Kazuhiro Sano

Differential effects of site-specific knockdown of estrogen receptor α in the medial amygdala, medial pre-optic area, and ventromedial nucleus of the hypothalamus on sexual and aggressive behavior of male mice.

Testosterone is known to play an important role in the regulation of male‐type sexual and aggressive behavior. As an aromatised metabolite of testosterone, estradiol‐induced activation of estrogen receptor α (ERα) may be crucial for the induction of these behaviors in male mice. However, the importance of ERα expressed in different nuclei for this facilitatory action of testosterone has not been determined. To investigate this issue, we generated an adeno‐associated virus vector expressing a small hairpin RNA targeting ERα to site‐specifically knockdown ERα expression. We stereotaxically injected either a control or ERα targeting vector into the medial amygdala, medial pre‐optic area (MPOA), or ventromedial nucleus of the hypothalamus (VMN) in gonadally intact male mice. Two weeks after injection, all mice were tested biweekly for sexual and aggressive behavior, alternating between behavior tests each week. We found that suppressing ERα in the MPOA reduced sexual but not aggressive behavior, whereas in the VMN it reduced both behaviors. Knockdown of ERα in the medial amygdala did not alter either behavior. Additionally, it was found that ERα knockdown in the MPOA caused a parallel reduction in the number of neuronal nitric oxide synthase‐expressing cells. Taken together, these results indicate that the testosterone facilitatory action on male sexual behavior requires the expression of ERα in both the MPOA and VMN, whereas the testosterone facilitatory action on aggression requires the expression of ERα in only the VMN.

Activation of the GPR30 Receptor Promotes Lordosis in Female Mice

Background/Aims: Estrogens are important effectors of reproduction and are critical for upregulating female reproductive behavior or lordosis in females. In addition to the importance of transcriptional regulation of genes by 17β-estradiol-bound estrogen receptors (ER), extranuclear signal transduction cascades such as protein kinase A (PKA) are also important in regulating female sexual receptivity. GPR30 (G-protein coupled receptor 30), also known as GPER1, a putative membrane ER (mER), is a G protein-coupled receptor that binds 17β-estradiol with an affinity that is similar to that possessed by the classical nuclear ER and activates both PKA and extracellular-regulated kinase signaling pathways. The high expression of GPR30 in the ventromedial hypothalamus, a region important for lordosis behavior as well as kinase cascades activated by this receptor, led us to hypothesize that GPR30 may regulate lordosis behavior in female rodents. Method: In this study, we investigated the ability of G-1, a selective agonist of GPR30, to regulate lordosis in the female mouse by administering this agent prior to progesterone in an estradiol-progesterone priming paradigm prior to testing with stud males. Results: As expected, 17β-estradiol benzoate (EB), but not sesame oil, increased lordosis behavior in female mice. G-1 also increased lordosis behavior in female mice and decreased the number of rejective responses towards male mice, similar to the effect of EB. The selective GPR30 antagonist G-15 blocked these effects. Conclusion: This study demonstrates that activation of the mER GPR30 stimulates social behavior in a rodent model in a manner similar to EB.

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

Significance Testosterone stimulation during the pubertal period is necessary for the full expression of male-type social behaviors. However, it is not known whether estrogen receptor (ER) α may be involved in pubertal organizational action of testosterone on the regulation of male-type social behaviors. In this study, we showed that the prepubertal knockdown of ERα in the medial amygdala (MeA) of gonadally intact male mice reduced both sexual and aggressive behaviors as well as the number of MeA neurons later in adulthood. These results indicate that not only aromatization but also ERα expression in the MeA during the pubertal period is required for organizational action of testosterone to fully masculinize neural circuitry responsible for the expression of male-type social behaviors. 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.

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.

Effects of Prepubertal or Adult Site-Specific Knockdown of Estrogen Receptor β in the Medial Preoptic Area and Medial Amygdala on Social Behaviors in Male Mice.

Abstract Testosterone, after being converted to estradiol in the brain, acts on estrogen receptors (ERα and ERβ) and controls the expression of male-type social behavior. Previous studies in male mice have revealed that ERα expressed in the medial preoptic area (MPOA) and medial amygdala (MeA) are differently involved in the regulation of sexual and aggressive behaviors by testosterone action at the time of testing in adult and/or on brain masculinization process during pubertal period. However, a role played by ERβ in these brain regions still remains unclear. Here we examined the effects of site-specific knockdown of ERβ (βERKD) in the MPOA and MeA on male social behaviors with the use of adeno-associated viral mediated RNA interference methods in ICR/Jcl mice. Prepubertal βERKD in the MPOA revealed that continuous suppression of ERβ gene expression throughout the pubertal period and adulthood decreased aggressive but not sexual behavior tested as adults. Because βERKD in the MPOA only in adulthood did not affect either sexual or aggressive behaviors, it was concluded that pubertal ERβ in the MPOA might have an essential role for the full expression of aggressive behavior in adulthood. On the other hand, although neither prepubertal nor adult βERKD in the MeA had any effects on sexual and aggressive behavior, βERKD in adulthood disrupted sexual preference of receptive females over nonreceptive females. Collectively, these results suggest that ERβ in the MPOA and MeA are involved in the regulation of male sexual and aggressive behavior in a manner substantially different from that of ERα.

The Role of Estrogen Receptor β in the Dorsal Raphe Nucleus on the Expression of Female Sexual Behavior in C57BL/6J Mice

17β-Estradiol (E2) regulates the expression of female sexual behavior by acting through estrogen receptor (ER) α and β. Previously, we have shown that ERβ knockout female mice maintain high level of lordosis expression on the day after behavioral estrus when wild-type mice show a clear decline of the behavior, suggesting ERβ may be involved in inhibitory regulation of lordosis. However, it is not identified yet in which brain region(s) ERβ may mediate an inhibitory action of E2. In this study, we have focused on the dorsal raphe nucleus (DRN) that expresses ERβ in higher density than ERα. We site specifically knocked down ERβ in the DRN in ovariectomized mice with virally mediated RNA interference method. All mice were tested weekly for a total of 3 weeks for their lordosis expression against a stud male in two consecutive days: day 1 with the hormonal condition mimicking the day of behavioral estrus, and day 2 under the hormonal condition mimicking the day after behavioral estrus. We found that the level of lordosis expression in ERβ knockdown (βERKD) mice was not different from that of control mice on day 1. However, βERKD mice continuously showed elevated levels of lordosis behavior on day 2 tests, whereas control mice showed a clear decline of the behavior on day 2. These results suggest that the expression of ERβ in the DRN may be involved in the inhibitory regulation of sexual behavior on the day after behavioral estrus in cycling female mice.

Roles of estrogen receptor α and β in the regulation of body weight and blood glucose level in male mice

bined anterograde tracing with BDA and immunohistochemistry for MCH and VGLUT2, we finally indicated that bouton-like varicosities of the BMA and ACo fibers apposed to somata and dendrites of the MCH–IR LH neurons were immunoreactive for VGLUT2. These data suggest that the BMA and ACo of the amygdala may exert excitatory influence upon the MCH-containing LH neurons for the regulation of feeding behavior.

Proteomics analysis of changes in proteins in rat sexually dimorphic hypothalamic nucleus during the critical period

Scaffold attachment factor (SAFB) 1 and 2 are implicated in various cellular processes (e.g. chromatin organization), and they are also known as corepressors of estrogen receptor alpha (ER ). Although their mRNAs are expressed ubiquitously, little is known about the regional difference of SAFB proteins in the brain. In this study, we examined localization of SAFB proteins in adult rat brain. SAFB1/2 immunoreactivity (ir) was observed in almost all the region, and particularly intense labeling was seen in the hippocampus, arcuate nucleus of the hypothalamus and medial amygdala, where ER is expressed. In vitro analysis showed that SAFB1-ir was expressed in the nucleus of cultured neurons, astrocytes and oligodendrocytes, whereas SAFB2-ir was detected in both nucleus and cytoplasm of these cells. These results suggest that SAFB1 is broadly coexpressed with SAFB2 in the brain and their interaction with ER is under investigation.

Short- and long-term estrogen depletions produce sex dependent changes in food intake and body weight

P3-f09 The effect of hardness of food on histamine release in rat amygdala Tomoko Ishizuka1, Noritaka Sako2, Tomotaka Murotani3, Mitsuko Shinohara1, Atsushi Yamatodani3, Kiyoshi Ohura1 1 Department of Pharmacology, Osaka Dental University, Osaka, Japan; 2 Department of Oral Physiology, Asahi University School of Dentistry, Gifu, Japan; 3 Division of Health Sciences, Osaka University Graduate School of Medicine, Osaka, Japan