Two-Step Actions of Testicular Androgens in the Organization of a Male-Specific Neural Pathway from the Medial Preoptic Area to the Ventral Tegmental Area for Modulating Sexually Motivated Behavior.

The medial preoptic area (MPOA) is a sexually dimorphic region of the brain that regulates social behaviors. The sexually dimorphic nucleus (SDN) of the MPOA has been studied to understand sexual dimorphism, although the anatomy and physiology of the SDN is not fully understood. Here, we characterized SDN neurons that contribute to sexual dimorphism and investigated the mechanisms underlying the emergence of such neurons and their roles in social behaviors. A target-specific neuroanatomical study using transgenic mice expressing Cre recombinase under the control of Calb1, a gene expressed abundantly in the SDN, revealed that SDN neurons are divided into two subpopulations, GABA neurons projecting to the ventral tegmental area (VTA), where they link to the dopamine system (CalbVTA neurons), and GABA neurons that extend axons in the MPOA or project to neighboring regions (CalbnonVTA neurons). CalbVTA neurons were abundant in males, but were scarce or absent in females. There was no difference in the number of CalbnonVTA neurons between sexes. Additionally, we found that emergence of CalbVTA neurons requires two testicular androgen actions that occur first in the postnatal period and second in the peripubertal period. Chemogenetic analyses of CalbVTA neurons indicated a role in modulating sexual motivation in males. Knockdown of Calb1 in the MPOA reduced the intromission required for males to complete copulation. These findings provide strong evidence that a male-specific neural pathway from the MPOA to the VTA is organized by the two-step actions of testicular androgens for the modulation of sexually motivated behavior.SIGNIFICANCE STATEMENT The MPOA is a sexually dimorphic region of the brain that regulates social behaviors, although its sexual dimorphism is not fully understood. Here, we describe a population of MPOA neurons that contribute to the sexual dimorphism. These neurons only exist in masculinized brains, and they project their axons to the ventral tegmental area, where they link to the dopamine system. Emergence of such neurons requires two testicular androgen actions that occur first in the postnatal period and second in the peripubertal period. These MPOA neurons endow masculinized brains with a neural pathway from the MPOA to the ventral tegmental area and modulate sexually motivated behavior in males.

In ovo o,p'-DDT exposure induces malformation of reproductive organs and alters the expression of genes controlling sexual differentiation in Japanese quail embryo.

In ovo exposure to o,p'-dichloro-diphenyl-trichloroethane (o,p'-DDT) impairs reproduction by inducing malformation of the reproductive organs in birds, although the mechanism remains unclear. Here, we examined the effects of o,p'-DDT on the development of the reproductive organs, the expression of genes controlling sexual differentiation, and the plasma concentrations of testosterone and estradiol in Japanese quail embryos. o,p'-DDT-containing sesame oil was injected into the yolk sac on Embryonic Day (E) 3 at a dose of 500, 2,000, or 8,000 µg per egg. On E15, the reproductive organs were observed; the gonads and Müllerian ducts (MDs) were sampled to measure the mRNA of steroidogenic enzymes, sex steroid receptors, anti-Müllerian hormone (AMH), and AMH receptor 2 (AMHR2); blood samples were collected to assay plasma testosterone and estradiol levels; and the gonads were used for histological analysis. o,p'-DDT dose-dependently increased the prevalence of hypertrophic MDs in females and residual MDs in males. In female MDs, o,p'-DDT dose-dependently decreased estrogen receptor (ER) α, ERß, and AMHR2 mRNA expression. o,p'-DDT dose-dependently induced left-biased asymmetry of testis size, and ovary-like tissue was found in the left testis after exposure to 8,000 µg per egg o,p'-DDT, although asymmetric gene expression did not occur. o,p'-DDT did not affect ovarian tissue but did decrease 17α-hydroxylase/C17-20 lyase mRNA expression and dose-dependently increased ERß mRNA expression. o,p'-DDT decreased plasma testosterone concentrations in females. These findings suggest that o,p'-DDT induces hypertrophy of the MDs and ovarian tissue formation in the left testis. Abnormal MD development may be linked to altered gene expression for sensing estrogens and AMH signals.

Sexually dimorphic expression of sexual differentiation genes in the internal genital organs of Japanese quail embryos.

Japanese quail (Coturnix japonica) is an avian model used to evaluate the reproductive and developmental toxicity of chemicals. The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail, NIES-L, which may be a better model because of its highly inbred characteristics. To understand sexual differentiation of the reproductive organs and the value of using NIES-L quails for avian toxicity assessment, we profiled estradiol and androgen plasma levels by enzyme-linked immunosorbent assay; the mRNA levels of estrogen receptor-α (ERα), ERß, and androgen receptor (AR) in the gonads, Müllerian ducts, Wolffian ducts; and the mRNA levels of steroidogenic enzymes, cholesterol side chain cleavage enzyme (P450scc), 17α-hydroxylase/C17-20 lyase (P45017α, lyase), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and aromatase (P450arom), anti-Müllerian hormone (AMH), and AMH receptor type 2 (AMHR2) in the gonads of NIES-L Japanese quails on embryonic days 9, 12, and 15 using a real-time quantitative PCR method. The plasma estradiol concentration was higher in females than males on these embryonic days, but no sex difference was found in the plasma androgens. The mRNA levels of all examined steroidogenic enzymes were significantly higher in female than male embryos. In particular, the P450arom mRNA levels showed a striking sex difference: P450arom was expressed in female but not male gonads. In contrast, the AMH and AMHR2 mRNA levels in the gonads were higher in males than females. The ERα, ERß, and AR mRNA levels increased in the left female gonad and peaked on embryonic day 15, but not in the left and right male gonads; therefore, there was a female-biased sex difference. The ERα, ERß, and AR mRNA levels in the left Müllerian duct, but not in the right Müllerian duct, of females increased and peaked on embryonic day 15, which resulted in asymmetric mRNA levels. The Wolffian ducts expressed ERα, ERß, and AR in both sexes, and no sex difference or asymmetry of mRNA levels was found. The information obtained from this study helps elucidate the molecular endocrinological basis of sexual dimorphism formation of reproductive organs and clarify the value of NIES-L quails for toxicity assessment.

Perinatal Exposure to Diesel Exhaust-Origin Secondary Organic Aerosol Induces Autism-Like Behavior in Rats.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social communication, poor social interactions, and repetitive behaviors. We aimed to examine autism-like behaviors and related gene expressions in rats exposed to diesel exhaust (DE)-origin secondary organic aerosol (DE-SOA) perinatally. Sprague-Dawley pregnant rats were exposed to clean air (control), DE, and DE-SOA in the exposure chamber from gestational day 14 to postnatal day 21. Behavioral phenotypes of ASD were investigated in 10~13-week-old offspring using a three-chambered social behavior test, social dominance tube test, and marble burying test. Prefrontal cortex was collected to examine molecular analyses including neurological and immunological markers and glutamate concentration, using RT-PCR and ELISA methods. DE-SOA-exposed male and female rats showed poor sociability and social novelty preference, socially dominant behavior, and increased repetitive behavior. Serotonin receptor (5-HT(5B)) and brain-derived neurotrophic factor (BDNF) mRNAs were downregulated whereas interleukin 1 ß (IL-ß) and heme oxygenase 1 (HO-1) mRNAs were upregulated in the prefrontal cortex of male and female rats exposed to DE-SOA. Glutamate concentration was also increased significantly in DE-SOA-exposed male and female rats. Our results indicate that perinatal exposure to DE-SOA may induce autism-like behavior by modulating molecules such as neurological and immunological markers in rats.

Pubertal activation of estrogen receptor α in the medial amygdala is essential for the full expression of male social behavior in mice.

Testosterone plays a central role in the facilitation of male-type social behaviors, such as sexual and aggressive behaviors, and the development of their neural bases in male mice. The action of testosterone via estrogen receptor (ER) α, after being aromatized to estradiol, has been suggested to be crucial for the full expression of these behaviors. We previously reported that silencing of ERα in adult male mice with the use of a virally mediated RNAi method in the medial preoptic area (MPOA) greatly reduced sexual behaviors without affecting aggressive behaviors whereas that in the medial amygdala (MeA) had no effect on either behavior. It is well accepted that testosterone stimulation during the pubertal period is necessary for the full expression of male-type social behaviors. However, it is still not known whether, and in which brain region, ERα is involved in this developmental effect of testosterone. In this study, we knocked down ERα in the MeA or MPOA in gonadally intact male mice at the age of 21 d and examined its effects on the sexual and aggressive behaviors later in adulthood. We found that the prepubertal knockdown of ERα in the MeA reduced both sexual and aggressive behaviors whereas that in the MPOA reduced only sexual, but not aggressive, behavior. Furthermore, the number of MeA neurons was reduced by prepubertal knockdown of ERα. These results indicate that ERα activation in the MeA during the pubertal period is crucial for male mice to fully express their male-type social behaviors in adulthood.

Strain differences in intermale aggression and possible factors regulating increased aggression in Japanese quail.

The National Institute for Environmental Studies (NIES) of Japan established a strain of Japanese quail (Coturnix japonica) known as NIES-L by rotation breeding in a closed colony for over 35years; accordingly, the strain has highly inbred-like characteristics. Another strain called NIES-Brn has been maintained by randomized breeding in a closed colony to produce outbred-like characteristics. The current study aimed to characterize intermale aggressive behaviors in both strains and to identify possible factors regulating higher aggression in the hypothalamus, such as sex hormone and neuropeptide expression. Both strains displayed a common set of intermale aggressive behaviors that included pecking, grabbing, mounting, and cloacal contact behavior, although NIES-Brn quail showed significantly more grabbing, mounting, and cloacal contact behavior than did NIES-L quail. We examined sex hormone levels in the blood and diencephalon in both strains. Testosterone concentrations were significantly higher in the blood and diencephalon of NIES-Brn quail compared to NIES-L quail. We next examined gene expression in the hypothalamus of both strains using an Agilent gene expression microarray and real-time RT-PCR and found that gene expression of mesotocin (an oxytocin homologue) was significantly higher in the hypothalamus of NIES-Brn quail compared to NIES-L quail. Immunohistochemistry of the hypothalamus revealed that numbers of large cells (cell area>500µm2) expressing mesotocin were significantly higher in the NIES-Brn strain compared to the NIES-L strain. Taken together, our findings suggest that higher testosterone and mesotocin levels in the hypothalamus may be responsible for higher aggression in the NIES-Brn quail strain.

Molecular Cloning of Ghrelin and Characteristics of Ghrelin-Producing Cells in the Gastrointestinal Tract of the Common Marmoset (Callithrix jacchus).

Ghrelin was first isolated from human and rat as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In the present study, we determined the ghrelin cDNA sequence of the common marmoset (Callithrix jacchus), a small-bodied New World monkey, and investigated the distribution of ghrelin-producing cells in the gastrointestinal tract and localization profiles with somatostatin-producing cells. The marmoset ghrelin cDNA coding region was 354 base pairs, and showed high homology to that in human, rhesus monkey, and mouse. Marmoset ghrelin consists of 28 amino acids, and the N-terminal region is highly conserved as found in other mammalian species. Marmoset preproghrelin and mature ghrelin have 86.3% and 92.9% homology, respectively, to their human counterparts. Quantitative RT-PCR analysis showed that marmoset ghrelin mRNA is highly expressed in the stomach, but it is not detected in other tissues of the gastrointestinal tract. In addition, a large number of ghrelin mRNA-expressing cells and ghrelin-immunopositive cells were detected in the mucosal layer of the stomach, but not in the myenteric plexus. Moreover, all the ghrelin cells examined in the stomach were observed to be closed-type. Double staining showed that somatostatin-immunopositive cells were not co-localized with ghrelin-producing cells; however, a subset of somatostatin-immunopositive cells is directly adjacent to ghrelin-immunopositive cells. These findings suggest that the distribution of ghrelin cells in marmoset differs from that in rodents, and thus the marmoset may be a more useful model for the translational study of ghrelin in primates. In conclusion, we have clarified the expression and cell distribution of ghrelin in marmoset, which may represent a useful model in translational study.

Role of Environmental Chemical Insult in Neuronal Cell Death and Cytoskeleton Damage.

Environmental influences, such as chemical exposure, have long been considered potential risk factors for neurodegenerative disorders, including neuromuscular diseases. However, no definitive links between environmental chemical exposure and a pathogenic mechanism of neurodegenerative disease has yet been established. In this study, we describe that exposure to arsenic, an environmental pollutant naturally found in drinking water, induces neuronal cell death and alteration of morphology, particularly neurite outgrowth and in the cytoskeleton of neurons. Since progressive cell loss accompanied by the alteration of neuronal structures and cytoskeleton is considered the major pathologic feature of neurodegenerative disorders, arsenic-induced neurotoxicity might contribute to an etiologic mechanism of some neurodegenerative diseases. Further, we discuss the importance of in vitro assay, particularly an embryonic toxicity test, for assessing the neurotoxicity of chemicals, because most of chemicals found in our environment remain to be evaluated regarding their neurotoxicity risk for neurodegenerative diseases.

Ethynylestradiol feminizes gene expression partly in testis developing as ovotestis and disrupts asymmetric Müllerian duct development by eliminating asymmetric gene expression in Japanese quail embryos.

In avian embryos, xenoestrogens induce abnormalities in reproductive organs, particularly the testes and Müllerian ducts (MDs). However, the molecular mechanisms remain poorly understood. We investigated the effects of ethynylestradiol (EE2) exposure on gene expression associated with reproductive organ development in Japanese quail embryos. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the left testis containing ovary-like tissues following EE2 exposure highly expressed the genes for steroidogenic enzymes (P450scc, P45017α, lyase, and 3ß-HSD) and estrogen receptor-ß, compared to the right testis. No asymmetry was found in these gene expression without EE2. EE2 induced hypertrophy in female MDs and suppressed atrophy in male MDs on both sides. RNA sequencing analysis of female MDs showed 1,366 differentially expressed genes between developing left MD and atrophied right MD in the absence of EE2, and these genes were enriched in Gene Ontology terms related to organogenesis, including cell proliferation, migration and differentiation, and angiogenesis. However, EE2 reduced asymmetrically expressed genes to 21. RT-qPCR analysis indicated that genes promoting cell cycle progression and oncogenesis were more highly expressed in the left MD than in the right MD, but EE2 eliminated such asymmetric gene expression by increasing levels on the right side. EE2-exposed males showed overexpression of these genes in both MDs. This study reveals part of the molecular basis of xenoestrogen-induced abnormalities in avian reproductive organs, where EE2 may partly feminize gene expression in the left testis, developing as the ovotestis, and induce bilateral MD malformation by canceling asymmetric gene expression underlying MD development.

Visualisation and characterisation of oestrogen receptor α-positive neurons expressing green fluorescent protein under the control of the oestrogen receptor α promoter.

Oestrogen receptor (ER)α plays important roles in the development and function of various neuronal systems through activation by its ligands, oestrogens. To visualise ERα-positive neurons, we generated transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the ERα promoter. In three independent tg lines, GFP-positive neurons were observed in areas previously reported to express ERα mRNA, including the lateral septum, bed nucleus of the stria terminalis, medial preoptic nucleus (MPO), hypothalamus, and amygdala. In these areas, GFP signals mostly overlapped with ERα immunoreactivity. GFP fluorescence was seen in neurites and cell bodies of neurons. In addition, the network and detailed structure of neurites were visible in dissociated and slice cultures of hypothalamic neurons. We examined the effect of oestrogen deprivation by ovariectomy on the structure of the GFP-positive neurons. The area of ERα-positive cell bodies in the bed nucleus of the stria terminalis and MPO was measured by capturing the GFP signal and was found to be significantly smaller in ovariectomy mice than in control mice. When neurons in the MPO were infected with an adeno-associated virus that expressed small hairpin RNA targeting the ERα gene, an apparent induction of GFP was observed in this area, suggesting a negative feedback mechanism in which ERα controls expression of the ERα gene itself. Thus, the ERα promoter-GFP tg mice will be useful to analyse the development and plastic changes of the structure of ERα-expressing neurons and oestrogen and its receptor-mediated neuronal responses.

Differential mRNA expression of neuroimmune markers in the hippocampus of infant mice following toluene exposure during brain developmental period.

Toluene, a volatile organic compound with a wide range of industrial applications, can exert neurotoxic and immunotoxic effects. However, the effects of toluene exposure on developmental immunotoxicity in the brain have not yet been characterized. To investigate the susceptible window to toluene exposure during development and the effects of fetal and neonatal toluene exposure on the neuroimmune markers, gestational day (GD) 14 pregnant mice, postnatal day (PND) 2 and PND 8 male offspring were exposed to filtered air (control; 0 ppm), or 5 or 50 ppm toluene for 6 h per day for five consecutive days. The neuroimmune markers in the hippocampus of PND 21 were examined using a real-time RT-PCR and immunohistochemical analysis. Mice exposed to 50 ppm toluene on PND 2-6 showed significantly increased levels of nerve growth factor (NGF) and tumor necrosis factor (TNF)-α mRNAs. In contrast, NGF and brain-derived neurotrophic factor (BDNF) and proinflammatory cytokines TNF-α, CCL3, NF-κB, toll-like receptor (TLR)-4, astrocyte marker glial fibrillary acidic protein (GFAP), and microglia marker ionized calcium binding adapter molecule (Iba)-1 mRNAs were increased significantly in mice exposed to 5 ppm toluene on PND 8-12. These results indicate that low-level toluene exposure during the late postnatal period (PND 8-12) might induce neuroinflammatory mediators via TLR4-dependent NF-κB pathway in the hippocampus of PND 21 male mice. Among the three developmental phases, PND 8-12 seems to be most sensitive to toluene exposure. This is the first study to show developmental phase- and dose-specific changes in neuroimmune markers in infant mice following toluene exposure.

Effects of aromatase or estrogen receptor gene deletion on masculinization of the principal nucleus of the bed nucleus of the stria terminalis of mice.

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is a sexually dimorphic nucleus, and the male BNSTp is larger and has more neurons than the female BNSTp. To assess the roles of neuroestrogen synthesized from testicular androgen by brain aromatase in masculinization of the BNSTp, we performed morphometrical analyses of the adult BNSTp in aromatase knockout (ArKO), estrogen receptor-α knockout (αERKO), and estrogen receptor-ß knockout (ßERKO) mice and their respective wild-type littermates. In wild-type littermates, the BNSTp of males had a larger volume and greater numbers of neuronal and glial cells than did that of females. The volume and neuron number of the BNSTp in ArKO and αERKO males and glial cell number of the BNSTp in αERKO males were significantly smaller than those of wild-type male littermates, and they were not significantly different from those in female mice with either gene knockout. In contrast, there was no significant morphological difference in the BNSTp between ßERKO and wild-type mice. Next, we examined the BNSTp of ArKO males subcutaneously injected with estradiol benzoate (EB) on postnatal days 1, 2, and 3 (1.5 µg/day). EB-treated ArKO males had a significantly greater number of BNSTp neurons than did oil-treated ArKO males. The number of BNSTp neurons in EB-treated ArKO males was comparable to that in wild-type males. These findings suggested that masculinization of the BNSTp in mice involves the actions of neuroestrogen that was synthesized by aromatase and that this estrogen mostly binds to ERα during the postnatal period.

Neuronal activation of the sexually dimorphic nucleus of the preoptic area in female and male rats during copulation and its sex differences.

The medial preoptic area, which plays an essential role in the control of sexual behavior in rats, contains a sexually dimorphic nucleus that consists of neurons expressing calbindin-D28 K (Calb) that is referred to as the CALB-SDN. The CALB-SDN is larger and contains more Calb neurons in males than in females. The physiological functions of the CALB-SDN are not fully understood; however, CALB-SDN neurons are activated during sexual behavior in males, suggesting that the male CALB-SDN is involved in regulation of sexual behavior. However, no information exists about the physiological functions of the female CALB-SDN. In the present study, we performed an immunohistochemical analysis of c-Fos, a neuronal activity marker, in the CALB-SDN of female and male rats that had copulated with conspecifics of the opposite sex to determine whether neurons of the female CALB-SDN are activated during copulation and whether the neuronal activity of the CALB-SDN differs between sexes. The numbers of c-Fos-immunoreactive cells with or without Calb-immunoreactivity (c-Fos+/Calb+ and c-Fos+/Calb- cells) were greater in the CALB-SDN of rats that had copulated than in rats that had not copulated in each sex. Although the number of Calb+ cells in the CALB-SDN was smaller in females than in males, the increase in the number of c-Fos+/Calb+ cells in the female CALB-SDN with copulation was comparable to that in the male CALB-SDN with copulation. The increase in the number of c-Fos+/Calb- cells in the CALB-SDN with copulation was more prominent in males than in females. These results suggest that CALB-SDN neurons are activated during copulation in both sexes. The patterns of neuronal activation in the CALB-SDN during copulation may differ between sexes.

The role of central corticotrophin-releasing factor receptor signalling in plasma glucose maintenance through ghrelin secretion in calorie-restricted mice.

Under severe calorie restriction (CR), the ghrelin-growth hormone axis in mice is involved in the maintenance of plasma glucose levels. Ghrelin, a stomach-derived acylated peptide, is up-regulated by the sympathetic nerve in the negative energy status. Central corticotrophin-releasing factor receptor (CRF-R) signalling stimulates the sympathetic tone. The present study aimed to examine the effect of central CRF-R signalling on the maintenance of plasma glucose concentrations in severe calorie-restricted mice with the involvement of ghrelin. Intracerebroventricular injections of urocorin-1 and urocorin-2, which are natural ligands for CRF-R1 and CRF-R2, elevated plasma ghrelin concentrations and ghrelin elevation with an i.c.v. injection of urocorin-1 was cancelled by atenolol (ß1 adrenergic receptor antagonist) administration. We then established a mice model of 60% CR and found that the administration of [d-Lys3]-GHRP-6 (a ghrelin receptor antagonist) in mice under 60% CR reduced the plasma glucose concentration more compared to the vehicle mice. Similarly, the atenolol injection in mice under 60% CR significantly reduced the plasma glucose concentration, which was rescued by the co-administration of ghrelin. An i.c.v. injection of the alpha helical CRH, a non-selective corticotrophin-releasing factor receptor antagonist, in mice under 60% CR significantly reduced the plasma glucose concentration, although the co-administration of α-helical CRH with ghrelin maintained plasma glucose levels. These results suggest that central CRF-R signalling is involved in the maintenance of plasma glucose levels in mice under severe CR via the sympathetic-ghrelin pathway.

Effect of D-cycloserine on spatial learning performance and memory function-related gene expression in mice following toluene exposure.

D-cycloserine (DCS), a partial N-methyl-D-aspartate (NMDA) receptor agonist, is a well-known cognitive enhancer. To investigate the effect of DCS in cognitive function following toluene exposure, eight-week-old male C3H/HeN mice were exposed to filtered air (0 ppm) or 50 ppm toluene for 6 h a day on 5 consecutive days a week for 6 weeks. The day after the day of final exposure, a spatial learning task was performed using a Morris water maze apparatus. During the learning task, some mice were treated with DCS intraperitoneally (20 mg/kg) 30 min before the first trial of the acquisition phase and probe trial. After completion of the spatial learning task, the hippocampus was collected from each mouse to examine memory function-related gene expression using the real-time RT-PCR method. During the acquisition phase, on day 3 and 4, toluene-exposed mice with DCS treatment showed significantly better learning performance than corresponding saline treated groups. Moreover, the toluene-exposed mice with DCS treatment also showed significantly improved memory retention during the probe trial and up-regulation of hippocampal NMDA receptor subunit 2B mRNA expression compared with the saline treated groups. Our findings indicate that a subunit-specific modulation of hippocampal NMDA receptor mRNA expression by DCS contributes to improvement of spatial learning performance in mice following toluene exposure.

Sexually Dimorphic Formation of the Preoptic Area and the Bed Nucleus of the Stria Terminalis by Neuroestrogens.

Testicular androgens during the perinatal period play an important role in the sexual differentiation of the brain of rodents. Testicular androgens transported into the brain act via androgen receptors or are the substrate of aromatase, which synthesizes neuroestrogens that act via estrogen receptors. The latter that occurs in the perinatal period significantly contributes to the sexual differentiation of the brain. The preoptic area (POA) and the bed nucleus of the stria terminalis (BNST) are sexually dimorphic brain regions that are involved in the regulation of sex-specific social behaviors and the reproductive neuroendocrine system. Here, we discuss how neuroestrogens of testicular origin act in the perinatal period to organize the sexually dimorphic structures of the POA and BNST. Accumulating data from rodent studies suggest that neuroestrogens induce the sex differences in glial and immune cells, which play an important role in the sexually dimorphic formation of the dendritic synapse patterning in the POA, and induce the sex differences in the cell number of specific neuronal cell groups in the POA and BNST, which may be established by controlling the number of cells dying by apoptosis or the phenotypic organization of living cells. Testicular androgens in the peripubertal period also contribute to the sexual differentiation of the POA and BNST, and thus their aromatization to estrogens may be unnecessary. Additionally, we discuss the notion that testicular androgens that do not aromatize to estrogens can also induce significant effects on the sexually dimorphic formation of the POA and BNST.

The glutamatergic system in the preoptic area is involved in the retention of maternal behavior in maternally experienced female rats.

Maternally experienced female rats show high maternal behavior performance for a long time after acquisition of maternal experience, although the mechanisms responsible for the retention of maternal behavior are not well understood. The medial preoptic area (MPOA) plays an important role in the onset and maintenance of maternal behavior in female rats. We aimed to determine whether maternal experience affects the glutamatergic system in the MPOA for the retention of maternal behavior in female rats. First, to determine the effects of maternal experience in the postpartum period on dendritic spines, which are the postsynaptic component of excitatory glutamatergic neurotransmission, we examined the number of dendritic spines on MPOA neurons of primiparous mothers that had experienced mothering until weaning (sufficiently experienced mothers) and of primiparous mothers that were separated from their pups on the day of parturition (insufficiently experienced mothers). The number of mushroom spines, but not other types of spine, was significantly greater in the sufficiently experienced mothers compared with that in the insufficiently experienced mothers. Next, to determine the effects of maternal experience in the postpartum period on the expression of ionotropic glutamate receptors, we measured the mRNA levels of AMPA receptor subunits (GluA1-A4) and NMDA receptor subunits (GluN1, GluN2A-2D) in the MPOA of primiparous female rats that were kept with pups until brain sampling. As a result, we found that the mRNA levels of GluA3 and GluN2B were significantly higher in primiparous females on the day of weaning compared with those in primiparous females on the day of parturition. Additionally, we examined the effects of CNQX, an AMPA receptor antagonist, and MK-801, an NMDA receptor antagonist, injected into the MPOA on maternal behavior in maternally experienced primiparous female rats. Maternal behavioral activity was significantly reduced when CNQX or MK-801 was injected into the MPOA. These findings indicate that long-term maternal experience in the postpartum period up-regulates glutamatergic neurotransmission by increasing the number of mushroom spines and glutamate receptor expression, which may be involved in the retention of maternal behavior in maternally experienced female rats.

Actions of Peripubertal Gonadal Steroids in the Formation of Sexually Dimorphic Brain Regions in Mice.

The calbindin-sexually dimorphic nucleus (CALB-SDN) and calbindin-principal nucleus of the bed nucleus of the stria terminalis (CALB-BNSTp) show male-biased sex differences in calbindin neuron number. The ventral part of the BNSTp (BNSTpv) exhibits female-biased sex differences in noncalbindin neuron number. We previously reported that prepubertal gonadectomy disrupts the masculinization of the CALB-SDN and CALB-BNSTp and the feminization of the BNSTpv. This study aimed to determine the action mechanisms of testicular androgens on the masculinization of the CALB-SDN and CALB-BNSTp and whether ovarian estrogens are the hormones that have significant actions in the feminization of the BNSTpv. We performed immunohistochemical analyses of calbindin and NeuN, a neuron marker, in male mice orchidectomized on postnatal day 20 (PD20) and treated with cholesterol, testosterone, estradiol, or dihydrotestosterone during PD20-70, female mice ovariectomized on PD20 and treated with cholesterol or estradiol during PD20-70, and PD70 mice gonadectomized on PD56. Calbindin neurons number in the CALB-SDN and CALB-BNSTp in males treated with testosterone or dihydrotestosterone, but not estradiol, was significantly larger than that in cholesterol-treated males. Noncalbindin neuron number in the BNSTpv in estradiol-treated females was significantly larger than that in cholesterol-treated females. Gonadectomy on PD56 had no significant effect on neuron numbers. Additionally, an immunohistochemical analysis revealed the expression of androgen receptors in the CALB-SDN and CALB-BNSTp of PD30 males and estrogen receptors-α in the BNSTpv of PD30 females. These results suggest that peripubertal testicular androgens act to masculinize the CALB-SDN and CALB-BNSTp without aromatization, and peripubertal ovarian estrogens act to feminize the BNSTpv.

Estrogenic regulation of social behavior and sexually dimorphic brain formation.

It has long been known that the estrogen, 17ß-estradiol (17ß-E), plays a central role for female reproductive physiology and behavior. Numerous studies have established the neurochemical and molecular basis of estrogenic induction of female sexual behavior, i.e., lordosis, in animal models. In addition, 17ß-E also regulates male-type sexual and aggressive behavior. In males, testosterone secreted from the testes is irreversibly aromatized to 17ß-E in the brain. We discuss the contribution of two nuclear receptor isoforms, estrogen receptor (ER)α and ERß to the estrogenic regulation of sexually dimorphic brain formation and sex-typical expression of these social behaviors. Furthermore, 17ß-E is a key player for social behaviors such as social investigation, preference, recognition and memory as well as anxiety-related behaviors in social contexts. Recent studies also demonstrated that not only nuclear receptor-mediated genomic signaling but also membrane receptor-mediated non-genomic actions of 17ß-E may underlie the regulation of these behaviors. Finally, we will discuss how rapidly developing research tools and ideas allow us to investigate estrogenic action by emphasizing behavioral neural networks.

Detection and Characterization of Estrogen Receptor Beta Expression in the Brain with Newly Developed Transgenic Mice.

Two types of nuclear estrogen receptors, ERα and ERß, have been shown to be differentially involved in the regulation of various types of behaviors. Due to a lack of tools for identifying ERß expression, detailed anatomical distribution and neurochemical characteristics of ERß expressing cells and cellular co-expression with ERα remain unclear. We have generated transgenic mice ERß-RFPtg, in which RFP was inserted downstream of ERß BAC promotor. We verified RFP signals as ERß by confirming: (1) high ERß mRNA levels in RFP-expressing cells collected by fluorescence-activated cell sorting; and (2) co-localization of ERß mRNA and RFP proteins in the paraventricular nucleus (PVN). Strong ERß-RFP signals were found in the PVN, medial preoptic area (MPOA), bed nucleus of the stria terminalis, medial amygdala (MeA), and dorsal raphe nucleus (DRN). In the MPOA and MeA, three types of cell populations were identified; those expressing both ERα and ERß, and those expressing exclusively either ERα or ERß. The majority of PVN and DRN cells expressed only ERß-RFP. Further, ERß-RFP positive cells co-expressed oxytocin in the PVN, and tryptophan hydroxylase 2 and progesterone receptors in the DRN. In the MeA, some ERß-RFP positive cells co-expressed oxytocin receptors. These findings collectively suggest that ERß-RFPtg mice can be a powerful tool for future studies on ERß function in the estrogenic regulation of social behaviors.