The 17 and 17? Isomers of Estradiol Both Induce Rapid Spine Synapse Formation in the CA1 Hippocampal Subfield of Ovariectomized Female Rats
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内分泌学杂志 2005年第1期
Center for Neural Recovery and Rehabilitation Research (N.J.M.), Helen Hayes Hospital, New York, New York 10993; Departments of Obstetrics, Gynecology and Reproductive Sciences (T.H., C.L.) and Neurobiology (C.L.), Yale University School of Medicine, New Haven, Connecticut; Department of Psychology (V.N.L.), Hunter College of City University of New York, New York, New York; and Laboratory of Molecular Neurobiology (T.H.), Biological Research Center, Hungarian Academy of Sciences, H6726 Szeged, Hungary
Address all correspondence and requests for reprints to: Neil J. MacLusky, Ph.D., Center for Neural Recovery and Rehabilitation Research, Helen Hayes Hospital, West Haverstraw, New York, New York 10993. E-mail: macluskyn@helenhayeshosp.org.
Abstract
Previous studies have demonstrated that estradiol-17? and estradiol-17 both induce short-latency effects on spatial memory in rats, estradiol-17 being at least as potent as its 17? isomer. To determine whether the mechanisms underlying these behavioral responses might include effects on hippocampal synaptic plasticity, CA1 pyramidal spine synapse density (PSSD) was measured in ovariectomized rats within the first few hours after sc estrogen injection. PSSD increased markedly (by 24%) 4.5 h after the administration of 45 μg/kg estradiol-17?. The PSSD response was significantly greater (44% above control) 30 min after estradiol-17? injection and was markedly dose dependent; a 3-fold lower estradiol-17? dose (15 μg/kg) did not significantly affect CA1 PSSD at either 30 min or 4.5 h. Estradiol-17 was a more potent inducer of PSSD than estradiol-17?. Dose-response analysis determined an ED50 for the effect of estradiol-17 on PSSD of 8.92 ± 1.99 μg/kg, with a maximal response at 15 μg/kg. These results demonstrate that high doses of estradiol induce rapid changes in CA1 PSSD. CA1 spine synapse formation appears to be more sensitive to estradiol-17 than to estradiol-17?, paralleling previous data on the effects of these two steroids on spatial memory. Rapid remodeling of hippocampal synaptic connections may thus contribute to the enhancement of spatial mnemonic processing observed within the first few hours after estrogen treatment. The potency of estradiol-17 suggests that hormone replacement therapy using this steroid might be useful clinically in ameliorating the impact of low endogenous estrogen production on the development and progression of neurodegenerative disorders involving the hippocampus.
Introduction
THE HIPPOCAMPAL FORMATION is believed to be involved in the mechanisms mediating the formation of memory, particularly memory that uses spatial cues (spatial memory). This region of the brain is also remarkable because it contains receptors for the principal steroid hormones, a variety of trophic factors, as well as a rich innervation from cholinergic, serotonergic, catecholaminergic, and glutamatergic systems (1, 2). A wide variety of hormones and drugs that affect these systems have also been shown to affect performance of spatial memory tasks (3).
Estrogens are potent regulators of mnemonic function. Low estrogen is associated with poor performance of spatial and other memory tasks in rats, whereas estrogen replacement enhances performance (4). Correlations between memory and circulating gonadal hormone levels have also been demonstrated in human beings (5, 6, 7). Although the mechanisms underlying these effects remain largely unknown, there is growing evidence to support the hypothesis that these effects may involve remodeling of the hippocampal circuitry. Estrogens have been shown to alter the density of pyramidal cell dendritic spines and apical spine synapses in the CA1 subfield of the hippocampus (8, 9, 10, 11, 12, 13). Paralleling these morphological changes, Sandstrom and Williams (14, 15) have demonstrated enhancement of a working memory version of the Morris water maze spatial memory task, within the time frame of previously reported estrogen-induced increases in spine density. Because changes in hippocampal-dependent trace conditioning are accompanied by effects on spine density (16), these results suggest that steroid-dependent changes in synaptic or spine density in the CA1 area may at least partially be responsible for hormonal effects on cognitive functions mediated by the hippocampus.
Behavioral and CA1 structural responses to gonadal steroids have typically been studied within 24–48 h after hormone treatment. Recently, however, we found that performance of a spatial memory task, object placement, could be enhanced in ovariectomized (OVX) rats within a much shorter interval (4–4.5 h) after estrogen administration (17). Moreover, a significantly enhanced response was elicited by estradiol-17 at lower doses than by estradiol-17?, contrasting with transcriptional estrogen responses, which are usually more sensitive to the 17? isomer (17, 18). Because previous studies have shown that CA1 dendritic structure is modulated within only a few hours of the changes in ovarian steroid levels occurring at proestrus (8), we postulated that the mnemonic effects of both isomers of estradiol might involve rapid alterations in CA1 pyramidal spine synapse density (PSSD). The present study was designed to test this hypothesis, by measuring CA1 PSSD within the first few hours after injection of the same doses of estradiol used in our previous behavioral studies.
Materials and Methods
Animals
Adult female Sprague Dawley rats (250–300 g; Charles River Laboratories, Wilmington, MA) were used throughout this study. Animals were kept under standard laboratory conditions, with tap water and regular rat chow ad libitum, under a 12-h light, 12-h dark cycle. All experiments conformed to National Institutes of Health and international guidelines on the ethical use of animals in experiments. Experimental protocols were approved by the Institutional Animal Care and Use Committee of Yale University Medical School.
Surgery and hormonal manipulations
Experiment 1.
Nine rats (three treatment groups each containing three animals) were anesthetized using a ketamine-xylazine cocktail (3 ml/kg im, containing 25 mg ketamine, 1.2 mg xylazine, and 0.03 mg acepromazine in 1 ml saline) and OVX. All animals were housed individually after surgery. Seven days later, the animals were treated with varying doses of estradiol-17? (0–45 μg/kg) dissolved in sesame oil (200 μl) via sc injection, 4.5 h before being killed.
Experiment 2.
Twelve rats (four treatment groups each containing three animals) were OVX as described above and, 1 wk later, injected sc with different doses of estradiol-17? (0–60 μg/kg) in sesame oil (200 μl) 30 min before being killed
Experiment 3.
Five rats were OVX as described above. The animals were injected sc with 45 μg/kg estradiol-17? in sesame oil (200 μl). At varying time intervals thereafter (10, 20, and 60 min), the rats were briefly sedated by exposure to CO2 gas and small samples of blood (100–200 μl) were withdrawn from the tail vein. At 270 min after injection, the rats were killed with CO2, the chest cavity was opened, and mixed venous blood was sampled directly from the right ventricle. The blood samples were allowed to clot at room temperature, and serum was separated and assayed for estradiol-17? using a commercially available RIA kit (Coat-A-Count kit, catalog item KE2D1; Diagnostic Products Corp., Los Angeles, CA).
Experiment 4.
Sixteen rats were OVX as described above, 1 wk before the experiments. In one group, nine rats (three groups of three animals) were injected sc with 0, 15, or 45 μg/kg estradiol-17 in sesame oil (200 μl) 30 min before being killed. In another group, seven rats were injected sc 30 min before being killed with a range of estradiol-17 doses (0–20 μg/kg) to generate a dose-response curve for the effects of this steroid on PSSD.
Tissue processing
For morphological studies, at the appropriate time intervals after estradiol or vehicle injection, rats were killed under deep ether anesthesia by transcardial perfusion of heparinized saline followed by a fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.35). Brains were removed and postfixed overnight in the same fixative without glutaraldehyde. The hippocampi were dissected out, and vibratome sections (100 μm) were cut perpendicular to the longitudinal axis of the hippocampus. Sections were postfixed in 1% osmium tetroxide (30 min), dehydrated in ethanol (the 70% ethanol contained 1% uranyl acetate for 30 min), and flat embedded in Araldite.
Synapse counts
PSSD was calculated according to our standard protocol using unbiased stereological methods (11, 12, 19). Briefly, to assess possible changes in the volume of the tissue, a correction factor was first calculated assuming that the treatments did not alter the total number of pyramidal cells (20). Thus, in all hippocampi, six to seven disector pairs (pairs of adjacent 2-μm toluidine blue-stained semithin sections mounted on slides) were analyzed using the technique of Braendgaard and Gundersen (21). The pyramidal cell density value (D) was calculated using a formula D = N/sT, where N is the mean disector score across all sampling windows, T is the thickness of the sections (2 μm), and s stands for the length of the window. Based on these values, a dimensionless volume correction factor kv was introduced: kv = D/D1, where D1 is the mean density across the groups of hippocampi.
Thereafter, disector pairs of consecutive serial ultrathin sections (reference and look-up) were cut from vibratome sections taken from all parts of the hippocampus along its septo-temporal axis and collected on formvar-coated single-slot grids. Subsequently, digitized images were taken at a magnification of x11,000 in a Tecnai 12 transmission electron microscope furnished with an AMT Advantage 4.00 HR/HR-B CCD camera system from an area located between the upper and middle third of the CA1 stratum radiatum (300–500 μm from the pyramidal cell layer; for an illustration of the precise hippocampal area sampled, see Ref. 22). Identical regions in reference and look-up sections were identified using landmarks such as myelinated fibers, large dendrites, or blood vessels that were not changed significantly between neighboring sections because of their size. Areas occupied by potentially interfering structures such as blood vessels, large dendrites, or glial cells were subtracted from the measured areas using the NIH Scion Image software.
To obtain a comparable measure of synaptic numbers, unbiased for possible changes in synaptic size, the disector technique was used (23). The digitized electron micrographs were printed out using a laser printer. Before data analysis, the printed pictures were coded, and the code was not broken until the analysis was completed. Only those spine synapses were counted that were present in the reference section but not in the look-up section. To increase the efficiency of spine synapse counting, the analysis was performed treating each reference section as a look-up section and vice versa (10).
PSSD was calculated with the help of a reference grid superimposed on the electron microscopic prints. The disector volume (volume of reference) was the unit area of the reference grid multiplied by the distance between the upper faces of the reference and look-up sections (21). Section thickness (average, 0.075 μm) was determined using the electron scattering technique. The measured synaptic density values were divided by the volume correction factor kv. This correction provided a synaptic density estimate normalized with respect to the density of pyramidal cells and also accounted for possible changes in hippocampal volume.
Statistical analysis
For synapse counts, at least 10 neuropil field-pairs were photographed on each electron microscopic grid. With at least three grids (containing a minimum of two consecutive ultrathin sections) prepared from each vibratome section (cut from the three portions of the hippocampus along its septo-temporal axis), each animal provided at least 3 x 3 x 10 x 2 = 180 neuropil fields for evaluation. PSSD for each animal was determined independently by two different investigators who were blinded to the identity of the treatment groups, and the results were cross-checked to preclude systematic analytical errors. Average PSSD values for each animal were used to calculate mean synapse densities (± SEM) for each treatment group. Results were analyzed by means of ANOVA, followed by the Scheffé test for comparison of individual group means. A criterion for statistical confidence of P < 0.05 (two-tailed) was adopted. PSSD dose-response data were analyzed by least-squares regression analysis, using a commercially available computer program (Sigmaplot 5.0; SPSS Inc., Chicago, IL).
Results
Initial experiments examined the effects of estradiol-17? on PSSD in the CA1 stratum radiatum at 4.5 h after estradiol administration, corresponding to the time interval at which we previously found estrogen to enhance spatial memory performance (17). Injection of 15 μg/kg estradiol-17? did not significantly affect PSSD measured 4.5 h later, compared with OVX vehicle-injected rats (Fig. 1). However, injection of a higher dose of estradiol-17? (45 μg/kg) resulted in a statistically significant (24%) increase in CA1 PSSD (Fig. 1).
FIG. 1. Density of pyramidal cell spine synapses in the CA1 stratum radiatum of the hippocampi of OVX rats 4.5 h after estradiol-17? (E2) injection. Rats were injected with either 15 or 45 μg/kg estradiol-17? in sesame oil (200 μl, sc) or the injection vehicle alone. Statistical analysis by one-way ANOVA: F = 31.0; df 2,6; P = 0.0007. Letters above the histogram bars indicate the results of the Scheffé post hoc test (P < 0.05 level). Groups labeled with the same letter are not significantly different from one another.
The effects of estradiol-17? were also studied at a shorter time (30 min) after administration of different doses of estradiol-17?. Results are shown in Fig. 2. Mean PSSD at 30 min after injection of 15 μg/kg estradiol-17? was slightly, but not significantly, elevated compared with that of vehicle-injected OVX females. However, at 30 min after 45 μg/kg estradiol-17?, CA1 PSSD was markedly increased (44%) compared with vehicle-injected controls. This response was further augmented, to approximately 65% above control, after administration of 60 μg/kg estradiol-17? (Fig. 2).
FIG. 2. Density of pyramidal cell spine synapses in the CA1 stratum radiatum of the hippocampi of OVX rats 30 min after estradiol-17? injection. Rats were injected with either different doses (15, 45, or 60 μg/kg) of estradiol-17? in sesame oil (200 μl, sc) or the injection vehicle alone. Statistical analysis by one-way ANOVA: F = 120.7; df 3,8; P < 0.0001. Letters above the histogram bars indicate the results of the Scheffé post hoc test (P < 0.05 level). Groups labeled with the same letter are not significantly different from one another.
To determine the circulating levels of estradiol-17? produced by the estradiol injections, serum concentrations of the hormone were measured at intervals over the first hour, as well as at 4.5 h, after sc injection of 45 μg/kg estradiol-17?. Results are presented in Table 1. Within 10 min of injection, serum estradiol-17? levels rose to more than 500 pg/ml. Thereafter, the concentrations of the steroid continued to increase, reaching more than 1 ng/ml at 4.5 h after treatment.
TABLE 1. Serum estradiol concentrations measured by RIA at different times after sc injection of OVX rats with estradiol (45 μg/kg) dissolved in 200 μl sesame oil
Rapid enhancement of object placement performance is observed after either estradiol-17? or estradiol-17 (17), despite the latter’s low affinity for estrogen receptors (24). Therefore, additional experiments were performed to determine whether estradiol-17 treatment also increases PSSD. Administration of 45 μg/kg estradiol-17 had a rapid and dramatic effect on CA1 PSSD, apparent even from inspection of individual micrographs (Fig. 3). Quantitative analysis of CA1 PSSD at 30 min after administration of 15 or 45 μg/kg estradiol-17 is summarized in Fig. 4A. Injection of 45 μg/kg estradiol-17 increased CA1 PSSD by 62%. Injection of a lower dose of the steroid (15 μg/kg) elicited an even larger increase in PSSD, to 81% above control. The PSSD response to 15 μg/kg estradiol-17 was greater than that observed at the same time interval after injection of 3- to 4-fold higher doses of estradiol-17? (Fig. 4A, cf. Fig. 2). To determine the dose dependency of the response to estradiol-17, OVX rats were injected with a range of doses of estradiol-17 between 0 and 20 μg/kg. PSSD was measured 30 min later. Results are shown in Fig. 4B. The data for PSSD vs. estradiol-17 dose were fitted by a four-parameter logistic dose-response curve. At 15 μg/kg, the response was maximal, within the range of the measurements obtained in OVX rats injected with 15 or 45 μg/kg estradiol-17 (Fig. 4A), close to the mean PSSD observed in animals treated with 60 μg/kg estradiol-17? (Fig. 2). The ED50 for the PSSD response to estradiol-17, calculated from the logistic curve fit, was 8.92 ± 1.99 μg/kg.
FIG. 3. Electron micrographs taken from the CA1 stratum radiatum of an OVX rat that received a sc injection of 45 μg/kg estradiol-17, dissolved in sesame oil, 30 min before perfusion-fixation (A) compared with an OVX rat that was killed the same length of time after injection of the oil vehicle alone (B). Note the higher density of spine synapses (arrows) in the estradiol-17-treated animal (A). D, dendrite; bar scale, 1 μm.
FIG. 4. Effects of estradiol-17 on the density of pyramidal cell spine synapses in the CA1 stratum radiatum of OVX rats. A, Rats were injected with either different doses (15 or 45 μg/kg) of estradiol-17 (E2-17) or the injection vehicle (sesame oil) alone and killed 0.5 h after injection. Statistical analysis by one-way ANOVA: F = 208.5; df 2,6; P < 0.0001. Letters above the histogram bars indicate the results of the Scheffé post hoc test (P < 0.05 level). Groups labeled with different letters are significantly different from one another. B, Dose-response curve for the induction of CA1 spine synapses by estradiol-17. OVX rats were injected with increasing doses of estradiol-17 in sesame oil vehicle, 30 min before perfusion-fixation. The mean densities of CA1 spine synapses in each animal are plotted against estradiol-17 dose. The line represents the best-fit four-parameter logistic regression calculated from the data. The ED50 calculated from the curve fit is indicated on the graph ± SE of the estimate, determined from the regression analysis.
Discussion
Previous studies have demonstrated that estradiol-17? increases both the number of dendritic spines and the density of spine synaptic contacts (PSSD) on CA1 pyramidal neurons (8, 9, 10, 11, 12, 13). These responses have been postulated to contribute to the effects of estradiol-17? on hippocampal function, including spatial memory (4, 25, 26). Recent work, however, indicated that estradiol-17? can enhance performance on a test of spatial memory, object placement, within 4 h of exposure to the hormone (17), much more rapidly than previously reported effects of estrogen on hippocampal PSSD. These results raised the question of whether changes in hippocampal synapse density occur with sufficient speed to contribute to the earliest behavioral effects of the hormone. The present studies were designed to examine this question, by measuring PSSD after treatment paradigms identical with those used in the behavioral studies. In our previous object placement behavioral tests (17), performance was enhanced by either estradiol-17? or estradiol-17, when sufficient doses were administered 30 min before the sample trial (4.5 h before the recognition/retention trial. We hypothesized that if CA1 PSSD contributes to rapid estrogen-mediated enhancement of spatial memory, then treatments that enhance object placement performance should also induce a significant increase in PSSD. Conversely, behaviorally ineffective hormone treatments should not significantly alter PSSD.
In OVX rats, PSSD was significantly higher 4.5 h after administration of 45 μg/kg estradiol-17?, but not after 15 μg/kg of the hormone. These observations are consistent with the hypothesis that increased CA1 PSSD may be associated with enhancement of object placement performance, because they parallel the behavioral data (17). Place memory was not significantly affected by 15 μg/kg estradiol-17? but was enhanced after higher doses (30–60 μg/kg) of the steroid, comparable to those required to induce increased PSSD. By contrast, visual recognition memory was enhanced by the lower dose of estrogen, 15 μg/kg (17). The differential association of increased PSSD with estradiol enhancement of place, as opposed to object recognition, memory may reflect the relative importance of the hippocampal circuitry in the acquisition of different types of mnemonic information. In rats, the hippocampus plays an essential role in place memory, whereas other regions of the brain appear to be more important for processing of visual recognition information (27, 28).
Unexpectedly, the data show that the increase in PSSD at 30 min after estradiol administration is even larger than that at 4.5 h. On the basis of previous studies of hippocampal pyramidal dendritic structure after estrogen exposure (8, 9, 10, 29), we anticipated that synaptic responses might not be observed until at least several hours after estradiol-17? injection. Clearly, however, CA1 PSSD can be modulated by estrogen within a much shorter time frame. At 30 min after injection of 45–60 μg/kg estradiol-17?, PSSD is already within the range of previous data obtained after 2 days of estrogen treatment (11). A possible explanation for both the rapid onset of the increase and subsequent decline in PSSD is provided by the data on serum estradiol levels. After sc injection in sesame oil, circulating estradiol-17? levels increase rapidly (30) (Table 1), consistent with the view that increases in PSSD may be initiated almost immediately after exposure of the hippocampus to elevated estrogen concentrations. The response is not sustained, however, because by 4.5 h PSSD falls substantially, despite the rising levels of estradiol in the circulation. There are two possible explanations for these data. The initial rapid induction of PSSD may be only a transient response. Alternatively, the decline in PSSD at 4.5 h may reflect down-regulation of the response mechanism, as estradiol-17? levels continue to increase. The latter hypothesis is supported by the data obtained with estradiol-17. PSSD at 30 min after a dose of 15 μg/kg estradiol-17 was significantly higher than after 45 μg/kg of this steroid, suggesting that the dose-response relationship between estrogen dose and synapse density may be bell shaped, with increases in circulating estrogen levels above maximal resulting in a diminished effect. After sc injection of estradiol-17? at 45 μg/kg, circulating hormone levels may rapidly rise to the point at which PSSD is maximally increased, the response then being reversed as serum hormone concentrations continue to climb (Table 1).
A second important conclusion suggested by the serum estradiol measurements is that short-latency effects on PSSD may be observed only with supraphysiological levels of the hormone. A significant increase in PSSD was observed only at 45 μg/kg, not at 15 μg/kg, of estradiol-17?. Subcutaneous injection of 45 μg/kg estradiol-17? results in circulating estradiol concentrations that are at least 10-fold higher than those observed at any stage of the reproductive cycle in normal female rats (31). Although circulating estradiol-17? was not measured after the lower, ineffective dose (15 μg/kg) of the steroid, it is a reasonable presumption that this probably also resulted in high estradiol concentrations during the first few hours after injection. These observations contrast with the situation in normal females, in which the considerably lower levels of estradiol released during the estrous cycle are known to induce a significant increase in CA1 PSSD (10). Although additional work will be necessary to precisely define the dose and time dependency of changes in PSSD after systemic estrogen administration, the available data suggest that estrogen-mediated induction of hippocampal spine synapses may involve mechanisms that can respond with differing latencies and time courses, depending on the circulating hormone concentrations. Rapid induction of both increases in CA1 PSSD (the present study) and hippocampal-mediated enhancement of object placement memory (17) may be observed only when estradiol-17? levels are above the normal physiological range.
The cellular mechanisms responsible for these effects remain to be elucidated. A reasonable hypothesis, however, is that the immediate and delayed responses to estradiol may reflect activation of different estrogen response mechanisms. Biological effects of estrogens include transcriptional responses mediated via activation of the nuclear estrogen receptor proteins, ER and ER?, as well as more rapid responses mediated via membrane receptor sites (32). In addition to the speed of the response, the potency of estradiol-17 raises the possibility that rapid induction of PSSD formation may specifically reflect the activation of membrane receptor systems. Membrane-associated ERs have been shown to exhibit enhanced sensitivity to estradiol-17, reflecting either the lipid-rich environment of the receptors in the membrane or the presence of unique membrane-associated receptor isoforms (33, 34). The present data indicate that rapid increases in PSSD can be activated by either estradiol-17? or estradiol-17, the latter being considerably more potent. By contrast, nuclear ER-mediated responses, such as uterine growth, typically are more than 100-fold less sensitive to estradiol-17 (17, 18, 24). Although in vivo potency does not necessarily reflect receptor affinity, because of the potential for contributions from hormone metabolism and clearance, the disparity between the present results and previous observations on nuclear ER-mediated responses is such that it would seem unlikely that they are mediated via identical receptor mechanisms.
There are several ways in which activation of membrane ERs could potentially be translated into effects on synaptogenesis. Membrane-associated ERs rapidly modulate important intracellular signaling pathways (reviewed in Ref. 32). Induction of ERK phosphorylation has been implicated in membrane receptor-mediated responses to estrogen (35, 36) and regulation of hippocampal synaptic plasticity (35, 36) as well as in learning processes mediated via the hippocampus (37). Estradiol also activates phosphatidylinositol 3-kinase, leading to phosphorylation of Akt, in the developing cerebral cortex (38) and in neurally derived cell lines (39) as well as in CA1 dendrites (40). Akt-like proteins have been implicated in cellular chemotaxis (41, 42, 43), whereas phosphorylation of Akt has been demonstrated to regulate transcription-independent synthesis of proteins involved in glutamatergic synapse formation (39, 44). These observations have led to a proposed mechanism for the effects of estrogen on CA1 dendritic spine density, involving estrogen-induced changes in synaptic protein synthesis and dendritic filopodial extension (45), which could also explain the rapid effects of estrogen on synaptogenesis, reported here. High concentrations of estradiol for a short time, or lower concentrations of the hormone over a longer period (10, 46), may activate membrane-associated signaling cascades, thereby altering the regulation of axonal and/or dendritic growth processes to increase the probability of spine synapse formation.
Regardless of the underlying mechanisms, the fact that the short-latency trophic effects of estrogen on the hippocampus can be mimicked by estradiol-17 has important implications for the potential development of novel forms of hormone replacement therapy (HRT). Numerous studies have demonstrated neurotrophic and neuroprotective effects of estrogen (reviewed in Refs. 35 and 47), consistent with clinical data suggesting that estrogen-based HRT may slow the progression of neurodegenerative diseases (48, 49). Long-term postmenopausal HRT, however, increases the risk of strokes, as well as breast and endometrial carcinoma (50, 51). Our data suggest that estradiol-17, a relatively weak estrogen in the tissues of the reproductive tract, is even more potent than estradiol-17? in terms of the rapid regulation of PSSD (Fig. 2, cf. Fig. 4) and at least as potent as estradiol-17? as an enhancer of short-term working memory (17). These findings parallel previous results on estrogen regulation of neuronal survival (52), the processing of amyloid precursor protein (53), and the expression of apolipoprotein E [a cofactor for estrogen-activated neurotrophic responses (54, 55, 56)], all of which exhibit sensitivity to estradiol-17 as well as estradiol-17?. Taken together, these data raise the possibility that HRT using estradiol-17 or structurally related estrogens might be capable of reproducing the central neuroprotective and neurotrophic effects of circulating estradiol-17?, while minimizing the potential for aberrant trophic responses in the peripheral reproductive target organs.
Acknowledgments
We are indebted to Klara Szigeti-Buck and Gladis Thomas for excellent technical assistance.
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Address all correspondence and requests for reprints to: Neil J. MacLusky, Ph.D., Center for Neural Recovery and Rehabilitation Research, Helen Hayes Hospital, West Haverstraw, New York, New York 10993. E-mail: macluskyn@helenhayeshosp.org.
Abstract
Previous studies have demonstrated that estradiol-17? and estradiol-17 both induce short-latency effects on spatial memory in rats, estradiol-17 being at least as potent as its 17? isomer. To determine whether the mechanisms underlying these behavioral responses might include effects on hippocampal synaptic plasticity, CA1 pyramidal spine synapse density (PSSD) was measured in ovariectomized rats within the first few hours after sc estrogen injection. PSSD increased markedly (by 24%) 4.5 h after the administration of 45 μg/kg estradiol-17?. The PSSD response was significantly greater (44% above control) 30 min after estradiol-17? injection and was markedly dose dependent; a 3-fold lower estradiol-17? dose (15 μg/kg) did not significantly affect CA1 PSSD at either 30 min or 4.5 h. Estradiol-17 was a more potent inducer of PSSD than estradiol-17?. Dose-response analysis determined an ED50 for the effect of estradiol-17 on PSSD of 8.92 ± 1.99 μg/kg, with a maximal response at 15 μg/kg. These results demonstrate that high doses of estradiol induce rapid changes in CA1 PSSD. CA1 spine synapse formation appears to be more sensitive to estradiol-17 than to estradiol-17?, paralleling previous data on the effects of these two steroids on spatial memory. Rapid remodeling of hippocampal synaptic connections may thus contribute to the enhancement of spatial mnemonic processing observed within the first few hours after estrogen treatment. The potency of estradiol-17 suggests that hormone replacement therapy using this steroid might be useful clinically in ameliorating the impact of low endogenous estrogen production on the development and progression of neurodegenerative disorders involving the hippocampus.
Introduction
THE HIPPOCAMPAL FORMATION is believed to be involved in the mechanisms mediating the formation of memory, particularly memory that uses spatial cues (spatial memory). This region of the brain is also remarkable because it contains receptors for the principal steroid hormones, a variety of trophic factors, as well as a rich innervation from cholinergic, serotonergic, catecholaminergic, and glutamatergic systems (1, 2). A wide variety of hormones and drugs that affect these systems have also been shown to affect performance of spatial memory tasks (3).
Estrogens are potent regulators of mnemonic function. Low estrogen is associated with poor performance of spatial and other memory tasks in rats, whereas estrogen replacement enhances performance (4). Correlations between memory and circulating gonadal hormone levels have also been demonstrated in human beings (5, 6, 7). Although the mechanisms underlying these effects remain largely unknown, there is growing evidence to support the hypothesis that these effects may involve remodeling of the hippocampal circuitry. Estrogens have been shown to alter the density of pyramidal cell dendritic spines and apical spine synapses in the CA1 subfield of the hippocampus (8, 9, 10, 11, 12, 13). Paralleling these morphological changes, Sandstrom and Williams (14, 15) have demonstrated enhancement of a working memory version of the Morris water maze spatial memory task, within the time frame of previously reported estrogen-induced increases in spine density. Because changes in hippocampal-dependent trace conditioning are accompanied by effects on spine density (16), these results suggest that steroid-dependent changes in synaptic or spine density in the CA1 area may at least partially be responsible for hormonal effects on cognitive functions mediated by the hippocampus.
Behavioral and CA1 structural responses to gonadal steroids have typically been studied within 24–48 h after hormone treatment. Recently, however, we found that performance of a spatial memory task, object placement, could be enhanced in ovariectomized (OVX) rats within a much shorter interval (4–4.5 h) after estrogen administration (17). Moreover, a significantly enhanced response was elicited by estradiol-17 at lower doses than by estradiol-17?, contrasting with transcriptional estrogen responses, which are usually more sensitive to the 17? isomer (17, 18). Because previous studies have shown that CA1 dendritic structure is modulated within only a few hours of the changes in ovarian steroid levels occurring at proestrus (8), we postulated that the mnemonic effects of both isomers of estradiol might involve rapid alterations in CA1 pyramidal spine synapse density (PSSD). The present study was designed to test this hypothesis, by measuring CA1 PSSD within the first few hours after injection of the same doses of estradiol used in our previous behavioral studies.
Materials and Methods
Animals
Adult female Sprague Dawley rats (250–300 g; Charles River Laboratories, Wilmington, MA) were used throughout this study. Animals were kept under standard laboratory conditions, with tap water and regular rat chow ad libitum, under a 12-h light, 12-h dark cycle. All experiments conformed to National Institutes of Health and international guidelines on the ethical use of animals in experiments. Experimental protocols were approved by the Institutional Animal Care and Use Committee of Yale University Medical School.
Surgery and hormonal manipulations
Experiment 1.
Nine rats (three treatment groups each containing three animals) were anesthetized using a ketamine-xylazine cocktail (3 ml/kg im, containing 25 mg ketamine, 1.2 mg xylazine, and 0.03 mg acepromazine in 1 ml saline) and OVX. All animals were housed individually after surgery. Seven days later, the animals were treated with varying doses of estradiol-17? (0–45 μg/kg) dissolved in sesame oil (200 μl) via sc injection, 4.5 h before being killed.
Experiment 2.
Twelve rats (four treatment groups each containing three animals) were OVX as described above and, 1 wk later, injected sc with different doses of estradiol-17? (0–60 μg/kg) in sesame oil (200 μl) 30 min before being killed
Experiment 3.
Five rats were OVX as described above. The animals were injected sc with 45 μg/kg estradiol-17? in sesame oil (200 μl). At varying time intervals thereafter (10, 20, and 60 min), the rats were briefly sedated by exposure to CO2 gas and small samples of blood (100–200 μl) were withdrawn from the tail vein. At 270 min after injection, the rats were killed with CO2, the chest cavity was opened, and mixed venous blood was sampled directly from the right ventricle. The blood samples were allowed to clot at room temperature, and serum was separated and assayed for estradiol-17? using a commercially available RIA kit (Coat-A-Count kit, catalog item KE2D1; Diagnostic Products Corp., Los Angeles, CA).
Experiment 4.
Sixteen rats were OVX as described above, 1 wk before the experiments. In one group, nine rats (three groups of three animals) were injected sc with 0, 15, or 45 μg/kg estradiol-17 in sesame oil (200 μl) 30 min before being killed. In another group, seven rats were injected sc 30 min before being killed with a range of estradiol-17 doses (0–20 μg/kg) to generate a dose-response curve for the effects of this steroid on PSSD.
Tissue processing
For morphological studies, at the appropriate time intervals after estradiol or vehicle injection, rats were killed under deep ether anesthesia by transcardial perfusion of heparinized saline followed by a fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.35). Brains were removed and postfixed overnight in the same fixative without glutaraldehyde. The hippocampi were dissected out, and vibratome sections (100 μm) were cut perpendicular to the longitudinal axis of the hippocampus. Sections were postfixed in 1% osmium tetroxide (30 min), dehydrated in ethanol (the 70% ethanol contained 1% uranyl acetate for 30 min), and flat embedded in Araldite.
Synapse counts
PSSD was calculated according to our standard protocol using unbiased stereological methods (11, 12, 19). Briefly, to assess possible changes in the volume of the tissue, a correction factor was first calculated assuming that the treatments did not alter the total number of pyramidal cells (20). Thus, in all hippocampi, six to seven disector pairs (pairs of adjacent 2-μm toluidine blue-stained semithin sections mounted on slides) were analyzed using the technique of Braendgaard and Gundersen (21). The pyramidal cell density value (D) was calculated using a formula D = N/sT, where N is the mean disector score across all sampling windows, T is the thickness of the sections (2 μm), and s stands for the length of the window. Based on these values, a dimensionless volume correction factor kv was introduced: kv = D/D1, where D1 is the mean density across the groups of hippocampi.
Thereafter, disector pairs of consecutive serial ultrathin sections (reference and look-up) were cut from vibratome sections taken from all parts of the hippocampus along its septo-temporal axis and collected on formvar-coated single-slot grids. Subsequently, digitized images were taken at a magnification of x11,000 in a Tecnai 12 transmission electron microscope furnished with an AMT Advantage 4.00 HR/HR-B CCD camera system from an area located between the upper and middle third of the CA1 stratum radiatum (300–500 μm from the pyramidal cell layer; for an illustration of the precise hippocampal area sampled, see Ref. 22). Identical regions in reference and look-up sections were identified using landmarks such as myelinated fibers, large dendrites, or blood vessels that were not changed significantly between neighboring sections because of their size. Areas occupied by potentially interfering structures such as blood vessels, large dendrites, or glial cells were subtracted from the measured areas using the NIH Scion Image software.
To obtain a comparable measure of synaptic numbers, unbiased for possible changes in synaptic size, the disector technique was used (23). The digitized electron micrographs were printed out using a laser printer. Before data analysis, the printed pictures were coded, and the code was not broken until the analysis was completed. Only those spine synapses were counted that were present in the reference section but not in the look-up section. To increase the efficiency of spine synapse counting, the analysis was performed treating each reference section as a look-up section and vice versa (10).
PSSD was calculated with the help of a reference grid superimposed on the electron microscopic prints. The disector volume (volume of reference) was the unit area of the reference grid multiplied by the distance between the upper faces of the reference and look-up sections (21). Section thickness (average, 0.075 μm) was determined using the electron scattering technique. The measured synaptic density values were divided by the volume correction factor kv. This correction provided a synaptic density estimate normalized with respect to the density of pyramidal cells and also accounted for possible changes in hippocampal volume.
Statistical analysis
For synapse counts, at least 10 neuropil field-pairs were photographed on each electron microscopic grid. With at least three grids (containing a minimum of two consecutive ultrathin sections) prepared from each vibratome section (cut from the three portions of the hippocampus along its septo-temporal axis), each animal provided at least 3 x 3 x 10 x 2 = 180 neuropil fields for evaluation. PSSD for each animal was determined independently by two different investigators who were blinded to the identity of the treatment groups, and the results were cross-checked to preclude systematic analytical errors. Average PSSD values for each animal were used to calculate mean synapse densities (± SEM) for each treatment group. Results were analyzed by means of ANOVA, followed by the Scheffé test for comparison of individual group means. A criterion for statistical confidence of P < 0.05 (two-tailed) was adopted. PSSD dose-response data were analyzed by least-squares regression analysis, using a commercially available computer program (Sigmaplot 5.0; SPSS Inc., Chicago, IL).
Results
Initial experiments examined the effects of estradiol-17? on PSSD in the CA1 stratum radiatum at 4.5 h after estradiol administration, corresponding to the time interval at which we previously found estrogen to enhance spatial memory performance (17). Injection of 15 μg/kg estradiol-17? did not significantly affect PSSD measured 4.5 h later, compared with OVX vehicle-injected rats (Fig. 1). However, injection of a higher dose of estradiol-17? (45 μg/kg) resulted in a statistically significant (24%) increase in CA1 PSSD (Fig. 1).
FIG. 1. Density of pyramidal cell spine synapses in the CA1 stratum radiatum of the hippocampi of OVX rats 4.5 h after estradiol-17? (E2) injection. Rats were injected with either 15 or 45 μg/kg estradiol-17? in sesame oil (200 μl, sc) or the injection vehicle alone. Statistical analysis by one-way ANOVA: F = 31.0; df 2,6; P = 0.0007. Letters above the histogram bars indicate the results of the Scheffé post hoc test (P < 0.05 level). Groups labeled with the same letter are not significantly different from one another.
The effects of estradiol-17? were also studied at a shorter time (30 min) after administration of different doses of estradiol-17?. Results are shown in Fig. 2. Mean PSSD at 30 min after injection of 15 μg/kg estradiol-17? was slightly, but not significantly, elevated compared with that of vehicle-injected OVX females. However, at 30 min after 45 μg/kg estradiol-17?, CA1 PSSD was markedly increased (44%) compared with vehicle-injected controls. This response was further augmented, to approximately 65% above control, after administration of 60 μg/kg estradiol-17? (Fig. 2).
FIG. 2. Density of pyramidal cell spine synapses in the CA1 stratum radiatum of the hippocampi of OVX rats 30 min after estradiol-17? injection. Rats were injected with either different doses (15, 45, or 60 μg/kg) of estradiol-17? in sesame oil (200 μl, sc) or the injection vehicle alone. Statistical analysis by one-way ANOVA: F = 120.7; df 3,8; P < 0.0001. Letters above the histogram bars indicate the results of the Scheffé post hoc test (P < 0.05 level). Groups labeled with the same letter are not significantly different from one another.
To determine the circulating levels of estradiol-17? produced by the estradiol injections, serum concentrations of the hormone were measured at intervals over the first hour, as well as at 4.5 h, after sc injection of 45 μg/kg estradiol-17?. Results are presented in Table 1. Within 10 min of injection, serum estradiol-17? levels rose to more than 500 pg/ml. Thereafter, the concentrations of the steroid continued to increase, reaching more than 1 ng/ml at 4.5 h after treatment.
TABLE 1. Serum estradiol concentrations measured by RIA at different times after sc injection of OVX rats with estradiol (45 μg/kg) dissolved in 200 μl sesame oil
Rapid enhancement of object placement performance is observed after either estradiol-17? or estradiol-17 (17), despite the latter’s low affinity for estrogen receptors (24). Therefore, additional experiments were performed to determine whether estradiol-17 treatment also increases PSSD. Administration of 45 μg/kg estradiol-17 had a rapid and dramatic effect on CA1 PSSD, apparent even from inspection of individual micrographs (Fig. 3). Quantitative analysis of CA1 PSSD at 30 min after administration of 15 or 45 μg/kg estradiol-17 is summarized in Fig. 4A. Injection of 45 μg/kg estradiol-17 increased CA1 PSSD by 62%. Injection of a lower dose of the steroid (15 μg/kg) elicited an even larger increase in PSSD, to 81% above control. The PSSD response to 15 μg/kg estradiol-17 was greater than that observed at the same time interval after injection of 3- to 4-fold higher doses of estradiol-17? (Fig. 4A, cf. Fig. 2). To determine the dose dependency of the response to estradiol-17, OVX rats were injected with a range of doses of estradiol-17 between 0 and 20 μg/kg. PSSD was measured 30 min later. Results are shown in Fig. 4B. The data for PSSD vs. estradiol-17 dose were fitted by a four-parameter logistic dose-response curve. At 15 μg/kg, the response was maximal, within the range of the measurements obtained in OVX rats injected with 15 or 45 μg/kg estradiol-17 (Fig. 4A), close to the mean PSSD observed in animals treated with 60 μg/kg estradiol-17? (Fig. 2). The ED50 for the PSSD response to estradiol-17, calculated from the logistic curve fit, was 8.92 ± 1.99 μg/kg.
FIG. 3. Electron micrographs taken from the CA1 stratum radiatum of an OVX rat that received a sc injection of 45 μg/kg estradiol-17, dissolved in sesame oil, 30 min before perfusion-fixation (A) compared with an OVX rat that was killed the same length of time after injection of the oil vehicle alone (B). Note the higher density of spine synapses (arrows) in the estradiol-17-treated animal (A). D, dendrite; bar scale, 1 μm.
FIG. 4. Effects of estradiol-17 on the density of pyramidal cell spine synapses in the CA1 stratum radiatum of OVX rats. A, Rats were injected with either different doses (15 or 45 μg/kg) of estradiol-17 (E2-17) or the injection vehicle (sesame oil) alone and killed 0.5 h after injection. Statistical analysis by one-way ANOVA: F = 208.5; df 2,6; P < 0.0001. Letters above the histogram bars indicate the results of the Scheffé post hoc test (P < 0.05 level). Groups labeled with different letters are significantly different from one another. B, Dose-response curve for the induction of CA1 spine synapses by estradiol-17. OVX rats were injected with increasing doses of estradiol-17 in sesame oil vehicle, 30 min before perfusion-fixation. The mean densities of CA1 spine synapses in each animal are plotted against estradiol-17 dose. The line represents the best-fit four-parameter logistic regression calculated from the data. The ED50 calculated from the curve fit is indicated on the graph ± SE of the estimate, determined from the regression analysis.
Discussion
Previous studies have demonstrated that estradiol-17? increases both the number of dendritic spines and the density of spine synaptic contacts (PSSD) on CA1 pyramidal neurons (8, 9, 10, 11, 12, 13). These responses have been postulated to contribute to the effects of estradiol-17? on hippocampal function, including spatial memory (4, 25, 26). Recent work, however, indicated that estradiol-17? can enhance performance on a test of spatial memory, object placement, within 4 h of exposure to the hormone (17), much more rapidly than previously reported effects of estrogen on hippocampal PSSD. These results raised the question of whether changes in hippocampal synapse density occur with sufficient speed to contribute to the earliest behavioral effects of the hormone. The present studies were designed to examine this question, by measuring PSSD after treatment paradigms identical with those used in the behavioral studies. In our previous object placement behavioral tests (17), performance was enhanced by either estradiol-17? or estradiol-17, when sufficient doses were administered 30 min before the sample trial (4.5 h before the recognition/retention trial. We hypothesized that if CA1 PSSD contributes to rapid estrogen-mediated enhancement of spatial memory, then treatments that enhance object placement performance should also induce a significant increase in PSSD. Conversely, behaviorally ineffective hormone treatments should not significantly alter PSSD.
In OVX rats, PSSD was significantly higher 4.5 h after administration of 45 μg/kg estradiol-17?, but not after 15 μg/kg of the hormone. These observations are consistent with the hypothesis that increased CA1 PSSD may be associated with enhancement of object placement performance, because they parallel the behavioral data (17). Place memory was not significantly affected by 15 μg/kg estradiol-17? but was enhanced after higher doses (30–60 μg/kg) of the steroid, comparable to those required to induce increased PSSD. By contrast, visual recognition memory was enhanced by the lower dose of estrogen, 15 μg/kg (17). The differential association of increased PSSD with estradiol enhancement of place, as opposed to object recognition, memory may reflect the relative importance of the hippocampal circuitry in the acquisition of different types of mnemonic information. In rats, the hippocampus plays an essential role in place memory, whereas other regions of the brain appear to be more important for processing of visual recognition information (27, 28).
Unexpectedly, the data show that the increase in PSSD at 30 min after estradiol administration is even larger than that at 4.5 h. On the basis of previous studies of hippocampal pyramidal dendritic structure after estrogen exposure (8, 9, 10, 29), we anticipated that synaptic responses might not be observed until at least several hours after estradiol-17? injection. Clearly, however, CA1 PSSD can be modulated by estrogen within a much shorter time frame. At 30 min after injection of 45–60 μg/kg estradiol-17?, PSSD is already within the range of previous data obtained after 2 days of estrogen treatment (11). A possible explanation for both the rapid onset of the increase and subsequent decline in PSSD is provided by the data on serum estradiol levels. After sc injection in sesame oil, circulating estradiol-17? levels increase rapidly (30) (Table 1), consistent with the view that increases in PSSD may be initiated almost immediately after exposure of the hippocampus to elevated estrogen concentrations. The response is not sustained, however, because by 4.5 h PSSD falls substantially, despite the rising levels of estradiol in the circulation. There are two possible explanations for these data. The initial rapid induction of PSSD may be only a transient response. Alternatively, the decline in PSSD at 4.5 h may reflect down-regulation of the response mechanism, as estradiol-17? levels continue to increase. The latter hypothesis is supported by the data obtained with estradiol-17. PSSD at 30 min after a dose of 15 μg/kg estradiol-17 was significantly higher than after 45 μg/kg of this steroid, suggesting that the dose-response relationship between estrogen dose and synapse density may be bell shaped, with increases in circulating estrogen levels above maximal resulting in a diminished effect. After sc injection of estradiol-17? at 45 μg/kg, circulating hormone levels may rapidly rise to the point at which PSSD is maximally increased, the response then being reversed as serum hormone concentrations continue to climb (Table 1).
A second important conclusion suggested by the serum estradiol measurements is that short-latency effects on PSSD may be observed only with supraphysiological levels of the hormone. A significant increase in PSSD was observed only at 45 μg/kg, not at 15 μg/kg, of estradiol-17?. Subcutaneous injection of 45 μg/kg estradiol-17? results in circulating estradiol concentrations that are at least 10-fold higher than those observed at any stage of the reproductive cycle in normal female rats (31). Although circulating estradiol-17? was not measured after the lower, ineffective dose (15 μg/kg) of the steroid, it is a reasonable presumption that this probably also resulted in high estradiol concentrations during the first few hours after injection. These observations contrast with the situation in normal females, in which the considerably lower levels of estradiol released during the estrous cycle are known to induce a significant increase in CA1 PSSD (10). Although additional work will be necessary to precisely define the dose and time dependency of changes in PSSD after systemic estrogen administration, the available data suggest that estrogen-mediated induction of hippocampal spine synapses may involve mechanisms that can respond with differing latencies and time courses, depending on the circulating hormone concentrations. Rapid induction of both increases in CA1 PSSD (the present study) and hippocampal-mediated enhancement of object placement memory (17) may be observed only when estradiol-17? levels are above the normal physiological range.
The cellular mechanisms responsible for these effects remain to be elucidated. A reasonable hypothesis, however, is that the immediate and delayed responses to estradiol may reflect activation of different estrogen response mechanisms. Biological effects of estrogens include transcriptional responses mediated via activation of the nuclear estrogen receptor proteins, ER and ER?, as well as more rapid responses mediated via membrane receptor sites (32). In addition to the speed of the response, the potency of estradiol-17 raises the possibility that rapid induction of PSSD formation may specifically reflect the activation of membrane receptor systems. Membrane-associated ERs have been shown to exhibit enhanced sensitivity to estradiol-17, reflecting either the lipid-rich environment of the receptors in the membrane or the presence of unique membrane-associated receptor isoforms (33, 34). The present data indicate that rapid increases in PSSD can be activated by either estradiol-17? or estradiol-17, the latter being considerably more potent. By contrast, nuclear ER-mediated responses, such as uterine growth, typically are more than 100-fold less sensitive to estradiol-17 (17, 18, 24). Although in vivo potency does not necessarily reflect receptor affinity, because of the potential for contributions from hormone metabolism and clearance, the disparity between the present results and previous observations on nuclear ER-mediated responses is such that it would seem unlikely that they are mediated via identical receptor mechanisms.
There are several ways in which activation of membrane ERs could potentially be translated into effects on synaptogenesis. Membrane-associated ERs rapidly modulate important intracellular signaling pathways (reviewed in Ref. 32). Induction of ERK phosphorylation has been implicated in membrane receptor-mediated responses to estrogen (35, 36) and regulation of hippocampal synaptic plasticity (35, 36) as well as in learning processes mediated via the hippocampus (37). Estradiol also activates phosphatidylinositol 3-kinase, leading to phosphorylation of Akt, in the developing cerebral cortex (38) and in neurally derived cell lines (39) as well as in CA1 dendrites (40). Akt-like proteins have been implicated in cellular chemotaxis (41, 42, 43), whereas phosphorylation of Akt has been demonstrated to regulate transcription-independent synthesis of proteins involved in glutamatergic synapse formation (39, 44). These observations have led to a proposed mechanism for the effects of estrogen on CA1 dendritic spine density, involving estrogen-induced changes in synaptic protein synthesis and dendritic filopodial extension (45), which could also explain the rapid effects of estrogen on synaptogenesis, reported here. High concentrations of estradiol for a short time, or lower concentrations of the hormone over a longer period (10, 46), may activate membrane-associated signaling cascades, thereby altering the regulation of axonal and/or dendritic growth processes to increase the probability of spine synapse formation.
Regardless of the underlying mechanisms, the fact that the short-latency trophic effects of estrogen on the hippocampus can be mimicked by estradiol-17 has important implications for the potential development of novel forms of hormone replacement therapy (HRT). Numerous studies have demonstrated neurotrophic and neuroprotective effects of estrogen (reviewed in Refs. 35 and 47), consistent with clinical data suggesting that estrogen-based HRT may slow the progression of neurodegenerative diseases (48, 49). Long-term postmenopausal HRT, however, increases the risk of strokes, as well as breast and endometrial carcinoma (50, 51). Our data suggest that estradiol-17, a relatively weak estrogen in the tissues of the reproductive tract, is even more potent than estradiol-17? in terms of the rapid regulation of PSSD (Fig. 2, cf. Fig. 4) and at least as potent as estradiol-17? as an enhancer of short-term working memory (17). These findings parallel previous results on estrogen regulation of neuronal survival (52), the processing of amyloid precursor protein (53), and the expression of apolipoprotein E [a cofactor for estrogen-activated neurotrophic responses (54, 55, 56)], all of which exhibit sensitivity to estradiol-17 as well as estradiol-17?. Taken together, these data raise the possibility that HRT using estradiol-17 or structurally related estrogens might be capable of reproducing the central neuroprotective and neurotrophic effects of circulating estradiol-17?, while minimizing the potential for aberrant trophic responses in the peripheral reproductive target organs.
Acknowledgments
We are indebted to Klara Szigeti-Buck and Gladis Thomas for excellent technical assistance.
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