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The Journal of Neuroscience, May 1, 2002, 22(9):3608-3614
Estrogen and Aging Affect the Subcellular Distribution of
Estrogen Receptor- in the Hippocampus of Female Rats
Michelle M.
Adams1,
Susan E.
Fink1,
Ravi A.
Shah1,
William G. M.
Janssen1,
Shinji
Hayashi2,
Teresa A.
Milner3,
Bruce S.
McEwen4, and
John H.
Morrison1
1 Kastor Neurobiology of Aging Laboratories, Fishberg
Research Center for Neurobiology, and Henry L. Schwartz Department of
Geriatrics and Adult Development, Mount Sinai School of Medicine, New
York, New York 10029, 2 Laboratory of Endocrinology,
Graduate School of Integrated Science, Yokohama City University,
Yokohama 236-0027, Japan, 3 Department of Neurology, Weill
Medical College of Cornell University, New York, New York 10021, and
4 Harold and Margaret Milliken Hatch Laboratory of
Neuroendocrinology, The Rockefeller University, New York, New York
10021
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ABSTRACT |
Estrogen replacement increases both the number of dendritic spines
and the density of axospinous synapses in the hippocampal CA1 region in
young rats, yet this is attenuated in aged rats. The estrogen
receptor- (ER-) is localized within select spines of CA1
pyramidal cells in young animals and thus may be involved locally in
this process. The present study investigated the effects of estrogen on
the ultrastructural distribution of ER- in the CA1 of young (3-4
months) and aged (22-23 months) Sprague Dawley rats using
postembedding immunogold electron microscopy. Within dendritic spines,
most ER- immunoreactivity (IR) was seen in plasmalemmal and
cytoplasmic regions of spine heads, with a smaller proportion within 60 nm of the postsynaptic density. In presynaptic terminals, ER--IR was
clustered and often associated with synaptic vesicles. Significant
effects of both aging and estrogen were observed. Quantitative analysis
revealed that nonsynaptic pools of ER--IR within the presynaptic and
postsynaptic compartments were decreased (35 and 27%, respectively) in
the young estrogen-replaced animals compared with those that received
vehicle. Such localized regulation of ER- in response to circulating
estrogen levels might directly affect synaptic signaling in CA1
pyramidal cells. No estrogen treatment-related differences were
observed in the aged animals. However, 50% fewer spines contained
ER- in the aged compared with young hippocampus. These data suggest
that the decreased responsiveness of hippocampal synapses to estrogen in aged animals may result from age-related decrements in ER- levels
and its subcellular localization vis-à-vis the synapse. Such a
role for spinous ER- has important implications for age-related attenuation of estrogen-induced hippocampal plasticity.
Key words:
CA1; ovariectomy; postembedding immunogold; electron
microscopy; synaptic plasticity
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INTRODUCTION |
The role of estrogen in controlling
the reproductive axis at the level of the hypothalamus has been studied
for many years and has been characterized in great detail (Fink, 1986 ).
However, estrogens also affect synaptic communication in brain regions involved in cognitive processing, such as the hippocampus (Woolley, 1998), and these effects may be of particular importance in the context
of aging, when both circulating estrogen levels change and
hippocampal-dependent functions decline (Sherwin, 2000). Our current
understanding of the effects of estrogen on synaptic plasticity in the
hippocampus is based primarily on data from young animals. For example,
dendritic spine density in CA1 pyramidal cells is sensitive to
naturally occurring estrogen fluctuations in young animals (Woolley et
al., 1990) as well as experimentally induced estrogen depletion and
replacement (Gould et al., 1990; Woolley and McEwen, 1992, 1993;
Woolley et al., 1996).
For the hypothalamus, it is well documented that aged animals do not
have the same reaction to estrogen deprivation as young animals (Wise
and Ratner, 1980; Steger et al., 1983; Gee et al., 1984; Mobbs et al.,
1984; Rubin et al., 1985; Belisle et al., 1990; Hwang et al., 1990;
Joshi et al., 1995). In addition, in the hippocampal formation,
dendritic spines of dentate granule cells respond differently to
estrogen replacement in middle-aged and young animals (Miranda et al.,
1999). Recently, we reported that the increase in spine density after
estrogen treatment in the CA1 of young animals is blunted in aged
animals (Adams et al., 2001a). Together, these results suggest that the
aged synapse may be fundamentally different from the young synapse in
its capacity for plasticity, particularly in response to estrogen.
The expression of estrogen receptor- (ER-) and ER- in the
brain, in particular in the hippocampal formation, provides a potential
target and mechanism for effects of estrogen in brain regions and
circuits mediating cognitive processes such as memory (Li et al., 1997;
Shughrue et al., 1997, 1998; Weiland et al., 1997; Pau et al., 1998;
Petersen et al., 1998; Register et al., 1998; Shughrue and
Merchenthaler, 2000; Brinton, 2001). Recent evidence suggests that some
of the effects of estrogen may not occur through regulating
transcription but instead are mediated by membrane-bound ERs, resulting
in activation of signal transduction cascades and second messenger
systems (Toran-Allerand et al., 1999; Toran-Allerand, 2000; Brinton,
2001; Kelly and Levin, 2001). Interestingly, it was reported recently
that ER- is localized within select dendritic spines of CA1
pyramidal cells (Milner et al., 2001), suggesting that estrogen may be
mediating its effects on synapses locally rather than through
regulation of nuclear transcription. Therefore, the present study was
designed to examine the subcellular localization of ER- and the
effects of estrogen on the synaptic and perisynaptic distribution of
ER- in CA1 hippocampal pyramidal cells. The analyses revealed
fundamental age-related differences in ER- and estrogen-induced
synaptic plasticity that have important implications for estrogen
replacement in the context of age when estrogen levels and
hippocampal-dependent functions decline.
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MATERIALS AND METHODS |
Animals. A total of 21 female rats were used in the
present study. Eleven young (3-4 months; ~225 gm) and 10 aged
(22-23 months; ~350 gm) female Sprague Dawley rats were purchased
from Harlan Sprague Dawley (Indianapolis, IN). Young rats were virgins,
and aged rats were either virgins or retired breeders. Animals were housed in a temperature-controlled room (12 hr light/dark cycle; lights
on at 7:00 AM). Food and water were available ad libitum. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of
Experimental Animals using protocols approved by the Institutional
Animal Care and Use Committee at Mount Sinai School of Medicine.
Surgical procedures. Bilateral ovariectomy was performed
under isoflurane anesthesia. After 7 d, a SILASTIC (Dow Corning, Midland, MI) capsule (capsule dimensions: inner diameter, 1.96 mm;
outer diameter, 3.18 mm) filled with either 17- estradiol (10% in
cholesterol) or cholesterol was implanted subcutaneously under
anesthesia and remained for 2 d. Young animals received an implant that
was 1 cm long, and aged animals received an implant that was 2 cm long.
Different implant lengths were used for animals of different ages to
account for differences in body weights (Lauber et al., 1990; Funabashi
et al., 1998), presumably resulting in comparable estrogen levels in
young and aged rats. Moreover, our previous study used a similar
estrogen replacement paradigm in a longer-term ovariectomy in aged
animals and observed a similar uterine response, and the circulating
estrogen levels were within a physiological range (Adams et al.,
2001b). We determined that the vehicle and estrogen regimens after
ovariectomy were effective by examining the uterus from each animal. In
both young and aged rats treated with vehicle, uteri were very
small and atrophied, and those that received estrogen displayed
uterine hypertrophy. This estrogen-replacement and
ovariectomy-interval regimen in the present study was chosen to
relate our findings to previous studies that have used a similar time
course (Weiland, 1992; Woolley and McEwen, 1992, 1993, 1994; Gazzaley
et al., 1996; Woolley et al., 1996, 1997; Adams et al., 2001a). It
should also be noted that the tissue blocks used in the present study
were also examined in a previous study (Adams et al., 2001a) in which
it was demonstrated that aged animals have a blunted spine response
compared with young animals; however, the aged rats are still able to
change the synaptic distribution of NMDA receptors after estrogen
treatment. Thus, using the present technique, one can examine multiple
aspects of receptor distribution in the same sets of animals and
minimize interexperimental variability.
In addition to the estrogen-manipulated young and aged rats, one young
rat was perfused on proestrus, and blocks from the CA1 region were
processed and postembedding immunogold for ER- was performed as
described below. This pilot study was performed to equate the labeling
pattern evident with postembedding immunogold with the reported
observations based on pre-embedding diaminobenzidine-based localization
(Milner et al., 2001). Our postembedding labeling had similar patterns
to the pre-embedding labeling described by Milner et al. (2001) (see
Results and Fig. 1).
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Figure 1.
Distribution of ER--IR within axospinous
synapses in the stratum radiatum of the hippocampal CA1 region. Gold
particles were observed to be localized both presynaptically
(A) and postsynaptically (B, C).
In the axon terminal (A), gold particles were
associated with small synaptic vesicles. In dendritic spines, gold
particles were found within the head of the spine (B, C)
and affiliated within the postsynaptic density. Particles within the
postsynaptic density were usually found in the more lateral portion.
ax, Axon; sp, spine. Scale bar,
100 nm.
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Tissue processing and perfusion. Two days after the capsules
were implanted, animals were anesthetized with 30% chloral hydrate and
perfused transcardially with 2% dextran in 0.1 M
phosphate buffer (PB; 50 ml/min), pH 7.4, for 1 min, followed with 4%
paraformaldehyde and 0.125% glutaraldehyde in PB for 10-15 min. The
carcasses were examined to confirm complete removal of both ovaries and
the presence of the subcutaneous implant. The uterus of each animal
also was examined to determine whether the estrogen replacement was
effective, with uterine hypertrophy seen only for estrogen-treated
animals. No animals in the present study had obvious pituitary tumors, incomplete removal of ovaries, or lack of implant. The brains were
removed and post-fixed overnight. Two blocks from the dorsal hippocampus (~1 mm thick) were randomly selected from each animal and
processed for postembedding immunogold.
Postembedding immunogold. Freeze substitution and
low-temperature embedding of the specimens was performed as described
previously (Van Lookeren Campagne et al., 1991; Hjelle et al., 1994;
Chaudhry et al., 1995). Slices were cryoprotected by immersion in
increasing concentrations of glycerol in PB (10, 20, and 30%) and were
plunged rapidly into liquid propane cooled by liquid nitrogen
( 190°C) in a Universal Cryofixation System KF80 (Reichert-Jung,
Vienna, Austria). The samples were immersed in 1.5% uranyl acetate
(for en bloc fixation) in anhydrous methanol (90°C, 24 hr) in a
cryosubstitution Automated Freeze Substitution unit (Leica, Vienna,
Austria). The temperature increased in steps of 4°C/hr from 90°C
to 45°C. The samples were washed with anhydrous methanol and
infiltrated with Lowicryl HM20 resin (Electron Microscopy Sciences, Ft.
Washington, PA) at 45°C with a progressive increase in the ratio of
resin to methanol for 1 hr each, followed with pure Lowicryl
(overnight). Polymerization was performed with ultraviolet light (360 nm) at 45°C for 48 hr, followed by 24 hr at room temperature.
The stratum radiatum of the CA1 region (~150-200 µm from the cell
bodies) was identified and sectioned. Pairs of ultrathin sections
(~70-80 nm thick as determined by interference colors) were cut by
diamond knife on a Reichert-Jung ultramicrotome and mounted on nickel
mesh grids for immunogold analysis. The mesh grids with ultrathin
sections for the immunolabeling studies were treated with a saturated
solution of NaOH in absolute ethanol, rinsed, and incubated in the
following solutions at room temperature: 0.1% sodium borohydride and
50 mM glycine, and then Tris-buffered saline containing 2%
human serum albumin. Single immunolabeling was performed, and sections
were incubated with primary antibody (Okamura et al., 1992) (dilution
of 1:2500) in the above diluent overnight, washed, and incubated
in secondary gold tagged (10 nm) antibody in Tris-buffered saline (2%
human serum albumin and polyethyleneglycol 20,000 Da; 5 mg/ml).
Sections were washed and dried, counterstained with 1% uranyl acetate
and Reynolds lead citrate, and viewed on a Jeol (Tokyo, Japan) 1200EX
electron microscope. Images were captured using the Advantage CCD
camera (Advanced Microscopy Techniques Corporation, Danvers, MA).
Control experiments omitting the primary antibody were performed, and
no immunogold labeling was observed. The specificity of this primary
antibody has been demonstrated previously in numerous experimental
paradigms (Okamura et al., 1992; Alves et al., 1998; Milner et al.,
2001).
Analysis of percentage of label of ER-. An analysis was
performed of the percentage of synaptic profiles that contained ER- immunoreactivity (IR). All synapses visible in an area of ~3000 µm2 were counted in the present
analysis. Any profile that contained at least two gold particles per
postsynaptic profile (i.e., in the synaptic cleft, postsynaptic
density, or spine head) was considered positive for ER-. At least
100 synapses were counted for each animal.
Synaptic bin analysis of ER-. The immunogold particle
density and distribution was analyzed using software developed in our laboratory (SynBin) (Adams et al., 2001a), based on principles regarding proximity to membranes articulated by Ottersen, Blackstad, and colleagues (Blackstad et al., 1990; Ruud and Blackstad, 1999). Accordingly, the position of each gold particle was determined as it
relates to the postsynaptic and presynaptic membrane structures. The
program analyzes the resulting data map and objectively assigns each
gold particle to a given bin, with bin sizes and targeted synaptic
domains established prospectively. The bin sizes are established on the
basis of the lateral resolution of the electron microscopy techniques
and to optimally separate synaptic and nonsynaptic pools of receptors.
Through this process, a precise gold particle/bin density emerges that
is an accurate reflection of gold particle distribution and density in
different compartments of the synaptic complex.
Gold particle analysis was done on 35-50 randomly chosen spines per
animal. Synapses cut obliquely that lacked clear visualization and
delineation of classic synaptic structures such as presynaptic and
postsynaptic membranes, a synaptic cleft, and postsynaptic density were
excluded from the quantitative analysis. For the present analysis, 30 nm was chosen for the bin width because it assures high resolution yet
comfortably accommodates the theoretical limit of resolution (i.e., 25 nm). The following zones were defined for each synapse: (1) two
postsynaptic bins, the first one 0-30 nm from the inner leaflet of the
postsynaptic membrane and the second 30-60 nm from the postsynaptic
membrane; (2) side bins that were 15 nm lateral to both of the
postsynaptic bins; (3) the synaptic cleft; (4) a cytoplasmic bin that
included gold particles >60 nm from the postsynaptic membrane; (5) two
presynaptic zones, one extending 0-30 nm from the inner border of the
presynaptic membrane and the second extending 30-60 nm from the inner
border of the presynaptic membrane; and (6) a presynaptic bin that
included gold particles >60 nm from the presynaptic membrane. With
such a design, gold particles in the 0-30 nm postsynaptic density bin are unquestionably synaptic in location, whereas all other postsynaptic bins may include particles representing nonsynaptic pools of ER-. In
addition, the lateral bins were used to establish a "buffer zone"
at the lateral edge of the synapse to account for gold particles at the
edge (i.e., within 15 nm) that might be labeling proteins associated
with the postsynaptic density.
Statistical analysis. Statistical analyses were performed
using StatView 5.0 (Abacus Concepts, Inc., Berkeley, CA). Potential group differences in percentage of labeled synapses and number of gold
particles per synaptic compartment between young ovariectomized vehicle- and estrogen-treated animals, as well as aged ovariectomized vehicle- and estrogen-treated rats, were evaluated by unpaired t tests. ANOVA was performed to determine overall
effects of age. Significance was set at p < 0.05.
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RESULTS |
Distribution of ER- using postembedding immunogold
electron microscopy
A pre-embedding immunoelectron microscopic analysis demonstrated
that ER--IR is localized in both terminals and dendritic spines of
young proestrus rats (Milner et al., 2001). In the present study, we
observed a pattern of staining with postembedding immunogold that was
qualitatively similar to that described with pre-embedding immunoelectron microscopy in all of the young and aged rats analyzed and included in the quantitative analysis (Fig. 1). Gold particles were
localized within the postsynaptic density and the membrane of the
perisynaptic region and were clustered within the cytoplasm of the head
of the spine, perhaps representing a spinous pool of the receptor. In
addition, gold particles were present in axon terminals, often
affiliated with synaptic vesicles. In some cases, ER- labeling was
seen in the presynaptic and postsynaptic compartments of the same
synaptic profile; however, most synaptic profiles had either
presynaptic or postsynaptic labeling.
ER- in young and aged estrogen- and vehicle-treated rats
To determine whether the number of synapses that contained
ER--IR in the CA1 pyramidal cells of young and aged female rats changed with estrogen treatment, the percentage of synaptic profiles within a 3000 µm2 area that were labeled
for ER- was analyzed. Only synapses that contained two or more gold
particles within the postsynaptic profile were counted. In both
estrogen- and vehicle-treated young animals, ~30% of the synapses
contained ER--IR (30 and 34%, respectively) (Fig.
2), with no significant difference in the
number of labeled synapses between these two groups
(p > 0.31) (Fig. 2). In addition, no difference
was observed in the number of ER--labeled synapses in the CA1
pyramidal cells between aged estrogen- or vehicle-treated rats (17 and
16%, respectively, p > 0.80) (Fig. 2). Although there was no difference in the number of labeled synapses across estrogen treatment groups of the same age, we did observe a striking overall effect of age (p < 0.0001) (Fig. 2). Our
results demonstrated an ~50% reduction in the percentage of synapses
that contained ER--IR in the aged groups.
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Figure 2.
Percentage of ER--IR-labeled synapses in young
and aged rats. The graphs illustrate that the percentage of ER--IR
synapses decreases (50%) with age (*p < 0.0001); however, there is no effect of estrogen treatment in
either group (both p values > 0.31).
n = 10 for each age group. OVX,
Ovariectomized; E2, estrogen-treated;
Veh, vehicle-treated.
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In addition to assessing whether the number of synapses that contain
ER- are altered in young and aged estrogen-treated rats, we
determined whether the amount of ER--IR per synapse was responsive to estrogen. To do this, we performed an analysis of the amount of
ER--IR as measured by the number of gold particles per synaptic profile. To quantify the synaptic distribution of ER--IR, we used
the computer-assisted method (i.e., SynBin) that was described in a
previous study examining NR1 levels, the obligatory subunit for a
functional NMDA receptor (Adams et al., 2001a). The present analysis
revealed that labeled synapses in young animals had significantly less
ER- after estrogen replacement than those treated with vehicle, and
these effects were confined to the presynaptic and postsynaptic compartments that were >60 nm from their respective membranes (both
p values < 0.05) (Fig. 3 and Table 1). In this
material, it was not possible to determine decisively the
degree to which the spinous pool of
ER- was associated with membranous
specializations (e.g., caveolas or spine
apparatus). However, in the presynaptic terminals, the ER- was
associated primarily with vesicles in which it could potentially affect
neurotransmitter release. The difference between the estrogen- and
vehicle-treated groups was ~35% presynaptically and 27%
postsynaptically. However, we observed no difference in any compartment
of the synapse between the aged ovariectomized rats given estrogen
compared with those that received vehicle (all p values > 0.16) (Fig. 3 and Table 1). The trend suggested that the effect seen
presynaptically in the young animals may also be occurring in the aged
animals. However, because of the variability in the aged
estrogen-treated groups, this effect was not significant.
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Figure 3.
Levels of ER--IR per synapse in young and aged
vehicle-treated (Veh) and estrogen-treated
(E2) rats. The amount of ER--IR
particles per synapse in young rats significantly decreases with
estrogen treatment compared with vehicle both presynaptically
(A) and postsynaptically
(B) (*both p values < 0.05).
No differences were observed in the estrogen- and vehicle-treated aged
groups (both p values > 0.16).
n = 5 for each treatment group. OVX,
Ovariectomized.
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DISCUSSION |
Our results demonstrated that synaptic and spinous ER--IR in
CA1 pyramidal cells is sensitive to both aging and estrogen in female
rats (Fig. 4). We found that the number
of synapses that contain ER--IR is decreased in aged animals
compared with young rats. However, the percentage of ER--IR-labeled
synapses is not affected by estrogen in either young animals or aged
animals. Although the number of synapses with ER--IR is not
different between young rats that received vehicle or estrogen, the
amount of ER--IR per labeled synapse is decreased in young
estrogen-treated rats compared with those that received vehicle.
However, no such regulation by estrogen was observed in aged animals.
The present findings regarding ER- may explain our previous
observation that synapse number in aged CA1 is not increased by
estrogen, although it is increased in CA1 of young female rats (Adams
et al., 2001a).
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Figure 4.
Schematic diagram illustrating the proposed
mechanisms of estrogen-induced plasticity of ER- in young
(A) and aged (B) animals.
Estrogen treatment either decreases the amount of ER- per synapse in
young animals or leads to a redistribution of the receptor resulting
from the formation of new dendritic spines and alterations in
presynaptic terminal shape. ER- levels are unchanged in aged animals
in both presynaptic and postsynaptic compartments, but there is
an overall decrease in the percentage of terminals that express
ER--IR. Small black dots represent immunogold
particles labeling ER-, open circles represent
synaptic vesicles, and the gray zones represent the
postsynaptic density.
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Synaptic and perisynaptic ER- is regulated by circulating
estrogen in CA1 of young females
Many studies have demonstrated that in both young animals and cell
lines exposed to estrogen, ER- and its mRNA are reduced (Osterlund
et al., 1998; Patisaul et al., 1999; Taleghany et al., 1999; Funabashi
et al., 2000; Nephew et al., 2000; Schreihofer et al., 2000), and it is
presumed in these cases that the reduction in ER- is a critical
element in regulating the transcriptional effects of estrogen.
Interestingly, the present results show that ER- protein is reduced
in a very specific manner in the presynaptic pool associated with
vesicles and the spinous pool, demonstrating a highly localized
regulation in response to decreased estrogen that would presumably
affect any local effects of estrogen on CA1 synapses. Such an effect is
consistent with the notion of classic denervation supersensitivity,
whereby the levels of a receptor are increased when the levels of the
ligand of that protein are reduced (Miledi and Potter, 1971). An
alternative explanation to the idea of classic denervation
supersensitivity is that the ER- is redistributed within the
terminals and spines. Estrogen replacement in young animals has been
shown to increase the density of spines and axospinous synapses in the
CA1 region of the hippocampus (Gould et al., 1990; Woolley and McEwen,
1992, 1993; Woolley et al., 1996; Adams et al., 2001a). In addition, in
the same young animals as used in the present study, estrogen treatment
increased the density of axospinous synapses (Adams et al., 2001a).
Therefore, it is also possible that as the number of spines increases
with estrogen treatment, the amount of ER- remains the same but is redistributed among the new spines and then effectively diluted over
the short term. In addition, estrogen replacement has been shown to
increase the number of multisynaptic contacts and alter the shape of
synaptic boutons (Woolley et al., 1996; Yankova et al., 2001), and, in
particular, it is the spine that grows out to make contact with the
pre-existing axospinous synapse. In this connection, the use of
radioimmunocytochemistry has demonstrated estrogen induction of IR for
spinophilin, a spine-specific protein, in the apical and basal
dendritic fields of CA1 where the new spine synapses are found (Brake
et al., 2001). Therefore, the present result in young animals with
estrogen treatment showing a decrease in ER- may result from a
redistribution of ER--IR within the affected terminals and spines,
as well as or in lieu of a direct effect on transport or local
synthesis of ER-.
These pools of ER- in the terminals and spines of CA1 have only
recently been appreciated (Milner et al., 2001), and the function of
such synaptic and perisynaptic ER- remains to be elucidated,
although multiple, rapid, nongenomic effects of estrogen have been
described in both neural and non-neural tissue (Toran-Allerand et al.,
1999; Brake et al., 2001; Brinton, 2001; Kelly and Levin, 2001). The
presynaptic pool of ER- is notably associated with vesicles and thus
may be related to effects on neurotransmitter release (Becker, 1990)
and reuptake (O'Malley et al., 1987) that have been described for
estrogen. Postsynaptically, multiple molecular targets for mediation of
rapid, local effects have been identified, particularly with respect to
second messenger systems, such as cAMP response element-binding
protein, phosphatidylinositol 3-kinase (PI3-kinase) and Akt, and
G-protein-coupled receptors (Kelly et al., 1999; Razandi et al., 1999;
Brinton, 2001) (K. T. Akama, S. E. Alves, W. G. Brake,
S. J.Lee, B. S. McEwen, T. A. Milner, V. Znamensky,
unpublished observations). One target of these actions appears to
be the regulation of translation of mRNAs into proteins involving
the PI3-kinase/Akt pathway, which also strongly implicates ER-
(Simoncini et al., 2000; Brinton, 2001) (Akama, McEwen, Milner, Znamensky, unpublished observations). Some of this translational control by estrogens might involve the dendritic ER- and the mRNA
that are targeted to dendrites in hippocampal and other neurons (McEwen, 2001). Other effects might be mediated by ER- as well as
ER- and possibly by a putative novel receptor that is highly homologous to ER-, referred to as ER-X (Toran-Allerand, 2000).
Synaptic and spinous ER- is decreased in CA1 of
aged females
Our previous study demonstrated that the increase in CA1
axospinous synapses in young animals is attenuated in aged females (Adams et al., 2001a). That analysis of axospinous synapse density was
performed using the same animals as those used in the present study.
Although the mechanism of action of estrogen regarding increased
synapse number in CA1 is not resolved, there is significant support for
the effect of estrogen being local in this case, rather than occurring
through regulation of transcription in the nucleus (McEwen, 2001). If
the spine response to estrogen in the hippocampus relies on ER-
levels in the axospinous synaptic junction and the levels of ER- are
decreased in the CA1 spines of aged females, then one would predict
that the spine increase would be attenuated with age, which is
precisely what occurs (Adams et al., 2001a). This phenomenon of an
impaired response to ER- is consistent with a study demonstrating
that ER- levels in the hypothalamus are decreased by estrogen in
young but not aged rats (Funabashi et al., 2000). The decreased number
of synapses that contain ER--IR in aged animals as well as the
decreased regulation of ER--IR by estrogen in these same animals
suggest that age-related shifts in ER- may render the aged brain
less plastic, which is consistent with other studies supporting a
decrease in synaptic plasticity in the aging hippocampus (Cotman and
Scheff, 1979; Scheff et al., 1980; Hoff et al., 1982a,b; West, 1984;
Schauwecker et al., 1995; Stone et al., 2000).
In the present study, only one postovariectomy interval and
estrogen-replacement regimen was examined. For the hypothalamus, it is
well documented that aged animals do not have the same reaction to
estrogen deprivation as young animals (Wise and Ratner, 1980; Steger et
al., 1983; Gee et al., 1984; Mobbs et al., 1984; Rubin et al., 1985;
Belisle et al., 1990; Hwang et al., 1990; Joshi et al., 1995). In
addition, in the hippocampal formation, dendritic spines of dentate
granule cells respond differently to estrogen replacement in
middle-aged and young animals (Miranda et al., 1999). Thus, it is
possible that a longer post-ovariectomy interval or estrogen
replacement may change the response of ER- in aged spines.
The potential functional role of ER- in spines
This study not only confirms a previous study (Milner et al.,
2001) demonstrating that ER--IR is present within the presynaptic terminal and spines of CA1 pyramidal neurons but also indicates that it
is responsive to estrogen in young animals and decreases with aging.
Interestingly, most of the ER--IR is within extrasynaptic membranes
or the spine cytoplasm or is associated with presynaptic vesicles,
rather than within the postsynaptic density. It will be of great
importance to determine the localization and potential colocalized
proteins of the spinous ER--IR with greater precision. For example,
it will be important to determine the degree to which the ER--IR is
sequestered in membranous caveola-like structures, as has been
hypothesized (Toran-Allerand, 2000), because such a localization would
position ER- to interact with the multiple signal transduction
pathways that have been hypothesized to function within such an
organelle in non-nervous system tissues (Schlegel et al., 1999). A
critical role for ER- in such caveolas was demonstrated recently in
endothelial cells, in that caveolar ER- was functionally and
biochemically linked to nitric oxide synthase in a critical signaling
module capable of regulating the local calcium levels. Such a link
between ER- and nitric oxide synthase could have profound local
effects on synaptic plasticity in CA1. However, the substrate for such
a complex is hypothetical at present, because caveolin and
caveolin-containing structures do not appear to be present in the
brain, although there may be a structural and functional homolog
involving flotillin-anchored caveolas that may be a site for ER- or
a putative homologous ER (Kelly and Levin, 2001), which may be
recognized by ER- antibodies referred to by Toran-Allerand (2000) as ER-X. Although such a scenario is compelling, given the rich environment that it would offer regarding local effects of
ER- or its homolog, its characterization in the nervous system in
general and in CA1 in particular will require extensive additional ultrastructural and biochemical analyses.
| |
FOOTNOTES |
Received Nov. 8, 2001; revised Jan. 15, 2002; accepted Jan. 28, 2002.
This work was supported by National Institute on Aging Grant
P01AG16765. We thank T. Oung, N. J. Riley, and A. P. Leonard for expert technical assistance and Dr. A. C. Gore for assistance with experimental design and helpful comments regarding this manuscript.
Correspondence should be addressed to John H. Morrison, Neurobiology of
Aging Laboratories, Mount Sinai School of Medicine, Box 1639, New York,
NY 10029-6574. E-mail: john.morrison{at}mssm.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
