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.

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.

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.

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.

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.

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.

siRNA silencing of estrogen receptor-α expression specifically in medial preoptic area neurons abolishes maternal care in female mice.

The medial preoptic area has been shown to be intricately involved in many behaviors, including locomotion, sexual behavior, maternal care, and aggression. The gene encoding estrogen receptor-α (ERα) protein is expressed in preoptic area neurons, and a very dense immunoreactive field of ERα is found in the preoptic region. ERα knockout animals show deficits in maternal care and sexual behavior and fail to exhibit increases in these behaviors in response to systemic estradiol treatment. In the present study, we used viral-vector mediated RNA interference to silence ERα expression specifically in the preoptic area of female mice and measured a variety of behaviors, including social and sexual aggression, maternal care, and arousal activity. Suppression of ERα in the preoptic area almost completely abolished maternal care, significantly increasing the latency to pup retrieval and significantly reducing the time the moms spent nursing and licking the pups. Strikingly, maternal aggression toward a male intruder was not different between control and preoptic ERα-silenced mice, demonstrating the remarkably specific role of ERα in these neurons. Reduction of ERα expression in preoptic neurons significantly decreased sexual behavior in female mice and increased aggression toward both sexual partners and male intruders in a seminatural environment. Estrogen-dependent increases in arousal, measured by home cage activity, were not mediated by ERα expression in the preoptic neurons we targeted, as ERα-suppressed mice had increases similar to control mice. Thus, we have established that a specific gene in a specific group of neurons is required for a crucially important natural behavior.

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 17ß-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 17ß-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, 17ß-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.

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.

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.

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.

Distinct behavioral phenotypes in male mice lacking the thyroid hormone receptor α1 or ß isoforms.

Thyroid hormones influence both neuronal development and anxiety via the thyroid hormone receptors (TRs). The TRs are encoded by two different genes, TRα and TRß. The loss of TRα1 is implicated in increased anxiety in males, possibly via a hippocampal increase in GABAergic activity. We compared both social behaviors and two underlying and related non-social behaviors, state anxiety and responses to acoustic and tactile startle in the gonadally intact TRα1 knockout (α1KO) and TRß (ßKO) male mice to their wild-type counterparts. For the first time, we show an opposing effect of the two TR isoforms, TRα1 and TRß, in the regulation of state anxiety, with α1 knockout animals (α1KO) showing higher levels of anxiety and ßKO males showing less anxiety compared to respective wild-type mice. At odds with the increased anxiety in non-social environments, α1KO males also show lower levels of responsiveness to acoustic and tactile startle stimuli. Consistent with the data that T4 is inhibitory to lordosis in female mice, we show subtly increased sex behavior in α1KO male mice. These behaviors support the idea that TRα1 could be inhibitory to ERα driven transcription that ultimately impacts ERα driven behaviors such as lordosis. The behavioral phenotypes point to novel roles for the TRs, particularly in non-social behaviors such as state anxiety and startle.

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.

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.

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.

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.

Estrogen-induced sexual incentive motivation, proceptivity and receptivity depend on a functional estrogen receptor alpha in the ventromedial nucleus of the hypothalamus but not in the amygdala.

The display of copulatory behaviors usually requires the presence of a mate and is, therefore, preceded by a search for and approach to a potential partner. The intensity of approach behaviors is determined by a process labeled sexual incentive motivation. Although it is known that female sexual motivation depends on estrogens, their site of action within the brain is unknown. In the present experiment, we obtained data relevant to this issue. An shRNA encoded within an adeno-associated viral (AAV) vector directed against the estrogen receptor alpha (ERalpha) gene (or containing a nonsense base sequence as a control treatment) was injected bilaterally into the ventromedial nucleus of the hypothalamus (VMN) or the posterodorsal amygdala (MePDA) of female rats. After an 80% reduction of the number of ERalpha in the VMN, sexual incentive motivation was absent after treatment with estradiol and progesterone. Proceptivity and receptivity were also much reduced, while the number of rejections was enhanced. Suppression of the ERalpha in the MePDA lacked these effects. Likewise, the inactive control AAV vector failed to modify any behavior. Thus, the ERalpha in the VMN, but not in the MePDA, is important for proceptivity and receptivity as well as for sexual incentive motivation. These results show that ERalpha in the VMN is crucial for the entire sequence of behavioral events from the processes leading to the establishment of sexual contact until the accomplishment of copulatory 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.

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.

Estrogen receptors in the medial amygdala inhibit the expression of male prosocial behavior.

Studies using estrogen receptor alpha (ERalpha) knock-out mice indicate that ERalpha masculinizes male behavior. Recent studies of ERalpha and male prosocial behavior have shown an inverse relationship between ERalpha expression in regions of the brain that regulate social behavior, including the medial amygdala (MeA), and the expression of male prosocial behavior. These studies have lead to the hypothesis that low levels of ERalpha are necessary to "permit" the expression of high levels of male prosocial behavior. To test this, viral vectors were used to enhance ERalpha in male prairie voles (Microtus ochrogaster), which display high levels of prosocial behavior and low levels of MeA ERalpha. Adult male prairie voles were transfected with ERalpha in the MeA (MeA-ERalpha) or the caudate-putamen (ERalpha control) or luciferase (MeA-site-specific control), and 3 weeks later tested for spontaneous alloparental behavior and partner preference. Enhancing ERalpha in the MeA altered/reduced male prosocial behavior. Only one-third of MeA-ERalpha males, compared with all control males, were alloparental. MeA-ERalpha males also displayed a significant preference for a novel female. This is a critical finding because the manipulations of neuropeptides, oxytocin and vasopressin, can inhibit the formation of a partner preference, but do not lead to the formation of a preference for a novel female. The results support the hypothesis that low levels of ERalpha are necessary for high levels of male prosocial behavior, and provide the first direct evidence that site-specific ERalpha expression plays a critical role in the expression of male prosocial behavior.