuron, which is retrogradely labeled with FG from the cervical
spinal cord (D; blue) and expresses NeuN (E; green). (Bar, 10 ?m.) (F) Overlay
showing BrdUrd??FG??NeuN? neuron colocalization. (G) Higher-magnifi-
cation overlay of the same neuron from C–F. (H) A separate example of an
adult-born neuron with a projection to the spinal cord. Laser-scanning con-
focal images were combined to produce 3D reconstructions of newborn
neurons. Viewing a BrdUrd??FG? newborn neuron along its x (H?), y (H??),
and z axes (H) unequivocally demonstrates the colocalization of BrdUrd and
FG. (I) Quantification of BrdUrd??FG? adult-born CSMN extending spinal
projections from 12 to 56 weeks after induction of original CSMN apoptosis.
Each point indicates the number of adult-born BrdUrd??FG? neurons per
mm3in an individual animal; each bar indicates the mean.
A subset of newborn layer V cortical neurons extends axons to the
Chen et al. PNAS ?
November 16, 2004 ?
vol. 101 ?
no. 46 ?
that carry the targeting chromophore, and because none of the
BrdUrd? cells we observed contained nanospheres, these Br-
dUrd??Dcx? or BrdUrd??NeuN? neurons are not preexisting
neurons. Conversely, none of the targeted chromophore-
containing neurons that survived were BrdUrd?. Third, control
mice receiving injections of chromophore-conjugated nanospheres
without photoactivation did not contain BrdUrd? neurons in the
cortex, confirming that injection and?or axotomy alone does not
lead to BrdUrd incorporation or neuron birth. Fourth, the Brd-
Urd? neurons we observed in experimental mice possess dense
relatively uniform nuclear BrdUrd staining, not light punctate
not during DNA repair. In contrast to BrdUrd integrated during
cell division, BrdUrd integrated during DNA repair yields a light
punctate pattern of BrdUrd staining. Fifth, the BrdUrd??NeuN?
neurons survive for ?1 year, indicating that they did not integrate
to sometimes precede the apoptosis of mature neurons (38–40).
Sixth, the BrdUrd??Dcx? cells we observed possess migratory
morphology in the corpus callosum and layer VI underlying tar-
geted region of the cortex; thus, these cannot be pathological
preexisting neurons misexpressing Dcx. Taken together, it is ex-
tremely unlikely that the BrdUrd? neurons we observed incorpo-
rated BrdUrd while being damaged or undergoing apoptosis. This
evidence identifies the BrdUrd? neurons reported here as adult-
Although we observed a significant number of new neurons, the
BrdUrd labeling almost certainly underestimates this number,
hours after administration. Although the small number of adult-
born neurons observed by using these methods would not be
sufficient for functional restoration, it is also possible that the
number of recruited CSMN could be increased by enhancing their
survival through a critical period until they establish supportive
target innervation over weeks to months. In future experiments, it
CSMN survival and enhancing axon outgrowth to targets.
Of particular interest for potential future therapeutic strategies
are the possibilities that immature adult-born neurons may be
fully mature preexisting neurons. The ability of adult-born neurons
to extend appropriate long-distance connections indicates that the
adult brain remains capable of supporting axon outgrowth, in
contrast to the long-held idea that the adult CNS is absolutely
to reform long-distance projections in these experiments may be in
part due to the absence of a glial scar and of up-regulation of
inhibitory extracellular matrix proteins, because the local microen-
vironment was physically unperturbed (e.g., by invasive transplan-
tation techniques) (41). It is also possible that some newborn
neurons extend axons to spinal cord by following the existing
corticospinal axon fascicles or by extending axons through empty
axon paths vacated by axons of neurons induced to undergo
apoptosis. It is likely, however, that the ability of newborn neurons
axon extension (42) and a relative lack of receptors to myelin-
associated growth-inhibitory molecules (43). It might be of interest
to examine whether immature adult-born neurons can extend their
axons through the regions of glial scarring associated with spinal
cord injury better than mature preexisting neurons.
It may be possible to use growth factors affecting proliferation,
differentiation, and?or survival to increase the efficiency of the
neurogenesis reported here. Growth factor enhancement of pro-
liferation of endogenous neural precursors might increase the
number of adult-born neurons initially generated, but simply in-
creasing the number of neurons generated would not be sufficient
to achieve functional neuronal circuit repair. Newly generated
the survival of adult-born neurons via specific growth factor ma-
nipulation might allow neurons more time to form appropriate
connections from which they might receive target-derived factors
necessary for their survival. Consistent with this idea is our finding
decreases with time until they reach the cervical spinal cord,
suggesting that adult-born neurons that do not receive proper
survival signals undergo apoptosis, just as occurs during initial
Taken together, these results demonstrate that endogenous
projections to the adult mouse spinal cord. Further understanding
the potential of endogenous neural precursors and controls over
their lineage-specific differentiation may allow the development of
more efficient neuronal replacement therapies that do not depend
on transplantation of exogenous cells.
This work was partially supported by grants from the National Institutes
of Health and the Christopher Reeve Paralysis Foundation (to J.D.M.).
Some confocal imaging was performed in facilities supported by the
Harvard Center for Neurodegeneration and Repair.
1. Gage, F. H. (2000) Science 287, 1433–1438.
2. Temple, S. (2001) Nature 414, 112–117.
3. Temple, S. & Alvarez-Buylla, A. (1999) Curr. Opin. Neurobiol. 9, 135–141.
4. Reynolds, B. A. & Weiss, S. (1992) Science 255, 1707–1710.
5. Richards, L. J., Kilpatrick, T. J. & Bartlett, P. F. (1992) Proc. Natl. Acad. Sci. USA 89,
6. Doetsch, F., Garcia-Verdugo, J. M. & Alvarez-Buylla, A. (1997) J. Neurosci. 17, 5046–5061.
7. Van Praag, H., Schinder, A. F., Christie, B. R., Toni, N., Palmer, T. D. & Gage, F. H. (2002)
Nature 415, 1030–1034.
8. Kempermann, G. & Gage, F. H. (2002) Brain Res. Dev. Brain Res. 134, 1–12.
9. Van Praag, H., Kempermann, G. & Gage, F. H. (1999) Nat. Neurosci. 2, 266–270.
10. Kempermann, G., Kuhn, H. G. & Gage, F. H. (1997) Nature 386, 493–495.
11. Shingo, T., Gregg, C., Enwere, E., Fujikawa, H., Hassam, R., Geary, C., Cross, J. C. & Weiss,
S. (2003) Science 299, 117–120.
12. Parent, J. M. & Lowenstein, D. H. (2002) Prog. Brain Res. 135, 121–131.
13. Parent, J. M., Yu, T. W., Leibowitz, R. T., Geschwind, D. H., Sloviter, R. S. & Lowenstein,
D. H. (1997) J. Neurosci. 17, 3727–3738.
14. Kempermann, G. & Kronenberg, G. (2003) Biol. Psychiatry 54, 499–503.
15. Kuhn, H. G., Dickinson-Anson, H. & Gage, F. H. (1996) J. Neurosci. 16, 2027–2033.
16. Magavi, S. S., Leavitt, B. R. & Macklis, J. D. (2000) Nature 405, 951–955.
17. Nakatomi, H., Kuriu, T., Okabe, S., Yamamoto, S., Hatano, O., Kawahara, N., Tamura, A.,
Kirino, T. & Nakafuku, M. (2002) Cell 110, 429–441.
18. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z. & Lindvall, O. (2002) Nat. Med. 8, 963–970.
19. Parent, J. M., Vexler, Z. S., Gong, C., Derugin, N. & Ferriero, D. M. (2002) Ann. Neurol.
20. Zhao, M., Momma, S., Delfani, K., Carlen, M., Cassidy, R. M., Johansson, C. B., Brismar, H.,
Shupliakov, O., Frisen, J. & Janson, A. M. (2003) Proc. Natl. Acad. Sci. USA 100, 7925–7930.
21. Scharff, C., Kirn, J. R., Grossman, M., Macklis, J. D. & Nottebohm, F. (2000) Neuron 25,
22. Sheen, V. L. & Macklis, J. D. (1995) J. Neurosci. 15, 8378–8392.
23. Macklis, J. D. (1993) J. Neurosci. 13, 3848–3863.
24. Sheen, V. L. & Macklis, J. D. (1994) Exp. Neurol. 130, 67–81.
25. Guillery, R. W. & Herrup, K. (1997) J. Comp. Neurol. 386, 2–7.
26. Fricker-Gates, R. A., Shin, J. J., Tai, C. C., Catapano, L. A. & Macklis, J. D. (2002)
J. Neurosci. 22, 4045–4056.
27. Shin, J. J., Fricker-Gates, R. A., Perez, F. A., Leavitt, B. R., Zurakowski, D. & Macklis, J. D.
(2000) J. Neurosci. 20, 7404–7416.
28. Gleeson, J. G., Lin, P. T., Flanagan, L. A. & Walsh, C. A. (1999) Neuron 23, 257–271.
29. Francis, F., Koulakoff, A., Boucher, D., Chafey, P., Schaar, B., Vinet, M. C., Friocourt, G.,
McDonnell, N., Reiner, O., Kahn, A., et al. (1999) Neuron 23, 247–256.
30. Wang, Y., Sheen, V. L. & Macklis, J. D. (1998) Exp. Neurol. 154, 389–402.
31. Mullen, R. J., Buck, C. R. & Smith, A. M. (1992) Development (Cambridge, U.K.) 116, 201–211.
32. Winner, B., Cooper-Kuhn, C. M., Aigner, R., Winkler, J. & Kuhn, H. G. (2002) Eur.
J. Neurosci. 16, 1681–1689.
33. Petreanu, L. & Alvarez-Buylla, A. (2002) J. Neurosci. 22, 6106–6113.
34. Hernit-Grant, C. S. & Macklis, J. D. (1996) Exp. Neurol. 139, 131–142.
35. Parent, J. M. (2003) Neuroscientist 9, 261–272.
36. Alvarez-Dolado, M., Pardal, R., Garcia-Verdugo, J. M., Fike, J. R., Lee, H. O., Pfeffer, K.,
Lois, C., Morrison, S. J. & Alvarez-Buylla, A. (2003) Nature 425, 968–973.
37. Weimann, J. M., Charlton, C. A., Brazelton, T. R., Hackman, R. C. & Blau, H. M. (2003)
Proc. Natl. Acad. Sci USA 100, 2088–2093.
38. Yang, Y., Geldmacher. D. S. & Herrup, K. (2003) J. Neurosci. 21, 2661–2668.
39. Konishi, Y & Bonni, A. (2003) J. Neurosci. 23, 1649–1658.
40. Katchanov, J. Katchanov, J., Harms, C., Gertz, K., Hauck, L., Waeber, C., Hirt, L., Priller,
J., von Harsdorf, R., Bruck, W., et al. (2001) J. Neurosci. 21, 5045–5053.
41. Grimpe, B. & Silver, J. (2004) J. Neurosci. 24, 1393–1397.
42. Tessier-Lavigne, M. & Goodman, C. S. (1996) Science 274, 1123–1133.
43. Oertle, T. & Schwab, M. E. (2003) Trends Cell Biol. 13, 187–194.
www.pnas.org?cgi?doi?10.1073?pnas.0406795101Chen et al.