Estrous cycle-dependent modulation of sexual receptivity in female mice by estrogen receptor beta-expressing cells in the dorsal raphe nucleus.

The sexual receptivity of female mice, shown as lordosis response, is mainly regulated by estradiol action on estrogen receptor alpha (ERα) and beta (ERß), depending on the day of the estrous cycle. Previous studies revealed that ERα in the ventromedial nucleus of the hypothalamus (VMH) plays an essential role in the induction of lordosis on the day of estrus (Day 1). However, the mechanisms of the transition to non-receptive states on the day after estrus (Day 2) are not completely understood. In the present study, we investigated the possible role of ERß, which is highly expressed in the dorsal raphe nucleus (DRN), in lordosis expression. We found that ERß-Cre female mice, which were ovariectomized and primed with estradiol and progesterone to mimic the estrous cycle, showed high levels of lordosis on Day 2 when ERß-expressing DRN (DRN-ERß+) neuronal activity was chemogenetically suppressed. This finding suggests that excitation of DRN-ERß+ neurons is necessary for the decline of lordosis on Day 2. Fiber photometry recordings during female-male behavioral interactions revealed that DRN-ERß+ neuronal activation in response to male intromission was significantly more prolonged on Day 2 compared to Day 1. Chemogenetic over-stimulation of DRN-ERß+ neurons induced c-Fos expression in brain areas known to be inhibitory for lordosis expression, even though they did not express anterogradely labeled fibers of DRN-ERß+ cells. These findings collectively suggest that DRN-ERß+ neuronal excitation serves as an inhibitory modulator and is responsible for the decline in receptivity during non-estrus phases.Significance Statement In females, switching from a sexually receptive state to a non-receptive phase during the estrous cycle is essential for effective behavioral interactions with males and successful reproduction. Here, we delineate the possible brain regions that regulate the decline in receptive behavioral responses, such as lordosis, from those shown on the day of estrus to those on the day after estrus. We found that excitation of neurons expressing estrogen receptor (ER) ß in the midbrain dorsal raphe nucleus is crucial for the suppression of lordosis during the day after estrus. This is contrasted with the facilitatory action of ERα, another type of ER, and provides new insights for understanding the neural basis of the adaptive expression of female reproductive behavior.

Estrogen Receptor ß in the Lateral Septum Mediates Estrogen Regulation of Social Anxiety-like Behavior in Male Mice.

17ß-estradiol (E2) regulates various forms of social behavior through the activation of two types of estrogen receptors, ERα and ERß. The lateral septum (LS) is thought to be one of the potential target sites of E2, but the role played by ERα and ERß in this brain area remains largely unknown. In the present study, we first analyzed the distribution of ERα and ERß with double fluorescent immunohistochemistry in a transgenic mouse line in which red fluorescent protein (RFP) signal has been a reliable marker of ERß expression. The overall number of ERß-RFP-expressing cells was significantly higher (about 2.5 times) compared to ERα-expressing cells. The distribution of the two types of ERs was different, with co-expression only seen in about 1.2% of total ER-positive cells. Given these distinctive distribution patterns, we examined the behavioral effects of site-specific knockdown of each ER using viral vector-mediated small interference RNA (siRNA) techniques in male mice. We found ERß-specific behavioral alterations during a social interaction test, suggesting involvement of ERß-expressing LS neurons in the regulation of social anxiety and social interest. Further, we investigated the neuronal projections of ERα- and ERß-expressing LS cells by injecting an anterograde viral tracer in ERα-Cre and ERß-iCre mice. Dense expression of green fluorescence protein (GFP) in synaptic terminals was observed in ERß-iCre mice in areas known to be related to the modulation of anxiety. These findings collectively suggest that ERß expressed in the LS plays a major role in the estrogenic control of social anxiety-like behavior.

Activity of estrogen receptor ß expressing neurons in the medial amygdala regulates preference toward receptive females in male mice.

The processing of information regarding the sex and reproductive state of conspecific individuals is critical for successful reproduction and survival in males. Generally, male mice exhibit a preference toward the odor of sexually receptive (RF) over nonreceptive females (XF) or gonadally intact males (IM). Previous studies suggested the involvement of estrogen receptor beta (ERß) expressed in the medial amygdala (MeA) in male preference toward RF. To further delineate the role played by ERß in the MeA in the neuronal network regulating male preference, we developed a new ERß-iCre mouse line using the CRISPR-Cas9 system. Fiber photometry Ca2+ imaging revealed that ERß-expressing neurons in the postero-dorsal part of the MeA (MeApd-ERß+ neurons) were more active during social investigation toward RF compared to copresented XF or IM mice in a preference test. Chemogenetic inhibition of MeApd-ERß+ neuronal activity abolished a preference to RF in "RF vs. XF," but not "RF vs. IM," tests. Analysis with cre-dependent retrograde tracing viral vectors identified the principal part of the bed nucleus of stria terminalis (BNSTp) as a primary projection site of MeApd-ERß+ neurons. Fiber photometry recording in the BNSTp during a preference test revealed that chemogenetic inhibition of MeApd-ERß+ neurons abolished differential neuronal activity of BNSTp cells as well as a preference to RF against XF but not against IM mice. Collectively, these findings demonstrate for the first time that MeApd-ERß+ neuronal activity is required for expression of receptivity-based preference (i.e., RF vs. XF) but not sex-based preference (i.e., RF vs. IM) in male mice.

Steroid hormone-mediated regulation of sexual and aggressive behaviour by non-genomic signalling.

Sex and aggression are well studied examples of social behaviours that are common to most animals and are mediated by an evolutionary conserved group of interconnected nuclei in the brain called the social behaviour network. Though glucocorticoids and in particular estrogen regulate these social behaviours, their effects in the brain are generally thought to be mediated by genomic signalling, a slow transcriptional regulation mediated by nuclear hormone receptors. In the last decade or so, there has been renewed interest in understanding the physiological significance of rapid, non-genomic signalling mediated by steroids. Though the identity of the membrane hormone receptors that mediate this signalling is not clearly understood and appears to be different in different cell types, such signalling contributes to physiologically relevant behaviours such as sex and aggression. In this short review, we summarise the evidence for this phenomenon in the rodent, by focusing on estrogen and to some extent, glucocorticoid signalling. The use of these signals, in relation to genomic signalling is manifold and ranges from potentiation of transcription to the possible transduction of environmental signals.

Sex and age differences in the distribution of estrogen receptors in mice.

Estrogen receptors (ERα and ERß) are crucial for the regulation of socio-sexual behaviors and the organization of sex-specific neural networks in the developing brain. However, how the distribution patterns of ERα and ERß change throughout life is unclear. Using genetically modified ERß-RFPtg mice, we investigated the distribution of ERα, ERß, and their colocalization in the ventromedial nucleus of the hypothalamus (VMH), anteroventral periventricular nucleus (AVPV), and bed nucleus of stria terminalis (BNST) from postnatal days (PD) 0 to 56. ERα expression was higher in females that showed an increase after PD14 in all brain regions, whereas ERß-RFP expression pattern was markedly different among the regions. In the VMH, ERß-RFP was highly expressed on PD0 and PD7 but decreased drastically to very low expression afterward in both sexes. In contrast, ERß-RFP expression was higher in females compared to males in the AVPV but lower in the BNST throughout life especially late- and post-pubertal periods. Our results demonstrating that ERα and ERß-RFP expression changed in a sex-, age- and region-specific manner contribute to further clarification of the mechanisms underlying estrogen-dependent organization of the brain in both sexes.

Lateral habenula glutamatergic neurons projecting to the dorsal raphe nucleus promote aggressive arousal in mice.

The dorsal raphe nucleus (DRN) is known to control aggressive behavior in mice. Here, we found that glutamatergic projections from the lateral habenula (LHb) to the DRN were activated in male mice that experienced pre-exposure to a rival male mouse ("social instigation") resulting in heightened intermale aggression. Both chemogenetic and optogenetic suppression of the LHb-DRN projection blocked heightened aggression after social instigation in male mice. In contrast, inhibition of this pathway did not affect basal levels of aggressive behavior, suggesting that the activity of the LHb-DRN projection is not necessary for the expression of species-typical aggressive behavior, but required for the increase of aggressive behavior resulting from social instigation. Anatomical analysis showed that LHb neurons synapse on non-serotonergic DRN neurons that project to the ventral tegmental area (VTA), and optogenetic activation of the DRN-VTA projection increased aggressive behaviors. Our results demonstrate that the LHb glutamatergic inputs to the DRN promote aggressive arousal induced by social instigation, which contributes to aggressive behavior by activating VTA-projecting non-serotonergic DRN neurons as one of its potential targets.

Neuromodulatory effect of interleukin 1ß in the dorsal raphe nucleus on individual differences in aggression.

Heightened aggressive behavior is considered as one of the central symptoms of many neuropsychiatric disorders including autism, schizophrenia, and dementia. The consequences of aggression pose a heavy burden on patients and their families and clinicians. Unfortunately, we have limited treatment options for aggression and lack mechanistic insight into the causes of aggression needed to inform new efforts in drug discovery and development. Levels of proinflammatory cytokines in the periphery or cerebrospinal fluid were previously reported to correlate with aggressive traits in humans. However, it is still unknown whether cytokines affect brain circuits to modulate aggression. Here, we examined the functional role of interleukin 1ß (IL-1ß) in mediating individual differences in aggression using a resident-intruder mouse model. We found that nonaggressive mice exhibit higher levels of IL-1ß in the dorsal raphe nucleus (DRN), the major source of forebrain serotonin (5-HT), compared to aggressive mice. We then examined the effect of pharmacological antagonism and viral-mediated gene knockdown of the receptors for IL-1 within the DRN and found that both treatments consistently increased aggressive behavior of male mice. Aggressive mice also exhibited higher c-Fos expression in 5-HT neurons in the DRN compared to nonaggressive mice. In line with these findings, deletion of IL-1 receptor in the DRN enhanced c-Fos expression in 5-HT neurons during aggressive encounters, suggesting that modulation of 5-HT neuronal activity by IL-1ß signaling in the DRN controls expression of aggressive behavior.

Limb-clasping, cognitive deficit and increased vulnerability to kainic acid-induced seizures in neuronal glycosylphosphatidylinositol deficiency mouse models.

Posttranslational modification of a protein with glycosylphosphatidylinositol (GPI) is a conserved mechanism exists in all eukaryotes. Thus far, >150 human GPI-anchored proteins have been discovered and ~30 enzymes have been reported to be involved in the biosynthesis and maturation of mammalian GPI. Phosphatidylinositol glycan biosynthesis class A protein (PIGA) catalyzes the very first step of GPI anchor biosynthesis. Patients carrying a mutation of the PIGA gene usually suffer from inherited glycosylphosphatidylinositol deficiency (IGD) with intractable epilepsy and intellectual developmental disorder. We generated three mouse models with PIGA deficits specifically in telencephalon excitatory neurons (Ex-M-cko), inhibitory neurons (In-M-cko) or thalamic neurons (Th-H-cko), respectively. Both Ex-M-cko and In-M-cko mice showed impaired long-term fear memory and were more susceptible to kainic acid-induced seizures. In addition, In-M-cko demonstrated a severe limb-clasping phenotype. Hippocampal synapse changes were observed in Ex-M-cko mice. Our Piga conditional knockout mouse models provide powerful tools to understand the cell-type specific mechanisms underlying inherited GPI deficiency and to test different therapeutic modalities.

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.

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.

Estrogen receptors α and ß in the central amygdala and the ventromedial nucleus of the hypothalamus: Sociosexual behaviors, fear and arousal in female rats during emotionally challenging events.

Estrogens receptors (ER) are involved in several sociosexual behaviors and fear responses. In particular, the ERα is important for sexual behaviors, whereas ERß modulates anxiolytic responses. Using shRNA directed either against the ERα or the ERß RNAs (or containing luciferase control) encoded within an adeno-associated viral vector, we silenced these receptors in the ventromedial nucleus of the hypothalamus (VMN) and the central amygdala (CeA). We exposed ovariectomized female rats, sequentially treated with estradiol benzoate and progesterone, to five stimuli, previously reported to elicit positive and negative affect. The subjects were housed in groups of 4 females and 3 males in a seminatural environment for several days before hormone treatment. We analyzed the frequency of a large number of behavior patterns. In addition, we performed analyses of co-occurrence in order to detect changes in the structure of behavior after infusion of the vectors. Silencing the ERα in the VMN disrupted lordosis and showed some anxiolytic properties in aversive situations, whereas silencing of the ERß in this structure had no effect. This was also the case after silencing the ERα in the CeA. Silencing of the ERß in this structure increased risk assessment, an expression of anxiety, and increased olfactory exploration of the environment. We hypothesize that the ERß in the CeA has an important role in the well-established anxiolytic effects of estrogens, and that it may modulate arousal level. Furthermore, it seems that the ERα in the VMN is anxiogenic in aversive or threatening situations, in agreement with other studies.

Hippocampal functional organization: A microstructure of the place cell network encoding space.

A clue to hippocampal function has been the discovery of place cells, leading to the 'spatial map' theory. Although the firing attributes of place cells are well documented, little is known about the organization of the spatial map. Unit recording studies, thus far, have reported a low coherence between neighboring cells and geometric space, leading to the prevalent view that the spatial map is not topographically organized. However, the number of simultaneously recorded units is severely limited, rendering construction of the spatial map nearly impossible. To visualize the functional organization of place cells, we used the activity-dependent immediate-early gene Zif268 in combination with behavioral, pharmacological and electrophysiological methods, in mice and rats exploring an environment. Here, we show that in animals confined to a small part of a maze, principal cells in the CA1/CA3 subfields of the dorsal hippocampus immunoreactive (IR) for Zif268 adhere to a 'cluster-type' organization. Unit recordings confirmed that the Zif268 IR clusters correspond to active place cells, while blockade of NMDAR (which alters place fields) disrupted the Zif268 IR clusters. Contrary to the prevalent view that the spatial map consists of a non-topographic neural network, our results provide evidence for a 'cluster-type' functional organization of hippocampal neurons encoding for space.

The Role of Estrogen Receptor ß (ERß) 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., 17ß-estradiol, but precise role played by ER ß (ERß) is still unclear. In the present study, we investigated effects of ERß gene disruption on social rank establishment in male mice. Adult male ERß knockout (ßERKO) 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 ßERKO 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 ßERKO pairs was not as apparent as that in WT pairs. Moreover, ßERKO mice showed lower levels of aggressive behavior than WT mice in social interaction tests. These findings collectively suggest that ERß may play a significant role in the establishment and maintenance of hierarchical social relationships among male mice.

VGF in the Medial Preoptic Nucleus Increases Sexual Activity Following Sexual Arousal Induction in Male Rats.

The central part of the medial preoptic nucleus (MPNc) is associated with sexual arousal induction in male rats. However, it is largely unclear how males are sexually aroused and achieve their first copulation. We previously reported that more MPNc neurons activate during the first copulation than the second copulation. In this study, to explore the molecules responsible for sexual arousal induction, we performed DNA microarray of the MPNc in sexually naive males and males after they copulated for their first and second times. We then performed quantitative PCR analyses to validate the results of the DNA microarray. Six genes were identified. Their expression increased following copulation and was higher in males after they copulated for the first time than after the second time. The genes encode transcription factors (Fos, Nfil3, and Nr4a3), a serine/threonine kinase (Sik1), an antioxidant protein (Srxn1), and a neuropeptide precursor VGF (Vgf), which may be the candidate genes responsible for sexual arousal induction. We examined the effects of Vgf knockdown in the MPNc on sexual partner preference and sexual behavior in sexually inexperienced and experienced males to determine the role of VGF in sexual arousal induction. A preference for estrous female rats was reinforced, and the latency of mount and intromission became short after sexually inexperienced males copulated for the first time. However, Vgf knockdown disrupted these phenomena. Vgf knockdown did not have any significant effect in sexually experienced males. VGF-derived neuropeptides presumably serve as an effector molecule to increase sexual activity following sexual arousal induction.

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

17ß-Estradiol (E2) regulates the expression of female sexual behavior by acting through estrogen receptor (ER) α and ß. Previously, we have shown that ERß 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 ERß may be involved in inhibitory regulation of lordosis. However, it is not identified yet in which brain region(s) ERß may mediate an inhibitory action of E2. In this study, we have focused on the dorsal raphe nucleus (DRN) that expresses ERß in higher density than ERα. We site specifically knocked down ERß 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 ERß knockdown (ßERKD) mice was not different from that of control mice on day 1. However, ßERKD 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 ERß in the DRN may be involved in the inhibitory regulation of sexual behavior on the day after behavioral estrus in cycling female mice.

Estrogen and oxytocin involvement in social preference in male mice: a study using a novel long-term social preference paradigm with aromatase, estrogen receptor-α and estrogen receptor-ß, oxytocin, and oxytocin receptor knockout male mice.

Certain aspects of social behavior help animals make adaptive decisions during encounters with other animals. When mice choose to approach another conspecific, the motivation and preference behind the interaction is not well understood. Estrogen and oxytocin are known to influence a wide array of social behaviors, including social motivation and social preference. The present study investigated the effects of estrogen and oxytocin on social preference using aromatase (ArKO), estrogen receptor (ER) α (αERKO), ERß (ßERKO), oxytocin (OTKO), oxytocin receptor (OTRKO) knockout and their respective wild-type (WT) male mice. Mice were presented with gonadally-intact versus castrated male (IC), intact male versus ovariectomized female (IF), or intact male versus empty cage (IE) stimuli sets for 5 days. ArWT showed no preference for either stimuli in IC and IF and intact male preference in IE, but ArKO mice preferred a castrated male or an ovariectomized female, or had no preference for either stimulus in IC, IF and IE stimuli sets, respectively, suggesting reduced intact male preference. α and ß WT mice preferred a castrated male, showed no preference, and preferred an intact male in IC, IF and IE, respectively. αERKO mice displayed similar modified social preference patterns as ArKO, whereas the social preference of ßERKO mice remained similar to ßWT. OTWT preferred a castrated male whereas OTKO, OTRWT and OTRKO mice failed to show any preference in IC and none showed preference for either stimuli in IF. Collectively, these findings suggest that estrogen regulates social preference in male mice and that impaired social preference in oxytocin-deficient mice may be due to severe deficits in social recognition.

Fear memory consolidation in sleep requires protein kinase A.

It is well established that protein kinase A (PKA) is involved in hippocampal dependent memory consolidation. Sleep is also known to play an important role in this process. However, whether sleep-dependent memory consolidation involves PKA activation has not been clearly determined. Using behavioral observation, animals were categorized into sleep and awake groups. We show that intrahippocampal injections of the PKA inhibitor Rp-cAMPs in post-contextual fear conditioning sleep produced a suppression of long-term fear memory, while injections of Rp-cAMPs during an awake state, at a similar time point, had no effect. In contrast, injections of the PKA activator Sp-cAMPs in awake state, rescued sleep deprivation-induced memory impairments. These results suggest that following learning, PKA activation specifically in sleep is required for the consolidation of long-term memory.

A Sexually Dimorphic Area of the Dorsal Hypothalamus in Mice and Common Marmosets.

We found a novel sexually dimorphic area (SDA) in the dorsal hypothalamus (DH) of mice. The SDA-DH was sandwiched between 2 known male-biased sexually dimorphic nuclei, the principal nucleus of the bed nucleus of the stria terminalis and the calbindin-sexually dimorphic nucleus, and exhibited a female-biased sex difference in neuronal cell density. The density of neurons in the SDA-DH was increased in male mice by orchidectomy on the day of birth and decreased in female mice by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. These findings indicate that the SDA-DH is defeminized under the influence of testicular testosterone, which acts via both directly by binding to the androgen receptor, and indirectly by binding to the estrogen receptor after aromatization. We measured the activity of SDA-DH neurons with c-Fos, a neuronal activity marker, in female mice during maternal and sexual behaviors. The number of c-Fos-expressing neurons in the SDA-DH of female mice was negatively correlated with maternal behavior performance. However, the number of c-Fos-expressing neurons did not change during female sexual behavior. These findings suggest that the SDA-DH contains a neuronal cell population, the activity of which decreases in females exhibiting higher performance of maternal behavior, but it may contribute less to female sexual behavior. Additionally, we examined the brain of common marmosets and found an area that appears to be homologous with the mouse SDA-DH. The sexually dimorphic structure identified in this study is not specific to mice and may be found in other species.

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.