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Sex Differences in Serotonergic But Not -Aminobutyric Acidergic (GABA) Projections to the Rat Ventromedial Nucleus of the Hypothalamus

Department of Zoology (H.B.P.), North Carolina State University, Raleigh, North Carolina 27695; CIIT Centers for Health Research (H.B.P., A.E.F., E.K.P.), Research Triangle Park, North Carolina 27709; Department of Epidemiology (A.E.F.), University of North Carolina, Chapel Hill, North Carolina 27519; and Department of Physiology and Biophysics (E.K.P.) and Specialized Neuroscience Research Program (E.K.P.), Howard University College of Medicine, Washington, DC 20059

Address all correspondence and requests for reprints to: Dr. Heather Patisaul, Department of Zoology, North Carolina State University, Raleigh, North Carolina 27695. E-mail: heather_patisaul{at}ncsu.edu.

	Abstract

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Hormonal conditions that elicit lordosis in female rats are ineffective in males, suggesting that this behavior is actively suppressed in males. Previous studies theorize that serotonergic and -aminobutyric acidergic (GABA) inputs to the ventrolateral division of the ventromedial nucleus of the hypothalamus (VMNvl) may contribute to lordosis inhibition in males. Using triple-label immunofluorescent techniques, the present studies explored potential sex differences in the density of these projections within three hypothalamic sites: the VMNvl, the arcuate nucleus (ARC), and the dorsomedial nucleus of the hypothalamus. Antibodies directed against HuC/D, estrogen receptor (ER)- and either serotonin (5-HT) or the -aminobutyric acid synthetic enzyme glutamic acid decarboxylase-65 were used to compare the densities of glutamic acid decarboxylase (GAD)-65- and 5-HT-containing fibers in each brain area, the percentage of VMNvl HuC/D immunoreactive (ir) neurons that contained ER, and the percentage of HuC/D and ER double-labeled cells receiving apparent contacts from 5-HT fibers between adult, gonadectomized male and female rats. The densities of VMNvl and ARC 5-HT immunolabeled fibers were significantly higher in the males, and the percentage of VMNvl HuC/D-ir neurons containing ER was significantly higher in the females. The percentage of HuC/D-ir cells contacted by 5-HT fibers was significantly higher in the males, compared with the females, but there was no sex difference in the proportion of those cells receiving contacts that were ER-ir. Neonatal administration of estradiol but not genistein masculinized 5-HT content in the adult female VMNvl, but the percentage of HuC/D-ir cells colabeled with ER was not significantly affected by treatment. A similar, but not statistically significant, pattern was observed in the ARC. These findings suggest that the development of serotonergic inputs to the male VMNvl is orchestrated by neonatal estradiol exposure. The hormone-dependent organization of these 5-HT projection patterns may be an important developmental mechanism accounting for sex-specific behaviors in adulthood.

	Introduction

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THE VENTROMEDIAL NUCLEUS of the hypothalamus (VMN) comprises diverse cell groups that are implicated in multiple behavioral and neuroendocrine systems. The ventrolateral division of the VMN (VMNvl) is critically involved in the regulation of the lordosis display, a reflexive behavior indicative of female sexual receptivity in rodents (1). Lordosis is facilitated and inhibited by both endocrine and neurochemical systems. In females, the steroid hormones 17β-estradiol (E2) and progesterone play an essential role in facilitating the display of this behavior, but neurotransmitters, including serotonin (5-HT) and -aminobutyric acid (GABA) also influence lordosis (2, 3, 4). By interacting with steroid hormones and each other (5, 6, 7), 5-HT and GABA can act to either facilitate or suppress lordosis.

In contrast to females, males rarely display lordosis behavior, even when administered large central or peripheral doses of E2 (8, 9). However, lordosis behavior can be induced in males after lesions to key areas within the forebrain and lower brain stem, suggesting that the lordosis response is actively suppressed in males through an inhibitory neurochemical mechanism. This suppression appears to be provided largely by two distinct inhibitory pathways. In the first, serotonergic projections originating from the dorsal raphe nucleus in the brain stem (DRN) to forebrain nuclei, including the VMN, inhibit lordosis in males (10, 11, 12). GABA may also be working through this pathway to influence lordosis in both males and females (13). A separate inhibitory pathway, projecting through the lateral septum to the midbrain central gray has also been shown to suppress lordosis in males (11, 14, 15, 16, 17, 18), but it is not yet known which neurotransmitters may be acting within this system to inhibit lordosis. Previous work has demonstrated that neither pathway is sufficient on its own to completely suppress lordosis in males (11), so both appear to be necessary and working in concert to exert their effects.

Serotonergic afferents from the DRN project directly to the VMN (19, 20) as well as the lateral septum (21). However, it is not known whether there are sex differences in the densities of 5-HT and/or GABAergic projections to the VMN or in the proportion of those fibers that make contact with hormone-responsive VMNvl neurons. We hypothesized that there would be increased serotonergic and GABAergic innervation of the VMNvl in males, compared with females, providing a tonic inhibitory influence to this critical behavioral nucleus. We further predicted that many of these fibers would make contact with neurons containing the -isoform of estrogen receptor (ER)-. The present studies investigated these possibilities by quantifying sex differences in the densities of 5-HT- and glutamic acid decarboxylase (GAD)-65-containing fibers projecting to ER-expressing VMNvl neurons in adult males and females.

For these experiments, ER, the cytoplasmic neuronal marker HuC/D and either 5-HT or GAD65 were labeled and quantified in the male and female VMNvl using triple-label immunofluorescent techniques. ER is abundant in the VMN but is restricted to the cell nucleus, so labeling for HuC/D was used to visualize the cell bodies of VMNvl neurons. We also examined 5-HT and GAD65 fiber density in the arcuate nucleus (ARC) and the dorsomedial nucleus of the hypothalamus (DMN). These regions were selected as control areas because of their proximate location to the VMN and their respective functional roles in the regulation of sexual behavior. The ARC is thought to play a role in the negative feedback regulation of gonadotropin secretion (22, 23) and therefore may have an indirect role in the coordination of sexual behavior. In contrast, the DMN is thought to be responsible for integrating autonomic, endocrine, and behavioral responses unrelated to reproduction, including sleep, feeding, and emotional stress (24, 25, 26). Accordingly, ER is abundant in the ARC but absent in the DMN (27, 28). To eliminate any possible influence of circulating gonadal hormones, animals in this first experiment were gonadectomized in adulthood.

We also sought to determine whether the serotonergic and GABAergic innervation of the VMNvl could be affected by the manipulation of the neonatal hormone environment. It is well established that neonatal estrogens, aromatized from testicular androgens, are required for the proper masculinization of the male rodent hypothalamus (29, 30). Blocking neonatal estrogens, either through castration or by the manipulation of aromatization or ERs, prevents the proper masculinization of the hypothalamus. Similarly, administration of aromatizable androgens or estrogens to females during this time period permanently masculinizes the hypothalamus, rendering them infertile and incapable of generating a lordosis response (31, 32, 33, 34). The number of neurons sending axons from the lateral septum to the midbrain central gray has previously been shown to be sexually dimorphic and influenced by steroid hormones neonatally (35, 36), suggesting that the neurotransmitter systems regulating sexual behavior are organized by estrogens during the neonatal period. We therefore hypothesized that exposure to E2 or the estrogen-like compound genistein (GEN) during the neonatal period could alter the density of serotonergic projections in the VMNvl. GEN is an isoflavone phytoestrogen found at high concentrations in soy-based foods. We previously demonstrated that neonatal exposure to GEN can alter the normal sexual differentiation of the anteroventral periventricular nucleus of the hypothalamus (AVPV) (37, 38).

For this second set of experiments, animals were treated neonatally with E2 or GEN and raised to adulthood. The neonatally treated animals were then gonadectomized (GNX) as adults and sequentially administered estradiol benzoate (EB) and progesterone. This treatment paradigm has previously been shown to effectively restore lordosis behavior in GNX females but not females masculinized by neonatal exposure to estradiol or in males (1, 31, 32, 33, 34, 39, 40). We therefore hypothesized that any observed sex differences in GABAergic or serotonergic projections would be disrupted by neonatal exposure to E2 and/or GEN but not adult hormone administration.

	Materials and Methods

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Animal care
Animals were housed in a humidity- and temperature-controlled room with a 12-h light, 12-h dark cycle (lights on from 0700 to 1900 h) at 23 C and 50% humidity at the CIIT Centers for Health Research. All animals were fed a soy-free, phytoestrogen-free diet ad libitum (Diet 5K96; Purina, Richmond, IN) on arrival and for the duration of the experiment. For the first experiment, adult male (n = 10) and female (n = 10) Sprague Dawley rats (Charles River, Raleigh, NC) arrived GNX on postnatal day (PND) 75 and were killed 8 d later by transcardial perfusion without further treatments. For the second experiment, pups were born in-house, cross-fostered at birth, and treated with E2, GEN, or a control vehicle during the neonatal period, as previously described (37, 38). Animals were then reared to adulthood, gonadectomized, given adult hormone replacement as described below, and killed by transcardial perfusion. Animal care and maintenance were conducted in accordance with the applicable portions of the Animal Welfare Act and the U.S. Department of Health and Human Services Guide for the Care and Use of Laboratory Animals. All experimental procedures involving animals were approved by the CIIT Centers for Health Research Institutional Animal Care and Use Committee.

Treatment preparation and administration
Tissue for the second experiment was derived from animals used in a previously published experiment (38). As described therein, on arrival the pregnant dams (n = 5; Charles River) were acclimated to the vivarium and fed a soy-free, phytoestrogen-free diet ad libitum. On the day of birth (PND 0), each pup was cross-fostered and randomly assigned to one of three treatment groups: sesame oil (n = 8 males, 8 females), E2 (n = 8 females), or GEN (n = 8 males, 8 females). E2 and GEN were dissolved in 10% EtOH in sesame oil. GEN was sonicated with low heat to facilitate complete dissolution. Once prepared, the solutions were kept at 4 C and wrapped in foil to prevent photodegradation. Treatments were administered by sc injection (0.05 ml) every 12 h for 48 h, beginning on the morning of PND 1 for a total of four injections. Care was taken to ensure that the injection site did not leak before returning the pup to the dam. Each injection contained 50 µg E2 (Steraloids, Newport, RI), 250 µg GEN (Indofine Chemical Co. Inc., Somerville, NJ), or sesame oil (OIL; Sigma, St. Louis, MO). The dose of 50 µg E2 every 12 h (100 µg/d) has previously been shown by our laboratory to effectively masculinize other sexually dimorphic nuclei in the hypothalamus (37). We also previously reported that the dose of 250 µg GEN every 12 h is sufficient to alter a number of sex differences in the AVPV (37, 38). The pups were weaned on PND 21 and maintained on the soy-free diet until adulthood.

On PND 85 the animals were gonadectomized under isoflurane anesthesia. To provide adequate time for endogenous hormone levels to diminish, the animals were allowed 11 d to recover. The GNX males and females were then sc injected with 10 µg EB dissolved in sesame oil at 0900 h, followed 48 h later (PND 98) by a sc injection of 500 µg progesterone dissolved in the same vehicle. This treatment paradigm has been consistently shown to stimulate lordosis behavior in females but not males or females masculinized by the neonatal administration of estrogens (1, 31, 32, 33, 34, 39, 40). Animals were killed by transcardial perfusion 6–8 h after progesterone administration.

Tissue collection and preparation
For both experiments, the animals were deeply anesthetized with sodium pentobarbital (Henry Schein, Inc., Port Washington, NY) and transcardially perfused with ice-cold 0.9% NaCl followed by 400 ml 4% paraformaldehyde in 0.05 M sodium phosphate buffer (pH 7.4). Brains were removed and postfixed in 20% sucrose in 4% paraformaldehyde for 3–4 h and then cryoprotected overnight at 4 C in 0.02 M potassium PBS (KPBS) containing 20% sucrose. Blocks of tissue containing the caudal hypothalamus, including the VMN, ARC, and DMN, were isolated, rapidly frozen in crushed dry ice, and stored at –80 C. Brains were sliced into 30-µm coronal sections on a frozen sliding microtome, and the sections from each brain were divided into two alternating series. Free-floating sections were then stored in an antifreeze solution (20% glycerol, 30% ethylene glycol in KPBS) at –20 C until immunohistochemical processing. For all animals, one set of tissue was used for immunolabeling of 5-HT, ER, and HuC/D. For the first experiment, the second anatomically matched series of sections was used for immunolabeling of GAD65, ER, and HuC/D.

5-HT immunohistochemistry
The sections were rinsed in a series of six 5-min washes of KPBS buffer with mild shaking before each step of immunohistochemical processing. The sections were preincubated overnight at 4 C in 2% normal donkey serum (Colorado Serum Co., Denver, CO) and 0.3% Triton X-100 in KPBS (LKPBS). They were then incubated for 72 h with mild shaking at 4 C in a cocktail of primary antibodies directed against 5-HT (goat antiserotonin, 1:6000; ImmunoStar, Hudson, WI), ER (rabbit polyclonal anti-ER C1355, 1:20,000; Upstate Biotechnology, Waltham, MA), and HuC/D (mouse anti-HuC/HuD 16A11, 1:500; Invitrogen, Carlsbad, CA) in LKPBS. HuC/D is a protein expressed in neuronal cytoplasm and for the purposes of our experiments was used to visualize the borders of neuronal cell bodies (41). After incubation and rinsing, the sections were then placed for 2 h in a cocktail of donkey secondary antibodies generated against goat, rabbit, and mouse IgGs conjugated to fluorophores excited at 488, 568, and 647 nm, respectively (Alexa-Fluor donkey antigoat 488, Alexa-Fluor donkey antirabbit 568, Alexa-Fluor donkey antimouse 647; Invitrogen). All secondary antibodies were diluted 1:200 in LKPBS. After secondary antibody incubation, sections were counterstained with Hoechst 33258 (Invitrogen) rinsed, mounted onto slides (Superfrost, Fisher, Pittsburgh, PA), and coverslipped using a standard glycerol mountant.

Specificity of the 5-HT staining was confirmed by incubating the primary antibody with the 5-HT antigen before using it for immunofluorescent staining. For this procedure, a set of hypothalamic sections containing the VMNvl were labeled using either the 5-HT antibody alone (1:6000) or antibody that had been preincubated with 10 µg/ml serotonin/BSA conjugate (ImmunoStar) at room temperature for 1 h. Upon visualization with a fluorescently tagged secondary antibody, bright serotonergic fibers were observed in control tissue sections. However, this staining was completely eliminated when the diluted antibody was preincubated with the antigen.

GAD65 Immunohistochemistry
Anatomically matched sections from the second series of tissue obtained in experiment 1 were used for immunolabeling of GAD65. As above, the sections were rinsed in a series of six 5-min washes of KPBS buffer with mild shaking before each step of immunohistochemical processing. The sections were first preincubated overnight at 4 C in LKPBS. The sections were then incubated in a cocktail of primary antibodies directed against GAD65 [ms anti-GAD65, 1:2000, a generous gift of Ake Lernmark (42)] and ER for 72 h with mild shaking at 4 C. After incubation and rinsing, the sections were placed in a cocktail of secondary antibodies generated against mouse and rabbit IgGs conjugated to fluorophores excited at 488 and 568 nm, respectively (Alexa-Fluor goat antimouse 488, Alexa-Fluor goat antirabbit 568; Invitrogen). The secondary antibodies were diluted 1:200 in LKPBS. After washing, the tissue was incubated in a biotinylated primary antibody directed against HuC/D (ms biotinylated anti-HuC/HuD; Invitrogen; 1:200 in LKPBS) for 72 h with mild shaking at 4 C. The sections were then rinsed and incubated with a streptavidin-conjugated fluorophore excited at 647 nm (Streptavidin Alexa Fluor 647 conjugate; Invitrogen) at 1:200 in LKPBS for 2 h with shaking. Finally, the sections were rinsed, counterstained with Hoechts 33258 (Invitrogen), mounted onto slides (Superfrost; Fisher), and coverslipped using glycerol mountant as described above.

Confocal microscopy and image analysis
5-HT, HuC/D, and ER triple-immunofluorescent label was visualized in the VMNvl and ARC in all animals from both experiments and in the DMN of the GNX adults in the first experiment. GAD65, HuC/D, and ER immunostaining was visualized in the VMNvl of all animals in both experiments. Anatomical identification of each section was made by comparing the counterstained sections to a brain atlas (43). Because ER, 5-HT, and GAD65 immunostaining was observed to be consistent and evenly distributed throughout the VMNvl, ARC, and DMN, a single, representative midlevel section from each region was selected for each animal. Immunolabeling of all triple-labeled sections was visualized using a Zeiss LSM 510 Meta confocal microscope (housed at the CIIT Centers for Health Research) that was fitted with a x63 oil-corrected objective lens. For imaging of GAD65-immunoreactive (ir) puncta, an optical magnification factor of x2 was used.

Anatomically matched, midlevel sections from each nucleus were selected for imaging of 5-HT or GAD65, ER, and HuC/D. For each scan, a set of serial image planes (z-step distance = 0.75 µm) was collected through the entire thickness of the section. Individual images were acquired sequentially for light emitted from each fluorophore and parsed into separate stacks of images for analysis using the Image J software package [National Institutes of Health (NIH), Bethesda, MD]. To control for variations in tissue thickness that would result in unequal numbers of image planes, substacks of consecutive image planes that excluded the rostral and caudal edges of the tissue sections were created for each set of scans. Substacks consisted of 25 image planes for 5-HT labeling in the VMNvl of the GNX adult and neonatally treated rats and 15 image planes for 5-HT labeling in the ARC and DMN of all sampled animals. Substacks of 15 planes were also used to analyze GAD65 labeling. Only data from sections with consistent staining throughout the entire thickness were included in the analysis.

Quantification of 5-HT and GAD65 fiber density
GAD65-ir has been shown previously to be localized to the axon terminals of GABAergic neurons (44, 45). Accordingly, GAD65 label in the present studies was observed to be in punctate structures that surrounded VMNvl cell bodies (see Fig. 2). In contrast, 5-HT-ir was localized to extended lengths of fibers rather than discrete puncta (see Fig. 1). To quantify the densities of 5-HT- and GAD65-containing structures, substacks of images depicting fluorescence emitted from the AlexaFluor-488 fluorophore were isolated for analysis. All analyses were performed using the Image J software package (NIH). Using methods consistent with those described previously (46), individual images contained within each substack were binarized to a threshold selected to optimize visualization of the signal and to minimize the inclusion of background fluorescence. Single pixels were removed to further reduce background influence. In the case of 5-HT labeling, fibers were then skeletonized to a thickness of one pixel to compensate for differences in individual fiber thickness. The number of bright pixels in each plane of the substack was then quantified using the Image J Voxel Counter plug-in (NIH). The voxel counts were then averaged within the substack to obtain a single measure that was used as a quantitative representation of the average density of labeling within the volume sampled.

View larger version (47K): [in this window] [in a new window]   FIG. 2. GAD65 labeling did not significantly differ between the sexes (A). GAD65 labeling was observed to be within punctuate structures, suggestive of axon terminals, surrounding the HuC/D-labeled neuronal cell bodies of the VMNvl in both the male (B) and female (C). Many of these labeled structures appeared to be in direct contact (white arrow) with the HuC/D-labeled neurons. Scale bar (red), 20 µm.

View larger version (81K): [in this window] [in a new window]   FIG. 1. Confocal images (single optical plane) depicting the higher 5-HT fiber content (green) in the VMNvl of the males (A), compared with the females (B). 5-HT labeling was readily observed within extended lengths of fibers, many of which appeared to be in close apposition to HuC/D-stained (blue) neuronal cell bodies (white arrow). ER labeling (red) was exclusively observed within the nuclei of HuC/D-labeled neuronal cell bodies neurons and was abundant in both the male (A) and female (B) VMNvl. 5-HT immunoreactivity was significantly higher in the males than females in both the VMNvl (C) and ARC (D) but not the DMN (E). *, P 0.03. Scale bar (red), 20 µm.

Quantification of HuC/D and ER immunoreactivity in the VMNvl

Quantification of 5-HT fibers apposing HuC/D- and ER-immunolabeled cells in the VMNvl
This analysis was similar to that used to quantify HuC/D and ER immunolabeling, but the criteria for selecting cells were necessarily more selective. Because 5-HT-ir fibers appeared to run along the periphery of the cell body, only cells for which the entire soma could be visualized within the image substack were quantified. As described above, substacks of 40 image planes were generated from the tissue triple labeled for 5-HT, ER, and HuC/D, and only substacks depicting consistent staining in every image plane were used for quantification (n = 7 females, 6 males). Image planes depicting the 5-HT, ER, and HuC/D immunolabel were merged using the Image J software package for analysis. The percentages of HuC/D-ir cells that were immunopositive or immunonegative for ER and that did or did not have apparent sites of contact with 5-HT-containing fibers were quantified using a modified stereological approach that used the optical fractionator probe of the StereoInvestigator software package (MBF Bioscience), as described in the previous section (47). Section thickness, dissector height, and counting frame dimensions were identical with those used to count HuC/D and ER immunoreactivity. Cells were counted by advancing through the individual image planes within each confocal substack and marking the cells of interest within each dissector. For each counting frame, separate markers were used to quantify cells that were ER immunopositive and received apparent fiber contacts (ER+/5-HT+), cells that were ER immunonegative and 5-HT+ (ER–/5-HT+), cells that were ER+ and received no apparent contacts from labeled fibers (ER+/5-HT–), and cells that were ER– and 5-HT– (ER–/5-HT). In most cases in which cells were determined to receive apparent contacts from 5-HT-ir fibers, extended lengths of labeled fiber were clearly observed to be in close apposition with HuC/D-labeled cell bodies, often appearing to be partially wrapped around the soma (see Fig. 1). For cells in which appositions were less easily identifiable or in which nearby fibers appeared to course in the z-plane of the substack, three dimensional reconstructions of the cell and its surrounding fibers were generated using the Volume Viewer plug-in for the Image J image analysis program (NIH). Reconstructions were then manually rotated to confirm that apparent contacts were maintained irrespective of the angle from which the cell was viewed.

Data analysis
All measures were analyzed separately for the GNX adults from experiment 1 and the neonatally treated adults from experiment 2. For experiment 1, differences between groups were assessed using a t test (separate variance, Bonferroni adjusted). For the neonatally treated animals in experiment 2, because there was no E2 treatment group among the males, it was inappropriate to analyze the data with treatment and sex as factors. Therefore, each treatment group was assigned a group number and the differences between groups were assessed using a two-tailed, one-way ANOVA followed by Fisher’s post hoc tests. All effects were considered to be statistically significant when P 0.05.

	Results

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5-HT fiber density was sexually dimorphic in the adult VMNvl and ARC but not the DMN
Nine males and nine females with consistent staining throughout the entire thickness of the VMNvl were selected for the analysis of 5-HT-ir. 5-HT-ir was localized to extended lengths of fibers, many of which appeared to be in close apposition to the HuC/D-labeled neuronal cell bodies (Fig. 1). Average 5-HT fiber density within the VMNvl was significantly higher in the males, compared with the females (P 0.0001). 5-HT fiber density was also significantly higher in the ARC of the males (n = 10), compared with the females (n = 6, P 0.03, Fig. 1). 5-HT-ir was appreciable in the DMN but no significant sex difference was observed (P = 0.55, Fig. 1).

GAD65-ir in the adult VMNvl did not differ between the sexes
Six males and seven females with consistent staining throughout the entire thickness of the VMNvl were selected for the analysis of GAD65-ir (Fig. 2). In contrast to the 5-HT-ir, GAD65-ir was observed in discrete punctuate structures surrounding the VMNvl cell bodes (Fig. 2, white arrow). This is consistent with previous reports of GAD65-ir in the brain, which demonstrated that GAD65 is localized to axon terminals (44, 45). GAD65 immunoreactive structures were observed in the VMNvl of both sexes, but there was no significant sex difference in the density of GAD65 immunolabeling (P = 0.26).

The percentage of HuC/D-ir neurons in the VMNvl that were colabeled for ER was higher in females than males
HuC/D and ER immunostaining was readily observed in the VMNvl (Figs. 1 and 2). All cells immunoreactive for ER were also immunopositive for HuC/D, and no extranuclear ER labeling was observed. The total number of HuC/D-labeled cells, regardless of ER content, did not statistically differ between the sexes (Fig. 3A). The percentage of HuC/D-ir cells that contained ER labeling was significantly higher in the females than males (P 0.0001, Fig. 3B).

View larger version (8K): [in this window] [in a new window]   FIG. 3. The total number of HuC/D-ir cells within the sampled region of the VMNvl did not significantly differ between the sexes (A). The percentage of HuC/D-ir VMNvl neurons that were also immunopositive for ER was significantly higher in the females, compared with the males (B). *, P 0.0001.

The percentage of HuC/D-immunolabeled cells apposed by 5-HT-ir fibers was higher in males than females, but the percentage of 5-HT contacts on ER-immunolabeled cells did not differ between the sexes.

View larger version (7K): [in this window] [in a new window]   FIG. 4. The percentage of 5-HT-ir fibers in contact with HuC/D-ir cells, regardless of ER-ir, was significantly higher in the males than females (A). The percentage of these contacted HuC/D-ir cells that were colabeled for ER did not differ between the sexes (B). *, P 0.01.

View larger version (22K): [in this window] [in a new window]   FIG. 5. Postnatal E2 treatment significantly increased 5-HT density in the female VMNvl, compared with the control females (A). A trend for decreased 5-HT fiber content in the control females, compared with the control males, was observed, but this effect did not reach statistical significance. The percentage of VMNvl neurons immunopositive for both ER and HuC/D was significantly higher in the control females, compared with the control males (B). Double labeling was unaffected by postnatal GEN treatment in either sex or postnatal E2 treatment in the females. *, P 0.04; , P 0.08.

Neonatal treatment did not significantly affect the colabeling of VMNvl ER and HuC/D

No significant effect of neonatal treatment with either E2 or GEN on 5-HT was observed in the ARC
In the ARC, the two-tailed ANOVA revealed no significant differences among the different groups [Fig. 6; F(4, 23) = 2.337, P 0.086], but the pattern of 5-HT average fiber density in the oil-treated control males and females was consistent with what was observed in the first experiment. The females neonatally treated with E2, but not GEN, had 5-HT levels more consistent with the control males than the control females, but this trend failed to reach statistical significance (P = 0.065). Finally, 5-HT average fiber density was slightly decreased in the males neonatally treated with GEN, compared with the control males, but this effect was also not statistically significant (P = 0.16). A t test was then performed to compare the oil-treated males and females. In this comparison, there was a significant sex difference in 5-HT fiber density in the ARC of the neonatally treated animals (Fig. 6; P 0.045).

View larger version (19K): [in this window] [in a new window]   FIG. 6. In the second experiment, the pattern of 5-HT fiber content in the ARC was similar to that observed in the animals from the first experiment. No main effect of treatment was found, but a trend for increased 5-HT fiber content in the ARC of the E2-treated females, compared with the oil-treated control females, was observed. When directly compared in a t test, 5-HT fiber content in the ARC was significantly lower in the control females, compared with the control males. *, P 0.05; , P 0.065.

	Discussion

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A sex difference was also observed in the percentage of VMNvl cells expressing ER. Whereas overall numbers of VMNvl neurons, as identified by the HuC/D label, were equivalent between males and females, these cells were significantly more likely to be ER immunopositive in the female. This finding is consistent with previous reports demonstrating that both estrogen binding (49) and ER immunoreactivity in the VMH (50) are increased in female rats, compared with males.

The observed sex differences in 5-HT fiber density and ER expression in the VMNvl is congruous with this site’s functional role in mediating lordosis. The VMNvl has long been accepted as a critical component of the neuroendocrine system regulating the lordosis display because lesions to this nucleus severely impair or eliminate this sexually receptive posture (1, 51, 52, 53, 54). Moreover, the facilitatory effects of estrogens on the lordosis response appear to be exerted exclusively through the -form of the ER because female ER knockout mice do not display lordosis, even after steroid hormone administration (55). Therefore, the observation in the present study that the percentage of HuC/D and ER double label was higher in the female rat VMNvl is consistent with the hypothesized activational role for ER in the elicitation of lordosis.

Whereas circulating estrogens play an essential role in stimulating lordosis in females (1, 56, 57, 58, 59), males rarely display lordosis behavior, even after EB administration (8). However, a lordosis response can be elicited in males after lesions to key areas within the forebrain and lower brain stem, indicating an active neurochemical suppression of lordosis in males. Our findings support the hypothesis that increased serotonergic input to the VMNvl in males may be one mechanism by which lordosis behavior is suppressed in males. This possibility is supported by previous work, describing two distinct inhibitory circuits that contribute to lordosis inhibition in males (10, 11, 12, 14, 15, 16, 17, 18). The primary inhibitory pathway appears to originate from the DRN, the principal site of origin of 5-HT projections (10, 11, 60). Therefore, there is a strong possibility that the sex difference in 5-HT-ir fibers observed in the present studies reflect a dimorphic serotonergic projection from the DRN. However, neuroanatomical tract tracing studies are required to definitively determine whether the 5-HT-ir fibers observed in the VMNvl originate from the DRN and whether the sex difference in fiber density reflects a difference in the number of 5-HT cells in the DRN or represents increased axonal branching, leading to the formation of denser terminal fields in the VMNvl of males. Increased 5-HT fiber density in the male VMNvl also suggests that 5-HT release is enhanced in the VMNvl of males, compared with females. This possibility is supported by the report that extracellular VMN 5-HT levels are significantly higher in intact males, compared with estrous females (61).

Although the density of 5-HT-ir fibers was appreciably greater in the male VMNvl, almost 40% of cells in the female VMNvl also appeared to receive contacts from 5-HT-containing fibers. Therefore, whereas serotonergic systems likely exert more robust inhibitory effects in the male, 5-HT also likely contributes to lordosis inhibition in females. Consistent with this premise, previous studies have reported that 5-HT release within the VMN varies across the estrous cycle in females and that extracellular VMN 5-HT levels are lowest on estrus, when females are sexually receptive (61, 62). The fluctuation of hypothalamic 5-HT is hypothesized to facilitate the female’s ability to regulate reproductive behavior such that mating occurs under physiological and environmental conditions that maximize reproductive success (4).

The modulation of this inhibitory serotonergic input within the VMNvl may be a mechanism by which estrogens regulate the lordosis response in females, with increasing levels of estrogens decreasing the inhibitory influence of 5-HT. Inputs from 5-HT fibers appeared to contact ER-ir cells in both sexes, suggesting that serotonergic and estrogenic influences converge on individual VMNvl neurons to modulate their activity. This observation supports previous evidence that estrogens interact directly with serotonergic systems in the VMNvl. For example, EB administration has previously been shown to decrease 5-HT-ir in the VMN of ovariectomized females (63), whereas estrogen supplementation increases serotonin reuptake transporter binding in the VMN (64). In addition, 5-HT receptors play a significant role in the intensity of the lordosis response in females, with the 5-HT_1A receptor exerting an inhibitory influence (65), whereas the 5-HT_2A/2C and 5-HT₃ receptor systems are thought to play a facilitatory role (4, 66, 67). Accordingly, EB administration reduces the potency of 5-HT_1A receptor agonists, thereby reducing the inhibitory effect of these compounds on lordosis (68). E2 has also been shown to modulate GABA_A receptor binding in a region-specific manner (69, 70). Functionally, this interaction between estrogens and neurotransmitter systems allows for the generation of lordosis responses that are appropriate for the animal’s sex, hormonal milieu, and environmental circumstances (70, 71, 72, 73).

The percentage of HuC/D-ir cells receiving apparent contacts from 5-HT-ir fibers was significantly greater in males than females. However, of those cells, the proportion that were also ER-ir was equivalent in both sexes and was similar to the percentage of all HuC/D that expressed ER, regardless of 5-HT inputs. The finding that 5-HT fibers did not appear to preferentially target ER-ir cells in either sex suggests that 5-HT may also impact hormone-independent functions mediated by the VMNvl. However, 5-HT-ir fibers were observed to be diffusely distributed, and the majority of the label lay in the intercellular space. This suggests that many contacts between 5-HT-labeled fibers and VMNvl cells were not axosomatic. Rather, serotonergic inputs to VMNvl neurons may be axoaxonal or may contact neuronal dendrites. It is also possible that 5-HT-containing fibers may make contacts with nonneuronal cells, such as astrocyte glia cells. Thus, the possibility remains that serotonergic inputs to other specific cell groups may be sexually dimorphic and influenced by neonatal estrogens. Future studies will be necessary to explore this possibility.

In contrast to 5-HT, we observed no appreciable sex difference in GAD65 content within the VMNvl. This finding was unexpected because previous studies suggest that the interaction of GABA and 5-HT is crucial for the precise control of lordosis behavior in males and females (5, 6, 13), and sex dimorphisms in hypothalamic GABAergic systems are well documented (41, 74, 75). For example, sexually dimorphic projections from the principal subdivision of the bed nucleus of the stria terminalis to the AVPV, a pathway implicated in sex-specific gonadotropin responses, provide GABAergic inputs in males that are 10 times denser than those seen in females (41). In the case of the VMNvl, however, it appears that sex-specific GABAergic effects may result primarily from sex differences in GABA receptor activity rather than differences in the densities of GABAergic inputs. Sex differences in the expression of GABA receptors and the genes for their subunits within the VMNvl have been reported previously (72), and functional studies support the premise that GABA receptor activity is involved in modulating lordosis. For example, GABA_B receptor agonists have been shown to decrease the lordosis response in females (76, 77), and a recent study demonstrated that administration of the GABA_B receptor agonist baclofen also decreased lordosis behavior in males with septal lesions (13). In contrast, the role of GABA_A receptors is less clear. Although activation of GABA_A receptors is generally thought to facilitate lordosis (68, 78), it is speculated that the activation of VMNvl GABA_A receptors may act to suppress lordosis (6, 79). Finally, prior studies have also demonstrated that after gonadectomy, GABA turnover in the VMNvl diminishes substantially in females but only transiently in males (80). Therefore, it is possible that in the present study, an appreciable sex difference in GAD content may have been attenuated by gonadectomy or that more sensitive histological methods are needed to detect subtle sex differences in GAD staining.

It is well established that neonatal administration of E2, or synthetic estrogens such as EB, functionally masculinizes the rodent brain, rendering females incapable of generating lordosis responses in adulthood (1, 81, 82, 83). One mechanism by which this might occur is through increased inhibitory serotonergic input to the VMNvl. Indeed, in the present studies, neonatal administration of E2 masculinized adult female 5-HT-ir in the VMNvl, although sex differences in 5-HT content did not reach statistical significance in the neonatally treated animals. Surprisingly, sex differences in ER expression within the VMNvl were only marginally affected by neonatal E2 administration, despite a previously demonstrated role for estrogens and/or aromatizable androgens in organizing this dimorphism (84, 85). The lack of significant neonatal E2 effects seen in the present studies may reflect the dose and timing of E2 administration used. We previously demonstrated that the neonatal E2 treatments used in the present studies are sufficient to masculinize nuclear volume and cell phenotypes in the AVPV (37). However, varying thresholds for sensitivity to neonatal hormone treatments may exist between brain regions and cell types, such that exposures are differentially effective in organizing specific dimorphisms within the brain. It is also possible that hormone replacement with EB and progesterone in adulthood, regardless of sex, may have minimized the sex difference. Previous studies have shown that within the VMN, dendritic spine density, dendrite length, and the number of terminal branches are significantly increased by the administration of estrogens, particularly in the VMNvl (86, 87, 88). Thus, in the second experiment, the administration of EB may have suppressed 5-HT fiber density to some degree in the males, thereby minimizing the sex difference seen in the first experiment. An alternative possibility is that additional components besides developmental estradiol exposure are required for the complete differentiation of this phenotype. For example, sex differences in VMNvl expression may result from a combination of hormonal and genetic contributions (89, 90).

We also observed a significant sex difference in the density of 5-HT-containing fibers in the ARC. In contrast to the VMNvl, the ARC is not considered to be critically involved in the display of lordosis. Thus, the functional significance of the observed sex differences in 5-HT inputs to the ARC is likely not to be related to a role in sexual behavior. However, the ARC contributes substantially to the regulation of other sexually dimorphic neuroendocrine systems. For example, neurons in the ARC have been shown to both influence GnRH neurons (22, 91) and directly regulate prolactin secretion from the anterior pituitary (92, 93). In rats, prolactin secretory responses are sexually dimorphic and influenced by both circulating estrogens and serotonergic activity (94, 95). Thus, in the present studies, the observed sex differences in 5-HT fiber density within the ARC may represent a sexually differentiated component of reproductive neuroendocrine systems not directly related to reproductive behavior. In contrast, in the DMN, a region not thought to contribute to lordosis behavior and devoid of ER, 5-HT-ir fibers were relatively sparse and no significant sex difference in 5-HT-ir was observed.

A number of sexually dimorphic cellular phenotypes and synaptic systems in the ARC appear to be organized during the early neonatal period (23, 96, 97, 98). However, whereas the effect of neonatal treatment on 5-HT fiber density in the ARC was pronounced in the present studies, it did not reach statistical significance under the two-tailed ANOVA. As with the VMNvl, dose and timing of neonatal E2 treatments may not have been optimal to completely disrupt the sexual differentiation of the ARC, and the adult hormone treatments may also have attenuated measurable sex differences. Moreover, some ARC dimorphisms appear to be organized by androgens around the time of puberty (99); therefore, it is possible that the neonatal treatment paradigm used was inadequate to fully impact the organization of 5-HT projections to the ARC. However, when only the control males and females from the second experiment were compared, significant sex differences in the ARC were detected. Thus, the inclusion of multiple groups in the initial two-tailed ANOVA analysis may have effectively minimized the statistical power required to detect this significance.

No effect of neonatal treatment with the endocrine-disrupting compound, GEN, on 5-HT density or ER immunoreactivity was observed in either the VMNvl or the ARC. The ineffectiveness of neonatal GEN treatments may be related to the dose and/or timing of administration. Previous studies have demonstrated that injection of 1 mg GEN daily for the first 5 d of life suppresses lordosis (100). However, a single injection of 1 mg GEN on the fourth day after birth is insufficient to impair lordosis (83). Collectively, these data suggest that the critical period for an effect of GEN on lordosis lies within the first few days of life. In the present studies, our treatments were confined to that critical period, but our dose was half of that used in the previous studies. Therefore, the dose may have been too low to generate an effect. We previously reported that this dose of GEN is sufficient to disrupt sexual differentiation within the AVPV (37). Thus, as was seen for E2, it appears that sexually dimorphic cell groups may exhibit differential sensitivity to the disruptive influences of neonatal GEN exposures. Identifying the doses of GEN that disrupt development of sexually dimorphic brain regions is critical for the generation of appropriate human risk assessment models. The dose of 500 µg/d used in the present study is lower than those that have been used in previous studies (83, 100) but closer to what is typically considered physiological for human infants (101, 102, 103).

It remains unclear which ER subtype mediates the neonatal masculinization of serotonergic inputs to the VMNvl. Although a definitive role for ER in the lordosis response has been well established, recent evidence suggests that ERβ may be essential for the proper development and maintenance of the lordosis inhibiting circuits in males. It was recently demonstrated that male mice lacking ERβ can be stimulated by the sequential administration of E2 and progesterone to engage in a low level of lordosis (104), a behavioral response not seen in wild-type animals. This observation suggests that the lordosis inhibiting circuit may not be properly masculinized in these animals and that ERβ may play a critical role in the development and function of this circuit. In mice, the raphe nuclei, particularly the DRN, contain appreciable levels of ERβ. More than 90% of these ERβ-ir neurons are colocalized with tryptophan hydroxylase, the rate-limiting enzyme for 5-HT synthesis (105). In contrast, coexpression of ER and tryptophan hydroxylase is far less abundant in this region. Therefore, it appears that ERβ may play the dominant role both during the development of serotonergic lordosis-inhibiting circuits and in the functional activity of these circuits during adulthood.

Our finding that GEN did not impact sexual differentiation of serotonergic projections to the VMNvl is consistent with previous data suggesting that GEN is primarily active through ERβ, whereas ER is required for the estrogen-dependent masculinization of this nucleus in rats (106). However, this finding initially appears to be inconsistent with the aforementioned studies in ER and ERβ knockout mice that demonstrate an important role for ERβ in the defeminization of sexually dimorphic circuits (104, 107). With respect to serotonergic systems, species differences in the ER expression patterns of 5-HT cells may contribute to this discrepancy because the raphe nuclei of the adult rat appears to contain very little ER and little to no ERβ, regardless of sex (108). It is also possible that organizational hormones may act primarily on cells in the target nucleus rather than the nucleus of origin to generate sex differences in axonal projections. Recent studies using organotypic cocultures to investigate the mechanisms by which neonatal estrogens masculinize sexually dimorphic projections from the principal subdivision of the bed nucleus of the stria terminalis to the AVPV suggest that organizational hormones may act primarily on cells in the AVPV by altering the release of chemotropic factors that impact axon guidance and neuronal targeting (109). Thus, hormone-mediated mechanisms within VMNvl target cells themselves may act similarly to preferentially guide DRN projections to the VMNvl in males.

The present experiments demonstrate pronounced sex differences in 5-HT, but not GAD65, fiber density in the VMNvl. Our findings further demonstrate that neonatal E2 administration masculinizes this neurochemical input in females, suggesting that the lordosis-inhibiting pathways are organized by hormone exposure during the classic, neonatal critical period of brain sexual differentiation. These findings expand on previous models demonstrating that estrogen-induced plasticity in VMN synaptic organization mediates behavioral responses (110) by revealing a permanent, neonatally organized circuit that is resistant to circulating estrogens in adulthood. By using triple-label immunofluorescent techniques, we have also demonstrated anatomically that estrogenic and serotonergic signals converge on individual cells in the VMNvl in a sexually dimorphic pattern. Our findings shed new light on the mechanisms by which hormones and neurotransmitters interact to control sex-specific behavioral and physiological functions.

	Acknowledgments

 
The authors gratefully acknowledge Victoria Wong for her assistance with the confocal microscopy as well as Tim Shepard, Paul Ross, and the entire animal care staff at the CIIT Centers for Health Research for their outstanding support with animal husbandry and maintenance. We also thank Amy C. Drew (Specialized Neuroscience Research Program at Howard University College of Medicine) for her contributions to the data collection and analysis of 5-HT fiber contacts onto VMNvl cells.

	Footnotes

 
This work was supported by American Chemistry Council Grant EKPLRI601 (to E.K.P.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 18, 2007

Abbreviations: ARC, Arcuate nucleus; AVPV, anteroventral periventricular nucleus of the hypothalamus; DMN, dorsomedial nucleus of the hypothalamus; DRN, dorsal raphe nucleus in the brain stem; E2, 17β-estradiol; EB, estradiol benzoate; ER, estrogen receptor; GABA, -aminobutyric acid; GAD, glutamic acid decarboxylase; GEN, genistein; GNX, gonadectomized; 5-HT, serotonin; ir, immunoreactive; KPBS, potassium PBS; LKPBS, Triton X-100 in KPBS; PND, postnatal day; VMN, ventromedial nucleus; VMNvl, ventromedial nucleus of the hypothalamus.

Received May 18, 2007.

Accepted for publication October 11, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Pfaff D 1999 Drive. Cambridge, MA: MIT Press
2. Rissman EF, Wersinger SR, Fugger HN, Foster TC 1999 Sex with knockout models: behavioral studies of estrogen receptor . Brain Res 835:80–90[CrossRef][Medline]
3. Frye CA 2001 The role of neurosteroids and non-genomic effects of progestins and androgens in mediating sexual receptivity of rodents. Brain Res Brain Res Rev 37:201–222[CrossRef][Medline]
4. Uphouse L 2000 Female gonadal hormones, serotonin, and sexual receptivity. Brain Res Brain Res Rev 33:242–257[CrossRef][Medline]
5. Luine V, Cowell J, Frankfurt M 1991 GABAergic-serotonergic interactions in regulating lordosis. Brain Res 556:171–174[CrossRef][Medline]
6. Guptarak J, Selvamani A, Uphouse L 2004 GABAA-5-HT1A receptor interaction in the mediobasal hypothalamus. Brain Res 1027:144–150[CrossRef][Medline]
7. Luine VN, Wu V, Hoffman CS, Renner KJ 1999 GABAergic regulation of lordosis: influence of gonadal hormones on turnover of GABA and interaction of GABA with 5-HT. Neuroendocrinology 69:438–445[CrossRef][Medline]
8. Yamanouchi K, Aria Y 1976 Heterotypical sexual behavior in male rats: individual difference in lordosis response. Endocrinol Jpn 23:179–182[Medline]
9. Satou M, Yamanouchi K 1999 Effect of direct application of estrogen aimed at lateral septum or dorsal raphe nucleus on lordosis behavior: regional and sexual differences in rats. Neuroendocrinology 69:446–452[CrossRef][Medline]
10. Kakeyama M, Umino A, Nishikawa T, Yamanouchi K 2002 Decrease of serotonin and metabolite in the forebrain and facilitation of lordosis by dorsal raphe nucleus lesions in male rats. Endocr J 49:573–579[CrossRef][Medline]
11. Kakeyama M, Yamanouchi K 1994 Two types of lordosis-inhibiting systems in male rats: dorsal raphe nucleus lesions and septal cuts. Physiol Behav 56:189–192[CrossRef][Medline]
12. Moreines J, Kelton M, Luine VN, Pfaff DW, McEwen BS 1988 Hypothalamic serotonin lesions unmask hormone responsiveness of lordosis behavior in adult male rats. Neuroendocrinology 47:453–458[Medline]
13. Kakeyama M, Yamanouchi K 1996 Inhibitory effect of baclofen on lordosis in female and male rats with dorsal raphe nucleus lesion or septal cut. Neuroendocrinology 63:290–296[Medline]
14. Nance DM, Phelps C, Shryne JE, Gorski RA 1977 Alterations by estrogen and hypothyroidism in the effects of septal lesions on lordosis behavior of male rats. Brain Res Bull 2:49–53[CrossRef][Medline]
15. Gordon JH, Nance DM, Wallis CJ, Gorski RA 1979 Effects of septal lesions and chronic estrogen treatment on dopamine, GABA and lordosis behavior in male rats. Brain Res Bull 4:85–89[CrossRef][Medline]
16. Kondo Y, Shinoda A, Yamanouchi K, Arai Y 1990 Role of septum and preoptic area in regulating masculine and feminine sexual behavior in male rats. Horm Behav 24:421–434[CrossRef][Medline]
17. Tsukahara S, Yamanouchi K 2001 Neurohistological and behavioral evidence for lordosis-inhibiting tract from lateral septum to periaqueductal gray in male rats. J Comp Neurol 431:293–310[CrossRef][Medline]
18. Nance DM, Shryne J, Gorski RA 1974 Septal lesions: effects on lordosis behavior and pattern of gonadotropin release. Horm Behav 5:73–81[CrossRef][Medline]
19. van de Kar LD, Lorens SA 1979 Differential serotonergic innervation of individual hypothalamic nuclei and other forebrain regions by the dorsal and median midbrain raphe nuclei. Brain Res 162:45–54[CrossRef][Medline]
20. Willoughby JO, Blessing WW 1987 Origin of serotonin innervation of the arcuate and ventromedial hypothalamic region. Brain Res 418:170–173[CrossRef][Medline]
21. Azmitia EC, Segal M 1978 An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J Comp Neurol 179:641–667[CrossRef][Medline]
22. Smith JT, Clifton DK, Steiner RA 2006 Regulation of the neuroendocrine reproductive axis by kisspeptin-GPR54 signaling. Reproduction 131:623–630[Abstract/Free Full Text]
23. Leedom L, Lewis C, Garcia-Segura LM, Naftolin F 1994 Regulation of arcuate nucleus synaptology by estrogen. Ann NY Acad Sci 743:61–71[CrossRef][Medline]
24. Horiuchi J, McDowall LM, Dampney RA 2006 Differential control of cardiac and sympathetic vasomotor activity from the dorsomedial hypothalamus. Clin Exp Pharmacol Physiol 33:1265–1268[CrossRef][Medline]
25. DiMicco JA, Samuels BC, Zaretskaia MV, Zaretsky DV 2002 The dorsomedial hypothalamus and the response to stress: part renaissance, part revolution. Pharmacol Biochem Behav 71:469–480[CrossRef][Medline]
26. Bellinger LL, Bernardis LL 2002 The dorsomedial hypothalamic nucleus and its role in ingestive behavior and body weight regulation: lessons learned from lesioning studies. Physiol Behav 76:431–442[CrossRef][Medline]
27. Shughrue PL, Lane MV, Merchenthaler I 1997 Comparative distribution of estrogen receptor and β mRNA in the rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
28. Simerly RB, Chang C, Muramatsu M, Swanson LW 1990 Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp Neurol 294:76–95[CrossRef][Medline]
29. Whales RE, Gladue BA, Olsen KL 1986 Lordotic behavior in male rats: genetic and hormonal regulation of sexual differentiation. Horm Behav 20:73–82[CrossRef][Medline]
30. Gorski RA 1985 Sexual dimorphisms of the brain. J Anim Sci 61(Suppl 3):38–61
31. Sodersten P 1978 Effects of anti-oestrogen treatment of neonatal male rats on lordosis behaviour and mounting behaviour in the adult. J Endocrinol 76:241–249[Abstract/Free Full Text]
32. Feder HH, Goy RW 1983 Effects of neonatal estrogen treatment of female guinea pigs on mounting behavior in adulthood. Horm Behav 17:284–291[CrossRef][Medline]
33. Hendricks SE 1972 Androgen modification of behavioral responsiveness to estrogen in the male rat. Horm Behav 3:47–54[Medline]
34. Crossley DA, Swanson HH 1968 Modification of sexual behaviour of hamsters by neonatal administration of testosterone propionate. J Endocrinol 41:13–14[Medline]
35. Tsukahara S, Ezawa N, Yamanouchi K 2003 Neonatal estrogen decreases neural density of the septum-midbrain central gray connection underlying the lordosis-inhibiting system in female rats. Neuroendocrinology 78:226–233[CrossRef][Medline]
36. Tsukahara S, Yamanouchi K 2002 Sex difference in septal neurons projecting axons to midbrain central gray in rats: a combined double retrograde tracing and ER-immunohistochemical study. Endocrinology 143:285–294[Abstract/Free Full Text]
37. Patisaul HB, Fortino AE, Polston EK 2006 Neonatal genistein or bisphenol-A exposure alters sexual differentiation of the AVPV. Neurotoxicol Teratol 28:111–118[CrossRef][Medline]
38. Patisaul HB, Fortino AE, Polston EK 2007 Differential disruption of nuclear volume and neuronal phenotype in the preoptic area by neonatal exposure to genistein and bisphenol-A. Neurotoxicology 28:1–12[CrossRef][Medline]
39. Patisaul HB, Dindo M, Whitten PL, Young LJ 2001 Soy isoflavone supplements antagonize reproductive behavior and ER- and ERβ-dependent gene expression in the brain. Endocrinology 142:2946–2952[Abstract/Free Full Text]
40. Moreines J, McEwen B, Pfaff D 1986 Sex differences in response to discrete estradiol injections. Horm Behav 20:445–451[CrossRef][Medline]
41. Polston EK, Gu G, Simerly RB 2004 Neurons in the principal nucleus of the bed nuclei of the stria terminalis provide a sexually dimorphic GABAergic input to the anteroventral periventricular nucleus of the hypothalamus. Neuroscience 123:793–803[CrossRef][Medline]
42. Hampe CS, Lundgren P, Daniels TL, Hammerle LP, Marcovina SM, Lernmark A 2001 A novel monoclonal antibody specific for the N-terminal end of GAD65. J Neuroimmunol 113:63–71[CrossRef][Medline]
43. Swanson L 1992 Brain maps: structure of the rat brain. Amsterdam: Elsevier
44. Esclapez M, Tillakaratne NJ, Kaufman DL, Tobin AJ, Houser CR 1994 Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J Neurosci 14:1834–1855[Abstract]
45. Oertel WH, Mugnaini E 1984 Immunocytochemical studies of GABAergic neurons in rat basal ganglia and their relations to other neuronal systems. Neurosci Lett 47:233–238[CrossRef][Medline]
46. Polston EK, Simerly RB 2003 Sex-specific patterns of galanin, cholecystokinin, and substance P expression in neurons of the principal bed nucleus of the stria terminalis are differentially reflected within three efferent preoptic pathways in the juvenile rat. J Comp Neurol 465:551–559[CrossRef][Medline]
47. Glaser JR, Glaser EM 2000 Stereology, morphometry, and mapping: the whole is greater than the sum of its parts. J Chem Neuroanat 20:115–126[CrossRef][Medline]
48. Matsumoto A, Arai Y 1986 Male-female difference in synaptic organization of the ventromedial nucleus of the hypothalamus in the rat. Neuroendocrinology 42:232–236[Medline]
49. Brown TJ, Hochberg RB, Zielinski JE, MacLusky NJ 1988 Regional sex differences in cell nuclear estrogen-binding capacity in the rat hypothalamus and preoptic area. Endocrinology 123:1761–1770[Abstract/Free Full Text]
50. Yokosuka M, Okamura H, Hayashi S 1997 Postnatal development and sex difference in neurons containing estrogen receptor- immunoreactivity in the preoptic brain, the diencephalon, and the amygdala in the rat. J Comp Neurol 389:81–93[CrossRef][Medline]
51. Pfaff DW, Sakuma Y 1979 Deficit in the lordosis reflex of female rats caused by lesions in the ventromedial nucleus of the hypothalamus. J Physiol 288:203–210[Abstract/Free Full Text]
52. Mathews D, Greene SB, Hollingsworth EM 1983 VMN lesion deficits in lordosis: partial reversal with pergolide mesylate. Physiol Behav 31:745–748[CrossRef][Medline]
53. Gibson BM, Floody OR 1998 Time course of VMN lesion effects on lordosis and ultrasound production in hamsters. Behav Neurosci 112:1236–1246[CrossRef][Medline]
54. Nance DM, Christensen LW, Shryne JE, Gorski RA 1977 Modifications in gonadotropin control and reproductive behavior in the female rat by hypothalamic and preoptic lesions. Brain Res Bull 2:307–312[CrossRef][Medline]
55. Ogawa S, Eng V, Taylor J, Lubahn DB, Korach KS, Pfaff DW 1998 Roles of estrogen receptor- gene expression in reproduction-related behaviors in female mice. Endocrinology 139:5070–5081[Abstract/Free Full Text]
56. Pfaff DW, Sakuma Y 1979 Facilitation of the lordosis reflex of female rats from the ventromedial nucleus of the hypothalamus. J Physiol 288:189–202[Abstract/Free Full Text]
57. Lisk RD 1969 Progesterone: biphasic effects on the lordosis response in adult or neonatally gonadectomized rats. Neuroendocrinology 5:149–160[Medline]
58. Davidson JM, Bloch GJ 1969 Neuroendocrine aspects of male reproduction. Biol Reprod 1(Suppl 1):67–92
59. Komisaruk BR, Diakow C 1973 Lordosis reflex intensity in rats in relation to the estrous cycle, ovariectomy, estrogen administration and mating behavior. Endocrinology 93:548–557[Abstract/Free Full Text]
60. Kakeyama M, Yamanouchi K 1992 Lordosis in male rats: the facilitatory effect of mesencephalic dorsal raphe nucleus lesion. Physiol Behav 51:181–184[CrossRef][Medline]
61. Gundlah C, Simon LD, Auerbach SB 1998 Differences in hypothalamic serotonin between estrous phases and gender: an in vivo microdialysis study. Brain Res 785:91–96[CrossRef][Medline]
62. Luine VN 1993 Serotonin, catecholamines and metabolites in discrete brain areas in relation to lordotic responding on proestrus. Neuroendocrinology 57:946–954[Medline]
63. Lu H, Yuri K, Ito T, Yoshimoto K, Kawata M 1998 The effects of oestrogen and progesterone on serotonin and its metabolite in the lateral septum, medial preoptic area and ventromedial hypothalamic nucleus of female rats. J Neuroendocrinol 10:919–926[CrossRef][Medline]
64. Krajnak K, Rosewell KL, Duncan MJ, Wise PM 2003 Aging, estradiol and time of day differentially affect serotonin transporter binding in the central nervous system of female rats. Brain Res 990:87–94[CrossRef][Medline]
65. Uphouse L, Montanez S, Richards-Hill R, Caldarola-Pastuszka M, Droge M 1991 Effects of the 5-HT1A agonist, 8-OH-DPAT, on sexual behaviors of the proestrous rat. Pharmacol Biochem Behav 39:635–640[CrossRef][Medline]
66. Wolf A, Caldarola-Pastuszka M, Uphouse L 1998 Facilitation of female rat lordosis behavior by hypothalamic infusion of 5-HT(2A/2C) receptor agonists. Brain Res 779:84–95[CrossRef][Medline]
67. Maswood N, Caldarola-Pastuszka M, Uphouse L 1998 Functional integration among 5-hydroxytryptamine receptor families in the control of female rat sexual behavior. Brain Res 802:98–103[CrossRef][Medline]
68. Jackson A, Uphouse L 1998 Dose-dependent effects of estradiol benzoate on 5-HT1A receptor agonist action. Brain Res 796:299–302[CrossRef][Medline]
69. O’Connor LH, Nock B, McEwen BS 1988 Regional specificity of gamma-aminobutyric acid receptor regulation by estradiol. Neuroendocrinology 47:473–481[Medline]
70. Maggi A, Perez J 1984 Progesterone and estrogens in rat brain: modulation of GABA (-aminobutyric acid) receptor activity. Eur J Pharmacol 103:165–168[CrossRef][Medline]
71. Frankfurt J, McKittrick C, Mendelson S, McEwen B 1994 Effect of 5,7-dihydroxytryptamine, ovariectomy and gonadal steroids on serotonin receptor binding in rat brain. Neuroendocrinology 59:245–250[Medline]
72. Clark AS, Myers M, Robinson S, Chang P, Henderson LP 1998 Hormone-dependent regulation of GABAA receptor gamma subunit mRNAs in sexually dimorphic regions of the rat brain. Proc Biol Sci 265:1853–1859[Abstract/Free Full Text]
73. Herbison AE, Fenelon VS 1995 Estrogen regulation of GABAA receptor subunit mRNA expression in preoptic area and bed nucleus of the stria terminalis of female rat brain. J Neurosci 15:2328–2337[Abstract]
74. Davis AM, Grattan DR, Selmanoff M, McCarthy MM 1996 Sex differences in glutamic acid decarboxylase mRNA in neonatal rat brain: implications for sexual differentiation. Horm Behav 30:538–552[CrossRef][Medline]
75. Searles RV, Yoo MJ, He JR, Shen WB, Selmanoff M 2000 Sex differences in GABA turnover and glutamic acid decarboxylase [GAD(65) and GAD(67)] mRNA in the rat hypothalamus. Brain Res 878:11–19[CrossRef][Medline]
76. Agmo A, Soria P, Paredes R 1989 GABAergic drugs and lordosis behavior in the female rat. Horm Behav 23:368–380[CrossRef][Medline]
77. Kow LM, Harlan RE, Shivers BD, Pfaff DW 1985 Inhibition of the lordosis reflex in rats by intrahypothalamic infusion of neural excitatory agents: evidence that the hypothalamus contains separate inhibitory and facilitatory elements. Brain Res 341:26–34[CrossRef][Medline]
78. McCarthy MM, Malik KF, Feder HH 1990 Increased GABAergic transmission in medial hypothalamus facilitates lordosis but has the opposite effect in preoptic area. Brain Res 507:40–44[CrossRef][Medline]
79. Canteras NS, Simerly RB, Swanson LW 1994 Organization of projections from the ventromedial nucleus of the hypothalamus: a Phaseolus vulgaris-leucoagglutinin study in the rat. J Comp Neurol 348:41–79[CrossRef][Medline]
80. Yoo MJ, Searles RV, He JR, Shen WB, Grattan DR, Selmanoff M 2000 Castration rapidly decreases hypothalamic -aminobutyric acidergic neuronal activity in both male and female rats. Brain Res 878:1–10[CrossRef][Medline]
81. Gorski RA 1963 Modification of ovulatory mechanisms by postnatal administration of estrogen to the rat. Am J Physiol 205:842–844[Abstract/Free Full Text]
82. Gerall AA 1967 Effects of early postnatal androgen and estrogen injections on the estrous activity cycles and mating behavior of rats. Anat Rec 157:97–104[CrossRef][Medline]
83. Kouki T, Okamoto M, Wada S, Kishitake M, Yamanouchi K 2005 Suppressive effect of neonatal treatment with a phytoestrogen, coumestrol, on lordosis and estrous cycle in female rats. Brain Res Bull 64:449–454[CrossRef][Medline]
84. Kuhnemann S, Brown TJ, Hochberg RB, MacLusky NJ 1995 Sexual differentiation of estrogen receptor concentrations in the rat brain: effects of neonatal testosterone exposure. Brain Res 691:229–234[CrossRef][Medline]
85. Bakker J, Pool CW, Sonnemans M, van Leeuwen FW, Slob AK 1997 Quantitative estimation of estrogen and androgen receptor-immunoreactive cells in the forebrain of neonatally estrogen-deprived male rats. Neuroscience 77:911–919[CrossRef][Medline]
86. Madeira MD, Ferreira-Silva L, Paula-Barbosa MM 2001 Influence of sex and estrus cycle on the sexual dimorphisms of the hypothalamic ventromedial nucleus: stereological evaluation and Golgi study. J Comp Neurol 432:329–345[CrossRef][Medline]
87. Calizo LH, Flanagan-Cato LM 2000 Estrogen selectively regulates spine density within the dendritic arbor of rat ventromedial hypothalamic neurons. J Neurosci 20:1589–1596[Abstract/Free Full Text]
88. Calizo LH, Flanagan-Cato LM 2002 Estrogen-induced dendritic spine elimination on female rat ventromedial hypothalamic neurons that project to the periaqueductal gray. J Comp Neurol 447:234–248[CrossRef][Medline]
89. Becker JB, Arnold AP, Berkley KJ, Blaustein JD, Eckel LA, Hampson E, Herman JP, Marts S, Sadee W, Steiner M, Taylor J, Young E 2005 Strategies and methods for research on sex differences in brain and behavior. Endocrinology 146:1650–1673[CrossRef][Medline]
90. Arnold AP, Xu J, Grisham W, Chen X, Kim YH, Itoh Y 2004 Minireview: sex chromosomes and brain sexual differentiation. Endocrinology 145:1057–1062[Abstract/Free Full Text]
91. Horvath TL, Garcia-Segura LM, Naftolin F 1997 Control of gonadotropin feedback: the possible role of estrogen-induced hypothalamic synaptic plasticity. Gynecol Endocrinol 11:139–143[Medline]
92. Demaria JE, Nagy GM, Lerant AA, Fekete MI, Levenson CW, Freeman ME 2000 Dopamine transporters participate in the physiological regulation of prolactin. Endocrinology 141:366–374[Abstract/Free Full Text]
93. Bjorklund A, Moore RY, Nobin A, Stenevi U 1973 The organization of tubero-hypophyseal and reticulo-infundibular catecholamine neuron systems in the rat brain. Brain Res 51:171–191[CrossRef][Medline]
94. Hou Y, Yang SP, Voogt JL 2003 Changes in estrogen receptor- expression in hypothalamic dopaminergic neurons during proestrous prolactin surge. Endocrine 20:131–138[CrossRef][Medline]
95. Van de Kar LD, Rittenhouse PA, Li Q, Levy AD 1996 Serotonergic regulation of renin and prolactin secretion. Behav Brain Res 73:203–208[Medline]
96. Smith JT, Popa SM, Clifton DK, Hoffman GE, Steiner RA 2006 Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J Neurosci 26:6687–6694[Abstract/Free Full Text]
97. Kauffman AS, Gottsch ML, Roa J, Byquist AC, Crown A, Clifton DK, Hoffman GE, Steiner RA, Tena-Sempere M 2007 Sexual differentiation of Kiss1 gene expression in the brain of the rat. Endocrinology 148:1774–1783[Abstract/Free Full Text]
98. Matsumoto A, Arai Y 1981 Effect of androgen on sexual differentiation of synaptic organization in the hypothalamic arcuate nucleus: an ontogenetic study. Neuroendocrinology 33:166–169[Medline]
99. Ciofi P, Lapirot OC, Tramu G 2007 An androgen-dependent sexual dimorphism visible at puberty in the rat hypothalamus. Neuroscience 146:630–642[CrossRef][Medline]
100. Kouki T, Kishitake M, Okamoto M, Oosuka I, Takebe M, Yamanouchi K 2003 Effects of neonatal treatment with phytoestrogens, genistein and daidzein, on sex difference in female rat brain function: estrous cycle and lordosis. Horm Behav 44:140–145[CrossRef][Medline]
101. Setchell KD 2000 Absorption and metabolism of soy isoflavones-from food to dietary supplements and adults to infants. J Nutr 130:654S–655S[Free Full Text]
102. Setchell KD, Zimmer-Nechemias L, Cai J, Heubi JE 1998 Isoflavone content of infant formulas and the metabolic fate of these phytoestrogens in early life. Am J Clin Nutr 68:1453S
103. Setchell KDR, Zimmer-Nechemias L, Cai J, Heubi JE 1997 Exposure of infants to phyto-oestrogens from soy-based infant formula. Lancet 350:23–27[CrossRef][Medline]
104. Kudwa AE, Bodo C, Gustafsson JA, Rissman EF 2005 A previously uncharacterized role for estrogen receptor β: defeminization of male brain and behavior. Proc Natl Acad Sci USA 102:4608–4612[Abstract/Free Full Text]
105. Nomura M, Akama KT, Alves SE, Korach KS, Gustafsson JA, Pfaff DW, Ogawa S 2005 Differential distribution of estrogen receptor (ER)- and ER-β in the midbrain raphe nuclei and periaqueductal gray in male mouse: Predominant role of ER-β in midbrain serotonergic systems. Neuroscience 130:445–456[CrossRef][Medline]
106. Patisaul HB, Melby M, Whitten PL, Young LJ 2002 Genistein affects ERβ- but not ER-dependent gene expression in the hypothalamus. Endocrinology 143:2189–2197[Abstract/Free Full Text]
107. Kudwa AE, Michopoulos V, Gatewood JD, Rissman EF 2006 Roles of estrogen receptors and β in differentiation of mouse sexual behavior. Neuroscience 138:921–928[CrossRef][Medline]
108. Sheng Z, Kawano J, Yanai A, Fujinaga R, Tanaka M, Watanabe Y, Shinoda K 2004 Expression of estrogen receptors (, β) and androgen receptor in serotonin neurons of the rat and mouse dorsal raphe nuclei; sex and species differences. Neurosci Res 49:185–196[CrossRef][Medline]
109. Ibanez MA, Gu G, Simerly RB 2001 Target-dependent sexual differentiation of a limbic-hypothalamic neural pathway. J Neurosci 21:5652–5659[Abstract/Free Full Text]
110. Flanagan-Cato LM, Calizo LH, Daniels D 2001 The synaptic organization of VMH neurons that mediate the effects of estrogen on sexual behavior. Horm Behav 40:178–182[CrossRef][Medline]

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