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Laboratory of Neuroendocrinology (W.G.B., S.E.A., J.C.D., S.J.L., K.B., B.S.M.) and Laboratory of Molecular and Cellular Neuroscience (P.B.A., P.G.), The Rockefeller University, New York, New York 10021
Address all correspondence and requests for reprints to: Wayne Brake, Ph.D., Laboratory of Neuroendocrinology, Box 165, The Rockefeller University, 1230 York Avenue, New York, New York 10021. E-mail: brakew{at}mail.rockefeller.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CI628 is a nonsteroidal SERM that blocks estrogen receptor (ER)
transcriptional activation of ER
and ER
via the classical
estrogen response element pathway (7, 8). Although SERMs
can have both agonist and antagonist properties, it has been shown that
CI628 predominantly acts as an antiestrogen by blocking
estrogen-induced progesterone receptor induction (9, 10)
PRL release (11) and lordosis behavior (9).
Thus, it should not be surprising that CI628 blocks estrogen-induced
formation of hippocampal dendritic spines as measured by Golgi staining
(6).
Despite progress in characterizing estrogen regulation of hippocampal synaptogenesis, the mechanism and function of such estrogen action has not been fully elucidated. This may be due, in part, to difficulties with the techniques involved. Studies have traditionally employed Golgi impregnation, electron microscopy, and confocal microscopy to examine estrogen’s effects on synaptic plasticity, restricting such studies to labs where these anatomical techniques are available. Determining the level of proteins associated with synapses may be another means to examine changes in synaptogenesis as well as identify molecular changes associated with this process. However, techniques such as Western blotting are unable to examine protein levels within discrete hippocampal laminae and traditional immunocytochemistry techniques measuring light density of DAB (3,3'-diaminobenzidine) staining or immunofluorescence are unreliable for determining relative protein levels.
The purpose of the present study was to examine in OVX rats the effects of estrogen, and the estrogen antagonist, CI628, on proteins associated with pre- and postsynaptic structures using a rapid and sensitive method of examining site-specific changes in relative protein levels, viz. radioimmunocytochemistry (RICC). This was carried out by using immunocytochemistry with primary antibodies directed against pre- and postsynaptic proteins followed by a radiolabeled secondary antibody for detection. Western immunoblotting was also employed to determine whether such protein changes could be detected in whole hippocampal tissue. Antibodies directed against synaptophysin, syntaxin, and spinophilin were employed in this study. Synaptophysin is a vesicular protein whereas syntaxin is a presynaptic membrane-bound protein; both proteins are implicated in vesicular docking and are considered to be reliable markers of synaptogenesis (12, 13, 14). Spinophilin is a recently characterized protein found predominantly in dendritic spines and implicated in spine homeostasis (15, 16).
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Materials and Methods |
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-(4-pyrrolidinethoxyl)phenyl-4-methoxy--nitrostilbene]; 10
mg/kg sc; Parke-Davis, Ann Arbor, MI) or sterile distilled
H2O vehicle (H2O). On the
third and fourth days, rats were injected with either estradiol
benzoate (EB; 10 µg/kg sc; Sigma, St. Louis, MO) or
sesame oil vehicle (Oil). That is, all animals received three days of
CI638 or H2O and two days of either EB or oil
(n = 6–8/group). For RICC, animals were deeply anesthetized 24 h following the final treatment, and transcardially perfused with 0.9% saline followed by freshly prepared 4% paraformaldehyde in 0.1 M PBS, pH 7.4, containing 0.03% glutaraldehyde. The brains were then postfixed in the perfusate overnight and sliced at 40 µm thickness along the coronal plane using a microtome. Sections were then stored in cryoprotectant (30% glycerol and 30% ethylene glycol in 0.1 M PB) at -20 C.
For Western immunoblotting, OVX animals (n = 5/group) were decapitated 24 h following 2 days of treatment with either EB or oil as described above. Animals treated with CI628 were not included in the immunoblot experiment. The brains were removed and the hippocampus was rapidly dissected out on a saline-rinsed chuck sitting in wet ice and then snap frozen in dry ice and stored at -80 C until used.
RICC
All antibodies employed in this study were previously titrated
to determine concentrations exhibiting maximum signal to minimum
background binding. Free-floating sections were serially washed (5
x 5 min) in 0.05 M phosphate buffer (PB), pH 7.4, to
remove cryoprotectant. Sections were then mounted onto Vectabond-coated
slides (Vector Laboratories, Inc. Burlingame, CA) and
allowed to dry for 1 h. Tissue was then washed (3 x 5 min)
in 0.1 M PBS, pH 7.4, at room temperature (RT) and then
submitted to treatment depending on the antibody examined. For
immunoreactivity (IR) to synaptophysin and syntaxin, tissue was blocked
with 2% normal goat serum in PBS containing 0.1% Triton x100 for
1 h at RT. Tissue was then incubated overnight at 4 C in primary
antibody (1:1000) diluted in PBS (monoclonal anti-synaptophysin raised
in mouse, monoclonal antisyntaxin raised in mouse, both are IgG1
isotype; Sigma). Sections incubated without primary
antibody were also included as negative controls. All sections were
then washed in PBS and incubated with secondary antibody (antimouse Ig
whole antibody raised in sheep with [35S]
label, specific activity = 900 Ci/mmol, Amersham Pharmacia Biotech, Piscataway, NJ) in PBS (1:100) for 1 h at
RT.
For spinophilin IR, sections were blocked in 2% normal horse serum containing 0.1% Triton X-100 for 1 h at RT. Sections were then incubated overnight in primary antibody (polyclonal anti-spinophilin raised in rabbit; 1:2000; see 1 for antibody purification methods) in PBS overnight at 4 C. Sections were then washed in PBS and incubated with secondary antibody (antirabbit Ig whole antibody raised in donkey with [35S] label, specific activity = 1466 Ci/mmol, Amersham Pharmacia Biotech) in PBS (1:100) for 1 h at RT. Following washes (3 x 15 min) in ice-cold PBS and a distilled H2O rinse, all sections included in this study were left to air-dry overnight. All slides were incubated collectively in the same dish at each step except the no primary controls which were kept separate from other slides throughout the experiment. Slides were then apposed to 3H-Hyperfilm (Amersham Pharmacia Biotech) alongside microscale-calibrated [14C] standard strips (Amersham Pharmacia Biotech), which were included with every film cassette. Multiple exposure times were carried out to determine the optimal density for each primary antibody (viz. 2–3 days).
Western blots
Membranes were extracted from hippocampal samples by
homogenizing tissue in ice-cold buffer containing 0.32 M
sucrose, 2 mM EDTA, 2 mM EGTA, and 20
mM HEPES along with protease inhibitors (trypsin inhibitor,
1 mg/ml; aprotinin, 7 mg/ml; pepstatin, 4 mg/ml). Protein tissue was
centrifuged at 4 C (500 x g for 10 min) and the
supernatant was again centrifuged at 4 C (32,000 x g
for 30 min). The pellet was resuspended in 100 mM
PBS (pH, 7.4) and centrifuged at 4 C (31,000 x g for
30 min) and the resultant pellet was resuspended in 100
mM PBS and stored at -80 C.
After estimation of protein concentration with a Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay, aliquots containing 2 µg of protein from each animal were separated by NuPAGE 4–20% Bis-Tris gel (NEN Life Science Products) and transferred onto a PVDF membrane (Amersham Pharmacia Biotech). The membranes were then blocked with TBS-T (25 mM Tris-base, pH 7.4, 137 mM NaCl, and 0.1% Tween 20) containing 5% nonfat milk for 1 h at room temperature. Membranes were washed in TBS-T and probed with 1:5000 antisynaptophysin, 1:5000 antisyntaxin, and 1:10,000 antispinophilin antibodies overnight at 4 C. Membranes were again washed in TBS-T and then incubated with either antimouse or antirabbit (depending on how primary Ab was raised) IgG HRP-labeled secondary Ab (1:10,000) for 2 h. All antibodies used for Western blots were titrated to determine maximum signal to minimum background binding to determine at the concentrations listed here. The antibody-reactive bands were visualized using chemiluminescence (ECL Western detection kit; Amersham Pharmacia Biotech).
Data format and analysis
For analysis of RICC, optical density measures were blindly
taken from every twelfth section of dorsal hippocampus (between -2.5
and -6.1 mm from bregma) of each rat using computerized image-analysis
software (MCID-M4, Imaging Research, Inc., St. Catherines,
Ontario, Canada). Density of the corpus callosum (which should contain
no pre- or postsynaptic protein) was measured as background and
subtracted from the density of each hippocampal subregion. It was
considered important for binding densities to be consistent between
different films. Consequently, the convention of expressing optical
densities relative to standard strips (reported as fmol/mg) as is used
in quantitative receptor autoradiography was chosen instead of
expressing the data as relative optical densities, which can vary
greatly from film to film and also vary with subtle differences in
exposure time. Thus, binding densities were converted to fmol/mg of
tissue based on the [14C] standard calibration
and the specific activity of the radiolabeled secondary antibody. Yet,
an important caveat should be noted. Considering the limitations of
antibody penetration into tissue, these values are intended to indicate
relative changes in IR rather than absolute protein levels. For each
primary antibody, mean IR was analyzed using a mixed factorial ANOVA
with treatment as a between measure and hippocampal region as a within
measure. Post hoc examination was carried out using
Scheffé’s F test.
For analysis of immunoblots, relative optical density was measured from the band migrating at the appropriate weight for each antibody, although each antibody produced only one major band. Optical densities were obtained from 5 lanes per group (each lane contained tissue from a single rat). Thus, analysis (one-way ANOVA) was carried out on n = 5 rats/group for each antibody.
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Results |
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shows that the antirabbit
secondary antibody displayed no specific binding when compared with
sections incubated with primary antibody. Similar results were
seen with the antimouse secondary antibody. The synaptophysin and
syntaxin monoclonal antibodies used here are commercially available. To
determine their specificity, these antibodies were tested using Western
blots on hippocampal tissue. The synaptophysin and syntaxin antibodies
recognized one major band each migrating at 38 kDa and 35 kDa,
respectively (data not shown), which are the molecular weights of
synaptophysin and syntaxin, indicating specificity for their respective
proteins. The spinophilin antibody employed in this study has been
tested for specificity elsewhere (15). To further test
antibody specificity, Western blots were carried out on hippocampal
tissue here (see Fig. 4) as well as by others using spinophilin
knockout mice. The same spinophilin antibody used here produced no
major bands in tissue from these knockout mice (16).
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. There was a significant interaction
between hippocampal region and treatment group for synaptophysin
(F(12,96) = 5.340, P <
0.0001). Post hoc analysis revealed higher synaptophysin IR
in animals treated with EB only (H2O+EB group)
when compared with other groups (P < 0.05) in both CA1
stratum radiatum and stratum oriens. No other synaptophysin IR
differences were observed. Similarly, differences in syntaxin IR were
observed only in CA1 stratum radiatum
(F(3,22) = 3.105, P =
0.0493) and stratum oriens (F(3,22) =
4.085, P = 0.0190). Syntaxin IR was highest in these
two CA1 subregions in the H2O+EB rats when
compared with CI628+EB (P < 0.05) and
H2O+Oil (P < 0.05) but not
CI628+Oil-treated rats.
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and 3) revealed a significant interaction
between treatment group and hippocampal region
(F(12,96) = 3.762; P <
0.0001). In the case of spinophilin, however, greater spinophilin IR
was observed in the estrogen-treated group
(EB+H2O) in comparison to all three other groups
in CA1 and the hilar region of the dentate gyrus (P <
0.05). Rats that received EB+H2O treatment also
showed greater spinophilin IR in CA3 in comparison to those treated
with CI628+EB (P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogen is shown to increase the synaptic proteins examined here by
approximately 20–30% in the CA1 region of hippocampus (Fig. 2). This
effect is in accordance with previous studies demonstrating such an
increase in CA1 spine number (5, 6) and excitatory
synapses (3) in response to estrogen. Further, these are
the first findings to demonstrate estrogen-induced changes in synaptic
markers in the hilus of the dentate gyrus and CA3 suggesting that
target sites for estrogen’s action in the hippocampus is more
widespread than originally thought. Moreover, the nonsteroidal estrogen
antagonist, CI628, prevented these effects. This too complies with
previous anatomical findings, which demonstrate that CI628 at the dose
used here inhibits estrogen-induced dendritic spine induction on CA1
pyramidal neurons (6). It is not surprising that Western
blots on whole hippocampal tissue did not reveal any group differences
in these proteins. Such findings underscore the importance of using a
technique which allows for the evaluation of protein changes in
discrete regions while maintaining anatomical integrity.
That presynaptic proteins examined here (viz. synaptophysin
and syntaxin) were affected by estrogen treatment suggests that
afferents to CA1 pyramidal neurons may either be indirectly
compensating for estrogen-induced changes in CA1 pyramidal cells,
interneurons, or astrocytes or they are directly responding to estrogen
treatment themselves. Indeed, ascending basal forebrain cholinergic
neurons and locus coeruleus norepinephrine neurons both innervate this
site and both express nuclear ERs (22, 23, 24, 25, 26). In addition,
Shaffer collaterals arising from CA3 neurons innervate CA1
pyramidal cells. Although neither CA1 nor CA3 cells have been
shown to exhibit nuclear ER either by immunocytochemistry
(27), or by autoradiography (28), these cells
have been reported to exhibit some (albeit little) binding of
125I-estrogen (29) and express
messenger RNA for both ER and ER (30).
Murphy and colleagues (31) originally posited that the
actions of estrogen on CA1 hippocampal plasticity may be mediated
through inhibitory interneurons. There is suggestive evidence that GABA
(
-aminobutyric acid) interneurons contain nuclear ER
(27) and it has been shown in vitro that
estrogens inhibit GABA and glutamic acid decarboxylase (the
rate-limiting enzyme in GABA synthesis) IR in these estrogen-sensitive
cells (31). This may confer a period of disinhibition of
excitatory synapses and may increase NMDA
(N-methyl-D-aspartate) receptor
expression on pyramidal neurons. This finding is relevant because it is
thought that estrogen-induced synaptogenesis is mediated via NMDA
receptors. NMDA receptors are up regulated on CA1 pyramidal neurons by
estrogen treatment (27, 32, 33) and NMDA receptor blockade
prevents estrogen-induced CA1 synaptogenesis (34).
It has been postulated that the rapid effects of estrogens may occur
outside the nucleus via membrane-associated ERs, which are thought
capable of initiating signal transduction (35, 36, 37). A
recent ultrastructural analysis of ER IR has determined that, in
addition to location in the nuclei in some nonprinciple cells,
extranuclear ER is present in the axons and axon terminals as well
as dendritic spines of the hippocampus (38). The majority
of ER-labeled terminals are seen in the dentate gyrus and CA1 region
of the hippocampus. These authors also observed extranuclear ER IR
in processes prominent near dendritic spines that are presumed to be
from astrocytes. These findings allow the possibility that estrogen may
act locally upon ERs at the excitatory synapses in CA1.
The fact that CI628 blocked estrogen up-regulation of synaptic and spine protein IR just as it had blocked estrogen induction of spines (6), is consistent with the action of estrogens via a nuclear ER. However, membrane-associated ER may also be involved, because the ability of such nonnuclear ER to stimulate second messenger systems (37) is blocked by a number of estrogen antagonists, including tamoxifen (Levin, E. R., personal communication; May, 24, 2000), which is similar in its mode of action to CI628. Thus, until the actions of extranuclear ERs at the CA1 synaptic site are determined, target sites for estrogen’s actions could possibly be, either uniquely or collectively, any of the CA1 inputs, astrocytes, inhibitory interneurons, and/or the CA1 dendritic spines themselves.
That estrogen increases spinophilin IR in CA1, where
dendritic spine induction has been demonstrated by structural studies
(4, 5, 6) supports the use of spinophilin as a marker for
examining changes in spine number. However, estrogen was shown here to
also increase spinophilin in the hilar region of the dentate gyrus. How
might these observations be reconciled? One possibility is that these
data may be revealing previously unidentified sites of estrogen
modulation of spine structure. An alternative possibility is that
spinophilin levels are representative of synaptic activity. Because
spinophilin acts functionally as a cytoskeletal scaffolding protein (it
serves to bundle F-actin filaments in vitro; 39), it is
potentially involved in regulating the dense F-actin-based cytoskeleton
found in dendritic spines. In addition, spinophilin serves as an anchor
for protein phosphatase-1 (15), an enzyme that
participates not only in regulation of the F-actin-based cytoskeleton
(40), but also in the regulation of the activity of
several ion channels and neurotransmitter receptors found in dendritic
spines (41). Thus, spinophilin levels may be dictated not
only by spine density per se, but also by the level of
ongoing and previous synaptic activity. It is becoming increasingly
apparent that spine morphology and biochemical content are responsive
to altered synaptic activity (42), and it will be of
interest to further examine the role of estrogen in this context. Thus,
our data are consistent with the idea that estrogen may be inducing
changes in spines located in hippocampal regions outside CA1 such as
the dentate gyrus. Interestingly, ER has been detected surrounding
the spine apparatus in select spines in both CA1 and the dentate gyrus
(38) and, in these regions at least, it may be colocalized
with spinophilin.
In summary, these data identify the involvement of three synaptic proteins in the estrogen-mediated regulation of synaptic plasticity in the hippocampus. They also uncover previously unidentified sites (viz. hilar region of the dentate gyrus and CA3) for estrogen actions in the dorsal hippocampus. Furthermore, these findings demonstrate that RICC is a relatively rapid and sensitive technique for examining synaptic changes in distinct brain regions without compromising anatomical integrity.
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Footnotes |
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Received September 11, 2000.
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and ER at AP1 sites. Science 277:1508–1510 and mRNA in the
rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
and ER expressed in Chinese hamster
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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,
Significance (P < 0.05; Scheffé’s)
vs. CI628+EB and H20+EB groups only.