Mariko Nakata

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-β knockout (βERKO) 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 βERKO 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.

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.

Modification of female and male social behaviors in estrogen receptor beta knockout mice by neonatal maternal separation.

Maternal separation (MS) is an animal model mimicking the effects of early life stress on the development of emotional and social behaviors. Recent studies revealed that MS stress increased social anxiety levels in female mice and reduced peri-pubertal aggression in male mice. Estrogen receptor (ER) β plays a pivotal role in the regulation of stress responses and anxiety-related and social behaviors. Behavioral studies using ERβ knockout (βERKO) mice reported increased social investigation and decreased social anxiety in βERKO females, and elevated aggression levels in βERKO males compared to wild-type (WT) mice. In the present study, using βERKO and WT mice, we examined whether ERβ contributes to MS effects on anxiety and social behaviors. βERKO and WT mice were separated from their dam daily (4 h) from postnatal day 1–14 and control groups were left undisturbed. First, MS and ERβ gene deletion individually increased anxiety-related behaviors in the open field test, but only in female mice. Anxiety levels were not further modified in βERKO female mice subjected to MS stress. Second, βERKO female mice showed higher levels of social investigation compared with WT in the social investigation test and long-term social preference test. However, MS greatly reduced social investigation duration and elevated number of stretched approaches in WT and βERKO females in the social investigation test, suggesting elevated levels of social anxiety in both genotypes. Third, peri-pubertal and adult βERKO male mice were more aggressive than WT mice as indicated by heightened aggression duration. On the other hand, MS significantly decreased aggression duration in both genotypes, but only in peri-pubertal male mice. Altogether, these results suggest that βERKO mice are sensitive to the adverse effects of MS stress on subsequent female and male social behaviors, which could then have overrode the ERβ effects on female social anxiety and male aggression.

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.

Analyses of running wheel activity (RWA) in aromatase-knockout (ArKO) mice

It is unclear how our brain predicts reward and punishing outcome on perception of ambiguous stimuli in environment. To investigate relationship between perceptually ambiguous stimuli and reward processing on them, we examined reward-predicting brain activity on high and low coherency random dot motion stimuli by using fMRI. First, we trained subjects to establish a contingency between particular direction of motion stimuli and delivery of reward. Then, we presented high and low coherence motion stimuli, and asked subjects to judge direction of the motion. Juice or saliva was followed by stimulus direction associated with reward or neutral outcome, respectively. The data at the time of cue presentation showed that activation in the caudate was correlated with reward prediction based on stimulus direction that was influenced by coherence level, whereas reward-predicting activation based on subject’s performance was observed in the putamen. The results suggested that stimulus-based and perception-based reward prediction could be dissociated in the basal ganglia.

The Role of Estrogen Receptor β (ERβ) in the Establishment of Hierarchical Social Relationships in Male Mice

Acquisition of social dominance is important for social species including mice, for preferential access to foods and mates. Male mice establish social rank through agonistic behaviors, which are regulated by gonadal steroid hormone, testosterone, as its original form and aromatized form. It is well known that estrogen receptors (ERs), particularly ER α (ERα), mediate effects of aromatized testosterone, i.e., 17β-estradiol, but precise role played by ER β (ERβ) is still unclear. In the present study, we investigated effects of ERβ gene disruption on social rank establishment in male mice. Adult male ERβ knockout (βERKO) mice and their wild type (WT) littermates were paired based on genotype- and weight-matched manner and tested against each other repeatedly during 7 days experimental period. They underwent 4 trials of social interaction test in neutral cage (homogeneous set test) every other day. Along repeated trials, WT but not βERKO pairs showed a gradual increase of agonistic behaviors including aggression and tail rattling, and a gradual decrease of latency to social rank determination in tube test conducted after each trial of the social interaction test. Analysis of behavioral transition further suggested that WT winners in the tube test showed one-sided aggression during social interaction test suggesting WT pairs went through a process of social rank establishment. On the other hand, a dominant-subordinate relationship in βERKO pairs was not as apparent as that in WT pairs. Moreover, βERKO mice showed lower levels of aggressive behavior than WT mice in social interaction tests. These findings collectively suggest that ERβ may play a significant role in the establishment and maintenance of hierarchical social relationships among male 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.

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

Effects of neonatal maternal separation (MS) on peri-pubertal onset of aggressive behavior in male mice

To evaluate the role of hippocampal zinc in exploratory behavior, the response of extracellular zinc in the hippocampus was examined in novel environments. Rats were placed for 50 min in a novel environment once a day for 8 days. Extracellular glutamate in the hippocampus was increased significantly during exploratory behavior on day 1, whereas extracellular zinc was decreased significantly. Extracellular zinc returned to the basal level during exploratory behavior on day 8. When exploratory behavior was observed during perfusion with 1 mM CaEDTA, a membraneimpermeable zinc chelator, locomotor activity in the novel environment was decreased. The decrease in extracellular zinc and the increase in extracellular glutamate in exploratory period were abolished by perfusion with CaEDTA. These results suggest that zinc uptake by hippocampal cells is linked to exploratory activity and required for the activation of glutamatergic neurotransmitter system.