Peter S. Eriksson1, 4, Ekaterina Perfilieva1, Thomas Björk-Eriksson2, Ann-Marie Alborn1, Claes Nordborg3, Daniel A. Peterson4
& Fred H. Gage4
1 Department of Clinical Neuroscience, Institute of Neurology,
Sahlgrenska University Hospital, 41345 Göteborg
, Sweden
2 Department of Clinical Neuroscience, Department of
Oncology, Sahlgrenska University Hospital, 41345 Göteborg
, Sweden
3 Department of Clinical Neuroscience, Department of
Pathology, Sahlgrenska University Hospital, 41345 Göteborg
, Sweden
4 Laboratory of Genetics, The Salk Institute for Biological
Studies, 10010 North Torrey Pines Road, La Jolla
, California 92037, USA
Correspondence should be addressed to Fred H. Gage
The genesis of new cells, including neurons, in the adult human brain
has not yet been demonstrated. This study was undertaken to investigate whether
neurogenesis occurs in the adult human brain, in regions previously identified
as neurogenic in adult rodents and monkeys. Human brain tissue was obtained
postmortem from patients who had been treated with the thymidine analog, bromodeoxyuridine
(BrdU), that labels DNA during the S phase. Using immunofluorescent labeling
for BrdU and for one of the neuronal markers, NeuN, calbindin or neuron specific
enolase (NSE), we demonstrate that new neurons, as defined by these markers,
are generated from dividing progenitor cells in the dentate gyrus of adult
humans. Our results further indicate that the human hippocampus retains its
ability to generate neurons throughout life.
Loss of neurons is thought to be irreversible in the adult human brain,
because dying neurons cannot be replaced. This inability to generate replacement
cells is thought to be an important cause of neurological disease and impairment.
In most brain regions, the generation of neurons is generally confined to
a discrete developmental period. Exceptions are found in the dentate gyrus
and the subventricular zone of several species that have been shown to generate
new neurons well into the postnatal and adult period1,
2,
3,
4,
5,
6.
Granule neurons are generated throughout life from a population of continuously
dividing progenitor cells residing in the subgranular zone of the dentate
gyrus in the rodent brain5. 'Newborn' neurons generated from
these progenitor cells migrate into the granule cell layer, differentiate,
extend axons and express neuronal marker proteins7,
8,
9,
10.
We examined whether progenitor cells reside in the adult human hippocampus
and whether new neurons are born within the dentate gyrus of the adult human
brain. Postmortem tissue from the hippocampus and the subventricular zone
of caudate nucleus was obtained from cancer patients (n = 5) who received
one intravenous infusion (250 mg; 2.5 mg/ml, 100 ml) of bromodeoxyuridine
(BrdU) for diagnostic purposes11. One patient diagnosed with
a similar type and location of cancer, but without BrdU treatment, was included
as a control. A thymidine analog, BrdU is incorporated into the DNA of dividing
cells and can be detected immunohistochemically in their progeny5,
12,
13.
Cell genesis and survival in the adult human dentate gyrus The number of surviving labeled, proliferating progenitors was quantified
using immunohistochemical staining for BrdU and unbiased counting techniques14,
15,
16. BrdU-labeled cells were quantified in the granule cell
layer and the subgranular zone of the dentate gyrus and in the hilus (area
CA4; Fig. 1a). In all BrdU-treated patients,
the granule cell layer contained BrdU-positive, round- to oval-shaped nuclei
with the typical morphology of granule cell neuron nuclei (
Fig. 1b−d). The morphological
appearance and localization of these nuclei were similar to those of the nuclei
of cells found in the dentate gyrus of adult mouse, rat and marmoset monkey5,
6,
17. No BrdU-immunoreactive profiles could be detected in the
control patient that had not received BrdU. The presence of BrdU-positive
nuclei in a brain region that is neurogenic in many other mammalian and primate
species indicates that proliferating neural progenitor cells are present in
the adult human dentate gyrus.
Figure 1. Newly generated cells can be detected in the adult human brain in patients
previously treated with BrdU.
a, The hippocampal region of the adult human brain immunoperoxidase-stained
for the neuronal marker NeuN. b, The hippocampal dentate gyrus granule
cell layer (GCL) visualized with immunoperoxidase staining for NeuN.
c, Differential interference contrast photomicrograph showing BrdU-labeled
nuclei (arrows) in the dentate granule cell layer (GCL). d, Differential
interference contrast photomicrograph showing a BrdU-labeled nucleus (arrow)
in the human dentate GCL. BrdU-positive nuclei have a rounded appearance and
resemble the chromatin structure of mature granule cells and are found within
the granule cell layer. e, Differential interference contrast photomicrograph
showing BrdU-positive cells (arrows) adjacent to the ependymal lining in the
subventricular zone of the human caudate nucleus. Cells with elongated nuclei
resembling migrating cells are in the rat subventricular zone (SVZ).
f, Differential interference contrast photomicrograph showing BrdU-positive
cells (arrows) with round to elongated nuclei in the subventricular zone of
the human caudate nucleus. All scale bars represent 50 m.
The number of BrdU-positive cells in the granule cell layer, the subgranular
zone and hilus varied between individuals, as may be expected because of the
varying post-infusion intervals and different ages of the patients (Fig. 2a−c). There is an apparent decline
in the number of cells that are detected in the patients that had the longest
interval between BrdU treatment and histological assessment. This decline
may indicate a progressive death of the newly generated cells over time. However,
the small sample weakens any quantitative conclusion.
Figure 2. Quantitation of newly generated cells in the adult human hippocampus.
The density of BrdU immunoperoxidase-stained cells in the subgranular zone
(SGC) (a) and the granule cell layer (GCL) (b) and the hilus
(c) was determined in 5 to 7 sections per patient (mean number of BrdU-positive
cells per mm3 sample volume s.e.m.) The corresponding
patient's age at the time of death and interval as BrdU infusion are given
for each BrdU-treated patient (n = 5).
BrdU-labeled cells co-express neuronal markers To determine the cell fate of the BrdU-immunoreactive cells, we did triple
immunofluorescent labeling for BrdU and cell-specific markers, including glial
fibrillary acidic protein (GFAP), a marker for astroglia, and one of the neuronal
markers, NeuN18,
19, calbindin20 or neuron specific
enolase21 (NSE). Confocal microscopy was used to determine the
phenotype of the BrdU-positive cells (Figs. 3 and 4). No evidence for cross-reactivity between GFAP-immunoreactivity
and any of the three neuronal markers was detected in these sections. GFAP-immunopositive
processes were observed to surround neurons and their processes, but did not
co-express in the same cell (Fig. 3e−
h). Autofluorescence was observed in this tissue, but the nonspecific
emission artifact was easily detected by its contribution to multiple wavelengths
(Fig. 3a−d).
We took care to evaluate potential sources of autofluorescence in the human
tissue by directly imaging untreated sections and sections from the control
patient both with and without the pretreatment for BrdU-immunohistochemical
detection in the absence of antibodies against BrdU. The autofluorescence
was present even in completely untreated sections and was restricted to the
cytoplasm of neuronally marked cells or to endothelial cells and red blood
cells and could be clearly distinguished from BrdU-immunoreactive profiles
(Fig. 3). The majority of the NeuN-positive neurons
that double-labeled with BrdU were located within or near the granule cell
layer and had the morphological characteristics of granule cell neurons: round
or oval nuclei with small- to medium-sized cell bodies (Fig.
3). Confocal microscopic Z-series analyses of BrdU-positive nuclei
unambiguously identified double-labeling with NeuN and demonstrated the existence
of newly generated (BrdU-labeled) granule cell neurons in the dentate gyrus
of the adult human brain (Fig. 3d−h). The phenotypic expression of these BrdU-positive
neurons in adult humans was equivalent to the expression found in adult rodents5,
10,
12,
17 (Fig. 3i−
l). The average fraction of BrdU-NeuN double-labeled cells in the
granule cell layer was 22.0 2.4% among the BrdU-treated patients.
Figure 3. Newly generated cells in the adult human dentate gyrus can express
a neuronal phenotype.
Simultaneous detection of immunofluorescent labels for NeuN (a;
scale bar represents 25 m), BrdU (b) and GFAP (c) for
detection of astrocytes and a merge of these signals in x-, y- and z-registration
(d) examined by confocal microscopy showed that BrdU-labeled nuclei could
specifically co-express NeuN without expressing GFAP (arrows in a−d
). In addition to specific BrdU-labeling, some neurons contained nonspecific
fluorescence in the green and red emission, reflecting their accumulation
of lipofuchsin granules (arrowheads in a−d). Red blood cells
and endothelial cells, present in several small blood vessels, also emit non-specific
green and red fluorescence (small blood vessel indicated by arrowheads, larger
blood vessels indicated by asterisks in e−h). The specificity
of BrdU-NeuN co-expression in three-dimensions is demonstrated by a series
of focal planes above (e,f) and below (g, h) the focal plane
shown in d (arrows indicate the same cell as in d). The appearance
of adult BrdU-positive neurons in the human dentate gyrus is equivalent to
BrdU-labeled neurons in the adult rat dentate gyrus (i−l; scale bar
represents 25 m). The rat tissue (i−l) is at a different
magnification than the human tissue (a−h).
Figure 4. Newly generated cells in the adult human dentate gyrus can express
additional neuronal phenotypes.
The calcium-binding protein, calbindin, is expressed by certain neuronal
populations, including dentate granule neurons, in vivo. a,
Fluorescent labeling of calbindin (red) and GFAP (blue) discriminated between
granule neurons and astrocytes. Scale bar represents 10 m. The arrow indicates
a newly generated, BrdU-labeled calbindin-positive neuron shown with the label-colors
merged in b. c, Another calbindin-positive neuron (arrow) that co-expresses
BrdU (d). Newly generated cells may also differentiate into astrocytes
(c,d; arrowheads). BrdU-labeled cells can also express neuron specific
enolase (NSE, shown in red; arrows in e and f) without expressing
GFAP (blue).
Neurons double-labeled with BrdU and NSE could also be detected in all
BrdU-treated subjects (Fig. 4e−
f). The average fraction of BrdU-labeled cells that were also NSE-labeled
in the granule cell layer was 22.7 2.8%. Cells that double-labeled
for BrdU and calbindin were also observed and provided further support for
the occurrence of neurogenesis in the adult SGZ, GCL and hilus (
Fig. 4a−d). The average fraction
of BrdU-labeled cells that were also calbindin-positive in the granule cell
layer was 7.9 2.2% among the BrdU-treated patients. The dentate gyrus
from all individuals contained a fraction of BrdU-positive cells that were
also GFAP-positive, 18.1 1.8%, and had the typical morphology of
star-shaped glial cells with small, irregularly shaped nuclei and small cell
bodies with thin, GFAP-positive processes (Fig. 4c−d). The dentate gyrus from all subjects contained BrdU-positive
cells that were immunonegative for both neuronal and glial markers. These
immunonegative cells likely represent quiescent undifferentiated cells, newborn
cells of a phenotype not examined here and/or a pool of asymmetrically dividing
progenitor cells. These cells were characterized by small, round-to-oval BrdU-positive
nuclei and the absence of cell-specific immunoreactivity.
To further confirm the presence of neurogenesis, we double-labeled using
antibodies against BrdU and either NSE, calbindin or NeuN with chromagens
for brightfield optics (alkaline phosphatase (Vector Blue) for BrdU and 3-amino-9-ethylcarbazole
(AEC) for the neuronal markers). Although confocal microscopy could not be
used for imaging, the brightfield chromagens had the advantage of not fading
with examination and not contributing autofluorescence to the image. Examination
of brightfield staining confirmed that BrdU-positive cells in the adult human
hippocampus could express a neuronal phenotype (Fig. 5a
−e) morphologically indistinguishable
from adult rodent BrdU-positive neurons (Fig. 5f
and g), strongly supporting our conclusion that
neurogenesis occurs in the dentate granule cell layer of the adult human brain.
Figure 5. Brightfield double-immunohistochemical demonstration of neuronal phenotype.
a, Staining with the neuronal marker NeuN labels both dentate
granule cells (top inset, shown in b) and hilar neurons (bottom inset,
shown in c). Scale bar represents 100 m. Combining NeuN staining
with differential interference contrast optics demonstrated that NeuN labeling
included the entire nucleus and perikaryal cytoplasm and extended into proximal
portions of major dendrites (arrowheads) in both granule cells (b;
scale bar represents 20 m) and hilar neurons (c, scale bar represents
20 m). Newly generated cells could be found in the dentate granule layer
when detected with antibodies against BrdU in conjunction with NeuN staining
(d, scale bar represents 25 m). Dark blue-stained BrdU-labeled
nuclei can co-express the neuronal marker NeuN shown in red (e, scale
bar represents 10 m). The appearance of BrdU-labeled cells in adult human
dentate gyrus is equivalent to that of the adult rat dentate gyrus (f
, scale bar represents 25 m; and g, scale bar represents
10 m.).
BrdU labeling in the subventricular zone of adult human brain Another neurogenic region, the subventricular zone (SVZ) adjacent to the
caudate nucleus, was examined for the presence of BrdU-positive cells. Tissue
samples from all BrdU-treated patients contained BrdU-positive cells within
the SVZ (Fig. 1e−f).
BrdU-positive cells did not co-express the cell-specific markers GFAP and
NeuN (data not shown). The morphology of BrdU-labeled nuclei within the SVZ
was small and round-to-oval, resembling that of progenitor cells in the rat
SVZ (ref. 5). This finding supports the idea that
the human SVZ contains progenitor cells and that these cells are required
to migrate from the SVZ before they differentiate22. We were
unable to detect any BrdU-immunoreactive cells in tissue from the control
patient who had not received BrdU treatment, supporting the interpretation
that the BrdU staining we report here reflects the persistence in the adult
human brain of cell genesis.
Discussion Our study demonstrates that cell genesis occurs in human brains and that
the human brain retains the potential for self-renewal throughout life. Although
earlier studies in adult primates have been unsuccessful in showing neurogenesis
in the dentate gyrus23,
24, a recent report has demonstrated
neurogenesis in three-year-old marmoset monkeys6. Although the
number of BrdU-labeled cells entering the neuronal lineage seems to be lower
in the human hippocampus than in marmosets, those monkeys6 were
considerably younger, even in relative terms, than the humans examined here
(average age of 64.4 2.9 years). Therefore, we conclude that, as
in rodents5,
25, neurogenesis in the human dentate gyrus continues
throughout life.
Although our results demonstrate that cells in the adult brain undergo
cell division and that some of the newly generated cells can survive and differentiate
into cells with morphological and phenotypic characteristics of neurons, we
have not proven that these newly generated cells are functional. We also do
not yet know the biological significance of cell genesis in the adult human
brain. However, this does provide a basis to investigate a newly discovered
type of 'neuroplasticity' in humans, one based on addition of neurons, that
has not been previously considered. Studies in rodents have shown that the
adult hippocampus contains progenitor cells that can be expanded in vitro
and grafted back into the adult brain, where they can respond to regional
cues by differentiation into site-specific phenotypes, including neurons26,
27. The presence of progenitor cells in the human dentate gyrus,
reported here, indicates that these cells also may be used for in vitro
and in vivo studies of cell differentiation and possibly subsequent
transplantation studies. Furthermore, environmental stimulation can influence
the rate of neurogenesis in the adult and senescent rodent dentate gyrus12,
17. The potential to regulate human neurogenesis should prove
to be an interesting area of investigation.
Methods Autopsy material. Human hippocampal tissue was obtained
at autopsy with the full consent of each family. All patients were diagnosed
with squamous cell carcinomas at the base of the tongue, in the larynx or
in the pharynx. All patients received bromodeoxyuridine (BrdU) (250 mg) dissolved
in saline and given as an intravenous infusion (2.5 mg/ml, 100 ml). The BrdU
was given to the patients to assess the proliferative activity of the tumor
cells, expressed as BrdU-labeling index. No signs of macroscopic or microscopic
metastases were found in autopsy material from the cerebrum in any of the
patients. No anti-cancer therapy was administered before or during BrdU administration
to any of the patients.
Tissue preparation. The hippocampal formation and ventricular
zone were dissected out and post-fixed in 4% paraformaldehyde for 24 hours
and then transferred into 30% sucrose solution until equilibrated. The hippocampi
were sectioned (slices 40 m in thickness) in the coronal plane on a sliding
microtome and stored at −20 °C in a cryoprotecting buffer containing
25% ethylene glycol, 25% glycerin and 0.05 M phosphate buffer. For comparison,
sections derived from BrdU-injected adult rats and mice that had been intracardially
perfused with 4% paraformaldehyde were processed in parallel with the human
tissue.
Histology. Immunohistochemical detection of BrdU requires
a pre-treatment of the sections to denature DNA (5). All staining was done on free-floating sections and the blocking
buffer contained both 3% normal donkey serum and 3% normal human serum (Sigma).
For sections stained only for BrdU, a mouse-anti-BrdU antibody (Boehringer;
diluted 1:400) was used in combination with the avidin-biotin complex method
using a biotinylated donkey anti-mouse-IgG antibody (Vector Laboratories,
Burlingame, California; diluted 1:167) and reacted with a diaminobenzidine
(DAB) chromagen.
Immunofluorescent double- and triple-labeling was done as described5,
12. For multiple immunostaining, BrdU was detected with a rat anti-BrdU
antibody (Harlan Sera-Lab, Loughborough, England; diluted 1:500) and visualized
with a FITC-conjugated secondary donkey anti-rat antibody (Jackson ImmunoResearch,
West Grove, Pennsylvania; diluted 1:250). For neuronal phenotype markers,
sections were incubated with one of the following antisera: rabbit anti-calbindin
antiserum (SWant, Bellinzona, Switzerland; diluted 1:1,000), or mouse anti-NeuN
antiserum (from R. Mullen18; diluted 1:20), or rabbit anti-NSE
antiserum (Polysciences, Warrington, Pennsylvania; diluted 1:800). These neuronal
markers were visualized by using the species-appropriate Cy3-conjugated secondary
antibody (Jackson ImmunoResearch, West Grove, Pennsylvania; diluted 1:250).
GFAP was detected in the same sections using a guinea pig anti-GFAP antiserum
(Advanced Immunochemicals, Long Beach, California; diluted 1:250) and visualized
using a Cy5-conjugated donkey anti-guinea pig antibody (Jackson ImmunoResearch,
West Grove, Pennsylvania; diluted1:250).
For double immunostaining using brightfield chromagens, sections were incubated
with a pooled solution of rat anti-BrdU (Harlan Sera-Lab, Loughborough, England;
diluted 1:500) and mouse anti-NeuN (from R. Mullen18; diluted
1:100) antisera. After being rinsed and blocked, sections were incubated first
with a biotinylated donkey anti-rat antibody (Jackson ImmunoResearch, West
Grove, Pennsylvania; diluted 1:250), followed by incubation with an alkaline
phosphatase avidin−biotin substrate and then reaction with the blue
chromagen (Vector Blue; Vector Laboratories, Burlingame, California). After
being rinsed and blocked further, the sections then were incubated with a
biotinylated donkey anti-mouse antibody (Jackson ImmunoResearch, West Grove,
Pennsylvania; diluted 1:250) followed by incubation with a peroxidase avidin-biotin
substrate and then reaction with the red chromagen (3-amino-9-ethylcarbazole
or AEC; Vector Laboratories, Burlingame, California). Sections were mounted
and coverslipped with an aqueous mounting medium.
Fluorescent signals were imaged using a confocal laser scanning microscope
equipped with a krypton/argon mixed gas laser (Bio-Rad MRC1024; Bio-Rad, Richmond,
California) using individual collection of wavelengths to minimize artifactual
detection of non-specific fluorescent emission. At least 20 BrdU-positive
cells within multiple immunofluorescently stained sections from each patient
were examined on the confocal microscope to determine the phenotype of BrdU-labeled
cells. Brightfield images were derived from conventional photomicrographs
digitized by using a slide scanner. Figures were composed by using Adobe Photoshop
(Adobe Systems, Mountain View, California) with image adjustments confined
to creating an equivalent signal distribution between panels.
Quantitation. The numbers of BrdU-positive cells in
the granule cell layer, the subgranular layer and the hilus, and their corresponding
sample volumes were determined in five to seven immunoperoxidase-stained coronal
sections 40 m in thickness, at least 240 m apart, from each patient.
Area estimations were done on an IBAS image analysis system. The section thickness
of 40 m (microtome setting) was used in the dissector estimation of volume.
The number of BrdU-positive cells was counted within the granule cell layer
(GCL) and two cell diameters below the granule cell layer, ignoring the cells
in the uppermost focal plane and focusing through the thickness of the section
(optical disector principle; refs. 14,15,16) to avoid oversampling errors.
The subgranular zone (SGZ) was defined as the area from one cell diameter
within the GCL from the hilus-GCL border and two cell diameters below the
hilus-GCL border (Fig. 1). Quantitative analysis of
BrdU immunoperoxidase-stained sections was done on a Nikon microscope equipped
with a video camera. The results are expressed as BrdU-positive cells per
sample volume per section.
Received 9 September 1998; Accepted 13 October 1998
REFERENCES
Altman, J. &. Das, G.D. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. >J. Comp. Neurol.124, 319−335 (1965). | PubMed | ISI | ChemPort |
Altman, J. & Das, G.D. Postnatal neurogenesis in the guinea-pig. Nature214, 1098−1101 (1967). | PubMed | ISI | ChemPort |
Caviness, V.S. Time of neuron origin in the hippocampus and dentate gyrus of normal and reeler mutant mice: an autoradiographic analysis. J. Comp. Neurol.151, 113−120 (1973). | PubMed | ISI |
Gueneau, G., Privat, A., Drouet, J. & Court, L. Subgranular zone of the dentate gyrus of young rabbits as a secondary matrix. A high-resolution autoradiographic study. Dev. Neurosci.5, 345−358 (1982). | PubMed | ISI | ChemPort |
Kuhn, H.G., Dickinson-Anson, H. & Gage, F.H. Neurogenesis in the dentate gyrus of the adult rat: Age-related decrease of neuronal progenitor proliferation. J. Neurosci.16, 2027−2033 (1996). | PubMed | ISI | ChemPort |
Gould, E., Tanapat, P., McEwen, B.S., Flugge, G. & Fuchs, E. Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress. Proc. Natl. Acad. Sci. USA 95, 3168−3171 (1998). | Article | PubMed | ChemPort |
Kaplan, M.S. & Bell, D.H. Mitotic neuroblasts in the 9-day-old and 11-month-old rodent hippocampus. J. Neurosci.4, 1429−1441 (1984). | PubMed | ISI | ChemPort |
Kaplan, M.S. & Hinds, J.W. Neurogenesis in the adult rat: electron microscopic analysis of light radioautographs. Science197, 1092−1094 (1977). | PubMed | ISI | ChemPort |
Stanfield, B.B. & Trice, J.E. Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. Exp. Brain Res.72, 399−406 (1988). | PubMed | ISI | ChemPort |
Cameron, H.A., Woolley, C.S., McEwen, B.S. & Gould, E. Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat. Neuroscience56, 337−344 (1993). | Article | PubMed | ISI | ChemPort |
Dolbeare, F. Bromodeoxyuridine: a diagnostic tool in biology and medicine, Part I: Historical perspectives, histochemical methods and cell kinetics. Histochem. J.27, 339−369 (1995). | Article | PubMed | ISI | ChemPort |
Kempermann, G., Kuhn, H.G. & Gage, F.H. Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc. Natl. Acad. Sci. USA 94, 10409−10414 (1997). | Article | PubMed | ChemPort |
del Rio, J.A. & Soriano, E. Immunocytochemical detection of 5'-bromodeoxyuridine incorporation in the central nervous system of the mouse. Dev. Brain Res.49, 311−317 (1989). | Article | ChemPort |
Gundersen, H.J. et al. The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS96, 857−881 (1988). | PubMed | ISI | ChemPort |
West, M.J. Regionally specific loss of neurons in the aging human hippocampus. Neurobiol. Aging14, 287−293 (1993). | Article | PubMed | ISI | ChemPort |
Coggeshall, R.E. & Lekan, H.A. Methods for determining numbers of cells and synapses: a case for more uniform standards of review. J. Comp. Neurol.364, 6−15 (1996). | Article | PubMed | ISI | ChemPort |
Kempermann, G., Kuhn, H.G. & Gage, F.H. More hippocampal neurons in adult mice living in an enriched environmen Nature386, 493−495 (1997). | Article | PubMed | ISI | ChemPort |
Mullen, R.J., Buck, C.R. & Smith, A.M. NeuN, a neuronal specific nuclear protein in vertebrates. Development116, 201−211 (1992). | PubMed | ISI | ChemPort |
Wolf, H.K. et al. NeuN: A useful neuronal marker for diagnostic histopathology. J. Histochem. Cytochem.44, 1167−1171 (1996). | PubMed | ISI | ChemPort |
Sloviter, R.S. Calcium-binding protein (calbindin-D28k) and parvalbumin immunocytochemistry: localization in the rat hippocampus with specific reference to the selective vulnerability of hippocampal neurons to seizure activity. J. Comp. Neurol.280, 183−196 (1989). | Article | PubMed | ISI | ChemPort |
Schmechel, D., Marango, P.J., Zis, A.P., Brightman, M. & Goodwin, F.K. Brain enolases as specific markers of neuronal and glial cells. Science199, 313−315 (1978). | PubMed | ISI | ChemPort |
Kirschenbaum, B. et al. In vitro neuronal production and differentiation by precursor cells derived from the adult human forebrain. Cereb. Cortex4, 576−589 (1994). | PubMed | ISI | ChemPort |
Rakic, P. Limits of neurogenesis in primates. Science227, 1054−1056 (1985). | PubMed | ISI | ChemPort |
Eckenhoff M.F. & Rakic, P. Nature and fate of proliferative cells in the hippocampal dentate gyrus during the life span of the rhesus monkey. J. Neurosci.8, 2729−2747 (1988). | PubMed | ISI | ChemPort |
Kempermann, G., Kuhn, H.G. & Gage, F.H. Experience-induced neurogenesis in the senescent dentate gyrus. J. Neurosci.18, 3206−3212 (1998). | PubMed | ISI | ChemPort |
Gage, F.H. et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc. Natl. Acad. Sci. USA 92, 11879−11883 (1995). | PubMed | ChemPort |
Suhonen, J.O., Peterson, D.A., Ray, J. & Gage, F.H. Differentiation of adult hippocampus-derived progenitors into olfactory neurons in vivo. Nature383, 624−627 (1996). | Article | PubMed | ISI | ChemPort |
Acknowledgments We thank G. Kempermann, T. Palmer, E. Brandon, M.-C. Senut, K. Sakurada,
L. Chehabeddine and M. L. Gage for their comments, and H. van Praag for the
contribution of rat tissue. In addition, we thank L. Kitabayashi for technical
assistance. This study was supported by grants from the Swedish Medical Research
Council (project no. K98-12X-12535-01A), Faculty of Medicine, University of
Göteborg, the Gunvor and Josef Anérs Stiftelse, the John and Brit
Wennerströms Stiftelse for Neurologisk Forskning, the Rune and Ulla Amlövs
Stiftelse for Neurologisk and Reumatologisk Forskning, NHR-fonden, Stiftelsen
Göteborgs MS förenings forsknings och byggnadsfond, Stiftelsen Handlanden
Hjalmar Svenssons Forskningsfond, Göteborgs Läkaresällskap,
Hjärnfonden, The Swedish Society of Medicine, Stiftelsen Lars Hiertas
Minne, Stiftelsen Assar Gabrielssons Fond and the Edit Jacobssons Fond and
from NIA and NINDS and the Alzheimer's Association (F.H.G.) and the American
Federation for Aging Research (D.A.P.).
m.
s.e.m.) The corresponding
patient's age at the time of death and interval as BrdU infusion are given
for each BrdU-treated patient (n = 5).
