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The Journal of Neuroscience, July 15, 1999, 19(14):5792-5801
Estrogen Stimulates a Transient Increase in the Number of New
Neurons in the Dentate Gyrus of the Adult Female Rat
Patima
Tanapat,
Nicholas B.
Hastings,
Alison J.
Reeves, and
Elizabeth
Gould
Department of Psychology, Princeton University, Princeton, New
Jersey 08544
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ABSTRACT |
To determine whether a sex difference exists in the production of
hippocampal cells during adulthood, we examined proliferating cells and
their progeny in adult rats using the thymidine analog bromodeoxyuridine (BrdU) combined with immunohistochemistry for markers
of neurons and glia. Additionally, to determine whether ovarian
hormones affect cell proliferation, we examined the numbers of
BrdU-labeled cells at different estrous cycle stages and after ovarian
steroid manipulation. Stereological analyses of the numbers of
BrdU-labeled cells revealed that females produced more cells than males
in the dentate gyrus but not in the subventricular zone. The production
of new hippocampal cells in females appears to be affected by ovarian
hormone levels; ovariectomy diminished the number of BrdU-labeled
cells, an effect reversed by estrogen replacement. A natural
fluctuation in cell proliferation was also noted; females produced more
cells during proestrus (when estrogen levels are highest) compared with
estrus and diestrus. Many of these cells acquired neuronal
characteristics, including the formation of dendrites and expression of
Turned-On-After-Division 64 kDa, a marker of immature granule neurons,
and the calcium-binding protein calbindin, a marker of mature granule
neurons. However, examination of the numbers of pyknotic cells and the
numbers of BrdU-labeled cells at longer survival times revealed that
many new cells in the dentate gyrus eventually degenerate. Consistently the number of labeled cells in females is no longer higher than that
observed in males by 2 weeks after the last BrdU injection. These
findings suggest that estrogen-enhanced cell proliferation during
proestrus results in more immature neurons in the hippocampal formation
of females compared with males and present the possibility that these
new cells exert an important influence on hippocampal function.
Key words:
neurogenesis; dentate gyrus; granule neuron; sex
differences; estrous cycle; estrogen
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INTRODUCTION |
Granule neurons are produced in the
dentate gyrus throughout life. In adulthood, precursor cells located in
the hilus and subgranular zone (sgz) of the dentate gyrus divide and
produce daughter cells that ultimately differentiate into mature
granule neurons (Altman and Das, 1965 ; Bayer, 1982; Cameron et al.,
1993; Gould et al., 1997). Stereological analyses have demonstrated that thousands of new hippocampal granule cells are produced daily in
adult rodents (Gould et al., 1999). Moreover, recent studies have
demonstrated that this phenomenon also occurs in many different mammalian species (Gould et al., 1997, 1998, 1999a,b), including humans (Eriksson et al., 1998). The relatively high number of new
neurons produced in adulthood and the conservation of this process
across mammalian taxa suggest that these cells play an important role
in hippocampal function.
Several previous studies have reported sex differences in
hippocampal function. Specifically, sex differences have been observed in hippocampal long-term potentiation (Maren et al., 1994), as well as
in the performance of various hippocampal-dependent tasks (Roof and
Havens, 1992; Roof et al., 1993; Galea et al., 1996; Kavaliers et al.,
1996). In addition, sex differences in hippocampal structure have been
reported. With respect to the dentate gyrus, previous studies have
demonstrated that males have a greater total number of granule neurons,
as well as more mossy fiber synapses in the hilus than females (Madeira
and Paula-Barbosa, 1993). Females, on the other hand, exhibit
a greater number of mossy fiber synapses in the CA3 region (Madeira
et al., 1991).
As in other brain regions, it is likely that many observed sex
differences in hippocampal structure and function are dependent on the
levels of circulating gonadal hormones during either development or
adulthood (Arnold and Breedlove, 1985; McEwen et al., 1995). During
development, estrogen treatment has been shown to affect the volume of
the CA1 and CA3 regions, as well as spatial navigation behavior (Isgor
and Sengelaub, 1998). Similarly, previous studies have demonstrated
dramatic effects of the ovarian hormone estrogen during adulthood on
the number of hippocampal synapses (Woolley and McEwen, 1992), the
strength of hippocampal LTP (Cordoba Montoya and Carrer, 1997), and
hippocampal-dependent learning (Daniel et al., 1997; Packard and
Teather, 1997a,b; Luine et al., 1998). These findings raise the
possibility that sex differences exist in adult neurogenesis in the
dentate gyrus and furthermore that estrogen may play an important role.
To determine whether a sex difference exists in adult granule
neuron production and survival, we examined proliferating cells and
their progeny using the thymidine analog bromodeoxyuridine (BrdU) and
immunohistochemistry for markers of neurons and glia, as well as the
numbers of pyknotic cells, in adult male and female rats. In addition,
to establish whether the production of new hippocampal cells is
affected by changing levels of ovarian steroids in adulthood, we also
characterized the production and survival of new hippocampal cells
during different stages of the estrous cycle and after experimental
manipulations of ovarian steroids.
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MATERIALS AND METHODS |
Animal treatments. Adult male (320-350 gm) and
female (200-280 gm) Sprague Dawley rats of the same age from the
breeding colony at Princeton University were used in all experiments.
The animals were group housed and maintained on a 12/12 hr light/dark
cycle (lights on 7:00 A.M.), and provided with ad
libitum access to food and water. In each experiment, stage
of estrous was determined by examining vaginal cytology (Stockard and
Papanicolaou, 1917; Shors et al., 1998) for a minimum of two weeks
before treatment to insure that all female rats were cycling regularly.
The rat estrous cycle lasts 4 or 5 d and consists of three stages:
proestrus which lasts 12 hr, estrus which lasts 36 hr, and diestrus
which may last either 48 or 72 hr (Long and Evans, 1922). In general, ~98% of the animals were judged to be cycling normally. For
perfusion, all of the animals were deeply anesthetized with an overdose
of sodium pentobarbital [Nembutal; 100 mg/kg body weight (b wt)] and
then perfused transcardially with 4.0% paraformaldehyde in 0.1 M phosphate buffer. All animal experimentation was
conducted in accordance with University guidelines and with The
National Institutes of Health Guide for the Care and Use of
Laboratory Animals.
Experiment 1. To establish whether a sex difference exists
in the number of adult-generated hippocampal cells, adult female and
male rats were injected with BrdU (100 mg/kg b wt, i.p.), a marker of
proliferating cells and their progeny (Nowakowski et al., 1989), at
2:00 P.M. for 5 consecutive days. The animals were given daily
injections for 5 d to eliminate potential variability caused by
the stage of estrous of individual females at the time of injection. To
insure that each female rat would experience the same amount of time in
each stage of the estrous cycle during the injection period, only
female rats that were in diestrus on the first day of injection were
used. Groups of male and female rats (n = 4-6) were
then perfused either 2 or 14 d after the last injection. The
brains were removed, post-fixed, and processed immunohistochemically
for BrdU and BrdU combined with one of several markers including
Turned-On-After-Division 64 kDa (TOAD-64), a marker of immature neurons
(Minturn et al., 1995); the calcium binding protein calbindin, a marker
of mature granule neurons (Kempermann et al., 1997); and glial
fibrillary acidic protein (GFAP), an astroglial marker (Cameron et al.,
1993).
To determine whether potential sex differences in the numbers of
proliferating cells may be attributable to sex differences in the
numbers of progenitor cells in the adult dentate gyrus, adjacent
sections were also processed for Ki67 immunohistochemistry. Ki67 is a
protein that is expressed by cells throughout the cell cycle with the
exception of a short period at the beginning of G1 (Lopez et al.,
1991).
Experiment 2. To determine whether the proliferation of
granule cell precursors and the production of new neurons are affected by estrous cycle stage, adult female rats in either proestrus, estrus,
or diestrus were injected with BrdU (200 mg/kg b wt, i.p.) at 2:00 P.M.
and perfused after a 2 hr survival. For purposes of comparison, male
rats were injected with BrdU and perfused 2 hr later (n = 5-6). The 2 hr survival was chosen because it is sufficient for the
uptake of BrdU into proliferating cells but not for mitosis or
migration to occur (Nowakowski et al., 1989). Additional female rats
were injected with BrdU during either proestrus, a time of maximal
estrogen levels, or estrus, a time of low estrogen levels, and
subsequently perfused after the following survival times: 4, 7, 14, and
21 d (n = 3). After perfusion, all of the brains
were removed, post-fixed, and processed immunohistochemically for BrdU.
In the case of animals that had a survival time >2 hr, the brains were
also processed for combined immunohistochemistry for BrdU and either
TOAD-64, calbindin, or GFAP. Additionally, sections processed for
BrdU staining alone were stained with cresyl violet for analysis of the
numbers of pyknotic cells.
Experiment 3. To investigate the possibility that estrogen
regulates the proliferation of granule cell precursors, adult female rats were anesthetized with Nembutal (50 mg/kg b wt) and either ovariectomized or sham ovariectomized. Six days after surgery, some of
the ovariectomized rats were injected with 17- -estradiol in sesame
oil vehicle (10 µg/rat, s.c.) while the remaining rats were injected
with vehicle alone (n = 4-5). This dose was chosen because it results in circulating levels of estradiol (Viau and Meaney,
1991; Sohrabji et al., 1994) in the proestrus range. Two hours later,
all of the rats were injected with BrdU (200 mg/kg b wt, i.p.) and
perfused after a 2 hr survival. The brains were removed, post-fixed,
and processed immunohistochemically for BrdU. The sections were then
stained with cresyl violet for analysis of the numbers of pyknotic cells.
Histological procedures. For each brain, 40 µm sections
through the entire dentate gyrus were cut in a bath of 0.1 M PBS with an oscillating tissue slicer. Unless
otherwise indicated, all washes and incubations were performed in PBS
for peroxidase immunohistochemistry and in 0.1 M
Tris-buffered saline for fluorescence immunohistochemistry.
For peroxidase BrdU immunolabeling, the sections were incubated in 0.1 M citric acid for 5 min at 90°C, rinsed, incubated in
0.6% H2O2 for 30 min, rinsed, digested in
trypsin (0.001-0.05%) in Tris buffer containing 0.1%
CaCl2 for 10 min, rinsed, denatured in 2N HCl for 30 min,
rinsed (pH 6.0), blocked in 3.0% normal horse serum for 30 min,
and incubated overnight at 4°C in mouse monoclonal antibody against
BrdU (1:200 + 0.5% Tween 20; Novocastra, Newcastle Upon Tyne, UK). On
the next day, the sections were rinsed several times, incubated in
biotinylated mouse secondary antisera (1:50; Vector Laboratories,
Burlingame, CA) for 60 min, rinsed, incubated in
avidin-biotin-horseradish peroxidase (AB; 1:50; Vector Laboratories)
for 60 min, rinsed again, and reacted for 5 min in 0.02%
diaminobenzidine (DAB) with 0.003% H2O2 or in
DAB containing 0.1% nickel ammonium sulfate and then rinsed. For
combined labeling, sections were incubated in polyclonal antisera
against one of the following: (1) rabbit anti-TOAD-64 (a gift from Dr.
Susan Hockfield, 1:7500); (2) rabbit anti-calbindin (1:750
and 0.5% Tween 20; Chemicon, Temecula, CA); or (3) goat
anti-GFAP (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA). On the
following day, the sections were incubated in biotinylated secondary
antisera for 60 min, rinsed, incubated in AB for 60 min, rinsed, and
reacted in DAB for 5 min. The sections were then mounted onto coated
slides and dried, counterstained using cresyl violet, dehydrated, and coverslipped under Permount.
For fluorescence BrdU immunolabeling, sections were pretreated as
above, incubated in primary antibody against BrdU overnight, rinsed,
incubated in Cy3-conjugated mouse secondary antisera (1:200; Sigma, St.
Louis, MO) for 15 min, rinsed several times, and then incubated in
polyclonal antisera against either TOAD-64, calbindin, or GFAP. On the
next day, sections incubated in anti-TOAD-64 or anti-calbindin were
incubated in Alexa 488-conjugated rabbit secondary antisera (1:1000;
Molecular Probes, Eugene, OR) for 30 min. Sections that were incubated
in polyclonal antisera against GFAP were incubated in biotinylated
secondary goat antisera for 60 min, rinsed, and then incubated in
Cy2-conjugated avidin (1:1000; Amersham, Arlington Heights, IL) for 30 min. After rinsing, all of the sections were mounted, dried, and then
coverslipped under 30% glycerol in TBS.
Additional sections processed for Ki67 peroxidase immunohistochemistry
were incubated in 0.1 M citric acid at 90°C for 5 min, rinsed, and then incubated overnight in mouse monoclonal antisera against Ki67 (1:750; Novocastra). On the following day, the sections were rinsed, incubated in biotinylated mouse secondary antisera for 60 min, rinsed, incubated in AB for 60 min, rinsed and then reacted in DAB
for 5 min. The sections were mounted onto coated slides, dried,
counterstained using cresyl violet, dehydrated, and coverslipped under Permount.
Data analysis. Prior to data analysis all of the slides were
coded, and the code was not broken until the analysis was completed. Stereological estimates of the number of immunolabeled cells in the
entire dentate gyrus were determined for brains processed for
peroxidase BrdU or Ki67 immunolabeling and in the subventricular zone
(svz) for brains processed for peroxidase BrdU immunolabeling. Because
adult-generated cells that become neurons ultimately migrate out of the
hilus, the numbers of BrdU-labeled cells in the hilus and sgz and gcl
(combined) were initially examined separately for animals surviving
longer than 2 hr after BrdU injection. In addition, stereological
estimates of the number of pyknotic cells in the sgz and gcl were also
determined. Pyknotic cells were characterized by lack of nuclear
membrane, pale or absent cytoplasm, and condensed, darkly stained
spherical chromatin (Gould et al., 1991). The total number of pyknotic
or immunolabeled cells was determined using a modified version of the
optical fractionator method (West et al., 1991). All pyknotic or
BrdU-labeled cells in the dentate gyrus were counted on every 12th
section throughout the dentate gyrus at 1000×, omitting cells located
in the outermost plane of focus, using an Olympus BX-60 light
microscope attached to an Optronics color video camera and a Dell
OptiPlex computer. For sections processed for combined
immunohistochemistry, 4-6 anatomically matched sections from each
brain were analyzed. The numbers of BrdU-labeled cells in the hilus and
in the gcl and sgz (combined) that were immunoreactive or
nonimmunoreactive for either TOAD-64, calbindin, or GFAP were then
determined. Because BrdU-labeled cells in the hilus rarely express
TOAD-64, calbindin, or GFAP (our unpublished observations), they
are likely to primarily represent a progenitor population and were
therefore analyzed for double-labeling separately from those located in
the gcl and sgz. A minimum of 300-400 BrdU-labeled cells were analyzed
per brain, and the numbers of double-labeled cells were expressed as
percentages of the total number of BrdU-labeled cells in the respective
regions. For fluorescence immunolabeling, tissue was examined first
with a conventional fluorescence microscope (Olympus BX-60).
Double-labeled cells were then verified using a confocal laser scanning
microscope (Zeiss Axiovert 510 LSM). Z-sectioning at 1 µm intervals
was performed for analysis, and optical stacks of 5-6 images were
produced for figures. Bilateral volume estimates of the gcl were made
in experiments that compared the numbers of BrdU-labeled cells in male
and female rats. These estimates were made by applying Cavalieri's
Principle (Gundersen et al., 1988) to cross sectional area measurements
of the gcl obtained via ImagePro software (Media Cybernetics). In
addition, the optical fractionator method (West et al., 1991) was used
to attain bilateral estimates of the total number of granule neurons in
the dentate gyrus in experiments comparing males and females. An
unbiased counting grid was superimposed on the video image of the
section and granule cells in a 15 × 15 × 40 µm sample
volume were counted. Total cell counts were analyzed using one-way or
two-way ANOVAs followed by Tukey-HSD post hoc
comparisons. Cell percentages were analyzed using distribution-free
methods; the Kruskal-Wallis ANOVA by ranks followed by pairwise
comparisons using the Mann-Whitney U test.
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RESULTS |
Sex differences in adult-generated cells in the dentate gyrus
Female rats exhibited ~45% more BrdU-labeled cells in the
dentate gyrus compared with male rats 2 d after the last BrdU
injection (F(1,11) = 7.49;
p = 0.023) (Figs.
1A,
2A,B). Whereas the numbers of
BrdU-labeled cells in the sgz and gcl were higher in females, a sex
difference in the numbers of BrdU-labeled cells in the hilus was not
observed. In both males and females, the majority of BrdU-labeled cells
were located in the gcl or sgz and expressed the morphological characteristics of granule cell precursors, i.e., medium-sized, round,
or oval nuclei. The remaining BrdU-labeled cells had the morphological
characteristics of glial precursors, i.e., small-sized, triangular or
irregular-shaped cell bodies. Additionally, the percentage of
BrdU-labeled cells that were immunoreactive for either TOAD-64, GFAP,
or calbindin (Fig.
2C-E) did not
differ between sexes. At this time point, ~80% of BrdU-labeled cells in the gcl and sgz of males and females were immunoreactive for TOAD-64. These cells had the morphological characteristics of granule
cells, i.e., a small amount of cytoplasm in the cell body and dendrites
extending toward the molecular layer (Fig. 2C). In contrast,
<1% of BrdU-labeled cells were immunoreactive for calbindin, and
~10% were immunoreactive for GFAP. In the hilus, no BrdU-labeled
cells immunoreactive for either calbindin or TOAD-64 were observed and
<1% were immunoreactive for GFAP.
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Figure 1.
Stereological estimates of the total numbers of
BrdU-labeled cells in the dentate gyrus of adult male and female rats.
A, Two days after a period of five daily BrdU
injections, female rats had significantly more BrdU-labeled cells in
the dentate gyrus compared with age-matched males. B, By
14 d after the last BrdU injection, this sex difference was no
longer evident. No significant differences in the granule cell layer
volume (C) or total number of granule neurons
(D) were observed between males and females. Bars
represent mean + SEM, each obtained from four to six animals.
Asterisk indicates a significant difference
(p < 0.05).
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Figure 2.
Confocal laser-scanning images of BrdU-labeled
cells (arrowheads) in the dentate gyrus of female
(A) and male (B) rats
injected with BrdU five times and perfused 2 d after the last
injection. More BrdU-labeled cells were observed in females compared
with males. C, Confocal image of BrdU-labeled cells
colabeled with TOAD-64, a marker of immature neurons
(arrow), in the dentate gyrus of an adult female.
Arrowheads indicate TOAD-64-labeled cells not labeled
with BrdU. D, Light microscopic image of a BrdU-labeled
cell (arrow) that is not colabeled with GFAP, a marker
of astroglia, in the dentate gyrus of an adult female.
Arrowheads indicate GFAP-labeled cells not labeled with
BrdU. E, BrdU-labeled cell (arrow) that
is colabeled with calbindin, a marker of mature neurons, in the dentate
gyrus of an adult female. Arrowheads indicate
calbindin-labeled cells not labeled with BrdU. F,
Ki67-labeled cells (arrowheads) in the subgranular zone
of the dentate gyrus. These cells were similar in morphology and
location to BrdU-labeled cells at early time points. BrdU-labeled cells
(arrowheads) at 4 d (G),
7 d (H), and 21 d
(I) after BrdU injection in the dentate
gyrus of the adult female. With increasing time after BrdU injection,
many BrdU-labeled cells become incorporated into the granule cell layer
and express the morphological characteristics of mature granule
neurons. More BrdU-labeled cells (arrowheads) were
observed in animals injected during proestrus
(J) compared with those injected during
estrus (K). These animals were perfused 2 hr after injection with BrdU. L, Pyknotic cell
(arrowhead) in the dentate gyrus of an adult
female. These cells were more numerous in females than males during the
majority of the estrous cycle, g, Granule cell layer;
h, hilus. Scale bars: B, 25 µm (applies
to A, B); C, 10 µm;
E, 10 µm (applies to D,
E); F, 10 µm; I, 10 µm
(applies to G-I); K, 25 µm (applies to
J, K); L, 10 µm.
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A sex difference in the numbers of BrdU-labeled cells was no longer
evident by 14 d after the last BrdU injection (Fig.
1B). By this time point, the majority of the
BrdU-labeled cells in both males and females were fully incorporated
into the gcl and were morphologically identical to neighboring mature
granule neurons. Additionally, the percentages of BrdU-labeled cells
that were immunoreactive for calbindin or GFAP were similar in both
groups. Approximately 50% of BrdU-labeled cells in the gcl and sgz
were immunoreactive for calbindin, whereas ~10% were immunoreactive for GFAP. In the hilus, no BrdU-labeled cells immunoreactive for calbindin were detected, and <1% of BrdU-labeled cells were
immunoreactive for GFAP. The total volume of the gcl and the total
numbers of granule neurons were not statistically different between
males and females (Fig. 1C,D).
In addition, no sex differences in the numbers of Ki67-labeled cells in
the dentate gyrus were observed (F(1,4) = 1.39; p = 0.301). In both males (9448 ± 561.5)
and females (10592 ± 789.9), Ki67-labeled cells were located in
clusters primarily in the sgz (Fig. 2F) and exhibited
the morphological characteristics and approximate distribution of
BrdU-labeled cells 2 hr after BrdU labeling (Fig. 2, compare
F to J, K).
Estrous cycle effects on granule cell production in the adult
dentate gyrus
The total number of BrdU-incorporating cells observed in the
dentate gyrus fluctuated across the estrous cycle. In animals perfused
2 hr after BrdU injection, the numbers of BrdU-labeled cells in females
that were injected during proestrus were 50% greater than those
observed in female rats that were injected during either estrus or
diestrus, as well as compared with those observed in age-matched males
(F(3,17) = 5.61; p = 0.0073) (Fig. 3A). Regardless
of the stage of estrous at the time of BrdU injection, the majority of
the BrdU-labeled cells were located in the sgz and expressed the
morphological characteristics of granule cell precursors, whereas the
remaining BrdU-labeled cells expressed the morphological
characteristics of glial precursors. In contrast, no differences
between either males and females, or females at any stage of the
estrous cycle were detected in the numbers of BrdU-labeled cells in the
svz (F(3,15) = 2.32; p = 0.117). The total numbers of pyknotic cells observed in these animals
were inversely correlated with the numbers of proliferating cells; that
is, the numbers of pyknotic cells were ~40% lower in females that
were in proestrus on the day of perfusion compared with females that
were in estrus or diestrus, and compared with age-matched males
(F(3,17) = 3.48; p = 0.042)
(Fig. 3B). No differences in the distribution of pyknotic
cells were observed among groups. Pyknotic cells were located in the
sgz and gcl and were characterized by the lack of a nuclear membrane,
pale or absent cytoplasm, and condensed, darkly stained, spherical
chromatin (Fig. 2L).
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Figure 3.
Stereological estimates of the total number of
BrdU-labeled cells in the dentate gyrus (A) and
pyknotic cells in the sgz (B) of adult female
rats injected with BrdU during diestrus, proestrus, or estrus and in
age-matched male rats. All animals were perfused 2 hr after BrdU
injection. Female rats injected during proestrus had significantly more
BrdU-labeled cells compared with females in estrus or diestrus, as well
as compared with age-matched males. Conversely, female rats in
proestrus on the day of perfusion had significantly fewer pyknotic
cells compared with females in estrus or diestrus, as well as compared
with age-matched males. Bars represent mean + SEM each obtained from
five or six animals. Asterisk indicates a significant
difference from all other means (p < 0.05).
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In female rats that were injected with BrdU during either estrus or
proestrus, the total number of BrdU-labeled cells in the dentate gyrus
increased between 2 hr and 4 d after BrdU labeling. The total
numbers of BrdU-labeled cells remained elevated up to 7 d after
BrdU labeling and then gradually decreased
(F(3,15) = 13.7; p = 0.0001). The numbers of BrdU-labeled cells in females injected during
proestrus remained higher than those observed in females injected
during estrus until 14 d after BrdU labeling (F(1,15) = 9.24; p = 0.0083) (Fig. 4). By this time, many
BrdU-labeled cells had become incorporated into the gcl, expressed the
morphological characteristics of mature granule neurons, and expressed
calbindin, a marker of mature granule neurons (~35%). By 21 d,
the majority of these cells were located deep in the gcl and were
indistinguishable in morphology from neighboring granule cells. At this
time point, the difference in the numbers of BrdU-labeled cells in
females injected during either proestrus or estrus was no longer
detectable. Although the numbers of BrdU-labeled cells in the sgz and
gcl varied, no difference in the numbers of BrdU-labeled cells in the
hilus was observed between females injected during proestrus or estrus,
or at any time point.
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Figure 4.
Stereological estimates of the total number of
BrdU-labeled cells in the dentate gyrus of adult female rats 4, 7, 14, and 21 d after a single BrdU injection administered during
proestrus (black bars) or estrus (white
bars). The numbers of BrdU-labeled cells decreased over time in
both groups but were greater in females injected during proestrus up to
14 d after BrdU labeling. By 21 d, this difference was no
longer detectable. Bars represent mean + SEM each obtained from three
animals. Asterisk indicates significant difference from
proestrus (p < 0.05).
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The percentage of BrdU-labeled cells in all regions examined that were
immunoreactive for TOAD-64, calbindin, or GFAP was similar between
females injected during either proestrus or estrus (Table
1). In both groups, the percentage of
BrdU-labeled cells in the gcl and sgz that were immunoreactive for
calbindin demonstrated a gradual but steady increase from 2.5% at
4 d to 68% at 21 d (p = 0.0003). In
contrast, the percentages of BrdU-labeled cells in the gcl and sgz
that were immunoreactive for either TOAD-64 (~60%) or GFAP
(~15%) did not change significantly over time in either group.
Likewise, the percentages of double-labeled cells did not change in the
hilus. In females injected during proestrus or estrus, no BrdU-labeled
cells immunoreactive for calbindin or TOAD-64 were detected at any time
point in the hilus, and the percentage of BrdU labeled cells in the
hilus that were immunoreactive for GFAP was consistently <1%.
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Table 1.
Percentages of BrdU-labeled cells immunoreactive for
calbindin, TOAD-64, or GFAP in the dentate gyrus of adult female rats
at 4, 7, 14, and 21 d after BrdU injection
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Estrogen effects on cell proliferation in the dentate gyrus of
adult female rats
Ovariectomy produced a 60% decrease in the numbers of
BrdU-labeled cells in the dentate gyrus of adult female rats compared with those observed in both sham-operated animals (the majority of
which were in proestrus on the day of BrdU injection), as well as those
replaced with estrogen after ovariectomy
(F(2,12) = 24.23; p = 0.0001) (Fig. 5A). Conversely,
ovariectomized female rats demonstrated a twofold increase in the
number of pyknotic cells in the gcl and sgz compared with those
observed in sham-operated females or ovariectomized females that were
replaced with estrogen (F(2,17) = 24.04;
p = 0.0001) (Fig. 5B).
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Figure 5.
Stereological estimates of the total number of
BrdU-labeled cells (A) and pyknotic cells
(B) in the dentate gyrus of sham ovariectomized,
ovariectomized, and estrogen-replaced ovariectomized adult female rats.
Ovariectomy produced a significant decrease in the numbers of
BrdU-labeled cells in the dentate gyrus of adult female rats compared
with both sham-operated controls (the majority of which were in
proestrus at the time of BrdU injection) and estrogen-replaced,
ovariectomized animals. Conversely, ovariectomy doubled the numbers of
pyknotic cells in the dentate gyrus of adult female rats compared with
both sham-operated controls and estrogen-replaced, ovariectomized
animals. Bars represent mean + SEM each obtained from four or five
animals. Asterisk indicates significant difference from
other means (p < 0.05).
|
|
| |
DISCUSSION |
These findings demonstrate a sex difference in the production of
new cells in the dentate gyrus resulting from a higher rate of cell
proliferation in females during proestrus. The majority of new cells in
males and females were located in the gcl or sgz, exhibited
morphological characteristics of granule neurons, and expressed a
marker of immature granule neurons (TOAD-64) but not a marker of
astroglia (GFAP). However, our results indicate that under standard
laboratory conditions, a greater number of cells degenerate in females
than in males. This conclusion is consistent with our observations that
(1) a sex difference in the number of BrdU-labeled cells was no longer
detectable 2 weeks after BrdU injection; (2) females exhibited more
pyknotic cells in the sgz and gcl compared with males during the
majority of the estrous cycle; and (3) there was no sex difference in
the total number of granule neurons in the dentate gyrus.
Our results demonstrate a positive correlation between circulating
estrogen levels and cell proliferation across the estrous cycle, and
after experimental hormone manipulation. Conversely, we observed a
negative correlation between circulating estrogen levels and the
numbers of pyknotic cells. This suggests that estrogen not only
stimulates cell proliferation, but also exerts important survival
effects, a finding consistent with results of studies in avian systems
(Burek et al., 1995; Hidalgo et al., 1995). Although the population of
cells that degenerates in response to decreased estrogen levels is
undetermined, the presence of pyknotic cells in the sgz suggests that
some of these cells are new neurons.
The total number of new cells produced when estrogen was high
(proestrus) compared with when estrogen was low (estrus) remained elevated until 14 d after BrdU labeling. At this time, many new cells generated during estrus or proestrus expressed a marker of mature
granule neurons (calbindin) and were morphologically indistinguishable
from neighboring granule cells. However, a difference in the number of
new neurons produced during proestrus versus estrus was not detectable
21 d after labeling, presumably as a result of cell death.
Collectively, our observations indicate that proestrus-associated
increases in estrogen stimulate cell proliferation, which produces a
transient increase in the pool of new neurons in the dentate gyrus.
Given that new cells extend axons into the CA3 region 4-10 d after
BrdU incorporation (Hastings and Gould, 1999), these cells may have a
functional impact, even at an immature stage. Thus, sex differences in
immature neuron production may have a significant implications for
hippocampal structure and function.
Sex differences in hippocampal structure and function
Several studies have reported sex differences in hippocampal
structure, including a sex difference in the pattern of mossy fiber
innervation. Although the overall number of mossy fiber synapses does
not differ, male rats have more mossy fiber synapses in the hilus
(Parducz and Garcia-Segura, 1993), whereas females have more mossy
fiber synapses in the CA3 region (Madeira et al., 1991). Previous
studies have suggested that developmentally younger granule cells have
more restricted projection patterns (Gaarskjaer, 1985). Thus,
differences in granule cell production during adulthood may account for
sex differences in mossy fiber distribution. Although we found a sex
difference favoring females in adult granule cell production, we did
not observe sex differences in total granule cell number or gcl volume.
These findings differ from a previous report of more granule neurons
and larger gcl volume in males compared with females (Madeira and
Paula-Barbosa, 1993). This discrepancy may be caused by age differences
(the rats examined in our study were younger) and different methods of
determining cell number. We used the fractionator method, a
stereological method not confounded by volumetric differences between
groups, whereas the previous study used a method that involves
multiplying numerical density by gcl volume of two separate sets of animals.
Numerous studies have demonstrated sex differences in
hippocampal-dependent function and specifically, in
hippocampal-dependent learning. In general, these
studies report augmented learning in males compared with females.
For example, it has been shown that males perform better than females
on the Morris water maze spatial task in several rodent species,
including deer mice (Kavaliers et al., 1996), voles (Galea et al.,
1996), and rats [although conflicting reports exist concerning rats
(Roof and Havens, 1992; Roof et al., 1993; Bucci et al., 1995)].
However, a recent study has demonstrated that the sex difference in
rats favoring males in spatial learning on this task can be reversed
with previous familiarization to the testing apparatus (Perrot-Sinal et
al., 1996). Females learn spatial tasks better than males once the novelty of the task is removed. Similarly, another paradigm less prone
to performance confounds has also found a sex difference favoring
females in hippocampal-dependent learning. This study reported that
females learn trace eyeblink conditioning, a classical conditioning
paradigm that requires the hippocampus for acquisition, faster than
males (Wood et al., 1998). At present, the extent to which these sex
differences in hippocampal-dependent behaviors are related to sex
differences in adult cell production remains unknown.
Estrogen effects on hippocampal structure and function
Our findings demonstrate a novel effect of estrogen on the adult
hippocampal formation, the stimulation of new cell production. This
effect appears to be mediated by circulating estrogen levels during
adulthood; estrogen removal during adulthood decreased cell
proliferation, whereas estrogen treatment rapidly reversed this effect.
Additionally, no sex difference in the numbers of Ki67-labeled cells
was detected, suggesting that estrogen-induced changes in cell
proliferation did not result from differences in precursor cell number.
In other non-neuronal systems, estrogen has been shown to stimulate
mitosis (Watters et al., 1997; Lee and Eghbali-Webb, 1998), possibly by
regulation of the G1/S transition of the cell cycle (Geum et al., 1997;
Hong et al., 1998). The magnitude and speed of observed increases in
cell proliferation in the dentate gyrus suggest that estrogen may
disinhibit a population of precursor cells that are normally
prevented from progressing through the cell cycle at the G1/S
transition. Alternatively, estrogen may stimulate precursor cells to
divide at a faster rate by shortening G1.
Numerous studies have focused on ovarian steroid effects on hippocampal
structure and function in adulthood. Several of these have demonstrated
a positive correlation between estrogen and dendritic spine and synapse
density in the hippocampal CA1 region after experimental manipulations
and in response to natural fluctuations (Woolley et al., 1990; Woolley
and McEwen, 1992; Desmond and Levy, 1997). These observations have
prompted studies designed to investigate how differences in hippocampal
physiology associated with the estrous cycle, or with estrogen
treatment, affect performance of hippocampal-dependent tasks. Although
estrogen treatment has generally been found to enhance learning (Daniel
et al., 1997; Packard and Teather, 1997a,b; Luine et al., 1998;
Packard, 1998), the results of estrous cycle studies have been
conflicting (Warren et al., 1995; Stackman et al., 1997; Warren and
Juraska, 1997), most likely because of differences in estrous stage
performance and the tasks examined.
With regard to how the present findings relate to hippocampal-dependent
learning, it is important to note that estrogen-induced changes in cell
proliferation are unlikely to result in immediate changes in
hippocampal function. Because new cells require time to become
integrated into existing circuitry, the period after changes in cell
proliferation is likely to be the most functionally relevant.
Additionally, estrogen-induced stimulation of cell proliferation is
likely to have important functional consequences when estrogen levels
are elevated or diminished chronically, as during pregnancy or with aging.
In previous work, we have demonstrated that adrenal steroids inhibit
granule cell proliferation in male rats (Gould et al., 1992; Cameron
and Gould, 1994). Likewise, we have shown that stressful experiences,
which increase circulating levels of glucocorticoids, also suppress
cell proliferation, and ultimately, the production of new granule
neurons (Gould et al., 1997, 1998). Given that the
hypothalamic-pituitary-adrenal (HPA) axis is known to suppress gonadal steroid levels (Kamel and Kubajak, 1987; Rabin et al., 1988;
Laatikainen, 1991), the impact of estrogen-induced changes in cell
proliferation is likely to be complicated by interactions with
glucocorticoids. Moreover, numerous studies have demonstrated sex
differences in the HPA axis (Handa et al., 1994; Chisari et al., 1995)
and in the effects of stress (Kavaliers and Galea, 1995; Wood et al.,
1998), which are also likely to impact estrogen effects on cell proliferation.
Functional significance of adult-generated hippocampal neurons
The functional significance of granule neurons produced in
adulthood remains enigmatic. Recent studies have demonstrated that a
substantial number of new neurons are produced in adulthood in many
mammals (Cameron et al., 1993; Gould et al., 1997, 1998, 1999a,b),
including humans (Eriksson et al., 1998). Additionally, our
observations that new cells extend axons 4-10 d after mitosis (Hastings and Gould, 1999) suggest that they may be rapidly integrated into functional circuitry and thus, may play an important role in
hippocampal function.
The hippocampal formation has been implicated in certain types of
learning and memory. One theory proposes that the hippocampal formation
plays a transient role in memory storage (Squire and Zola, 1998). If
the hippocampal formation is necessary for temporary processing of
information that is sent elsewhere for storage, then a rejuvenating
population of neurons capable of rapidly forming synaptic connections
may be well suited to participate in such a function, a possibility
previously suggested for the bird song system (Nottebohm, 1989).
Further evidence for a possible role of adult-generated cells in
learning comes from studies of birds and mice demonstrating that
environmental complexity enhances the number of adult-generated
hippocampal neurons (Barnea and Nottebohm, 1994; Kempermann et al.,
1997). Our recent results have shown that hippocampal-dependent
learning increases the number of new granule neurons by enhancing their
survival, suggesting an important association between learning and
adult-generated neurons (Gould et al., 1999a). Comparisons
between sexes have not been performed for either enriched environment
or learning effects on new neurons. The results of the present report
demonstrating that females produced more new cells that acquire
characteristics of immature neurons than males, but that many of these
cells die in deprived laboratory conditions, present the intriguing
possibility that conditions of enhanced environmental complexity and
increased learning opportunities may have a greater impact on the
hippocampal structure in females than in males.
| |
FOOTNOTES |
Received Feb. 22, 1999; revised April 19, 1999; accepted April 27, 1999.
This work was supported by National Institutes of Health Grants MH52423
and MH59740 (E.G.).
Correspondence should be addressed to Patima Tanapat, Department of
Psychology, Princeton University, Green Hall, Princeton, NJ 08544.
| |
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TOP
ABSTRACT
INTRODUCTION
