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Endocrinology, doi:10.1210/en.2004-0477
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Endocrinology Vol. 145, No. 9 4154-4161
Copyright © 2004 by The Endocrine Society

Effects of Dehydroepiandrosterone and Flutamide on Hippocampal CA1 Spine Synapse Density in Male and Female Rats: Implications for the Role of Androgens in Maintenance of Hippocampal Structure

Neil J. MacLusky, Tibor Hajszan and Csaba Leranth

Departments of Obstetrics, Gynecology, and Reproductive Sciences (N.J.M., T.H., C.L.) and Neurobiology (C.L.), Yale University School of Medicine, New Haven, Connecticut 06520; Laboratory of Molecular Neurobiology (T.H.), Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Hungary; and Center for Reproductive Sciences (N.J.M.), Columbia University Medical School, New York, New York 10032

Address all correspondence and requests for reprints to: Csaba Leranth, M.D., Ph.D., Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, FMB 313, New Haven, Connecticut 06520-8063. E-mail: csaba.leranth{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of androgens and the androgen antagonist, flutamide, on the density of dendritic spine synapses in the CA1 subfield of the hippocampus were studied in gonadectomized male and female rats. Treatment of orchidectomized male rats with dehydroepiandrosterone (DHEA; 2 d, 1 mg/d sc) increased the density of CA1 spine synapses observed 2 d later, by 106%, without significantly affecting ventral prostate weight. The hippocampal response to DHEA was unaffected by blockade of intracerebral estrogen biosynthesis using the aromatase inhibitor, letrozole. By contrast, flutamide alone (2 d; 5 mg/d, sc) increased CA1 spine synapse density by 66%, whereas in combination the effects of flutamide and DHEA were additive rather than inhibitory. Additive effects on CA1 synapse density were also observed in males using combinations of flutamide with 5{alpha}-dihydrotestosterone (2 d, 500 µg/d, sc). At the same doses, flutamide had no effect on prostate weight and completely blocked the effects on the prostate of treatment with 5{alpha}-dihydrotestosterone. Treatment of ovariectomized females with DHEA increased CA1 spine synapse density to a level similar to that observed in the male. As in males, flutamide in females increased CA1 spine synapse formation and further augmented the response to DHEA. These results demonstrate that flutamide and DHEA have positive effects on hippocampal CA1 spine synapse density in both sexes. They also suggest that conventional measures of androgen agonist or antagonist activity, exemplified by ventral prostate growth, may not be indicative of effects on hippocampal CA1 synaptogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE DENSITY OF pyramidal cell dendritic spine synapses in the CA1 subfield of the hippocampus is modulated by circulating gonadal steroids. In female rats, estrogens (1, 2, 3) and androgens (4) both increase the number of CA1 spine synapses. In males, maintenance of CA1 spine synapses is dependent on continued testicular androgen secretion. Thus, castration reduces whereas testosterone replacement therapy increases CA1 spine synapse density (5). The mechanisms underlying these effects remain unknown.

In primates, one of the most abundant circulating androgens, dehydroepiandrosterone (DHEA), is a product of adrenal steroidogenesis. In both men and women, adrenal production of DHEA increases before puberty, plateaus in adulthood but then declines between the third an seventh decades of life (6). Several studies have demonstrated that DHEA has effects on cognitive function in human beings as well as laboratory animals (7). Recently we reported in female rats that DHEA reproduces the effects of testosterone on CA1 spine synapse density (8). The effects of DHEA were completely blocked by administration of the aromatase inhibitor, letrozole, suggesting that the response was mediated via intracerebral conversion of DHEA to estradiol (8). Because hippocampal spine synapse formation has been implicated in memory (9), the ability of DHEA to induce CA1 synapses provides a potential cellular mechanism to explain the behavioral effects of this steroid. It also provides further support for the concept that DHEA replacement therapy may have beneficial cognitive effects in aging, particularly in cases of low endogenous DHEA secretion (7, 10). A key remaining question, however, is whether DHEA is capable of exerting comparable effects in both sexes (11). Because estrogen does not significantly increase CA1 spine synapse density in males (5), the dependence of the DHEA response in females on estrogen biosynthesis (8) raises the possibility that this response may be sexually differentiated.

The present studies were performed to test this hypothesis. The effects of DHEA on CA1 spine synapse density were examined in gonadectomized male rats using the same experimental paradigm used previously in females (8). The effects of DHEA were compared with those of the nonaromatizable androgen, 5{alpha}-dihydrotestosterone (DHT). In addition, to test the potential role of hippocampal androgen receptors (12, 13, 14), the effects of DHEA were examined after treatment with either letrozole or the antiandrogen, flutamide. Our data indicate that the male hippocampus responds to DHEA with an increase in CA1 spine synapse density that is comparable in magnitude with that observed in females. The response mechanism differs from that in females, however, in that induction of spine synapses by DHEA in males is unaffected by inhibition of aromatase activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male (280–300 g) and female (240–270 g) Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were kept in individual cages on a 12-h light, 12-h dark cycle and provided with unlimited access to water and rat chow. All animal protocols used in this study were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory animals and approved by the Institutional Animal Care and Use Committee of Yale University.

Surgery and hormonal manipulations
Animals were deeply 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 gonadectomized. Treatments in all rats were initiated 1 wk after gonadectomy. In experiment I, 18 orchidectomized (ORCH) males were divided into six groups, each containing three rats. Three of the groups (1D, E, and F) were injected sc with DHEA (1 mg/200 µl sesame oil per day, 2 d). The other three groups (1A, B, and C) received the oil vehicle. Groups 1C and F were pretreated with flutamide (5 mg/d, sc in sesame oil) 1 h before DHEA or vehicle injection. Groups 1B and E were pretreated with the aromatase inhibitor, letrozole (Novartis AG, Basel, Switzerland; 1 mg/d sc dissolved in 200 µl 2.5% carboxymethylcellulose) 1 h before injection of DHEA or vehicle. In experiment II, 12 ovariectomized (OVX) female rats were divided into two treatment groups, each containing six animals. Group 2A was injected sc with the sesame oil vehicle (200 µl/d, 2 d). Group 2B received DHEA (1 mg/200 µl sesame oil per day, sc, 2 d). Three of the six animals in each group were pretreated with flutamide (5 mg/d, sc, 2 d) 1 h before the injections of oil or DHEA. In experiment III, an additional 12 ORCH male rats were divided into two groups. Group 3A was injected sc with the sesame oil injection vehicle (200 µl daily, 2 d) alone. Group 3B received DHT dissolved in sesame oil (200 µl) via two sc injections (500 µg/injection) separated by 24 h. Three of the six animals in each group were pretreated with flutamide (5 mg/d, sc, 2 d) 1 h before the injections of oil or DHT.

Tissue processing
Two days after the last 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). During the perfusion, the descending aorta was firmly clamped to prevent access of the fixative to the lower half of the body. Brains were removed and postfixed overnight in the same fixative without glutaraldehyde. The ventral prostates were removed from the males, dissected free of adhering connective tissue, and weighed. The hippocampi were dissected out, divided into three pieces (septal, temporal, and midportions), and vibratome sections (100 µm) cut perpendicular to the longitudinal axis of each tissue block. The 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
Spine synapse density was calculated in all animal groups according to our standard protocol using unbiased stereological methods (3, 15, 16). 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 (17). 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 (18). 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 unit area of the window. Based on these values, a dimensionless volume correction factor (kv) was introduced: kv = D/D1, where D1 is the mean pyramidal cell 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 septotemporal 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.4). 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 due to 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 synapse size, the disector technique was used (19). The digitized electron micrographs were printed 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 that were present in the reference section but not in the look-up section were counted (Fig. 1Go). To increase the efficiency of spine synapse counting, the analysis was performed treating each reference section as a look-up section and vice versa (1).



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  		FIG. 1. Electron micrographs taken from adjacent ultrathin sections prepared from the outer third of the stratum radiatum of the CA1 area of a DHEA-treated OVX rat. Only those spine synapses (long arrows) that were present only in the reference (A), but not on the look-up (B) section and vice versa were counted. Spine synapses that are seen on both sections were not counted and are labeled with arrowheads. Bar scale, 1 µm.

 
Spine synapse density 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 (18). Section thickness (average 0.075 µm) was determined using the electron scattering technique. The measured synaptic density values were divided by the 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 of synaptic density data
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 longitudinal septotemporal axis), each animal provided at least 3 x 3 x 10 x 2 = 180 neuropil fields for evaluation. Spine synapse density for each animal was determined independently by two different investigators who were blinded to the identity of the treatment groups and the results cross-checked to preclude systematic analytical errors. Average spine synapse density 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effects of DHEA on CA1 synapse density in ORCH males are shown in Fig. 2Go. ORCH males were treated with DHEA alone, DHEA + flutamide, or DHEA + the aromatase inhibitor letrozole. At the dose of DHEA used (1 mg/d sc), there was no significant effect on ventral prostate weight (control vehicle-injected ORCH rats 36.7 ± 3.0 mg; DHEA-injected rats 33.0 ± 7.0 mg). However, DHEA more than doubled CA1 spine synapse density, compared with the vehicle-injected controls (Fig. 2Go; vehicle injected 0.450 ± 0.001 synapses/µm3; DHEA treated 0.930 ± 0.026 synapses/µm3). Pretreatment with flutamide (5 mg/d sc) further increased the CA1 spine synapse density observed after DHEA to a level (1.207 ± 0.023 synapses/µm3) higher than that observed after treatment of males with either testosterone or DHT. Pretreatment with letrozole, at a dose previously shown to abolish induction of CA1 spine synapses by DHEA in females (8), had no significant effect on the response to DHEA in ORCH males (Fig. 2Go).



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  		FIG. 2. Effect of DHEA treatment ± flutamide or letrozole on the density of pyramidal cell spine synapses in the CA1 stratum radiatum of ORCH male rats. DHEA increased spine synapse density, a response that was augmented by pretreatment with flutamide but unaffected by pretreatment with letrozole. Statistical analysis: two-way ANOVA: DHEA effect, F = 766.1; df 1,12; P < 0.0001; inhibitor effect, F = 131.2; df 2,12; P < 0.0001; DHEA x inhibitor interaction, F = 0.371; df 2,12; P > 0.5. Results of individual group comparisons (Scheffé test; P < 0.05 level) are presented as letters above the histogram bars, in which bars with the same letter represent results that are not significantly different from one another. Different letters denote statistically significant differences in mean synapse density.

 
Treatment of OVX females with DHEA increased spine synapse density by approximately 65%. As in the male, this response was further augmented by pretreatment of the females with flutamide. Flutamide alone, in OVX females, induced an increase in CA1 spine synapse density that was almost equal to the response observed with DHEA (Fig. 3Go).



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  		FIG. 3. Effect of DHEA treatment ± flutamide on the density of pyramidal cell spine synapses in the CA1 stratum radiatum of OVX female rats. DHEA increased spine synapse density, a response that was augmented by pretreatment with flutamide. Statistical analysis: two-way ANOVA: DHEA effect, F = 201.7; df 1,8; P < 0.0001; flutamide effect, F = 171.6; df 1,8; P < 0.0001; DHEA x flutamide, F =1.903; df 1,8; P > 0.2. Results of individual group comparisons (Scheffé test; P < 0.05 level) are presented as letters above the histogram bars. Different letters denote statistically significant differences in mean synapse density.

 
The changes in CA1 synapse density in ORCH males after DHT treatment with and without flutamide are shown in Fig. 4Go (upper panel). As expected from our previous results (5), DHT increased synapse density by more than 100%, compared with vehicle-injected ORCH rats. Flutamide treatment also increased CA1 synapse density but to a significantly lesser extent (66%) than DHT. Consistent with the previous results with DHEA, flutamide did not inhibit the synaptogenic effects of DHT. Responses to flutamide and the androgen appeared to be additive, rather than inhibitory. Thus, flutamide increased synapse density to approximately the same extent in both in vehicle- and DHT-treated ORCH males.



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  		FIG. 4. Effects of DHT alone or in combination with flutamide on hippocampal synapse density and ventral prostate weight in ORCH male rats. Rats were ORCH and 1 wk later steroid and flutamide treatments were initiated. Top panel, Density of pyramidal cell spine synapses in the CA1 stratum radiatum. Administration of flutamide (5 mg) sc 1 h before the steroid or vehicle injections significantly increased synapse density. Statistical analysis: two-way ANOVA: DHT effect, F= 623.1; df 1,8; P < 0.0001; flutamide effect, F = 168.9; df 1,8; P < 0.0001; DHT x flutamide, F = 0.144; df 1,8; P > 0.5. Lower panel, Ventral prostate weight. In the prostate, flutamide alone had no significant effect but completely blocked the response to DHT. Statistical analysis: two-way ANOVA: DHT effect, F = 22.7; df 1,8; P = 0.0014; flutamide effect, F = 43.4; df 1,8; P = 0.0002; DHT x flutamide, F = 47.9; df 1,8; P = 0.0001. Results of individual group comparisons (Scheffé test; P < 0.05 level) are presented as letters above the histogram bars. Different letters denote statistically significant differences in mean synapse density and prostate weight.

 
Mean ventral prostate weights in rats treated with vehicle, DHT, or flutamide are shown in Fig. 4Go (lower panel). In ORCH males, ventral prostate weight was reduced to 35–40 mg, approximately 20% of the weight of this organ in intact males (20, 21). Treatment with DHT for 2 d increased prostate weight more than 2-fold, a response that was completely blocked by flutamide. Flutamide alone had no significant effect on prostate weight.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data reported in this paper demonstrate that: 1) DHT and DHEA both induce large increases in CA1 spine synapse density in ORCH rats; 2) the androgen receptor antagonist, flutamide, also increases CA1 spine synapse density; 3) in both sexes, flutamide does not inhibit the effects of the androgens on synapse density; and 4) the effects of DHEA, DHT, and flutamide on CA1 synapse density do not correlate with the androgenic bioactivity of these agents, measured in terms of ventral prostate weight.

Relative potencies of DHT and DHEA: sex differences in the mechanism of androgen action
We have reported elsewhere that androgens induce a rise in CA1 spine synapse density in OVX females as well as ORCH males (4, 5). The present data confirm and extend these observations, demonstrating that treatment of ORCH males with DHEA for 2 d restores CA1 spine synapse density to levels similar to those reported for intact males (5). Because DHEA is extensively metabolized in vivo to steroids with androgenic and estrogenic bioactivity (6, 22), we anticipated that DHEA might have some effect on CA1 spine synapse density. That the effects of DHEA might be comparable with those of DHT, however, was not anticipated because estradiol has no effect on CA1 spine synapses in males (5) and the effects of DHEA on spine synapse density in females are almost entirely dependent on aromatization (8). In the ventral prostate, DHEA is a considerably less potent androgen than either testosterone or DHT (21, 23). Our data for prostate weight confirm the difference in androgenic potency of these two steroids: with this relatively short-term treatment paradigm, there was no effect of DHEA on the prostate, whereas DHT increased ventral prostate organ weight more than 2-fold. The apparent androgen receptor dependence of CA1 spine synapse regulation in males (5) led us, therefore, to suspect that DHEA might have a more limited impact on spine synapse density in males than females. In fact, the data show that comparable increases in CA1 spine synapse density are observed after DHEA in both sexes.

The explanation for these findings may lie in the relative potencies of androgens, compared with estrogens, in males and females. The response to DHT is smaller in females than it is in males (4, 5), suggesting that the female may be less sensitive to the effects of androgen. However, the female is clearly more sensitive than the male to the effects of locally synthesized estrogen. Thus, in females, letrozole completely blocks the effects of DHEA (8) and almost completely blocks the effects of testosterone (4) on CA1 spine synapse numbers. In males, by contrast, aromatization does not appear to play a significant role: the synaptic effects of DHEA in males are completely unaffected by letrozole administration (Fig. 2Go). This is consistent with our previous observation that estradiol has no significant effect on CA1 spine synapse numbers in ORCH rats (5). Thus, although DHEA induces quantitatively similar numbers of CA1 spine synapses in males and females, different mechanisms mediate the responses. In males, aromatization is not involved, whereas in females the DHEA-induced increase in CA1 spine synapse density is mediated almost completely via intracerebral estrogen biosynthesis.

The hippocampus is sexually differentiated during development via the actions of androgens secreted in perinatal life as well as at around the time of puberty (24, 25). In adulthood, the hippocampus remains a target for the effects of gonadal steroids, containing populations of androgen (12, 26) and estrogen (27) receptor-expressing neurons as well as aromatase (28). The pyramidal neurons of CA1 appear to express particularly high levels of the androgen receptor (12), consistent with the hypothesis that androgens may directly regulate dendritic structure in these cells. It is also possible that hippocampal synapse formation may be regulated indirectly because in both males and females, the effects of gonadal steroids on CA1 spine synapse density are critically dependent on ascending subcortical connections from the basal forebrain (29, 30). Previous work on the hormonal regulation of choline acetyltransferase in the forebrain has demonstrated considerable sex differences in response to both estrogen and androgen (31, 32). Therefore, sex differences in the regulation of CA1 spine synapse density by gonadal steroids could also arise indirectly as a result of differences between males and females in the effects of the steroids on the basal forebrain cholinergic system.

The effects of DHEA are of considerable potential importance because of the role of this steroid in primate endocrinology. DHEA is the most abundant androgen secreted by the human adrenal gland (6). It is also synthesized to a limited extent within the brain itself (33). Previous work has shown that DHEA has neuroprotective and neurotrophic properties (34), consistent with the hypothesis that the dramatic decline in DHEA levels that occurs between the ages of 30 and 65 yr (6, 35) may contribute to age-related neurodegenerative processes. It remains to be determined, however, whether the present data can be extrapolated to the situation in human beings. The adult rat produces very little adrenal DHEA (36), so the present data reflect effects of DHEA injection against a low endogenous background of this steroid, in contrast to the situation in normal healthy human beings. The DHEA dose used here is fairly high, approximately 5- to 8-fold higher, per kilogram body weight, than the replacement doses recommended for men and women (10). The present data could reflect the effects of relatively short-term treatment (2 d) as opposed to the prolonged periods of exposure that are used for human hormone replacement. The route of steroid administration may also play a role in terms of differences in the patterns of DHEA metabolism observed after injection vs. oral treatment. Additional work will be required to better define the effects on hippocampal synaptogenesis of DHEA treatment via different routes of administration in primate as well as rodent experimental models.

Flutamide does not block androgen-induction of CA1 spine synapses: potential mechanisms
The discordance between the apparent potencies of DHT and DHEA on the prostate, compared with CA1 synapse formation, is further heightened by the data from the flutamide-pretreated animals. In both sexes, flutamide not only failed to block the effects of DHT and DHEA on CA1 spine synapse density, but it also significantly enhanced synapse formation when given either by itself or in combination with androgen. Whereas the underlying mechanisms remain unknown, previous work in other systems has identified a number of potential signaling pathways by which androgens and antiandrogens might exert apparently anomalous effects.

One possibility is that the effects of the androgens on hippocampal synaptogenesis may involve membrane-associated receptor mechanisms. In androgen receptor-negative prostate cancer cells, hydroxyflutamide, the principal bioactive metabolite of flutamide, has been shown to activate the MAPK pathway (37). Immunocytochemical studies demonstrated extranuclear localization of both androgen (38) and estrogen (39, 40) receptors in neurons, consistent with the possibility of nongenomic effects of aromatizable and nonaromatizable androgens on the brain. Membrane receptor-activated kinase cascades, including the MAPK pathway, have been implicated in the mechanisms mediating effects of estradiol on hippocampal spine synapse density (41). The effects of flutamide and the gonadal steroids could thus conceivably be mediated via convergence onto common intracellular signaling pathways, activated via membrane-associated receptor systems.

Membrane receptors are not necessarily involved, however, because effects of antiandrogens such as flutamide are also subject to regulation at the cell nuclear level. Previous work in androgen-sensitive cancer cells has demonstrated that flutamide can act under some circumstances as a partial androgen agonist, activating androgen receptor-dependent gene transcription (42). The same kind of response could contribute to the synaptic effects of flutamide, noted here. Transcriptional responses to androgens are modulated to a considerable extent by receptor-associated coactivator and corepressor proteins (43). In prostate cancer cells, the level of expression of the androgen receptor coactivator ARA-70 has a major impact on the androgen agonist activity of androgen (DHT) and antiandrogens such as hydroxyflutamide and bicalutamide (44). It is conceivable that differences between hippocampal neurons and prostatic epithelial cells in the expression patterns of nuclear coactivator proteins, including ARA-70, could alter the sensitivity of hippocampal cell nuclear androgen receptors to DHEA and flutamide, enhancing the ability of both of these agents to induce CA1 spine synapses. Sex differences in the patterns of intracranial metabolism of DHEA (to DHT, compared with estradiol) and/or the expression levels of nuclear receptor-associated coactivator proteins could, likewise, determine the extent to which the synaptogenic effects of DHEA depend on aromatase-dependent vs. aromatase-independent pathways in males and females.

The possibility also exists that androgens and antiandrogens may influence hippocampal synaptogenesis via mechanisms that do not involve gonadal steroid receptors at all. A recent report indicates that flutamide has weak benzodiazepine-like activity in mice, exerting inhibitory effects on pentylenetetrazole-induced convulsions (45). Whether such effects play a role in the responses observed in the present study is questionable: the median dose of flutamide required to elicit anticonvulsive effects in mice is 67 mg/kg (45), approximately 3-fold higher than the dose of flutamide used here. Moreover, the available evidence suggests that potentiation of GABAergic responses might diminish, rather than enhance, hippocampal synapse formation. Woolley (46) proposed that the effects of estrogen on hippocampal synaptogenesis may involve disinhibition of pyramidal cell input, mediated via estrogen-regulated suppression of GABAergic input. If this hypothesis is correct, then further potentiation of GABAergic responses might be expected to reduce, not increase, CA1 spine synapse formation. Nevertheless, the idea that androgen effects might at least in part be mediated via interactions with the GABA-benzodiazepine receptor complex is attractive, in view of the evidence linking androgen action to this receptor system. Both DHT and DHEA can be converted to metabolites that have the potential to interact with steroid recognition sites on GABA receptors (6, 47, 48). Behavioral effects of androgens in rodents have been linked to actions on GABAergic function (49, 50). DHEA itself has been reported to exert effects opposite those of benzodiazepines in the adult male rat brain, antagonizing GABA-mediated chloride uptake (51). Such a DHEA effect could perhaps mimic the actions of estrogen in the female (46), disinhibiting the pyramidal cells via a reduction in GABAergic transmission.

Pathological and therapeutic implications
The data presented in this paper have important implications for the physiological role of androgens as well as the potential development of novel hormone-replacement therapies (HRT) in aging men and women. The effects of estrogens on CA1 spine synapse density have been postulated to contribute to the positive effects of these hormones on hippocampally mediated cognitive behavior (52, 53, 54) and clinical observations on the incidence of Alzheimer’s disease (AD) in patients receiving estrogen-based HRT (55). Androgens, like estrogens, have been demonstrated to enhance cognitive function in human beings (56, 57) and experimental animals (58). By analogy to the effects of estrogens, we have postulated that induction of hippocampal spine synapses may contribute to androgen-mediated enhancement of cognitive performance (4, 5). Serum total testosterone and DHEA levels tend to be lower in cases of AD, suggesting that reduced circulating androgen concentrations could either accompany or precede the onset of this disease (59, 60, 61). Consistent with this hypothesis, a recent study (62) demonstrated that older men with low levels of free circulating testosterone appear to be at increased risk for developing AD, compared with men with higher serum levels of this hormone.

If androgen is important for maintenance of normal cognitive function, then removal of endogenous sources of androgen and/or blockade of androgen receptors could have adverse effects on cognitive performance. This is a potential concern for men with prostate cancer because of the widespread use of GnRH analog-based suppression of testicular androgen synthesis combined with oral flutamide therapy for treatment of this disease. The present data suggest, however, that combined GnRH/flutamide therapy probably does not eliminate central androgenic responses because of the ability of flutamide to partially reverse the effects of testicular androgen withdrawal. In flutamide-treated rats, we observed a CA1 spine synapse density that was markedly higher than in ORCH controls (Fig. 4Go), approximately 85% of that observed in intact males (5). The available clinical data are consistent with the hypothesis that flutamide treatment may not seriously impair androgen-sensitive cognitive responses: a study on the effects of 9 months of GnRH/flutamide combination therapy for prostate cancer reported only mild impairment of spatial cognitive function, combined with enhancement of verbal memory (63).

The present results also provide further support for the hypothesis (4, 5) that the effects of diminished endogenous gonadal steroid production on hippocampal structure may be reversible using androgen-based replacement therapy. After treatment with DHEA + flutamide, ORCH males and OVX females both exhibit extraordinarily high CA1 spine synapse densities, approximately 20% higher than the CA1 synapse densities we previously reported for intact males, ORCH males treated with testosterone (5), or females treated with estradiol (3). Whereas the functional consequences of this increase in CA1 spine synapse density remain to be determined, it is tempting to speculate that selective HRT incorporating low doses of androgen, or partial androgen agonist/antagonist combinations, might be sufficient to counteract changes in the brain resulting from declining gonadal hormone secretion. In aging men (62, 64) as well as women (65, 66), rates of cognitive decline and susceptibility to neurodegenerative disorders appear to be inversely related to circulating free gonadal steroid levels. Steroid replacement therapy, however, has significant long-term disadvantages as a result of the potential for the hormones to increase the risk of other diseases, such as cardiovascular disease and carcinoma of the reproductive organs (67, 68, 69). The present data indicate that androgenic effects on the hippocampus do not necessarily have to be accompanied by systemic androgen responses. It may therefore be possible, using androgen-based hormone replacement regimens, to prevent the loss of hippocampal dendritic spine synapses that normally accompanies declining rates of gonadal steroid secretion, with minimal systemic side effects. Because the hippocampal circuitry is vitally important for some aspects of memory (70, 71, 72), such targeted therapy might offer a valuable adjunct to other forms of therapy in treatment of neurodegenerative disorders involving deficits in hippocampal function, without the risks associated with conventional estrogen-based HRT (67, 68, 69).


   Acknowledgments
 
We are indebted to Dr. Richard Hochberg for helpful discussions in the design and conduct of these studies and Klara Szigeti-Buck and Gladis Thomas for excellent technical assistance.


   Footnotes
 
This work was supported by National Institutes of Health Grants MH60858 and NS42644.

Abbreviations: AD, Alzheimer’s disease; DHEA, dehydroepiandrosterone; DHT, 5{alpha}-dihydrotestosterone; HRT, hormone replacement therapy; kv, volume correction factor; ORCH, orchidectomized; OVX, ovariectomized.

Received April 14, 2004.

Accepted for publication May 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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