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Endocrinology Vol. 143, No. 1 285-294
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Sex Difference in Septal Neurons Projecting Axons to Midbrain Central Gray in Rats: A Combined Double Retrograde Tracing and ER-Immunohistochemical Study

Shinji Tsukahara and Korehito Yamanouchi

Advanced Research Center for Human Sciences (S.T., K.Y.) and Department of Basic Human Sciences (K.Y.), Laboratory of Neuroendocrinology, School of Human Sciences, Waseda University, Mikajima, Tokorozawa, Saitama 359-1192, Japan

Address all correspondence and requests for reprints to: Korehito Yamanouchi, Laboratory of Neuroendocrinology, Department of Basic Human Sciences, School of Human Sciences, Waseda University, 2-579-15, Mikajima, Tokorozawa, Saitama 359-1192, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sex difference in the number of neurons projecting axons from the lateral septum (LS) to the midbrain central gray (MCG) that are concerned with the lordosis-inhibiting system was investigated by injection of Fluoro-Gold (FG), a retrograde tracer, into the rostral MCG on the right side in male and female rats. Immunohistochemistry for ER-{alpha} and -ß was also performed with or without combination with FG immunostaining. All animals were gonadectomized. Lordosis was observed after treatment with E2 in some animals. In the results, lordosis was rare in males, compared with females. FG-immunoreactive (ir) cells were concentrated in the intermediate LS on the right side, and its number in the females was significantly higher than that in the males. There was no sex difference in the distribution and number of ER{alpha}-ir and ERß-ir cells in the LS. Furthermore, the number of ERs-ir cells was not influenced by E2 in either males or females. Double FG-ERß-ir cells were less than 20% of total FG-ir cells in the LS in both males and females. These data suggest that the LS-MCG connection is sexually dimorphic but that there is no sex difference in the expression of ERs in the LS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LORDOSIS, A CHARACTERISTIC female sexual behavior, is regulated by facilitatory (1) and inhibitory (2) neural systems in the forebrain. The ventromedial hypothalamic nucleus exerts an estrogen-dependent lordosis-facilitating influence (3, 4, 5). On the contrary, an inhibitory influence on lordosis exists in the septum because destruction of the septum (6, 7) or transection of the ventral output fibers of the septum (8, 9) decreases the threshold of estrogen to induce lordosis in female rats. The lordosis inhibition in the septum is released by estrogen in female rats because direct implantation of estrogen into the lateral septum (LS) enhances lordosis in subthreshold doses of estrogen-treated ovariectomized rats (10). In addition, implants of dihydrotestosterone into the LS are reported to prevent estrogen- induced lordosis behavior in female rats (11).

In male rats, the incidence of lordosis is very low even when castrated and treated with large doses of estrogen (12), whereas lesions of the septum (13, 14, 15) or ventral cuts of the septum (16) induce lordosis. These data strongly suggest that the lordosis-inhibitory system in the septum is concerned with sex differences in the regulation of female sexual behavior. On the basis of the result indicating that implantation of estrogen into the LS facilitates lordosis in female rats but not in male rats (10), response to estrogen in the LS is related to sex differences of the lordosis-inhibitory system in the septum. In the rat LS, the existence of ER-{alpha} and -ß has been demonstrated (17, 18, 19), although sex difference in the receptors has not yet been clearly determined.

Our recent study suggests that inhibition of lordosis in male rats is produced by the neuronal cells in the intermediate part of the LS and that the cells directly project their axons to the midbrain central gray (MCG) (15). The MCG also integrates the lordosis-facilitating influence in the ventromedial hypothalamic nucleus (1).

In the present study, as one step to clarify sexual differentiating mechanisms in the LS, sex differences in the numbers of neurons projecting axons to the MCG and in expression of ER{alpha} and ERß in the LS were determined. Furthermore, sex differences in the number of ERß-containing septal neurons projecting axons to the MCG was investigated by a combination of retrograde tracing and ERß-immunohistochemical analyses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In the first experiment, a retrograde tracer was injected into the MCG and the tracer-labeled neuronal cells were detected immunohistochemically to clarify sex differences in the neuronal projection from the LS to the MCG. In the second experiment, single ER{alpha} or ERß immunostaining was performed to examine sex differences in the distribution and number of ER{alpha}- or ERß-containing cells in the LS. In addition, double immunostaining for ERß and the tracer was carried out to investigate differences in the number of ERß-containing septal neurons that project the axons to the MCG between males and females.

Animals
Adult male (210–320 g) and female (210–280 g) Wistar rats (Takasugi Animal Farm, Saitama, Japan) were used according to the Guidelines for the Care and Use of Laboratory Animals in the Human Science Department of Waseda University. The animals were kept in a light (14 h light/10 h dark, lights on at 0700 h)- and temperature (23–25 C)- controlled room with free access to water and food. All males and females were gonadectomized under ether anesthesia.

Exp 1
Two weeks after gonadectomy, five males and five females were sc implanted with two silicon tubes (inner diameter 1.57 mm; outer diameter 3.18 mm; length 30 mm., Kaneka Medix Co., Osaka, Japan) containing E2 crystalline (Sigma, St. Louis, MO) while they were under ether anesthesia. The E2-treated gonadectomized rats were subjected to the tests for lordosis. Two days after E2 treatment, the test was started and three tests in total were carried out every other day. On the day following the last behavioral test, Fluoro-Gold (FG, Fluoro-Chrome Inc., Englewood, CO), a retrograde tracer, was injected into the right side of the MCG. Four days after FG injection, the brain was fixed and brain sections were made. The forebrain sections were immunostained for FG and the number of FG-immunoreactive (FG-ir) cells in the LS and the cingulate cortex (CgCx) was counted. To determine the FG injection area, the midbrain sections were also immunostained for FG, and the location and volume of the area was examined.

Exp 2
Single ER{alpha} or ERß immunohistochemical analysis was carried out in animals with or without E2 treatment. Double immunohistochemistry for FG and ERß was performed in non-E2-treated rats. In E2-treated females and males (five rats for each), the forebrain sections prepared for Exp 1 were used. Three weeks after gonadectomy, non-E2-treated males and females (five rats for each) were injected with FG into the right side of the MCG, followed by the same histological process as in Exp 1.

After single immunostaining for either ER{alpha} or ERß, the number of ER{alpha}-ir or ERß-ir cells in the LS and the medial preoptic nucleus (MPO) was counted. In double FG- and ERß-immunostained forebrain sections of non-E2-treated rats, the numbers of double FG- and ERß-immunoreactive (FG-ERß-ir) cells and single FG-ir cells in the LS were counted. To determine the FG injection area in the non-E2-treated rats, the midbrain sections were immunostained for FG and the location and volume of the area was examined.

General procedures
Behavioral test

An experimental rat was placed in a plastic observation cage with two vigorous male rats. The lordosis quotient (LQ; number of lordosis reflexes/10 mounts x 100) in each animal was recorded.

Retrograde tracer injection
Animals anesthetized by ether were placed in a stereotaxic instrument with the bregma and lambda at the same dorsoventral level. FG (8% solution dissolved in distilled water) was injected iontophoretically into the right side of the MCG through a glass micropipette (tip diameter, 40–50 µm). The tip of the micropipette was lowered to a point 6.5 mm caudal to the bregma, 4.5 mm below the dura, and 0.5 mm to the right side to the midline, and then a positive current of 2 µA was applied for 8 min continuously.

Tissue preparation for immunohistochemical analyses
The animals were deeply anesthetized by sodium pentobarbital (20–25 mg per animal) and were perfused intracardially with 50 mM PBS (pH 7.4) followed by 4% paraformaldehyde-50 mM phosphate buffer (pH 7.4). Brains were postfixed with the same fixative for 2 h and immersed in 30% sucrose-50 mM phosphate buffer for 4–5 d at 4 C. Serial coronal brain sections (50 µm) were made with a cryostat and collected from the septal region to the inferior colliculus as five series of sections. Each series of the sections was used for each immunohistochemical analysis for FG and/or ERs.

In all animals, one series of the sections was stained with cresyl fast violet. They were used for histological determination of the brain regions according to the rat brain atlas (20).

Preparation of primary antibodies
Anti-FG. A polyclonal rabbit anti-FG antibody (1:9000, Chemicon International, Inc., Temecula, CA), which was diluted in 5% normal goat serum (NGS, Chemicon International, Inc.)-0.4% Triton X-100-50 mM PBS, was used. The specificity of this antibody had been confirmed by preabsorption with the antigen (15). In the present study, some sections were processed without the anti-FG antibody as an immunohistochemical negative control. Immunoreactivities were not observed in any of the control sections (data not shown).

Anti-ER{alpha}. A polyclonal rabbit anti-ER{alpha} antibody (catalog no. PA1-308, Affinity BioReagents, Inc., Golden, CO) was employed. According to the instructions, the antibody was generated by immunizing rabbits with a synthetic peptide corresponding to the N-terminal residues 1–21 of human ER{alpha} conjugated to keyhole limpet hemocyanin and showed no cross-reactivity with ERß. The peptide sequence is completely conserved in rats. This antibody was diluted in 5% NGS-0.4% Triton X-100-50 mM PBS at a concentration of 1 µg/ml for use in immunostaining. To confirm the specificity of the antibody, anti-ER{alpha} solution was preabsorbed with the immunizing peptide (catalog no. PEP-036, Affinity BioReagents, Inc.) at a final concentration of 25 µg/ml at 4 C overnight. Then the preabsorbed solution was used for immunostaining for ER{alpha}. Some sections were processed without the primary antibody. As a result, ER{alpha} immunoreactivities were prevented when the antibody was preabsorbed with the antigen, and no immunoreactivity was observed in the section processed without the antibody (data not shown).

Anti-ERß. A polyclonal rabbit anti-ERß antibody (catalog no. sc-8974, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), which was raised against a recombinant protein corresponding to the N-terminal residues 1–150 of human ERß, was used for the present experiments. According to the instructions, the antibody was reacted with ERß of rat origin without cross-reactivity to ER{alpha}. However, before usage of this antibody for immunohistochemistry, an immunoblot analysis was carried out to check whether the antibody detects a substance having the same molecular mass as that of rat ERß.

For the immunoblot analysis, samples were prepared from the septal region including the LS and the preoptic region including the MPO. Samples of the cerebellar region were also prepared as a positive control because the existence of ERß mRNA and the protein was reported (17, 21, 22, 23). Three boiled homogenate samples of each region of each intact female or male rat (15 µl at a final concentration of 1 µg of wet weight of tissue/µl Laemmli buffer) were electrophoresed on a 10% polyacrylamide gel and electrotransferred to a polyvinylidene difluoride membrane. A mixture of biotinylated standard molecules was also boiled and loaded. Briefly, the membrane was blocked with 5% nonfat dry milk (NDM) in 40 mM Tris-buffered saline (TBS, pH 7.5) for 1 h at room temperature, incubated with 1 µg/ml rabbit anti-ERß antibody (sc-8974, Santa Cruz Biotechnology Inc.) in 5% NDM-0.1% Tween-20-40 mM TBS for 72 h at 4 C, and incubated with peroxidase-labeled goat antirabbit IgG (1:200, Vector Laboratories, Inc., Burlingame, CA) and peroxidase-conjugated streptavidin (1:3000, DAKO Corp., Carpinteria, CA) in 5% NDM-0.1% Tween-20-40 mM TBS for 2 h at room temperature. Immunoreactive signals were visualized on an x-ray file by using ECL Plus Western blotting detection reagents (Amersham Pharmacia Biotech, Little Chalfont, UK). As a result, a dense immunoreactive band with a molecular mass of approximately 54 kDa was detected in all samples. The molecular mass detected by our immunoblot analysis corresponded to the 54.2-kDa protein encoded by ERß cDNA isolated from rat prostate (24). A faint band with a molecular mass of approximately 36 kDa was also detected, but the relative amount of the 36-kDa band was apparently slighter than that of the 54-kDa band.

Based on the results of the immunoblot analysis, the rabbit anti-ERß antibody (sc-8974, Santa Cruz Biotechnology Inc.) was diluted in 5% NGS-0.4% Triton X-100-50 mM PBS (final concentration: 1 µg/ml) and employed for immunohistochemistry. To establish that a severe immunoreactive condition minimized any nonspecific reaction, the diluent was reacted with extra brain sections for 72 h at 4 C, and then the forebrain sections were processed by the diluent according to the following procedure. Sections of the rat cerebellum were made and immunostained for ERß as a positive control. In the cerebellar sections, most of immunoreactivities were seen in the Purkinje cells, as previously reported by in situ hybridization or immunohistochemical studies (17, 21, 23) (data not shown). In addition, some forebrain and cerebellar sections were processed without the anti-ERß antibody for a negative control, and no immunoreactivity was observed in these sections (data not shown).

Single immunostaining for FG, ER{alpha}, or ERß
Free-floating sections were incubated with 0.6% H2O2-50 mM PBS for 30 min at room temperature before and after rinsing with 50 mM PBS. The sections were incubated with 5% NGS-0.4% Triton X-100-50 mM PBS for 1 h at room temperature and then with the primary antibody for each FG, ER{alpha}, or ERß containing 5% NGS-0.4% Triton X-100-50 mM PBS for 72 h at 4 C. After washing with 50 mM PBS, the sections were reacted with goat antirabbit immunoglobulins conjugated to peroxidase labeled-dextran polymer in Tris-HCl buffer (EnVision Plus, DAKO Corp.) for 30 min at room temperature. The sections were rinsed with 100 mM Tris-HCl buffer (pH 7.2) and then reacted with 0.05% 3, 3'-diaminobenzidine (DAB), 0.01% H2O2, and 0.08% ammonium nickel sulfate containing 100 mM Tris-HCl for visualization of FG-, ER{alpha}-, or ERß-ir cells. FG injection sites in the MCG were determined by using a Vector SG substrate kit (Vector Laboratories, Inc.).

Immunostained sections mounted on slides were dehydrated through a graded series of ethanols, cleared by xylene, and then coverslipped with an embedding. These sections were examined by light microscopy.

Double immunostaining for FG and ERß
First, free-floating sections were processed according to the same protocol as for the single immunostaining for ERß, except for the chromogenic reaction. The sections were reacted with 0.05% DAB and 0.01% H2O2 containing 100 mM Tris-HCl. After rinsing in 50 mM PBS, they were incubated in 100 mM glycine-HCl buffer (pH 2.2) for 90 min to dissociate antibody-antigen complex. The buffer was replaced with a fresh one every 30 min. The sections were then treated with 0.6% H2O2-50 mM PBS for 30 min at room temperature. Before and after washing with 50 mM PBS, the sections were incubated with rabbit anti-FG antibody (1:9000, Chemicon) containing 5% NGS-0.4% Triton X-100-50 mM PBS for 72 h at 4 C. Then they were incubated with EnVision Plus reagent (DAKO Corp.) for 30 min at room temperature. After rinsing with 100 mM Tris-HCl buffer, FG-immunoreactivities were visualized by an SG substrate kit (Vector Laboratories, Inc.).

For immunohistochemical control, some ERß-immunostained sections were processed according to the above-mentioned protocol, except the step for incubation with anti-FG antibody. Instead of anti-FG antibody incubation, sections were incubated with 5% NGS-0.4% Triton X-100-50 mM PBS for 72 h at 4 C. In the control sections, FG-ir signals were completely blocked (data not shown). In addition, ERß-ir signals produced by DAB reactions were still apparent, and no additional chromogenic reaction was raised from an SG (Vector Laboratories, Inc.).

Quantitative analysis of FG-ir cells in the LS and the CgCx
In one series of the forebrain sections, the LS was included in seven to nine sections, and all of these sections were used for counting the number of FG-ir cells in each rat. The number of cells in the CgCx was also measured by using the same sections.

Photomicrographic digital images of the sections were taken (final magnification x28, 250 pixels/inch). Each pixel of these images was scaled from 0 (black) to 255 (white). After calibration of a scale of the background level in each image, signals brighter than the background were removed. Then signals darker than the background level were scaled again, with the scale of the background being 255.

Measurement of the number of FG-ir cells in the LS and the CgCx was carried out by using NIH Image version 1.61 (NIH, Bethesda, MD). As criteria of determination of FG-ir cells, signals composed of 4–28 pixels and darker than 110 of 256 scales were counted. The number of FG-ir cells in the LS and the CgCx of each animal was shown as the average in all sections measured. Then the mean number of each group was calculated.

Quantitative analysis of ER{alpha}-ir or ERß-ir cells in the LS and the MPO
Numbers of ER{alpha}-ir or ERß-ir nuclei in the right side of the LS and the MPO were measured. Ten areas selected from the LS at three levels from rostral to caudal were examined to count ER{alpha}-ir or ERß-ir cells in each rat (Fig. 3Go). In the MPO, areas chosen from three sections at the level from rostral to caudal were examined (Fig. 4Go). Each area was 300 µm x 200 µm.



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  		Figure 3. A, Number of ER{alpha}- or ERß-immunoreactive cells in the LS of non-E2-treated or E2-treated males and females. Five animals were used for each group. Values are the mean ± SEM. {dagger} or {dagger}{dagger}, P < 0.05 or P < 0.01 vs. ER{alpha}-immunoreactive cells of the same hormonal conditioned group. B, Boxed black areas (300 µm x 200 µm) indicate areas in which the number of the cells was measured at three levels of the LS from rostral (1 ) to caudal (3 ). LSd, Dorsal part of the LS; LSi, intermediate part of the LS; LSv, ventral part of the LS.

 


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  		Figure 4. Number of ER{alpha}- or ERß-immunoreactive cells in the MPO of non-E2-treated or E2-treated males and females. Five animals were used for each group. *, P < 0.05 vs. non-E2-treated female; {dagger} or {dagger}{dagger}, P < 0.05 or P < 0.01 vs. ER{alpha}-immunoreactive cells of the same hormonal conditioned group. B, Boxed black areas (300 µm x 200 µm) indicate areas in which the number of the cells was measured at three levels of the MPO from rostral (1 ) to caudal (3 ).

 
Photomicrographic digital images in each area (final magnification, x280; 200 pixels/inch) were taken. After transforming these images according to the processes described above, particles (minimum particle size, 20 pixels; maximum particle size, 610 pixels) darker than 110 of 256 scales were counted as ER{alpha}-ir or ERß-ir cell nuclei by using NIH Image. In each animal, the number of ER{alpha}- or ERß-ir cells was shown by the average of the number in the areas of the LS or MPO. Then the mean number of each group was calculated.

Quantitative analysis of FG-ir and FG-ERß-ir cells in the LS
In the one series of double FG- and ERß-immunostained sections, the LS was included in 7–10 sections. In the right side of the LS of these sections, the numbers of FG-ir cells and FG-ERß-ir cells were measured.

To distinguish FG-ir signals in the cytoplasm with or without ERß-ir signals in the nucleus, photomicrographic color prints of the LS were taken (final magnification x200) and then montage photomicrographs were made. FG-ir cell bodies (blue) and ERß-ir cell nuclei (brown) in all parts of the LS on the right side were counted with the naked eye. When the color of the cytoplasm was blue and the nucleus was darker brown than that of the background, the cell was identified as a FG-ERß-ir cell. When the cell whose cytoplasm was blue and the color of whose nucleus was not different from that of the background, the cell was determined as a FG-ir cell. To avoid possible bias, all photomicrographs were coded. Two persons who did not know the source of the material counted the cell numbers, and the average number was used as data for each rat.

In each animal, the number of FG-ir and FG-ERß-ir cells in the right side of the LS were shown by the average of the number in all LS sections. Then the mean number of each group was calculated. The percentage of the number of FG-ERß-ir cells in all FG-ir cells was also calculated in each animal and then averaged in each group.

Volume of FG injection area in the MCG
Photomicrographic digital images of the MCG were taken to measure the volume of the FG injection area (final magnification x28; 250 pixels/inch). After being processed by the same transformation as that for counting FG-ir cells, an area darker than 110 of 256 scales was measured as the FG penetrated area by using NIH Image. The volume of the FG injection area was estimated by the calculation: Total of FG injection area in one series of sections x thickness of section x 5 = number of series of sections. Then the mean volume of the FG injection area of each group was calculated.

Statistical analyses
Differences of the mean LQ were analyzed by ANOVA with repeated measures. When comparing the difference in the number of FG-ir and FGERB-ir cells and the volume of FG injection area between groups, the unpaired t test was used. The right- and left-side differences in the number of FG-ir cells of each group were analyzed by the paired t test. Two-way ANOVA was used to test for significant differences in the number of ER{alpha}-ir or ERß-ir cells of the LS or the MPO among groups. When a significant difference among groups was detected, the unpaired t test was used to assess differences between groups. The test was also used to compare the number of ERß-ir cells with that of ER{alpha}-ir cells in the LS or the MPO between the same hormonal conditioned groups. The percentage of numbers of FG-ERß-ir cells in the total number of FG-ir cells was analyzed by the Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1
Sex difference in lordosis behavior. In females, all animals showed lordosis and the LQ gradually increased according to three behavioral tests. The mean LQs in the females were 14.0 ± 11.6, 54.0 ± 14.0, and 66.0 ± 11.2 at 2, 4, and 6 d after E2 treatment. In contrast, only three males displayed lordosis thought the test, and the LQ was very low. The mean LQs in the male group were 0, 2.0 ± 2.0, and 6.0 ± 4.0 in three tests. There were significant differences in the mean LQ between the male and the female by ANOVA with repeated measures [F(2, 16) = 7.34, P < 0.01].

Distribution of FG-ir cells in the forebrain of male and female rats.There was no difference in the distribution of FG-ir cells in the LS and CgCx of E2-treated male and female rats (Fig. 1Go). In the septal region, many FG-ir neuronal cell bodies were found scattered over the LS, especially the intermediate part of the LS on the side ipsilateral to the FG injection site (the right side) in both males and females. However, the medial septum and the septofimbrial nucleus had only a few FG-ir cells. On the left side of the LS, a few FG-ir cells were seen in the intermediate part. In the anteroposterior axis, the majority of FG-ir cells was observed from the rostral end of the LS to the anterior end of the septofimbrial nucleus. The number of FG-ir cells was drastically decreased in the caudal LS. Several FG-ir cells were also localized in the ventral part of the rostral LS on the right side. However, the dorsal part of the LS had few FG-ir cells on both sides in both males and females.



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  		Figure 1. Photomicrographs of the CgCx (A1 and B1) and LS (A2 and B2) in FG-immunostained sections of E2-treated male and female rats that received the tracer injection into the midbrain central gray on the right side. A3 and B3 show a high magnification of the intermediate part of the LS (LSi) in A2 and B2, respectively. Scale bars in A1, A2, B1 and B2 indicate 500 µm, and those in A3 and B3, 100 µm. cc, Corpus callosum; LV, lateral ventricle.

 
In the limbic cortex in the forebrain sections, a large number of FG-ir pyramidal cells was found in the CgCx on the right side, but only a few cells on the left side in both E2-treated males and females. Besides the CgCx, the agranular insular cortex, the motor cortex and the forelimb and hindlimb regions of the primary somatosensory cortex also had several FG-ir pyramidal cells on the right side. Only a few FG-ir pyramidal cells were seen in the contralateral side of the agranular insular cortex.

In addition to the septum and the limbic cortex, the prominent location of FG-ir cells were in the bed nucleus of the stria terminalis, the medial and lateral preoptic area, and the medial and median preoptic nuclei. In these regions, more FG-ir cells were found on the right side than on the left side.

Sex difference in the number of FG-ir cells in the LS. The number of FG-ir cells in the LS of E2-treated females was significantly (P < 0.05) larger than that of E2-treated males on both sides (Fig. 2Go). In each sex, the numbers of FG-ir cells in the LS on the side ipsilateral to the FG injection site (the right side) were significantly (P < 0.05) higher than those on the left side.



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  		Figure 2. Number of FG-ir cells in the LS and CgCx of E2-treated males and females on sides ipsilateral (right) and contralateral (left) to FG injection site. Values are mean ± SEM. Numbers in parentheses indicate the number of animals used. {dagger}, P < 0.05 vs. left side; *, P < 0.05 vs. male.

 
In the CgCx, there was no sex difference in the number of FG-ir pyramidal cells (Fig. 2Go). The number of FG-ir cells on the right side was significantly (P < 0.05) higher than that on the left side in both male and female groups.

Exp 2
Sex difference in the number of ER{alpha}-ir cells in the LS and the MPO. Most ER{alpha}-ir signals were localized in the nucleus but only a few in the cytoplasm. The distribution of ER{alpha}-ir cells had no striking difference among groups. ER{alpha}-ir cells were found scattered over the LS, but the population was very small and the immunoreactive signals were not strong. In the analysis counting the number of ER{alpha}-ir cells, two-way ANOVA indicated no sex differences in the number of cells in the LS [F(1, 16) = 1.19, P = 0.29] (Fig. 3Go). There was also no significant difference for the effect of E2 treatment [F(1, 16) = 0.42, P = 0.53] or the effect of interaction between sex and E2 treatment [F(1, 16) = 0.57, P = 0.46].

A large number of ER{alpha}-ir cells was concentrated in the MPO, especially in the region neighboring the periventricular hypothalamic nucleus. There was no significant difference in the number of ER{alpha}-ir cells in the MPO between males and females [F(1, 16) = 1.92, P = 0.18] (Fig. 4). Significant effects of E2 treatment on the number of ER{alpha}-ir cells in the MPO were detected [F(1, 16) = 10.2, P < 0.01]. The number of ER{alpha}-ir cells in non-E2-treated females was significantly (P < 0.05) higher than that in E2-treated females. Although the number of cells in the non-E2-treated males tended to be higher than that in E2-treated males, the difference was not detected as significant. However, two-way ANOVA indicated no interactive effect of E2 treatment on sex difference in the number of ER{alpha}-ir cells [F(1, 16) = 1.38, P = 0.26], suggesting that E2 treatment decreases in number in both male and female groups.

Sex difference in the number of ERß-ir cells in the LS and the MPO. Most ERß-ir signals were observed in the nucleus of cells in the LS and the MPO. In several cells having strong ERß-ir signals in the nucleus in the LS, ERß-ir signals were also found in the cytoplasm.

ERß-ir cells were observed all over the LS. No apparent difference in the distribution of ERß-ir cells was seen in the LS between females and males. As for the results of counting the number of ERß-ir cells in the LS, there were no sex differences [F(1, 16) = 0.11, P = 0.75] (Fig. 3Go). The number of ERß-ir cells of the E2-treated group did not differ from that of the non-E2-treated group [F(1, 16) = 0.14, P = 0.71]. In addition, no interactive effect between sex and E2 treatment was recognized statistically [F(1, 16) = 0.04, P = 0.84]. When compared between the numbers of ERß-ir and ER{alpha}-ir cells in the LS, that of ERß-ir cells was significantly higher than that of ER{alpha}-ir cells in non-E2-treated males (P < 0.01), E2-treated males (P < 0.05), non-E2-treated females (P < 0.05), and E2-treated females (P < 0.01).

In the MPO, numerous cells having strong ERß-ir signals in the nucleus were seen. There was no significant effect of sex [F(1, 16) = 0.41, P = 0.53], E2 treatment [F(1, 16) = 0.12, P = 0.74], or interaction between sex and E2 treatment [F(1, 16) = 0.60, P = 0.45] on the number of ERß-ir cells in the MPO (Fig. 4Go). The number of ERß-ir cells in the MPO was significantly greater than that of ER{alpha}-ir cells in E2-treated males (P < 0.05) and E2-treated females (P < 0.01). However, such a difference was not seen in non-E2-treated groups.

Sex difference in the number of FG-ERß- ir cells in the LS.Double immunohistochemistry for FG and ERß was performed only in non-E2-treated males and non-E2-treated females because there was no difference in the distribution and number of ERß-ir cells between non-E2-treated and E2-treated groups by single ERß immunostaining.

In double FG-ERß-ir cells, FG-ir (blue) and ERß-ir (brown) signals were able to be distinguished from each other with different colors (Fig. 5Go). The distribution and amount of FG-ir cells in the LS of non-E2-treated rats seemed to be the same as that of E2-treated rats in Exp 1. In addition, the distribution of ERß-ir cells in the LS was the same as that in the results of the single immunostaining for ERß described above.



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  		Figure 5. Representative photomicrographs of the LS in double FG-ERß-immunostained sections of non-E2-treated males (A) and females (B). Arrows or arrowheads indicate double FG-ERß-immunoreactive cells or single FG-immunoreactive cells, respectively. Scale bars indicate 50 µm.

 
Most FG-ERß-ir cells were seen in the intermediate and ventral parts of the LS on the right side but only a few on the left side in both the females and males. Although the number of FG-ir cells became smaller at the caudal levels in the anteroposterior axis, FG-ERß-ir cells were equally observed in the axis. In the measurement of the number of FG-ir cells and FG-ERß-ir cells in the right side of the LS, the total number of FG-ir cells in females was significantly (P < 0.01) larger than that in males (Fig. 6Go). The number of FG-ERß-ir cells in the females was also significantly (P < 0.05) greater than that in the males. However, the percentage of the number of the FG-ERß-ir cells in total of the FG-ir cells was 13.3 ± 2.7% or 18.8 ± 4.8% in the males or females, respectively. There was no significant difference in the value between groups.



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  		Figure 6. Number of FG-ir and FG-ERß-ir cells in the right side of the LS in non-E2-treated males and females that received FG injection into the midbrain central gray on the right side. Values are the mean ± SEM. Numbers in parentheses indicate the number of animals used. *, P < 0.05 vs. male; **, P < 0.01 vs. male.

 
FG injection site in the MCG in Exps 1 and 2
In all animals used in this study, most of the FG injection area was located on the right side in the rostral part of the MCG (Fig. 7Go). In four of five E2-treated males and three of five E2-treated females used in Exp 1, and four of five non-E2-treated males and four of five non-E2-treated females used in Exp 2, the location of the tip of a micropipette was established as being in the central part of the MCG on the right side. In one each of E2-treated females, E2-treated males, and non-E2-treated males, the tip was located more dorsal to the center of the right side of the MCG. In the remaining one each of non-E2-treated and E2-treated females, the tip was located more ventral. Although the location of the tip in the MCG had a slight difference among animals, there was no apparent difference in the FG-penetrated area. Furthermore, the mean volume of the FG injection site was 0.60 ± 0.05 or 0.70 ± 0.15 mm3 in E2-treated males or E2-treated females in Exp 1, respectively. No significant difference in volume was detected between groups. In non-E2-treated males or females of Exp 2, the volume of the FG injection area was 0.59 ± 0.15 or 0.58 ± 0.05 mm3, respectively, having no significant difference between the groups.



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  		Figure 7. A representative photomicrograph of the FG injection site in the rostral MCG on the right side. Scale bar shows 1 mm. Aq, Aqueduct.

 
In the rostral MCG, FG penetrated all of the right-side regions in all animals. In the anteroposterior axis, the FG-penetrated area was seen between the levels of the anterior end of the medial longitudinal fasciculus and the levels of the posterior end of the deep mesencephalic nucleus in all animals except two E2-treated males. The injection area of the two males was observed between the levels of the anterior end of the dorsal raphe nucleus and the levels of the posterior end of the ventral tegmental nucleus. In the dorsoventral axis, a small peripheral part of the dorsal or ventral MCG was partially intact in all animals. The area surrounding the MCG, such as the deep white and gray layers of the superior colliculus, intercollicular nucleus, deep mesencephalic nucleus, supraoculomotor cap, supraoculomotor periaqueductal gray, and dorsal raphe nucleus were partially penetrated by FG in all rats.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sexual dimorphism of neurons in the LS
In the first experiment, FG-ir neuronal cells were seen mainly in the intermediate part of the LS on the right side in both female and male rats that received FG injection into the right side of the rostral MCG. This confirms our previous report demonstrating a direct ipsilateral projection from the intermediate LS to the rostral MCG in male rats (15). In the female and male forebrain, besides the LS, many FG-ir cells were also found in the right-side region of the preoptic area (POA) and the CgCx. Because the LS, CgCx, and POA are involved in the inhibitory system for regulation of female sexual behavior (1, 2), these inhibitory signals are thought to be sent to a lordosis-integrating center in the MCG on the ipsilateral side.

The present results counting the number of FG-ir cells demonstrated that the number of FG-ir cells in the LS of females showing high LQ was greater than that in males with low levels of LQ. On the other hand, the number of FG-ir cells in the CgCx of the females was comparable to that of the males. These suggest that neurons in the LS directly connecting with the MCG are sexually dimorphic but not in the CgCx-MCG connection. Because inhibition for lordosis in the LS is responsible for causing sex differences in regulating lordosis (2) and the LS-MCG neural tract is involved in lordosis-inhibition in male rats (15), the sex difference in the LS-MCG connection may be reflected on the sex difference in the lordosis-inhibiting function of the LS. Structural sexually dimorphic areas had been reported in the forebrain, such as the POA, amygdala, and arcuate nucleus (25). Although no structural sex difference has been reported yet in the LS, a sexually dimorphic synaptic response to E, increasing in the synaptic number of females but not of males by estrogen, has been reported in the rat LS (26). From this report and the present experiment, it can be speculated that neural inputs and outputs in the female LS are larger than those in the male LS.

In the present experiment, the number of FG-ir cells in the LS was not different under conditions with and without estrogen in both female and male rats. This suggests that the quantity of the LS-MCG connection is not influenced by steroid hormones in adulthood. Morphological sex differences of the brain are formed during the critical period for the sex differentiation of the brain under the influence of androgen (27, 28). Sexual dimorphism of the amounts of neurons in the LS may also be formed during the critical period. During the critical period, androgen is changed to estrogen by an aromatizing enzyme resulting in the development of the male brain in mammals (29). Apoptotic cell death is reported to be involved in a process of sexual differentiation of the rat brain (30, 31). One report has suggested that the effect of estrogen on apoptosis is caused by binding with ER (32). The reduction of number of the male LS neurons may be induced by steroid under such mechanism.

ER and sex differences
Because lordosis inhibition of the female LS is released by estrogen but not in males (10), the difference of sensitivity to estrogen causes a sex difference in the lordosis-regulating system in the LS. The neurons in the LS are known to contain ER{alpha} (17, 18, 33). In this experiment, ER{alpha} was also seen in the LS in both female and male rats. However, ER{alpha}-containing cells were very scarce in both sexes. Furthermore, the sex difference in the number of ER{alpha}-containing cells in the LS could not be detected in this experiment. According to these results, it is likely that the number of ER{alpha}-containing cells in LS is not concerned with sex differences of the lordosis-inhibiting system in the LS. However, ER{alpha} knockout female (34, 35, 36) and male (37, 38, 39) mice have been reported to show abnormalities of sexual behaviors and gonadotropin secretion. The possibility that ER{alpha} contributes to release the inhibition of lordosis in the female LS cannot be excluded.

In contrast to the LS, the POA including the MPO contained a large number of ER{alpha}-ir cells in the present study, as described in many reports (18, 40, 41, 42). The POA is important not only for the regulation of male (43) and female (1) sexual behavior but also for the control of gonadotropin secretion (44, 45). Furthermore, the POA is well known to include a sexually dimorphic nucleus (46, 47). Therefore, the POA is thought to play a key role in sex differences in the reproduction-regulating system.

The present study showed that ER{alpha}-ir cell numbers in the MPO of E2-treated females was less than that of non-E2-treated females. This is consistent with the previous paper demonstrating a reduction of ER{alpha}-ir cells in the female MPO by estrogen (40). There was a tendency toward a decrease in the number of ER{alpha}-ir cells in the male MPO by estrogen, although there was no statistical difference in this experiment. A similar result has been reported, indicating that the number of ER{alpha} mRNA-containing cells in the MPO of castrated male rats was comparable to that of intact rat, although the amount of ER{alpha} mRNA in the MPO was increased by castration (42). Thus, there remains the possibility of existence of the downregulating system for ER{alpha} by estrogen in the male POA as well as in the female POA. Although sex difference in the number of ER{alpha}-ir cells was not observed in this experiment, it can be speculated that the control system of ER{alpha} is sexually differentiated in reproductive functions in the POA.

Precise distribution of ERß-immunoreactivities in the rat brain has been shown in the report by Shughrue and Merchenthaler (19). ERß mRNA and the protein have been also reported in the LS and POA of rats (17, 19, 23). The present experiment also showed the existence of ERß-ir cells in the LS and the MPO in both female and male rats. In addition, there was no sex differences in the distribution and number of ERß-ir cells. The present double FG-ERß-immunohistochemical results suggest that less than 20% of LS neurons projecting axons to the MCG had ERß in both male and female rats.

In the LS and the MPO, the number of ERß-ir cells was not changed by treatment with estrogen in both gonadectomized female and male rats in the present experiment. This is confirmed partially by the report that ERß mRNA expression in the MPO was not influenced by estrogen in female rats (48). On the other hand, it is necessary to note that down-regulation of ERß by estrogen has been shown in the paraventricular hypothalamic nucleus (48).

ERß knockout mice show normal sexual behavior (49) and have normal fertile ability (50). In this context, ERß-ir cells in the LS seem not to contribute to the regulation of lordosis. However, there is a possibility that the ER{alpha} system overcomes effects of the deficiency of the ERß system in regulating reproductive functions in ERß knockout mice. ERß has been reported to influence the modulation of ER{alpha} by estrogen (51). There is evidence of colocalization of ERß and ER{alpha} in neurons of the several forebrain regions (52). Thus, the possibility that ERß-containing neurons play some role in the lordosis-inhibiting system in the LS cannot be excluded.

ERß mRNA expression in the hypothalamus/preoptic region has been reported to be sexually dimorphic during perinatal and postnatal development but not in adulthood (53). Furthermore, the brains of ERß knockout mice show morphological abnormalities in the brain (54). Thus, ERß may play an important role in the sexual differentiation during the critical period, the development of the brain and its implicated functions. Further experiments are needed to clarify the role of ER-containing neurons in the LS for the sex difference in female sexual behavior-regulating system.


   Acknowledgments
 
We are grateful to H. Yurino for his technical assistance.


   Footnotes
 
This work was supported in part by research grants from Waseda University to S.T. (2001A-178) and K.Y. (2001A-611), Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (12760189 to S.T. and 11640669 to K.Y.), and the Promotion and Mutual Aid Corporation for Private Schools of Japan (to K.Y.).

Abbreviations: CgCx, Cingulate cortex; DAB, 3, 3'-diaminobenzidine; FG, fluoro-gold; FG-ERß-ir, FG- and ERß-immunoreactive; FG-ir, FG-immunoreactive; LQ, lordosis quotient; LS, lateral septum; MCG, midbrain central gray; MPO, medial preoptic nucleus; NDM, nonfat dry milk; NGS, normal goat serum; POA, preoptic area; TBS, Tris-buffered saline.

Received June 26, 2001.

Accepted for publication September 19, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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