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Dihydrotestosterone Increases Hippocampal N-Methyl-D-Aspartate Binding But Does Not Affect Choline Acetyltransferase Cell Number in the Forebrain or Choline Transporter Levels in the CA1 Region of Adult Male Rats

Laboratories of Neuroendocrinology (R.D.R., B.S.M.) and Neurobiology and Behavior (A.M.J.), The Rockefeller University, New York, New York 10021; Neuroscience Program (D.S., J.E.T.), Oberlin College, Oberlin, Ohio 44704; and Department of Psychology (I.N.K.), Columbia University, New York, New York 10027

Address all correspondence and requests for reprints to: Russell D. Romeo, Laboratory of Neuroendocrinology, The Rockefeller University, Box 165, New York, New York 10021. E-mail: romeor{at}rockefeller.edu.

	Abstract

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Testosterone, acting through its androgenic metabolite 5-dihydrotestosterone (DHT), can increase dendritic spine density in the CA1 region of the male rat hippocampus. The mechanisms mediating this increase in spines are presently unknown. In female rats, estrogen (E) has been shown to increase spine density, which is in part mediated by increases in N-methyl-D-aspartate (NMDA) receptors in the CA1 region and cholinergic forebrain inputs to the hippocampus. Whether similar mechanisms are responsible for the DHT-induced increase in spines in the male remains to be determined. In the first experiment, we used [³H]glutamate NMDA receptor binding autoradiography to assess whether DHT-treated males had higher NMDA receptor levels in the CA1 region of the hippocampus, compared with oil-treated males. In the second set of experiments, we used choline acetyltransferase (ChAT) in situ hybridization and immunohistochemistry to assess whether DHT could affect ChAT cell number in the forebrain. We also investigated the effect of DHT on hemicholinium-3-sensitive choline transporter levels in the CA1 region of the male hippocampus. We found that DHT significantly increased NMDA receptor binding in the CA1 region of males but had no effect on ChAT cell number in the forebrain or hemicholinium-3-sensitive choline transporter protein levels in the CA1 region. These data indicate that, similar to E-induced spinogenesis in females, DHT-induced increases in spine formation in males may require increases in NMDA receptors. However, unlike E-treated females, these data suggest that DHT does not influence cholinergic inputs to the hippocampus.

	Introduction

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IN SEVERAL SPECIES, estrogen (E) has been shown to increase dendritic spine density, pre- and postsynaptic proteins, and synaptic connectivity in the CA1 region of the female hippocampus (1, 2, 3, 4, 5, 6, 7, 8). In parallel with these morphological and biochemical changes, E-treated females also exhibit superior performance on certain hippocampal-dependent learning and memory tasks (7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) and enhanced long-term potentiation (LTP) (19, 20, 21). Interestingly, there is a sex difference in the effect of E such that E increases spine density and proteins in females but not males (1, 22, 23).

In contrast to the inability of E to influence hippocampal structure in males, testosterone, acting through its androgenic metabolite 5-dihydrotestosterone (DHT), has recently been reported to increase dendritic spine synapses in the CA1 region of the male rat hippocampus (22). The spine-promoting effects of DHT are not completely sexually dimorphic because female rats treated with DHT also show a slight, but significant, increase in CA1 spine synapse density (24). Male mice also experience an increase in CA1 hippocampal spine density during puberty, when endogenous testosterone levels rise. This pubertal increase in dendritic spines can be blocked by prepubertal castration (25). The mechanisms through which androgens promote hippocampal synaptic connectivity in males are presently unknown.

In the female hippocampus, the ability of E to increase CA1 spine synapses is mediated by the nuclear estrogen receptor (ER) (26). However, CA1 hippocampal pyramidal cells are devoid of appreciable levels of nuclear ERs (but do contain some extranuclear ERs) (27). Instead, the effects of E in females appear to be predominantly mediated transsynaptically through multiple pathways that do possess nuclear ERs (28, 29). E-induced spinogenesis is mediated by N-methyl-D-aspartate (NMDA) receptors in the CA1 region (30), which increase significantly on E treatment (31, 32). Importantly, E enhances acetylcholine production and choline acetyltransferase (ChAT) cell number in the forebrain in females (33, 34, 35, 36, 37, 38, 39) and acetylcholine release into the hippocampus (40), an effect that does not occur in males (37, 39, 41). This increase in cholinergic inputs to the hippocampus in response to E appears to mediate the increase in NMDA receptors on the CA1 pyramidal cells (31). Indeed, this cholinergic input is critical to the ability of E to increase hippocampal spine density in females such that if this projection is disrupted, E is ineffective in augmenting hippocampal spine density (42, 43).

The purpose of the present study was to elucidate whether similar mechanisms are responsible for androgen-induced spinogenesis in males. Specifically, we addressed whether DHT could increase NMDA receptors in the CA1 pyramidal region of the hippocampus, as measured by receptor autoradiography. A recent study (44) indicated that transection of the cholinergic forebrain pathway partially inhibits the ability of testosterone to increase spine synapse density in males. Thus, we also investigated whether DHT influences cholinergic inputs to the male hippocampus. Using in situ hybridization and immunohistochemistry, we assessed ChAT-positive cell number in the medial septum (MS), vertical limb of the diagonal band of Broca (VDB), and horizontal limb of the diagonal band of Broca (HDB), forebrain nuclei replete with ChAT that project to the hippocampus (e.g. MS and VDB) (45). Furthermore, we investigated whether DHT could influence postsynaptic hemicholinium-3-sensitive choline transporter (CHT) protein levels in the CA1 region of males because CHT levels have been shown to be positively correlated with acetylcholine synthesis and release (46, 47, 48, 49).

	Materials and Methods

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Animals and housing
For all experiments, adult (at least 90 d of age) male Sprague Dawley rats were commercially obtained from Charles River Laboratories (Harlan, NY). Animals were housed two to three per cage in clear polycarbonate cages with wood chip bedding. All animals were maintained on a 12-h light, 12-h dark schedule, and the temperature was kept at 21 ± 2 C. All animals had ad libitum access to food and tap water. All procedures were carried out in accordance with the guidelines established by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Experimental design and tissue processing
Two experiments were performed. Experiment 1 was conducted to measure NMDA receptor binding in the hippocampus of DHT- and oil-treated adult males. Experiment 2 was conducted to assess ChAT-positive cell number in the forebrain and CHT protein levels in the CA1 region of DHT- and oil-treated males.

In experiment 1, all males were castrated under sodium pentobarbital anesthesia (50 mg/kg, ip). One week after castration, animals received two sc injections of 500 µg 5-androstan-17ß-ol-3-one propionate (DHTP n = 15; Steraloids, Inc., Newport, RI) or the sesame oil vehicle (n = 15) 24 h apart and were killed 48 h after the last injection. This dose and injection regimen has been used previously to show DHT-induced increases in spine synapse number in adult male rats (22). After killing with an overdose of sodium pentobarbital (130 mg/kg, ip), brains were removed and snap frozen on powdered dry ice and stored at –70 C until sectioning or micropunching (see below). The androgen-sensitive seminal vesicles were also removed and weighed after expulsion of the seminal fluid. For a subset of brains (n = 9), coronal sections were made on a cryostat (20 µm), thaw mounted on Fisher Plus slides (Fisher Scientific, Pittsburgh, PA), and stored at –70 C until receptor autoradiography and in situ hybridization (experiment 2) were performed (see below). The remaining DHT- and oil-treated brain (n = 6) were left whole at –70 C until micropunches were performed for experiment 2 (see below).

For experiment 2, additional animals were treated in an identical manner as in experiment 1, except all animals were perfused after the overdose of sodium pentobarbital. For perfusion, DHTP- (n = 5) and oil-treated (n = 6) males were transcardially perfused with 150 ml of 0.9% heparinized saline followed by 200 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were then postfixed for 4 h in 4% paraformaldehyde in PB and then stored in 30% sucrose in PB for 24–48 h. Coronal brain sections were made on a vibratome (40 µm) and stored at –20 C in cryoprotectant until processed for immunohistochemistry (see below).

Receptor autoradiography
In experiment 1, NMDA receptor binding was determined using a previously published protocol (31, 32). Briefly, slide-mounted sections were thawed and dried. Slides were then preincubated in 50 mM Tris-acetate buffer (TAB; pH 7.4) for 45 min at room temperature and dried. Six sections per brain were incubated in 150 nM [³H]glutamic acid (56.0 Ci/mmol; Amersham Biosciences, Buckinghamshire, UK) in TAB, and six alternate sections were incubated in TAB containing 150 nM [³H]glutamic acid plus 1 mM NMDA (Sigma, St. Louis, MO). Sections were then rinsed four times for 5 sec, each in ice-cold TAB, and dried. Slides were apposed to Kodak MS film (Amersham) for 30 d and then developed for 4 min in Kodak D-19 developer and fixed for 5 min with Kodak rapid-fix.

In situ hybridization histochemistry
To determine ChAT mRNA containing cells in experiment 2, we constructed a ChAT probe using PCR on rat cDNA isolated from the medial septum with the following primers: forward, 5'-CAAGACACCAATGACCAGC-3'; reverse, 5'-CAACATCCAAGACAAAGAACTG-3'. These primers yielded a single band of 277 bp corresponding to nucleotides 352–628 of rat ChAT cDNA (50). The fragment was highly specific and homologous to other mammalian cDNA for ChAT, showing 95% homology with mouse ChAT cDNA. The fragment was gel purified and rapidly TA-ligated into the PCR*2.1-TOPO plasmid according to the manufacturer’s instructions (Invitrogen, San Diego, CA). The resulting clone was reanalyzed for size and presence of unique restrictions sites. The plasmid was linearized using appropriate restriction enzymes and used as a template to prepare antisense and sense cRNA probes labeled by in vitro transcription with digoxigenin (DIG).

Only a random subset of animals (n = 6 per group) that were previously sectioned (experiment 1) were processed for in situ hybridization histochemistry in experiment 2. ChAT mRNA was measured using a previously published DIG-labeled in situ hybridization protocol with slight modifications (51). In brief, slide-mounted sections were thawed and fixed in 3.8% formaldehyde for 10 min. Sections were then processed with proteinase K [1 mg/ml, 0.1 M Tris buffer (pH 8.0); 50 mM EDTA; 10 min] at 37 C and 0.25% acetic anhydride in 0.1 M triethanolamine for 10 min. Before hybridization, slides were rapidly dehydrated in a graded series of ethanols (70, 95, and 100%). Sections were incubated in hybridization buffer [60% formamide; 10% dextran sulfate; 10 mM Tris-HCl (pH 8.0); 1 mM EDTA (pH 8.0); 0.6 M NaCl; 0.2% N-laurylsarcosine, 500 mg/ml; 200 mg/ml tRNA; 1x Denhardt’s; 0.25% sodium dodecyl sulfate (SDS); and 10 mM dithiothreitol] containing DIG-labeled ChAT antisense cRNA probes for 16 h at 60 C. After a high-stringency posthybridization wash, sections were treated with RNase A and were then further processed for immunodetection with a nucleic acid detection kit (Roche Molecular Biochemicals, Indianapolis, IN). Sections were incubated in 1.0% blocking reagent in buffer 1 [100 mM Tris-HCl buffer, 150 mM NaCl (pH 7.5)] for 1 h at room temperature and then incubated at 4 C in an alkaline phosphatase-conjugated DIG antibody (Roche Applied Science, Indianapolis, IN) diluted 1:3500 in buffer 1 for 3 d. On the following day, sections were washed in buffer 1 twice (5 min each) and incubated in buffer 3 [100 mM Tris-HCl buffer (pH 9.5), containing 100 mM NaCl and 50 mM MgCl₂] for 5 min. They were then incubated in a solution containing nitroblue tetrazolium salt (0.34 mg/ml) and 5-bromo-4-chrolo-3-indolyl phosphate toluidinium salt (0.18 mg/ml; Roche Applied Science) for 8 h. The colorimetric reaction was halted by immersing the sections in buffer 4 [10 mM Tris-HCl containing 1 mM EDTA (pH 8.0)]. Tissue hybridized with the sense probe resulted in no specific labeling.

Immunohistochemistry
In experiment 2, free-floating fixed sections were washed 3 x 5 min with 0.1 M PBS, blocked with 2% normal rabbit serum in PBS for 1 h and then incubated in goat anti-ChAT primary antibody (1:6500; Chemicon International, Temecula, CA, AB144P) in 0.1 M PBS with 2% normal rabbit serum and 0.05% Triton X-100 for 3 d at 4 C. This primary antibody has previously been validated for the detection of ChAT in rat brain (35). Sections were then incubated in rabbit antigoat IgG secondary antibody (1:200; Vector Laboratories, Burlingame, CA) in 0.1 M PBS for 1 h, followed by avidin-biotin-complex (1:100; Vector Elite kit) in 0.1 M PB for 1 h and then 3,3'-diaminobenzidine for 5 min (0.2 mg 3,3'-diaminobenzidine in 0.18 M sodium acetate, with 25 mg/ml nickel sulfate and 0.003% hydrogen peroxide). All incubations were done at room temperature, and sections were washed in 3 x 5 min with 0.1 M PBS before and after each incubation. Sections were mounted on gelatin-coated slides and air dried. Slides were then dipped into distilled H₂O (dH₂O) and dehydrated with a series of ethanols (70, 95, and 100% each for 2 min), cleared with Histoclear (Lamb, Ltd., East Sussex, UK), and coverslipped with permount (Fisher Scientific). Tissue incubated without primary antiserum resulted in no specific immunostaining.

Micropunches and Western blots
To measure CHT protein levels in the CA1 region, a subset of the brains obtained from experiment 1 (n = 6) were cut on a freezing, sliding microtome, and tissue punches were made by using a 0.5-mm-diameter sample corer tool (Fine Science Tools, Foster City, CA). Coronal sections (400 µm thick) were punched through the CA1 hippocampal region and medial preoptic region of the hypothalamus. Hypothalamic punches were made to assess aromatase protein levels to verify the effectiveness of the DHT treatment. Punches were immediately snap frozen on powdered dry ice and stored at –70 C until protein assays and Western blots were conducted.

To prepare lysates for protein assays and Western blots, the tissues were homogenized by sonication in lysis buffer (1% SDS in dH₂O with Roche complete, mini, EDTA-free protease inhibitor cocktail 1836170, Roche Diagnostics, Basel, Switzerland) and then incubated in a boiling water bath for 10 min. Lysates were spun in a microfuge (13,000 rpm for 30 sec) to remove insoluble material. The protein concentration of the cleared supernatant was determined by the BCA method (Pierce, Rockford, IL). Lysates were subjected to SDS gel electrophoresis, blotted to a nitrocellulose membrane (Invitrogen), and probed with either the CHT (1:1500; polyclonal, affinity purified; gift of Dr. Randy D. Blakely, Vanderbilt University Medical Center, Nashville, TN) or aromatase (1:5000; polyclonal, affinity purified; BP278; Novus Biologicals, Littleton, CO) antibodies. Proteins were visualized by chemiluminescence (Pierce) according to the manufacturer’s instructions. After membranes were probed for CHT and aromatase, the membranes were stripped using SDS/2-mercapto-ethanol for 20 min at 50 C and reprobed with actin (1:50,000, monoclonal; Sigma) as a loading control. After stripping, membranes were washed and incubated with chemiluminescence to make sure the original signal was removed.

Autoradiographic, histochemical, and Western blot analyses
For NMDA receptor binding, relative ODs (RODs) of the exposed film (black values) were measured using computerized image analysis software (MCID-M4, Imaging Research, Inc., St. Catherines, Ontario, Canada). Measurements were obtained for the stratum oriens and radiatum of the hippocampal CA1 and CA3 region and dentate gyrus. Measurements were obtained from the stratum oriens and radiatum of the CA1 and CA3 region because these areas are the dendritic fields of the hippocampal pyramidal cells and contain the majority of the hippocampal NMDA receptors. The dentate gyrus was analyzed as a control area because an earlier study had shown no changes in NMDA binding in this area on steroid treatment (32). Bilateral measurements were made from four to six sections and averaged. Only sections corresponding to plates 31–33 of Paxinos and Watson (52) for the hippocampus were analyzed to ensure the same anatomical sites within a brain area were sampled for each animal.

The areal density (cells per unit area) of ChAT mRNA containing cells and ChAT-immunoreactive (ir) cells were determined for the MS, VDB, and HDB. These areas were chosen for analysis because these nuclei contain ChAT activity and project to the hippocampus (e.g. MS and VDB) (45). Furthermore, long-term testosterone treatment has been reported to increase ChAT protein in these forebrain regions in adult male rats (53). Each brain region was centered under x10 magnification, and the magnification was then increased to x100. ChAT-positive cells that fell within a superimposed ocular grid were counted (1000 µm²; the area analyzed was smaller than the nucleus). Brain sections were anatomically matched across animals and sections corresponding only to plates 15–17 (MS and VDB) and plates 19 and 20 (HDB) of the Paxinos and Watson rat brain atlas (52) were analyzed to ensure the same anatomical sites within a brain area were sampled for each animal. Two bilateral counts were made for each nucleus and averaged. All data are expressed as mean number of ChAT mRNA-containing or ir cells per 1000 µm². The experimenter making the anatomical analyses was blind to the experimental condition of the animals.

For Western blot analyses, the RODs of the ir bands were measured using computerized image analysis software (MCID-M4). The experimenter making the density measures was blind to the experimental condition of the samples.

Data analysis
The data from each experiment were analyzed using two-tailed t tests. Differences were considered significant with P < 0.05. All data are presented as mean ± SEM.

	Results

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Effectiveness of DHTP treatments
DHTP treatment significantly increased the wet weight of the androgen-sensitive seminal vesicles (t₁₈ = –11.013, P < 0.05; Fig. 1A). Furthermore, micropunches of the medial preoptic region showed that 500 µg DHTP significantly increased hypothalamic aromatase protein levels (t₁₂ = –0.986, P < 0.05; Fig. 1B), an androgen-regulated event (54). Taken together, these data suggest that the 500-µg dose of DHTP and injection regimen used in this and a previous experiment (22) induce physiological changes in both the periphery and central nervous system.

View larger version (21K): [in this window] [in a new window]   FIG. 1. DHTP significantly increased seminal vesicle weights (A) and hypothalamic aromatase protein levels (B) in adult males. Asterisk indicates a significant difference. All values are mean ± SEM.

View larger version (15K): [in this window] [in a new window]   FIG. 2. DHTP significantly increased NMDA binding in both the stratum oriens and radiatum of the hippocampal CA1 region but not the dentate gyrus. Asterisks indicate significant differences. All values are mean ± SEM.

View larger version (149K): [in this window] [in a new window]   FIG. 3. Autoradiograms of oil-treated (A and B) or DHTP-treated (C and D) males. A and C are from tissue sections incubated with [3H]glutamate and NMDA; B and D are from sectioned incubated with [3H]glutamate only. Arrowheads delineate the extent of the CA1 stratum oriens and radiatum measured.

View larger version (17K): [in this window] [in a new window]   FIG. 4. The areal density of ChAT mRNA containing cells per 1000 µm2 (A) and ChAT-ir cells per 1000 µm2 (B) in the MS, VDB, and HDB in DHTP- and oil-treated adult males. All values are mean ± SEM.

View larger version (94K): [in this window] [in a new window]   FIG. 5. Photomicrographs of ChAT mRNA-containing cells (A) and ChAT-ir cells (B) in the MS of a DHT-treated male. Bar, 300 µm.

View larger version (38K): [in this window] [in a new window]   FIG. 6. DHTP had no effect on CHT protein levels in the CA1 region of the male hippocampus. Bottom panels are representative CHT and actin (loading control) immunoblots from oil- and DHTP-treated males. All values are mean ± SEM.

	Discussion

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NMDA receptors are found predominantly in the postsynaptic specialization and are involved in mediating excitatory neurotransmission (56). Therefore, our data suggest that DHT-treated males have a more excitable hippocampus. DHT has been shown to increase the duration of action potentials in the CA1 region and decrease the amplitude of the afterhyperpolarization, suggesting that DHT increases hippocampal excitability (57). Interestingly, DHT has been reported to decrease LTP in the CA1 region of the hippocampus in adult male rats (58). It should be noted, however, that these males experienced chronic DHT treatment for up to 43 d. It is possible that the more acute treatment protocol used in the present study, which has been shown to increase spine synapses (22), may result in enhanced LTP. Future experiments will need to address this possibility.

The role of androgens, and DHT specifically, in hippocampal-dependent learning and memory is unclear. For example, DHT has been shown to increase cognitive performance on hippocampal-dependent tasks in male rodents and humans (59, 60, 61), whereas another study (62) reported that DHT had no effect on hippocampal-dependent working memory in aged male rats. Future studies will need to establish whether the dose of DHT and injection regimen used in this, and a previous study showing increases in spine synapses (22), affect performance on hippocampal-dependent learning and memory tasks.

Our ChAT in situ hybridization and immunocytochemistry analyses do not support a role for DHT in modulating ChAT cell number in the MS, VDB, or HDB of the forebrain in adult males. Furthermore, our CHT protein data from the CA1 micropunches suggest that DHT treatment does not affect acetylcholine release into the CA1 region because CHT levels have been shown to be positively correlated with acetylcholine synthesis and release (46, 47, 48, 49). Thus, unlike the ability of E to increase cholinergic cell number in the forebrain of females (33, 34, 35, 36, 37, 38, 39) and acetylcholine release into the hippocampus (40), DHT does not appear to increase cholinergic activity in males. These data suggest that DHT-induced increases in spine synapses (22) and NMDA receptor binding (present study) are independent of changes in forebrain cholinergic activity or acetylcholine release into the CA1 region. A recent study reported that the ability of testosterone to increase hippocampal spine synapses was partially inhibited in males that had fimbria/fornix transections (44), which would disrupt cholinergic inputs to the hippocampus. However, our present data suggest that the contribution of the subcortical inputs to testosterone-mediated increases in hippocampal spine synapses may be independent of the cholinergic projections included in the fimbria/fornix pathway.

Testosterone has been previously reported to increase ChAT protein in the MS of adult males (53). However, it was not established whether the testosterone-induced increase in forebrain ChAT protein was due to the androgenic (e.g. DHT) or estrogenic (e.g. estradiol) metabolites of testosterone. Furthermore, the hormone treatment in the previous study was considerably longer (e.g. 28 d), compared with the treatment paradigm used in the present study (i.e. 72 h). Thus, our data suggest that if androgens, or locally synthesized estrogens, can influence cholinergic activity in the forebrain of males, the hormonal stimulation must be present for longer than 72 h.

In females, the E-induced increase in NMDA receptors appears to be dependent on the increase in cholinergic inputs to the hippocampus (31). Our data suggest the DHT-induced increase in hippocampal NMDA receptors is independent of changes in forebrain cholinergic cell number and CA1 hippocampal CHT levels in males. It is important to note that males have androgen receptors in the pyramidal cells of the CA1 hippocampal region (63, 64, 65). Thus, the ability of DHT to increase NMDA receptors in the dendritic fields of the CA1 region may be directly mediated at the level of the pyramidal cells. This would indicate that androgen-induced increases in spine synapses may be more dependent on the direct actions of the steroid on the pyramidal neurons than the trans-synaptic mechanisms required for E to increase hippocampal spine density in females. Furthermore, we observed no change in NMDA receptor levels in response to DHT treatment in the dentate gyrus or CA3 region of the hippocampus, areas with a relative paucity of androgen receptors, lending further support to this conjecture.

In conclusion, our data indicate that DHT treatment previously shown to increase spine synapses also increases NMDA receptor binding in the CA1 stratum oriens and radiatum of the adult male hippocampus. This effect is similar to the E-induced increase in NMDA receptors in the female hippocampus (31, 32), which underlies the increase in dendritic spines on E treatment (30). Our results also show that the DHT-mediated increase in hippocampal NMDA receptors is not accompanied by changes in ChAT-containing cell number, an effect dissimilar to that observed in E-treated females (33, 34, 35, 36, 37, 38, 39). Thus, the present set of experiments indicate that the mechanisms mediating steroid-induced hippocampal synaptic plasticity in males may operate through mechanisms different from those hypothesized to mediate estrogen-induced synaptic plasticity in females.

	Acknowledgments

 
We thank Dr. Randy Blakely for his generous gift of CHT antibody.

	Footnotes

 
This work was supported by National Institutes of Health Grants NS 07080 (to B.S.M.) and MH 065749 (to R.D.R.).

First Published Online January 20, 2005

Abbreviations: ChAT, Choline acetyltransferase; CHT, hemicholinium-3-sensitive choline transporter; dH₂O, distilled H₂O; DHT, 5-dihydrotestosterone; DHTP, 5-androstan-17ß-ol-3-one propionate; DIG, digoxigenin; E, estrogen; ER, estrogen receptor; HDB, horizontal limb of the diagonal band of Broca; ir, immunoreactive; LTP, long-term potentiation; MS, medial septum; NMDA, N-methyl-D-aspartate; PB, phosphate buffer; ROD, relative OD; SDS, sodium dodecyl sulfate; TAB, Tris-acetate buffer; VDB, vertical limb of the diagonal band of Broca.

Received July 12, 2004.

Accepted for publication October 25, 2004.

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