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Estradiol Increases Pre- and Post-Synaptic Proteins in the CA1 Region of the Hippocampus in Female Rhesus Macaques (Macaca mulatta)

Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University (J.M.C., Z.R.), New York, New York 10021; Laboratory of Neuroendocrinology, Rockefeller University (R.D.R., B.S.M.), New York, New York 10021; Department of Psychology, University of California (W.G.B.), Santa Barbara, California 93106; and Divisions of Reproductive Sciences and Neuroscience, Oregon National Primate Research Center (C.L.B.), Beaverton, Oregon 97006

Address all correspondence and requests for reprints to: Dr. Bruce S. McEwen, Laboratory of Neuroendocrinology, Rockefeller University, 1230 York Avenue, Box 165, New York, New York 10021. E-mail: mcewen{at}rockefeller.edu.

	Abstract

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The role of estrogen (E) in promoting learning and memory in females has been well studied in both rodent and primate models. In female rats, E increases dendritic spine number, synaptogenesis, and synaptic proteins in the CA1 region of the hippocampus, an area of the brain that mediates learning and memory. In the present study, we used radioimmunocytochemistry to examine whether E and progesterone were capable of modulating the levels of pre- and postsynaptic proteins in the CA1 region of the female nonhuman primate hippocampus. It was found that E increased syntaxin, synaptophysin (presynaptic), and spinophilin (postsynaptic) levels in the stratum oriens and radiatum of the CA1 region, whereas combined E and progesterone treatment decreased these synaptic proteins to the levels found in untreated, spayed controls. Furthermore, progesterone treatment alone significantly increased synaptophysin levels in the stratum oriens and radiatum of the CA1 region. The levels of these synaptic proteins were unaltered by hormone treatment in the dentate gyrus, suggesting that this steroid-induced plasticity is hippocampal region specific. As these synaptic proteins are important components of the synaptic apparatus and reliable markers of synaptogenesis, it appears that E-induced increases in cognitive function of higher order mammals may be mediated in part by the effect of E on hippocampal synaptogenesis and synaptic plasticity.

	Introduction

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THE ROLE OF estrogen (E) in promoting learning and memory in females has been well studied in both rodent and primate models. For instance, behavioral studies in rats and mice have demonstrated improvements in a number of learning and memory tasks in adult females treated with E compared with their ovariectomized controls (1, 2, 3, 4). Similarly, nonhuman primates treated with E show improved cognitive function (5, 6), as do postmenopausal women receiving E replacement therapy (7, 8, 9). The mechanisms by which E mediates these beneficial effects on cognition are not well understood. However, E affects the structure and function of the hippocampus (10), an area of the brain that mediates learning and memory (11). Thus, one hypothesis is that E mediates these effects on memory and cognition in part by acting on the hippocampus.

In rats, E has been shown to increase dendritic spine number and synaptic density in the CA1 region of the female hippocampus (10, 12). Furthermore, radioimmunocytochemistry (RICC) has been used to show significant increases in syntaxin, synaptophysin, and spinophilin in the CA1 pyramidal cells of females (13). These proteins are important components of the pre- and postsynaptic apparatus and are reliable markers of synaptogenesis (14, 15). In addition to these morphological and biochemical changes, E facilitates the induction of hippocampal long-term potentiation, a putative electrophysiological correlate of learning and memory (16).

A recent Golgi study in African green monkeys (Cercopithecus aethiops sabaeus) showed that spine density in the CA1 pyramidal cells of E-treated females was significantly higher than that in ovariectomized, control females (17). In the present study we wanted to extend these morphological observations by elucidating whether E was capable of modulating the levels of pre- and postsynaptic proteins in the CA1 region of the female nonhuman primate hippocampus. Furthermore, clinical studies suggest that progesterone (P) might counteract beneficial effects of E on cognition (7, 18), and rat studies indicate that P in the presence of E decreases spine density and synaptogenesis (19). Thus, the role of P in modulating the levels of synaptic proteins in the primate hippocampus in the presence or absence of E was also investigated in the current experiment.

	Materials and Methods

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Experimental animals
Approval for the animals used in this experiment was obtained from the Oregon National Primate Research Center animal care and use committee. Three to 6 months before the start of this experiment, adult female rhesus macaques (Macaca mulatta), between the ages of 7–15 yr (i.e. young to middle-aged), were ovariectomized and hysterectomized (spayed) according to accepted veterinary surgery protocol. All animals were born in Oregon and were maintained in good health, with weights ranging from 4–8 kg. Animals were maintained in accordance with the NIH Guide for Care and Use of Laboratory Animals.

Hormone treatments and tissue preparation
Treatment and tissue preparation of these animals have previously been described in detail (20). Briefly, 20 adult female spayed rhesus macaques were obtained. Five monkeys were assigned to each of 4 treatment groups where empty SILASTIC brand capsules (placebo; Dow Corning Corp., Midland, MI) or hormone-impregnated capsules were sc inserted into the animals. All capsules were inserted in the periscapular area under ketamine anesthesia. Monkeys in the control group received empty SILASTIC capsules for the treatment period. E-treated monkeys received a 4.5-cm crystalline estradiol (E₂; 1,3,5,10-estratrien-3,17ß-diol; Steraloids, Wilton, NH)-filled SILASTIC capsule for the 28-d treatment period. Monkeys in the P-treated group each received an empty SILASTIC capsule for the first 14 d of treatment, followed by a 6-cm SILASTIC capsule filled with crystalline P (4-pregnen-3,20-dione; Steraloids) for the last 14 d. An E₂-filled capsule was used for the first 14 d of treatment in the E₂+P group, followed by the addition of a P-filled capsule for the last 14 d. The steroid concentrations achieved and treatment durations were chosen to mimic the differentiation of the uterine endometrium during the normal 28-d menstrual cycle (21). Furthermore, this treatment paradigm allows for direct comparison of E₂+P to E₂ only and P only in a physiologically relevant time frame for primates. The hormone levels experienced by these animals have been previously reported (20). Specifically, E levels were within the range of those reported in the mid to late follicular phase, whereas P levels were within the range of those reported in the midluteal phase of the primate menstrual cycle.

At the end of the treatment period, animals were killed according to methods recommended by the Panel on Euthanasia of the American Veterinary Association. Each animal was sedated with ketamine, then given an overdose of pentobarbital (25 mg/kg, iv), and exasanguinated by severing the descending aorta. The left ventricle of the heart was cannulated, and the head of each animal was perfused with 1 liter saline, followed by 7 liters 4% paraformaldehyde in 3.8% borate, pH 9.5. The brain was removed and dissected. Tissue blocks were postfixed in 4% paraformaldehyde for 3 h, then transferred to 0.02 M potassium PBS containing 10%, followed by 20%, glycerol and 2% dimethylsulfoxide at 4 C for 3 d to cryoprotect the tissue. After infiltration, the block was frozen in isopentene cooled to -55 C and stored at -80 C until sectioning. Coronal sections of the left temporal lobe were cut on a sliding microtome at a thickness of 25 µm and were mounted on Vectabond-coated slides (Vector Laboratories, Inc., Burlingame, CA). Slides were dried at room temperature for 2 h and stored at -80 C until the RICC was performed (see below). One animal from the E group was excluded from all analyses due to inadequate fixation.

RICC
All antibodies used in this experiment were tested for their specificity (see below) and titrated to attain concentrations at which maximal antibody binding occurred with minimal background signaling. The techniques used for RICC have been previously described (13). Briefly, to evaluate immunoreactivity to syntaxin and synaptophysin, slides were rinsed in 0.1 M PBS (three times, 5 min each time), then blocked in 1% BSA in PBS for 1 h at room temperature. Slides were then incubated with the primary antibody (1:500) diluted in 1% BSA in PBS (monoclonal antisyntaxin and monoclonal antisynaptophysin raised in mice, both IgG1 isotype; Sigma-Aldrich Corp., St. Louis, MO) overnight at 4 C. Slides incubated without primary antibody were used as negative controls. Slides were then rinsed in PBS (three times, 5 min each time), incubated with secondary antibody (1:100) in PBS (antimouse Ig whole antibody sheep-raised with ³⁵S label; Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at room temperature, rinsed in ice-cold PBS (three times, 5 min each time), then dipped in ice-cold distilled water (dH₂O), and left to air-dry at room temperature overnight.

For spinophilin immunoreactivity, slides were rinsed in PBS with 0.1% Triton X-100 (nine times, 5 min each time), then left to incubate in PBS with 0.1% Triton overnight at room temperature. Slides were then blocked in 2% BSA in PBS with 0.1% Triton for 1 h at room temperature, rinsed in PBS for 5 min, then incubated with primary antibody (1:500) diluted in 2% BSA in PBS (polyclonal antispinophilin raised in rabbit; Upstate Biotechnology, Inc., Lake Placid, NY) for 4 d. Sections incubated without primary antibody were used as negative controls. Slides were then rinsed in PBS (three times, 5 min each time) and incubated with secondary antibody (1:100) in PBS (antirabbit Ig whole antibody raised in donkey with ³⁵S label; Amersham Pharmacia Biotech) for 1 h at room temperature. After rinses in ice-cold PBS (three times, 5 min each time) and dH₂O, slides were left to air-dry at room temperature overnight. Dried slides were then placed on Kodak Biomax film (Eastman Kodak Co., New Haven, CT) with microscale-calibrated ¹⁴C standard strips (Amersham Pharmacia Biotech) and exposed for 2 d. The omission of the syntaxin, synaptophysin, or spinophilin primary antibody resulted in no specific staining in the CA1 region or dentate gyrus of the hippocampus. In addition to the primary antibody omissions and Western blots (see below), we determined the specificity of the spinophilin antibody by preadsorbing the antibody with purified spinophilin protein (2 µg/ml; gift from Dr. Paul Greengard, Rockefeller University) at 4 C for 24 h. This preadsorption reduced the specific spinophilin signal in the CA1 region and dentate gyrus of the hippocampus. Preadsorptions were not run on the syntaxin and synaptophysin antibodies, because these antibodies have been shown to cross-react with primate tissue (information supplied by the manufacturer, Sigma-Aldrich Corp.).

Antibody specificity: Western blots
To determine whether each antibody employed here was specific and selective in primates, Western blots were used to examine whole cell protein extracts from hippocampal tissue from a single macaque. Proteins were extracted from hippocampal samples by homogenizing tissue in ice-cold lysate buffer containing 0.4 M NaCl, 1 mM EDTA, and 10% glycerol, along with protease inhibitors (1 µg/ml pepstatin, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 2 mM dithiothreitol). The homogenate was immediately centrifuged at 4 C (105,000 x g for 30 min), and the supernatant was stored at -20 C.

The protein concentration was estimated with a protein assay (Bio-Rad Laboratories, Inc., Richmond, CA). Aliquots containing 40 µg protein were separated using NuPAGE 4–20% Bis-Tris gel (Invitrogen, Carlsbad, CA) and were transferred onto a nitrocellulose membrane. The membranes were then processed for signal using the Western Breeze chemiluminescent immunodetection kit (Invitrogen, San Diego, CA). Membranes were blocked for 30 min at room temperature using a Hammessten casein blocking agent and were probed with antibodies diluted in blocking agent (antisyntaxin, 1:8000; antisynaptophysin, 1:5000; antispinophilin, 1:2000) overnight at 4 C. Membranes were washed and then incubated in antimouse (syntaxin and synaptophysin) and antirabbit (spinophilin) IgG alkaline phosphatase-conjugated secondary antibody for 30 min. The antibody-reactive bands were visualized using chemiluminescence. Antibodies were titrated to determine maximum signal to minimum background binding to determine the concentrations listed here.

Two of the three primary antibodies used in this study recognize a single protein band migrating at the appropriate mass in the macaque hippocampus. Specifically, the antisynaptophysin antibody recognizes only one band migrating at the mass of synaptophysin (38 kDa), whereas the antispinophilin antibody recognizes the 120-kDa spinophilin band. The antisyntaxin antibody recognized a double band around the appropriate mass (syntaxin, 35 kDa) along with a secondary minor band that migrates in the 60–70 kDa range. This staining pattern for syntaxin is also observed after blotting rat hippocampus (Brake, W. G., unpublished observation).

OD measurements
For RICC analysis, OD measurements were taken using computerized image analysis software (MCID-M4, Imaging Research, Inc., St. Catherines, Canada). The stratum oriens and stratum radiatum of the CA1 region and the molecular and polymorphic layers of the dentate gyrus were analyzed. The density of the temporal lobe axonal tracts (which contain no pre- or postsynaptic proteins) was measured as background and subtracted from the density of each hippocampal subregion. The experimenter measuring the ODs was blinded to the treatment condition of the animals. Due to weak signal, one animal each from the E₂, E₂+P, and P alone groups were excluded from the syntaxin analyses, whereas one animal from the E₂ group was excluded from the synaptophysin analyses. Criterion for exclusion was any value at least 3 SD below the mean for that experimental group.

Statistical analyses
All data were analyzed using one-way ANOVAs. Significant differences were probed with Student-Newman-Keuls post hoc tests. Differences were considered significant at P < 0.05. All data are presented as the mean ± SEM.

	Results

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We examined the effects of E₂ on two presynaptic markers, syntaxin and synaptophysin (Fig. 1). A one-way ANOVA revealed that syntaxin levels were significantly elevated in the stratum radiatum of E₂-treated females [F(1, 12) = 4.775; P < 0.05; Fig. 1], with a trend for syntaxin to be elevated in the stratum oriens. Syntaxin levels in monkeys receiving E₂+P or P alone were not different from those in control, spayed monkeys. For synaptophysin, E₂ alone and P alone significantly increased synaptophysin levels in both the stratum oriens and radiatum [F(1, 14) = 7.812 and 4.986, respectively; P < 0.05; Fig. 1]. However, E₂+P treatment caused synaptophysin to return to levels observed in control, spayed monkeys.

View larger version (20K): [in this window] [in a new window]   FIG. 1. The relative ODs of syntaxin (top), synaptophysin (middle), and spinophilin (bottom) in the stratum oriens and radiatum in the CA1 region of spayed female rhesus macaques treated with placebo (spayed), E2, E2+P, or P alone. Asterisks indicate significant differences from bars without asterisks. All data are presented as the mean ± SEM.

View larger version (92K): [in this window] [in a new window]   FIG. 2. A representative autoradiogram of the spinophilin signal in the stratum oriens and radiatum in the CA1 region and molecular and polymorphic layers of the dentate gyrus of spayed, E2, E2+P-treated, and P-treated monkeys. Note the increased spinophilin signal in the stratum oriens and radiatum in the CA1 region of the E-treated monkey. Arrowheads indicate the layers analyzed. The bottom left panel is a spinophilin primary deletion control. DG, Dentate gyrus; mDG, molecular layer of the dentate gyrus; pDG, polymorphic layer of the dentate gyrus; SO, stratum oriens; SR, stratum radiatum.

	Discussion

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These data are consistent with prior results found in rats and mice (4, 10, 13), while expanding the role of E in its synaptogenic effects to higher order mammals. Our findings also support a recent Golgi study which showed that E treatment increased spine density in the hippocampus of ovariectomized adult female African green monkeys (17). Furthermore, our results are in agreement with a recent study demonstrating the ability of E to increase spinophilin immunoreactivity in the hippocampus of ovariectomized rhesus macaques (23). That we were able to show an increase in both pre- and postsynaptic proteins suggests that the increased number of spines in the above-mentioned studies form synapses as well.

The ability of E to increase synapses within the hippocampus suggests that this hormone may play a role in certain cognitive functions. Numerous clinical studies in postmenopausal women have demonstrated that E replacement therapy may improve certain aspects of memory and cognitive function (7, 8, 9, 24). Thus, increased synaptic connectivity might be one mechanism of action for E’s cognitive enhancing effects. Precisely how E mediates these effects still needs to be delineated. Prior studies using macaque hippocampus have been unable to demonstrate the presence of E receptor message or protein (Kohama, S., and C. L. Bethea, unpublished observation), although E receptor ß mRNA has been detected in this region (25). Future experiments using specific E receptor antagonists need to be performed to define the pathway by which E induces alterations in the nonhuman primate synapse. Moreover, the involvement of possible trans-synaptic effects of E need to be examined, such as the estrogenic regulation of hippocampal inhibitory interneurons and cholinergic inputs to the hippocampus.

The effects of P on synaptogenesis have been less well studied in rodents and not at all in nonhuman primates. Prior studies in rats have shown that P administered concomitantly with E results in decreased spine density in the CA1 region (19). Our results parallel these findings, as the E₂+P group had lower levels of syntaxin, synaptophysin, and spinophilin levels compared with the E₂ alone group. Indeed, the levels of synaptic proteins in the E₂+P group were similar to those found in untreated, spayed females. Taken together, these data suggest that the decrease in cognitive function observed in postmenopausal women treated with E and P (7, 18) may be due in part to decreases in hippocampal synaptogenesis.

Interestingly, the female monkeys in our study that received P alone showed an increase in synaptophysin levels. The effect of P alone on synaptic density is relatively unknown. Certain forms of P have been reported to be neurotrophic and neuroprotective in primary hippocampal cultures (26). Moreover, a recent study in mouse cerebral cortical explants showed that treatment with either E₂ or P alone was able to stimulate the phosphorylation of Akt (27). Akt is part of a signaling cascade shown to induce the translation of PSD-95 mRNA (28), a key postsynaptic protein. Thus, our data suggest that P alone may be capable of stimulating similar growth-promoting and neuroprotective effects as E and may be operating through similar mechanisms. However, the specific mechanisms for the P effect must be different for synaptophysin, as the other protein markers, syntaxin and spinophilin, did not show an effect of P alone. It should be noted that although syntaxin and synaptophysin are both presynaptic proteins, they need not show identical increases to affect synaptogenesis. Indeed, synaptophysin and syntaxin exhibit different temporal patterns of expression during synaptogenesis in development and aging (29).

What is needed are morphological (i.e. electron microscopy) studies that can more specifically enumerate synaptic connections after hormonal treatment. Moreover, studies have shown that aged subjects react differently to estrogenic stimulation at the synaptic level. For instance, E appears to increase the concentration of N-methyl-D-aspartate receptors per synapse in aged female rats, but not the absolute number of synapses (30). Aged rhesus macaques do show increased spinophilin immunoreactivity in response to E₂ treatment, although to a lesser degree than their young adult counterparts (23). The subjects used in our study were all young to middle-aged adults. Thus, it would be interesting to examine the effects of E alone on presynaptic proteins as well as E and P effects on the levels of pre- and postsynaptic proteins in the aged female primate hippocampus.

In summary, our findings indicate that E increases syntaxin, synaptophysin, and spinophilin levels in the CA1 region of the female nonhuman primate hippocampus. In conjunction with previous studies, it appears that E-induced increases in cognitive function of higher order mammals may be influenced by the effect of E on hippocampal synaptogenesis and synaptic plasticity.

	Acknowledgments

 
We thank Dr. Susan Lee for valuable technical assistance.

	Footnotes

 
This work was supported by NIH Grants NS07080 (to B.S.M.), MH62677 (to C.L.B.), and RR-00163 (for operation of Oregon National Primate Research Center).

Abbreviations: E, Estrogen; E₂, estradiol; P, progesterone; RICC, radioimmunocytochemistry.

Received February 18, 2003.

Accepted for publication August 7, 2003.

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