Testosterone and Dihydrotestosterone, but not

Estradiol, Enhance Survival of New Hippocampal

Neurons in Adult Male Rats
Mark D. Spritzer, Liisa A.M. Galea
Department of Psychology, The University of British Columbia, Vancouver,
British Columbia, Canada V6T 1Z4

Received 17 November 2006; revised 11 January 2007; accepted 5 February 2007

ABSTRACT: Past research suggested that androgens

may play a role in the regulation of adult neurogenesis
within the dentate gyrus. We tested this hypothesis by
manipulating androgen levels in male rats. Castrated or
sham castrated male rats were injected with 5-Bromo20 deoxyuridine (BrdU). BrdU-labeled cells in the dentate gryus were visualized and phenotyped (neural or
glial) using immunohistochemistry. Castrated males
showed a signicant decrease in 30-day cell survival
within the dentate gyrus but there was no signicant
change in cell proliferation relative to control males,
indicating that androgens positively affect cell survival,
but not cell proliferation. To examine the role of testosterone on hippocampal cell survival, males were
injected with testosterone s.c. for 30 days starting the

INTRODUCTION
Older men with low levels of testosterone generally
perform poorly at cognitive tasks and have increased
incidence of neurodegenerative diseases compared to
older men with higher levels of testosterone (Baum,
2005; Hogoverst et al., 2005). Exogenous testosterone in older males can reduce the risk of Alzheimers
disease and improve cognition (Cherrier et al., 2005;
Hogoverst et al., 2005). Testosterone can be metaCorrespondence to: Dr. M. D. Spritzer (mspritze@middlebury.
edu)..
Contract grant sponsor: Canadian Institute for Health Research
(CIHR); contract grant number: MOP62929.
' 2007 Wiley Periodicals, Inc.
Published online 20 March 2007 in Wiley InterScience (www.
interscience.wiley.com).
DOI 10.1002/dneu.20457

day after BrdU injection. Higher doses (0.5 and 1.0 mg/
kg) but not a lower dose (0.25 mg/kg) of testosterone
resulted in a signicant increase in neurogenesis relative to controls. We next tested the role of testosterones two major metabolites, dihydrotestosterone
(DHT), and estradiol, upon neurogenesis. Thirty days
of injections of DHT (0.25 and 0.50 mg/kg) but not estradiol (0.010 and 0.020 mg/kg) resulted in a signicant
increase in hippocampal neurogenesis. These results
suggest that testosterone enhances hippocampal neurogenesis via increased cell survival in the dentate gyrus
through an androgen-dependent mechanism.
' 2007
Wiley Periodicals, Inc. Develop Neurobiol 67: 13211333, 2007

Keywords: adult neurogenesis; androgen; dentate gyrus;

male rats; testosterone

bolized to estradiol, and thus many of its neuroprotective effects are via estradiol. Nonetheless, testosterone, independent of the estradiol pathway, has
been shown to have some neuroprotective properties
(Frye and McCormick, 2000).
The dentate gyrus region of the hippocampus is
a site of adult neurogenesis within the mammalian
brain (van Praag et al., 2002; Jessberger and
Kempermann, 2003; Zhao et al., 2006). Two major
components of neurogenesis are generally measured:
the number of newly proliferated cells produced
(i.e. cell proliferation) and the number of cells surviving to different time points (i.e. cell survival). Both
these components of adult hippocampal neurogenesis
appear to be regulated by multiple factors, including
sex hormones (Galea et al., 2006). A reduction of
adult neurogenesis in the hippocampus has been asso1321

1322

Spritzer and Galea

ciated with impaired long-term memory in adult rats

(Snyder et al., 2005; Winocur et al., 2006), while a
decline in spatial memory in aged rats corresponds
with reduced survival of new neurons (Drapeau et al.,
2003; Wati et al., 2006 but see Bizon et al., 2004).
Therefore, a better understanding of the physiological
pathways that regulate adult neurogenesis may provide insights for treating age-related dementia.
Growing evidence indicates that male sex hormones (androgens) inuence adult neurogenesis. Testosterone implants increased neuron survival but had
no effect on cell proliferation in the HVC of female
canaries (Rasika et al., 1994; Louissaint et al., 2002).
Similarly, testosterone implants increased cell survival in the ventricular zone of male European starlings (Absil et al., 2003). Ormerod and Galea (2003)
found that compared to reproductively inactive male
voles, reproductively active males, with higher testosterone levels, have increased survival of newly proliferated cells but showed no signicant difference in
cell proliferation, which suggests that elevated androgen levels during the breeding season may enhance
hippocampal cell survival. Similarly, testosterone
implants had no effect on hippocampal cell proliferation in castrated male voles (Fowler et al., 2003).
Androgens, therefore, seem to increase survival of
new neurons but have no effect on cell proliferation;
however studies are needed to conrm this and determine the physiological mechanisms involved.
Estradiol is a major metabolite of testosterone
(Chung and Hu, 2002); therefore, testosterone could
inuence neurogenesis in males indirectly through its
aromatization to estradiol. Estradiol can enhance or
suppress hippocampal cell proliferation in adult
female rats depending on the dose and timing of estradiol administered (Ormerod et al., 2003a; Tanapat
et al., 2005). Estradiol enhanced hippocampal cell
survival in male voles, but only when administered
during the period of axon extension and not during
other stages of new neuron development (Ormerod
et al., 2004). These studies suggest that testosterone
could enhance survival of hippocampal neurons via
conversion to estradiol.
In the following experiments, we examined the
effects of androgens, and their metabolites on cell
proliferation and survival in the dentate gyrus in adult
male rats.

METHODS
Animals
Adult male Sprague Dawley rats (*60 days old) were purchased from at the University of British Columbia animal
Developmental Neurobiology. DOI 10.1002/dneu

care facility. Animals were housed individually in opaque

polyurethane bins with aspen-chip bedding in a temperature-controlled room [(21 6 1)8C] with a 12:12 h light/dark
cycle (lights on at 0700 h). Tap water and rodent chow
(Lab Diet No. 5012; Jamieson) were provided ad libitum
throughout the experiments. All experiments were conducted in accordance with the ethical guidelines set by the
Canadian council on animal care.

Surgery
Surgeries were conducted one week after rats arrived in the
colony using aseptic procedures. Rats weighed 270360 g
on the day of surgeries. Under halothane anesthesia (4% in
oxygen during induction, 2% in oxygen during maintenance), males were either bilaterally castrated or received
sham-castrations. For castrations, both testes were extracted
through a small incision made at the posterior tip of the
scrotum and were ligated with a silk suture. Sham operations involved incisions into the skin and muscle layers of
the scrotum that were sutured without removing the testes.
Immediately after surgery, Emla cream (2.5% lidocaine,
2.5% prilocaine) was applied to the incision site and each
rat was given a s.c. injection of Ketoprofen (5 mg/kg body
mass) as analgesics. One week was allowed for full recovery from surgery prior to further experimentation.

Experiment 1
Experiment 1 was designed to determine the effects of
castration on cell proliferation and survival in the dentate
gyrus. Males were given an i.p. injection of the thymidine
analog BrdU (5-Bromo-20 deoxyuridine; 200 mg/kg) to label
dividing cells and their progeny. BrdU (Sigma-Aldrich,
St. Louis, MO, USA) was prepared prior to injection by
dissolving 20 mg/mL in warm 0.9% saline buffered with
7 lL 2 N NaOH/mL saline. To determine the effects of
castration on cell proliferation, castrates (N 5) and shams
(N 5) were perfused 24 h after BrdU injection, as this is
sufcient for a single mitotic division (Cameron and
McKay, 2001). Cell survival was assessed by perfusing
castrates (N 5) and shams (N 5) 30 days after BrdU
injection.

Experiment 2
Experiment 2 was designed to determine the effects of
testosterone on 30-day cell survival in the dentate gyrus.
Castrated males were divided into four groups (N 5 per
group) that received 29 days of s.c. injections (0.1 mL) of
either sesame oil or one of three doses of testosterone
propionate (0.25 mg, 0.50 mg, or 1.00 mg/0.1 mL sesame
oil; Sigma-Aldrich). All subjects received an i.p. injection
of BrdU (200 mg/kg) 24 h prior to the rst day of testosterone injections, which allowed for one mitotic division prior
to starting testosterone injections and therefore any effects
of hormone treatment were because of changes in cell
survival independent of initial cell proliferation. Subjects

Androgens Enhance Adult Neurogenesis

1323

Figure 1 Photomicrographs of BrdU-labeled cells in the dentate gyrus of adult male rats. (A)
Photomicrograph (31000 magnication) of a cluster of newly proliferated cells located in the
subgranular zone between the granule cell layer (GCL) and hilus in the dentate gyrus of a shamcastrated adult male 24 h after BrdU injection. (B) Photomicrgraph (31000 magnication) of a
surviving BrdU-labeled cell in the subgranular zone in a castrated male rat 30 days after BrdU
injection and following 29 days of testosterone injections (1.0 mg). Arrows in A and B point to
BrdU-labeled cells. (C) Confocal image (3630 magnication) of the subgranular zone in a
castrated male rat 30 days after BrdU injection and following 29 days of testosterone injections
(0.5 mg). The arrow in C points to a cell that is double-labeled with BrdU and NeuN. Scale bars
represent 10 lm for each image.

were perfused 30 days after BrdU injection and 24 h after

the last testosterone injection.

Experiment 3
Experiment 3 was designed to determine the effects of
testosterone metabolites, dihydrotestosterone (DHT) and
estradiol, on cell survival. Castrated males were divided
into ve groups (N 7 per group) that received 29 days of
s.c. injections (0.1 mL) of either sesame oil, one of two
doses of DHT (0.25 or 0.50 mg/0.1 mL oil), or one of two
doses of 17b-estradiol benzoate (EB; 0.010 or 0.020 mg/0.1
mL oil). Hormones (Sigma-Aldrich) were dissolved in sesame oil and thoroughly mixed for each day of injections.
As for Experiment 2, all subjects received an i.p. injection
of BrdU (200 mg/kg) 24 h prior to the rst day of hormone
injections, and subjects were perfused 30 days after the
BrdU injection (24 h after the last hormone injections).

Histology
Rats were anesthetized with a lethal dose of sodium pentobarbital (Euthanyl; Bimeda-MTC, Cambridge, ON,
Canada) and perfused transcardially with 0.9% saline
followed by 4% paraformaldehyde. Brains were extracted
and postxed with 4% paraformaldehyde (48C) for 24 h.
Brains were then cryoprotected with 30% sucrose in 0.1 M
TBS (0.08 M Tris-HCl, 0.02 M Tris-base, 0.9% saline, pH
7.4) and stored at 48C until slicing. Brains were sliced into
40 lm coronal sections through the extent of dentate gyrus
in a bath of TBS using a vibratome (Leica VT1000S).

Tissue was collected and stored in antifreeze solution

(0.05 M TBS, 30% ethylene glycol, and 20% glycerol) at

208C until immunohistochemical processing.
Every 5th section was used for all staining procedures
and cell counting. For all experiments, peroxidase immunohistochemstry was performed on sections to visualize
BrdU-labeled cells. For Experiments 2 and 3, uorescence
immunohistochemistry was performed on sections from
three randomly selected subjects from each treatment group
in order to phenotype BrdU-labeled cells.

Immunohistochemistry
Peroxidase immunohistochemistry was performed on
free-oating tissue sections to visualize BrdU-labeled cells
[Fig. 1(A,B)]. The sections were rinsed three times for
10 min between steps with TBS (pH 7.4) unless otherwise
stated. Tissue was initially incubated for 30 min in 0.6%
H2O2 to eliminate endogenous peroxidase activity. DNA
was denatured by applying 2 N HCl for 30 min at 378C, and
this step was immediately followed by a 10 min incubation
in 0.1 M borate buffer (pH 8.5) to neutralize the acid.
Sections were blocked for 30 min with 3.0% normal donkey
serum (NDS; Chemicon, Temecula, CA, USA) in TBS and
then incubated 48 h at 48C in mouse monoclonal antibody
against BrdU (1:200 in TBS, 3% NDS and 0.1% Triton-X;
Roche Diagnostics, Laval, Quebec, Canada). Sections were
next incubated for 4 h at room temperature in donkey
anti-mouse secondary antibodies (1:100 in TBS; Vector
Laboratories, Burlington, ON, Canada). Sections were
incubated for 2 h in avidin-biotin horseradish peroxidase
Developmental Neurobiology. DOI 10.1002/dneu

1324

Spritzer and Galea

complex (ABC Elite Kit; 1:50; Vector Laboratories).

Sections were reacted for *5 min in a solution of 0.02%
diaminobenzidine (DAB; Sigma-Aldrich) and 0.003%
H2O2 in 0.1 M TBS. The sections were mounted onto Superfrost/Plus slides (Fisher Scientic, Nepean, ON, Canada)
and dried overnight. Finally, sections were counterstained
with cresyl violet acetate, dehydrated with ethanol, cleared
with xylene, and coverslipped with Permount (Fisher
Scientic).
Immunouorescent labeling was performed on freeoating tissue to determine the cellular phenotypes of BrdUlabeled cells [Fig. 1(C)]. Sections were triple stained for
expression of BrdU, neuronal nuclei (NeuN), and glial brillary acidic protein (GFAP). NeuN is expressed by mature
neurons (Cameron et al., 1993), and GFAP is expressed by
astroglial cells (Debus et al., 1983). The sections were
rinsed three times for 10 min between steps with TBS
(pH 7.4) unless otherwise stated. For each blocking step,
we used 0.1 M TBS with 3% NDS and 0.3% Triton-X 100.
All antibodies were diluted in 0.1 M TBS with 3% NDS
and 0.1% Triton-X 100. Sections were rst blocked for
30 min, followed by a 48 h incubation at 48C in a cocktail
of mouse monoclonal anti-NeuN (1:250; Chemicon) and
rabbit polyclonal anti-GFAP (1:500; Chemicon). Sections
were blocked for 30 min, followed by an overnight incubation at 48C in a cocktail of secondary antibodies:
donkey anti-mouse FITC (1:200; Jackson Immunoresearch,
West Gove, PA, USA) to visualize NeuN and donkey antirabbit Alexa647 (1:200; Molecular Probes, Eugene, OR,
USA) to visualize GFAP. Sections were xed in 4% paraformaldehyde for 10 min and then rinsed twice with 0.9%
saline. DNA was denatured by incubating sections in 2 N
HCl for 30 min at 378C. Sections were next rinsed three
times with 0.9% saline and blocked for 30 min. Sections
were next incubated 48 h at 48C with rat monoclonal antiBrdU (1:250; Oxford Biotechnology, Oxfordshire, UK).
Sections were blocked again for 30 min, followed by an
overnight incubation at 48C with donkey anti-rat Cy3
(1:500; Jackson Immunoresearch) to visualize BrdU-labeled
cells. Sections were mounted on Superfrost/Plus slides,
coverslipped with the antifading agent diazobicyclooctane
(TBS, 2.5% DABCO, 10% polyvinyl alcohol and 20%
glycerol), and stored at
208C.

cell body morphology to eliminate cells that were clearly

nonneuronal (Cameron et al., 1993; Ormerod and Galea,
2003). A stereological estimate of the total number of
BrdU-labeled cells in the granule cell layer and the hilus
was obtained for each rat by multiplying the number of
cells counted by 10 (i.e. inverse of the sampling ratio). For
each section, the area of the granule cell layer and the hilus
was determined using Simple PCI (version 5.1, Compix,
Imaging Systems). For each subject, the volume (mm3) of
each granule cell layer and hilus was then estimated using
Cavalieris method (Gundersen et al., 1998). Specically,
volume was calculated using the formula:
Volume 10sampling ratio 3

X

section area in mm2

3 0:040 mm section thickness

For Experiments 1 and 2, no signicant differences in
the volume of the dentate gyrus were observed among treatment groups, and therefore the density of BrdU-labeled
cells was not analyzed. However, for Experiment 3, there
was a nonsignicant trend for differences between treatments in dentate gyrus volumes, and therefore the density
of BrdU-labeled cell was also analyzed. Cell density was
calculated for each subject as the total number of BrdUlabeled cells within a sub-region of the dentate gyrus
(granule cell layer or hilus) divided by the volume (mm3)
of that sub-region.
Phenotypes of 25 BrdU-labeled cells/brain were determined by examining cells at 4003 magnication using a
uorescent microscope (Nikon Eclipse 600). The percentage of BrdU-labeled cells colabeled with NeuN and GFAP
was determined. Phenotypes were veried for a subset
(5 cells/brain) of BrdU-labeled cells using a confocal
microscope (BioRad 2000) at 6303 magnication (Ernst
and Christie, 2005). Z-sections at 0.4 lm intervals were
taken and optical stacks of 10 images were created with
NIH Image for PC (Scion Corporation, Frederick, Maryland) and imported into Adobe Photoshop (Adobe Systems,
San Jose, California) for channel merging. Cell images
were rotated in orthogonal planes to verify double labeling.

Hormone Assays
Cell Counting
The experimenter was blind to the treatment groups during
cell counting. For all sections, the granule cell layer and
hilus were scored separately. All BrdU-labeled cells in the
granule cell layer and hilus were counted exhaustively
for one side (chosen randomly) of every 5th section through
the entire rostrocaudal extent of the dentate gyrus (24
26 sections/brain) at 10003 magnication using a light
microscope (Nikon Eclipse 600). Cells observed within
20 lm of the inner edge of the granule cell layer (subgranular zone) were combined with the granule cell layer
counts. Cells were considered BrdU-labeled if they were
intensely stained and exhibited medium-sized round or oval
Developmental Neurobiology. DOI 10.1002/dneu

Testosterone and estradiol levels were assayed for all subjects. Blood was collected from the chest cavity at the time
of perfusion and samples were stored overnight at 48C.
Samples were centrifuged at 10,000 rpm for 15 min, and serum was decanted and stored at
208C. Serum testosterone
was assayed using an ImmuChem Coated Tube RIA Kit
(MP Biomedicals, Costa Mesa, CA, USA), and estradiol
was assayed using a Coat-A-Count RIA kit (Diagnostic
Products Corporation, Los Angeles, CA, USA). The lower
limit of detection was 0.2 ng/mL for testosterone and 8 pg/
mL for estradiol. The testosterone antibody had some crossreactivity with DHT (7.8%), 5a-androsterone-3b, 17b-diol
(2.2%), and 11-oxytestosterone (2.0%), but had no crossreactivity with progesterone, estrogens, or glucocorticoids

Androgens Enhance Adult Neurogenesis

1325

(all <0.01%). The estradiol antibody had moderate crossreactivity with estrone (10.0%), but no cross-reactivity with
testosterone, DHT, progesterone, or glucocorticoids (all
<0.01%). The intra-assay coefcients of variation were
8.2% for testosterone and 7.2% for estradiol. The interassay coefcient of variation for testosterone was 9.5% (estradiol was measured using a single assay).

Statistical Analyses
Total number of BrdU-labeled cells and dentate gyrus
volumes were each analyzed using repeated-measures
analysis of variance (ANOVA) with brain region (granule
cell layer, hilus) as the within-subjects factor and treatment
(castrate, sham-castrate, or hormone injection groups) as
the between-subjects factor. Hormone levels were compared
among treatment groups using t-tests (Experiment 1) or
ANOVAs (Experiments 2 and 3). Subjects that had hormone
levels below the detection limit of the assay were assigned
a concentration of 0 for statistical analyses. Post-hoc
comparisons were made using Neuman-Keuls procedure.
Statistica 6.1 (Statsoft, Tulsa, OK, USA) was used for all
analyses, and a signicance level of a 0.05 was used.

RESULTS
Experiment 1: Castration Reduces Cell
Survival but not Cell Proliferation
Castration had no signicant effect on cell proliferation in the granule cell layer or hilus but resulted in a
reduction in cell survival in the granule cell layer
after 30 days. Twenty-four hours after BrdU injection, castrated and sham-castrated males showed no
signicant difference in the total number of BrdUlabeled cells within the dentate gyrus [region 3 treatment interaction, p 0.31; treatment effect, p
0.41; Fig. 2(A)], indicating there was no signicant
effect of androgen status on cell proliferation. Signicantly more BrdU-labeled cells were found in the
granule cell layer than in the hilus [main effect of
region, F(1, 8) 29.5, p < 0.0001]. In contrast,
castration had a signicant effect on the total number
of BrdU-labeled cells in the dentate gyrus 30 days
after BrdU injection [region 3 treatment interaction,
F(1, 7) 17.3, p 0.004; Fig. 2(B)]. Castrated males
had signicantly fewer BrdU-labeled cells in the
granule cell layer (p < 0.001) but not in the hilus
(p 0.87) than did sham-castrated males.
Castration had no signicant effects on the volumes of the granule cell layer or the hilus (Table 1).
For the 30-day cell survival groups, there was a signicant region 3 treatment interaction for the volume
of the dentate gyrus [F(1, 7) 6.72, p 0.036], but
post-hoc tests revealed no signicant differences

Figure 2 Total number of BrdU-labeled cells (mean 6

S.E.M.) within the granule cell layer (white bars) and hilus
(black bars) of the dentate gyrus of castrated and shamcastrated male rats. (A) Castration had no effect on cell proliferation measured 24 h after BrdU injection but (B) castration caused a signicant reduction in the number of cells
surviving 30 days after BrdU injection. *Signicant difference between groups (p < 0.05).

between castrated and sham-castrated males in the

volume of the hilus (p 0.09) or the granule cell
layer (p 0.14). For the cell proliferation groups,
there was no signicant region 3 treatment interaction (p 0.18) or treatment effect (p 0.33).
Serum testosterone levels were signicantly higher
among sham-castrated males than among castrated
males for both the cell proliferation groups [t(7)
7.62, p 0.0001] and the 30-day cell survival groups
[t(7) 3.69, p 0.008; Table 2]. For the cell proliferation groups, serum estradiol levels were not
signicantly different between the castrated and
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Spritzer and Galea

1326

Table 1 Volumes (mean 6 SEM) of Granule Cell Layer (GCL) and Hilus Within the Dentate Gyri
for Males Used in Three Different Experiments
Experiment

Treatment

GCL Volume (mm3)

Hilus Volume (mm3)

Proliferation shams
Proliferation castrates
Survival shams
Survival castrates
Oil
0.25 mg T
0.50 mg T
1.00 mg T
Oil
0.25 mg DHT
0.50 mg DHT
0.01 Mg EB
0.02 Mg EB

5
4
4
5
5
5
5
4
7
7
7
7
7

2.82 6 0.17
2.97 6 0.24
3.20 6 0.14
2.95 6 0.13
3.30 6 0.11
2.83 6 0.05
3.37 6 0.12
3.17 6 0.24
3.51 6 0.21
3.55 6 0.10
3.48 6 0.22
3.19 6 0.18
3.01 6 0.13

7.09 6 0.48
7.96 6 0.93
7.50 6 0.21
7.84 6 0.29
7.02 6 0.42
6.38 6 0.31
7.35 6 0.43
6.90 6 0.19
7.92 6 0.44
7.92 6 0.33
7.56 6 0.59
7.21 6 0.42
6.59 6 0.51

There were no signicant differences among groups, although estradiol-treated males tended to have smaller volumes than controls
(p 0.055). T, testosterone; DHT, dihydrotestosterone; EB, 17b-estradiol benzoate.

sham-castrated males (p 0.94; Table 2). Perhaps

surprisingly, for the 30-day cell survival groups,
castrated males had signicantly higher estradiol levels than did sham-castrated males [t(7) 3.85, p
0.006]. However, note that the difference in the mean
estradiol levels between these two groups was only
4.5 pg/mL (Table 2).

injected control animals, and the level of neurogenesis in testosterone-injected castrates was consistent
with that observed among the sham-castrated males
of Experiment 1. Specically, testosterone signicantly increased the total number of BrdU-labeled
cells surviving for 30 days in the granule cell layer
compared with the oil-injected control group [region
3 treatment interaction, F(3, 15) 5.04, p 0.013;
Fig. 3]. Males injected with 0.5 or 1.0 mg testosterone
had signicantly more BrdU-labeled cells in the granule cell layer than did oil-injected controls [p
0.0013 and p 0.0022, respectively]. Males injected
with 0.25 mg testosterone showed a non-signicant
trend for more BrdU-labeled cells in the granule cell

Experiment 2: Testosterone Increases

Neurogenesis via Cell Survival Within
the Granule Cell Layer
Testosterone injections signicantly increased neurogenesis within the granule cell layer relative to oil-

Table 2 Hormone Concentrations (mean SEM) in Serum Collected at the Time of Perfusion
for Males Used in Experiment 13
Experiment

Treatment

T (ng/ml)

Estradiol (pg/ml)

Proliferation shams
Proliferation castrates
Survival shams
Survival castrates
Oil
0.25 mg T
0.50 mg T
1.00 mg T
Oil
0.25 mg DHT
0.50 mg DHT
0.01 mg EB
0.02 mg EB

5
4
4
5
5
5
5
4
7
7
7
7
7

1.57 6 0.18
<0.20a
1.93 6 0.60
<0.20a
<0.20
2.33 6 0.24
4.30 6 0.57
6.33 6 0.97
<0.20a
<0.20a
<0.20a
<0.20a
<0.20a

12.4 6 1.3
11.5 6 1.4
10.3 6 0.8
14.8 6 0.8
10.7 6 1.6
11.1 6 1.1
16.9 6 4.2
18.8 6 2.0
13.5 6 1.1
14.1 6 1.3
12.5 6 1.3
137.1 6 17.4
526.7 6 59.3

Testosterone levels were signicantly higher in the sham castrate groups compared to castrates in Experiment 1. Testosterone levels were
signicantly higher in the testosterone-treated groups compared to controls and each other group in Experiment 2. Testosterone levels were
undetectable and estradiol levels were signicantly higher in the EB groups compared to controls in Experiment 3.
a
Indicates all samples below the detection limit of the assay. T, testosterone; DHT, dihydrotestosterone; EB, 17b-estradiol benzoate.

Developmental Neurobiology. DOI 10.1002/dneu

Androgens Enhance Adult Neurogenesis

1327

treatment effect, p 0.11) and *70% of BrdUlabeled cells were colabeled with the neuronal marker
NeuN (Table 3).
As expected, higher doses of testosterone resulted
in a signicant increase in serum testosterone levels
at the time of perfusion [F(2, 11) 17.87, p 0.003;
Table 2], and post-hoc comparisons showed that all
groups differed signicantly from one another (all
p < 0.05). Serum estradiol levels also showed a weak
tendency to increase with higher injected doses of testosterone, but these differences were not signicant
(p 0.11; Table 2).
Figure 3 Total number of BrdU-labeled cells (mean 6
S.E.M.) within the granule cell layer (white bars) and hilus
(black bars) of the dentate gyrus of castrated male rats following injections with vehicle (sesame oil) or testosterone
propionate for 30 days. The low dose of testosterone
(0.25 mg) had no signicant effect on cell survival, while
the two higher doses (0.5 and 1.0 mg) caused a signicant
increase in cell survival. *Signicantly different from the
oil control group (p < 0.05).

layer than among control males (p 0.053). There

were no signicant differences between groups in
total number of BrdU-labeled cells in the hilus (all
p > 0.30).
Testosterone injections had no signicant effects
on dentate gyrus volumes [region 3 treatment interaction, p 0.86; treatment effect, p 0.09; Table 1],
but as expected there was a signicant main effect of
area (p < 0.0001), indicating that the hilus had a
larger volume than the granule cell layer. The percentage of BrdU-labeled cells that colabeled with
NeuN and GFAP did not differ signicantly among
groups (phenotype 3 treatment interaction, p 0.71;

Experiment 3: DHT, but not EB, Increases

Neurogenesis via Cell Survival Within
Granule Cell Layer
DHT, but not EB, signicantly increased neurogenesis within the granule cell layer relative to oil-injected
controls. Furthermore, the level of neurogenesis in
the DHT-injected groups was consistent with the levels observed in the testosterone-injected males in
Experiment 2 and the sham-castrated males of
Experiment 1. Specically, the total number of BrdUlabeled cells surviving for 30 days within the granule
cell layer was signicantly greater in males injected
with 0.25 or 0.50 mg of DHT than in oil-injected controls [region 3 treatment interaction, F(4,30) 6.99,
p 0.0004; post-hoc tests, all p < 0.0005; Fig. 4].
There was also a trend for males injected with estradiol to have a smaller dentate gyrus volume than did
males in the other treatment groups (treatment effect,
p 0.055; region 3 treatment interaction, p 0.28;
Table 1). Because of this trend, the density of BrdUlabeled cells (cells/mm3) was also analyzed. As for
total number of BrdU-labeled cells, the density of
BrdU labeled cells was signicantly different among

Table 3 Percentage (mean SEM) of BrdU-Labeled Cells in Granule Cell Layer of the Dentate Gyrus that
were Colabeled with NeuN, Colabeled with GFAP, or Labeled with BrdU Only for Experiments 2 and 3
Treatment
Oil
0.25 mg T
0.50 mg T
1.00 mg T
Oil
0.25 mg DHT
0.50 mg DHT
0.01 mg EB
0.02 mg EB

Experiment

BrdU/NeuN (%)

BrdU/GFAP (%)

BrdU only (%)

2
2
2
2
3
3
3
3
3

70.7 6 6.1
73.3 6 2.3
69.3 6 3.5
66.7 6 7.4
78.7 6 1.3
72.0 6 4.0
77.3 6 1.3
76.0 6 2.3
77.3 6 5.3

13.3 6 5.8
4.0 6 2.3
4.0 6 2.3
4.0 6 2.3
8.0 6 2.3
5.3 6 3.5
8.0 6 2.3
5.3 6 2.7
9.3 6 3.5

16.0 6 4.0
22.7 6 1.3
26.7 6 1.3
29.3 6 5.3
13.3 6 1.3
22.7 6 1.3
14.7 6 1.3
18.7 6 4.8
13.3 6 3.5

In both studies, there was a greater percentage of BrdU-labeled cells that colabeled with NeuN. Percentages were calculated using
25 BrdU-labeled cells per brain and 3 subjects per treatment group. T, testosterone; DHT, dihydrotestosterone; EB, 17b-estradiol benzoate.

Developmental Neurobiology. DOI 10.1002/dneu

1328

Spritzer and Galea

Figure 4 Total number of BrdU-labeled cells (mean 6 SEM) within the granule cell layer (white
bars) and hilus (black bars) of the dentate gyrus of castrated male rats following injections with vehicle (sesame oil), DHT, or estradiol for 30 days. Both the 0.25 mg and 0.50 mg doses of DHT
caused a signicant increase in cell survival, while both doses of estradiol had no signicant effect
on cell survival. *signicantly different from the oil control group (p < 0.05).

treatment groups [region 3 treatment interaction:

F(4,30) 5.03, p 0.003], with the two DHTinjected groups, but not the estradiol-injected groups,
signicantly greater than the control group.
The percentage of BrdU-labeled cells that colabeled with NeuN and GFAP did not differ signicantly among groups (phenotype 3 treatment interaction, p 0.98; treatment effect, p 0.17) and 70
80% of BrdU-labeled cells were colabeled with the
neuronal marker NeuN (Table 3).
Serum testosterone levels were below the detection limit of the assay (0.20 ng/mL) for all rats. Serum estradiol levels differed signicantly among
groups [F(4, 30) 64.5, p < 0.0001; Table 2]. The
two groups injected with estradiol had signicantly
higher estradiol levels than all other groups (all p <
0.02), and the 0.02 mg estradiol group had a signicantly higher estradiol level than did the 0.01 mg estradiol group (p 0.0001). The two DHT injected
groups did not differ signicantly from each other or
the oil injected group in estradiol level (all p > 0.95).

DISCUSSION
In the present study we found that castration had no
signicant effect on hippocampal cell proliferation
but caused a decrease in hippocampal cell survival in
adult male rats, indicating that androgens positively
affect new cell survival. High levels of testosterone
increased hippocampal neurogenesis likely via cell
survival compared to control males. The testosterone
metabolite DHT, but not estradiol, increased hippocampal neurogenesis likely via cell survival compared to control males. These results suggest that tesDevelopmental Neurobiology. DOI 10.1002/dneu

tosterone enhances hippocampal neurogenesis via

increased cell survival in the dentate gyrus through
an androgen-dependent, but not an estrogen-dependent,
mechanism.

Androgens Enhance Adult Hippocampal

Neurogenesis
Our nding that androgens increase neuron survival
but not cell proliferation within the dentate gyrus is
supported by previous studies. Reproductively active
and reproductively inactive male meadow voles
showed no difference in levels of cell proliferation in
the dentate gyrus (Galea and McEwen, 1999;
Ormerod and Galea, 2003). However, reproductively
active male voles had higher levels of cell survival in
the dentate gyrus than did reproductively inactive
males (Ormerod and Galea, 2003). These results parallel our ndings for castrated versus intact male rats,
given that reproductively active male voles have
higher circulating androgen levels than do reproductively inactive males (Galea and McEwen, 1999).
Fowler et al. (2003) found that implants of testosterone or DHT had no effect on cell proliferation within
the dentate gyri of castrated male meadow voles, further supporting the conclusion that androgens do not
signicantly inuence hippocampal cell proliferation.
Similarly, testosterone implants given to birds
increased cell survival but not cell proliferation in
various brain regions (Rasika et al., 1994; Absil et al.,
2003). Interestingly, Brown et al. (1993) observed no
effect of testosterone implants on 7-day cell survival
in the HVC of female canaries, whereas Rasika et al.
(1994) found that testosterone implants caused an

Androgens Enhance Adult Neurogenesis

increase in 60-day cell survival in the HVC of male

and female canaries. This suggests that the effects of
androgens on the survival of new neurons may be
more pronounced over longer time periods like the
30-day survival period used for the present experiment.
We found that the number of BrdU-labeled cells
24 h after BrdU injection was not affected by castration but that the number of BrdU-labeled cells 30
days after BrdU injection was affected by both castration as well as injection of testosterone or DHT. We
have interpreted this to indicate that androgens inuence cell survival but not cell proliferation within the
dentate gyrus. In Experiments 2 and 3, hormone
injections were not given until 24 h after BrdU injection to test the effects of hormones on cell survival.
However, some BrdU-labeled progenitor cells may
have undergone subsequent divisions 24 h after BrdU
injection, and we may have observed some effects of
hormones on cell proliferation even with our 30-day
time scale. Using a mouse model, Zhao et al. (2006)
recently demonstrated that only 18% of cells within
the dentate gyrus are still proliferating at 3 days after
initial labeling, and this number drops to 3.5% by 7
days after initial labeling. This suggests our observed
effects at 30 days post labeling are indeed primarily
because of the effects of hormones on cell survival.
Furthermore, we did not nd an effect of androgen removal on initial cell proliferation (i.e. 24 h after
BrdU injection), which indicates that hormonal
effects on subsequent cell divisions are unlikely.
Finally, by 30 days post injection, a majority of cells
expressed mature cell markers and were located in
the granule cell layer, not in the proliferative subgranular zone (Fig. 1; Table 3).
Brannvall et al. (2005) found that adult male rats
injected with the anabolic steroid nandrolone (19-nortestosterone) had decreased cell survival (5-day and
14-day) within the dentate gyrus. This result seems to
contradict our nding that testosterone and DHT
enhanced survival of new neurons. The difference in
results may have been due to differences in the hormones used (nandrolone vs testosterone or DHT) or
because of the relatively high dose nandrolone used
(*4 mg/rat). Although nandrolone is a testosterone
analogue, there is some evidence that at high doses it
alters brain functions and behavior in ways that differ
from physiological levels of testosterone (Linfqvist
et al., 2002; Kindlundh et al., 2003). Furthermore, in
vitro studies have shown that low doses of testosterone can induce neurite outgrowth (Estrada et al.,
2006a), whereas high doses induce apoptosis in neurons (Estrada et al., 2006b). Hence, the effects of
androgens on hippocampal neurogenesis may be

1329

dose-dependent, with lower androgen levels enhancing neurogenesis, and higher androgen levels suppressing neurogenesis.
Estradiol has been previously shown to inuence
neurogenesis in the adult dentate gyrus of female
rodents. Numerous studies have shown that acute
administration of estradiol increases cell proliferation
among female rodents (Tanapat et al., 1999, 2005;
Banasr et al., 2001; Ormerod et al., 2003a,b). We did
not observe a signicant effect of castration on cell
proliferation among male rats, but we also did not
observe a signicant change in estradiol levels among
these animals. However, circulating estrogen levels
in males in the present study may have been too low
to inuence cell proliferation. Relatively few studies
to date have examined the effects of estradiol on cell
survival in the dentate gyrus in adult rodents.
Ormerod et al., 2004 found that ve days of estradiol
injections (0.01 mg) increased 16-day cell survival
among male meadow voles. However, this effect was
only observed if males were injected with estradiol
during the axon-extension phase of neurogenesis
(i.e. 610 days following BrdU injection) and not
during other phases of neurogenesis. Therefore, estradiol may be effective in increasing neurogenesis
among male rodents only if administered during specic time periods in cell maturation cycle rather than
continuously as was done in our study.

Possible Mechanisms for Androgen

Effects on Neurogenesis
Androgens could bind to receptors on newly proliferated cells within the adult hippocampus to inuence
their survival directly. Androgen receptors and androgen receptor mRNAs have been localized within the
CA1 sub-region of the hippocampus of the adult male
rats (Kerr et al., 1995; Xiao and Jordan 2002). Although at lower levels than in the CA1, androgen
receptors have also been detected in the dentate gyrus
(Brannvall et al., 2005; Tabori et al., 2005). Interestingly, androgen receptors have been found in neural
stem cell cultures developed from cells from the subventricular zone of adult female rats (Brannvall et al.,
2005). Hence, androgens could increase hippocampal
neurogenesis by binding to androgen receptors on
newly proliferated cells.
After passing through the cell membrane, androgens generally bind to the cytoplasmic androgen
receptors to act as a transcription factors (Heinlein
and Chang, 2002), and changes in gene transcription
could explain the effects of androgens on neurogenesis. Besides this classical pathway, androgens have
Developmental Neurobiology. DOI 10.1002/dneu

1330

Spritzer and Galea

been shown to act through a variety of other pathways

(Heinlein and Chang, 2002), and one of these alternative pathways may be involved in the increased neurogenesis induced by testosterone and DHT. For
example, 5a-androstane-3b, 17b-diol (3b-diol) is a
metabolite of DHT, and it can bind to neuronal estrogen receptors to have genomic effects (Pak et al.,
2005). Hence, although DHT cannot be aromatized to
estradiol, it can act on estrogen receptors via 3b-diol.
However, the fact that estradiol administration in the
present study had no signicant effect on the survival
of new neurons in Experiment 3 suggests that estrogen receptor binding is not involved in the survivalenhancing effects of DHT.
Androgens may also inuence adult neurogenesis
indirectly by altering levels of neurotrophic factors
within the brain. For example, female canaries given
testosterone implants showed increased levels of
brain-derived neurotrophic factor (BDNF), and
BDNF infusions increased neuron survival within the
HVC (Rasika et al., 1999). Vascular endothelial
growth factor (VEGF) seems to play an intermediary
role between testosterone and BDNF among canaries
(Louissaint et al., 2002). Specically, testosterone
implants resulted in increased VEGF, which in turn
led to increased proliferation of capillary epithelial
cells within the HVC. These new epithelial cells were
shown to synthesize BDNF, which promoted neurogenesis within the HVC. Some evidence suggests that
similar interactions between testosterone and trophic
factors inuence neurogenesis within the rodent hippocampus. Both BDNF and VEGF have been associated with increased survival of newly proliferated
neurons within the dentate gyrus (Lee et al., 2002;
Cao et al., 2004; Scharfman et al., 2005). Male rats
have higher levels of BDNF within the dentate gyrus
than do females (Franklin and Perrot-Sinal, 2006),
suggesting that BDNF levels may be regulated by sex
hormones. Testosterone, BDNF and VEGF all decline
with increasing age among male rats (Wang et al.,
1993; Chen et al., 1994; Hattiangady et al., 2005;
Shetty et al., 2005), and aged males show reduced
hippocampal neurogenesis relative to young males
(Heine et al., 2004; McDonald and Wojtowicz,
2005). Surprisingly, Bimonte-Nelson et al. (2003)
found no effect of DHT or testosterone implants upon
BDNF levels within the hippocampi of aged male
rats, perhaps because of a reduction in hippocampal
androgen receptors with age.
Androgens can have neuroprotective effects
against a wide range of damage including kainate
lesions (Ramsden et al., 2003b), oxidative stress
(Ahlbom et al., 1999, 2001), hyperphosphorylation of
s protein (Papasozomenos and Shanavas, 2002), and
Developmental Neurobiology. DOI 10.1002/dneu

b-amyloid toxicity (Gouras et al., 2000; Pike, 2001;

Ramsden et al., 2003a; Zhang et al., 2004). Although
some of the neuroprotective properties of testosterone
may occur through aromatization to estradiol (e.g.
Heyer et al., 2005), growing evidence indicates that
neuroprotection can also occur via androgen-dependent pathways (Pike, 2001; Hammond et al., 2001;
Ahlbom et al., 2001). Among castrated male rats,
DHT implants caused a signicant reduction in neuronal loss in the hippocampus following kainite
lesions (Ramsden et al., 2003b). Some recent studies
have shown that the neuroprotective properties of
androgens involve activation of a mitogen-activated
protein kinase (MAPK) pathway (Nguyen et al.,
2005; Gatson et al., 2006), and MAPK pathways
have been shown to increase neuron survival in a
wide range of contexts (reviews: Johnson and Lapadat, 2002; Cavanaugh, 2004; Hetman and Godz,
2004). Therefore, androgens may activate the MAPK
pathway to increase hippocampal neurogenesis via
neuron survival.
In addition to proximate mechanisms, there are
also a number of evolutionary explanations for our
results. Testosterone levels uctuate naturally during
the lifespan of a male rodent, and presumably the survival of new granule neurons will be enhanced during
periods of elevated testosterone. For example, male
meadow voles show enhanced neurogenesis during
the breeding season when testosterone levels are elevated (Ormerod and Galea, 2003). Furthermore, male
meadow voles expand their home ranges during the
breeding season, (Gaulin and FitzGerald, 1989), and
males with better spatial ability range more widely
and locate more mates (Spritzer et al., 2005). Thus,
elevated testosterone during the breeding season may
promote neuron survival and better spatial ability,
which in turn allows males to more effectively locate
mates. As a second example, both testosterone levels
and cell survival are elevated in socially dominant
male rats relative to subordinates (Schuurman, 1980;
Kozorvitskiy and Gould, 2004). Although speculative, this may be because dominant individuals
engage in more social interactions than subordinates
and therefore must remember more social relationships. In support of this idea, there is some evidence
that better memory of social interactions correlates
with increased neurogenesis (Olariu et al., 2005).

Implications
This study demonstrated that androgens enhance neurogenesis via an androgen-dependent pathway. Some
evidence indicates that age-related dementia is also

Androgens Enhance Adult Neurogenesis

related to androgen levels. Although our study was

not conducted with aged males, it may provide some
insights into the potential link between testosterone
levels and age-related dementia. As in rodents, testosterone levels decline with increasing age in men
(Harman et al., 2001), and older men with higher testosterone levels perform better at memory tasks
(Moffat et al., 2002; Yaffe et al., 2002; Driscoll et al.,
2005). Furthermore, free testosterone levels were
found to be lower in men who later developed
Alzheimers disease (Moffat et al., 2004). Adult neurogenesis may provide a link between reduced testosterone and impaired cognitive abilities among elderly
men. Among rats, there is a dramatic decrease in hippocampal neurogenesis with increasing age (Heine
et al., 2004; McDonald and Wojtowicz, 2005).
Drapeau et al. (2003) found that aged males with
impaired performance on the Morris water maze had
lower levels of survival of new neurons in the dentate
gyrus compared to aged males with unimpaired performance in the Morris water maze. However, the
survival of newly proliferated neurons seems to be
specically important in long-term memory formation rather than task acquisition in the water maze
(Ambrogini et al., 2004; Snyder et al., 2005). Wati
et al. (2006) found that aged males showed both
impaired contextual memory and decreased cell survival within the dentate gyrus relative to young
males. Thus, these ndings, coupled with the ndings
from the present study suggest that testosterone
enhances the survival of new hippocampal neurons,
and that maintaining testosterone levels into old age
could have similar promoting effects on hippocampal
neurogenesis and possibly learning and memory. To
test this hypothesis, future studies should examine the
effects of androgens on neurogenesis in aged subjects.
The authors thank Stephanie Lieblich for assistance with
experimental procedures. We thank Alice Chan, Anne
Cheng, Pralle Kriengwatana, and Wayne Yu for their assistance. LAMG is a Michael Smith Senior Scholar.

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Developmental Neurobiology. DOI 10.1002/dneu